method for degrading or hydrolyzing a polysaccharide, for producing soluble saccharides and for prod

专利摘要:
METHOD FOR DEGRADING OR HYDROLYZING A POLYSACARIDE, TO PRODUCE SOLUBLE SACARIDES, TO PRODUCE AN ORGANIC SUBSTANCE, TO PRODUCE A FERMENTATION PRODUCT, AND, USE OF AN HYDROLYTIC OXIDE ENZYME. The invention provides a method of degrading or hydrolyzing a polysaccharide, preferably cellulose or chitin, comprising bringing said polysaccharide into contact with one or more oxide-hydrolytic enzymes, preferably a protein of the CBM33 family (preferably CBP21) or a protein of the GH61 family, wherein said degradation or hydrolysis is carried out in the presence of at least one reducing agent and at least one divalent metal ion. A method for producing an organic substance comprising said method is also provided.
公开号:BR112013002377B1
申请号:R112013002377-5
申请日:2011-08-05
公开日:2020-12-29
发明作者:Gustav Vaaje-Kolstad；Bjorge Westereng；Vicente G. H. Eijsink；Svein J. Horn；Morten Sorliie；Zarah Forsberg
申请人:Novozymes A/S；Novozymes Inc.；
IPC主号:

专利说明:

Reference to a string listing
[001] This application contains a sequence listing in computer readable form, which is incorporated by reference here. Fundamentals of the Invention Field of the Invention
[002] The present invention relates to methods of degrading or hydrolyzing a polysaccharide, such as chitin or cellulose, comprising bringing said polysaccharide into contact with an oxide-hydrolytic enzyme, such as a CBP21 or GH61 protein, wherein said degradation or hydrolysis is carried out in the presence of at least one reducing agent and at least one divalent metal ion. The invention also concerns the use of additional saccharolytic enzymes, such as hydrolases and beta-glucosidases, to increase the level or extent of degradation, and the fermentation of the resulting sugars to generate an organic substance such as an alcohol, preferably ethanol, which can be used as a biofuel. Description of the Related Art
[003] Efficient enzymatic conversion of crystalline polysaccharides is important for an economically and environmentally sustainable bioeconomy, but remains unfavorably inefficient.
[004] The transition to a greener economy stimulated research with enzymes capable of efficiently degrading recalcitrant carbohydrates, such as cellulose and chitin (Fig. 1A), for the production of biofuels (Himmel et al., 2007, Science 315: 804). Cellulose is the most abundant organic molecule on Earth and offers a renewable and apparently inexhaustible raw material for the production of fuels and chemicals. Chitin is a common constituent of fungal cell walls, crustacean shells and insect exoskeletons. It is the second most abundant polymer in nature and each year more than one billion tons of chitin are produced in the biosphere, mainly by insects, fungi, crustaceans and other marine organisms. Chitin is abundantly available as a by-product of aquaculture, one of the fastest growing bioproduction industries on Earth.
[005] The conversion of cellulosic raw materials into ethanol has the advantages of the immediate availability of large quantities of raw material, the need to avoid burning or landfilling the materials and the purity of ethanol fuel. Wood, agricultural residues, herbaceous crops and municipal solid residues are considered as raw materials for the production of ethanol. These materials mainly consist of cellulose, hemicellulose and the polysaccharide not associated with lignin. Once cellulose is converted to glucose, glucose is easily fermented by yeast in ethanol.
[006] A variety of microorganisms exist to ferment polysaccharide hydrolysis products, to yield desirable end products such as alcohol. The selection of appropriate microorganisms allows the hydrolysis products of cellulose, chitin and other polysaccharides to be fermented to yield the products used, such as alcohol.
[007] The predominant polysaccharide in the primary cell wall of the biomass is cellulose, the second most abundant is hemicellulose and the third is pectin. The secondary cell wall, produced after the cells stop growing, also contains polysaccharides and is reinforced by polymeric lignin cross-linked with hemicellulose. Cellulose is an anhydrocelobiosis homopolymer and thus a linear beta- (1-4) -D-glucan, although hemicelluloses include a variety of compounds, such as xylans, xyloglucans, arabinoxylans and mannans in complex structures branched with a spectrum substituents. Although generally polymorphic, cellulose is found in plant tissue, primarily as an insoluble crystalline matrix of parallel glycan chains. Hemicelluloses in general have hydrogen bound to cellulose, as well as other hemicelluloses, which helps to stabilize the cell wall matrix.
[008] Bacteria and fungi are involved in complex enzymatic systems that enable their growth in cellulose-rich plant material, but these organisms typically require weeks, months or even years to decompose a fallen trunk or a cultivated corn stalk. Likewise, microorganisms contain enzyme systems to degrade chitin. Bacterial chitinase helps to provide a carbon source for bacterial growth. Insects produce chitinase to digest their cuticle at each seedling. In plants, chitinase is known to provide a protective role against parasitic fungi. For the production of chemicals or fuel, from these same materials containing cellulose and chitin, the industry requires chemical products or enzyme systems at an affordable price, which can carry out the work in hours or days.
[009] In a traditional way, enzyme systems capable of degrading such carbohydrates are considered to consist of two types of hydrolytic enzymes called glycoside hydrolases: endo-acting enzymes that randomly cut into the carbohydrate chain and exo-processing enzymes (chito or cellobiohydrolases), which degrade the polymers at the ends of the chain (Figure 1B). Although this model is generally accepted, it remains difficult to understand how, for example, an endoglucanase may be able to drag a simple polysaccharide chain from its crystalline environment and force the chain productively into the fissure of its active site (Figure 1B).
[0010] After cellulases gained the interest of biochemists, speculation occurred about the possible existence of a factor that interrupts the substrate, which can make the crystalline substrate more accessible to the hydrolytic enzyme (Reese et al., 1950, J Bacteriol. 59 : 485). Recently, it was discovered that microorganisms that break down chitin actually produce a protein that increases substrate accessibility and potentiates hydrolytic enzymes (Vaaje-Kolstad et al., 2005, J. Biol. Chem. 280: 28492; Figs. 1C and D). The first example was a single domain protein called CBP21 (CBP for chitin-binding protein) produced by the chitinolytic bacteria Serratia marcescens. This protein was classified as a carbohydrate binding module (CBM) and belongs to the CBM33 family, as defined by the CAZy nomenclature (Boraston et al., 2004, Biochem. J. 382: 769, Henrissat, 1991, Biochem. J. 280 (Pt 2): 309). Another example concerns two proteins containing CBM33 from Thermobifida fusca, called E7 and E8, which enhance the hydrolysis of chitin by chitinase and the hydrolysis of cellulose by cellulases (Moser et al., 2008, Biotechnol. Bioeng. 100 (6) : 1066-77). Like CBP21, E7 is a single domain protein that comprises only one CBM33 domain. E8 is a three-domain protein, which means that it carries two domains in addition to a CBM33 domain.
[0011] It has recently been observed that proteins currently classified as glycoside hydrolases of the family 61 (GH61) in the CAZy nomenclature act synergistically with cellulases (Harris et al., 2010, Biochemistry 49: 3305), and are structurally similar to CBM33 proteins ( Harris et al., 2010, supra; Karkehabadi et al., 2008, J. Mol. Biol. 383: 144; Figure 1E). While CBM33 and GH61 show little sequence similarity, structural similarity is evident (Figures 1D and 1E), including a completely conserved diagnostic arrangement of the N-terminal amino group, an N-terminal histidine and another histidine residue (Figure 1F ; Karkehabadi et al., 2008, supra) that forms a promiscuous metal binding site (see below). Based on data from the available literature, including a recent detailed study of several GH61 proteins, it seems very unlikely that GH61 proteins are endoglucanases, in the way originally known (Harris et al., 2010, supra). Like CBM33 proteins, GH61 proteins do not have a slit or pocket connecting to the substrate, nor do they have a characteristic arrangement of acidic amino acids that may indicate glycoside hydrolase activity. In contrast, both types of proteins exhibit an almost flat substrate-binding surface (Figures 1D and 1E; Harris et al., 2010, supra; Vaaje-Kolstad et al., 2005, J. Biol. Chem. 280: 11313 ). In general, these results and observations show that CBM33 and GH61 proteins have similar functions, that is, enhancing the efficiency of known hydrolytic enzymes (glycoside hydrolases) that act on crystalline polysaccharides. Furthermore, these results and observations strongly suggest that these proteins thus employ the same type of mechanism. So far, this mechanism remains undefined.
[0012] The present invention provides methods of degrading or hydrolyzing a polysaccharide, such as chitin or cellulose, comprising bringing said polysaccharide into contact with an oxide-hydrolytic enzyme in the presence of at least one reducing agent and at least one divalent metal ion. Summary of the Invention
[0013] The present invention relates to methods of degrading or hydrolyzing a polysaccharide, comprising putting said polysaccharide in contact with one or more oxidohydrolytic enzymes, wherein said degradation or hydrolysis is carried out in the presence of at least one reducing agent and at least one divalent metal ion.
[0014] The present invention also relates to methods of producing soluble saccharides, wherein said method comprises degrading or hydrolyzing a polysaccharide by the method defined above, wherein said degradation or hydrolysis releases said soluble saccharides, and optionally isolates said soluble saccharides.
The present invention also relates to methods of producing an organic substance comprising the steps of: (i) degrading or hydrolyzing a polysaccharide by the method defined above to produce a solution comprising soluble saccharides; (ii) fermenting said soluble saccharides to produce said organic substance as the fermentation product and, optionally, (iii) recovering said organic substance.
[0016] The present invention also relates to a process of producing a fermentation product comprising: (a) saccharifying a cellulosic material with an enzymatic composition comprising an endoglucanase, a cellobiohydrolase, a beta-glucosidase, a GH61 polypeptide with better cellulolytic activity , and a CBM33; (b) fermenting the saccharified cellulosic material with one or more fermenting microorganisms to synthesize the fermentation product; and (c) recovering the fermentation product from the fermentation. Brief Description of the Figures
[0017] Figure 1 shows (a) repeated units of disaccharide in cellulose and chitin. (B) A schematic representation of the degradation of chitin or cellulose by glycoside hydrolases with endo action (upper part) and (processing) exo action (lower part). (C) The effect of CBP21 on chitinase efficiency; chitinase is chitinase C (ChiC), an endoquitinase of the 18 family of S. marcescens (data from Vaaje-Kolstad et al., 2005, J. Biol. Chem. 280: 28492). (D) The crystal structure of CBP21 (Vaaje-Kolstad et al., 2005, J. Biol. Chem. 280: 11313). The side chains of the conserved histidine residues on the flat binding surface are shown in stick representation. (E) The crystal structure of a GH61E of Thielavia terrestris (Harris et al., 2010, Biochemistry 49: 3305). The side chains of the conserved histidine residues on the binding surface are shown in stick representation. (F) A detailed view of the conserved arrangement of the two histidines and the N-terminal amino group in CBP21 (dark gray) and GH61E (light gray), overlaid using PyMol. Figures D, E and F were generated using PyMol.
[0018] Figure 2 shows (a) A MALDI-TOF MS spectrum that exhibits oxidized soluble chito-oligosaccharides generated by CBP21 that acts on beta-chitin nanocrystals. This represents the first time that such oxidized products have been detected. (B) A chitin chain with a GlcNAcA unit. (C) 2.0 mg / mL beta-chitin treated with 1.0 μM CBP21 in the presence (bottle on the left) or absence (bottle on the right) of 1.0 mM ascorbic acid. After incubation with or without ascorbic acid, the tubes were treated in an identical manner: they were shaken and then left for one minute for the chitin material to settle / sediment. The figure shows that the broken material in the left flask settles much more slowly. This figure shows the great potential of CBP21 to break down crystalline chitin when applied under ideal conditions, that is, in the presence of a reducing agent and a divalent metal ion. (D) A MALDI-TOF MS spectrum of products obtained after incubation of beta-chitin (2.0 mg / mL) with 1.0 μM AnCDA (an overexpressed and purified chitin deacetylase from Aspergillus nidulans), CBP21 1.0 μM and 1.0 mM ascorbic acid for 16 hours, showing deacetylated oxidized chito-oligosaccharides (all major products contain two acetylated sugars). A control reaction without CBP21 did not yield soluble products (results not shown). (E) A MALDI-TOF MS spectrum of products obtained after incubating beta-chitin (2.0 mg / mL) with 1.0 μM AnCDA and 0.5 μM ChiC for 8 hours, showing deacetylated chitosoligosaccharides (all major products contain an acetylated sugar). The peaks of MS are marked by the observed atomic mass and the degree of polymerization (DP) of the oligosaccharide. The marks often refer to the peak of greatest intensity in the respective group (which comprises several adducts; see figure 3). "Ox" indicates the presence of a GlcNAcA at the end of the chain opposite the non-reducing end. Asterisks indicate a deacetylated product. 100% relative intensity represents 2.5x104, 1.1x103 and 5.4x104 of arbitrary units (a.u.), in panels A, D and E, respectively. See figure 3 for additional experiments with product verification.
[0019] Figure 3 shows product identity controls by MALDI-TOF. (a) products containing CBP21-degraded beta-chitin GlcNAcA are in equilibrium with 1.5 δ-lactone, and at low pH, this balance can be made observable (Pocker & Green, 1973, J. Am. Chem. Soc 95: 113). A sample with hexameric products was adjusted to pH ~ 3.0 with acetic acid, and incubated for four hours at room temperature. The sample was then analyzed by MALDI-TOF MS. The spectrum clearly shows both the acid form and the 1.5 δ-lactone, which differ by 18 units of atomic mass (amu), representing a water molecule. The balance between the two forms will eventually lead to the exchange of both oxygen added during the reaction with oxygen from the water solvent. In fact, when the samples of the product obtained after carrying out the reaction in H218O (see examples) were adjusted to low pH, it was observed not only a lactone and an acid that exhibits a mass increase of 2 amu compared to the reactions carried out in H216O , but also, after some time, an acid that shows a mass increase of 4 amu (results not shown). (B) Details of the mass spectrum for DP6ox. The fact that the difference in mass between Na + and K + corresponds to the mass of an oxygen atom can complicate the interpretation. However, similar ratios between simple sodium and potassium adducts, and their corresponding sodium / potassium salt adducts (often observed for acid sugars), indicate that the product is in fact an oxidized hexamer. A second verification of the identity of the products observed by MALDI-TOF MS was carried out by adding LiCl, in a final concentration of 33 mM, to the sample before the MALDI-TOF MS analysis to generate new diagnostic adducts. The products that correspond to the Li adduct of the acid, and the Li adduct of the Li salt of the acid, were observed (C), thus confirming the identity of Na and K adducts observed in (B). The peaks in all MALDI-TOF MS spectra are marked according to their observed atomic mass and the degree of polymerization (DP) of the oligosaccharide. "Ox" symbolizes oxidized. (D) PSD spectra overlaid with the hexamer fraction obtained by treatment with beta-chitin CBP21 in H216O (black) and H218O (gray). The inserts provide detailed views of parts of the spectrum. The data show that Y ions differ by 2 amu, while B ions show identical masses regardless of the type of water used. The data are completely compatible with the chemical structure shown above, the mass spectrum, and show that the heavy oxygen atom is introduced into the oxidized reducing end. Note that the chemical structure shown has been simplified for clarification: many hydroxyl groups, as well as C6, are absent. 100% of the relative intensity represents 4.6x104, 1.1x104, 0.8x103 and 1.8x104 a.u. in panels A, B, C and D, respectively.
[0020] Figure 4 shows the effect of ascorbic acid (a reducer) on the potentiation effect of CBP21 on chitinase efficiency. The efficiency of beta-chitin degradation (0.1 mg / mL) by ChiC 0.5 μM was analyzed in the presence (upper gray line in filled triangles) or absence (lower gray line in filled diamonds) of 1.0 µM CBP21 and 1.0 mM ascorbic acid at pH 8.0. A parallel reaction containing beta-chitin, ChiC and CBP21 (the same concentrations as above), but no ascorbic acid was run as a control (central dark gray line in filled squares). The results show that CBP21 promotes chitin degradation by ChiC considerably better in the presence of ascorbic acid (all chitin is degraded after 2.5 hours), compared to conditions where ascorbic acid is absent. A parallel reaction containing beta-chitin and ChiC (the same concentrations above), in the absence of ascorbic acid, can yield a result similar to that represented by the gray line in filled diamonds (results not shown, but the experiment is included and shown in figure 10B ), meaning that ascorbic acid alone had no effect on ChiC efficiency.
[0021] Figure 5 shows the analysis of soluble reaction products from the CBP21 catalysis. (a) After 4 days of incubation of 2 mg / mL of beta-chitin with CBP21 1.0 μM and 1.0 mM ascorbic acid at pH 8.0, the soluble products are dominated by short, even-numbered oligosaccharides, with ends acid reducing agents. Due to the low solubility of chitin oligosaccharides, larger oligosaccharides are not observed (see examples for the detection of larger products). (B) Each acid oligosaccharide species was observed as H, Na and K adducts, and Na and K adducts of the acid oligosaccharide Na salt. 100% of the relative intensity represents 3.5x103 a.u.
[0022] Figure 6 shows the UHPLC separation of oxidized oligosaccharides with acidic (GlcNAcA) ends. A semiquantitative analysis of soluble products generated by CBP21 was performed by separating the oxidized oligosaccharides by UHPLC. (a) Spectrum of the typical product that shows the frequency of products with even and odd numbering, which was also observed in the MALDI-TOF MS analysis (for example, figures 2D and 5). Note that the alpha and beta anomers of unoxidized oligosaccharides can be separated under these chromatographic conditions. The fact that only single peaks are observed confirms the modification of the reducing end. Peak identities were determined using MALDI-TOF MS (results not shown). (B) Speeds of the initial reaction of CBP21 mediated by solubilization of chitin in hexameric products (complete lines), or pentameric products (dotted lines), in the presence of ascorbic acid 5,0 (gray lines, circular data points), 1, 0 (dark gray lines, square data points) and 0.2 mM (black lines, triangular data points). These results show that the reaction speed depends on the concentration of ascorbic acid, and that odd-numbered products are generated at a slower rate than even-numbered products.
[0023] Figure 7 shows the MALDI-TOF MS analysis of the products detected after treating 2.0 mg / mL of beta-chitin with CBP21 1.0 μM and 1.0 mM ascorbic acid, in Tris buffered with H218O pH 8, 0 (a), or in Tris buffered with H216O pH 8.0, saturated with 18O2 (B). All major products show an increase in mass of 2 amu, compared to reactions carried out in solutions that do not contain water or isotope-labeled molecular oxygen (see figures 2A and 5). (C) Adducts of the oxidized hexameric product shown in panel (B). Note the small amount of product marked with a non-isotype (indicated by the arrow), which most likely results from the initial stage of the reaction where 16O2 was still present (before saturation with 18O2; see materials and methods). 100% of the relative intensity represents 6.8x103 and 2.0x103 a.u. in panels A and B and C, respectively. (D) Scheme for the enzymatic reaction catalyzed by CBP21. In the final oxidized product, one oxygen comes from molecular oxygen and one from water.
[0024] Figure 8 shows the effect of several potentially inhibitory factors on CBP21 activity. Sodium dithionite (SD) is a well-known oxygen scavenger, used routinely to create an oxygen-free (anaerobic) environment for enzymatic reactions. (a) MALDI-TOF MS spectrum of a reaction mixture containing 2.0 mg / mL beta-chitin, 1.0 μM CBP21, 1.0 mM ascorbic acid and 10 mM sodium dithionite, incubated for 16 hours at 37 ° C ° C, under anaerobic conditions (all vials were degassed with a Schlenk line). The spectrum shows noise at the baseline and no noticeable peak intensity; in other words, there are no detectable quantities of the generated CBP21 products. 100% of the relative intensity represents 0.9x102 a.u. Panel (B) shows that the potentiating effect of CBP21 on chitinase (ChiC) is eliminated by adding sodium dithionite (SD). Reaction conditions: 0.1 mg / mL beta-chitin, 0.1 μM ChiC, 1.0 mM ascorbic acid, 10 mM sodium dithionite, 1.0 μM CBP21, incubated for 16 hours at 37 ° C under conditions anaerobic (all vials were degassed with a Schlenk line). The quantities of the final product were analyzed by HPLC. Panel (C) shows additional control experiments, showing product development during the incubation of 0.45 mg / mL beta-chitin with 0.5 μM ChiC and reduced 1 mM glutathione (which has the same effect as ascorbic acid see figure 11), in 20 mM Tris-HCl pH 8.0. Additional additions / conditions were CBP21 1.0 M (standard conditions for maximum activity; dark lines with filled diamonds), CBP21 1.0 μM in a solution with reduced oxygen concentration obtained by gas exchange (line with positive signs; note that the results shown in panel C of figure 7, which shows the presence of 16O2 even after intense gas exchange in the Schlenk line, show that really anaerobic conditions were not obtained), CBP21 1.0 μM and 2 mM potassium cyanide, an imitation well-known O2 (line with filled squares) and no CBP21, none of the additional conditions (standard control experiment; line with filled circles). The addition of 2.0 mM sodium azide, a known heme protein inhibitor, did not inhibit CBP21 activity and the curves were similar to the curve with filled diamonds. CBP21 was also completely inhibited by Oxyrace (Oxyrace Inc., Mansfield, OH), a bacterial oxidase that uses lactate as a hydrogen donor in order to create an anaerobic environment (results not shown). Panel (D) shows the production of GlcNAc3GlcNAcA (ie, an oxidized tetramer) during a reaction of 0.45 mg / mL of beta-chitin, 1.0 µM CBP21 and 1.0 mM reduced glutathione in Tris-HCl 20 mM pH 8.0, in the absence (line with filled diamonds) or presence (line with filled squares) of 2.0 mM potassium cyanide. This result shows that cyanide inhibits the oxidation reaction. The data shown in panels B, C and D are +/- SD mean (N = 3); error bars (not visible for each point) indicate SD.
[0025] Figure 9 shows the effect of divalent cations on CBP21 activity. The figure shows the ChiC activity, monitored by measuring the concentration of (GlcNAc) 2 produced by HPLC. The reaction mixtures contained 2.0 mg / mL of beta-chitin in 1.0 mM ascorbic acid, 5 mM EDTA, 0.5 μM ChiC in 20 mM Tris pH 8.0, in the presence or absence of CBP21 without metal 1 , 0 μM. Solid lines indicate reactions with CBP21 without metal; The dotted lines indicate reactions without CBP21. Halfway through the reaction (indicated by the arrow), MgCl2 (final concentration 25 mM), ZnCl2 (25 mM) or buffer was added to one of the two (parallel) reaction mixtures. Squares, MgCl2 added; circles, ZnCl2 added; triangles, buffer added. The results clearly demonstrate that divalent cations are essential for CBP21 function; controls show that cations do not affect ChiC activity.
[0026] Figure 10 shows the importance of conserved His114. CBP21 and GH61 proteins contain two very conserved histidines (H28 and H114 in CBP21), one of which is the N-terminal histidine of the mature secreted protein. In order to investigate the importance of His114, the H114A mutant variant of CBP21 was analyzed. Panel (a) shows that incubating CBP21 1.0 μMH114A with 2.0 mg / mL beta-chitin, 1.0 mM ascorbic acid, in 20 mM Tris pH 8.0 for 16 hours at 37 ° C, did not take to the production of soluble oxidized oligosaccharides. 100% of the relative intensity represents 3.5x104 au Panel (B) shows the release of the product by degrading 0.1 mg / mL of beta-chitin with 0.5 μM ChiC in 1.0 mM ascorbic acid, 20 mM Tris pH 8.0, in the presence or absence of CBP21WT (upper gray line in filled triangles and dark gray line with filled diamonds, respectively), and in the presence of CBP21H114A (gray line with filled squares). Dark gray lines with filled circles show a reaction without CBP21 and without ascorbic acid. The data clearly shows that His114 is essential for CBP21 function, since CBP21H114A is unable to enhance chitinase activity, even in the presence of ascorbic acid.
[0027] Figure 11 shows the effect of other reducers on the activity of CBP21. Shows the reaction products obtained by incubating 2.0 mg / mL beta-chitin for 16 hours at 37 ° C, in the presence of 1.0 μM CBP21 and (a) 1.0 mM reduced glutathione or (B) in 1.0 mM Fe (II) SO4, in 20 mM Tris pH 8.0. Note that the profiles of the observed product are very similar to those obtained in the presence of ascorbic acid (Figure 5). Figures 5 and 6 show that CBP21 activity is greater in the presence of ascorbic acid. Reducers, such as reduced glutathione and Fe (II) SO4, have similar effects on the degradation reaction kinetics (results not shown). 100% of the relative intensity represents 1.5x104 and 0.8x103 a.u. in panels A and B, respectively.
[0028] Figure 12 shows the effect of ascorbic acid on beta-chitin. It is well established that the reducers undergo self-oxidation in oxygenated solutions, thus generating reactive oxygen species that can possibly oxidize biomolecules present in the surrounding environment. The figure shows the MALDI-TOF MS analysis of a reaction mixture obtained after incubating a sample containing 2.0 mg / mL of beta-chitin and 1 mM ascorbic acid, in 20 mM Tris pH 8.0, for four days at 37 ° C. The spectrum shows no sign of soluble oxidized oligosaccharides. 100% of the relative intensity represents 4.4x104 a.u.
[0029] Figure 13 shows the effect of CBP21 on natural soluble oligosaccharides. Previous studies (Vaaje-Kolstad et al., 2005, J. Biol. Chem. 280: 11313; Suzuki et al., 1998, Biosci. Biotechnol. Biochem. 62: 128) show that CBP21 binds specifically to beta-chitin and not soluble or amorphous forms of chitin. With the results presented here in mind, the ability of CBP21 to oxidize and / or hydrolyze chitin's natural oligosaccharides was investigated. A solution containing 100 μM GlcNAc6, in 20 mM Tris pH 8.0, was incubated for 16 hours in the presence of only buffer, 1.0 mM ascorbic acid (AA) or 1.0 mM ascorbic acid and 1.0 μM CBP21, and the reaction products were analyzed by MALDI-TOF MS (panels UM, B and C, respectively) and quantified by HPLC (panel D). No degradation or oxidation of GlcNAc6 was observed. Similar experiments were carried out with polymeric and soluble chitin derived from chitin; again, none of the degradation or oxidation products can be detected (results not shown). 100% of the relative intensity represents 5.7x103, 1.4x104 and 4.0x104 a.u. for panels A, B and C, respectively.
[0030] Figure 14 shows the possible effect of Fenton's chemistry on the solubilization of beta-chitin. It can be determined that the observed CBP21-mediated oxide-hydrolytic cleavage of beta-chitin is related to the generation of conditions that lead to Fenton-type chemistry (analogous to what has been proposed for cellulose degradation by cellobiose dehydrogenases (Hammel et al. , 2002, Enzyme. Microb. Tech. 30: 445; Henriksson et al., 2000, J. Biotechnol. 78: 93; Hyde & Wood, 1997, Microbiol-Uk 143: 259). Although Fenton's chemistry is not particularly efficient in the pH used in this study (8.0), it was found that the Fenton type process can occur.The Fenton chemistry is well known for depolymerizing polysaccharides, and can yield oxidized products similar to those generated by CBP21. 2.0 mg / mL of beta-chitin were incubated for 16 hours with 0.03% H2O2 and 5.0 mM Fe (II) SO4, in 20 mM Tris pH 8.0 at 37 ° C. The soluble reaction phase was analyzed by MALDI-TOF MS The mass spectrum shown in the figure did not reveal the formation of soluble products. As controls, the MALDI experiments -TOF were repeated using various dilutions of the original sample; the reaction samples where the H2O2 concentration was 0.3% or 0.003% were also analyzed. Soluble products have never been observed (results not shown). 100% of the relative intensity represents 1.2x104 a.u.
[0031] Figure 15 shows: (a) EfCBM33 crystals (Uniprot ID: Q838S1; EF0362; uniprot.org/uniprot/Q838S1), obtained by vapor drop diffusion experiments, which were used to collect the data of 0.95Â. (B) Shows the side chain of the exposed solvent Trp, modeled on the 2Fo-Fc map. (C) Overlapping of the main chains of CBP21 and EfCBM33; the side chains of conserved histidines and an exposed surface of aromatic amino acid (Tyr in CBP21, Trp in EfCBM33) are shown. (D) MALDI-TOF MS spectrum of soluble products obtained after treating 2.0 mg / mL of beta-chitin with "washed" crystals of EfCBM33, dissolved in 20 mM Tris pH 8.0; like CBP21, the product spectrum is dominated by oxidized oligosaccharides with even numbering, clearly demonstrating the oxide-hydrolytic activity. (E) Degradation of 2.0 mg / mL of alpha-chitin (from shrimp shells) by 0.3 μM of Enterococcus feacalis chitinase (protein name (EF0361), in the presence or absence of EfCBM33 0.3 μM and 1.0 mM reducer (R: reduced glutathione), incubated at 37 ° C with agitation at 900 rpm, a reinforcement of chitinase activity is evident in the presence of EfCBM33 and reducer.
[0032] Figure 16 shows the speed and degree of oxidative cleavage by CBP21 activity. Panel (a) shows the production of unoxidized dimers (top line with diamonds), oxidized trimers (line with triangles) and oxidized tetramers (dark line with squares) synthesized during the incubation of 0.45 mg / mL beta-chitin , 1.0 μM CBP21, 0.5 μM ChiC and 1.0 mM reduced glutathione in 20 mM Tris-HCl pH 8.0 (ChiC oxidized oligosaccharide hydrolysis produces only minimal amounts of oxidized dimers; the three products shown represent the vast majority of oligomers produced under these conditions). After five hours, approximately 4.9% of total sugars (theoretical number based on chitin concentration) are oxidized *. Panel (B) shows the production of oxidized sugars during the incubation of 0.45 mg / mL of beta-chitin, 1.0 µM CBP21 and 1.0 mM reduced glutathione in 20 mM Tris-HCl pH 8.0. The degree of oxidation was determined by rapid conversion of chitin treated with a large dose of a chitinase cocktail, followed by UHPLC detection of oxidized dimers (filled diamonds) and trimers (filled triangles), as described in the materials and methods section. The linear part of the reaction represents an oxidation rate of approximately 1 per minute **. Maximum levels are reached after 2-3 hours and represent an oxidation level of 7.6% of total sugars (theoretical number based on chitin concentration) ***. The data in both panels are +/- SD average (N = 3); error bars (not visible for each point) indicate SD.
[0033] * Final concentrations of trimer and oxidized tetramer are 53 and 55 μM, respectively, increasing by a total of 108 μM GlcNAcA. The molar concentration of GlcNAc in the solution is 2,217 μM. Thus, the degree of oxidation is 108 / 2,217; in other words, 4.9% of the sugars are GlcNAcA. ** The calculated rates for oxidized dimer and oxidized trimer are 0.68 and 0.60 μM / min. When added, the rate of oxidized products generated is 1.28 μM / min, and when taking into account the concentration of CBP21 (1.0 μM), the oxide-hydrolysis rate is 1.28 per minute. *** At the maximum levels reached, CBP21 produced oxidized dimer and oxidized trimer at 93 μM and 75 μM, respectively, which were added up to 168 μM GlcNAcA. The molar concentration of GlcNAc in the solution is 2,217 μM. Thus, the degree of oxidation is 168 / 2,217; in other words, 7.6% of the sugars are GlcNAcA.
[0034] Figure 17 shows the MALDI-TOF MS analysis of soluble products generated by a CBM33 protein (CelS2 1 μM) incubated with microcrystalline cellulose (2.0 mg / mL AVICEL®) in 20 mM Tris-HCl pH 8 buffer , 0, in the presence of 1.0 mM ascorbic acid (external electron donor), incubated for 20 hours at 50 ° C, with horizontal agitation at 250 rpm. The main peaks are noted with molecular weight and degree of polymerization (DP). All peaks noted are Na adducts of oxidized cell-oligosaccharides (hence, the subscript “ox”). The most detailed analysis of the adduct groupings is shown in figure 18.
[0035] Figure 18 shows the MALDI-TOF MS analysis of the hexamer adduct cluster of the oxidized oligosaccharides generated by CelS2 (from the same sample described in the legend of figure 17). The peaks are noted with molecular weight, degree of polymerization (DP); “Ox” indicates oxidation. The spectrum shows small amounts of the natural hexameric oligosaccharide (m / z = 1,013.14). Natural oligosaccharides can originate from the cleavage of the CelS2 substrate near the reducing end, or from release from the substrate breaking (see also figure 19). The dominant peak represents the Na adduct of the oxidized hexamer (m / z = 1,029.14). In addition, peaks representing the K adduct of the oxidized hexamer (m / z = 1,045.08) and the Na adduct of the Na salt of the oxidized hexamer (m / z = 1,051.13) can also be observed.
[0036] Figure 19 shows the HPAEC analysis of soluble products generated after incubation of 1.0 μM CelS2 with 10 mg / mL AVICEL® in 50 mM Tris-HCl pH 8.0, 1 mM MgCh, at 50 ° C , 900 rpm (horizontal agitation) in the presence (upper line "pink") or absence (lower line "dark purple") of reduced glutathione 0.5 mM (external electron donor). In the absence of reduced glutathione, only small amounts of natural cell-oligosaccharides are observed (Glc3-Glc6), while higher amounts of both natural cell-oligosaccharides and oxidized cell-oligosaccharides are observed when reduced glutathione is present. The DP of oxidized cell oligosaccharides is indicated by n, where n = 3 (the smallest marked oxidized cell oligosaccharide has a DP of three (= n)). The presence of small amounts of natural cell oligosaccharides was also observed in the control reaction without added CelS2 (10 mg / mL AVICEL® in 50 mM Tris-HCl pH 8.0, in the presence and absence of reduced 0.5 mM glutathione; results not shown). Thus, the substrate itself contains some shorter soluble non-oxidized cell-oligomers, which are released by incubation, even without CelS2. It is well known in the literature that AVICEL® has a relatively low degree of polymerization (Wallis et al., 1992, Carbohydrate Polymers 17: 103-110). In addition, the cleavage of the chain by CelS2 near the reducing end of a cellulose chain will increase such products. The peak observation was performed by comparing the chromatogram with the chromatogram obtained by a chemically prepared mixture of oxidized cell-oligosaccharides with sizes ranging from DP1 to DP10. Note the periodicity of oxidized cell oligosaccharides also observed in the MALDI-TOF MS analysis (Figure 17).
[0037] Figure 20 shows the total sugar (g / L) ([Glc] + cellobiose, which is reported as [Glc]), after incubation of CelS2 or E7 1 μM and 0.05 μL / mL of CELLUCLAST ™ ( “CC”) with 10 mg / mL of filter paper for 24 or 120 hours, in the presence or absence of reduced 1 mM glutathione (“RG”). The reactions were run in Bis-tris / HCl 50 mM pH 6.5, 1 mM MgCl2, at 50 ° C and 900 rpm (horizontal stirring). The control reactions, containing the cellulosic substrate suspended in the same buffer used for the enzymatic assays, did not provide any detectable signal for either glucose or cellobiosis.
[0038] Figure 21 shows the concentration of cellobiosis (g / L) after 18 or 40 hours of incubation of 1.0 μM CelS2 and 5 μg / mL of Cel7A (“Cel7A”), with 10 mg / mL of paper filter, in the presence or absence of 1 mM reduced glutathione (“RG”). These reactions were carried out in 50 mM sodium acetate pH 5.5, 1 mM MgCh, at 50 ° C, 900 rpm (horizontal incubation). Only the concentration of cellobiose was quantified, since the amount of glucose was lower (less than 5% of the total sugar). The control reactions, containing the cellulosic substrate suspended in the same buffer used for the enzymatic assays, did not provide any detectable signal for either glucose or cellobiosis.
[0039] Figure 22 shows the HPAEC profiles that show products obtained after incubating CelS2 (1.0 μM) with microcrystalline cellulose (10 mg / mL AVICEL®) in 20 mM Tris-HCl pH 8.0 buffer, in the presence of 1.0 mM ascorbic acid (external electron donor), in the presence (lanyard marked “pink”, lower line) or absence (line marked “green”, upper line) of 2.0 mM potassium cyanide. The products were analyzed after incubation for 24 hours at 50 ° C, with vertical shaking at 900 rpm. The remaining two lines (bottom) represent the same conditions observed previously (CelS2 + cyanide; line "blue", buffer + cyanide; line "brown"), in the absence of ascorbic acid. The DP of oxidized cell-oligosaccharides is indicated (n = 3), that is, the less visible oxidized cell-oligosaccharide has a DP of three (GlcNAc-GclNAc-GlcNAcA).
[0040] Figure 23 shows the MALDI-TOF MS analysis of soluble products generated by CelS2-WT 1 μM (panel A) or CelS2-H144 1 μM (panel B), by incubation with microcrystalline cellulose (10 mg / mL AVICEL ®) in 20 mM Tris-HCl pH 8.0 buffer, in the presence of 1.0 mM ascorbic acid (external electron donor) for 24 hours at 50 ° C with horizontal agitation at 250 rpm. The main peaks are observed with molecular weight and degree of polymerization (DP). All peaks observed are Na adducts of natural cell-oligosaccharides, oxidized cell-oligosaccharides (called “ox”) or the sodium salts of oxidized cell-oligosaccharides. Neither natural nor oxidized cell oligosaccharides can be observed in the reaction with CelS2-H144 (panel B). The low intensity peak observed at 1,199.55 m / z does not correspond to any oligosaccharide that results from cellulose oxidation or hydrolysis, and is probably a background component. The presence of natural cell oligosaccharides is probably the result of the substrate's low mean SD (AVICEL®; Wallis et al., 1992, Carbohydrate Polymers 17: 103-101); chain cleavage by CelS2 near the reducing end of a cellulose chain will also increase such products. See figure 19 caption for further discussion.
[0041] Figure 24 shows the effect of CelS2 (1.0 μM) on the degradation of poplar sawdust from the steam explosion (SEP; 2.0 mg / mL) by CELLUCLAST ™ (0.016 μl / ml), in 20 mM sodium acetate buffer pH 5.5, 1.0 mM MgCl2, 1.0 mM reduced glutathione (RG; external electron donor) incubated for 20 hours at 50 ° C, 250 rpm (horizontal shaking). The figure shows the release of cellobiose after 20 hours.
[0042] Figure 25 shows the cellulose degradation of high molecular weight filter paper (10 mg / mL), in 20 mM sodium acetate buffer pH 5.5, by 0.8 μg / mL of CELLUCLAST ™ (CC ), in the presence or absence of 40 μg / mL of CelS2 and reduced glutathione 0.5 mM (RG), as shown by the increase in soluble cell oligosaccharides (Glc and Glc2; converted to total Glc) over time. In reactions where no CelS2 was present, 40 μg / mL of purified BSA was added in order to maintain an identical protein load. Under these conditions, reactions with CelS2 alone did not yield detectable amounts of Glc or Glc2 (not shown). CBM33 active with chitin, CBP21, did not affect CC efficiency (not shown). RG did not have an effect on reactions only with CC; only one of the two overlapping curves is shown. The data are +/- average SD (N = 3); the error bars indicate SD.
[0043] Figure 26 shows cellulose degradation in a reaction of 10 mg / mL filter paper with a combination of 40 μg / mL CelS2 (or 40 μg / mL BSA to compensate for the protein load in reactions without CelS2) and 5 μg / mL of HjCel7A purified in 1 mM MgCl2, 20 mM sodium acetate buffer pH 5.5. By far, the main products of these reactions are cell-oligosaccharides released by cellulases (whose activity is enhanced by CelS2). In the case of HjCel7A (Panel C), the main product is cellobiose, and the formation of this product is shown. RG indicates the presence of a reducer, in this case reduced glutathione. The “+/- RG” mark indicates that the production curves only for Cel7A (without the presence of CelS2) with and without RG were essentially identical. The reactions were run at 50 ° C.
[0044] Figure 27 shows the HPAEC profiles of products obtained after incubating AVICEL® with the CBM33 N-terminal domain of CelS2, in the absence of reducer (lower line) and in the presence of reduced glutathione (2nd lower line), gallic acid ( darker upper lines) or ascorbic acid (upper lighter lines), all of which function as an external electron donor. 1 μM of the CBS33 N-terminal domain of CelS2 was incubated with 10 mg / mL of AVICEL® in the presence of 0.8 mM of one of the reducers, or in the absence of any reducer. The reactions were run in 50 mM succinate buffer pH 5.5, 1 mM MgCh, at 50 ° C and 900 rpm (vertical agitation) for 20 hours.
[0045] Figure 28 shows the total sugar (g / L) [Glc] (glucose and cellobiose reported as [Glc]) released after incubation of 0.08 μL / mL of CELLUCLAST ™ (CC), in the presence or absence of E7 1 μM and reduced glutathione 2 mM (RG) as external electron donors, with 2 mg / mL filter paper cellulose. The reactions were incubated in 20 mM sodium acetate pH 5.5, 1 mM MgCh, at 50 ° C and 900 rpm (horizontal shaking) for up to 50 hours. RG showed no effect on reactions with CC alone; only one of the two overlapping curves is shown. Control reactions with filter paper suspended in the same buffer used in the enzymatic assays, or E7 in the absence of cellulases, did not yield detectable amounts of Glc or Glc2 (not shown).
[0046] Figure 29 shows MALDI-TOF MS analysis of soluble products generated by 1 μM E7 incubated with 10 mg / mL of AVICEL® in 20 mM Tris-HCl pH 8.0 and 1 mM MgCl2, in the presence of ascorbic acid 1 mM as an external electron donor, incubated for 20 hours at 50 ° C with vertical shaking at 900 rpm. The oxidized oligosaccharides (Glc3- 7GlcA) are observed as sodium adducts and as sodium adducts of the oligosaccharide sodium salts, and are observed with their molecular weights (m / z); Natural cell-oligosaccharides are also present, but have been excluded from observation. Such natural oligosaccharides may be the result of cleavage of CBM33 near the reducing end of the substrate.
[0047] Figure 30 shows HPAEC profiles of products generated by CelS2 1 μM in full size, in the presence (upper gray line) or in the absence (lower dark gray line) of 1 mM ascorbic acid (AA), or by the CBM33 N domain -CelS2 terminal in the presence of AA (second dark upper line), or by the C-terminal CBM2 domain of CelS2 in the presence of AA (lower dark second line), by incubation with 10 mg / mL of AVICEL®. The products were analyzed after 20 hours of incubation in 20 mM ammonium acetate buffer pH 5.0, 1 mM MgCL at 50 ° C and 900 rpm (vertical agitation). The chromatogram is enlarged to emphasize the oxidized cell oligosaccharides, produced by the full-size CelS2 and the N-terminal CBM33 domain of CelS2.
[0048] Figure 31 shows the reactivation of activity by adding metals to the CBN33 N-terminal domain of CelS2 treated with EDTA. 0.8 mg / mL of CBM33 was incubated with 400 μM EDTA for 3 hours at 20 ° C. This EDTA-treated enzyme (40 μg / mL) was incubated with 10 mg / mL of AVICEL® in 50 mM MES pH 6.6 buffer, containing 1.7 mM reduced glutathione and one of 6 different metal ions (10 μM; Mg2 + , Fe3 +, Zn2 +, Co2 +, Ca2 +, Cu2 +). The remaining EDTA concentration in these reaction mixtures was 20 μM. The HPAEC overlapping chromatograms show products released after 20 hours of incubation at 50 ° C. Only Cu2 + (upper gray line) reactivated the enzyme under these conditions and during the use of these low metal concentrations.
[0049] Figure 32 shows A) Alignment of GH61 sequences using ClustalW. The HjGH61A sequences were aligned in a profile created by structurally aligning TtGH61E and HjGH61B. The N-terminal signal peptides, present in the products of the natural main gene, were removed from the sequences before producing the alignment. B) The structural overlap of TtGH61E (green) and HjGH61B (orange) is observed. The side chains of six conserved active sites and the surface residues stained red in alignment (a) are shown in magenta in (B). An insert present in HjGH61B is stained yellow in (a) (shaded section) and (B) (3 helix turns).
[0050] Figure 33 shows the structure of CBP21 (a) compared with a structural model of E7 (B) and the structure of TtGH61E (C). Note the similarity between E7 and TtGH61E (Met and Val at positions 207 and 161, respectively, are both hydrophobic residues; the corresponding residue in CBP21, T183, is more polar). CBP21 does not have a histidine (CBP21 has D182), tyrosine (CBP21 has F187) and a glutamine (CBP21 has E60), which are conserved in the two GH61s shown. Residues are numbered as they appear in the primary gene product, that is, a protein with an N-terminal signal peptide. Currently processed mature proteins start with a histidine, which can then be histidine 1 (as shown in alignment in (a)). H28, H37 and H19 correspond to this histidine 1 in panels A, B and C, respectively.
[0051] Figure 34 shows the HPAEC chromatograms that show the production of oligosaccharides from microcrystalline cellulose (AVICEL®) by TtGH61E (upper curve, with larger peaks) and TaGH61A (lower curve). GH61 proteins (140 μg / mL) were incubated at 50 ° C with 10 mg / mL of Avicel in MES buffer 50 mM pH 6.6, containing 2.4 mM ascorbic acid (a reducer), for 24 hours, with shaking horizontal at 700 rpm. Figure 35 shows chromatograms (Rezex RFQ-Fast Fruit H + column) that show glucose production from AVICEL® and filter paper by Cellic ™ CTec2 (final concentration of added protein was 1.1 μg / mL), in the presence or absence of ascorbic acid. The enzymes were incubated at 50 ° C with 10 mg / mL of substrate (AVICEL® or filter paper), in 50 mM MES buffer pH 6.6, with or without 0.5 mM ascorbic acid, for 24 hours, with shaking horizontal at 700 rpm. Detailed Description of the Invention
[0052] Surprisingly, the inventors now observe that CBM33 and GH61 proteins, such as oxide hydrolases, use molecular oxygen and water to introduce chain breaks on the surfaces of crystalline polysaccharides, that is, on the surface of a solid phase, to open the polysaccharide material inaccessible for hydrolysis by normal glycoside hydrolases. Although not wishing to be bound by theory, it is believed that the carbohydrate chain is oxidized by molecular oxygen and the chain cleavage is carried out by concomitant hydrolysis (Figure 7D). In view of this mechanism, enzymes can be referred to as oxide-hydrolases. However, these enzymes may alternatively be referred to here as enzymes that degrade polysaccharides (or simply enzymes), in view of their ability to perform or assist in the cleavage of glycosidic bonds into polysaccharides. The enzymatic cleavage of crystalline polysaccharides in a reaction dependent on reactive oxygen species has not been described so far.
[0053] These enzymes have flat surfaces that bind to the well-organized, solid and flat surfaces of the crystalline material, and that catalyze chain breaks. The disruption in the chain will result in the interruption of the crystalline envelope and greater accessibility to the substrate, an effect that can be increased by modifying one of the new ends of the chain. At the cleavage point, one of the new ends is a normal non-reducing end (indicated by R-OH in figure 7D). The other new end may be a new reducing end if cleavage is performed by a normal glycoside hydrolase. However, in this case the product is different and the last sugar is oxidized to 2- (acetylamino) -2-deoxy-D-gluconic acid (Figure 7D). This unprecedented “acid chain end” will interfere with the normal crystalline envelope, as it will not have the normal conformation of the sugar ring chain and as a result of carrying a load. Effects on non-crystalline substrates are also possible.
[0054] The genes encoding these oxide hydrolases (such as genes encoding elements of the CBM33 or GH61 families) are abundant in microorganisms that degrade chitin and cellulose. As assessed by gene sequences, CBM33 and GH61 proteins are found both as single-domain proteins (that is, that consist of only one CBM33 or GH61 domain) and as multi-domain proteins (that is, that consist of at least one more domain, often a domain that is likely to be involved in substrate binding). When the CBM33 or GH61 domain containing proteins has more than one domain, the additional domains are generally coupled to the C terminus of the CBM33 or GH61 domain, because the N termination of the CBM33 or GH61 domain is essential for oxide-hydrolytic activity. The knowledge of the mode of action of these enzymes allowed their catalytic efficiency to be optimized to achieve more efficient enzymatic conversion of biomass into sugars that can be used in fermentation.
[0055] In view of the identification of the role of molecular oxygen in catalysis, it is now observed that the efficiency of the reaction can be improved by the addition of reducers that can act as an electron donor and / or generate reactive oxygen species. In the presence of divalent metal ions, the reducers improve the enzymatic conversion of recalcitrant polysaccharides.
[0056] Thus, in a first aspect, the present invention provides a method of degrading or hydrolyzing a polysaccharide comprising bringing said polysaccharide into contact with one or more oxidohydrolytic enzymes, wherein said degradation or hydrolysis is carried out in the presence of at least one reducing agent and at least one divalent metal ion.
[0057] In the manner referred to here, "degrading" said polysaccharide refers to degradation by interrupting the glycosidic bonds that connect the sugar monomers in the polysaccharide polymer.
[0058] The degradation of said polysaccharide is improved by the use of said reducing agents and metals with respect to the performance of said method without these means, thus, the rate or degree of interruption of the glycosidic bonds that connect the sugar monomers is greater. This can be easily determined by measuring product formation, for example, at certain defined time points, or by measuring the amount of undegraded polysaccharide substrate that remains, for example, at certain defined time points. This can be accomplished using methods that are well known in the art based, for example, on the determination of released reducing sugars (Horn et al., 2004, Carbohydrate Polymers 56 (1): 35-39 and their references) or fragment determination released, for example, fragments of cellulose or chitin, for example, by quantitative analysis of chromatograms obtained with high performance liquid chromatography (Hoell et al., 2005, Biochim. Biophys. Acta 1748 (2): 180-190). Preferably, said degradation measurement is evaluated in the presence of one or more relevant saccharolytic enzymes, as described hereinafter.
[0059] If the rate of degradation (for example, hydrolysis), that is, the number of broken bonds (for example, hydrolysed) in a certain period of time was higher when the substrate was exposed to the oxide-hydrolytic enzyme in the presence of instead of the absence of reducing agents and metal ions, then the degradation rate is considered to be higher. Preferably, the use of reducing agents and metal ions reduces the time spent on degradation (both complete and at the same level of partial degradation, for example, when additional saccharolytic enzymes are used, see here later) by at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times. Alternatively expressed, the use of reducing agents and metal ions increases the rate of degradation by at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times.
[0060] "Hydrolyzate" refers to the chemical reaction in which water reacts with a compound to produce other compounds and involves the separation of a bond and the addition of the hydrogen cation and the hydroxide anion from the water. In the case of polysaccharide hydrolysis, glycosidic bonds are cleaved by hydrolysis. Hydrolysis of polysaccharides to soluble sugars is referred to as "saccharification". Hydrolysis of polysaccharides in the manner referred to herein results in degradation of the polysaccharide into smaller polysaccharides, including oligosaccharides and saccharide monomers, such as glucose.
[0061] The hydrolysis of the polysaccharide can be partial or complete. In the case of complete hydrolysis, complete saccharification is achieved, that is, only soluble sugars (for example, mono- and disaccharides) remain. In partial hydrolysis, in addition to soluble sugars, the oligosaccharides and larger polysaccharides remain. As described herein, the methods of the invention include methods in which only oxidohydrolytic enzymes are used for degradation, or in which both oxidohydrolytic enzymes and saccharolytic enzymes are used for degradation. In the latter case, preferably at least 0.05-10%, for example, 0.05 to 5%, preferably 0.1 to 1% of the glycosidic bonds of the initial polysaccharide are degraded (i.e., interrupted, for example, hydrolyzed) in oligosaccharides that can be separated from the polysaccharide substrate, or can remain associated despite cleavage. In the latter case, in which saccharolytic enzymes are also used, preferably at least 50% (especially preferably 60, 70, 80, 90, 95, 96, 97, 98, 99 or 100%) of the glycosidic bonds of the initial polysaccharide they are degraded, for example, hydrolyzed. Alternatively expressed, in the latter case, preferably at least 50% (especially preferably 60, 70, 80, 90, 95, 96, 97, 98, 99 or 100%) of the initial polysaccharide is hydrolyzed to mono- or disaccharides.
[0062] With respect to cellulose, the level of degradation can be assessed by determining the increase in the level of cellobiose and / or glucose.
[0063] As mentioned here, the so-called "polysaccharide" has a polymeric carbohydrate structure, formed of repeated units (either mono- or disaccharides) joined by glycosidic bonds and with the general formula (C6HioO5) n, for example, in which 40 <n <3,000. Preferably said polysaccharide is at least partially crystalline, that is, it is in a crystalline form or has crystalline portions, that is, a shape or portion that shows a three-dimensional repetition pattern of atoms, ions or molecules with fixed distances between the parts constituents.
[0064] Preferably, said polysaccharide is cellulose, hemicellulose or chitin and can be in isolated form or can be present in impure form, for example, in a material containing cellulose, hemicellulose or chitin (i.e., a material containing polysaccharide), which can optionally contain other polysaccharides, for example, in case cellulose, hemicellulose and / or pectin are also present.
[0065] As an example, the material containing cellulose can be trunks, leaves, bark, straw and ears of plants or leaves, branches and woods of trees. The cellulose-containing material can be, but is not limited to, herbaceous material, agricultural waste, forest waste, municipal solid waste, paper and pulp waste, and ground paper waste. The cellulose-containing material can be any type of biomass including, but not limited to, wood sources, municipal solid waste, paper waste, agricultural crops and agricultural waste (see, for example, Wiselogel et al., 1995, in “Handbook on Bioethanol ”(Charles E. Wyman, editor), pp. 105-118). Preferably, the cellulose-containing material is in the form of lignocellulose, for example, a plant cell wall material containing lignin, cellulose and hemicellulose in a mixed matrix.
[0066] In a preferred aspect, the cellulose-containing material is corn straw. In another preferred aspect, the cellulose-containing material is corn fiber, corn cobs, millet husks or rice straw. In another preferred aspect, the cellulose-containing material is the processed waste paper and pulp. In another preferred aspect, the cellulose-containing material is woody or herbaceous plants. In another preferred aspect, the cellulose-containing material is bagasse.
[0067] "Cellulose" is a simple sugar polymer glucose covalently linked by beta-1,4 bonds. Cellulose is a straight-chain polymer: unlike starch, no spiral or branching occurs and the molecule assumes an extended and somewhat rigid rod-like conformation, aided by the equatorial conformation of glucose residues. The multiple hydroxyl groups in glucose from one chain form hydrogen bonds with oxygen molecules in the same chain or in a nearby chain, keeping the chains tightly together side by side and forming microfibrils with high tensile strength.
[0068] Compared to starch, cellulose is also much more crystalline. Whereas starch undergoes a transition from crystalline to amorphous when heated beyond 60-70 ° C in water (as in cooking), cellulose requires a temperature of 320 ° C and a pressure of 25 MPa to become amorphous in Water.
[0069] Several different crystalline structures of cellulose are known, corresponding to the location of the hydrogen bonds between and within the tapes. Natural cellulose is cellulose I, with structures Iα and Iβ. Cellulose produced by bacteria and algae is enriched in Iα, while cellulose from larger plants consists mainly of Iβ. The cellulose in regenerated cellulose fibers is cellulose II. The conversion of cellulose I to cellulose II is not reversible, suggesting that cellulose I is metastable and cellulose II is stable. With various chemical treatments it is possible to produce cellulose III and cellulose IV structures.
[0070] "Hemicellulose" is derived from various sugars in addition to glucose, especially xylose, but also including mannose, galactose, rhamnose and arabinose. Hemicellulose consists of shorter chains than cellulose; around 200 units of sugar. Furthermore, hemicellulose is branched, while cellulose is unbranched.
[0071] "Chitin" is defined herein as any polymer containing beta- (1-4) linked N-acetylglucosamine residues, which are bound in a linear manner. Crystalline chitin in alpha form (where chains run antiparallel), beta form (where chains run in parallel) or gamma form (where there is a mixture of parallel and antiparallel chains), amorphous chitin, colloidal chitin, chitin forms in which part (for example, up to 5, 10, 15 or 20%) of the N-acetylglucosamine sugars are deacetylated, are all included in the definition of this term.
[0072] Other forms of chitin that are found in nature include copolymers with proteins and these copolymers, which include matrices of protein chitin that are found in insects and shells of crustaceans and any other naturally occurring or synthetic copolymers comprising chitin molecules, as defined herein, are also included in the definition of “chitin”.
[0073] The term "chitin" thus includes preparations of purified alpha, beta and gamma crystalline chitin, or chitin obtained or prepared from natural sources, or chitin which is present in natural sources. Examples of such natural sources include squid feather, shrimp shells, crab shells, insect cuticles and fungal cell walls. Examples of commercially available chitins are those available from sources such as France Chitin, Hov-Bio, Sigma, Sekagaku Corp, among others.
[0074] In the manner referred to here, "putting" said polysaccharide in contact with an oxide-hydrolytic enzyme refers to leaving the two entities together, in an appropriate manner, to allow the catalytic properties of the enzyme to be efficient.
[0075] The exact kinetics of the reaction between the oxide-hydrolytic enzyme and the polysaccharide will depend on many factors, such as the type of polysaccharide to be degraded, the amount of enzyme present, the temperature and the pH. The type of polysaccharide and its degree of amorphism will vary with the substrate source and the isolation / purification process, but can be evaluated, for example, by measuring the degree of crystallinity of the substrate (which is a method known in the art).
[0076] Taking these considerations into account, appropriate incubation periods and conditions can be determined to maximize degradation (for example, hydrolysis with glycoside hydrolases). Exemplary methods are discussed below.
[0077] Thus, the polysaccharide and oxide-hydrolytic enzyme are mixed together or placed in contact with each other to allow their interaction. This may simply involve mixing the solutions of the different components directly, or applying the enzyme to the material containing polysaccharide.
[0078] As referred to herein, "one or more" preferably means 2, 3, 4, 5 or 6 or more of the cited enzymes. When more than one of the enzymes is used, they can be selected in alignment with the substrate to be used, for example, to provide complementary or synergistic action. Thus, for example, the oxido-hydrolytic enzymes that can be combined are efficient in different regions of the substrate, for example, different crystalline faces. Preferred combinations are described here later.
[0079] As used herein, an “oxide-hydrolytic enzyme” is an enzyme that uses molecular oxygen, or an activated form of it (“reactive oxygen species”), to cleave glycoside bonds into polysaccharides, preferably chitin or cellulose . The newly generated ends of the chain are a normal non-reducing end and an oxidized “acid” end which in the case of chitin is a 2- (acetylamino) -2-deoxy-D-gluconic acid, and in the case of cellulose it is a gluconic acid .
[0080] Preferably said enzyme has a metal binding site and requires the presence of a divalent metal ion for complete activity. The structural environment of this metal ion is the diagnosis (and the union) for enzymes CBM33 and GH61. The metal is attached to at least three ligands that are completely conserved in both families: (1) a histidine that is at position 1 of the mature protein (i.e., the N-terminal residue of the protein after the signal peptide for secretion is cleaved ); (2) the N-terminal amino group of the mature protein; and (3) another histidine residue that is completely conserved.
[0081] Oxide-hydrolases that belong to the CBM33 or GH61 family can be identified by analyzing the gene sequences (and the corresponding predicted amino acid sequences of the gene products) using standard bioinformatics methods. For example, you can use an existing multiple sequence alignment of CBM33 or GH61 enzymes, for example, represented by a Hidden Markov model, to search for homologous sequences in sequence databases. The sequences recovered by such searches may very likely be active oxide-hydrolases. They can most certainly be obtained (1) by checking whether the gene encodes a protein with a signal peptide for secretion, using, for example, the SignalP program; (2) checking whether the N-terminal residue after cleavage of the signal peptide (cleavage site to be predicted using, for example, SignalP) is a histidine; (3) checking if there is another histidine in the protein sequence that aligns with a histidine completely or almost completely (> 90%) conserved in the multiple sequence alignment; (4) using homology model construction, using automated servers such as Swiss-Model, to verify that this second histidine is probably located near the N-terminus and the N-terminal histidine.
[0082] Those skilled in the art can easily determine by experiment whether a protein is an oxide-hydrolytic enzyme, according to the definition described above, determining whether it can cleave glycoside bonds and whether this process becomes more efficient in the presence of molecular oxygen , a reducer and a divalent metal ion. Furthermore, it can be tested whether the alleged oxide-hydrolase works synergistically with known saccharolytic enzyme preparations, and whether the magnitude of this synergistic effect depends on the presence of reducers and divalent metal ions. Experiments such as those conducted in the examples can be used, thus, the effect of reducers and metal ions on enzymatic activity can be evaluated.
[0083] Preferably, said oxide-hydrolytic enzyme contains at least one domain that, based on the sequence similarity analyzed, for example, in the current databases Pfam or CAZy, is classified as a protein of the family CBM33 or GH61. When CBM33 or GH61 containing proteins have more than one domain, the additional domains are usually coupled to the C terminus of the CBM33 or GH61 domain, because the N termination of the CBM33 or GH61 domain is essential for oxide-hydrolytic activity (see below ). The CBM33 family was classified by the CAZy system (Active Carbohydrate-Enzymes) as a family that modulates the carbohydrate bond, implying the absence of enzymatic activity. GH61 proteins were classified as glycoside hydrolases. However, no classification is correct in view of the results presented by the inventors, and evidently the CAZy classification of these two protein families needs to be corrected. The CAZy classification is based on sequence similarity, which groups protein domains that share a certain minimum level of sequence similarity in a family. The CBM33 and GH61 domains share similar functions and share a similar structural fold, the core of which is a twisted beta-sandwich sandwich fold, similar to that found in the type-III fibronectin domains (Figures 1D, E). They can also share a structural element located on the diagnostic surfaces, completely conserved (Figure 1F), which consists of a histidine at position 1 of the mature protein (that is, the protein after the signal peptide that directs secretion has been cleaved), another completely conserved histidine residue, which is known to be important for catalytic activity and an N-terminal amino group that acts with these two histidines to bind a metal ion. Despite these many characteristics of union and diagnosis, the two families share little sequence identity and, therefore, will most likely remain in two different families even after correcting the CAZy classification.
[0084] The oxide-hydrolytic enzyme is preferably a class of GH61 protein. Thus, the oxide-hydrolytic enzyme of the invention can contain, consist or consist essentially of a GH61 domain, or GH61 protein, or a biologically active fragment thereof. In this context, “essentially consists of” indicates that additional amino acids may be present in the protein, in addition to those that constitute the GH61 domain or protein. Preferably, there are 1-3, 1-5, 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90 or 90-100 or more additional amino acids present. These additional amino acids are generally present at the C-terminus of the GH61 domain.
[0085] As previously mentioned, the oxide-hydrolytic enzyme can comprise a GH61 domain or protein. Additional modules or domains can thus be present in the protein, which, when present, are preferably at the C-termination.
[0086] In a preferred characteristic, a natural GH61 domain or protein, or a biologically active fragment thereof, is used, although variants of the natural form can be used, some of which are described here later.
[0087] Oxido-hydrolytic enzymes that comprise or consist of a GH61 domain or protein, or fragments or variants thereof, are here referred to collectively as GH61 proteins or elements or proteins of the GH61 family.
[0088] Examples of suitable natural proteins in this family are provided in Table 1 below which provides the relevant database access numbers, which are incorporated by reference here. Table 1: GH61 proteins and their accession numbers




[0089] Phanerochaete chrysosporium GH61 proteins are also preferred (see Vanden Wymelenberg et al., 2009, Appl Environ Microbiol. 75 (12): 4058-68; Hori et al., 2011, FEMS Microbiol Lett. 321 (1) : 14-23).
[0090] The preferred GH61 proteins are from fungi, in particular from Thielavia, especially preferably from Thielavia terrestris or Thielavia aurantiacus. Especially preferably, said GH61 protein is GH61A from Thielavia aurantiacus or GH61B, GH61C, GH61D, GH61E or GH61G from Thielavia terrestris, as previously described. Other preferred GH61 proteins include Hypocrea jecorin GH61A and B (SEQ ID NOs: 15 and 16).
[0091] The GH61 protein can thus be, or correspond to, or comprise a naturally occurring GH61 protein, which is found in nature, or a biologically active fragment of it. Alternatively, the GH61 protein may be an unnatural variant in the manner disclosed hereinafter.
[0092] In a preferred alternative characteristic, the oxide-hydrolytic enzyme is a class of protein in the CBM33 family. The CBM33 family comprises a carbohydrate binding module (CBM) that is defined as a contiguous amino acid sequence in a carbohydrate binding protein, with a discrete fold with carbohydrate binding activity. For example, chitinases that are known to contain one or more chitin binding modules, in addition to catalytic regions. ChiA from Serratia marcescens contains a fibronectin type III - CBM type, ChiB from Serratia marcescens contains a 5 CBM family and ChiC from Serratia marcescens contains a family 12 and a fibronectin type III - type CBM. See Bourne & Henrissat, 2001, Curr. Opin. Struct. Biol. 11: 593 for domain nomenclature. Likewise, many cellulases contain CBMs that bind to cellulose. Proteins that bind to chitin and that contain CBMs, which stimulate binding, can be, for example, structural or signaling molecules, or they can be enzymes and the complete function of the protein can be determined by domains that are present, in addition to carbohydrate-binding molecule. CBMs for use in the methods of the invention, however, must have oxide-hydrolytic activity in the manner previously defined. So far, such oxide-hydrolytic activity has been detected only in one CBM family, called CBM 33 family. This is exemplified by the function of the chitin-binding protein (CBP) CBP21.
[0093] The elements of the 33 family of the carbohydrate binding modules (CBM33) can be identified according to the CAZY classification system (cazy.org/CAZY/fam/acc_CBM.html), which is based on the sequence similarities (Davies & Henrissat, 2002, Biochem Soc T 30: 291-297 and Bourne & Henrissat, 2001, supra). The proteins in this family are known to bind chitin, but binding to other polysaccharides, including cellulose, has also been observed (Moser et al., 2008, Biotechnol. Bioeng. 100 (6): 1066-77). For some of these proteins, it is observed that they act synergistically with chitinases and cellulases in the degradation of chitin and cellulose, respectively (Vaaje-Kolstad et al., 2005, J. Biol. Chem. 280 (31): 28492-7; Vaaje -Kolstad et al., 2009, FEBS J. 276 (8): 2402-15; Moser et al., 2008, supra), in the manner described in the examples.
[0094] Studies of the action of the chitin binding protein CBP21 (and other CBM33 proteins) now lead to the identification of CBM33 proteins as oxide hydrolases.
[0095] As described here, all elements of the CBM 33 family contain a carbohydrate binding module from the 33 family. In several cases, the CBM33 module constitutes the complete protein, that is, the protein consists or essentially consists of a CBM of the simple 33 family, which is synthesized in nature and secreted as such. However, some CBMs in the 33 family can be fused to one or more additional non-catalytic carbohydrate binding modules (for example, CBM family 2 modules, CBM family 3 and CBM family 5). These proteins are bi or multidominium proteins. There is also a known example of a carbohydrate binding module of the 33 family, which is present as an individual module, in a much larger catalytic protein. This is the beta-1,4-mannanase protein from Caldibacillus cellulovorans (Sunna et al., 2000, Appl. Environ. Micro. 66 (2): 664-670). CBMs of the 33 family are generally approximately 150-250 amino acids, for example, 160-240, 170-230, 180-220, 190-210 amino acids in size, and have a molecular weight of approximately 20 kDa, preferably 19-21 kDa, 18-21 kDa, 19-22 kDa or 18-20 kDa in size, although CBM33 domains, as large as 300-400 amino acids with a molecular weight of approximately 30-40 kDa, can also be used. The size of a protein can be easily determined by standard methods that are known in the art.
[0096] Preferably, the oxide-hydrolytic enzyme consists of a single family CBM 33, or essentially consists of a family 33 CBM.
[0097] If the said oxide-hydrolytic enzyme "essentially consists of" a CBM of the 33 family, it means that additional amino acids may be present in the protein, in addition to those that constitute the CBM of the 33 family. Preferably, there are 1-3, 1 -5, 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90 or 90-100 or more additional amino acids present. These additional amino acids are generally present in the C-terminus of the CBM of the 33 family.
[0098] Alternatively, the oxide-hydrolytic enzyme can comprise a CBM of the family 33. The additional modules or domains can thus be present in the protein. Examples of such modules are the CBM family 2 modules, CBM family 3 and CBM family 5. If additional domains or modules are present, they are generally found in the C-terminal region of the CBM family 33.
[0099] Thus, in a preferred aspect, the oxide-hydrolytic enzyme may contain, consist or consist essentially of a naturally occurring CBM of the 33 family (or protein of the CBM33 family), such as CBP21 (or a homologue thereof of a another species), or a biologically active fragment of it. Alternatively, it may contain, consist or consist essentially of a variant of a naturally occurring 33 CBM (or CBM33 family protein), or a biologically active fragment thereof.
[00100] The oxido-hydrolytic enzymes that comprise or consist of a CBM module of the family 33, or the complete CBM protein of the family 33 (which comprises the CBM module of the family 33), or fragments or variants thereof, are referred to here, collectively, as CBM33 proteins or elements or proteins of the CBM33 family.
[00101] CBM33 naturally occurring proteins, which can be used in the invention, include CBM33 microbial (for example, bacterial), eukaryotic (for example, Dictyostelium) or viral proteins. Bacterial CBM33 proteins, however, are preferred.
[00102] Examples of known CBM33 proteins, which can be used in methods of the invention, and the relevant database access numbers (which are incorporated herein by reference) are shown in table 2: Table 2: CBM33 proteins and their numbers access



Other preferred bacterial CBM33 proteins include:


[00103] CBM33 bacterial proteins can be from any appropriate source, but are preferably from a genus selected from the group consisting of Bacillus, Chromobacterium, Enterococcus, Francisella, Hahella, Lactobacillus, Lactococcus, Legionella, Listeria, Oceanobacillus, Photobacterium, Photothabdus, Proteus, Pseudoalteromonas, Pseudomonas, Rickettsia, Saccharophagus, Salinvibrio, Serratia, Shewanella, Sodalis, Streptomyces, Thermobifida, Vibrio and Yersini and optionally Cellulomonas and Cellvibrio.
[00104] Preferably, said CBM33 protein is a CBP21 described in U.S. patent application 2007/0218046, which is incorporated herein by reference. For example, CBP21 from Serratia marescens (SEQ ID NO: 4) is preferred. Alternatively, EfCBM33 from Enterococcus faecalis (SEQ ID NO: 5), E7 from Thermobifida fusca (SEQ ID NO: 6), CelS2 from Streptomyces coelicolor A3 (2) (SEQ ID NO: 7), Cfla_0175 from Cellulomonas flavigena DSM 20109) (SEQ ID NO: 8), Cfla_0172 from Cellulomonas flavigena DSM 20109) (SEQ ID NO: 9), Cfla_0316 from Cellulomonas flavigena DSM 20109) (SEQ ID NO: 10), Cfla_0490 from Cellulomonas flavigena DSM 20109) (SEQ ID NO: 11), CJA_2191 (Cbp33A) from Cellvibrio japonicus Ueda107 (SEQ ID NO: 12), CJA_3139 (Cbp33 / 10B) from Cellvibrio japonicus Ueda107 (SEQ ID NO: 13) and SCO1734 from Streptomyces coelicolar A3 (2) : 14) can be used. ChbA from B. amyloliquefaciens (Chu et al., 2001, Microbiology 147 (Pt 7): 1793-803) CHB1, 2 & 3 from Streptomyces (Svergun et al., 2000, Biochemistry 39 (35): 10677-83, Zeltins et al., 1997, Eur. J. Biochem. 246 (2): 557-64, Zeltins et al., 1995, Anal. Biochem. 231 (2): 287-94, Schnellmann et al., 1994, Mol. Microbiol. 13 (5): 807-19; Kolbe et al., 1998, Microbiology 144 (Pt 5): 1291-7; Saito et al., 2001, Appl. Environ. Microbiol. 67 (3): 1268-73 ) and Alteramonas CBP1 (Tsujibo et al., 2002, Appl. Environ. Microbiol. 68: 263-270) are also preferred CBM33 proteins for use in the invention. All of these references are incorporated by reference here.
[00105] The oxide-hydrolytic enzyme can thus be, or correspond to, or comprise a naturally occurring CBM33 family protein (such as CBP21, EfCBM33, ChbA, CHB1, 2 & 3 and CBP1 or E7, CelS2, Cfla_0175, Cfla_0172 , Cfla_0316, Cfla_0490, CJA_2191 (Cbp33A), CJA_3139 (Cbp33 / 10B) and SCO1734) or protein from the GH61 family, in which it is found in nature or a biologically active fragment thereof. Alternatively, the oxide-hydrolytic enzyme can be an unnatural variant in the manner disclosed hereinafter.
[00106] As previously mentioned, oxidohydrolytic enzymes can be natural proteins, or biologically active fragments thereof, or molecules containing these enzymes. Furthermore, unnatural proteins can be derived from a naturally occurring protein, for example, from a protein in the GH61 or CBM33 family.
Such fragments preferably have at least 200, 300 or 400 amino acids in size, and preferably comprise simple and short deletions of N from the C-terminus, for example, a terminal C deletion of 1, 2, 3, 4 or 5 amino acids.
[00108] All such variants or fragments must maintain the functional property of the protein from which they are derived, in such a way that they are "biologically active". Thus, they have to maintain the oxide-hydrolytic activity, for example, under the conditions described in the examples (for example, they exhibit better activity when used in the presence of a reducing agent, and one or more saccharolytic enzymes, when compared to carrying out the method without the reducing agent, see, for example, Figure 4). Furthermore, said biologically active fragments and variants must be able to improve degradation in the manner described herein, that is, to improve the degradation of the polysaccharide substrate (for example, when said degradation is carried out in the presence of one or more saccharolytic enzymes) when used in the presence of a reducing agent and a divalent metal ion, with respect to degradation that omits the reducing agent and the divalent metal ion. Some loss of activity is contemplated, for example, the biologically active fragment or variant can present at least 50, 60, 70, 80, 90 or 95% of the oxide-hydrolytic activity of the full-size sequence, in which said activity can be evaluated in terms of the extent or level of degradation, for example, hydrolysis, achieved over an adjusted period of time, for example, in the manner assessed by the synthesis of reaction products such as oligo and / or disaccharides.
[00109] Variants include or comprise naturally occurring variants of the oxido-hydrolytic enzymes described above, such as comparable or homologous proteins found in another species, or more particularly variants found in other microorganisms, which exhibit functional properties of the enzymes in the manner described previously.
[00110] Naturally occurring oxido-hydrolytic enzyme variants, as defined herein, can also be generated synthetically, for example, using standard molecular biology techniques that are known in the art, for example, standard mutagenesis techniques such as mutagenesis targeted or random site. Such variants further include or comprise proteins with at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% similarity or sequence identity to an occurring oxide-hydrolytic enzyme natural at the amino acid level.
[00111] Thus, in a preferred aspect, the oxide-hydrolytic enzyme for use in the methods described herein is a polypeptide comprising an amino acid sequence shown in any one of SEQ ID NOs: 1 to 14 (for example, SEQ ID NOs: 1-4 or 1-5) and / or 15 to 16 (optionally with or without the main peptide, where present), or a sequence of at least 30, 40, 50, 60, 70, 80, 90, 95, 97 , 98 or 99% sequence identity thereof, or a biologically active fragment thereof comprising at least 100 amino acids (preferably at least 200 or 300 amino acids) of said sequence.
[00112] In the following sequences, the main peptides, where present, are underlined. SEQ ID NO: 1 - GH61E T. terrestris (Acc No. ACE10234.) MLANGAIVFLAAALGVSGHYTWPRVNDGADWQQVRKADNWQDNG YVGDVTSPQIRCFQATPSPAPSVLNTTAGSTVTYWANPDVYHPGPVQ FYMARVPDGEDINSWNGDGAVWFKVYEDHPTFGAQLTWPSTGKSSF AVPIPPCIKSGYYLLRAEQIGLHVAQSVGGAQFYISCAQLSVTGGGSTE PPNKVAFPGAYSATDPGILINIYYPVPTSYQNPGPAVFSCMLANGAIVF LAAALGVSGHYTWPRVNDGADWQQVRKADNWQDNGYVGDVTSPQ IRCFQATPSPAPSVLNTTAGSTVTYWANPDVYHPGPVQFYMARVPDG EDINSWNGDGAVWFKVYEDHPTFGAQLTWPSTGKSSFAVPIPPCIKSG YYLLRAEQIGLHVAQSVGGAQFYISCAQLSVTGGGSTEPPNKVAFPG AYSATDPGILINIYYPVPTSYQNPGPAVFSC SEQ ID NO: 2 - T. aurantiacus GH61A (Acc No. ABW56451.) MSFSKIIATAGVLASASLVAGHGFVQNIVIDGKKYYGGYLVNQYPYM SNPPEVIAWSTTATDLGFVDGTGYQTPDI- ICHRGAKPGALTAPVSPGGTVELQWTPWPDSHHGPVINYLAPCNGDC STVDKTQLEFFKIAESGLINDDNPPGIWASDNLIAANNSWTVTIPTTIAP GNYVLRHEIIALHSAQNQDGAQNYPQCINLQVTGGGSDNPAGTLGTA LYHDTDPGILINIYQKLSSYIIPGPPLYTG SEQ ID NO: 3 - GH61B T. terrestris (Acc No. ACE10231.) MKSFTIAALAALWAQEAAAHATFQDLWIDGVDYGSQCVRLPASNSP VTNVASDDIRCNVGTSRPTVKCPVKAGSTVTIEMHQQPGDRSCANEA IGGDHYGPVMV YMSKVDDAVTADGSSGWFKVFQDSWAKNPSGSTG DDDYWGTKDLNSCCGKMNVKIPEDIEPGDYLLRAEVIALHVAASSGG AQFYMSCYQLTVTGSGSATPSTVNFPGAYSASDPGILINIHAPMSTYV VPGPTVYAGGSTKSAGSSCSGCEATCTVGSGPSATLTQPTSTATATSA PGGGGSGCTAAKYQQCGGTGYTGCTTCASGSTCSAVSPPYYSQCL SEQ ID NO: 4 - Serratia marcescens CBP21 MNKTSRTLLSLGLLSAAMFGVSQQANAHGYVESPASRAYQCKLQLN TQCGSVQYEPQSVEGLKGFPQAGPADGHIASADKSTFFELDQQTPTR WNKLNLKTGPNSFTWKLTARHSTTSWRYFITKPNWDASQPLTRASFD LTPFCQFNDGGAIPAAQVTHQCNIPADRSGSHVILAVWDIADTANAFY QAIDVNLSK
[00113] In the previous sequence (SEQ ID NO: 4), amino acid residues 1 to 27 correspond to the main peptide, which is necessary for the secretion of the protein in a natural system, and amino acids 28-196 correspond to the mature protein. Using Pfam to discover domain / module (“The Pfam protein families database” by Finn et al., 2010, Nucleic Acids Research Database Issue 38: D211-222), for residues 28-194 of SEQ ID NO: 4, that is , essentially the complete mature protein, are classified as CBM33. Similarly, with respect to the sequence shown in SEQ ID NO: 5, the mature protein starts at position 29 (H) below. SEQ ID NO: 5 - Enterococcus faecalis EfCBM33 of (Acc No. Q838S1.) MKKSLLTIVLAFSFVLGGAALAPTVSEAHGYVASPGSRAFFGSSAGG NLNTNVGRAQWEPQSIEAPKNTFITGKLASAGVSGFEPLDEQTATRW HKTNITTGPLDITWNLTAQHRTASWDYYITKNGWNPNQPLDIKNFDK IASIDGKQEVPNKVVKQTINIPTDRKGYHVIYAVWGIGDTVNAFYQAI DVNIQ SEQ ID NO: 6 - E7 Thermobifida fusca (Acc No. Q47QG3.) MHRYSRTGKHRWTVRALAVLFTALLGLTQWTAPASAHGSVINPATR NYGCWLRWGHDHLNPNMQYEDPMCWQAWQDNPNAMWNWNGLY RDWVGGNHRAALPDGQLCSGGLTEGGRYRSMDAVGPWKTTDVNNT FTIHLYDQASHGADYFLVYVTKQGFDPTTQPLTWDSLELVHQTGSYP PAQNIQFTVHAPNRSGRHVVFTIWKASHMDQTYYLCSDVNFV SEQ ID NO: 7 - Streptomyces coelicolor A3 CelS2 (2) (Acc. No. Q9RJY2) MVRRTRLLTLAAVLATLLGSLGVTLLLGQGRAEA HGVAMMPGSRTYLCQLDAKTGTGALDPTNPACQAALDQSGATALY NWFAVLDSNAGGRGAGYVPDGTLCSAGDRSPYDFSAYNAARSDWP RTHLTSGATIPVEYSNWAAHPGDFRVYLTKPGWSPTSELGWDDLELI QTVTNPPQQGSPGTDGGHYYWDLALPSGRSGDALIFMQWVRSDSQE NFFSCSDVVFDGGNGEVTGIRGSGSTPDPDPTPTPTDPTTPPTHTGSCM AVYSVENSWSGGFQGSVEVMNHGTEPLNGWAVQWQPGGGTTLGGV WNGSLTSGSDGTVTVRNVDHNRVVPPDGSVTFGFTATSTGNDFPVDS IGCVAP
[00114] The signal peptide for proteins in SEQ ID NOs: 1, 2, 3, 4, 5, 6 and 7 is underlined. The two histidines conserved in the metal binding motif of these proteins are shown in bold format. Signal peptides and histidines are similarly known in SEQ ID Nos: 8-14, below. SEQ ID NO: 8 - Cfla_0175 Cellulomonas flavigena DSM 20109 MPRHRSTRRALAGLAATAVVTTALVTVPTVAQAHGGLTNPPTRTYA CYQDGLAGGAAAGEAGNIRPRNAACVNAFDNEGNYSFYNWYGNLL GTIAGRHETIADGKVCGPDARFASYNTPSSAWPTTKVTPGQTMTFQY AAVARHPGWFTTWITKDGWNQNEPIGWDDLEPAPFDRVLDPPLREG GPAGPEYWWNVKLPSNKSGKHVLFNIWERTDSPESFYNCVDVDFGG GGTVTPSPTPSVTPTRTPTPSPTPSVTPSPTPSVTPTPTPTPTPTPSPTPTL TVTPTPTPTSVPGDSVCELEVDTSSAWPGGFQGTVTVFNATMEPVNG WQVSWKFTNGETIAQSWSGVTSQSGSTVTVKNADWNSTIAHHNAVN FGFIGURASGTPKAVTDATLNGKPCIVR SEQ ID NO: 9 - Cfla_0172 Cellulomonas flavigena DSM 20109 MFIPTRSRFGRLARLALAVPLALAATGIVATSASAHGSVTDPPSRNYG CWEREGGTHMDPAMAQRDPMCWQAFQANPNTMWNWNGNFREGV GGRHEQVIPDDQLCSAGKTQNGLYASLDTPGPWIMKTVPHNFTLTLT DGAMHGADYMRIYVSKAGYDPTTDPLGWDDIELIKETGRYGTTGLY QADVSIPSNRTGRAVLFTIWQASHLDQPYYICSDININGTAPTQQPTQQ PTQQPTQQPTQQPTQQPTQQPTQQPTQQPTQQPTQNPGTGACTATVK AASTWGNGWQGEVTVTAGSSAINGWKVTVGGASITQAWSGSYSGG TFSNAEWNGKLAAGASTTAGFIASGTPGTLTATCTAA SEQ ID NO: 10 - Cfla_0316 Cellulomonas flavigena DSM 20109 MSRISPLRRVAAACGALAIGAATVVGSIALAAPA SAHGAVSDPPSRIY GCWERWASNFTDPAMATSDPQCWDAWQSEPQAMWNWNGMFKEG AAGQHEQSIPDGKLCSADNPLYAAADDPGPWRTTPVDHDFRLTLHDP SNHGADYLKIYVTKQGYDARSEALTWADLELVKTTGRYATSSPYVT DVSVPRDRTGHHVVFTIWQASHLDQPYYQCSDVTFGGGGTPTTSPTT PAPTPTTPAPTTPAPTPTTPAPTTPAPTTPAPTTPAPTTPAPTQPADGAC TAAIEVVSAWQGGYQATVTVTAGSGGLDGWTVTVPGATITQAWNGT ATGSTITAAGWNGTVAAGGTAGVGFLGSGSPDGLTATCAAA SEQ ID NO: 11 - Cfla_0490 DSM 20109 Cellulomonas flavigena MRSHALPRSARPTPGRLLLSVLAVIALAFAVLTVAPAPSAQAHGWISD PPSRQDLCYTGAVSNCGPVMYEPWSVEAKKGSMQCSGGGRFTELDN ESRSWPRQNLKTNQVFTWDIVANHSTSTWEYFVDGRLHTTIDDKGAL PPNRFTHTINNLPEGNHKIFVRWNIADTVNAFYQCIDAYITPGGTPGPT QQPTQQPTQQPTQQPTQQPTQQPTQQPTQQPGNGACTATFKTNNAW GNGYQGEITVTAGSSAIRGWKVTVNGATITQAWSSQLSGSTLSNASW NGSLNAGASTTLGFIANGTPSGVTATCAAA SEQ ID NO: 12 - CJA_2191 (Cbp33A) of Cellvibrio japonicus Ueda107 MFNTRHLLAGVSQLVKPASMMILAMASTLAIHEASAHGYVSSPKSRV IQCKENGIENPTHPACIAAKAAGNGGLYTPQEVAVGGVRDNHDYYIP DGRLCSANRANLFGMDLARNDWPATSVTPGAREFVWTNTAAHKTK YFRYYITPQGYDHSQPLRWSDLQLIHDSGPADQEWVSTHNVILPYRT GRHIIYSIWQRDWDRDAAEG FYQCIDVDFGNGTGTGSSSSVASSVVSS VTSSSVASSVASSLSNDTCATLPSWDASTVYTNPQQVKHNSKRYQAN YWTQNQNPSTNSGQYGPWLDLGNCVTSGGSSSVASSSVASSVASSVT SSVASSVVSGNCISPVYVDGSSYANNALVQNNGSEYRCLVGGWCTV GGPYAPGTGWAWANAWELVRSCQ SEQ ID NO: 13 - CJA_3139 (Cbp33 / 10B) Cellvibrio japonicus Ueda107 MNNKFVKMGGMGALLLAFSALSFGHGFVDSPGARNYFCGAVTKPD HVMNGVARYPECAGAFANDFNGGYSYMSVLTHHQGRKVLGPVARN VCGFDSETWNGGKTPWDNAINWPVNNINSGTLTFSWDISNGPHFDDT SDFRYWITKPGFVYQVGRELTWADFEDQPFCDLAYNDDNPGAYPNV RADKPNTHFHTTCTVPARTGRHVIYAEWGREPPTYERFHGCIDVQIGG GSNSSVPVSSSSSSRSSSSSSLAPSSSSRSSSSSSSVSSSRSSSSSVVSSSSS SRPASSSSSSTGGSTEYCNWYGWQVAICKNTTSGWSNENQQTCIGRD TCNAPR SEQ ID NO: 14 - SCO1734 Streptomyces coelicolor A3 (2) MPAPSASRRAAAVAVAGLAPLALTTLAAAPASAHGSMGDPVSRVSQ CHAEGPENPKSAACRAAVAAGGTQALYDWNGIRIGNAAGKHQELIP DGRLCSANDPAFKGLDLARADWPATGVSSGSYTFKYRVTAPHKGTF KVYLTKPGYDPSKPLGWGDLDLSAPVATSTDPVASGGFYTFSGTLPE RSGKHLLYAVWQRSDSPEAFYSCSDVTFGGDGDGDGDGGSGSGAAT GDDTASGDAEAGAAPAPEASAPSEEQLAAAAEKSTIEHHGHGDQDA ATTTDPTDPAAAPEEAPGTAAEPHQVKAAGGGTENLAETGGDSTTPY IA VGGAAALALGAAVLFASVRRRATTGGRHGH
[00115] SEQ ID NOs: 15 and 16 are shown without a major peptide. The two histidines conserved in the metal binding motif of these proteins are shown in bold format. SEQ ID NO: 15 - HjGH61A of Hypocrea jecorina HGHINDIVINGVWYQAYDPTTFPYESNPPIVVGWTAADLDNGFVSPD AYQNPDI- ICHKNATNAKGHASVKAGDTILFQWVPVPWPHPGPIVDYLANCNGD CETVDKTTLEFFKIDGVGLLSGGDPGTWASDVLISNNNTWVVKIPDN LAPGNYVLRHEIIALHSAGQANGAQNYPQCFNIAVSGSGSLQPSGVLG TDLYHATDPGVLINIYTSPLNYIIPGPTVVSGLPTSVAQGSSAATATAS ATVPGGGSGPTSRTTTTARTTQASSRPSSTPPATTSAPAGGPTQTLYGQ CGGSGYSGPTRCAPPATCSTLNPYYAQCLN SEQ ID NO: 16 - HjGH61 of Hypocrea jecorina HGQVQNFTINGQYNQGFILDYYYQKQNTGHFPNVAGWYAEDLDLGF ISPDQYTTPDIVCHKNAAPGAISATAAAGSNIVFQWGPGVWPHPYGPI VTYVVECSGSCTTVNKNNLRWVKIQEAGINYNTQVWAQQDLINQGN KWTVKIPSSLRPGNYVFRHELLAAHGASSANGMQNYPQCVNIAVTGS GTKALPAGTPATQLYKPTDPGILFNPYTTITSYTIPGPALW
[00116] When the variants are generated, it can be noted that the appropriate residues for modification depend on the properties that are being sought in a variant like this. In the case where a variant with the same oxide-hydrolytic activity as the natural parent molecule being sought, the residues are generally those residues that are not involved in the catalytic reaction or interaction of the enzyme with the substrate chitin. However, these residues can be targeted, alternatively, to develop variants with better reactivity. This can be achieved by standard protein genetic engineering techniques, or by techniques based on random mutagenesis followed by selection, in which all techniques are well known in the technology. Attempts to improve the function of the oxido-hydrolytic enzymes may include improving the binding and catalytic capacity of the enzyme, for example, to act on other substrates, for example, carbohydrate containing copolymers, for example, protein-carbohydrate copolymers.
[00117] Those skilled in the art will recognize the potential of using the structure of natural proteins to create variants that are optimized by another insoluble polymeric polysaccharide substrate (for example, other forms of chitin or cellulose), or copolymers containing insoluble carbohydrates.
[00118] In the case of GH61 proteins presented in SEQ ID NOs: 1, 2 and 3 (and 15-16), preferably residues at positions 19, 86, 169, 171 and 210 of SEQ ID NO: 1 are conserved (see Harris et al., 2010, Biochemistry 49: 3305-3316, where His-1 of the mature protein appears at position 19) or the corresponding residues in other GH61 proteins. Such corresponding residues can be easily found by sequence alignment.
[00119] In the case of CBP21, several residues have been shown to be important in binding CBP21 to chitin and, more specifically, in the ability of CBP21 to improve chitin degradation (Vaaje-Kolstad et al., 2005, J. Biol. Chem. 280 : 11313-11319 and 28492-28497). Several mutations have been shown not to affect binding, but they do affect CBP21's ability to improve chitin breakdown. These results can be predicted for other CBM33 proteins, such as EfCBM33, E7, CelS2, Cfla_0175, Cfla_0172, Cfla_0316, Cfla_0490, CJA_2191 (Cbp33A), CJA_3139 (Cbp33 / 10B) and SCO1734. These residues are preferably unmodified with respect to the wild type CBP21 sequence shown in SEQ ID NO: 4 (or any of SEQ ID NOs: 5 to 14, for example, SEQ ID NO: 5), if the goal is to modify, for example, the stability of CBP (for example, in process conditions), but these residues can be targeted if the objective is to improve or change the functional properties of CBP21.
[00120] Preferred variants of CBP21 retain one or more, and preferably all of: a tyrosine residue at position 54, a glutamic acid residue at position 55, a glutamic acid residue at position 60, a histidine residue at position 114, an aspartic acid residue at position 182, and an asparagine at position 185 (sequence numbering according to SEQ ID NO: 4).
[00121] With respect to amino acid sequences, "sequence similarity", preferably "sequence identity", refers to the sequences that have the declared value when evaluated, for example, using the SWISS- protein sequence database PROT, using FASTA pep-cmp with a variable pam factor and gap creation penalty set to 12.0, and gap length penalty set to 4.0, and a 2-amino acid window. The sequence identity at a particular residue is intended to include identical residues that were simply derived. Sequence identity assessments are performed with respect to the full length sequence of the cited sequence that is used for comparison.
[00122] Preferred "variants" include those in which, instead of the naturally occurring amino acid, the amino acid that appears in the sequence is a structural, for example, unnatural analog thereof. The amino acids used in the sequences can also be derived or modified, for example, labeled, glycosylated or methylated, providing the function of the oxide-hydrolytic enzyme that is not significantly affected in an adverse way.
[00123] Additional preferred variants are those in which, with respect to the amino acid sequences described above, the amino acid sequence has been modified by substitution, addition and / or elimination or chemical modification of single or multiple amino acids (for example, in 1 to 10, for example, 1 to 5, preferably 1 or 2 residues), including deglycosylation or glycosylation, but which still maintains functional activity as they bind to the polysaccharide substrate and improve its degradation, particularly when used together with a or more saccharolytic enzymes.
[00124] In the meaning of "addition", amino and / or carboxyl terminal fusion protein or polypeptide variants are included, comprising an additional protein or polypeptide, or other molecule fused to the enzyme sequence. Terminal carboxyl fusions are preferred. It can be guaranteed that any such fusion to the enzyme does not adversely affect the functional properties required for use in the methods of the invention, as presented herein elsewhere.
[00125] The "substitution" variants preferably involve the replacement of one or more amino acids with the same number of amino acids, and which makes substitutions conservative.
[00126] Such functionally equivalent variants mentioned above include in particular naturally occurring biological variations (for example, found in other microbial species) and derivatives prepared using known techniques. In particular, the functionally equivalent variants of the oxido-hydrolytic enzymes described herein extend to enzymes that are functional (or present), or derived from different genera or species, in addition to those specific molecules mentioned herein.
[00127] Variants, such as those described above, can be generated in any appropriate manner using techniques that are known and described in the technology, for example, using standard recombinant DNA technology.
[00128] Preferably, the variants or fragments described herein are derived from the natural sequences presented above, particularly those from any one of SEQ ID NOs: 1 to 14 (for example, SEQ ID NOs: 1-4 or 1-5) and / or 15 to 16.
[00129] As mentioned here, a “reducing agent” is an element or compound in a redox reaction (reduction-oxidation) that reduces another species, and that becomes oxidized and is, therefore, the electron donor in the reaction redox. Preferably, the reducing agent is non-enzymatic. In this particular invention, the reduced compound is oxygen which, by reduction, becomes activated, improving the oxide-hydrolytic function, for example, of GH61 or CBM33 proteins. The reducing agent can function as an electron donor in the enzymatic process, and it is possible that the electron donation occurs through the generation of reactive oxygen species such as O2-. The reducing agent promotes the donation of electrons and / or the generation of reactive oxygen. Preferably, said reducing agent is ascorbic acid, reduced glutathione or Fe (II) SO4. The additionally preferred reducing agents are LiAlH4 and NaBH4. Other preferred reducing agents include organic acids (such as succinic acid, gallic acid, coumaric acid, humic acid and ferulic acid) and reducing sugars (such as glucose, glucosamine and N-acetylglycosamine). Alternatively, lignin containing reducing groups, or fragments thereof, can be used as the reducing agent. As previously noted, Fe (II) SO4 can be used as a reducing agent and thus will also contribute to the required divalent metal ion. Although a simple compound can supply both the reducing agent and the metal ion, it is preferable that these characteristics are provided by different compounds, that is, that the reducing agent and the metal ion are separate compounds.
[00130] More than one of such agents can be used in alignment with the methods of the invention, and can be selected according to the substrate and conditions used (for example, pH and temperature). It will be understood that the efficiency and stability of reducing agents can vary between these agents and depends on the pH. Thus, the pH and the reducing agent can be optimized with respect to the oxide-hydrolytic enzyme to be used.
[00131] Preferably, said divalent metal ion is Ca, Co, Mg, Mn, Ni or Zn. In an alternative embodiment, the divalent metal ion is Cu. Thus, for example, salts such as MgCl2, ZnCl2 or CoCl2 (or alternatively CuCl2) can be used.
[00132] The following description presents conditions that can be used to perform the method of the invention, but it can be noted that any of the appropriate conditions can be used.
[00133] Before placing the material containing polysaccharide in contact with the oxide-hydrolytic enzyme, the material containing polysaccharide can be pre-treated.
[00134] The material containing polysaccharide can be pretreated, for example, to disrupt the components of the plant cell wall, using conventional methods known in the art. Prior to pretreatment, where appropriate, the material containing polysaccharide can be subjected to pre-soaking, humidification, or conditioning using methods known in the art. Physical pretreatment techniques include, for example, various types of grinding, irradiation, vapor vaporization / explosion and hydrothermolysis; chemical pretreatment techniques can include dilute acid, alkaline (eg, pretreatment with lime), organic solvent (such as pretreatment with organosolv), ammonia treatments (eg, ammonia percolation (APR) and ammonia fiber / freeze explosion (AFEX)), sulfur dioxide, carbon dioxide, wet oxidation and pH-controlled hydrothermolysis; and biological pretreatment techniques can apply microorganisms that solubilize lignin (see, for example, Hsu, 1996, Pre-treatment of biomass, in “Handbook on Bioethanol: Production and Utilization”, Wyman, ed., Taylor & Francis, Washington, DC, 179-212; Ghosh & Singh, 1993, Adv. Appl. Microbiol. 39: 295-333; McMillan, 1994, Pretreating lignocellulosic biomass: a review, in “Enzymatic Conversion of Biomass for Fuels Production”, Himmel et al. eds., ACS Symposium Series 566, American Chemical Society, Washington, DC, Chapter 15; Gong et al., 1999, Advances in Biochemical Engineering / Biotechnology, Scheper, ed., Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Olsson & Hahn-Hagerdal, 1996, Enz. Microb. Tech. 18: 312-331; and Vallander & Eriksson, 1990, Adv. Biochem. Eng./Biotechnol. 42: 63-95). Additional pretreatments include ultrasound, electroporation, microwave, supercritical CO2, supercritical H2O and ammonia percolation.
[00135] Pre-treated corn straw is a material containing cellulose derived from corn straw, for example, by treatment with heat and diluted acid.
[00136] After the optional pretreatment, the material containing polysaccharide (the substrate) can be exposed to the oxide-hydrolytic enzyme, in vitro, in any appropriate vessel, for example, by mixing the substrate and the enzyme together in an appropriate medium ( for example, a solution, such as an aqueous solution) or by applying the enzyme to the substrate (for example, applying the enzyme in a solution to a substrate).
[00137] In a preferred embodiment, the oxide-hydrolytic enzyme is present in a buffer such as a phosphate buffer, for example, a sodium phosphate buffer, or Tris buffer. The appropriate concentration ranges for such a buffer are 1-100 mM. The oxide-hydrolytic enzyme can be supplied as a purified preparation (as described hereinafter), or it can be present in a composition, in which it can be a major component, preferably comprising at least 20, 30, 40, 50, 60 or 70% w / w dry weight in the composition, or it may be a minor component (for example, in a mixture with one or more saccharolytic enzymes), preferably comprising at least 1, 2, 5 or 10%, for example 1- 5% w / w dry weight in the composition.
[00138] The enzyme can be present in the solution at any suitable concentration, such as a concentration of 0.001-1.0 mg / ml, for example, 0.01-0.1 mg / ml or 0.05-0.5 mg / ml.
[00139] The polysaccharide substrate is present in the reaction mixture in any suitable concentration that will depend, to some extent, on the purity of the polysaccharide in the material that contains it. Conveniently, however, the polysaccharide itself is present in a concentration of 0.1 to 200 mg / ml, preferably 0.2 to 20 mg / ml or 0.5 to 50 mg / ml, or more preferably 25 to 150 mg / ml , especially preferably at least 25 mg / ml. Preferably, the polysaccharide is present in the material containing the polysaccharide at a level of> 50%, for example,> 60, 70, 80 or 90%, w / w dry weight in the material.
[00140] Preferably, the polysaccharide substrate is exposed to the enzyme, for example, by incubation together, for a period of 4, 6, 12 or 24 hours or more, such as 4-24 or 6-24 hours, for example, 36 or 48 hours or more, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more. In a preferred aspect, the incubation is 6-24 hours. This incubation is generally carried out at or near 50 ° C, although temperatures appropriate to optimize the improvement of polysaccharide degradation can be easily determined by those skilled in the art. For example, the temperature can be in the range of 20-65 ° C, for example, 30-60 ° C, preferably 50-55 ° C.
[00141] It will be understood that the required incubation periods, pH, temperature, substrate and enzyme concentrations are not independent of each other. Thus, a wide range of conditions can be contemplated, which can be easily assessed. Oxido-hydrolytic enzymes work to improve degradation by saccharolytic enzymes and, thus, can allow the use of lower concentrations of the longer or shorter reaction periods.
[00142] Preferably, a pH in the range of 4 to 9 is used. Preferably, the pH is in the range of 5 to 8. The preferred pH is about pH 5-6.
[00143] Where a reducing agent is used, the reducing agent is preferably added for the duration of the degradation reaction, although it can be added after this reaction has started and can be present only as long as the oxide-hydrolytic enzyme is present or active. The reducing agents are preferably added in a final concentration range of 0.1 to 100 mM, preferably 0.5 to 20 mM, especially preferably 1-5 mM. The reducing agents may be present in the polysaccharide substrate, for example, lignin present in a lignocellulosic biomass, but preferably said reducing agents are added to the reaction mixture.
[00144] As in the reducing agent, the metal ion can be added at the beginning or during the degradation reaction. However, it may not always be necessary to add metal ions to the reaction mixture, since some material containing substrate may contain sufficient metal ions for the reaction to proceed satisfactorily. However, in a preferred aspect, metal ions are added to the reaction mixture. In one embodiment, the metal ion can be added to the oxide-hydrolytic enzyme, during its production or isolation, or to the enzyme before its addition to the reaction mixture, in such a way that the enzyme is “pre-charged” with the metal ion relevant. The metal ions are preferably added to a final concentration range of 0.001 to 50 mM, for example, 0.01 to 50 mM, preferably 0.1 to 5 mM. The concentration to be used can be easily determined for the particular metal ion to be used in the reaction mixture. For example, the lowest concentrations of Cu2 + (for example, 0.001 to 0.1 mM) may be appropriate in relation to the concentration required for other metal ions. It will be understood that the ideal concentrations of metal ion to some degree depend on the concentrations of enzyme and substrate used in the enzymatic conversion reactions that are established.
[00145] As observed here, it is believed that oxidohydrolytic enzymes catalyze hydrolysis by oxidation with molecular oxygen. Therefore, it is imperative (as noted in the examples) that molecular oxygen is available for use in the reaction. As such, any of the conditions that result in an oxygen-free (anaerobic) environment should be avoided.
[00146] Thus, in a preferred aspect, the method comprises bringing said polysaccharide into contact with an oxide-hydrolytic enzyme, and adding at least one reducing agent and preferably at least one divalent metal ion to the reaction mixture.
[00147] Preferably, the incubation is performed with shaking, particularly when a cellulose-containing material is used.
[00148] In a preferred aspect, the oxide-hydrolytic enzyme is used in a concentration of 0.01 to 0.5 mg / mL and the polysaccharide substrate in 25 to 150 mg / mL (when calculated according to the content of target substrate, and not taking into account the additional material that may be present with the substrate), and the reaction is carried out at pH 5-8 for 6 to 24 hours at 50 to 55 ° C.
[00149] In methods in which the degradation or hydrolysis is carried out only with the oxide-hydrolytic enzyme, the result of said reaction is the incomplete degradation of the polysaccharide to yield long and very insoluble oligosaccharides and smaller fractions of soluble oligosaccharides, including perhaps very fractions smaller disaccharides. Preferably, said degradation or hydrolysis is further improved or terminated by the use of appropriate additional degradative glycoside hydrolases.
[00150] Thus, in a further preferred aspect, the present invention provides a method of degrading or hydrolyzing a polysaccharide comprising: a) bringing said polysaccharide into contact with one or more oxido-hydrolytic enzymes, wherein said degradation or hydrolysis is performed in the presence of at least one reducing agent and at least one divalent metal ion, and b) placing said polysaccharide (or its degradation or hydrolysis product) in contact with one or more saccharolytic enzymes selected from a cellulose hydrolase or chitin hydrolase.
[00151] Obviously, in carrying out the method, the oxide-hydrolytic enzyme and the saccharolytic enzyme have to be selected according to the polysaccharide substrate, for example, GH61 and a cellulose hydrolase for cellulose and CBP21, or another protein from the CBM33 family and a chitin hydrolase for chitin. Cross-reaction between different substrates may also be possible, for example, proteins of the CBM33 family can be efficient as oxido-hydrolytic enzymes in cellulose, for example, SEQ ID NO: 5 (EfCBM33) and other CBM33 proteins described here can be used in methods of the invention carried out with cellulose. Similarly, the elements of the GH61 family can be used on substrates other than cellulose, for example, chitin or hemicellulose.
[00152] So far, the enzymatic activity of elements of the CBM33 family has been reported only for chitin as a substrate (for example, CBP21 of Serratia marcescens). However, the present invention demonstrates that elements of the CBM33 family, for example, E7 and CelS2, work with cellulose. The enzymatic function implies hydrolysis (cleavage) and oxidation of cellulose chains to insoluble cellulose crystals, which enable a faster deconstruction of cellulose by cellulases. CBM33 enzymes that act on cellulose work optimally in the presence of an external electron donor (eg, a reducing agent) and divalent metal ions. These enzymatic characteristics are very similar to those previously observed for CBM33 enzymes that act on chitin. In the experiments described here, a single CBM33 domain of Thermobifida fusca (Uniprot ID: Q47QG3; E7) and a CBM33 multidomain (Uniprot ID: Q9RJY2; a CBM33 with a CBM2 attached to the C-terminal side of the protein; CelS2) of Streptomyces coelicolor A3 (2) were expressed and purified, and their ability to enhance the enzymatic activity of degrading cellulose was observed. CBM33 proteins for the cellulose reaction are preferably obtained from cellulolytic bacteria, for example, bacteria of the genera Cellulomonas, Cellvibrio, Thermobifida or Streptomyces, for example, bacteria of the species Celllulomonas flavigena, Cellvibrio japonicus, Thermobifida fusca or Streptomyces spp. (preferably E7 and CelS2 aqio revealed, and Cfla_0175, Cfla_0172, Cfla_0316, Cfla_0490, CJA_2191 (Cbp33A), CJA_3139 (Cbp33 / 10B) and SCO1734), and / or have one or more cellulose-binding modules (for example, family CBM 2) attached to the C-terminal end.
[00153] The natural or genetically modified variants of these oxido-hydrolytic enzymes, with altered substrate specificity (for example, from chitin to cellulose), can be combined with other substrates and saccharolytic enzymes.
[00154] It will be obvious to professionals in the field that polysaccharides such as chitin and, especially, cellulose, can occur in complex copolymeric matrices, for example, hemicelluloses, in the case of plant cell wall material. Since cellulose and hemicelluloses interact very well, it is possible that loosening the cellulose structure by an oxide hydrolase could make not only cellulose, but also hemicellulose, more accessible to attack by appropriate saccharolytic enzymes. Thus, oxide hydrolases such as proteins of the GH61 and CBM33 family can also be used concomitantly, for example, with hemicellulases or other enzymes that target polymers without chitin and without cellulose in complex copolymeric materials containing chitin or cellulose, in order to increase the saccharolytic efficiency of these enzymes.
[00155] As referred to herein, a "saccharolytic enzyme" is an enzyme that is able to cleave glycosidic bonds between saccharide monomers or dimers in a polysaccharide, using a standard hydrolytic mechanism employed by many enzymes classified in the glycoside hydrolase (GH) families ), in the CAZy database. These enzymes include cellulose hydrolases, chitin hydrolases and beta-glucosidases.
[00156] As referred to herein, a "cellulose hydrolase" is an enzyme that hydrolyzes cellulose or intermediate breakdown products. Preferably, the hydrolase is a cellulase. Cellulases are classified as glycosyl hydrolases (GH) in families that are based on their degree of identity and are in families GH 1, 3, 5-9, 12, 44, 45, 48 and 74. Good basis on the mechanism, they can be grouped into exo-1,4-beta-D-glucanases or cellobiohydrolases (CBHs, EC 3.2.1.91), endo-1,4-beta-D-glucanases (EGs, EC 3.2.1.4) and beta-glucosidases ( BGs, EC 3.2.1.21). EGs cleave glycosidic bonds in cellulose microfibrils, which preferentially act in amorphous cellulose regions. EGs fragment cellulose chains to generate reactive ends for CBHs, which act that act “processively” to degrade cellulose, including crystalline cellulose, from both the reducing (CBH1) and non-reducing (CBHII) ends, to generate mainly cellobiosis. Cellobiosis is a water-soluble dimer, linked to beta-1,4, of glucose. Beta-glucosidases hydrolyze cellobiose into glucose. GH61 enzymes were previously classified as weak endoglucanases, based on the activity of a family member, although this is not now considered correct. In the manner mentioned above, it is observed that the present inventors consider GH61 enzymes as oxide-hydrolytics.
[00157] The ability of cellulose hydrolases to hydrolyze cellulose can be assessed using methods known in the art, including methods in which unmodified cellulose is used as a substrate. The activity is then evaluated by measuring the released products, using both HPLC-based methods and methods that determine the number of newly formed reducing ends (eg, Zhang et al., 2009, Methods Mol. Biol. 581: 213- 31; Zhang et al., 2006, Biotechnol. Adv. 24 (5): 452-81). Alternatively, the efficiency of cellulose hydrolase can be assessed using an appropriate substrate and determining whether the viscosity of the incubation mixture decreases during the reaction. The reduction that results in viscosity can be determined by a vibration viscometer (for example, MIVI 3000 from Sofraser, France). The determination of cellulase activity, measured in terms of cellulase viscosity unit (CEVU), quantifies the amount of catalytic activity present in a sample, measuring the sample's ability to reduce the viscosity of a substrate solution.
[00158] Cellulases can be obtained from commercial sources, that is, companies such as Novozymes, Danisco and Biocatalysts. Examples of commercial cellulases include, for example, CELLIC ™ CTec (Novozymes A / S), CELLIC ™ CTec2 (Novozymes A / S), CELLUCLAST ™ (Novozymes A / S), NOVOZYM ™ 188 (Novozymes A / S), CELLUZYME ™ (Novozymes A / S), CEREFLO ™ (Novozymes A / S), and ULTRAFLO ™ (Novozymes A / S), ACCELERASE ™ (Genencor Int.), LAMINEX ™ (Genencor Int.), SPEZYME ™ CP (Genencor Int. ), FILTRASE® NL (DSM); METHAPLUS® S / L 100 (DSM), ROHAMENT ™ 7069 W (Rohm GmbH), FIBREZYME® LDI (Dyadic International, Inc.), FIBREZYME® LBR (Dyadic International, Inc.), or VISCOSTAR® 150L (Dyadic International, Inc .).
[00159] Alternatively, cellulases can be produced using standard recombinant techniques for protein expression. The scientific literature contains several examples of cloning, overexpression, purification and subsequent application of all types of cellulases, for example, endoglucanases, cellobiohydrolases and beta-glucosidases.
[00160] Examples of bacterial endoglucanases that can be used in the methods of the present invention include, but are not limited to, an Acidothermus cellulolyticus endoglucanase (WO 91/05039; WO 93/15186; US patent 5,275,944; WO 96/02551; US patent 5,536,655, WO 00/70031, WO 05/093050); endoglucanase III from Thermobifida fusca (WO 05/093050) and endoglucanase V and Thermobifida fusca (WO 05/093050).
[00161] Examples of fungal endoglucanases that can be used in the present invention include, but are not limited to, a Trichoderma reesei endoglucanase I (Penttila et al., 1986, Gene 45: 253-263; Trichoderma reesei endoglucanase Cel7B; number access to BANCO DE GENES ™ M15665); endoglucanase II from Trichoderma reesei (Saloheimo, et al., 1988, Gene 63: 11-22); endoglucanase II from Trichoderma reesei Cel5A; BANCO DE GENES ™ accession number M19373); endoglucanase III from Trichoderma reesei (Okada et al., 1988, Appl. Environ. Microbiol. 64: 555-563; BANCO DE GENES ™ accession number AB003694); endoglucanase V from Trichoderma reesei (Saloheimo et al., 1994, Molecular Microbiology 13: 219-228; accession number to BANCO DE GENES ™ Z33381); endoglucanase from Aspergillus aculeatus (Ooi et al., 1990, Nucleic Acids Research 18: 5884); endoglucanase from Aspergillus kawachii (Sakamoto et al., 1995, Current Genetics 27: 435-439); endoglucanase from Erwinia carotovara (Saarilahti et al., 1990, Gene 90: 9-14); Fusarium oxysporuma endoglucanase (BANCO DE GENES ™ accession number L29381); endoglucanase from Humicola grisea var. thermoidea (BANCO DE GENES ™ accession number AB003107); Melanocarpus albomyces endoglucanase (accession number to BANCO DE GENES ™ MAL515703); endoglucanase of Neurospora crassa (accession number to BANCO DE GENES ™ XM_324477); endoglucanase V from Humicola insolens; Myceliophthora thermophila CBS 117.65 endoglucanase; basidiomycete endoglucanase CBS 495.95; basidiomycete endoglucanase CBS 494.95; endoglucanase from Thielavia terrestris NRRL 8126 CEL6B; endoglucanase from Thielavia terrestris NRRL 8126 CEL6C; endoglucanase from Thielavia terrestris NRRL 8126 CEL7C; endoglucanase from Thielavia terrestris NRRL 8126 CEL7E; endoglucanase from Thielavia terrestris NRRL 8126 CEL7F; Cladorrhinum foecundissimum endoglucanase ATCC 62373 CEL7A; and Trichoderma reesei strain VTT-D-80133 (BANCO DE GENES ™ M15665 accession number) endoglucanase.
[00162] Examples of cellobiohydrolases used in the present invention include, but are not limited to, Trichoderma reesei cellobiohydrolase I; cellobiohydrolase II from Trichoderma reesei; cellobiohydrolase I of Humicola insolens; cellobiohydrolase II from Myceliophthora thermophila; cellobiohydrolase II of Thielavia terrestris (CEL6A); cellobiohydrolase I from Chaetomium thermophiluma and cellobiohydrolase II from Chaetomium thermophiluma.
[00163] Examples of beta-glucosidases used in the present invention include, but are not limited to, Aspergillus oryzae beta-glucosidase; beta-glucosidase from Aspergillus fumigatus; beta-glucosidase from Penicillium brasilianum IBT 20888; beta-glucosidase from Aspergillus niger and beta-glucosidase from Aspergillus aculeatus.
[00164] Cellulase mixtures can be used, for example, a cellulase mixture comprising at least one endoglucanase, for example, which belongs to the GH family 5, 7 or 12, a cellobiohydrolase that moves to the reducing end, for example example, which belongs to the GH 6 family, a cellobiohydrolase that moves to the non-reducing end, for example, which belongs to the GH 7 family, and a beta-glucosidase. More preferably, more complex mixtures are used, in particular mixtures containing various endoglucanases with different substrate specificities (for example, which act on different faces of the cellulose crystals). The appropriate cellulases can be easily identified, taking into account the substrate to be degraded.
[00165] As referred to herein, a "chitin hydrolase" is an enzyme that hydrolyzes chitin or intermediate breaking products. Preferably, said chitin hydrolase is a chitinase, chitosanase or lysozyme. The degradation can be complete or partial. For example, the activity of some chitin hydrolase, for example, chitinases on chitin substrates is not strong enough to result in complete degradation of the substrate. This is particularly the case for chitinases, such as ChiG from Streptomyces coelicolor, which does not have its own CBM, or chitinases such as ChiB from S. marcescens. In this case, the use of an oxide-hydrolytic enzyme that acts on chitin, according to the present invention, can result in better degradation of chitin and preferably result in complete degradation that was not previously possible. Other chitinases, such as ChiC from S. marcescens, are able to completely degrade chitin, but the speed of this process is increased by the addition of an oxide-hydrolytic enzyme such as CBP21.
[00166] Chitinase enzymes are found in plants, microorganisms and animals. Chitinases have been cloned from various species of microorganisms and have been categorized into two very distinct families, determined GH18 family and GH19 family of glycoside hydrolases, based on sequence similarities (Henrissat and Bairoch, 1993, Biochem, J. 293: 781 -788). These enzymes are referred to here collectively as chitin hydrolases.
[00167] There are several ways to measure chitinase activity, which are well known in the art, including methods in which unmodified chitin is used as a substrate. Activity in unmodified chitin is assessed by measuring released products, using both HPLC-based methods and methods that determine the number of newly formed reducing ends.
[00168] Chitinases can be obtained from commercial sources, that is, companies such as Sigma. Alternatively, chitinases can be produced using standard recombinant techniques for protein expression. The specific literature contains several examples of cloning, overexposure, purification and subsequent application of all types of chitinases (for example, Horn et al., 2006, FEBS J. 273 (3): 491-503 and their references).
[00169] Other hydrolytic enzymes suitable for hydrolyzing additional non-cellulose-associated polysaccharides (or not associated with chitin) include hemicellulases, such as acetylxylan esterases, arabinofurosidases, feruloyl esterases, glucuronidases, mannanases, xylanases and xylosidases.
[00170] Cellulase mixtures can also be used in conjunction with hemicellulases. Hemicellulases can also be obtained from commercial sources. Examples of commercial hemicellulases include, for example, SHEARZYME ™ (Novozymes A / S), CELLIC ™ HTec (Novozymes A / S), CELLIC ™ HTec2 (Novozymes A / S), VISCOZYME® (Novozymes A / S), ULTRAFLO® (Novozymes A / S), PULPZYME® HC (Novozymes A / S), MULTIFECT® Xylanase (Genencor), ACCELLERASE® XY (Genencor), ACCELLERASE® XC (Genencor), ECOPULP® TX-200A (AB Enzymes), HSP 6000 Xylanase (DSM), DEPOL ™ 333P (Biocatalysts Limited, Wales, UK), DEPOL ™ 740L. (Biocatalysts Limited, Wales, UK), and DEPOL ™ 762P (Biocatalysts Limited, Wales, UK).
[00171] Examples of xylanases used in the methods of the present invention include, but are not limited to, Aspergillus aculeatus xylanase (GeneSeqP: AAR63790; WO 94/21785), Aspergillus fumigatus xylanases (WO 2006/078256) and Thielavia terrestris NRRL xylanases 8126 (WO 2009/079210).
[00172] Examples of beta-xylosidases used in the methods of the present invention include, but are not limited to, Trichoderma reesei beta-xylosidase (UniProtKB / TrEMBL accession number Q92458), Talaromyces emersonii (SwissProt accession Q8X212) and Neurospora crassa ( SwissProt access number Q7SOW4).
[00173] Examples of acetylxylan esterases used in the methods of the present invention include, but are not limited to, Hypocrea jecorina acetylxylan esterase (WO 2005/001036), Neurospora crassa acetylxylan esterase (UniProt access number q7s259), Thielavia terris acetylxylan esterase NRRL 8126 (WO 2009/042846), Chaetomium globosum acetylxylane esterase (UniProt accession number Q2GWX4), Chaetomium gracile acetyl esterase (GeneSeqP accession number AAB82124), acetylxylane esterase of Phaeosphaeria nodorum (uniPriloxyneiline) Humicola insolens DSM 1800 (WO 2009/073709).
[00174] Examples of ferulic acid esterases used in the methods of the present invention include, but are not limited to, Humicola insolens DSM 1800 feruloyl esterase (WO 2009/076122), Neurospora crassa feruloyl esterase (UniProt access number Q9HGR3) and feruloyl esterase de Neosartorya fischeri (UniProt Access number A1D9T4).
[00175] Examples of arabinofuranosidases used in the methods of the present invention include, but are not limited to, Humicola insolens DSM 1800 arabinofuranosidase (WO 2009/073383) and Aspergillus niger arabinofuranosidase (GeneSeqP accession number AAR94170).
[00176] Examples of alpha-glucuronidases used in the methods of the present invention include, but are not limited to, Aspergillus clavatus alpha-glucuronidase (UniProt access number alcc12), Trichoderma reesei alpha-glucuronidase (UniProt access number Q99024), alpha - Talaromyces emersonii glucuronidase (accession number UniProt Q8X211), alpha-glucuronidase from Aspergillus niger (accession number UniProt Q96WX9), alpha-glucuronidase from Aspergillus terreus (accession number SwissProt Q0CJP9) and alpha-glucuronic acid number SwissProt Q4WW45 access).
[00177] Although the use of natural saccharolytic enzymes is preferred, the variants defined according to the properties described here for the oxide-hydrolytic enzyme variants can also be used.
[00178] Preferably, when said polysaccharide is cellulose, said saccharolytic enzyme is an endo-1,4-beta-D-glucanase, optionally used in combination with other 1,4-beta-D-glucanases, such as cellobiohydrolases and / or beta-glucosidases.
[00179] Thus, the enzymes to be used in the methods of the invention can be selected based on the polysaccharide substrate to be hydrolyzed. For example, CBP21 binds only to beta-chitin and can therefore be an oxide-hydrolytic enzyme suitable for use if the methods of the invention are applied to beta-chitin. Similarly, ChbB of B amyloliquifaciens, described in Chu et al. (supra), can be applied to beta-chitin.
[00180] CHB1, CHB2 and CHB3 were all isolated from S olivaceovirides (Svergun et al., Zeltins et al., Schnellman et al., Kolbe et al., Saito et al., Supra). The binding preferences of these three proteins have been determined and CHB1 and CHB2 preferentially bind to alpha-chitin, whereas CHB3 binds to both alpha and beta-chitin. Alteromonas CBP1, described by Tsujibo et al., Binds to both alpha and beta-chitin, with a preference for the alpha form.
Alternatively, elements of the GH61 family, as described herein, can be used to aid in the degradation of chitin in the methods of the invention.
[00182] Similarly, when the substrate is chitin, the saccharolytic enzyme can be selected in this way. Chitinase properties have been documented (for example, Hollis et al., 1997, Arch. Biochem. Biophys. 344: 335-342 and Suzuki et al., 1998, Biosci. Biotech. Bioch. 62: 128-135; Horn et al., 2006).
[00183] Preferred combinations for chitin beta-hydrolysis are CBP21 (or variants or fragments thereof) with one or more of ChiA, ChiB, ChiC and ChiG. Preferred combinations for chitin alpha-hydrolysis are CHB1 or CHB2 (or variants or fragments thereof) with one or more of ChiA, ChiB, ChiC and ChiG. Alternatively, elements of the GH61 family, as described herein, can be used with appropriate chitinases.
[00184] When the substrate is cellulose, the oxide-hydrolytic enzyme is preferably a protein of the GH61 family (as described here), known for its ability to act on cellulose, and proteins of the CBM33 family can also be used. The appropriate saccharolytic enzymes can be selected from known enzymes, for example, cellulases described herein.
[00185] In a preferred aspect, two or more oxidohydrolytic enzymes are employed in the methods of the invention. In view of their specific substrate specificities, better degradative effects can be expected when used together. Thus, for example, two or more proteins of the CBM33 family and / or proteins of the GH61 family can be used (as described here), for example, two or more proteins of the CBM33 family, two or more proteins of the GH61 family or one combination of one or more of each of the proteins of the CBM33 family and proteins of the GH61 family in the methods (for example, at least one protein of the CBM33 family and at least one protein of the GH61 family). Thus, for example, chitin (or cellulose) can be brought into contact with a protein of the CBM33 family and a protein of the GH61 family, for example, preferably selected from the proteins described here (for example, polypeptides comprising a sequence of amino acids shown in any of SEQ ID NOs: 4 and / or 5 and / or 6 to 14, or related sequences or fragments described herein (proteins of the CBM33 family) and polypeptides comprising an amino acid sequence shown in any of SEQ ID NOs: 1 to 3 and / or 15 to 16, or related sequences or fragments described here (proteins of the GH61 family).
[00186] Enzymes suitable for use according to the invention can be determined to use selection techniques, to evaluate hydrolysis in vitro, for example, in the manner described in the examples.
[00187] To identify oxido-hydrolytic enzymes that can be used in combination, the enzymes can be evaluated to determine whether their activity will achieve better effects on the substrate. For example, during the degradation of biomass, elements of the CBM33 / GH61 families can be combined, which are known from experiments such as those described here, to present different specificities for the various forms of chitin (for example, alpha-chitin or beta-chitin) or cellulose (for example, various types of cellulose fibers, cellulose pulps, filter paper, microcrystalline cellulose, AVICEL®, carboxymethylcellulose) that occur in nature, pre-treated biomass and / or biomass obtained by chemical modification, many of these forms being easily accessible for experimentation. In biomass, chitin and cellulose often occur as heteropolymers, containing other polysaccharides often referred to as hemicelluloses or even proteins. It can be expected that the different elements of the CBM33 / GH61 families show different activities on these different substrates. Biomass is often heterogeneous, both in nature and as a result of the biomass being mixed during the development of the process in the factory. By mixing the elements of the CBM33 / GH61 families with known differences in biomass preferences, more efficient processes can be achieved. Additional synergies can be obtained using elements from the CBM33 / GH61 families, which preferably act on different polysaccharides in the biomass, such as xylan.
[00188] Oxido-hydrolytic enzymes with different activities can also be identified by examining the periodicity of the reaction products (see examples here). Thus, a combination can be performed between the oxido-hydrolytic enzymes (for example, members of the CBM33 / GH61 families) with different periodicities. The periodicity for CBP21 is shown in figures 2D, 6A and 7B; the frequency for EfCBM33 is shown in figure 15D; the periodicity for CelS2 is shown in figures 17, 19, 23A, 27 and 31; and the periodicity for E7 is shown in figure 29. A possible explanation for the variation in periodicity is that crystalline cellulose has several shapes and that the crystals have different faces (that is, types of surface; see, for example, Carrard et al ., 2000, Proc Natl Acad Sci USA 97 (19): 10342-7), and that different elements of the CBM33 and GH61 families attack different faces. In view of the different activity of the enzymes, combinations of enzymes (which can achieve synergistic effects) can be performed. So, for example, you can combine a GH61 with CelS2.
[00189] In the methods described above, which use both an oxide-hydrolytic enzyme and a saccharolytic enzyme, the step with the oxide-hydrolytic enzyme is performed under conditions that allow the enzyme to interact or bind to the polysaccharide in the manner described here. The same conditions and considerations are applied to the additional step using the additional saccharolytic enzymes (hydrolases), the step of which can be carried out simultaneously or subsequently in the first step. In total, the incubation can be conducted for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more, but is typically performed preferably for about 8 to about 96 hours, more preferably about 8 to about 72 hours, and most preferably about 8 to about 48 hours or 4 to 24 hours.
[00190] Preferably, aqueous solutions of the enzymes are used and, preferably, enzymatic hydrolysis is carried out in a suitable aqueous environment, under conditions that can be easily determined by those skilled in the art.
[00191] Each enzyme used in the methods can be supplied as a purified preparation (in the manner described hereinafter), or it can be present in a composition, (for example, including the other enzymes for use in the methods) preferably in at least 1, 2, 5 or 10%, preferably 1-5% w / w dry weight in the composition.
[00192] For the methods described here, hydrolysis can be carried out as a fed or continuous batch process, where the material containing polysaccharide (substrate), which can be pre-treated, is gradually fed, for example, in a solution of enzyme-containing hydrolysis.
[00193] Saccharification is generally carried out in agitated tank reactors or fermenters, under controlled conditions of pH, temperature and mixture discussed here. The appropriate conditions of process time, temperature and pH can be easily determined by those skilled in the art and are discussed here, and may depend on the substrate and enzymes used and their concentrations, and whether the substrate has been pre-treated and whether a fermenting organism is included, see here below.
[00194] The dry solids content is in the range of preferably about 5 to about 50% by weight, more preferably about 10 to about 40% by weight, and above all preferably about 15 to about 30% in weight.
[00195] Each enzyme used in the reaction can be present in the solution in any suitable concentration, such as a concentration of 0.001-1.0 mg / mL, for example, 0.01-0.1 mg / mL or 0.05- 0.5 mg / ml. Alternatively expressed, the enzymes can be used at a concentration of 0.1-100 mg of enzyme / g of polysaccharide substrate, for example, 1-50 mg / g of substrate. The appropriate concentration can be determined depending on the substrate and the material containing the substrate, and the reaction conditions, for example, temperature, pH and duration.
[00196] The steps in which the oxide-hydrolytic enzyme and the saccharolytic enzyme (s) are brought into contact with the polysaccharide substrate can be carried out separately or together, or a combination of these, for example, oxide-hydrolytic enzyme can be added and after an initial incubation period the saccharolytic enzyme (s) can be added. Alternatively, the oxide-hydrolytic enzyme can be removed before the saccharolytic enzyme is added. Any step in which the oxide-hydrolytic enzyme is not present (for example, a step in which only one saccharolytic enzyme is used) needs to be carried out in the presence of a reducing agent and / or metal ion.
[00197] Other enzymes may also be added in addition to, or as an alternative to, the hydrolytic enzymes of chitin or cellulose discussed above, depending on the nature of the substrate to be degraded. For example, if the polysaccharide to be degraded is a protein-containing copolymer, proteases can also be added. Suitable examples include alkalase, neutrase, papain and other proteolytic enzymes of broad specificity. At each experimental establishment, the suitability of the proteases will need to be checked, especially if other enzymes (for example, chitinases or cellulases), which can be destroyed by some of the available proteases, are present simultaneously. If the polysaccharide is a copolymer with hemicelluloses, hemicellulolytic enzymes can be added.
[00198] Furthermore, the product resulting from the use of the enzyme described above oxide-hydrolytic and saccharolytic enzymes may include small soluble oligosaccharides (particularly disaccharides). Since dimeric products inhibit glycoside hydrolases, and since monomers are the most desirable product resulting from the degradation / hydrolysis process for further processing (see here below), additional enzymes called beta-glucosidases are also preferably used in the methods of the invention.
[00199] Thus, in a preferred aspect, said method of degrading or hydrolyzing a polysaccharide further comprises bringing said polysaccharide (or its degradation or hydrolysis product) into contact with one or more beta-glucosidases. Such enzymes can be identified and used in the specified manner (for example, with respect to their concentration) for other saccharolytic enzymes. With respect to cellulose, a beta-glucosidase (s) can be used, and with respect to chitin, a beta-N-acetylglucosaminidase (s) can be used ( s).
[00200] The steps in which the oxide-hydrolytic enzyme, saccharolytic enzyme (s) and beta-glucosidase (s) are brought into contact with the polysaccharide substrate can be carried out separately or together, or a combination of these, by For example, the oxide-hydrolytic enzyme can be added and after an initial incubation period, the sucrolytic enzyme (s) and beta-glucosidase (s) can be added, or the last two enzymes can be added sequentially . Alternatively, the oxide-hydrolytic enzyme can be removed before the other enzymes are added. Any step in which the oxide-hydrolytic enzyme is not present (for example, a step in which only one sucrolytic enzyme (s) and / or beta-glucosidase (s) are used) needs to be conducted in presence of a reducing agent and / or metal ion.
[00201] The oxido-hydrolytic enzymes and saccharolytic enzymes for use in the methods of the invention can be isolated, extracted or purified from several different sources, or can be synthesized by several different means. In the manner mentioned above, enzymes can be supplied in purified preparations or in the presence of other components.
[00202] Chemical syntheses can be carried out by methods well known in the art and which involve, in the case of peptides, cyclic adjustments of deprotection reactions of the selection of the functional groups of a terminal amino acid, and coupling of selectively protected amino acid residues, followed finally it can complete deprotection of all the functional groups. The synthesis can be carried out in solution or on a solid support, using suitable solid phases known in the art, such as the well-known procedure of Merrifield solid phase synthesis.
[00203] Preferably, the enzymes for use in the invention are substantially purified, for example, without pyrogen, for example, more than 70%, especially preferably more than 90% pure (in the manner evaluated, for example, in the case of peptides or proteins, by an appropriate technique such as peptide mapping, sequencing, or chromatography or gel electrophoresis). Purification can be carried out, for example, by chromatography (for example, HPLC, size exclusion, ion exchange, affinity, hydrophobic interaction, reverse phase) or capillary electrophoresis.
[00204] Recombinant protein expression is also known in the art, and an appropriate nucleic acid sequence can be used to express the enzymes used here for subsequent expression and optional purification, using techniques that are well known in the art. For example, an appropriate nucleic acid sequence can be operably linked to a promoter for the expression of the enzyme to be used in bacterial cells, for example, E. coli, which can then be isolated or, if the enzyme is secreted, the medium of culture or the host that expresses the enzyme can be used as the source of the enzyme.
[00205] The methods described above have applications in numerous different fields, in which hydrolysis of polysaccharides forms one of the steps of the method, or in which the products of such hydrolysis are used.
[00206] Thus, in a further aspect, the present invention provides a method of producing soluble saccharides, wherein said method comprises degrading or hydrolyzing a polysaccharide by putting said polysaccharide in contact with one or more oxido-hydrolytic enzymes, wherein the said degradation or hydrolysis is carried out in the presence of at least one reducing agent and at least one divalent metal ion, and said degradation or hydrolysis releases said soluble saccharides.
[00207] In a preferred alternative aspect, the invention provides a method of producing soluble saccharides, wherein said method comprises degrading or hydrolyzing a polysaccharide: a) bringing said polysaccharide into contact with one or more oxido-hydrolytic enzymes, wherein said degradation or hydrolysis is carried out in the presence of at least one reducing agent and at least one divalent metal ion, b) placing said polysaccharide (or its degradation or hydrolysis product) in contact with one or more saccharolytic enzymes selected from a cellulose hydrolase or chitin hydrolase and, optionally, c) placing said polysaccharide (or its degradation or hydrolysis product) in contact with one or more beta-glucosidases; wherein said degradation or hydrolysis releases said soluble saccharides.
[00208] The result of complete hydrolysis is soluble sugars. In general, a mixture of monomeric sugars and higher-order oligosaccharides (for example, disaccharides) is generated. In the manner discussed above, beta-glucosidases are preferably used to produce monomeric sugars. The partially or completely degraded polysaccharide-containing material is preferably recovered by further processing, for example, fermentation. The soluble products of degradation of the material containing polysaccharide can be separated from the insoluble material using technology well known in the art, such as centrifugation, filtration and gravity adjustment.
[00209] Preferably, said soluble saccharides are isolated or recovered after said degradation or hydrolysis process. Preferably, the soluble saccharides that are isolated or recovered are chitobiose and / or N-acetylglycosamine (from chitin) or cellobiose and / or glucose (from cellulose) and / or oligosaccharides thereof.
[00210] N-acetylglycosamine and N-acetylglycosamine oligosaccharides have numerous commercial uses, including use as a food supplement. Chitin fragments are useful in several applications, including use as immune stimulants (Aam et al., 2010, Drugs 8 (5): 1482-517).
[00211] Soluble saccharides that result from cellulose hydrolysis have several applications, particularly for use as an energy source in fermentation reactions.
[00212] Preferably, the saccharide mixture released after hydrolysis containing monomeric sugars is fermented to generate an organic substance such as an alcohol, for example, ethanol.
[00213] Thus, the present invention further provides a method of producing an organic substance, preferably an alcohol, comprising the steps of: i) degrading or hydrolyzing a polysaccharide by a method comprising: a) bringing said polysaccharide into contact with one or more oxido-hydrolytic enzymes, in which said degradation or hydrolysis is carried out in the presence of at least one reducing agent and at least one divalent metal ion, and b) putting said polysaccharide (or its degradation or hydrolysis product) in contact with one or more saccharolytic enzymes selected from a cellulase or chitinase and, optionally, c) putting said polysaccharide (or its degradation or hydrolysis product) in contact with one or more beta-glucosidases; to produce a solution comprising soluble saccharides; ii) fermenting said soluble saccharides, preferably with one or more fermenting microorganisms, to produce said organic substance as the fermentation product and, optionally, iii) recovering said organic substance.
[00214] Optionally, said soluble saccharides produced in step (i) can be isolated or purified from said solution.
[00215] The organic substance thus produced forms an additional aspect of the invention.
[00216] As referred to herein, "soluble saccharides" include monosaccharides, disaccharides and oligonucleotides that are soluble in water, preferably mono and / or disaccharides. Preferably, said soluble saccharides are fermentable, for example, glucose, xylose, xylulose, arabinose, maltose, mannose, galactose and / or soluble oligosaccharides. "Fermentation" refers to any fermentation process or any process comprising a fermentation step.
[00217] The above method may further comprise the use of one or more additional enzymes, such as esterases (for example, lipases, phospholipases and / or cutinases), proteases, laccases and peroxidases.
[00218] The hydrolysis (saccharification) and fermentation steps can be carried out separately and / or simultaneously and include, but are not limited to, separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), saccharification and cofermentation (SSCF) , hybrid hydrolysis and fermentation (HHF), separate hydrolysis and cofermentation (SHCF), hybrid hydrolysis and cofermentation (HHCF) and direct microbial conversion (DMC). Conveniently, any method known in the art, comprising pretreatment, enzymatic hydrolysis (saccharification), fermentation or a combination of these, can be used in the practice of the foregoing methods.
[00219] Conveniently, a conventional apparatus may include a batch fed agitated reactor, a batch agitated reactor, a continuous flow agitated reactor with ultrafiltration and / or a continuous plug-flow column reactor (by Castilhos Corazza et al. , 2003, Acta Scientiarum. Technology 25: 33-38; Gusakov & Sinitsyn, 1985, Enz. Microb. Technol. 7: 346-352), a friction reactor (Ryu & Lee, 1983, Biotechnol. Bioeng. 25: 53 - 65), or a reactor with intensive agitation induced by an electromagnetic field (Gusakov et al., 1996, Appl. Biochem. Biotechnol. 56: 141- 153). Additional types of reactor include, for example, fluidized bed, covered, immobilized reactors and extruders for hydrolysis and / or fermentation.
[00220] Pre-treatments that can be used have been discussed here and apply to all methods of the invention. Polysaccharide-containing material can be pre-treated prior to hydrolysis and / or fermentation. Pre-treatment is preferably carried out before the hydrolysis step. Alternatively, the pretreatment can be carried out simultaneously with hydrolysis, as well as simultaneously treating the material containing polysaccharide with the enzymes used in the methods (i.e., oxidohydrolytic and saccharolytic enzymes) to release fermentable sugars, such as glucose and / or cellobiosis. In many cases, the pre-treatment stage itself results in some conversion of biomass into fermentable sugars (even in the absence of enzymes).
[00221] The fermentable sugars obtained by the method of the invention can be fermented by one or more fermenting microorganisms, capable of fermenting the sugars directly or indirectly in a desirable fermentation product. The fermentation conditions depend on the desirable fermentation product and fermentation organism, and can be easily determined by those skilled in the art.
[00222] In the fermentation stage, the sugars released from the substrate are fermented in a product, for example, ethanol, by a fermenting organism, such as yeast. The polysaccharide substrate to be used in the method can be selected based on the desired fermentation product.
[00223] The "fermenting microorganism" refers to any microorganism, including bacterial and fungal organisms, suitable for use in the fermentation process to synthesize a fermentation product. The fermenting organism can be C6 and / or C5 fermenting organisms, or a combination thereof. Both C6 and C5 fermenting organisms are well known in the art. Suitable fermenting microorganisms are capable of fermenting, that is, converting sugars, such as glucose, xylose, xylulose, arabinose, maltose, mannose, galactose, or oligosaccharides directly or indirectly into the desired fermentation product.
[00224] Examples of bacterial and fungal fermenting organisms that produce ethanol are described by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69: 627-642.
[00225] Examples of fermenting microorganisms that can ferment C6 sugars include bacterial and fungal organisms, such as yeasts. Yeasts include strains of Saccharomyces spp., Preferably Saccharomyces cerevisiae.
[00226] Examples of fermenting organisms that can ferment C5 sugars include bacterial and fungal organisms, such as yeasts. Preferred C5 fermenting yeasts include strains of Pichia, preferably Pichia stipitis, such as Pichia stipitis CBS 5773; strains of Candida, preferably Candida boidinii, Candida brassicae, Candida sheatae, Candida diddensii, Candida pseudotropicalis or Candida utilis.
[00227] Other fermenting organisms include strains of Zymomonas, such as Zymomonas mobilis; Hansenula, like Hansenula anomala; Klyveromyces, such as K. fragilis; Schizosaccharomyces, such as S. pombe, and E. coli, especially strains of E. coli that have been genetically modified to improve ethanol yield.
[00228] In a preferred aspect, the yeast is a Saccharomyces spp. In a more preferred aspect, the yeast is Saccharomyces cerevisiae, Saccharomyces distaticus, Saccharomyces uvarum. In another preferred aspect, the yeast is a Kluyveromyces, for example, Kluyveromyces marxianus or Kluyveromyces fragilis.
[00229] Other yeasts that can be used include Clavispora, for example, Clavispora lusitaniae or Clavispora opuntiae; Pachysolen, for example, Pachysolen tannophilus; and Bretannomyces, for example, Bretannomyces clausenii.
[00230] Bacteria that can efficiently ferment hexose and pentose in ethanol include, for example, Zymomonas, such as Zymomonas mobilis and Clostridium, such as Clostridium thermocellum.
[00231] Commercially available yeasts suitable for ethanol production include, for example, ETANOL RED ™ yeast (available from Fermentis / Lesaffre, USA), FALI ™ (available from Fleischmann's Yeast, USA), SUPERSTART ™ fresh yeast and THERMOSACC ™ ( available from Ethanol Technology, Wl, USA), BIOFERM ™ AFT and XR (available from NABC - North American Bioproducts Corporation, GA, USA), GERT STRAND ™ (available from Gert Strand AB, Sweden) and FERMIOL ™ (available from DSM Specialties ).
[00232] The fermentor microorganism (s) is (are) typically added to the material with degraded polysaccharide and fermentation is carried out for about 8 to about 96 hours, such as about 24 to about 60 hours. the temperature is typically between about 26 ° C to about 60 ° C, in particular about 32 ° C to 50 ° C and at about pH 3 to about pH 8, such as around pH 4-5, 6, or 7. The above conditions will certainly depend on several factors, including the fermenting microorganism that is used.
[00233] The fermentor microorganism (s) is preferably applied in amounts of approximately 105 to 1012, preferably approximately 107 to 1010, especially approximately 2 x 108 viable cell counts per ml of fermentation broth.
[00234] For the production of ethanol, after fermentation, the fermented sludge is distilled to extract the ethanol. The ethanol obtained according to the methods of the invention can be used, for example, as an ethanol-type fuel, ethanol for drinking, i.e., potable neutral spirits, or industrial ethanol.
[00235] A fermentation stimulator can be used in combination with any of the enzymatic processes described here to improve the fermentation process and, in particular, the performance of the fermenting microorganism, such as improving the ethanol rate and yield. A "fermentation stimulator" refers to stimulators for the growth of fermenting microorganisms, in particular, yeasts. Preferred fermentation stimulators for growth include vitamins and minerals. Examples of vitamins include multivitamins, biotin, pantothenate, nicotinic acid, meso-inositol, thiamine, pyridoxine, para-aminobenzoic acid, folic acid, riboflavin and vitamins A, B, C, D and E.
[00236] The organic substance, which is the fermentation product, can be any substance derived from fermentation. The fermentation product can be, without limitation, an alcohol (for example, arabinitol, butanol, ethanol, glycerol, methanol, 1,3-propanediol, sorbitol or xylitol); an organic acid (for example, acetic acid, acetonic acid, adipic acid, ascorbic acid, citric acid, 2,5-diceto-D-gluconic acid, formic acid, fumaric acid, gluconic acid, gluconic acid, glucuronic acid, glutaric acid , 3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid, propionic acid, succinic acid or xylonic acid); a ketone (for example, acetone); an aldehyde (for example, formaldehyde); an amino acid (for example, aspartic acid, glutamic acid, glycine, lysine, serine or threonine); or a gas (for example, methane, hydrogen (H2), carbon dioxide (CO2) or carbon monoxide (CO)). The fermentation product can also be an alkane, a cycloalkane, an alkene, isoprene or polyketide. The fermentation product can also be protein.
[00237] In a preferred aspect, the fermentation product is an alcohol. It will be understood that the term "alcohol" includes a substance that contains one or more hydroxyl fractions. Preferably, the alcohol is arabinitol, butanol, ethanol, glycerol, methanol, 1,3-propanediol, sorbitol or xylitol. Ethanol is the preferred product.
[00238] The fermentation product (s) can be recovered from the fermentation medium using any method known in the art including, but not limited to, chromatography (eg, ion exchange, affinity, hydrophobic, isoelectric and size exclusion focusing), electrophoretic procedures (eg preparatory isoelectric focusing), differential solubility (eg ammonium sulfate precipitation), distillation or extraction. For example, ethanol is separated from fermented material containing cellulose, and is purified by conventional distillation methods. Ethanol with a purity of up to about 96 vol. % It can be obtained.
[00239] The present invention is further described by the following examples which are not to be construed as limiting the scope of the invention. EXAMPLES
[00240] Example 1: Effects of the oxido-hydrolytic enzymes CBP21, EfCBM33, CelS2 and E7 on chitin or cellulose substrates. Materials and methods Reagents
[00241] The pure squid feather beta-chitin powder (80 # mesh) was obtained from France Chitin (Marseille, France). H218O (containing 97% 18O) and 18O2 (containing 99% 18O) was obtained from Cambridge Isotope Laboratories Inc. (Andover, MA). 2,5-di-idroxy-benzoic acid (DHB) was obtained from Bruker Daltonics (Bremen, Germany). Dithionite, ascorbic acid, reduced glutathione, Fe (II) SO4, Cu (I) acetate, MgCl2, ZnCl2, CoCl2, LiCl, acetonitrile, Trisma-Base, HCl, EDTA and H2O2 (30% v / v) were all obtained from Sigma-Aldrich Inc. The Schlenk line was produced at the University of Oslo and used with an internal supply of N2 (99.999% pure). The N2 gas was obtained from YARA PRAXAIR (Oslo, Norway). N-acetyl-D-glucosamine oligosaccharides that vary from dimer to hexamer have been obtained from Seikagaku (Tokyo, Japan). Chitin microspheres for protein purification were obtained from New England Biolabs.
[00242] Beta-chitin monocrystalline nanofibers were prepared according to what was described in (Fan et al., 2008, Biomacromolecules 9: 1919), by sonicating 3.0 mg / mL beta-chitin particles suspended in 0.2 M acetic acid using a Vibracell ultrasonic processor, equipped with a 3 mm sonication probe (Sonics, Newtown, CT), in four one-minute intervals with 30-second breaks between each interval. Before use, the buffer in the chitin-monocrystalline fiber suspension was changed to 20 mM Tris, pH 8.0, by dialysis. These monocrystalline fibers were used for the experiment shown in figure 2A, where a larger surface area was needed to enable the detection of CBP21 activity in the absence of additional reductants and enzymes. All other experiments were carried out with untreated beta-chitin. Cloning, expression and purification of recombinant proteins Chitin-binding protein 21 (CBP21) from Serratia marcescens
[00243] CBP21 was cloned, produced and purified in the manner previously described (Vaaje-Kolstad et al., 2005, J. Biol. Chem. 280: 11313). Briefly, the E. coli BL21 DE3 strain that carries the pRSET-B vector containing the cbp21 gene was grown overnight and collected. The periplasmic content of cells containing CBP21 was extracted by cold osmotic shock, filtered through a 0.2 micron syringe filter and maintained at 4 ° C. Additionally, CBP21 was purified from the periplasmic extract by chitin affinity chromatography, using chitin microspheres (NEB) as a chromatographic medium. CBP21 was ligated to the column using 20 mM Tris pH 8.0 and 1.0 M (NH4) 2SO4 as binding buffer. After the unbound protein passed through the column, the binding buffer ran through a column volume before eluting CBP21 with 20 mM acetic acid. The fraction containing CBP21 was concentrated using a filter unit for Amicon ultracentrifuge with a molecular weight cut of 10 kDa, dialyzed in 20 mM Tris-HCl pH 8.0 and stored at 4 ° C until use. Protein purity was assessed by SDS-PAGE (generally> 99% pure) and protein concentration was determined by the Bio-Rad protein assay (Bio-Rad Laboratories, Inc., USA), according to the instructions provided by the manufacturer. CBP21 single-site mutants were prepared in the manner previously described (Vaaje-Kolstad et al., 2005, J. Biol. Chem. 280: 11313), using the QuickChange site-directed mutagenesis kit (Stratagene). Chitinase C (ChiC) from Serratia marcescens
[00244] ChiC was cloned and produced in the manner previously described (Synstad et al., 2008, Biosci. Biotechnol. Biochem. 72: 715). Briefly, E. coli BL21 DE3 cells, which carry a pREST-B vector containing the chic gene in the control of the T7 promoter, were grown in OD 0.6 and were induced in 0.4 mM IPTG. The enzyme was extracted from the cells by periplasmic extraction with cold osmotic shock. In addition, ChiC was purified from the periplasmic extract by chitin affinity chromatography, using chitin microspheres (NEB) as column material. Using Tris-HCl pH 8.0 20 M as running buffer, the periplasmic extract containing ChiC passed through the column at 2.5 mL / min, allowing the chitinase to bind to the chitin microspheres. After the unbound proteins in the extract had passed through the column, a column volume of running buffer was run through the column before eluting the bound chitinase with 20 mM acetic acid. After elution, the purified protein was concentrated using an Amicon ultrafiltration device (Millipore), and was finally dialyzed in Tris-HCl, pH 8.0, 20 mM and stored at 4 ° C before use. Protein purity was routinely assayed by SDS-PAGE and protein concentration was determined by the Bio-Rad method. Chitin deacetylase (AnCDA) from Aspergillus nidulans
[00245] Aspergillus nidulans FGSC A4 (obtained from FGSC) was grown at 37 ° C in solid YAG medium (5 g / L yeast extract, 20 g / L glucose, 20 g / L agar, 1 mL / L of Cove trace elements, 1.2 g / L of MgSO4.7H2O) for 24-48 hours to provide an inoculum, and in liquid YG medium, typically for 16-24 hours with a lot of agitation (250-300 rpm). Genomic DNA was isolated with the SP Fungi DNA Mini kit (Omega Bio-tek, USA). The gene (Gene Bank Accession Number EAA66447.1) was amplified from the genomic DNA Aspergillus nidulans FGSC A4, using extension and overlapping polymerase chain reactions, excluding introns, as well as the part of the gene encoding the signal peptide. The initiator oligonucleotides for the reactions were P1f (BglII): cga aga tct acg cct ctg cct ttg gtt c (SEQ ID NO: 17), P2r: gag acg tgg tcg tat gta tgt gcg ccg act tga tg (SEQ ID NO: 18 ); P3f: caa gtc ggc gca cat aca tac gac cac gtc tcc ctc c (SEQ ID NO: 19); P4r: cca aca gtc gta gct toc aac cct cga gca tta ac (SEQ ID NO: 20); and P5r (Hindi-iI): cag aag ctt tca atg until cca cgc aat ctc tcc until acc gag aca up to acc aac agt cgt agc tat caa c (SEQ ID NO: 21). A BglII and a Hindi-iI site were incorporated at the beginning of the end of the AnCDA gene to produce a His N-terminal fused construct in alignment in the pBAD / HisB vector (s). This vector is a variant of the commercial vector pBAD / HisB (Invitrogen, USA), where the region between the N-terminal polyhistidine tail and the multiple cloning site has been decreased (Kallio et al., 2006, J. Mol. Biol. 357: 210). The resulting plasmids were transformed into Escherichia coli TOP10 cells (Invitrogen), and the inserted gene was sequenced at the sequencing facility of the Department of Chemistry, Biotechnology and Food Science at Norwegian University of Life Sciences.
[00246] For protein expression, the transformed E. coli strain was grown at 37 ° C in 2xTY medium (16 g / L tryptone, 10 g / L yeast extract, 5 g / L NaCl) containing 100 mg ampicillin per liter until OD600 = 0.6, and subsequently induced with 0.02% (w / v; final concentration) arabinose before additional incubation at 28 ° C overnight. The cells were collected by centrifugation, and the protein was homogeneously purified by Ni2 + affinity chromatography. Protein concentrations were determined using the Bio-Rad protein assay, with bovine serum albumin as a standard. E7 of Thermobifida fusca xy (Q47QG3)
[00247] The sequence of the gene encoding the mature Q47QG3 variant of T. fusca xy (E7; residues 37-222) was cloned by amplifying the corresponding region of the T. fusca xy genomic DNA gene (obtained from ATCC) using primer oligonucleotides determined according to the In-Fusion cloning protocol (Clontech). The resulting PCR product was inserted into a modified vector pRSETB (Invitrogen), using In-Fusion ™ technology (Clonetech) in alignment with the signal peptide to direct the protein product in the periplasm, by expression in E. coli. The modified vector pRSETB shows the region containing His tag replaced by the signal sequence encoding the region of the Serratia marcescens cbp gene (Vaaje-Kolstad et al., 2005, J. Biol. Chem. 280: 11313). Inserting the genes of interest in alignment with the signal sequence, the gene product will be transported to the periplasm by expression in E. coli, and the exported protein will have a natural N-termination, which means that the protein sequence begins with a histidine. The satisfactory constructs were sequenced for verification, and were transformed into E. coli BL21 DE (3) for protein expression. The cultures were grown overnight at 37 ° C. The cells were collected by centrifugation and subjected to periplasmic extraction by cold osmotic shock. The mature protein E7 was purified by chitin affinity chromatography, using the chitin microspheres to capture the protein. The capture buffer contained 20 mM Tris-HCl pH 8.0 and 1.0 M ammonium sulfate. The protein was eluted from the column using 20 mM acetic acid. The peaks containing pure protein were grouped and concentrated using 10 kDa Sartorius Vivaspin devices. Using the same protein concentration device, the buffer was changed to 20 mM Tris pH 8.0. CelS2 of Streptomyces coelicolor A3 (2) (Q9RJY2)
[00248] A gene encoding the mature Q9RJY2 form of S. coelicolor A3 (2) (CelS2; residues 35-364) has been cloned by amplifying the corresponding gene region of the S. coelicolor A3 (2) genomic DNA (obtained from ATCC), using primer oligonucleotides designed according to the LIC cloning protocol (Novagen), which places a hexahistidine tag on the N-terminus of the protein that can be removed using factor Xa protease, leaving no unnatural amino acid in the N-termination of the protein. The PCR product was inserted into the pET-32 LIC vector according to the instructions provided by the manufacturer (Novagen). The satisfactory constructs were sequenced for verification and were transformed into E. coli Rosetta DE (3) for protein expression. Expression of soluble target protein was obtained by growing a pre-culture of 5 ml of transformed Rosetta DE cells (3) overnight at 37 ° C, which were used the next day to inoculate a volume of 300 ml of medium LB, and the growth continued with agitation at 250 rpm at 37 ° C. The gene expression was induced by adding IPTG in a final concentration of 0.1 mM when the cell density reached an O.D. of 0.6, followed by immediate transfer of the culture to an incubator with shaking at 20 ° C and to continue cultivation overnight. The next day, the cells were collected by centrifugation. Cell precipitates were resuspended in sonication buffer (20 mM Tris-HCl pH 8.0, 100 M PMSF, lysozyme and DNAse), followed by sonication using a Vibra cell ultrasonic processor, equipped with a 3 mm sonication probe (Sonics ) in order to release cytoplasmic proteins. Cell debris was removed by centrifugation, and His-labeled Q9RJY2 was purified by standard IMAC purification protocols (immobilized metal affinity chromatography) using Nickel-NTA IMAC resin (Qiagen). The purified protein was concentrated using 10 kDa Sartorius Vivaspin protein concentration devices, which were also used concurrently to change the buffer with a buffer suitable for the removal of His tag factor Xa (100 mM NaCl, CaCl2 5 , 0 mM, 50 mM Tris pH 8.0). His tags were cleaved by adding factor Xa and incubating overnight at room temperature, followed by removal of His tag using standard IMAC chromatography. The flow through the protein fraction containing the processed Q9RJY2 protein and the Xa factor was collected and concentrated using 10 kDa Sartorius Vivaspin® protein concentration devices. Finally, factor Xa was removed using Xarrest agarose microspheres, according to the manufacturer's instructions (Novagen). The pure protein buffer was changed to 20 mM Tris pH 8.0 using Sartorius Vivaspin® protein concentration devices with a 10 kDa cut. The correct processing of His tag was verified by SDS-PAGE analysis.
[00249] The protein concentration was quantified using the Bio-Rad Bradford micro assay (Bio-Rad) and the protein purity was validated by SDS-PAGE.
[00250] Site-directed mutagenesis of the gene encoding the CelS2 protein was performed using the Quickchange® mutagenesis kit (Stratagene), and according to the instructions provided by the manufacturer. The mutated protein was expressed and purified using methods identical to those used for wild type protein. Purification of Cel7A from Trichoderma reesei / Hypocrea jecorina
[00251] Cel7 cellulase The endo-acting GH7 family of Hypocrea jecorina has been purified from commercially available H. jecorina CELLUCLAST ™ extract (Novozymes), using purification protocols described by Jager et al., 2010, Biotechnology for Biofuels 3 ( 18). In summary, the H. jecorina extract was adjusted to 10 mM AmAc, pH 5.0, and the enzyme was purified using a DEAE-sepharose column attached to an Àcta purifier, running 10 mM AmAc pH 5.0 as a mobile phase. . The relevant fractions were grouped and concentrated using Sartorius Vivaspin protein concentration devices with a 10 kDa cut. Purity was assessed using SDS-PAGE analysis. Product analysis by mass spectrometry (MS) Flight time by matrix-assisted laser desorption / ionization (MALDI-TOF)
[00252] Two microliters of a 9 mg / mL mixture of 2,5-dihydroxybenzoic acid (DHB) in 30% acetonitrile were applied to a MTP 384 steel bottom TF target plate (Bruker Daltonics). One microliter samples were then mixed into the DHB drop and dried in a stream of air. The samples were analyzed with an Ultraflex MALDI-TOF / TOF instrument (Bruker Daltonics GmbH, Bremen, Germany) with a 337 nm nitrogen laser beam. The instrument worked in the positive acquisition dome and was controlled by the FlexControl 3.3 software package. All spectra were obtained using the reflector mode with an acceleration voltage of 25kV, a reflective acceleration of 26, and pulsed ion extraction of 40 ns in the positive ion mode. The acquisition range used was m / z 0 to 7,000. The data were collected from an average of 400 laser shots, with the lowest laser energy needed to obtain sufficient signal for the noise proportions. The peak lists were generated from the MS spectra using the Bruker FlexAnalysis software (Version 3.3). Post-source drop spectra (PSD) using the Bruker Daltonics LIFT system were recorded at 8 kV precursor ion acceleration voltage and fragment acceleration (19 kV LIFT voltage). Reflective voltages 1 and 2 were adjusted to 29 and 14.5 kV, respectively. Product analysis by HPLC and UHPLC High performance liquid chromatography (HPLC)
[00253] The isocratic HPLC was run on a Dionex Ultimate 3000 HPLC system established with a 4.6x250 mm Amide-80 column (Tosoh Bioscience, Montgomeryville, PA, USA), with an Amide-80 guard column. The mobile phase it consisted of 70% acetonitrile: 30% H2O MilliQ and the flow rate was 0.7 mL / min. The eluted oligosaccharides were monitored recording absorption at 190 nm. Chromatograms were recorded, integrated and analyzed using the Chromeleon 6.8 (Dionex) chromatography software. The main chitin degradation product by ChiC is (GlcNAc) 2 (> 95% of the total amount of degradation products on a molar basis), thus, only the peaks (GlcNAc) 2 were subjected to data analysis and were used to quantification of the extent of chitin degradation. A standard solution containing 0.10 mM (GlcNAc) 2 was analyzed at regular intervals during the sample series, and the resulting mean values (which exhibit less than 3% standard deviations) were used for calibration.
[00254] Because of the experimental simplicity and the yield, the degradation of chitin in reactions with ChiC and CBP21 was evaluated quantitatively by measuring only the concentration of the dominant product, (GlcNAc) 2. As a result of this simplification, product levels with respect to chitin degraded to the maximum tend to be up to 25% below expectations, based on the initial concentration of chitin. This "loss" is due to the non-detection of the following products: (1) oligomers and larger monomers that can be quantified to an estimate of 5% by weight of the total product mixture, (2) partially deacetylated products; the chitin that is used contains a small fraction of deacetylated sugars (about 8%), (3) oxidized sugars. Conditions that reinforce CBP21 activity to the maximum were used. The amounts of undetected oxidized sugars can be quantified in as high as 10-15% of the starting material (Figure 16). High performance liquid ultracromatography (UHPLC)
[00255] The UHPLC was run on an Agilent 1290 Infinity UHPLC system equipped with a diode array detector, established with a Waters Acquity UPLC BEH amide column (2.1x150 mm with a 2.1 x 30 mm pre-column, both with a column particle size material of 1.7 μm) using 5 μL sample injections. The separation of the oxidized oligosaccharides was obtained at a column temperature of 30 ° C and a flow rate of 0.4 mL / min starting at 72% ACN, (a): 28% Tris-HCl pH 8.0 15 mM, ( B) for 4 minutes, followed by an 11-minute gradient at 62% A: 38% B, which was maintained for three minutes. The reconditioning of the column was obtained by a gradient of two minutes, in initial conditions and subsequent run in initial conditions for 5 minutes. The eluted oligosaccharides were monitored by recording the absorption at 205 nm. The chromatograms were recorded, integrated and analyzed using the ChemStation rev. B.04.02 (Agilent Technologies). The identity of the eluted oligosaccharides was verified by MALDI-TOF MS analysis, according to the protocol described above. Degradation and sampling reactions General reaction conditions
[00256] Typical reactions started by mixing beta-chitin (0.5 to 2 mg / mL) with CBP21 (0.1 to 1 μM), ChiC (0.5 μM) or AnCDA (1 μM) or combinations of these enzymes, in a total volume of 0.5 mL in 1.5 mL plastic reaction tubes (Axygen Scientific Inc, CA), or in 1.8 mL borosilicate glass flasks with screw caps and rubber septa coated with TEFLON® . All reactions were performed in 20 mM Tris-HCl, pH 8.0, and incubated at 37 ° C with agitation at 1,000 rpm in an Eppendorf thermal mixer, unless otherwise stated. All reactions used for quantification were run in triplicates. All reactions used for qualitative purposes were repeated at least three times. Reactions with CBP21
[00257] Chitin solubilization by CBP21 was investigated by adding 1.0 μM CBP21 to a reaction solution containing 2.0 mg / mL beta-chitin, and 5.0, 1.0 or 0.2 mM ascorbic acid in 20 mM Tris-HCl pH 8.0. The reactions were incubated at 37 ° C and samples were obtained at regular time intervals for analysis by MALDI-TOF MS and UHPLC. In order to investigate the effects of reducing agents on CBP21 function, ascorbic acid was exchanged for both 1 mM reduced glutathione and 1 mM Fe (II) SO4 in some reactions.
[00258] The effect of CBP21 on chitinase activity was studied by adding 0.5 μM ChiC and 1.0 μM CBP21 to a reaction solution containing 2.0 mg / mL beta-chitin and 1.0 mM ascorbic acid in Tris -20 mM HCl pH 8.0. The reaction was incubated at 37 ° C and sampled at regular time intervals. Chitin degradation was measured by determining the concentration of (GlcNAc) 2 by HPLC. Control experiments, where CBP21 and / or ascorbic acid were excluded from the reaction solution, were performed in the same way.
[00259] To investigate whether CBP21 was able to cleave and / or oxidize soluble substrates, a 500 μL reaction solution containing 1.0 μM CBP21, (GlcNAc) 6 100 μM (0.12 mg / mL) and ascorbic acid 1 , 0 mM, all dissolved in 20 mM Tris pH 8.0, was incubated for 16 hours at 37 ° C before analysis of the product by MALDI-TOF MS. The same was done for control reactions, where both CBP21 and ascorbic acid, or both, were excluded from the reaction solution.
[00260] An experiment designed to visualize the range of polymeric products, generated by CBP21, was carried out combining CBP21 1.0 μM and AnCDA 1.0 μM in a reaction solution containing 2.0 mg / mL of beta-chitin, acid ascorbic 1.0 mM and 10 μL of CoCl2 (necessary for complete AnCDA activity) in 20 mM Tris-HCl pH 8.0. Control reactions were performed where CBP21 was excluded from the reaction solution, and / or replaced by 0.5 μM ChiC. The reactions were incubated for 16 hours at 37 ° C, followed by product analysis with MALDI-TOF MS. Reaction without molecular oxygen and related control reactions
[00261] In order to obtain a reaction solution without di-oxygen, all components of the reaction, except the enzyme or enzymes, were mixed in a closed glass bottle with a screw cap containing a rubber septum, and degassed using a Schlenk line. The enzyme was added to a separate flask, which was treated identically to the flask containing the reaction mixture. Before beginning the degassing procedure, a freshly prepared 1.0 M dithionite solution was added to the reaction solution to yield a final concentration of 10 mM and ensure total removal of molecular oxygen in the solution. The degassing procedure was performed by penetrating the rubber septum of the sealed bottle with a needle connected to the Schlenk line, followed by five cycles of degassing of 5 minutes (under vacuum) and 1 minute of N2 saturation. The final cycle leaves the bottles slightly pressurized by N2. After degassing both the reaction solution and the enzyme solution, a syringe was used to withdraw an appropriate amount of the enzyme solution, which was then immediately injected into the flask containing the reaction solution, in order to initiate the reaction, although injection of air bubbles has been avoided. The effect of an environment without molecular oxygen was evaluated by analyzing the activity of CBP21 1.0 μM in 2.0 mg / mL beta-chitin, in the presence of 1.0 mM ascorbic acid in 20 mM Tris-HCl pH 8.0 , by MALDI-TOF MS. In addition, the degradation of 0.1 mg / mL of beta-chitin by ChiC 0.5 μM in the presence of CBP21 1.0 μM and ascorbic acid 1.0 mM, in 20 mM Tris-HCl pH 8.0, was analyzed in the same environment with no oxygen. The samples were analyzed by HPLC after 16 hours of incubation at 37 ° C.
[00262] These last experiments were also carried out in the absence of sodium dithionite and in a higher concentration of chitin (0.45 mg / mL), but with other identical reaction conditions. It can be noted that, although every precaution has been taken to prevent oxygen from entering the reaction solution, the removal of di-oxygen is not 100% efficient. This can be observed by studying the result of the 18O2 experiment (see figure 7C), where the products that result from oxidation by 16O2 are detected.
[00263] Additional control experiments were carried out running from experiments where 0.45 mg / mL beta-chitin, CBP21 1.0 μM, ChiC 0.5 μM and reduced glutathione 1.0 mM, in Tris-HCl 20 mM pH 8.0, were run in the presence of both 2.0 mM sodium azide and 2.0 mM potassium cyanide. The reactions were incubated at 37 ° C, sampled in 30, 60 and 90 minutes, and the products were analyzed by UHPLC. Reactions under metal chelation conditions
[00264] The divalent cations were removed by chelation, through dialysis of a 10 mg / mL solution of CBP21, in a buffer containing 20 mM Tris-HCl and 5 mM EDTA. The protein solution was present in a Slide-A-Lyzer cassette (Pierce) with a dialysis membrane with a 10 kDa MW cut. Dialysis was carried out for 16 hours at 4 ° C with a protein due to buffer volume of 1: 1,000, with moderate magnetic stirring. The reactions with CBP21 without metal were carried out in the manner described above, except that EDTA was added to the buffer reaction at a final concentration of 5 mM. The reactivation of CBP21 without metal was achieved by adding both ZnCl2 and MgCl2 to the reaction mixture, in a final concentration of 25 mM. The reactions were run for 180 minutes and sampled at 30-minute intervals. For the reactivation experiment, divalent cations were added to the appropriate reaction solutions, immediately after the third sampling (90 minutes). Chitin degradation was measured by determining the concentration of (GlcNAc) 2 by HPLC. Reactions in buffered H218O
[00265] Beta-chitin and ascorbic acid were each suspended / dissolved in H218O to yield concentrations of 2 mg / mL and 1.0 M, respectively. In order to reach the correct pH in the H218 ° reaction solution, 10 μL of 1.0 M Tris-HCl pH 8.0 were transferred to a glass flask, which was heated with dry air at 60 ° C, until all the liquid evaporated. 498 μL of the beta-chitin suspension was transferred to the same glass vial, reaching the desired pH for the reaction. Concomitantly, 0.5 μL of ascorbic acid (dissolved in H218O) and 0.75 μL of a 660 μM solution of CBP21 (dissolved in H216O) were added to the solution to initiate the reaction, yielding final concentrations of 1 mM for ascorbic acid and 1 μM for CBP21. The glass flask was sealed with a screw cap with a rubber lining coated with TEFLON® to ensure the least possible contamination of H216O from the air phase in the reaction solution. After incubation for 16 hours at 37 ° C, the reaction products were analyzed by MALDI-TOF MS. Reactions in a saturated solution of 18O2 gas
[00266] In a glass vial containing a reaction mixture of 2.0 mg / mL of beta-chitin and 1.0 mM ascorbic acid, in 20 mM Tris pH 8.0, CBP21 was added to yield a final concentration 1.0 μM. Immediately after starting the reaction, a screw cap containing a rubber septum coated with TEFLON® was used to close the flask, and the Schlenk line was used to remove dissolved molecular oxygen and fill the free space with N2 (according to the procedure described in the title “reaction without molecular oxygen”). After the five cycles of degassing and filling with N2 were completed, a gas cylinder containing compressed 18O2 gas was connected to the vial by pushing a needle through the vial's septum. The flask was then placed in a vacuum, removing atmospheric gas that remained in the tube and in the free space of the flask. After isolating the vial and the 18O2 gas cylinder from the rest of the Schlenk line, properly closing the open valves, the free space of the vial was filled with 18O2 gas by slowly opening the gas cylinder regulator. The flask was then removed from the needle connections and, after incubation at 37 ° C for 16 hours, the reaction products were analyzed by MALDI-TOF MS. Reaction of beta-chitin with Fenton chemistry
[00267] In order to determine whether Fenton's chemistry (Fe2 + and H2O2 combined in a saturated oxygen solution to yield reactive hydroxyl radicals; Sawyer et al., 1996, Acc. Chem. Res. 29: 409) can yield soluble products from chitin, 2 mg / mL of beta-chitin suspended in 20 mM Tris-HCl pH 8.0 were incubated for 16 hours with 10 mM Fe (II) SO4 and 0.3, 0.03 or 0.003% of ( v / v) H2O2, in plastic sample tubes with perforated caps (to release gas generated during the reaction). The samples were analyzed by MALDI-TOF MS. Control experiment with another identified CBM33 extracting the CBM33 (EF0362) genome from Enterococcus feacalis
[00268] The gene encoding the mature CBM of the 33 family of Enterococcus faecalis, EfCBM33 (Uniprot ID: Q838S1; EF0362; uniprot.org/uniprot/Q838S1), without its natural main peptide, was cloned into the vector pRSET-B-CBP21 in alignment with the main CBP21 peptide, replacing the gene encoding CBP21. The protein was expressed in E. coli BL21 DE3 cells, collected from the periplasmic fraction by cold osmotic shock, and purified with homogeneity by chitin affinity chromatography. Thus, this protein was expressed and purified in exactly the same way as in CBP21, using the main CBP21 peptide to direct secretion. Fractions containing pure protein (assessed by SDS-PAGE) were pooled and concentrated using an Amicon centrifugal concentrator with a 10 kDa cut to yield a 20 mg / mL solution. The protein was crystallized by drop diffusion vapor diffusion experiments using a crystallization liquor containing 1.0 MK / Na tartrate, 0.1 M imidazole pH 8.0 and 0.2 M NaCl. The crystals in the pyramidal form and measuring approximately 0.2 mm in width (see figure 15A) were obtained after 48 hours of incubation at room temperature. A 0.95 Â data set was collected from a single crystal and the structure was calculated by molecular substitution using CBP21 (PDB ID 2BEM) as a template. The refinement is complete, but not published. The quality of the data is illustrated by panel B of figure 15. Panel C of this figure 15 shows a structural overlap of CBP21 and EfCBM33.
[00269] For use in chitin degradation experiments, eight crystals were collected from a 2 μL drop, using a nylon loop, and were transferred to a 4 μL drop containing the crystallization liquor from the reservoir of plug. The crystals were mixed together in order to "wash" potential contaminants. After the first wash, the crystals were transferred to a new drop of 4 μL containing the crystallization liquor in a second wash cycle. Finally, all crystals were transferred and dissolved in a 4 μL drop containing 20 mM Tris-HCl pH 8.0. The resulting solution was diluted by adding it to a test tube containing 46 μL of 20 mM Tris-HCl, pH 8.0. For reactions with beta-chitin, 5 μL of the EfCBM33 solution was mixed with a 95 μL solution containing 2 mg / mL of beta-chitin and 2 mM ascorbic acid in 20 mM Tris-HCl pH 8.0; the reaction mixture was then incubated for 90 minutes at 37 ° C, in a test tube incubator that rotates at 1,400 rpm. The products soluble in the reaction supernatant were analyzed by MALDI-TOF, using the same methods as those used to test CBP21 activity. Additionally, the ability of EfCBM33 to reinforce alpha-chitin degradation was investigated by conducting an experiment where 2.0 mg / mL of alpha-chitin (shrimp shells) was incubated with 0.3 μM of Enterococcus feacalis chitinase (name of protein (EF0361)) in the presence or absence of 0.3 μM EfCBM33 and 1.0 mM reducer (R: reduced glutathione), incubated at 37 ° C with shaking at 900 rpm. An increase in chitinase activity is evident in the presence of EfCBM33 and reducing agent. Determination of the reaction speed of CBP21 and degree of substrate oxidation
[00270] Using the UHPLC method to separate the oxidized chitosoligosaccharides, pure samples of GlcNAc3GlcNAcA and GlcNAc4GlcNAcA were obtained by fractionation of samples of beta-chitin treated by CBP21 in the presence of ascorbic acid. The fractions were vacuum dried (SpeedyVac), and resuspended in 50 μL of MilliQ water. Purity was checked by MALDI-TOF MS. Isolated GlcNAc3GlcNAcA or GlcNAc4GlcNAcA were each incubated for 2 hours at 37 ° C with 7.0 μM of a recombinant pure chitinase from family 19 (ChiG of Streptomyces coelicolor (Hoell et al., 2006, FEBS J. 273: 4889)) , resulting in the production of equimolar amounts of GlcNAc2 and GlcNAcGlcNAcA or GlcNAc2GlcNAcA, respectively. The amount of GlcNAc2 that results from hydrolysis was estimated using a predetermined standard curve. The response factors for GlcNAcA containing oligosaccharides were obtained by determining the peak area ratios of GlcNAcGlcNAcA / GlcNAc2 and GlcNAc2GlcNAcA / GlcNAc2 and found to be 0.71 and 0.81, respectively. A response factor for GlcNAc3GlcNAcA was approximately 0.88, by extrapolating the two response factors determined experimentally. Using the determined response factors, the GlcNAcGlcNAcA and GlcNAc2GlcNAcA peaks can be quantified using GlcNAc2 for calibration. In experiments for the simultaneous detection and quantification of GlcNAc2 and oxidized oligomers, 1m0 mM of reduced glutathione was used as a reducer instead of ascorbic acid, due to the latter interfering with the chromatographic analysis. In addition, the reactions contained 0.45 mg / mL beta-chitin, CBP21 1.0 μM and 0.5 ChiC μM in 20 mM Tris-HCl pH 8.0.
[00271] The reactions were incubated at 37 ° C in an Eppendorf thermal mixer with shaking at 1,000 rpm, and sampled in 30, 60, 120 and 300 minutes. All samples were mixed 1: 1 with 100% acetonitrile in order to stop the reaction and the soluble products were analyzed by UHPLC. The separation of the oxidized oligosaccharides and GlcNAc2 was achieved using a column temperature of 30 ° C and a flow of 0.4 mL / min, with a gradient start at 80% ACN (a): 20% Tris-HCl pH 8.0 15 mM, (B) for 4.5 minutes, followed by an 11-minute gradient at 63% A: 37% B, which was maintained for 3.5 minutes. The reconditioning of the column was achieved by a gradient of two minutes in initial conditions, and subsequent running in initial conditions of 5 minutes. The eluted oligosaccharides were monitored by recording the absorption at 205 nm. The chromatograms were recorded, integrated and analyzed using the ChemStation rev. B.04.02 (Agilent Technologies).
[00272] In order to approximate the rate of CBP21 oxide-hydrolytic activity, reactions containing 0.45 mg / mL of beta-chitin, CBP21 1.0 μM and 1.0 mM reduced glutathione in 20 mM Tris-HCl pH 8.0 were incubated at 37 ° C and sampled at 10, 15, 30.45, 60 and 300 minutes. Instead of stopping the reaction with acetonitrile, a cocktail of purified recombinant chitinases containing 28 μM ChiC (see above), 71 μM ChiG (Hoell et al., 2006, supra), 63 μM ChiB (Brurberg et al., 1995, Microbiology 141: 123, Brurberg et al., 1996, Microbiology 142: 1581) and ChiA 15 μM (Brurberg et al., 1996, supra, Brurberg et al., 1994, FEMS Microbiol. Lett. 124: 399) was added to the sample (0.1 volume) in order to obtain complete and rapid degradation of chitin. In these conditions, the insoluble chitin disappears completely in 30 minutes. The amounts of the oxidized products (exclusively GlcnAcGlcNAcA and GlcNAc2GlcNAcA) were determined using the UHPLC method highlighted above. Analysis and quantification of glucose and cellobiosis by HPLC (E7 and CelS2)
[00273] The samples containing glucose and cellobiose were analyzed by isocratic HPLC, run on a Dionex Ultimate 3000 HPLC system established with a 7.8x100 mm Rezex RFQ-Fast Fruit H + column (Phenomonex) heated to 80 ° C. The mobile phase consisted of 5 mM sulfuric acid and the flow rate used was 1.0 mL / min. The eluted glucose and cellobiose were monitored by recording a refractive index. Quantification was obtained by running glucose and cellobiose patterns. Chromatograms were recorded, integrated and analyzed using the Chromeleon 6.8 (Dionex) chromatography software. Analysis of natural and oxidized cell oligosaccharides using HPAEC (E7 and CelS2)
[00274] The separation of natural and oxidized cell-oligosaccharides was achieved using a Dionex Bio-LC equipped with a CarboPack PA1, a column temperature of 30 ° C and a flow rate of 0.25 mL / min, with initial conditions, ie ie, 0.1 M NaOH. A stepwise linear gradient with increasing amounts of sodium acetate was applied, increased by 0.1 M NaOH and 0.1 M sodium acetate in 10 minutes, then in 0.1 M NaOH and 0.3 M sodium acetate for 25 minutes, then increasing to 0.1 M NaOH and 1.0 M sodium acetate in 30 minutes, which was maintained for 10 minutes. The reconditioning of the column was achieved by a gradient of one minute in initial conditions, and a subsequent run in initial conditions for 14 minutes. The eluted oligosaccharides were monitored for detection of PAD. Chromatograms were recorded and analyzed using Chromeleon 7.0. Peak identification was achieved by a procedure including the following steps: the oxidized cell-oligosaccharides were separated using a Dionex Ultimate 3000 UHPLC system that carries a 150x2.1 mm Hypercarb column (Thermo Scientific), running a water gradient / 0 , 1% TFA and acetonitrile / 0.1% TFA (from 20 to 80% acetonitrile), according to the method developed by Westphal et al., 2010, Journal of Chromatography A 1217: 689-695. The eluted peaks were manually fractionated, freeze-dried, redissolved in MilliQ water and identified using MALDI-TOF MS. The pure oxidized cell oligosaccharides with known identity were then analyzed using the HPEAC method described above, in order to establish the identity of the oxidized cell oligosaccharides generated by CelS2 and E7. Cellulose degradation experiments (E7 and CelS2)
[00275] The tests performed to evaluate the function of E7 and CelS2 were established using a variety of cellulosic substrates (AVICEL®, filter paper and wood chips from the explosion of poplar vapor), both at pH 5.5 ( 20 mM sodium acetate buffer), 6.5 (20 mM Bis-Tris buffer) and 8.0 (20 mM Tris buffer), in reaction mixtures containing 1.0 mM MgCl2. The effect of the presence of an external electron donor was investigated by adding 1.0 mM or 0.5 mM reduced glutathione, or ascorbic acid, to the reaction mixture (see captions for details). Results
[00276] It is noted here that CBP21, a single domain protein comprising a CBM33 domain, is in fact an enzyme that catalyzes an oxide-hydrolytic cleavage of glycosidic bonds in crystalline chitin, thereby opening up the inaccessible polysaccharide material for glycolide hydrolysis normal hydrolases. This enzymatic activity was first discovered when traces of unnatural chito-oligosaccharides were detected by incubating monocrystalline beta-chitin nanofibers with CBP21 (Figure 2A). The products were identified as chitin oligosaccharides with a 2- (acetylamino) -2-deoxy-D-gluconic acid (GlcNAcA) at the reducing end (Figures 2B and 3). It is observed that the addition of reducers greatly increases the efficiency of the reaction (Figures 4 and 6), allowing the destruction of large crystalline beta-chitin particles with CBP21 only (Figure 2C), which releases a range of oxidized products ( Figure 5 and below) and reinforces the efficiency of chitinase at much higher levels than previously observed (compare figure 1C with figure 4).
[00277] If CBP21 has acted randomly on crystalline surfaces, the generation of larger oligosaccharides is expected, which are difficult to detect due to their poor solubility. Most of the soluble products generated by CBP21 in the presence of a stronghold had a DP below 10 (Figure 2A; Figure 5). To visualize larger products, a newly cloned chitin deacetylase from Aspergillus nidulans (AnCDA) was explored to increase the solubility of larger chitin fragments by deacetylation. This approach actually revealed the formation of chitin fragments with higher DP, both with CBP21 (Figure 2D) and with an endoquitinase (ChiC; Figure 2E). Both CBP21 and ChiC generated long products, indicative of a type of “endo” activity.
[00278] Two important characteristics stand out. First, during the use of CBP21, all detected products are oxidized (that is, they contain a fraction of GlcNAcA), confirming the observation that CBP21 catalyzes the oxidative hydrolysis of glycosidic bonds. In the second, while the products released by ChiC represent a continuous sequence of sizes, the products released by CBP21 are dominated by oligosaccharides in even numbers (Figures 2D and 6). This shows that ChiC tends to cleave any glycosidic bond, whereas CBP21 shows a strong preference for cleaving each second glycosidic bond. Keeping in mind the periodicity of the disaccharide in the substrate (Figure 1A), this important observation implies that ChiC approaches single polymer chains from “any side”, while CBP21 has to approach the substrate from a fixed side. The latter situation can be achieved if the polysaccharide chain cleaved by CBP21 is part of an intact crystalline structure. Evidently, the endoquitinase ChiC and CBP21 have different roles in the hydrolysis of chitin.
[00279] The CBP21-mediated cleavage mechanism has been further investigated by isotope labeling. The H218O experiments showed that one of the oxygen atoms introduced at the new oxidized chain end originates from water (Figure 7A). The only possible source for the second oxygen is molecular oxygen, and this was confirmed by experiments carried out under 18O2 saturation conditions (Figures 7B and C). The removal of dissolved molecular oxygen or in the reaction solution inhibited CBP21 activity (Figures 8A, B and C), confirming the requirement for molecular oxygen for catalysis. The strong inhibition by cyanide, a common imitation of molecular oxygen, helps in the crucial role of the oxidative stage (Figures 8C and D). Thus, the reaction catalyzed by CBP21 comprises a hydrolytic step and an oxidation step, as summarized in figure 7D. Enzymatic oxidohydrolysis of polysaccharides has not been described so far. CBP21 is referred to herein as a "chitin oxide hydrolase". Likewise, GH61 proteins are referred to as “cellulose oxidohydrolases”.
[00280] It was observed that CBP21 catalysis is dependent on the presence of a divalent cation (Figure 9), which can bind to the conserved histidine motif (Figure 1F). Interestingly, the activity of GH61 proteins also depends on divalent cations (Harris et al., 2010, Biochemistry 49: 3305). Structural studies of both CBP21 (Vaaje-Kolstad et al., 2005, J. Biol. Chem. 280: 11313) and GH61 proteins (Harris et al., 2010, Biochemistry 49: 3305; Karkehabadi et al., 2008, J Mol. Biol. 383: 144) show considerable structural plasticity at the metal bonding site, explaining why the metal bonding site is promiscuous and why the need for divalent cations is very nonspecific (as shown in figure 9 and in Harris et al., 2010, Biochemistry 49: 3305). The mutation of the second histidine (His114 in CBP21 and His89 in GH61E of Thielavia terrestris) in the metal binding motif neutralized the activity of both proteins (Figure 10 and Harris et al., 2010, Biochemistry 49, 3305, respectively). It is possible that the binding of the metal ion directly assists in the binding / stabilization / activation of reactive oxygen and / or hydrolytic water species during catalysis. Alternatively, the metal ion may be essential to stabilize the structure of the protein's active site region, without directly interacting with oxygen or water.
[00281] The reducers reinforced the oxide-hydrolytic activity of CBP21 at extreme levels (Figures 2C, 4, 6, 11), which may well be beyond what is always achieved in nature. Reducers are likely to act as electron donors in the oxidative enzymatic process, but the exact flow of electrons from the reducer to molecular oxygen is still unclear. One option is for electron donation to occur through the generation of reactive oxygen species such as O2-. Another option is that the electrons are somehow channeled into a complex involving CBP21, the substrate and O2, which implies that reactive oxygen species, such as O2-, emerge only in the substrate and will not be, or will be, little , present in the solution. More interestingly, and of biotechnological importance, these data show that the activity of these new enzymes can be enhanced considerably by simply adjusting the reaction conditions.
[00282] Several control experiments were conducted, in which all confirmed the conclusion that the formation of oxidized products occurs only in the presence of CBP21 and crystalline substrate. The presence of reducers alone did not yield oxidized products (Figure 12) and did not potentiate or inhibit the action of chitinase (Figures 4 and 10B). When incubated under ideal conditions with hexameric N-acetylglycosamine, neither degradation products nor oxidized oligosaccharides were observed (Figure 13). We also studied the possibility that CBP21 can function without directly interacting with the cleaved bond itself, since it is possible to contemplate some type of “destruction” mechanism induced by CBP21, making the substrate more accessible for the action of species of reactive oxygen generated in an independent way from CBP21, for example, by Fenton chemistry. However, none of the soluble products can be detected by subjecting beta-chitin to Fenton's chemistry (Figure 14). All of these experiments confirm that CBP21 actively participates in the oxide-hydrolytic cleavage reaction.
[00283] As shown in figure 8, CBP21 activity is greatly inhibited by cyanide, a known imitation of O2, but not by azide, a known inhibitor of heme proteins. Several reducers capable of functioning as electron donors reinforced CBP21 activity (Figures 2C and 11). The experimental data indicate that the oxidation stage catalyzed by CBP21 is an independent cofactor and depends on an external electron donor. Oxygenases as an independent cofactor have been described previously, but it is known that these enzymes normally use conjugated carbanions in the substrates as electron donors (Fetzner and Steiner, 2010, Appl. Microbiol. Biotechnol. 86: 791), a mechanism that is probably not the case of a polysaccharide substrate. If the oxidation step occurs first, this may imply that CBP21 catalyzes the cofactor regardless of the oxygenation of a saturated carbon, which is unprecedented and, perhaps, not very likely. On the other hand, a mechanism like this can yield an intermediate product, for example, an ester bond, which may be more prone to hydrolysis than the original glycosidic bond. Alternatively, the hydrolytic step may occur first, which may imply that CBP21 is capable of hydrolyzing glycosidic bonds in a crystalline environment, using a mechanism unknown at the moment. A hydrolytic step like this may require some degree of substrate dissortion (Davies et al., 2003, Mapping the conformational itinerary of beta-glycosidases by X-ray crystallography. Biochem. Soc. Trans. 31: 523 and Vocadlo and Davies, 2008 , Mechanistic insights into glycosidase chemistry. Curr. Opin. Chem. Biol. 12: 539), which looks like a challenge in a crystalline envelope. However, in favor of this mechanism, the subsequent oxidation of the resulting sugar and aldehyde ("reducing end") is more direct than the oxidation of a saturated carbon.
[00284] CBP21 introduces chain breaks, which are probably the most inaccessible and rigid parts of the crystalline polysaccharide substrates, and its mode of action differs fundamentally from the mode of action of glycoside hydrolases. The key difference is that glycoside hydrolases are designed to host a chain of simple “soluble” polysaccharides in their slits or catalytic pockets, and that their affinity and proximity to the crystalline substrate tend to be mediated by non-hydrolytic binding domains. In contrast, the CBP21 and GH61 enzymes have flat surfaces (Figures 1D and E) that bind to the well-ordered, solid and flat surfaces of the crystalline material, and catalyze chain breaks by an oxide-hydrolytic mechanism. The break in the chain will result in the interruption of the crystalline envelope and greater accessibility to the substrate, an effect that can be increased by modifying one of the moving ends of the chain. At the cleavage point, one of the new ends is a normal non-reducing end (indicated by R-OH in figure 7D). The other new end may be a new reducing end if the cleavage has been carried out by a normal glycoside hydrolase. However, in this case, the product is different and the last sugar is oxidized to become 2- (acetylamino) -2-deoxy-D-gluconic acid (Figures 2B and 7D; GlcNAcA). This new “acid chain end” will interfere with the normal crystalline envelope, as it will not have the normal chain shape of the sugar ring, and as a result of carrying a load.
[00285] The enzyme activity demonstrated in this study is difficult to discover, due to the dependence on the detection of products with little solubility, and with a potentially high tendency to remain attached to the crystalline material. In this sense, working with chitin is easier than working with cellulose, because the product's solubilities are slightly higher, and because the crystalline envelope is less compact (Eijsink et al., 2008, Trends Biotechnol. 26: 228). The experiment with chitin deacetylase (Figures 2D and E) provided essential ideas, but this approach cannot simply be used for cellulose. Looking for the activities of breaking the chain of GH61 proteins with a commonly used reducing end, the assays obviously will not work due to the oxidative mechanism.
[00286] Using methods to quantify oxidized products, it was possible to estimate the speed and the degree of oxidation in various conditions (Figure 16). When CBP21 acts alone in beta-chitin, under ideal conditions, the oxidation rate is in the order of 1 per minute and the maximum extent of oxidation is about 7.6% of the sugars. The simultaneous degradation of beta-chitin with CBP21 and ChiC under ideal conditions leads to the oxidation of approximately 4.9% of the sugars. It should be noted that, in nature, enzymes such as CBP21 act normally and simultaneously with at least one and, in the case of S. marcescens, three chitinases (Suzuki et al., 1998, Biosci. Biotechnol. Biochem. 62: 128 ).
[00287] Experiments conducted with the CBM33 EfCBM33 family protein revealed that this protein is functionally similar to CBP21. The results of the MS analysis, represented in panel D of figure 15, are similar to those obtained with CBP21, clearly showing the formation of oxidized products and the oxide-hydrolytic properties of this protein. It is interesting to note that EfCBM33 not only works with beta-chitin (Figure 15D), but also with alpha-chitin (Figure 15E).
[00288] The efficiency of CBM33 proteins in cellulose as the substrate was examined. E7 mature wild type, CelS2 and a cellulase classified as belonging to the GH 7 family and originating from Trichoderma reesei, called Cel7A (Harjunpaa et al., 1999, FEBS Letters 443: 149-153), were purified in ~ 95 % purity using the cloning, expression and purification strategies described in the material and methods section. Finally, a cellulase mixture called CELLUCLAST ™, which is a commercial product readily available and known from Novozymes, was used.
[00289] To determine whether CelS2 and / or E7 have the ability to release soluble sugars from crystalline cellulose, CelS2 or E7 was incubated with AVICEL® in the presence or absence of an external electron donor (reduced glutathione or ascorbic acid). Possible reaction products were analyzed using MALDI-TOF MS for the qualitative detection of product types and HPAEC (high pressure ion exchange chromatography), a chromatographic method that allows product identification and, in principle, quantification. In fact, soluble oligomeric products were observed with both proteins (shown only for CelS2; figures 17-19) and with both detection methods. Natural cell oligosaccharides were observed both in conditions with and without external electron donor, but in substantially greater quantities in the latter condition. Oxidized cell-oligosaccharides were observed only in experiments where an external electron donor was present.
[00290] More specifically, the analysis of MALDI-TOF MS revealed the presence of cell-oligosaccharides with an oxidized reducing end (that is, cell-oligosaccharides with the reducing glucose fraction replaced by a gluconic acid fraction; figures 17 and 18 ). The degree of polymerization (DP) of oxidized, soluble and released oligosaccharides, visible in figure 17, varied from DP4 to DP7, and the product signs showed an alternative intensity / quantity depending on the cell-oligosaccharide that is numbered as odd or even in terms DP (called even-numbered cell oligosaccharides). The HPAEC analysis of the same samples reflected the results provided by the MALDI-TOF MS analysis, showing that the presence of peaks with alternating intensities, which represents oxidized cell-oligosaccharides (Figure 19).
[00291] The even numbered oligosaccharide domain is a logical consequence of the fact that CBM33 enzymes attack the polysaccharide chains in their crystalline environment. Since the cellulose and chitin repeat unit is a dimer, only every second sugar / glycosidic bond in the crystalline polysaccharide chain is prone to cleavage by the CBM33 enzyme, which means that the released products may tend to have a numbered DP pair. (NB. Acid hydrolysis of both crystalline cellulose and chitin provides an even distribution of odd and even numbered oligosaccharides).
[00292] Figure 19 shows that incubation with CelS2, in the presence of a reducing agent, leads to the cleavage of cellulose chains in the crystal in an oxide-hydrolytic manner, releasing soluble oxidized cell-oligosaccharides (light gray, upper line). This also leads to the further release of unoxidized cell oligosaccharides, which is probably partly the result of the interruption of the AVICEL® crystalline surface, which facilitates the release of smaller oligomers that are captured in the crystal. In addition, the cleavage of the chain by CelS2 near the reducing end of a cellulose chain will increase in such products.
[00293] The reactions containing crystalline cellulose (filter paper) and cellulases (both a cellulase mixture called CELLUCLAST ™, and a single component cellulase, Cel7A) were monitored to release cellobiose and glucose in the presence and absence of CelS2 / E7, and / or an external electron donor.
[00294] When CELLUCLAST ™ was incubated with filter paper in the presence of CelS2 or E7, the glucose yield was actually higher than when the filter paper incubated with CELLUCLAST ™ alone (Figure 20; ~ 3.0 fold increase for CelS2 and E7 after 24 hours incubation, and ~ 9 times increase for CelS2, and ~ 3.5 times increase for E7 after 120 hours incubation). When both CelS2 and an external electron donor were added, glucose yields were even higher (Figure 20; ~ 5-fold increase for CelS2, and 3.5-fold increase for E7 after 24-hour incubation, and ~ 11-fold increase times for CelS2 and an increase of ~ 4.5 times for E7 after 120 hours incubation), indicating that the presence of an external electron donor is really important to reinforce the effect of CelS2 and E7.
[00295] Since the CELLUCLAST ™ product contains a complex mixture of hydrolytic activities that can complicate the interpretation of results, a monocomponent cellulase (Cel7A) was purified from CELLUCLAST ™ (Novozymes) and used to investigate the efficiency of the reinforcement of CelS2. Effects similar to those observed during the combination of CELLUCLAST ™ and CelS2, in the presence and absence of an external electron donor, were also observed during the incubation of Cel7A with CelS2 (Figure 21). In the absence of an external electron donor, CelS2 improved the activity of Cel7A (in terms of cellobiose yield) 5.5 and 7.3 times after 18 and 40 hours of incubation, respectively. In the presence of an external electron donor, CelS2 improved the activity of Cel7A 6.7 and 9.8 times after 18 and 40 hours of incubation, respectively.
[00296] Figures 20 and 21 show that, in addition to enhancing cellulose activity, the presence of CelS2 in both cases also helped to maintain cellulase activity over time. While the activity of cellulases incubated in the presence or absence of an external electron donor stops almost completely in the first 18 - 24 hours, cellulase activity continues in samples incubated with CelS2. This effect may be a partial explanation for the synergistic effects that CBM33 proteins have on the degradation of chitin or cellulose by hydrolytic enzymes (chitinase and cellulases, respectively).
[00297] The oxidized products, generated by CelS2 in the presence of an external electron donor, exist in a balance of two forms, the glucono delta-lactone that gains in population in pH slightly acidic pH, and the form of gluconic acid that dominates at slightly alkaline pH. At slightly alkaline pH (for example, pH 8.0) it is likely that the charge that develops on the crystalline surface of the cellulose, due to the activity of CelS2, may assist in the desorption and interruption / solubilization of the cellulose crystal and, thus, increases the accessibility of the substrate for cellulases. However, it is possible that oxidized oligosaccharides and or oxidized ends of the chain in cellulose crystals may also inhibit certain cellulases (eg exo-acting enzymes), probably causing the degree of CelS2 / CBM33 activity to be carefully adjusted for optimal reinforcement efficiency.
[00298] During the probing of the cellulose reinforcing properties, from CelS2 at various pHs, using Cel7A monocomponent as the hydrolytic component of cellulose, pH 5.5 becomes the ideal pH for the activity. Less activity was observed at pH 6.5 and no activity can be detected at pH 8.0 (data not shown). The most obvious reason for the decrease in cellulose hydrolysis increasing pH is the pH stability and the efficiency of Cel7A. Cellulase is thick to inactivate at pH 8.0, which is a common feature of fungal cellulases in general (Garg & Neelakantan, 1981, Biotechnology and Bioengineering 23: 1653-1659 and Wood, 1985, Biochemical Society Transactions 13: 407-410 .). CelS2 is a bacterial enzyme that originates from Streptomyces species that are known to grow ideally on cellulosic substrates, under approximately neutral pH conditions (Kontro et al., 2005, Letters in Applied Microbiology 41: 32-38.). The data shown in figure 19 show that CelS2 is active at pH 8.0.
[00299] Due to the pH dependent properties of cellulases, the synergy experiments reported here were performed at slightly acidic pH, which may be sub-ideal for the particular CBM33 used (mainly CelS2 of Streptomyces). Thus, it is possible that the observed effects of CelS2 are less than that which can occur under conditions optimized for CelS2 activity.
[00300] More generally, one skilled in the art will know that natural enzymes vary in terms of their pH and ideal temperatures for the activity. Those skilled in the art will know that this will also apply to hydrolytic enzymes, such as cellulases and chitinases, and oxido-hydrolytic enzymes, such as CBP21 and CelS2. It is obvious that to obtain the ideal complete reaction efficiency, and / or to maximize the reinforcing effect of a CBM33, it is necessary to take into account the ideal pH and the ideal temperature of both CBM33 and hydrolytic enzymes. Enzymes with varying ideal pH and temperature can be obtained by selecting appropriate enzymes from nature, or by modifying properties such as ideal pH of natural enzymes, using types of technology to genetically modify the protein.
[00301] It is possible that the pH affects the performance of a type (s) of CBM33 + hydrolase (s) of the enzyme system, due to the pH affecting the balance between lactone and the acidic form of the oxidized products, which can again affect the efficiency of hydrolytic enzymes. The pH can also affect the reducer.
[00302] These experiments show that some CBMs of the 33 family, such as CelS2 and E7, are active in crystalline cellulose, reinforcing cellulase activity both in the absence and in the presence of an external electron donor, but show substantially reinforcing activity / effect larger with the electron donor present. These observations, as well as the results previously obtained by the action of CBP21 in chitin, indicate that the oxidation of one of the ends of the recently generated chain is important for the function of these CBM enzymes33. That oxidation, which is in fact also part of the mechanism for CBM33s to act on cellulose, is further aided by data showing that cyanide, a well-known imitation of oxygen, inhibits the generation of oxidized cell oligosaccharides when CelS2 is incubated with AVICEL® in the presence of an external electron donor (Figure 22).
[00303] Additionally, a CelS2 variant was generated containing a mutation of a possibly essential conserved residue, histidine 144, in alanine. This histidine is one of the most conserved residues in the metal binding motif, characteristic of CBMs in the family 33, and corresponds to His114 in CBP21 (Figures 1D and F; Figure 10; Vaaje-Kolstad et al., 2010, Science 330: 219- 222). Figure 23 shows that CelS2-H144A was not able to generate oxidized cell-oligosaccharides in the presence of an external electron donor (Figure 23).
[00304] It is important to emphasize once again that the experiments were carried out at just one pH, which probably may not be ideal for one of the two components of the enzyme (CBM33 or the hydrolytic enzyme), in the manner previously discussed. The cellulases used work ideally in the acid range of the pH scale, while CBM33s that oxidize cellulose are more active in the basic range of the pH scale. Those skilled in the art will understand that different and better results can be obtained when adapting the pH of the reaction, and / or selecting enzyme variants that are more suitable for the pH used here, and work together at this particular pH. The full potential of the oxidative reinforcement effect, therefore, may not be seen in these experiments.
[00305] Finally, other cellulosic variants (filter paper and aspen sawdust from the steam explosion) were also investigated as substrates for CelS2. Oxidized and soluble cell oligosaccharides were not observed by MALDI-TOF MS analysis (results not shown). This is most likely due to the high DP of cellulose in these substrates; cleavages on crystalline surfaces cannot easily lead to the release of soluble cell-oligosaccharides (= short) when the complete SD is very high. AVICEL® is special, which is a form of microcrystalline cellulose that has a very low DP (~ 60-100; Wallis et al., 1992, Carbohydrate Polymers 17: 103-110 and Mormann and U, 2002, Carbohydrate Polymers 50: 349-353) compared to that of the other tested substrates.
[00306] However, when the CBM33s cellulase reinforcing effect, in this case CelS2, was investigated with a more "natural" substrate (represented by poplar sawdust from the steam explosion), the CelS2 effect was actually present , as shown by a ~ 2-fold increase; Figure 24). The data shown for the effect enhanced by CELLUCLAST ™ on sawdust from poplar from the steam explosion, which represent a more likely substrate, which is used in industrial biomass degradation applications, also show that the activity of CelS2 does not depend on the substrate is of high purity. Of course, CelS2 also acts on cellulose present in a more natural environment (that is, inserted into a plant / plant cell wall matrix).
[00307] In addition to showing completely new enzymatic activity to modify the solid surfaces of the polysaccharide, these results point in new directions for the enzymatic conversion of recalcitrant polysaccharides. Evidently, proteins of the CBM33 family (such as CBP21, EfCBM33 (Figure 15), E7 and CelS2 (Figures 17-24)) and proteins of the GH61 family (such as the GH61 proteins found in T. terrestris, Phanerochaete chrysosporium or Hypocrea jecorina) can greatly increase the efficiency of hydrolytic enzyme mixtures for chitin and cellulose, and a first look at the potential of GH61 proteins for cellulose conversion has actually been made very recently (Harris et al., 2010, Biochemistry 49: 3305 ). The present invention shows how the beneficial effects of CBM33 and proteins of the GH61 family can be enhanced by adjusting the reaction conditions, that is, the combined presence of an appropriate metal ion and agents that are reducing, and / or that generate reactive oxygen species . Evidently, the dependence of these oxide-hydrolases in the presence of molecular oxygen and reducers that act as external electron donors provides parameters to determine the process.
[00308] Example 2: Effects of CelS2 on cellulose degradation by cellulases, in the presence of reduced glutathione.
[00309] Figures 20 and 21 show that one of the effects of CelS2 and E7 is to increase cellulase activity. Although reactions with CELLUCLAST ™ or Cel7A alone can hardly produce any additional soluble glucose after the first time point (24 hours in figure 20, 18 hours in figure 21), the release of soluble glucose continues in many cases where a protein CBM33 is present, and this effect is greater in the presence of a reducer. To further analyze this, a time course experiment was conducted to examine the effect of CelS2 on CELLUCLAST ™ activity over time. Materials and methods
[00310] As in example 1. Additional experimental details, including minor deviations from the standard protocols described in example 1, are provided in the figure captions, where necessary, for this example and the following examples. Results
[00311] The results (Figure 25) clearly show that the production of soluble glucose by CELLUCLAST ™ decreases, while the production of soluble glucose continues if CelS2 is present. This effect is greatest if a reducing agent is also present. It is possible that CelS2 somehow prevents cellulases from becoming irreversibly and non-productively linked to the substrate, modifying the surface of the cellulosic substrate, a phenomenon that is generally considered to reduce the efficiency of cellulase (Jalak and Valjamae, 2010, Biotechnology and Bioengineering 106: 871-883).
[00312] Example 3: Effects of CelS2 and Cel7A on cellulose degradation by cellulases in the presence of reduced glutathione.
[00313] To examine the functionality of CelS2, including its effect on prolonging cellulase activity, in more detail, the effect of CelS2 on the efficiency of a single-component cellulase was studied. The monocomponent enzyme was HjCel7A, obtained by purifying a mixture of cellulases from Hypocrea jecorina (Trichoderma reesei). Materials and methods
[00314] As in example 1. Results
[00315] The results, shown in figure 26, clearly show that CelS2 reinforces the cellulase activity, Cel7A, and that this reinforcing effect is greater in the presence of a reducer. The results also show that the presence of CelS2 prolongs the activity of Cel7A.
[00316] Example 4: Effects of different reducers in the efficiency studies of CBM33, with CBM33 N-terminal domain produced recombinantly from CelS2.
[00317] To verify the functionality of different reducers, the CBM33 N-terminal domain produced recombinantly from CelS2 was incubated with 0.8 mM of reduced glutathione, gallic acid or ascorbic acid and the release of oxidized oligosaccharides from AVICEL® was monitored . Materials and methods
[00318] As in example 1. Results
[00319] Figure 27 shows that all the tested reducers reinforced the activity of the CBM33 protein, although to a different extent. The choice of the ideal reducer will depend, among other things, on the pH, the substrate and the type of protein GH61 / CBM33.
[00320] Example 5: Effects of the reducer on the E7 reinforcing effect on cellulase activity.
[00321] According to the bioinformatics analysis Pfam (pfam.org), E7 is a single domain CBM33 protein (Uniprot ID: Q47QG3; E7), while CelS2 (Uniprot ID: Q9RJY2) comprises a CBM33 domain with a CBM2 ( carbohydrate binding module 2; see cazy.org) attached to the C-terminal side of the protein. It is important to note that single domain proteins, such as E7, are active on their own, as shown in figure 20. To further illustrate this, figure 28 that has been prepared shows cellulose degradation by CELLUCLAST ™, or a combination of CELLUCLAST ™ and E7 in the presence or absence of a reducer. Materials and methods
[00322] As in example 1. Results
[00323] It is evident in figure 28 that E7 acts in synergy with CELLUCLAST ™, and that the presence of reducers has a reinforcing effect in E7. Also note that figure 28 shows that one of the effects of E7 is to prolong cellulase activity over time, similar to what was observed with CelS2, in the manner previously described. Figure 29 shows a MALDI spectrum of oxidized products produced by E7, by incubation with AVICEL® and a reducer.
[00324] Example 6: Effects of additional CBMs (the CBM33 N-terminal domain of CelS2 and the CBM2 C-terminal domain of CelS2).
[00325] As noted in example 6, E7 is a single domain CBM33 protein, while CelS2 comprises a CBM33 domain with a CBM2 attached to the C-terminal side of the protein. It is important to note that single-domain proteins, such as E7, are active on their own, as illustrated in Figure 28. To further illustrate this, Figure 30 was prepared to show the products released from cellulose by full size CelS2, the recombinantly expressed CBS33 N-terminal domain of CelS2, and the recombinantly expressed CBS2 C-terminal domain of CelS2. Materials and methods
[00326] zterminal is reproduced recombinantly: HGVAMMPGSRTYLCQLDAKTGTGALDPTNPACQAALDQSGATALY NWFAVLDSNAGGRGAGYVPDGTLCSAGDRSPYDFSAYNAARSDWP RTHLTSGATIPVEYSNWAAHPGDFRVYLTKPGWSPTSELGWDDLELI QTVTNPPQQGSPGTDGGHYYWDLALPSGRSGDALIFMQWVRSDSQE NFFSCSDVVFDGG (SEQ ID NO: 22). Results
[00327] As expected, the CBM2 domain alone showed no activity on cellulose (Figure 30). However, the CBM33 domain shows activity in AVICEL® without its CBM2, although less than the activity of the complete protein.
[00328] These data clearly show that CBM33 domains alone can act synergistically with cellulases, and that the activity of these unique domains is stimulated with respect to the presence of reducers (see also figure 27). The data also shows that the presence of additional CBM domains, such as CBM2 in CelS2, may be beneficial for CBM33 activity. By analogy with what is known in the scientific literature about the effects of adding various types of CBMs to various types of active carbohydrate-enzymes, it will be understood that the activity and efficiencies of naturally occurring CBM33 and GH61 proteins can be manipulated by removing, adding or changing Additional CBMs that are merged into the CBM33 or GH61 domain.
[00329] Example 7: CelS2 metallic activation.
[00330] To identify which metal is preferred for CelS2, the activity of the N-terminal CBM33 domain of CelS2 was inhibited by EDTA, and different metals were tested to reactivate the protein. Materials and methods
[00331] As in example 1. Results
[00332] Figure 31 clearly shows that, under the conditions used in this experiment, including very low metallic concentrations (compared, for example, to the concentrations used in figure 9), only the copper of the tested metals was able to reactivate CelS2 (metal concentration 10 μM in the presence of 20 μM EDTA). Thus, copper ions may be the preferred metal ion for CelS2. It can be noted, albeit in somewhat higher (but still low) concentrations, that many other metal ions can also work, at least for practical purposes (Vaaje-Kolstad et al., 2010, supra; Harris et al., 2010, supra; Figure 9).
[00333] Exactly which metal ion is used for CBM33s and GH61s remains somewhat uncertain, but for practical purposes, several metal ions will work at low concentrations (1 mM range). If CBM33s and GH61 prefer copper, the fact that other divalent metal ions have been observed to activate both the CBM33 and GH61 enzyme (for example, Figure 9 and Harris et al., 2010, supra) may be due to the following: ions of Cu2 + naturally present in very low amounts in the reaction mixtures (for example, as part of the substrate) may be inaccessible to the enzyme, due to their binding, for example, to the substrate. By adding other metal ions that bind to the same "binding sites", the bound copper ions can be released and thus become available to the enzymes (they are "non-competitive" from the binding sites by the divalent metals added) . At concentrations in the range of 1 mM, which work well for other metals, Cu2 + inhibits the action of CBM33, which may be due to the non-specific binding (not shown).
[00334] Example 9: Sequence and activity comparisons.
[00335] GH61 enzymes, which are known to be active in cellulose (TtGh61E, Harris et al., 2010, supra) or which are likely to be active in cellulose by analogy, such as the two GH61 proteins encoded in the Hypocrea jecorina genome ( or Trichoderma reesei; HjGH61A & HjGH61B), share several conserved residues, in addition to the two histidines that constitute the metal binding site (Figure 32). Interestingly, these same residues are converted to active E7 in cellulose, while several of them are absent in CBP21 active in chitin (Figure 33).
[00336] Example 10: Cleavage of cellulose by GH61 proteins in the presence of reducing agents.
[00337] The proteins GH61 TtGH61E (SEQ ID NO: 1) and TaGH61A (SEQ ID NO: 2) were incubated with cellulose and ascorbic acid to demonstrate that these proteins can cleave cellulose and yield oxidized products in the presence of ascorbic acid. The two proteins were cloned and produced in the manner described in Harris et al., 2010, supra. Figure 34 clearly shows that TtGH61E cuts the cellulose, producing oxidized and natural cell-oligosaccharides in a similar way to CBM33 proteins. Although the standard of the product is similar to that obtained with CBM33s, it is not identical. The patterns typically obtained with CBM33s, which act on cellulose in the presence of reducing agents (Figures 19, 22, 27, 30, and 31), show different periodicities (that is, relative abundances of the various products, both oxidized and natural oligosaccharides). This may indicate that TtGH61E acts on the substrate in a slightly different way, for example, attacking another side of the crystal. Evidently, however, the general result of the reaction of TtGH61E in the presence of the reducer is similar to the result of the reactions of CBM33s in cellulose in the presence of the reducer. Figure 34 also shows that TaGH61A is much less active than TtGH61E, at least on this substrate, the production of oxidized sugars being very low. Notably, this enzyme produces relatively more natural cell-oligosaccharides, that is, a product pattern that is evidently different from the product patterns obtained with TtGH61E and CBM33s, which may again indicate little variation in the substrate binding specificity.
[00338] Example 11: Effect of reducers on Cellic ™ CTec2, a cellulase preparation containing GH61.
[00339] Since the commercially available cellulase preparation, Cellic ™ CTec2, contains GH61 proteins, the effect of ascorbic acid on the efficiency of this enzyme preparation has been tested. In this experiment, the effect of ascorbic acid on the release of glucose from cellulose, by Cellic ™ CTec2, was investigated. Figure 35 shows that the presence of ascorbic acid increased the release of glucose from both filter paper and AVICEL®, by about 30%, demonstrating the beneficial effects of ascorbic acid.

权利要求:
Claims (18)
[0001]
1. Method for degrading or hydrolyzing a polysaccharide, characterized by the fact that it comprises putting said polysaccharide in contact with chitinase or cellulase and one or more oxido-hydrolytic enzymes, in which said polysaccharide is chitin, cellulose or chitin and cellulose, in whereas said degradation or hydrolysis is carried out in the presence of at least one reducing agent selected from the group of ascorbic acid, cumaric acid, ferulic acid, gallic acid, glucose, glucosamine, N-acetylglycosamine, reduced glutathione, humic acid, succinic acid , Fe (II) SO4, LiAlH4, NaBH4, and lignin or its fragments and at least one divalent metal ion selected from the group of Ca2 +, Co2 +, Cu2 +, Mg2 +, Mn2 +, Ni2 +, and Zn2 +, where the one or more oxido-hydrolytic enzymes is one or more proteins of the carbohydrate binding module of the family 33 (CBM33), one or more glycoside hydrolase proteins of the family 61 (GH61) or a combination of one or more proteins of the carbohydrate binding module arbohydrate of the family 33 (CBM33) and one or more glycoside hydrolase proteins of the family 61 (GH61), and where the degradation of the polysaccharide is increased by the presence of one or more oxido-hydrolytic enzymes in combination with at least one reducing agent and by minus one divalent metal ion compared to without the combination.
[0002]
2. Method according to claim 1, characterized by the fact that two or more oxido-hydrolytic enzymes are used in said method.
[0003]
Method according to claim 2, characterized by the fact that one of said oxido-hydrolytic enzymes is a protein of the CBM33 family, and another of said oxido-hydrolytic enzymes is a protein of the GH61 family.
[0004]
Method according to any one of claims 1 to 3, characterized in that said GH61 protein consists of an amino acid sequence presented in any one of SEQ ID NOs: 1, 2, 3, 15 and 16.
[0005]
Method according to any one of claims 1 to 4, characterized in that said CBM33 protein consists of an amino acid sequence presented in any one of SEQ ID NOs: 4-14.
[0006]
Method according to any one of claims 1 to 5, characterized in that it additionally comprises: (a) putting said polysaccharide or its degradation or hydrolysis product in contact with one or more saccharolytic enzymes selected from a cellulose hydrolase or chitin hydrolase, and (b) optionally bringing said polysaccharide or its degradation or hydrolysis product into contact with one or more beta-glucosidases, wherein said enzymes are brought into contact with said polysaccharide simultaneously with said oxide enzyme -hydrolytic.
[0007]
7. Method according to claim 6, characterized by the fact that said saccharolytic enzyme is chitinase or cellulase.
[0008]
8. Method for producing soluble saccharides, characterized in that said method comprises degrading or hydrolyzing a polysaccharide by a method as defined in any one of claims 1 to 7, wherein said degradation or hydrolysis releases said soluble saccharides, and optionally isolating said soluble saccharides, and wherein said soluble saccharides are chitobiose, and / or N-acetylglycosamine, and / or oligosaccharides thereof, or cellobiose, and / or glucose, and / or oligosaccharides thereof.
[0009]
9. Method for producing an organic substance, characterized in that it comprises the steps of: (i) degrading or hydrolyzing a polysaccharide by a method as defined in any one of claims 1 to 8, to produce a solution comprising soluble saccharides, in whereas said soluble saccharides are selected from the group consisting of chitobiosis, N-acetylglycosamine, cellobiose, glucose, and chitobiose oligosaccharides, N-acetylglycosamine, cellobiose, and glucose; (ii) fermenting said soluble saccharides to produce said organic substance as the fermentation product and, optionally, (iii) recovering said organic substance.
[0010]
10. Method according to claim 9, characterized by the fact that said organic substance is an alcohol.
[0011]
11. Method for producing a fermentation product, characterized by the fact that it comprises: (a) degrading or hydrolyzing a cellulosic material with an enzymatic composition comprising a GH61 polypeptide and one or more enzymes selected from the group consisting of a cellulase, a hemicellulase , an esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swolenin, in which said degradation or said hydrolysis is carried out in the presence of at least one reducing agent selected from the group ascorbic acid, cumaric acid, ferulic acid, gallic acid, glucose, glucosamine, N-acetylglucosamine, reduced glutathione, humic acid, succinic acid, Fe (II) SO4, LiAlH4, NaBH4, and lignin or its fragments and at least one ion divalent metallic selected from the group of Ca2 +, Co2 +, Cu2 +, Mg2 +, Mn2 +, Ni2 +, and Zn2 +, in which the degradation or hydrolysis of cellulosic material is increased by the presence of GH61 protein in comb inhalation with at least one reducing agent and at least one metal ion divalent in relation to without the combination; (b) fermenting the degraded cellulosic material with one or more fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.
[0012]
12. Method according to claim 11, characterized by the fact that cellulase is one or more enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase and a beta-glucosidase.
[0013]
13. Method for producing a fermentation product, characterized by the fact that it comprises: (a) saccharifying a cellulosic material with an enzymatic composition comprising a GH61 protein and one or more enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, a beta-glucosidase, and a CBM33, in which saccharification is performed in the presence of at least one reducing agent selected from the group consisting of ascorbic acid, cumaric acid, ferulic acid, gallic acid, glucose, glucosamine, N-acetylglycosamine, reduced glutathione , humic acid, succinic acid, Fe (II) SO4, LiAlH4, NaBH4, and lignin or its fragments and at least one divalent metal ion selected from the group of Ca2 +, Co2 +, Cu2 +, Mg2 +, Mn2 +, Ni2 +, and Zn2 +, wherein the saccharification of the cellulosic material is increased by the presence of the GH61 protein in combination with at least one reducing agent and at least one divalent metal ion compared to without the combination; (b) fermenting the saccharified cellulosic material with one or more fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.
[0014]
Method according to claim 13, characterized in that the enzyme composition additionally comprises one or more enzymes selected from the group consisting of an esterase, an expansin, a hemicellulase, a laccase, a ligninolytic enzyme, a pectinase, an peroxidase, a protease and a swolenin.
[0015]
Method according to any one of claims 11 to 14, characterized in that steps (a) and (b) are carried out simultaneously in simultaneous saccharification and fermentation.
[0016]
16. Method according to any one of claims 11 to 15, characterized in that the fermentation product is an alcohol, an alkane, a cycloalkane, an alkene, an amino acid, a gas, isoprene, a ketone, an organic acid , or polyketide.
[0017]
17. Method according to any one of claims 11 to 16, characterized in that the cellulosic material is pre-treated.
[0018]
18. Method according to any of claims 11 to 17, characterized by the fact that steps (a) and (b) are carried out simultaneously.

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CN113564135B|2021-09-26|2021-12-07|广东省科学院微生物研究所|Lyase monooxygenase mutant with improved enzyme activity and stability, genetically engineered bacterium and application|

法律状态:
2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according art. 34 industrial property law|

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2019-05-21| B06T| Formal requirements before examination|

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2019-10-15| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application according art. 36 industrial patent law|

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2020-10-13| B09A| Decision: intention to grant|

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2020-12-29| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 05/08/2011, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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GB1013317.1|2010-08-06|

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GBGB1013317.1A|GB201013317D0|2010-08-06|2010-08-06|Method|

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GBGB1016858.1A|GB201016858D0|2010-10-06|2010-10-06|Method|

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GB1016858.1|2010-10-06|

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GB1105062.2|2011-03-25|

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GBGB1105062.2A|GB201105062D0|2011-03-25|2011-03-25|Method|

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PCT/US2011/046838|WO2012019151A1|2010-08-06|2011-08-05|Methods of degrading or hyrolyzing a polysaccharide|

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