method for treating cellulosic material, polypeptide, isolated nucleic acid molecule, recombinant ex

专利摘要:
METHOD TO TREAT CELLULOSIC MATERIAL, POLYPEPTIDE, ISOLATED NUCLEIC ACID MOLECULE, RECOMBINANT EXPRESSION VEROR, HOST CELL, PROCESSES FOR PRODUCING A POLYPEPTIDE, AND FOR OBTAINING AN ENZYME PREPARATION, ENZYME PREPARATION AND ENZYME PREPARATION The present invention concerns the production of sugar hydrolysates from cellulosic material. The method can be used, for example, for the production of fermentable sugars for the production of bioethanol from lignocellulosic material. Cellulolytic enzymes and their production by recombinant technology are described, as well as uses of enzymes and enzyme preparations.
公开号:BR112012016320B1
申请号:R112012016320-5
申请日:2010-12-30
公开日:2021-04-20
发明作者:Terhi Puranen；Sauli Toikka；Kim Langfelder；Jari Vehmaanperã
申请人:Roal Oy；
IPC主号:

专利说明:

FIELD OF THE INVENTION
[001] The present invention relates to a method for treating cellulosic material with a fungal CBHII/Cel6A cellobiohydrolase enzyme or an enzyme preparation comprising said enzyme. The enzyme is useful in a number of industrial applications, particularly in the production of biofuels, where the production of fermentable sugars from lignocellulosic material in moderate to high temperature ranges is advantageous. The invention further relates to CBHII/Cel6A fungal polypeptides and isolated nucleic acid molecules encoding said enzymes, a recombinant vector, host cells for producing said enzymes, enzyme compositions comprising said enzymes as well as a process for the preparation of such compositions. The invention also relates to various uses of said enzymes or enzyme compositions, where enzymatic conversion of cellulosic or lignocellulosic material is desired. FUNDAMENTALS OF THE INVENTION
[002] The limited resources of fossil fuels and increasing amounts of CO2 released from them and which cause the phenomenon of the greenhouse effect have increased a need towards the use of biomass as a renewable and clean energy source. A promising alternative technology is the production of biofuels such as ethanol, butanol or propanol from cellulosic materials. In the transport sector, biofuels are just being an option, which can reduce CO2 emissions by an order of magnitude. Ethanol can be used in existing vehicles and distribution systems and thus does not require costly infrastructure investments. Sugars derived from cellulosic and lignocellulosic renewable raw materials can also be used as raw materials for a variety of chemicals that can replace oil-based chemicals.
[003] Most carbohydrates in plants are in the form of lignocellulose, which consists essentially of cellulose, hemicellulose and lignin. In a conventional lignocellulose-to-ethanol process, the lignocellulosic material is first chemically or physically pretreated, using acid hydrolysis, steam explosion, ammonia fiber expansion, alkaline wet oxidation or pretreatment with ozone, to make the cellulose fraction more accessible to enzymatic hydrolysis. The cellulose fraction is then hydrolyzed to obtain sugars that can be fermented by yeast into ethanol and distilled to obtain pure ethanol. Lignin is obtained as a major co-product that can be used as a solid fuel. In this separate hydrolysis and fermentation (SHF) process the temperature of the enzymatic hydrolysis is typically higher than that of fermentation. The use of thermostable enzymes in hydrolysis offers potential benefits such as higher reaction rates at elevated temperatures, reduced enzyme load due to higher specific activity and lifetime of the enzymes, increased flexibility with respect to process configuration and better hygiene.
[004] There is ongoing research to make the bioethanol production process more economical. One of the options is the simultaneous saccharification and fermentation (SSF) process. The main benefits are reduced end-product inhibition of enzymatic hydrolysis and reduced investment costs. The challenges lie in verifying favorable conditions, eg temperature and pH, for both enzymatic hydrolysis and fermentation. In the consolidated bioprocess (CBP), the amount of externally added enzymes can be significantly reduced by exploiting a fermentative organism or ethanologen, which is capable of producing a series of lignocellulolytic enzymes.
[005] In recent years, the metabolic engineering of microorganisms used in ethanol production has shown significant progress. In addition to Saccharomyces cerevisisiae, microorganisms such as bacterial species Zymomonas and Escherichia coli and yeasts such as Pichia stipitis and Kluyveromyces fragilis were targeted to produce ethanol from cellulose. In the SSF process, inhibitor and temperature tolerance as well as the ability to utilize multiple sugars are important properties of the fermentation microorganism. Engineered yeasts were developed being able to ferment pentose, xylose and arabinose sugars in addition to glucose. Thermophilic microbes such as Thermoanaerobacterium saccharolyticum or Clostridium thermocellum are designed to ferment sugars, including xylose, to ethanol at elevated temperatures of 50°C to 60°C (thermophilic SSF or TSSF). Such fermentative organisms also have potential as CBPs (Shaw et al. 2008).
[006] Enzymatic hydrolysis is considered the most promising technology for converting cellulosic biomass into fermentable sugars. However, enzymatic hydrolysis is only used in a limited amount on an industrial scale and especially when using heavily lignified material, such as wood and agricultural waste, the technology is not satisfactory. Efforts have been made to improve the efficiency of enzymatic hydrolysis of cellulosic material (Badger 2002; Kurabi et al., 2005).
[007] WO2001060752 (Forskningscenter Riso, DK) describes a continuous process for converting solid lignocellulosic biomass into flammable combustible products. After pretreatment by wet oxidation or steam explosion, the biomass is partially separated into cellulose, hemicellulose and lignin and is then subjected to partial hydrolysis using one or more carbohydrase enzymes (EC 3.2).
[008] WO2002024882 (Iogen Bio-Products Corp., CA) refers to a method of converting cellulose to glucose by treating a pretreated lignocellulosic substrate with an enzyme mixture comprising cellulase and a modified cellobiohydrolase I (CBHI) obtained by inactivation of its cellulose binding domains (CBD). US2004/0005674 (Athenix Corp., US) describes new enzyme mixtures that can be used directly on the lignocellulose substrate. The synergistic enzyme contains a cellulase and an auxiliary enzyme, such as cellulase, xylanase, ligninase, amylase, protease, lipidase or glucuronidase or any combination thereof. Cellulase is considered to include endoglycanase (EG), beta-glycosidase (BG) and cellobiohydrolase (CBH) enzymes. US20050164355 (Novozymes Biotech Inc., US) describes a method for degrading lignocellulosic material with one or more cellulolytic enzymes selected from EG, BG and CBH and in the presence of at least one surfactant. Additional enzymes such as hemicellulases, esterase, peroxidase, protease, laccase or mixtures thereof can also be used.
[009] The best investigated and most widely applied cellulolytic enzymes of fungal origin were derived from Trichoderma reesei (the anamorph of Hypocrea jecorina). Lesser known fungal cellulases were also disclosed.
[0010] Hong et al. (2003a and 2003b) characterize Thermoascus aurantiacus EG and CBHI and its production in yeast. Tuohy et al. (2002) describe three forms of cellobiohydrolases, including CBHI and CBHII from Talaromyces emersonii.
[0011] The use of cellobiohydrolase I (CBHI), a member of the family 7 of glycosyl hydrolases in the enzymatic conversion of cellulosic material is known, for example, from WO03/000941 (Novozymes A/S, DK), which concern enzymes CBHI obtained from various fungi. WO2005074656 (Novozymes Inc., US) discloses polypeptides having cellulolytic activity derived, for example, from Thermoascus aurantiacus.
[0012] WO2007071818 (Roal Oy, FI) describes the production of sugar hydrolysates from cellulosic material by enzymatic conversion and enzyme preparations comprising said enzymes. Enzymes useful in the method include thermostable cellobiohydrolase, endoglycanase, betaglycosidase and optionally xylanase which is derived from Thermoascus aurantiacus, Acremonium thermophilum or Chaetomium thermophilum.
[0013] Cellobiohydrolases II have been disclosed in several applications. WO2004056981 (Novozymes A/S, DK) describes polypeptides having cellobiohydrolase II activity and polynucleotides encoding the polypeptides as well as methods for producing and using the polypeptides in applications such as in ethanol production. Full-length DNA sequences are disclosed from Aspergillus tubigensis, Chaetomium thermophilum, Myceliophtora thermophila, Thielavia species, Acremonium thermophilum, Trichophaea saccata, Stibella annualata and Malbrancheae cinnamonea. EP1578964 B1 (Novozymes A/S, DK) describes the full-length amino acid sequence of C. thermophilum CBHII and a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions with a fragment of the nucleotide sequence encoding said enzyme.
CN1757709 (Shandong Agricultural Univ., CN) describes the nucleotide sequence of a thermophilic CBHII enzyme from Chaetomium thermophilum CT2 and its expression in the yeast Pichia pastoris. The enzyme is able to convert the rejected fiber material.
WO2006074005 (Genencor Int., Inc., US) describes a CBHII/Cel6A enzyme variant of Hypocrea jecorin (Trichoderma reesei). The variant enzyme is useful, for example, in the production of bioethanol.
WO2007094852 (Diversa Corp., US; Verenium Corp., US) describes cellulolytic enzymes, nucleic acids encoding them and methods for their production and use. The enzymes can be an endoglycanase, a cellobiohydrolase, a beta-glycosidase, a xylanase, a mannanase, a betaxylosidase, an arabinofuranosidase or an oligomerase. Enzyme and enzyme mixtures are useful, for example, in the manufacture of fuel or bioethanol.
[0017] WO2008095033 (Syngenta, CH, Verenium Corp., US) describes enzymes having lignocellulolytic activity, including cellobiohydrolases useful, for example, in the manufacture of fuels and processing of biomass materials. WO2009045627 (Verenium Corp., US) describes methods for breaking down hemicellulose using enzymes having xylanase, mannanase and/or glycanase activity and increased activity and stability at increased pH and temperature.
WO2009089630 (Iogen Energy Corp., CA) describes a family 6 cellulase variant with reduced glucose inhibition comprising one or more amino acid substitutions.
[0019] WO2009059234 (Novozymes Inc., US) describes methods of producing cellulosic material reduced to a redox active metal ion useful in degrading or converting a cellulosic material and producing a fermentation product. The application discloses, for example, a CBHII polypeptide from Chaetomium thermophilum.
[0020] WO2009085868 (Novozymes A/S, DK) describes polypeptide, polynucleotides encoding the enzyme and a method for producing a fermentation product, which comprises saccharification of a cellulosic material with a cellulolytic enzyme composition comprising said polypeptide. Such cellobiohydrolases can be derived from Trichoderma reesei, Humicola insolens, Myceliophtora thermophila, Thielavia terrestris and Chaetomium therinophilum. WO2006074435 and US2006218671 (Novozymes Inc., US) describe nucleotide and amino acid sequences of Thielavia terrestris Cel6A cellobiohydrolase. US 7220565 (Novozymes Inc., US) describes polypeptides having cellulolytic enhancing activity and identity to the mature amino acid sequence of Myceliophtora thermophila CBHII.
US20070238155 and US20090280105 (Dyadic Int., Inc., US) describe enzymatic compositions comprising novel enzymes from Chrysosporium lucknowense, which comprise, for example, the enzymes CBHIIa and CBHIIb designated to family 6 of glycosyl hydrolases. Enzymatic compositions are effective in hydrolyzing lignocellulosic material.
[0022] The genomic sequence of Neurospora crassa OR74A is disclosed in Galagan et al. (2003), including an exoglycanase 2 precursor sequence. Collins et al. (2003) describe the coding sequence of Talaromyces emersonii CBHII/Cel6A.
[0023] The market for biofuels such as renewable fuels for transport is expected to increase considerably in the near future. As a result, there is rapidly growing interest in using alternative feed stocks for biofuel production. Fermentation of cellulosic biomass in plants and wood or municipal waste to ethanol and other alcohols is an attractive avenue for fuels that complement fossil fuels. A barrier to the production of cellulosic and lignocellulosic biomass biofuels is the robustness of the cell wall and the presence of sugar monomers in the form of inaccessible polymers that require a large amount of processing to manufacture the sugar monomers available to microorganisms that they are typically used for the production of alcohol by fermentation. Thus, there is a continuing need for new methods, as well as new enzymes and enzyme mixtures, that enhance the efficiency of degradation of cellulosic and lignocellulosic substrates. Particularly, enzymes and enzyme mixtures are required being able to attack the different glycosidic bonds of the crystalline cellulosic material and in this way provide almost complete hydrolysis of variant materials to be treated. There is also a need for enzymes, which are stable at high process temperatures, thus allowing the use of high biomass consistency and leading to high sugar and ethanol concentrations. This method can lead to significant savings in energy costs and investments. The high temperature reduces the risk of contamination during hydrolysis. The present invention aims to satisfy at least part of these needs. SUMMARY OF THE INVENTION
[0024] The aim of the present invention is to provide new enzymes and enzyme compositions to enhance the efficiency of cellulose hydrolysis. Cellobiohydrolases and particularly cellobiohydrolases II obtainable from Acremonium thermophilum, Melanocarpus albomyces, Chaetomium thermophilum or Talaromyces emersonii are useful in the hydrolysis and degradation of cellulosic material. Enzymes are kinetically very effective over a wide range of temperatures and although they have high activity at high temperatures, they are also very effective at standard hydrolysis temperatures. This makes it extremely well suited for varying variant cellulosic substrate hydrolysis processes carried out at both conventional and elevated temperatures.
[0025] The present invention relates to a method for treating cellulosic material with a CBHII/Cel6A polypeptide or an enzyme preparation comprising said polypeptide or a fermentative microorganism that produces said polypeptide, said method comprising the steps: i) production of a CBHII/Cel6A polypeptide of the invention or an enzyme preparation comprising said polypeptide or a fermentative microorganism that produces said polypeptide; ii) reacting the cellulosic material with the CBHII/Cel6A polypeptide of the invention or the enzyme preparation comprising said polypeptide or the fermentative microorganism that produces said polypeptide and iii) obtaining partially or fully hydrolyzed cellulosic material. The CBHII/Cel6A polypeptide useful in said method has cellobiohydrolase activity and comprises an amino acid sequence having at least 76% identity with the full-length polypeptide of SEQ ID NO:12, at least 76% identity with the polypeptide of full length SEQ ID NO:14, at least 95% identity to the full length polypeptide of SEQ ID NO:15, or at least 91% identity to the full length polypeptide of SEQ ID NO:16. The CBHII/Cel6A polypeptide can also be a fragment or variant having similar properties, such as similar substrate specificity and pH and temperature dependence or stability. The cellobiohydrolase CBHII/Cel6A useful in the method is a cellobiohydrolase II enzyme from the family 6 of glycosyl hydrolases and has both a conserved fold and stereochemistry of the hydrolysis reaction.
The CBHII/Cel6A cellobiohydrolases applicable in the method can be obtained from a genus of Acremonium, Melanocarpus, Chaetomium or Talaromyces, more preferably from A. thermophilum, M. albomyces, C. thermophilum or T. emersonii, more preferably from T. emersonii of the deposited strain A. thermophilum CBS 116240, M. albomyces CBS 685.95, C. thermophilum CBS 730.95 or T. emersonii DSM 2432.
[0027] The cellobiohydrolase CBHII/Cel6A applicable in the method is capable of hydrolyzing different cellulosic material at moderate to high temperatures, particularly in combination with other enzymes used in the hydrolysis of various cellulosic or lignocellulosic materials.
[0028] The cellobiohydrolase CBHII/Cel6A of the invention is applicable in various uses, particularly in the production of biofuel.
[0029] The present invention also relates to novel CBHII/Cel6A cellobiohydrolases, having cellobiohydrolase activity and comprises an amino acid sequence having at least 76% identity with the full length polypeptide of SEQ ID NO:12, at least 76 % identity to the full-length polypeptide of SEQ ID NO:14, at least 95% identity to the full-length polypeptide of SEQ ID NO:15, or at least 91% identity to the full-length polypeptide of SEQ ID NO:16. Said CBHII/Cel6A polypeptide can also be a fragment or variant having similar properties, such as similar substrate specificity and pH and temperature dependence or stability. Said cellobiohydrolase CBHII/Cel6A is capable of hydrolyzing cellulosic material at moderate to high temperatures.
Said enzyme is encoded by an isolated nucleic acid molecule, which comprises a polynucleotide sequence encoding a polypeptide of the invention. Preferably, said nucleic acid molecule comprises the polynucleotide sequence defined in SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:9 or SEQ ID NO:10 or a subsequence thereof.
The cellobiohydrolase CBHII/Cel6A of the invention can be encoded by an isolated nucleic acid molecule, which hybridizes under stringent conditions to a polynucleotide sequence included in SEQ ID NO:11, SEQ ID NO:13, SEQ ID N°:9, SEQ ID N°:10, SEQ ID N°:7, SEQ ID N°:8 or a subsequence thereof.
Said enzyme is encoded by an isolated polynucleotide included in plasmid pALK2582 deposited in Escherichia coli under accession number DSM 22946, plasmid pALK2581 deposited in E.coli under accession number DSM 22945, plasmid pALK2904 deposited in E.coli under accession number DSM 22947 or plasmid pALK3006 deposited in E.coli under accession number DSM 23185.
The cellobiohydrolase CBHII/Cel6A of the invention can be produced from a recombinant expression vector that comprises the nucleic acid molecule or nucleotide sequence encoding said cellobiohydrolase. Said cellobiohydrolase polypeptide can be produced in a heterologous host, preferably in a microbial host.
The invention also relates to an isolated nucleic acid molecule comprising a polynucleotide sequence encoding a CBHII/Cel6A fungal polypeptide selected from the group consisting of: (a) a nucleic acid molecule or polynucleotide sequence encoding a polypeptide having cellobiohydrolase activity and comprising a full-length amino acid sequence as described in SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16 or a fragment or variant thereof having similar properties; (b) a nucleic acid molecule or polynucleotide sequence encoding a polypeptide having cellobiohydrolase activity and at least 76% identity to the full length amino acid sequence of SEQ ID NO:12, at least 76% identity to the full-length amino acid sequence of SEQ ID NO:14, at least 95% identity to the full-length amino acid sequence of SEQ ID NO:15, or at least 91% identity to the long amino acid sequence the entirety of SEQ ID NO:16 or a fragment or variant thereof having similar properties; (c) a nucleic acid molecule comprising the coding sequence for the polynucleotide sequence described as SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:9 or SEQ ID NO:10; (d) a nucleic acid molecule comprising the coding sequence for the polynucleotide sequence contained in DSM 22946, DSM 22945, DSM 22947 or DSM 23185; (e) a nucleic acid molecule whose coding sequence differs from the coding sequence of a nucleic acid molecule according to any one of (c) to (d) due to the degeneration of the genetic code and (f) a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule contained in DSM 22946, DSM 22945, DSM 22947 or DSM 23185 and which encodes a polypeptide having cellobiohydrolase activity and an amino acid sequence that shows at least 76% identity with the sequence of full-length amino acid as described in SEQ ID NO:12, at least 76% identity with the full-length amino acid sequence of SEQ ID NO:14, at least 95% identity with the full-length amino acid sequence of SEQ ID NO:15 or at least 91% identity with the full length amino acid sequence of SEQ ID NO:16 or a fragment or variant thereof having similar properties.
[0035] The invention further relates to a recombinant expression vector comprising the nucleic acid molecule or polynucleotide sequence of the invention operatively linked to regulatory sequences capable of directing the expression of the gene encoding the cellobiohydrolase CBHII/Cel6A of the invention and production of said CBHII/Cel6A cellobiohydrolase in a suitable host.
[0036] The invention also relates to a host cell comprising the recombinant expression vector as described above. Preferably, the microbial host, such as a filamentous fungal host.
The preferred hosts are Trichoderma, Aspergillus, Fusarium, Humicola, Chrysosporium, Neurospora, Rhizopus, Penicillium and Mortiriella. More preferably, the host is Trichoderma or Aspergillus, most preferably a filamentous fungus T. reesei.
[0038] The present invention relates to a process for producing a polypeptide of the invention having cellobiohydrolase activity, said process comprising the steps of culturing the host cell of the invention and recovering the polypeptide. There is also within the invention a polypeptide having cellobiohydrolase activity encoded by a nucleic acid molecule of the invention where it is obtainable by the process described above.
[0039] The invention also relates to a process for obtaining an enzyme preparation comprising the steps of culturing a host cell of the invention and preparing the total culture broth or separating the cells from the used culture medium and obtaining the supernatant. There is also within the invention an enzyme preparation obtainable by the process described above. The invention also relates to an enzyme preparation, which comprises the cellobiohydrolase CBHII/Cel6A of the invention.
[0040] The enzyme preparation may further comprise other enzymes selected from the group: cellobiohydrolase, endoglycanase, beta-glycosidase, beta-glycanase, xyloglycanase, xylanase, beta-xylosidase, mannanase, beta-mannosidase, α-glucuronidase, acetyl xylan esterase, α-arabinofuranosidase, α-galactosidase, pectinase, enveloping endo- and exo-α-L-arabinases, agalactosidase, endo- and exo-galactoronase, endopectinlyase, pectine lyase and pectinesterase, phenol esterase, peroxidase-dependent ligninase involving ligninase manganese, H2O2 generating enzyme and laccase with or without a mediator.
[0041] The enzyme preparation may be in the form of total culture broth or used culture medium. This can be in the form of liquid, powder or granules.
[0042] There is also within the invention, the use of the CBHII/Cel6A polypeptide or enzyme preparation of the invention for the production of biofuels, for detergents, for the treatment of fibers, for the treatment of feed and food, for pulp and paper, for beverages or for any applications involving the hydrolysis or modification of cellulosic material.
[0043] Particularly, the CBHII/Cel6A polypeptide or enzyme composition comprising said polypeptide is useful for the production of biofuels. BRIEF DESCRIPTION OF THE DRAWINGS
[0044] Figure 1 schematically shows the expression cassettes used in the transformation of Trichoderma reesei protoplasts for the overproduction of recombinant CBHII/Cel6A proteins. The cbh2/cel6A genes were under the control of the T. reesei cbhl/cel7A promoter (p cbhl) and transcription termination was ensured using the T. reesei cbhl/cel7A terminator sequence (t cbhl). The amdS gene was included as a transformation marker.
[0045] Figure 2 shows the determination of Ph dependence for enzyme compositions (100 µg of protein in the reaction) comprising the recombinant cellobiohydrolase CBHII/Cel6As from Acremonium thermophilum CBS 116240, Melanocarpus albomyces CBS 685.95; Chaetomium thermophilum CBS 730.95 and Talaromyces emersonii DSM 2432. Hydrolysis was performed on Avicel Ph 101 cellulose within a pH range of 3 to 10 to 50°C for 21 hours. The formation of reducing sugars was determined by the para-hydroxybenzoic acid hydrazide (PAHBAH) method (Lever, 1972) using a standard cellobiose curve.
[0046] Figure 3 shows the determination of thermal stability for enzyme compositions (100 μg protein in the reaction) comprising the recombinant cellobiohydrolases CBHII/Cel6As from Acremonium thermophilum CBS 116240, Melanocarpus albomyces CBS 685.95; Chaetomium thermophilum CBS 730.95 and Talaromyces emersonii DSM 2432. Hydrolysis was performed on Avicel Ph 101 cellulose within a temperature range of 40°C to 80°C at optimum pH of the enzyme compositions for 21 hours. The formation of reducing sugars was determined by the para-hydroxybenzoic acid hydrazide (PAHBAH) method (Lever, 1972) using a standard cellobiose curve.
[0047] Figure 4 shows hydrolysis results of steam-blasted hardwood material carried out with enzymatic mixtures comprising the cellobiohydrolase CBHII/Cel6A of the invention. The hardwood substrate was hydrolyzed using different enzyme mixtures at a dosage of 5 mg protein per g total solids at both 55°C and 37°C. The composition of the thermophilic enzyme mixture (MIXTURE 2) and the mesophilic enzyme mixtures (MIXTURE T. REESEI ENZYMES and MIXTURE ACC), which comprises At_ALKO4245_Cel6A, Ma_ALKO4237_Cel6A or Ct_ALK04265_Cel6A are described in more detail in Example 4. Duplicate shakes were removed after a 72 hour hydrolysis period and quantified by HPLC, where the concentration of glucose and xylose was determined. The results of substrate voids, containing buffer instead of the enzyme sample, were subtracted from the results obtained with the enzyme mixtures. The combined concentration of glucose and xylose is shown.
[0048] Figure 4A shows the results of steam blasted hardwood hydrolysis performed at 55°C with a thermophilic enzyme mixture (MIX 2) supplemented with At_ALK04245_Cel6A (MIX 2_AT), MaALK04237_Cel6A (MIX 2_MA) or Ct_ALK04265_Cel6C (MIX 2_CTA) ).
[0049] Figure 4B shows the results of steam-blasted hardwood performed at 37°C with a mixture of mesophilic enzyme (MIXTURE T. REESEI ENZYMES) supplemented with At_ALKO4245_Cel6A (MIX TR_AT), MaALK04237_Cel6A (MIX TR_MA) or Ct_ALK04265_Cel6 TR_CT).
[0050] Figure 4C shows the results of steam blasted hardwood hydrolysis performed at 37°C with a mesophilic enzyme mixture (ACC MIX) supplemented with At_ALKO4245_Cel6A (ACC_AT MIX), MaALKO4237_Cel6A (ACC_MA MIX) or Ct_ALK04265_Cel6A (MIX ACC_Cel6A) ACC_CT).
[0051] Figure 5 shows material results of steam exploded corn cobs performed with enzyme mixtures comprising the cellobiohydrolase CBHII/Cel6A of the invention. The corn cob substrate was hydrolyzed using different enzyme mixtures at a dosage of 5 mg protein per g total solids at 55°C. The composition of the thermophilic enzyme mixture (MIXTURE 2) comprising the At_ALKO4245_Cel6A polypeptide of the invention (MIXTURE 2_AT) is described in more detail in Example 5. Samples from duplicate shake flasks were taken after a hydrolysis period of 72 hours and quantified by HPLC, in which the concentration of glucose and xylose was determined. The results of substrate voids, containing buffer instead of the enzyme sample, were subtracted from the results obtained with the enzyme mixtures. The combined concentration of glucose and xylose is shown. SEQUENCE LISTING SEQ ID NO:1 Oligonucleotide Primer Sequence CBH_1S SEQ ID NO:2 Oligonucleotide Primer Sequence CBH_1AS SEQ ID NO:3 Oligonucleotide Primer Sequence CBH8 SEQ ID NO:4 Oligonucleotide Primer Sequence SEQ ID NO:5 Sequence of oligonucleotide primer Te_CBH_A SEQ ID NO:6 Sequence of oligonucleotide primer Te_CBH_B SEQ ID NO:7 Sequence of PCR fragment obtained from Acremonium thermophilum ALK04245 (CBS 116240) using CBH_1S and CBH_1AS initiators. SEQ ID NO:8 Sequence of the PCR fragment obtained from Melanocarpus albomyces ALK04237 (CBS 685.95) using primers CBH_1S and CBH_1AS. SEQ ID NO:9 the nucleotide sequence of the cbh2/cel6A gene from Chaetomium thermophilum ALK04265 (CBS 730.95). SEQ ID NO:10 is the nucleotide sequence of the cbh2/cel6A gene from Talaromyces emersonii RF8069 (DSM 2432). SEQ ID NO:11 the nucleotide sequence of the cbh2/cel6A gene from Acremonium thermophilum ALK04245 (CBS 116240). SEQ ID NO:12 The deduced amino acid sequence of CBHII/Cel6A from Acremonium thermophilum ALK04245 (CBS 116240). SEQ ID NO:13 is the nucleotide sequence of the cbh2/cel6A gene from Melanocarpus albomyces ALK04237 (CBS 685.95). SEQ ID NO:14 The deduced amino acid sequence of the CBHII/Cel6A Melanocarpus albomyces ALK04237 (CBS 685.95). SEQ ID NO:15 The deduced amino acid sequence of CBHII/Cel6A from Chaetomium thermophilum ALK04265 (CBS 730.95). SEQ ID NO:16 The deduced amino acid sequence of the CBHII/Cel6A from Talaromyces emersonii RF8069 (DSM 2432) CBHII/Cel6A. DEPOSITS
Acremonium thermophilum ALK04245 has been deposited with Centraalbureau Voor Schimmelcultures at Upsalalaan 8, 3584 CT, Utrecht, the Netherlands on September 20, 2004 and accession number CBS 116240.
[0053] Chaetomium thermophilum ALKO4265 has been deposited with Centraalbureau Voor Schimmelcultures at Oosterstraat 1, 3742 SK BAARN, the Netherlands on November 8, 1995 and accession number CBS 730.95. After the end of the current filing period, samples will be stored under authorizations in order to make the strain available beyond the mandatory patent period.
Melanocarpus albomyces ALKO4237 has been deposited with Centraalbureau Voor Schimmelcultures at Oosterstraat 1, 3740 AG BAARN, the Netherlands on October 11, 1995 and accession number CBS 685.95. After the end of the current filing period, samples will be stored under authorizations in order to make the strain available beyond the mandatory patent period.
The Escherichia coli RF8175 strain including plasmid pALK2582 has been deposited at Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ), Inhoffenstrasse 7B, D-38124 Braunschweig, Germany on September 9, 2009 and designated accession number DSM 22946.
The E.coli strain RF8174 including plasmid pALK2581 has been deposited at Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ), Inhoffenstrasse 7B, D-38124 Braunschweig, Germany on September 9, 2009 and accession number designated DSM 22945 .
The E.coli strain RF8214 including plasmid pALK2904 has been deposited at Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ), Inhoffenstrasse 7B, D-38124 Braunschweig, Germany on September 9, 2009 and accession number designated DSM 22947 .
[0058] E.coli strain RF8333 including plasmid pALK3006 has been deposited at Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ), Inhoffenstrasse 7B, D-38124 Braunschweig, Germany on December 10, 2009 and accession number designated DSM 23185 . DETAILED DESCRIPTION OF THE INVENTION
[0059] The present invention provides a method for treating cellulosic material with a CBHII/Cel6A cellobiohydrolase polypeptide or an enzyme composition comprising said polypeptide. The invention also provides fungal cellobiohydrolase CBHII/Cel6A polypeptides and enzyme compositions comprising said CBHII/Cel6A polypeptides, which polypeptides exhibit broad substrate specificity and are stable over wide pH and temperature ranges. These perform well at both moderate and high temperatures. Particularly, CBHII/Cel6A cellobiohydrolase polypeptides are stable for at least 21 hours up to 70°C, preferably over a temperature range of 40°C to 60°C. The polypeptides and enzyme compositions comprising said polypeptides are ideal in different applications that require efficient hydrolysis of complex cellulosic or lignocellulosic materials, where cellulose is one of the main components. Polypeptides and enzyme compositions are useful, for example, in the hydrolysis of cellulosic material to produce sugar monomers from the polymeric starting material, which can then be fermented by microorganisms in the production of biofuel. Thus, the present invention provides alternative CBHII/Cel6A cellobiohydrolases for use in biofuel and other applications. CBHII/Cel6A fungal cellobiohydrolases can be produced in high-yielding fungal hosts with or without downstream processing, for example, separation of fermentation broth and mycelia is easy to accomplish or in fermentation organisms.
[0060] "Cellulose" or "cellulosic material" as used herein refers to any material that comprises cellulose as a significant component. Cellulose is a major structural component of higher plants. It provides plant cells with high tensile strength that helps them withstand mechanical stress and osmotic pressure. Cellulose is a 3-1,4-glycan composed of straight chains of glucose residues joined by 6-1,4-glycosidic bonds. Cellobiose is the smallest repeating unit of cellulose. Examples of cellulosic material include textile fibers derived, for example, from cotton, linen, hemp, jute and man-made cellulosic fibers such as modal, viscose, lyocel.
[0061] The term "cellulose" or "cellulosic material" also refers to "lignocellulose" or "lignocellulosic material" which comprises cellulose as a significant component.
[0062] In cell walls, cellulose is wrapped in variously oriented sheets, which are embedded in a matrix of hemicellulose, pectin and/or phenol propanol polymer units such as lignin to form the "lignocellulosic material" or "lignocellulose". Lignocellulose is physically hard, dense and inaccessible. Lignocellulose-containing materials include, for example, plant materials such as wood, including hardwood and softwood, hardwood chips and softwood, wood pulp, sawdust, and forestry and industrial wood waste; herbaceous crops, agricultural biomass such as cereal straw, sugar beet pulp, corn fiber, forage and corn cobs, cereal beta-glycans, sugarcane bagasse, stalks, leaves, husks, and others; waste products such as municipal solid waste, newspaper and waste office papers, waste fiber, paper sludge, shredding waste eg grain; crops dedicated to energy (eg, willow, poplar, grass or yellow grass, and others).
[0063] The cellulosic and lignocellulosic material is degraded in nature by several of several organisms including bacteria and fungi that produce enzymes capable of hydrolyzing carbohydrate polymers. Degradation usually requires the action of many enzymes that typically act sequentially or simultaneously. The biological conversion of cellulose to glucose in general requires the three main types of hydrolytic enzymes: (1) Endoglycanases (EG) that cut beta-1,4-glycosidic bonds mainly in the amorphous regions of cellulose; (2) Exoglycanases or cellobiohydrolases (CBH) which cut the disaccharide cellobiose from the reducing or non-reducing end of the crystalline cellulose polymer chain; (3) Beta-1,4-glycosidases (BG) that hydrolyze cellobiose and other cello-oligosaccharides short to glucose. Glucose and cellobiose act as end-product inhibitors of the hydrolysis reaction.
[0064] "Cellulase" or "cellulolytic enzyme" is an enzyme having "cellulase activity" or "cellulolytic activity", which means that it is capable of hydrolyzing cellulosic material. One of the most studied cellulolytic enzyme systems is that of the filamentous fungus Trichoderma reesei which is known to have at least 2 CBHs, 8 EGs and 5 BGs.
[0065] The "lignocellulolytic enzymes" are enzymes having "lignocellulase activity" or "lignocellulolytic activity", which means that they are capable of hydrolyzing lignocellulosic material such as celluloses, hemicelluloses or derivatives thereof into smaller saccharides.
The "beta-glycans" are (1-*3),(1-*4)-beta-D-glycans of mixed bonding. These are common in, for example, cereals.
[0067] "Beta-glycanase" as used herein refers to enzymes that can at least partially cleave bonds in beta-glycan.
[0068] "Celobiohydrolase" or "CBH" as used herein refers to enzymes that cleave 1,4-beta-D-glycosidic bonds of polymers such as reducing or non-reducing end cellulose and primarily produce cellobiose. These are also called exoglycanases or 1,4-beta-D-glycan cellobiohydrolases or cellulose 1,4-beta-cellobiosidases. CBHs have a modular structure consisting of distinct domains, such as a catalytic domain and an N- or C-terminal cellulose binding domain (CBD). There are naturally occurring cellobiohydrolases that need CBD. CBHs can also have additional domains of unknown function. The different domains are usually joined by an O-glycosylated linker or joining peptide rich in glycine, proline, serine or threonine.
[0069] By "cellobiohydrolase II" or "cellobiohydrolase CBHII" or "cellobiohydrolase CBHII/Cel6A" or "cellulase CBHII/Cel6A" or "polypeptide CBHII/Cel6A" or "enzyme CBHII/Cel6A" is meant in connection with this invention 1,4-8-D-glycan cellobiohydrolase enzyme classified as EC 3.2.1.91 by the Nomenclature of the International Union of Biochemistry and Molecular Biology (IUBMB; see http://www.iubmb.org). based on their structural similarities, the cellobiohydrolases II or cellobiohydrolases CBHII/Cel6A of the present invention are classified into the family 6 of glycosyl hydrolases (GH6) having similar amino acid sequences and three-dimensional structures (Henrissat 1991; Henrissat and Bairoch 1993, 1996; Henrissat et al. al. 1998; see also http://www.cazy.org/fam/GH6.html). Because there is a direct relationship between sequence and fold similarities, such a classification is believed to reflect the structural characteristics of enzymes better than their substrate specificity alone, helps to reveal the evolutionary relationships between the enzymes, and provides a convenient tool to conduct mechanistic information.
[0070] By the term "cellobiohydrolase activity" or "CBH activity" as used in the invention is understood hydrolytic activity that acts on 1,4-beta-D-glycosidic bonds in cellulose, cellotriosis, cellotetraose or any glucose polymer bound by beta-1,4 that primarily releases cellobiose from the reducing or non-reducing end of the polymer chain. The enzymatic cleavage of a glycosidic bond is a stereoselective process, in which the configuration about the anomeric center (carbon C1) can be inverted or retained. Both mechanisms contain a pair of carboxylic acid residues arranged on either side of the bond to be cleaved. Inversion enzymes use a single substitution mechanism, since enzyme retention involves a double substitution reaction (Sinnott, 1990; Withers and Aebersold, 1995). The stereochemical course of hydrolysis is usually determined by proton NMR, in which the anomeric α- and β- protons give different chemical changes (Withers et al., 1986).
[0071] By the term "cellobiohydrolase II activity" or "CBHII/Cel6A activity" as used in the invention is understood hydrolytic activity that acts on 1,4-beta-D-glycosidic bonds in cellulose, cellotriose, cellotetraose or in any polymer bound by beta-1,4 which releases primarily cellobiose from the non-reducing end of the polymer chain. The CBHII/Cel6A cellobiohydrolases reaction mechanism is inversion.
[0072] Methods to analyze cellulase activity are well known in the literature and are referred to, for example, by Ghose (1987), Tomme et al. (1988) and van Tilbeurgh et al. (1988). Total cellulase activity is commonly measured as filter paper degrading activity (FPU). Cellobiohydrolase activity can be analyzed using small soluble cellodextrins and their chromogenic glycosides, such as 4-methylumbelliferyl-beta-D-glycosides. CBHII/Cel6A cellobiohydrolases can be identified based on the response of the hydrolysis reaction; CBHII/Cel6A enzymes cannot cleave the heterosidic bond of small chromogenic oligosaccharides (van Tilbeurgh et al., 1988). Cellobiohydrolase activity can also be analyzed in microcrystalline cellulose, such as Avicel Ph 101, as used in Example 3. The formation of soluble reducing sugars after hydrolysis can be determined by the para-hydroxybenzoic acid hydrazide (PAHBAH) method (Lever , 1972) using a cellobiose standard curve, the Somogyi-Nelson method (Somogyi, 1952), alkaline ferricyanide method (Robyt and Whelan, 1972), the 2,2'-bichinconinate method (Waffenschmidt and Jaenicke, 1987) or the dinitrosalicylic (DNS) method of Miller (1959). Other cellulosic substrates include, for example, Solka floc cellulose or phosphoric acid swollen cellulose (Karlsson et al., 2001).
[0073] Cellobiohydrolase II can also be identified in a Western assay or ELISA using polyclonal or monoclonal antibodies raised against the purified protein CBHII/Cel6A.
[0074] The term "cellobiohydrolase CBHII/Cel6A" thus means cellobiohydrolase II enzymes that are members of the 6 family of glycosyl hydrolases, having both a conserved fold and a stereochemical hydrolysis reaction.
[0075] The term "moderate temperature" or "conventional temperature" in the context of the present invention means temperatures commonly used in the hydrolysis of cellulose and corresponding to the optimum temperatures or thermal stabilities of the enzymes used in such processes. In this way, the terms refer to temperature ranges from 30°C to 45°C.
[0076] The term "high temperature" or "high temperature" refers to temperature ranges from 45°C to 70°C. In short-term hydrolysis processes, enzymes can be effective even up to 80°C. Enzymes active or stable in such high temperature ranges are also called "thermostable" or "thermophilic" enzymes.
[0077] The strains of microorganisms capable of producing CBHII/Cel6A cellobiohydrolase polypeptide or CBHII/Cel6A cellobiohydrolase activity can be evaluated on different substrates. First, the chosen strains are grown in a suitable medium. After a sufficient amount of a cellobiohydrolase of interest is produced, the enzyme can be isolated or purified and its properties can be more carefully characterized. Alternatively, genes encoding cellobiohydrolases or cellobiohydrolase CBHII/Cel6A in various organisms can be isolated and an amino acid sequence encoded by the genes can be compared to the amino acid sequence of cellobiohydrolases CBHII/Cel6A isolated and characterized in Example 1.
[0078] The cellobiohydrolase enzymes produced, particularly the CBHII/Cel6A cellobiohydrolase enzymes can be purified using conventional methods of enzyme chemistry, such as salt preparation, ultrafiltration, ion exchange chromatography, affinity chromatography, gel filtration and chromatography of hydrophobic interaction. Purification can be monitored by protein determination, enzyme activity assays, and SDS polyacrylamide gel electrophoresis. The enzyme activity and stability of the purified enzyme at various temperature and pH values as well as the molecular mass and isoelectric point can be determined. alternatively, the properties of CBHII/Cel6A cellobiohydrolases of the invention can be identified by production of the enzymes in a recombinant host and purification and characterization of an enzyme from recombinant CBHII/Cel6A cellobiohydrolases. Also, the properties of the recombinant enzyme preparation comprising the cellobiohydrolase CBHII/Cel6A of the invention as one of the main enzyme components can be characterized as described in Example 3.
[0079] The molecular mass of purified cellobiohydrolase CBHII/Cel6A can be determined by mass spectrometry or on SDS-PAGE according to Laemmli (1970). Molecular mass can also be predicted from an enzyme amino acid sequence using the pI/MW tool on the ExPASy server (http://expasy.orp/tools/pi tool.html; Gasteiger et al., 2003).
[0080] The dependence and thermostability of a CBHII/Cel6A cellobiohydrolase enzyme can be determined in a suitable buffer at different temperatures using, for example, Avicel cellulose as a substrate as described in Example 3 or using other substrates and buffer systems in literature. The determination of pH dependence and pH stability can be carried out in a suitable buffer at different pH values following activity on a cellulosic substrate.
[0081] The pI can be determined by isoelectric focusing on an immobilized pH gradient gel composed of polyacrylamide, starch or agarose or by stimulating pI from an amino acid sequence, for example, using the pI/MW tool on the server ExPASy (http://expasy.org/tools/pi tool.html; Gasteiger et al., 2003).
The N-terminus of purified recombinant CBHII/Cel6A enzyme as well as internal peptides can be sequenced according to Edman degradation chemistry (Edman and Begg, 1967) or by prediction of the cleavage site of the secretion signal sequence of the amino acid sequence, for example, using the SignalP V3.0 program (Nielsen et al., 1997; Nielsen and Krogh, 1998; Bendtsen et al., 2004) as described in Example 1 or other methods described in the literature.
[0083] The term "full length" means the form of enzyme translated from the coding of the DNA sequence, starting with the ATG start codon, which encodes the first methionine in an amino acid sequence and ends the stop codon of TGA, TAG or TAA.
[0084] The term "mature" means the form of enzyme which, after removal, of the signal sequence (secretory signal peptide or pre-peptide) comprises an essential amino acid for enzymatic or catalytic activity. In filamentous fungi, this is in secreted form into the culture medium as a result of, for example, N-terminal processing of the signal sequence and other N-terminal processing and post-translational glycosylation. Furthermore, the mature form means an enzyme that has been cleaved from its carrier protein in fusion constructs.
[0085] Many of the bacterial and fungal cellobiohydrolases are produced as modular enzymes (Srisodsuk et al., 1993; Suurnakki et al., 2000). In addition to a catalytic or core domain expressing cellulolytic activity, these enzymes may comprise one or more cellulose binding domains (CBDs), also referred to as carbohydrate binding domains/modules (CBD/CBM), which may be located at the N or C terminals of the catalytic domain. CBDs have carbohydrate binding activity and they mediate cellulase enzyme binding to crystalline cellulose but have little or no effect on the cellulase hydrolytic activity of the enzyme on soluble substrates. These two domains are typically connected via a flexible and highly glycosylated linker or hinge region as is evident from the amino acid sequence of SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16.
"Fragment" as used in the invention means a polypeptide which requires one or more N- and/or C-terminal amino acid residues of the full-length polypeptide of SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16, such as the mature form of the polypeptide or other enzymatically active portion of the polypeptide. The fragment still has the essential catalytic activity or cellobiohydrolase activity of the full-length CBHII/Cel6A cellobiohydrolase and substantially similar properties such as pH and temperature dependence and stability and substrate specificity. The polypeptides of the invention disclosed in SEQ ID NO:12, SEQ ID NO:14, SEQ ID:15 and SEQ ID NO:16 naturally contain an N-terminal CBD and a linker. These native linkers or regions of CBD can be replaced, for example, by a CBD from a species of Trichoderma or Chaetomium. The CBHII/Cel6A enzymes of the invention can be used in applications also without a signal sequence and/or CBD or the signal sequence and/or CBD can be derived from different enzymes of the above microorganisms or different microorganism or be synthetically or recombinantly incorporated to the catalytic domain of the above enzymes.
[0087] The term "identity" as used herein means the identity between the two amino acid sequences compared to each other within the corresponding sequence region having approximately the same amount of amino acids. For example, the identity of a full-length or mature sequence of the two amino acid sequences can be compared. Also, the identity of a full-length or mature sequence that needs the N-terminal CBD of the two amino acid sequences can be compared. In this way, the comparison of, for example, CBHII/Cel6A sequences including CBD and/or signal sequences with sequences that need those elements is not within the context of the invention. The identity of the full length sequences can be measured using, for example, ClustalW alignment (eg www.ebi.ac.uk/Tool/ClustalW) as a matrix as follows: BLOSUM, Open Slit: 10, Slit Extension: 0.5, or using a Clone Manager program (version 9) (Scientific and Educational Software, Cary, USA), including the "Compare Two Sequences/Global/Compare" sequence functions as amino acids/registration matrix BLOSUM62 as described in Example 1.
[0088] The amino acid sequence of the two molecules to be compared may differ in one or more positions, which, however, do not alter the biological function or structure of the molecules. Such "variants" can occur naturally because of different host organisms or mutations in the amino acid sequence, for example, as an allelic variant within the same strain, species or genus or these can be targeted by specific mutagenesis. These may comprise amino acid substitutions, deletions, combinations or insertions of one or more positions in the amino acid sequence, but they function in a substantially similar manner to the enzymes defined in SEQ ID NO:12, SEQ ID NO:14, SEQ ID No.:15 or SEQ ID No.:16, that is, these comprise a variant having cellulolytic activity.
[0089] The present invention relates to a method for treating cellulosic material, also including lignocellulosic material with a CBHII/Cel6A polypeptide or an enzyme composition comprising said polypeptide or as in a consolidated bioprocess, the cellulosic material can be treated as a fermentative organism that produces said polypeptide, wherein the CBHII/Cel6A polypeptide exhibits cellobiohydrolase activity and comprises an amino acid sequence having at least 76% identity with the full-length polypeptide of SEQ ID NO:12, at least 76 % identity to the full-length polypeptide of SEQ ID NO:14, at least 95% identity to the full-length polypeptide of SEQ ID NO:15, or at least 91% identity to the full-length polypeptide of SEQ ID NO:16 or a fragment or variant thereof having similar properties. Said enzyme is capable of hydrolyzing cellulosic material including lignocellulose at moderate to high temperatures. Said method comprises the following steps: i) producing a CBHII/Cel6A polypeptide of the invention or an enzyme composition comprising said polypeptide or a fermentative microorganism that produces said polypeptide; ii) reacting the cellulosic material with the CBHII/Cel6A polypeptide of the invention or the enzyme composition comprising said polypeptide or the fermentative microorganism that produces said polypeptide; and iii) obtaining partially or fully hydrolyzed cellulosic material, including hydrolyzed lignocellulosic material.
CBHII/Cel6A cellobiohydrolase enzymes useful for treating or hydrolyzing cellulosic material are "obtainable from" any organism including plants. Preferably, the CBHII/Cel6A enzymes originate from microorganisms, for example bacteria or fungi. The bacteria can be, for example, from a genus selected from Bacillus, Azospirillum, Streptomyces and Pseudomonas. More preferably, the enzyme originates from fungi (including filamentous fungi and yeasts), for example, from a genus selected from the group consisting of Schizosaccharomyces, Kluyveromyces, Pichia, Saccharomyces, Candida and Yarrowia or filamentous fungi Achaetomium, Acremonium, yeasts. Aspergillus, Aureobasidium, Botrytis, Chaetomium, Chrysosporium, Cryptococcus, Collybia, Hungers, Fusarium, Humicola, Hypocrea, Lentinus, Magnaporthea, Melanocarpus, Mucor, Myceliophthora, Myriococcum, Peachlimastix, Penicilomyleurospora, Polyporus, Pycnoporus, Rhizoctonia, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Trametes and Trichoderma. Preferably, the CBHII/Cel6A enzymes are derived from Acremonium thermophilum, Melanocarpus albomyces, Chaetomium thermophilum or Talaromyces emersonii.
According to a preferred embodiment of the invention, the enzymes are obtainable from a filamentous fungus strain ALK04245 deposited as CBS 116240 and presently classified as Acremonium thermophilium strain, Melanocarpus albomyces ALK04237 deposited as CBS 685.95, Talaromyces emersonii strain DSM 2432 (in applicant's culture collection under number RF8069) or Chaetomium thermophilum ALK04265 strain deposited as CBS 730.95.
[0092] The celobioidrolases CBHII / Cel6A the present invention are marked At_ALK04245_Cel6A, Ma_ALKO4237_Cel6A, Ct_ALK04265_Cel6A and Te_RF8069_Cel6A, one celobioidrolase CBHII / Cel6A that originates from the strain Acremonium thermophilum CBS 116240, Melanocarpus albomyces CBS 685.95, Chaetomium thermophilum CBS 730.95 or Talaromyces emersonii DSM 2432 and members of the 6 family of glycoside hydrolases. Table 1: The CBHII/Cel6A cellobiohydrolases of the invention

[0093] According to a preferred embodiment of the invention, the fungal cellobiohydrolase enzyme CBHII/Cel6A useful in the method is a polypeptide having cellobiohydrolase activity and comprising an enzyme from At_ALK04245_Cel6A having the full length amino acid sequence of SEQ ID No.:12 or an amino acid sequence having at least 76% identity with the amino acid sequence SEQ ID No.:12. Preferred enzymes show at least 78%, 80% or 82%, preferably at least 84%, 86% or 88%, more preferably at least 90%, even more preferably at least 92% identity. Even more preferably, the amino acid sequence shows at least 94% or at least 96% or 97%, more preferably at least 98%, more preferably 99% identity with the amino acid sequence of SEQ ID NO:12. The identities of the two enzymes are compared within the corresponding sequence regions, that is, within the full-length region of the cellobiohydrolase CBHII/Cel6A.
[0094] Another preferred embodiment is a fungal CBHII/Cel6A cellobiohydrolase enzyme useful in the method having cellobiohydrolase activity and comprising a Ma_ALKO4237_Cel6A enzyme having the full length amino acid sequence of SEQ ID NO:14 or a sequence of amino acid having at least 76% identity with the amino acid sequence SEQ ID NO:14. Preferred enzymes show at least 78%, 80% or 82%, preferably at least 84%, 86% or 88%, more preferably at least 90%, even more preferably at least 92% identity. Even more preferably, the amino acid sequences show at least 94% or at least 96% or 97%, more preferably at least 98%, more preferably 99% identity with the amino acid sequence of SEQ ID NO:14. The identities of the two enzymes are compared within the two enzymes are compared within the corresponding sequence regions, that is, within the full length region of a CBHII/Cel6A cellobiohydrolase.
[0095] Still in a preferred embodiment of the invention is a fungal cellobiohydrolase CBHII/Cel6A enzyme useful in the method having cellobiohydrolase activity and comprising the Ct_ALKO4265_Cel6A enzyme having the full length amino acid sequence of SEQ ID NO:15 or an amino acid sequence having at least 95% identity with the amino acid sequence SEQ ID NO:15. Preferred enzymes show at least 96%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% identity with the amino acid sequence of SEQ ID NO:15. The identities of two enzymes are compared to the corresponding sequence regions, i.e. within the full length region of the cellobiohydrolase CBHII/Cel6A.
[0096] Yet a further preferred embodiment of the invention is a fungal CBHII/Cel6A cellobiohydrolase enzyme useful in the method having cellobiohydrolase activity and comprising the enzyme of TeRF8069_Cel6A having the full length amino acid sequence of SEQ ID NO:16 or an amino acid sequence having at least 91% identity with the amino acid sequence SEQ ID NO:16. Preferred enzymes show at least 92%, more preferably at least 93%, even more preferably at least 94% identity. Even more preferably the amino acid sequence shows at least 95% or at least 96% or 97%, more preferably at least 98%, more preferably 99% identity with the amino acid sequence of SEQ ID NO:16. The identities of two enzymes are compared to the corresponding sequence regions, i.e. within the full length region of the cellobiohydrolase CBHII/Cel6A.
[0097] The CBHII/Cel6A fungal cellobiohydrolases of the invention are active or stable over a wide pH range of at least pH 3 to pH 10 and more preferably over a pH range of at least pH 3 to pH 7 when subjected to the test at 50°C for 21 hours using Avicel Cellulose as a substrate, as described in Example 3.
[0098] In particular, At_ALK04245_Cel6A is active between pH 3 and pH 7, preferably between pH 4 and pH 6 and more preferably between pH 4 and pH 5. the maximum activity of At_ALKO4245_Cel6A is at pH 5 when tested at 50 °C for 21 hours using Avicel Cellulose as a substrate.
[0099] Ma_ALK04237_Cel6A is active in a pH range between pH 3 and pH 10, preferably between pH 4 and pH 8, more preferably between pH 4 and pH 7, even more preferably between pH 4 and pH 6 and even more preferably between pH 4 and pH 5. The maximum activity of Ma_ALK04237_Cel6A is at pH 4 when tested at 50°C for 21 hours using Avicel cellulose as a substrate.
[00100] Ct_ALK04265_Cel6A is active in a pH range between pH 3 and pH 10, preferably between pH 3 and pH 8, more preferably between pH 3 and pH 7, even more preferably between pH 4 and pH 7 and even more preferably between pH 4 and pH 6. The maximum activity of Ct_ALK04265_el6A is at pH 5 when tested at 50°C for 21 hours using Avicel cellulose as a substrate.
[00101] Te_RF8089_Cel6A is active in a pH range between pH 3 and pH 7, preferably between pH 3 and pH 6 and more preferably between pH 4 and pH 6. The maximum activity of Te_RF8089_Cel6A is at pH 4 when subjected to the test at 50°C for 21 hours using Avicel Cellulose as a substrate.
[00102] The enzymes of the invention are effective in degrading cellulosic or lignocellulosic material over a wide range of temperature. The cellobiohydrolase CBHII/Cel6As of the invention are active or stable for up to 21 hours over a temperature range between 40°C and 70°C when tested at the optimum pH of enzymes using Avicel cellulose as a substrate, as described in Example 3 .
[00103] At_ALK04245_Cel6A cellobiohydrolase is active between 40°C and 70°C, preferably between 40°C and 60°C, more preferably between 50°C and 60°C. At the optimum pH the maximum activity of At_ALK04245_Cel6A is at 60°C when using incubation time for 21 hours and Avicel cellulose as a substrate.
[00104] Ma_ALK04237_Cel6A cellobiohydrolase is active in a temperature range between 40°C and 60°C. The enzyme shows maximum activity at 50°C when incubated for 21 hours at the optimum pH using Avicel cellulose as a substrate. Ct_ALK04265_Cel6A cellobiohydrolase is active in a temperature range between 40°C and 70°C, preferably in the range of 50°C to 60°C. At optimal pH the maximum activity is at 60°C when using incubation time for 21 hours and Avicel cellulose as a substrate. Te_RF8089_Cel6A cellobiohydrolase is active between 40°C and 70°C, preferably between 40°C and 60°C, more preferably between 50°C and 60°C. The maximum activity is at 60°C when incubated for 21 hours at the optimum pH using Avicel Cellulose as a substrate.
[00105] Cellulolytic enzymes already in use in hydrolysis of cellulosic material and production of fermentable sugars by, for example, applications of bioethanol derived mainly from well-studied microorganisms, such as the filamentous fungus Trichoderma reesei (for example, a Accellerase® product line, Genencor Int., Inc., US). Cellulolytic enzymes are conventionally used at temperatures ranging from 30°C to 45°C. The cellobiohydrolase CBHII/Cel6As of the present invention are efficient at these temperatures as well, but in addition they work extremely still at temperatures up to 70°C, such as between 40°C and 70°C, for example, between 40°C and 70°C or between 40°C and 60°C or between 50°C and 60°C as shown in Example 3. Short hydrolysis time enzyme compositions can be functional up to 80°C. For longer incubation times full hydrolysis is required and therefore lower temperatures are normally used. This makes a cellobiohydrolase CBHII/Cel6As of the invention extremely well suited for varying cellulosic substrate hydrolysis processes performed at both conventional and moderate temperatures and at elevated temperatures.
[00106] In Examples 4 and 5, experiments performed on various cellulosic materials such as hardwood and corn cobs are described. From Figure 4 it is evident that the performance of enzyme blends supplemented with the fungal cellobiohydrolases CBHII/Cel6A At_ALKO4245_Cel6A, Ma_ALK04237_Cel6A or Ct_ALK04265_Cel6A in hydrolysis of steam-blasted hardwood is by far better than the performance of the blends without each supplementation. In the experiments carried out at 55°C (Fig. 4A), the amount of sugars released from the hardwood substrate was observed to increase 12%, 14% and 26% supplemented with enzymes Ma_ALKO4237_Cel6A, Ct_ALKO4265_Cel6A or At_ALK04245_Cel6A in MIX 2, respectively. The enzyme Acremonium thermophilum ALK04245 was observed to be the best performing CBHII/Cel6A studied in this one. At_ALK04245_Cel6A shows the increased hydrolysis also at 37°C added in the technique-established Trichoderma mixture (T.REESEI ENZYME MIXTURE) (Fig. 4B) or in the commercial product (ACC MIX) (Fig. 4C).
[00107] Similar results were also obtained in the steam-blasted corn cobs (Fig. 5). Thermophilic enzyme BLEND 2 was supplemented with the enzyme At_ALKO4245_Cel6A of the invention. The combined amount of glucose and xylose after 72 hours of hydrolysis was remarkably higher than without such supplementation.
[00108] According to a preferred embodiment of the invention the method for treating lignocellulosic or cellulosic material, such as lignocellulosic material including hemicellulose, pectin and lignin involves the use of one or more of the CBHII/Cel6A cellobiohydrolases of the invention as a "enzyme composition" or "enzyme preparation" which comprises at least one additional enzyme capable of hydrolyzing said material. Additional enzymes can be selected from the group of cellobiohydrolase, endoglycanase, beta-glycosidase, beta-glycanase, xyloglycanase, xylanase, beta-xylosidase, mannanase, beta-mannosidase, α-glucuronidase, acetyl xylan esterase, α-arabinofurases galactosidase, pectinase, endo- and exo-α-L-arabinases enveloping, α-galactosidase, endo- and exo-galactosidase, endopectinlyase, pectinesterase, pectate lyase, phenol esterase, ligninase involving lignin peroxidase, peroxidase-dependent generator of H2O2 and laccase with or without a mediator.
[00109] The enzyme preparation or composition comprises at least one of the enzymes defined above. It can contain the enzymes at least in partially purified or isolated form. This can essentially still consist of the desired enzymes. Alternatively, the preparation can be used or filtered culture medium containing one or more of the desired enzymes. Preferably the enzyme preparation is used culture medium. The "used culture medium" refers to the host culture medium which comprises the enzymes produced. Preferably host cells are separated from the medium after production. The enzyme preparation or composition can also be a "whole culture broth" obtained, optionally after killing the production hosts or microorganisms without any further downstream processing or purification of the desired cellulolytic enzymes. In the "consolidated bioprocess" the enzyme composition or at least some of the enzymes in the enzyme composition can be produced by the fermentative micro-organisms.
[00110] The "isolated polypeptide" in the present context may simply be that the cells and cell debris were removed from the culture medium containing the polypeptide. The polypeptides are conveniently isolated, for example, by adding the anionic and/or cationic polymers to the culture medium used to enhance precipitation of cells, cell debris and some enzymes that have secondary activities. The medium is then filtered using an inorganic filtering agent and a filter to remove precipitants formed. After this the filtrate is further processed using a semi-permeable membrane to remove excess salts, sugars and metabolic products.
[00111] In addition to the enzymatic activity, the preparation may contain additives such as mediators, stabilizers, buffers, preservatives, surfactants and/or components of the culture medium. Preferred additives are such as are commonly used in enzyme preparations intended for a particular application. The enzyme preparation can be in liquid, powder or granule form.
[00112] According to an embodiment of the invention the enzyme preparation comprises the mixture of CBHs, EGs and BGs, optionally together with the hemicellulose degradation enzymes in combination with the cellobiohydrolase CBHII/Cel6As of the invention. Different enzyme mixtures and combinations can be used to suit different substrate materials and process conditions. For example, if the degradation process is to be carried out at a high temperature, thermostable enzymes are chosen.
[00113] The "hemicellulose" is a heterogeneous group of carbohydrate polymers containing mainly different glycans, xylans and mannans. Hemicellulose consists of a linear structure with β-1,4-linked residues substituted with short side chains usually containing acetyl acid, 4-O-glucuronic acid, L-arabinose and galactosyl groups. Hemicellulose can be chemically cross-linked to lignin and cellulose. "Xylane degrading enzymes" or "xylanases" include both exohydrolytic and endohydrolytic enzymes such as endo-1,4-beta-D-xylanase (EC 3.2.1.8) or exo-1,4-beta-D-xylosidase (EC 3.2.1.37), which breaks hemicellulose to xylose. Glyco and galactomannans are hydrolyzed by endo-1,4-beta-mannanases (EC3.2.1.78) and beta-mannosidase (EC 3.2.1.25) to produce beta-D-mannose. Enzymes capable of removing secondary chain substituents include α-glucuronidases, acetyl xylan esterases, α-arabinofuranosidases, and α-galactosidases that act cooperatively with structure-degrading enzymes (Biely et al. 1997; Sundberg and Poutanen, 1991; Stalbrand; Stalbrand). et al., 1995).
[00114] The enzymes involved in the degradation of "pectin" involve endo- and exo-α-L-arabinases and α-galactosidase, endo- and exo-galactoronases, endopectinliases and pectinesterases (Del Canizo et al., 1994).
[00115] "Lignin" is a cross-linked polymer of the complex of differently substituted p-hydroxyphenylpropane units. Its hydrolysis involves lignin peroxidases, phenol esterases, manganese-dependent peroxidase, H2O2 generating enzyme and laccases (Cullen and Kersten, 2004).
[00116] The enzymes of the enzyme composition can be added to the lignocellulosic material simultaneously or sequentially.
[00117] According to a preferred embodiment of the invention, the method is applicable on various cellulose, lignocellulose and beta-glycan containing materials such as plant materials, for example wood, including hardwood and softwood, hardwood and softwood chips, wood pulp, sawdust and forestry and industrial wood residues; herbaceous crops, agricultural biomass such as cereal straw, beet pulp, corn fiber, corn forage and corn cobs, cereal beta-glycans, sugarcane bagasse, stalks, leaves, husks, and others; waste products such as waste municipal solid, newspaper and waste office paper, waste fiber, paper sludge, waste milling eg grains; dedicated energy crops (eg, willow, poplar, grass or yellow grass and others).
[00118] According to an embodiment of the invention, the method is applicable for the production of biofuel such as ethanol, propanol and butanol and others from cellulosic material.
[00119] Within the context of the invention is a method, wherein the CBHII/Cel6A polypeptide used in the hydrolysis of cellulosic material derived from the Acreinonium thermophilum CBS 116240 strain and has the amino acid sequence of SEQ ID NO:12 or at least 78% , 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO:12 or a fragment or variant thereof having cellobiohydrolase activity.
The present invention also relates to a fungal CBHII/Cel6A polypeptide having cellobiohydrolase activity and comprising an amino acid sequence having at least 76% identity with the full-length polypeptide of SEQ ID NO:12, at least 76% identity to the full-length polypeptide of SEQ ID NO:14, at least 95% identity to the full-length polypeptide of SEQ ID NO:15, or at least 91% identity to the full-length polypeptide of SEQ ID NO:16 or a fragment or variant thereof having similar properties.
[00121] The CBHII/Cel6A polypeptide can be obtained from the filamentous fungal genus Acremonium, Melanocarpus, Chaetomium or Talaromyces. Preferred species include Acremonium thermophilum, Melanocarpus albomyces, Chaetomium thermophilum or Talaromyces emersonii.
According to the preferred embodiment of the invention the enzymes are obtained from a filamentous fungal strain ALK04245 deposited as CBS 116240 then classified as Acremonium thermophilium strain, Melanocarpus albomyces ALK04237 deposited as CBS 685.95, Chaetomium thermophilum strain ALK04265 deposited as CBS 730.95 or Talaromyces emersonii DSM 2432 strain (in candidate culture collection under number RF8069).
[00123] According to the preferred embodiment of the invention the CBHII/Cel6A fungal cellobiohydrolase enzyme is a polypeptide having cellobiohydrolase activity and comprising the At_ALK04245_Cel6A enzyme having the full length amino acid sequence of SEQ ID NO:12 .
[00124] Another preferred embodiment of the invention is a fungal cellobiohydrolase enzyme CBHII/Cel6A having cellobiohydrolase activity and comprising the enzyme of Ma_ALKO4237_Cel6A with the full length amino acid sequence of SEQ ID NO:14.
[00125] Still in a preferred embodiment of the invention is a fungal cellobiohydrolase enzyme CBHII/Cel6A having cellobiohydrolase activity and comprising the Ct_ALKO4265_Cel6A enzyme with the full length amino acid sequence of SEQ ID NO:15.
[00126] Yet a further preferred embodiment of the invention is a fungal CBHII/Cel6A cellobiohydrolase enzyme having cellobiohydrolase activity and comprising the Te_RF8069_Cel6A enzyme with the full length amino acid sequence of SEQ ID NO:16.
[00127] According to a preferred embodiment of the invention, the cellobiohydrolase enzyme CBHII/Cel6A is capable of hydrolyzing the cellulosic material at moderate to high temperatures. The cellobiohydrolases CBHII/Cel6A of the invention are active or stable further at temperatures up to 70°C, such as between 40°C and 70°C, for example between 40°C and 60°C or between 50°C and 60°C , when tested at the optimum pH of enzymes using Avicel Ph101 cellulose as a substrate, as described in Example 3. For short hydrolysis time enzyme compositions may be functional up to 80°C. For longer incubation times full hydrolysis is required and therefore lower temperatures are normally used. This makes the CBHII/Cel6A cellobiohydrolases of the invention extremely well suited for varying cellulosic substrate hydrolysis processes performed at both conventional and moderate temperatures and at elevated temperatures requiring thermostability of enzyme temperatures.
[00128] According to the preferred embodiment of the invention the fungal CBHII/Cel6A is encoded by an isolated nucleic acid molecule which comprises a polynucleotide sequence, which encodes a polypeptide which comprises the amino acid sequence characterized in SEQ ID No. :12, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16. Thus, within the scope of the invention is the cellobiohydrolase enzyme CBHII/Cel6A or polypeptide comprising the amino acid sequence of the full-length form of the enzyme At_ALKO4245_Cel6A characterized in SEQ ID NO:12, enzyme Ma_ALKO4237_Cel6A characterized in SEQ ID NO: 14, Ct_ALKO4265 characterized in SEQ ID NO:15 or TeRF8069_Cel6A characterized in SEQ ID NO:16.
[00129] Furthermore, within the scope of the present invention are the polypeptides encoded by the nucleic acid molecules encoding a CBHII/Cel6A polypeptide having cellobiohydrolase activity and at least 76% identity with the full length amino acid sequence of SEQ ID No.:12, at least 76% identity to the full-length amino acid sequence of SEQ ID NO:14, at least 95% identity to the full-length amino acid sequence of SEQ ID NO:15, or at least minus 91% to the full-length amino acid sequence of SEQ ID NO:16. The identities of two enzymes are compared within the corresponding sequence regions, i.e. within the full-length region of the CBHII/Cel6A polypeptide.
[00130] Within the scope of the invention is a polypeptide sequence, which is encoded by a nucleic acid molecule encoded by a fragment of the polypeptide, that the polypeptide fragment requires one or more amino acid residues from the N-terminus and /or C of the full length polypeptide of SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16. The fragment still has the essential catalytic activity or cellobiohydrolase activity of full-length CBHII/Cel6A cellobiohydrolase and substantially similar properties such as pH and temperature dependence and substrate specificity. The fragment may, for example, be an enzyme that requires the secretion signal sequence or carbohydrate or CBD binding domain.
[00131] Also included are natural or synthetic variants of cellobiohydrolases CBHII/Cel6A, which have the properties similar to the polypeptides defined in SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO. :16. Variation can result from deletion, substitution, insertion, addition or combination of one or more positions in the amino acid sequence.
A preferred embodiment of the invention is a cellobiohydrolase CBHII/Cel6A which is encoded by an isolated nucleic acid molecule comprising a polynucleotide sequence included in SEQ ID NO:11, SEQ ID NO:13, SEQ ID No.:9 or SEQ ID No.:10 or a subsequence thereof.
Within the context of the invention is a cellobiohydrolase CBHII/Cel6A, which is encoded by a nucleic acid molecule or polynucleotide sequence that hybridizes under stringent conditions to a polynucleotide sequence or a subsequence thereof included in SEQ ID NO. :11, SEQ ID NO:13, SEQ ID NO:9 or SEQ ID NO:10. Also, the preferred embodiment is a polypeptide encoded by a nucleic acid molecule or polynucleotide sequence hybridizing with a probe prepared using PCR, such as the PCR fragment included in SEQ ID NO:7 or SEQ ID NO:8 .
[00134] Standard biology methods can be used in isolating cDNA or a genomic DNA from the host organism, for example, methods described in molecular biology textbooks such as Sambrook and Russell, 2001. cDNA or a genomic gene that encoding the CBHII/Cel6A cellobiohydrolase of the invention can be isolated using the DNA probes, which were prepared based on the N-terminal amino acid sequence or tryptic peptides of the purified CBHII/Cel6A enzyme. Alternatively, the probe can be designed based on the known nucleotide or amino acid sequences of homologous cellobiohydrolases. CBHII/Cel6A clones can also be evaluated based on activity on plates containing a specific substrate by the enzyme or by the use of specific antibodies by CBHII/Ce6A cellobiohydrolase.
[00135] Hybridization with a DNA probe, such as for example, SEQ ID NO:7 consists of more than 100-200 nucleotides, is usually carried out under "high stringency" conditions, i.e. hybridization at one temperature, which is 20-25°C below the calculated melting temperature (Tm) of a perfect hybrid, the calculated Tm according to Bolton and McCarthy (1962) and post-hybridization washes at the lower salt concentration. Usually prehybridization and hybridization are carried out at least 65°C in 6xSSC (or 6xSSPE), 5xDenhardt reagent, 0.5% (w/v) SDS, 100 μg/ml denatured, fragmented salmon sperm DNA. Addition of 50% formamide lowers prehybridization and hybridization temperatures at 42°C. High stringency washes are carried out in low salt concentration, eg in 2xSSC-0.1% SDS (w/v) at room temperature and finally in 0.1xSSC-0.1% SDS (w/v ) at least 65°C, for example at 68°C.
[00136] In the present invention the genes At_ALKO4245cel6A, MaALKO4237cel6A, Ct_ALKO4265cel6A and TeRF8069cel6A were isolated with a probe prepared by PCR using stringent hybridization conditions as described in Example 1. The genomic cbh2/cel6A genes were isolated by using oligonucleotide primers based on published nucleotide sequences or use of degenerate oligonucleotide primers designed based on alignment of previously known amino acid sequences of CBHII/Cel6A proteins.
[00137] According to a preferred embodiment of the invention the fungal cellobiohydrolase enzyme CBHII/Cel6A is encoded by an isolated nucleic acid molecule, which comprises the nucleotide sequence of SEQ ID NO:11 which encodes a form of length total enzyme At_ALKO4245Cel6A of SEQ ID NO:12. Another preferred embodiment of the invention is a fungal CBHII/Cel6A cellobiohydrolase encoded by an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:13, which encodes the full length form of the enzyme MaALK04237_Cel6A having the sequence of amino acid of SEQ ID NO:14. Another preferred embodiment of the invention is a fungal CBHII/Cel6A cellobiohydrolase encoded by an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:9, which encodes the full length form of the enzyme Ct_ALKO4265_Cel6A having the sequence of amino acid of SEQ ID NO:15. A further preferred embodiment of the invention is a fungal CBHII/Cel6A cellobiohydrolase encoded by an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:10, which encodes the full length form of the enzyme TeRF8069Cel6A having the sequence of amino acid of SEQ ID NO:16.
[00138] According to another preferred embodiment of the invention the fungal cellobiohydrolase CBHII/Cel6A is encoded by the polynucleotide sequence included in plasmid pALK2582 deposited in Escherichia coli RF8175 under accession number DSM 22946, plasmid pALK2581 deposited in Escherichia coli RF8174 accession number DSM 22945, plasmid pALK2904 deposited in Escherichia coli RF8214 under accession number DSM 22947, or plasmid pALK3006 deposited in Escherichia coli RF8333 under accession number DSM 23185.
[00139] An embodiment of the invention is the cellobiohydrolase CBHII/Cel6A produced from the recombinant expression vector that comprises the nucleic acid molecule encoding the fungal cellobiohydrolase CBHII/Cel6A as characterized above, operatively linked to regulatory sequences capable of direct the expression of a gene encoding said cellobiohydrolase enzyme CBHII/Cel6A in a suitable host. The construction of said recombinant expression vector and use of said vector is described in more detail in Example 2.
Suitable hosts for the production of fungal cellobiohydrolase CBHII/Cel6A are homologous or heterologous hosts, such as microbial hosts including bacteria, yeasts and fungi.
Filamentous fungi, such as Trichoderma, Aspergillus, Fusarium, Humicola, Chrysosporium, Neurospora, Rhizopus, Penicilliurn and Mortiriella, are preferred production hosts due to ease of downstream processing and recovery of the enzyme product. Suitable hosts include species such as type T. reesei, A. niger, A. oryzae, A. soybeane, A. awamori or A. japonicus strains, F. venenatum or F. oxysporum, H. insolens or H. lanuginosa, N. crassa and C. lucknowense, some of which are listed as enzyme-producing host organisms in, for example, AMFEP 2007 list of commercial enzymes (http://www.amfep.org/list.html). More preferably, the enzyme is produced in a filamentous fungal host of the Trichoderma or Aspergillus genus, such as T. reesei, or A. niger, A. oryzae or A. awamori. According to the most preferred embodiment of the invention the fungal cellobiohydrolase enzyme CBHII/Cel6A is produced in T. reesei.
According to the preferred embodiment of the invention the CBHII/Cel6A polypeptide is At_ALKO4245Cel6A deriving from Acremonium thermophilum CBS 116240 and having the amino acid sequence of SEQ ID NO:12.
The present invention also relates to an isolated nucleic acid molecule comprising a polynucleotide sequence encoding a fungal CBHII/Cel6A cellobiohydrolase selected from the group consisting of: (a) a nucleic acid molecule or polynucleotide sequence which encodes a polypeptide having cellobiohydrolase activity and which comprises a full-length amino acid sequence as described in SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16 or a fragment or variant thereof having similar properties; (b) a nucleic acid molecule or polynucleotide sequence encoding a polypeptide having cellobiohydrolase activity and at least 76% identity to the full length amino acid sequence of SEQ ID NO:12, at least 76% identity to the full-length amino acid sequence of SEQ ID NO:14, at least 95% identity to the full-length amino acid sequence of SEQ ID NO:15, or at least 91% identity to the long amino acid sequence the entirety of SEQ ID NO:16 or a fragment or variant thereof having similar properties; (c) a nucleic acid molecule which comprises the coding sequence of the nucleotide sequence as described in SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:9 or SEQ ID NO:10; (d) a nucleic acid molecule comprising the coding sequence for the polynucleotide sequence contained in DSM 22946, DSM 22945, DSM 22947 or DSM 23185; (e) a nucleic acid molecule whose coding sequence differs from the coding sequence of a nucleic acid molecule according to any one of (c) to (d) due to the degeneracy of the genetic code; and (f) a nucleic acid molecule that hybridizes under stringent conditions to a nucleic acid molecule contained in DSM 22946, DSM 22945, DSM 22947 or DSM 23185 or a subsequence thereof and encoding a polypeptide having cellobiohydrolase activity and a amino acid sequence showing at least 76% identity to the full-length amino acid sequence as described in SEQ ID NO:12, at least 76% to the full-length amino acid sequence of SEQ ID NO:14, at least 95% to the full length amino acid sequence of SEQ ID NO:15 or at least 91% to the full length amino acid sequence of SEQ ID NO:16.
[00144] The nucleic acid molecule of the invention can be RNA or DNA, where the DNA can consist of genomic DNA or cDNA.
[00145] Standard molecular biology methods can be used in the isolation and enzyme treatments of the polynucleotide sequence encoding the fungal cellobiohydrolase CBHII/Cel6A of the invention, including isolation of genomic or plasmid DNA, digestion of DNA to produce the DNA fragments , sequencing, E.coli transformations etc. Basic methods are described in standard molecular biology textbooks, eg Sambrook and Russell, 2001.
[00146] The isolation of the At_ALK04245_cel6A, Ma_ALK04237_cel6A, Ct_ALK04265_cel6A and TeRF8069cel6A gene encoding the polypeptides At_ALKO4245_Cel6A, Ma_ALKO4237_Cel6A, Ct_ALKO4265_cel6A and TeRF8069B fragment is described in Brief PCR ID No. 1,6031 and 6bp PCR Example No. 1031 SEQ. (SEQ ID NO:8) obtained by using the sequences of the degenerate oligonucleotide primers (SEQ ID NO:1 and SEQ ID NO:2) were used to isolate the At_ALK04245_cel6A gene from Acremonium thermophilum ALK04245 and the Ma_ALKO4237_cel6A gene from Melanocarpus albomyces ALK04237 in the pCR®4Blunt-TOPO® vector. The full-length A. thermophilum cel6A gene in plasmid pALK2582 deposited in E.coli to the DSMZ culture harvest under accession number DSM 22946. The full-length Malbomyces cel6A gene was included in plasmid pALK2581 deposited in E.coli by collecting E.coli. DSMZ culture under the DSM accession number 22945.
The Ct_ALK04265_cel6A gene was isolated using the primer pairs of SEQ ID N°:3 and SEQ ID N°:4 and the TeRF8069_cel6A gene was isolated using the primer pairs of SEQ ID N°:5 and SEQ ID N°:6 as described in Example 1. PCR fragments containing the full length cel6A genes from Chaetomium thermophilum ALK04265 and Talaromyces emersonii RF8069 were included in plasmids pALK2904 and pALK3006 deposited in E.coli for the DSMZ culture harvest under accession numbers DSM 22947 and DSM 23185, respectively.
The deduced amino acid sequences of cellobiohydrolases CBHII/Cel6A were analyzed from the DNA sequences as described in Example 1.
[00149] The length of the gene At_ALK04245_cel6A (SEQ ID NO:11), which encodes Acremonium thermophilum cellobiohydrolase CBHII/Cel6A (SEQ ID NO:12), is 1830bp (including the stop codon). Four putative ions were observed to have lengths of 79, 72, 117 and 126bps. Thus, the coding region of At_ALK04245cel6A is 1434bp (stop codon not included) and the deduced protein sequence consists of 478 amino acids including an 18 amino acid predicted signal sequence (SignalP V3.0; Nielsen et al., 1997; Nielsen and Krogh, 1998 and Bendtsen et al., 2004). The deduced amino acid sequence has homology to published CBHII/Cel6A sequences when analyzed using the BLAST program, version 2.2.9 to NCBI, National Center for Biotechnology Information; Altschul et al., 1990). The predicted molecular mass of the mature enzyme excluding the signal sequence was 48,918 Da and the predicted pI was 4.82. These predictions were made using the Compute pUMW tool on the ExPASy server (Gasteiger et al., 2003). The identity values of the full length sequence At_ALKO4245_Cel6A and the corresponding regions of the homologous sequences were obtained by using a Clone Manager Program (version 9) including the functions "Compare two Sequences/Global/Compare sequences as amino acids/BLOSUM62 scoring matrix". Values (%) show identity with the other CBHII/Cel6A cellobiohydrolases of the present invention is shown in Table 6. Identity with the published CBHII/Cel6A amino acid sequences is shown in Table 7 and Table 8.
[00150] The At_ALKO4245_Cel6A of the invention shows the greatest homology to the full length polypeptide of Thielavia terrestris NRRL 8126 (SEQ ID NO:2 in US 7,220,565, Novozymes Inc., US and SEQ ID NO:49 in W02009085868, Novozymes A/S, DK), the unnamed protein product of Podospora anserina DSM 980 (EMBL Accession No. XP_001903170) and the deduced endoglycanase 2 precursor of Neurospora crassa OR74A (XM 955677). The identity with the T. terrestris proteins is within the 75% full-length polypeptide. Identities with P. anserina and N. crassim polypeptides were 69%.
The length of the gene Ma_ALK04237_cel6A (SEQ ID NO:13), which encodes Melanocarpus albomyces cellobiohydrolase CBHII/Cel6A (SEQ ID NO:14), is 1607bp (including the stop codon). Two putative introns were observed to be 93 and 95bps long. Thus, the coding region of Ma_ALK04237_cel6A is 1416bp (stop codon not included) and the deduced protein sequence consists of 472 amino acids including the 17 amino acid predicted signal sequence (SignalP V3.0; Nielsen et at., 1997; Nielsen and Krogh, 1998 and Bendtsen et al., 2004). The predicted molecular mass of the mature enzyme excluding the signal sequence was 48,627 Da and the predicted pI was 4.50. These predictions were made using the Compute pI/MW tool on the ExPASy server (Gasteiger et at., 2003). The Ma_ALKO4237_Cel6A of the invention shows the greatest homology to the full-length polypeptide from an uncharacterized organism, disclosed as an amino acid sequence SEQ ID NO:413 in W02008095033 (Syngenta Inc., US) (72%), to the Thielavia CBHII polypeptide terrestris NRRL 8126 (SEQ ID NO:49 in W02009085868, Novozymes A/S, DK) (71%) and the hypothetical protein CHGG10762 from Chaetomium globosum CBS 148151 (EMBL Accession No. XP001226029) (75% identity).
[00152] The length of the Ct_ALK04265_cel6A gene (SEQ ID NO:9), which encodes the Chaetomium thermophilum cellobiohydrolase CBHII/Cel6A (SEQ ID NO:15) is 1757bp (including the stop codon). Three putative ions were observed having lengths of 77, 196 and 56bps. Thus, the coding region of Ct_ALK04265cel6A is 1425bp (stop codon not included) and the deduced protein sequence consists of 475 amino acids including the 17 amino acid predicted signal sequence (SignalP V3.0; Nielsen et at., 1997; Nielsen and Krogh, 1998 and Bendtsen et al., 2004). The predicted molecular mass of the mature enzyme excluding the signal sequence was 49 408 Da and the predicted pI was 5.31. The Ct_ALK04265Cel6A of the invention shows the major homology to cellobiohydrolase deduced from Chaetomium thermophilum CT2 family 6 (EMBL Accession No. AY861348; CN 1757709, Shandong Agricultural University, CN), to the C. thermophilum polypeptide CGMCCO859 having cellobiohydrolase activity (SEQ II) ID NO:2 in EP1578964B1, Novozymes A/S, DK) and the Chaetomium thermophilum CBHII of SEQ ID NO:36 (the amino acid sequence of the sequence listing) or SEQ ID NO:46 (the amino acid sequence of the description) or SEQ ID NO:45 (the nucleotide sequence of the description) in W02009059234, Novozymes Inc., US. The identities within the full length polypeptides were 94%.
The length of the TeRF8069cel6A gene (SEQ ID NO:10), which encodes Talaromyces emersonii cellobiohydrolase CBHII/Cel6A (SEQ ID NO:16), is 1754bp (including the stop codon). Seven putative introns were observed having lengths of 50, 44, 52, 56, 53, 59 and 60bps. Thus, the coding region of TeRF8069cel6A is 1377bp (stop codon not included) and the deduced protein sequence consists of 459 amino acids including the 19 amino acid predicted signal sequence (SignalP V3.0; Nielsen et at., 1997; Nielsen and Krogh, 1998 and Bendtsen et al., 2004). The predicted molecular mass of the mature enzyme excluding the signal sequence was 46.618 Da and the predicted pI was 4.27. The TeRF8069Cel6A of the invention shows major homology to the polypeptide from Talaromyces emersonii (Q8N1B5 in Figure 3A-C of W02006074005, Novozymes Inc., US) and to cellobiohydrolase II from Talaromyces emersonii (AY075018). Identities within the full-length amino acid sequences were 90%.
[00154] Thus, within the scope of the invention is an isolated polynucleotide sequence or isolated nucleic acid molecule encoding a cellobiohydrolase enzyme CBHII/Cel6A or polypeptide comprising the amino acid sequence of the full length form of the characterized At_ALKO4245Cel6A enzyme in SEQ ID N°:12, MaALKO4237_Cel6A enzyme characterized in SEQ ID N°:14, Ct_ALK04265 characterized in SEQ ID N°:15 or Te_RF8069_Cel6A characterized in SEQ ID N°:16.
[00155] Still within the scope of the present invention are nucleic acid molecules or polynucleotide sequence encoding a CBHII/Cel6A polypeptide having cellobiohydrolase activity and at least 76%, preferably at least 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96% or 97%, more preferably at least 98%, more preferably 99% identity with the full length amino acid sequence of SEQ ID NO. :12. The identities of two enzymes are compared within corresponding sequence regions, i.e. within the full-length region of the CBHII/Cel6A polypeptide.
[00156] Another preferred embodiment of the invention is a fungal cellobiohydrolase enzyme CBHII/Cel6A showing at least 76%, preferably at least 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92 %, 94%, 96% or 97%, more preferably at least 98%, more preferably 99% identity to the full length amino acid sequence of SEQ ID NO:14.
Further, preferred CBHII/Cel6A cellobiohydrolases show at least 95%, preferably at least 96% or at least 97%, even more preferably at least 98% identity, more preferably at least 99% identity with the amino acid sequence of full length SEQ ID NO:15.
[00158] Other preferred CBHII/Cel6A polypeptides show at least 91%, preferably at least 92%, 93%, 94%, 95%, 96% or 97%, more preferably at least 98% and most preferably 99% identity with the full-length amino acid sequence of SEQ ID NO:16.
[00159] Within the scope of the invention is an isolated nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide, which has the amino acid sequence of the full length cellobiohydrolase CBHII/Cel6A of the invention as well as natural or synthetic fragment or variants of the polypeptides of the invention that have cellobiohydrolase activity and properties similar to the full-length polypeptide. Such a fragment may, for example, be an enzyme that requires the secretion signal sequence or carbohydrate or CBD binding domain.
A preferred embodiment of the invention is an isolated nucleic acid molecule comprising the "coding sequence" of the polynucleotide sequence included in SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:9 or SEQ ID NO:10 or a subsequence thereof. According to a preferred embodiment of the invention the polypeptide is encoded by a nucleic acid molecule having the nucleotide sequence SEQ ID NO:11 which comprises the coding sequence by the enzyme At_ALK04245Cel6A. The term "coding sequence" means a nucleotide sequence that starts from the translation start codon (ATG) and stops at the translation stop codon (TAA, TAG or TGA) and may comprise the intron regions. The translated full-length polypeptide usually starts with methionine.
According to another preferred embodiment of the invention the isolated nucleic acid molecule comprises a sequence coding for a polynucleotide sequence included in plasmid pALK2582 deposited in Escherichia coli RF8175 under accession number DSM 22946, plasmid pALK2581 deposited in Escherichia coli RF8174 under accession number DSM 22945, plasmid pALK2904 deposited in Escherichia coli RF8214 under accession number DSM 22947, or plasmid pALK3006 deposited in Escherichia coli RF8333 under accession number DSM 23185, said polynucleotide sequence coding for Atel642 , Ma_ALKO4237_Cel6A, Ct_ALK04265Cel6A and TeRF8069Cel6A cellobiohydrolases, respectively.
The nucleic acid molecule of the invention may also be an analogue of the nucleotide sequence characterized above. By "degeneration" is meant those analogs of the nucleotide sequence, which differ by one or more nucleotides or codons, but which encode the recombinant CBHII/Cel6A of the invention.
The nucleic acid molecule may also be a nucleic acid molecule or polynucleotide sequence which hybridizes under stringent conditions to a polynucleotide sequence contained in plasmids pALK2582, pALK2581, pALK2904 or pALK3006 deposited in E.coli under the numbers of accession DSM 22946, DSM 22945, DSM 22947 or DSM 23185 or a subsequence thereof and encoding a polypeptide having cellobiohydrolase activity and an amino acid sequence in which within the region of the corresponding sequence shows at least 76% identity with the sequence of full-length amino acid as described in SEQ ID NO:12, at least 76% identity with the full-length amino acid sequence of SEQ ID NO:14, at least 95% identity with the full-length amino acid sequence of SEQ ID NO:15 or at least 91% to the full length amino acid sequence of SEQ ID NO:16. The hybridization DNA can originate from a fungus belonging to the genus Acremonium, Melanocarpus, Chaetomium or Talaromyces or it can originate from other fungal species.
[00164] According to the preferred embodiment of the invention the fungal cellobiohydrolase enzyme CBHII/Cel6A is encoded by an isolated nucleic acid molecule, which comprises the nucleotide sequence of SEQ ID NO:11 which encodes a form of length total enzyme At_ALKO4245_Cel6A of SEQ ID NO:12.
[00165] The present invention also relates to a recombinant expression vector or recombinant expression construct, which can be used to propagate or express the nucleic acid molecule or polynucleotide sequence encoding the choice of cellobiohydrolase CBHII/Cel6A in a suitable eukaryotic or prokaryotic host. The recombinant expression vector comprises DNA or nucleic acid sequences that facilitate or direct the expression and secretion of the CBHII/Cel6A polypeptide sequence encoding or gene in a suitable host, such as promoters, enhancers, terminators (including translation termination signals or transcription) and signal sequences operably linked to the polynucleotide sequence encoded by said polypeptide. An expression vector may further comprise marker genes for selection of the transforming strains or the selection marker may be introduced into the cell in another vector construct by co-transformation. Said regulatory sequences can be homologous or heterologous to the production organism and these can originate from the organism, from which the gene encoding the CBHII/Cel6A polypeptide is isolated.
[00166] Examples of promoters for the expression of CBHII/Cel6A of the invention in filamentous fungal hosts are the promoters of A. oryzae TAKA amylase, alkaline protease ALP and triose phosphate isomerase, Rhizopus miehei lipase, Aspergillus niger or A. awamorglai glucoamylase (A. awamorglai glucoamylase). ), Fusarium oxysporum trypsin-like protease, promoter 1 Chrysosporium lucknowense cellobioidrolase, Trichoderma reesei cellobioidrolase I (Ce17A) etc.
[00167] In yeast, for example, promoters from S. cerevisiae enolase (ENO-1), galactokinase (GAL1), alcohol dehydrogenase (ADH2) and 3-phosphoglycerate kinase can be used to provide expression.
[00168] Examples of promoter sequences for the direction of transcription of the CBHII/Cel6A polypeptide of the invention in a bacterial host are the Escherichia coli lac operon promoter, the Streptomyces coelicolor agarase dagA promoter, the B. licheniformis alpha-amylase promoter ( amyL), the promoter of the B. stearothennophilus maltogenic amylase gene (ainyM), the promoters of the B. sublitis xylA and xylB genes, etc.
Suitable terminators include those from the genes mentioned above or any of the characterized terminator sequences.
[00170] Suitable transformation or selection markers include those that complement a defect in or add a new trait, eg enzyme activity to the host, eg the dal genes of B. subtilis or B. licheniformis or Aspergillus amdS and niaD. Selection can also be based on a marker that confers antibiotic resistance, such as resistance to ampicillin, kanamycin, chloramphenicol, tetracycline, phleomycin, or hygromycin.
Extracellular production of CBHII/Cel6A of the invention is preferable. Thus, the recombinant vector comprises sequences that facilitate secretion in the selected host. The CBHII/Cel6A cellobiohydrolase signal sequence of the invention or the presequence or prepeptide can be included in the recombinant expression vector or the natural signal sequence can be replaced with another signal sequence capable of facilitating expression in the selected host. Thus, the chosen signal sequence can be homologous or heterologous in an expression host.
[00172] Examples of suitable signal sequences are those from fungal or yeast organisms, e.g. signal sequences from well expressed genes. Such signal sequences are well known from the literature.
[00173] The recombinant vector may further comprise sequences that facilitate the integration of the vector into the host chromosomal DNA to obtain stable expression. The vector can also be a fusion construct and comprises sequences that encode a loading polypeptide that is genetically fused to the same coding sequence as the coding sequence of the protein of interest and that enhances secretion of the polypeptide in a heterologous or host organism that facilitates protein purification after production. Such carriers include, for example, proteins produced in high amounts by the natural host, such as cellulase from T. reesei or glycoamylases from Aspergillus species. The carrier polypeptide can also be an intact domain of the secretable protein, such as the CBD. The carrier protein and protein of interest can remain as a fusion after secretion, or are separated by proteolytic processing during the protein export process in the host, or are chemically or biochemically separated after secretion into the supernatant.
The cellobiohydrolases At_ALKO4245Cel6A, MaALKO4237Cel6A, Ct_ALKO4265Cel6A and Te_RF8069_Cel6A of the invention were expressed with the signal sequence itself from the Trichoderma reesei cbhl promoter (cel7A) as described in Example 2. The T. reei expression construct used to transform the host. also the cbhl terminator and amdS tag for selecting transformants from non-transformed cells.
[00175] The present invention also relates to host cells comprising the recombinant expression vector as described above. Suitable hosts for the production of fungal CBHII/Cel6A cellobiohydrolase are homologous or heterologous hosts, such as microbial hosts including bacteria, yeasts and fungi. Production systems in plant or mammalian cells are also possible.
Filamentous fungi, such as Trichoderma, Aspergillus, Fusarium, Humicola, Chrysosporium, Neurospora, Rhizopus, Penicilliurn and Mortiriella, are preferred production hosts due to the ease of downstream processing and recovery of the enzyme product. Suitable expression and production host systems are, for example, the production system developed by the host of the filamentous fungus Trichoderma reesei (EP 244234), or Aspergillus production system, such as A. oryzae or A. niger (WO 9708325, US 5,843 .745, US 5,770,418), A. awamori, A. soybeane and A. japonicus type strains, or the production system developed by Fusarium, such as F. oxysporum (Malardier et at., 1989) or F. venenatum and by Neurospora crassa, Rhizopus rniehei, Mortiriella alpinis, H. lanuginosa or H. insolens or by Chrysosporium lucknowense (US 6,573,086). Suitable production systems developed for yeasts are systems developed by Saccharomyces, Schizosaccharomyces or Pichia pastoris. Suitable production systems developed for the bacterium are a production system developed by Bacillus, for example, by B. subtilis, B. licheniformis, B. amyloliquefaciens, by E.coli, or by actinomycete Streptomyces. Preferably the cellobiohydrolase CBHII/Cel6A of the invention is produced in a filamentous fungal host of the genus Trichoderma or Aspergillus, such as T. reesei, or A. niger, A oryzae, A. soybeane, A. awamori or the type A. japonicus strains . According to the most preferred embodiment of the invention the fungal cellobiohydrolase CBHII/Cel6A is produced in T. reesei.
The production host cell may be homologous or heterologous to the cellobiohydrolase CBHII/Cel6A of the invention. Preferably the recombinant host is modified to express the secreted cellulolytic enzymes as its main activity or one of its main activities. This can be done by deleting more homologous secreted genes, for example, the four major cellulases from Trichoderma and by heterologous site-targeting genes that have been modified to ensure high expression and production levels. For example, the host may be free of homologous cellobiohydrolases due to removal of said cellobiohydrolases by inactivating or removing one or more host cellobiohydrolases, for example, by deleting the genes encoding such homogeneous or homologous cellobiohydrolases.
[00178] The present invention also relates to a process for the production of a CBHII/Cel6A polypeptide having cellobiohydrolase activity, said process comprising the steps of cultivating the natural or recombinant host cell carrying out the recombinant expression vector by cellobiohydrolase CBHII /Cel6A of the invention under the appropriate conditions and optionally isolating said enzyme. The production medium may be a medium suitable for the development of the host organism and containing the inducers for efficient expression. Suitable means are well known from the literature.
[00179] The invention relates to a polypeptide having cellobiohydrolase activity, said polypeptide being encoded by the nucleic acid molecule of the invention in which it is obtainable by the process described above.
[00180] The invention further relates to a process to obtain an enzyme preparation comprising a CBHII/Cel6A polypeptide, which has cellobiohydrolase activity, said process comprising the steps of cultivating a host cell performing an expression vector of the invention and preparing the total culture broth or separating the cells from the used culture medium and obtaining the supernatant having cellobiohydrolase activity.
[00181] The present invention also relates to an enzyme preparation, which comprises the CBHII/Cel6A enzyme characterized above. The enzyme preparation or composition has cellobiohydrolase activity and is obtainable by the process according to the invention.
[00182] Within the invention is an enzyme preparation comprising the fungal cellobiohydrolase CBHII/Cel6A of the invention.
Said enzyme preparation may further comprise different types in addition to the CBHII/Cel6A cellobiohydrolase of this invention, for example, another cellulase including cellobiohydrolase, endoglycanase and beta-glycanase, beta-glycanase, an amylase, a lipase, cutinase, a protease, xylanase, beta-xylosidase, mannanase, beta-mannosidase, α-glucuronidase, acetyl xylan esterase, α-arabinofuranosidase, α-galactosidase, pectinase, endo- and exo-al-arabinases, enveloping α-galactosidase, endo exo-galactoronase, xyloglycanase, endopectinlyase, pectin lyase, and pectinesterase, phenol esterase, ligninase involving lignin peroxidase, manganese-dependent peroxidase, H2O2 generating enzyme and/or an oxidase such as a laccase or peroxidase with or without a mediator. These enzymes are expected to enhance the performance of the inventive CBHII/Cel6A enzyme by removing carbohydrates, proteins and oils or fats present in the material to be handled. Said enzymes can be natural or recombinant enzymes produced by the host strain or can be added to the culture supernatant after the production process. Enzyme compositions or enzyme preparations can contain any combination of these enzymes. CBHII/Cel6A cellobiohydrolases can also be used in combination with commercially available enzyme preparations.
[00184] The enzymes necessary for the hydrolysis of cellulosic material according to the invention can be added in an enzymatically effective amount simultaneously, for example, in the form of an enzyme mixture, or sequentially, or as a part of the simultaneous saccharification and process of fermentation (SSF) or these can be produced by the fermentative microorganism in the consolidated bioprocess.
[00185] The enzyme preparation or composition may contain the enzymes in at least partially purified and isolated form. It can still essentially consist of the desired enzyme or enzymes. Alternatively the preparation can be a whole culture broth or used or filtered culture medium containing one or more desired enzymes. In a consolidated bioprocess, enzymes can be produced by the fermentative microorganism used in the process. In addition to the enzymatic activity, the preparation may contain additives such as mediators, stabilizers, buffers, preservatives, surfactants and/or culture medium components.
[00186] Preferred additives are like, which are commonly used by enzyme preparations intended for a particular application. surfactants are useful in emulsifying grease and wetting surfaces. The surfactant can be a nonionic including semipolar and/or anionic and/or cationic and/or zwitterionic. Buffers can be added to an enzyme preparation to modify the pH or affect the performance or stability of other ingredients. Suitable stabilizers include polyols such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or boric acid derivatives, peptides, etc. The bleaching agent is used to oxidize and degrade organic compounds. Examples of suitable chemical bleaching systems are H2 O2 sources, such as perborate or percarbonate with or without peracid forming bleach activators such as tetra-acetylethylenediamine, or alternatively peroxyacids, for example, amide, imide or sulfone type. Chemical oxidants can be partially or completely replaced by the use of oxidizing enzymes, such as laccases or peroxidases. Many laccases do not work effectively in the absence of mediators. Complexing or building agents include substances such as zeolite, diphosphate, triphosphate, carbonate, citrate, etc. The enzyme preparation may further comprise one or more polymers such as carboxymethylcellulose, poly(ethylene glycol), poly(vinyl alcohol), poly(vinylpyrrolidone) etc. Also, softeners, caustics, preservatives to avoid wasting other ingredients, abrasives and substances modified for foam and viscosity properties can be added.
[00187] According to a preferred embodiment of the invention said enzyme preparation is in the form of used culture medium, liquid, powder or granules. Preferably the enzyme preparation is used culture medium.
[00188] The present invention also relates to various uses of the fungal cellobiohydrolase CBHII/Cel6A of the invention, where hydrolysis or modification of the cellulosic material is desired. Such uses include any application where cellulolytic enzymes are conventionally used, such as in the fuel, textile, detergent, pulp and paper, feed, or food or beverage industry. The addition of the inventive fungal cellobiohydrolase CBHII/Cel6A to an enzyme composition comprising other cellulose degrading enzymes such as cellobiohydrolase I, endoglycanases and beta-glycosidases greatly enhances hydrolysis and leads to near-total hydrolysis of the polymeric cellulose structure to the monomers of glucose. The major product of CBHII/Cel6A action is cellobiose composed of two glucose units.
[00189] A preferred embodiment is the use of the method of the invention in applications requiring the stability/performance of cellobiohydrolase CBHII/Cel6A at moderate/conventional or elevated temperatures, i.e. requiring thermophilic or thermostable enzymes. Elevated temperatures are shown to enhance the hydrolysis of crystalline cellulose present in the cellulosic or cellulosic lignomaterial, thereby reducing the total amount of enzymes needed in hydrolysis or reducing the required hydrolysis time. Also, since at elevated temperatures the viscosity of the lignocellulosic substrate is lowered, thermostable enzymes make it possible to work at the higher solid loads and avoid investment cost.
[00190] Another preferred embodiment is the use of the CBHII/Cel6A polypeptide of the invention or the preparation of enzyme comprising said polypeptide in the hydrolysis of cellulosic material for the production of biofuel comprising ethanol, propanol, butanol and others. In biofuel production the cellobiohydrolases CBHII/Cel6A of the invention are similar to other cellulolytic enzymes especially suitable for the production of glucose monomers from polymeric cellulosic material which can then be fermented by yeast strains into ethanol and used as fuel.
[00191] The cellulosic lignomaterial can be pretreated before enzymatic hydrolysis to break down the fiber structure of the cellulosic substrates and make the cellulose fraction more accessible to cellulolytic enzymes. Current pretreatments include mechanical, chemical or thermal processes and combinations thereof. The material can be, for example, pretreated by steam explosion or acid hydrolysis. The saccharification process, ie production of sugar monomers and fermentation by the yeast strains, can be carried out separately or simultaneously in the same reactor. The cellobiohydrolase CBHII/Cel6A of the invention has a greater advantage over commercial enzymes as it is also stable at elevated temperatures, i.e. thermostable. The hydrolysis of cellulosic or cellulosic lignomaterial is known to intensify at elevated temperatures and produce more efficient sugar monomers.
[00192] The use of CBHII/Cel6As polypeptide of the invention enables the use of high biomass consistency and leads to high sugar and ethanol concentrations. This method can lead to significant savings in energy and investment costs. The high temperature also decreases the risk of contamination during hydrolysis.
[00193] Sugar hydrolysates can also serve as raw material by other non-microbial processes, for example, for enrichment, isolation and purification of high value sugars or various polymerization processes.
[00194] Glucose monomers can also be used as intermediates or raw materials for the production of various chemical or building blocks for chemical industry processes, for example, called biorefinery.
[00195] In the pulp and paper industry, polypeptides can be used to modify cellulosic fiber, for example, in the treatment of brown paper pulp, mechanical pulp or recycled paper.
[00196] In the textile industry cellobiohydrolases CBHII/Cel6A observes applications in softening and/or improving the feel of cotton fabrics and removal of indigo pigments in replacement of stone washing.
[00197] In the food industry the cellobiohydrolases CBHII/Cel6A of the invention can be used in the degradation of cellulosic or hemicellulosic materials present in feed stocks.
[00198] In the detergent industry the cellobiohydrolase CBHII/Cel6A can be used in the hand washing machine or machine or dish washing compositions for the removal of cellulosic dyes.
[00199] The invention is illustrated by the following non-limiting examples. From the experimental results it can be concluded that the fungal cellobiohydrolase CBHII/Cel6A of the invention is able to satisfy the widest variation of demand from different industry requiring the efficient hydrolysis of the cellulosic material present in the variation of feed stocks. EXAMPLE 1. Cloning of cellobiohydrolase 2 (cbh2) genes
[00200] Standard molecular biology methods have been used in DNA enzyme isolation and treatment (eg plasmid DNA isolation, DNA digestion to produce DNA fragments), E.coli transformations, sequencing etc. The basic methods used were as described by the enzyme, reagent or kit manufacturer or as described in standard molecular biology textbooks, eg Sambrook and Russell (2001). Isolation of genomic DNA was performed as described in detail by Raeder and Broda (1985).
[00201] Four thermophilic fungal strains from the collection of Roal Oy culture were selected for cloning based on previous verifications that the strains produce thermostable cellulases (W02007071818; Maheshwari et al., 2000; Murray et al., 2003; Miettinen- Oinonen et al., 2004). Probes for cloning Acremonium thermophilum ALK04245 and Melanocarpus albomyces ALK04237 cbh2 were synthesized by PCR. Degenerate oligos were designed based on the alignment of previously published amino acid sequences of cellobiohydrolase II (CBHII) proteins. The sequences of the homologous primers for cloning the cbh2 genes from the Chaetomium thermophilum ALK4265 and Talaromyces emersonii RF8069 strains were obtained from the published nucleotide sequences (AY861348; DQ020255; CQ838150; Murray et al., 2003; AY07365018; AF43). The sequences of the primers are shown in Table 2 (SEQ ID NO:1-6).
Table 2. Oligonucleotides used as PCR primers to amplify the probes for evaluating the cbh2 genes from Acremonium thermophilum ALK04245 and Melanocarpus albomyces ALK04237 and to amplify the full length cbh2 genes from Chaetomium thermophilum ALK04265 and Talaromyces emersonii RF8069.

[00203] The probes were amplified by PCR with primers described in Table 2 using genomic DNA as a template in the reactions. PCR mixtures of Acremonium thermophilum ALK04245 and Melanocarpus albomyces ALK04237 contain 10 mM Tris-HC1, pH 8.8, 50 mM KCl, 0.1% Triton X-100, 1.5 mM MgCl2, 0.2 mM of dNTPs, 5 μM of each primer and 1-2 units of Dynazyme EXT DNA polymerase (Finnzymes, Finland) and 0.5-1 μg of the corresponding genomic DNA. The conditions for the PCR reactions were as follows: 5 min initial denaturation at 95°C, followed by 30 cycles of 1 min at 95°C, 1 min annealing at 60°C (±5°C gradient), 2 min extension at 72°C and a final extension at 72°C for 10 min. For PCR cloning of cbh2 genes from Chaetomium thermophilum ALK04265 and Talaromyces emersonii RF8069 strains, the reactions contained 1x Phusion GC and 1x Phusion HF buffers, respectively, with 0.2 mM dNTPs, 5 μM each primer and 1-2 units of Phusion DNA polymerase (Finnzymes, Finland) and 0.5-1 μg of the corresponding genomic DNA. The conditions for the PCR reactions were as follows: 3 min initial denaturation at 98°C, followed by 30 cycles of 30 seconds at 98°C, 30 seconds with annealing at 55°C (±5°C gradient), 1- 2 minute extension at 72°C and a final extension at 72°C for 10 minutes.
The primer combinations described in Table 2 produced the specific DNA product having the expected size (according to calculations based on published cbh2 sequences). The DNA products were isolated and purified from the PCR reaction mixtures and cloned into the pCR®4Blunt-TOPO® vector according to the manufacturer's instructions (Invitrogen, USA). Insertions were characterized by sequencing and performing Southern blot hybridizations to genomic DNAs digested with various restriction enzymes. The PCR fragments, which were chosen to be used as probes for the cloning of the gene from the Acremonium thermophilum ALK04245 and Melanocarpus albomyces ALK04237 strains are shown in Table 3. In addition, the table describes the PCR fragments containing the full-length cbh2 genes of Chaetomium thermophilum ALK04265 and Talaromyces emersonii RF8069 strains. Table 3. Primers used in PCR reactions, probes chosen for the evaluation of cbh2 genes from Acremonium thermophilum ALR04245 and Melanocarpus albomyces ALR04237 and DNA fragments containing the full length cbh2 genes from Chaetomium thermophilum ALK04265 and Talaromyces emersonii RF8069. The genomic template DNA and the name of the plasmid containing a probe fragment are shown.

The deduced amino acid sequences from all these PCR fragments have homology to published CBHII/Cel6A sequences (BLAST program, version 2.2.9 to NCBI, National Center for Biotechnology Information; Altschul et al., 1990). The 1757bp PCR fragment in plasmid pALK2904 (SEQ ID NO:9) and the 1788bp PCR fragment in plasmids pALK3006 (SEQ ID NO:10) containing the full length cbh2/Cel6A genes from Chaetomium thermophilum ALK04265 and Talaromyces emersonii RF8069, respectively . The gene encoding a Chaetomium thermophilum ALK04265 was named as the Ct_ALKO4265_Cel6A gene and Talaromyces emersonii RF8069 was named as Te_RF8069_Cel6A. The E.coli strains of RF8214 (=pALK2904) and RF8333 (=pALK3006) were deposited with the DSM collection under accession numbers DSM 22947 and DSM 23185, respectively.
The genomic DNAs Acremonium thermohilum ALK04245 and Melanocarpus albomyces ALK04237 were digested with various restriction enzymes for Southern blot analysis. The probes for the hybridizations were the EcoRI 1032 bp (SEQ ID NO:7) and 831 bp (SEQ ID NO:8) fragments, cut from plasmids pALK2580 and pALK2576, respectively. The above probes were labeled using digoxigenin according to the manufacturer's instructions (Roche, Germany). Hybridizations were performed overnight at 68°C. After hybridization the filters were washed 2 x 5 minutes at room temperature using 2 x SSC - 0.1% SDS followed by 2 x 15 minutes at 68°C using 0.1 x SSC - 0.1% SDS.
From the genomic DNA of Acremonium thermophilum ALK04245, approximate 8 kb XbaI digested fragment was hybridized using dioxygenin labeled 1032bp EcoRI fragment of pALK2580 as a probe. Correspondingly, about 4.5 kb BamHI digested fragment was hybridized with 831bp EcoPJ fragment labeled by dioxygenin from pALK2576 from the genomic DNA of Melanocarpus albomyces ALK04237. Hybridization of genomic DNA fragments were isolated from the group of digested genomic fragments based on their size. Genomic fragments were isolated from the agarose gel and cloned into pBluescript II KS+ vectors (Stratagene, USA) cleaved with XbaI (Acremonium thermophilum ALK04245) or BamHI (Melanocarpus albomyces ALK04237). Ligation mixtures were transformed into Escherichia coli XL10-Gold cells (Stratagene) and placed on LB plates (Luria-Bertani) containing 50-100 µg/ml ampicillin. E.coli colonies were screened for positive clones using colonial hybridization with inserts pALK2580 and pALK2576 as probes under hybridization conditions corresponding to that described above by Southern blot analysis, except using the hybridization temperature of 65°C instead of 68° Ç. Several positive clones were collected from the plates. These were shown by restriction digestion to contain inserts of the expected sizes. The full-length gene encoding Acremonium thermohilum ALK04245 CBHII/Cel6A (At_ALK04245_Cel6A, SEQ ID NO:11) was sequenced from the 7 kb Xbal insert and the plasmid containing its insert was named pALK2582. The E.coli RF8175 strain including plasmid pALK2582 has been deposited to the DSM collection under accession number DSM 22946. The gene encoding a protein Acremonium thermohilum ALK04245 has been named as At_ALK04245_cel6A. Correspondingly, the full-length gene encoding a Melanocarpus albomyces ALK04237 CBHII/Cel6A (Ma_ALK04237_Cel6A, SEQ ID NO:12) was sequenced from the 5 kb BamHI insert and the plasmid containing this insert was named pALK2581. The E.coli strain RF8174 including plasmid pALK2581 has been deposited to the DSM collection under accession number DSM 22945. The gene encoding a Melanocarpus albomyces ALK04237 has been named as Ma_ALK04237_cel6A. Relevant information on genes and deduced protein sequence (SEQ ID NO: 9-16) are summarized in Table 4 and Table 5, respectively. Table 4. Summary on cbh2/cel6A genes isolated from Acremonium thermophilum ALK04245, Melanocarpus albomyces ALK04237, Chaetomium thermophilum ALK04265 and Talaromyces emeraonii RF8069.
Table 5. Amino acid sequence summary deduced from the cbh2/cel6A gene sequences of Acremonium thermophilum ALK04245, Melanocarpus albomyces ALK04237, Chaetomium thermophilum ALK04265 and Talaromyces emersonii RF8069.

Comparison of the deduced CBHII/Cel6A sequences of Acremonium thermophilum ALK04245, Melanocarpus albomyces ALK04237, Chaetomium thermophilum ALK04265 and Talaromyces emersonii DSM 2432 (RF8069) in each other is shown in Table 6. A Clone Manager program (version 9) the functions "Compare Two Sequences/Global/Compare sequences as amino acids/BLOSUM62 scoring matrix" were used to determine the degree of identity. Table 6. Identity values (%) obtained from alignment of deduced CBHII/Cel6A amino acid sequences of Acremonium thermophilum ALK04245, Melanocarpus albomyces ALK04237, Chaetomium thermophilum ALK04265 and Talaromyces emersonii RF8069. The full-length amino acid sequences including the signal sequence were aligned. A Clone Manager 9 program (Compare Two Sequences/Global/Compare sequences as amino acids/BLOSUM62 scoring matrix) was used to determine the degree of identity.

Comparison of the deduced CBHII/Cel6A sequences from Acremonium thermophilum ALK04245, Melanocarpus albomyces ALK04237, Chaetomium thermophilum ALK04265 and Talaromyces emersonii RF8069 to the sequences observed from the databases are shown in Tables 7 and 8. Table 7. greater identity with the deduced CBHII/Cel6A amino acid sequences of Acremonium thermophilum ALK04245, Melanocarpus albomyces ALK04237, Chaetomium thermophilum ALK04265 and Talaromyces emersonii RF8069. The full-length amino acid sequences including the signal sequence were aligned. The database search was performed using BLAST (tblastn, nr/nt database) and the Clone Manager 9 program (Compare Two Sequences/Global/Compare sequences as amino acids/BLOSUM62 scoring matrix) was used to determine the degree of identity.
Table 8. Patent sequences of greater identity are the deduced CBHII/Cel6A amino acid sequences of Acremonium thermophilum ALK04245, Melanocarpus albomyces ALK04237, Chaetomium thermophilum ALK04265 and Talaromyces emersonii RF8069. The full-length amino acid sequences including the signal sequences were aligned. Searches of Chemical Abstracts Service (CAS) Registry System and Patented Protein Sequences NCBI databases were performed using BLAST and Clone Manager 9 program (Compare Two Sequences/Global/Compare sequences as amino acids/BLOSUM62 scoring matrix) was used for the determination of degree of identity.
*) Description w0200959234 refers to SEQ ID NO:45 (DNA) or SEQ ID NO:46 (protein). Sequence listing W0200959234 refers to SEQ ID NO:35 (DNA) or SEQ ID NO:36 (protein). EXAMPLE 2. Production of CBHII/Cel6A recombinant proteins in TRICHODERMA REESEI
Expression plasmids were constructed by the production of recombinant CBHII/Cel6A proteins from Acremonium thermophilum ALK04245, Melanocarpus albomyces ALK04237, Chaetomium thermophilum ALK04265 and Talaromyces emersonii RF8069 in Trichoderma reesei. The constructed expression plasmids are listed in Table 9. The recombinant cbh2/cel6A genes, including their own signal sequence, were exactly fused to the T. reesei cbh1/cel7A promoter by PCR. The transcription termination was guaranteed by the terminator T. reesei cbhl/cel7A and the marker gene A. nidulans amdS was used for the selection of transformants as described in Paloheimo et al. (2003). Linear expression cassettes (Fig. 1) were isolated from vector structures after EcoRI or EcoRI-SpeI digestion and were transformed into T. reesei protoplasts. The host strain used does not produce any of the four major T. reesei cellulases (CBHI, CBHII, EGI, EGII). The transformations were performed in Penttila et al. (1987) with the modifications described in Karhunen et al. (1993), selecting acetamide as a single nitrogen source (amdS marker gene). Transformants were purified on selection plates by simple conidia before sporylation of it to PD. Table 9. Expression cassettes constructed to produce recombinant proteins CBBII/Cel6A from Acremonium thermophilum ALK04245, Melanocarpus albomyces ALK04237, Chaetomium thermophilum ALK04265 and Talaromyces emersonii RF8069 in Trichoderma reesei. The total structure of the expression cassettes was as described in Fig. 1. The cloned cbh2/Cel6A genes were exactly fused to the T. reesei cbhl/cel7A promoter.
(a The expression cassette for the T. reesei transformation was isolated from the vector backbone by using EcoRI or EcoRI-SpeI digestion. (b The number of nucleotides after the stop codon of the cloned recombinant gene that was included in a expression cassette. The restriction site at the 3' end of the genomic gene fragment that was used in the construction of the expression cassette is indicated in parenthesis. The Ct_ALK04265_cel6A gene fragment was labeled 3' extram by EcoRI (a site present in the pCR 4Blunt-TOPO vector.) Correspondingly, the Te_RP8069_cel6A gene fragment was tagged from the 3' end by BamHI (a site created after the stop codon in PCR). This does not carry the original Ct_ALK04265_cel6A or Te_RF8069_Cel6A terminator in the constructs before the terminator sequences cbhl.
CBHII/Cel6A production of transformants was analyzed from culture supernatants from shake flask cultures. Transformants were inoculated from the PD slopes to shake flasks containing 50 ml of cellulase based lactose complex inducing medium (Joutsjoki et al., 1993) buffered with 5% KH2PO4. The CBHII/Cel6A protein production of the transformants was analyzed by the culture supernatants after it had grown for 7 days at 30°C, 250 rpm. The heterologous production of recombinant proteins was analyzed by SDSPAGE with subsequent Coomassive staining. The genotypes of the chosen transformants were confirmed using Southern blot analyzes in which the various genomic digests were included and the respective expression cassette was used as a probe.
[00212] The best production transformants were chosen to be cultivated in laboratory scale bioreactors. The transformants were cultured in lab bioreactors at 28°C in cellulase induction complex medium for 3-4 days with a pH control of 4.4 ± 0.2 (NH3/H3PO4) to obtain the material for application tests . Supernatants were recovered by centrifugation and filtration through Seitz-K 150 and EK filters (Pall SeitzSchenk Filter systems GmbH, Bad Kreuznach, Germany). EXAMPLE 3. Hydrolysis of crystalline cellulose (Avicel) by recombinant enzymes CBHII/Cel6A
[00213] Recombinant CBHII/Cel6A enzyme preparations were characterized in terms of pH dependence and thermal stability using crystalline cellulose (Avicel) as a substrate. The pH dependence and thermal stability for recombinant CBHII/Cel6A proteins from Acremonium thermophilum ALK04245, Melanocarpus albomyces ALK04237, Chaetomium thermophilum ALK04265 and Talaromyces emersonii RF8069 were determined within a pH range of 3.0-10.0 and a range of temperature 40°C - 80°C, respectively. Crystalline cellulose hydrolysis assays (Ph 101, Avicel; Fluka, Bucsh, Switzerland) were performed in 2.0 ml tube scale in a 50 mM sodium acetate pH 6.0. To determine the optimum pH, substrate solutions (Avicel, 50 mg/ml in sodium acetate, pH 6.0) in the pH range from 3 to 10 were stirred with the enzyme preparation (100 μg of protein in the reaction) at 50°C and the final volume of the reaction mixture was 650 µl. Hydrolysis was continued for 21 hours and stopped by the addition of 326 µl of stop reagent containing 9 vol of 94% ethanol and 1 vol of 1 M glycine (pH 11). The solution was filtered through a Millex GV13 0.22 µm filter unit (Millipore, Billerica, MA, USA). The formation of soluble reducing sugars in the supernatant was determined by the parahydroxybenzoic acid hydrazide (PAHBAH) method (Lever, 1972) using a standard cellobiose curve (200 μM to 1200 μM cellobiose). A freshly made 0.1 M PAHBAH (Sigma-Aldrich, St. Louis, MO, USA) in 0.5 M NaOH solution (200 μl) was added to 300 μl of the filtered sample and boiled for 10 minutes after which time the solution was cooled to room temperature. Absorbance at 405 nm was measured from duplicate samples by Multiskan EX (Thermo Labsystems, Franklin, MA, USA). Correspondingly, the thermal stability of CBHII/Cel6A recombinant proteins was determined in Avicel substrate solutions in the temperature range of 40°C to 80°C at the optimum pH with a reaction time of 21 hours. The results indicate that the optimum pH for Talaromyces emersonii RF8069 CBHII/Cel6A and Melanocarpus albomyces ALK04237 CBHII/Cel6A is 4.0, considering the enzymes Chaetomium thermophilum ALK04265 and Acremonium thermophilum ALK04245 having an optimum pH at 5.0 (Fig. 2). Thermal stability was observed to be substantially greater by Acremonium thermophilum ALK04245, Chaetomium thermophilum ALK04265 and Talaromyces emersonii RF8069 CBHII/Cel6A as compared to that of the Melanocarpus albomyces protein ALK04237 (Fig. 3). EXAMPLE 4. Hydrolysis of hardwood substrate with enzyme preparations comprising recombinant cellobiohydrolase CBHII/Cel6A
The steam-blasted hardwood was resuspended in 0.05 M and sodium acetate buffer, pH 4.8. The final weight of the hydrolysis mixture was 20 g of which the total solid concentrations were 2% (w/w). The substrate was hydrolyzed using the different enzyme mixtures at a dosage of 5 mg protein per g total solids in 50 ml shake flasks. The protein contents of the enzyme components and mixtures were determined using the Pierce BCA Assay Kit (Thermo Scientific, Product Number 23227) with bovine serum albumin (Thermo Scientific, Product Number 23209) as a standard. The shake flasks were shaken in a linear shaking water bath set to different temperatures. For each sample point, a 1 ml sample was taken from duplicate shake flasks and boiled for 10 minutes to terminate enzymatic hydrolysis, centrifuged and the supernatant analyzed for reaction products from hydrolysis. Voids containing only substrate (only buffer added instead of enzymes) were prepared identically to other samples.
[00215] Three separate blend combinations were prepared (a thermophilic MIXTURE 2, a mesophilic ACC MIXTURE and a T.REESEI ENZYME MIXTURE) with different Cel6A/CBHII substitutions.
[00216] A mixture of the thermostable cellulases was prepared using the following components:
[00217] Thermophilic Ce17A/CBHI preparation containing thermoascus aurantiacus ALK04242 Ce17A with genetically linked CBD from T. reesei CBHI/Ce17A (W02007071818).
[00218] Thermophilic endoglycanase preparation containing Acrernonium thermophilum ALK04245 Ce145A endoglycanase (At EG40/Ce145A, W02007071818).
[00219] Thermophilic β-glycosidase preparation containing thermoascus aurantiacus ALK04242 β-glycosidase (Ta_13G_81/Ce13A, W02007071818).
[00220] Thermophilic xylanase preparation containing Nonornurea flexuosa Xyn11A (AM24, W02005100557, enzymes AB Oy, FI)).
[00221] All cellulases were heterologously produced as monocomponents in the host strain Trichoderma reesei having cellulase free foundation (the genes encoding the four major cellulases Ce17A/CBHI, Cel6A/CBHII, Ce17B/EGI and Ce15A/EGII were deleted). Crude culture supernatants were used in the mixture. The enzyme components were combined as follows (per 10 ml of the mixture): CBHI/Ce17A preparation 330 mg (71.2%), endoglycanase preparation 105 mg (22.7%), β-glycosidase preparation 7.5 mg ( 1.6%) and 21 mg xylanase preparation (4.5%). The volume was made up to 10 ml with tap water. The final protein concentration of the mixture was 46.35 mg/ml. This enzyme mixture was denoted MIX 2.
[00222] For the Cel6A/CBHII performance test in hydrolysis with MIXTURE 2, 15% (49.5 mg) of the CBHI/Ce17A component of MIXTURE 2 was replaced by Ma_ALK04237_Cel6A (MIX_2MA), Ct_ALKO4265_Cel6A (MIX 2_CT), or At_ALKOA4245_C 2_AT), respectively.
[00223] A mixture established in the art was prepared by combining with the following components (per 10 ml of the mixture): ECONASE® CE (Roal Oy, a classic T. reesei enzyme product) 470 mg (94%), β preparation -glycosidase (At βG_101/Ce13A, W02007071818) 20 mg (4%) and xylanase preparation (Ta_ XYN_30, W02007071818) 10 mg (2%). The volume was made up to 10 ml with tap water. The final protein concentration in this mixture was 50 mg/ml. This enzyme mixture was denoted as T. REESEI ENZYME MIXTURE. At βG_101/Ce13A and Ta XYN30 enzymes were heterologously produced as monocomponents in the host strain Trichoderma reesei having cellulase free foundation.
[00224] For the Cel6A/CBHII performance test in hydrolysis with T. REESEI ENZYME MIXTURE, 15% (70.5 mg) of the ECONASE® CE component of the T. REESEI ENZYME MIXTURE was replaced by MaALKO4237Cel6A (TR_MA MIXTURE), Ct_ALKO4265_Cel6A (MIX TR_CT), or At_ALK04245_Cel6A (MIX TR_AT), respectively.
The ACC MIXTURE was prepared from the commercial product Accellerase® 1000 (from Genencor International/Danisco A/S) (per 10 ml): protein 400 mg Accellerase® 1000 (100%). The volume was made up to 10 ml with tap water. The final protein concentration in this mixture was 40 mg/ml.
[00226] For the Cel6A/CBHII performance test in hydrolysis with ACC MIX, 15% (60 mg) of the Accellerase® 1000 component of the ACC MIX was replaced by Ma_ALKO4237_Cel6A (ACC_MA MIX), CtALKO4265_Cel6A (ACC_CTMISTURA MIX), or Atel_ALK04245 ACC_AT), respectively.
[00227] For the MIXTURE 2 combinations, the hydrolysis was performed at 55°C, while the hydrolysis temperature for the ENZYME MIXTURE T. REESEI ENZYMES and ACC MIXTURE experiments was 37°C. Samples were taken from hydrolysis after 72 hours, quantified by HPLC and glucose and xylose concentrations were determined. The results from the substrate voids were subtracted from the samples with the enzymes and the combined glucose and xylose concentration is shown in Fig. 4A-C.
[00228] The results clearly show the best performance of MIXTURE 2 with Cel6A/CBHII enzymes thermostable at 55°C. The amount of sugars released from the hardwood substrate was observed to increase 12%, 14% and 26% by supplementation with Ma_ALKO4237_Cel6A, Ct_ALKO4265_Cel6A or At_ALK04245_Cel6A enzymes in blend 2, respectively. The enzyme Acremonium thermophilum ALK04245 was observed to be the best performing Cel6A/CBHII in this study (Fig. 4A). At_ALK04245_Cel6A shows increased hydrolysis at 37°C added to the technique-established Trichodermuma mixture (T.REESEI ENZYMES ENZYME MIXTURE) (Fig. 4B) or to the commercial product (ACC MIXTURE) (Fig. 4C). Example 5. Corn cob hydrolysis with enzyme preparations comprising a recombinant CBHII/Cel6A cellobiohydrolase
[00229] Steam-blasted corn cobs were resuspended in 0.05 M sodium acetate buffer, pH 4.8. The final weight of the hydrolysis mixture was 20 g of which the total solids was 2% (w/w). The substrate was hydrolyzed using the different enzyme mixtures at a dosage of 5 mg protein per g total solids in 50 ml shake flasks. The protein contents of the enzyme components and mixtures were determined using the Pierce BCA Assay Kit (Thermo Scientific, Product Number 23227) with bovine serum albumin (Thermo Scientific, Product Number 23209) as a standard. The shake flasks were shaken in a linear shake water bath set to 55°C. For each sample point, a 1 ml sample was taken from the duplicate shake flasks and boiled for 10 minutes to terminate enzymatic hydrolysis, centrifuged and the supernatant analyzed for reaction products from hydrolysis. Voids containing only substrate (only buffer added instead of enzymes) were prepared identically to the other samples.
[00230] A mixture of thermostable cellulases was prepared using the following components:
[00231] Thermophilic Ce17A/CBHI preparation containing thermoascus aurantiacus ALK04242 Ce17A with genetically linked CBD from T. reesei CBHI/Ce17A (W02007071818).
[00232] Thermophilic endoglycanase preparation containing Acremonium thermophilum ALK04245 Ce145A endoglycanase (At EG_40/Ce145A, W02007071818).
Thermophilic β-glycosidase preparation containing thermoascus aurantiacus ALK04242 β-glycosidase (Ta βG_81/Cel3A, W02007071818).
[00234] Thermophilic xylanase preparation containing Nonomurea flexuosa Xyn11A (AM24, W02005100557).
[00235] All cellulases were heterologously produced as monocomponents in the host strain Trichoderma reesei having cellulase free foundation (the genes encoding the four major cellulases Ce17A/CBHI, Cel6A/CBHII, Ce17B/EGI and Ce15A/EGII were deleted). Crude culture supernatants were used in the mixture. The enzyme components were combined as follows (per 10 ml of the mixture): CBHUCe17A preparation 330 mg (71.2%), endoglycanase preparation 105 mg (22.7%), β-glycosidase preparation 7.5 mg (1 .6%) and 21 mg xylanase preparation (4.5%). The volume was made up to 10 ml with tap water. The final protein concentration of the mixture was 46.35 mg/ml. This enzyme mixture was denoted MIX 2.
[00236] For the performance test At_ALK04245Cel6A in the hydrolysis with MIXTURE 2, 15% (49.5 mg) of the CBHI/Ce17A component of MIXTURE 2 was replaced by At_ALKO4245_Cel6A (MIXTURE 2 AT), respectively.
[00237] Samples were taken from the hydrolysis after 72 hours, quantified by HPLC and the concentration of glucose and xylose were determined. The results from the substrate voids were subtracted from the enzyme samples and the combined glucose and xylose concentration is shown in Fig. 5.
[00238] Similar to that described in Example 4, the results clearly show the best performance of MIX 2 with the enzyme At_ALK04245Cel6A at 55°C for the corn cob substrate. REFERENCES Altschul SF, W Gish, W Miller, EW Myers and DJ Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410. AMFEP, 2007. Association of Manufacturers and Formulators of Enzyme products, List of commercial enzymes at http://www.amfep.org/list.html (updated 30 November 2007). Badger, PC. 2002. Ethanol from cellulose: a general review. In Trends in new crops and new uses. J Janick and A Whipkey (eds.). ASHS Press, Alexandria, VA, USA, pp. 17-21. Bendtsen JD, H Nielsen, G von Heijne and S Brunak. 2004. Improved prediction of signal peptides: SignalP 3.0. J. Mol. Biol. 340:783795. Biely P, M Vrsanska, M Tenkanen and D Kluepfel. 1997. Endo-beta-1,4-xylanase families: differences in catalytic properties. Journal of Biotechnology 57: 151-166. Bolton ET and BJ McCarthy. 1962. The general method for the isolation of RNA complementary to DNA. Proc. Nat. Sci.USA 48:1390-1397. Collins CM, PG Murray, S Denman, A Grassick, TT Teeri, L Byrnes and MG Tuohy. 2003. Molecular cloning of a cellobiohydrolase genes of Talaromyces emersonii. Biochem Biophys Res. Comm. 301:280-286. Cullen D and PJ Kersten. 2004. Enzymology and molecular biology of lignin degradation. In: The Mycota III. Biochemistry and molecular biology. R Brambl and GA Marzluf (eds.). 2nd edition, Springer-Verlag, Berlin-Heidelberg, pages 249-273. Del Caiiizo AN, RA Hours, MV Miranda, 0 Cascone. 1994. Fractionation of fungal pectic enzymes by immobilized metal ion affinity chromatography. J. Sci. Food Agricultural. 64:527-531. Edman P and G Begg. 1967. The Protein Sequenator. Eur. J. Biochem. 1:80-91. Galagan JE, SE Calvo, KA Borkovich, US Selker, ND Read, D Jaffe, W FitzHugh, LJ Ma, S Smirnov, S Purcell, B Rehman, T Elkins, R Engels, S Wang, CB Nielsen, J Butler, M Endrizzi , D Thu, P Ianakiev, D Bell-Pedersen, MA Nelson, M Werner-Washburne, CP Selitrennikoff, JA Kinsey, EL Braun, A Zelter, U Schulte, GO Kothe, G Jedd, W Mewes, C Staben, E Marcotte, D Greenberg, A Roy, K Foley, J Naylor, N Stange-Thoman, R Barrett, S Gnerre, M Kamal, M Kamvysselis, E Mauceli, C Bielke, S Rudd, D Frishman, S Krystofova, C Rasmussen, RL Metzenberg, DD Perkins, S Kroken, C Cogoni, G Macino, D Catcheside, W Li, RJ Pratt, SA Osmani, CP DeSouza, L Glass, MJ Orbach, JA Berglund, R Voelker, 0 Yarden, M Plaman, S Seiler, J Dunlap , A Radford, R Aramayo, DO Natvig, LA Alex, G Manhaupt, DJ Ebbole, M Freitag, I Paulsen, MS Sachs, ES Lander, C Nusbaum, B Birren. 2003. The genome sequence of the filamentous fungus Neurospora crassa. Nature 422:859-868. Gasteiger E, A Gattiker, C Hoogland, I Ivanyi, RD Appel and A Bairoch. 2003. ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res. 31: 3784-3788. Ghose TK. 1987. International Union of Pure and Applied Chemistry. Measurement of cellulase activities. Pure and Appl. Chem. 59: 257268. Henrissat B. 1991. A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J. 280: 309-316. Henrissat B and A Bairoch. 1993. New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J. 293: 781-788. Henrissat B and A Bairoch. 1996. Updating the sequence-based classification of glycosyl hydrolases. Biochem. J. 316: 695-696 Henrissat B, TT Teeri and RAJ Warren. 1998. A scheme for designating enzymes that hydrolyse the polysaccharides in the cell wall of plants. FEBS Letters 425: 352-354. Hong J, H Tamaki, K Yamamoto and H Kumagai. 2003a. Cloning of a gene encoding a thermo-stabile endo-(3-1,4-glycanase from Thermoascus aurantiacus and its expression in yeast. Biotechnol. Letters 25: 657-661. Hong J, H Tamaki, K Yamamoto and H Kumagai. 2003b. Cloning of a gene encoding thermostable cellobiohydrolase from Thermoascus aurantiacus and its expression in yeast. Appl. Microbiol. Biotechnol. 63: 42-50. Joutsjoki VV, TK Torkkeli and KMH Nevalainen. 1993. Transformation of Trichoderma reesei with the Hormoconise resin glucoamy. P (gamP) gene: production of a heterologous glucoamylase by Trichoderma reesei, Curr. Genet. 24: 223-228. Karhunen T, A Mantyla, KMH Nevalainen and PL Suominen, 1993. High frequency one-step gene replacement in Trichoderma reesei. I. Endoglycanase I overproduction. Mol. Gen. Genet. 241:515-522. Karlsson J, M Saloheimo, M Siika-aho, M Tenkanen, M Penttila and F Tjerneld. 2001. Homologous expression and characterization of Cell61A (EGIV) of Trichoderma reesei Eur J Biochem 268:6498-6507 Kurabi A, A Berlin, N Gi lkes, Kil Kilburn, Markov, Skomarovsky, Gusakov, Okunev, Sinitsyn, Gregg, D Xie and J Saddler. 2005. Enzymatic hydrolysis of steam-exploded and organosolv-pretreated Douglas-Fir ethanol by novel and commercial fungal cellulases. Appl. Biochem and Biotechnol. Vol 121-124: 219-229. Laemmli UK. 1970. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227:680-685. Lever M. 1972. A new reaction for colorimetric determination of carbohydrates. Anal. Biochem., 47: 276-279. Maheshwari R, G Bharadwaj and MK Bhat. 2000. Thermophilic fungi: their physiology and enzymes. Microbiol. Mol. Biol. Rev. 64:461-488. Malardier L, MJ Daboussi, J Julien, F Roussel, C Scazzocchio and Y Brygoo. 1989. Cloning of the nitrate reductase gene (niaD) of Aspergillus nidulans and its use for transformation of Fusarium oxysporum. Gene 78:147156. Miettinen-Oinonen A, J Londesborough, V Joutsjoki, R Lantto, J Vehmaanpera. 2004. Three cellulases from Melanocarpus albomyces with application in textile industry. Microb Enzyme Technol. 34:332-341. Miller GL. 1959. Use of dinitrosalisylic acid reagent for determination of reducing sugars. Anal. Chem. 31:426-428. Murray PG, CM Collins, A Grassick and MG Tuohy. 2003. Molecular cloning, transcriptional, and expression analysis of the first cellulase gene (cbh2), encoding cellobioidrolase II, from the moderately thermophilic fungus Talaromyces emersonii, and structure prediction of the gene product. Biochem. Biophys. Common Res. 301:280-286. Nielsen H, J Engelbrecht, S Brunak and G von Heijne. 1997. Identification of prokaryotic and eykaryotic signal peptides and prediction of their cleavage sites. Protein Engineering 10:1-6. Nielsen H and A Krogh. 1998. Prediction of signal peptides and signal anchors by a hidden Markov model. In: Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology (ISMB 6), AAAI Press, Menlo Park, California, pp. 122-130. Paloheimo M, A Mantyla, J Kallio, and P Suominen. 2003. High-yield production of a bacterial xylanase in the filamentous fungus Trichoderma reesei requires a carrier polypeptide with an intact domain structure. Appl. Env. Microbiol. 69:7073-7082. Penttila M, H Nevalainen, M Ratto, E Salminen and J Knowles. 1987. A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei. Gene 61:155-164. Raeder U and P Broda. 1985. Rapid preparation of DNA from filamentous fungi. Left. Appl. Microbiol. 1:17-20. Robyt JF and WJ Whelan. 1972. Reducing value methods for maltodextrins: I. Chain length dependence of alkaline 3,5-dinitrosalisylate and chain-length independence of alkaline copper. Anal. Biochem. 45:510-516. Sambrook J and DW Russell. 2001. Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, New York, US. Shaw AJ, KK Podkaminer, SG Desai, JS Bardsley, SR Rogers, PG Thorne, DA Hogsett and LR Lynd. 2008. Metabolic engineering of a thermophilic bacterium to produce ethanol at high yield. Proc. Natl. Academic Sci. USA 105: 13769-13774. Sinnott ML. 1990. Catalytic mechanisms of enzymic glycosyl transfer. Chem. Rev. 90: 1171-1202. Somogyi M. 1952. Notes on sugar determination. J. Biol. Chem. 195:19-23. Srisodsuk M, T Reinikainen, M Penttila and T Teeri. 1993. Role of the interdomain linker peptide of Trichoderma reesei cellobioidrolase I in its interaction with crystalline cellulose. J. Biol. Chem. 268(28): 20756-61. Stalbrand H, A Saloheimo, J Vehmaanpera, B Henrissat and M Penttila. 1995. Cloning and expression in Saccharomyces cerevisiae of a Trichoderma reesei 13-mannanase gene containing a cellulose binding domain. Appl. Environ. Microbiol. 61:1090-1097. Sundberg M and K Poutanen. 1991. Purification and properties of two acetylxylan esterases of Trichoderma reesei. Biotechnol. Appl. Biochem. 13: 1-11. Suurnakki A, M Tenkanen, M Siika-aho, M-L Niku-Paavola, L Viikari and J Buchert. 2000. Trichoderma reesei cellulases and their core domains in the hydrolysis and modification of chemical pulp. Cellulose 7: 189209. van Tilbeurgh H, F Loonties, C de Bruyne and M Claeyssens. 1988. Fluorogenic and chromogenic glycosides as substrates and ligands of carbohydrases. Methods Enzymol. 160:45-59. Tomme P, S McRae, T Wood and M Claeyssens. 1988. Chromatographic separation of cellulolytic enzymes. Methods in Enzymol. 160: 187-192. Tuohy M, J Walsh, P Murray, M Claeyssens, M Cuffe, A Savage and M Coughan. 2002. Kinetic parameters and mode of action of cellobiohydrolases produced by Talaromyces emersonii. Biochem. Biophys. Minutes 1596: 366-380. Waffenschmidt S and L Jaenicke. 1987. Assay of reducing sugars in the nanomole range with 2,2'bicinchoninate. Anal. Biochem. 165:337-340. Withers SG, D Dombroski, LA Berven, DG Kilburn, RC Miller Jr, RAJ Warren and NR Gilkes. 1986. Direct 1H NMR determination of the stereochemical course of hydrolases catalyzed by glycanase components of the cellulose complex. Biochem. Biophys. Common Res. 139:487-494. Withers SG and Aebersold R. 1995. Approaches to labeling and identification of active site residues in glycosidases. Protein Sci. 4:361-372.

权利要求:
Claims (17)
[0001]
1. Enzyme preparation, characterized in that it comprises a CBHII/Cel6A fungal polypeptide having cellobiohydrolase activity and consisting of an amino acid sequence of SEQ ID NO: 12 SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO : 16 and additives selected from the group consisting of mediators, stabilizers, buffers, preservatives, surfactants and/or culture medium components, wherein the CBHII/Cel6A polypeptide is active at a temperature between 50°C and 70°C.
[0002]
2. Enzyme preparation according to claim 1, characterized in that said polypeptide is obtained from a genus of Acremonium, Melanocarpus, Chaetomium or Talaromyces, more preferably from species A. thermophilum, M albomyces, C. thermophilum or T. emersonii, more preferably from A. thermophilum CBS 116240, M. albomyces CBS 685.95, C. thermophilum CBS 730.95 or T. emersonii DSM 2432.
[0003]
3. Enzyme preparation according to claim 1 or 2, characterized in that said polypeptide of the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16 are encoded by a nucleic acid molecule comprising a polynucleotide sequence included in SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:9 or SEQ ID NO:10 or their degenerate sequences encoding the same polypeptide, respectively.
[0004]
4. Enzyme preparation according to any one of claims 1 to 3, characterized in that said preparation comprises other enzymes selected from the group of cellobiohydrolase, endoglycanase, beta-glucosidase, beta-glycanase, xyloglycanase, xylanase, beta-xylosidase , mannanase, beta-mannosidase, α-glucuronidase, acetyl xylan esterase, α-arabinofuranosidase, α-galactosidase, pectinase, endo- and exo-α-L-arabinases involved, α-galactosidase, endo and exo-pectactorinase, endopectonase lyase and pectinesterase, phenol esterase, ligninase involving lignin peroxidase, manganese-dependent peroxidase, H2O2 generating enzyme and laccase with or without a mediator.
[0005]
5. Enzyme preparation according to any one of claims 1 to 4, characterized in that said enzyme preparation is in the form of total culture broth, used culture medium, liquid, powder or granules.
[0006]
6. Recombinant expression vector, characterized in that it comprises a nucleic acid molecule as defined in SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 9 or SEQ ID NO: 10 or its degenerate coding sequences the same polypeptide of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively, operably linked to heterologous regulatory sequences capable of directing cellobiohydrolase CBHII/Cel6A gene expression and production of said cellobiohydrolase CBHII/Cel6A in a suitable host.
[0007]
7. Host cell, characterized in that it comprises the expression vector as defined in claim 6, in which the host cell is selected from the genera Trichoderma, Aspergillus, Fusarium, Humicola, Chrysosporium, Neurospora, Rhizopus, Penicillium or Mortiriella.
[0008]
8. Host cell according to claim 7, characterized in that the host cell is Trichoderma or Aspergillus, preferably Trichoderma reesei.
[0009]
9. Process for producing a polypeptide having cellobiohydrolase activity, characterized in that it comprises the steps of culturing the host cell as defined in claim 7 or 8 and recovering the polypeptide.
[0010]
10. Process for obtaining an enzyme preparation, characterized in that it comprises the steps of culturing a host cell as defined in claim 7 or 8, and preparing the total culture broth or separating the cells from the used culture medium and obtaining the supernatant.
[0011]
11. Method for treating cellulosic material with an enzyme preparation as defined in any one of claims 1 to 5, characterized in that it comprises the following steps: i) producing said enzyme preparation comprising said polypeptide or a micro- fermentative organism that produces said polypeptide; ii) reacting the enzyme preparation comprising said polypeptide or the fermentative microorganism that produces said polypeptide and iii) obtaining partially or fully hydrolyzed cellulosic material.
[0012]
12. Method according to claim 11, characterized in that the CBHII/Cel6A polypeptide is capable of hydrolyzing the cellulosic material at a temperature ranging between 40 and 70°C.
[0013]
13. Method according to claim 11 or 12, characterized in that the cellulosic material is treated with an enzyme composition as defined in any one of claims 1 to 5 in combination with at least one additional enzyme capable of hydrolyzing said cellulosic material selected from a group of cellobiohydrolase, endoglycanase, beta-glycosidase, beta-glycanase, xyloglycanase, xylanase, beta-xylosidase, mannanase, beta-mannosidase, α-glucuronidase, acetyl xylan esterase, α-arabinofuranosidase, α-arabinofuranosidase , endo- and exo-α-L-arabinases enveloping, α-galactosidase, endo- and exo-galactosidase, endopectinlyase, pectate lyase, and pectinesterase, phenol esterase, ligninase involving lignin peroxidase, manganese-dependent peroxidase, H2O2 generating enzyme and lactase with or without mediators.
[0014]
14. Method according to claim 12, characterized in that the enzymes are added to the cellulosic material simultaneously or sequentially.
[0015]
15. Method according to any one of claims 11 to 13, characterized in that the cellulosic material is selected from the group consisting of plant materials, agricultural biomass, waste products and crops dedicated to energy.
[0016]
16. Use of the CBHII/Cel6A polypeptide, characterized by the fact that it is for the production of biofuels, for detergents, for the treatment of fibers, for the treatment of feed and food, for pulp and paper, for beverages or for any applications involving the hydrolysis or modification of cellulosic material, wherein the polypeptide has an amino acid sequence selected from SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16.
[0017]
17. Use of an enzyme preparation as defined in any one of claims 1 to 5, characterized in that it is for the production of biofuels, for detergents, for the treatment of fibers, for the treatment of feed and food, for pulp and paper , for beverages or for any applications involving the hydrolysis or modification of cellulosic material.

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Class et al.2014|Patent application title: Treatment of Cellulosic Material and Enzymes Useful Therein Inventors: Jari Vehmaanperä | Jari Vehmaanperä | Marika Alapuranen | Terhi Puranen | Terhi Puranen | Matti Siika-Aho | Jarno Kallio | Jarno Kallio | Satu Hooman | Sanni Voutilainen | Teemu Halonen | Liisa Viikari | Assignees: Roal OY

同族专利:

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公开号 | 公开日

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EP2519630B1|2016-11-02|

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FI122937B|2012-09-14|

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CA2785944C|2019-07-16|

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US20110159544A1|2011-06-30|

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EP2519630A2|2012-11-07|

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CN102712917A|2012-10-03|

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CA2785944A1|2011-07-07|

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ES2612512T3|2017-05-17|

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WO2011080317A3|2011-10-13|

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CN102712917B|2015-09-23|

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US20140065693A1|2014-03-06|

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AP2012006392A0|2012-08-31|

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FI20096412A0|2009-12-30|

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BR112012016320A2|2017-02-21|

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DK2519630T3|2017-02-06|

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US8580552B2|2013-11-12|

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FI20096412A|2011-07-01|

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US9200267B2|2015-12-01|

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WO2011080317A2|2011-07-07|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

GB8610600D0|1986-04-30|1986-06-04|Novo Industri As|Transformation of trichoderma|

---

ES2153037T3|1994-06-03|2001-02-16|Novo Nordisk Biotech Inc|SCYTALIDIUM PURIFIED LACQUES AND NUCLEIC ACIDS CODING THEM.|

---

US5770418A|1994-06-24|1998-06-23|Novo Nordisk A/S|Purified polyporus laccases and nucleic acids encoding same|

---

US6008029A|1995-08-25|1999-12-28|Novo Nordisk Biotech Inc.|Purified coprinus laccases and nucleic acids encoding the same|

---

US7883872B2|1996-10-10|2011-02-08|Dyadic International , Inc.|Construction of highly efficient cellulase compositions for enzymatic hydrolysis of cellulose|

---

DE69922978T2|1998-10-06|2005-12-08|Emalfarb, Mark Aaron, Jupiter|TRANSFORMATION SYSTEM IN FILAMENTOUS FUNGICIDES CHRYSOSPORIUM HOST CELLS|

---

DK1259466T3|2000-02-17|2009-01-05|Univ Denmark Tech Dtu|Process for the preparation of lignocellulosic material|

---

CA2422558C|2000-09-25|2012-02-14|Iogen Energy Corporation|Method for glucose production with a modified cellulase mixture|

---

DK2295544T3|2001-06-26|2016-09-26|Novozymes As|Polypeptides with cellobiohydrolase I activity and the same coding polynucleotides|

---

US20040005674A1|2002-04-30|2004-01-08|Athenix Corporation|Methods for enzymatic hydrolysis of lignocellulose|

---

EP1578964B2|2002-12-20|2013-09-04|Novozymes A/S|Polypeptides having cellobiohydrolase ii activity and polynucleotides encoding same|

---

ES2357037T3|2004-01-16|2011-04-15|Novozymes, Inc.|METHODS FOR DEGRADING LIGNOCELLULOSIC MATERIALS.|

---

WO2005074656A2|2004-02-06|2005-08-18|Novozymes, Inc.|Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same|

---

PL1737951T3|2004-04-16|2010-10-29|Ab Enzymes Oy|Method and dna constructs for increasing the production level of carbohydrate degrading enzymes in filamentous fungi|

---

EP2292747B1|2004-12-30|2017-01-25|Danisco US Inc.|Variant hypocrea jecorina cbh2 cellulases|

---

DE602006019050D1|2005-01-06|2011-02-03|Novozymes Inc|POLYPEPTIDES WITH CELLOBIOHYDROLASE ACTIVITY AND POLYNUCLEOTIDES THAT CODE|

---

CN1757709A|2005-07-15|2006-04-12|山东农业大学|Saccharomyce engineering strain for expressing cbh2 gene and its construction method|

---

FI120045B|2005-12-22|2009-06-15|Roal Oy|Treatment of cellulose materials and enzymes useful therein|

---

EP2420570B1|2006-02-10|2013-12-04|Verenium Corporation|Arabinofuranosidase enzymes, nucleic acids encoding them and methods for making and using them|

---

JP2010516296A|2007-01-30|2010-05-20|ヴェレニウムコーポレイション|Enzymes for treating lignocellulosic substances, nucleic acids encoding the same and methods for producing and using the same|

---

US7923236B2|2007-08-02|2011-04-12|Dyadic International , Inc.|Fungal enzymes|

---

EP2708602B1|2007-10-03|2019-02-27|BP Corporation North America Inc.|Xylanases, nucleic acids encoding them and methods for making and using them|

---

WO2009059234A2|2007-11-01|2009-05-07|Novozymes, Inc.|Methods of reducing the inhibitory effect of a redox active metal ion on the enzymatic hydrolysis of cellulosic material|

---

US8455233B2|2007-12-19|2013-06-04|Novozymes A/S|Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same|

---

BRPI0821048A2|2007-12-19|2015-06-16|Novozymes As|Isolated polypeptide and polynucleotide, nucleic acid construct, recombinant host cell, methods for making the polypeptide, a precursor cell mutant, a protein and a fermentation product, to inhibit expression of a polypeptide, to degrade or convert a cellulosic material , and to ferment a cellulosic material, transgenic plant, plant part or plant cell, and inhibitory rna melecule|

---

EP2245148A4|2008-01-18|2011-08-24|Iogen Energy Corp|Cellulase variants with reduced inhibition by glucose|US7407788B2|2002-11-21|2008-08-05|Danisco A/S, Genencor Division|BGL7 beta-glucosidase and nucleic acids encoding the same|

---

MX2012009282A|2010-02-11|2012-09-07|Dsm Ip Assets Bv|Polypeptide having cellobiohydrolase activity and uses thereof.|

---

WO2011143632A2|2010-05-14|2011-11-17|Codexis, Inc.|Cellobiohydrolase variants|

---

DK2668265T3|2011-01-26|2017-10-16|Novozymes As|Hitherto unknown glycoside hydrolases from thermophilic fungi|

---

MX2013007720A|2011-01-26|2013-08-09|Novozymes As|Polypeptides having cellobiohydrolase activity and polynucleotides encoding same.|

---

WO2012149403A1|2011-04-27|2012-11-01|Codexis, Inc.|Cellobiohydrolase variants|

---

WO2013028278A1|2011-08-23|2013-02-28|Codexis, Inc.|Cellobiohydrolase variants|

---

CN104053666A|2011-11-18|2014-09-17|诺维信股份有限公司|Polypeptides Having Endoglucanase Activity And Polynucleotides Encoding Same|

---

US9708591B2|2011-11-18|2017-07-18|Novozymes Inc.|Polypeptides having endoglucanase activity and polynucleotides encoding same|

---

US9441256B2|2012-02-23|2016-09-13|Purdue Research Foundation|Lignases and aldo-keto reductases for conversion of lignin-containing materials to fermentable products|

---

WO2013166312A1|2012-05-02|2013-11-07|The Trustees Of Columbia University In The City Of New York|Biofuel production enzymes and uses thereof|

---

US8975058B2|2012-05-24|2015-03-10|Roal Oy|Endoglucanases for treatment of cellulosic material|

---

FI124477B|2012-06-07|2014-09-15|Roal Oy|New proteins for cellulosic material processing|

---

US9458440B2|2012-06-07|2016-10-04|Roal Oy|Proteins for the treatment of cellulosic material|

---

US9434929B2|2012-10-26|2016-09-06|Roal Oy|Esterases useful in the treatment of cellulosic and lignocellulosic material|

---

US9850512B2|2013-03-15|2017-12-26|The Research Foundation For The State University Of New York|Hydrolysis of cellulosic fines in primary clarified sludge of paper mills and the addition of a surfactant to increase the yield|

---

CN103343111B|2013-07-05|2015-08-26|山东尤特尔生物科技有限公司|A kind of mesophilic protein and its preparation method and application|

---

CN103484390A|2013-09-17|2014-01-01|山东农业大学|Pichia pastoris engineering strains expressing nine-family glucoside hydrolase gene CtAA9b|

---

CN103509727A|2013-09-17|2014-01-15|山东农业大学|Pichia pastoris engineering strain capable of expressing glycosyl hydrolase family 9 gene CtAA9c|

---

CN103525709B|2013-09-27|2015-09-09|中国科学院成都生物研究所|A kind of common Fusarium, the method preparing viscosity-reduction enzyme and application thereof|

---

CN103556474B|2013-10-23|2016-01-06|浙江省纺织测试研究院|Cellulose fibre ferment treatment absorption method in a kind of waste textile|

---

US9951363B2|2014-03-14|2018-04-24|The Research Foundation for the State University of New York College of Environmental Science and Forestry|Enzymatic hydrolysis of old corrugated cardboardfines from recycled linerboard mill waste rejects|

---

US20170233707A1|2014-09-30|2017-08-17|Danisco Us Inc|Compositions comprising beta-mannanase and methods of use|

---

CN106190874B|2015-05-06|2020-06-26|中国科学院天津工业生物技术研究所|Method for enhancing production of filamentous fungal protein|

---

CN106282142B|2016-08-05|2019-05-31|南京林业大学|A kind of preparation method for the alpha-galactosidase that beta-Mannosidase content is low|

---

CN106243391B|2016-08-05|2018-12-11|南京林业大学|The preparation method of transparent timber|

---

US20180265910A1|2017-03-17|2018-09-20|New England Biolabs, Inc.|Cleavage of Fucose in N-Glycans|

---

CN108976302B|2018-08-17|2021-04-13|中国科学院青岛生物能源与过程研究所|Cellulosomal enzyme formulations for catalyzing the saccharification of lignocellulose|

---

CN110755459B|2019-11-22|2021-06-29|广西南亚热带农业科学研究所|Extraction process of annona squamosa leaf polyphenol|

法律状态:
2017-11-07| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-07-16| B07A| Technical examination (opinion): publication of technical examination (opinion) [chapter 7.1 patent gazette]|

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2020-12-08| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

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2021-04-06| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-04-20| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 10 (DEZ) ANOS CONTADOS A PARTIR DE 20/04/2021, OBSERVADAS AS CONDICOES LEGAIS. |

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2021-05-18| B16B| Notification of grant cancelled|Free format text: ANULADA A PUBLICACAO CODIGO 16.1 NA RPI NO 2624 DE 20/04/2021 POR TER SIDO INDEVIDA. |

优先权:

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申请号 | 申请日 | 专利标题

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FI20096412A|FI122937B|2009-12-30|2009-12-30|Method for treating cellulosic material and CBH II / Cel6A enzymes useful herein|

---

FI20096412|2009-12-30|

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PCT/EP2010/070927|WO2011080317A2|2009-12-30|2010-12-30|Method for treating cellulosic material and cbhii/cel6a enzymes useful therein|

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