mannanase variant, nucleic acid molecule, method for preparing the modified mannanase, composition a

专利摘要:
MANANASE VARIANT, MANANASE, NUCLEIC ACID MOLECULE, VECTOR, HOST CELL, METHOD FOR THE PREPARATION OF MODIFIED MANANASE, COMPOSITION, AND, USE OF MODIFIED MANANASE. The present disclosure provides novel mannanase variants that have an amino acid sequence that varies from that of the precursor/wild-type Trichoderma reesei mannanase and that have one or more advantageous properties such as improved thermostability; temperature/activity profile; pH/activity profile; specific activity and pH/protease sensitivity. The new mannanase variants are useful and used in alcohol fermentation processes and/or productions, for coffee extraction and coffee residue processing, as a food and feed supplement, for enzyme-aided bleaching of paper pulps. , as a bleaching and/or desizing agent in the textile industry, to stimulate gas and oil wells with worse hydraulic bills, as cooking ingredients, for the removal of biofilms and in release systems, for grain processing or for the processing of renewable resources intended for the production of biofuels and fabrics, oil drilling, cleaning, laundry, detergent and cellulose fiber processing industries.
公开号:BR112012017292B1
申请号:R112012017292-1
申请日:2010-11-24
公开日:2021-05-18
发明作者:Oliver Kensch
申请人:Basf Se；
IPC主号:

专利说明:

DISCLOSURE FIELD
[001] The technology provided in this relates to improved variants of microbial mannanases, more specifically to microbial enzymes presenting the mannanase activity as its enzymatic activity; nucleic acid molecules encoding said mannanases, vectors, host cells containing the nucleic acids and methods for producing the mannanases; compositions comprising said mannanases; methods for the preparation and production of such enzymes and methods for the use of such enzymes for the processing of food and feed, for the extraction of coffee and the processing of coffee residue, as a supplement to food and feed, by aided bleaching by enzyme from paper pulps, as a bleaching and/or desizing agent in the textile industry, for the stimulation of oil and gas by hydraulic fracture, as a detergent, as cooking ingredients, for the removal of biofilms and in release systems, for grain processing or by processing renewable resources intended for the production of biofuels and in the textile processing, oil drilling, cleaning, laundry, detergent and cellulose fiber industries. FOUNDATION
[002] Endo-β-1,4-D-mannanase (β-mannanase; EC 3.2.1.78) catalyzes the random hydrolysis of manno-glycoside bonds in mannan-based polysaccharides. More oligosaccharides degrade β-mannanases below DP4 (Biely and Tenkanen (1998) Enzymology of hemicellulose degradation, pages 25-47. In Harman and Kubiceck (ed) Trichoderma and Gliocladium, vol.2, Taylor and Francis Ltd. London), however , residual activity was demonstrated in mannotriosis, indicating at least four subsites for binding of mannose in the protein. The main end products of hydrolysis are often mannobiose and mannotriosis, although significant amounts of mannose are also produced. Some β-mannanases are capable of degrading crystalline mannan. In addition to hydrolysis, several β-mannanases including β-mannanase from Trichoderma reesei, have been shown to form transglycosylation products with mannose or mannobiose as the glycosidic bond acceptor.
[003] The β-mannanases have been isolated from a wide range of organisms including bacteria, fungi, plants and animals. Although more extracellular, some β-mannanases appear to be cell-associated. Its expression is often induced by development in mannan or galactomannan, however, β-mannanase from T. reesei can also be induced by cellulose, while its expression is suppressed by glucose and other monosaccharides. Often multiple mannanases with different isoelectric points are observed in the same organism, representing different gene products or different products from the same gene, respectively.
[004] In general, β-mannanases have excellent moderate temperature between 40°C and 70°C, except some thermophilic β-mannanases (Politz et al. (2000) a thermostable endo-1,4-β-mannanase highly A thermostable from the marine bacterium Rhodothermus marinus; Appl. Microbiol. Biotechnol. 53:715-721). The optimal pH is in the neutral or acidic region, eg pH 5.0 for β-mannanase from T. reesei (Arisan-Atac et al. (1993) Purification and characterization of a β-mannanoase from Trichoderma reesei C-30; Appl. Microbiol. Biotechnol. 39:58-62). The molecular weights of the enzyme ranges between 30 kD and 80 kD.
[005] WO 2008009673 discloses variants of Trichoderma reesei mannanases improved in thermal stability and low pH/pepsin resistance for use in the hydrolysis of galactomannan containing plant material, e.g. palm seed sludge (PKE) and for the use in animal feed.
[006] For example, thermostability is required by feed additives that are incorporated into feed mixtures prior to a granulation procedure comprising high temperatures. Additionally, mannanases apply as feed additives needed at low pH and stable pepsin and will be active at low pH in order to be able to work efficiently in the stomach of eg monogastric animals.
[007] However, the direction in the feed industry is to increase the granulation temperatures still. The enzyme stability currently around 90°C to 95°C at the granulation temperature is targeted to be able to use the enzymes throughout all industrially relevant feed production plants. Therefore, the availability of a mannanase with improved thermal stability should be highly advantageous, as it should allow the use of the enzyme also in plants with high operating temperature. SUMMARY OF THE INVENTION
[008] In a first aspect, embodiments of the invention provide mannanase variants that have an amino acid sequence that varies from that of the wild type/Trichoderma reesei mannanase precursor (SEQ ID NO: 1) and that do not have a or more advantageous properties. Such properties may include but are not limited to favorable: thermostability; temperature/activity profile; pH/activity profile; specific activity; and pH/protease sensitivity.
[009] In a further aspect, the embodiments of this invention relates to a mannanase variant comprising a mannanase that contains a substitution to one or more positioned positions of the group consisting of: 66, 215 or 259, wherein each position corresponding to the position of the Trichoderma reesei mannanase wild-type/precursor amino acid sequence (SEQ ID NO: 1).
[010] In a further aspect, embodiments of this invention relate to mannanase variants having an amino acid sequence ranging from that wild type/Trichoderma reesei mannanase precursor (SEQ ID NO: 1), comprises the variation 201S, 207F and 274L and at least one variation at one or more positions corresponding to position 66, 215 or 259 compared to the amino acid sequence of SEQ. ID No.: 1.
[011] In a still further aspect, embodiments of this invention provide the nucleic acids encoding the mannanase variants as disclosed herein, as well as vectors and host cells comprising such nucleic acids. In other embodiments, the sequences are used in the processes that produce the mannanase variants.
[012] Furthermore, the embodiments of this invention generally concern the use of the mannanase variants for the digestion of galactomannan, in particular catalyzing the random hydrolysis of manno-glycoside linkages into mannan-based polysaccharides. Advantageously, the mannanase variants of this invention can be used in industrial applications including, for example, methods for liquefying starch and for enhancing the digestion of galactomannan in food and animal feed. Advantageously, mannanase variants according to embodiments of the present invention are useful and used in alcoholic fermentation processes and/or productions, for coffee extraction and coffee residue processing, as a supplement to food and feed. , for enzyme-aided bleaching of paper pulps, as bleaching and/or desizing agents in the textile industry, for the stimulation of oil and gas by hydraulic fracture, as detergents, as cooking ingredients, for the removal of biofilms and in release systems, for grain processing or by processing renewable resources intended for the production of biofuels and in the textile processing, oil drilling, cleaning, laundry, cellulose fiber detergent industries.
[013] In other aspects, this invention relates to enzyme compositions comprising the mannanase variant as described herein, wherein the enzyme composition is useful for, or used in, commercial applications. In one embodiment, the enzyme composition can be an animal feed composition. In other embodiments, the enzyme composition can be used in starch hydrolysis processes (e.g., liquefaction). In an advantageous embodiment, the variants and/or the enzyme composition can be used in alcohol fermentation processes. In still further embodiments, an enzyme composition comprising a mannanase encompassed by the invention will include additional enzymes such as phytases, glucoamylases, alpha amylases, protease, cellulases, hemicellulases and combinations thereof.
[014] In a further aspect, embodiments of this invention concern methods for producing the mannanase variants in a host cell by transforming the host cell with a DNA construct, advantageously including a promoter having transcriptional activity in the cell host cell by culturing the transformed host cell in a suitable culture medium to allow expression of said mannanase and produce mannanase. The method can also include the recovery of produced mannanase. In one embodiment, the host cell is a Trichoderma-like fungus, such as T. reesei, a yeast, a bacterium, or a plant cell. In an advantageous embodiment of this invention, the mannanase variant has the sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 or variants, forms modified, homologs, fusion proteins, functional equivalents or fragments thereof.
[015] Before the invention is described in detail, it is understood that this invention is not limited to the particular component parts of the described devices or process steps of the described methods as such devices are the methods may vary. It will also be understood that the terminology used is for the purpose of describing the particular embodiments only and is not intended to be limiting. It should be noted that, as used in the specification and the appended claims, the singular forms “a”, “an” and “the” include the singular and/or plural referents otherwise the context clearly dictates otherwise. It will further be understood that, in cases where parameter ranges are data which are delimited by numerical values, the ranges are considered to include these limiting values. BRIEF DESCRIPTION OF THE DRAWINGS
[016] FIG. 1 shows an amino acid sequence of wild type Trichoderma reesei mannanase, (SEQ ID NO: 1).
[017] FIG. 2 shows the amino acid sequence of Trichoderma reesei mannanase V-31 variant disclosed in WO 2008/009673 (SEQ ID NO: 2).
[018] FIG. 3 shows the amino acid sequence of an additional mannanase variant Trichoderma reesei V-31/S3R disclosed in WO 2008/009673 (SEQ ID NO: 3).
[019] FIG. 4 shows the amino acid sequence of a TM-1 mannanase variant advantageous in accordance with the present invention (SEQ ID NO: 4).
[020] FIG. 5 shows the nucleic sequence of the TM-1 mannanase variant (SEQ ID NO: 4) according to the present invention (SEQ ID NO: 5).
[021] FIG. 6 shows the amino acid sequence of a further advantageous TM-100 mannanase variant in accordance with the present invention (SEQ ID NO: 6).
[022] FIG. 7 shows the amino acid sequence of a further advantageous TM-108 mannanase variant in accordance with the present invention (SEQ ID NO: 7).
[023] FIG. 8 shows the amino acid sequence of a further advantageous TM-CBD-148 mannanase variant in accordance with the present invention (SEQ ID NO: 8).
[024] FIG. 9 shows the amino acid sequence of the additional advantageous TM-144 mannanase variant according to the present invention (SEQ ID NO: 9).
[025] FIG. 10 shows the comparison of wild-type/precursor Trichoderma reesei mannanase and the S3R variant with respect to stability and thermal activity.
[026] FIG. 11 shows the amino acid sequence of a cellulose binding domain (CBD). DETAILED DESCRIPTION OF THIS INVENTION
[027] Disclosed herein are variants of Trichoderma reesei mannanases (EC 3.2.1.78) and nucleic acid encoding mannanases that can be used in industrial applications including methods for protein hydrolysis, biomass degradation and for enhancing galactomannan digestion contained in food and/or animal feed.
[028] In particular, mannanase variants according to the present invention show particular improved thermal stability, pH/pepsin stability at the same time retained or improved specific activities compared to the precursor mannanase enzyme used. These characteristics make them specifically useful for the industrial application of animal feed, food and for the degradation of galactomannan in plant material in general.
[029] The present invention discloses enzymes with an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 1 or variants, modified forms, homologues, fusion proteins, functional equivalents or fragments thereof, or comprise one or more insertions , deletions or mutations or any combination thereof. A homologous mannanase according to the present invention comprises any enzyme having a sequence identity of at least 70% or preferably at least 80%, 85%, 90%, 95%, 97% or 99%, preferably to SEQ ID NO: 1, more preferably SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.
[030] The mannanase variants of this invention have mannanase activity and an amino acid sequence that varies from the wild-type/precursor Trichoderma reesei mannanase amino acid sequence (SEQ ID NO: 1) comprising one or more variations (including substitutions , insertions or cancellations).
[031] In an advantageous embodiment, the amino acid sequence of the mannanase variants comprises at least the 201S, 207F and 274L variation and at least one variation in one or more positions corresponding to position 66, 215 or 259 compared to the amino acid sequence of SEQ. ID No.: 1. For example, advantageously the variation in mannanase variants can be selected from the group consisting of: 66P, 215T and 259R.
[032] An advantageous embodiment of the invention is a mannanase variant according to SEQ ID NO: 4 or variants, modified forms, homologs, fusion proteins, functional equivalents or fragments thereof, or comprises one or more insertions, deletions or mutations or any combination thereof and the mannanase that has at least the minimum percent sequence identity and/or percent homology to the mannanase of SEQ ID NO: 4, wherein the minimum percent identity and/or homology is at least 50% , at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 96%, at least 97%, at least 98% or at least 99% .
[033] In additional advantageous embodiments, the mannanase variants according to the present invention comprise the additional variations 201S, 207F and 274L and to a variation in one or more positions corresponding to position 66, 215 or 259 a variation in position 3 and/or 181 corresponding to the position of the amino acid sequence of SEQ ID NO: 1. For example, the variation at position 3 is 3R, the variation at position 181 is 181A or 181H (181/A/H).
[034] In further advantageous embodiments, the mannanase variants according to the present invention comprise ad variations 201S, 207F and 274L and at least the variations in position 66, 215 and 259 compared to the amino acid sequence of SEQ. ID No.: 1. In an advantageous example the variations in positions 66, 215 and 259 respectively are 66P, 215T and 259R.
[035] In yet one embodiment, the mannanase variants further comprise the variations 201S, 207F and 274L and at position 66, 215 and 259 the variation in position, preferably 181A/H compared to the amino acid sequence of the SEQ . ID No.: 1. In an advantageous embodiment, the mannanase variants further comprise a variation in position 3, similar to 3R.
[036] In an advantageous embodiment, the amino acid sequence of the mannanase variants according to the present invention comprises at least the variation 201S, 207F and 274L and at least one variation in one or more positions corresponding to position 66, 215 or 259 and one or more additional variations, wherein the position of variation is 31, 97, 113, 146, 149, 173, 181, 280, 282, 331 or 344 compared to the amino acid sequence of SEQ. ID No.: 1. For example, the ranges are 31Y, 97R, 113Y, 146Q, 149K, 173H/T, 181H/A, 280S/L/R, 282D, 331S or 344D.
[037] Advantageous embodiments of the invention are mannanase variants that have at least the minimum percentage sequence identity and/or percent homology to the mannanases according to the present invention, wherein the minimum percentage identity and/or homology is at least 50%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 96%, at least 97%, at least 98 % or at least 99%.
[038] Further advantageous embodiments are mannanase variants having mannanase activity and an amino acid sequence that varies from the amino acid sequence of wild-type/precursor Trichoderma reesei mannanase (SEQ ID NO: 1), wherein an amino acid of the mannanase variant comprises variations 201S, 207F and 274L and at least variations selected from the group consisting of: 1) F31Y/S66P/Q97R/N113Y/N173H/V181H/A215T/Q259R/Q280R/ N282D/N331S 2) S66P/Q97R/N113Y/N173H/V181A/A215T/Q259R/Q280S/ N331S 3) F31Y/S66P/Q97R/N173H/V181H/A215T/Q259R/Q280R/ N282D 4) F31Y/S66P/Q97R/N173H/V181 A215T/Q259R/Q280S/ N282D/N331S 5) F31Y/S66P/Q97R/N113Y/N173H/V181H/A215T/Q259R/ Q280R/ N282D 6) F31Y/S66P/Q97R/Q149K/N173H/VQ18H/A215T ) S66P/Q97R/N113Y/N173H/V181A/A215T/Q259R 8) F31Y/S66P/Q97R/N113Y/N173H/V181H/A215T/Q259R/Q280S/ N282D/N331S 9) S66P/N113Y/HN113Y/V 10) F31Y/S66P/Q97R/N113Y/K146Q/N173H/V181H/A215T/ Q259R/ Q280L/N282D 11) F31Y/S66P /Q97R/N113Y/K146Q/N173H/V181A/A215T/ Q259R/ Q280L/N282D/N331S 12) F31Y/S66P/Q97R/N113Y/K146Q/N173H/V181H/A215T/Q259R/Q280L 13) F173/HQ97/S66 /V181H/A215T/Q259R/N282D 14) F31Y/S66P/Q97R/N113Y/K146Q/N173H/V181A/A215T/ Q259R/Q280R/N282D 15) S66P/N113Y/V181H/A215T/Q259R 16) S66NP/Q97 N173H/V181H/A215T/Q259R/Q280L/ N282D 17) F31Y/S66P/Q97R/N113Y/K146Q/N173H/V181A/A215T/Q259R/Q280L/N282D 18) F31Y/S66P/N173H/V181H/A215T/N173H/V181A/A215T/Q259R/Q280L/N282D 18) F31Y/S66P/N173H/V181H/A215T/N ) F31Y/S66P/Q97R/N173H/V181H/A215T/Q259R/Q280L 20) S66P/Q97R/N113Y/N173H/V181H/A215T/Q259R/Q280R/ N282D 21) F31Y/S66P/Q97R/N113Y/N173T /Q259R/ Q280R/N282D 22) F31Y/S66P/Q97R/N173T/V181H/A215T/Q259R/Q280R/ N282D 23) F31Y/S66P/Q97R/N173H/V181H/A215T/Q259R/Q280S/ N282D 24) S66 N173H/V181H/A215T/Q259R/Q280S/N282D 25) S66P/Q97R/N113Y/N173H/V181A/A215T/Q259R/Q280S/ N282D/N331S 26) S66P/Q97R/N113Y/V181H/A282D/QL2 ) S66P/N113Y/N173H/V181H/A215T/Q259R/N331S 28) F31Y/S66P/Q97R/N113Y/K146Q/N173H/V181A/A215T/Q259R/Q280R/N282D/ N331S 29) F31Y/S66P/Q97R/N113Y/K146Q/V181H/A215T/Q259R/Q280S/N282D/N331S 30) S66P/Q97R/N113Y/N173T/V181A/A215T/Q259R 31) F31Y/S66NQ3/Q97 /N173H/V181A/A215T/ Q259R/Q280S/N331S 32) F31Y/S66P/Q97R/N113Y/V181H/A215T/Q259R 33) S66P/Q97R/N113Y/N173H/V181A/A215T/Q259R/N331S 3466) F Q97R/N113Y/V181H/A215T/Q259R/Q280L 35) F31Y/S66P/Q97R/N113Y/K146Q/V181H/A215T/Q259R/ Q280L 36) F31Y/S66P/Q97R/K146Q/V181H/A28280DR/Q259R/Q2 ) S66P/N113Y/V181H/A215T/Q259R/N282D 38) F31Y/S66P/Q97R/V181H/A215T/Q259R/N282D 39) S66P/N113Y/N173H/V181H/A215T/Q259R/Q280S/N33Y1S Q97R/N113Y/K146Q/N173H/V181H/A215T/ Q259R/Q280S/N331S 41) S66P/V181H/A215T/Q259R/N282D 42) F31Y/S66P/Q97R/N113Y/K146Q/V181H/A215T/QL259R ) S66P/Q97R/N113Y/N173H/V181H/A215T/Q259R/N282D 44) S66P/Q97R/N113Y/V181H/A215T/Q259R/N282D 45) S66P/V181H/A215T/Q259R 46) S66P/Q97R/Q97R A215T/Q259R/Q280R/N282D 47) F31Y/S66P/N173T/V181H/A215T/Q259R/N282D 48) F31Y/S66P/N113Y/V181H/A215T/Q259R/Q280R/N344D 49) F31Y/S66P/Q97R/N113Y/N173H/V181H/A215T/Q259R/Q280R/N282D 50) F31Y/S66P/Q97R/N113Y/N173H/V181H/A215T/Q259R/ Q280S/N282D/N282D 51) S N173H/V181H/A215T/Q259R/Q280S 52) S66P/Q97R/N113Y/N173H/V181A/A215T/Q259R 53) S66P/Q97R/N113Y/N173H/V181A/A215T/Q259R/Q280S 54) S66HN173/N113 /A215T/Q259R/Q280S/N282D 55) F31Y/S66P/Q97R/N173H/V181H/A215T/Q259R/Q280R/ N282D 56) S66P/N113Y/N173H/V181H/A215T/Q259R 57) S66P/Q97R/N V181A/A215T/Q259R/N331S 58) F31Y/S66P/Q97R/N173H/V181H/A215T/Q259R/Q280S/ N282D/N331S 59) S66P/N113Y/N173H/V181H/A215T/Q259R/Q280S/N33 /N113Y/N173H/V181A/A215T/Q259R/Q280S/ N331S 61) F31Y/S66P/Q97R/Q149K/N173H/V181H/A215T/Q259R/ Q280S/N331S 62) S66P/A215T/ Q259R 63)P/3R215T66 Q259R
[039] Embodiments of this invention also include variants of any of the mannanases shown in sequences 1) to 63), which have mannanase activity and an amino acid sequence having a percent sequence identity and/or percent homology of at least 50%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99% compared to each of the mannanase variants shown in sequences 1) to 63).
[040] Further embodiments of the invention are nucleic acid molecules selected from the group consisting of a) a nucleic acid molecule encoding the mannanase variants according to the present invention; b) a nucleic acid molecule encoded by the derivative of the mannanase variants according to the present invention, preferably wherein the derivative of one or more amino acid residues are conservatively substituted; c) a nucleic acid molecule which is a fraction, variant, homolog, derivative or fragment of the nucleic acid molecule shown as SEQ ID NO: 5; d) a nucleic acid molecule which is capable of hybridizing to any one of the nucleic acid molecules of a) - c) under the stringent conditions e) a nucleic acid molecule which is capable of hybridizing the complement of any one of the molecules of nucleic acid from a) - c) under the stringent conditions f) a nucleic acid molecule having a sequence identity of at least 95% with any of the nucleic acid molecules from a) - e) and encoded by mannanase, g) a nucleic acid molecule having a sequence identity of at least 70% with any of the nucleic acid molecules of a) - e) and encoded by mannanase, h) or a complement of any of the nucleic acid molecules of a) - g).
[041] Further embodiments of the invention are vectors and host cells comprising the nucleic acid molecules encoding the mannanase variants according to the present invention.
[042] Further, embodiments are the methods for preparing the mannanase variants according to the invention, comprising culturing the transformed host cell and isolating the modified mannanase from the culture.
[043] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994) and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provides a skilled person with a general dictionary of many of the terms used in this invention.
[044] This invention is not limited by the exemplary methods and materials disclosed herein and any methods and materials similar or equivalent to those described herein may be used in practice or testing in the embodiments of this invention. Numeric Ranges are inclusive of the numbers defined in the range. Unless otherwise indicated, nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written from left to right in amino to carboxy orientation, respectively.
[045] The headings provided herein are not limiting of various aspects or embodiments of this invention which may be referred to as a total of the specification. Consequently, the terms immediately defined below are more fully defined by reference to the specification as a total.
[046] In one embodiment of the present invention, the mannanase enzymes show particularly improved thermal stability and in the same period improved or specific activities retained compared to the mannanase enzymes disclosed in WO 2008/009673. These characteristics make them especially useful for the industrial application of animal feed, food and for the degradation of galactomannan in plant material in general.
[047] Therefore, the present invention is also directed to a method for the production of mannanases in Trichoderma reesei, active at low pH values as present in the stomach and upper intestine of animals and the crop, stomach and upper intestine of poultry. It is already another object of the present invention to provide a mannanase that can be added to animal feed prior to granulation in order to allow accurate and reproducible enzyme dosing and to avoid an additional spraying step in the feed preparation.
[048] The term "mannanase" refers to any enzyme capable of hydrolyzing the polyose chains that are composed of mannose units (mannopolymers or polymannoses). "Mananase", therefore, comprises endomannanases and exomannanases that cleave the mannopolymers internally or from the polymer terminal ends, respectively.
[049] The term "functional equivalent of mannanase" or "functional equivalent of this" means that the enzyme has about the same functional characteristics as that of Trichoderma reesei mannanase.
[050] The term "modified form" or "variant" means that the enzyme has been modified from its original form (precursor/wild-type, weight) but retains the same functional enzymatic characteristics as that of Trichoderma reesei mannanase.
[051] The term "fusion proteins" comprises all proteins derived from the precursor mannanase or any variant thereof by covalently fusing the additional amino acid sequences at the C- and/or N-terminus. The source and composition of the additional amino acid sequence is natural from any living organism or virus or unnatural.
[052] The term "functional fragment" or "effective fragment" means a fragment or portion of Trichoderma reesei mannanase or derivative thereof that retains about the same enzymatic function or effect.
[053] The term "homologous mannanase" according to the present invention comprises any enzyme with a sequence identity of at least 70% or preferably at least 80%, 85%, 90%, 95%, 97% or 99% to precursor mannanase.
[054] The term "polynucleotide" corresponding to any genetic material of any length and any sequence, comprising single-stranded and double-stranded DNA and RNA molecules, including regulatory elements, structural genes, gene clusters, plasmids, total genomes and fragments of these.
[055] The term "position" in a polynucleotide or polypeptide refers to specific single base or amino acids in a polynucleotide or polypeptide sequence, respectively.
[056] The term "polypeptide" comprises proteins such as enzymes, antibodies and others, medium length polypeptides such as peptide inhibitors, cytokines and others, as well as short peptides below an amino acid sequence below ten in length, such as peptide receptor ligands, peptide hormones and others.
[057] The term "mannanase variants" means any mannanase molecule obtainable by random or site-directed mutagenesis, insertion, deletion, recombination and/or any other method of protein engineering, which leads to differing mannanases in its amino acid sequence from the precursor mannanase. The terms "wild-type mannanase", "wild-type enzyme", or "wild-type" according to the invention describe a mannanase enzyme with an amino acid sequence observed in nature or a fragment thereof.
[058] The "precursor mannanase" can be an isolated wild-type mannanase or a fragment thereof, or one or more mannanase variants selected from the mannanase library.
[059] The term "mannanase library" describes at least one mannanase variant or a mixture of mannanases wherein each single mannanase, each mannanase resp. variant, is encoded by a different polynucleotide sequence.
[060] The term “gene library” indicates a library of polynucleotides that encodes the mannanase library.
[061] The term "isolated" describes any molecule separated from its natural source.
[062] The term "mutation" refers to the substitution or replacement of single or multiple nucleotide triplets, nucleotide triplets, insertions or deletions of one or more codons, homologous or heterologous recombination between different genes, fusion of additional coding sequences at the end of the coding sequence, or insertion of additional coding sequences, or any combination of these methods, which result in a polynucleic acid sequence that encodes a desired protein. Thus, the term "mutations" also refers to all changes in the polypeptide sequence encoded by the polynucleic acid sequence modified by one or more of the changes described above. Amino acid residues are abbreviated according to the following Table 1 in one or three letter code.
[063] The term "nucleic acid molecule" or "nucleic acid" is intended to indicate any single or double stranded nucleic acid molecule of cDNA, genomic DNA, synthetic DNA or RNA, PNAS or LNA origin.
[064] The term "stringent conditions" refers to conditions under which a probe will hybridize to its target subsequence, but no other than the sequences. Stringent conditions are sequence dependent and will be different under different circumstances. The longer sequences will specifically hybridize at higher temperatures. Generally, stringent conditions are selected from about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic length and pH. The Tm is the temperature (under defined ionic length, pH and nucleic acid concentration) at which 50% of the probes complementary to the probes complementary to the target sequence hybridizes to the target sequence at equilibrium. (As target sequences are usually present in excess, at Tm, 50% of the probes are occupied at equilibrium). Typically, stringent conditions will be those where the salt concentration is less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion (or other salts) at pH 7.0 at 8m3 and the temperature is at least about 30°C for short probes (eg 10 to 50 nucleotides) and at least about 60°C for long probes. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide and others.
[065] The term "nucleic acid molecule fragment" is intended to indicate a nucleic acid which comprises a subset of a nucleic acid molecule according to any of the claimed sequences.
[066] The same is applicable to the term “nucleic acid molecule fraction”.
[067] The term "variant nucleic acid molecule" herein refers to a nucleic acid molecule that is substantially similar in structure and biological activity to a nucleic acid molecule according to any of the claimed sequences.
[068] The term "nucleic acid molecule homologue" refers to a nucleic acid molecule of the sequence which has one or more nucleotides added, deleted, substituted or otherwise chemically modified in comparison to a nucleic acid molecule of according to any of the claimed sequences, provided whenever the homologue retains substantially the same binding properties as the latter.
[069] The term "derivative," as used herein, refers to a nucleic acid molecule that has similar binding characteristics to the target nucleic acid sequence as a nucleic acid molecule according to any of the claimed sequences. TABLE 1: AMINO ACID ABBREVIATIONS


[070] Mutations or variations are described by using the following nomenclature: position; substituted amino acid residues. According to this nomenclature, the substitution of, for example, an Alanine residue for a glycine residue at position 20 is indicated as 20G. When an amino acid residue at a given position is substituted with two or more alternative amino acid residues these residues are separated by a comma or a slash. For example, replacing Alanine at position 30 with glycine or glutamic acid is indicated as 20G/E, or 20G, 20E.
[071] In addition, the following nomenclature should also be used: amino acid residue in protein structure; position; substituted amino acid residues. According to this nomenclature, the substitution of, for example, an Alanine residue for a glycine residue at position 20 is indicated as Ala20Gly or A20G, or 20G. Alanine override at the same position is shown as Ala20* or A20*. The insertion of an additional amino acid residue (eg glycine) is indicated as Ala20AlaGly or A20AG. Nullification of consecutive length of amino acid residues (for example, between Alanine at position 20 and glycine at position 21) is indicated as Δ(Ala20-Gly21) or Δ(A20-G21). When a sequence contains a knockout compared to the precursor protein used for numbering, an insertion at such a position (eg, an Alanine at position 20 knockout) is indicated as *20Ala or *20A. Multiple mutations are separated by plus the sign or a slash. For example, two mutations at positions 20 and 21 replacing Alanine and glutamic acid for glycine and Serine, respectively, are indicated as A20G+E21S or A20G/E21S. When an amino acid residue at a given position is replaced with two or more alternative amino acid residues these residues are separated by a comma or a slash. For example, substitution of Alanine at position 30 with glycine or glutamic acid is indicated as A20G, E or A20G/E, or A20G, A20E. When the proper position for the modification is identified therein without any specific modification being suggested, it is understood that any amino acid residue may be substituted for the amino acid residue present in the position. Thus, for example, when a modification of an Alanine at position 20 is mentioned but not specified, it is understood that Alanine can be deleted or substituted by any other amino acid residue (i.e. any one of R, N, D, C , Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V).
[072] The terms "conservative mutation", or "conservative substitution", respectively, refer to a mutation of amino acids that a person skilled in the art should consider a conservative in a first mutation. "Conservative" in this context means an amino acid similar in terms of amino acid characteristics. If, for example, a mutation leads to a specific position in a substitution of a non-aliphatic amino acid residue (eg Ser) with an aliphatic amino acid residue (eg Leu) then a substitution at the same position with a different aliphatic amino acid (eg Ile or Val) is referred to as a conservative mutation. Further amino acid characteristics include residue size, hydrophobicity, polarity, charge, pK value and other amino acid characteristics known in the art. Consequently, a conservative mutation can include substitution such as basic to basic, acid to acid, polar to polar, etc. These series of amino acids thus derived are similar in being conserved for structural reasons. These series can be described in the form of a Venn diagram (Livingstone CD and Barton GJ (1993) “Protein sequence alignments: a strategy for the hierarchical analysis of residue conservation” Comput.Appl Biosci. 9:745-756; Taylor WR (1986) “The classification of amino acid conservation” J.Theor.Biol. 119; 205-218). Conservative substitutions can be made, for example, in accordance with the table below which describes an acceptable Venn diagram grouping of amino acids. TABLE 2: AMINO ACIDS FROM THE VENN DIAGRAM GROUP

[073] The term “catalytic activity” or “activity” quantitatively describes the conversion of a given substrate under defined reaction conditions. The term "residual activity" is defined as the ratio of the enzyme's catalytic activity under a certain set of conditions to the catalytic activity under a different set of conditions. Therefore the residual activity ai is given by ai=vi/v0 where v indicates any measure of the catalytic activity and ai*100 is the relative activity in percentage. The term "specific activity" quantitatively describes the catalytic activity per quantity of the enzyme under defined reaction conditions.
[074] The term "thermostability", "temperature stability" or "thermal stability" describes the property of a protein to withstand limited heat exposure without losing its activity at lower temperatures, for example, at the temperature where its activity may be measure.
[075] The term "pH stability" describes the property of a protein to withstand limited exposure to pH values significantly deviating from the pH where its stability is optimal, eg more than one pH unit above or below the optimal pH, without losing its activity under conditions where its activity can be measured.
[076] The term "proteolytic stability" describes the property of a protein to resist limited exposure to proteases under conditions where the proteases are active without losing activity under conditions where their activity can be measured.
[077] The term "plasmid", "vector system" or "expression vector" means a construct capable of expression in vivo or in vitro. In the context of the present invention, these constructs can be used to introduce the enzymes encoding the encoding genes into host cells.
[078] The term "host cell" in relation to the present invention includes any cell which comprises the nucleic acid molecule or an expression vector as described above and which is used in the recombinant production of an enzyme having the specific properties as defined herein or in the methods of the present invention.
[079] The "inactivation temperature" is defined as the temperature at which the residual activity of the mannanase enzyme after incubation for a certain duration and subsequent cooling to room temperature is 50% of the residual activity of the same mannanase enzyme incubated for the same duration under the same conditions at room temperature.
[080] The term “renewable resources” refers to the biomass substrates that are developed and collected, similar crops, straw, wood and wood products. The term "biological fuel" refers to the solid, liquid or gaseous fuel that is made from or derived from biomass, similar to biodiesel, Biogas, vegetable oil, Bioethanol, BioHydrogen, Bio-Dimethyl ether, Biomethanol, BTL (" Biomass to liquid") - fuel, GTL ("Gas to liquid") - fuel and others.
[081] The term "functional equivalent of this" means that the enzyme has about the same functional characteristics as those of Trichoderma reesei mannanase. The term "modified form" or "variant" means that the enzyme has been modified from its original form but retains the same functional enzymatic characteristics as those of Trichoderma reesei mannanase. In particular, the terms "variant" or "modified form" encompass mannanase enzymes having an amino acid sequence derived from the precursor/wild-type mannanase amino acid sequence and having one or more amino acid substitutions, insertions, deletions or any combination thereof. , which together are referred to as the mutations.
[082] "Fusion proteins" comprise all proteins derived from precursor mannanase or any variant thereof by covalently fusing an additional amino acid sequence to the C- and/or N-terminus of the precursor mannanase.
[083] The "percentage of sequence identity", with respect to two amino acid or polynucleotide sequences, refers to the percentage of residues that are identical in the two sequences when the sequences are optionally aligned. Thus, 80% amino acid sequence identity means that 80% of the amino acids in two optionally aligned polypeptide sequences are identical. Percent identity can be determined, for example, by directly comparing sequence information between two molecules by aligning the sequences, counting the exact number of points between their aligned sequences, divided by the length of the shortest sequence, and multiplying the result by 100. Readily available computer programs can be used to aid analysis, such as ALIGN, Dayhoff, MO in "Atlas of Protein Sequence and Structure", M.O. Dayhoff et., Suppl. 3:353358, National Biomedical Research Foundation, Washington, DC, which adapts to the local homology algorithm of Smith and Waterman (1981) Advances in Appl. Math. 2:482-489 for peptide analysis. Programs for determining sequence nucleotide identity are available in Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, WI), e.g. BESTFIT, FASTA, and GAP programs, which also rely on Smith and Waterman algorithm. These programs are readily used with the manufacturer's recommended default 5 parameters and described on the Wisconsin Sequence Analysis package referenced above. An example of an algorithm that is suitable for determining sequence similarity is the BLAST algorithm, which is described in Altschul, et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). Likewise, computer programs for determining percent homology are also readily available.
[084] A "feed" and a "food," respectively, means any natural or artificial diet, flour or other or components of such flours intended or suitable to be eaten, absorbed, digested, by an animal and a human being, respectively. .
[085] A "food or feed additive" is a compound or multi-component composition suitable for being added to food or feed. It may, but is not required to comprise one or more compounds such as vitamins, minerals or feed enhancing enzymes and suitable carriers and/or excipients and is usually supplied in a suitable form which can be added to the animal feed.
[086] Modified forms or variants may present the characteristics of altered enzyme compared to the precursor enzyme. Preferably, the modified forms or variants have one or more of the following enhanced phenotypes: increased thermostability; and/or increased proteolytic stability (eg against pepsin); and/or increased specific activity and/or improved stability at low pH. The term "functional" or "effective" fragment means a fragment or portion of Trichoderma reesei mannanase that retains about the same enzymatic function or effect.
[087] It is also understood that the present invention comprises all molecules that are derived from the precursor mannanase and all variants thereof described in this application, by post-translational processing compared to the genetically modified amino acid sequence. These post-translational modifications comprise, but are not limited to, proteolytic cleavage of N-terminal sequences such as prosequences and/or leader, proteolytic removal of C-terminal extensions, N- and/or O-glycosylation, lipidation, acylation , deamidation, pyroglutamate formation, phosphorylation and/or others, or any combination thereof, as these occur during production/expression by the natural host or any suitable expression host. These post-translational modifications may or may not have an influence on the physical or enzymatic properties of the enzymes as explored herein.
[088] Preferably, said changes lead to improved enzyme properties such as greater thermostability and/or greater specific activity and/or improved stability at low pH and/or greater resistance against proteolytic cleavage by protease such as pepsin; and/or high residual activity at low pH.
[089] In preferred embodiments of the present invention, the modified mannanase has a substitution at one or more of positions 201, 207, 274, 66, 215, 259, 31, 97, 113, 146, 149, 173, 181, 280, 282, 331 or 344, relating to the precursor/wild-type manase numbering given in SEQ ID NO: 1. These positions are characterized in that the mutagenesis of the enzyme at these positions leads to improvement in the characteristics of the desired enzyme.
[090] Already basically, several amino acid substitutions with respect to wild-type/precursor mannanase have rendered beneficial in terms of thermostability, either by itself and/or in combination with others. These substitutions are shown in Table 3. In addition, various amino acid substitutions also turned out to be very beneficial in terms of pH stability, stability against proteases (particularly pepsin) and/or specific activity. These replacements are shown in Table 4.
[091] In a still further aspect, the invention relates to a nucleic acid molecule and use of a nucleic acid molecule selected from the group consisting of (a) a nucleic acid molecule encoded a modified mannanase according to the above description, (b) a nucleic acid molecule encoded by the modified mannanase derivative according to the above description, wherein the derivative one or more amino acid residues are conservatively substituted; (c) the nucleic acid molecule shown as SEQ ID NO:5; (d) a nucleic acid molecule which is a variant, homolog, derivative or fragment of the nucleic acid molecule shown as SEQ ID NO:5; (e) a nucleic acid molecule that is the complement of the nucleic acid molecule shown in SEQ ID NO:5; (f) a nucleic acid molecule that is the complement of a variant, homologue, derivative or fragment of the nucleic acid molecule shown as SEQ ID NO:5; (g) a nucleic acid molecule that is capable of hybridizing to the nucleic acid molecule shown in SEQ ID NO: 5; (h) a nucleic acid molecule that is capable of hybridizing to a variant, homologue, derivative or fragment of the nucleic acid molecule shown as SEQ ID NO:5; (i) a nucleic acid molecule that is the complement of a nucleic acid molecule that is capable of hybridizing to the nucleic acid molecule shown in SEQ ID NO:5; (j) a nucleic acid molecule that is the complement of a nucleic acid molecule that is capable of hybridizing to a variant, homologue, derivative or fragment of the nucleic acid molecule shown as SEQ ID NO:5; (k) a nucleic acid molecule that is capable of hybridizing the complement of the nucleic acid molecule shown in SEQ ID NO: 5; (l) a nucleic acid molecule which is capable of hybridizing the complement of the variant, homologue, derivative or fragment of the nucleic acid molecule shown as SEQ ID NO: 5. (m) a nucleic acid molecule having a sequence identity of at least 95% with any of the nucleic acid molecules of a) - l) and encoded by mannanase, (n) a nucleic acid molecule having a sequence identity of at least 70% with any of the nucleic acid molecules of a) - b) and encoded by mannanase, and/or (o) a fraction or a complement of any of the nucleic acid molecules of a) - n).
[092] A nucleotide or nucleic acid is considered to hybridize to one or more of the above nucleotides if it is capable of hybridizing under conditions of medium stringency, more preferably under high stringency, even more preferably under conditions of very high stringency.
[093] To prepare a hybridization blot, standard molecular biology protocols for blotting can be used (eg, Southern blotting for DNA hybridizations). The amount of target DNA depends on the relative abundance of the target sequence. If a pure target sequence is to be used, between 1 and 5 picograms of DNA per kilobase of polynucleotides are preferred. Typically, the detection limit is about 0.5 pg DNA for a radioactive probe with the specific activity of 109 dpm/mg which is equivalent to a single copy gene 500 bp in length in 3.3 mg of complex genomic DNA of the genome (eg, human). In practice one will approximate 10 mg of genomic DNA - for example, to screen organisms, such as micro-organisms, that contain a mannanase encoding polynucleotide of the invention. If the target DNA is bacterial or a plasmid it will have to dilute the DNA accordingly to avoid overexposure. The target DNA is blotted, for example, by spot blotting, or by means of gel blotting from electrophoresis. Preferred conditions are described in Membrane Transfer and Detection Methods, Amersham International plc, UK.- PI/162/85/1) Hybond N+ positively charged nylon membrane is preferably used (Amersham Life Science). The probe is preferably prepared in accordance with Pharmacia's 'Ready to Go DNATM labeling kit' to prepare a probe of > 1 x 109 dpm/micrograms. The probe is used in Hybridization Buffer at a concentration of 1 x106 dpm milliliter of Hybridization Buffer. Blots are preferably prehybridized in hybridization buffer (6 x SSC, 5 x Reinhardt's solution and 0.5% SDS and denatured salmon sperm DNA to 100 mg/ml buffer) for one hour at 65°C, followed by hybridization in hybridization buffer containing the denatured labeled probe with 12 hour shaking at 65°C. The blots are then washed with an appropriate volume wash buffer (typically 50 ml) in 2 x SSC, 0.1% SDS for 30 minutes at 65°C, followed by a second wash in a suitable volume wash buffer (typically 50 ml) in the same wash buffer (2 x SSC, 0.1% SDS) for the medium stringency wash, or 0.1% x SSC, 0.1% SDS for 10 minutes at 65°C (high stringency), the second wash can be repeated at 70°C by the very high stringency wash.
[094] The nucleic acid molecule of the present invention may comprise the nucleotide sequences encoding SEQ ID NO: 1 or an effective fragment thereof or a variant, modified form, homologue or derivative thereof.
[095] In particular, the invention provides a plasmid or vector system which comprises a nucleic acid sequence encoding mannanase as described herein or a homologue or derivative thereof. Preferably, the plasmid or vector system comprises the nucleic acid sequence encoded by the amino acid SEQ ID NO: 4 or the sequence which is at least 75% homologous thereof or an effective fragment thereof, or any of the derivatives of SEQ ID NO: 1 described in this. Suitably the plasmid or vector system is an expression vector for the expression of any of the enzymes encoded by the nucleic acid sequence shown in any one of SEQ ID NO: 4 or the sequence which is at least 75% homologous (identical) therein in a microorganism. Suitable expression vectors are described here. Furthermore, the invention provides a plasmid or vector system for the expression of any of the modified enzymes or variants or functional fragment described herein. Suitable expression vectors are described here.
[096] Improvements in mannanase characteristics in accordance with the present invention are intended for use in a variety of technical processes such as, but not limited to, use as an additive for food and feed products, for food and feed processing, pulp and paper production, as well as for oil/gas stimulation by hydraulic fracturing, generation of slow release formulations in drugs or in detergents, in particular in the removal of bacterial biofilms. In particular, the improvements are directed to enzyme stability under conditions of these or other applications and/or stability during stomach transit in case of an additive for food or feed and/or activity or stability in human or animal stomach and/ or intestinal under the acidic conditions of the upper gastrointestinal tract. Such improvements comprise, among other parameters, increased stability at elevated temperatures, preferably at temperatures above 60°C and/or increased stability against proteolytic digestion, preferably against digestive tract proteases and/or increased stability at low pH and/or the activity at low pH values and/or the overall efficiency of releasing mannose and/or oligomannoses from broad polymannose containing the carbohydrates.
[097] The increase in stability at elevated temperatures is quantified by the inactivation temperature of the enzyme. The inactivation temperature is defined as the temperature at which the residual activity of the mannanase enzyme after incubation for a certain duration and subsequent cooling to room temperature is 50% of the residual activity of the same mannanase enzyme incubated for the same duration under the same conditions under room temperature.
[098] Thermostability differences are the differences in °C between the inactivation temperatures of two enzymes. In the preferred embodiment of the invention the mannanase variants are applied in processes at elevated temperatures, making the mannanase variants with a desirable higher inactivation temperature.
[099] When compared to wild-type mannanase, mannanases of the invention are characterized by greater residual activity after a thermal incubation at temperatures above the wild-type mannanase inactivation temperature, providing greater process stability.
[0100] Cloning of T. reesei mannanase: In addition Trichoderma reesei mannanase as shown in SEQ ID NO: 1 an additional Trichoderma reesei mannanase was cloned having a sequence of SEQ ID NO: 1 with the substitution of Serine to Arginine in position 3 (S3R mutation). This mannanase variant was compared to a Trichoderma reesei mannanase according to SEQ ID NO: 1 with respect to thermal stability and catalytic activity in releasing mannose from a substrate containing polymannose. The results shown in Figure 10 demonstrate that the S3R substitution has no effect on properties relevant to the invention and is therefore a neutral mutation (see also WO 2008/009673). Therefore, in the context of this invention the term "by weight" or "mannanase by weight" "wild type mannanase" or "Trichoderma reesei mannanase" is understood to comprise the mannanases according to SEQ ID NO: 1 and the mannanase according to with SEQ ID NO: 1 further having the neutral mutation S3R.
[0101] Thermostability in buffer: In the preferred embodiment of the invention, mannanase variants have an increased residual activity and/or inactivation temperature when incubated at temperatures >60°C for >30 minutes. In a most preferred embodiment the increased residual activity and/or inactivation temperature is obtained after incubation in an acetate buffer for 45 minutes. Preferably, the inactivation temperature of the mannanase variant is >68°C, more preferably >70°C or >72°C or >74°C, or more preferably >84°C. The specific inactivation temperature was given in Table 3 in conjunction with their respective mutations.
[0102] Fusion proteins: It is also understood that the amino acid sequence disclosed in SEQ ID NO: 1 and derivatives thereof described therein for use in accordance with the present invention can be produced as an N- and/or-terminal fusion protein or C, for example, to aid extraction, detection and/or purification and/or to add functional properties to the mannanase molecule. The fusion protein partner can be any protein or peptide including any polypeptide sequence derived from the natural host, any other naturally occurring amino acid sequence as well as synthetic sequences. Examples of fusion protein partners include, but are not limited to, glutathione-S-transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains), FLAG-, MYC tags or other well known tags in any person skilled in the art. It should also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of the fusion protein sequences. Preferably, the fusion protein will not impede the activity of the protein sequence of interest.
[0103] In the preferred embodiment of the invention the mannanase variants are fused to functional domains including leader peptides, propeptides, binding domains or catalytic domains.
[0104] Binding domains can include, but are not limited to, carbohydrate binding domains of various specificities, providing increased affinity to carbohydrate components present during mannanase application. It is also envisioned that the fusion partner domain may comprise enzymatically active domains, such as activities that support the action of mannanase in production of the desired product providing activity on one or more substrate components and/or any product of a catalytic reaction of mannanase. Non-limiting examples of catalytic domains include: cellulases, hemicellulases such as xylanase, exo-mannanases, glucanases, arabinases, galactosidases, pectinases, and/or other activities such as proteases, lipases, acid phosphatases and/or others or functional fragments thereof.
[0105] Linkers: Fusion proteins may optionally be linked to mannanase via a linker sequence comprising preferably less than 100 amino acids, more preferably less than 50 amino acids, less than 30 amino acids or less than 20 amino acids. The linker may simply join the mannanase and the fusion domain without significantly affecting the properties of the component, or it may optionally have functional importance for the intended application due to its amino acid composition, structure and/or post-translational modification occurring during expression in the natural host or any suitable heterologous host. The source of the linker sequence can be an amino acid sequence from any organism or any synthetic peptide sequence.
[0106] Additional proteins: the mannanases described herein for use in accordance with the present description can also be used in conjunction with one or more additional proteins of interest (POIs) or nucleotide sequences of interest (NOIs). Non-limiting examples of POIs include: phytases, hemicellulases, alpha-galactosidases, beta-galactosidases, lactases, beta-glucanases, endo-beta-1,4-glucanases, cellulases, xylosidases, xylanases, xyloglucanases, xylan acetyl esterases, galactanases, exomannanases, pectinases, pectin lyases, pectinesterases, polygalacturonases, arabinases, rhamnogalacturonases, laccases, reductases, oxidases, phenoloxidases, ligninases, proteases, amylases, phosphatases, lipolytic enzymes, and/or other cutinases. These enzymes include that, for example, modulate the viscosity of the substrate/suspension solution or increase the accessibility and/or solubility of the polymannose substrate. The NOI can still be an antisense sequence for any of those sequences. As described above, POI can still be a fusion protein. The POI can still be fused into a secretion sequence. In an advantageous embodiment, the mannanase variant according to the present invention is used in conjunction with at least the phytases.
[0107] Other sequences can also facilitate the secretion or increase the yield of secreted POI. Such sequences should encode such sequences should code for chaperone proteins such as, for example, the Aspergillus niger cyp B gene product described in UK Patent Application 9821198.0.
[0108] The POI encoded NOI may be designed to alter its activity for a number of reasons, including, but not limited to, alterations that modify the processing and/or expression of the expression product thereof. By way of additional example, the NOI can also be modified to optimize expression in a particular host cell. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites.
[0109] The NOI encoded by the POI can include within synthetic or modified nucleotides- such as methylphosphonate and phosphorothioate backbones.
[0110] The NOI encoded by the POI can be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of the sequences flanking the 5' and/or 3' end ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiester bonds within the structure. of the molecule.
[0111] Expression of mannanase genes: In order to produce the mannanase enzyme, the DNA encoding the enzyme can be chemically synthesized from published sequences or directly obtained from host cells harboring the gene (for example, by screening cDNA library or PCR amplification). The mannanase gene can be included in an expression cassette and/or cloned into a suitable expression vector by standard molecular cloning techniques. Such expression cassettes or vectors often contain the sequences that aid in the initiation and termination of transcription (for example, promoters and terminators) and may contain the selectable markers. Cassettes may also comprise more or less of the mRNA strand and their expression may or may not include an amplification step prior to mRNA production. The mannanase gene to be expressed may or may not contain certain protein domains, such as binding polymer domains (e.g., carbohydrate binding domains) of various specificities. The expression cassette or vector can be introduced into a suitable expression host cell which will then express the corresponding mannanase gene. Particularly acceptable expression hosts are the bacterial expression host genus including Escherichia (for example Escherichia coli), Pseudomonas (for example P. fluorescens or P. stutzerei), Proteus (for example Proteus mirabilis), Ralstonia (for example , Ralstonia eutropha), Streptomyces, Staphylococcus (for example S. carnosus), Lactococcus (for example L. lactis), lactic acid bacteria or Bacillus (subtilis, megaterium, licheniformis, etc.). Also particularly suitable are yeast expression hosts such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Hansenula polymorpha, Kluyveromyces lactis or Pichia pastoris. Especially suitable are fungal expression hosts such as Aspergillus niger, Chrysosporium lucknowense, Aspergillus (for example A. oryzae, A. niger, A. nidulans, etc.) or Trichoderma reesei. Also suitable are mammalian expression hosts such as mouse (e.g., NS0), Chinese hamster ovary (CHO) or baby hamster kidney (BHK) cell lines, transgenic mammalian systems such as rabbit, goat or cattle, other eukaryotic hosts, such as insect cells or viral expression systems such as M13-like bacteriophages, T7 or Lambda phage, or viruses such as Baculovirus or plant expression systems.
[0112] Mannanase genes are introduced into expression host cells by a number of transformation methods including, but not limited to, electroporation, lipid helper transformation or transfection ("lipofection"), chemically mediated transfection (eg, CaCl and/or CaP), lithium acetate mediated transformation (eg of host cell protoplasts), biolistic "gene gun" transformation, PEG mediated transformation (eg of host cell protoplasts), protoplast fusion (for example, using bacterial or eukaryotic protoplasts), liposome-mediated transformation, Agrobacterium tumefaciens, adenovirus or other phage or viral transformation or translation.
[0113] Alternatively, enzyme variants are expressed intracellularly. Optionally, after intracellular expression of the enzyme variants, or secretion into the periplasmic space using signal sequences such as those mentioned above, the permeabilization or lysis step can be used to release the mannanase enzyme into the supernatant. Disruption of the membrane barrier can be effected by the use of mechanical means such as ultrasonic waves, pressure treatment (French pressure), cavitation or use of membrane digestive enzymes such as lysozyme or enzyme mixtures. As an additional alternative, the genes encoding the mannanase enzyme are expressed cell-free by use of an appropriate cell-free expression system. For example, S30 extract from Escherichia coli cells was used for this purpose or commercially available systems (eg, CECF technology by Roche Applied Science, Inc.). In cell-free systems, the gene of interest was typically transcribed with the assistance of a promoter, but ligation to form a circular expression vector is optional. RNA can also be exogenously added or generated without transcription and translated in cell-free systems. The configurations of the expression constructs for in vitro expression and running all of the above expression systems are well within the ability of the skilled artisan.
[0114] The above methods of cloning and expression of the Trichoderma reesei mannanase gene are suitable both for industrial scale expression and for use in high-end evaluations for the evaluation of mutated variants.
[0115] Purification: As described above, mannanase proteins can be expressed in a variety of expression systems and consequently appropriate downstream processing and purification procedures will be selected. The protein of interest can be secreted in the extracellular or periplasmic space or expressed intracellularly. In an advantageous embodiment of the invention the mannanase variant is expressed in a microbial host and the protein is secreted in the periplasmic or extracellular space. Cells expressing the mannanase variants are preserved by methods well known to anyone skilled in the art, such as but not limited to cryo stocks. Expression organism cultures are prepared in an appropriate volume with standard fermentation methods. In the preferred embodiment, cultures for protein expression are inoculated from the cryo stock and the culture volume successively increased in the appropriate vessels. In the preferred embodiment the cells are grown in a fermentor and optionally growing conditions such as pH, temperature, oxygen and/or nutrient supply are controlled. A first purification step comprises separating the cells from the supernatant using one or more of several techniques, such as sedimentation, microfiltration, centrifugation, flocculation or the like. In the preferred embodiment the method applied is microfiltration. In the case of intracellular expression, cells are subjected to treatments that result in a release of the protein from the intracellular space. These treatments may comprise for example pressure, enzymatic, osmotic shock, freezing, ultrasonic or other treatment to produce a cell extract which may or may not be subjected to further purification.
[0116] In an advantageous embodiment of the invention the protein is secreted into the supernatant in an optional purification step comprising concentrating the supernatant by ultrafiltration. Purification of additional protein from the supernatant or concentrated supernatant can be carried out with one or more of several methods comprising extraction or fractionation methods such as ammonium sulfate or ethanol or acid precipitation, or chromatographic methods including but not limited to ion exchange, hydrophobic interaction, hydroxylapatite, size fractionation by gel filtration, phosphocellulose or lectin chromatography, and affinity chromatography or any combination thereof. In a more preferred method the affinity-labeled protein is purified by metal-chelate affinity chromatography to obtain a protein of high purity.
[0117] The preferred purification method produces a protein purity of >30%, in a more preferred method the purity is >50%, >60%, >70%, or >80%. Still in a more preferred method the purity is >90%, in a more preferred method the purity is >95% and in a more preferred method the purity is >98%.
[0118] In another advantageous embodiment of the invention the supernatant or the supernatant partially purified by ultrafiltration or the concentrated and/or diafiltered supernatant is dried by any of several technical methods such as, but not limited to, spray drying, lyophilization , evaporative drying, thin layer evaporation, centrifugal evaporation, conveyor drying or any combination thereof.
[0119] Yet in an advantageous embodiment of the invention the fermented cell suspension including the expressed variants of mannanase is dried as a whole using processes such as, but not limited to, fluid bed drying, carrier drying, spray drying or drum drying or any combination thereof.
[0120] Formulations: In general, mannanase compositions or any derivative described herein may be liquid or dry. Liquid compositions can comprise mannanase alone or in combination with other proteins or enzymes and can contain additives that support the stability and/or activity of mannanase or other proteins or enzymes in the composition. This includes, but is not limited to, glycerol, sorbitol, propylene glycol, salts, sugars, preservatives, pH buffers and carbohydrates. Typically, the liquid composition is an aqueous or oily based paste, suspension or solution.
[0121] Dry compositions can be generated from the liquid composition including the fermentation supernatant or cell suspension or cell extract by spray drying, lyophilization, evaporation drying, thin layer evaporation, centrifugal evaporation, carrier drying or any combination thereof . The dry compositions can be granulated to the appropriate size to be compatible with additional downstream applications such as food or feed processing or to quantify as a component for food or animal feed.
[0122] Before drying a bulking agent can be added to the liquid composition which, after drying, effectively enhances the properties of the dry composition such as providing high heat stability due to the enzyme's protection from environmental factors by the reagent. volume, more technical handling properties and others.
[0123] Once a dry preparation is obtained, the agglomeration granules can be prepared using agglomeration techniques, for example, in a shear mixer during which a filler material and the enzyme are co-agglomerated to form the granules . Absorption granules are prepared by having cores of a carrier material to absorb or coat with the enzyme. Typical filler materials include sodium sulfate, kaolin, talc, magnesium aluminum silicate and cellulose fibers. Optionally, binders such as dextrins are also included in the pellet agglomeration. Typical carrier materials include starch, for example in the form of cassava, corn, potato, rice and wheat, or salts may be used.
[0124] Optionally the granules are coated with a coating mixture. Such a mixture comprises coating agents, preferably hydrophobic coating agents, such as hydrogenated palm oil and beef tallow and, if desired, other additives such as calcium carbonate and kaolin.
[0125] In a particularly preferred embodiment the compositions comprising the mannanases of the invention are intended for applications in food and feed processing or as a supplement to food and feed. In this case, the mannanase compositions may additionally contain other substituents such as coloring agent, flavor compounds, stabilizers, vitamins, minerals, other food or feed enhancing enzymes and others. This applies in particular to so-called premixes. Food additives in accordance with the present invention can be combined with other food components to produce processed food products. Other such food components include one or more others, preferably thermostable, enzyme supplements, vitamin food additives and mineral food additives. The resulting combined food additives, possibly including several different types of compounds, can then be mixed in an appropriate amount with the other food components such as cereal or vegetable proteins to form a processed food product. Processing these components into a food product can be accomplished using any of the methods currently available.
[0126] In an advantageous embodiment of the invention the mannanase composition further comprises an effective amount of one or more food or feed enhancing enzymes, in particular selected from the group consisting of, but not limited to, phytases, hemicellulases, alpha-galactosidases, beta-galactosidases, lactases, beta-glucanases, endo-beta-1,4-glucanases, cellulases, xylosidases, xylanases, xyloglucanases, xylan acetyl esterases, galactanases, exo-mannanases, pectinases, pectinases, pectinesterases, polygalacturonases, arabinases, rhamnogalacturonases, laccases, reductases, oxidases, phenoloxidases, ligninases, proteases, amylases, phosphatases, lipolytic enzymes, cutinases and/or others.
[0127] The mannanase formulations of the invention are used for processing and/or manufacturing food or animal feed.
[0128] Technical applications: covered is the use of mannanase derivatives as food additives or digestive aids that promote the degradation of oligomannose containing the food material, thereby releasing potentially beneficial oligomannoses or their derivatives.
[0129] Another application in the field of food and feed processing is the production of mannooligosaccharides as important prebiotics from PKE for food or feed. By treating PKE or other components containing galactomannan with mannooligosaccharides mannanase and D-mannose are produced. Mannooligosaccharides are used as prebiotic components for food and feed: Mannooligosaccharides promote the development of probiotics (eg Bifidobacterium and Lactobacillus sp), inhibit the development of Salmonella enterobacteria, neutralize the anti-nutritional properties of lectins and observe the applications in the pharmaceutical industry. Furthermore, mannooligosaccharides and especially mannose are suspected of being immune to the stimulating components in the food material.
[0130] Another application in the field of food and feed processing is the cleavage of mannan containing the components in the cell wall of fruits for the improvement of recovery, for example, by the addition of said enzymes to pineapples, lemons, oranges, lemon- Galician, grapefruits, before the compression procedure. An advantageous application is the use of the mannanase variants according to the present invention in baking processes, for example for biscuits, breads etc.
[0131] Another application in the field of food and feed processing is the use of mannanase according to the present invention to produce the improvement in palm seed oil extraction. The oily content remains in the palm seed extruder which is between 5-12% after pressure. This remnant can still be reduced by chemical extraction to about 3%. For application of the mannanase according to the invention, the release actually must be rapidly increased, thus providing an improved process. Additionally the resultant palm kernel blaster would be of higher quality due to the reduced fiber content of galactomannan which are known to be anti-nutritive components in the feed.
[0132] Yet another application in the field of food and feed processing is the release of D-mannose from PKE or other components containing galactomannan. Palm seed meal contains about 20% mannose and binds with the fibers of galactomannan. Treatment of PKE, copra or other galactomannan containing crude substances with mannanases causes the release of D-mannose. Mannose and its derivatives are ingredients used in food (eg, low-calorie dietary food product), pharmaceuticals (mannose cures more than 90% of all urinary tract infections), cosmetics, textiles and in polymer manufacturing. Because of limited supply mannose is very expensive to present compared to other more common hexose sugars and its supply is rare. D-Mannose can also be used as a raw material for the production of mannitol. Mannitol itself is derived from mannose through reduction with much higher yield and fewer by-products than from fructose conversion. Mannitol is a polyol widely used in the food and pharmaceutical industries because of these unique functional properties: mannitol is used as a sweetener, for pharmaceutical formulations (chewable tablets and granulated powders), in the production of chewable gum, as an embodying agent, texturing and anti-solidification for food, as osmoactive pharmaceutical and diabetic food component.
[0133] Furthermore, in the above context of food and feed processing the use of mannanase according to the invention for the partial hydrolysis of galactomannans by incubation of guar gum or carob bean gum is provided. The resulting hydrolysates are used in the food and brewery industry as texturing components for pharmaceutical applications.
[0134] Production of sugars: The mannanase enzymes described in the present invention are in particular useful for the production of sugars or oligosaccharides from polymannose containing plant material such as palm seed, coconut, konjac, carob bean gum, guar gum and soy. Preferred is plant material similar to palm seed meal, palm seed blaster, copra flour, copra granules and soy husks.
[0135] In a particular preferred embodiment the mannanase enzymes according to the present invention are applied for the production of mannose and mannopolymers such as mannobiose, mannotriose, mannotetraose, mannopentaose, mannoexaose, mannoeptaose, mannooctaose, mannonose and larger polymers of mannose and/or derivatives thereof. Also preferred are the galactosyl mannooligosaccharides thereof with the different ratios between galactose and mannose ranging from 1 to 0.05.
[0136] Yet in a preferred embodiment of the present invention sugars are composed of mannose and glucose and are referred to as glucomannans. These polyols should be composed of 2, 3, 4, 5, 6, 7, 8, 9 or more mannose and/or glucose monomers with a mannose content of 1/15, 1/14, 1/13, 1/ 12, 1/11, 1/10, 1/9, 1/8, 1/7, 1/6, 1/5, 1/4, 1/3, 1/2, 1, 2/3, 3/ 4, 3/5, 4/5, 5/6, 2/7, 3/7, 4/7, 5/7, 6/7, 3/8, 5/8, 7/8, 2/9, 4/9, 3/10, 2/11, 4/11, 3/12, 2/13 or 1/14. Also particularly preferred are the galactosyl glucomannooligosaccharides thereof with the different ratios between galactose and mannose ranging from 1 to 0.05.
[0137] Still in a preferred embodiment of the present invention mannanase is used in combination with other glucanase-like carbohydrases, and/or xylanase, and/or alpha-galactosidase and/or cellulase for the hydrolysis of plant material in order to generate the sugars.
[0138] In a most preferred embodiment of the present invention the hydrolysis of polymannose containing the plant material leads to sugars having a prebiotic functionality. These sugars are generated to promote the development of probiotics, bacteria that are known to support a healthy immune system. Examples of such bacteria are bifidobacteria. The known bifidoabteria are B. adolescensis, B. angulatum, B. animalis, B. asteroides, B. bifidum, B. bifidum, B. boum, B. breve, B. catenulatum, B. choerinum, B. coryneforme, B. cuniculi, B. dentium, B. gallicum, B. gallinarum, B. indicum, B. infantile, B. longum, B. magnum, B. merycicum, B. minimum, B. pseudocatenulatum, B. pseudolongum, B. pullorum, B. ruminantium, B. saeculare, B. scardovii, B. subtile, B. thermacidophilum and B. thermophilum.
[0139] Coffee extraction: The mannanase enzymes described according to the present invention are useful for the hydrolysis of galactomannan that is present in liquid coffee extracts. In the preferred embodiment of the invention mannanase is used to inhibit the formation of gels as these occur during freeze drying of liquid coffee extracts. The reduced viscosity of the extract reduces energy consumption during drying. In yet a more preferred embodiment of the invention the mannanase enzymes are applied in an immobilized form which reduces enzyme consumption and prevents contamination of the coffee extract.
[0140] In this context, another application of interest is the use of mannanase enzymes according to the present invention for the production of mannose or manno-oligosaccharides from the coffee residue, in order to receive the products of high values. As described before, mannanase releases mannose or oligosaccharides from the coffee residue which are high value functional foods and food components. In the coffee beverage industry, spent coffee grounds are generally used as fuel or treated as an industrial waste). Roasted coffee contains 1.8 to 4.4% mannan. Therefore, spent coffee grinders contain ample amount of β-mannan, which can be converted to mannooligosaccharides by enzymatic hydrolysis. Mannooligosaccharides obtained from coffee mannan are said to reduce serum lipid levels in humans (Jpn J food eng 6 (2005)).
[0141] Animal feed: Several anti-nutritional factors limit the use of specific plant material in the preparation of animal feed and food for humans. Plant material containing mannan-like oligomannans, galactomannan, glucomannan and galactoglucomannan is described to reduce the digestibility and absorption of mineral-like nutritional compounds, vitamins, sugars and fats by animals. The negative effects are in particular due to the high viscosity of manopolymers and the ability of manopolymers to absorb nutritional compounds. These effects can be eliminated/reduced through the use of mannopolymer degrading enzymes, termed mannanase enzymes which then left a much higher proportion of mannopolymer containing cheap plant material in the feed and therefore a cost reduction. Additionally, through the activity of the mannanase mannopolymers enzymes are broken down to monosaccharides which can be readily assimilated and provide additional energy.
[0142] In order to use an enzyme as an effective feed supplement eg monogastric animals similar to poultry or pig this was stable in the stomach. This means being stable at low pH (approximately pH 2-3) and additionally it was resistant against pepsin at this low pH. Furthermore such enzymes need to be active at low pH (approximately pH 3.0) to be effective in the stomach. The mannanase enzymes provided in the present invention fulfill all these criteria unlike other mannanase enzymes like for example the wild type mannanase from Trichoderma reesei which is not stable at low pH, in particular not stable against pepsin at low pH. Therefore the mannanase enzymes provided in the present invention are especially well suited for feed applications where the enzyme is active in the animal.
[0143] The mannanase enzymes according to the present invention are useful as feed additives for monogastric animals such as poultry and pig, as well as for human food. The feed, however, can also be supplied to ducks, goose, as well as bovine, canine, goat, feline equine, as well as crustaceans and fish. Mannanase enzymes can also be used to pre-treat the feed instead of adding it to the feed.
[0144] In an advantageous embodiment of the invention mannanase enzymes are added to the feed for weaning pigs, pig nursery, piglets, fattening pigs, growing pigs, killing pigs, laying hens, grilling chickens, turkey.
[0145] Still in an advantageous embodiment of the invention mannanase enzymes are additives to the ration of compounds of plant material similar to palm seed, coconut, konjac, locust bean gum, guar gum, soy, barley, oats , flax, wheat, corn, linseed, citrus pulp, cottonseed, crushed walnut, rapeseed, sunflower, peas, lupine and vitamins as well as minerals. In a still more preferred embodiment of the invention the mannanase enzymes are additives to the feed partially composed of palm seed flour, palm seed sludge, copra flour, copra granules and/or soy husks.
[0146] Yet in an advantageous embodiment of the invention mannanase enzymes are used in combination with other enzymes selected from the group consisting of, but not limited to, phytases, alpha-galactosidases, beta-galactosidases, pectinases, xylanases , arabinoxylanases, proteases, beta-glucanases, cellulases, galactanases, endoglucanases, xylosidases, cutinases, lipases and/or phospholipases for the preparation of feed. Mannanase enzymes with or without additional enzymes can also be used in combination with minerals, vitamins and other typical feed supplements.
[0147] Since the mannanase enzymes according to the present invention are thermostable enzymes these can be subjected to heat without losing significant activity. Therefore mannanase enzymes can be used in pelleted feed production processes where heat is applied to the feed mixture prior to the granulation step, as is the case in more commercial pellet mills. The mannanase enzyme can be added to other feed ingredients in advance of the granulation step or after the granulation step to already formed feed granules.
[0148] Yet in a preferred embodiment of the present invention mannanase enzymes are used in animal feed that is specially fed to animals under circumstances where no antibiotic is desired.
[0149] In an advantageous embodiment the mannanase enzymes are used in animal feed partially composed of palm seed meal, palm seed expellers, copra flour, copra granules and/or soy husks. In a more preferred embodiment mannanase enzymes are used in animal feed for grilling chickens which is partially composed of palm seed meal, palm seed blasters, copra meal, copra granules and/or soy husks.
[0150] Paper pulp industry: The mannanase enzymes according to the present invention are useful in enzyme-aided bleaching of paper pulps similar to chemical pulps, semi-chemical pulp, brown paper pulps, mechanical pulps or prepared pulps by the sulfide method. The pulps must also be totally chlorine-free pulps bleached with oxygen, ozone, peroxide or peroxyacids.
[0151] In an advantageous embodiment of the present invention mannanase enzymes are used by the enzyme-aided whitening of pulps produced by modified or continuous pulp methods that exhibit a low lignin content.
[0152] Still in an advantageous embodiment of the present invention the mannanase enzymes in such applications can be applied alone or preferably in combination with xylanase and/or endoglucanase and/or alpha-galactosidase and/or cellobiohydrolase enzymes.
[0153] The bleaching and/or desizing agent in the textile industry: The mannanase enzymes according to the present invention are also useful for the bleaching of cellulosic fibers of no other than cotton, wool yarn or fabric comprising linen, jute , ramie or linen by incubating the fiber, yarn or fabric with the mannanase in accordance with the present invention for a given period of time and under conditions suitable to produce a bleaching of the fiber, yarn or fabric. Degradation of hemicellulose improves the fabric bleaching process.
[0154] In textile printing using a paste containing a pigment and a biological polymer (eg guar gum) as a thickener, removal of the thickener and excess pigment is made much more efficient by washing the printed fabric in the presence of mannanase. The enzymatic breakdown of the thickener reduces the process time as well as the amount of energy and water needed to achieve satisfactory tissue quality.
[0155] The mannanase enzymes according to the present invention are useful in desizing fabrics made of for example synthetic fibers where often galactomannans similar to guar gum or carob bean gum are used as sizing agents.
[0156] Gas and oil stimulation by hydraulic fracturing: The mannanase enzymes according to the present invention are useful in a hydraulic fracturing method used in gas or oil stimulation. Whereas mannanase enzymes act as liquefying agents in a hydraulic fluid that is based on or composed of a mannopolymer and usually contains sand.
[0157] The mannanase enzymes according to the present invention are thermostable enzymes these are preferably used in hydraulic fracturing applications that are carried out at high temperatures.
[0158] In another advantageous embodiment of the invention the liquefaction activity of mannanase enzymes in a hydraulic fracture application is controlled (inhibited or promoted) by environmental conditions similar to pH and temperature.
[0159] Detergents: The mannanase enzymes according to the present invention can be used in detergent compositions in order to facilitate the removal of mannopolymer containing stains / soils. In the preferred embodiment of the present invention mannanase enzymes are used in detergent compositions in combination with other enzymes from the group of amylases, cellulases, lipases, pectinases, protease and endoglucanases.
[0160] Removal of biofilms: The mannanase enzymes described in the present invention are useful for the removal of mannopolymer containing biofilms. Preferably, for such an application the mannanase enzymes are used in combination with detergents and/or other enzymes from the group of alpha-galactosidases, pectinases, xylanases, arabinoxylanases, proteases, beta-glucanases, cellulases, galactanases, endoglucanases, xylosidases, cutinases and lipases.
[0161] Release systems: The mannanase enzymes according to the present invention can be used for the time-dependent and/or targeted release of matter. This is achieved through the use of systems that are based on the mannopolymer gels that contain and transport matter. The function of the mannanase enzyme in such a system is the controlled release of matter by partial or complete degradation of the gel, due to specific change in the gel's environment, for example, the pH and/or temperature which activates the mannanase enzymes.
[0162] In an advantageous embodiment of the present invention mannanase enzymes are used for the targeted release of a drug in a pharmaceutical application.
[0163] Renewable resources, ie biomass substrates that are developed and collected, similar crops, straw, wood and wood products, are received more and more attention as these are the suitable substrates for the production of biofuels, ie solid, liquid, or fuel gas similar to Biodiesel, Biogas, Vegetable Oil, Bioethanol, Biobutanol, BioHydrogen, Bio-Dimethyl Ether, Biomethanol, BTL ("Liquid Biomass")-fuel, GTL ("Liquid Gas")-fuel and others. In the 1st generation of biofuels, said plants were converted using methods established from the food industry, i.e. they were compressed in order to obtain vegetable oil or amide containing grain, was converted to sugar and subsequently fermented with yeast in order to get Bioethanol. This means that the energy reservoirs (ie fat and/or starch) of said plants were exclusively used. This leads to low energy yields, or low biofuel production amounts per acre. In the 2nd generation biological fuels, not only the energy reservoirs of said plants are being used, but the method tends to use the complete biomass of the plant.
[0164] In this context, the mannanase according to the invention can be used to convert plant biomass containing hemicellulose into sugars, which can be metabolized by specific yeast (for example, Saccharomyces sp.) or bacterial strains and other microorganisms in order to produce the fermentation products. These fermentation products can be fuels similar to Bioethanol, Biobutanol but can also be similar building block molecules to 3-Hydroxy propionic acid, aspartic acid, xylitol and gluconic acid. For more building block molecules that can be derived from sugars see (Werpy and Petersen (2004) Top Value Added Chemicals from Biomass: Volume 1-Results of Screening for Potential Candidates from Sugars and Synthesis Gas. National Renewable Energy Laboratory Report NREL/TP-510-35523, Figure 3 and Table 8).
[0165] Other potential uses comprise the catalytic processing of the products that was obtained from the renewable resources that help from mannanases according to the invention. This comprises the processing of glucose and/or fructose, both obtained with the help of a mannanase according to the invention, into 2,5-dimethylfuran, a heterocyclic compound which is supposed to have many fuel properties than bioethanol, as it has a higher density. 40% higher energy, it is chemically stable and insoluble in water.
[0166] Said methods realize great benefits from the improved properties of mannanases according to the invention, particularly from the enhanced heat stability. This means that the respective biomass to sugar conversion can take place under high temperature conditions, which speed up the respective processes and thus render these economically more efficient.
[0167] In the inventors' recent experiments, palm seed expeller substrates (PKE) were used to feed the yeast (Saccharomyces cerevisiae). Said substrates contain about 37% of galactomannan. It is provided that PKE treated with two mannanases according to the invention (ie variant B, variant C and/or variant 31) leads to the release of a large amount of sugars and thus resulted in an improved yeast development in comparison to untreated PKE. This is a clear suggestion towards the above postulation, ie that the mannanases according to the invention can be useful tools to intensify the yield in, for example, Bioethanol production from renewable resources (see example 19 in WO 2008/009673) .
[0168] All said uses of mannanase according to the invention have in common that these methods realize substantial benefit from the improved properties of mannanase according to the invention, particularly in terms of enhanced thermostability and enhanced resistance against low pH values and protease enzymes.
[0169] This is mainly due to the fact that most of said uses take place in environments with unfavorable conditions, similar to mammalian digestive tracts, where the prevailing low pH values, or at high temperatures that are applied accelerate, facilitate and economically optimize hydrolysis processes similar to converting renewable resources to sugars as described above.
[0170] The following methods and examples are offered for illustrative purposes and are not intended to limit the scope of the present invention in any way. METHODS AND EXAMPLES
[0171] In the following examples, materials and methods of the present invention are provided including the determination of catalytic properties of enzymes obtainable by the method. It is to be understood that these examples are for illustrative purposes only and are not to be construed as limiting this invention in any way. All publications, Patents and Patent Applications cited herein are, therefore, incorporated by reference in their entirety for all purposes. EXAMPLE 1: PURIFICATION OF MANANASE ENZYMES THROUGH HISTAG
[0172] Purification of mannanase enzymes without carbohydrate binding domain (CBD) was performed using a C-terminal 6xHisTag fused to the mannanase enzymes.
[0173] S. saccharomyces, transformed with a plasmid encoded by mannanase labeled by 6xHis, was cultured in shake flasks at 30°C for 72 hours in SC-galactose culture medium. Cells from 2 liters of culture medium were removed by centrifugation and the supernatant subjected to a 40-fold concentration by ultrafiltration using a 5 kDa cut-off membrane. The concentrate was subsequently diafiltered with the same cutoff for buffer exchange (50 mM NaH2PO4, 300 mM NaCl, pH 5.0) and concentration to a final volume of 1/40 of the culture volume. The concentrate was filtered through a 0.45 µm filter and pH adjusted to 8.0 before loading onto a metallic affinity column (BD-Talon, BD-Bioscience). The column was washed with several bed volumes of diafiltration buffer pH 8.0. Mannanase was eluted with a gradient from 0% to 100% buffer B, whereas buffer B contains 50 mM NaH2PO4, 300 mM NaCl and 250 mM imidazole at pH 6.0. The eluted protein samples were analyzed by SDS PAGE. Fractions containing mannanase were pooled and dialyzed against buffer containing 50 mM NaOAc, pH 5.0. The purity of the mannanase samples was controlled with reverse phase chromatography using absorption at 280 nm for protein detection. EXAMPLE 2: PURIFICATION OF MANANASE ENZYMES WITH CBD TERMINAL C
[0174] Purification of mannanase enzymes with a C-terminal CBD was performed using anion exchange and hydrophobic interaction chromatography. In detail, S. saccharomyces, transformed with a plasmid encoded by the respective mannanase enzyme, was cultivated in shake flasks at 30°C for 72 hours in SC-galactose culture medium. Cells from 5 liters of culture medium were removed by centrifugation and the supernatant was subjected to concentration and buffer exchange (buffer A: 20 mM Tris(hydroxymethyl)-aminomethane, pH 8.6) by ultrafiltration using a membrane 10 kDa cutoff. Finally 200 ml of mannanase concentrate in buffer A was generated. The solution was applied to a 6.3 ml TSKgel SuperQ-5PW(30) column (Tosoh Bioscience) equilibrated with buffer A. The column was washed with several column volumes of buffer A. Subsequently the mannanase enzyme was eluted with a linear gradient from 0 to 100% buffer B (20 mM Tris(hydroxymethyl)-aminomethane, pH 8.6, 1M NaCl) in 120 ml. The eluted protein samples were analyzed by SDS PAGE. The mannanase-containing fractions were pooled, diluted 1:1 with a 3M ammonium sulfate solution and loaded into a balance (buffer C: 50 mM NaOAc, pH 5.0, 1.5 M ammonium sulfate) 8.8 ml of TSK Gel phenyl-5PW column (20) (Tosoh Bioscience). The column was washed with buffer C. Mannase elution was performed with a linear gradient from 0 to 100% buffer D (50 mM NaOAc, pH 5.0) in 100 ml. The eluted protein samples were analyzed by SDS PAGE. Fractions containing mannanase were pooled and dialyzed against buffer D. Purity of the mannanase samples was controlled with reverse phase chromatography using absorption at 280 nm for protein detection. EXAMPLE 3: GENERATION AND CHARACTERIZATION OF MANANASE
[0175] The mannanase variants were generated using the different methods by mutagenesis of the DNA encoding the cassette-like mannanase proteins or PCR mutagenesis or other well known technical mutagenesis methods. These methods comprise such methods as disclosed in Morinaga et al., Biotechnology 2:646-649 (1984) and in Nelson and Long, Analytical Biochemistry 180:147-151(1989); or the Error Threshold Mutagenesis protocol described in WO 92/18645. For mutagenic PCR another suitable method is disclosed by Cadwell and Joyce, PCR Methods Appl. 3:136-140(1994).
[0176] The mannanase variants were heterologously expressed in one or more of the following expression hosts: Saccharomyces cerevisiae, Bacillus subtilis and Escherichia coli. EXAMPLE 4: DETERMINATION OF TEMPERATURE STABILITY
[0177] The temperature stability of mannanase variants is characterized by their inactivation temperature. The inactivation temperature was determined by measuring the residual activity of mannanase enzymes after incubation at different temperatures. Residual activities were determined by measuring mannanase activities with or without prior temperature challenge of the mannanase samples. In more detail, mannanase samples were incubated in 50 mM NaOAc buffer, pH 5.0 and 0.025% Triton-X-100 for 45 minutes at various temperatures. Subsequently mannanase activities were determined using AZCL-galactomannan (carob, Megazyme) as a substrate. For these mannanase samples and enzyme from mannanase calibration series (mannanase purified according to Seq. ID No. 3 with a C-terminal 6xHisTag) were incubated with 1 mg/ml AZCL-galactomannan, 50 mM NaOAc , pH 5.0 and 0.1% Triton-X-100 for 60 minutes at 37°C. Supernatants from the AZCL-galactomannan assay were subsequently transferred into a 96-well microtiter plate and absorption was determined at 590 nm in a standard plate reader. Absorption data for the mannanase enzyme calibration series were plotted against enzyme concentration. The activities of other mannanase samples were calculated using the equations that were generated by appropriate curve fitting the data to the standard mannanase enzyme series. Therefore, the activities of the mannanase samples as expressed as equivalent activities of the mannanase enzyme calibration series.
[0178] The mannanase enzyme inactivation temperature is defined as the temperature at which the residual mannanase activity is 50% compared to the residual activity of the same mannanase after incubation under the same conditions but at room temperature. Where appropriate extrapolations and interpolations from the activity data were made in order to determine the temperature corresponding to 50% residual activity. Differences in temperature stability (TD) in [°C] were calculated by subtracting the inactivation temperature of two enzymes in each other.
[0179] Table 3: Stability of temperature differences (TD) in [°C] for the mannanase variants. The variants shown are based on Seq. ID No. 3 and carries the C-terminus of a 6xHisTag or carbohydrate binding domain (CBD, Seq. ID No. 10, Fig.11). The substitutions presented were introduced in Seq. ID No. 3. Temperature difference stability (TD) is defined as (variant inactivation temperature) - (inactivation temperature of Seq. ID No. 3) with both variant and enzyme with Seq. ID No. 3 having identical C-terminal label. The enzyme with Seq. ID No. 3 performs with a C-terminal CBD exhibits an inactivation temperature of 74.6°C. The enzyme with Seq. ID #3 performs a C-terminal 6xHisTag displays an inactivation temperature of 75.7°C.


EXAMPLE 5: SPECIFIC ACTIVITY
[0180] The specific activity of the mannanase enzymes was determined using the purified enzymes according to examples 1 and 2. The mannanase activity was defined as reducing sugar release from galactomannan. Mannanase protein was determined by optical density (OD) measurements at 280 nm.
[0181] In detail, purified mannanase samples were diluted in 50 mM NaOAc, pH 5.0. Galactomannan carob solution (low viscosity, Megazyme) was added to total yield concentrations of 0.7% (w/v) galactomannan, 50 mM NaOAc, pH 5.0 and approximately 10 µg/ml mannanase protein. The solutions were incubated for 16 hours at 37°C.
[0182] Subsequently the amount of sugar reduction was determined as follows. One part of the assay of galactomannan or defined mannose solutions was mixed with one part of a solution containing 1% (w/v) dinitrosalicylic acid (DNSA), 30% (w/v) potassium sodium tartrate and 0.4 M of NaOH. The mixture was incubated for 10 minutes at 99°C and 5 minutes at 4°C. Finally the absorption was measured at 540 nm. Reduction of sugar equivalents (as mannose equivalents) were calculated by plotting the absorption data for the standard mannose samples against the mannose concentration. The amount of sugar equivalent reduction for the samples was calculated using the equations that were generated by appropriate curve fitting of the data for the standard mannose samples.
[0183] The concentrations of mannanase were calculated from the optical density of the preparations at 280 nm and the extinction coefficient for each variant of mannanase. Extinction coefficients were calculated on the basis of the amino acid composition of the proteins according to the method provided by Gill and von Hippel, Analytical Biochemistry 182:319-326 (1989).
[0184] The specific activity of the mannanase enzymes according to the present invention is expressed in nkat per mg of mannanase protein in the galactomannan carob substrate, as described above. An activity of an nkat is defined as the release of nanomol reduces sugars per second.
[0185] Table 4: Specific activity of mannanase variants. The variants shown based on Seq. ID No. 3 and carry the C-terminus a 6xHisTag or carbohydrate binding domain (CBD, Seq. ID No. 10). The substitutions presented were introduced in Seq. ID No. 3. Specific activity values are defined as (variant-specific activity)/(reference-specific activity). The reference in this case is the mannanase of Seq. ID No. 3 with the same C-terminal label as present in the respective variant. The reference with a C-terminal 6xHisTag has a specific activity of 1228 nkat/mg and the reference with a C-terminal CBD has a specific activity of 535nkat/mg.
EXAMPLE 6: LOW PH / PEPSIN STABILITY
[0186] For the determination of low pH/pepsin stability of mannanase enzymes, S. saccharomyces, transformed with a plasmid encoded by the respective mannanase enzyme, was cultivated in shake flasks at 30°C for 72 hours in culture medium SC-galactose. Cells were removed by centrifugation and the supernatant separated and concentrated, for example, 10 times by ultrafiltration with 10 kDa cut-off membranes. Concentrated surfactants were diluted, for example, 10 times in an autoclaved solution containing 30 g/l of potato fruit water, 30 g/l of liquid substance from corn steep liquor, 5 g/l of sulphate ammonium, 15 g/l of KH2PO4, 10 g/l of carob bean gum and 20 g/l of cellulose (Avicell). The diluted mannanase samples were mixed 1:1 with pepsin assay (200 mM glycine-HCl, pH 1.5, 5 mg/ml BSA predigestive pepsin, 2 mM CaCl2 and 0.5 mg/ml pepsin ) and incubated for 2 hours at 37°C. The pH of the mixture was pH 2.45. In addition, a control sample was generated. For this same diluted mannanase sample, it was mixed 1:1 with a control assay (100 mM NaOAc, pH 5.2, 5 mg/ml pre-digested pepsin BSA and 2 mM CaCl2) and incubated for 2 hours at 37°C °C. The pH of the mixture was pH 5.2.
[0187] For the determination of mannanase activity, 1 part of the above mixtures or mannanase enzyme calibration series (mannanase purified according to Seq. ID No. 3 with a C-terminal 6xHisTag) are mixed with 14 parts of the AZCL-galactomannan assay (200 mM NaOAc, pH 5.0, 0.1% Triton-X-100, 1% AZCL-galactomannan carob (Megazyme)) and incubated for 60 minutes at 37°C. Samples are centrifuged and supernatants analyzed by absorption at 590 nm. Absorption data for the mannanase enzyme calibration series were plotted against enzyme concentration. The activities of other mannanase samples were calculated using the equations that were generated by appropriate curve fitting the data to the mannanase enzyme calibration series. Therefore, the activities of the mannanase samples are expressed as activity equivalents of the mannanase enzyme calibration series.
[0188] Residual activities of mannanase enzymes are calculated as the following ratio:
[0189] (Mananase activity after incubation in the pepsin assay)/(mannanase activity after incubation in the control assay).
[0190] Table 5: Low pH / pepsin stability of mannanase variants. The variants shown are based on Seq. ID No. 3 and carry the C-terminus a carbohydrate binding domain (CBD, Seq. ID No. 10). The substitutions presented were introduced in Seq. ID No. 3. The reference mannanase according to Seq. ID #1 with a C-terminal CBD exhibits a residual activity of 39%.
EXAMPLE 7: COMPARISON OF TRICHODERMA REESEI MANANASE AND THE S3R VARIANT
[0191] The temperature stability and mannose production of Trichoderma reesei mannanase as shown in SEQ ID NO: 1 was compared to the mannanase variant derived from SEQ ID NO: 1 by introducing the S3R substitution (Serine to arginine in position 3) . The experiment and results are described in detail in WO 2008/009673 (example 8, p.100101; Fig. 1B) and in Fig. 10.
[0192] Sequence listing, free text SEQ ID NO 1: Trichoderma reesei mannanase wild type fragment / amino acid SEQ ID NO 2: mannanase V-31 variant disclosed in WO 2008/009673 / amino acid SEQ ID NO 3: mannanase variant V-31/S3R disclosed in WO 2008/009673 / amino acid SEQ ID NO 4: TM-1 mannanase variant / amino acid SEQ ID NO 5: TM-1 mannanase variant / DNA SEQ ID NO 6: TM-100 mannanase / amino acid SEQ ID NO 7: TM-108 mannanase variant / amino acid SEQ ID NO 8: TM-CBD-148 mannanase variant / amino acid SEQ ID NO 9: TM-144 mannanase variant / amino acid SEQ ID NO 10: CBD / amino acid

权利要求:
Claims (6)
[0001]
1. MANANASE VARIANT, characterized in that it consists of an amino acid sequence of SEQ ID NO: 1 of wild-type/precursor Trichoderma reesei mannanase, wherein the amino acid of the mannanase variant comprises the variations 3R, 201S, 207F and 274L, and variations selected from the group consisting of: 1) F31Y/S66P/Q97R/N113Y/N173H/V181H/A215T/Q259R/Q280R/ N282D/N331S 2) S66P/Q97R/N113Y/N173H/V181A/A215T/Q259R/ Q280S/N331S 3) F31Y/S66P/Q97R/N173H/V181H/A215T/Q259R/Q280R/N282D 4) F31Y/S66P/Q97R/N173H/V181H/A215T/Q259R/Q280S/N282D/ N331S 5) F31QY/S /N113Y/N173H/V181H/A215T/Q259R/Q280R/ N282D 6) F31Y/S66P/Q97R/Q149K/N173H/V181H/A215T/Q259R/Q280L 7) S66P/Q97R/N113Y/N173H/VQ259R/A215T) F31Y/S66P/Q97R/N113Y/N173H/V181H/A215T/Q259R/Q280S/ N282D/N331S 9) S66P/N113Y/N173H/V181H/A215T/Q259R 10) F31Y/S66P/Q97R/N113/N331S/K146 A215T/Q259R/ Q280L/N282D 11) F31Y/S66P/Q97R/N113Y/K146Q/N173H/V181A/A215T/Q259R/ Q280L/N282D/N331S 12) F31Y/S66P/Q97R/N113Y/K146VQ/N173H 15T/Q259R/Q280L 13) F31Y/S66P/Q97R/N173H/V181H/A215T/Q259R/N282D 14) F31Y/S66P/Q97R/N113Y/K146Q/N173H/V181A/A215T/Q259R/ Q280R/N282D 15) /V181H/A215T/Q259R 16) S66P/Q97R/N113Y/N173H/V181H/A215T/Q259R/Q280L/N282D 17) F31Y/S66P/Q97R/N113Y/K146Q/N173H/V181A/A215T/Q259R/QL) F31Y/S66P/N173H/V181H/A215T/Q259R/N282D 19) F31Y/S66P/Q97R/N173H/V181H/A215T/Q259R/Q280L 20) S66P/Q97R/N113Y/N173H/V181H/A215T/Q259NR/Q259NR ) F31Y/S66P/Q97R/N113Y/N173T/V181H/A215T/Q259R/Q280R/ N282D 22) F31Y/S66P/Q97R/N173T/V181H/A215T/Q259R/Q280R/N282D 23) F31Y/S66P/HQ97R /A215T/Q259R/Q280S/N282D 24) S66P/N113Y/N173H/V181H/A215T/Q259R/Q280S/N282D 25) S66P/Q97R/N113Y/N173H/V181A/A215T/Q259R/Q280S/N282D/N282D) Q97R/N113Y/V181H/A215T/Q259R/Q280L/N282D 27) S66P/N113Y/N173H/V181H/A215T/Q259R/N331S 28) F31Y/S66P/Q97R/N113Y/K146Q/N173H/VQ181/A215T N282D/N331S 29) F31Y/S66P/Q97R/N113Y/K146Q/V181H/A215T/Q259R/Q280S/ N282D/N331S 30) S66P/Q97R/N113Y/N173T/V181A/A215T /Q259R 31) F31Y/S66P/Q97R/N113Y/K146Q/N173H/V181A/A215T/Q259R/ Q280S/N331S 32) F31Y/S66P/Q97R/N113Y/V181H/A215T/Q259R 33) S66P/Q97R/N11 V181A/A215T/Q259R/N331S 34) F31Y/S66P/Q97R/N113Y/V181H/A215T/Q259R/Q280L 35) F31Y/S66P/Q97R/N113Y/K146Q/V181H/A215T/Q259R/Q280L/Q280L 36) F31 /K146Q/V181H/A215T/Q259R/Q280R/N282D 37) S66P/N113Y/V181H/A215T/Q259R/N282D 38) F31Y/S66P/Q97R/V181H/A215T/Q259R/N282D 39) S66P/NH113VY A215T/Q259R/Q280S/N331S 40) F31Y/S66P/Q97R/N113Y/K146Q/N173H/V181H/A215T/Q259R/ Q280S/N331S 41) S66P/V181H/A215T/Q259R/N282D 42) F31QY/S66 /K146Q/V181H/A215T/Q259R/Q280L/ N331S 43) S66P/Q97R/N113Y/N173H/V181H/A215T/Q259R/N282D 44) S66P/Q97R/N113Y/V181H/A215T/Q259R/H662D 45) A215T/Q259R 46) S66P/Q97R/N113Y/V181H/A215T/Q259R/Q280R/N282D 47) F31Y/S66P/N173T/V181H/A215T/Q259R/N282D 48) F31Y/S66P/N113Y/VQ181H/A215R /N344D 49) F31Y/S66P/Q97R/N113Y/N173H/V181H/A215T/Q259R/Q280R/ N282D 50) F31Y/S66P/Q97R/N113Y/N173H/V181H/A215T/Q259R/Q 280S/ N282D/N331S 51) S66P/N113Y/N173H/V181H/A215T/Q259R/Q280S 52) S66P/Q97R/N113Y/N173H/V181A/A215T/Q259R 53) S66P/Q97R/N113Y/Q259H215T /Q280S 54) S66P/N113Y/N173H/V181H/A215T/Q259R/Q280S/N282D 55) F31Y/S66P/Q97R/N173H/V181H/A215T/Q259R/Q280R/N282D 56) S66P/N113Y/N173H215/V181 Q259R 57) S66P/Q97R/N113Y/N173H/V181A/A215T/Q259R/N331S 58) F31Y/S66P/Q97R/N173H/V181H/A215T/Q259R/Q280S/N282D/ N331S 59) S66P/N113VY/N181 /Q259R/Q280S/N331S 60) S66P/Q97R/N113Y/N173H/V181A/A215T/Q259R/Q280S/N331S 61) F31Y/S66P/Q97R/Q149K/N173H/V181H/A215T/Q259R/Q280S/N331S /A215T/Q259R.
[0002]
2. MANANASE VARIANT according to claim 1, characterized by having mannanase activity and an amino acid sequence that varies from the amino acid sequence of wild type/precursor Trichoderma reesei mannanase (SEQ ID NO: 1), wherein the amino acid sequence of the mannanase variant is SEQ ID NO: 8 and comprises the 331S variation.
[0003]
3. NUCLEIC ACID MOLECULE, characterized by being of SEQ ID NO: 5.
[0004]
4. METHOD FOR THE PREPARATION OF MODIFIED MANANASE, as defined in any one of claims 1 to 2, characterized in that it comprises the isolation of the modified mannanase from the culture.
[0005]
5. COMPOSITION, characterized in that it comprises modified mannanase as defined in any one of claims 1 to 2, wherein the composition additionally comprises an effective amount of one or more food or feed enhancing enzymes selected from the group consisting of phytases, hemicellulases, alpha-galactosidases, beta-galactosidases, lactases, beta-glucanases, endo-beta-1,4-glucanases, cellulases, xylosidases, xylanases, xyloglucanases, xylan acetyl esterases, galactanases, exo-mannanases, pectinases lyases, pectinesterases, polygalacturonases, arabinases, rhamnogalacturonases, laccases, reductases, oxidases, phenoloxidases, ligninases, proteases, amylases, phosphatases, lipolytic enzymes and cutinases.
[0006]
6. USE OF MODIFIED MANANASE, as defined in any one of claims 1 to 2, characterized in that it is for food and feed processing, for coffee extraction, for processing coffee residue, as a supplement to food and feed, for bleaching aided by the enzyme from paper pulps, as bleaching and/or desizing agents in the textile industry, for the stimulation of oil and gas by hydraulic fracture, as a detergent, for the removal of biofilms, in release systems, and/or for the processing of renewable resources intended for the production of biofuels.

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法律状态:
2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-12-31| B07A| Technical examination (opinion): publication of technical examination (opinion) [chapter 7.1 patent gazette]|

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2020-09-01| 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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2020-12-15| 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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2021-03-16| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-04-06| B09X| Decision of grant: republication|

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2021-05-18| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 24/11/2010, OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF |

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