CN113302294A - Polypeptides having xylanase activity and their use for improving the nutritional quality of animal feed - Google Patents

Polypeptides having xylanase activity and their use for improving the nutritional quality of animal feed Download PDF

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CN113302294A
CN113302294A CN202080009202.6A CN202080009202A CN113302294A CN 113302294 A CN113302294 A CN 113302294A CN 202080009202 A CN202080009202 A CN 202080009202A CN 113302294 A CN113302294 A CN 113302294A
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seq
polypeptide
xylanase
amino acids
alpha
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J·弗里克曼
K·詹森
E·弗拉森科
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Novozymes AS
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Novozymes AS
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/248—Xylanases
    • C12N9/2482—Endo-1,4-beta-xylanase (3.2.1.8)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
    • A23K10/38—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material from distillers' or brewers' waste
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/189—Enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
    • Y02P60/87—Re-use of by-products of food processing for fodder production

Abstract

The present invention relates to polypeptides having xylanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides. The invention also relates to a method of using a polypeptide having xylanase activity in a method for improving the nutritional quality of distillers Dried Grains (DGS) or distillers dried grains with solubles (DDGS) produced as a byproduct of a fermentation product production process, a method for producing a fermentation product, and the use of an enzyme blend comprising a polypeptide having xylanase activity in these methods.

Description

Polypeptides having xylanase activity and their use for improving the nutritional quality of animal feed
Reference to sequence listing
This application contains a sequence listing in computer readable form, which is incorporated herein by reference.
Technical Field
The present invention relates to polypeptides having xylanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides. The invention also relates to a method of using a polypeptide having xylanase activity in a method for improving the nutritional quality of distillers Dried Grains (DGS) or distillers dried grains with solubles (DDGS) produced as a byproduct of a fermentation product production process, a method for producing a fermentation product, and the use of an enzyme blend comprising a polypeptide having xylanase activity in these methods.
Background
Methods for producing fermentation products (e.g., ethanol) from starch-or lignocellulose-containing material are well known in the art. The preparation of starch-containing material such as corn for such fermentation processes typically begins with grinding the corn in a dry or wet milling process. The wet milling process involves fractionating corn into different fractions, with only the starch fraction going to the fermentation process. The dry milling process involves milling corn kernels into meal and mixing the meal with water and enzymes. Two different types of dry milling methods are commonly used. The most commonly used process, often referred to as the "conventional process", involves milling the starch-containing material, then liquefying the gelatinized starch at elevated temperatures, typically using a bacterial alpha-amylase, followed by Simultaneous Saccharification and Fermentation (SSF) in the presence of a glucoamylase and a fermenting organism. Another well-known process, often referred to as the "raw starch hydrolysis" process (RSH process), involves grinding starch-containing material and then simultaneously saccharifying and fermenting granular starch at a temperature below the initial gelatinization temperature, typically in the presence of an acid fungal alpha-amylase and a glucoamylase.
In processes for producing ethanol from corn, a liquid fermentation product is recovered from a beer (often referred to as "beer mash") following the SSF or RSH process, for example by distillation to separate the desired fermentation product (e.g., ethanol) from other liquids and/or solids. The remaining fraction is referred to as "whole stillage". Whole stillage typically contains about 10% to 20% solids. The whole stillage is separated into a solid fraction and a liquid fraction, for example by centrifugation. The separated solid fraction is called "wet cake" (or "wet grain"), while the separated liquid fraction is called "thin stillage". The wetcake and thin stillage contained about 35% and 7% solids, respectively. The wet cake (with optional additional dewatering) is used as a component in animal feed or is dried to provide "distillers dried grains" (DDG) for use as a component of animal feed. The thin stillage is typically evaporated to provide evaporator condensate and slurry, or alternatively may be recycled to the slurry tank as "counterflow". The evaporator condensate may be sent to the methanator before being discharged, and/or may be recycled to the slurry tank as "boil-off water". The slurry can be blended into DDG or added to the wet cake before or during the drying process (which may in turn comprise one or more dryers) to produce DDGs (distillers dried grains with solubles). The slurry typically contains about 25% to 35% solids. Oil may also be extracted from the thin stillage and/or syrup, as a by-product (for biodiesel production), as a feed or food additive or product, or other biorenewable product.
Distillers grains with solubles (DGS) and distillers dried grains with solubles (DDGS) are by-products of the grain ethanol industry, which are used in animal feed. DGS and DDGS are rich in fiber, so the highest feasible inclusion rate of monogastric animals (such as poultry and swine, for example) is lower than that of ruminants (such as cattle). Glycosyl hydrolases such as, for example, endoxylanases, are added to the feed blend to increase the digestibility of the fiber-rich feed blend. However, there are some challenges associated with the role of enzymes added to feed blends; for example, uniform mixing of enzymes into the feed blend, thermal stability of the enzyme protein during feed pelleting, stability of the enzyme protein during passage through the low pH stomach, relatively short residence time in the intestinal tract of some animal species.
Disclosure of Invention
The present invention overcomes the above challenges by adding a polypeptide having xylanase activity of the present disclosure, or an enzyme blend comprising a polypeptide having xylanase activity and/or a cellulolytic composition, upstream during a fermentation product production process, e.g., during a Simultaneous Saccharification and Fermentation (SSF) step, wherein a free-flowing slurry is mixed continuously, the temperature is stable (e.g., between 30 ℃ and 35 ℃), the pH is stable (e.g., between about pH 4 and pH 5), and the residence time is typically in the range of 54 to 80 hours.
The invention more particularly relates to the addition of a polypeptide having xylanase activity, or an enzyme blend comprising a polypeptide having xylanase activity, of the present disclosure during an SSF process to produce a DDGS product or a DGS product with higher digestibility for an animal (e.g., a monogastric animal). Without wishing to be bound by theory, it is believed that when fiber (e.g., corn) is solubilized, encapsulation of nutrients (e.g., proteins, oils, and residual starch) is reduced, thereby making these nutrients more readily available, and the solubilized fiber can be fermented by the gut microbiome into metabolizable products (e.g., fatty acids). Furthermore, the dissolved fibres have the potential to have a positive effect on the intestinal health by acting as a substrate for the beneficial intestinal flora.
The polypeptides having xylanase activity of the present disclosure are members of the G98 family (collectively referred to herein as "GH 98 xylanases"). GH98 xylanase is known to produce larger oligosaccharides than GH30 xylanase (Rogowski et al, 2015). It is believed that a larger oligosaccharide profile may have a positive impact on gut health, feed value, and DDGS color (e.g., due to reduced formation of scorch and Maillard (Maillard) products during drying).
In one aspect, the invention relates to a polypeptide having xylanase activity, selected from the group consisting of:
(a) a polypeptide having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO 1, 5, or 7;
(b) a polypeptide encoded by a polynucleotide that hybridizes under very high stringency conditions to (i) the mature polypeptide coding sequence of SEQ ID NO:2, 6, or 8, or (ii) the full-length complement of (i) or (ii);
(c) a polypeptide encoded by a polynucleotide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO. 2, 6, or 8; and
(d) a fragment of the polypeptide of (a), (b), or (c), which fragment has xylanase activity.
The invention also relates to polynucleotides encoding the polypeptides of the invention, nucleic acid constructs, recombinant expression vectors, recombinant host cells comprising the polynucleotides, and methods for producing the polypeptides.
In one aspect, the invention relates to a GH98 xylanase or an enzyme blend comprising a GH98 xylanase. In an embodiment, the enzyme blend further comprises a cellulolytic composition. In an embodiment, the cellulolytic composition is present in the blend in a ratio of xylanase to cellulolytic composition of about 5:95 to about 95: 5. In an embodiment, the ratio of xylanase and cellulolytic composition in the blend is about 10: 90. In an embodiment, the ratio of xylanase and cellulolytic composition in the blend is about 20: 80. In an embodiment, the ratio of xylanase and cellulolytic composition in the blend is about 50: 50.
In embodiments, the enzyme blend comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or at least 100% xylanase. In an embodiment, the enzyme blend comprises at least 5%, at least 10% xylanase, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95% cellulolytic composition.
In embodiments, the xylanase is from the genus Microbacterium (Microbacterium) or Paenibacillus (Paenibacillus). In an embodiment, the xylanase is a GH98 xylanase selected from the group consisting of: (i) 1 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto;
(ii) 5 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto; and
(iii) 7 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto.
In embodiments, the cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) cellobiohydrolase I; (ii) cellobiohydrolase II; (iii) a beta-glucosidase; and (iv) a GH61 polypeptide having cellulolytic enhancing activity.
In embodiments, the cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) aspergillus fumigatus (Aspergillus fumigatus) cellobiohydrolase I; (ii) aspergillus fumigatus cellobiohydrolase II; (iii) aspergillus fumigatus beta-glucosidase; and (iv) a Penicillium emersonii (Penicillium emersonii) GH61A polypeptide having cellulolytic enhancing activity.
In an embodiment, the cellulolytic composition comprises: (i) cellobiohydrolase I comprising amino acids 27 to 532 of SEQ ID No. 15 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 27 to 532 of SEQ ID No. 15; (ii) a cellobiohydrolase II comprising amino acids 20 to 454 of SEQ ID No. 16, or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 20 to 454 of SEQ ID No. 16; (iii) a β -glucosidase or variant thereof comprising amino acids 20 to 863 of SEQ ID No. 17, having at least one substitution selected from the group consisting of F100D, S283G, N456E, and F512Y and having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 20 to 863 of SEQ ID No. 17; and/or (iv) a GH61A polypeptide having cellulolytic enhancing activity comprising amino acids 26 to 253 of SEQ ID No. 18 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 26 to 253 of SEQ ID No. 18.
In an embodiment, the cellulolytic composition comprises: (i) cellobiohydrolase I comprising amino acids 27 to 532 of SEQ ID No. 15 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 27 to 532 of SEQ ID No. 15; and (ii) a β -glucosidase or variant thereof comprising amino acids 20 to 863 of SEQ ID No. 16, having at least one substitution selected from the group consisting of F100D, S283G, N456E, and F512Y and having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 20 to 863 of SEQ ID No. 16.
In embodiments, the cellulolytic composition further comprises an endoglucanase.
In an embodiment, the cellulolytic composition is derived from a strain selected from the group consisting of Aspergillus (Aspergillus), penicillium (penicillium), Talaromyces (Talaromyces), and Trichoderma (Trichoderma), optionally wherein: (i) the aspergillus strain is selected from the group consisting of: aspergillus flavus (Aspergillus aurantiacus), Aspergillus niger (Aspergillus niger), and Aspergillus oryzae (Aspergillus oryzae); (ii) the penicillium strain is selected from the group consisting of: penicillium emersonii and penicillium oxalicum (penicillium oxalicum); (iii) the Talaromyces strain is selected from the group consisting of: talaromyces aurantiacaus and Talaromyces emersonii; and (iv) the Trichoderma strain is Trichoderma reesei (Trichoderma reesei). In an embodiment, the cellulolytic composition comprises a trichoderma reesei cellulolytic composition.
In another aspect, the present invention relates to a method of producing a fermentation product comprising the steps of: (a) saccharifying a starch-containing material with an alpha-amylase, a glucoamylase, and a GH98 xylanase or an enzyme blend or composition comprising the GH98 xylanase at a temperature below the initial gelatinization temperature; and (b) fermenting using a fermenting organism.
In another aspect, the present invention relates to a process for producing a fermentation product from starch-containing material, the process comprising the steps of: (a) liquefying a starch-containing material with an alpha-amylase; (b) saccharifying the liquefied material obtained in step (a) with a glucoamylase and a GH98 xylanase of the invention or an enzyme blend or composition comprising the GH98 xylanase; and (c) fermenting using a fermenting organism.
In the examples, saccharification and fermentation are carried out simultaneously. In embodiments, the starch-containing material comprises maize, corn, wheat, rye, barley, triticale, sorghum, switchgrass, millet, pearl millet, millet. In embodiments, the fermentation product is an alcohol, in particular ethanol. In an embodiment, the fermenting organism is a yeast, in particular a Saccharomyces species (Saccharomyces sp.), more in particular Saccharomyces cerevisiae (Saccharomyces cerevisiae).
In another aspect, the invention relates to a method for improving the nutritional quality of distillers Dried Grains (DGS) or distillers dried grains with solubles (DDGS) produced as a byproduct of a fermentation product production process, the method comprising performing the method of producing a fermentation product of the invention, and recovering the fermentation product to produce DGS or DDGS as a byproduct, wherein the DGS or DDGS produced has improved nutritional quality.
In embodiments, the TME of the DGS or DDGS is increased by at least 5%, at least 10%, at least 15%, or at least 20% compared to the true metabolic energy of the DGS or DDGS produced in the absence of a GH98 xylanase of the invention, or an enzyme blend or composition comprising the GH98 xylanase, during a saccharification step, a fermentation step, and/or a simultaneous saccharification and fermentation step of a method of producing a fermentation product of the invention.
In an embodiment, the animal is a monogastric animal.
In embodiments, the DGS or DDGS produced after drying is not blackened compared to that produced in the absence of a GH98 xylanase of the invention, or an enzyme blend or composition comprising the GH98 xylanase, during the saccharification step, fermentation step, and/or simultaneous saccharification and fermentation step of the methods of the invention.
In another aspect, the invention relates to the use of the GH98 xylanase of the invention, or an enzyme blend or composition comprising the GH98 xylanase, to improve the nutritional quality of DGS or DDGS produced as a byproduct of a fermentation product production process, preferably without causing the DDG or DDGS to darken.
In another aspect, the invention relates to the use of a GH98 xylanase of the invention, or an enzyme blend or composition comprising the GH98 xylanase, for solubilizing fibers, preferably for solubilizing xylose and arabinose.
Drawings
Figure 1 shows the average DP4+ yield (g/L) of each GH98 enzyme blend compared to a control cellulolytic composition not supplemented with xylanase.
FIG. 2 shows the average glucose yield (g/L) for each GH98 enzyme blend compared to a control cellulolytic composition not supplemented with xylanase.
Overview of sequence listing
SEQ ID NO 1 is the amino acid sequence of GH98 xylanase from Microbacterium oxydans.
SEQ ID NO. 2 is a polynucleotide sequence encoding the GH98 xylanase of SEQ ID NO. 1.
SEQ ID NO 3 is the amino acid sequence of GH98 xylanase from Microbacterium hydrocarbonoxydans (Microbacterium hydrocarbonoxydans).
SEQ ID NO 4 is the amino acid sequence of GH98 xylanase from Microbacterium species SA 39.
SEQ ID NO 5 is the amino acid sequence of GH98 xylanase from Bacillus glucanolyticus.
SEQ ID NO. 6 is a polynucleotide sequence encoding the GH98 xylanase of SEQ ID NO. 5.
SEQ ID NO 7 is the amino acid sequence of the mature GH98 xylanase from Paenibacillus terreus.
SEQ ID NO 8 is a polynucleotide sequence encoding the GH98 xylanase of SEQ ID NO 7.
SEQ ID NO 9 is the amino acid sequence of GH98 xylanase from Paenibacillus terreus.
SEQ ID NO 10 is the amino acid sequence of the mature GH98 xylanase from Paenibacillus species DMB 20.
SEQ ID NO 11 is the amino acid sequence of a glucoamylase from Talaromyces emersonii.
SEQ ID NO 12 is the amino acid sequence of a glucoamylase from Gloeophyllum sepiarium.
SEQ ID NO 13 is the amino acid sequence of a glucoamylase from Pleurotus densatus (Gloeophyllum trabeum).
SEQ ID NO:14 is the amino acid sequence of Rhizomucor pusillus alpha-amylase having the following substitutions G128D + D143N, having an Aspergillus niger glucoamylase linker and a Starch Binding Domain (SBD).
SEQ ID NO 15 is the amino acid sequence of the full-length cellobiohydrolase I from Aspergillus fumigatus.
SEQ ID NO 16 is the amino acid sequence of the full-length cellobiohydrolase II from Aspergillus fumigatus.
SEQ ID NO 17 is the amino acid sequence of the full length beta-glucosidase from Aspergillus fumigatus.
18 is the amino acid sequence of the full length GH61 polypeptide from penicillium emersonii.
SEQ ID NO 19 is the amino acid sequence of the full length alpha-amylase from Bacillus stearothermophilus.
SEQ ID NO:20 is the amino acid sequence of the full length GH10 xylanase from Geotrichum thermophilum (Dictyoglobllus thermophilum).
SEQ ID NO 21 is the amino acid sequence of the full length GH11 xylanase from Geotrichum thermophilum.
SEQ ID NO:22 is the amino acid sequence of the full length GH10 xylanase from Talaromyces myceliophthora (Rasamsonia byssochlamydes).
SEQ ID NO:23 is the amino acid sequence of the full length GH10 xylanase from Talaromyces leycettanus.
SEQ ID NO 24 is the amino acid sequence of the full length GH10 xylanase from Aspergillus fumigatus.
SEQ ID NO 25 is the amino acid sequence of the full-length endoglucanase from Talaromyces reesei.
SEQ ID NO 26 is the amino acid sequence of the full-length endoglucanase from Penicillium capsulatum.
SEQ ID NO:27 is the amino acid sequence of the full-length endoglucanase from Trichophaea fuliginosa (Trichophaea saccharocata).
SEQ ID NO 28 is the amino acid sequence of the full length GH45 endoglucanase from Chaetomium faecalis (Sordaria fimicola).
SEQ ID NO:29 is the amino acid sequence of the full length GH45 endoglucanase from Thielavia terrestris.
SEQ ID NO 30 is the amino acid sequence of a full length glucoamylase from Penicillium oxalicum (Penicillium oxalicum).
SEQ ID NO 31 is the amino acid sequence of a full-length protease from Pyrococcus furiosus.
SEQ ID NO 32 is the amino acid sequence of the full-length protease from Thermoascus aurantiacus (Thermoascus aurantiacus).
33 is a codon optimized polynucleotide sequence of a GH98 xylanase from Microbacterium oxydans.
SEQ ID NO 34 is the amino acid sequence of the secretion signal of Bacillus clausii (Bacillus clausii).
SEQ ID NO 35 is the amino acid sequence of a polyhistidine tag.
Definition of
Allelic variants: the term "allelic variant" means any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation and can lead to polymorphism within a population. Gene mutations can be silent (no change in the encoded polypeptide) or can encode polypeptides with altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.
Alpha-amylases (alpha-1, 4-glucan-4-glucanohydrolase, EC 3.2.1.1) are a group of enzymes that catalyze the hydrolysis of starch and other straight and branched chain 1, 4-glucosidic oligo-and polysaccharides.
Animals: the term "animal" refers to all animals except humans. Examples of animals are non-ruminants and ruminants. Ruminants include, for example, animals such as sheep, goats, cattle (e.g., beef, dairy, and calf), deer, yaks, camels, llamas, and kangaroos. Non-ruminant animals include monogastric animals, such as pigs or live pigs (including but not limited to piglets, growing pigs and sows); poultry, such as turkeys, ducks, and chickens (including but not limited to broiler chickens and layer chickens); horses (including but not limited to hot, cold and warm blooded horses), calves; fish (including but not limited to amber, giant slippery tongue fish, fish, sea bass, blue fish, sebastes (bocachico), Cyprinus carpio, catfish, kamao (cachama), carp, catfish, capelin, galoshes, pongamus albomaculatus, cobia, cod, dolichos, bream, chufa, eels, gobies, flounder, grouper, yellowtail, parfel, silverfish, mudfish, mullet, parpa (paco), marmot (pearl), Pagrub (jery), jejuniper (jemerrey), jewfish, pike, butterfish, salmonster, salmons, salpers (salper), salmons, salsa, salmons, salsa, salmons, salsa, salmons, salsa, Sweet fish (sweet fish), bungarus parvus, trout (terror), tilapia, trout, tuna, turbot, white trout, white fish, and white fish); and crustaceans (including but not limited to shrimp and prawn).
Animal feed: the term "animal feed" refers to any compound, formulation or mixture suitable or intended for ingestion by an animal. Animal feed for monogastric animals typically comprises the concentrate together with vitamins, minerals, enzymes, direct fed microorganisms (direct fed microorganisms), amino acids and/or other feed ingredients (as in a premix), while animal feed for ruminants typically comprises forage (including roughage and silage), and may also comprise the concentrate together with vitamins, minerals, enzymes, direct fed microorganisms, amino acids and/or other feed ingredients (as in a premix).
Beta-glucosidase: the term "beta-glucosidase" means a beta-D-glucoside glucohydrolase (e.c.3.2.1.21) which catalyzes the hydrolysis of terminal non-reducing beta-D-glucose residues and releases beta-D-glucose.
For the purposes of the present invention, according to Venturi et al, 2002, excellular beta-D-glucosidase from Chaetomium thermophilum var. coprophilum: production, purification and sodium biochemical [ Extracellular beta-D-glucosidase from Chaetomium thermophilum coprophilum: production, purification and some Biochemical Properties ]J.basic Microbiol]42:55-66 procedure beta-glucosidase activity was determined using p-nitrophenyl-beta-D-glucopyranoside as substrate. One unit of beta-glucosidase is defined as containing 0.01% at 25 deg.C, pH 4.8
Figure BDA0003162048240000121
20 (polyoxyethylene sorbitan monolaurate) in 50mM sodium citrate produced 1.0 micromoles of p-nitrophenol anion per minute from 1mM p-nitrophenyl-beta-D-glucopyranoside as substrate.
Weight gain: the term "body weight gain" means the increase in live weight of an animal over a given period of time, for example from day 1 to day 21.
cDNA: the term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial primary RNA transcript is a precursor of mRNA that is processed through a series of steps, including splicing, and then rendered into mature spliced mRNA.
Cellobiohydrolase: the term "cellobiohydrolase" means a 1, 4-beta-D-glucan cellobiohydrolase (E.C.3.2.1.91), which catalyses the hydrolysis of 1, 4-beta-D-glycosidic bonds in cellulose, cellooligosaccharides, or any polymer containing beta-1, 4-linked glucose, releasing cellobiose from the reducing or non-reducing end of the chain (Teeri, 1997, Crystalline cellulose degradation: New knowledge of cellobiohydrolase function ], Trends in Biotechnology [ biotechnological Trends ]15: 160.
Cellobiohydrolase activity was determined according to the procedures described in the following documents: lever et al, 1972, anal. biochem. [ assay biochemistry ]47: 273-; van Tilbeurgh et al, 1982, FEBS Letters [ Provisions of European Association of Biochemical society ]149: 152-; van Tilbeurgh and Claeussensens, 1985, FEBS Letters [ European Association of biochemistry Association ]187: 283-; and Tomme et al, 1988, Eur.J.biochem. [ J.Eur. Biochem., 170: 575-581. In the present invention, the method of Tomme et al can be used for determining cellobiohydrolase activity.
Cellulolytic enzyme, cellulolytic composition, or cellulase: the terms "cellulolytic enzyme", "cellulolytic composition", or "cellulase" mean one or more (e.g., several) enzymes that hydrolyze a cellulosic material. Such enzymes include one or more endoglucanases, one or more cellobiohydrolases, one or more beta-glucosidases, or a combination thereof. Two basic methods for measuring cellulolytic activity include: (1) measurement of Total cellulolytic Activity, and (2) measurement of individual cellulolytic activities (endoglucanase, cellobiohydrolase, and beta-glucosidase), such as Zhang et al, Outlook for cellulose improvement: Screening and selection strategies [ prospects for cellulase improvement: screening and selection strategies 2006, Biotechnology Advances [ Advances in Biotechnology ]24: 452-481. The total cellulolytic activity is typically measured using insoluble substrates including Whatman No. 1 filter paper, microcrystalline cellulose, bacterial cellulose, algal cellulose, cotton, pretreated lignocellulose, etc. The most common measurement of total cellulolytic activity is a filter paper measurement using Whatman No. 1 filter paper as substrate. This assay was established by the International Union of Pure and Applied Chemistry (IUPAC) (Ghose,1987, Measurement of cellulase Activity, Pure Applied. chem. [ Pure and Applied Chemistry ]59: 257-68).
Determining cellulolytic enzyme activity by measuring the increase in hydrolysis of cellulosic material by one or more cellulolytic enzymes under the following conditions: 1-50mg cellulolytic enzyme protein/g cellulose in pretreated corn stover ("PCS") (or other pretreated cellulosic material) at a suitable temperature (e.g., 50 ℃, 55 ℃, or 60 ℃) for 3-7 days, as compared to a control hydrolysis without added cellulolytic enzyme protein. Typical conditions are: 1ml of reacted, washed or unwashed PCS, 5% insoluble solid, 50mM sodium acetate (pH5), 1mM MnSO450 ℃, 55 ℃ or 60 ℃, for 72 hours, by
Figure BDA0003162048240000141
Column HPX-87H (Bio-Rad Laboratories, Inc., Hercules, Calif.) for sugar analysis.
A coding sequence: the term "coding sequence" means a polynucleotide that directly specifies the amino acid sequence of a variant. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon (e.g., ATG, GTG, or TTG) and ends with a stop codon (e.g., TAA, TAG, or TGA). The coding sequence may be genomic DNA, cDNA, synthetic DNA, or a combination thereof.
And (3) control sequence: the term "control sequences" means nucleic acid sequences necessary for expression of a polynucleotide encoding a variant of the invention. Each control sequence may be native (i.e., from the same gene) or foreign (i.e., from a different gene) to the polynucleotide encoding the variant, or native or foreign with respect to one another. Such control sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, and a transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding the variant.
Endoglucanase: the term "endoglucanase" means an endo-1, 4- (1, 3; 1,4) - β -D-glucan 4-glucanohydrolase (e.c.3.2.1.4) which catalyzes the endo-hydrolysis of β -1,4 linkages in cellulose, cellulose derivatives (such as carboxymethyl cellulose and hydroxyethyl cellulose), 1,4- β -D-glucosidic linkages in lichenin, mixed β -1,3 glucans such as cereal β -D-glucans or xyloglucans, and other plant materials containing cellulosic components. Endoglucanase activity may be determined by measuring a decrease in the viscosity of the substrate or an increase in the reducing end as determined by a reducing sugar assay (Zhang et al, 2006, Biotechnology Advances [ Biotechnology Advances ]24: 452-481). For the purposes of the present invention, endoglucanase activity was determined according to the procedure of Ghose,1987, Pure and appl. chem [ Pure and applied chemistry ]59:257-268, using carboxymethylcellulose (CMC) as substrate at pH 5, 40 ℃.
Expressing: the term "expression" includes any step involved in the production of a variant, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
Expression vector: the term "expression vector" means a linear or circular DNA molecule comprising a polynucleotide encoding a variant and operably linked to control sequences that provide for its expression.
Family 61 glycoside hydrolases: the term "family 61 glycoside hydrolase" or "family GH 61" or "GH 61" means a polypeptide belonging to glycoside hydrolase family 61 according to Henrissat B.,1991, A classification of glycosyl hydrolases based on amino acid sequence similarity, biochem.J. [ J. Biochem.280: 309. and Henrisit B., and Bairoch A.,1996, Updating the sequence-based classification of glycosyl hydrolases [ more recent sequence-based classification of glycosyl hydrolases ], biochem.J. [ J. Biochem.316: 695. sup. 696-. Enzymes in this family were originally classified as glycoside hydrolases based on measurements of very weak endo-1, 4- β -D-glucanase activity in one family member. The structure and mode of action of these enzymes are not normative, and they cannot be considered as true glycosidases. However, they are retained in the CAZy classification based on their ability to enhance the breakdown of lignocellulose when used in combination with a cellulase or a mixture of cellulases.
Feed conversion rate: the term "feed conversion ratio" refers to the amount of feed that is fed to an animal to increase the weight of the animal by a specified amount. Improved feed conversion ratio means lower feed conversion ratio. By "lower feed conversion ratio" or "improved feed conversion ratio" is meant that the use of the feed additive composition in the feed results in a reduction in the amount of feed required to feed the animal to increase its weight by the same amount as compared to the amount of feed required to increase the animal's weight to a specified amount when the feed does not contain the feed additive composition.
Feed efficiency: the term "feed efficiency" means the amount of weight gain per unit of feed when an animal is fed arbitrarily or a specified amount of food over a period of time. By "increased feed efficiency" is meant that the use of the feed additive composition according to the invention in a feed results in an increased weight gain per unit feed intake compared to animals fed the feed in the absence of the feed additive composition.
Fragment (b): the term "fragment" means a polypeptide lacking one or more (e.g., several) amino acids from the amino and/or carboxy terminus of the major portion of the mature polypeptide; wherein the fragment has enzymatic activity. In one aspect, a fragment contains at least 85% (e.g., at least 90%, or at least 95%) of the amino acid residues of the mature polypeptide of the enzyme.
Glucoamylases (glucan 1, 4-alpha-glucosidase, EC 3.2.1.3) are a group of enzymes that catalyze the sequential hydrolysis of terminal (1 → 4) -linked alpha-D-glucose residues from the nonreducing end of the chain and release beta-D-glucose.
Hemicellulolytic or hemicellulase: the term "hemicellulolytic enzyme" or "hemicellulase" means one or more (e.g., several) enzymes that hydrolyze hemicellulosic material. See, for example, Shallom and Shoham,2003, Microbial hemicellulases, Current Opinion In Microbiology 6(3) 219-. Hemicellulases are key components in the degradation of plant biomass. Examples of hemicellulases include, but are not limited to: acetyl mannan esterase, acetyl xylan esterase, arabinanase, arabinofuranosidase, coumaroyl esterase, feruloyl esterase, galactosidase, glucuronidase, mannanase, mannosidase, xylanase, and xylosidase. The substrates of these enzymes (hemicelluloses) are a heterogeneous population of branched and linear polysaccharides that bind via hydrogen bonds to cellulose microfibrils in the plant cell wall, thereby cross-linking them into a robust network. Hemicellulose is also covalently attached to lignin, forming a highly complex structure with cellulose. The variable structure and organization of hemicellulose requires the synergistic action of many enzymes to completely degrade it. The catalytic module of hemicellulases is a Glycoside Hydrolase (GH) which hydrolyzes glycosidic linkages, or a Carbohydrate Esterase (CE) which hydrolyzes ester linkages of the acetate or ferulate side groups. These catalytic modules can be assigned to GH and CE families by numerical labeling based on their primary sequence homology. Some families with overall similar folds may be further grouped into alphabetically labeled clans (e.g., GH-a). Information and updated classifications of these and other carbohydrate active enzymes are available in carbohydrate active enzyme (CAZy) databases. Hemicellulase activity may be measured at a suitable temperature, for example, 50 ℃, 55 ℃, or 60 ℃ according to Ghose and Bisaria,1987, Pure & Appl. Chern [ Pure and applied chemistry ]59: 1739-.
Host cell: the term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.
Separating: the term "isolated" means a substance in a form or environment not found in nature. Non-limiting examples of isolated substances include (1) any non-naturally occurring substance; (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide, or cofactor, which is at least partially removed from one or more or all of the naturally occurring components associated with its property; (3) any substance that is modified by man relative to substances found in nature; or (4) any substance that is modified by increasing the amount of the substance relative to other components that are intrinsically associated with the substance (e.g., multiple copies of a gene encoding the substance, using a promoter that is stronger than the promoter intrinsically associated with the gene encoding the substance). The isolated material may be present in a sample of fermentation broth.
Mature polypeptide: the term "mature polypeptide" means a polypeptide that is in its final form following translation and any post-translational modifications such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, and the like.
In one aspect, the mature polypeptide of the Microbacterium oxydans xylanase is amino acids 33 to 884 of SEQ ID NO:1, and amino acids 1 to 32 of SEQ ID NO:1 are signal peptides. In one aspect, the mature polypeptide of the microbacterium hydrocarbonoxydans xylanase is amino acids 35 to 890 of SEQ ID NO:3, and amino acids 1 to 34 of SEQ ID NO:3 are signal peptides. In one aspect, the mature polypeptide of the microbacterium species SA39 xylanase is amino acids 35 to 890 of SEQ ID No. 4, and amino acids 1 to 34 of SEQ ID No. 4 are signal peptides. In one aspect, the mature polypeptide of the Bacillus glucanase of Gluconobacter xylanases is amino acids 31 to 826 of SEQ ID NO 5 and amino acids 1 to 30 of SEQ ID NO 5 are signal peptides. In one aspect, the mature polypeptide of Paenibacillus terrae xylanase is amino acids 35 to 831 of SEQ ID NO. 7, and amino acids 1 to 34 of SEQ ID NO. 7 are signal peptides. In one aspect, the mature polypeptide of Paenibacillus terrae xylanase is amino acids 35 to 831 of SEQ ID NO:9, and amino acids 1 to 34 of SEQ ID NO:9 are signal peptides. In one aspect, the mature polypeptide of the Paenibacillus species DMB20 xylanase is amino acids 1 to 794 of SEQ ID NO 10. In one aspect, the mature polypeptide of Aspergillus fumigatus cellobiohydrolase I is amino acids 27 to 532 of SEQ ID NO. 15, and amino acids 1 to 26 of SEQ ID NO. 15 are signal peptides. In another aspect, the mature polypeptide of Aspergillus fumigatus cellobiohydrolase II is amino acids 20 to 454 of SEQ ID NO 16, and amino acids 1 to 19 of SEQ ID NO 16 are signal peptides. In another aspect, the mature polypeptide of Aspergillus fumigatus beta-glucosidase is amino acids 20 to 863 of SEQ ID NO:17, and amino acids 1 to 19 of SEQ ID NO:17 are signal peptides. In another aspect, the mature polypeptide of the Penicillium species (Penicillium sp.) GH61 polypeptide is amino acids 26 to 253 of SEQ ID No. 18, and amino acids 1 to 25 of SEQ ID No. 18 are signal peptides.
It is known in the art that host cells can produce a mixture of two or more different mature polypeptides (i.e., having different C-terminal and/or N-terminal amino acids) expressed from the same polynucleotide. It is also known in the art that different host cells process polypeptides differently, and thus one host cell expressing a polynucleotide may produce a different mature polypeptide (e.g., having a different C-terminal and/or N-terminal amino acid) when compared to another host cell expressing the same polynucleotide.
Mature polypeptide coding sequence: the term "mature polypeptide coding sequence" means a polynucleotide that encodes a mature polypeptide.
Mutant: the term "mutant" means a polynucleotide encoding a variant.
Nucleic acid construct: the term "nucleic acid construct" means a nucleic acid molecule, either single-or double-stranded, that is isolated from a naturally occurring gene or modified to contain segments of nucleic acids in a manner not otherwise found in nature, or that is synthetic, that contains one or more control sequences.
Nutrient digestibility: the term "nutrient digestibility" means the fraction of a nutrient that disappears from the gastrointestinal tract or a designated segment of the gastrointestinal tract (e.g., the small intestine). Nutrient digestibility may be measured as the difference between the nutrient administered to the subject and the nutrient excreted in the stool of the subject, or the difference between the nutrient administered to the subject and the nutrient retained in the digest over a designated segment of the gastrointestinal tract (e.g., the ileum).
Nutrient digestibility as used herein can be measured by: the difference between the intake of nutrients over a period of time and the excreted nutrients obtained by total collection of the excreta; or using inert markers that are not absorbed by the animal and allow the researcher to calculate the amount of nutrients that disappear throughout the gastrointestinal tract or a segment of the gastrointestinal tract. Such inert markers may be titanium dioxide, chromium oxide, or acid insoluble ash. Digestibility may be expressed as the percentage of nutrients in the feed, or as the mass unit of digestible nutrients/mass unit of nutrients in the feed. Nutrient digestibility as used herein encompasses starch digestibility, fat digestibility, protein digestibility and amino acid digestibility.
Energy digestibility, as used herein, means the total energy of feed consumed minus the total energy of feces, or the total energy of feed consumed minus the total energy of remaining digesta on a specified segment of the animal's gastrointestinal tract (e.g., ileum).
Metabolizable energy as used herein refers to the apparent metabolizable energy and means the total energy of feed consumed minus the total energy contained in feces, urine and digested gas products. Energy digestibility and metabolic energy can be measured as the difference between total energy intake and total energy in digesta excreted in feces or present in a designated segment of the gastrointestinal tract, suitably corrected for nitrogen excretion to calculate the metabolic energy of the feed using the same method as measuring nutrient digestibility.
Operatively connected to: the term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs the expression of the coding sequence.
Percent dissolved xylan: the term "percent solubilized xylan" means the amount of xylose measured in the supernatant after incubation with the enzyme compared to the total amount of xylose present in the substrate prior to enzyme incubation. For the purposes of the present invention, the percent of xylan solubilized can be calculated using defatted, de-starched maize (DFDSM) as the substrate. DFDSM was prepared in the experimental part according to "preparation of defatted, de-starchy maize (DFDSM)". The percent solubilized xylan from defatted, de-starched maize (DFDSM) can be determined as described herein in the "xylose solubilization assay" using reaction conditions of 20 μ g enzyme/g DFDSM and incubation at 40 ℃, pH 5 for 2.5 hours. Thus the term "performed under reaction conditions of 20 μ g xylanase variant/g defatted, de-starched maize (DFDSM) and incubation at 40 ℃, pH 5 for 2.5 hours" is understood to mean calculating the percentage of solubilized xylan as described herein in the "xylose solubilization assay".
In a more detailed example, in 100mM sodium acetate, 5mM CaCl2A2% (w/w) DFDSM suspension was prepared (pH 5) and allowed to hydrate for 30min at room temperature with gentle stirring. After hydration, 200 μ l of the substrate suspension was pipetted into a 96-well plate and mixed with 20 μ l of enzyme solution to obtain a final enzyme concentration of 20PPM (20 μ g enzyme/g substrate) relative to the substrate. The enzyme/substrate mixture was hydrolyzed by standing at 40 ℃ for 2.5h in a plate incubator with gentle agitation (500 RPM). After enzymatic hydrolysis, the enzyme/substrate plate was centrifuged at 3000RPM for 10min and 50 μ l of the supernatant was mixed with 100 μ l of 1.6M HCl and transferred to a 300 μ l PCR tube and left to hydrolyze acid for 40min at 90 ℃ in a PCR instrument. Samples were neutralized with 125 μ l 1.4M NaOH after acid hydrolysis and loaded onto HPAE-PAD for monosaccharide analysis.
Polypeptide having cellulolytic enhancing activity: the term "polypeptide having cellulolytic enhancing activity" means a GH61 polypeptide that catalyzes the enhancement of hydrolysis of a cellulosic material by an enzyme having cellulolytic activity. For the purposes of the present invention, cellulolytic enhancing activity is determined by measuring the increase in reducing sugars or the increase in the total amount of cellobiose and glucose from the hydrolysis of a cellulosic material by a cellulolytic enzyme under the following conditions: 1-50mg total protein per gram of cellulose in PCS, wherein the total protein comprises 50-99.5% w/w of a cellulolytic enzyme protein and 0.5-50% w/w of a GH61 polypeptide having cellulolytic enhancing activity The protein, at appropriate temperature (e.g., 50 ℃, 55 ℃, or 60 ℃) and pH (e.g., 5.0 or 5.5 ℃), for 1-7 days, compared to an equivalent total protein loading control hydrolysis without cellulolytic enhancing activity (1-50mg cellulolytic protein/g cellulose in PCS). In one aspect, the Aspergillus oryzae beta-glucosidase (recombinantly produced in Aspergillus oryzae according to WO 02/095014) is loaded in the presence of cellulase proteins at 2% to 3% total protein weight Aspergillus oryzae beta-glucosidase (recombinantly produced in Aspergillus oryzae as described in WO 2002/095014) or at 2% to 3% total protein weight Aspergillus fumigatus beta-glucosidase (recombinantly produced in Aspergillus oryzae)
Figure BDA0003162048240000201
1.5L (Novozymes A/S), Denmark Baggesverde (R) ((R))
Figure BDA0003162048240000202
Denmark)) was used as a source of cellulolytic activity.
A GH61 polypeptide having cellulolytic enhancing activity enhances hydrolysis of a cellulosic material catalyzed by a enzyme having cellulolytic activity by reducing the amount of cellulolytic enzyme required to achieve the same degree of hydrolysis, preferably by at least 1.01-fold, e.g., by at least 1.05-fold, at least 1.10-fold, at least 1.25-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or at least 20-fold.
Sequence identity: the degree of relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity". For The purposes of The present invention, The sequence identity between two amino acid sequences is determined using The Needman-Wunsch algorithm (Needman-Wunsch algoritm) (Needleman and Wunsch,1970, J.Mol.biol. [ J.M. 48: 443-) as implemented in The Nidel program of The EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al 2000, Trends Genet. [ genetic Trends ]16: 276-) (e.g., version 5.0.0 or later). The parameters used are gap opening penalty of 10, gap extension penalty of 0.5, and EBLOSUM62 (EMBOSS version of BLOSUM 62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the non-reduced option) is used as the percent identity and is calculated as follows:
(same residue x 100)/(alignment Length-total number of vacancies in alignment)
For The purposes of The present invention, The sequence identity between two deoxynucleotide sequences is determined using The Needman-Wolsch algorithm (Needleman and Wunsch,1970, supra) as implemented in The Nidel program of The EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al, 2000, supra) (e.g., version 5.0.0 or later). The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC 4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the non-reduced option) is used as the percent identity and is calculated as follows:
(identical deoxyribonucleotides x 100)/(alignment length-total number of vacancies in alignment)
Variants: the term "variant" means a polypeptide having an enzyme or enzyme-enhancing activity that comprises an alteration (i.e., a substitution, insertion, and/or deletion) at one or more (e.g., several) positions. Substitution means that an amino acid occupying a certain position is substituted with a different amino acid, deletion means that the amino acid occupying a certain position is removed, and insertion means that an amino acid is added next to and immediately after the amino acid occupying a certain position.
Wild-type xylanase: the term "wild-type" xylanase means a xylanase expressed by a naturally occurring microorganism (e.g., a bacterium, yeast, or filamentous fungus) found in nature.
Xylanase: the term "xylanase" means an endo-1, 4-beta-xylanase (e.c.3.2.1.8) which catalyzes the internal hydrolysis of 1, 4-beta-D-xylosidic bonds in xylan. For the purposes of the present invention, xylanase activity may be determined using 0.2% AZCL-arabinoxylan as substrate in 0.01% TRITON (R) X-100 and 200mM sodium phosphate buffer (pH 6) at 37 ℃ or 0.2% AZCL-xylan as substrate in 0.01% TRITON (R) X-100 and 20mM sodium acetate buffer (pH 5.0) at 50 ℃ (see example 17). One unit of xylanase activity is defined as producing 1.0 micromole of azurin per minute at 37 ℃, pH 6, with 0.2% AZCL-arabinoxylan as substrate in 200mM sodium phosphate (pH 6), or at 50 ℃, pH 5, with 0.2% AZCL-xylan as substrate in 20mM sodium acetate (pH 5). Alternatively, xylanase activity may be determined according to the assays described in the materials and methods section.
Variant naming conventions
For the purposes of the present invention, any of SEQ ID NOs 1, 3-5, 7, or 9-10 can be used to determine the corresponding amino acid residue in another xylanase. The amino acid sequence of The other xylanase is aligned to any of SEQ ID NO 1, 3-5, 7 or 9-10 and based on this alignment The amino acid position corresponding to any of SEQ ID NO 1, 3-5, 7 or 9-10 is determined using The Needman-Wunsch algorithm (Needman-Wunsch algoritm) (Needleman and Wunsch,1970, J.Mol.biol. [ J.Biol. [ J.Mbiol. ]48: 443) as implemented in The Nidel (Needle) program of The EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al 2000, Trends Genet. [ 16: 276. sup. ] 277) (e.g. version 5.0.0.0.0 or later). The parameters used are gap opening penalty of 10, gap extension penalty of 0.5, and EBLOSUM62 (EMBOSS version of BLOSUM 62) substitution matrix.
Another identification of corresponding amino acid residues in a xylanase can be determined by alignment of multiple polypeptide sequences using their corresponding default parameters using several computer programs including, but not limited to, MUSCLE (by log-desired multiple sequence comparison; version 3.5 or more; Edgar,2004, Nucleic Acids Research [ Nucleic acid Research ]32:1792-1794), MAFFT (version 6.857 or more; Katoh and Kuma,2002, Nucleic Acids Research [ Nucleic acid Research ]30: 3059-3066; Katoh et al, 2005, Nucleic Acids Research [ Nucleic acid Research ]33: 511-518; Katoh and Toh,2007, Bioinformatics [ Bioinformatics ]23: 372-374; Katoh et al, 2009, method in Molecular Biology [ Molecular Biology ] 39-537; version 2010, and Biopsoats et al; Biopsoatic information: 2010: 26; 2010, and optionally, 1994, Nucleic Acids Research [ Nucleic Acids Research ]22: 4673-4680).
Other pairwise sequence comparison algorithms can be used when other enzymes deviate from the polypeptide of any of SEQ ID NOs 1, 3-5, 7, or 9-10 such that conventional sequence-based comparison methods cannot detect their relationship (Lindahl and Elofsson,2000, J.mol.biol. [ J.Mol. [ J.Mol ]295:613- & 615). Higher sensitivity in sequence-based searches can be obtained using search programs that utilize probabilistic representations (profiles) of polypeptide families to search databases. For example, the PSI-BLAST program generates multiple spectra by iterative database search procedures and is capable of detecting distant homologues (Atschul et al, 1997, Nucleic Acids Res. [ Nucleic Acids research ]25: 3389-. Even greater sensitivity can be achieved if a family or superfamily of polypeptides has one or more representatives in a protein structure database. Programs such as GenTHREADER (Jones,1999, J.mol.biol. [ journal of molecular biology ]287: 797-815; McGuffin and Jones,2003, Bioinformatics [ Bioinformatics ]19:874-881) use information from a variety of sources (PSI-BLAST, secondary structure prediction, structural alignment profiles, and solvation potentials) as input to neural networks that predict the structural folding of query sequences. Similarly, the method of Gough et al, 2000, J.mol.biol. [ J. Mol. ]313: 903-. These alignments can in turn be used to generate homology models for polypeptides, and the accuracy of such models can be assessed using a variety of tools developed for this purpose.
For proteins of known structure, several tools and resources are available for searching and generating structural alignments. For example, the SCOP superfamily of proteins has been aligned structurally, and those alignments are accessible and downloadable. Two or more Protein structures may be aligned using a variety of algorithms such as distance alignment matrices (Holm and Sander,1998, Proteins [ Protein ]33:88-96) or combinatorial extensions (Shindyalov and Bourne,1998, Protein Engineering [ Protein Engineering ]11: 739-.
In describing variations of the invention, the nomenclature described below is adapted for ease of reference. Accepted IUPAC single letter or three letter amino acid abbreviations are used.
Substitution. For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Accordingly, substitution of threonine at position 226 with alanine is denoted as "Thr 226 Ala" or "T226A". Multiple mutations are separated by a plus sign ("+"), e.g., "Gly 205Arg + Ser411 Phe" or "G205R + S411F" represents the substitution of glycine (G) and serine (S) at positions 205 and 411 with arginine (R) and phenylalanine (F), respectively.
Absence of. For amino acid deletions, the following nomenclature is used: original amino acid, position,*. Accordingly, the deletion of glycine at position 195 is denoted as "Gly 195" or "G195". Multiple deletions are separated by a plus sign ("+"), e.g., "Gly 195 + Ser 411" or "G195 + S411".
Insert into. For amino acid insertions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Accordingly, insertion of a lysine after the glycine at position 195 is denoted as "Gly 195 GlyLys" or "G195 GK". The insertion of multiple amino acids is denoted as [ original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid # 2; etc. of]. For example, the insertion of lysine and alanine after glycine at position 195 is denoted as "Gly 195 GlyLysAla" or "G195 GKA". In such cases, the inserted one or more amino acid residues are numbered by adding a lower case letter to the position number of the amino acid residue preceding the inserted one or more amino acid residues. In the above example, it was shown that,the sequence would thus be:
parent strain: variants:
195 195 195a 195b
G G-K-A
multiple variations . Variants that include multiple alterations are separated by a plus sign ("+"), e.g., "Arg 170Tyr + Gly195 Glu" or "R170Y + G195E" representing substitutions of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively.
Different changes. Where different changes can be introduced at one position, the different changes are separated by a comma, e.g., "Arg 170Tyr, Glu" represents the substitution of arginine at position 170 with tyrosine or glutamic acid. Thus, "Tyr 167Gly, Ala + Arg170Gly, Ala" denotes the following variants:
"Tyr 167Gly + Arg170 Gly", "Tyr 167Gly + Arg170 Ala", "Tyr 167Ala + Arg170 Gly", and "Tyr 167Ala + Arg170 Ala".
Detailed Description
The present invention relates to a novel GH98 xylanase, an enzyme blend or composition comprising the novel GH98 xylanase, and its use in a process for improving the nutritional quality of Distillers Dried Grains (DDG) or solubles-containing Distillers Dried Grains (DDGs) produced as a byproduct of a fermentation product production process, a process for producing a fermentation product, and a process for solubilizing fiber, preferably for solubilizing xylose and arabinose.
DDGS is typically fed to cattle because high fiber content limits the nutritional value of monogastric animals (e.g., poultry and swine). Therefore, there is a need for a solution that specifically improves the nutritional value of DDGS for monogastric animals. By dissolving part of the fiber, the nutritional value for monogastric animals can be increased. One solution to dissolve the fiber is to add enzymes to the feed blend, however, the shorter residence time and less than ideal in vivo conditions limit the efficacy of the enzymes added to the feed.
The work described herein shows that upstream addition of a GH98 xylanase of the disclosure or an enzyme blend comprising the GH98 xylanase during a fermentation product production process (e.g., during simultaneous saccharification and fermentation) can significantly increase the degree of fibrolysis. Unexpectedly, as an added benefit, the GH98 xylanase of the disclosure, or an enzyme blend or composition comprising the GH98 xylanase, significantly increases the degree of fiber solubilization without causing the DDGS to darken during the drying process.
I. Polypeptides having xylanase activity
In embodiments, the invention relates to polypeptides having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID No. 1, which polypeptides have xylanase activity. In one aspect, the polypeptide differs from the mature polypeptide of SEQ ID NO:1 by up to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids. In embodiments, the polypeptide having xylanase activity comprises, consists of, or consists essentially of the mature polypeptide of SEQ ID NO: 1.
In embodiments, the invention relates to polypeptides having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID No. 3, which polypeptides have xylanase activity. In one aspect, the polypeptide differs from the mature polypeptide of SEQ ID NO:3 by up to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids. In embodiments, the polypeptide having xylanase activity comprises, consists of, or consists essentially of the mature polypeptide of SEQ ID No. 3.
In embodiments, the invention relates to polypeptides having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID No. 3, which polypeptides have xylanase activity. In one aspect, the polypeptide differs from the mature polypeptide of SEQ ID No. 4 by up to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids. In embodiments, the polypeptide having xylanase activity comprises, consists of, or consists essentially of the mature polypeptide of SEQ ID No. 4.
In particular embodiments, the invention relates to a polypeptide having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID No. 1, and wherein the polypeptide has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% xylanase activity of the mature polypeptide of SEQ ID No. 1.
In particular embodiments, the invention relates to a polypeptide having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID No. 3, and wherein the polypeptide has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% xylanase activity of the mature polypeptide of SEQ ID No. 1. In particular embodiments, the invention relates to a polypeptide having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID No. 3, and wherein the polypeptide has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% xylanase activity of the mature polypeptide of SEQ ID No. 3.
In particular embodiments, the invention relates to a polypeptide having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID No. 4, and wherein the polypeptide has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% xylanase activity of the mature polypeptide of SEQ ID No. 1.
In particular embodiments, the invention relates to a polypeptide having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID No. 4, and wherein the polypeptide has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% xylanase activity of the mature polypeptide of SEQ ID No. 4.
The polynucleotide of SEQ ID NO. 2 or subsequences thereof, and the polypeptide of SEQ ID NO. 2 or fragments thereof, can be used to design nucleic acid probes according to methods well known in the art to identify and clone DNA encoding polypeptides having protease activity from strains of different genera or species. In particular, standard southern blotting procedures can be followed, using this The probe-like probes are hybridized with the genomic DNA or cDNA of the target cell to identify and isolate the corresponding gene therein. Such probes may be significantly shorter than the complete sequence, but should be at least 15, such as at least 25, at least 35, or at least 70 nucleotides in length. Preferably, the nucleic acid probe is at least 100 nucleotides in length, e.g., at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in length. Both DNA and RNA probes may be used. The probes are typically labeled (e.g., with)32P、3H、35S, biotin, or avidin) to detect the corresponding gene. Such probes are encompassed by the present invention.
Genomic DNA or cDNA libraries prepared from such other strains may be screened for DNA that hybridizes with the probes described above and encodes a polypeptide having protease activity. Genomic or other DNA from such other strains may be separated by agarose or polyacrylamide gel electrophoresis or other separation techniques. The DNA from the library or the isolated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. To identify clones or DNA hybridizing to SEQ ID NO 2 or subsequences thereof, vector material was used in southern blots.
For the purposes of the present invention, hybridization indicates that the polynucleotide hybridizes to a labeled nucleic acid probe corresponding to: (i) 2, SEQ ID NO; (ii) mature polypeptide coding sequence of SEQ ID NO. 2; (iii) the full-length complementary sequence thereof; or (iv) a subsequence thereof; hybridization is performed under very low to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X-ray film or any other detection means known in the art.
In one aspect, the nucleic acid probe is nucleotides 1 to 2655 of SEQ ID NO. 2. In another aspect, the nucleic acid probe is a polynucleotide encoding: 1, a mature polypeptide thereof, or a fragment thereof. In another aspect, the nucleic acid probe is SEQ ID NO 2. In another aspect, the nucleic acid probe is a polynucleotide encoding: 3, a mature polypeptide thereof, or a fragment thereof. In another aspect, the nucleic acid probe is a polynucleotide encoding: 4, a mature polypeptide thereof, or a fragment thereof.
In another embodiment, the invention relates to a polypeptide having xylanase activity encoded by a polynucleotide having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO. 2. In further embodiments, the polypeptide has been isolated.
In another embodiment, the invention relates to variants of the mature polypeptide of SEQ ID NO. 1 comprising substitutions, deletions, and/or insertions at one or more (e.g., several) positions. In embodiments, the number of amino acid substitutions, deletions, and/or insertions introduced into the mature polypeptide of SEQ ID No. 1 is up to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the invention relates to variants of the mature polypeptide of SEQ ID NO. 3 comprising substitutions, deletions, and/or insertions at one or more (e.g., several) positions. In embodiments, the number of amino acid substitutions, deletions, and/or insertions introduced into the mature polypeptide of SEQ ID No. 3 is up to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the invention relates to variants of the mature polypeptide of SEQ ID NO. 4 comprising substitutions, deletions, and/or insertions at one or more (e.g., several) positions. In embodiments, the number of amino acid substitutions, deletions, and/or insertions introduced into the mature polypeptide of SEQ ID No. 4 is up to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
In embodiments, the invention relates to polypeptides having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID No. 5, which polypeptides have xylanase activity. In one aspect, the polypeptide differs from the mature polypeptide of SEQ ID NO:5 by up to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids. In embodiments, the polypeptide having xylanase activity comprises, consists of, or consists essentially of the mature polypeptide of SEQ ID No. 5.
In particular embodiments, the invention relates to a polypeptide having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID No. 5, and wherein the polypeptide has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% xylanase activity of the mature polypeptide of SEQ ID No. 5.
The polynucleotide of SEQ ID NO. 6 or subsequences thereof, and the polypeptide of SEQ ID NO. 5 or fragments thereof can be used to design nucleic acid probes according to methods well known in the art to identify and clone DNA encoding a polypeptide having protease activity from strains of different genera or species. In particular, such probes can be used to hybridize to genomic DNA or cDNA of a cell of interest following standard southern blotting procedures in order to identify and isolate the corresponding gene therein. Such probes may be significantly shorter than the complete sequence, but should be at least 15, such as at least 25, at least 35, or at least 70 nucleotides in length. Preferably, the nucleic acid probe is at least 100 nucleotides in length, e.g., at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in length. Both DNA and RNA probes may be used. The probes are typically labeled (e.g., with) 32P、3H、35S, biotin, or avidin) to detect the corresponding gene. Such probes are encompassed by the present invention.
Genomic DNA or cDNA libraries prepared from such other strains may be screened for DNA that hybridizes with the probes described above and encodes a polypeptide having protease activity. Genomic or other DNA from such other strains may be separated by agarose or polyacrylamide gel electrophoresis or other separation techniques. The DNA from the library or the isolated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. To identify clones or DNA hybridizing to SEQ ID NO 6 or subsequences thereof, vector material was used in southern blots.
For the purposes of the present invention, hybridization indicates that the polynucleotide hybridizes to a labeled nucleic acid probe corresponding to: (i) 6, SEQ ID NO; (ii) mature polypeptide coding sequence of SEQ ID NO 6; (iii) the full-length complementary sequence thereof; or (iv) a subsequence thereof; hybridization is performed under very low to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X-ray film or any other detection means known in the art.
In one aspect, the nucleic acid probe is nucleotides 1 to 2481 of SEQ ID NO 6. In another aspect, the nucleic acid probe is a polynucleotide encoding: 5, a mature polypeptide thereof, or a fragment thereof. In another aspect, the nucleic acid probe is SEQ ID NO 6.
In another embodiment, the invention relates to a polypeptide having xylanase activity encoded by a polynucleotide having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO 6. In further embodiments, the polypeptide has been isolated.
In another embodiment, the invention relates to variants of the mature polypeptide of SEQ ID NO. 5 comprising substitutions, deletions, and/or insertions at one or more (e.g., several) positions. In embodiments, the number of amino acid substitutions, deletions, and/or insertions introduced into the mature polypeptide of SEQ ID No. 5 is up to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the invention relates to variants of the mature polypeptide of SEQ ID NO. 5 comprising substitutions, deletions, and/or insertions at one or more (e.g., several) positions. In embodiments, the number of amino acid substitutions, deletions, and/or insertions introduced into the mature polypeptide of SEQ ID No. 5 is up to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
In embodiments, the invention relates to polypeptides having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID No. 7, which polypeptides have xylanase activity. In one aspect, the polypeptide differs from the mature polypeptide of SEQ ID No. 7 by up to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids. In embodiments, the polypeptide having xylanase activity comprises, consists of, or consists essentially of the mature polypeptide of SEQ ID No. 7.
In embodiments, the invention relates to polypeptides having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID No. 9, which polypeptides have xylanase activity. In one aspect, the polypeptide differs from the mature polypeptide of SEQ ID NO:9 by up to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids. In embodiments, the polypeptide having xylanase activity comprises, consists of, or consists essentially of the mature polypeptide of SEQ ID No. 9.
In embodiments, the invention relates to polypeptides having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID No. 10, which polypeptides have xylanase activity. In one aspect, the polypeptide differs from the mature polypeptide of SEQ ID NO 10 by up to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids. In embodiments, the polypeptide having xylanase activity comprises, consists of, or consists essentially of the mature polypeptide of SEQ ID NO: 10.
In particular embodiments, the invention relates to a polypeptide having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID No. 7, and wherein the polypeptide has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% xylanase activity of the mature polypeptide of SEQ ID No. 7.
In particular embodiments, the invention relates to a polypeptide having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID No. 9, and wherein the polypeptide has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% xylanase activity of the mature polypeptide of SEQ ID No. 7.
In particular embodiments, the invention relates to a polypeptide having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID No. 9, and wherein the polypeptide has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% xylanase activity of the mature polypeptide of SEQ ID No. 9.
In particular embodiments, the invention relates to a polypeptide having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID No. 10, and wherein the polypeptide has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% xylanase activity of the mature polypeptide of SEQ ID No. 7.
In particular embodiments, the invention relates to a polypeptide having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID No. 10, and wherein the polypeptide has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% xylanase activity of the mature polypeptide of SEQ ID No. 10.
The polynucleotide of SEQ ID NO. 8 or subsequences thereof, and the polypeptide of SEQ ID NO. 8 or fragments thereof, can be used to design nucleic acid probes according to methods well known in the art to identify and clone DNA encoding polypeptides having protease activity from strains of different genera or species. In particular, such probes can be used to hybridize to genomic DNA or cDNA of a cell of interest following standard southern blotting procedures in order to identify and isolate the corresponding gene therein. Such probes may be significantly shorter than the complete sequence, but should be at least 15, such as at least 25, at least 35, or at least 70 nucleotides in length. Preferably, the nucleic acid probe is at least 100 nucleotides in length, e.g., at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in length. Both DNA and RNA probes may be used. The probes are typically labeled (e.g., with) 32P、3H、35S, biotin, or avidin) to detectThe corresponding gene. Such probes are encompassed by the present invention.
Genomic DNA or cDNA libraries prepared from such other strains may be screened for DNA that hybridizes with the probes described above and encodes a polypeptide having protease activity. Genomic or other DNA from such other strains may be separated by agarose or polyacrylamide gel electrophoresis or other separation techniques. The DNA from the library or the isolated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. To identify clones or DNA hybridizing to SEQ ID NO 8 or subsequences thereof, vector material was used in southern blots.
For the purposes of the present invention, hybridization indicates that the polynucleotide hybridizes to a labeled nucleic acid probe corresponding to: (i) 8 in SEQ ID NO; (ii) mature polypeptide coding sequence of SEQ ID NO. 2; (iii) the full-length complementary sequence thereof; or (iv) a subsequence thereof; hybridization is performed under very low to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X-ray film or any other detection means known in the art.
In one aspect, the nucleic acid probe is nucleotides 1 to 2496 of SEQ ID NO. 8. In another aspect, the nucleic acid probe is a polynucleotide encoding: 7, a mature polypeptide thereof, or a fragment thereof. In another aspect, the nucleic acid probe is SEQ ID NO 8. In another aspect, the nucleic acid probe is a polynucleotide encoding: a polypeptide of SEQ ID NO 9, a mature polypeptide thereof, or a fragment thereof. In another aspect, the nucleic acid probe is a polynucleotide encoding: 10, a mature polypeptide thereof, or a fragment thereof.
In another embodiment, the invention relates to a polypeptide having xylanase activity encoded by a polynucleotide having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO. 8. In further embodiments, the polypeptide has been isolated.
In another embodiment, the invention relates to variants of the mature polypeptide of SEQ ID NO. 7 comprising substitutions, deletions, and/or insertions at one or more (e.g., several) positions. In embodiments, the number of amino acid substitutions, deletions, and/or insertions introduced into the mature polypeptide of SEQ ID No. 7 is up to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the invention relates to variants of the mature polypeptide of SEQ ID NO 9 comprising substitutions, deletions, and/or insertions at one or more (e.g., several) positions. In embodiments, the number of amino acid substitutions, deletions, and/or insertions introduced into the mature polypeptide of SEQ ID No. 9 is up to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the invention relates to variants of the mature polypeptide of SEQ ID NO 10 comprising substitutions, deletions, and/or insertions at one or more (e.g., several) positions. In embodiments, the number of amino acid substitutions, deletions, and/or insertions introduced into the mature polypeptide of SEQ ID No. 10 is up to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
Amino acid changes can be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; typically a small deletion of 1-30 amino acids; small amino-terminal or carboxy-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by altering the net charge or another function (e.g., a polyhistidine segment, an epitope, or a binding domain).
Examples of conservative substitutions are within the following groups: basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions which do not normally alter specific activity are known in The art and are described, for example, by H.Neurath and R.L.Hill,1979, in The Proteins, Academic Press, N.Y.. Common substitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly.
Essential amino acids in polypeptides can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells,1989, Science 244: 1081-1085). In the latter technique, a single alanine mutation is introduced at each residue in the molecule, and the resulting molecule is tested for protease activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al, 1996, J.biol.chem. [ J.Biol ]271: 4699-4708. The active site of an enzyme or other biological interaction can also be determined by physical analysis of the structure, as determined by techniques such as: nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, along with mutating putative contact site amino acids. See, e.g., de Vos et al, 1992, Science [ Science ]255: 306-); smith et al, 1992, J.mol.biol. [ J.Mol.224: 899-); wlodaver et al, 1992, FEBS Lett. [ Provisions of the European Association of biochemistry ]309: 59-64. The identity of the essential amino acids can also be inferred from alignment with the relevant polypeptide.
Single or multiple amino acid substitutions, deletions and/or insertions can be made and tested using known mutagenesis, recombination and/or shuffling methods, followed by relevant screening procedures such as those described by Reidhaar-Olson and Sauer,1988, Science [ Science ]241: 53-57; bowie and Sauer,1989, Proc. Natl. Acad. Sci. USA [ Proc. Natl. Acad. Sci. ]86: 2152-2156; WO 95/17413; or those disclosed in WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al, 1991, Biochemistry [ Biochemistry ]30: 10832-.
The mutagenesis/shuffling approach can be combined with high throughput, automated screening methods to detect the activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al, 1999, Nature Biotechnology [ Nature Biotechnology ]17: 893-896). Mutagenized DNA molecules encoding active polypeptides can be recovered from the host cells and rapidly sequenced using methods standard in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.
The polypeptides may be hybrid polypeptides in which a region of one polypeptide is fused at the N-terminus or C-terminus of a region of another polypeptide.
The polypeptide may be a fusion polypeptide or a cleavable fusion polypeptide in which another polypeptide is fused at the N-terminus or C-terminus of the polypeptide of the invention. Fusion polypeptides are produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the invention. Techniques for producing fusion polypeptides are known in the art and include ligating the coding sequences encoding the polypeptides such that they are in frame and expression of the fusion polypeptide is under the control of one or more of the same promoter and terminator. Fusion polypeptides can also be constructed using intein technology, where the fusion polypeptide is produced post-translationally (Cooper et al, 1993, EMBO J. [ J. European society of molecular biology ]12: 2575-.
The fusion polypeptide may further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved, thereby releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in the following documents: martin et al, 2003, J.Ind.Microbiol.Biotechnol. [ journal of Industrial microorganism Biotechnology ]3: 568-576; svetina et al 2000, J.Biotechnol. [ J.Biotechnology ]76: 245-; Rasmussen-Wilson et al 1997, appl. environ. Microbiol. [ application and environmental microbiology ]63: 3488-; ward et al, 1995, Biotechnology [ Biotechnology ]13: 498-503; and Contreras et al, 1991, Biotechnology [ Biotechnology ]9: 378-; eaton et al, 1986, Biochemistry [ Biochemistry ]25: 505-512; Collins-Racie et al, 1995, Biotechnology [ Biotechnology ]13: 982-; carter et al, 1989, Proteins: Structure, Function, and Genetics [ Proteins: structure, function, and genetics ]6: 240-; and Stevens,2003, Drug Discovery World 4: 35-48.
In one aspect, a signal peptide of a polypeptide having xylanase activity is replaced with another signal peptide to enhance expression of the polypeptide. In one embodiment, the signal peptide is from Bacillus, e.g., the Bacillus clausii signal peptide of SEQ ID NO: 34. In the examples, the signal peptide of the polypeptide of SEQ ID NO. 1 is replaced by the signal peptide of SEQ ID NO. 34. In the examples, the signal peptide of the polypeptide of SEQ ID NO. 3 is replaced by the signal peptide of SEQ ID NO. 34. In the examples, the signal peptide of the polypeptide of SEQ ID NO. 4 is replaced by the signal peptide of SEQ ID NO. 34. In the examples, the signal peptide of the polypeptide of SEQ ID NO. 5 is replaced by the signal peptide of SEQ ID NO. 34. In the examples, the signal peptide of the polypeptide of SEQ ID NO. 7 is replaced by the signal peptide of SEQ ID NO. 34. In the examples, the signal peptide of the polypeptide of SEQ ID NO. 9 is replaced by the signal peptide of SEQ ID NO. 34. In the examples, the signal peptide of the polypeptide of SEQ ID NO. 10 is replaced by the signal peptide of SEQ ID NO. 34.
In embodiments, the polypeptide having xylanase activity comprises a tag, e.g., to facilitate purification of the polypeptide. Purification tags are well known in the art. For example, the polypeptide may include a polyhistidine tag. In an embodiment, the polypeptide having xylanase activity has a polyhistidine tag comprising or consisting of the amino acid sequence shown in SEQ ID No. 35. In an embodiment, the mature polypeptide of SEQ ID NO. 1 or a variant thereof has a polyhistidine tag comprising or consisting of the amino acid sequence shown in SEQ ID NO. 35. In an embodiment, the mature polypeptide of SEQ ID NO. 3 or a variant thereof has a polyhistidine tag comprising or consisting of the amino acid sequence shown in SEQ ID NO. 35. In an embodiment, the mature polypeptide of SEQ ID NO. 4 or a variant thereof has a polyhistidine tag comprising or consisting of the amino acid sequence shown in SEQ ID NO. 35. In an embodiment, the mature polypeptide of SEQ ID No. 5 or a variant thereof has a polyhistidine tag comprising or consisting of the amino acid sequence shown in SEQ ID No. 35. In an embodiment, the mature polypeptide of SEQ ID NO. 7 or a variant thereof has a polyhistidine tag comprising or consisting of the amino acid sequence shown in SEQ ID NO. 35. In an embodiment, the mature polypeptide of SEQ ID NO. 9 or a variant thereof has a polyhistidine tag comprising or consisting of the amino acid sequence shown in SEQ ID NO. 35. In an embodiment, the mature polypeptide of SEQ ID NO. 10 or a variant thereof has a polyhistidine tag comprising or consisting of the amino acid sequence shown in SEQ ID NO. 35.
Sources of Polypeptides having protease Activity
The polypeptide having a xylanase activity of the present invention can be obtained from a microorganism belonging to the genus Microbacterium. In one aspect, the polypeptide having xylanase activity is a microbacterium oxydans polypeptide, e.g., a microbacterium oxydans xylanase of SEQ ID NO:1 or a variant thereof, which variant has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto. In embodiments, the variant is a Microbacterium hydrocarbonoxydans polypeptide of SEQ ID NO 3. In an embodiment, the variant is a Microbacterium species SA39 polypeptide of SEQ ID NO. 4.
The polypeptide having a xylanase activity of the present invention can be obtained from a microorganism of the genus paenibacillus. In one aspect, the polypeptide having xylanase activity is a bacillus glucanase polypeptide, e.g., a bacillus glucanase polypeptide of SEQ ID No. 5, or a variant thereof, which variant has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto. In another aspect, the polypeptide having xylanase activity is a paenibacillus terrae polypeptide, e.g., a paenibacillus terrae polypeptide of SEQ ID No. 7 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto. In an embodiment, the variant is the Paenibacillus terrae polypeptide of SEQ ID NO 9. In an embodiment, the variant is the Paenibacillus species DMB20 polypeptide of SEQ ID NO 10.
Strains of these species are readily available to the public at many Culture collections, such as the American Type Culture Collection (ATCC), the German Culture Collection of microorganisms (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ), the Dutch cultures Collection (CBS), and the Northern Regional Research Center of the American Agricultural Research Service Culture Collection (NRRL).
The above-mentioned probes can be used to identify and obtain the polypeptide from other sources, including microorganisms isolated from nature (e.g., soil, compost, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, compost, water, etc.). Techniques for the direct isolation of microorganisms and DNA from natural habitats are well known in the art. The polynucleotide encoding the polypeptide can then be obtained by similarly screening a genomic DNA or cDNA library or a mixed DNA sample of another microorganism. Once a polynucleotide encoding a polypeptide has been detected using one or more probes, the polynucleotide can be isolated or cloned by using techniques known to those of ordinary skill in the art (see, e.g., Sambrook et al, 1989, supra).
Polynucleotides of
The invention also relates to polynucleotides encoding the polypeptides of the invention, as described herein. In embodiments, polynucleotides encoding the polypeptides of the invention have been isolated.
Techniques for isolating or cloning polynucleotides are known in the art and include isolation from genomic DNA or cDNA or a combination thereof. Cloning of polynucleotides from genomic DNA can be accomplished, for example, by detecting cloned DNA fragments with shared structural features using the well-known Polymerase Chain Reaction (PCR) or antibody screening of expression libraries. See, e.g., Innis et al, 1990, PCR: A Guide to Methods and Application [ PCR: method and application guide ], Academic Press, New York. Other nucleic acid amplification procedures such as Ligase Chain Reaction (LCR), Ligation Activated Transcription (LAT) and polynucleotide-based amplification (NASBA) can be used. The polynucleotide may also be cloned from a strain of the genus Microbacterium (in particular Microbacterium oxydans, Microbacterium hydrocarbonoxydans, Microbacterium species SA39) or a related organism and may thus, for example, be an allelic or species variant of the polypeptide coding region of the polynucleotide. The polynucleotide may be cloned from a strain of paenibacillus (e.g. bacillus glucanotrys, paenibacillus terrestris, paenibacillus species DMB20) or a related organism and thus, for example, may be an allelic or species variant of the polypeptide coding region of the polynucleotide.
Nucleic acid constructs
The invention also relates to nucleic acid constructs comprising a polynucleotide of the invention operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences. In particular embodiments, at least one control sequence is heterologous to the polynucleotide encoding a variant of the invention. Thus, the nucleic acid construct is not visible in nature.
Polynucleotides can be manipulated in a number of ways to provide for expression of a polypeptide. Depending on the expression vector, it may be desirable or necessary to manipulate the polynucleotide prior to its insertion into the vector. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art. The control sequence may be a promoter, i.e., a polynucleotide recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention. The promoter comprises transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any polynucleotide that exhibits transcriptional activity in the host cell, including variant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
Examples of suitable promoters for directing transcription of the nucleic acid construct of the invention in a bacterial host cell are promoters obtained from the following genes: bacillus amyloliquefaciens (Bacillus amyloliquefaciens) alpha-amylase Gene (amyQ), Bacillus licheniformis (Bacillus licheniformis) alpha-amylase Gene (amyL), Bacillus licheniformis penicillinase Gene (penP), Bacillus stearothermophilus (Bacillus stearothermophilus) maltogenic amylase Gene (amyM), Bacillus subtilis (Bacillus subtilis) levansucrase Gene (sacB), Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis cryIIIA Gene (Agaissse and Lereclus,1994, Molecular Microbiology [ Molecular Microbiology ]13:97-107), Escherichia coli (E.coli) lac operon, Escherichia coli trc promoter (Egon et al, 1988, Gene [ Gene ]69: 301), Streptomyces cyaneus (Streptomyces amyloliquefaciens) Gene (Acidac), Escherichia coli strain A Gene (beta. lactase Gene) 3775, Nature, USA 31. delta. 20, Natales. alpha. strain, Nature, USA 31. alpha. lactamase, Natales. alpha. lactamase, Nature strain 3775, USA. 20. alpha. lactamase, Nature, USA, 19727. alpha. lactamase, Nature, 19727. alpha. lactamase, Nature, S. kola, S. acidla, S. kola, S. ko, And the tac promoter (DeBoer et al, 1983, Proc. Natl. Acad. Sci. USA [ Proc. Natl. Acad. Sci. ]80: 21-25). Other promoters are described in Gilbert et al, 1980, Scientific American [ Scientific Americans ]242:74-94, "Useful proteins from recombinant bacteria ]; and Sambrook et al, 1989, supra. Examples of tandem promoters are disclosed in WO 99/43835.
The control sequence may also be a transcription terminator which is recognized by a host cell to terminate transcription. The terminator is operably linked to the 3' -terminus of the polynucleotide encoding the polypeptide. Any terminator which is functional in the host cell may be used in the present invention.
Preferred terminators for bacterial host cells are obtained from the following genes: bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).
The control sequence may also be an mRNA stability region downstream of the promoter and upstream of the coding sequence of the gene, which increases the expression of the gene.
Examples of suitable mRNA stabilizing regions are obtained from the following genes: bacillus thuringiensis cryIIIA gene (WO 94/25612) and Bacillus subtilis SP82 gene (Hue et al, 1995, Journal of Bacteriology 177: 3465-.
The control sequence may also be a leader sequence, a nontranslated region of an mRNA which is important for translation by the host cell. The leader sequence is operably linked to the 5' -terminus of the polynucleotide encoding the polypeptide. Any leader sequence that is functional in the host cell may be used.
The control sequence may also be a signal peptide coding region that codes for a signal peptide linked to the N-terminus of the polypeptide and directs the polypeptide into the cell's secretory pathway. The 5' end of the coding sequence of the polynucleotide may itself contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence encoding the polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. In cases where the coding sequence does not naturally contain a signal peptide coding sequence, an exogenous signal peptide coding sequence may be required. Alternatively, the foreign signal peptide coding sequence may simply replace the native signal peptide coding sequence to enhance secretion of the polypeptide. However, any signal peptide coding sequence that directs an expressed polypeptide into the secretory pathway of a host cell may be used.
Effective signal peptide coding sequences for use in bacterial host cells are those obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alpha-amylase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Other signal peptides are described by Simonen and Palva,1993, Microbiological Reviews [ Microbiological review ]57:109- & 137. In an embodiment, the signal peptide comprises or consists of the B.clausii signal peptide of SEQ ID NO 34.
The control sequence may also be a propeptide coding sequence that codes for a propeptide positioned at the N-terminus of a polypeptide. The resulting polypeptide is called a pro-enzyme (proenzyme) or propolypeptide (or zymogen in some cases). A propolypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence may be obtained from the following genes: bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei (Rhizomucor miehei) aspartic protease, and Saccharomyces cerevisiae alpha-factor.
In the case where both a signal peptide sequence and a propeptide sequence are present, the propeptide sequence is positioned next to the N-terminus of a polypeptide and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence.
V. expression vector
The present invention also relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals. The polynucleotide and control sequence may be joined together to produce a recombinant expression vector which may contain one or more convenient restriction sites to allow insertion or substitution of the polynucleotide encoding the polypeptide at such sites. In particular embodiments, at least one control sequence is heterologous to the polynucleotide of the invention. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector such that the coding sequence is operably linked with the appropriate control sequences for expression.
The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.
The vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for ensuring self-replication. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the genome and replicated together with the chromosome or chromosomes into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell may be used, or a transposon may be used.
The vector preferably contains one or more selectable markers that allow for convenient selection of transformed cells, transfected cells, transduced cells, and the like. A selectable marker is a gene the product of which provides biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
Examples of bacterial selectable markers are the Bacillus licheniformis or Bacillus subtilis dal genes, or markers that confer antibiotic resistance (e.g., ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance).
The selectable marker may be a dual selectable marker system as described in WO 2010/039889. In one aspect, the dual selectable marker is an hph-tk dual selectable marker system.
The vector preferably contains one or more elements that allow the vector to integrate into the genome of the host cell or the vector to replicate autonomously in the cell, independently of the genome.
For integration into the host cell genome, the vector may rely on the polynucleotide sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or nonhomologous recombination. Alternatively, the vector may contain additional polynucleotides for directing integration by homologous recombination into the host cell genome at one or more precise locations in one or more chromosomes. To increase the likelihood of integration at a precise location, the integrational elements should contain a sufficient number of nucleic acids, e.g., 100 to 10000 base pairs, 400 to 10000 base pairs, and 800 to 10000 base pairs, which have a high degree of sequence identity with the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding polynucleotides. Alternatively, the vector may be integrated into the genome of the host cell by non-homologous recombination.
For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicon mediating autonomous replication that functions in a cell. The term "origin of replication" or "plasmid replicon" means a polynucleotide that enables a plasmid or vector to replicate in vivo.
Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184, which allow replication in E.coli, and the origins of replication of plasmids pUB110, pE194, pTA1060, and pAM β 1, which allow replication in Bacillus.
More than one copy of a polynucleotide of the invention may be inserted into a host cell to increase production of the polypeptide. An increased copy number of the polynucleotide may be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide, wherein cells comprising amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, may be selected for by culturing the cells in the presence of the appropriate selectable agent.
Procedures for ligating the elements described above to construct the recombinant expression vectors of the invention are well known to those of ordinary skill in the art (see, e.g., Sambrook et al, 1989, supra).
VI. host cell
The present invention also relates to recombinant host cells comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of a polypeptide of the present invention. In one embodiment, the one or more control sequences are heterologous to the polynucleotide of the invention. The construct or vector comprising the polynucleotide is introduced into a host cell such that the construct or vector is maintained as a chromosomal integrant or as an autonomously replicating extra-chromosomal vector, as described earlier. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of host cell will depend to a large extent on the gene encoding the polypeptide and its source.
The host cell may be any cell useful in the recombinant production of a polypeptide of the invention, e.g., a prokaryote or a eukaryote.
The prokaryotic host cell may be any gram-positive bacterium. Gram-positive bacteria include, but are not limited to: bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Bacillus coli, Staphylococcus, Streptococcus and Streptomyces. Gram-negative bacteria include, but are not limited to: campylobacter (Campylobacter), Escherichia coli, Flavobacterium (Flavobacterium), Clostridium (Fusobacterium), Helicobacter (Helicobacter), Clavibacterium (Ilyobacter), Neisseria (Neisseria), Pseudomonas (Pseudomonas), Salmonella (Salmonella), and Ureabasma (Ureapasma).
The bacterial host cell may be any Bacillus cell including, but not limited to, Bacillus alkalophilus (Bacillus alkalophilus), Bacillus amyloliquefaciens, Bacillus brevis (Bacillus brevis), Bacillus circulans (Bacillus circulans), Bacillus clausii, Bacillus coagulans (Bacillus coagulousns), Bacillus firmus (Bacillus firmus), Bacillus lautus (Bacillus lautus), Bacillus lentus (Bacillus lentus), Bacillus licheniformis, Bacillus megaterium (Bacillus megaterium), Bacillus pumilus (Bacillus pumilus), Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.
Introduction of DNA into bacillus cells can be achieved by: protoplast transformation (see, e.g., Chang and Cohen,1979, mol.Gen. Genet. [ molecular and general genetics ]168: 111-. The introduction of DNA into E.coli cells can be achieved by: protoplast transformation (see, e.g., Hanahan,1983, J.mol.biol. [ J.Biol. ]166: 557-. The introduction of DNA into Streptomyces cells can be achieved by: protoplast transformation, electroporation (see, e.g., Gong et al, 2004, Folia Microbiol. (Praha) [ leaf-line microbiology (Bragg) ]49: 399-. The introduction of DNA into a Pseudomonas cell can be achieved by: electroporation (see, e.g., Choi et al, 2006, J. Microbiol. methods [ journal of microbiological methods ]64: 391-. The introduction of DNA into Streptococcus cells can be achieved by: natural competence (natural competence) (see, e.g., Perry and Kuramitsu,1981, infection. immun. [ infection and immunity ]32: 1295-. However, any method known in the art for introducing DNA into a host cell may be used.
Production method
The present invention also relates to methods of producing a polypeptide of the present invention, comprising (a) culturing a cell that produces the polypeptide in its wild-type form under conditions conducive for production of the polypeptide; and optionally (b) recovering the polypeptide.
The present invention also relates to methods of producing a polypeptide of the present invention, comprising (a) cultivating a recombinant host cell of the present invention under conditions conducive for production of the polypeptide; and optionally (b) recovering the polypeptide.
The host cells are cultured in a nutrient medium suitable for the production of the polypeptide using methods known in the art. For example, the cell may be cultured by shake flask culture, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. Culturing occurs in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions, for example, in catalogues of the American Type Culture Collection. If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from the cell lysate.
The polypeptide can be recovered using methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures, including but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. In one aspect, a fermentation broth comprising the polypeptide is recovered.
The polypeptide can be purified by a variety of procedures known in the art, including, but not limited to, chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic chromatography, focus chromatography, and size exclusion chromatography), electrophoretic procedures (e.g., preparative isoelectric focusing electrophoresis), differential solubilization (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson and Ryden editors, VCH Publishers [ VCH Publishers ], new york, 1989), to obtain a substantially pure polypeptide.
In an alternative aspect, the polypeptide is not recovered, but rather a host cell of the invention expressing the polypeptide is used as a source of the polypeptide.
Fermentation broth formulation or cell composition
The invention also relates to fermentation broth formulations or cell compositions comprising the polypeptides of the invention. The fermentation broth product further comprises additional components used in the fermentation process, such as, for example, cells (including host cells containing a gene encoding a polypeptide of the invention, which host cells are used to produce the polypeptide of interest), cell debris, biomass, fermentation medium, and/or fermentation product. In some embodiments, the composition is a cell-killed whole broth containing one or more organic acids, killed cells and/or cell debris, and culture medium.
The term "fermentation broth" as used herein refers to a preparation produced by fermentation of a cell that has not undergone or has undergone minimal recovery and/or purification. For example, a fermentation broth is produced when a microbial culture is grown to saturation by incubation under carbon-limited conditions that allow protein synthesis (e.g., expression of an enzyme by a host cell) and secretion of the protein into the cell culture medium. The fermentation broth may contain an unfractionated or fractionated content of the fermented material obtained at the end of the fermentation. Typically, the fermentation broth is unfractionated and comprises spent culture medium and cell debris present after removal of microbial cells (e.g., filamentous fungal cells), e.g., by centrifugation. In some embodiments, the fermentation broth contains spent cell culture medium, extracellular enzymes, and viable and/or non-viable microbial cells.
In embodiments, the fermentation broth formulations and cell compositions comprise a first organic acid component (comprising at least one organic acid of 1-5 carbons and/or salt thereof) and a second organic acid component (comprising at least one organic acid of 6 or more carbons and/or salt thereof). In particular embodiments, the first organic acid component is acetic acid, formic acid, propionic acid, a salt thereof, or a mixture of two or more of the foregoing; and the second organic acid component is benzoic acid, cyclohexane carboxylic acid, 4-methyl pentanoic acid, phenylacetic acid, a salt thereof, or a mixture of two or more of the foregoing.
In one aspect, the composition contains one or more organic acids, and optionally further contains killed cells and/or cell debris. In one embodiment, the killed cells and/or cell debris are removed from the cell-killed whole broth to provide a composition free of these components.
The fermentation broth formulation or cell composition may further comprise preservatives and/or antimicrobial (e.g., bacteriostatic) agents including, but not limited to, sorbitol, sodium chloride, potassium sorbate, and other agents known in the art.
The cell-killed whole broth or composition may contain unfractionated contents of the fermented material obtained at the end of the fermentation. Typically, a cell-killing whole broth or composition comprises spent medium and cell debris present after microbial cells (e.g., filamentous fungal cells) are grown to saturation, incubated under carbon limiting conditions to allow protein synthesis. In some embodiments, the cell-killing whole broth or composition contains spent cell culture medium, extracellular enzymes, and killed filamentous fungal cells. In some embodiments, methods known in the art may be used to permeabilize and/or lyse microbial cells present in a cell-killed whole broth or composition.
The whole broth or cell composition as described herein is typically a liquid, but may contain insoluble components, such as killed cells, cell debris, media components, and/or one or more insoluble enzymes. In some embodiments, insoluble components may be removed to provide a clear liquid composition.
The whole broth formulations and cell compositions of the invention may be produced by the methods described in WO 90/15861 or WO 2010/096673.
IX. enzyme composition
The invention also relates to compositions comprising the polypeptides of the invention. These compositions may comprise the GH98 xylanase of the invention as the major enzyme component, e.g. a monocomponent composition.
Alternatively, the compositions may comprise enzymatic activities, such as one or more (e.g., several) enzymes selected from the group consisting of: a hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase, e.g., an alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase, beta-galactosidase, beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.
In an embodiment, the composition comprises a xylanase of the invention and a glucoamylase. In embodiments, the compositions comprise a xylanase of the invention and a glucoamylase derived from Talaromyces emersonii (e.g., SEQ ID NO: 11). In embodiments, the compositions comprise a trehalase of the invention and a glucoamylase derived from a genus mucorales (Gloeophyllum), such as mucorales fragilis (g.sepiarium) (e.g., SEQ ID NO:12) or mucorales densicola (g.trabeum) (e.g., SEQ ID NO: 13). In an embodiment, the composition comprises a trehalase of the invention, a glucoamylase, and an alpha-amylase. In an embodiment, the composition comprises a trehalase of the invention, a glucoamylase and an alpha-amylase derived from a strain of Rhizomucor, preferably Rhizomucor pusillus, such as a Rhizomucor pusillus alpha-amylase hybrid having a linker (e.g., from aspergillus niger) and a starch binding domain (e.g., from aspergillus niger). In an embodiment, the composition comprises a trehalase of the invention, a glucoamylase, an alpha-amylase, and a cellulolytic enzyme composition. In an embodiment, the composition comprises a trehalase of the invention, a glucoamylase, an alpha-amylase, and a cellulolytic enzyme composition, wherein the cellulolytic composition is derived from trichoderma reesei. In an embodiment, the composition comprises a trehalase of the invention, a glucoamylase, an alpha-amylase, and a protease. In an embodiment, the composition comprises a trehalase of the invention, a glucoamylase, an alpha-amylase, and a protease. The protease may be derived from Thermoascus aurantiacus. In an embodiment, the composition comprises a trehalase of the invention, a glucoamylase, an alpha-amylase, a cellulolytic enzyme composition, and a protease. In embodiments, the compositions comprise a trehalase, glucoamylase (e.g., from Talaromyces emersonii, Gloeophyllum fragrans, or Gloeophyllum trabeum), alpha-amylase (e.g., from Rhizomucor pusillus, particularly having a linker and a starch binding domain, particularly from Aspergillus niger, particularly with substitutions G128D + D143N (numbering using SEQ ID NO: 14)); a cellulolytic enzyme composition derived from trichoderma reesei, and a protease (e.g., derived from thermoascus aurantiacus or grifola gigantea (Meripilus giganteus)).
Examples of specifically contemplated minor enzymes, e.g., a glucoamylase from basket mussenia as set forth in SEQ ID No. 11 herein or a glucoamylase having, e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with SEQ ID No. 11 herein, can be found in the "enzymes" section below.
The composition may be prepared according to methods known in the art, and may be in the form of a liquid or dry composition. The composition may be stabilized according to methods known in the art.
Examples of preferred uses of the compositions of the present invention are given below. The dosage of the composition and other conditions under which the composition is used can be determined based on methods known in the art.
Enzyme blend
In one aspect, the invention relates to an enzyme blend comprising a xylanase and/or cellulolytic composition. The enzyme blend can be used, for example, to solubilize fibers (e.g., corn fibers, such as arabinose, xylose, etc.), preferably without blackening DDG or DDGs produced as a byproduct of a fermentation product production process (e.g., ethanol) during the SSF step (or pre-saccharification step) of the fermentation product production process. When the cellulolytic composition is included in the blend, the ratio of xylanase and cellulolytic composition can be optimized to increase the fiber solubilization of any particular substrate (e.g., corn fiber) and minimize or prevent blackening of downstream DDG or DDGs.
In one aspect, the invention relates to an enzyme blend comprising a xylanase. In one aspect, the invention relates to an enzyme blend comprising a xylanase and a cellulolytic composition, wherein the ratio of xylanase and cellulolytic composition in the blend is from about 5:95 to about 95: 5. In an embodiment, the ratio of xylanase to cellulolytic composition is 10: 90. In the examples, the ratio of xylanase to cellulolytic composition is 15: 85. In the examples, the ratio of xylanase to cellulolytic composition is 20: 80. In the examples, the ratio of xylanase to cellulolytic composition is 25: 75. In the examples, the ratio of xylanase to cellulolytic composition is 30: 70. In an embodiment, the ratio of xylanase to cellulolytic composition is 35: 65. In an embodiment, the ratio of xylanase to cellulolytic composition is 40: 60. In the examples, the ratio of xylanase to cellulolytic composition is 45: 55. In the examples, the ratio of xylanase to cellulolytic composition is 50: 50. In the examples, the ratio of xylanase to cellulolytic composition is 55: 45. In an embodiment, the ratio of xylanase to cellulolytic composition is 60: 40. In an embodiment, the ratio of xylanase to cellulolytic composition is 65: 35. In the examples, the ratio of xylanase to cellulolytic composition is 70: 30. In the examples, the ratio of xylanase to cellulolytic composition is 75: 25. In the examples, the ratio of xylanase to cellulolytic composition is 80: 20. In the examples, the ratio of xylanase to cellulolytic composition is 85: 15. In the examples, the ratio of xylanase to cellulolytic composition is 90: 10.
Xylanase
The present invention encompasses the use of any xylanase that, when optionally blended together with cellulolytic compositions in various ratios, is capable of dissolving fiber (e.g., arabinose, xylose, etc.) in a fermentation product production process (such as ethanol, among others), preferably without causing the DDGS to darken upon drying.
In one embodiment, the xylanase is from the order of taxonomic microbacteriales (Micrococcales) or Bacillales (Bacillales), or preferably from the family taxonomic Microbacteriaceae (Microbacteriaceae), Bacillaceae (Bacillaceae), or Paenibacillaceae (Paenibacillaceae), or more preferably from the genus taxonomic microbacterium, bacillus, or Paenibacillus, or even more preferably from the species taxonomic microbacterium oxydans, or microbacterium species SA39, bacillus amyloliquefaciens, bacillus subtilis, bacillus glucanotungensis, bacillus foddensis (Paenibacillus pabuli), or bacillus terreus. In one embodiment, the xylanase has at least 70%, e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID No. 1, SEQ ID No. 3, or SEQ ID No. 4 and is obtained or obtainable from the order microbacteriales, or preferably from the family microbacteriaceae, or more preferably from the genus microbacterium, or even more preferably from the species microbacterium oxydans, microbacterium hydrocarbonoxydans, or microbacterium species SA 39. In one embodiment, the xylanase has at least 70%, e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, or SEQ ID No. 10 and can be obtained from the order bacteroidales, or preferably bacteroides, or more preferably from the order bacteroides, or even more preferably from the order bacillus subtilis, bacillus amyloliquefaciens, bacillus licheniformis, bacillus licheniformis, or bacillus Bacillus glucanase, paenibacillus foraging, or paenibacillus terrestis. In one embodiment, the xylanase is a GH98 family xylanase (referred to herein as a GH98 xylanase).
The xylanase can be (a) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID No. 1, which polypeptide has xylanase activity. In one aspect, the xylanase amino acid sequence differs from SEQ ID No. 1 by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In one embodiment, the xylanase enzyme comprises or consists of the amino acid sequence of SEQ ID NO:1, is a fragment of SEQ ID NO:1, wherein the fragment has xylanase activity or comprises the amino acid sequence of SEQ ID NO:1 and N-terminal and/or C-terminal extensions or deletions of up to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids).
The xylanase can be (a) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID No. 3, which polypeptide has xylanase activity. In one aspect, the xylanase amino acid sequence differs from SEQ ID No. 3 by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In one embodiment, the xylanase enzyme comprises or consists of the amino acid sequence of SEQ ID NO. 3, is a fragment of SEQ ID NO. 2, wherein the fragment has xylanase activity or comprises the amino acid sequence of SEQ ID NO. 3 and N-terminal and/or C-terminal extensions or deletions of up to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids).
The xylanase can be (a) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID No. 4, which polypeptide has xylanase activity. In one aspect, the xylanase amino acid sequence differs from SEQ ID No. 4 by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In one embodiment, the xylanase enzyme comprises or consists of the amino acid sequence of SEQ ID NO. 4, is a fragment of SEQ ID NO. 4, wherein the fragment has xylanase activity or comprises the amino acid sequence of SEQ ID NO. 4 and N-terminal and/or C-terminal extensions or deletions of up to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids).
The xylanase enzyme can be (a) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID No. 5, which polypeptide has xylanase activity. In one aspect, the xylanase amino acid sequence differs from SEQ ID No. 5 by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In one embodiment, the xylanase enzyme comprises or consists of the amino acid sequence of SEQ ID NO. 5, is a fragment of SEQ ID NO. 5, wherein the fragment has xylanase activity or comprises the amino acid sequence of SEQ ID NO. 5 and N-terminal and/or C-terminal extensions or deletions of up to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids).
The xylanase enzyme can be (a) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID No. 7, which polypeptide has xylanase activity. In one aspect, the xylanase amino acid sequence differs from SEQ ID No. 7 by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In one embodiment, the xylanase enzyme comprises or consists of the amino acid sequence of SEQ ID NO. 7, is a fragment of SEQ ID NO. 7, wherein the fragment has xylanase activity or comprises the amino acid sequence of SEQ ID NO. 7 and N-terminal and/or C-terminal extensions or deletions of up to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids).
The xylanase enzyme can be (a) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID No. 9, which polypeptide has xylanase activity. In one aspect, the xylanase amino acid sequence differs from SEQ ID No. 9 by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In one embodiment, the xylanase enzyme comprises or consists of the amino acid sequence of SEQ ID NO. 9, is a fragment of SEQ ID NO. 9, wherein the fragment has xylanase activity or comprises the amino acid sequence of SEQ ID NO. 9 and N-terminal and/or C-terminal extensions or deletions of up to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids).
The xylanase can be (a) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID No. 10, which polypeptide has xylanase activity. In one aspect, the xylanase amino acid sequence differs from SEQ ID No. 10 by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In one embodiment, the xylanase enzyme comprises or consists of the amino acid sequence of SEQ ID NO:10, is a fragment of SEQ ID NO:10, wherein the fragment has xylanase activity or comprises the amino acid sequence of SEQ ID NO:10 and N-terminal and/or C-terminal extensions or deletions of up to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids).
In embodiments, the xylanase comprises a variant xylanase having one or more substitutions described in EP application No. 17177304.7 (which is incorporated herein by reference in its entirety).
In embodiments, the xylanase comprises a variant xylanase having one or more substitutions described in international patent application No. PCT/EP2017/065336 (which is incorporated herein by reference in its entirety).
The polypeptides may be hybrid polypeptides in which a region of one polypeptide is fused at the N-terminus or C-terminus of a region of another polypeptide.
The xylanase can be a fusion polypeptide or a cleavable fusion polypeptide in which another polypeptide is fused at the N-terminus or C-terminus of the polypeptide of the invention. Fusion polypeptides are produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the invention. Techniques for producing fusion polypeptides are known in the art and include ligating the coding sequences encoding the polypeptides such that they are in frame and expression of the fusion polypeptide is under the control of one or more of the same promoter and terminator. Fusion polypeptides can also be constructed using intein technology, where the fusion polypeptide is produced post-translationally (Cooper et al, 1993, EMBO J. [ J. European society of molecular biology ]12: 2575-.
The fusion polypeptide may further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved, thereby releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in the following documents: martin et al, 2003, J.Ind.Microbiol.Biotechnol. [ journal of Industrial microorganism Biotechnology ]3: 568-576; svetina et al 2000, J.Biotechnol. [ J.Biotechnology ]76: 245-; Rasmussen-Wilson et al 1997, appl. environ. Microbiol. [ application and environmental microbiology ]63: 3488-; ward et al, 1995, Biotechnology [ Biotechnology ]13: 498-503; and Contreras et al, 1991, Biotechnology [ Biotechnology ]9: 378-; eaton et al, 1986, Biochemistry [ Biochemistry ]25: 505-512; Collins-Racie et al, 1995, Biotechnology [ Biotechnology ]13: 982-; carter et al, 1989, Proteins: Structure, Function, and Genetics [ Proteins: structure, function, and genetics ]6: 240-; and Stevens,2003, Drug Discovery World 4: 35-48.
The xylanase may be obtained from a microorganism of any genus. For the purposes of the present invention, the term "obtained from … …" as used herein in connection with a given source shall mean that the parent encoded by the polynucleotide is produced by that source or by a strain into which a polynucleotide from that source has been inserted. In one aspect, the parent is secreted extracellularly.
The polypeptide may be a bacterial polypeptide. In one embodiment, the polypeptide is a bacterium from the class bacillus (Bacilli), such as from the order bacillus, or preferably from the family paenibacillaceae, or more preferably from the genus taxonomic genus bacillus or paenibacillus, or even more preferably from the species taxonomic species bacillus subtilis, bacillus amyloliquefaciens, bacillus glucanohydrolus, bacillus licheniformis, paenibacillus foddensis, or paenibacillus terrestris.
In one embodiment, the polypeptide is from a bacterium of the class actinomycetes (actinobacilla), e.g. from the order Microsphales, or preferably from the family Classification Microbacteriaceae or Micrococcaceae, or more preferably from the genus Classification Microbacterium, Micrococcus, or Micrococcum (Microcos), or even more preferably from the species Microbacterium oxydans, or Microbacterium species SA 39.
In one aspect, the xylanase is a bacillus glucanase, a paenibacillus saccharolyticus, a paenibacillus foraging, or a paenibacillus terrestris xylanase.
In one aspect, the xylanase is a microbacterium oxydans, microbacterium hydrocarbonoxydans, or microbacterium species SA39 xylanase.
In a preferred aspect, the xylanase is a Microbacterium oxydans xylanase, e.g., a xylanase having an amino acid sequence of SEQ ID NO: 1.
In a preferred aspect, the xylanase is a Bacillus glucanase, e.g., a xylanase having an amino acid sequence of SEQ ID NO. 5.
In a preferred aspect, the xylanase is a Paenibacillus terrestris xylanase, e.g., a xylanase having an amino acid sequence of SEQ ID NO. 7.
It is to be understood that for the aforementioned species, the invention encompasses complete and incomplete stages (perfect and perfect states), and other taxonomic equivalents (equivalents), such as anamorphs, regardless of their known seed names. Those skilled in the art will readily recognize the identity of appropriate equivalents.
Strains of these species are readily available to the public at many Culture collections, such as the American Type Culture Collection (ATCC), the German Culture Collection of microorganisms (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ), the Dutch cultures Collection (CBS), and the Northern Regional Research Center of the American Agricultural Research Service Culture Collection (NRRL).
The above-mentioned probes can be used to identify and obtain the xylanases from other sources, including microorganisms isolated from nature (e.g., soil, compost, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, compost, water, etc.). Techniques for the direct isolation of microorganisms and DNA from natural habitats are well known in the art. The polynucleotide encoding the parent can then be obtained by similarly screening a genomic DNA or cDNA library of another microorganism or mixed DNA sample. Once a polynucleotide encoding a parent has been detected using one or more probes, the polynucleotide can be isolated or cloned by utilizing techniques known to those of ordinary skill in the art (see, e.g., Sambrook et al, 1989, supra).
In an embodiment, the xylanase is a paenibacillus GH98 xylanase. In an embodiment, the xylanase is a microbacterium GH98 xylanase. Exemplary GH98 xylanases for use in the enzyme blends and methods of the invention include those from the genera Classification of Bacteroides (Bacteroides), Cellvibrio (Cellvibrio), Clostridium (Clostridia), Cystobacter (Cystobacter), Bacillus, Dickaya (Dickeya), filamentous Bacillus (Fibrobacter), Geobacillus, Meloidogyne (Meloidogyne), Microbacterium, Micromonospora (Micromonospora), Myxobacterium (Mucilanisbacter), Bacteroides (Paludibacter), perforated nematode (Rapholus), Ruminococcus (Ruminococcus), Serratia (Serratia), Streptomyces, Verrucosispora (Verrucosispora), and Xanthomonas (Xanthomonas).
In an embodiment, the xylanase is a GH98 xylanase selected from the group consisting of: (i) 1 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% amino acid sequence identity thereto; (ii) 3 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% amino acid sequence identity thereto; (iii) 4 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% amino acid sequence identity thereto; (iv) 5 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% amino acid sequence identity thereto; (v) 7 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% amino acid sequence identity thereto; (vi) 9 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% amino acid sequence identity thereto; and (vii) the Paenibacillus species DMB20 xylanase of SEQ ID NO:10 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% amino acid sequence identity thereto.
In embodiments, a xylanase, e.g., a GH98 xylanase, e.g., a GH98 xylanase of SEQ NO:1, 3-5, 7, 8-10, or a variant thereof, is administered in the pre-saccharification, and/or simultaneous saccharification and fermentation at a concentration of between 0.0001-1mg EP (enzyme protein)/g DS, e.g., 0.0005-0.5mg EP/g DS, e.g., 0.001-0.1mg EP/g DS.
Cellulose decomposition composition
The present invention encompasses the use of any cellulolytic composition that, when blended together with various ratios of xylanase, is capable of solubilizing fibers (e.g., arabinose, xylose, etc.) in a fermentation product production process (such as ethanol, among others), without causing the DDGS to darken upon drying. The cellulolytic composition used in the enzyme blend or process of the invention for producing a fermentation product may be derived from any microorganism. As used herein, "derived from any microorganism" means that the cellulolytic composition comprises one or more enzymes expressed in the microorganism. For example, a cellulolytic composition derived from a strain of trichoderma reesei means that the cellulolytic composition comprises one or more enzymes expressed in trichoderma reesei.
In an embodiment, the cellulolytic composition is derived from a strain of aspergillus, such as a strain of aspergillus flavus, aspergillus niger, or aspergillus oryzae.
In an embodiment, the cellulolytic composition is derived from a strain of Chrysosporium (Chrysosporium), such as a strain of Chrysosporium lucknowense (Chrysosporium lucknowense).
In the examples, the cellulolytic composition is derived from a strain of Humicola (Humicola), such as a strain of Humicola insolens (Humicola insolens).
In an embodiment, the cellulolytic composition is derived from a strain of penicillium, such as a strain of penicillium emersonii or penicillium oxalicum.
In embodiments, the cellulolytic composition is derived from a strain of the genus Talaromyces, such as a strain of Talaromyces aureofaciens or Talaromyces emersonii.
In an embodiment, the cellulolytic composition is derived from a strain of trichoderma, such as a strain of trichoderma reesei.
In a preferred embodiment, the cellulolytic composition is derived from a strain of trichoderma reesei. In preferred embodiments, the trichoderma reesei cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) cellobiohydrolase I; (ii) cellobiohydrolase II; (iii) a beta-glucosidase; and (iv) a GH61 polypeptide having cellulolytic enhancing activity. In another preferred embodiment, the trichoderma reesei cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) aspergillus fumigatus cellobiohydrolase I; (ii) aspergillus fumigatus cellobiohydrolase II; (iii) aspergillus fumigatus beta-glucosidase; and (iv) a penicillium emersonii GH61A polypeptide having cellulolytic enhancing activity.
In preferred embodiments, the trichoderma reesei cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) cellobiohydrolase I or cellobiohydrolase II; (ii) a beta-glucosidase; and (iii) an endoglucanase. In another preferred embodiment, the trichoderma reesei cellulolytic composition comprises at least one, at least two, or at least three enzymes selected from the group consisting of: (i) aspergillus fumigatus cellobiohydrolase I; (ii) aspergillus fumigatus beta-glucosidase; and (iii) an endoglucanase.
In yet another preferred embodiment, the trichoderma reesei cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) cellobiohydrolase I comprising amino acids 27 to 532 of SEQ ID No. 15 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with amino acids 27 to 532 of SEQ ID No. 15; (ii) cellobiohydrolase II comprising amino acids 20 to 454 of SEQ ID No. 16, or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with amino acids 20 to 454 of SEQ ID No. 16; (iii) a β -glucosidase or variant thereof comprising amino acids 20 to 863 of SEQ ID No. 17, having at least one substitution selected from the group consisting of F100D, S283G, N456E, and F512Y, and having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with amino acids 20 to 863 of SEQ ID No. 17; and (iv) a GH61A polypeptide having cellulolytic enhancing activity or a variant thereof, the GH61A polypeptide comprising amino acids 26 to 253 of SEQ ID No. 18, the variant having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to amino acids 26 to 253 of SEQ ID No. 18.
In embodiments, the trichoderma reesei cellulolytic composition further comprises an endoglucanase.
In a preferred embodiment, the cellulolytic composition is derived from a strain of aspergillus flavus. In preferred embodiments, the aspergillus flavus cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) cellobiohydrolase I; (ii) cellobiohydrolase II; (iii) a beta-glucosidase; and (iv) a GH61 polypeptide having cellulolytic enhancing activity. In another preferred embodiment, the Aspergillus flavus cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) aspergillus fumigatus cellobiohydrolase I; (ii) aspergillus fumigatus cellobiohydrolase II; (iii) aspergillus fumigatus beta-glucosidase; and (iv) a penicillium emersonii GH61A polypeptide having cellulolytic enhancing activity.
In yet another preferred embodiment, the Aspergillus flavus cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) cellobiohydrolase I comprising amino acids 27 to 532 of SEQ ID No. 15 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with amino acids 27 to 532 of SEQ ID No. 15; (ii) cellobiohydrolase II comprising amino acids 20 to 454 of SEQ ID No. 16, or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with amino acids 20 to 454 of SEQ ID No. 16; (iii) a β -glucosidase or variant thereof comprising amino acids 20 to 863 of SEQ ID No. 17, having at least one substitution selected from the group consisting of F100D, S283G, N456E, and F512Y, and having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with amino acids 20 to 863 of SEQ ID No. 17; and (iv) a GH61A polypeptide having cellulolytic enhancing activity or a variant thereof, the GH61A polypeptide comprising amino acids 26 to 253 of SEQ ID No. 18, the variant having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to amino acids 26 to 253 of SEQ ID No. 18.
In an embodiment, the aspergillus flavus cellulolytic composition further comprises an endoglucanase.
In a preferred embodiment, the cellulolytic composition is derived from a strain of aspergillus niger. In a preferred embodiment, the aspergillus niger cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) cellobiohydrolase I; (ii) cellobiohydrolase II; (iii) a beta-glucosidase; and (iv) a GH61 polypeptide having cellulolytic enhancing activity. In another preferred embodiment, the aspergillus niger cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) aspergillus fumigatus cellobiohydrolase I; (ii) aspergillus fumigatus cellobiohydrolase II; (iii) aspergillus fumigatus beta-glucosidase; and (iv) a penicillium emersonii GH61A polypeptide having cellulolytic enhancing activity.
In yet another preferred embodiment, the aspergillus niger cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) cellobiohydrolase I comprising amino acids 27 to 532 of SEQ ID No. 15 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with amino acids 27 to 532 of SEQ ID No. 15; (ii) cellobiohydrolase II comprising amino acids 20 to 454 of SEQ ID No. 16, or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with amino acids 20 to 454 of SEQ ID No. 16; (iii) a β -glucosidase or variant thereof comprising amino acids 20 to 863 of SEQ ID No. 17, having at least one substitution selected from the group consisting of F100D, S283G, N456E, and F512Y, and having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with amino acids 20 to 863 of SEQ ID No. 17; and (iv) a GH61A polypeptide having cellulolytic enhancing activity comprising amino acids 26 to 253 of SEQ ID No. 18, or a variant thereof, having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to amino acids 26 to 253 of SEQ ID No. 18.
In an embodiment, the aspergillus niger cellulolytic composition further comprises an endoglucanase.
In a preferred embodiment, the cellulolytic composition is derived from a strain of aspergillus oryzae. In preferred embodiments, the aspergillus oryzae cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) cellobiohydrolase I; (ii) cellobiohydrolase II; (iii) a beta-glucosidase; and (iv) a GH61 polypeptide having cellulolytic enhancing activity. In another preferred embodiment, the aspergillus oryzae cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) aspergillus fumigatus cellobiohydrolase I; (ii) aspergillus fumigatus cellobiohydrolase II; (iii) aspergillus fumigatus beta-glucosidase; and (iv) a penicillium emersonii GH61A polypeptide having cellulolytic enhancing activity.
In yet another preferred embodiment, the aspergillus oryzae cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) cellobiohydrolase I comprising amino acids 27 to 532 of SEQ ID No. 15 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with amino acids 27 to 532 of SEQ ID No. 15; (ii) cellobiohydrolase II comprising amino acids 20 to 454 of SEQ ID No. 16, or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with amino acids 20 to 454 of SEQ ID No. 16; (iii) a β -glucosidase or variant thereof comprising amino acids 20 to 863 of SEQ ID No. 17, having at least one substitution selected from the group consisting of F100D, S283G, N456E, and F512Y, and having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with amino acids 20 to 863 of SEQ ID No. 17; and (iv) a GH61A polypeptide having cellulolytic enhancing activity or a variant thereof, the GH61A polypeptide comprising amino acids 26 to 253 of SEQ ID No. 18, the variant having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to amino acids 26 to 253 of SEQ ID No. 18.
In an embodiment, the aspergillus oryzae cellulolytic composition further comprises an endoglucanase.
In a preferred embodiment, the cellulolytic composition is derived from a strain of penicillium emersonii. In a preferred embodiment, the penicillium emersonii cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) cellobiohydrolase I; (ii) cellobiohydrolase II; (iii) a beta-glucosidase; and (iv) a GH61 polypeptide having cellulolytic enhancing activity. In another preferred embodiment, the penicillium emersonii cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) aspergillus fumigatus cellobiohydrolase I; (ii) aspergillus fumigatus cellobiohydrolase II; (iii) aspergillus fumigatus beta-glucosidase; and (iv) a penicillium emersonii GH61A polypeptide having cellulolytic enhancing activity.
In yet another preferred embodiment, the penicillium emersonii cellulose-decomposing composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) cellobiohydrolase I comprising amino acids 27 to 532 of SEQ ID No. 15 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with amino acids 27 to 532 of SEQ ID No. 15; (ii) cellobiohydrolase II comprising amino acids 20 to 454 of SEQ ID No. 16, or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with amino acids 20 to 454 of SEQ ID No. 16; (iii) a β -glucosidase or variant thereof comprising amino acids 20 to 863 of SEQ ID No. 17, having at least one substitution selected from the group consisting of F100D, S283G, N456E, and F512Y, and having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with amino acids 20 to 863 of SEQ ID No. 17; and (iv) a GH61A polypeptide having cellulolytic enhancing activity or a variant thereof, the GH61A polypeptide comprising amino acids 26 to 253 of SEQ ID No. 18, the variant having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to amino acids 26 to 253 of SEQ ID No. 18.
In embodiments, the penicillium emersonii cellulolytic composition further comprises an endoglucanase.
In a preferred embodiment, the cellulolytic composition is derived from a strain of penicillium oxalicum. In a preferred embodiment, the penicillium oxalicum cellulose decomposition composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) cellobiohydrolase I; (ii) cellobiohydrolase II; (iii) a beta-glucosidase; and (iv) a GH61 polypeptide having cellulolytic enhancing activity. In another preferred embodiment, the penicillium oxalicum cellulose decomposition composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) aspergillus fumigatus cellobiohydrolase I; (ii) aspergillus fumigatus cellobiohydrolase II; (iii) aspergillus fumigatus beta-glucosidase; and (iv) a penicillium emersonii GH61A polypeptide having cellulolytic enhancing activity.
In yet another preferred embodiment, the penicillium oxalicum cellulose decomposition composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) cellobiohydrolase I comprising amino acids 27 to 532 of SEQ ID No. 15 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with amino acids 27 to 532 of SEQ ID No. 15; (ii) cellobiohydrolase II comprising amino acids 20 to 454 of SEQ ID No. 16, or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with amino acids 20 to 454 of SEQ ID No. 16; (iii) a β -glucosidase or variant thereof comprising amino acids 20 to 863 of SEQ ID No. 17, having at least one substitution selected from the group consisting of F100D, S283G, N456E, and F512Y, and having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with amino acids 20 to 863 of SEQ ID No. 17; and (iv) a GH61A polypeptide having cellulolytic enhancing activity or a variant thereof, the GH61A polypeptide comprising amino acids 26 to 253 of SEQ ID No. 18, the variant having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with amino acids 26 to 253 of SEQ ID No. 18.
In embodiments, the penicillium oxalicum cellulose disintegration composition further comprises an endoglucanase.
In a preferred embodiment, the cellulolytic composition is derived from a strain of Talaromyces aurantiaca. In preferred embodiments, the golden basket fungus cellulose decomposing composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) cellobiohydrolase I; (ii) cellobiohydrolase II; (iii) a beta-glucosidase; and (iv) a GH61 polypeptide having cellulolytic enhancing activity. In another preferred embodiment, the golden basket fungus cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) aspergillus fumigatus cellobiohydrolase I; (ii) aspergillus fumigatus cellobiohydrolase II; (iii) aspergillus fumigatus beta-glucosidase; and (iv) a penicillium emersonii GH61A polypeptide having cellulolytic enhancing activity.
In yet another preferred embodiment, the golden basket fungus cellulose decomposition composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) cellobiohydrolase I comprising amino acids 27 to 532 of SEQ ID No. 15 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with amino acids 27 to 532 of SEQ ID No. 15; (ii) cellobiohydrolase II comprising amino acids 20 to 454 of SEQ ID No. 16, or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with amino acids 20 to 454 of SEQ ID No. 16; (iii) a β -glucosidase or variant thereof comprising amino acids 20 to 863 of SEQ ID No. 17, having at least one substitution selected from the group consisting of F100D, S283G, N456E, and F512Y, and having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with amino acids 20 to 863 of SEQ ID No. 17; and (iv) a GH61A polypeptide having cellulolytic enhancing activity comprising amino acids 26 to 253 of SEQ ID No. 18, or a variant thereof, having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to amino acids 26 to 253 of SEQ ID No. 18.
In embodiments, the golden basket fungus cellulolytic composition further comprises an endoglucanase.
In a preferred embodiment, the cellulolytic composition is derived from a strain of basket fungus emersonia. In preferred embodiments, the emersonia cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) cellobiohydrolase I; (ii) cellobiohydrolase II; (iii) a beta-glucosidase; and (iv) a GH61 polypeptide having cellulolytic enhancing activity. In another preferred embodiment, the emersonia cellulose decomposing composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) aspergillus fumigatus cellobiohydrolase I; (ii) aspergillus fumigatus cellobiohydrolase II; (iii) aspergillus fumigatus beta-glucosidase; and (iv) a penicillium emersonii GH61A polypeptide having cellulolytic enhancing activity.
In yet another preferred embodiment, the emersonia cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of: (i) cellobiohydrolase I comprising amino acids 27 to 532 of SEQ ID No. 15 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with amino acids 27 to 532 of SEQ ID No. 15; (ii) cellobiohydrolase II comprising amino acids 20 to 454 of SEQ ID No. 16, or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with amino acids 20 to 454 of SEQ ID No. 16; (iii) a β -glucosidase or variant thereof comprising amino acids 20 to 863 of SEQ ID No. 17, having at least one substitution selected from the group consisting of F100D, S283G, N456E, and F512Y, and having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with amino acids 20 to 863 of SEQ ID No. 17; and (iv) a GH61A polypeptide having cellulolytic enhancing activity comprising amino acids 26 to 253 of SEQ ID No. 18, or a variant thereof, having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to amino acids 26 to 253 of SEQ ID No. 18.
In embodiments, the emersonia cellulolytic composition further comprises an endoglucanase.
The cellulolytic composition may further comprise a plurality of enzyme activities, such as one or more (e.g., several) enzymes selected from the group consisting of: acetyl xylan esterase, acylglycerol lipase, amylase, alpha-amylase, beta-amylase, arabinofuranosidase, cellobiohydrolase, cellulase, ferulic acid esterase, galactanase, alpha-galactosidase, beta-glucanase, beta-glucosidase, glucan 1, 4-a-maltohydrolase, glucan 1, 4-a-glucosidase, glucan 1, 4-a-maltohydrolase, lysophospholipase, lysozyme, alpha-mannosidase, beta-mannosidase (mannanase), phytase, phospholipase A1, phospholipase A2, phospholipase D, protease, pullulanase, pectin esterase, triacylglycerol lipase, beta-glucosidase, arabinofuranosidase, and combinations thereof, A xylanase, a beta-xylosidase, or any combination thereof.
In embodiments, the cellulolytic composition comprises one or more formulations as disclosed herein, preferably one or more compounds selected from the list consisting of: glycerin, ethylene glycol, 1, 2-propylene glycol, or 1, 3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, kaolin, and cellulose.
In the examples, a cellulolytic composition, e.g.derived from a strain of Aspergillus, Penicillium, Talaromyces, or Trichoderma, such as a Trichoderma reesei cellulolytic composition, is administered in a pre-saccharification, and/or simultaneous saccharification and fermentation at a concentration of 0.0001-3mg EP/g DS, preferably 0.0005-2mg EP/g DS, preferably 0.001-1mg/g DS, more preferably 0.005-0.5mg EP/g DS, even more preferably 0.01-0.1mg EP/g DS.
Xi. Process for producing a fermentation product
The invention also relates to a process for producing a fermentation product from starch-containing material using a fermenting organism, wherein a polypeptide having xylanase activity (GH98 xylanase) or an enzyme blend or composition comprising a xylanase and a cellulolytic composition (e.g., derived from trichoderma reesei) is added before and/or during fermentation. One skilled in the art will appreciate that any polypeptide having a xylanase described in part I above, a composition described in part IX above, and/or an enzyme blend described in part X above or otherwise described herein can be used in the methods of the invention (including the methods of part XI).
Process for producing a fermentation product from a material containing ungelatinized starch
In one aspect, the invention relates to a process (often referred to as a "raw starch hydrolysis" process) for producing a fermentation product from starch-containing material without gelatinizing (i.e., not cooking) the starch-containing material, wherein a GH98 xylanase of the present disclosure, or an enzyme blend or composition comprising the xylanase and a cellulolytic composition, is added. A fermentation product, such as ethanol, can be produced without liquefying an aqueous slurry comprising starch-containing material and water. In one embodiment, the method of the present invention comprises: the (e.g., milled) starch-containing material (e.g., granular starch) is saccharified below the initial gelatinization temperature, preferably in the presence of an alpha-amylase and/or carbohydrate source-producing enzyme, to produce a variety of sugars that can be fermented into a fermentation product by a suitable fermenting organism. In this embodiment, the desired fermentation product, e.g., ethanol, is produced from ungelatinized (i.e., uncooked), preferably milled, grain such as corn.
Thus, in one aspect, the invention relates to a process for producing a fermentation product from starch-containing material, the process comprising simultaneously saccharifying and fermenting starch-containing material using a carbohydrate-source producing enzyme and a fermenting organism in the presence of a GH98 xylanase of the invention, or an enzyme blend or composition comprising the xylanase, at a temperature below the initial gelatinization temperature of said starch-containing material.
Exemplary enzyme blends used in this process are described in section X above referred to as "enzyme blends". Saccharification and fermentation may also be separate. Thus, in another aspect, the present invention relates to a process for producing a fermentation product, the process comprising the steps of:
(i) saccharifying a starch-containing material with a carbohydrate source-producing enzyme (e.g., glucoamylase) at a temperature below the initial gelatinization temperature; and
(ii) fermenting using a fermenting organism;
wherein steps (i) and/or (ii) are performed using at least one glucoamylase, and a GH98 xylanase of the invention or an enzyme blend or composition comprising the xylanase.
In embodiments, the GH98 xylanase of the invention or an enzyme blend or composition comprising the xylanase is added during saccharification step (i). In embodiments, the GH98 xylanase of the invention or an enzyme blend or composition comprising the xylanase is added during the fermentation step (ii).
In one embodiment, an alpha amylase, in particular a fungal alpha-amylase, is also added in step (i). Steps (i) and (ii) may be performed simultaneously. In embodiments, the GH98 xylanase of the invention, or an enzyme blend or composition comprising the xylanase, is added during Simultaneous Saccharification and Fermentation (SSF). In an embodiment, the fermenting organism is a yeast and the GH98 xylanase of the invention or an enzyme blend or composition comprising the xylanase is added during propagation of the yeast.
Method for producing a fermentation product from a material containing gelatinized starch
In one aspect, the present invention relates to processes for producing fermentation products, particularly ethanol, from starch-containing material, the processes comprising: a liquefaction step, and a saccharification and fermentation step performed sequentially or simultaneously. Accordingly, the present invention relates to a process for producing a fermentation product from starch-containing material, the process comprising the steps of:
(a) liquefying a starch-containing material in the presence of an alpha-amylase to form a liquefied mash;
(b) saccharifying the liquefied mash using a carbohydrate source producing enzyme to produce fermentable sugars; and
(c) fermenting the sugar using a fermenting organism under conditions suitable for production of the fermentation product;
Wherein a GH98 xylanase of the invention, or an enzyme blend or composition comprising the xylanase, is added before or during step (c).
The slurry is heated above the gelatinization temperature and the alpha-amylase variant may be added to start liquefaction (dilution). In an embodiment, the slurry may be jet cooked to further gelatinize the slurry prior to being subjected to the alpha-amylase in step (a). In an embodiment, the liquefaction may be performed as a three-step hot slurry process. The slurry is heated to between 60-95 ℃, preferably between 70-90 ℃, such as preferably between 80-85 ℃, at pH 4-6, in particular at pH 4.5-5.5, and the alpha-amylase variant, optionally together with protease, carbohydrate source producing enzyme (such as glucoamylase), phospholipase, phytase, and/or pullulanase, is added to start liquefaction (dilution). The liquefaction process is usually carried out at a pH of 4-6, in particular at a pH of from 4.5 to 5.5. The saccharification step (b) may be performed using conditions well known in the art. For example, the complete saccharification process may last from about 24 to about 72 hours, however, typically only a pre-saccharification of typically 40-90 minutes is performed at a temperature between 30 ℃ and 65 ℃, typically about 60 ℃, followed by a complete saccharification during fermentation in a simultaneous saccharification and fermentation process (SSF process). Saccharification is typically carried out at a temperature of from 20 ℃ to 75 ℃, particularly from 40 ℃ to 70 ℃, typically about 60 ℃ and at a pH between 4 and 5, generally at about pH 4.5. The most widely used process in the production of fermentation products, especially ethanol, is the Simultaneous Saccharification and Fermentation (SSF) process, in which saccharification is absent a holding stage, meaning that a fermenting organism (e.g. yeast) and an enzyme can be added together. SSF may typically be performed at a temperature of from 25 ℃ to 40 ℃, e.g. from 28 ℃ to 35 ℃, e.g. from 30 ℃ to 34 ℃, preferably about 32 ℃. In the examples, the fermentation is carried out for 6 to 120 hours, in particular 24 to 96 hours.
In embodiments, the GH98 xylanase of the invention or an enzyme blend or composition comprising the xylanase is added during saccharification step (b). In embodiments, the GH98 xylanase of the invention or an enzyme blend or composition comprising the xylanase is added during the fermentation step (c). Steps (b) and (c) may be performed simultaneously. In embodiments, the GH98 xylanase of the invention, or an enzyme blend or composition comprising the xylanase, is added during Simultaneous Saccharification and Fermentation (SSF). In an embodiment, the fermenting organism is a yeast and the GH98 xylanase of the invention or an enzyme blend or composition comprising the xylanase is added during propagation of the yeast.
Starch-containing material
Any suitable starch-containing starting material may be used in the process of the present invention. The starting materials are generally selected based on the desired fermentation product. Examples of starch-containing starting materials suitable for use in the process of the present invention include barley, legumes, manioc (cassava), cereals, corn, milo, peas, potatoes, rice, rye, sago, sorghum, sweet potatoes, cassava (tapioca), wheat, and whole grains, or any mixture thereof. The starch-containing material may also be corn and barley of the waxy or non-waxy type. In a preferred embodiment, the starch-containing material is corn. In a preferred embodiment, the starch-containing material is wheat.
Fermentation product
The term "fermentation product" means a product produced by a fermentation process or process that includes the use of a fermenting organism. Fermentation products include alcohols (e.g., ethanol, methanol, butanol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, succinic acid, gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gas (e.g. H)2And CO2) (ii) a Antibiotics (e.g., penicillin and tetracycline); an enzyme; vitamins (e.g. riboflavin, B)12Beta-carotene); and hormones. In preferred embodiments, the fermentation product is ethanol, e.g., fuel ethanol; drinking ethanol, i.e. neutral drinking ethanol; or industrial alcohols or products used in the consumable alcohol industry (e.g., beer and wine), dairy industry (e.g., fermented dairy products), leather industry, and tobacco industry. Preferred types of beer include ale (ale), stout, porter, lagoon (lager), bitter, malt (malt liquor), low malt (happoushu), high alcohol, low calorie or light beer. In an embodiment, the fermentation product is ethanol.
Fermenting organisms
The term "fermenting organism" refers to any organism suitable for producing a desired fermentation product, including bacterial and fungal organisms, such as yeast and filamentous fungi. Suitable fermenting organisms are capable of fermenting (i.e., converting) fermentable sugars (such as arabinose, fructose, glucose, maltose, mannose, or xylose) directly or indirectly to the desired fermentation product.
Examples of fermenting organisms include fungal organisms, such as yeast. Preferred yeasts include strains of Saccharomyces (Saccharomyces), in particular Saccharomyces cerevisiae or Saccharomyces uvarum (Saccharomyces uvarum); a strain of the genus Pichia (Pichia), in particular a strain of Pichia stipitis (Pichia stipitis), such as Pichia stipitis CBS 5773, or Pichia pastoris (Pichia pastoris); strains of the genus Candida, in particular Candida arabinofaciens, Candida boidinii, Candida didanosis, Candida shehatae, Candida sannaringitis, Candida pseudotropicalis, or Candida utilis. Other fermenting organisms include strains of Hansenula (Hansenula), in particular Hansenula anomala (Hansenula anomala) or Hansenula polymorpha (Hansenula polymorpha); a strain of the genus Kluyveromyces (Kluyveromyces), in particular Kluyveromyces fragilis (Kluyveromyces fragilis) or Kluyveromyces marxianus (Kluyveromyces marxianus); and strains of the genus Schizosaccharomyces (Schizosaccharomyces), particularly Schizosaccharomyces pombe (Schizosaccharomyces pombe).
In an embodiment, the fermenting organism is a C6 sugar fermenting organism, such as for example a strain of saccharomyces cerevisiae.
In an embodiment, the fermenting organism is a C5 sugar fermenting organism, such as for example a strain of saccharomyces cerevisiae.
Fermentation of
Fermentation conditions are determined based on, for example, the type of plant material, the fermentable sugars available, the fermenting organism or organisms, and/or the desired fermentation product. Suitable fermentation conditions can be readily determined by one of ordinary skill in the art. The fermentation can be carried out under the conditions conventionally used. The preferred fermentation process is an anaerobic process.
For example, fermentation may be carried out at temperatures up to 75 ℃, e.g., between 40 ℃ and 70 ℃, such as between 50 ℃ and 60 ℃. However, it is also known that bacteria have a significantly lower optimum temperature down to around room temperature (around 20 ℃). Examples of suitable fermenting organisms can be found in the "fermenting organisms" section above.
For ethanol production using yeast, the fermentation may last from 24 to 96 hours, in particular from 35 to 60 hours. In an embodiment, the fermentation is carried out at a temperature of between 20 ℃ and 40 ℃, preferably between 26 ℃ and 34 ℃, in particular around 32 ℃. In the examples, the pH is from pH 3 to 6, preferably around pH 4 to 5.
Recovery of fermentation products
After fermentation or SSF, the fermentation product may be separated from the fermentation medium. The slurry may be distilled to extract the desired fermentation product (e.g., ethanol). Alternatively, the desired fermentation product may be extracted from the fermentation medium by microfiltration or membrane filtration techniques. The fermentation product may also be recovered by steam stripping or other methods well known in the art. Typically, a fermentation product, e.g., ethanol, is obtained having a purity of up to, e.g., about 96 vol.% ethanol.
Thus, in one embodiment, the process of the invention further comprises distillation to obtain a fermentation product, e.g., ethanol. The fermentation and distillation may be carried out simultaneously and/or separately/sequentially; optionally, one or more process steps for further refining the fermentation product follow.
After the distillation process is complete, the remaining material is considered whole stillage. As used herein, the term "whole stillage" includes material remaining at the end of the distillation process after recovery of the fermentation product, e.g., ethanol. The fermentation product may optionally be recovered by any method known in the art.
Separating (dewatering) the whole distillers 'grains into distillers' grains water and wet cake
In one embodiment, the whole stillage is separated or partitioned into a solid phase and a liquid phase by one or more methods of separating the stillage water from the wet cake. Separation of the whole stillage into stillage and wet cake to remove a significant portion of the liquid/water can be accomplished using any suitable separation technique, including centrifugation, pressing, and filtration. In a preferred embodiment, the separation/dehydration is performed by centrifugation. In industry, the preferred centrifuge is a decanter centrifuge, preferably a high speed decanter centrifuge. One example of a suitable centrifuge is the NX 400 steep cone series from Alfa Laval (Alfa Laval), which is a high performance settler. In another preferred embodiment, other conventional separation equipment (e.g., plate/frame filter presses, belt presses, screw presses, gravity concentrators, and de-watering machines) or the like is used to perform the separation.
Processing of lees water
Thin stillage is the term used for the supernatant of whole stillage centrifugation. Typically, thin stillage contains 4-6% Dry Solids (DS) (mainly protein, soluble fiber, fines, and cell wall components) and is at a temperature of about 60-90 ℃. The stream of stillage water can be condensed by evaporation to provide two process streams including: (i) an evaporator condensate stream comprising condensate removed from the stillage during evaporation; and (ii) a slurry stream comprising a more concentrated stream of non-volatile dissolved and undissolved solids, such as non-fermentable sugars and oils remaining from the stillage water as a result of the removal of the evaporated water. Optionally, oil may be removed from the thin stillage or may be removed as an intermediate step in an evaporation process, which is typically carried out using a series of several evaporation stages. The serum and/or de-oiled serum can be introduced into the dryer along with the wet grains (from the whole stillage separation step) to provide a product known as distillers dried grains with solubles, which can also be used as animal feed.
In an embodiment, the serum and/or de-oiled serum is sprayed into one or more dryers to combine the serum and/or de-oiled serum with whole stillage to produce distiller's dried grain with solubles.
Between 5 vol-% and 90 vol-%, such as between 10% and 80%, such as between 15% and 70%, such as between 20% and 60%, of the thin stillage (e.g. optionally hydrolysed) may be recycled (as counter-current) to step (a). The recycled thin stillage (i.e. counter-current) may constitute about 1 vol-% to 70 vol-%, preferably 15 vol-% to 60 vol-%, especially from about 30 vol-% to 50 vol-% of the slurry formed in step (a).
In embodiments, the method further comprises recycling at least a portion of the stream of stillage water into the slurry, optionally after oil has been extracted from the stream of stillage water.
Drying of wet cake and production of distiller's dried grain and distiller's dried grain with solubles
After the wet cake containing about 25 wt% to 40 wt%, preferably 30 wt% to 38 wt% dry solids has been separated from the thin stillage (e.g., dewatered), it can be dried on a drum dryer, spray dryer, ring dryer, fluidized bed dryer, or the like to produce "distillers dried grains" (DDG). DDG is a valuable feed ingredient for animals such as livestock, poultry and fish. DDG is preferably provided at a moisture content of less than about 10 wt% to 12 wt% to avoid mold and microbial degradation and increase shelf life. In addition, high moisture content also makes it more expensive to transport DDG. The wet cake is preferably dried under conditions that do not denature the protein in the wet cake. The wet cake may be blended with a slurry isolated from thin stillage and dried into soluble-containing ddg (ddgs). The partially dried intermediate product, sometimes referred to as a modified wet distillers grain, can be produced by partially drying the wet cake, optionally adding a slurry before, during, or after the drying process.
Methods for improving the nutritional quality of DDG or DDGS
In another aspect, the invention relates to a method for improving the nutritional quality of distillers Dried Grains (DGS) or distillers dried grains with solubles (DDGS) produced as a byproduct of a fermentation product production process.
In an embodiment, a method for improving the nutritional quality of DGS or DDGS produced as a byproduct of a fermentation product production process comprises performing section XI above (e.g., an RSH process comprising a liquefaction step or a conventional cooking process) or a process for producing a fermentation product as described in the examples herein, and recovering the fermentation product to produce DGS or DDGS as a byproduct, wherein the DGS or DDGS produced have improved nutritional quality. The step of recovering the fermentation product to produce DGS or DDGS as a by-product may comprise any one or combination of the steps described above for recovering one or more fermentation products, such as by distillation, to produce whole stillage, separation of whole stillage into wet cake and thin stillage, processing of thin stillage, drying of wet cake and production of DDG or DDGS, and the like.
As used herein, "improved nutritional quality" means an increase in the True Metabolic Energy (TME) of a DDG or DDGs of at least 5% as compared to DDG or DDGs produced in a fermentation product production process (e.g., the RSH process set forth in section XI or a conventional cooking process comprising a liquefaction step) in which the GH98 xylanase of the disclosure or an enzyme blend or composition comprising the xylanase is not added during pre-saccharification, fermentation, and/or simultaneous saccharification and fermentation.
In embodiments, the GH98 xylanases of the invention, or enzyme blends and compositions comprising the same, and methods increase the TME of the DDG or DDGs by at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 26%, at least 27%, at least 28%, compared to DDG or DDGs produced in a fermentation product production process (e.g., the RSH process set forth in section XI, or a conventional cooking process comprising a liquefaction step) in which no GH98 xylanase of the disclosure, or enzyme blend or composition comprising the same, is added during pre-saccharification, fermentation, and/or simultaneous saccharification and fermentation) At least 29%, or at least 30%.
In embodiments, the GH98 xylanases of the invention, or enzyme blends or compositions comprising the xylanases, and methods improve the nutritional quality of DDG or DDGs without causing the DDG or DDGs to darken after drying. Those skilled in the art will appreciate that the degree of darkening of the DDG or DDGs after drying can be readily assessed during the fermentation product production process (e.g., during SSF for ethanol production using corn mash substrate) after addition of a GH98 xylanase of the invention or enzyme blend or composition comprising the xylanase, e.g., by measuring the DDG or DDGs color using the Hunter color scale (see examples herein).
XII enzyme
One or more of the enzymes and polypeptides described below are to be used in an "effective amount" in the blends or methods of the invention. The following should be read in the context of the enzyme disclosure in the "definitions" section above.
Cellulolytic compositions for use in the enzyme blends or processes and methods of the invention
The cellulolytic composition used in the process for producing a fermentation product of the invention may be derived from any microorganism. As used herein, "derived from any microorganism" means that the cellulolytic composition comprises one or more enzymes expressed in the microorganism. For example, a cellulolytic composition derived from a strain of trichoderma reesei means that the cellulolytic composition comprises one or more enzymes expressed in trichoderma reesei.
In an embodiment, the cellulolytic composition is derived from a strain of aspergillus, such as a strain of aspergillus flavus, aspergillus niger, or aspergillus oryzae.
In an embodiment, the cellulolytic composition is derived from a strain of Chrysosporium (Chrysosporium), such as a strain of Chrysosporium lucknowense (Chrysosporium lucknowense).
In the examples, the cellulolytic composition is derived from a strain of Humicola (Humicola), such as a strain of Humicola insolens (Humicola insolens).
In an embodiment, the cellulolytic composition is derived from a strain of penicillium, such as a strain of penicillium emersonii or penicillium oxalicum.
In embodiments, the cellulolytic composition is derived from a strain of the genus Talaromyces, such as a strain of Talaromyces aureofaciens or Talaromyces emersonii.
In an embodiment, the cellulolytic composition is derived from a strain of trichoderma, such as a strain of trichoderma reesei.
In a preferred embodiment, the cellulolytic composition is derived from a strain of trichoderma reesei.
The cellulolytic composition may comprise one or more of the following polypeptides (including enzymes): a GH61 polypeptide having cellulolytic enhancing activity, a β -glucosidase, CBHI and CBHII, or a mixture of two, three, or four thereof.
In a preferred embodiment, the cellulolytic composition comprises a beta-glucosidase having a relative ED50 loading value of less than 1.00, preferably less than 0.80, such as preferably less than 0.60, such as between 0.1-0.9, such as between 0.2-0.8, such as 0.30-0.70.
The cellulolytic composition may comprise some hemicellulases, such as for example xylanase and/or β -xylosidase. The hemicellulase may be derived from an organism that produces the cellulolytic composition or from another source, for example, the hemicellulase may be exogenous to an organism that produces the cellulolytic composition, such as trichoderma reesei, for example.
In a preferred embodiment, the hemicellulase content in the cellulolytic composition is less than 10 wt.%, such as less than 5 wt.% of the cellulolytic composition.
In an embodiment, the cellulolytic composition comprises a beta-glucosidase.
In embodiments, the cellulolytic composition comprises a GH61 polypeptide having cellulolytic enhancing activity and a beta-glucosidase.
In another embodiment, the cellulolytic composition comprises a β -glucosidase and CBH.
In another embodiment, the cellulolytic composition comprises a GH61 polypeptide having cellulolytic enhancing activity, a beta-glucosidase, and CBHI.
In another embodiment, the cellulolytic composition comprises β -glucosidase and CBHI.
In another embodiment, the cellulolytic composition comprises a GH61 polypeptide having cellulolytic enhancing activity, a beta-glucosidase, CBHI, and CBHII.
In another embodiment, the cellulolytic composition comprises a β -glucosidase, CBHI, and CBHII.
The cellulolytic composition may further comprise one or more enzymes selected from the group consisting of: cellulases, GH61 polypeptides having cellulolytic enhancing activity, esterases, patulin, laccases, ligninolytic enzymes, pectinases, peroxidases, proteases, and swollenins.
In embodiments, the cellulase is one or more enzymes selected from the group consisting of: endoglucanases, cellobiohydrolases, and beta-glucosidases.
In an embodiment, the endoglucanase is endoglucanase I.
In an embodiment, the endoglucanase is endoglucanase II.
Cellobiohydrolases I
In one embodiment, the cellulolytic composition used according to the invention may comprise one or more CBH I (cellobiohydrolase I). In one embodiment, the cellulolytic composition comprises a cellobiohydrolase I (cbhi), such as a cellobiohydrolase I derived from: a strain of Aspergillus, such as a strain of Aspergillus fumigatus, such as Cel7A CBHI disclosed in SEQ ID NO:6 in WO 2011/057140 or SEQ ID NO:15 herein, or a strain derived from Trichoderma, such as a strain of Trichoderma reesei.
In embodiments, the aspergillus fumigatus cellobiohydrolase I or homolog thereof is selected from the group consisting of:
(i) cellobiohydrolase I comprising the mature polypeptide of SEQ ID No. 15 herein;
(ii) cellobiohydrolase I comprising an amino acid sequence having at least 70%, e.g., 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the mature polypeptide of SEQ ID No. 15 herein;
(iii) Cellobiohydrolase I encoded by a polynucleotide comprising a nucleotide sequence having at least 70%, e.g., 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the mature polypeptide coding sequence of SEQ ID No. 1 in WO 2013/148993; and
(iv) cellobiohydrolase I encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g., very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO:1 of WO 2013/148993, or the full-length complement thereof.
Cellobiohydrolase II
In one embodiment, the cellulolytic composition used according to the invention may comprise one or more CBH II (cellobiohydrolase II). In one embodiment, the cellobiohydrolase II (cbhii) is a cellobiohydrolase II as derived from: a strain of an Aspergillus species, such as a strain of Aspergillus fumigatus, such as one of SEQ ID NO 16 herein; or a strain of Trichoderma, such as Trichoderma reesei; or a strain of the genus Thielavia, such as a strain of Thielavia terrestris, such as cellobiohydrolase II CEL6A from Thielavia terrestris.
In embodiments, the aspergillus fumigatus cellobiohydrolase II or homolog thereof is selected from the group consisting of:
(i) cellobiohydrolase II comprising the mature polypeptide of SEQ ID No. 16 herein;
(ii) cellobiohydrolase II comprising an amino acid sequence having at least 70%, e.g., 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the mature polypeptide of SEQ ID No. 16 herein;
(iii) cellobiohydrolase II encoded by a polynucleotide comprising a nucleotide sequence having at least 70%, e.g., 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the mature polypeptide coding sequence of SEQ ID No. 3 in WO 2013/148993; and
(iv) cellobiohydrolase II encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g., very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO:3 of WO 2013/148993, or the full-length complement thereof.
Beta-glucosidase
In one embodiment, the cellulolytic composition used according to the invention may comprise one or more beta-glucosidases. In one embodiment, the beta-glucosidase may be a beta-glucosidase derived from: a strain of aspergillus, such as aspergillus oryzae, such as one disclosed in WO 2002/095014 or a fusion protein with β -glucosidase activity disclosed in WO 2008/057637; or Aspergillus fumigatus, such as one disclosed in WO 2005/047499 or SEQ ID NO:17 herein, or Aspergillus fumigatus beta-glucosidase variants, such as one disclosed in WO 2012/044915 or co-pending PCT application PCT/US11/054185 (or U.S. provisional application No. 61/388,997), such as one having the following substitutions: F100D, S283G, N456E, F512Y.
In another embodiment, the beta-glucosidase is derived from a strain of Penicillium, such as the strain of Penicillium brasiliensis (Penicillium brasilianum) disclosed in WO 2007/019442; or a strain of Trichoderma, such as a strain of Trichoderma reesei.
In an embodiment, the beta-glucosidase is an aspergillus fumigatus beta-glucosidase or a homolog thereof selected from the group consisting of:
(i) a beta-glucosidase comprising the mature polypeptide of SEQ ID NO 17;
(ii) A beta-glucosidase comprising an amino acid sequence having at least 70%, e.g., 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the mature polypeptide of SEQ ID No. 17 herein;
(iii) a beta-glucosidase encoded by a polynucleotide comprising a nucleotide sequence that is at least 70%, e.g., 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the mature polypeptide coding sequence of SEQ ID No. 5 in WO 2013/148993; and
(iv) a beta-glucosidase encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g., very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO. 5 of WO 2013/148993, or the full-length complement thereof.
In embodiments, the beta-glucosidase is a variant comprising substitutions at one or more (several) positions corresponding to positions 100, 283, 456, and 512 of the mature polypeptide of SEQ ID NO:17 herein, wherein the variant has beta-glucosidase activity.
In embodiments, the parent beta-glucosidase of the variant is (a) a polypeptide comprising the mature polypeptide of SEQ ID NO:17 herein; (b) a polypeptide having at least 80% sequence identity to the mature polypeptide of SEQ ID NO 17 herein; (c) a polypeptide encoded by a polynucleotide that hybridizes under high or very high stringency conditions with (i) the mature polypeptide coding sequence of seq id no: (i) the mature polypeptide coding sequence of SEQ ID NO:5 in WO 2013/148993, (ii) the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO:5 in WO 2013/148993, or (iii) the full-length complementary strand of (i) or (ii); (d) a polypeptide encoded by a polynucleotide having at least 80% identity to the mature polypeptide coding sequence of SEQ ID No. 5 of WO 2013/148993 or a cDNA sequence thereof; or (e) a fragment of the mature polypeptide of SEQ ID NO 17 herein, which fragment has beta-glucosidase activity.
In embodiments, the β -glucosidase variant has at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% sequence identity to the amino acid sequence of a parent β -glucosidase.
In embodiments, the variant has at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% but less than 100% sequence identity to the mature polypeptide of SEQ ID No. 17 herein.
In embodiments, the beta-glucosidase is from a strain of aspergillus, such as a strain of aspergillus fumigatus, such as aspergillus fumigatus beta-glucosidase (SEQ ID NO:17 herein), comprising one or more substitutions selected from the group consisting of: L89M, G91L, F100D, I140V, I186V, S283G, N456E, and F512Y; such as variants thereof with the following substitutions:
-F100D+S283G+N456E+F512Y；
-L89M+G91L+I186V+I140V；
-I186V+L89M+G91L+I140V+F100D+S283G+N456E+F512Y。
in embodiments, the number of substitutions is between 1 and 4, such as 1, 2, 3, or 4 substitutions.
In an embodiment, a variant comprises a substitution at a position corresponding to position 100, a substitution at a position corresponding to position 283, a substitution at a position corresponding to position 456, and/or a substitution at a position corresponding to position 512.
In a preferred embodiment, the β -glucosidase variant comprises the following substitutions: phe100Asp, Ser283Gly, Asn456Glu, Phe512Tyr in SEQ ID NO 17 herein.
In a preferred embodiment, the beta-glucosidase has a relative ED50 load value of less than 1.00, preferably less than 0.80, such as preferably less than 0.60, such as between 0.1-0.9, such as between 0.2-0.8, such as 0.30-0.70.
GH61 polypeptides having cellulolytic enhancing activity
In one embodiment, a cellulolytic composition used according to the invention may comprise one or more GH61 polypeptides having cellulolytic enhancing activity. In one embodiment, the enzyme composition comprises a GH61 polypeptide having cellulolytic enhancing activity, such as one derived from a strain of thermoascus, such as thermoascus aurantiacus, e.g., as described in WO 2005/074656 as SEQ ID No. 2; or a strain derived from a Thielavia, such as Thielavia terrestris, such as the polypeptide described in WO 2005/074647 as SEQ ID NO. 7 and SEQ ID NO. 8; or a strain derived from Aspergillus, such as a strain of Aspergillus fumigatus, such as the polypeptide described in WO 2010/138754 as SEQ ID NO. 2; or a strain derived from Penicillium, such as one of the strains of Penicillium emersonii, such as one of the SEQ ID NOs 18 disclosed in WO 2011/041397 or herein.
In an embodiment, the penicillium species GH61 polypeptide or homologue thereof having cellulolytic enhancing activity is selected from the group consisting of:
(i) A GH61 polypeptide having cellulolytic enhancing activity comprising the mature polypeptide of SEQ ID No. 18 herein;
(ii) a GH61 polypeptide having cellulolytic enhancing activity comprising an amino acid sequence that is at least 70%, e.g., 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the mature polypeptide of SEQ ID No. 18 herein;
(iii) a GH61 polypeptide having cellulolytic enhancing activity encoded by a polynucleotide comprising a nucleotide sequence that is at least 70%, e.g., 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the mature polypeptide coding sequence of SEQ ID No. 7 of WO 2013/148993; and
(iv) a GH61 polypeptide having cellulolytic enhancing activity encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g., very high stringency conditions, to the mature polypeptide coding sequence of SEQ ID NO:7 of WO 2013/148993, or the full-length complement thereof.
Cellulose decomposition composition
As mentioned above, the cellulolytic composition may comprise a plurality of different polypeptides (e.g. enzymes).
In an embodiment, the cellulolytic composition comprises a trichoderma reesei cellulolytic composition, further comprising a thermoascus aurantiacus GH61A polypeptide having cellulolytic enhancing activity (WO 2005/074656) and an aspergillus oryzae beta-glucosidase fusion protein (WO 2008/057637).
In another embodiment, the cellulolytic composition comprises a Trichoderma reesei cellulolytic composition, further comprising an Thermoascus aurantiacus GH61A polypeptide (SEQ ID NO:2 in WO 2005/074656) and Aspergillus fumigatus beta-glucosidase (SEQ ID NO:2 of WO 2005/047499) having cellulolytic enhancing activity.
In another embodiment, the cellulolytic composition comprises a trichoderma reesei cellulolytic composition, further comprising a penicillium emersonii GH61A polypeptide having cellulolytic enhancing activity disclosed in WO 2011/041397, aspergillus fumigatus beta-glucosidase (SEQ ID NO:2 of WO 2005/047499), or a variant thereof having the following substitutions: F100D, S283G, N456E, F512Y.
The enzyme composition of the invention may be in any form suitable for use, such as, for example, a crude fermentation broth with or without cells removed, a cell lysate with or without cell debris, a semi-purified or purified enzyme composition, or a host cell (e.g., a trichoderma host cell) from which the enzyme is derived.
The enzyme composition may be a dry powder or granulate, a non-dusting granulate, a liquid, a stabilized liquid or a stabilized protected enzyme. The liquid enzyme composition may be stabilized according to established methods, for example by adding a stabilizer, such as a sugar, sugar alcohol or other polyol, and/or lactic acid or another organic acid.
In a preferred embodiment, the cellulolytic composition comprises a beta-glucosidase having a relative ED50 loading value of less than 1.00, preferably less than 0.80, such as preferably less than 0.60, such as between 0.1-0.9, such as between 0.2-0.8, such as 0.30-0.70.
In an embodiment, the cellulolytic enzyme composition (i.e. during saccharification in step ii) and/or fermentation or SSF in step iii)) is added at a dose of 0.0001-3mg EP/g DS, preferably 0.0005-2mg EP/g DS, preferably 0.001-1mg/g DS, more preferably from 0.005-0.5mg EP/g DS, even more preferably 0.01-0.1mg EP/g DS.
Alpha-amylase present and/or added during liquefaction
According to the invention, in the liquefaction, an alpha-amylase is optionally present and/or added together with hemicellulases, endoglucanases, proteases, carbohydrate source producing enzymes (such as glucoamylase), phospholipases, phytases, and/or pullulanases.
The alpha-amylase added during the liquefaction step i) may be any alpha-amylase. Preferred are bacterial alpha-amylases, such as in particular bacillus alpha-amylases, such as bacillus stearothermophilus alpha-amylase, which is stable at the temperatures used during liquefaction.
Bacterial alpha-amylases
The term "bacterial alpha-amylase" means any bacterial alpha-amylase classified under EC 3.2.1.1. The bacterial alpha-amylases for use according to the invention may for example be derived from a strain of bacillus (sometimes also referred to as geobacillus). In an embodiment, the bacillus alpha-amylase is derived from a strain of bacillus amyloliquefaciens, bacillus licheniformis, bacillus stearothermophilus, bacillus species TS-23, or bacillus subtilis, but may also be derived from other bacillus species.
Specific examples of bacterial alpha-amylases include Bacillus stearothermophilus alpha-amylase of SEQ ID NO:3 in WO 99/19467 or SEQ ID NO:19 herein, Bacillus amyloliquefaciens alpha-amylase of SEQ ID NO:5 in WO 99/19467, and Bacillus licheniformis alpha-amylase of SEQ ID NO:4 in WO 99/19467, and Bacillus species TS-23 alpha-amylase disclosed as SEQ ID NO:1 in WO 2009/061380 (all sequences are hereby incorporated by reference).
In embodiments, the bacterial alpha-amylase may be an enzyme having a degree of identity of at least 60%, e.g., at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to any of the sequences set forth in SEQ ID NOs 3, 4, or 5 in WO 99/19467 and SEQ ID No. 1 in WO 2009/061380, respectively.
In embodiments, the alpha-amylase can be an enzyme having a degree of identity of at least 60%, e.g., at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to any sequence as set forth in SEQ ID No. 3 in WO 99/19467, or SEQ ID No. 19 herein.
In a preferred embodiment, the alpha-amylase is derived from Bacillus stearothermophilus. The Bacillus stearothermophilus alpha-amylase may be a mature wild-type or a mature variant thereof. The mature Bacillus stearothermophilus alpha-amylase or variant thereof may be naturally truncated during recombinant production. For example, the mature Bacillus stearothermophilus alpha-amylase may be truncated at the C-terminus, so it is about 491 amino acids long (as compared to SEQ ID NO:3 in WO 99/19467 or SEQ ID NO:19 herein), such as from 480 to 495 amino acids long.
The bacillus alpha-amylase may also be a variant and/or a hybrid. Examples of such variants can be found in any of the following: WO 96/23873, WO 96/23874, WO 97/41213, WO 99/19467, WO 00/60059, WO 02/10355 and WO 2009/061380 (all documents are hereby incorporated by reference). Specific alpha-amylase variants are disclosed in U.S. patent nos. 6,093,562, 6,187,576, 6,297,038, and 7,713,723 (incorporated herein by reference) and include bacillus stearothermophilus alpha-amylase (often referred to as BSG alpha-amylase) variants having: deletion of one or two amino acids at any of positions R179, G180, I181, and/or G182, preferably the double deletion disclosed in WO 96/23873-see, e.g., page 20, lines 1-10 (hereby incorporated by reference), preferably corresponding to the deletion of positions I181 and G182 compared to the amino acid sequence of B.stearothermophilus alpha-amylase as set forth in SEQ ID NO:3 disclosed in WO 99/19467 or SEQ ID NO:19 herein, or the deletion of amino acids R179 and G180 using SEQ ID NO:3 in WO 99/19467 or SEQ ID NO:19 herein. Even more preferred are bacillus alpha-amylases, especially Bacillus Stearothermophilus (BSG) alpha-amylase having one or two amino acid deletions in the amino acid sequence corresponding to positions R179, G180, I181, and G182, preferably having a double deletion corresponding to R179 and G180, or preferably a deletion at positions 181 and 182 (denoted I181 + G182), and optionally further comprising a N193F substitution (denoted I181 + G182 + N193F), compared to the wild type BSG alpha-amylase amino acid sequence set forth in SEQ ID NO:3 disclosed in WO 99/19467 or SEQ ID NO:19 herein. The bacterial alpha-amylase may also have a substitution at a position corresponding to S242 variant of Bacillus licheniformis alpha-amylase as shown in SEQ ID NO:4 in WO 99/19467, or Bacillus stearothermophilus alpha-amylase of SEQ ID NO:3 in WO 99/19467, or S239 in SEQ ID NO:19 herein.
In embodiments, the variant is an S242A, E, or Q variant, preferably an S242Q, or A variant (numbered using SEQ ID NO:19 herein) of a Bacillus stearothermophilus alpha-amylase.
In the examples, the variant is a position E188 variant, preferably an E188P variant (numbered using SEQ ID NO:19 herein) of Bacillus stearothermophilus alpha-amylase.
Other contemplated variants are the Bacillus species TS-23 variants disclosed in WO 2009/061380, in particular the variants defined in claim 1 of WO 2009/061380 (hereby incorporated by reference).
Bacterial hybrid alpha-amylases
The bacterial alpha-amylase may also be a hybrid bacterial alpha-amylase, for example comprising the 445C-terminal amino acid residues of Bacillus licheniformis alpha-amylase (shown in SEQ ID NO:4 of WO 99/19467) and the 37N-terminal amino acid residues of alpha-amylase derived from Bacillus amyloliquefaciens alpha-amylase (shown in SEQ ID NO:5 of WO 99/19467). In preferred embodiments, the hybrid has one or more, especially all, of the following substitutions:
G48A + T49I + G107A + H156Y + A181T + N190F + I201F + A209V + Q264S (using Bacillus licheniformis numbering in SEQ ID NO:4 of WO 99/19467). Also preferred are variants having one or more of the following mutations (or corresponding mutations in other bacillus alpha-amylases): H154Y, A181T, N190F, A209V, and Q264S and/or the deletion of two residues between positions 176 and 179, preferably the deletion of E178 and G179 (position numbering using SEQ ID NO:5 of WO 99/19467).
In The examples, The bacterial alpha-amylase is The mature part of a chimeric alpha-amylase disclosed in Richardson et al, 2002, The Journal of Biological Chemistry 277(29), 267501-26507, referred to as BD5088 or variants thereof. The alpha-amylase is the same as shown in WO 2007134207 as SEQ ID NO. 2. The mature enzyme sequence begins after the initial "Met" amino acid at position 1.
Thermostable alpha-amylase
According to the invention, optionally an alpha-amylase is used in combination with a hemicellulase, preferably a xylanase, having a melting point (DSC) of greater than 80 ℃. Optionally, endoglucanases having a melting point (DSC) of more than 70 ℃, such as more than 75 ℃, in particular more than 80 ℃ may be included. The thermostable alpha-amylase (e.g.bacterial alpha-amylase) is preferably derived from Bacillus stearothermophilus or Bacillus species TS-23. In the examples, the alpha-amylase was assayed at pH 4.5, 85 ℃ and 0.12mM CaCl2The lower has a T1/2(min) of at least 10. In the examples, the alpha-amylase was assayed at pH 4.5, 85 ℃ and 0.12mM CaCl2The lower has a T1/2(min) of at least 15. In the examples, the alpha-amylase was assayed at pH 4.5, 85 ℃ and 0.12mM CaCl2The lower has a T1/2(min) of at least 20. In the examples, the alpha-amylase was assayed at pH 4.5, 85 ℃ and 0.12mM CaCl 2At the bottom, it has a T1/2(min) of at least 25. In the examples, the alpha-amylase was assayed at pH 4.5, 85 ℃ and 0.12mM CaCl2The lower has a T1/2(min) of at least 30. In the examples, the alpha-amylase was assayed at pH 4.5, 85 ℃ and 0.12mM CaCl2The lower has a T1/2(min) of at least 40. In the examplesAlpha-amylase at pH 4.5, 85 ℃ 0.12mM CaCl2The lower has a T1/2(min) of at least 50. In the examples, the alpha-amylase was assayed at pH 4.5, 85 ℃ and 0.12mM CaCl2The lower has a T1/2(min) of at least 60. In the examples, the alpha-amylase was assayed at pH 4.5, 85 ℃ and 0.12mM CaCl2The lower has a T1/2(min) between 10 and 70. In the examples, the alpha-amylase was assayed at pH 4.5, 85 ℃ and 0.12mM CaCl2The lower has a T1/2(min) between 15 and 70. In the examples, the alpha-amylase was assayed at pH 4.5, 85 ℃ and 0.12mM CaCl2The lower has a T1/2(min) between 20 and 70. In the examples, the alpha-amylase was assayed at pH 4.5, 85 ℃ and 0.12mM CaCl2The lower has a T1/2(min) between 25 and 70. In the examples, the alpha-amylase was assayed at pH 4.5, 85 ℃ and 0.12mM CaCl2The lower has a T1/2(min) between 30 and 70. In the examples, the alpha-amylase was assayed at pH 4.5, 85 ℃ and 0.12mM CaCl2The lower has a T1/2(min) between 40-70. In the examples, the alpha-amylase was assayed at pH 4.5, 85 ℃ and 0.12mM CaCl 2The lower has a T1/2(min) between 50 and 70. In the examples, the alpha-amylase was assayed at pH 4.5, 85 ℃ and 0.12mM CaCl2The lower has a T1/2(min) between 60-70.
In the examples, the alpha-amylase is a bacterial alpha-amylase, preferably derived from a strain of bacillus, especially bacillus stearothermophilus, as disclosed in WO 99/19467 as SEQ ID NO:3 or SEQ ID NO:19 herein, with one or two amino acid deletions at positions R179, G180, I181, and/or G182, especially R179 and G180 deletions, or with I181 and G182 deletions, with mutations in the following list of mutations. In a preferred embodiment, the bacillus stearothermophilus alpha-amylase has a double deletion I181+ G182, and optionally the substitution N193F, optionally further comprising a mutation selected from the list:
-V59A+Q89R+G112D+E129V+K177L+R179E+K220P+N224L+Q254S；
-V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S；
-V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+D269E+D281N；
-V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+I270L；
-V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+H274K；
-V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+Y276F；
-V59A+E129V+R157Y+K177L+R179E+K220P+N224L+S242Q+Q254S；
-V59A+E129V+K177L+R179E+H208Y+K220P+N224L+S242Q+Q254S；
-V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S；
-V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+H274K；
-V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+Y276F；
-V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+D281N；
-V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+M284T；
-V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+G416V；
-V59A+E129V+K177L+R179E+K220P+N224L+Q254S；
-V59A+E129V+K177L+R179E+K220P+N224L+Q254S+M284T；
-A91L+M96I+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S；
-E129V+K177L+R179E；
-E129V+K177L+R179E+K220P+N224L+S242Q+Q254S；
-E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+Y276F+L427M；
-E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+M284T；
-E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+N376*+I377*；
-E129V+K177L+R179E+K220P+N224L+Q254S；
-E129V+K177L+R179E+K220P+N224L+Q254S+M284T；
-E129V+K177L+R179E+S242Q；
-E129V+K177L+R179V+K220P+N224L+S242Q+Q254S；
-K220P+N224L+S242Q+Q254S；
-M284V；
-V59A+Q89R+E129V+K177L+R179E+Q254S+M284V。
in an embodiment, the alpha-amylase is selected from the group of bacillus stearothermophilus alpha-amylase variants:
-I181*+G182*；
-I181*+G182*+N193F；
preferably
-I181*+G182*+E129V+K177L+R179E；
-I181*+G182*+N193F+E129V+K177L+R179E；
-181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S；
-I181 x + G182 x + N193F + V59A + Q89R + E129V + K177L + R179E + Q254S + M284V; and
-I181 + G182 + N193F + E129V + K177L + R179E + K220P + N224L + S242Q + Q254S (numbering using SEQ ID NO:19 herein).
In embodiments, the bacterial alpha-amylase (e.g., a bacillus alpha-amylase, such as a bacillus stearothermophilus alpha-amylase) has at least 60%, such as at least 70%, such as at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature portion of the polypeptide of SEQ ID No. 19 herein.
In embodiments, a bacterial alpha-amylase variant (e.g., a bacillus alpha-amylase variant, such as a bacillus stearothermophilus alpha-amylase variant) has at least 60%, such as at least 70%, such as at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% identity to the mature portion of the polypeptide of SEQ ID No. 19 herein.
It will be appreciated that when reference is made to Bacillus stearothermophilus alpha-amylase and variants thereof, they are normally naturally produced in truncated form. In particular, the truncation is such that the B.stearothermophilus alpha-amylase shown in SEQ ID NO:3 in WO99/19467 or SEQ ID NO:19 herein or a variant thereof is truncated at the C-terminus and is typically about 491 amino acids in length, such as from 480 to 495 amino acids in length.
Thermostable hemicellulases present and/or added during liquefaction
According to the invention, an optional hemicellulase (preferably a xylanase) having a melting point (DSC) of greater than 80 ℃ is present in combination with an alpha-amylase, such as a bacterial alpha-amylase (described above), and/or is added to liquefaction step i).
The thermostability of the hemicellulase (preferably xylanase) can be determined by differential scanning calorimetry as described in the materials and methods sectiondThe assays described in "assays for endoglucanase and hemicellulase".
In embodiments, the hemicellulase, in particular the xylanase, in particular GH10 or GH11 xylanase, has a melting point (DSC) of greater than 82 ℃, such as greater than 84 ℃, such as greater than 86 ℃, such as greater than 88 ℃, such as greater than 90 ℃, such as greater than 92 ℃, such as greater than 94 ℃, such as greater than 96 ℃, such as greater than 98 ℃, such as greater than 100 ℃, such as between 80 ℃ and 110 ℃, such as between 82 ℃ and 110 ℃, such as between 84 ℃ and 110 ℃.
In a preferred embodiment, the hemicellulase, in particular the xylanase, in particular GH10 xylanase, has at least 60%, such as at least 70%, such as at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity with the mature part of the polypeptide of SEQ ID No. 20 herein, preferably originates from a strain of dictyococcus, such as a strain of dictyococcus thermophilus.
In a preferred embodiment, the hemicellulase, in particular the xylanase, in particular GH11 xylanase, has at least 60%, such as at least 70%, such as at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity with the mature part of the polypeptide of SEQ ID No. 21 herein, preferably derived from a strain of dictyococcus, such as a strain of dictyococcus thermophilus.
In a preferred embodiment, the hemicellulase, in particular the xylanase, in particular GH10 xylanase, has at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID No. 22 herein, preferably derived from a strain of the genus botrytis (Rasamsonia), such as a strain of botrytis cinerea.
In a preferred embodiment, the hemicellulase, in particular the xylanase, in particular the GH10 xylanase, has at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID No. 23 herein, preferably derived from a strain of the genus talaromyces, such as a strain of the species talaromyces reesei.
In a preferred embodiment, the hemicellulase, in particular the xylanase, in particular the GH10 xylanase, has at least 60%, such as at least 70%, such as at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity with the mature part of the polypeptide of SEQ ID No. 24 herein, preferably derived from a strain of aspergillus, such as a strain of aspergillus fumigatus.
Thermostable endoglucanases present and/or added during liquefaction
According to the invention, in the liquefaction step i), an optional endoglucanase ("E") having a melting point (DSC) of more than 70 ℃ (such as between 70 ℃ and 95 ℃) may be present and/or added in combination with an alpha-amylase, such as a thermostable bacterial alpha-amylase, and an optional hemicellulase, preferably a xylanase, having a melting point (DSC) of more than 80 ℃.
Thermostability of endoglucanases T can be determined by differential scanning calorimetry as in the materials and methods section of WO 2017/112540 (which is incorporated herein by reference in its entirety)dDetermination of endoglucanase and hemicellulase "determination described under the heading.
In embodiments, the endoglucanase has a melting point (DSC) of more than 72 ℃, such as more than 74 ℃, such as more than 76 ℃, such as more than 78 ℃, such as more than 80 ℃, such as more than 82 ℃, such as more than 84 ℃, such as more than 86 ℃, such as more than 88 ℃, such as between 70 ℃ and 95 ℃, such as between 76 ℃ and 94 ℃, such as between 78 ℃ and 93 ℃, such as between 80 ℃ and 92 ℃, such as between 82 ℃ and 91 ℃, such as between 84 ℃ and 90 ℃.
In a preferred embodiment, the endoglucanase used in the method of the invention or comprised in the composition of the invention is a glycoside hydrolase family 5 endoglucanase or a GH5 endoglucanase (see CAZy database at the website "www.cazy.org").
In the examples, the GH5 endoglucanase is from family EG II, an basket fungus endoglucanase as shown in SEQ ID NO:25 herein, a Penicillium capsulatum endoglucanase as shown in SEQ ID NO:26 herein, and a Trichophaea fusca (Trichophaea saccharocata) endoglucanase as shown in SEQ ID NO:27 herein.
In an embodiment, the endoglucanase is a family GH45 endoglucanase. In the examples, the GH45 endoglucanase is from family EG V, coprinus faecalis as shown in SEQ ID No. 28 herein or thielavia terrestris endoglucanase as shown in SEQ ID No. 29 herein.
In embodiments, the endoglucanase has at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID No. 25 herein. In embodiments, the endoglucanase is derived from a strain of the genus Talaromyces, such as a strain of Talaromyces reesei.
In embodiments, the endoglucanase has at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID No. 26 herein, preferably derived from a strain of the genus penicillium, such as a strain of penicillium capsulatum.
In embodiments, the endoglucanase has at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID No. 27 herein, preferably is derived from a strain of the species lachnum (Trichophaea), such as a strain of the species lachnum fusca.
In embodiments, the endoglucanase has at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID No. 28 herein, preferably is derived from a strain of coprinus, such as a strain of coprinus.
In embodiments, the endoglucanase has at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID No. 29 herein, preferably is derived from a strain of the genus thielavia, such as a strain of thielavia terrestris.
In the examples, the endoglucanase is added in the liquefaction step i) at a dose of 1-10000. mu.g EP (enzyme protein)/g DS, such as 10-1000. mu.g EP/g DS.
Enzymes for producing carbohydrate sources present and/or added during liquefaction
According to the invention, in the liquefaction, an optional carbohydrate source producing enzyme, in particular a glucoamylase, preferably a thermostable glucoamylase, may be present and/or added together with an alpha-amylase and optionally a hemicellulase (preferably a xylanase) with a melting point (DSC) of more than 80 ℃ and optionally an endoglucanase with a melting point (DSC) of more than 70 ℃ and optionally a pullulanase and/or optionally a phytase.
The term "carbohydrate source producing enzyme" includes any enzyme that produces fermentable sugars. The carbohydrate-source producing enzyme is capable of producing carbohydrates which can be used as an energy source by one or more fermenting organisms in question, for example when used in the process of the invention for producing a fermentation product, such as ethanol. The produced carbohydrates can be converted directly or indirectly into the desired fermentation product, preferably ethanol. According to the invention, mixtures of enzymes producing a carbohydrate source may be used. Specific examples include glucoamylase (for glucose producers), beta-amylase, and maltogenic amylase (for maltose producers).
In a preferred embodiment, the carbohydrate source producing enzyme is thermostable. The carbohydrate-source producing enzyme, particularly the thermostable glucoamylase, may be added with or separately from the alpha-amylase and thermostable protease.
In a specific and preferred embodiment, the carbohydrate-source producing enzyme is a thermostable glucoamylase, preferably of fungal origin, preferably a filamentous fungus, such as a strain from the genus Penicillium, especially a strain of Penicillium oxalicum, in particular the Penicillium oxalicum glucoamylase disclosed as SEQ ID NO:2 and shown in SEQ ID NO:30 herein in WO 2011/127802 (which is hereby incorporated by reference).
In embodiments, the thermostable glucoamylase has at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the mature polypeptide shown in SEQ ID No. 2 of WO 2011/127802 or SEQ ID No. 30 herein.
In the examples, the carbohydrate-source producing enzyme, particularly the thermostable glucoamylase, is a penicillium oxalicum glucoamylase shown herein in SEQ ID No. 30.
In a preferred embodiment, the carbohydrate-source producing enzyme is a variant of Penicillium oxalicum glucoamylase disclosed as SEQ ID NO:2 in WO 2011/127802 and shown herein in SEQ ID NO:30, with a K79V substitution (referred to as "PE 001") (using the mature sequence shown in SEQ ID NO:14 for numbering). As disclosed in WO 2013/036526 (which is hereby incorporated by reference), the K79V glucoamylase variant has reduced sensitivity to protease degradation relative to the parent.
Contemplated variants of the penicillium oxalicum glucoamylase are disclosed in WO 2013/053801 (which is hereby incorporated by reference).
In embodiments, the variants have reduced sensitivity to protease degradation.
In embodiments, the variants have improved thermostability compared to the parent.
More specifically, in embodiments, the glucoamylase has a K79V substitution (numbered using SEQ ID NO:30 herein) corresponding to the PE001 variant, and further includes at least one of the following substitutions or combinations of substitutions:
T65A；Q327F；E501V；Y504T；Y504*；T65A+Q327F；T65A+E501V；T65A+Y504T；T65A+Y504*；Q327F+E501V；Q327F+Y504T；Q327F+Y504*；E501V+Y504T；E501V+Y504*；T65A+Q327F+E501V；T65A+Q327F+Y504T；T65A+E501V+Y504T；Q327F+E501V+Y504T；T65A+Q327F+Y504*；T65A+E501V+Y504*；Q327F+E501V+Y504*；T65A+Q327F+E501V+Y504T；T65A+Q327F+E501V+Y504*；E501V+Y504T；T65A+K161S；T65A+Q405T；T65A+Q327W；T65A+Q327F；T65A+Q327Y；P11F+T65A+Q327F；
R1K + D3W + K5Q + G7V + N8S + T10K + P11S + T65A + Q327F; P2N + P4S + P11F + T65A + Q327F; P11F + D26C + K33C + T65A + Q327F; P2N + P4S + P11F + T65A + Q327W + E501V + Y504T; R1E + D3N + P4G + G6R + G7A + N8A + T10D + P11D + T65A + Q327F; P11F + T65A + Q327W; P2N + P4S + P11F + T65A + Q327F + E501V + Y504T; P11F + T65A + Q327W + E501V + Y504T; T65A + Q327F + E501V + Y504T; T65A + S105P + Q327W; T65A + S105P + Q327F; T65A + Q327W + S364P; T65A + Q327F + S364P; T65A + S103N + Q327F; P2N + P4S + P11F + K34Y + T65A + Q327F; P2N + P4S + P11F + T65A + Q327F + D445N + V447S; P2N + P4S + P11F + T65A + I172V + Q327F; P2N + P4S + P11F + T65A + Q327F + N502; P2N + P4S + P11F + T65A + Q327F + N502T + P563S + K571E; P2N + P4S + P11F + R31S + K33V + T65A + Q327F + N564D + K571S; P2N + P4S + P11F + T65A + Q327F + S377T; P2N + P4S + P11F + T65A + V325T + Q327W; P2N + P4S + P11F + T65A + Q327F + D445N + V447S + E501V + Y504T; P2N + P4S + P11F + T65A + I172V + Q327F + E501V + Y504T; P2N + P4S + P11F + T65A + Q327F + S377T + E501V + Y504T; P2N + P4S + P11F + D26N + K34Y + T65A + Q327F; P2N + P4S + P11F + T65A + Q327F + I375A + E501V + Y504T; P2N + P4S + P11F + T65A + K218A + K221D + Q327F + E501V + Y504T; P2N + P4S + P11F + T65A + S103N + Q327F + E501V + Y504T; P2N + P4S + T10D + T65A + Q327F + E501V + Y504T; P2N + P4S + F12Y + T65A + Q327F + E501V + Y504T; K5A + P11F + T65A + Q327F + E501V + Y504T; P2N + P4S + T10E + E18N + T65A + Q327F + E501V + Y504T; P2N + T10E + E18N + T65A + Q327F + E501V + Y504T; P2N + P4S + P11F + T65A + Q327F + E501V + Y504T + T568N; P2N + P4S + P11F + T65A + Q327F + E501V + Y504T + K524T + G526A; P2N + P4S + P11F + K34Y + T65A + Q327F + D445N + V447S + E501V + Y504T; P2N + P4S + P11F + R31S + K33V + T65A + Q327F + D445N + V447S + E501V + Y504T; P2N + P4S + P11F + D26N + K34Y + T65A + Q327F + E501V + Y504T; P2N + P4S + P11F + T65A + F80 + Q327F + E501V + Y504T; P2N + P4S + P11F + T65A + K112S + Q327F + E501V + Y504T; P2N + P4S + P11F + T65A + Q327F + E501V + Y504T + T516P + K524T + G526A; P2N + P4S + P11F + T65A + Q327F + E501V + N502T + Y504; P2N + P4S + P11F + T65A + Q327F + E501V + Y504T; P2N + P4S + P11F + T65A + S103N + Q327F + E501V + Y504T; K5A + P11F + T65A + Q327F + E501V + Y504T; P2N + P4S + P11F + T65A + Q327F + E501V + Y504T + T516P + K524T + G526A; P2N + P4S + P11F + T65A + V79A + Q327F + E501V + Y504T; P2N + P4S + P11F + T65A + V79G + Q327F + E501V + Y504T; P2N + P4S + P11F + T65A + V79I + Q327F + E501V + Y504T; P2N + P4S + P11F + T65A + V79L + Q327F + E501V + Y504T; P2N + P4S + P11F + T65A + V79S + Q327F + E501V + Y504T; P2N + P4S + P11F + T65A + L72V + Q327F + E501V + Y504T; S255N + Q327F + E501V + Y504T; P2N + P4S + P11F + T65A + E74N + Q327F + E501V + Y504T; P2N + P4S + P11F + T65A + G220N + Q327F + E501V + Y504T; P2N + P4S + P11F + T65A + Y245N + Q327F + E501V + Y504T; P2N + P4S + P11F + T65A + Q253N + Q327F + E501V + Y504T; P2N + P4S + P11F + T65A + D279N + Q327F + E501V + Y504T; P2N + P4S + P11F + T65A + Q327F + S359N + E501V + Y504T; P2N + P4S + P11F + T65A + Q327F + D370N + E501V + Y504T; P2N + P4S + P11F + T65A + Q327F + V460S + E501V + Y504T; P2N + P4S + P11F + T65A + Q327F + V460T + P468T + E501V + Y504T; P2N + P4S + P11F + T65A + Q327F + T463N + E501V + Y504T; P2N + P4S + P11F + T65A + Q327F + S465N + E501V + Y504T; or P2N + P4S + P11F + T65A + Q327F + T477N + E501V + Y504T.
In a preferred embodiment, the penicillium oxalicum glucoamylase variant has a substitution K79V numbered using SEQ ID NO:23 herein (PE001 variant) and further comprises one of the following mutations:
P11F+T65A+Q327F；
P2N+P4S+P11F+T65A+Q327F；
P11F+D26C+K33C+T65A+Q327F；
P2N+P4S+P11F+T65A+Q327W+E501V+Y504T；
P2N + P4S + P11F + T65A + Q327F + E501V + Y504T; or
P11F+T65A+Q327W+E501V+Y504T。
In embodiments, a glucoamylase variant (e.g., a penicillium oxalicum glucoamylase variant) has at least 60%, such as at least 70%, such as at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% identity to the mature polypeptide of SEQ ID No. 30 herein.
The carbohydrate source producing enzyme, particularly glucoamylase, may be added in an amount of 0.1-100. mu.g EP/gDS, such as 0.5-50. mu.g EP/g DS, such as 1-25. mu.g EP/g DS, such as 2-12. mu.g EP/g DS.
Pullulanase present and/or added during liquefaction
Optionally, during the liquefaction step i), pullulanase may be present and/or added together with alpha-amylase and optionally hemicellulase (preferably xylanase) having a melting point (DSC) of greater than 80 ℃. As mentioned above, proteases, carbohydrate source producing enzymes, preferably a thermostable glucoamylase may also optionally be present and/or added during liquefaction step i).
Pullulanase may be present and/or added during the liquefaction step i) and/or the saccharification step ii) or simultaneous saccharification and fermentation.
Pullulanases (e.c.3.2.1.41, pullulan 6-glucan-hydrolase) are debranching enzymes characterized by their ability to hydrolyze alpha-1, 6-glycosidic bonds in, for example, amylopectin and pullulan.
Pullulanases encompassed by the present invention include pullulanase from Bacillus amyloliquefaciens (Bacillus amyloderamificans) disclosed in U.S. Pat. No. 4,560,651 (incorporated herein by reference), pullulanase from Bacillus amyloderamificans (SEQ ID NO: 2) disclosed in WO 01/151620 (incorporated herein by reference), pullulanase from Bacillus amyloliquefaciens (Bacillus deramificans) disclosed in WO 01/151620 (incorporated herein by reference) as SEQ ID NO:4, and pullulanase from Bacillus amyloliquefaciens (Bacillus acidopulyticus) disclosed in WO 01/151620 (incorporated herein by reference) as SEQ ID NO:6, and also pullulanase described in FEMS Mic.Let.FEMS microbiology letters (1994)115, 97-106.
Further pullulanases encompassed according to the present invention include pullulanases from Pyrococcus woosenei (Pyrococcus woesei), in particular from Pyrococcus woosenei DSM No.3773 disclosed in WO 92/02614.
In an embodiment, the pullulanase is a family GH57 pullulanase. In an embodiment, the pullulanase comprises the X47 domain as disclosed in WO 2011/087836 (which is hereby incorporated by reference). More specifically, the pullulanase may be derived from strains of the genus Thermococcus (Thermococcus), including Thermococcus thermophilus (Thermococcus litoralis) and Thermococcus hydrothermalis (Thermococcus hydrothermalis), such as the Thermococcus hydrothermus pullulanase truncated at the X4 site immediately after the X47 domain shown in WO 2011/087836. The pullulanase may also be a Thermococcus thermophilus and Thermococcus hydrothermal pullulanase hybrid or a Thermococcus hydrothermal/Thermococcus thermophilus hybrid having a truncation site X4 as disclosed in WO 2011/087836, which is hereby incorporated by reference.
In another embodiment, the pullulanase is a pullulanase comprising the X46 domain disclosed in WO 2011/076123 (Novozymes).
According to the invention, pullulanase may be added in effective amounts, including preferred amounts of about 0.0001-10mg enzyme protein per gram DS, preferably 0.0001-0.10mg enzyme protein per gram DS, more preferably 0.0001-0.010mg enzyme protein per gram DS. The pullulanase activity can be determined as NPUN. Assays for determining NPUN are described in the materials and methods section below.
Suitable commercially available pullulanase products include PROMOZYME 400L, PROMOZYMETMD2 (Novozymes A/S, Denmark), OPTIMAX L-300 (Jenengke corporation (Genencor Int.), USA), and AMANO 8 (Annenghan corporation (Amano), Japan).
Phytase present and/or added during liquefaction
Optionally, in the liquefaction, the phytase may be present and/or added in combination with an alpha-amylase and optionally a hemicellulase (preferably xylanase) with a melting point (DSC) greater than 80 ℃.
The phytase used according to the invention may be any enzyme capable of releasing inorganic phosphate from phytic acid (phytate) or any of its salts (phytate). Phytases can be classified according to their specificity in the initial hydrolysis step, whereby the phosphate group is hydrolyzed first. The phytase used in the present invention may have any specificity, and may be, for example, a 3-phytase (EC 3.1.3.8), or a 6-phytase (EC 3.1.3.26), or a 5-phytase (no EC number). In embodiments, the phytase has a temperature optimum of greater than 50 ℃, such as in the range from 50 ℃ to 90 ℃.
The phytase may be derived from a plant or a microorganism, such as a bacterium or a fungus, e.g. a yeast or a filamentous fungus.
The plant phytase may be from wheat bran, maize, soybean or lily pollen. Suitable plant phytases are described in Thomlinson et al, Biochemistry [ Biochemistry ], 1(1962), 166-; barrientos et al, plant.Physiol. [ journal of plant physiology ],106(1994), 1489-; WO 98/05785; in WO 98/20139.
The bacterial phytase may be from Bacillus, Citrobacter (Citrobacter), Hafnia (Hafnia), Pseudomonas, Butterella (Buttiauxella), or Escherichia (Escherichia), in particular Bacillus subtilis, Citrobacter buchneri (Citrobacter braakii), Citrobacter freundii (Citrobacter freundii), Hafnia alvei (Hafnia alvei), Bulgax bucillus (Buttiauxella gaviniae), Butterus villagelis (Buttiauxella agrestis), Klebsiella noensis (Buttiauxella noackies), and Escherichia coli. Suitable bacterial phytases are described in Paver and Jagannathan,1982, Journal of Bacteriology 151: 1102-1108; cosgrove,1970, Australian Journal of Biological Sciences [ Journal of bioscience Australia ]23: 1207-1220; greiner et al, Arch.biochem.Biophys. [ Agrochemical biophysiology ],303,107-113, 1993; WO 1997/33976; WO 1997/48812, WO 1998/06856, WO 1998/028408, WO 2004/085638, WO 2006/037327, WO 2006/038062, WO 2006/063588, WO 2008/092901, WO 2008/116878, and WO 2010/034835.
The yeast phytase may be derived from Saccharomyces or Schwanniomyces, in particular from the species Saccharomyces cerevisiae or Schwanniomyces occidentalis. The foregoing enzymes have been described as suitable yeast phytases in Nayini et al, 1984, Lebensmittel Wissenschaft und Technie [ food science and technology ]17: 24-26; wodzinski et al, adv.appl.Microbiol. [ applied microbiological progress ],42, 263-303; AU-A-24840/95.
The phytase from filamentous fungi may be derived from ascomycetes (ascomycetes ) of the phylum mycomycota or Basidiomycota (Basidiomycota), such as Aspergillus, thermophilic fungi (thermolomyces) (also known as humicola), Myceliophthora (Myceliophthora), monascus (Manascus), penicillium, leucoderma (Peniophora), cephalospora (Agrocybe), pileus (Paxillus), or Trametes (Trametes), in particular Aspergillus terreus (Aspergillus terreus), Aspergillus niger, Aspergillus awamori (Aspergillus niger var. trawamori), Aspergillus ficus (Aspergillus ficus), Aspergillus fumigatus, Aspergillus oryzae, Myceliophthora (t.lanuginosus) (also known as mansonia (h.uginosa)), thermophilic sporotrichinosus, Aspergillus terreus (trichoderma), Aspergillus versicolor (trichoderma), Aspergillus niger or trichoderma versicolor (trichoderma), or trichoderma (trichoderma), trichoderma sp). Suitable fungal phytases are described in Yamada et al, 1986, Agric.biol.chem. [ agricultural and biochemical ]322: 1275-1282; piddington et al, 1993, Gene [ Gene ]133: 55-62; EP 684,313; EP 0420358; EP 0684313; WO 1998/28408; WO 1998/28409; JP 7-67635; WO 1998/44125; WO 1997/38096; in WO 1998/13480.
In a preferred embodiment, the phytase is derived from a Brucella species, such as Brucella Galvanica, Brucella townssa, or Brucella norathensis, such as those disclosed as SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6, respectively, in WO 2008/092901 (incorporated herein by reference).
In a preferred embodiment, the phytase is derived from Citrobacter, such as Citrobacter buchneri, as disclosed in WO 2006/037328 (hereby incorporated by reference).
The modified phytase or phytase variant may be obtained by methods known in the art, in particular by the methods disclosed in: EP 897010, EP 897985, WO 99/49022; WO 99/48330, WO 2003/066847, WO 2007/112739, WO 2009/129489, and WO 2010/034835.
Commercially available phytases containing products include BIO-FEED PHYTASETM、PHYTASE NOVOTMCT or L (both from Novozymes, Inc.), LIQMAX (DuPont), or RONOZYMETMNP、
Figure BDA0003162048240001061
HiPhos、
Figure BDA0003162048240001062
P5000(CT)、NATUPHOSTMNG 5000 (from DSM).
Carbohydrate source producing enzymes present and/or added during saccharification and/or fermentation
According to the invention, an enzyme producing a carbohydrate source, preferably a glucoamylase, is present and/or added during saccharification and/or fermentation.
In a preferred embodiment, the carbohydrate source producing enzyme is a glucoamylase of fungal origin, preferably from the genus aspergillus, preferably a strain of aspergillus niger, aspergillus awamori, or aspergillus oryzae; or a strain of Trichoderma, preferably Trichoderma reesei; or a strain of the genus Talaromyces, preferably a strain of Talaromyces emersonii.
Glucoamylase
According to the present invention, the glucoamylase present and/or added in the saccharification and/or fermentation may be derived from any suitable source, e.g., from a microorganism or a plant. Preferred glucoamylases are of fungal or bacterial origin and are selected from the group consisting of: aspergillus glucoamylases, in particular Aspergillus niger G1 or G2 glucoamylase (Boel et al, (1984), EMBO J. [ journal of the European society of molecular biology ]3(5), pp. 1097-1102), or variants thereof, such as those disclosed in WO 92/00381, WO 00/04136, and WO 01/04273 (from Novozymes, Denmark); aspergillus awamori glucoamylase as disclosed in WO 84/02921; aspergillus oryzae glucoamylase (Agric. biol. chem. [ agricultural and biochemical ] (1991),55(4), pp. 941-949), or variants or fragments thereof. Other aspergillus glucoamylase variants include variants with enhanced thermostability: G137A and G139A (Chen et al (1996), prot. Eng. [ protein engineering ]9, 499-505); D257E and D293E/Q (Chen et al, (1995), prot. Eng. [ protein engineering ]8, 575-; n182(Chen et al (1994), biochem. J. [ J. biochem ]301, 275-; disulfide bond, A246C (Fierobe et al, (1996), Biochemistry [ Biochemistry ],35: 8698-; and Pro residues were introduced at the A435 and S436 positions (Li et al, (1997), Protein Eng. [ Protein engineering ]10, 1199-.
Other glucoamylases include Athelia rolfsii (Athelia rolfsii) (previously designated as revolute bacteria (cornium rolfsii)) glucoamylase (see U.S. Pat. No. 4,727,026 and Nagasaka et al (1998) "Purification and properties of the raw-starch-degrading glucoamylases from cornium rolfsii [ Purification and properties of crude starch degrading glucoamylases from cornium, applied microbiology and biotechnology ]50: 323-. In a preferred embodiment, the glucoamylase used in the saccharification and/or fermentation process is the gram-senium glucoamylase disclosed in WO 99/28448.
Bacterial glucoamylases contemplated include those from the genus Clostridium (Clostridium), particularly Clostridium amyloliquefaciens (c.thermosaccharium) (EP 135,138) and Clostridium hydrosulfuricum (WO 86/01831).
Fungal glucoamylases contemplated include Trametes annulata (Trametes cingulata), Tricholoma papulosum (Pachykytospora papyracea), and Pachylomyces leucovora (Leucopaxillus giganteus), and Phanerochaete erythraea rubescens (Peniophora rufomarginata), all disclosed in WO 2006/069289, or mixtures thereof. Hybrid glucoamylases are also contemplated according to the invention. Examples include the hybrid glucoamylases disclosed in WO 2005/045018. Specific examples include the hybrid glucoamylases disclosed in table 1 and table 4 of example 1 (these hybrids are hereby incorporated by reference).
In the examples, the glucoamylase is derived from a strain of the genus Pycnoporus (Pycnoporus), in particular from a strain of the genus Pycnoporus as described in WO 2011/066576 (SEQ ID NO 2, 4, or 6); or a strain derived from the genus Aphyllophora (Gloephyllum), in particular a strain derived from the genus Aphyllophora as described in WO 2011/068803 (SEQ ID NO:2, 4, 6, 8, 10, 12, 14, or 16); or from a strain of the genus Oreoporus (Nigrosomes), in particular from a strain of the species Oreoporus as disclosed in WO 2012/064351 (SEQ ID NO:2) (all references hereby incorporated by reference). Also contemplated are glucoamylases that exhibit high identity with any of the above glucoamylases, i.e., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity with any of the mature portions of the above enzyme sequences.
In an example, glucoamylase may be added to saccharification and/or fermentation in the following amounts: 0.0001 to 20AGU/g DS, preferably 0.001 to 10AGU/g DS, in particular between 0.01 and 5AGU/g DS, for example 0.1 to 2AGU/g DS.
Commercially available compositions comprising glucoamylase include AMG 200L; AMG 300L; SANTMSUPER、SANTMEXTRA L、SPIRIZYMETMPLUS、SPIRIZYMETMFUEL、SPIRIZYMETMB4U、SPIRIZYMETMULTRA、SPIRIZYMETMEXCEL、SPIRIZYMETMACHIEVE, and AMGTME (from Novozymes A/S); OPTIDEXTM300. GC480, GC417 (from Genencor Int.); AMIGASETMAnd AMIGASETMPLUS (from Dismantman (DSM)); G-ZYMETMG900、G-ZYMETMAnd G990 ZR (from Danisco US, Danisco, usa).
Maltogenic amylase
The carbohydrate-source producing enzyme present and/or added during saccharification and/or fermentation may also be a maltogenic alpha-amylase. A "maltogenic alpha-amylase" (glucan 1, 4-alpha-maltohydrolase, E.C.3.2.1.133) is capable of hydrolyzing maltose in both amylose and amylopectin in the alpha-conformation. Maltogenic amylases from Bacillus stearothermophilus strain NCIB 11837 are commercially available from Novozymes corporation (Novozymes A/S). Maltogenic alpha-amylases are described in U.S. patent nos. 4,598,048, 4,604,355, and 6,162,628, which are hereby incorporated by reference. In a preferred embodiment, maltogenic amylase may be added in an amount of 0.05-5mg total protein/g DS or 0.05-5MANU/g DS.
Proteases present and/or added during liquefaction
In embodiments of the invention, in liquefaction, an optional protease (e.g., a thermostable protease) may be present and/or added with an alpha-amylase (e.g., a thermostable alpha-amylase), and a hemicellulase (preferably a xylanase) having a melting point (DSC) greater than 80 ℃, and optionally an endoglucanase, a carbohydrate-source producing enzyme (particularly a glucoamylase, optionally a branched chain amylase, and/or optionally a phytase).
Proteases are classified into the following groups according to their catalytic mechanism: serine proteases (S), cysteine proteases (C), aspartic proteases (A), metalloproteinases (M), and unknown or yet unclassified proteases (U), see Handbook of Proteolytic Enzymes [ Handbook of Proteolytic Enzymes ], A.J.Barrett, N.D.Rawlings, J.F.Wosener (ed), Academic Press [ Academic Press ] (1998), particularly in the summary section.
In a preferred embodiment, the thermostable protease used according to the invention is a "metalloprotease", defined as a protease belonging to EC 3.4.24 (metalloendopeptidase), preferably EC 3.4.24.39 (acidic metalloprotease).
To determine whether a given protease is a metalloprotease, reference is made to the above-mentioned "Handbook of Proteolytic Enzymes" and the guidelines indicated therein. Such a determination can be made for all types of proteases, whether they are naturally occurring or wild-type proteases, or genetically engineered or synthetic proteases.
Protease activity may be measured using any suitable assay, wherein a substrate is employed, which comprises peptide bonds relevant to the specificity of the protease in question. The determination of the pH value and the determination of the temperature likewise apply to the protease in question. Examples of measuring the pH value are pH 6, 7, 8, 9, 10, or 11. Examples of measurement temperatures are 30 ℃, 35 ℃, 37 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, or 80 ℃.
Examples of protease substrates are caseins, such as Azurine-Crosslinked Casein (azzurine-crossliked Casein) (AZCL-Casein). Two protease assays are described below in the "materials and methods" section of WO 2017/112540 (which is incorporated herein by reference), with the preferred assay being the so-called "AZCL-casein assay".
In the examples, the thermostable protease has a protease activity of at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 100%, of the JTP196 variant (example 2 from WO 2017/112540) or protease Pfu (SEQ ID NO:31 herein), as determined by the AZCL-casein assay described in the "materials and methods" section of WO 2017/112540.
There is no limitation on the source of the thermostable protease used in the method or composition of the present invention, as long as it satisfies the thermostability characteristics defined below.
In one embodiment, the protease is of fungal origin.
In a preferred embodiment, the thermostable protease is a variant of a metalloprotease as defined above. In an embodiment, the thermostable protease used in the method or composition of the invention is of fungal origin, such as a fungal metalloprotease derived from a strain of thermoascus, preferably a strain of thermoascus aurantiacus, especially thermoascus aurantiacus CGMCC No.0670 (classified as EC 3.4.24.39).
In embodiments, the thermostable protease is a variant of the mature part of the metalloprotease shown in SEQ ID NO:2 disclosed in WO 2003/048353 or a variant of SEQ ID NO:1 in WO 2010/008841 and the mature part shown herein as SEQ ID NO:32, further having mutations selected from the list of:
-S5*+D79L+S87P+A112P+D142L；
-D79L+S87P+A112P+T124V+D142L；
-S5*+N26R+D79L+S87P+A112P+D142L；
-N26R+T46R+D79L+S87P+A112P+D142L；
-T46R+D79L+S87P+T116V+D142L；
-D79L+P81R+S87P+A112P+D142L；
-A27K+D79L+S87P+A112P+T124V+D142L；
-D79L+Y82F+S87P+A112P+T124V+D142L；
-D79L+Y82F+S87P+A112P+T124V+D142L；
-D79L+S87P+A112P+T124V+A126V+D142L；
-D79L+S87P+A112P+D142L；
-D79L+Y82F+S87P+A112P+D142L；
-S38T+D79L+S87P+A112P+A126V+D142L；
-D79L+Y82F+S87P+A112P+A126V+D142L；
-A27K+D79L+S87P+A112P+A126V+D142L；
-D79L+S87P+N98C+A112P+G135C+D142L；
-D79L+S87P+A112P+D142L+T141C+M161C；
-S36P+D79L+S87P+A112P+D142L；
-A37P+D79L+S87P+A112P+D142L；
-S49P+D79L+S87P+A112P+D142L；
-S50P+D79L+S87P+A112P+D142L；
-D79L+S87P+D104P+A112P+D142L；
-D79L+Y82F+S87G+A112P+D142L；
-S70V+D79L+Y82F+S87G+Y97W+A112P+D142L；
-D79L+Y82F+S87G+Y97W+D104P+A112P+D142L；
-S70V+D79L+Y82F+S87G+A112P+D142L；
-D79L+Y82F+S87G+D104P+A112P+D142L；
-D79L+Y82F+S87G+A112P+A126V+D142L；
-Y82F+S87G+S70V+D79L+D104P+A112P+D142L；
-Y82F+S87G+D79L+D104P+A112P+A126V+D142L；
-A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L；
-A27K+Y82F+S87G+D104P+A112P+A126V+D142L；
-A27K+D79L+Y82F+D104P+A112P+A126V+D142L；
-A27K+Y82F+D104P+A112P+A126V+D142L；
-A27K+D79L+S87P+A112P+D142L；
-D79L+S87P+D142L。
in a preferred embodiment, the thermostable protease is a variant of a mature metalloprotease disclosed as: the variant, either as disclosed in WO 2003/048353 for the mature part of SEQ ID NO. 2 or as disclosed in WO 2010/008841 for the mature part of SEQ ID NO. 1 or SEQ ID NO. 32 herein, has the following mutations:
D79L+S87P+A112P+D142L；
D79L + S87P + D142L; or
A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L。
In embodiments, the protease variant has at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99% but less than 100% identity to the mature part of the polypeptide of SEQ ID No. 2 disclosed in WO 2003/048353 or the mature part of SEQ ID No. 1 disclosed in WO 2010/008841 or SEQ ID No. 32 herein.
The thermostable protease may also be derived from any bacterium, as long as the protease has the thermostability characteristics as defined according to the invention.
In the examples, the thermostable protease is derived from a strain of the bacterium Pyrococcus (Pyrococcus), such as a strain of Pyrococcus furiosus (pfu protease).
In the examples, the protease is one as shown in SEQ ID NO:1 of U.S. Pat. No. 6,358,726-B1 (Takara Shuzo Company), and SEQ ID NO:31 herein.
In embodiments, the thermostable protease is one of SEQ ID No. 31 disclosed herein or a protease with at least 80% identity, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to SEQ ID No. 1 in U.S. patent No. 6,358,726-B1 or SEQ ID No. 31 herein. Pyrococcus furiosus protease can be purchased from Nippon Takara Bio Inc. (Takara Bio, Japan).
Pyrococcus furiosus protease is a thermostable protease according to the invention. The commercial product Pyrococcus furiosus protease (Pfu S) was found to have a thermal stability of 110% (80 ℃/70 ℃) and 103% (90 ℃/70 ℃) at pH 4.5 (see example 5), determined as described in example 2 of WO 2017/112540.
In one embodiment, thermostable proteases have a thermostability value of more than 20% determined as relative activity at 80 ℃/70 ℃ as determined in example 2.
In embodiments, the protease has a thermostability determined to be more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 100%, such as more than 105%, such as more than 110%, such as more than 115%, such as more than 120% of the relative activity at 80 ℃/70 ℃.
In embodiments, the protease has a thermostability determined as a relative activity at 80 ℃/70 ℃ of between 20% and 50%, such as between 20% and 40%, such as 20% and 30%.
In embodiments, the protease has a thermostability determined as a relative activity at 80 ℃/70 ℃ of between 50% and 115%, such as between 50% and 70%, such as between 50% and 60%, such as between 100% and 120%, such as between 105% and 115%.
In the examples, the protease has a thermostability value of more than 10% determined as relative activity at 85 ℃/70 ℃ as determined as described in example 2 of WO 2017/112540.
In embodiments, the protease has a thermal stability of more than 10%, such as more than 12%, more than 14%, more than 16%, more than 18%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 100%, more than 110%, determined as relative activity at 85 ℃/70 ℃.
In embodiments, the protease has a thermostability determined as a relative activity at 85 ℃/70 ℃ of between 10% and 50%, such as between 10% and 30%, such as between 10% and 25%.
In embodiments, the protease has more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90% of the residual activity determined as at 80 ℃; and/or
In embodiments, the protease has more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90% of the residual activity determined as at 84 ℃.
The determination of "relative activity" as well as "residual activity" was carried out as described in example 2 of WO 2017/112540.
In an embodiment, the protease may have a thermostability at 85 ℃ of greater than 90, such as greater than 100, as determined using the Zein-BCA assay disclosed in example 3 of WO 2017/112540.
In an embodiment, the protease has a thermostability at 85 ℃ of more than 60%, e.g. more than 90%, e.g. more than 100%, e.g. more than 110%, as determined using a Zein-BCA assay.
In embodiments, the protease has a thermostability at 85 ℃ of between 60% -120%, such as between 70% -120%, such as between 80% -120%, such as between 90% -120%, such as between 100% -120%, such as 110% -120%, as determined using a Zein-BCA assay.
In the examples, the thermostable protease has at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 100% of the activity of JTP196 protease variant or protease Pfu as determined by the AZCL-casein assay described in the materials and methods section of WO 2017/112540.
Further aspects of the invention
In another aspect of the invention, it relates to the use of the enzyme blend of the invention for improving the nutritional quality of distillers Dried Grains (DGS) or distillers dried grains with solubles (DDGS) produced as a byproduct of the fermentation product production process of the invention, preferably without causing the DDG or DDGS to darken.
Any of the enzyme blends disclosed in section I herein may be used in this manner. In various embodiments in this regard, additional enzymes (such as the enzymes or enzyme compositions described under the "enzymes" section) can be used in combination with the enzyme blends of the present invention.
In embodiments, the enzyme blend is used to improve the nutritional quality of DGS or DDGs by increasing the TME of DDG or DDGs by at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20% when administered to an animal (e.g., a non-ruminant, e.g., a monogastric animal, e.g., a poultry, or a pig, etc.) as compared to the TME of DDG or DDGs produced as a byproduct when the enzyme blend is not present during the saccharification, fermentation, or simultaneous saccharification and fermentation steps of a fermentation product production process for producing DDG or DDGs byproducts. In embodiments, the enzyme blend is used to improve the nutritional quality of DGS or DDGs by increasing the TME of DDG or DDGs by at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, or at least 50% in an animal (e.g., a non-ruminant animal, e.g., a monogastric animal, e.g., a poultry, or a pig, etc.) compared to the TME of DDG or DDGs produced as a byproduct when the enzyme blend is not present during the saccharification, fermentation, or simultaneous saccharification and fermentation steps of a fermentation product production process for producing DDG or DDGs byproducts.
In yet another aspect of the invention, it relates to the use of the enzyme blend of the invention for increasing the dissolution of fibers present in the beer during the fermentation product production process of the invention, preferably without causing the DDG or DDGs to darken. In an embodiment, the enzyme blend is used to increase fiber dissolution during the production of alcohol (e.g., ethanol) from starch-containing material. In an embodiment, the enzyme blend is used to increase the solubilization of corn fiber in an ethanol production process (such as the RSH process or conventional cooking including a liquefaction step). In the examples, the enzyme blend was used to increase the solubilization of arabinose. In the examples, the enzyme blend was used to increase the dissolution of xylose.
Any of the enzyme blends disclosed in section I herein may be used in this manner. In various embodiments in this regard, additional enzymes (such as the enzymes or enzyme compositions described under the "enzymes" section) can be used in combination with the enzyme blends of the present invention.
In embodiments, the enzyme blend is used to increase the dissolution of fibers (e.g., arabinose, xylose, etc.) contacted with the enzyme blend by at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20% as compared to the dissolution of fibers not contacted with the enzyme blend of the invention. In embodiments, the enzyme blend is used to increase the solubilization of a fiber (e.g., arabinose, xylose, etc.) contacted with the enzyme blend by at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, or at least 50% as compared to a fiber not contacted with the enzyme blend.
The invention is further defined by the following numbered paragraphs:
1. a polypeptide having xylanase activity, selected from the group consisting of:
(a) a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a mature polypeptide of SEQ ID NO 1, 5, or 7;
(b) a polypeptide encoded by a polynucleotide that hybridizes under very high stringency conditions to the mature polypeptide coding sequence of SEQ ID NO. 2, 6, or 8, or any full-length complement thereof;
(c) a polypeptide encoded by a polynucleotide having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO 2, 6, or 8;
(d) a fragment of the polypeptide of (a), (b), or (c), which fragment has xylanase activity.
2. The polypeptide of paragraph 1 wherein:
(i) the mature polypeptide is amino acids 33 to 884 of SEQ ID NO. 1;
(ii) the mature polypeptide is amino acids 31 to 826 of SEQ ID NO 5; and
(iii) The mature polypeptide is amino acids 35 to 831 of SEQ ID NO 7.
3. An enzyme blend comprising the polypeptide of any of paragraphs 1-2.
4. The enzyme blend of paragraph 3, further comprising a cellulolytic composition.
5. The enzyme blend of paragraph 4, wherein the cellulolytic composition is present in the blend at a xylanase to cellulolytic composition ratio of about 5:95 to about 95:5, such as 5:95, 10:90, 20:80, 50:50, 80:20, 90:10, and 95: 5.
6. A polynucleotide encoding the polypeptide of any one of paragraphs 1-2.
7. A nucleic acid construct or recombinant expression vector comprising the polynucleotide of paragraph 6 operably linked to one or more heterologous control sequences that direct the production of the polypeptide in an expression host.
8. A recombinant host cell comprising the polynucleotide of paragraph 6 operably linked to one or more heterologous control sequences that direct the production of the polypeptide.
9. A method of producing a polypeptide having xylanase activity, comprising (a) culturing a host cell as described in paragraph 8 under conditions conducive for production of the polypeptide, and (b) optionally recovering the polypeptide.
10. A method of producing a fermentation product, the method comprising the steps of:
(a) saccharifying a starch-containing material with an alpha-amylase, a glucoamylase, and a GH98 xylanase or an enzyme blend comprising the GH98 xylanase at a temperature below the initial gelatinization temperature;
(b) fermenting using a fermenting organism to produce the fermentation product; and
(c) optionally recovering the by-products.
11. A process for producing a fermentation product from starch-containing material, the process comprising the steps of:
(a) liquefying a starch-containing material with an alpha-amylase;
(b) saccharifying the liquefied material obtained in step (a) with glucoamylase and GH98 xylanase or an enzyme blend comprising the GH98 xylanase;
(c) fermenting using a fermenting organism; and
(d) optionally recovering the by-products.
12. The method of paragraph 10 or 11, wherein saccharification and fermentation are performed simultaneously.
13. The method of any of paragraphs 10-12, wherein the starch-containing material comprises maize, corn, wheat, rye, barley, triticale, sorghum, switchgrass, millet, pearl millet, millet.
14. The method of any of paragraphs 10-13, wherein the fermentation product is an alcohol, particularly ethanol.
15. The method of any of paragraphs 10-14, wherein the byproduct is Distillers Dried Grains (DDG) or distillers dried grains with solubles (DDGS).
16. The process of any of paragraphs 10-15, wherein the DDG or DDGs has improved nutritional quality compared to DDG or DDGs recovered as a byproduct of a process for producing a fermentation product in which the GH98 xylanase or an enzyme blend comprising the GH98 xylanase is not present or added.
17. The method of paragraph 16, wherein the DDG or DDGS has increased fat content.
18. The method of any of paragraphs 10-17, wherein the TME of the DGS or DDGS is increased by at least 5%, at least 10%, at least 15%, or at least 20% compared to the true metabolic energy of DGS or DDGS produced in the absence of a GH98 xylanase or an enzyme blend comprising the GH98 xylanase during the saccharification step, fermentation step, and/or simultaneous saccharification and fermentation step of the process.
19. The method of paragraph 18, wherein the TME is for a monogastric animal.
20. The method of paragraph 15 or 16, wherein the DGS or DDGS produced after drying is not blackened compared to DGS or DDGS produced during a saccharification step, a fermentation step, and/or a simultaneous saccharification and fermentation step of the method of any one of paragraphs 10 to 19 in the absence of a polypeptide having xylanase activity as described in any one of paragraphs 1-2 or an enzyme blend as described in any one of paragraphs 3-5.
21. The method of any of paragraphs 10-20, wherein the fermenting organism is a yeast, particularly a saccharomyces species, more particularly saccharomyces cerevisiae.
22. The method of any of paragraphs 10-21, wherein the enzyme blend further comprises a cellulolytic composition.
23. The method of any of paragraphs 10-22, wherein the cellulolytic composition is present in the blend at a xylanase to cellulolytic composition ratio of about 5:95 to about 95:5, such as 5:95, 10:90, 20:80, 50:50, 80:20, 90:10, and 95: 5.
24. The enzyme blend of any of paragraphs 3-5 or the method of any of paragraphs 10-23, wherein the cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of:
(i) cellobiohydrolase I;
(ii) cellobiohydrolase II;
(iii) a beta-glucosidase; and
(iv) a GH61 polypeptide having cellulolytic enhancing activity.
25. The enzyme blend of any of paragraphs 3-5 or the method of any of paragraphs 10-24, wherein the cellulolytic composition comprises at least one, at least two, at least three, or at least four enzymes selected from the group consisting of:
(i) Aspergillus fumigatus cellobiohydrolase I;
(ii) aspergillus fumigatus cellobiohydrolase II;
(iii) aspergillus fumigatus beta-glucosidase; and
(iv) a Penicillium emersonii GH61A polypeptide having cellulolytic enhancing activity.
26. The enzyme blend of any of paragraphs 3-5 or the method of any of paragraphs 10-25, wherein the cellulolytic composition comprises:
(i) cellobiohydrolase I comprising amino acids 27 to 532 of SEQ ID No. 15 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 27 to 532 of SEQ ID No. 15;
(ii) a cellobiohydrolase II comprising amino acids 20 to 454 of SEQ ID No. 16, or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 20 to 454 of SEQ ID No. 16;
(iii) a β -glucosidase or variant thereof comprising amino acids 20 to 863 of SEQ ID No. 17, having at least one substitution selected from the group consisting of F100D, S283G, N456E, and F512Y and having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 20 to 863 of SEQ ID No. 17; and/or
(iv) A GH61A polypeptide having cellulolytic enhancing activity comprising amino acids 26 to 253 of SEQ ID No. 18 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 26 to 253 of SEQ ID No. 18.
27. The enzyme blend of any of paragraphs 3-5 or the process of any of paragraphs 10-26, wherein the cellulolytic composition further comprises an endoglucanase.
28. The enzyme blend of any of paragraphs 3-5 or the process of any of paragraphs 10-27, wherein the cellulolytic composition is derived from a strain selected from the group consisting of aspergillus, penicillium, talaromyces, and trichoderma, optionally wherein: (i) the aspergillus strain is selected from the group consisting of: aspergillus flavus (Aspergillus aurantiacus), Aspergillus niger (Aspergillus niger), and Aspergillus oryzae (Aspergillus oryzae); (ii) the penicillium strain is selected from the group consisting of: penicillium emersonii and penicillium oxalicum (penicillium oxalicum); (iii) the Talaromyces strain is selected from the group consisting of: talaromyces aurantiacaus and Talaromyces emersonii; and (iv) the Trichoderma strain is Trichoderma reesei (Trichoderma reesei).
29. The enzyme blend of any of paragraphs 3-5 or the method of any of paragraphs 10-28, wherein the cellulolytic composition comprises a trichoderma reesei cellulolytic composition.
30. The polypeptide of paragraph 1 or 2, the enzyme blend of any of paragraphs 3-5, or the method of any of paragraphs 10-29, wherein the xylanase is a GH98 family xylanase from the genus microbacterium or paenibacillus.
31. The method of any of paragraphs 10-30, wherein the xylanase is a GH98 xylanase selected from the group consisting of:
(i) 1 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% amino acid sequence identity thereto;
(ii) 5 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto; and
(iii) 7 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity thereto.
32. Use of a polypeptide having xylanase activity according to any of paragraphs 1-2, or an enzyme blend according to any of paragraphs 3-5, for improving the nutritional quality of DGS or DDGS produced as a by-product of a fermentation product production process according to any of paragraphs 1-31, preferably without causing the DDG or DDGS to darken.
33. Use of a polypeptide having xylanase activity according to any of paragraphs 1-2, or an enzyme blend according to any of paragraphs 3-5, for solubilising fibres, preferably for solubilising xylose and arabinose.
The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrative of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of the present invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In case of conflict, the present disclosure, including definitions, will control. Various references are cited herein, the disclosures of which are incorporated by reference in their entirety. The invention is further described by the following examples, which should not be construed as limiting the scope of the invention.
Materials and methods
Alpha-amylase 369(AA 369): a bacillus stearothermophilus alpha-amylase having the following mutations:
I181X + G182X + N193F + V59A + Q89R + E129V + K177L + R179E + Q254S + M284V (SEQ ID NO:19 herein), truncated to 491 amino acids.
Cellulolytic composition:a cellulolytic composition derived from trichoderma reesei comprising: aspergillus fumigatus Cel7A CBH1 disclosed as SEQ ID NO 6 and herein SEQ ID NO 15 in WO 2011/057140; aspergillus fumigatus CBH II disclosed in WO 2011/057140 as SEQ ID NO 18 and herein SEQ ID NO 16; aspergillus fumigatus beta-glucosidase (SEQ ID NO:2 in WO 2005/047499 or SEQ ID NO:17 herein) variants (F100D, S283G, N456E, F512Y) disclosed in WO 2012/044915 or co-pending PCT application PCT/US 11/054185; and GH61A polypeptide having cellulolytic enhancing activity derived from a strain of Penicillium emersonii (SEQ ID NO:2 in WO 2011/041397 or SEQ ID NO:18 herein).
E-SEP：Comprising a blend of Trichoderma reesei cellulose strains expressing a transgenic GH10 xylanase and expressing GH62 arabinofuranosidase.
Glucoamylase sa (gsa):a blend comprising: talaromyces emersonii glucoamylase disclosed as SEQ ID NO 34 in WO 99/28448, trametes annulatus glucoamylase disclosed as SEQ ID NO 2 in WO 06/69289, and Rhizomucor miehei alpha-amylase with the following substitutions G128D + D143N with the Aspergillus niger glucoamylase linker and Starch Binding Domain (SBD) disclosed in SEQ ID NO 14 herein (the activity ratio in AGU: AGU: FAU-F is about 20:5: 1).
Protease Pfu:the protease from Pyrococcus furiosus shown in SEQ ID NO 31 herein.
GH98#1: GH98 xylanase from Paenibacillus terreus having the amino acid sequence of SEQ ID NO. 7.
GH98#2: GH98 xylanase from Bacillus glucanolyticus having the amino acid sequence of SEQ ID NO: 5.
GH98#3：GH98 xylanase from Microbacterium oxydans having the amino acid sequence of SEQ ID NO: 1.
Yeast:ETHANOL REDTMfrom Red Star/Lesfre, USA.
Xylose dissolution assay
The activity of xylanase variants on defatted, de-starched maize (DFDSM) can be measured by high performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD). Can be in 100mM sodium acetate and 5mM CaCl2A 2% (w/w) DFDSM suspension was prepared (pH 5) and allowed to hydrate for 30 min at room temperature under mild stirring. After hydration, 200 μ l of substrate suspension can be pipetted into a 96-well plate and mixed with 20 μ l of enzyme solution to obtain a final enzyme concentration of 20PPM (20 μ g enzyme/g substrate) relative to substrate. The enzyme/substrate mixture can then be hydrolyzed at 40 ℃ within 2.5 hours under mild agitation (500RPM) in a plate incubator (Biosan PST-100 HL). After enzymatic hydrolysis, the enzyme/substrate plate can be centrifuged at 3000RPM for 10 minutes and 50 μ l of the supernatant (hydrolysate) is mixed with 100 μ l of 1.6M HCl and then transferred to a 300 μ l PCR tube and acid hydrolyzed in a PCR instrument at 90 ℃ for 40 minutes at rest. The purpose of the acid hydrolysis was to convert soluble polysaccharides released by xylanase variants into monosaccharides that can be quantified using HPAE-PAD. The sample was neutralized with 125. mu.l of 1.4M NAOH after acid hydrolysis and immobilized on HPAE-PAD for monosaccharide analysis (xylose, arabinose, and glucose) (Dionex ICS-3000 using a CarboPac PA1 column). A suitable calibration curve was made using a monosaccharide stock solution that underwent the same acid hydrolysis procedure as the sample. The percent xylose dissolved was calculated according to the following formula:
Figure BDA0003162048240001241
Where [ xylose ] represents the concentration of xylose in the supernatant as measured by HPAE-PAD, V represents the volume of the sample, MW represents the molecular weight of internal xylose in arabinoxylan (132g/mol), Xxyl represents the fraction of xylose in DFDSM (0.102), and Msub represents the mass of DFDSM in the sample.
Examples of the invention
EXAMPLE 1 cloning and expression of bacterial GH98 endo-. beta.1, 4-xylanase
GH98 endo-beta-1, 4-xylanases are derived from bacterial strains isolated from environmental samples by standard microbial isolation techniques. Isolated pure strains were identified and assigned to classification based on DNA sequencing of the 16S ribosomal gene (table 1).
Table 1:
strains or colonies Country of origin Mature proteins
Microbacterium oxydans USA SEQ:1
Bacillus subtilis for treating glucan USA SEQ:5
Paenibacillus terrae USA SEQ:7
Isolating chromosomal DNA from pure culture and use
Figure BDA0003162048240001242
Techniques perform whole genome sequencing. Base ofGenome sequencing, assembly of subsequent reads, and gene discovery (i.e., annotation of gene function) are known to those skilled in the art and this service is commercially available.
Genomic sequences of putative endo-beta-1, 4-xylanases from The family GH98 of The CAZY database (Lombard V, Golginda Ramulu H, Drula E, Coutinho PM, Henrisat B (2014) The Carbohydrate-active enzymes database (CAZy) in 2013[2013 Carbohydrate-active enzyme database (CAZy) ]Nucleic Acids Res [ Nucleic acid research]42: D490-D495.). This analysis identified 3 genes encoding putative GH98 endo-beta-1, 4-xylanase, which were subsequently removed as linear DNA fragments from
Figure BDA0003162048240001251
Ordered and expressed recombinantly in B.subtilis. The DNA encoding the putative GH98 endo-beta-1, 4-xylanase from Microbacterium oxydans was codon optimized for Bacillus subtilis prior to ordering (SEQ ID NO: 33).
The integration construct used was a plasmid-based product prepared from the fusion of the gene of interest between two Bacillus subtilis chromosomal regions, together with a strong promoter and a chloramphenicol resistance marker. The GH98 endo-beta-1, 4-xylanase gene was expressed under the control of a triple promoter system (as described in WO 99/43835) consisting of the promoter of the bacillus licheniformis alpha-amylase gene (amyL), the promoter of the bacillus amyloliquefaciens alpha-amylase gene (amyQ) and the bacillus thuringiensis cryIIIA promoter containing stabilizing sequences.
The gene was fused with DNA encoding a secretion signal of B.clausii (encoding the following amino acid sequence: MKKPLGKIVASTALLISVAFSSSIASA (SEQ ID NO:34)) instead of the natural secretion signal. Furthermore, the expression construct resulted in the addition of an amino-terminal polyhistidine tag consisting of the amino acid sequence HHHHHHPR (SEQ ID NO:35) to the mature GH98 endo-beta-1, 4-xylanase to facilitate easy purification by immobilized metal affinity chromatography.
The resulting plasmid was transformed into Bacillus subtilis and integrated into the pectin lyase locus on the chromosome by homologous recombination. The recombinant Bacillus subtilis clone containing the integrated expression construct is then grown in liquid culture. The broth was centrifuged (20000x g, 20min) and the supernatant carefully decanted from the pellet and used for purification of the enzyme, or alternatively the sterile filtered supernatant was used directly for the assay.
Purification of recombinant enzymes by immobilized metal affinity chromatography
The pH of the clear supernatant was adjusted to pH 8, filtered through a 0.2 μ M filter, and the supernatant was applied to 5ml of HisTrapTMOn excel columns. Before loading, the column had been equilibrated in 5 Column Volumes (CV) of 50mM Tris/HCl pH 8. To remove unbound material, the column was washed with 8CV of 50mM Tris/HCl pH 8 and elution of target was obtained with 50mM HEPES pH 7+10mM imidazole. Eluting the protein in HiPrepTM26/10 desalting was performed on desalting columns equilibrated with 3CV of 50mM HEPES (pH 7) +100mM NaCl. This buffer was also used for elution of the target and the flow rate was 10 ml/min. Relevant fractions were selected and pooled based on chromatogram and SDS-PAGE analysis.
Example 2
Example 2 demonstrates the effectiveness of various GH98 xylanases of the invention and enzyme mixtures comprising a 10:90 ratio of GH98 xylanase and a cellulolytic composition (listed in the materials and methods section) on corn fiber. High solids corn fiber was subjected to a 3 day saccharification assay at 15% DS, pH 5, and 32 ℃. Each xylanase was tested as a 10% replacement (of protein) in a cellulolytic composition ("VD") and the total protein loading was 0.25mg EP/g Dry Solids (DS) (the protein concentration of each xylanase in the assay was 0.00375 mg/ml). The incubations were supplemented with GSA (0.6AGU/g DS).
Figure 1 shows the average DP4+ yield (g/L) of each GH98 enzyme blend compared to a control cellulolytic composition not supplemented with xylanase. FIG. 2 shows the average glucose yield (g/L) for each GH98 enzyme blend compared to a control cellulolytic composition not supplemented with xylanase. FIGS. 1 and 2 show that the GH98 xylanase from Microbacterium oxydans (# 3; SEQ ID NO:1) performs better than the GH98 xylanase from Paenibacillus sp.
Figure IDA0003162048290000011
Figure IDA0003162048290000021
Figure IDA0003162048290000031
Figure IDA0003162048290000041
Figure IDA0003162048290000051
Figure IDA0003162048290000061
Figure IDA0003162048290000071
Figure IDA0003162048290000081
Figure IDA0003162048290000091
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Figure IDA0003162048290000111
Figure IDA0003162048290000121
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Figure IDA0003162048290000151
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Figure IDA0003162048290000181
Figure IDA0003162048290000191
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Figure IDA0003162048290000261
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Figure IDA0003162048290000471
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Figure IDA0003162048290000491
Figure IDA0003162048290000501
Figure IDA0003162048290000511
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Figure IDA0003162048290000551
Figure IDA0003162048290000561
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Figure IDA0003162048290000581
Figure IDA0003162048290000591
Figure IDA0003162048290000601
Figure IDA0003162048290000611
Figure IDA0003162048290000621

Claims (20)

1. A polypeptide having xylanase activity, selected from the group consisting of:
(a) a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a mature polypeptide of SEQ ID NO 1, 5, or 7;
(b) A polypeptide encoded by a polynucleotide that hybridizes under very high stringency conditions to the mature polypeptide coding sequence of SEQ ID NO. 2, 6, or 8, or any full-length complement thereof;
(c) a polypeptide encoded by a polynucleotide having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO 2, 6, or 8;
(d) a fragment of the polypeptide of (a), (b), or (c), which fragment has xylanase activity.
2. The polypeptide of claim 1, wherein:
(i) the mature polypeptide is amino acids 33 to 884 of SEQ ID NO. 1;
(ii) the mature polypeptide is amino acids 31 to 826 of SEQ ID NO 5; and
(iii) the mature polypeptide is amino acids 35 to 831 of SEQ ID NO 7.
3. An enzyme blend comprising the polypeptide of any one of claims 1-2.
4. The enzyme blend of claim 3, further comprising a cellulolytic composition.
5. The enzyme blend of claim 4, wherein the cellulolytic composition is present in the blend in a xylanase to cellulolytic composition ratio of about 5:95 to about 95:5, such as 5:95, 10:90, 20:80, 50:50, 80:20, 90:10, and 95: 5.
6. A polynucleotide encoding the polypeptide of any one of claims 1-2.
7. A nucleic acid construct or recombinant expression vector comprising the polynucleotide of claim 6 operably linked to one or more heterologous control sequences that direct the production of the polypeptide in an expression host.
8. A recombinant host cell comprising the polynucleotide of claim 6 operably linked to one or more heterologous control sequences that direct the production of the polypeptide.
9. A method of producing a polypeptide having xylanase activity, comprising (a) culturing the host cell of claim 8 under conditions conducive for production of the polypeptide, and (b) optionally recovering the polypeptide.
10. A method of producing a fermentation product, the method comprising the steps of:
(a) saccharifying a starch-containing material with an alpha-amylase, a glucoamylase, and a GH98 xylanase or an enzyme blend comprising the GH98 xylanase at a temperature below the initial gelatinization temperature;
(b) fermenting using a fermenting organism to produce the fermentation product; and
(c) Optionally recovering the by-products.
11. A process for producing a fermentation product from starch-containing material, the process comprising the steps of:
(a) liquefying a starch-containing material with an alpha-amylase;
(b) saccharifying the liquefied material obtained in step (a) with glucoamylase and GH98 xylanase or an enzyme blend comprising the GH98 xylanase;
(c) fermenting using a fermenting organism; and
(d) optionally recovering the by-products.
12. The method of claim 10 or 11, wherein saccharification and fermentation are performed simultaneously.
13. The method of any one of claims 10-12, wherein the starch-containing material comprises maize, corn, wheat, rye, barley, triticale, sorghum, switchgrass, millet, pearl millet, millet.
14. The method of any one of claims 10-13, wherein the fermentation product is an alcohol, particularly ethanol, more particularly fuel ethanol.
15. The method of any one of claims 10-14, wherein the byproduct is Distillers Dried Grains (DDG) or distillers dried grains with Solubles (DDGs).
16. The process of any one of claims 10-15, wherein the DDG or DDGs has improved nutritional quality compared to DDG or DDGs recovered as a byproduct of a process for producing a fermentation product in which the GH98 xylanase or enzyme blend comprising the GH98 xylanase is absent or not added.
17. The method of claim 16, wherein the DDG or DDGs has increased fat content.
18. The method of any one of claims 10-17, wherein the TME of the DGS or DDGS is increased by at least 5%, at least 10%, at least 15%, or at least 20% compared to the true metabolic energy of DGS or DDGS produced in the absence of a GH98 xylanase or an enzyme blend comprising the GH98 xylanase during a saccharification step, a fermentation step, and/or a simultaneous saccharification and fermentation step of the process.
19. The method of claim 18, wherein the TME is directed against a monogastric animal.
20. The method of claim 15 or 16, wherein the DGS or DDGS produced after drying is not blackened compared to that produced during a saccharification step, fermentation step, and/or simultaneous saccharification and fermentation step of the method of any one of claims 10-19 in the absence of the polypeptide having xylanase activity of any one of claims 1-2 or the enzyme blend of any one of claims 3-5.
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