Mannanase variants

Abstract

This specification relates to variants of mannanases, compositions comprising the variants, methods of their production, and methods of using the variants to degrade and modify mannan-containing materials.

Description

Invention Field

[0001] This invention relates to variants of mannanases. These variants are useful in industrial applications where the degradation or modification of mannans is desired, such as in laundry and cleaning applications, and in the animal, food, pulp and paper, and petroleum industries. The invention also provides useful mannanases, polynucleotides encoding these enzymes, enzyme compositions, and methods for their production and use.

[0002] background

[0003] Mannans are polysaccharides containing mannose found in various plants. Mannans have poor solubility in water, and their physicochemical properties cause viscous dispersion. Furthermore, mannans have a high water-binding capacity. All these properties cause problems in several industries, including brewing, baking, animal nutrition, and laundry and cleaning applications.

[0004] In plant-based diets, various β-mannans exist, and depending on their quantity and characteristics, they can impair nutrient digestion, microbial colonization, and growth performance. Enzymatic degradation of mannans reduces the digesta viscosity of highly water-soluble mannans and leads to the production of mannan oligosaccharides, which can form water-insoluble linear mannans found in legumes. Mannanases increase mean daily gain, feed efficiency, weight uniformity, and survival rate in all monogastric animals.

[0005] In animal feed applications (such as feed for monogastric animals with grain-based diets), mannans are a promoter of intestinal content viscosity, which negatively impacts feed digestibility and animal growth rate. For ruminants, mannans represent a major component of fiber intake, and more complete digestion of mannans contributes to higher feed conversion efficiency.

[0006] For laundry and cleaning applications, enzyme compositions containing mannanase can be used to degrade mannan. However, it is difficult to provide mannanases that are stable under varying storage and use conditions while still exhibiting good mannan-degrading activity.

[0007] The stability of industrial enzymes is an important characteristic because these enzymes are often used under conditions very different from their natural environment. Often, wild-type enzymes that show good performance in initial tests are not suitable for industrial-scale production, or are unstable under typical application or storage conditions.

[0008] N-linked glycosylation of proteins is a type of post-translational modification in which an oligosaccharide sugar molecule called a glycan attaches to the amide nitrogen group of an asparagine (Asn, N) residue in the protein. This type of linkage is important for both the structure and function of enzymes and other proteins.

[0009] One object of the present invention is to provide variants of mannanase that exhibit improved stability when applied in various industrial processes and demonstrate mannanase activity, as well as enzyme compositions for mannan degradation or modification.

[0010] Overview

[0011] According to the first aspect, a variant of mannanase is provided, which contains at least one amino acid substitution at positions corresponding to positions 123, 158, 180, 272, 307, or 316, wherein said variant has mannanase activity and is selected from:

[0012] 1) A polypeptide having at least 85% sequence identity with residues 27-331 of SEQ ID NO: 2;

[0013] 2) Variants encoded by polynucleotides that hybridize under highly stringent conditions to:

[0014] a) Nucleotides 79-993 of SEQ ID NO:1 (man7), or

[0015] b) The full-length complement of a); and

[0016] 3) A variant encoded by a polynucleotide, wherein the polynucleotide has at least 95% sequence identity with SEQ ID NO:1 or its genomic DNA sequence;

[0017] Furthermore, the amino acid number corresponds to the amino acid number of the full-length amino acid sequence of SEQ ID NO:2 (Man7) containing the signal sequence.

[0018] The mannanase variants of the present invention are advantageous in terms of good stability and mannanase activity. These variants exhibit improved stability in detergents, particularly at high temperatures. Therefore, the mannanase variants of the present invention can provide improved yields in production and better performance in use. The variants exhibit particularly good stability at temperatures typically used in detergents and in applications where mannan degradation is employed (such as in laundry detergents).

[0019] In one embodiment, the variant has at least two substitutions. This is advantageous for further improving stability in detergents and at high temperatures. Alternatively, two substitutions may be selected to improve another property of the variant in addition to stability.

[0020] According to a second aspect of the invention, an enzyme composition is provided comprising a variant of the mannanase of the first aspect and

[0021] a. At least one preservative, said preservative being selected, for example, from organic acids, citric acid, ascorbic acid, benzoic acid and its salts and derivatives, sodium benzoate, benzoate / ester, hydroxybenzoate / ester and derivative, sorbic acid, sodium sorbate, sorbate / ester, salts such as sodium chloride or potassium chloride, 1,2-benzisothiazolin-3-one (BIT) or combinations thereof;

[0022] b. Optionally, at least one stabilizer selected from polyols, propylene glycol, polyethylene glycol, hexanediol, glycerol, sugars, sugar alcohols, polysaccharides, lactic acid, boric acid, boric acid derivatives, aromatic borate esters, 4-formylphenylboronic acid, phenylboronic acid derivatives, peptides, surfactants, or combinations thereof.

[0023] c. Optionally, at least one enzyme selected from proteases, amylases, cellulases, lipases, xylanases, mannanases, cutinases, esterases, phytases, DNases, pectinsases, pectinases, pectate lyases, glycosylases, arabinogalactanases, galactanases, xanthan gumases, xylose glucanases, laccases, peroxidases, and oxidases, with or without a mediator, or a combination thereof; and

[0024] d. Optionally, at least one filler selected from maltodextrin, flour, sodium chloride, sulfate, sodium sulfate, or combinations thereof.

[0025] As demonstrated by the examples, the variants included in the enzyme compositions according to the invention have structures and properties that allow for production in recombinant host cells and make them available for industrial applications. The enzyme compositions are particularly favorable for detergent formulations because the mannanase variants exhibit good stability, detergency, and specific activity when used in laundry and washing applications to degrade mannan.

[0026] According to the third aspect, a detergent composition comprising a variant of the mannanase of the first aspect or an enzyme composition of the second aspect is provided.

[0027] The detergent composition of the present invention is advantageous because it is stable, effective and economical in removing stains containing mannan.

[0028] According to another aspect, the use of the enzyme composition of the present invention or a variant of the mannanase of the present invention in detergents and methods of using the enzyme composition of the present invention or a variant of the mannanase of the present invention in detergents are provided.

[0029] According to the fourth aspect, a recombinant host cell containing a genetic element is provided, said genetic element allowing the production of at least one recombinant polypeptide containing a variant of the mannanase of the first aspect.

[0030] According to the fifth aspect, a method for generating a recombinant polypeptide having mannanase activity is provided, and the method includes:

[0031] a. Culturing recombinant host cells for the fourth aspect, among which...

[0032] The genetic element contains at least one control sequence that controls the production of the recombinant polypeptide in the recombinant host cell;

[0033] The genetic element optionally includes at least one sequence encoding a signal sequence for transporting the recombinant polypeptide outside the host cell; and

[0034] Culture was carried out under conditions that allowed for the production of the recombinant polypeptide; and

[0035] b. Recover the recombinant polypeptide.

[0036] The method provides an efficient approach to producing recombinant polypeptides containing variants of mannanase. Because the mannanase variants are generated in recombinant host cells, a production system is provided that can be optimized, customized, and controlled in a desired manner. The mannanase variants produced by this method can differ from natural mannanase at both structural and functional levels.

[0037] According to another aspect, an enzyme preparation is provided comprising a recombinant polypeptide having mannanase activity and obtainable by using the host cells of the present invention.

[0038] The enzyme preparation or enzyme composition may further comprise other enzymes selected from proteases, amylases, cellulases, lipases, xylanases, mannanases, keratases, esterases, phytases, DNases, pectinases, pectic acid lyases, pectinases, glycosylases, arabinogalactanases, galactanases, xanthan gumases, xylose glucanases, laccases, peroxidases, and oxidases (with or without a medium), as well as suitable additives selected from stabilizers, buffers, surfactants, bleaching agents, media, corrosion inhibitors, detergent builders, anti-redeposition agents, optical brighteners, dyes, pigments, fragrances, corrosives, abrasives, and preservatives.

[0039] According to a sixth aspect, a method for degrading or modifying a material containing mannan is provided, comprising treating the mannan-containing material with an effective amount of the enzyme composition of the present invention or a variant of the mannanase of the present invention.

[0040] According to a seventh aspect, animal feed is provided comprising the enzyme composition of the present invention or a variant of the mannanase of the present invention, and at least one plant-derived protein source or product or byproduct containing mannan, and

[0041] a. Optionally, at least one enzyme selected from proteases, amylases, phytases, xylanases, endoglucanases, β-glucanases, or combinations thereof; and

[0042] b. Optionally, at least one filler selected from maltodextrin, flour, salt, sodium chloride, sulfate, sodium sulfate, or combinations thereof.

[0043] According to the eighth aspect, a feed supplement is provided comprising the enzyme composition of the present invention or a variant of the mannanase of the present invention; and

[0044] a. Optionally, at least one enzyme selected from proteases, amylases, phytases, xylanases, endoglucanases, β-glucanases, or combinations thereof; and

[0045] b. Optionally, at least one filler selected from maltodextrin, flour, salt, sodium chloride, sulfate, sodium sulfate, or combinations thereof.

[0046] Compared to feeds without the aforementioned variants, the feeds and feed supplements improve the nutritional value of the feeds. The enzyme compositions of the present invention contain variants of mannanase, which have improved stability. The enzyme compositions and variants of the present invention degrade mannan present in feeds, thereby making them more easily digestible by animals. In particular, for feeds containing soybean meal, the mannan-oligosaccharides produced by enzymatic digestion have a beneficial effect on the gut microbiota and therefore a beneficial effect on animal performance. The action of the mannanase variants can be enhanced by including xylanase to digest arabinoxylan present in corn-soybean-based diets. The variants of the present invention can also be used to modify the rheological properties of wet feeds.

[0047] In one embodiment, the feed may contain animal protein, such as meat meal or bone meal.

[0048] According to another aspect, the use of the animal feed or feed supplement of the present invention in the following and the method of using the animal feed or feed supplement of the present invention in the following are provided:

[0049] a. to feed animals;

[0050] b. Increase the weight gain of animals.

[0051] In one embodiment, the animal is a monogastric animal or a ruminant. In another embodiment, the animal is a broiler, laying hen, pig, turkey, or aquaculture organism such as a fish. In yet another embodiment, the animal is a ruminant.

[0052] According to the ninth aspect, the use of variants of the invention or enzyme compositions of the invention in oil drilling or hydraulic fracturing and methods of using variants of the invention or enzyme compositions of the invention in oil drilling or hydraulic fracturing are provided.

[0053] The enzyme compositions of the present invention and their variants are advantageous in altering the rheological properties of oil drilling fluids and hydraulic fracturing fluids and in improving oil recovery rates.

[0054] According to the tenth aspect, the use of variants of the invention or enzyme compositions of the invention in the treatment of coffee extracts, fruit juices, pineapple juices or soy milk is provided, as well as methods of using variants of the invention or enzyme compositions of the invention in the treatment of coffee extracts, fruit juices, pineapple juices or soy milk.

[0055] Using the variants of the present invention and the enzyme composition of the present invention is advantageous in processing coffee extracts because it reduces the viscosity of the coffee extracts.

[0056] The use of variants of the invention and the enzyme compositions of the invention is advantageous in the processing and production of fruit juices because they reduce viscosity and increase filtration rate, stability and facilitate the extraction of fruit components.

[0057] Using the variants of the present invention and the enzyme compositions of the present invention is advantageous in the processing and manufacture of soy milk because it improves the yield, color, protein content and taste of soy milk.

[0058] In another aspect, nucleic acid molecules encoding variants of the mannanase of the present invention are provided.

[0059] In another aspect, a carrier comprising the nucleic acid molecules of the present invention is provided.

[0060] In another aspect, a functional fragment of a variant of the mannanase of the present invention is provided, and a polynucleotide encoding thereof is provided.

[0061] In another aspect, the sequence information disclosed herein relating to the polynucleotide sequence encoding the mannanase of the present invention can be used as a tool for identifying other homologous mannanases. For example, polymerase chain reaction (PCR) can be used to amplify sequences encoding other homologous mannanases from various biological sources. Furthermore, genome mining methods can be used to identify sequences encoding other homologous mannanases from genome databases. Brief description of the attached diagram

[0063] Figure 1 A schematic diagram showing the vector pEV1 used for replication in the genus Bacillus.

[0064] Figure 2 The expression cassette used in the transformation of Trichoderma reesei is shown schematically.

[0065] Figure 3A -B describes the stain removal performance of the variant and wild-type Man7 (produced in Trichoderma) as an increase in brightness (the sum of ΔL* of the three stains) in the presence of 4.4 g / l of a commercial heavy-duty liquid detergent A at 40 °C, 16 °dH, 60 min, pH approximately 8.3, and with enzyme administered as active units per wash (MNU).

[0066] Figure 3A The variants TBH1, TBH2, TBH3, TBH4, TBH5, TBH6, TBH7, TBH8, TBH9 and the wild type are shown.

[0067] Figure 3B Variants TBH6, TBH10, TBH11 and wild type are shown.

[0068] Figure 4A -B describes the stain removal performance of the variant and wild-type Man7 (produced in Trichoderma) as an increase in brightness (the sum of ΔL* for 3 stains) in the presence of 3.8 g / l of commercial color detergent powder at 40 °C, 16 °dH, 60 min, pH approximately 10 and given as enzyme as active unit per wash (MNU).

[0069] Figure 4A The variants TBH1, TBH2, TBH3, TBH4, TBH5, TBH6, TBH7, TBH8, TBH9 and the wild type are shown.

[0070] Figure 4B Variants TBH6, TBH10, TBH11 and wild type are shown.

[0071] Figure 5 The stain removal performance of variants TBH1, TBH2, TBH3, TBH4, TBH5, TBH6, TBH7, TBH8, TBH9 and wild-type Man7 (produced in Trichoderma) as an increase in brightness (the sum of ΔL* for the three stains) was described at 40 °C, 16 °dH, 60 min, pH approximately 9.5 and enzyme administered as active units per wash (MNU), in the presence of 3.8 g / L commercial bleaching detergent powder.

[0072] Figure 6The stain removal performance of variants BH18, BH21, BH23, BH24, BH25 and wild-type Man7 (produced in Bacillus) as an increase in brightness (the sum of ΔL* for the three stains) is described at 40 °C, 16 °dH, 60 min, pH approximately 8.3 and enzyme administered as active units per wash (MNU), in the presence of 4.4 g / L of commercial heavy-duty liquid detergent A.

[0073] Figure 7 The stain removal performance of variants BH18, BH21, BH23, BH24, BH25 and wild-type Man7 (produced in Bacillus) as an increase in brightness (the sum of ΔL* for the three stains) is described at 40 °C, 16 °dH, 60 min, pH approximately 10 and enzyme administered as active units per wash (MNU), in the presence of 3.8 g / L commercial color detergent powder.

[0074] Figure 8 The stain removal performance of variants BH18, BH21, BH23, BH24, BH25 and wild-type Man7 (produced in Bacillus) as an increase in brightness (the sum of ΔL* for the three stains) is described at 40 °C, 16 °dH, 60 min, pH approximately 9.5 and enzyme administered as active units per wash (MNU), in the presence of 3.8 g / L commercial bleaching detergent powder.

[0075] Figure 9 The variant TBH6 was shown to be stable at 37°C in commercial heavy-duty liquid detergents compared to the wild-type enzyme produced in Trichoderma.

[0076] Figure 10 The variant BH25 was shown to be stable at 37°C in commercial heavy-duty liquid detergents compared to the wild-type enzyme produced in Bacillus spp.

[0077] Figure 11A -B indicates the stability of variants and wild types produced in the Trichoderma genus at 50°C for 7 days in commercial heavy-duty liquid detergents.

[0078] Figure 11A The variants TBH1, TBH2, TBH3, TBH4, TBH5, TBH6, TBH7, TBH8, TBH9 and the wild type are shown.

[0079] Figure 11B Variants TBH6, TBH10, TBH11 and wild type are shown.

[0080] Figure 12 The flowchart shown relates to the production of instant coffee using the mannanase variant of the present invention.

[0081] Figure 13 The combined variant TBH14 (a combination of TBH5 and TBH6) demonstrates stability in commercial heavy-duty liquid detergents at 50°C for 7 days, compared to variants TBH5 and TBH6 alone.

[0082] Preservation

[0083] In accordance with the Budapest Treaty on the international recognition of microbial deposits for patent proceedings purposes, the following strains shall be deposited:

[0084] Escherichia coli strain RF12379 (including plasmid pALK4434) was deposited on March 2, 2017 at the German Center for Microbiology and Cell Culture Collection (Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH, DSMZ), Inhoffenstrasse 7 b, D-38124 Braunschweig, Germany, with accession number DSM32425.

[0085] Escherichia coli strain RF12380 (including plasmid pALK4435) was deposited on March 2, 2017 at the German Center for Microbiology and Cell Culture Collection (Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH, DSMZ), Inhoffenstrasse 7 b, D-38124 Braunschweig, Germany, with accession number DSM32426.

[0086] Escherichia coli strain RF12381 (including plasmid pALK4436) was deposited on March 2, 2017 at the German Center for Microbiology and Cell Culture Collection (Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH, DSMZ), Inhoffenstrasse 7 b, D-38124 Braunschweig, Germany, with accession number DSM32427.

[0087] Escherichia coli strain RF12382 (including plasmid pALK4437) was deposited on March 2, 2017 at the German Center for Microbiology and Cell Culture Collection (Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH, DSMZ), Inhoffenstrasse 7 b, D-38124 Braunschweig, Germany, with accession number DSM32428.

[0088] Escherichia coli strain RF12383 (including plasmid pALK4438) was deposited on March 2, 2017 at the German Center for Microbiology and Cell Culture Collection (Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH, DSMZ), Inhoffenstrasse 7 b, D-38124 Braunschweig, Germany, with accession number DSM32429.

[0089] Escherichia coli strain RF12384 (including plasmid pALK4439) was deposited on March 2, 2017 at the German Center for Microbiology and Cell Culture Collection (Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH, DSMZ), Inhoffenstrasse 7 b, D-38124 Braunschweig, Germany, with accession number DSM32430.

[0090] Escherichia coli strain RF12385 (including plasmid pALK4440) was deposited on March 2, 2017 at the German Center for Microbiology and Cell Culture Collection (Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH, DSMZ), Inhoffenstrasse 7 b, D-38124 Braunschweig, Germany, with accession number DSM32431.

[0091] Escherichia coli strain RF12386 (including plasmid pALK4441) was deposited on March 2, 2017 at the German Center for Microbiology and Cell Culture Collection (Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH, DSMZ), Inhoffenstrasse 7 b, D-38124 Braunschweig, Germany, with accession number DSM32432.

[0092] Escherichia coli strain RF12387 (including plasmid pALK4442) was deposited on March 2, 2017 at the German Center for Microbiology and Cell Culture Collection (Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH, DSMZ), Inhoffenstrasse 7 b, D-38124 Braunschweig, Germany, with accession number DSM32433.

[0093] Escherichia coli strain RF12456 (including plasmid pALK4432) was deposited on May 18, 2017 at the German Center for Microbiology and Cell Culture Collection (Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH, DSMZ), Inhoffenstrasse 7 b, D-38124 Braunschweig, Germany, with accession number DSM32518.

[0094] Escherichia coli strain RF12457 (including plasmid pALK4433) was deposited on May 18, 2017 at the German Center for Microbiology and Cell Culture Collection (Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH, DSMZ), Inhoffenstrasse 7 b, D-38124 Braunschweig, Germany, with accession number DSM32519.

[0095] sequence list

[0096] SEQ ID NO: 1 man7 DNA sequence

[0097] Full-length amino acid sequence of SEQ ID NO: 2 Man7

[0098] The deduced amino acid sequence (mature) of SEQ ID NO: 3 Man7

[0099] The core amino acid sequence of SEQ ID NO: 4 Man7, without CMB.

[0100] SEQ ID NO: 5 variant tbh1 Synthetic gene sequence

[0101] Deduced amino acid sequence (mature) of variant TBH1, SEQ ID NO: 6

[0102] SEQ ID NO: 7 variant tbh2 Synthetic gene sequence

[0103] Deduced amino acid sequence (mature) of variant TBH2 of SEQ ID NO: 8

[0104] SEQ ID NO: 9 variant tbh3 Synthetic gene sequence

[0105] Deduced amino acid sequence (mature) of SEQ ID NO: 10 variant TBH3

[0106] SEQ ID NO: 11 variant tbh4 Synthetic gene sequence

[0107] Deduced amino acid sequence (mature) of SEQ ID NO: 12 variant TBH4

[0108] SEQ ID NO: 13 variant tbh5 Synthetic gene sequence

[0109] Deduced amino acid sequence (mature) of variant TBH5, SEQ ID NO: 14

[0110] SEQ ID NO: 15 variant tbh6 Synthetic gene sequence

[0111] Deduced amino acid sequence (mature) of variant TBH6, SEQ ID NO: 16

[0112] SEQ ID NO: 17 variant tbh7 Synthetic gene sequence

[0113] Deduced amino acid sequence (mature) of variant TBH7, SEQ ID NO: 18

[0114] SEQ ID NO: 19 variant tbh8 Synthetic gene sequence

[0115] Deduced amino acid sequence (mature) of variant TBH8, SEQ ID NO: 20

[0116] SEQ ID NO: 21 variant tbh9 Synthetic gene sequence

[0117] Deduced amino acid sequence (mature) of variant TBH9, SEQ ID NO: 22

[0118] SEQ ID NO: 23 variant tbh10 Synthetic gene sequence

[0119] Deduced amino acid sequence (mature) of variant TBH10, SEQ ID NO: 24

[0120] SEQ ID NO: 25 variant tbh11 Synthetic gene sequence

[0121] Deduced amino acid sequence (mature) of variant TBH11, SEQ ID NO: 26

[0122] SEQ ID NO:27 variant bh18 DNA sequence

[0123] Deduced amino acid sequence (mature) of SEQ ID NO:28 variant BH18

[0124] SEQ ID NO:29 variant bh21 DNA sequence

[0125] Deduced amino acid sequence (mature) of variant BH21, SEQ ID NO:30

[0126] SEQ ID NO:31 variant bh23 DNA sequence

[0127] Deduced amino acid sequence (mature) of variant BH23 of SEQ ID NO:32

[0128] SEQ ID NO:33 variant bh24 DNA sequence

[0129] Deduced amino acid sequence (mature) of variant BH24, SEQ ID NO:34

[0130] SEQ ID NO:35 variant bh25 DNA sequence

[0131] Deduced amino acid sequence (mature) of variant BH25, SEQ ID NO:36

[0132] The sequence of the oligonucleotide primer Man7_Var1 (SEQ ID NO:37)

[0133] The sequence of the oligonucleotide primer Man7_Var2, SEQ ID NO:38

[0134] The sequence of oligonucleotide primer Man7_Var3, SEQ ID NO:39

[0135] The sequence of oligonucleotide primer Man7_Var4 (SEQ ID NO:40)

[0136] The sequence of oligonucleotide primer Man7_Var5, SEQ ID NO:41

[0137] The sequence of oligonucleotide primer Man7_Var6, SEQ ID NO:42

[0138] The sequence of oligonucleotide primer Man7_Var7, SEQ ID NO:43

[0139] The sequence of oligonucleotide primer Man7_Var8, SEQ ID NO:44

[0140] The sequence of oligonucleotide primer Man7_Var9, SEQ ID NO:45

[0141] The sequence of oligonucleotide primer Man7_Var10, SEQ ID NO:46

[0142] The sequence of oligonucleotide primer Man7_Var11, SEQ ID NO:47

[0143] The sequence of the oligonucleotide primer Man7_Var12, SEQ ID NO:48.

[0144] Detailed Explanation

[0145] Mannans are polysaccharides composed of a mannose backbone linked by β-1,4-bonds and galactose side chains (attached to the backbone by α-1,6-bonds). Mannans are derived from plant-based materials such as guar gum and locust bean gum. Glucomannan is a polysaccharide with a roughly regularly alternating β-1,4-linked mannose and glucose backbone. Galactomannan and galactoglucomannan are mannans and glucomannans with α-1,6-linked galactose side branches, respectively.

[0146] The terms "functional fragment" or "effective fragment" refer to a fragment or portion of the variant of SEQ ID NO: 2 that retains approximately the same enzymatic function or effect.

[0147] The terms "mannanase variant" and "mannanase variant" refer to any mannanase molecule obtained through site-directed or random mutagenesis, insertion, substitution, deletion, recombination, and / or any other protein engineering method that results in the amino acid sequence of the mannanase differing from that of the parental mannanase (i.e., wild-type mannanase). The terms "wild-type mannanase," "wild-type enzyme," "wild-type," or "wt" as used in this disclosure describe a mannanase or fragment thereof having an amino acid sequence found in nature.

[0148] The term "catalytic activity" or "activity" quantitatively describes the transformation of a given substrate under defined reaction conditions. The term "residual activity" is defined as the ratio of the enzyme's catalytic activity under a specific set of conditions to its catalytic activity under different sets of conditions. Therefore, residual activity a i By a i =v i / v0 is given, where v represents any measure of catalytic activity, and a i *100 is relative activity as a percentage. The term "specific activity" quantitatively describes the catalytic activity per unit amount of enzyme under defined reaction conditions.

[0149] The term "protein hydrolytic stability" describes the property of a protein to withstand limited exposure to a protease while the protease is active, and to retain its activity while the protease's activity can be measured.

[0150] As used herein, the term "mannanase" or "galactomannanase" refers to a mannanase that, according to what is known in the art, is defined as mannan-1,4-β-mannanosidase, and has the alternative names β-mannanase and galactomannanase, and catalyzes the hydrolysis of 1,4-β-D-mannoside bonds in mannan, galactomannan, glucomannan, and galactoglucomannan. Mannanases are classified as EC 3.2.1.78 according to enzyme nomenclature.

[0151] As used herein, “isolated” means a substance in a form not found in nature or in an environment not found in nature. Non-limiting examples of isolated substances include (1) any substance not naturally occurring, (2) any substance from which one or more or all of its naturally occurring components associated with it in nature have been removed, including any enzyme, variant, nucleic acid, protein, peptide, or cofactor; (3) any substance artificially modified relative to a substance found in nature, such as a variant; or (4) any substance modified by increasing or decreasing the amount of the substance relative to other components naturally associated with it (e.g., recombinant production in a host cell; one or more copies of the gene encoding the substance; and the use of a promoter that is alternative to the promoter naturally associated with the gene encoding the substance). In one embodiment, the polypeptides, enzymes, variants, polynucleotides, host cells, or compositions of the present invention are isolated.

[0152] As used herein, the term “comprising” includes the broader meanings of “including,” “containing,” and “comprehending,” as well as the narrower expressions “consisting of” and “consisting of only.”

[0153] As used herein, "variant" means the insertion, substitution, or deletion of one or more nucleotides / amino acids or a chemically modified sequence or sequence fragment (nucleotide or amino acid). In one embodiment, the term variant also includes recombinant mannanase.

[0154] As used herein, "conservative amino acid substitution" is the substitution of an amino acid residue by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. In one embodiment, a conserved amino acid in this specification refers to an amino acid within the following group: hydrophobic (FWYHKMILVAGC); aromatic (FWYH); aliphatic (ILV); polar (WYHKREDCSTNQ); charged (HKRED); positively charged (HKR); negatively charged (ED); small (VCAGSPTND); tiny (AGS). Thus, when an amino acid is substituted by an amino acid within the same group, a conserved substitution occurs.

[0155] In one embodiment, the substitution is a substitution with at least one amino acid residue, such as a substitution with one, two, or three amino acids. In a further embodiment, the at least one amino acid is Ala.

[0156] As used herein, “non-conservative amino acid substitution” is an amino acid substitution in which an amino acid is replaced by an amino acid from a different group as defined above. Non-conservative substitution may result in one amino acid being replaced by another amino acid with different biochemical properties (such as charge, hydrophobicity, and / or size). In one embodiment, non-conservative substitution alters at least one property of the variant, such as stability, glycosylation pattern, folding, structure, activity, or affinity.

[0157] N-linked glycosylation occurs in eukaryotes but rarely in bacteria. Attachment of glycan residues to a protein requires recognition of a common sequence. N-linked glycans almost always attach to an asparagine (Asn) side chain that is part of the Asn-X-Ser / Thr common sequence (where X is any amino acid except proline). The inventors have also found that non-glycosylated Asn side chains structurally close to the active site of mannanase are important for obtaining variants with good mannan degradation performance. Unbound by any theory, the sugars in glycans are polar molecules, and when attached to Asn, they reside on the surface of the protein, causing glycosylation of the Asn and its vicinity, resulting in structural changes. Site-directed mutagenesis of the Asn or Ser / Thr residues in the Asn-Xaa-Thr (Ser) common sequence can be used to prevent glycosylation of the desired N-linked glycosylation site in variants of the first aspect of the invention.

[0158] In one embodiment, the amino acid of the variant is replaced by residues that prevent N-linked glycosylation of residue 283 when expressed in a host cell capable of N-linked glycosylation.

[0159] In one embodiment, a variant of the invention comprises at least one Asn-X-Ser / Thr common sequence.

[0160] In one embodiment, a variant of the invention comprises at least one Pro residue at position X of the Asn-X-Ser / Thr common sequence.

[0161] In one implementation, the substitution is either conservative or non-conservative.

[0162] As used herein, "peptide" and "polypeptide" are amino acid sequences comprising a plurality of sequentially aggregated amino acid residues. For the purposes of this invention, a peptide is a molecule comprising up to 20 amino acid residues, and a polypeptide comprises more than 20 amino acid residues. The peptide or polypeptide may include modified amino acid residues, naturally occurring amino acid residues not encoded by a codon, and non-naturally occurring amino acid residues. As used herein, "protein" may refer to a peptide or polypeptide of any size. A protein may be an enzyme, protein, antibody, membrane protein, peptide hormone, regulator, or any other protein.

[0163] The term "polynucleotide" refers to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to 3' ends. Polynucleotides include RNA and DNA and can be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules.

[0164] As used herein, in the context of polynucleotides, the terms "modification," "modified," and similar terms refer to modifications in the coding or non-coding regions of a polynucleotide (such as regulatory sequences, 5' untranslated regions, 3' untranslated regions, upregulated genetic elements, downregulated genetic elements, enhancers, repressors, promoters, exon or intron regions). In some embodiments, the modification may be structural only and have no effect on the biological effects, functions, or functions of the polynucleotide. In other embodiments, the modification is a structural modification that provides a change in the biological effects, functions, or functions of the polynucleotide. Such modifications can enhance, inhibit, or alter the biological function of the polynucleotide.

[0165] As used herein, “identity” refers to the percentage of exact amino acid residue matches between two aligned sequences relative to the number of positions of residues present in both sequences. When one sequence has residues that do not have corresponding residues in the other sequence, the alignment procedure allows for vacancies in the alignment, and these positions are not counted in the denominator of the identity calculation. Identity is measured using the EMBL-EBI website ( www.ebi.ac.uk / Tools / psa / emboss_needle / The value is determined by the pairwise sequence alignment tool EMBOSS Needle.

[0166] As used herein, low stringency means, for probes at least 100 nucleotides in length, the corresponding conditions are following a standard Southern blotting procedure: 55°C, in 5× SSC, 0.1% N-lauroyl sarcosine, 0.02% SDS, and 1% blocking reagent (Roche 11 096 176 001), for 12 to 24 hours of pre-hybridization and hybridization. The vector material is then washed 2–3 times for 15 minutes each at 55°C using 2X SSC and 0.1% SDS.

[0167] As used herein, highly stringent conditions mean that for probes at least 100 nucleotides in length, the conditions correspond to hybridization for 12 to 24 hours under pre-hybridization and hybridization conditions following a standard Southern blotting procedure at 65°C in 5× SSC, 0.1% N-lauroyl sarcosine, 0.02% SDS, and 1% blocking reagent (Roche 11 096 176 001). The vector material is then washed 2–3 times for 15 minutes each time at 65°C using 0.1X SSC and 0.1% SDS.

[0168] As used herein, "host cell" refers to any cell type that is readily transformed, transfected, transduced, mated, hybridized, etc., using nucleic acid constructs or expression vectors containing polynucleotides. The term "host cell" encompasses any progeny that differs due to mutations occurring during replication. A non-limiting example of a host cell is a fungal cell, specifically a filamentous fungal cell from the phylum Ascomycota. Ascomycota ), Subdivision Pezizomycotina Preferred sources are from the class Ficotyle (Class 1). Sordariomycetes ), Subclass Sarcotyle Hypocreomycetidae ), Sarcoptales ( Hypocreales ) and Microcystales ( Microascales ) and Aspergillus genus ( Aspergillus ), genus *Aureobasidium* Chrysosporium ), genus *Dermocytotrichum* Myceliophthora ) and humic molds ( Humicola A group composed of members of the family Hypocreaceae; more preferably from the family Hypocreaceae ( Hypocreacea ), family Ceratophyllum ( Nectriaceae Clavicipitaceae ( Clavicipitaceae ), Microcystis ( Microascaceae ) and Trichoderma ( Trichoderma (Asexual Sarcoptes genus) Hypocrea Fusarium ( )), Fusarium genus ( Fusarium ), Gibberella genus ( Gibberella ), genus *Cirsium* ( Nectria ), *Botrytis* genus ( Stachybotrys ), ergot ( Claviceps Metarhizium anisopliae Metarhizium ), Villosiclava Cordyceps genus ( Ophiocordyceps ), Cephalosporium ( Cephalosporium ) and Cetacea ( Scedosporium The group consists of ) and more preferably comes from Trichoderma reesei ( Trichoderma reesei ) (Red-brown fleshy fungus ( Hypocrea jecorina )), Lemon Trichoderma ( T. citrinoviridae ), Trichoderma longifolia ( T. longibrachiatum ), Trichoderma viride T. virens Trichoderma harzianum ( T. harzianum ), Trichoderma acicularis ( T. asperellum ), dark green Trichoderma ( T. atroviridae Trichoderma ginrylifolia ( T. parareesei Fusarium oxysporum ( Fusarium oxysporum ), Fusarium graminearum ( F. gramineanum ), Fusarium pseudograss ( F. pseudograminearum ), Fusarium moniliforme ( F. venenatum Fujikura gibberellin Gibberella fujikuroi ), gibberellinii ( G. moniliformis ), Maize Fusarium ( G. zeaea ), flagellated red scabies (blood red scabies) ( Nectria (Haematonectria) haematococca ), paper grape spike ( Stachybotrys chartarum ), S. chlorohalonata, Rye ergot ( Claviceps purpurea ), Metarhizium anisopliae ( Metarhizium acridum ), Metarhizium anisopliae ( M. anisopliae ), Rhizoctonia solani ( Villosiclava virens Cordyceps sinensis Ophiocordyceps sinensis ), Cephalosporium (Cephalosporium) ( Acremonium (Cephalosporium) chrysogenum ) and Cetacea tricuspidata ( Scedosporium apiospermum ) and Aspergillus niger ( Aspergillus niger ), Aspergillus awamori ( Aspergillus awamori Aspergillus oryzae ( Aspergillus oryzae ), Rudd's chlortetracycline ( Chrysosporium lucknowense ), thermophilic pyridamus ( Mycelium thermophilic ), specific humic mold ( Humicola insolens ) and gray humic mold ( Grey humicola The group consisting of *Trichoderma reesei* is preferred. Non-limiting examples of host cells are bacterial cells, preferably Gram-positive bacilli (e.g., *Bacillus subtilis*). Bacillus subtilis ), Bacillus licheniformis ( B. licheniformis ), Bacillus megaterium ( B. megatherium ), Bacillus amyloliquefaciens ( B. amyloliquefaciens ), Bacillus pumilus ( B. small Gram-negative bacteria (e.g., Escherichia coli) Escherichia coli Actinomycetes (e.g., Streptomyces genus) Streptomyces sp.)), and yeast (e.g., brewer's yeast) Saccharomyces cerevisiae Pichia pastoris () Shepherd's pie ), Yarrowia lipolytica ( Yarrowia lipolytica )).

[0169] In one embodiment, the host cell is a fungal cell, preferably a filamentous fungal cell, such as *Trichoderma* or *Trichoderma reesei*. In another embodiment, the host cell is a bacterial cell, preferably a Gram-positive bacillus cell, such as *Bacillus subtilis*. B. subtilis ), Bacillus licheniformis, Bacillus megaterium, Bacillus amyloliquefaciens, and Bacillus pumilus.

[0170] In one embodiment, the host cell is capable of N-linked glycosylation.

[0171] "Recombinant cell" or "recombinant host cell" refers to a cell or host cell that has been genetically modified or altered to include nucleic acid sequences that are not natural to the cell or host cell. Genetic modification may include the integration of polynucleotides into the genome of the host cell. The polynucleotides may also be exogenous in the host cell. In one embodiment, the host cell of the present invention is a recombinant host cell.

[0172] As used herein, "expression" includes any step involved in the production of a polypeptide in a host cell, including but not limited to transcription, translation, post-translational modification, and secretion. Expression may be followed by the harvesting (i.e., recycling) of the host cell or the expressed product.

[0173] The term "expression vector" refers to a linear or circular DNA molecule containing a segment encoding a target polypeptide, operatively linked to an additional segment that provides for its transcription. Such an additional segment may include promoter and terminator sequences and may optionally include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, vectors, etc. Expression vectors are typically derived from plasmid or viral DNA, or may contain elements of both. An expression vector can be any expression vector that facilitates recombinant DNA procedures, and the choice of vector will often depend on the host cell in which the vector is to be introduced. Thus, the vector can be a self-replicating vector, i.e., a vector existing as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid. Alternatively, the vector can be a vector that is integrated into the host cell genome upon introduction into the host cell and replicates along with the chromosome already integrated therein. In one embodiment, the vector of the present invention is an expression vector.

[0174] The term “recombinantly generated” or “recombinantly produced” used in connection with the generation of peptides or proteins is defined according to the standard definition in the art.

[0175] As used in this article in conjunction with the terms "derived from" and "available" in relation to a specific microbial source, it means that the polynucleotide is expressed by the specific source (homologous expression) or by a cell in which a gene from the source has been inserted (heterologous expression).

[0176] The term "enzyme composition" refers to a conventional enzymatic fermentation product that may be isolated and purified from a single species of microorganism, such a formulation typically containing a variety of different enzymatic activities; or a mixture of single-component enzymes (preferably derived from bacterial or fungal species using conventional recombinant techniques) that have been fermented and may be isolated and purified separately, and which may be derived from different species, preferably fungal or bacterial species or fermentation products of microorganisms that act as host cells for producing recombinant mannanase, but which also produce other enzymes.

[0177] The term "operably linked," when referring to DNA segments, means arranging said segments so that they function in coordination for their intended purpose, such as transcription starting in a promoter and proceeding through coding segments to a terminator.

[0178] The term "promoter" refers to a portion of a gene containing a DNA sequence that enables the binding of RNA polymerase and the initiation of transcription. Promoter sequences are typically found in the 5' uncoding region of a gene, but this is not always the case.

[0179] The term "secretion signal sequence" or "signal sequence" refers to the DNA sequence encoding a polypeptide ("secretion peptide") that, as a component of a larger polypeptide, guides the larger polypeptide through the secretory pathway of the host cell in which it is produced. The secretion signal sequence can be natural or can be replaced by a secretion signal sequence or vector sequence from another source. Depending on the host cell, the larger peptide may be cleaved to remove the secretion peptide during transport via the secretory pathway.

[0180] The term "core region" or "catalytic domain" refers to a domain of an enzyme that may or may not have been modified or altered, but which retains at least a portion of its original activity. The core region of the mannanase according to the invention corresponds to amino acids aligned with amino acids 27-331 of Man7 SEQ ID NO: 2.

[0181] The term "linker" or "spacer region" refers to a polypeptide containing at least two amino acids, which may be located between the domains of a multi-domain protein (e.g., an enzyme containing an enzyme core and a binding domain (such as a carbohydrate-binding module (CBM)) or any other enzyme hybrid), or between two proteins or polypeptides generated as a fusion polypeptide (e.g., a fusion protein containing two core enzymes). For example, an enzyme core-CBM fusion protein is provided by sequentially fusing a DNA sequence encoding an enzyme core, a DNA sequence encoding a linker, and a DNA sequence encoding CBM into an open reading frame and expressing that construct.

[0182] Effective amount refers to the amount sufficient to degrade mannose in the chosen application.

[0183] The following abbreviations are used for amino acids:

[0184] A. Ala alanine

[0185] C Cys cysteine

[0186] D Asp Aspartic Acid

[0187] E Glutamate

[0188] F Phe Phenylalanine

[0189] Glycine

[0190] H His histidine

[0191] I Ile Isoleucine

[0192] K Lysine

[0193] L-Leu Leucine

[0194] M Met Methionine

[0195] N Asn Asparagine

[0196] P Proline

[0197] Q Gln glutamine

[0198] R Arg Arginine

[0199] S Serine

[0200] T Threonine

[0201] V Valine

[0202] W Trp tryptophan

[0203] Y Tyr (tyrosine)

[0204] The following nomenclature is used to describe substitutions: amino acid residue in the protein scaffold; position; substituted amino acid residue. According to this nomenclature, for example, a serine residue at position 20 substituted with a glycine residue is denoted as Ser20Gly or S20G.

[0205] The terms "detergent composition" and "detergent," unless otherwise specified, include general-purpose or heavy-duty detergents in solid, granular, or powder form, particularly cleaning detergents; general-purpose detergents in liquid, gel, or paste form, particularly so-called heavy-duty liquid (HDL) types; liquid fabric detergents; hand or light dishwashing agents, particularly those with high foaming properties; machine dishwashing agents, including various tablet, granular, liquid, and rinse-aid types for household and institutional use; liquid cleaning and disinfecting agents, car or carpet cleaners, bathroom cleaners; metal cleaners; and cleaning aids such as bleaching additives and "stain-sticks" or pretreatment types. The terms "detergent," "detergent composition," and "detergent formulation" are used with reference to mixtures intended for use as a washing medium to clean contaminated objects. In some embodiments, the term is used with reference to cleaning fabrics and / or clothing (e.g., "laundry detergent"). In alternative embodiments, the term refers to other detergents, such as detergents for cleaning dishes, utensils, etc. (e.g., "dishwashing detergent"). This invention is not intended to be limited to any particular detergent formulation or composition. In addition to the mannanase according to the invention, the term is intended to cover detergents that may contain, for example, surfactants, builders, chelators or chelating agents, bleaching systems or bleaching components, polymers, fabric conditioners, foam promoters, soap bubble inhibitors, dyes, fragrances, tannish inhibitors, optical brighteners, bactericides, fungicides, soil suspending agents, preservatives, water-soluble agents, fabric colorants, dispersants, dye transfer inhibitors, fluorescent brighteners, soil-release polymers, anti-redeposition agents, anti-shrinkage agents, anti-wrinkle agents, bactericides, adhesives, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam conditioners, fragrances, pigments, SOD inhibitors, solvents, and for liquid detergents, structuring agents, structural elastic agents, enzyme inhibitors or stabilizers, enzyme activators, transferases, hydrolases, oxidoreductases, bluing agents and fluorescent dyes, antioxidants, and solubilizers.

[0206] The term "textiles" means any textile material, including yarns, yarn intermediates, fibers, nonwoven materials, natural materials, synthetic materials, and any other textile material, fabrics made from these materials, and products made from these fabrics (e.g., clothing, linens, and other items). Textiles or fabrics can take the form of knitted fabrics, woven fabrics, twill, nonwoven fabrics, felt, yarns, and terry cloth. Textiles can be cellulose-based, such as natural cellulose materials including cotton, flax / linen, jute, ramie, sisal, or coconut fiber, or man-made cellulose materials (e.g., derived from wood pulp), including viscose / rayon, ramie, cellulose acetate fiber, lyocell fiber, or blends thereof. Textiles or fabrics can also be based on non-cellulose fibers, such as natural polyamides, including wool, camel hair, cashmere, mohair, rabbit hair, and silk, or synthetic polymers, such as nylon, aramid, polyester, acrylic, polypropylene, and spandex / elastane, or blends thereof, as well as blends of cellulose-based and non-cellulose-based fibers. Examples of blends are cotton and / or rayon / viscose blends with one or more accompanying materials such as wool, synthetic fibers (e.g., polyamide fibers, acrylic fibers, polyester fibers, polyvinyl alcohol fibers, polyvinyl chloride fibers, polyurethane fibers, polyurea fibers, aramid fibers), and cellulose-containing fibers (e.g., rayon / viscose, ramie, flax / linen, jute, cellulose acetate fibers, lyocell fibers). Fabrics can be conventional washable garments, such as dyed household clothes. When the term fabric or garment is used, it is also intended to include the broader term textiles.

[0207] The term "stability" includes storage stability and stability during use, such as during the washing process (as defined by washing stability), and reflects the stability of the mannanase according to the invention as a function of time, for example, how much activity is retained when the mannanase is held in solution, particularly in detergent solution. Stability is affected by many factors, such as pH, temperature, detergent composition, such as proteases, stabilizers, builders, surfactants, etc. Mannanase stability can be measured using an "activity assay" as described in the examples.

[0208] As used herein, "mannanase activity" refers to the mannan-degrading activity of the polypeptide. Degradation or modification as used herein means the hydrolysis of mannan units from the mannan polysaccharide by mannanase. The mannan-degrading activity of the polypeptide according to the invention can be tested according to standard test procedures known in the art. Example 5 provides an example of a standard method for determining mannanase activity.

[0209] In one embodiment, a variant of the invention includes at least one substitution at positions 123, 158, 180, 272, 285, or 307 or combinations thereof.

[0210] In one embodiment, a variant of the invention includes at least one substitution at positions M123, A158, F180, G272, T285, or T307, or combinations thereof.

[0211] In one embodiment, a variant of the invention includes at least one additional substitution at positions M123, A158, F180, G272, T307, or L316, or combinations thereof. In another embodiment, the variant includes one, two, three, or four additional substitutions.

[0212] In one implementation, position 316 is not substituted. This facilitates the preservation of folding and interaction of residues near the site surrounding position 316. Furthermore, Figure 8 The results show that in commercial detergents, similar performance can be achieved with lower amounts of enzyme when using substituted or unsubstituted variants of 316.

[0213] In one implementation, the mannanase variant comprises a set of substitutions listed in Table 1.

[0214] In one embodiment, the substitution comprises replacing the amino acid at the said position with one of the following: Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.

[0215] In one embodiment, the variant contains at least one substitution or a set of substitutions at the following locations:

[0216] M123, A158, F180, G272; a single substitute for T307 or L316; or

[0217] M123 and G272; or

[0218] A158 and T307; or

[0219] L316; or

[0220] T307; or

[0221] M123, A158, and T307; or

[0222] M123 and L316;

[0223] A158, T307 and L316;

[0224] F180 and L316; or

[0225] M123, A158, G272; T307 and L316;

[0226] Or a combination thereof.

[0227] In one implementation, the substitution is a substitution that results in improved stability of the variant.

[0228] In one implementation, the variant includes at least one additional substitution selected from the following:

[0229] Substitutions to prevent the glycosylation of N283;

[0230] Replacement of 283 or 285; or

[0231] Replacement of N283, T285 or S285; or

[0232] T285 or S285 is replaced by residues other than T or S.

[0233] T285 or S285 is replaced by residue A.

[0234] In one embodiment, when generated in a host cell capable of N-linked glycosylation, the additional substitution results in a glycosylation alteration of the variant. In another embodiment, the glycosylation alteration is a reduction in the degree of glycosylation of the variant.

[0235] In one embodiment, the variant has mannanase activity and contains at least one additional amino acid substitution at positions corresponding to positions 123, 158, 180, 272, 307, or 316, wherein the variant having mannanase activity is selected from: 1) a variant having at least 85% sequence identity with residues 27-331 of SEQ ID NO: 2; 2) a variant encoded by a polynucleotide that hybridizes under highly stringent conditions to: a) nucleotides 79-993 of SEQ ID NO: 1 (man7), b) the full-length complement of a); and 3) a variant encoded by a polynucleotide that has at least 95% sequence identity with SEQ ID NO: 1 or its genomic DNA sequence; and wherein the amino acid number corresponds to the amino acid number of SEQ ID NO: 2 (man7).

[0236] In one embodiment, the variant contains a P residue at positions 284 and / or 286. Substitution with a P residue at any one or both of the above positions can cause a structural change in the variant that prevents N-linked glycosylation.

[0237] In one embodiment of a variant of the invention, position T285 or S285 is replaced with residues other than T or S.

[0238] In one embodiment of a variant of the invention, position T285 or S285 is replaced with alanine.

[0239] In one embodiment, when generated in a host cell capable of N-linked glycosylation, the variant has 85% sequence identity with residues 27-331 of SEQ ID NO: 2 and a non-glycosylated Asn residue at position 283.

[0240] In one embodiment, a variant of the invention comprises at least one additional glycosylated N site, wherein the position corresponding to N283 is not glycosylated. Such a variant can be obtained by generating a variant in a host cell capable of N-glycosylation, resulting in at least partial glycosylation of other N-glycosylation sites, but wherein N-glycosylation of N283 is inhibited.

[0241] In a further embodiment, variants of the invention do not contain any N-glycosylation sites. Such variants can be obtained by generating variants in host cells capable of N-glycosylation, resulting in the inhibition of glycosylation in the variants.

[0242] In one embodiment, excluding the signal sequence, the variant has a predicted molecular weight between 50,000 and 51,000, preferably between 50,800 and 50,970.

[0243] In one implementation, the predicted pI of the variant is between 4.5 and 4.8, preferably between 4.6 and 4.75.

[0244] The prediction of the pI or molecular weight of the variant can be carried out as described in Table 3.

[0245] In a further embodiment of the invention, the variant has mannanase activity. In one embodiment, when produced in a host cell capable of N-linked glycosylation, the variant has a non-glycosylated Asn residue at position 283, and when produced in a eukaryotic host cell, the variant has increased specific activity compared to wild-type mannanase.

[0246] In one embodiment of the enzyme composition of the present invention, the enzyme composition further comprises one or more additional enzymes selected from proteases, lipases, keratinases, amylases, glycosylases, cellulases, pectinases, pectic acid lyases, pectinases, esterases, phytases, mannanases, arabinogalactanases, galactanases, xylanases, oxidases, xanthan gumases, xylose glucanases, DNases, laccases, and / or peroxidases, preferably selected from proteases, amylases, cellulases, and lipases.

[0247] The enzyme compositions of the present invention, containing mannanase and additional enzymes, are advantageous in providing synergistic effects. Such additional enzymes are desirable when the enzyme compositions of the present invention, containing mannanase, are used in detergents, for example, when washing away stains. Particularly advantageous synergistic enzymes that work with mannanase in detergents are amylases, proteases, and cellulases, or combinations thereof, such as compositions containing mannanase, amylase, and proteases.

[0248] In one embodiment, the enzyme composition of the present invention is provided in the form of a liquid composition or a solid composition, such as a solution, dispersion, paste, powder, microparticle, granule, coated granule, tablet, cake, crystal, syrup, gel or pellet.

[0249] In one embodiment, the detergent composition of the present invention is in the form of a liquid detergent or a solid detergent, preferably in the form of strips, homogeneous tablets, tablets having two or more layers, pouches having one or more compartments, ordinary or dense powders, microparticles, granules, pastes, gels, or ordinary, dense, or concentrated liquids.

[0250] In one embodiment, the detergent composition of the present invention further comprises one or more additional enzymes selected from proteases, lipases, keratinases, amylases, glycoses, cellulases, pectinases, pectic acid lyases, pectinases, esterases, mannanases, arabinogalactanases, galactanases, xylanases, oxidases, xanthan gumases, xylose glucanases, laccases, DNases, and / or peroxidases, preferably selected from proteases, amylases, cellulases, and lipases.

[0251] The enzyme compositions of the present invention can also be used in detergents or enhancers added to the detergent during or before washing, and are, for example, in the form of liquid, gel, powder, granules, or tablets. The enzyme compositions and detergent components can also be impregnated in a carrier (such as textiles).

[0252] The present invention also relates to the use of enzyme compositions or detergent compositions as disclosed herein for the degradation of mannans and methods of using enzyme compositions or detergent compositions as disclosed herein for the degradation of mannans.

[0253] In a further embodiment, the present invention relates to the use of enzyme compositions or detergent compositions as disclosed herein in the laundry process and methods of using enzyme compositions or detergent compositions as disclosed herein in the laundry process.

[0254] The present invention also relates to a method for removing stains from a surface, comprising contacting the surface with an enzyme composition or detergent composition as disclosed herein.

[0255] The present invention also relates to a method for degrading mannan, comprising applying an enzyme composition or detergent composition as disclosed herein to the mannan, preferably wherein the mannan is on the surface of a textile or at least partially embedded in the textile.

[0256] Generally, the selected enzyme should be compatible with the selected detergent (i.e., pH-optimal, compatibility with other enzymatic and non-enzymatic components, etc.), and the enzyme should be present in an effective amount.

[0257] The composition for use in solid laundry detergents may, for example, include 0.000001%-5%, such as 0.000005%-2%, such as 0.00001%-1%, such as 0.00001%-0.1% of enzyme protein by weight of the composition.

[0258] The composition for use in laundry liquid may, for example, include 0.000001%-3%, such as 0.000005%-1%, such as 0.00001%-0.1% of enzyme protein by weight of the composition.

[0259] The composition for use in automatic dishwashers may, for example, include 0.000001%-5%, such as 0.000005%-2%, such as 0.00001%-1%, such as 0.00001%-0.1% of enzyme protein by weight of the composition.

[0260] The additional component ad in the second aspect of the invention provides improved properties to the enzyme composition of the invention. The enzyme composition is compatible with the additional component and enhances its suitability for various applications.

[0261] Salts, such as sodium chloride and sodium sulfate, function as drying aids.

[0262] The present invention also relates to various uses of the enzyme compositions of the invention, such as for the degradation of mannan and for use in the laundry process.

[0263] In one embodiment, the variant of the mannanase includes a core region.

[0264] In one embodiment, the variant of the mannanase comprises CBM.

[0265] Providing mannanases that remain active at temperatures above ambient is advantageous for applications where mannan degradation is required under such conditions. Furthermore, the mannanases according to the invention exhibit good stability and activity under alkaline conditions, which is advantageous in detergent applications and biomass treatment.

[0266] In one embodiment, the variant of the mannanase has an amino acid sequence that is at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identical to SEQ ID NO:2.

[0267] In one embodiment, the variant of the mannanase has an amino acid sequence that is at least 90% sequence identical to SEQ ID NO:2.

[0268] In one embodiment, the mannanase has an amino acid sequence that is not 100% identical to SEQ ID NO:2 [Man7].

[0269] In one embodiment of the third aspect, the host cell is selected from:

[0270] Fungal cells,

[0271] Filamentous fungal cells, which originate from the phylum Ascomycota (Division) Ascomycota ), Subdivision Pezizomycotina Preferred sources are from the class Ficotyle (Class 1). Sordariomycetes ), Subclass Sarcotyle Hypocreomycetidae ), Sarcoptales ( Hypocrisy ) and Microcystales ( Microascales ) and Aspergillus genus ( Aspergillus ), genus *Aureobasidium* Chrysosporium ), genus *Dermocytotrichum* Mycelium ) and humic molds ( Humicola A group consisting of members;

[0272] More preferably from the family Hypocreaceae ( Hypocreaceae ), family Ceratophyllum ( Nectaraceae Clavicipitaceae ( Clavicipitaceae ), Microcystis ( Microascaceae ) and Trichoderma ( Trichoderma (Asexual Sarcoptes genus) Hypocritical Fusarium ( )), Fusarium genus ( Fusarium ), Gibberella genus ( Gibberella ), genus *Cirsium* ( Nectarine ), *Botrytis* genus ( Stachybotrys ), ergot ( Harpsichord Metarhizium anisopliae Metarhizium ), Villosiclava Cordyceps genus ( Ophiocordyceps ), Cephalosporium ( Cephalosporium ) and Cetacea ( Scedosporium A group consisting of )

[0273] More preferably from Trichoderma reesei ( Trichoderma reesei ) (Red-brown fleshy fungus ( Hypocritical liver )), Lemon Trichoderma ( T. citrinoviridae ), Trichoderma longifolia ( T. longibrachiatum ), Trichoderma viride T. virens Trichoderma harzianum ( T. harzianum ), Trichoderma acicularis ( T. asperellum ), dark green Trichoderma ( T. atroviridae Trichoderma ginrylifolia ( T. parareesei Fusarium oxysporum ( Fusarium oxysporum ), Fusarium graminearum ( F. gramineanum ), Fusarium pseudograss ( F. pseudograminearum ), Fusarium moniliforme ( F. poisoned Fujikura gibberellin Gibberella fujikuroi ), gibberellinii ( G. moniliformis ), Maize Fusarium ( G. zeaea ), flagellated red scabies (blood red scabies) ( Nectria (Haematonectria) haematococci ), paper grape spike ( Stachybotrys paper ), S. chlorohalonata, Rye ergot ( Harpsichord purple ), Metarhizium anisopliae ( Metarhizium acridum ), Metarhizium anisopliae ( M. anisopliae ), Rhizoctonia solani ( Green sedge Cordyceps sinensis Ophiocordyceps sinensis ), Cephalosporium (Cephalosporium) ( Acremonium (Cephalosporium) chrysogenum ) and Cetacea tricuspidata ( Scedosporium apisperm ) and Aspergillus niger ( Aspergillus niger ), Aspergillus awamori ( Aspergillus awamori Aspergillus oryzae ( Aspergillus oryzae ), Rudd's chlortetracycline ( Chrysosporium lucknowense ), thermophilic pyridamus ( Myceliophthora thermophila ), specific humic mold ( Humicola insolens ) and gray humic mold ( Humicola grisea A group composed of )

[0274] Bacterial cells, preferably Gram-positive bacilli such as Bacillus subtilis ( B. Subtilis ), Bacillus licheniformis ( B. Licheniformis ), Bacillus megaterium ( B. megaterium ), Bacillus amyloliquefaciens ( B. amyloliquefaciens ), Bacillus pumilus ( B. pumilus Gram-negative bacteria such as Escherichia coli ( Escherichia coli Actinomycetes, such as Streptomyces ( Streptomyces sp.), and

[0275] Yeast, such as brewer's yeast ( Saccharomyces cerevisiae Pichia pastoris () Pichia pastoris ), Yarrowia lipolytica ( Yarrowia lipolytica ),

[0276] The best choice is either Trichoderma reesei or Bacillus.

[0277] In one embodiment, the host cell is a eukaryotic host cell capable of N-linked glycosylation. In a preferred embodiment, the host cell is *Trichoderma reesei*.

[0278] Recombinant host cells can be used to produce variants of mannanase and carry the polynucleotide encoding it. Recombinant host cells are also useful in the preparation of mannanase variants with different properties. For example, host cells can be selected that provide post-translational modifications favorable to stability or activity, or that facilitate the post-processing and formulation of mannanase variants produced in host cells.

[0279] In one embodiment, the enzyme composition of the present invention comprises a recombinant host cell from the second aspect.

[0280] In one embodiment, a variant of the mannanase of the present invention is a recombinant polypeptide, which is a fusion protein.

[0281] In one embodiment, the recombinant polypeptide of the present invention is a fusion protein, which further comprises at least one of the following:

[0282] The amino acid sequence that provides the secretion signal sequence;

[0283] Amino acid sequences that are easy to purify, such as affinity tags and His-tags;

[0284] Enhanced amino acid sequences, such as amino acid sequences used as carriers, such as CBM;

[0285] An amino acid sequence with enzymatic activity; and

[0286] Provide the amino acid sequence of the fusion protein with binding affinity, such as the carbohydrate-binding moiety.

[0287] CBM, the carbohydrate-bound portion, is advantageous as a carrier, for example, in the production of Trichoderma.

[0288] In one embodiment, the host cell is non-pathogenic. This is particularly advantageous for use in animal feed and in detergent applications, such as in household laundry detergents.

[0289] In one embodiment of the fifth aspect, the mannan-containing material is selected from plant-based materials, textiles, wastewater, sewage, oil, or combinations thereof.

[0290] In one embodiment, the material containing mannan is a textile material or fabric.

[0291] In another embodiment, the material containing mannan is recycled waste paper; mechanical pulp, chemical pulp, semi-chemical pulp, kraft paper or other papermaking pulp; fibers undergoing a dehydration process; or material containing guar gum or locust bean gum.

[0292] In another embodiment, the degradation or modification is carried out in an aqueous environment in which the mannanase exhibits activity.

[0293] In a preferred embodiment, the mannan-containing material, which is degraded or modified in the method, is applied to textiles or fabrics optionally with mannan stains. By degrading the mannan attached to the textiles or fabrics, dirt or grime bound to the mannan is released and cannot be re-bound to the mannan or mannan stains. The textiles or fabrics can be of any material, such as cotton, flax / linen, jute, ramie, sisal, or coconut fiber, or synthetic cellulose products (e.g., derived from wood pulp), including viscose / rayon, modal, cellulose acetate, lyocell, cupro, or blends thereof.

[0294] In one embodiment, the feed of the present invention comprises or consists of corn and soybean meal.

[0295] In one embodiment, the plant-derived protein source comprises soybean, cereals such as barley, wheat, rye, oats, or corn, or consists of soybean, cereals such as barley, wheat, rye, oats, or corn.

[0296] In one embodiment, the product or byproduct containing mannan comprises palm kernel, guar bean powder, or coconut kernel powder, or is composed of palm kernel, guar bean powder, or coconut kernel powder.

[0297] In one embodiment, the animal feed or feed supplement of the present invention is formulated in the form of a wet composition or a dry composition.

[0298] In one embodiment, a composition comprising at least one variant of mannanase is used in the pulp and paper industry, biobleaching, fiber modification, drainage improvement, and the petroleum industry, specifically in the oil drilling or oil supply industry, for hydraulic fracturing or controlling the viscosity of drilling fluids.

[0299] In one embodiment, a composition comprising at least one variant of mannanase is used in the textile and detergent industry, biomass processing and biomass hydrolysis, and preferably in the biofuel, starch, pulp and paper, food, baking, feed or beverage industry.

[0300] In one embodiment, a variant of mannanase randomly hydrolyzes the intra-β-1,4-mannoside bond.

[0301] In one implementation, the variant of mannanase, or the nucleotide sequence encoding the corresponding wild-type mannanase, may be obtained or derived from a bacterial source.

[0302] In one embodiment, a variant of mannanase is fused with at least one additional polypeptide to form a fusion polypeptide. In addition to those mannanases, the fusion polypeptide or the additional polypeptide may also have other catalytic or binding activities. In one embodiment, the additional polypeptide comprises or is composed of a carbohydrate-binding module, which optionally is a fragment of another protein or enzyme derived from the same or different organism as the mannanase.

[0303] In one implementation, a variant of mannanase is linked to another polypeptide via a linker.

[0304] In one embodiment, a method for mechanically treating a fabric is provided, the method comprising treating the fabric with a washing solution containing a variant of mannanase of the first aspect or an enzyme composition of the second aspect during a washing cycle of a machine washing process.

[0305] In one embodiment, the use of the enzyme composition of the second aspect or a variant of the mannanase of the first aspect, together with an enzyme, in a cleaning composition for fabric cleaning and / or fabric stain removal, said enzyme being selected from proteases, amylases, cellulases, lipases, xylanases, mannanases, keratinases, esterases, phytases, DNases, pectinases, pectinases, pectic acid lyases, glycosylases, arabinogalactanases, galactanases, xanthan gumases, xylose glucanases, laccases, peroxidases, and oxidases, with or without a mediator.

[0306] In one embodiment, a variant of the mannanase of the first aspect or an enzyme composition of the second aspect is provided for use with an enzyme in a cleaning composition for cleaning hard surfaces, such as floors, walls, bathroom tiles, etc., said enzyme being selected from proteases, amylases, cellulases, lipases, xylanases, mannanases, keratinases, esterases, phytases, DNases, pectinsases, pectinases, pectic acid lyases, glycosylases, arabinogalactanases, galactanases, xanthan gumases, xylose glucanases, laccases, peroxidases, and oxidases, with or without a mediator.

[0307] In one embodiment, a variant of the mannanase of the first aspect or an enzyme composition of the second aspect is provided for use with an enzyme in a cleaning composition for hand and machine dishwashing, said enzyme being selected from proteases, amylases, cellulases, lipases, xylanases, mannanases, keratinases, esterases, phytases, DNases, pectinases, pectinases, pectic acid lyases, glycosylases, arabinogalactanases, galactanases, xanthan gumases, xylose glucanases, laccases, peroxidases, and oxidases, with or without a mediator. Example

[0308] The following embodiments are provided to illustrate various aspects of the invention. They are not intended to limit the invention, which is defined by the appended claims.

[0309] Example 1. Variant Design

[0310] To improve the stability and specific activity of wild-type Man7 mannanase, variant sets were designed based on the structural analysis of the wild-type enzyme. Using Bioluminate software (Schrödinger LCC), a structural model of Man7 was created using coordinates from the β-D-1,4-mannanase (1WKY) of the Bacillus sp. species. The design included two or more specific mutations for each variant. Table 1 shows a list of variants. The amino acid numbers correspond to the amino acid numbers of the full-length amino acid sequence of SEQ ID NO:2 (Man7) containing the signal sequence.

[0311] Table 1. List of Man7 variants

[0312]

[0313] Example 2. Cloning of the synthetic mannanase variant gene

[0314] Standard molecular biology methods are used in DNA isolation and enzymatic treatment (e.g., plasmid DNA isolation, DNA digestion to produce DNA fragments), E. coli transformation, sequencing, etc. The basic methods used are as described by the enzyme, reagent, or kit manufacturer.

[0315] Ordering variants from GenScript tbh1 - tbh11 As a synthetic construct, it lacks its own signal peptide coding sequence and undergoes codon optimization for *Trichoderma reesei*. The gene obtained from GenScript will be... tbh1 - tbh11 The plasmid DNA was resuspended in sterile water and used according to the manufacturer's instructions. Nru I and Bam HI restriction enzyme (Thermo Fisher Scientific) digestion and cloning to use Nru I and Bam The ligation mixture was transformed into *E. coli* XL1-Blue or XL10-Gold cells (AH Diagnostics) and plated on LB (Luria-Bertani) plates containing 50–100 µg / ml ampicillin. Several *E. coli* colonies were collected from the plates, and DNA was isolated using the GenJet Plasmid Mini-Preparation Kit (Thermo Fisher Scientific). Positive clones were screened using restriction digestion to show that they contained inserts of the expected size. tbh1- tbh11 The fusion sites of the expression plasmids containing the mannanase gene were sequenced, and the plasmids were named pALK4415-pALK4423, pALK4430, and pALK4431, respectively (see Example 4 for details). The plasmids also included information from GenScript. thb The plasmid DNA of the gene was transformed into XL10-Gold Escherichia coli cells (Agilent) and deposited at the DSMZ strain depository. Information regarding the gene and its deduced amino acid sequence (SEQ ID NO: 5-36) is summarized in Tables 2 and 3, respectively. Escherichia coli strains RF12379-RF12387, RF12456, and RF12457, which respectively contain plasmids pALK4434-pALK4442, pALK4432, and pALK4433, have been deposited at the DSMZ Depository under accession numbers DSM 32425, DSM 32426, DSM 32427, DSM 32428, DSM 32429, DSM 32430, DSM 32431, DSM 32432, DSM 32433, DSM 32518, and DSM 32519.

[0316] Table 2. About genes encoding synthetic genes tbh1 - tbh11 Overview of mannanase variants

[0317]

[0318] (a This includes the stop codon.

[0319] Table 3. Overview of the amino acid sequence deduced from the gene sequence encoding the mannanase variant.

[0320]

[0321] (a Signal sequences were predicted using the program SignalP v3.0 and NN / HMM (Nielsen et al., 1997; Nielsen & Krogh, 1998; Bendtsen et al., 2004).

[0322] (b The predicted signal sequence is not included. Prediction was performed using Clone Manager Professional version 9 for Windows, Sci-Ed Software.

[0323] Example 3. man7 Site-directed mutagenesis of genes

[0324] Unless otherwise specified, standard molecular biology methods, including DNA manipulation and transformation, were used. Mutations were introduced by site-directed mutagenesis as described in Kunkel 1985. The resulting mutations are listed in Table 4. The amino acid numbers correspond to the amino acid numbers of SEQ ID NO:2 (Man7).

[0325] Table 4. List of mutations introduced in Man7 by site-directed mutagenesis

[0326]

[0327] For amplification, Pfx Accu Prime polymerase (Invitrogen) was used. PCR was performed according to the manufacturer's instructions. The following PCR conditions were used to construct the expression plasmid: initial denaturation at 94°C for 120 seconds, followed by 35 cycles of annealing at 94°C for 15 seconds, annealing at one of the following 50 / 55°C for 30 seconds, extension at 68°C for 110 / 290 seconds, and a final extension at 68°C for 10 minutes. pEV1 man 7 is used as a template for PCR. The sequences of the primers used for cloning are shown in Table 5. The overhangs used for hybridization are underlined.

[0328] Table 5. Used for generation man7 List of primer variants

[0329]

[0330] For cloning purposes, NEBuilder® Hifi DNA AssemblyMaster Mix (NEB, Frankfurt) was used according to the kit manufacturer's instructions. The resulting expression plasmid was transformed into competent cells induced in Bacillus subtilis SCK6, as described in Zhang & Zhang 2011. Figure 1 Transformed cells were plated onto LB (Luria-Bertani) plates supplemented with 10 mg / L kanamycin. The plates were incubated at 37°C for 20 h. Growing colonies were picked and plasmids were isolated using the QiaPrep MiniPrep kit (Qiagen, Germany). The isolation procedure was performed according to the manufacturer's recommendations for Gram-positive plasmid preparation. The inserts were sequenced by Sanger sequencing (GATC, Germany), revealing DNA sequences corresponding to the mature portions of variants 1 through 5. Sequence comparisons were performed using ClustalW sequence alignment (Thompson et al. 1994). Finally, the expression plasmid was transformed into Bacillus production strains via electroporation. The Bacillus production strains were grown in electroporation medium containing 20 g / L tryptone, 10 g / L yeast extract, 10 g NaCl, and 2 M sucrose, and 10 ml were harvested at 0.4 OD (600 nm). Cells were washed with electroporation buffer containing 0.272 M sucrose, 1 mM MgCl2, and 7 mM KH2PO4, and then resuspended in 250 μl of electroporation buffer. Electroporation was performed under the following conditions: 1.2 kV, 150 Ω, 50 μF. 1 ml of electroporation medium was then added, and the cells were incubated at 37°C for 3 h. Cells were plated on LB plates supplemented with 20 mg / L kanamycin and incubated at 37°C for 18 h. Cloning was validated as described above and used to generate materials for analytical testing. Therefore, the strain was inoculated with standard expression under protein-inducible conditions and incubated at 37°C for 30 h. The supernatant was harvested and used for analysis and application testing.

[0331] Example 4. Production of recombinant mannanase variant protein in Trichoderma reesei

[0332] Expression plasmids for producing recombinant mannanases TBH1-TBH11 (SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26) in *Trichoderma reesei* were constructed. The constructed expression plasmids are listed in Table 6. *Trichoderma reesei* was used... cel6 A / cbh 2. The CBM vector and adapter (followed by the Kex2 protease recognition site) were used to fuse the recombinant mannanase gene (without its own signal sequence) into Trichoderma reesei. cel7A / cbh1 Promoter. By Trichoderma reesei cel7A / cbh1 The terminator ensures transcription termination, and Aspergillus nidulans ( A. Nidulans ) amdS Marker genes are used for screening transformants, as described by Paloheimo et al. (2003). Linear expression cassettes ( Figure 2 )exist Not After digestion, the protoplasts separated from the vector backbone and transformed into *Trichoderma reesei* protoplasts. The host strain used did not produce any of the four major *Trichoderma reesei* cellulases (CBHI, CBHII, EGI, EGII). The transformation was performed as described by Penttilä et al. (1987) (with modifications as described by Karhunen et al. (1993)), selecting acetamase as the sole nitrogen source. amdS (Marker gene). Transformants are purified by single conidia on a selection plate and then spored on PD.

[0333] Table 6. Expression cassettes constructed to produce recombinant TBH1-TBH11 (SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26) proteins in *Trichoderma reesei*. The overall structure of the expression cassettes is shown in Table 6. Figure 2 As described in [the text].

[0334]

[0335] (a Expression cassettes for Trichoderma reesei transformation are used by... Not I digestion separates the vector backbone.

[0336] Mannanase production from transformants was analyzed from the culture supernatant of shake-flask cultures. Transformants were inoculated from PD slant culture into shake flasks containing 50 ml of compound lactosyl cellulase induction medium buffered with 5% KH2PO4 (Joutsjoki et al. 1993). After growth at 30°C and 250 rpm for 7 days, mannanase protein production was analyzed from the culture supernatant. Heterogeneous production of recombinant protein was analyzed by SDS-PAGE and subsequent Coomassie staining. The supernatant was also recovered by centrifugation for application testing.

[0337] Select the optimal production transformant for cultivation in a laboratory-scale bioreactor. Cultivate the transformant in the bioreactor in batches or via an additional feed type process under typical mesophilic fungal culture temperatures and weakly acidic conditions, under protein-inducible conditions. Continue cultivation until the sugar content of the culture medium is depleted or until a suitable yield is achieved. Recover the supernatant by centrifugation or filtration for application testing.

[0338] Example 5. Determination of galactomannanase activity by DNS method

[0339] Mannanase activity (MNU) was measured as the release of reducing sugars from galactomannan (0.3 w / w-%) over 5 min at 50 °C and pH 7.0. The amount of reduced carbohydrates released was determined spectrophotometrically using dinitrosalicylic acid.

[0340] The substrate (0.3 w / w-%) used in the assay is prepared as follows: At approximately 80°C, using a heated magnetic stirrer, 0.6 g of locust bean gum (Sigma G-0753) is placed in 50 mM sodium citrate buffer pH 7 (or citrate phosphate buffer pH 7) and heated to boiling point. The solution is cooled and dissolved overnight in a cold room (2–8°C) with continuous stirring, and insoluble residues are removed by centrifugation. The solution is then filled to 200 ml with buffer. The substrate is stored frozen and thawed in a boiling water bath to approximately 80°C before use, cooled to room temperature, and carefully mixed.

[0341] The DNS reagent used in this assay was prepared by dissolving 50 g of 3,5-dinitrosalicylic acid (Sigma D-550) in approximately 4 liters of water. 80.0 g of NaOH was gradually added and dissolved under continuous magnetic stirring. Then, 1500 g of Rochelle salt (K-Na-tartrate, Merck 8087) was added in small portions under continuous stirring. The solution, carefully heated to a maximum temperature of 45°C, was cooled to room temperature and filled to a final volume of 5000 ml. This solution was then filtered through Whatman 1 filter paper and stored in a black bottle at room temperature.

[0342] The reaction is initiated by adding 1.8 ml of substrate solution to each of two tubes and equilibrating at 50°C for 5 minutes. Then, 200 μl of appropriately diluted enzyme solution is added to one of the tubes, thoroughly mixed using a vortex mixer, and incubated precisely at 50°C for 5 minutes. The enzyme blank does not require equilibration or incubation. The reaction is stopped and mixed by adding 3.0 ml of DNS reagent to both tubes. 200 μl of sample solution is added to the enzyme blank tube. Both tubes are placed in a boiling water bath. After boiling for precisely 5 minutes, the tubes are placed in a cooling water bath and allowed to cool to room temperature. The absorbance of the sample against the enzyme blank is measured at 540 nm, and the activity is read from the calibration curve and multiplied by the dilution factor. Appropriate dilutions produce absorbance differences of 0.15–0.4.

[0343] A 20 mM mannose stock solution was prepared by dissolving 360 mg of mannose (SigmaM-6020, stored in a desiccator) in assay buffer, and then diluted to solutions containing 3, 6, 10, and 14 μmol / ml mannose to prepare standard curves. The standards were treated similarly to the samples, except for incubation at 50°C. The absorbance was measured at 540 nm against the reagent blank (a standard dilution containing buffer instead of mannose). Calibration curves were constructed for each series of assays.

[0344] One mannanase unit (MNU) is defined as the amount of enzyme that produces reducing carbohydrates (with a reducing power corresponding to 1 nmol of mannose) from galactomannan in one second under the test conditions (1 MNU = 1 nkat).

[0345] Example 6. Stain removal performance of mannanase variants produced in Trichoderma and commercial detergents.

[0346] The ability of cultured samples of the TBH1-TBH11 variants produced in *Trichoderma* (as described in Example 4) to remove mannanase-sensitive standard stains was tested using commercial detergents at 40°C and a water hardness of 16°dH, and compared with wild-type enzymes. The following artificially soiled test fabrics from Center for Testmaterial BV (Netherlands) were used: mannanase-sensitive chocolate pudding on cotton (E-165), locust bean gum and pigment on cotton (CS-73), and guar gum and carbon black on cotton (CS-43). Fabrics were cut into 6 cm x 6 cm samples, with two of each used in the test.

[0347] A commercial heavy-duty liquid detergent A containing all enzymes except mannanase was used at a concentration of 4.4 g / L of wash solution. Enzyme-free commercial color detergent powder was used at 3.8 g / L, and enzyme-free commercial bleaching detergent powder was used at 4.2 g / L. The wash solution containing the detergents was prepared in synthetic tap water with a hardness of 16°dH. Savinase® 16 L (0.5 w / w %) and Stainzyme® 12 L (0.4 w / w %) were added to the hard water along with the commercial color detergent powder and bleaching detergent powder. The liquid detergents already contained amylase and protease. The pH of the wash solution was approximately 8.3, with the pH of the color detergent powder approximately 10 and the pH of the bleaching detergent approximately 9.5.

[0348] Mannanase was administered at a concentration of 0.025 and / or 0.05 MNU per ml of wash solution. Activity was measured as described in Example 5. The control sample contained a wash solution without mannanase.

[0349] For synthetic tap water with a hardness of 16°dH, the following stock solutions are prepared in deionized water (Milli-Q or equivalent):

[0350] A stock solution with a calcium hardness of 1000°d: CaCl2 x 2 H2O (1.02382.1000, Merck KGaA, Germany) 26.22 g / l

[0351] A stock solution with a magnesium hardness of 200°d: MgSO4 x 7 H2O (1.05886.1000, Merck KGaA, Germany) 8.79 g / l H2O

[0352] NaHCO3 stock solution: NaHCO3 (1.06329.1000 Merck KGaA, Germany) 29.6 g / L

[0353] 13.3 ml of CaCl2 solution, 13.3 ml of MgSO4 solution, and 10.0 ml of freshly prepared NaHCO3 solution were added to a volumetric flask in the given order, and the volume was brought to 1 liter with deionized water and mixed. The water hardness was determined by complexometric titration and found to be correct.

[0354] The stain removal process was performed using the Atlas LP-2 Launder-Ometer as follows. The Launder-Ometer was first preheated to 40°C. Then, detergent, 250 ml of synthetic tap water with a hardness of 16°dH, and diluted enzyme (<1.0 ml) were added to a 1.2 L container. The stain was added, and the Launder-Ometer was run at 40°C and 42 rpm for 60 min. Afterward, the fabric sample was carefully rinsed under running water and dried overnight in room air on a sun-proof grid.

[0355] The stain removal effect was evaluated using a Konica Minolta CM-3610A spectrophotometer, with color as a reflectance value measured using L*a*b* color space coordinates (light source D65 / 10°, 420 nm cutoff). The fading of stains, indicating mannanase performance (stain removal efficiency), was calculated as ΔL* (delta L*), which is the photometric value L* of the enzyme-treated fabric minus the photometric value L* of the fabric treated with a mannanase-free wash (control). The final result (total stain removal effect) is shown as the sum of ΔL* for each of the three stains. The color value of each stain is the average of two fabric samples.

[0356] The results obtained using commercial liquid detergents are shown in Figure 3, the results using commercial color detergent powders are shown in Figure 4, and the results using commercial bleach detergents are shown in Figure 5. Figure 5 In medium concentrations, all variants (TBH1-TBH11) showed excellent stain removal performance with different types of commercial detergents. The performance of the variants was similar to that of the wild type.

[0357] Example 7. Stain removal performance of mannanase variants produced in Bacillus spp. and commercial detergents.

[0358] The ability of culture supernatants of Bacillus variants BH18, BH21, BH23, BH24, and BH2 (as described in Example 3) produced in the genus Bacillus to remove mannanase-sensitive standard stains was tested using commercial detergents at 40°C and a water hardness of 16°dH, and compared with wild-type enzymes. A test system similar to that described in Example 6 was used.

[0359] Results obtained using commercial liquid detergents are shown in Figure 6 In the results using commercially available colored detergent powder, the results are shown in Figure 7 In the middle, and the results of using commercial bleach detergents were shown in Figure 8 In the study, all variants (BH18, BH21, BH23, BH24, BH25) demonstrated excellent stain removal performance using different types of commercial detergents. The performance of the variants was similar to that of the wild type.

[0360] Example 8. Stability of mannanase variants in commercial liquid detergents at 37°C

[0361] The stability of culture supernatants of several mannanase variants produced by *Trichoderma* (as described in Example 4) or *Bacillus* (as described in Example 3) was tested in a commercial liquid heavy-duty detergent A containing proteases and all other enzymes except mannanase, and compared with wild-type enzymes. The mannanase preparation was added to the detergent at 4 w / w-%, and the samples were incubated in capped plastic tubes at 37°C for approximately 16 or 24 weeks. Activity was measured at specific intervals using the activity assay described in Example 5, except for a 30-min incubation time. The results were calculated as residual activity (%), obtained by dividing the activity of the sample collected at specific time points by the initial activity of the sample.

[0362] When stored at high temperatures, such as 37°C, for 24 weeks, variants TBH2, TBH3, TBH4, TBH5, and especially TBH6 showed improved stability compared to the wild-type enzyme Man7 produced in *Trichoderma*. The results of stability tests on TBH6 compared to the wild-type were shown in... Figure 9 middle.

[0363] When stored at high temperatures, such as 37°C, for several weeks, variants BH21, BH23, BH24, and especially BH25 showed improved stability compared to the wild-type enzyme Man7 produced in Bacillus spp. The results of stability tests on BH25 compared to the wild-type were shown in… Figure 10 middle.

[0364] Example 9. Stability of mannanase variants in commercial liquid detergents at 50°C

[0365] The stability of culture supernatants of the TBH1-TBH11 variant produced in *Trichoderma* (as described in Example 4) was tested under extreme conditions in a commercial liquid heavy-duty detergent A containing protease but not mannanase, and compared with that of the wild-type enzyme. The mannanase preparation was added to the detergent at 4 w / w-%, and the samples were incubated in capped plastic tubes at 50°C for 7 days. Activity was measured using the activity assay described in Example X, except for a 30-min incubation time. The results were calculated as residual activity (%), obtained by dividing the activity of the sample collected at a specific time point by the initial activity of the sample.

[0366] Based on the results shown in Figure 11, all variant enzymes exhibited better stability at 50°C for 7 days compared to the wild type. The stability of TBH1, TBH2, TBH4, TBH5, TBH6, TBH10, and TBH11 under extreme conditions was particularly significantly improved compared to the wild-type enzymes.

[0367] Some of the optimal production variants were cultured in a laboratory-scale bioreactor, and stability was retested using a similar testing system as described above (except that the amount of mannanase preparation in detergent was 1 w / w-%). When using laboratory-scale cultured material, the mannanase variants TBH5 and TBH6 also showed significantly better stability than the wild type (data not shown).

[0368] The results showed that the mannanase variants exhibited excellent stain removal performance and significantly improved stability compared to the wild-type enzyme, especially at high temperatures. They could also be produced more economically due to their higher specific activity and yield (data not shown).

[0369] Example 10. Combination of variants TBH5 and TBH6

[0370] To further improve the mannanase properties, mutant combinations (M123I, A158S, S229A, G272Q, T285A, T307R, and L316K) from the TBH5 and TBH6 variants were used. As described in Examples 2 and 4, the combined variant TBH14 was prepared as a synthetic construct, cloned into an expression vector, and generated in *Trichoderma reesei*. The stability of the combined variant TBH14 was tested at 50°C for 7 days, as described in Example 9. Variants TBH6 and TBH5 were used for comparison. Results are shown in... Figure 13 The stability of the combined variant TBH14 is at least as good as or better than that of variants TBH6 or TBH5, which have shown improved stability compared to the wild type in Example 9; that is, when stored at high temperatures such as 50°C for several days, the stability of TBH14 is also improved compared to the wild-type enzyme produced in Trichoderma. The combined variant TBH14 also shows excellent stain removal performance (similar to the wild type) with commercial detergents.

[0371] Example 10b. Efficiency study of mannanase alone and in combination with non-starch polysaccharide (NSP) degrading enzymes in broilers.

[0372] The effects of the recombinant mannanase variant of the present invention on broiler growth were investigated. The ultrafiltrate of a fermentation culture containing the recombinant mannanase was dried, and the target levels were applied to pelleted broiler diets, either alone or in combination with commercially available xylanase-based products.

[0373] Control diets were prepared by feeding patients with corn and hulled, solvent-extracted soybean meal that contained no enzymes, or by adding different levels of the recombinant mannanase of the present invention, either alone or in combination with standard doses of commercial xylanase.

[0374] The initial weight of the broilers was between 30 g and 50 g. The experiment lasted between 3 and 5 weeks. Each treatment consisted of at least 6 replicates (10 broilers per replicate). In each case, the moisture, crude protein, crude fiber, fat, ash, and enzyme protein of the diet were analyzed.

[0375] Prepare five types of meals:

[0376] 1) No supplementary control (BD)

[0377] 2) BD + Mannanase 1-500 mg / kg

[0378] 3) BD + Mannanase 1-1000 mg / kg

[0379] 4) BD + mannanase 1-500 mg / kg + xylanase 1-10 mg / kg

[0380] 5) BD + xylanase 1-10 mg / kg.

[0381] Animal health and mortality were examined daily by visual inspection. Body weight gain (BW), feed intake (FI), and feed conversion ratio (FCR) were measured at days 0, 14, and 35. FCR was calculated as the total amount of feed consumed during the same period divided by the weight gain. The role of the recombinant mannanase variant was determined based on comparisons with animals fed the same diet or the same diet supplemented with xylanase.

[0382] Example 11. Instant Coffee Production

[0383] Mannans are the main storage polysaccharide component of coffee endosperm, leading to their high viscosity, which negatively impacts the technical processing of instant coffee and increases energy consumption during drying. These effects are attributed to the formation of hard, insoluble crystalline structures in mannans. β-Mannanase, often added along with other enzymes such as pectinase and cellulase, during the concentration step in instant coffee production, reduces the viscosity of the coffee extract. Mannanase is also used to hydrolyze galactomannans present in liquid coffee extracts to inhibit gel formation during freeze-drying of instant coffee. Furthermore, due to the use of enzymatic treatment, coffee bean extracts can be concentrated through low-cost processes such as evaporation.

[0384] according to Figure 12 The following flowchart was tested at a temperature of 10°C and an enzyme dosage of 0.15% ds.

[0385] The mannanase variant of the present invention was tested in a mixture of different enzymes (such as pectinase and cellulase).

[0386] Under standard processing conditions, the viscosity of coffee extract increases significantly over time. However, using an enzyme mixture containing the mannanase variant of the present invention, the viscosity is significantly reduced, resulting in improved downstream processing, such as spray drying or freeze drying.

[0387] Example 12. Pineapple Processing

[0388] Specifically, mannanase can be used for the extraction and clarification of pineapple juice because pineapple contains a large amount of mannan, including glucomannan and galactomannan.

[0389] Mannanase helps improve the extraction of valuable fruit components, reduce the viscosity of juice before concentration, and improve filtration speed and the stability of the final product.

[0390] The pineapple was ground in a meat grinder, and 500 g of the paste was then placed in a 1000 ml beaker. Enzymes were applied at 21°C for 60 minutes. The paste was then pressed using a small Hafico press according to the following pressing protocol: 0 bar for 2 min, 50 bar for 2 min, 100 bar for 2 min, 150 bar for 2 min, 200 bar for 1 min, 300 bar for 1 min, and 400 bar for 1 min. The resulting juice was then centrifuged at 4500 rpm for 5 minutes, and its turbidity and viscosity were analyzed.

[0391] The mannanase variants of the present invention were tested in enzyme mixtures A, B and C (Table 7).

[0392] The enzyme was first diluted with tap water and then added to the pineapple paste.

[0393] Table 7. Enzyme Mixture

[0394] enzyme activity Dosage [ppm] 5 ml [% enzyme solution] blank 5 ml H2O Mixture A pectinase 50 0.50% Mixture B Pectinase + Arabinase 50 0.50% Mixture C Pectinase + Mannanase 50 0.50%

[0395] The application of the mannanase variant of the present invention resulted in increased juice yield and reduced turbidity in pineapple treatment.

[0396] Example 13. Mannanase treatment of soybeans for soymilk production

[0397] To enzymatically treat soybeans to obtain soymilk, a "heat treatment" is typically used. For hot soymilk treatment, dried soybeans are mixed and ground with boiling tap water at a ratio of 1:7 (soaked soybeans:water) in a mixer. The entire soybean slurry is cooled to 50-55°C before adding the enzyme. The pH level of the soybean slurry should be around pH 6.5 and can be adjusted with NaHCO3. Mannanase is added to the slurry at a dosage of 1 kg / t dried soybeans and stirred for 30 min. After the reaction time is complete, the slurry is pressed using a laboratory press to obtain the final product: soymilk. To ensure consistent pressing profiles, the pressure and corresponding pressing time are specified as shown in Table 8. In addition to the samples from the enzymatically reacted samples, a control sample without any enzyme is prepared, in which water is used instead of the enzyme solution.

[0398] Table 8. Suppression Scheme

[0399] Pressure [Bar] 0 50 100 300 Time [min] 2 2 2 1

[0400] After pressing, heat the soy milk in the microwave until it boils to stop the enzymatic reaction. Analysis of the soy milk:

[0401] Yield (in grams per hour)

[0402] Brix (°Brix) measured using a refractometer provides a direct correlation with the sugar content in soy milk.

[0403] The turbidity of the juice was measured using an NTU-photometer, which measures turbidity.

[0404] Brightness was measured using the LAB measurement method.

[0405] Protein content was determined using a CN- analyzer (combustion method).

[0406] Flavor.

[0407] Soy milk treated with the mannanase variant of the present invention showed increased yield, brighter color, increased Brix content, lower turbidity, higher protein content, and better taste (off-flavor removal).

[0408] Without limiting the scope and interpretation of the patent claims, certain technical effects of one or more aspects or embodiments disclosed herein are listed below: one technical effect is the degradation or modification of mannan. Another technical effect is providing a mannanase with good storage stability.

[0409] The foregoing description, through specific embodiments and non-limiting examples of implementations of the invention, has provided a complete and detailed description of the best mode contemplated by the inventors for carrying out the invention. However, it will be apparent to those skilled in the art that the invention is not limited to the details of the embodiments presented above, but can be implemented in other embodiments using equivalent methods without departing from the characteristics of the invention.

[0410] Furthermore, some features of the aspects and embodiments disclosed above in this invention can be used to achieve benefits without the need for corresponding use of other features. Therefore, the above description should be considered merely illustrative of the principles of the invention and not as limiting thereto. Consequently, the scope of the invention is limited only by the appended patent claims.

[0411] In one embodiment, at least one component of the composition of the present invention has different chemical, structural, or physical characteristics compared to the corresponding natural component, said at least one component being derived from said natural component. In one embodiment, said characteristics are at least one of uniform size, uniform dispersion, different isotypes, different codon degeneracy, different post-translational modifications, different methylation, different tertiary or quaternary structures, different enzyme activities, different affinities, different binding activities, and different immunogenicity.

[0412] References

[0413]

[0414]

Claims

1. A variant of mannanase, comprising the amino acid sequence of SEQ ID NO: 2 and having substitutions selected from the group consisting of: M123I, S229A, G272Q, T285A; A158S, S229A, T285A, T307R; S229A, T285A, L316K; M123I, A158S, S229A, G272Q, T285A, T307R; M123I, S229A, G272Q, T285A, L316K; A158S, S229A, T285A, T307R, L316K; A158S, S229A, T307R, L316K; A158S, T285A, T307R, L316K; M123I, S229A, G272Q, T285A, L316K, A158S, T307R; A158S, T307R; M123I, A158S, G272Q, T307R; M123I, G272Q, L316K; and A158S, T307R, L316K; The mannanase variants described therein possess mannanase activity, and the selected substitutions lead to increased stability of the variants. The amino acid number corresponds to the amino acid number of the full-length amino acid sequence of SEQ ID NO:2 containing the signal sequence.

2. An enzyme composition comprising the variant of claim 1 and At least one preservative selected from organic acids, citric acid, ascorbic acid, benzoic acid and its salts and derivatives, sodium benzoate, benzoate / ester, hydroxybenzoate / ester and derivative, sorbic acid, sodium sorbate, sorbate / ester, salt, sodium chloride or potassium chloride, 1,2-benzisothiazolin-3-one (BIT) or combinations thereof.

3. The enzyme composition of claim 2, wherein it is in the form of a liquid composition or a solid composition, solution, dispersion, paste, powder, microparticles, granules, coated granules, tablets, cakes, crystals, syrups, gels or pellets.

4. A detergent composition comprising a variant of the mannanase of claim 1 or an enzyme composition of any one of claims 2-3.

5. The detergent composition of claim 4, which is in the form of a liquid detergent or a solid detergent, in the form of a strip, a homogeneous tablet, a tablet having two or more layers, a sachet having one or more compartments, a plain or dense powder, microparticles, granules, a paste, a gel, or a plain, dense, or concentrated liquid.

6. The detergent composition of claim 4 or 5, further comprising one or more additional enzymes selected from proteases, lipases, keratases, amylases, glycoses, cellulases, pectinases, esterases, mannanases, arabinogalactanases, galactanases, xylanases, oxidases, xanthan gumases, xylose glucanases, laccases, DNases, and / or peroxidases.

7. A recombinant host cell containing a genetic element that allows the production of at least one recombinant polypeptide comprising a variant of claim 1.

8. The recombinant host cell of claim 7, wherein the host cell is selected from fungal cells and bacterial cells.

9. The recombinant host cell of claim 7, wherein the host cell is yeast.

10. The recombinant host cell of claim 7, wherein the recombinant polypeptide is a fusion protein, and in addition to having an amino acid sequence having mannanase activity, the fusion protein further comprises at least one of the following: The amino acid sequence that provides the secretion signal sequence; Amino acid sequences that facilitate purification, affinity tags or His-tags; The enhanced amino acid sequence, used as the carrier amino acid sequence, or CBM; An amino acid sequence with enzymatic activity; and Provide the fusion protein with a binding affinity amino acid sequence or a carbohydrate-binding moiety.

11. A method for producing a recombinant polypeptide containing a variant of mannanase, comprising: a. Culturing the recombinant host cell of any one of claims 7-10, wherein The genetic element includes at least one control sequence that controls the production of the recombinant polypeptide in the recombinant host cell; The genetic element comprises at least one sequence encoding a signal sequence for transporting the recombinant polypeptide outside the host cell; and Culture was carried out under conditions that allowed for the production of the recombinant polypeptide; and b. Recover the recombinant polypeptide.

12. A method for degrading or modifying a material containing mannan, comprising treating the mannan-containing material with an effective amount of an enzyme composition of any one of claims 2-3 or a variant of claim 1.

13. The method of claim 12, wherein the mannan-containing material is based on plant material, wastewater, oil, or a combination thereof.

14. Animal feed comprising an enzyme composition of any one of claims 2-3 or a variant of the mannanase of claim 1, and at least one plant-derived protein source or product or byproduct containing mannan, and a. At least one enzyme, said enzyme being selected from proteases, amylases, phytases, xylanases, endoglucanases, β-glucanases, or combinations thereof; and b. At least one filler selected from maltodextrin, flour, salt, or combinations thereof.

15. The animal feed of claim 14, wherein the filler is selected from sodium chloride and sulfate.

16. A feed supplement comprising an enzyme composition of any one of claims 2-3 or a variant of the mannanase of claim 1; and a. At least one enzyme, said enzyme being selected from proteases, amylases, phytases, xylanases, endoglucanases, β-glucanases, or combinations thereof; and b. At least one filler selected from maltodextrin, flour, salt, or combinations thereof.

17. The feed supplement of claim 16, wherein the filler is selected from sodium chloride and sulfate.

18. Use of a variant of claim 1 or an enzyme composition of any one of claims 2-3 in oil drilling or hydraulic fracturing.

19. Use of a variant of claim 1 or an enzyme composition of any one of claims 2-3 in the treatment of coffee extract, fruit juice or soy milk.

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