Variant polypeptides having glucanase activity

Abstract

The present application relates to a glucanase variant.

Description

Technical Field

[0001] The present application relates to modified polypeptides having glucanase activity. Background Art

[0002] 1,3-1,4-β-glucan is a polysaccharide that forms the cell walls of many plants and is particularly abundant in the cell walls of commercially valuable cereals such as barley, rye, sorghum, rice and wheat. However, glucan is an anti-nutritional factor that interferes with the digestion and absorption of plant-based feeds by animals. Especially for monogastric animals, it is difficult to degrade and utilize glucan due to the lack of glucanase (EC 3.2.1.73) that can degrade these anti-nutritional factors.

[0003] Therefore, effective nutritional intake of livestock may be possible by adding glucanase to feed, and for this purpose, it is necessary to develop glucanase suitable for feed (Murphy P, Bello FD, O'Doherty JV, Arendt EK, Sweeney T, Coffey A (2012) Effects of cereal β-glucans and enzyme inclusion on the porcine gastrointestinal tract microbiota. Anaerobe 18(6): 557–565). Summary of the invention

[0004] [Technical issues]

[0005] At the same time, heat resistance that can withstand high temperature processes is essential for feed enzymes.

[0006] [Technical solution]

[0007] One object of the present application is to provide a modified polypeptide having glucanase activity, wherein any one or more of the amino acids at positions 53, 199 and 200 corresponding to the N-terminus of SEQ ID NO: 1 is replaced by another amino acid.

[0008] Another object of the present application is to provide a polynucleotide encoding the polypeptide of the present application.

[0009] Another object of the present application is to provide a microorganism, which comprises one or more of the modified polypeptide of the present application; a polynucleotide encoding the modified polypeptide; and a vector containing the polynucleotide.

[0010] Another object of the present application is to provide a composition for degrading glucan, which comprises one or more of the polypeptide described in the present application and the microorganisms described in the present application.

[0011] Another object of the present application is to provide a method for degrading glucan, which comprises: reacting one or more of the polypeptide and the microorganism described in the present application with glucan.

[0012] Another object of the present application is to provide a feed additive composition comprising the polypeptide described in the present application and one or more of the microorganisms described in the present application.

[0013] Another object of the present application is to provide a method for preparing a feed product, comprising: mixing a feed additive composition with a feed component, wherein the feed additive composition comprises one or more of the polypeptide described in the present application and the microorganism described in the present application.

[0014] Another object of the present application is to provide a food composition; a composition for preparing food; a detergent composition; a fabric composition; and a paper processing composition, which comprises one or more of the polypeptide described in the present application and the microorganism described in the present application.

[0015] Another object of the present application is to provide a method for preparing a modified polypeptide having glucanase activity, comprising: culturing the microorganism described in the present application.

[0016] [Beneficial Effects]

[0017] The modified polypeptide having glucanase activity of the present application has improved heat resistance and can therefore be advantageously applied to various industrial fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 These are the results of evaluating the heat resistance of C, M1, M2, and M3 enzymes. DETAILED DESCRIPTION

[0019] Hereinafter, the present application will be described in detail. Meanwhile, each description and embodiment disclosed herein can be applied to other descriptions and embodiments respectively. That is to say, all combinations of various elements disclosed herein fall within the scope of the present application. In addition, the scope of the present application is not limited by the specific embodiments described below.

[0020] Furthermore, those skilled in the art will be able to recognize or ascertain using no more than routine experimentation many equivalents to the specific aspects of the invention described herein. Additionally, such equivalents are also intended to be encompassed by this application.

[0021] In addition, many papers and patent documents are cited in this specification. The contents of the cited papers and patent documents are incorporated herein in their entirety by reference, and the level of the technical field to which the present invention belongs and the contents of the present invention will be more clearly described.

[0022] One aspect of the present application provides a modified polypeptide having glucanase activity, wherein any one or more of the amino acids at positions 53, 199 and 200 corresponding to the N-terminus of SEQ ID NO: 1 is replaced by another amino acid.

[0023] As used herein, the term "modified polypeptide having glucanase activity" refers to any polypeptide or glucanase having glucanase activity, in which any one or more of the amino acids at positions 53, 199 and 200 corresponding to the N-terminus of SEQ ID NO: 1 are replaced with another amino acid variant.

[0024] As used herein, the term "glucanase" refers to an enzyme having an activity capable of cleaving a glycosidic bond connecting two glucose residues in a glucan. Non-limiting examples of glucanase activity include an endocellulase activity acting on a β-1,4 bond between D-glucose, and a lichenase or lichenase activity that cleaves a (1,4)-β-D-glycosidic bond of a β-D-glucan containing 1,3- and 1,4-bonds. As an embodiment, the glucanase of the present application may have an activity corresponding to a glucanase classified as EC 3.2.1.73.

[0025] In one embodiment, the reference glucanase intended to be mutated in the present application may be a protein having an amino acid sequence of SEQ ID NO: 1, or a protein comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or higher or 99% or higher homology or identity with the amino acid sequence of SEQ ID NO: 1. In addition, it is obvious that any protein having an amino acid sequence (wherein a portion of the sequence is deleted, modified, substituted or added), as long as the amino acid sequence has such homology or identity and exhibits the corresponding efficacy of the protein described in the present application, may also fall within the scope of the reference protein intended to be mutated in the present application.

[0026] Although the protein comprising the amino acid sequence of SEQ ID NO:1 has been defined as an example of a reference glucanase, it does not exclude the addition of meaningless sequences, possible naturally occurring mutations or silent mutations upstream or downstream of the amino acid sequence, and it is obvious to those skilled in the art that any protein having the same or corresponding activity as the protein consisting of the amino acid sequence of SEQ ID NO:1 may belong to the scope of the reference glucanase. Examples of other reference glucanases include glucanases derived from Orpinomyces sp., Neocallimastix sp., Piromyces sp., Clostridium sp., Rhodothermus sp. or Ruminococcus sp. Another example of a glucanase includes a wild-type glucanase derived from Orpinomyces sp.

[0027] As used herein, the term "variant" or "modified polypeptide" refers to a protein having one or more different amino acids compared to the sequence through conservative substitutions and / or modifications such that the function and properties of the protein are retained.

[0028] The variant is different from a sequence identified by substitution, deletion or addition of several amino acids. Such variants can generally be identified by modifying one or more of the above-mentioned amino acid sequences of the protein and evaluating the properties of the modified protein. That is, the ability of the variant can be enhanced, unchanged or reduced relative to the native protein. In addition, some variants may include one or more of those regions that have been removed, such as an N-terminal leader sequence or a transmembrane domain. In addition, other variants may include variants in which regions have been removed from the N- and / or C-terminal of the mature protein. The term "variant" or "modified polypeptide" can be used interchangeably with terms such as modification, modified protein, mutant, mutant protein, variant, variant, etc., without limitation, as long as these terms are used to represent changes.

[0029] As used herein, the term "conservative substitution" refers to the substitution of one amino acid with another amino acid having similar structure and / or chemical properties. Generally, conservative substitutions have little or no effect on the activity of the resulting polypeptide.

[0030] The variant may have, for example, one or more conservative substitutions while still retaining one or more biological activities. Such amino acid substitutions generally occur based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or the amphipathic nature of the residues.

[0031] Amino acids can be divided into the following groups:

[0032] In one example, positively charged (basic) amino acids include arginine, lysine and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; amino acids with non-polar side chains (non-polar amino acids) include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan and proline; amino acids with polar or hydrophilic side chains (polar amino acids) include serine, threonine, cysteine, tyrosine, asparagine and glutamine. In another example, amino acids with charged side chains include arginine, lysine, histidine, glutamic acid, aspartic acid; amino acids with uncharged amino acids (called neutral amino acids) include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine and glutamine. In another example, aromatic amino acids include phenylalanine, tryptophan and tyrosine. In another example, the branched-chain amino acids include valine, leucine and isoleucine. In another example, the 20 amino acids can be divided into 5 groups according to their size, starting with the relatively small amino acid group, i.e., glycine, alanine, serine; cysteine, proline, threonine, aspartic acid, asparagine; valine, histidine, glutamic acid, glutamine; isoleucine, leucine, methionine, lysine, arginine; and phenylalanine, tryptophan and tyrosine, but the classification of amino acids is not limited thereto.

[0033] In addition, the variants may also include deletions or additions of amino acids that have minimal effects on the properties and secondary structure of the polypeptide. For example, the polypeptide may be conjugated at the N-terminus with a signal (or leader) sequence that participates in the co-translational or post-translational transfer of proteins. In addition, the polypeptide may also be conjugated with another sequence or linker to identify, purify or synthesize the polypeptide.

[0034] In one embodiment, the modified polypeptide of the present application can be a modified polypeptide having glucanase activity, wherein in the above-mentioned glucanase, any one or more of the amino acids at positions 53, 199 and 200 at the N-terminus corresponding to SEQ ID NO: 1 are replaced by another amino acid.

[0035] In the present application, the term "replaced by another amino acid" is not limited as long as it is replaced by an amino acid other than the amino acid before replacement. Meanwhile, in the present application, when it is stated that "a specific amino acid has been replaced", it is obvious that the amino acid has been replaced by an amino acid different from the amino acid before replacement, even if it is not specifically stated that the amino acid has been replaced by a different amino acid.

[0036] In any of the above embodiments, the modified polypeptide of the present application may include replacing the amino acid corresponding to position 53 with cysteine ​​(C).

[0037] In any of the above embodiments, the modified polypeptide of the present application may include replacing the amino acid corresponding to position 199 with cysteine ​​(C).

[0038] In any of the above embodiments, the modified polypeptide of the present application may include replacing the amino acid corresponding to position 200 with proline (P).

[0039] In any of the above embodiments, the modified polypeptide of the present application may include any one or more substitutions selected from the following:

[0040] i) replacing the amino acid corresponding to position 53 with cysteine ​​(C);

[0041] ii) replacing the amino acid corresponding to position 199 with cysteine ​​(C); and

[0042] iii) Substituting the amino acid corresponding to position 200 with proline (P).

[0043] In any of the above embodiments, the modified polypeptide of the present application may include a substitution selected from the following:

[0044] (i) replacing the amino acid corresponding to position 200 with proline (P);

[0045] (ii) replacing the amino acid corresponding to position 53 with cysteine ​​(C), and replacing the amino acid corresponding to position 199 with cysteine ​​(C); or

[0046] (iii) Substitution of the amino acid corresponding to position 200 with proline (P), substitution of the amino acid corresponding to position 53 with cysteine ​​(C), and substitution of the amino acid corresponding to position 199 with cysteine ​​(C).

[0047] In any of the above embodiments, the modified polypeptide described in the present application may include replacement of the amino acid corresponding to position 53 with cysteine ​​(C), and replacement of the amino acid corresponding to position 199 with cysteine ​​(C), as well as modification of amino acid pairs (i.e., between cysteine ​​residues) to form disulfide bonds.

[0048] The "Nth position" described in the present application may include the Nth position and the amino acid position corresponding to the Nth position. For example, it may include the amino acid position corresponding to any amino acid residue in the mature polypeptide disclosed in the specific amino acid sequence. In one example, the specific amino acid sequence may be the amino acid sequence of SEQ ID NO: 1.

[0049] As used herein, the term "corresponding to" refers to an amino acid residue at a position in a peptide, or an amino acid residue that is similar, identical, or homologous to a residue in a peptide. Identifying an amino acid at a corresponding position may refer to determining a specific amino acid in a sequence that corresponds to a specific sequence. As used herein, "corresponding region" generally refers to a similar or corresponding position in a related protein or a reference protein.

[0050] In the present application, the specific numbering of the amino acid residue positions in the protein used herein can be adopted. For example, by aligning the sequence of the protein of the present application with the target protein to be compared, the amino acid residue positions of the protein of the present application can be renumbered to the corresponding positions.

[0051] For example, any amino acid sequence is aligned with SEQ ID NO: 1, and based on the alignment, each amino acid residue of the amino acid sequence can be numbered by reference to the numerical position of the amino acid residue corresponding to the amino acid residue of SEQ ID NO: 1. For example, a sequence alignment algorithm as described herein can identify the position of an amino acid, or the position at which a modification such as a substitution, insertion or deletion occurs, compared to a query sequence (also referred to as a "reference sequence").

[0052] Examples of the alignment can be determined by the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) and the Needleman program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), etc., but are not limited thereto, and sequence alignment programs such as pairwise sequence alignment algorithms known in the art can be appropriately used.

[0053] In one embodiment, the modified polypeptide described herein can be a polypeptide in which any one or more of the amino acids corresponding to positions 53, 199 and 200 at the N-terminus of SEQ ID NO: 1 are replaced by another amino acid, and includes the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more homology or identity with the amino acid sequence of SEQ ID NO: 1.

[0054] In any of the above embodiments, the modified polypeptide described in the present application can be a polypeptide in which any one or more of the amino acids corresponding to positions 53, 199 and 200 at the N-terminus of SEQ ID NO: 1 are replaced by another amino acid, and includes the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher homology or identity with the amino acid sequence of SEQ ID NO: 1.

[0055] In any of the above embodiments, the modified polypeptide of the present application may include any of the amino acid sequences in SEQ ID NOS: 2-4.

[0056] In any of the above embodiments, the modified polypeptide of the present application may be substantially composed of any of the amino acid sequences in SEQ ID NOS: 2-4, more particularly, composed of any of the amino acid sequences in SEQ ID NOS: 2-4, but not limited thereto.

[0057] In any of the above embodiments, the variant may include any one of the amino acid sequences in SEQ ID NOS: 2-4, or a sequence having at least 80% or higher homology or identity thereto, and the amino acids corresponding to positions 53, 199 and 200 in the amino acid sequence are fixed (i.e., in the amino acid sequence of the variant, the amino acids corresponding to positions 53, 199, and 200 are the same as the amino acids at positions 53, 199 and 200 of any one of the amino acid sequences in SEQ ID NOS: 2-4).

[0058] In addition, it is obvious that any protein having an amino acid sequence in which a portion of the sequence is deleted, modified, substituted or added, as long as the amino acid sequence has such homology or identity and exhibits an effect corresponding to the variant, such protein may also fall within the scope of the present application.

[0059] As used herein, the term "homology" or "identity" refers to the degree of similarity between two given amino acid sequences or nucleotide sequences, which can be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0060] The sequence homology or identity of conservative polynucleotides or polypeptides can be determined by standard comparison algorithms and can be used with the default gap penalty established by the program used. Basically, homology or identity sequences are usually expected to hybridize with all or part of the sequence under moderate or high stringency conditions. Obviously, hybridization with polynucleotides containing universal codons or degenerate codons in the hybridizing polynucleotide is also included.

[0061] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined, for example, by a known computer algorithm such as the "FASTA" program (Pearson et al., (1988) Proc. Natl. Acad. Sci. USA 85:2444) using default parameters. Alternatively, it can be determined by the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), which uses the Needleman program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (preferably, version 5.0.0 or later) (GCG program package (Devereux J et al., Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul SF et al., J MOLEC BIOL 215:403 (1990); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and CARILLO et al. (1988) SIAM J Applied Math 48:1073). For example, BLAST or ClustalW from the National Center for Biotechnology Information (NCBI) can be used to determine homology, similarity or identity.

[0062] Homology, similarity or identity of polynucleotides or polypeptides can be determined, for example, by comparing sequence information using, for example, the GAP computer program (e.g., Needleman et al., (1970), J Mol Biol. 48: 443), as disclosed in Smith and Waterman, Adv. Appl. Math (1981) 2: 482. In short, the GAP program defines homology, similarity or identity as the value obtained by dividing the number of similarly arranged symbols (i.e., nucleotides or amino acids) by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program may include: (1) a unary alignment matrix (containing a value of 1 for identity and a value of 0 for non-identity) and the weighted alignment matrix of Gribskov et al. (1986), Nucl. Acids Res. 14:6745 as disclosed in Schwartz and Dayhoff, ed., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353–358 (1979) (or the EDNAFULL substitution matrix (EMBOSS version of NCBI NUC4.4)); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap open penalty of 10 and a gap extension penalty of 0.5); and (3) no penalty for end gaps.

[0063] Thus, the terms "homology" or "identity" as used herein indicate the relatedness between sequences.

[0064] As used herein, "enzyme activity" or "glucanase activity" exhibits at least one catalytic activity. In particular, it may be primarily expressed as k cat / K m The conversion efficiency of the enzyme is, but not limited to,

[0065] When the enzyme is completely saturated with substrate, k cat It refers to the rate constant (catalytic constant) at which an enzyme converts a substrate into a product per unit time, also known as the turnover number. m The reaction rate is at its maximum value (V max ) at half the substrate concentration.

[0066] Examples of ways to express enzyme activity include specific activity (μmol × mg of substrate converted) -1 ×min -1 ) or volumetric activity (μmol×mL of converted substrate -1 ×min -1) etc. However, the definition of enzyme activity is not limited to the above, and enzyme activity can be defined and evaluated based on information known in the art.

[0067] As used herein, enzyme stability refers to the enzyme activity maintained during storage or reaction time. In order to measure this change in stability, the initial enzyme activity can be measured and compared under specified conditions at zero time (100%) and after a period of time (x%), thereby indicating the degree of enzyme activity loss or the level of enzyme stability.

[0068] Factors that affect enzyme activity include, for example, pH, heat, the presence of other substances (eg, oxidants, chelators), and the like.

[0069] As used herein, the term "thermostability" refers to the ability of a protein to function within a specific temperature range. In one embodiment, the variants provided herein may be active within a range of about 20°C to about 120°C, and in particular may be active within a range of about 60°C to about 100°C.

[0070] As used herein, the term "thermostability" refers to the ability of a protein to function after exposure to a specific temperature (e.g., high heat or low temperature). For example, thermotolerant proteins may not function at the temperature to which they are exposed, but may become functional when returned to the optimal temperature environment. In one embodiment, the modified polypeptides provided in the present application can retain functional capabilities after exposure to high temperatures in the range of about 60°C to about 100°C.

[0071] Compared to other enzymes (e.g., wild-type enzymes, parent enzymes, and / or other variants), improved stability or heat resistance may include: maintaining higher enzyme activity; increasing the pH range, temperature range, and / or time range within which the protein maintains its function, etc.

[0072] In one embodiment, the modified polypeptide provided in the present application may have improved heat resistance and / or thermal stability compared to the polypeptide of SEQ ID NO: 1.

[0073] In one embodiment, the modified polypeptides provided herein may have increased heat resistance and / or thermal stability compared to the wild-type polypeptide.

[0074] In one embodiment, the modified polypeptide provided in the present application may have improved heat resistance compared to the polypeptide of SEQ ID NO: 1.

[0075] In any of the above embodiments, the properties exhibited by the modified polypeptides provided in the present application may be suitable for or improve the application activity in various industrial fields (including feed, baking, pulp bleaching, etc.).

[0076] Another aspect of the present application provides a polynucleotide encoding the modified polypeptide described in the present application.

[0077] The "polynucleotide" used herein refers to a nucleotide polymer composed of nucleotide monomers linked into a long chain by covalent bonds, and is a DNA or RNA chain of at least a certain length.

[0078] The polynucleotide encoding the modified polypeptide described in the present application may include any polynucleotide encoding the modified polypeptide of the present application or a polypeptide having an activity corresponding to the modified polypeptide, without limitation.

[0079] Due to codon degeneracy or taking into account codon preference in the organism in which the polypeptide is to be expressed, the polynucleotide of the present application may undergo various modifications in the coding region without changing the amino acid sequence of the polypeptide.

[0080] In one embodiment, the polynucleotide encoding the modified polypeptide of the present application may have or include a nucleotide sequence having at least 50%, 60%, 70%, 75%, 80%, 85% or 90% or more and 100% or less homology or identity with the polynucleotide sequence of SEQ ID NO:5.

[0081] In any of the above embodiments, the polynucleotide encoding the glucanase variant of the present application may have or include a nucleotide sequence having at least 50%, 60%, 70%, 75%, 80%, 85% or 90% or more and 100% or less homology or identity with the polynucleotide sequence of SEQ ID NO: 5, and in the sequence having such homology or identity, the codon encoding the 53rd amino acid at the N-terminus of SEQ ID NO: 1 may be one of the codons encoding an amino acid other than arginine (R); the codon encoding the 199th amino acid may be one of the codons encoding an amino acid other than threonine (T); the codon encoding the 200th amino acid may be one of the codons encoding an amino acid other than asparagine (N); or a combination thereof.

[0082] In any of the above embodiments, the polynucleotide encoding the glucanase variant of the present application may have or include a nucleotide sequence having at least 50%, 60%, 70%, 75%, 80%, 85% or 90% or more and 100% or less homology or identity with the polynucleotide sequence of SEQ ID NO: 5, and in the sequence having such homology or identity, the codon encoding the 53rd and / or 199th amino acid corresponding to the N-terminus of SEQ ID NO: 1 may be one of the codons encoding cysteine ​​(C), and / or the codon encoding the amino acid corresponding to the 200th amino acid may be one of the codons encoding proline (P).

[0083] In addition, the polynucleotides of the present application may include probes that can be prepared from known gene sequences, for example, any sequence that can hybridize to all or part of the complementary sequence of the polynucleotide sequence of the present application under stringent conditions, without limitation.

[0084] "Stringent conditions" refer to conditions that allow specific hybridization between polynucleotides. These conditions are specifically described in the literature (J Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, stringent conditions may include conditions in which genes having a high homology or identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more hybridize to each other, and genes having a homology or identity lower than the above homology or identity do not hybridize to each other, or washing conditions for Southern hybridization, i.e., washing once, particularly twice or three times, at a salt concentration and temperature corresponding to 60°C, 1×SSC, 0.1% SDS, particularly 60°C, 0.1×SSC, 0.1% SDS, more particularly 68°C, 0.1×SSC, 0.1% SDS.

[0085] Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize to each other. For example, with respect to DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of the present application can include isolated nucleotide fragments that are complementary to the entire sequence and nucleic acid sequences that are substantially similar thereto.

[0086] Specifically, polynucleotides having homology or identity with the polynucleotides of the present application can be detected using hybridization conditions, wherein the hybridization conditions include the following conditions: m The hybridization step is 55°C. In addition, T m The value may be 60° C., 63° C., or 65° C., but is not limited thereto, and may be appropriately adjusted by those skilled in the art according to their purpose.

[0087] The appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotides and the degree of complementation, variables that are well known in the art (eg, Sambrook et al.).

[0088] Another aspect of the present application provides a vector comprising a polynucleotide encoding the modified polypeptide of the present application. The modified peptide and polynucleotide are as described in other aspects above.

[0089] As used herein, the term "vector" may include a DNA construct containing a nucleotide sequence of a polynucleotide encoding a target polypeptide, the polynucleotide being operably linked to a suitable expression control region (expression control sequence) so that the target polypeptide can be expressed in a suitable host cell. The expression control sequence may include a promoter capable of initiating transcription, any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for regulating transcription and translation termination. Once transformed into a suitable microorganism, the vector may replicate or function independently of the host genome or may be integrated into the host genome.

[0090] In one example, the polynucleotide encoding the target protein can be inserted into the chromosome by a vector for intracellular chromosome insertion. The polynucleotide can be inserted into the chromosome by any method known in the art, such as by homologous recombination, but is not limited thereto. The vector can also include a selection marker to confirm insertion into the chromosome. The selection marker is used to select cells transformed with the vector, i.e., to confirm whether the target nucleic acid molecule has been inserted, and a marker providing a selectable phenotype (e.g., drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface polypeptides) can be used. Only cells expressing the selection marker can survive or show different phenotypes in an environment treated with a selection agent, so transformed cells can be selected.

[0091] The vector used in the present application is not particularly limited, and any vector known in the art can be used.

[0092] Examples of vectors commonly used in prokaryotic cells include, as phage vectors or cosmid vectors, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A, etc.; as plasmid vectors, plasmid vectors based on pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET, etc. can be used. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, pDCM2 vectors, etc. can be used.

[0093] As the example of the carrier used in eukaryotic cells, yeast expression vectors can include yeast integration plasmids (YIp) and extrachromosomal plasmid vectors. Extrachromosomal plasmid vectors can include yeast additional plasmids (YEp), yeast replicating plasmids (YRp) and yeast centromere plasmids (YCp). In addition, yeast artificial chromosomes (YAC) can also be used as the carrier of the application. Specific examples of vectors that can be used may include pESCHIS, pESC-LEU, pESC-TRP, pESC-URA, GatewaypYES-DEST52, pAO815, pGAPZ A, pGAPZ B, pGAPZ C, pGAPαA, pGAPαB, pGAPαC, pPIC3.5K, pPIC6 A, pPIC6 B, pPIC6 C, pPIC6αA, pPIC6αB, pPIC6αC, pPIC9K, pYC2 / CT, pYD1 yeast display vector, pYES2, pYES2 / CT, pYES2 / NT A, pYES2 / NT B, pYES2 / NT C. pYES2 / CT, pYES2.1, pYES-DEST52, pTEF1 / Zeo, pFLD1, PichiaPinkTM, p427-TEF, p417-CYC, pGAL-MF, p427-TEF, p417-CYC, P TEF-MF, pBY011, pSGP47, pSGP46, pSGP36, pSGP40, ZM552, pAG303GAL-ccdB, pAG414GAL-ccdB, pAS404, pBridge, pGAD-GH, pGAD T7,pGBK T7, pHIS-2, pOBD2, pRS408, pRS410, pRS418, pRS420, pRS428, yeast micron type A, pRS403, pRS404, pRS405, pRS406, pYJ403, pYJ404, pYJ405 and pYJ406, but are not limited thereto.

[0094] As used herein, the term "transformation" refers to the introduction of a vector containing a polynucleotide encoding a target protein into a host cell or microorganism so that the protein encoded by the polynucleotide can be expressed in the host cell. As long as the converted polynucleotide can be expressed in the host cell, it is not important whether the converted polynucleotide is integrated into the chromosome of the host cell and located therein or located outside the chromosome, and both situations can be included. In addition, the polynucleotide may include DNA and / or RNA encoding the target protein. The polynucleotide may be introduced in any form as long as it can be introduced into the host cell and expressed therein. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a gene construct including all elements required for its autonomous expression. The expression cassette may generally include a promoter, a transcription terminator, a ribosome binding site, or a translation terminator operably connected to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. In addition, the polynucleotide may be introduced into the host cell as is and operably connected to a sequence required for expression in the host cell, but is not limited thereto.

[0095] In addition, as used herein, the term "operably linked" means that a gene sequence is functionally linked to a promoter sequence, which initiates and mediates the transcription of a polynucleotide encoding a target polypeptide of the present application.

[0096] The method for transforming the vector of the present application may include any method capable of introducing nucleic acid into cells, and may be transformed by selecting suitable standard techniques known in the art according to the host cell. For example, the method may include electroporation, calcium phosphate (CaPO 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method and lithium acetate-DMSO method, but are not limited thereto.

[0097] Another aspect of the present application provides a microorganism comprising one or more of the modified polypeptide of the present application; a polynucleotide encoding the modified polypeptide; and a vector containing the polynucleotide.

[0098] The microorganism may comprise the modified polypeptide, the polynucleotide encoding the variant, a nucleic acid construct and / or a vector containing the polynucleotide. The nucleic acid construct or vector may be integrated into the chromosome, or may be retained as a self-replicating extrachromosomal vector.

[0099] The microorganism of the present application can include any microorganism, as long as it can express the modified polypeptide of the present application, without restriction.For example, the microorganism can include any cell, such as prokaryotic organism or eukaryotic organism, that can be used for recombinant production of the modified polypeptide of the present application.In another example, the microorganism includes fungi and bacteria.

[0100] In one embodiment, the microorganism of the present application can be a microorganism belonging to the genus Escherichia, and can be Escherichia coli, Escherichia albertii, Escherichia fergusonii, Escherichia hermannii, Escherichia vulneris or Escherichia blattae. In any of the above embodiments, the microorganism can be Escherichia coli, but is not limited thereto.

[0101] Another aspect of the present application provides a composition for degrading glucan, which includes one or more of the modified polypeptide of the present application and a microorganism, wherein the microorganism contains one or more of the modified polypeptide, a polynucleotide encoding the modified polypeptide, and a vector containing the polynucleotide.

[0102] Another aspect of the present application provides a use of a composition for degrading glucan, wherein the composition comprises one or more of the modified polypeptide and microorganisms of the present application, and the microorganism comprises one or more of the modified polypeptide, a polynucleotide encoding the modified polypeptide, and a vector containing the polynucleotide.

[0103] Another aspect of the present application provides a method for degrading glucan, comprising: reacting one or more of the modified polypeptide and microorganism of the present application with glucan, wherein the microorganism comprises one or more of the modified polypeptide, a polynucleotide encoding the modified polypeptide, and a vector containing the polynucleotide.

[0104] The modified polypeptide of the present application and / or a microorganism comprising one or more of the modified polypeptide, the polynucleotide encoding the modified polypeptide and the vector containing the polynucleotide can be used to degrade glucan-containing materials.

[0105] In one example, in addition to the modified polypeptide of the present application, the composition for degrading glucan provided in the present application may also include naturally occurring materials or non-naturally occurring materials.

[0106] In one embodiment, the composition for degrading glucan provided in the present application may also include any constituent elements applicable to various industrial fields (including animal feed, baking, biomass saccharification, pulp bleaching, etc.).

[0107] Examples of substances that may be added include stabilizers, surfactants, builders, chelating agents, dispersants, enzymes, enzyme stabilizers, catalysts, activators, carriers, binders, lubricants, disintegrants, excipients, solubilizers, suspending agents, colorants, flavorings, buffers, preservatives, analgesics, solubilizers, isotonic agents, diluents, lubricants, and the like, but are not limited thereto.

[0108] Another aspect of the present application provides a feed additive composition, which comprises one or more of the following: the modified polypeptide of the present application and a microorganism, wherein the microorganism comprises one or more of the modified polypeptide, a polynucleotide encoding the modified polypeptide and a vector containing the polynucleotide.

[0109] Another aspect of the present application provides a method for preparing a feed product, comprising: mixing a feed additive composition and a feed component, wherein the feed additive composition comprises one or more of the following: a modified polypeptide of the present application and a microorganism, wherein the microorganism comprises one or more of the modified polypeptide, a polynucleotide encoding the modified polypeptide, and a vector containing the polynucleotide.

[0110] The modified polypeptide of the present application or the microorganism of the present invention can be used in any of the following applications:

[0111] a) additives in animal feed; and / or

[0112] b) feed supplements for animals; and / or

[0113] c) Decomposition of cereal-based material (eg this may be whole cereals or cereal parts).

[0114] The feed product of the present application may refer to any natural or artificial diet, single meal, etc., or a component of a single meal eaten, ingested and digested by an animal, or a component of a single meal suitable for eating, ingesting and digesting, and the feed product may be prepared in various forms known in the art.

[0115] The modified polypeptide having glucanase activity of the present application can be used in various industrial fields, such as food, paper, pulp, detergent, industrial waste treatment, biomaterials and bioenergy, etc. Regarding the application of glucanase in the industrial field, literature such as McCarthy TC, Lalor E, Hanniffy O, Savage AV, Tuohy MG, Comparison of wide-type and UV-mutantβglucanase-producing strains of Talaromyces emersonii with potential in brewing applications, J Ind Microbiol Biotechnol, 2005; Chaari F, Belghith-Fendri L, Blibech M, Driss D, Ellouzia SZ, Sameh M, Ellouz-Chaabouni S, Biochemical characterization of a lichenase from Penicillium occitanis Pol6 and its potential application in the brewing industry, Process Biochem, 2014; Maktouf S, Moulis C, Kamoun A, Chaari F, Chaabouni SE, Remaud-Simeon M, A laundrydetergent compatible lichenase: statistical optimization for production undersolid state fermentation on crude millet, Ind Crops Prod, 2013, etc. can be incorporated by reference.

[0116] Another aspect of the present application provides a food composition; or a composition for preparing food, which comprises a modified polypeptide with glucanase activity of the present application or a microorganism of the present application. In one example, glucanase can be used for brewing (beer, wine, etc.) and for producing fruit juice. Adding glucanase during saccharification can reduce the viscosity of wort, improve filterability, increase the dissolution rate of malt, prevent beer turbidity, and stabilize beer quality.

[0117] Another aspect of the application provides a kind of detergent composition, it comprises the modified polypeptide with glucanase activity of the application or the microorganism of the application.Detergent composition is the composition for removing unwanted compounds from articles to be cleaned such as fabric, tableware or hard surface.The example of detergent composition includes fabric detergent, textile detergent, cleaning agent for the hard surface of glass, wood, plastic, pottery, metal countertop and window etc., carpet cleaner, oven cleaner, fabric fragrance, fabric softener, dishwasher detergent etc.

[0118] Another aspect of the present application provides a kind of textile composition, it comprises the modified polypeptide with beta-glucanase activity of the present application or the microorganism of the present application.The beta-glucanase of the present application can be used for processing the fiber for making clothing.These processes include the process of removing dyestuff after bleaching, dyeing and bio-polishing of cotton fabric or cotton blended fabric.

[0119] Another aspect of the present application provides a paper processing composition, which comprises the modified polypeptide having glucanase activity of the present application or the microorganism of the present application. The modified polypeptide having glucanase activity of the present application can be used to improve wastewater in the pulp industry, remove ink or decolorize.

[0120] In any of the above embodiments, the composition provided in the present application may further include any constituent elements applicable to various industrial fields. In any of the above embodiments, the composition provided in the present application may further include any other enzymes applicable to the intended use.

[0121] In any of the above embodiments, the composition provided in the present application may contain the modified polypeptide having glucanase activity of the present application or the microorganism of the present application in an amount suitable for the intended use.

[0122] In any of the above embodiments, the composition provided in the present application can be provided in a form suitable for the intended use. As a non-limiting example, the composition can be in the form of a bar, a sheet, a powder, a granule, a paste or a liquid.

[0123] Another aspect of the present application provides a method for preparing a modified polypeptide having glucanase activity, comprising: culturing a microorganism comprising one or more of a modified polypeptide, a polynucleotide encoding the modified polypeptide, and a vector containing the polynucleotide.

[0124] The culturing may include growing the microorganism in a culture medium.

[0125] As used herein, the term "culture" refers to the growth of host cells under appropriately controlled environmental conditions. The culture process of the present application can be carried out under suitable culture media and culture conditions known in the art. Those skilled in the art can easily adjust this culture process for use according to the strain to be selected. Specifically, the culture can be batch culture, continuous culture and fed-batch culture, but is not limited thereto.

[0126] As used herein, the term "medium" refers to a mixture of substances containing nutrients required for culturing host cells as the main component, which provides nutrients and growth factors, as well as water necessary for survival and growth. Specifically, the culture medium and other culture conditions used to cultivate the host cells of the present application can be any culture medium for conventional culture of host cells, without any particular restrictions. However, the host cells of the present application can be cultured under aerobic conditions in a conventional culture medium containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids and / or vitamins, etc., while adjusting temperature, pH, etc.

[0127] In the present application, the carbon source may include carbohydrates such as glucose, sucrose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc. In addition, the carbon source may include natural organic nutrients such as starch hydrolysate, molasses, blackstrap molasses, rice bran, cassava, cane molasses, and corn syrup, etc. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) may be used, and in addition, various other carbon sources may be used in appropriate amounts without restriction. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0128] The nitrogen source may include an inorganic nitrogen source such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; amino acids such as glutamic acid, methionine, glutamine, etc.; and an organic nitrogen source such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its degradation product, defatted soybean cake or its degradation product, etc. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.

[0129] The phosphorus source may include monopotassium phosphate, dipotassium phosphate or corresponding sodium salts, etc. Examples of inorganic compounds may include sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc. In addition, amino acids, vitamins and / or suitable precursors may be included. These constituents or precursors may be added to the culture medium in batches or continuously, but these phosphorus sources are not limited thereto.

[0130] In addition, during the culture of the host cells, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. in an appropriate manner. In addition, the use of defoamers such as fatty acid polyethylene glycol esters during the culture process can prevent the formation of bubbles. In addition, oxygen or oxygen-containing gas can be injected into the culture medium to maintain aerobic conditions in the culture medium; or nitrogen, hydrogen or carbon dioxide can be injected to maintain anaerobic or microaerobic conditions, but gas may not be injected, but the type of gas is not limited thereto.

[0131] The temperature of the culture medium may be in the range of from 20° C. to 55° C., specifically from 25° C. to 40° C., but not limited thereto. The culture may be continued until a desired amount of useful substances is obtained, and may be specifically performed for 24 to 196 hours, but not limited thereto.

[0132] In one embodiment, the method for preparing the modified polypeptide of the present application may further include recovering the modified polypeptide of the present application expressed in the culturing step.

[0133] In another embodiment, the modified polypeptide expressed in the culture step can be recovered using methods known in the art to which the present application belongs. For example, the polypeptide can be recovered from the culture medium by conventional methods, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation or precipitation.

[0134] Regarding the recovery method, the modified polypeptide can be collected using a method for cultivating the microorganism of the present application, for example, according to batch culture, continuous culture or fed-batch culture method, using a suitable method known in the art. For example, centrifugation, filtration, treatment with a protein crystallization precipitant (salting out), extraction, ultrasonic crushing, ultrafiltration, dialysis, various chromatography (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, etc.), HPLC and a combination of these methods can be used, and the variant of the present application can be recovered from a culture medium or a host cell using a suitable method known in the art.

[0135] In another embodiment, the modified polypeptide expressed by the host cell in the culturing step may not be recovered. In this embodiment, the host cell expressing the modified polypeptide may be used as a source of the modified polypeptide.

[0136] Another aspect of the present application provides a composition, which includes one or more of the modified polypeptide and microorganisms of the present application; the microorganism includes one or more of the modified polypeptide, a polynucleotide encoding the modified polypeptide and a vector containing the polynucleotide.

[0137] As described above, the composition can be used for food, feed additives, food preparation, detergents, textiles or paper processing.Another aspect of the present application provides the use of the composition for food, feed additives, food preparation, detergents, textiles or paper processing, as described in the other aspects above.

[0138] [Mode for Carrying Out the Invention]

[0139] Hereinafter, the present application will be described in more detail by examples and experimental examples. However, these examples and experimental examples are given for illustrative purposes only, and the scope of the present application is not intended to be limited by these examples and experimental examples.

[0140] Example 1. Preparation of protein with glucanase activity

[0141] Example 1-1. Preparation of variant recombinant plasmids

[0142] Three variants, M1 (SEQ ID NO: 2), M2 (SEQ ID NO: 3) and M3 (SEQ ID NO: 4), were prepared based on the synthetic sequence C (SEQ ID NO: 1) prepared from a gene derived from genomic DNA of Rhizoctonia PC-2 strain.

[0143] PCR was performed under the following conditions to amplify the variant gene for cloning. Specifically, variants were prepared by PCR using template, primer and PCR premix (Taq 2X premix, Bioneer). M1 was prepared using M1-F and M1,R primers based on synthetic sequence C, M2 was prepared using M2-F1, M2-F2, M2-R1 and M2-R2 primers based on synthetic sequence C, and M3 was prepared using M3-F and M3R primers based on M2.

[0144] PCR was performed using an Eppendorf Mastercycler Nexus GX2 with the following reaction conditions:

[0145] Initial denaturation: 95°C, 2 min

[0146] Denaturation: 95℃, 20sec

[0147] Annealing: 50℃, 10sec

[0148] Extension: 72°C, 1 min (30 cycles from denaturation to extension)

[0149] Final extension: 72℃, 5min

[0150] The amplified gene was cloned into pET28a vector (Takara, catalog number 639650) using the In-Fusion HD Cloning Kit.

[0151] [Table 1]

[0152]

[0153]

[0154] The M1, M2 and M3 variants are mutated at the following positions: In M1, the amino acid asparagine at position 200 in SEQ ID NO: 1 is replaced by proline. In M2, the arginine at position 53 in SEQ ID NO: 1 is replaced by cysteine, and the threonine at position 199 is replaced by cysteine. In M3, the arginine at position 53 in SEQ ID NO: 1 is replaced by cysteine, the threonine at position 199 is replaced by cysteine, and the asparagine at position 200 is replaced by proline.

[0155] Example 1-2. Preparation of transformants having glucanase gene and enzyme expression

[0156] The recombinant plasmid prepared in Example 1-1 was transformed into Escherichia coli BL21. In addition, to produce the glucanase variant, the transformant was inoculated into 5 ml of LB medium and cultured at 37°C for 18 hours. For the main culture, 5 mL of the pre-culture medium was inoculated into 300 mL of LB medium and incubated at 37°C. Then, when the OD 600 When the value reached 0.4–0.6, 120 μL of 1 M IPTG was added and incubated for another 12 h. The cells were recovered by centrifugation and redispersed in 35 mL of lysis buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 10 mM imidazole).

[0157] Example 1-3. Purification of polypeptides having glucanase activity

[0158] The cells in the dispersion solution were broken using an ultrasonic disperser, and a crude enzyme solution, i.e., a supernatant, was obtained by centrifugation. The crude enzyme solution was adsorbed by passing through a Ni-NTA resin (Qiagen, catalog number 30230), and then passed through a washing buffer (containing only 20 mM imidazole in the lysis buffer composition) and an elution buffer (containing only 250 mM imidazole in the lysis buffer composition) in sequence to obtain a purified polypeptide having glucanase activity.

[0159] Example 2. Evaluation of thermotolerance of glucanase variants

[0160] Example 2-1. Measurement of residual activity of glucanase variants

[0161] To evaluate the thermotolerance of the variants, each purified enzyme was allowed to stand at 75°C and 80°C for 5 minutes. To measure the activity of the enzyme after heat treatment, 50 μL of 1% dextran, 5 μL of 1m buffer and an appropriate amount of enzyme were mixed before and after heat treatment, and three-grade distilled water was added to a final volume of 100 μL. Thereafter, the mixture was stirred well and reacted at 37°C, and the resulting reducing sugar was measured by the DNS method. The method for preparing the DNS solution is as follows:

[0162] 6.3 g of 3,5-dinitrosalicylic acid (samchus, D1267) was added to 500 mL of distilled water and dissolved at 50°C, while 21 g of sodium hydroxide (Daejung, 7571-4400) was added. Then 182 g of potassium sodium tartrate tetrahydrate (Daejung, 6618-4400) and 5 g of phenol (Sigma-Aldrich, P1037) were added, and 300 mL of distilled water was added. After adding 5 g of anhydrous sodium sulfite (Daejung, 7634-4405), the mixture was stirred to dissolve and cooled, and the final volume was adjusted to 1000 mL with distilled water, and then filtered to complete the preparation.

[0163] Example 3. Results of heat resistance of variants

[0164] In order to evaluate the heat resistance of proteins and variants having glucanase activity, the purified enzymes C, M1, M2, and M3 prepared in Examples 1-3 were placed at 75° C. and 80° C. for 5 minutes, respectively. Then, the residual activity was confirmed at 37° C. using the heat-treated enzymes.

[0165] As a result, all three variants (M1, M2, M3) showed improved heat resistance compared to C. When the enzyme activity at 37°C before heat treatment was set to 100%, it was found that under the temperature condition of 80°C, the heat resistance of M1 was 54% and the heat resistance of M2 was 70%, which was improved by more than 50% relative to 1% of C. In addition, M3 showed an excellent residual activity of 92% or more.

[0166] According to the foregoing, those skilled in the art to which the present application belongs will be able to understand that, without changing the technical concept or essential features of the present application, the present application can be implemented in other specific forms. In this regard, the exemplary embodiments disclosed herein are only for illustrative purposes and should not be construed as limiting the scope of the present application. On the contrary, the present application is intended to cover not only the exemplary embodiments, but also various substitutions, modifications, equivalents and other embodiments that may be included in the spirit and scope of the present application defined by the appended claims.

Claims

1. A modified polypeptide having glucanase activity, wherein any one or more of the amino acids at positions 53, 199 and 200 corresponding to the N-terminus of SEQ ID NO: 1 is replaced by another amino acid.

2. The modified polypeptide according to claim 1, wherein the modified polypeptide comprises any one or more substitutions selected from the group consisting of: i) replacing the amino acid corresponding to position 53 with cysteine ​​(C); ii) replacing the amino acid corresponding to position 199 with cysteine ​​(C); and iii) Substituting the amino acid corresponding to position 200 with proline (P).

3. The modified polypeptide of claim 1, wherein the modified polypeptide comprises a substitution selected from the group consisting of: (i) replacing the amino acid corresponding to position 200 with proline (P); (ii) replacing the amino acid corresponding to position 53 with cysteine ​​(C), and replacing the amino acid corresponding to position 199 with cysteine ​​(C); or (iii) Substitution of the amino acid corresponding to position 53 with cysteine ​​(C), substitution of the amino acid corresponding to position 199 with cysteine ​​(C), and substitution of the amino acid corresponding to position 200 with proline (P).

4. The modified polypeptide according to claim 1, wherein the modified polypeptide comprises substitution of the amino acid corresponding to position 53 with cysteine ​​(C) and substitution of the amino acid corresponding to position 199 with cysteine ​​(C), and modification of the amino acid pair to form a disulfide bond. 5 . The modified polypeptide according to claim 1 , wherein the modified polypeptide comprises any one amino acid sequence selected from SEQ ID NOS: 5-7. The modified polypeptide according to claim 1 , wherein the modified polypeptide has improved heat resistance compared to the polypeptide of SEQ ID NO:

1.

7. A polynucleotide encoding the modified polypeptide according to any one of claims 1 to 6.

8. A microorganism comprising one or more of the modified polypeptide of any one of claims 1 to 6; a polynucleotide encoding the modified polypeptide; and a vector containing the polynucleotide.

9. A composition for degrading glucan, comprising one or more of the following: a modified polypeptide as described in any one of claims 1 to 6; and a microorganism, the microorganism comprising one or more of a modified polypeptide as described in any one of claims 1 to 6, a polynucleotide encoding the modified polypeptide, and a vector containing the polynucleotide.

10. A method for degrading glucan, comprising: reacting one or more of the following with dextran: the modified polypeptide of any one of claims 1 to 6; and a microorganism comprising one or more of the modified polypeptide according to any one of claims 1 to 6, a polynucleotide encoding the modified polypeptide, and a vector containing the polynucleotide.

11. A composition comprising one or more of the following: a modified polypeptide as described in any one of claims 1 to 6; and a microorganism comprising a modified polypeptide as described in any one of claims 1 to 6, a polynucleotide encoding the modified polypeptide, and one or more of a vector containing the polynucleotide.

12. A method for preparing a feed product, comprising: A feed additive composition is mixed with a feed component, wherein the feed additive composition comprises one or more of the following: a modified polypeptide as described in any one of claims 1 to 6; and a microorganism comprising one or more of a modified polypeptide as described in any one of claims 1 to 6, a polynucleotide encoding the modified polypeptide, and a vector containing the polynucleotide.

13. The composition according to claim 11, wherein the composition is used in food, feed additive, food preparation, detergent, textile or paper processing.

14. A method for preparing a polypeptide having glucanase activity, comprising: Cultivating a microorganism comprising the modified polypeptide of any one of claims 1 to 6; a polynucleotide encoding the polypeptide; and one or more of a vector containing the polynucleotide.

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