Novel polypeptides having glucanase activity

Abstract

The present application relates to a polypeptide having glucanase activity.

Description

Technical Field

[0001] The present application relates to 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 polypeptide of SEQ ID NO: 4 having glucanase activity.

[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 comprising one or more of the polypeptide of SEQ ID NO: 4; a polynucleotide encoding the polypeptide; and a vector containing the polynucleotide.

[0010] Another object of the present application is to provide a composition for degrading glucan, comprising one or more of the following: a polypeptide of SEQ ID NO: 4 and a microorganism, wherein the microorganism comprises one or more of the polypeptide of SEQ ID NO: 4, a polynucleotide encoding the polypeptide, and a vector containing the polynucleotide.

[0011] Another object of the present application is to provide a feed additive composition, which comprises one or more of the following: a polypeptide of SEQ ID NO: 4 and a microorganism, wherein the microorganism comprises one or more of the polypeptide of SEQ ID NO: 4, a polynucleotide encoding the polypeptide, and a vector containing the polynucleotide.

[0012] Another object of the present application is to provide a method for degrading glucan, comprising: reacting one or more of the following with glucan: a polypeptide of SEQ ID NO: 4 and a microorganism, wherein the microorganism comprises one or more of a polypeptide of SEQ ID NO: 4, a polynucleotide encoding the polypeptide, and a vector containing the polynucleotide.

[0013] Another object of the present application is to provide a method for preparing a feed product, which comprises: mixing a feed additive composition with a feed component, wherein the feed additive composition comprises one or more of the following: a polypeptide of SEQ ID NO: 4 and a microorganism, wherein the microorganism comprises one or more of a polypeptide of SEQ ID NO: 4, a polynucleotide encoding the polypeptide, and a vector containing the polynucleotide.

[0014] Another object of the present application is to provide a food composition; a composition for preparing food; a detergent composition; a textile composition; and a paper processing composition, wherein the composition comprises one or more of the following: a polypeptide of SEQ ID NO: 4 and a microorganism, wherein the microorganism comprises one or more of a polypeptide of SEQ ID NO: 4, a polynucleotide encoding the polypeptide, and a vector containing the polynucleotide.

[0015] Another object of the present application is to provide a method for preparing a polypeptide having glucanase activity, which comprises culturing a microorganism comprising a polypeptide of SEQ ID NO: 4; a polynucleotide encoding the polypeptide; and one or more of a vector containing the polynucleotide.

[0016] [Beneficial Effects]

[0017] The polypeptide having glucanase activity of the present application has improved heat resistance and thermal stability, and thus can be advantageously applied in various industrial fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a graph confirming the activities of a glucanase reference sequence derived from Saccharomyces (SEQ ID NO: 2) and the novel glucanase of the present application (SEQ ID NO: 4) at different pH values.

[0019] Figure 2It is a graph confirming the activities of a glucanase reference sequence derived from Saccharomyces (SEQ ID NO: 2) and the novel glucanase of the present application (SEQ ID NO: 4) at different temperatures.

[0020] Figure 3 It is a graph confirming the residual activities of the reference sequence of the glucanase derived from Saccharomyces (SEQ ID NO: 2) and the novel glucanase of the present application (SEQ ID NO: 4) under different temperature changes. DETAILED DESCRIPTION

[0021] 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.

[0022] 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.

[0023] 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.

[0024] One aspect of the present application provides a polypeptide of SEQ ID NO: 4, which has glucanase activity. The polypeptide is a novel polypeptide having glucanase activity.

[0025] As used herein, the term "glucanase" refers to an enzyme with the activity of being able to cut the glycosidic bond connecting two glucose residues in a glucan. Non-limiting examples of glucanase activity include an endocellulase activity acting on the β-1,4 bonds between D-glucose, and a lichenase or lichenase activity that cuts the (1,4)-β-D-glycosidic bonds of the β-D-glucans containing 1,3- and 1,4-bonds. As an embodiment, the glucanase with glucanase activity of the application can have the activity corresponding to a glucanase classified as EC 3.2.1.73.

[0026] Although the novel polypeptide with glucanase activity provided in the present application has been defined as the polypeptide of SEQ ID NO:4, it does not exclude the addition of meaningless sequences, possible naturally occurring mutations or silent mutations upstream or downstream of SEQ ID NO:4, and it is obvious to those skilled in the art that any polypeptide with the same or corresponding activity as the protein composed of the amino acid sequence of SEQ ID NO:4 may fall within the scope of the polypeptide with glucanase activity provided in the present application.

[0027] That is to say, although described in the present application as "a protein or polypeptide having an amino acid sequence described by a specific sequence number" or "a protein or polypeptide comprising an amino acid sequence described by a specific sequence number", it is obvious that any protein having an amino acid sequence in which part of the sequence is deleted, modified, substituted or added can be used in the present application as long as it has the same or corresponding activity as a polypeptide consisting of the amino acid sequence of the corresponding sequence number.

[0028] In addition, by comparing the sequence of the polypeptide of SEQ ID NO:4 provided in the present application with the sequence of the reference glucanase polypeptide, and comparing the reference sequence with the sequence of the polypeptide provided in the present application, the polypeptide with glucanase activity obtained by introducing the modification corresponding to the polypeptide provided in the present application into the reference sequence of glucanase can also be included in the scope of the polypeptide provided in the present application. An example of such a reference sequence can include the amino acid sequence of SEQ ID NO:2 or a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto. The reference sequence can be used as a parent sequence for introducing a modification, or as a control for evaluating thermal stability and / or thermotolerance.

[0029] As another example of a reference sequence, a wild-type glucanase sequence can be used. Examples of wild-type glucanases include glucanases derived from Orpinomyces sp., Neocallimastix sp., Piromyces sp., or Ruminococcus sp. An example of a wild-type glucanase can include the amino acid sequence of NCBI sequence ID No.014412.1 or a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto.

[0030] The polypeptide of SEQ ID NO:4 provided in the present application can be a polypeptide of a wild-type glucanase known in the art (e.g., a wild-type glucanase derived from a species of the genus Rhizoctonia), specifically, compared to the "wild type", because one or more amino acids are different from the amino acid sequence of NCBI sequence ID No.014412.1, it can be referred to as a "modified polypeptide" or "variant".

[0031] In addition, compared with the amino acid sequence of SEQ ID NO: 4, one or more amino acids are different from the sequence by conservative substitution and / or modification, while retaining the function and properties of the protein. Modified polypeptides may also be included in the scope of the polypeptides provided in the present application. Such modifications may include, for example, modifications that remove a certain region from the N-terminus and / or C-terminus of the mature protein.

[0032] As used herein, the term "conservative substitution" refers to the replacement of an amino acid with another amino acid having similar structure and / or chemical properties. The polypeptides of the present application may have, for example, one or more conservative substitutions while still retaining one or more biological activities possessed by the polypeptide of SEQ ID NO: 4. Such amino acid substitutions may generally occur based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues.

[0033] In one embodiment, the novel polypeptide with glucanase activity provided in the present application may include SEQ ID NO: 4, consist essentially of SEQ ID NO: 4, or consist of SEQ ID NO: 4. In another embodiment, the novel polypeptide with glucanase activity provided in the present application may have at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more or 100% homology or identity with SEQ ID NO: 4. In addition, it may include, consist essentially of, or consist of sequences having the above homologies or identities.

[0034] As used herein, the term "homology" or "identity" refers to the degree of relatedness 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.

[0035] The sequence homology or identity of conservative polynucleotides or polypeptides can be determined by standard comparison algorithms and can be used together with the default gap penalty established by the used program. Basically, homology or identity sequences can usually hybridize with at least about 50%, 60%, 70%, 80% or 90% of the sequence length or the total length under moderate or high stringency conditions. Obviously, polynucleotide hybridizations that hybridize with polynucleotides containing universal codons or degenerate codons are also included.

[0036] 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) using 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.

[0037] 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 binary 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.

[0038] In addition, whether any two polynucleotide or polypeptide sequences have homology, similarity or identity to each other can be identified by aligning the sequences in a Southern hybridization experiment under defined stringent conditions. Defining appropriate hybridization conditions is within the skills of those skilled in the art and can be determined by methods well known to those skilled in the art (e.g., 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), but not limited thereto.

[0039] 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,

[0040] 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.

[0041] 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.

[0042] As used herein, enzyme stability refers to maintaining enzyme activity 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.

[0043] Factors that affect enzyme activity include, for example, pH, heat, the presence of other substances (eg, oxidants, chelators), and the like.

[0044] As used herein, the term "thermostability" refers to the ability of a protein to function within a specific temperature range. In one embodiment, the variant provided in the present application may be active in the range of about 20°C to about 120°C, and in particular may be active in the range of about 60°C to about 100°C, but is not limited thereto. In any of the above embodiments, the polypeptide having glucanase activity provided in the present application may be active at 50°C to 80°C, but the temperature range is not limited thereto.

[0045] 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.

[0046] Compared to other enzymes (e.g., wild-type enzymes, parent enzymes, and / or other modified polypeptides), the improved stability may include maintaining higher enzyme activity; increasing the pH range, temperature range, and / or time range over which the protein maintains its function, etc. In the present application, the improved stability can be compared with a wild-type glucanase, for example, the glucanase of SEQ ID NO: 2, but is not limited thereto.

[0047] In one embodiment, the polypeptide having glucanase activity provided in the present application may have one or more properties selected from heat resistance and thermal stability improved compared to the polypeptide of SEQ ID NO: 2.

[0048] In any of the above embodiments, the variant may maintain at least 20% or more relative activity at 50°C to 80°C, but is not limited thereto.

[0049] In any of the above embodiments, the properties exhibited by the polypeptide having glucanase activity provided in the present application may be suitable for or improve the application activity in various industrial fields (including feed, baking, pulp bleaching, etc.).

[0050] Another aspect of the present application provides a polynucleotide encoding a polypeptide having the glucanase activity of the present application.

[0051] 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.

[0052] The polynucleotide encoding the polypeptide having glucanase activity of the present application may include any polynucleotide encoding the polypeptide of SEQ ID NO: 4 or a polypeptide having corresponding activity, without limitation.

[0053] Due to codon degeneracy or in consideration of codon preference in an organism in which the polypeptide is to be expressed, the polynucleotide encoding the polypeptide having glucanase activity of the present application may undergo various modifications in the coding region within the range of not changing the amino acid sequence of the polypeptide.

[0054] For example, the polynucleotide encoding the polypeptide having glucanase activity of the present application may be the amino acid sequence of SEQ ID NO: 4 or a polynucleotide sequence encoding a polypeptide having homology or identity thereto.

[0055] In one embodiment, the polynucleotide encoding the polypeptide with glucanase activity 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 less than 100% homology or identity with the polynucleotide sequence of SEQ ID NO: 1. In another embodiment, the polynucleotide encoding the polypeptide with glucanase activity of the present application may consist of or be substantially composed of the sequence of SEQ ID NO: 3 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more homology or identity with the sequence of SEQ ID NO: 3, but is not limited thereto.

[0056] 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.

[0057] "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.

[0058] 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, for 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.

[0059] 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 performed at 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.

[0060] 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.).

[0061] Another aspect of the present application provides a vector comprising a polynucleotide encoding a polypeptide having the glucanase activity of the present application. The peptide and polynucleotide are as described in the other aspects above.

[0062] 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.

[0063] 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.

[0064] The vector used in the present application is not particularly limited, and any vector known in the art can be used.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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 (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method and lithium acetate-DMSO method, etc., but is not limited thereto.

[0070] Another aspect of the present application provides a microorganism, which includes one or more of the polypeptides having glucanase activity of the present application; a polynucleotide encoding the polypeptide; and a vector containing the polynucleotide.

[0071] The microorganism may include the above polypeptide, a polynucleotide encoding the polypeptide, 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.

[0072] The microorganism of the present application can comprise any microorganism, as long as it can express the polypeptide with the beta-glucanase activity of the present application, without restriction.For example, the microorganism can comprise any cell, for example prokaryotic organism or eukaryotic organism, that can be used for recombinant production to have the polypeptide of the beta-glucanase activity of the present application.In another example, the microorganism comprises fungi and bacterium.

[0073] 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.

[0074] Another aspect of the present application provides a composition for degrading glucan, which comprises one or more of the following: a polypeptide having the glucanase activity of the present application and a microorganism, wherein the microorganism comprises one or more of the polypeptide, a polynucleotide encoding the polypeptide and a vector containing the polynucleotide.

[0075] Another aspect of the present application provides the use of a composition for degrading glucan, wherein the composition comprises one or more of the following: a polypeptide having the glucanase activity of the present application and a microorganism, wherein the microorganism comprises one or more of the polypeptide, a polynucleotide encoding the polypeptide and a vector containing the polynucleotide.

[0076] Another aspect of the present application provides a method for degrading glucan, comprising: reacting one or more of the following with glucan: a polypeptide having the glucanase activity of the present application and a microorganism, wherein the microorganism comprises one or more of the polypeptide, a polynucleotide encoding the modified polypeptide and a vector containing the polynucleotide.

[0077] The polypeptide having the glucanase activity of the present application and / or a microorganism comprising one or more of the polypeptide, the polynucleotide encoding the modified polypeptide and the vector containing the polynucleotide can be used to degrade glucan-containing materials.

[0078] In one example, in addition to the 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.

[0079] 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.).

[0080] 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, etc., but are not limited thereto.

[0081] Another aspect of the present application provides a feed additive composition comprising one or more of the following: a polypeptide having the glucanase activity of the present application and a microorganism; the microorganism comprises one or more of the polypeptide, a polynucleotide encoding the modified polypeptide and a vector containing the polynucleotide.

[0082] 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 polypeptide having the glucanase activity of the present application and a microorganism, wherein the microorganism comprises one or more of a polypeptide, a polynucleotide encoding a modified polypeptide and a vector containing the polynucleotide.

[0083] The microorganism comprising the polypeptide having the glucanase activity of the present application, the polynucleotide encoding the modified polypeptide and / or the vector containing the polynucleotide can be used in any of the following applications:

[0084] a) additives in animal feed; and / or

[0085] b) feed supplements for animals; and / or

[0086] c) Decomposition of cereal-based material (eg this may be whole cereals or cereal parts).

[0087] 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.

[0088] The glucanase 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 applications 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 inbrewing applications, J Ind Microbiol Biotechnol, 2005; Chaari F, Belghith-FendriL, 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 laundry detergentcompatible lichenase: statistical optimization for production under solidstate fermentation on crude millet,Ind Crops Prod, 2013 et al., are incorporated by reference.

[0089] Another aspect of the present application provides a food composition; and a composition for preparing food, comprising the beta-glucanase of the present application or the microorganism of the present application. In one example, the beta-glucanase can be used for brewing (beer, wine, etc.) and for producing fruit juice. Adding beta-glucanase during saccharification can reduce the viscosity of wort, improve filterability, increase the dissolution rate of malt, prevent beer turbidity, and stabilize beer quality.

[0090] Another aspect of the application provides a detergent composition comprising the beta-glucanase of the application or the microorganism of the application.Detergent compositions are compositions for removing unwanted compounds from articles to be cleaned such as fabrics, dishes or hard surfaces. The example of detergent compositions comprises fabric detergents, textile detergents, cleaning agents for the hard surfaces of glass, timber, plastics, pottery, metal countertops and windows, carpet cleaners, oven cleaners, fabric fragrances, fabric softeners, dishwasher detergents etc.

[0091] Another aspect of the application provides a textile composition comprising the beta-glucanase of the application or the microorganism of the application. The beta-glucanase of the application can be used for processing the fiber for making clothing. These processings include the bio-polishing operation of removing dyestuff and cotton or cotton blended fabrics after bleaching, dyeing.

[0092] Another aspect of the present application provides a paper processing composition comprising the glucanase of the present application or the microorganism of the present application. The glucanase of the present application can be used to improve wastewater in the pulp industry, remove ink or decolorize.

[0093] In any of the above embodiments, the composition provided in the present application may also include any constituent elements applicable to various industrial fields. In any of the above embodiments, the composition provided in the present application may also include any other enzymes applicable to the intended use.

[0094] In any of the above embodiments, the composition provided in the present application may include the glucanase of the present application or the microorganism of the present application in an amount suitable for the intended use.

[0095] 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.

[0096] Another aspect of the present application provides a method for preparing a polypeptide having glucanase activity, comprising: culturing a microorganism comprising one or more of a polypeptide having glucanase activity of the present application, a polynucleotide encoding the polypeptide, and a vector containing the polynucleotide.

[0097] The culturing may include growing the microorganism in a culture medium.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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 the aerobic conditions of 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.

[0104] 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.

[0105] In one embodiment, the method for preparing the polypeptide having glucanase activity of the present application may further include recovering the polypeptide having glucanase activity of the present application expressed in the culturing step.

[0106] In another embodiment, the polypeptide with glucanase activity expressed in the culturing 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.

[0107] Regarding the recovery method, the polypeptide can be collected using the 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 polypeptide of the present application can be recovered from a culture medium or a host cell using a suitable method known in the art.

[0108] In another embodiment, the polypeptide expressed by the host cell during the culturing step may not be recovered. In this embodiment, the host cell expressing the polypeptide may be used as a source of the polypeptide.

[0109] Another aspect of the present application provides a composition comprising one or more of the following: a polypeptide having the glucanase activity of the present application and a microorganism, wherein the microorganism comprises one or more of the polypeptide, a polynucleotide encoding the polypeptide and a vector containing the polynucleotide.

[0110] 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.

[0111] [Mode for Carrying Out the Invention]

[0112] 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.

[0113] Example 1. Preparation of protein with glucanase activity

[0114] Example 1-1. Preparation of template

[0115] The reference gene corresponding to 1,3-1,4-β-glucanase in the genomic DNA of the Saccharomyces PC-2 strain was codon-optimized for expression in Escherichia coli (SEQ ID NO: 1). In addition, in order to improve heat resistance, a variant gene (in which multiple point mutations were introduced based on SEQ ID NO: 1 as a template) was codon-optimized (SEQ ID NO: 3). Finally, the optimized gene was synthesized by Cosmogenetech Co., Ltd.

[0116] The reference sequence was used as a control for the experiments, and the synthetic control and variant genes were each cloned into pET28a vectors (pET28-W, pET28-V) for expression.

[0117] Example 1-2. Preparation of transformants having glucanase gene and enzyme expression

[0118] The recombinant plasmids pET28-W and pET28-V were transformed with E. coli BL21. To generate the control and variants, the transformants were inoculated into 5 ml of LB medium and pre-cultured at 37°C for 18 h. 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).

[0119] Example 1-3. Purification of a protein having glucanase activity

[0120] 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 (only 20 mM imidazole in the lysis buffer composition) and an elution buffer (only 250 mM imidazole in the lysis buffer composition) in sequence to obtain purified controls and variants.

[0121] Example 2. Evaluation of properties of glucanase variants

[0122] Example 2-1. Measurement of glucanase variant activity

[0123] To evaluate the properties of the control and variants, 50 μL of 1% dextran, 5 μL of 1M buffer and an appropriate amount of enzyme were mixed 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 was as follows:

[0124] 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.

[0125] Example 2-2. Evaluation of optimal conditions for glucanase variants

[0126] To evaluate the optimal pH conditions, citrate buffer (pH 3-5), potassium phosphate buffer (pH 6-7), Tris-HCl buffer (pH 8-9) and glycine-NaOH buffer (pH 10-12) were used. In addition, 50 mM sodium acetate (pH 5.2) was used as a buffer to evaluate the optimal temperature conditions, and the activity was measured at 40°C, 50°C, 60°C, 70°C, 75°C and 80°C, respectively.

[0127] To assess the thermotolerance of the glucanase variants, appropriate amounts of enzyme were allowed to stand at 60°C, 70°C, 75°C and 80°C for 5 minutes each and then the activity was assessed at 37°C.

[0128] Example 3. Results of evaluation of the properties of glucanase variants

[0129] Example 3-1. Evaluation of optimal conditions

[0130] The optimal conditions for the glucanase were evaluated for each temperature and pH. As a result of the evaluation, both the control group and the variants showed higher relative activity at pH 5-7. The activity was highest at pH 6, and the activity was rapidly lost under alkaline conditions ( Figure 1 ).

[0131] However, the two enzymes differed significantly in temperature characteristics. Although similar characteristics were observed before 60°C, at 70°C, only 4% of the activity was retained in the control group, while the variant showed 100% activity. In addition, the variant showed 24% activity even at 80°C, based on which it was confirmed that the variant had enhanced activity at high temperatures ( Figure 2 ).

[0132] Example 3-2. Evaluation of heat resistance

[0133] To evaluate the thermotolerance of the glucanase, the purified control and variants were placed in various heat conditions ranging from 60°C to 80°C, and then the residual activity was evaluated at 37°C, and the activity was evaluated based on the activity at 37°C (100%) ( Figure 3 ).

[0134] As a result, it was confirmed that the control group retained 81% of activity at 60°C, but lost all activity at 70°C or higher. In contrast, the variant was confirmed to show 73% residual activity at 75°C, and no activity loss occurred at 70°C or lower.

[0135] Based on the above, it was confirmed that the novel glucanase of the present application has improved heat resistance and thermostability.

[0136] 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 polypeptide as shown in SEQ ID NO: 4, which has glucanase activity.

2. The polypeptide according to claim 1, wherein the polypeptide has improved one or more properties selected from the group consisting of heat resistance and thermal stability compared to the polypeptide of SEQ ID NO:

2.

3. A polynucleotide encoding the polypeptide shown in SEQ ID NO:

4.

4. A microorganism comprising one or more of a polypeptide as shown in SEQ ID NO: 4; a polynucleotide encoding the polypeptide; and a vector containing the polynucleotide.

5. A composition for degrading glucan, comprising one or more of the following: a polypeptide as shown in SEQ ID NO:4 and a microorganism, wherein the microorganism comprises one or more of the polypeptide as shown in SEQ ID NO:4, a polynucleotide encoding the polypeptide and a vector containing the polynucleotide.

6. A method for degrading glucan, comprising: One or more of the following are reacted with glucan: a polypeptide as shown in SEQ ID NO: 4 and a microorganism, wherein the microorganism comprises one or more of the polypeptide as shown in SEQ ID NO: 4, a polynucleotide encoding the polypeptide, and a vector containing the polynucleotide.

7. A composition comprising one or more of the following: a polypeptide as shown in SEQ ID NO:4 and a microorganism, wherein the microorganism comprises one or more of a polypeptide as shown in SEQ ID NO:4, a polynucleotide encoding the polypeptide, and a vector containing the polynucleotide.

8. 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 following: a polypeptide as shown in SEQ ID NO:4 and a microorganism, wherein the microorganism comprises one or more of a polypeptide as shown in SEQ ID NO:4, a polynucleotide encoding the polypeptide, and a vector containing the polynucleotide.

9. The composition according to claim 7, wherein the composition is used for food, feed additive, food preparation, detergent, textile or paper processing.

10. A method for preparing a polypeptide having glucanase activity, comprising: Cultivating a microorganism comprising the polypeptide shown in SEQ ID NO:4; One or more of a polynucleotide encoding the polypeptide; and a vector containing the polynucleotide.

Images