Enzyme preparations containing transglutaminase and their uses

Abstract

The present invention addresses the problem of providing: an enzyme agent containing a transglutaminase having high reactivity at a low temperature; and use of the enzyme agent. The present invention provides an enzyme agent that contains, as an active ingredient, a transglutaminase having an amino acid sequence that has a sequence identity of 90% or more with respect to the amino acid sequence of SEQ ID NO: 1.

Description

Technical Field

[0001] The present invention relates to an enzyme agent (transglutaminase agent) containing transglutaminase as an active ingredient and its uses.

Background Art

[0002] Transglutaminase (also referred to as TG) is an enzyme that catalyzes the acyl transfer reaction of the γ-carboxylamide group of glutamine residues within a peptide chain. When the ε-amino group of a lysine residue in a protein acts as an acyl acceptor, an ε-(γ-Gln)-Lys cross-linkage is formed intramolecularly or intermolecularly in the protein molecule.

[0003] Although various uses of transglutaminase in protein processing have been developed, few enzymes have been put into practical use. Many of those used in food applications are derived from the genus Streptomyces or its related species, and other than that, transglutaminase derived from the genus Bacillus is known. Patent Document 1 discloses transglutaminase derived from the genus Streptomyces, and Patent Document 2 discloses transglutaminase derived from Kutzneria albida.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] When transglutaminase is used in meat and seafood processing, it is required to react at low temperatures, but existing transglutaminases have insufficient reactivity at low temperatures. The problem that this invention aims to solve is to provide an enzyme preparation containing a transglutaminase with high reactivity at low temperatures, and its applications. [Means for solving the problem]

[0006] The inventors have discovered a novel transglutaminase derived from Longimycelium tulufanense and identified its amino acid sequence. The sequence identity between the amino acid sequence of the novel transglutaminase of the present invention and the amino acid sequence of the transglutaminase derived from the genus Streptomyces described in Patent Document 1 was 29%. Furthermore, the sequence identity between the amino acid sequence of the novel transglutaminase of the present invention and the amino acid sequence of the transglutaminase derived from Kutzneria albida described in Patent Document 2 was 65%. In addition, the inventors measured the reactivity of the novel transglutaminase identified above and found that it exhibits high reactivity at low temperatures. The present invention was completed based on the above findings.

[0007] The present invention provides the following: <1> An enzyme preparation containing a transglutaminase as an active ingredient, which has an amino acid sequence having more than 90% sequence identity with the amino acid sequence of Sequence ID No. 1. <2> Transglutaminase is derived from the genus Longimycelium. <1> The enzyme preparation described above. <3> An enzyme preparation containing transglutaminase as the active ingredient, derived from the genus Longimycelium, with a molecular weight of 28-30 kDa as measured by SDS-PAGE, and an enzyme activity at 20°C that is 20% or more of the enzyme activity at 50°C. <4> The optimal pH is pH 6.5 to 7.5. <3> The enzyme preparation described above. <5> The enzyme activity after heat treatment at 50°C for 40 minutes is 60% or more of the enzyme activity before heat treatment. <3> or <4> The enzyme preparation described above. <6> The enzyme activity at pH 4 is 50% or more of the enzyme activity at pH 8. <3> from <5> An enzyme preparation as described in one of the following. <7> <1> from <6> A method for modifying a protein or peptide, comprising acting an enzyme preparation described in any one of the above onto a substrate. <8> <1> from <6> A method for producing food, comprising applying an enzyme preparation described in any one of the following to food. <9> <1> from <6> Foods treated with any one of the enzyme preparations listed below. <10> A method for producing a transglutaminase comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, comprising the step of culturing a bacterium of the genus Longimycelium. <11> A method for producing transglutaminase, comprising the step of culturing the Longimycelium tulufanense NBRC107726 strain. [Effects of the Invention]

[0008] The enzyme preparation containing transglutaminase according to the present invention exhibits high reactivity at low temperatures and is suitable for use in the food and medical fields. [Brief explanation of the drawing]

[0009] [Figure 1] Figure 1 shows the results of confirming the molecular weight of transglutaminase derived from Longimycelium tulufanense. [Figure 2] Figure 2 shows a comparison of the optimal temperatures for transglutaminase derived from Longimycelium tulufanense and transglutaminase derived from S. mobaraensis. [Figure 3] Figure 3 shows a comparison of the optimal pH for transglutaminase derived from Longimycelium tulufanense and transglutaminase derived from S. mobaraensis. [Figure 4]Figure 4 shows a comparison of the thermal stability (50°C) of transglutaminase derived from Longimycelium tulufanense and transglutaminase derived from S. mobaraensis. [Figure 5] Figure 5 shows a comparison of the pH stability of transglutaminase derived from Longimycelium tulufanense and transglutaminase derived from S. mobaraensis. [Figure 6] Figure 6 shows a comparison of the cross-linking reactivity of transglutaminase derived from Longimycelium tulufanense and transglutaminase derived from S. mobaraensis. [Figure 7] Figure 7 shows a comparison of the cross-linking activity of transglutaminase derived from Longimycelium tulufanense and transglutaminase derived from S. mobaraensis in soybeans. [Figure 8] Figure 8 shows a comparison of the cross-linking activity of transglutaminase derived from Longimycelium tulufanense and transglutaminase derived from S. mobaraensis in peas. [Figure 9] Figure 9 shows a comparison of the cross-linking activity of transglutaminase derived from Longimycelium tulufanense and transglutaminase derived from S. mobaraensis in almonds. [Figure 10] Figure 10 shows a comparison of the cross-linking activity of transglutaminase derived from Longimycelium tulufanense and transglutaminase derived from S. mobaraensis in rye. [Figure 11] Figure 11 shows a comparison of the cross-linking activity of transglutaminase derived from Longimycelium tulufanense and transglutaminase derived from S. mobaraensis in wheat. [Figure 12] Figure 12 shows a comparison of the cross-linking activity of transglutaminase from Longimycelium tulufanense and transglutaminase from S. mobaraensis in coconut. [Figure 13] Figure 13 shows the comparison of the cross-linking activities of the transglutaminases derived from Longimycelium tulufanense and S. mobaraensis against thioseeds. [Figure 14] Figure 14 shows the results of each natural raw material shown in FIGS. 7 to 13 at the time of 21-hour reaction. The transglutaminase derived from Longimycelium tulufanense is shown on the left side of each column, and the transglutaminase derived from S. mobaraensis is shown on the right side of each column. [Figure 15] Figure 15 shows the results of each natural raw material shown in FIGS. 7 to 13 at the time of 21-hour reaction at a reaction temperature of 5°C. The transglutaminase derived from Longimycelium tulufanense is shown on the left side of each column, and the transglutaminase derived from S. mobaraensis is shown on the right side of each column. [Figure 16] Figure 16 shows the comparison of the strength of tofu when tofu is produced using the transglutaminase derived from Longimycelium tulufanense or the transglutaminase derived from S. mobaraensis. The transglutaminase derived from Longimycelium tulufanense is shown on the left side of each column, and the transglutaminase derived from S. mobaraensis is shown on the right side of each column. [Figure 17] Figure 17 shows the comparison of the strength of soy milk yogurt when soy milk yogurt is produced using the transglutaminase derived from Longimycelium tulufanense or the transglutaminase derived from S. mobaraensis. The transglutaminase derived from Longimycelium tulufanense is shown on the left side of each column, and the transglutaminase derived from S. mobaraensis is shown on the right side of each column.

Embodiments for Carrying Out the Invention

[0010] <00(...)​​The enzyme preparation of the present invention contains a transglutaminase as an active ingredient, which consists of the amino acid sequence of Sequence ID No. 1 or an amino acid sequence equivalent to said amino acid sequence. An "equivalent amino acid sequence" refers to an amino acid sequence that differs in part from the reference amino acid sequence (the amino acid sequence of SEQ ID NO: 1), but where this difference does not substantially affect the protein's function (in this case, transglutaminase activity). Therefore, an enzyme with an equivalent amino acid sequence catalyzes the enzymatic reaction by transglutaminase. The degree of activity is not particularly limited as long as it can perform its function as a transglutaminase. However, it is preferable that the activity is at or above the level of an enzyme consisting of the reference amino acid sequence (having the amino acid sequence of SEQ ID NO: 1).

[0011] The amino acid sequence of Sequence ID No. 1 is the amino acid sequence (mature) of transglutaminase derived from Longimycelium tulufanense. The full-length amino acid sequence of transglutaminase derived from Longimycelium tulufanense is shown in Sequence ID No. 2.

[0012] A "partial difference in the amino acid sequence" can result from, for example, the deletion or substitution of one or more amino acids in the amino acid sequence, the addition or insertion of one or more amino acids to the amino acid sequence, or any combination thereof. A partial difference in the amino acid sequence is acceptable as long as transglutaminase activity is maintained (some variation in activity is acceptable). As long as this condition is met, the location of the amino acid sequence difference is not particularly limited. Furthermore, the amino acid sequence difference may occur at multiple locations.

[0013] The number of amino acids that result in some differences in the amino acid sequence is, for example, less than about 30%, less than about 20%, or less than about 10% of the total amino acids that make up the amino acid sequence, preferably less than about 8%, more preferably about 6%, even more preferably less than about 4%, even more preferably less than about 2%, and most preferably less than about 1%. Accordingly, the equivalent protein has, for example, about 70% or more, about 80% or more, or about 90% or more, preferably about 92% or more, more preferably about 94% or more, even more preferably about 96% or more, even more preferably about 98% or more, and most preferably about 99% or more identity with the reference amino acid sequence.

[0014] One typical example of a "partial difference in amino acid sequence" is a mutation (change) in the amino acid sequence resulting from the deletion or substitution of 1 to 40 (preferably 1 to 30, more preferably 1 to 10, even more preferably 1 to 7, even more preferably 1 to 5, and even more preferably 1 to 3) amino acids among the amino acids that make up the amino acid sequence, the addition or insertion of 1 to 40 (preferably 1 to 30, more preferably 1 to 10, even more preferably 1 to 7, even more preferably 1 to 5, and even more preferably 1 to 3) amino acids to the amino acid sequence, or a combination thereof.

[0015] Preferably, an equivalent amino acid sequence is obtained by conserved amino acid substitutions occurring at amino acid residues that are not essential for transglutaminase activity. Here, "conservative amino acid substitution" refers to the substitution of a certain amino acid residue with an amino acid residue having a side chain of similar properties. Amino acid residues are classified into several families based on their side chains, such as basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Conservative amino acid substitutions are preferably substitutions between amino acid residues within the same family.

[0016] The identity (%) of two amino acid sequences can be determined, for example, by the following procedure. First, the two sequences are aligned to allow for optimal comparison (for example, a gap may be introduced in the first sequence to optimize the alignment with the second sequence). When a molecule (amino acid residue) at a specific position in the first sequence is the same as the molecule at the corresponding position in the second sequence, the molecules at that position are said to be identical. The identity of the two sequences is a function of the number of identical positions common to the two sequences (i.e., identity (%) = number of identical positions / total number of positions × 100), and preferably, the number and size of gaps required for alignment optimization are also taken into consideration.

[0017] The comparison and determination of identity between two sequences can be achieved using mathematical algorithms. A concrete example of a mathematical algorithm usable for sequence comparison is the algorithm described in Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, which was modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77, but is not limited to this. Amino acid sequence identity can be obtained, for example, using blastp (protein-protein BLAST) from the National Center for Biotechnology Information (NCBI). Default parameters can be used, but for example, using the BLOSUM62 matrix and setting Gap Costs to Existence: 11 and Extension: 1 is recommended.

[0018] The transglutaminase, which is the active ingredient of the enzyme preparation of the present invention, may be part of a larger protein (e.g., a fusion protein). Examples of sequences to be added to the fusion protein include sequences useful for purification, such as multiple histidine residues, and sequences to ensure stability during recombinant production.

[0019] The transglutaminase in this invention (hereinafter also referred to as "this enzyme") can be obtained by culturing a microorganism that produces the transglutaminase (a transglutaminase-producing strain), such as a bacterium of the genus Longimycelium, for example, Longimycelium tulufanense. The transglutaminase-producing strain may be a wild-type strain or a mutant strain (for example, a mutant strain can be obtained by ultraviolet irradiation). A specific example of a transglutaminase-producing strain is Longimycelium tulufanense (NBRC107726). Longimycelium tulufanense (NBRC107726) is a strain stored at the NBRC (National Institute of Technology and Evaluation, Biotechnology Center), and can be obtained by following prescribed procedures.

[0020] This enzyme can be prepared from the culture medium and / or cells of a microorganism that produces this enzyme. The culture conditions and methods are not particularly limited, as long as the enzyme is produced. That is, given that the enzyme is produced, methods and conditions suitable for culturing the microorganism used can be appropriately set. Either liquid culture or solid culture may be used, but liquid culture is preferred. The culture conditions for liquid culture will be explained as an example.

[0021] The culture medium is not particularly limited as long as it is a medium on which the microorganisms used can grow. For example, a carbon source such as glucose, sucrose, genthiobiose, soluble starch, glycerin, dextrin, molasses, or organic acids, a nitrogen source such as ammonium sulfate, ammonium carbonate, ammonium phosphate, ammonium acetate, or gelatin, peptone, yeast extract, corn steep liquor, casein hydrolysate, bran, or meat extract, or an inorganic salt such as potassium salt, magnesium salt, sodium salt, phosphate, manganese salt, iron salt, or zinc salt can be added. Vitamins, amino acids, etc., may be added to the culture medium to promote the growth of the microorganisms used. The pH of the culture medium should be adjusted to, for example, about 3 to 8, preferably about 4 to 7, and the culture temperature should be usually about 20 to 40°C, preferably about 25 to 35°C, and the culture should be carried out under aerobic conditions for 1 to 20 days, preferably about 3 to 10 days. As for the culture method, for example, the shaking culture method or the aerobic deep culture method using a jar fermenter can be used.

[0022] After culturing under the above conditions, the target enzyme is recovered from the culture medium or bacterial cells. When recovering from the culture medium, for example, insoluble matter can be removed by filtering or centrifugation of the culture supernatant, and then the enzyme can be obtained by separating and purifying it using a combination of methods such as ultrafiltration, salting out by ammonium sulfate precipitation, dialysis, or various chromatography methods using ion exchange resins. On the other hand, when recovering from bacterial cells, for example, the bacterial cells can be crushed by pressurization or sonication, and then the enzyme can be obtained by separating and purifying them in the same manner as above. Alternatively, the bacterial cells can be recovered from the culture medium beforehand by filtration or centrifugation, and then the above series of steps (crushing, separation, and purification of bacterial cells) can be performed.

[0023] This enzyme can also be easily prepared by genetic engineering techniques. For example, it can be prepared by transforming a suitable host cell (e.g., E. coli) with the DNA encoding this enzyme and recovering the protein expressed in the transformant. The recovered protein can then be purified as appropriate according to the purpose. Obtaining the target enzyme as a recombinant protein in this way allows for various modifications. For example, by inserting the DNA encoding this enzyme and another suitable DNA into the same vector and using that vector to produce recombinant proteins, this enzyme can be obtained as a recombinant protein to which any peptide or protein is linked. Furthermore, modifications such as the addition of glycans and / or lipids, or modifications that result in N-terminal or C-terminal processing, may be performed. Such modifications make it possible to simplify the extraction and purification of recombinant proteins, or to add biological functions.

[0024] Normally, gene expression and the recovery of the expression product (the enzyme) are carried out using a suitable host-vector system as described above, but a cell-free synthesis system may also be used. Here, a "cell-free synthesis system (cell-free transcription system, cell-free transcription / translation system)" refers to the in vitro synthesis of mRNA and proteins encoded by nucleic acids (DNA or mRNA) from a template, using ribosomes and transcription / translation factors derived from living cells (or obtained by genetic engineering techniques), rather than using living cells. In a cell-free synthesis system, a cell extract obtained by purifying a cell lysate as needed is generally used. The cell extract generally contains ribosomes, various factors such as initiation factors, and various enzymes such as tRNA, which are necessary for protein synthesis. When synthesizing proteins, various amino acids, energy sources such as ATP and GTP, and other substances necessary for protein synthesis, such as creatine phosphate, are added to this cell extract. Of course, ribosomes, various factors, and / or various enzymes prepared separately may be supplemented as needed during protein synthesis.

[0025] The development of transcription / translation systems that reconstitute each molecule (factor) necessary for protein synthesis has also been reported (Shimizu, Y. et al.: Nature Biotech., 19, 751-755, 2001). In this synthesis system, genes for 31 factors, consisting of three initiation factors, three elongation factors, four factors involved in termination, 20 aminoacyl-tRNA synthetases that bind each amino acid to tRNA, and methionyl-tRNA formyltransferase, which constitute the bacterial protein synthesis system, are amplified from the E. coli genome, and the protein synthesis system is reconstituted in vitro using these. The present invention may also utilize such a reconstituted synthesis system.

[0026] The term "cell-free transcription / translation system" is used interchangeably with "cell-free protein synthesis system," "in vitro translation system," or "in vitro transcription / translation system." In an in vitro translation system, RNA is used as a template to synthesize proteins. Template RNA can include total RNA, mRNA, or in vitro transcripts. In contrast, in vitro transcription / translation systems use DNA as a template. The template DNA should contain a ribosome-binding region and preferably a suitable terminator sequence. In an in vitro transcription / translation system, conditions are set up with the necessary factors added to ensure that the transcription and translation reactions proceed sequentially.

[0027] The purified enzyme obtained as described above can also be provided in powder form by methods such as freeze-drying, vacuum drying, or spray drying. In this case, the purified enzyme may be pre-dissolved in acetate buffer, phosphate buffer, triethanolamine buffer, Tris-HCl buffer, or GOOD's buffer. Preferably, acetate buffer, phosphate buffer, or triethanolamine buffer can be used. Examples of GOOD's buffers include PIPES, MES, or MOPS.

[0028] The degree of purity of the enzyme is not particularly limited. Furthermore, the final form may be liquid or solid (including powder).

[0029] Through our investigations, the properties of the transglutaminase derived from Longimycelium tulufanense, consisting of the amino acid sequence of SEQ ID NO: 1, were determined as follows (see the examples below for details). Therefore, this enzyme can also be identified by the following enzymatic chemical properties. Details of the measurement conditions and procedures for transglutaminase activity when evaluating each enzymatic chemical property are shown in the examples below.

[0030] (1) Effect This enzyme is a transglutaminase.

[0031] (2) Optimal temperature and low-temperature reactivity When comparing temperatures at 10, 20, 30, 40, 50, 60, and 70°C, the optimal temperature for this enzyme is approximately 50°C. If the enzyme activity of this enzyme at a reaction temperature of 50°C is taken as 100%, the relative enzyme activity of this enzyme at a reaction temperature of 20°C is 20% or more (preferably 30% or more, more preferably 40% or more, and even more preferably 50% or more). When the enzyme activity of this enzyme at a reaction temperature of 50°C is taken as 100%, preferably the relative enzyme activity of this enzyme at a reaction temperature of 5°C is 10% or more (preferably 15% or more, more preferably 20% or more), the relative enzyme activity of this enzyme at a reaction temperature of 10°C is 10% or more (preferably 20% or more, more preferably 30% or more), the relative enzyme activity of this enzyme at a reaction temperature of 30°C is 40% or more (preferably 50% or more, more preferably 60% or more, even more preferably 70% or more), and the relative enzyme activity of this enzyme at a reaction temperature of 40°C is 70% or more (preferably 80% or more, more preferably 90% or more).

[0032] (3) Optimal pH When comparing pH levels of 5, 6, 7, 8, 9, and 10, the optimal pH for this enzyme is pH 7.

[0033] (4) Thermal stability (50℃) The enzyme activity of this enzyme after heat treatment at 50°C for 40 minutes is 60% or more (preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more) of the enzyme activity before heat treatment. Preferably, this enzyme does not experience a substantial decrease in activity even after heat treatment at 50°C for 10 minutes. Preferably, this enzyme does not experience a substantial decrease in activity even after heat treatment at 50°C for 20 minutes. Preferably, the enzyme activity after heat treatment at 50°C for 80 minutes is 50% or more (preferably 60% or more, more preferably 70% or more, and even more preferably 80% or more) of the enzyme activity before heat treatment. Preferably, the enzyme activity of the enzyme after heat treatment at 50°C for 120 minutes is 40% or more (preferably 50% or more, more preferably 60% or more) of the enzyme activity before heat treatment.

[0034] (5)pH stability This enzyme exhibits an enzymatic activity at pH 4 that is 50% or more (preferably 60% or more, more preferably 70% or more, and even more preferably 80% or more) of its enzymatic activity at pH 8. Preferably, the enzyme activity at pH 5, pH 6, and pH 7 is 80% or more (preferably 85% or more, more preferably 90% or more) of the enzyme activity at pH 8. Preferably, the enzyme activity at pH 3 is 30% or more of the enzyme activity at pH 8 (preferably 40% or more, more preferably 50% or more, even more preferably 60% or more, and particularly preferably 70% or more). Preferably, the enzyme activity at pH 2 is 20% or more (preferably 30% or more, more preferably 40% or more, and even more preferably 50% or more) of the enzyme activity at pH 8. Preferably, the enzyme's activity at pH 11 is 80% or more (preferably 85% or more, more preferably 90% or more) of its activity at pH 8.

[0035] Furthermore, the molecular weight of this enzyme, as measured by SDS-PAGE (SDS-polyacrylamide gel electrophoresis), is 28-30 kDa. On the other hand, the molecular weight calculated from the amino acid sequence of Sequence ID No. 2 is 30 kDa.

[0036] The content of the active ingredient (this enzyme) in the enzyme preparation of the present invention is not particularly limited, but for example, the content of the active ingredient can be set or adjusted so that the transglutaminase activity per gram of this enzyme preparation is 0.1 U to 5000 U, preferably 1 U to 500 U, and more preferably 10 U to 300 U. The definition of the unit of transglutaminase activity (1 U) is as described in the examples below.

[0037] The enzyme preparation of the present invention may contain, in addition to the active ingredient (modified enzyme of the present invention), excipients, buffers, suspending agents, stabilizers, preservatives, antiseptics, physiological saline, various proteins, various protein decomposition products, various extracts, various salts, various antioxidants, cysteine, glutathione, sodium glutamate, sodium inosinate, sodium guanylate, calcined seashell calcium, silicon dioxide, etc. Excipients that can be used include starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, sucrose, glycerol, etc. Buffers that can be used include phosphates, citrates, acetates, etc. Stabilizers that can be used include propylene glycol, ascorbic acid, etc. Preservatives that can be used include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, etc. Antiseptics that can be used include ethanol, benzalkonium chloride, parahydroxybenzoic acid, chlorobutanol, etc. Examples of proteins include soy protein, wheat protein, corn protein, milk protein, and animal-derived protein. Examples of extracts include meat extract, plant extract, and yeast extract. Examples of salts include chlorides, phosphates, polyphosphates, pyrophosphates, citrates, lactates, and carbonates. Examples of antioxidants include L-ascorbate and sodium bisulfite. The form of the enzyme preparation of the present invention is not particularly limited and may be, for example, powder, granules, liquid, or capsule.

[0038] 2. Genes The gene encoding this enzyme consists of DNA encoding a protein containing the amino acid sequence of SEQ ID NO: 1. Specific examples of this embodiment are DNA consisting of the nucleotide sequence shown in SEQ ID NO: 3 and DNA consisting of the nucleotide sequence shown in SEQ ID NO: 4. The DNA of SEQ ID NO: 3 encodes only the amino acid sequence of the mature protein (SEQ ID NO: 1), while the DNA of SEQ ID NO: 4 encodes the signal peptide and pro-sequence in addition to the mature protein (the amino acid sequence of SEQ ID NO: 1).

[0039] The gene encoding this enzyme is typically used for the preparation of the enzyme. Genetic engineering methods using this enzyme-encoding gene allow for the acquisition of a more homogeneous enzyme. Furthermore, this method is suitable for preparing large quantities of the enzyme. However, the uses of the gene encoding this enzyme are not limited to its preparation. For example, the nucleic acid can be used as an experimental tool for elucidating the mechanism of action of this enzyme, or as a tool for designing or creating variants (modified versions) of this enzyme.

[0040] In this specification, "the gene encoding this enzyme" refers to the nucleic acid from which this enzyme can be obtained when that gene is expressed. This includes not only nucleic acids having a base sequence corresponding to the amino acid sequence of this enzyme, but also nucleic acids obtained by adding a sequence that does not encode an amino acid sequence to such a nucleic acid. Codon degeneracy is also taken into consideration.

[0041] Nucleic acids can be prepared in an isolated state by standard genetic engineering, molecular biological, biochemical, chemical synthesis, PCR (e.g., overlap PCR), or a combination thereof, with reference to the sequence information disclosed herein or in the accompanying sequence listings.

[0042] Nucleic acids (hereinafter also referred to as "equivalent nucleic acids," and the base sequence defining an equivalent nucleic acid, also referred to as the "equivalent base sequence") may be used, whose function is equivalent to that of the gene encoding this enzyme, but whose base sequence differs in some respects. An example of an equivalent nucleic acid is DNA that encodes a protein having characteristic enzymatic activity (i.e., transglutaminase activity) based on the base sequence of the nucleic acid encoding this enzyme, and which consists of a base sequence containing one or more base substitutions, deletions, insertions, additions, or inversions. Base substitutions and deletions may occur at multiple sites. The term "multiple" here varies depending on the position and type of amino acid residues in the three-dimensional structure of the protein encoded by the nucleic acid, but is for example 2 to 40 bases, preferably 2 to 20 bases, and more preferably 2 to 10 bases. The equivalent nucleic acid has sequence identity of, for example, 70% or more, 80% or more, 85% or more, or 90% or more with respect to the reference base sequence (SEQ ID NO: 3 or SEQ ID NO: 4), preferably 92% or more, more preferably 94% or more, even more preferably 96% or more, even more preferably about 98% or more, and most preferably 99% or more.

[0043] Equivalent nucleic acids like those described above can be obtained, for example, by restriction enzyme treatment, treatment with exonucleases or DNA ligases, or by introducing mutations using site-directed mutagenesis (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York) or random mutagenesis (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York). Equivalent nucleic acids can also be obtained by other methods such as ultraviolet irradiation.

[0044] Nucleic acids having a nucleotide sequence complementary to the nucleotide sequence of the gene encoding this enzyme may be used. Nucleic acids having a nucleotide sequence that is at least approximately 90%, 92%, 94%, 96%, 98%, or 99% identical to the nucleotide sequence of the gene encoding this enzyme, or a nucleotide sequence complementary thereto, may be used.

[0045] Nucleic acids having a base sequence that hybridizes under stringent conditions to a base sequence complementary to the base sequence of the gene encoding this enzyme or its equivalent base sequence may be used. "Stringent conditions" refer to conditions under which so-called specific hybrids are formed and nonspecific hybrids are not formed. Such stringent conditions are known to those skilled in the art and can be set by referring, for example, to Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York) or Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987). As stringent conditions, for example, one can incubate the samples at approximately 50°C using a hybridization solution (50% formamide, 10×SSC (0.15M NaCl, 15mM sodium citrate, pH 7.0), 5×Denhardt solution, 1% SDS, 10% dextran sulfate, 10 μg / ml denatured salmon sperm DNA, 50mM phosphate buffer (pH 7.5)), followed by washing at approximately 65°C with 0.1×SSC and 0.1% SDS. Even more preferred stringent conditions include, for example, using a hybridization solution consisting of 50% formamide, 5×SSC (0.15M NaCl, 15mM sodium citrate, pH 7.0), 1×Denhardt solution, 1% SDS, 10% dextran sulfate, 10 μg / ml denatured salmon sperm DNA, and 50mM phosphate buffer (pH 7.5).

[0046] Recombinant DNA containing the gene encoding this enzyme may be used. Recombinant DNA is provided, for example, in the form of a vector. In this specification, the term "vector" means a nucleic acid molecule that can transport the nucleic acid inserted therein into a target such as a cell.

[0047] An appropriate vector is selected depending on the intended use (cloning, protein expression) and the type of host cell. Examples of vectors that use E. coli as a host include M13 phage or its variants, λ phage or its variants, pBR322 or its variants (pB325, pAT153, pUC8, etc.), examples of vectors that use yeast as a host include pYepSec1, pMFa, pYES2, examples of vectors that use insect cells as a host include pAc, pVL, examples of vectors that use mammalian cells as a host include pCDM8, pMT2PC, etc.

[0048] The vector is preferably an expression vector. An "expression vector" is a vector that can introduce the inserted nucleic acid into a target cell (host cell) and express it within that cell. Expression vectors usually contain promoter sequences necessary for the expression of the inserted nucleic acid, as well as enhancer sequences that promote expression. Expression vectors containing selection markers can also be used. When such expression vectors are used, the selection marker can be used to confirm whether (and to what extent) the expression vector has been introduced.

[0049] Insertion of nucleic acids into vectors, insertion of selection marker genes (if necessary), insertion of promoters (if necessary), etc., can be performed using standard recombinant DNA techniques (see, for example, Molecular Cloning, Third Edition, 1.84, Cold Spring Harbor Laboratory Press, New York, which are well-known methods using restriction enzymes and DNA ligases).

[0050] For host cells, it is preferable to use microorganisms such as Escherichia coli, filamentous fungi (Aspergillus oryzae), actinomycetes (Streptomyces genus, Streptomyces lividan, Streptomyces mobaraensis), Bacillus subtilis, and budding yeast (Saccharomyces cerevisiae) due to their ease of handling. However, any host cell capable of replicating recombinant DNA and expressing the gene for this enzyme can be used. Examples of Escherichia coli include E. coli BL21(DE3)pLysS when using the T7 promoter, and E. coli JM109 otherwise. Examples of budding yeast include SHY2, AH22, or INVSc1 (Invitrogen).

[0051] Microorganisms possessing recombinant DNA (i.e., transformants) can be used. These microorganisms can be obtained by transfection or transformation using the vector of the present invention described above. For example, the calcium chloride method (Journal of Molecular Biology (J.Mol. Biol.), Vol. 53, p. 159 (1970)), the Hanahan method (Journal of Molecular Biology, Vol. 166, p. 557 (1983)), the SEM method (Gene, Vol. 96, p. 23 (1990)), the Chung et al. method (Proceedings of the National Academy of Sciences of the USA, Vol. 86, p. 2172 (1989)), calcium phosphate coprecipitation method, electroporation (Potter, H. et al., Proc. Natl. Acad. Sci. USA 81, 7161-7165 (1984)), lipofection (Felgner, PL et al., Proc. Natl. This can be carried out according to Acad. Sci. USA 84,7413-7417 (1984), etc. The microorganisms mentioned above can be used to produce this enzyme.

[0052] 3. Uses of enzyme preparations The present invention further relates to the uses of the enzyme preparation of the present invention. In other words, by acting the enzyme preparation of the present invention on a substrate (protein or peptide), the protein or peptide can be modified. Specific examples of its use include the production of food or food materials (protein-containing food materials) by acting the enzyme preparation of the present invention on food or food materials (protein-containing food materials). The quality of food can be improved by acting the enzyme preparation of the present invention on food. According to the present invention, food treated with the enzyme preparation of the present invention is provided. Furthermore, the enzyme preparation of the present invention can be used not only for the production of food or food materials (protein-containing food materials) but also for the production of industrial materials and pharmaceutical raw materials.

[0053] Specific examples of food products include, but are not limited to, processed meat products (e.g., processed meat products such as ham and bacon; processed meat products such as sausages, hamburgers, and meatballs), processed seafood products (e.g., fish sausage, kamaboko, chikuwa, satsuma-age, etc.), rice products (e.g., cooked white rice, sekihan, pilaf, takikomi gohan, porridge, risotto, onigiri, etc.), noodles (e.g., udon, pasta, soba, Chinese noodles, yakisoba, instant noodles, etc.), grain flours (e.g., wheat flour, barley flour, corn flour, buckwheat flour, etc.) and their processed products, dairy products (yogurt, cheese, butter, ice cream, etc.), tofu and its processed products, and plant-based protein foods (plant-based milk, plant-based yogurt (e.g., soy milk yogurt, etc.), meat substitutes, etc.). Food ingredients are not particularly limited, but include casein, soybeans, peas, chickpeas, broad beans, mung beans, almonds, oats, rye, wheat, coconut, chia seeds, and eggs. Soybeans, peas, rye, wheat, coconut, and chia seeds are particularly preferred as food ingredients.

[0054] The method for producing food using this enzyme is not particularly limited, but examples include a method that includes the following steps (a) and (b). (a) A step of applying the enzyme to food or food material, and (b) A step of inactivating or removing the enzyme contained in the obtained enzyme-treated food or enzyme-treated food material. [Examples]

[0055] Example 1: Preparation of transglutaminase derived from Longimycelium tulufanense Cells of Longimycelium tulufanense (obtained from the National Institute of Technology and Evaluation (NITE)) (NBRC107726) were cultured in trypto-sawyer broth medium (manufactured by Nissui Pharmaceutical Co., Ltd.) at 37°C for 4 days, and the supernatant was collected. The obtained supernatant was dialyzed with 20 mM phosphate buffer (pH 6) according to a standard procedure. The dialyzed sample was subjected to ion exchange chromatography using a cation exchange column (SP Sepharose FF, manufactured by GE Health Care) equilibrated with the same buffer, and the active fraction was desalted and concentrated to obtain a crude purified enzyme sample. The active fraction was confirmed by the method described in Example 3 below. The sample used in Example 3 was obtained by further gel filtration of the obtained crude purified enzyme sample using Superdex 200 HR.

[0056] Example 2: Sequence confirmation of transglutaminase derived from Longimycelium tulufanense. N-terminal analysis of the purified enzyme yielded the result "ATSLPPIAPPLPRGVQSKS". Based on this sequence, the protein was estimated from genomic information and found to match the amino acid sequence of a protein of unknown function (WP_189053072.1). Using this amino acid sequence as a reference, the region including the upstream and downstream regions was amplified from the genome by PCR, the base sequence was confirmed, and the amino acid sequence was identified.

[0057] Amino acid sequence (mature plant sequence only) (SEQ ID NO: 1) ATSLPPIAPPLPRGVQSKSWSVPDYIAAWEKQHGRPMTAEERYHLARGCIGVTVVNLDREDAPNPPLNLSFGTYQRAMEVQAALNEIVATRPSPREYAEQVRKHPALQGVQNLV RAFPTFIDPANLHAAIFSKRFYSKQDPNWTDEQAAEMYRPNPRTGQVDMSTYRYRARPGYVNFDYGWYDEQTNNWWHANHAEPGMKVYQSTLRYYSRPLLDFDEQVFTVAFARVA

[0058] Amino acid sequence (full length) *Underlined portion is the mature plant sequence (SEQ ID NO: 2) MKKWLPRALVALFVLLGLPAGLAGTAHAAVVAHAAV ATSLPPIAPPLPRGVQSKSWSVPDYIAAWEKQHGRPMTAEERYHLARGCIGVTVVNLDREDAPNPPLNLSFGTYQRAMEVQAALNEIVATRPSPREYAEQVRKHPALQGVQNLV RAFPTFIDPANLHAAIFSKRFYSKQDPNWTDEQAAEMYRPNPRTGQVDMSTYRYRARPGYVNFDYGWYDEQTNNWWHANHAEPGMKVYQSTLRYYSRPLLDFDEQVFTVAFARVA

[0059] Base sequence (mature) (SEQ ID NO: 3) gccacgtcgctgccaccgatcgcaccgccgcttccccggggcgtgcagagcaagagctggtcggtgccggactacattgccgcctgggagaagcaacacggtaggccgatgacggccgaggagcggtaccacctcgcccggggctgcatcggcgtcaccgtggtcaacctcgaccgggaggacgcgccgaacccgccgctcaacctgtcgttcggcacttaccagcgggccatggaagtgcaggccgcgctgaacgagatcgtcgcgacccggccgtcgccgcgggagtacgccgagcaggtgcgcaagcacccggcgctgcagggcgtgcagaacctggtgcgggcgttccccacgttcatcgacccggcgaacctgcacgccgccatcttctccaagcggttctactcgaagcaggacccgaactggaccgacgagcaggcggccgagatgtaccggccgaatccgcggaccggccaggtcgacatgagcacctaccgctaccgggcgcggccgggttacgtgaacttcgactacggttggtacgacgagcagacgaacaactggtggcacgccaaccacgcggagccgggcatgaaggtctaccagagcacgttgcggtactactcccggccgctgctggatttcgacgagcaggttttcaccgtggcgttcgcgcgggtcgcctga

[0060] Base sequence (full length) (SEQ ID NO: 4) gtgaagaagtggttgccgcgcgccctggtcgcgctgttcgtgctcctggggttacccgccggcctggccggaacggcacatgccgcggtcgtcgcgcacgccgccgtcgccacgtcgctgccaccgatcgcaccgccgcttccccggggcgtgcagagcaagagctggtcggtgccggactacattgccgcctgggagaagcaacacggtaggccgatgacggccgaggagcggtaccacctcgcccggggctgcatcggcgtcaccgtggtcaacctcgaccgggaggacgcgccgaacccgccgctcaacctgtcgttcggcacttaccagcgggccatggaagtgcaggccgcgctgaacgagatcgtcgcgacccggccgtcgccgcgggagtacgccgagcaggtgcgcaagcacccggcgctgcagggcgtgcagaacctggtgcgggcgttccccacgttcatcgacccggcgaacctgcacgccgccatcttctccaagcggttctactcgaagcaggacccgaactggaccgacgagcaggcggccgagatgtaccggccgaatccgcggaccggccaggtcgacatgagcacctaccgctaccgggcgcggccgggttacgtgaacttcgactacggttggtacgacgagcagacgaacaactggtggcacgccaaccacgcggagccgggcatgaaggtctaccagagcacgttgcggtactactcccggccgctgctggatttcgacgagcaggttttcaccgtggcgttcgcgcgggtcgcctga

[0061] Example 3: Confirmation of the molecular weight of the transglutaminase derived from Longimycelium tulufanense Transglutaminase derived from Longimycelium tulufanense (gel filtration purified sample) was electrophoresed on NuPAGE® Novex 4-12% Bis-Tris Gel w / MES and stained with Coomassie® Blue R-250. Mark12 was used as a molecular weight marker. TM An unstained standard was used. The results are shown in Figure 1. A band for this enzyme was observed at the 28-30 kDa position.

[0062] Example 4: Confirmation of various properties of the enzyme (Enzymes used) Transglutaminase derived from S. mobaraensis (control product) Transglutaminase derived from Longimycelium (the present invention)

[0063] (Activity measurement method) The enzyme was dissolved in 200 mM Tris-HCl pH 6.0 containing 0.4% cysteine ​​and treated at 30°C for 1 hour. The solution was then diluted to an appropriate concentration with 200 mM Tris-HCl pH 6.0 (sample solution). 100 μL of substrate solution (R-1) was added to 10 μL of the sample solution and mixed. The mixture was then reacted at 37°C for 10 minutes. 100 μL of color development solution (R-2) was added to stop the reaction and form an Fe complex. The absorbance at 525 nm was then measured. As a control, the absorbance of a similar reaction using pre-heat-inactivated enzyme solution was measured, and the absorbance difference from the sample solution was determined. Separately, a calibration curve was created using L-glutamic acid-γ-monohydroxamic acid instead of the enzyme solution, and the amount of hydroxamic acid produced was determined from the absorbance difference. Enzyme activity producing 1 μmol of hydroxamic acid per minute was defined as 1 unit (1 U).

[0064] (Substrate solution (R-1)) Dissolve 2.42 g of 2-amino-2-hydroxymethyl-1,3-propanediol, 0.70 g of hydroxylammonium hydrochloride, 0.31 g of reduced glutathione, and 1.01 g of Z-Gln-Gly (benzyloxycarbonyl-L-glutaminylglycine) in distilled water to make a total volume of 100 mL (pH 6.0).

[0065] (Substrate solution (R-2)) Mix 30 mL of 3M hydrochloric acid solution, 30 mL of 12% trichloroacetic acid solution, and 30 mL of 5% iron(III) chloride solution.

[0066] (Method for confirming properties) <Method for confirming the optimal temperature> For the measurement, the temperature was changed to each temperature and the activity measurement was carried out. <Method for confirming the optimal pH> The substrate solution used for the activity measurement was adjusted with 20 mM Britton-Robinson buffer (pH 5 - 10), and the measurement was carried out. <Method for confirming the thermal stability> The sample was heat-treated at 50 °C, and the remaining activity was measured. <Method for confirming the pH stability> The enzyme sample was diluted 2-fold with 20 mM Britton-Robinson buffer (pH 2 - 10), treated at 37 °C for 1 hour, and the remaining activity was measured.

[0067] (Results) The measurement results of the optimal temperature are shown in Table 1 and Figure 2. The measurement results of the optimal pH are shown in Table 2 and Figure 3. The measurement results of the thermal stability are shown in Table 3 and Figure 4. The measurement results of the pH stability are shown in Table 4 and Figure 5.

[0068] [Table 1]

[0069] [Table 2]

[0070] [Table 3]

[0071] [Table 4]

[0072] (Consideration) As shown in Tables 1-4 and Figures 2-5, the transglutaminase of the present invention was confirmed to have high reactivity at low temperatures and high thermal stability compared to existing transglutaminases.

[0073] Example 5: Confirmation of polymer crosslinking activity We confirmed that high molecular weight proteins are crosslinked by transglutaminase, using casein as a substrate. (method) 20 μl of 5% casein solution (prepared in 50 mM phosphate buffer (pH 7)) was mixed with 10 μl of purified enzyme solution (0.1 mg / mL), and the reaction was carried out at 5°C and 37°C. Samples were taken periodically, diluted 50-fold, and then SDS-PAGE sample buffer was added and the mixture was boiled to stop the reaction. The mixture was then subjected to SDS-PAGE.

[0074] (result) Figure 6 shows the results of confirming the cross-linking reactivity of transglutaminase. Figure 6 shows, from left to right, the SDS-PAGE results for samples with reaction times of 0 hours, 1 hour, 3 hours, and 5 hours.

[0075] (Consideration) The results shown in Figure 6 confirm that, for the same weight, the enzyme derived from L. tulufanese exhibits high activity even at low temperatures. This demonstrates that the transglutaminase of the present invention is a transglutaminase with high low-temperature reactivity.

[0076] Example 6: Confirmation of crosslinking activity for natural raw materials (SDS-PAGE) We confirmed that transglutaminase cross-links proteins derived from natural raw materials, using the following raw materials as substrates.

[0077] [Table 5]

[0078] (method) 90 μL of 1% substrate solution (prepared in 50 mM phosphate buffer (pH 7)) was mixed with 10 μL of purified enzyme solution at each concentration (0.01 mg / mL, 0.1 mg / mL, 1 mg / mL), and the mixture was reacted overnight at 5°C and 37°C. SDS-PAGE sample buffer was added and the mixture was boiled to stop the reaction, and then subjected to SDS-PAGE.

[0079] (result) The crosslinking reactivity of the transglutaminase was confirmed regardless of which natural raw material was used as the substrate. This demonstrates that the transglutaminase of the present invention can catalyze crosslinking reactions with various natural raw materials.

[0080] Example 7: Confirmation of crosslinking activity for natural raw materials (quantification of free ammonia content) The objective was to quantify the crosslinking reaction of proteins derived from natural raw materials, specifically by determining the amount of ammonia released by the crosslinking reaction. The raw materials listed in the table of Example 6 were used as substrates.

[0081] (method) 1.35 mL of 1% substrate solution (prepared in 50 mM phosphate buffer (pH 7)) was mixed with 0.15 mL of 1 mg / mL purified enzyme solution, and the reaction was carried out at 5°C and 37°C for 2 and 21 hours, respectively. After the reaction, an equal volume of trichloroacetic acid was added to stop the reaction. The amount of free ammonia was measured using Ammonia-Test Wako. The amount of free ammonia before the reaction (0 hours) was set to 0 mM, and the amount of free ammonia over time was quantified.

[0082] (result) The measurement results are shown in Figures 7-13. Furthermore, Figure 14 shows the results of each natural raw material after a 21-hour reaction, as shown in Figures 7-13. Regardless of the raw material used as the substrate, an increase in ammonia release over time was observed. In particular, for soybeans, peas, rye, wheat, coconut, and chia seeds, the amount of ammonia released was higher compared to existing transglutaminases, suggesting a possible high cross-linking reactivity. Furthermore, Figure 15 shows the results of a 21-hour reaction at a reaction temperature of 5°C for each of the natural raw materials shown in Figures 7 to 13. In all cases, the amount of ammonia released was equivalent to or greater than that of existing transglutaminases, suggesting the possibility of high crosslinking reactivity at low temperatures.

[0083] Example 8: Tofu production The effect of this enzyme in tofu production was confirmed.

[0084] (method) 20 mL of soy milk (organic soy milk, unsweetened, Sujata Meilaku, protein content 5%) was treated at 55°C for 5 minutes. 0.6 mL of 20% magnesium chloride hexahydrate solution and 1 mL of purified enzyme solution diluted to achieve enzyme concentrations of 25 μg, 50 μg, 100 μg, and 250 μg per gram of soy milk protein were added, and the mixture was stirred for 5 seconds. After reacting at 55°C for 1 hour, tofu was prepared by storing at 4°C for 2 hours. For comparison, tofu prepared without enzyme addition was also prepared. The strength (Firmness (N); specifically, compressive strength) of the obtained tofu was measured using a rheometer (COMPAC-100II, Sun Science Co., Ltd.). The measurement conditions were: Mode: 20, Adapter: No. 13, Repeat: 1, Indentation distance: 5 mm.

[0085] (result) The measurement results are shown in Figure 16. We confirmed that using this enzyme improved the strength of tofu. Furthermore, we confirmed that it was more effective than existing transglutaminases.

[0086] Example 9: Production of soy milk yogurt The effect of this enzyme in soy milk yogurt production was confirmed.

[0087] (method) 50 mL of soy milk (organic soy milk, unsweetened, Sujata Meilaku, protein content 5%) was added to a 100 mL beaker, and 0.3 g of starter culture (King's Yogurt Starter Culture, Ohta's Isan) (Lactobacillus cremoris BRF (DM-1, DM-2 strains), Streptococcus thermophilus (KI-1 strain)) was added. After adding 1 mL of purified enzyme solution diluted to achieve enzyme amounts of 50 μg, 100 μg, and 250 μg per gram of soy milk protein, the mixture was thoroughly stirred. The beaker was covered with plastic wrap and left to stand at 28°C for 20 hours, then stored at 4°C to prepare soy milk yogurt. For comparison, soy milk yogurt was also prepared without adding enzymes. Soy milk yogurt We also prepared the following. Soy milk yogurt The strength (Firmness (N); specifically, compressive strength) was measured using a rheometer (COMPAC-100II, Sun Science Co., Ltd.). The measurement conditions were: Mode: 20, Adapter: No. 13, Repeat: 1, Indentation distance: 5 mm.

[0088] (result) The measurement results are shown in Figure 17. Using this enzyme, we confirmed an improvement in the strength of soy milk yogurt. [Industrial applicability]

[0089] The enzyme preparation of the present invention contains transglutaminase, which has high low-temperature reactivity, as its active ingredient. Therefore, it is suitable for use in the food and medical fields and has high industrial value.

[0090] The present invention is not limited in any way to the embodiments and examples described above. Various modifications are also included in this invention, provided they do not depart from the scope of the claims and are easily conceivable by those skilled in the art. All content of papers, published patent gazettes, and other patent publications explicitly mentioned herein shall be cited by reference.

Claims

1. It contains as an active ingredient a transglutaminase consisting of an amino acid sequence having more than 90% sequence identity with the amino acid sequence of Sequence ID No.

1. An enzyme preparation in which the enzyme activity at 20°C is 20% or more of the enzyme activity at 50°C.

2. The enzyme preparation according to claim 1, wherein the transglutaminase is derived from the genus Longimycelium.

3. The enzyme preparation according to claim 1, wherein the molecular weight measured by SDS-PAGE is 28 to 30 kDa.

4. The enzyme preparation according to claim 1, wherein the optimal pH is pH 6.5 to 7.

5.

5. The enzyme preparation according to claim 1, wherein the enzyme activity after heat treatment at 50°C for 40 minutes is 60% or more of the enzyme activity before heat treatment.

6. The enzyme preparation according to claim 1, wherein the enzyme activity at pH 4 is 50% or more of the enzyme activity at pH 8.

7. A method for modifying a protein or peptide, comprising acting an enzyme preparation according to any one of claims 1 to 6 on a substrate.

8. A method for producing food, comprising applying an enzyme preparation according to any one of claims 1 to 6 to food.

9. A method for producing transglutaminase, comprising the steps of culturing a bacterium of the genus Longimycelium and purifying transglutaminase, wherein the transglutaminase has an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, and its enzyme activity at 20°C is 20% or more of its enzyme activity at 50°C.

10. A method for producing transglutaminase, comprising the steps of culturing the Longimycelium tulufanense NBRC107726 strain and purifying the transglutaminase.

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