Enzyme agent for protein deamidation and protein crosslinking and use thereof

Abstract

The problem addressed by the present invention is to provide an enzyme agent for protein deamidation and protein crosslinking containing an enzyme having protein deamidation activity and transglutaminase activity, and the use thereof. The present invention provides an enzyme agent for protein deamidation and protein crosslinking that contains as an active ingredient an enzyme having protein deamidation activity and transglutaminase activity comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1.

Description

Enzyme preparation for protein deamidation and protein cross-linking and its use

[0001] The present invention relates to an enzymatic agent (enzymatic agent for protein deamidation and protein cross-linking) containing as an active ingredient an enzyme having protein deamidation activity and transglutaminase activity, and uses thereof.

[0002] Protein deamidases (e.g., protein glutaminase (EC number: 3.5.1.44)) are enzymes that act on macromolecular proteins to catalyze their deamidation. Protein deamidases deamidate glutamine residues in proteins, generating negatively charged carboxyl groups, thereby causing various changes in the properties of proteins. For example, an increase in hydration force and an increase in electrostatic repulsion due to a decrease in the isoelectric point of a protein reduces protein-protein interactions (i.e., reduces association), thereby increasing the solubility and water dispersibility of the protein. Furthermore, the exposure of internal hydrophobic regions due to changes in the higher-order structure of a protein confers surface activity to the protein, thereby improving the emulsifying power, emulsion stability, foaming ability, and foam stability of the protein. Because protein deamidases can significantly change the properties of proteins in this way, they have dramatically increased the uses of proteins. For this reason, protein deamidases are highly useful and have attracted great interest in the technical field.

[0003] Meanwhile, transglutaminase (EC number: 2.3.2.13) (also referred to as TG) is an enzyme that catalyzes the acyl transfer reaction of the γ-carboxylamide group of glutamine residues in peptide chains. When the ε-amino group of a lysine residue in a protein acts as an acyl acceptor, it forms an ε-(γ-Gln)-Lys crosslink within or between protein molecules. The activity of transglutaminase can be utilized to modify proteins or peptides, and it is therefore used in the production of meat binding, sausages, tofu, bread, and noodles. Furthermore, the use of transglutaminase is being investigated not only in the food industry but also in the textile, medical, and cosmetic fields.

[0004] Protein deamidating enzymes and transglutaminase have in common the fact that they act on glutamine residues, but are different enzymes with different EC numbers. Transglutaminase catalyzes the deamidation of glutamine residues in the absence of an acyl acceptor, but hardly catalyzes the deamidation in the presence of an acyl acceptor such as a protein. Therefore, when it is desired to catalyze both protein deamidation and transglutaminase reaction, it has been necessary to use separately prepared enzymes in combination. For example, Patent Document 1 discloses a method for modifying proteins by allowing protein glutaminase and transglutaminase to act on the proteins, and describes that the smoothness of the produced foods is improved by treating them with protein glutaminase and transglutaminase in the production of yogurt using milk, tofu using soy milk, and noodles using wheat.

[0005] WO 2009 / 113628

[0006] As described above, it is expected that the combined use of protein glutaminase and transglutaminase will have a protein modification effect, but the enzymes available for use are limited. Furthermore, in conventional techniques, two types of enzymes must be used in combination to promote both the protein deamidation reaction and the transglutaminase reaction, which complicates the production process. Therefore, the problem that the present invention aims to solve is to provide a novel enzyme preparation containing, as an active ingredient, an enzyme having both protein deamidation activity and transglutaminase activity.

[0007] The present inventors have now newly discovered that a polypeptide derived from Longimycelium tulfanense has both protein deamidation activity and transglutaminase activity. The present invention was completed based on the above findings.

[0008] The present invention provides the following inventions. <1> An enzymatic preparation for protein deamidation and protein cross-linking, comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 and containing, as an active ingredient, an enzyme having protein deamidation activity and transglutaminase activity. <2> The enzymatic preparation for protein deamidation and protein cross-linking according to <1>, wherein the enzyme is derived from the genus Longimycelium. <3> A method for deamidating and cross-linking a protein or peptide, comprising allowing the enzymatic preparation for protein deamidation and protein cross-linking according to <1> or <2> to act on the protein or peptide. <4> A method for deamidating and cross-linking a protein-containing food or drink, comprising allowing the enzymatic preparation for protein deamidation and protein cross-linking according to <1> or <2> to act on the protein-containing food or drink. <5> A method for producing a food or drink, comprising allowing the enzymatic preparation for protein deamidation and protein cross-linking according to <1> or <2> to act on the protein-containing food or drink. <6> A food or drink treated with the enzymatic preparation for protein deamidation and protein cross-linking according to <1> or <2>. <7> A method for producing a protein deamidating and protein cross-linking enzyme comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, the method comprising the step of culturing bacteria of the genus Longimycelium. <8> An enzymatic preparation for protein deamidating and protein cross-linking, comprising, as an active ingredient, an enzyme having protein deamidating activity and transglutaminase activity, the enzyme having an amino acid sequence having 65% or more sequence identity with the amino acid sequence of SEQ ID NO: 6. <9> The enzymatic preparation for protein deamidating and protein cross-linking according to <8>, wherein the enzyme is derived from the genus Crossiella. <10> A method for deamidating and cross-linking a protein or peptide, the method comprising allowing the enzymatic preparation for protein deamidating and protein cross-linking according to <8> or <9> to act on the protein or peptide. <11> A method for deamidating and cross-linking a protein-containing food or drink, the method comprising allowing the enzymatic preparation for protein deamidating and protein cross-linking according to <8> or <9> to act on the protein-containing food or drink. <12> A method for producing a food or drink, the method comprising allowing the enzymatic preparation for protein deamidating and protein cross-linking according to <8> or <9> to act on the protein-containing food or drink.<13> A food or drink treated with the enzymatic agent for protein deamidation and protein cross-linking according to <8> or <9>. <14> A method for producing a protein deamidating and protein cross-linking enzyme comprising an amino acid sequence having 65% or more sequence identity with the amino acid sequence of SEQ ID NO: 6, comprising a step of culturing a bacterium of the genus Crossiella. <15> An enzymatic agent for protein deamidation and protein cross-linking, comprising, as an active ingredient, an enzyme having protein deamidating activity and transglutaminase activity, comprising an amino acid sequence having 65% or more sequence identity with the amino acid sequence of SEQ ID NO: 10. <16> The enzymatic agent for protein deamidation and protein cross-linking according to <15>, wherein the enzyme is derived from the genus Krasilnikovia. <17> A method for deamidating and cross-linking a protein or peptide, comprising allowing the enzymatic agent for protein deamidation and protein cross-linking according to <15> or <16> to act on the protein or peptide. <18> A method for deamidating and cross-linking a protein-containing food or drink, comprising allowing the enzymatic agent for protein deamidation and protein cross-linking according to <15> or <16> to act on the protein or peptide. <19> A method for producing a food or drink, comprising allowing the enzymatic agent for protein deamidation and protein cross-linking according to <15> or <16> to act on a protein-containing food or drink. <20> A food or drink treated with the enzymatic agent for protein deamidation and protein cross-linking according to <15> or <16>. <21> A method for producing a protein deamidating and protein cross-linking enzyme comprising an amino acid sequence having 65% or more sequence identity with the amino acid sequence of SEQ ID NO: 10, comprising a step of culturing bacteria of the genus Krasilnikovia.

[0009] Use of an enzyme agent for protein deamidation and protein crosslinking, wherein the enzyme agent consists of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 and contains, as an active ingredient, an enzyme having protein deamidation activity and transglutaminase activity.

[0010] The enzymatic preparation for protein deamidation and protein cross-linking, which contains the enzyme having protein deamidation activity and transglutaminase activity according to the present invention, is suitable for use in the fields of food and medical applications.

[0011] FIG. 1 shows the results of the adhesiveness evaluation of enzyme-treated casein proteins in Examples and Comparative Examples.

[0012] 1. Enzymatic preparation for protein deamidation and protein cross-linking The enzymatic preparation for protein deamidation and protein cross-linking according to a first embodiment of the present invention comprises, as an active ingredient, an enzyme having protein deamidation activity and transglutaminase activity, which comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence equivalent to said amino acid sequence (hereinafter also referred to as "protein deamidation and protein cross-linking enzyme" or "the enzyme").

[0013] The enzymatic agent for protein deamidation and protein cross-linking according to the second embodiment of the present invention comprises, as an active ingredient, an enzyme having protein deamidation activity and transglutaminase activity, which comprises the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence equivalent to said amino acid sequence (hereinafter also referred to as "protein deamidation and protein cross-linking enzyme" or "the enzyme").

[0014] The enzymatic agent for protein deamidation and protein cross-linking according to the third embodiment of the present invention comprises, as an active ingredient, an enzyme having protein deamidation activity and transglutaminase activity, which comprises the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence equivalent to said amino acid sequence (hereinafter also referred to as "protein deamidation and protein cross-linking enzyme" or "the enzyme").

[0015] The present invention may also relate to an enzymatic preparation for protein deamidation and protein cross-linking, which contains, as an active ingredient, an enzyme having protein deamidation activity and transglutaminase activity, which consists of the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence equivalent to said amino acid sequence (hereinafter also referred to as "protein deamidation and protein cross-linking enzyme" or "the enzyme") (fourth embodiment).

[0016] The protein deamidating and protein cross-linking enzyme of the present invention is an enzyme that exhibits the action of degrading or cross-linking amide group-containing side chains of proteins. Specifically, the protein deamidating and protein cross-linking enzyme of the present invention has the activity of deamidating glutamine residues in proteins and converting them to glutamic acid (protein glutaminase activity), and also catalyzes the acyl transfer reaction of the γ-carboxylamide group of glutamine residues in proteins. This allows for effective protein modification.

[0017] An "equivalent amino acid sequence" refers to an amino acid sequence that is partially different from a reference amino acid sequence (the amino acid sequence of SEQ ID NO: 1, 6, 10, or 14), but the difference does not substantially affect the function of the protein (here, protein deamidation activity and transglutaminase activity). Therefore, an enzyme having an equivalent amino acid sequence catalyzes an enzymatic reaction by an enzyme having protein deamidation activity and transglutaminase activity. The level of activity is not particularly limited as long as it can exhibit the function of an enzyme having protein deamidation activity and transglutaminase activity. However, it is preferable that the level of activity is the same as or higher than that of an enzyme consisting of the reference amino acid sequence (having the amino acid sequence of SEQ ID NO: 1, 6, 10, or 14).

[0018] In the first embodiment, the protein deamidating and protein cross-linking enzyme is preferably derived from the genus Longimycelium, and more preferably derived from Longimycelium tulfanense. The amino acid sequence of SEQ ID NO: 1 is the amino acid sequence (mature form) of the protein deamidating and protein cross-linking enzyme derived from Longimycelium tulfanense. The full-length amino acid sequence of the protein deamidating and protein cross-linking enzyme derived from Longimycelium tulfanense is shown in SEQ ID NO: 2.

[0019] In the second embodiment, the protein deamidating and protein cross-linking enzyme is preferably derived from the genus Crossiella, and more preferably derived from Crossiella cryophila. The amino acid sequence of SEQ ID NO: 6 is the amino acid sequence (mature form) of the protein deamidating and protein cross-linking enzyme derived from Crossiella cryophila. The full-length amino acid sequence of the protein deamidating and protein cross-linking enzyme derived from Crossiella cryophila is shown in SEQ ID NO: 7.

[0020] In the third embodiment, the protein deamidating and protein cross-linking enzyme is preferably derived from the genus Krasilnikova, and more preferably derived from Krasilnikova cinnamomea. The amino acid sequence of SEQ ID NO: 10 is the amino acid sequence (mature form) of the protein deamidating and protein cross-linking enzyme derived from Krasilnikova cinnamomea. The full-length amino acid sequence of the protein deamidating and protein cross-linking enzyme derived from Krasilnikova cinnamomea is shown in SEQ ID NO: 11.

[0021] In the fourth embodiment, the protein deamidating and protein cross-linking enzyme is preferably derived from the genus Longispora, and more preferably derived from Longispora fulva. The amino acid sequence of SEQ ID NO: 14 is the amino acid sequence (mature form) of the protein deamidating and protein cross-linking enzyme derived from Longispora fulva. The full-length amino acid sequence of the protein deamidating and protein cross-linking enzyme derived from Longispora fulva is shown in SEQ ID NO: 15.

[0022] A "partial difference in the amino acid sequence" occurs, for example, by deletion or substitution of one or more amino acids among the amino acids constituting the amino acid sequence, 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 permissible as long as the protein deamidation activity and transglutaminase activity are maintained (although some variation in activity is acceptable). As long as this condition is met, the position of the difference in the amino acid sequence is not particularly limited. Furthermore, differences in the amino acid sequence may occur at multiple positions (locations).

[0023] The number of amino acids that result in a partial difference in the amino acid sequence is, for example, a number corresponding to less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% of all amino acids that make up the amino acid sequence, preferably a number corresponding to less than about 8%, more preferably a number corresponding to less than about 6%, even more preferably a number corresponding to less than about 4%, even more preferably a number corresponding to less than about 2%, and most preferably a number corresponding to less than about 1%. Thus, an equivalent protein has, for example, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 81% or more, about 82% or more, about 83% or more, about 84% or more, about 85% or more, about 86% or more, about 87% or more, about 88% or more, about 89% or more, about 90% or more, about 91% or more, about 92% or more, preferably about 93% or more, more preferably about 94% or more, even more preferably about 95% or more, even more preferably about 96% or more, even more preferably about 97% or more, even more preferably about 98% or more, and most preferably about 99% or more identity to a reference amino acid sequence.

[0024] A typical example of a "partial difference in the amino acid sequence" is a mutation (change) in the amino acid sequence due to 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 constituting the amino acid sequence, 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.

[0025] Preferably, an equivalent amino acid sequence is obtained by conservative amino acid substitution at an amino acid residue that is not essential for protein deamidation activity and transglutaminase activity. Here, "conservative amino acid substitution" refers to the substitution of an amino acid residue with an amino acid residue having a side chain with 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), uncharged polar 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.

[0026] The percent identity of two amino acid sequences can be determined, for example, by the following procedure. First, the two sequences are aligned to enable optimal comparison (for example, gaps may be introduced into the first sequence to optimize alignment with the second sequence). When a molecule (amino acid residue) at a specific position in the first sequence is the same as a molecule at the corresponding position in the second sequence, the molecules at that position are considered to be identical. The identity of two sequences is a function of the number of identical positions shared by the two sequences (i.e., percent identity = number of identical positions / total number of positions × 100), and preferably takes into account the number and size of gaps required for optimal alignment.

[0027] Comparison of two sequences and determination of identity can be achieved using a mathematical algorithm. A specific example of a mathematical algorithm that can be used for sequence comparison is the algorithm described in Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68 and 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, for example, using the BLOSUM62 matrix and setting Gap Costs to Existence: 11 and Extension: 1.

[0028] The protein deamidating and protein cross-linking enzyme, which is an active ingredient of the enzymatic preparation of the present invention, may be a part of a larger protein (e.g., a fusion protein). Examples of sequences added to the fusion protein include sequences that are useful for purification, such as multiple histidine residues, and additional sequences that ensure stability during recombinant production.

[0029] The protein deamidating and protein cross-linking enzyme in the first embodiment can be obtained by culturing a microorganism that produces the protein deamidating and protein cross-linking enzyme (the present enzyme-producing strain), for example, a bacterium of the genus Longimycelium, such as Longimycelium tulufanense. That is, the present invention may relate to a method for producing a protein deamidating and protein cross-linking enzyme comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, the method comprising the step of culturing a bacterium of the genus Longimycelium. The present enzyme-producing strain may be a wild-type strain or a mutant strain (e.g., a mutant strain obtained by ultraviolet irradiation). A specific example of the present enzyme-producing strain is Longimycelium tulufanense (NBRC107726). Longimycelium tulfanense (NBRC107726) is a strain stored at NBRC (National Institute of Technology and Evaluation, Biotechnology Center), and can be purchased by following the prescribed procedures.

[0030] The protein deamidating and protein cross-linking enzyme in the second embodiment can be obtained by culturing a microorganism that produces the protein deamidating and protein cross-linking enzyme (the enzyme-producing strain), for example, a bacterium of the genus Crossiella, such as Crossiella cryophila. That is, the present invention may relate to a method for producing a protein deamidating and protein cross-linking enzyme comprising an amino acid sequence having 65% or more sequence identity with the amino acid sequence of SEQ ID NO: 6, which method comprises the step of culturing a bacterium of the genus Crossiella. The enzyme-producing strain may be a wild-type strain or a mutant strain (a mutant strain can be obtained by, for example, ultraviolet irradiation).

[0031] The protein deamidating and protein cross-linking enzyme in the third embodiment can be obtained by culturing a microorganism that produces the protein deamidating and protein cross-linking enzyme (the enzyme-producing strain), for example, a bacterium of the genus Krasilnikovia, such as Krasilnikovia cinnamomea. That is, the present invention may relate to a method for producing a protein deamidating and protein cross-linking enzyme comprising an amino acid sequence having 65% or more sequence identity with the amino acid sequence of SEQ ID NO: 10, which method comprises the step of culturing a bacterium of the genus Krasilnikovia. The enzyme-producing strain may be a wild-type strain or a mutant strain (a mutant strain can be obtained by, for example, ultraviolet irradiation).

[0032] The protein deamidating and protein cross-linking enzyme in the fourth embodiment can be obtained by culturing a microorganism that produces the protein deamidating and protein cross-linking enzyme (the enzyme-producing strain), for example, a bacterium of the genus Longispora, such as Longispora fulva. That is, the present invention may relate to a method for producing a protein deamidating and protein cross-linking enzyme comprising an amino acid sequence having 65% or more sequence identity with the amino acid sequence of SEQ ID NO: 14, which comprises the step of culturing a bacterium of the genus Longispora. The enzyme-producing strain may be a wild-type strain or a mutant strain (a mutant strain can be obtained by, for example, ultraviolet irradiation).

[0033] The present enzyme can be prepared from the culture medium and / or cells of a microorganism that produces the present enzyme. The culture conditions and method are not particularly limited as long as the present enzyme is produced. That is, a method and conditions suitable for culturing the microorganism to be used can be appropriately set, provided that the present enzyme is produced. The culture method may be either liquid culture or solid culture, but liquid culture is preferably used. The culture conditions will be explained using liquid culture as an example.

[0034] The medium is not particularly limited as long as it allows the growth of the microorganisms used. For example, a medium containing a carbon source such as glucose, sucrose, gentiobiose, soluble starch, glycerin, dextrin, molasses, or organic acid, a nitrogen source such as ammonium sulfate, ammonium carbonate, ammonium phosphate, or 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, may be used. Vitamins, amino acids, and the like may be added to the medium to promote the growth of the microorganisms used. The pH of the medium is adjusted to, for example, about 3 to 8, preferably about 4 to 7, and the culture temperature is usually about 20 to 40°C, preferably about 25 to 35°C, under aerobic conditions for 1 to 20 days, preferably about 3 to 10 days. Examples of culture methods that can be used include shaking culture and aerobic submerged culture using a jar fermenter.

[0035] 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, the culture supernatant is filtered, centrifuged, or the like to remove insoluble matter, and then the enzyme can be obtained by separating and purifying the enzyme using an appropriate combination of methods such as concentration using an ultrafiltration membrane, salting out using ammonium sulfate precipitation, dialysis, and various types of chromatography using ion exchange resins. On the other hand, when recovering from bacterial cells, the enzyme can be obtained by disrupting the bacterial cells, for example, by pressure treatment or ultrasonic treatment, and then separating and purifying the enzyme in the same manner as above. Note that the bacterial cells may be recovered from the culture medium in advance by filtration, centrifugation, or the like, and then the above series of steps (disruption, separation, and purification of the bacterial cells) may be carried out.

[0036] The present enzyme can also be easily prepared by genetic engineering techniques. For example, it can be prepared by transforming a suitable host cell (e.g., Escherichia coli) with DNA encoding the present enzyme and recovering the protein expressed in the transformant. The recovered protein is then purified as appropriate depending on the purpose. Obtaining the desired enzyme as a recombinant protein in this way allows for various modifications. For example, by inserting the DNA encoding the present enzyme and other appropriate DNA into the same vector and producing a recombinant protein using the vector, it is possible to obtain the present enzyme as a recombinant protein to which any peptide or protein is linked. Furthermore, modifications such as the addition of sugar chains and / or lipids, or modifications that cause N- or C-terminal processing, may also be performed. These modifications can simplify the extraction and purification of the recombinant protein, or impart biological functions to the protein.

[0037] Typically, gene expression and recovery of the expression product (the present enzyme) are carried out using an appropriate host-vector system as described above. However, cell-free synthesis systems may also be used. Here, "cell-free synthesis systems (cell-free transcription systems, cell-free transcription / translation systems)" refer to in vitro synthesis of mRNA and proteins encoded by template nucleic acids (DNA and mRNA) using ribosomes and transcription / translation factors derived from living cells (or obtained by genetic engineering techniques), rather than using living cells. Cell-free synthesis systems typically use cell extracts obtained by purifying cell lysates as needed. Cell extracts generally contain ribosomes, various factors such as initiation factors, and various enzymes such as tRNA, all of which are necessary for protein synthesis. During protein synthesis, various amino acids, energy sources such as ATP and GTP, and other substances necessary for protein synthesis, such as creatine phosphate, are added to the cell extract. Of course, separately prepared ribosomes, various factors, and / or various enzymes may be supplemented as needed during protein synthesis.

[0038] The development of a transcription / translation system in which each molecule (factor) required for protein synthesis is reconstituted has also been reported (Shimizu, Y. et al.: Nature Biotech., 19, 751-755, 2001). In this synthesis system, the genes for 31 factors that make up the bacterial protein synthesis system, including 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, were amplified from the E. coli genome, and the protein synthesis system was reconstituted in vitro using these genes. Such a reconstituted synthesis system may be used in the present invention.

[0039] 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. The template RNA can be total RNA, mRNA, or an in vitro transcription product. On the other hand, in an in vitro transcription / translation system, DNA is used as a template. The template DNA should contain a ribosome binding domain and preferably contains an appropriate terminator sequence. In an in vitro transcription / translation system, conditions are established in which factors required for each reaction are added so that the transcription and translation reactions proceed sequentially.

[0040] The purified enzyme obtained as described above can be provided as a powder by, for example, freeze-drying, vacuum drying, or spray-drying. In this case, the purified enzyme may be dissolved in advance 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 buffer include PIPES, MES, and MOPS.

[0041] The degree of purification of the enzyme is not particularly limited, and the final form may be liquid or solid (including powder).

[0042] The present enzyme catalyzes both the deamidation of the γ-amide group of glutamine residues in proteins and the acyl transfer reaction between the γ-amide group of glutamine residues and the ε-amino group of lysine residues in proteins. The content of the active ingredient (the present enzyme) in the enzyme preparation of the present invention is not particularly limited, but the content of the active ingredient can be set or adjusted so that the protein deamidation activity per gram of the present enzyme preparation is 0.01 U to 5000 U, preferably 0.1 U to 2000 U, and more preferably 1 U to 1000 U. Furthermore, the content of the active ingredient can be set or adjusted so that the transglutaminase activity per gram of the present enzyme preparation is 0.01 U to 5000 U, preferably 0.1 U to 2000 U, and more preferably 1 U to 1000 U.

[0043] The following method can be used to measure transglutaminase activity. The enzyme is dissolved in 200 mM Tris-HCl pH 6.0 containing 0.4% cysteine, treated at 30°C for 1 hour, and then diluted to an appropriate concentration with 200 mM Tris-HCl pH 6.0 (sample solution). 100 μL of substrate solution (R-1) obtained by the method described below is added to 10 μL of the sample solution, mixed, and allowed to react at 37°C for 10 minutes. 100 μL of color-developing solution (R-2) obtained by the method described below is added to terminate the reaction and form an Fe complex, after which the absorbance at 525 nm is measured. As a control, the absorbance of a sample prepared in the same manner using a heat-inactivated enzyme solution is measured, and the absorbance difference with the sample solution is determined. A calibration curve is separately prepared using L-glutamic acid-γ-monohydroxamic acid instead of the enzyme solution, and the amount of hydroxamic acid produced is determined from the absorbance difference. The enzyme activity that produces 1 μmol of hydroxamic acid per minute is defined as 1 unit (1 U).

[0044] (Substrate solution (R-1)) 2.42 g of 2-amino-2-hydroxymethyl-1,3-propanediol, 0.70 g of hydroxyammonium chloride, 0.31 g of reduced glutathione, and 1.01 g of Z-Gln-Gly (benzyloxycarbonyl-L-glutaminylglycine) were dissolved in distilled water to a total volume of 100 mL (pH 6.0).

[0045] (Substrate solution (R-2)) Mix 30 mL of 3 M hydrochloric acid solution, 30 mL of 12% trichloroacetic acid solution, and 30 mL of 5% iron (III) chloride solution.

[0046] The protein deamidation activity can be measured using the following method using a synthetic substrate. 0.1 mL of a sample solution containing protein deamidase is added to 1 mL of 0.2 M phosphate buffer (pH 6.5) containing 30 mM Z-Gln-Gly, and the mixture is left to stand at 37°C for 10 minutes. Then, 1 mL of 0.4 M TCA (trichloroacetic acid) solution is added to terminate the reaction. As a blank, 1 mL of 0.4 M TCA solution is added to 1 mL of 0.2 M phosphate buffer (pH 6.5) containing 30 mM Z-Gln-Gly, and 0.1 mL of the sample solution containing protein deamidase is further added, followed by standing at 37°C for 10 minutes.

[0047] The amount of ammonia generated in the reaction solution obtained above is measured using an Ammonia Test Wako (Fujifilm Wako Pure Chemical Industries). The ammonia concentration in the reaction solution is determined from a calibration curve showing the relationship between ammonia concentration and absorbance (630 nm) prepared using an ammonia standard solution (ammonium chloride).

[0048] The activity of protein deamidase is calculated from the following formula, where 1 unit (1 U) is the amount of enzyme that produces 1 μmol of ammonia per minute. In the formula, the volume of the reaction solution is 2.1, the volume of the enzyme solution is 0.1, Df is the dilution factor of the enzyme solution, and 17.03 is the molecular weight of ammonia.

[0049]

[0050] On the other hand, when measuring protein deamidation activity on a protein substrate and calculating the ratio of protein deamidation activity to transglutaminase activity, the ratio can be calculated from the ratio of the amount of deamidated insulin B chain produced to the amount of insulin B chain decreased when insulin B chain is used as a substrate. Details of the measurement conditions, measurement procedures, etc. will be described later in Examples.

[0051] The protein deamidation activity of the enzyme preparation of the present invention is not particularly limited. For example, in an evaluation method using insulin B chain, the lower limit of the deamidation rate is 4% or more, 5% or more, 7% or more, preferably 10% or more, and more preferably 15% or more, and the upper limit of the deamidation rate is 99% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, preferably 30% or less, and more preferably 20% or less.

[0052] The transglutaminase activity of the enzyme preparation of the present invention is not particularly limited. For example, in an evaluation method using insulin B chain, the lower limit of the cross-linking rate is 10% or more, 20% or more, 30% or more, 40% or more, preferably 50% or more, and more preferably 60% or more, and the upper limit of the cross-linking rate is 95% or less, 92% or less, 90% or less, preferably 88% or less, and more preferably 85% or less.

[0053] The ratio of protein deamidation activity to transglutaminase activity in the present enzyme preparation is not particularly limited, and examples thereof include "protein deamidation activity / transglutaminase activity" in an evaluation method using insulin B chain of 0.04 or more, 0.05 or more, 0.06 or more, 0.07 or more, 0.08 or more, 0.09 or more, 0.10 or more, 0.11 or more, 0.12 or more, 0.13 or more, 0.14 or more, 0.15 or more, 0.16 or more, 0.17 or more, 0.18 or more, 0.19 or more, and 0.20 or more.

[0054] Furthermore, the protein deamidation activity and transglutaminase activity of the enzyme preparation of the present invention can also be evaluated by detecting the transition of peaks in an HPLC analysis of the reaction-stopped solution after the reaction with a synthetic substrate, as described above. Details of the measurement conditions, measurement procedures, etc. will be described later in the Examples.

[0055] The enzyme preparation of the present invention may contain, in addition to the active ingredient (the protein deamidating and protein cross-linking enzyme of the present invention), excipients, buffers, suspending agents, stabilizers, preservatives, antiseptics, physiological saline, various proteins, various protein hydrolysates, various extracts, various salts, various antioxidants, cysteine, glutathione, sodium glutamate, sodium inosinate, sodium guanylate, calcined shell calcium, silicon dioxide, etc. Examples of excipients that can be used include starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, sucrose, glycerol, etc. Examples of buffers that can be used include phosphates, citrates, acetates, etc. Examples of stabilizers that can be used include propylene glycol, ascorbic acid, etc. Examples of preservatives that can be used include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, etc. Examples of preservatives that can be used include ethanol, benzalkonium chloride, parahydroxybenzoic acid, chlorobutanol, etc. Examples of proteins include soybean 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 chloride, phosphate, polyphosphate, pyrophosphate, citrate, lactate, and carbonate. 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.

[0056] 2. Gene In a first embodiment, the gene encoding the present enzyme consists of DNA encoding a protein comprising 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 mature amino acid sequence (SEQ ID NO: 1), while the DNA of SEQ ID NO: 4 encodes the mature amino acid sequence (amino acid sequence of SEQ ID NO: 1) as well as a signal peptide and a pro-sequence.

[0057] In a second embodiment, the gene encoding the present enzyme consists of DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 6. Specific examples of this embodiment are DNA consisting of the nucleotide sequence shown in SEQ ID NO: 8 and DNA consisting of the nucleotide sequence shown in SEQ ID NO: 9. The DNA of SEQ ID NO: 8 encodes only the mature amino acid sequence (SEQ ID NO: 6), while the DNA of SEQ ID NO: 9 encodes the mature amino acid sequence (SEQ ID NO: 6) as well as a signal peptide and a pro-sequence.

[0058] In a third embodiment, the gene encoding the present enzyme consists of DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 10. Specific examples of this embodiment are DNA consisting of the nucleotide sequence shown in SEQ ID NO: 12 and DNA consisting of the nucleotide sequence shown in SEQ ID NO: 13. The DNA of SEQ ID NO: 12 encodes only the mature amino acid sequence (SEQ ID NO: 10), while the DNA of SEQ ID NO: 13 encodes the mature amino acid sequence (SEQ ID NO: 10) as well as a signal peptide and a pro-sequence.

[0059] In a fourth embodiment, the gene encoding the present enzyme consists of DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 14. Specific examples of this embodiment are DNA consisting of the nucleotide sequence shown in SEQ ID NO: 16 and DNA consisting of the nucleotide sequence shown in SEQ ID NO: 17. The DNA of SEQ ID NO: 16 encodes only the mature amino acid sequence (SEQ ID NO: 14), while the DNA of SEQ ID NO: 17 encodes the mature amino acid sequence (SEQ ID NO: 14) as well as a signal peptide and a pro-sequence.

[0060] The gene encoding the present enzyme is typically used to prepare the present enzyme. A genetic engineering preparation method using the gene encoding the present enzyme makes it possible to obtain the present enzyme in a more homogeneous state. This method is also suitable for preparing large quantities of the present enzyme. The use of the gene encoding the present enzyme is not limited to the preparation of the present enzyme. For example, the nucleic acid can also be used as an experimental tool for elucidating the mechanism of action of the present enzyme, or as a tool for designing or creating mutants (modified forms) of the present enzyme.

[0061] As used herein, the term "gene encoding the present enzyme" refers to a nucleic acid that, when expressed, gives the present enzyme, and includes not only a nucleic acid having a base sequence corresponding to the amino acid sequence of the present enzyme, but also a nucleic acid in which a sequence that does not encode an amino acid sequence is added to such a nucleic acid. Codon degeneracy is also taken into consideration.

[0062] Nucleic acids can be prepared in an isolated state by standard genetic engineering techniques, molecular biological techniques, biochemical techniques, chemical synthesis, PCR (e.g., overlap PCR), or a combination thereof, using the sequence information disclosed in this specification or the attached sequence listing as a reference.

[0063] Nucleic acids that, when compared with the nucleotide sequence of the gene encoding the present enzyme, have the same function as the protein they encode but differ in part from the nucleotide sequence (hereinafter also referred to as "equivalent nucleic acids"; a nucleotide sequence specifying an equivalent nucleic acid is also referred to as "equivalent nucleotide sequence"). Examples of equivalent nucleic acids include DNAs that have a nucleotide sequence containing one or more base substitutions, deletions, insertions, additions, or inversions based on the nucleotide sequence of the nucleic acid encoding the present enzyme, and that encode a protein having enzymatic activity characteristic of the present enzyme (i.e., protein deamidation activity and transglutaminase activity). Base substitutions or deletions may occur at multiple sites. Here, "multiple" refers to, for example, 2 to 40 bases, preferably 2 to 20 bases, and more preferably 2 to 10 bases, although this varies depending on the position and type of amino acid residues in the three-dimensional structure of the protein encoded by the nucleic acid. Equivalent nucleic acids have sequence identity to the reference base sequence (SEQ ID NO: 3, 4, 8, 9, 12, 13, 16, or 17), for example, 50% or more, 60% or more, 65% or more, 70% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, preferably 93% or more, more preferably 94% or more, even more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably about 98% or more, and most preferably 99% or more.

[0064] Such equivalent nucleic acids can be obtained, for example, by restriction enzyme treatment, treatment with exonuclease or DNA ligase, 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.

[0065] A nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of the gene encoding the present enzyme may be used, or a nucleic acid having a nucleotide sequence that is at least about 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of the gene encoding the present enzyme or a nucleotide sequence complementary thereto.

[0066] Nucleic acids having a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the gene encoding the present enzyme or a nucleotide sequence complementary to the equivalent nucleotide sequence may also 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 well known to those skilled in the art and can be determined with reference to, for example, 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). An example of stringent conditions includes incubation at approximately 50°C using a hybridization solution (50% formamide, 10x SSC (0.15 M NaCl, 15 mM sodium citrate, pH 7.0), 5x Denhardt's solution, 1% SDS, 10% dextran sulfate, 10 μg / ml denatured salmon sperm DNA, 50 mM phosphate buffer (pH 7.5)), followed by washing at approximately 65°C using 0.1x SSC and 0.1% SDS. More preferred stringent conditions include, for example, a hybridization solution containing 50% formamide, 5x SSC (0.15 M NaCl, 15 mM sodium citrate, pH 7.0), 1x Denhardt's solution, 1% SDS, 10% dextran sulfate, 10 μg / ml denatured salmon sperm DNA, and 50 mM phosphate buffer (pH 7.5).

[0067] Recombinant DNA containing a gene encoding the present enzyme may be used. The recombinant DNA may be provided, for example, in the form of a vector. As used herein, the term "vector" refers to a nucleic acid molecule that can transport an inserted nucleic acid into a target such as a cell.

[0068] An appropriate vector is selected depending on the intended use (cloning, protein expression) and the type of host cell. Examples of vectors using E. coli as a host include M13 phage or its modified forms, λ phage or its modified forms, and pBR322 or its modified forms (pB325, pAT153, pUC8, etc.), vectors using yeast as a host include pYepSec1, pMFa, pYES2, etc., vectors using insect cells as a host include pAc and pVL, and vectors using mammalian cells as a host include pCDM8 and pMT2PC.

[0069] The vector is preferably an expression vector. An "expression vector" refers to a vector that can introduce a nucleic acid inserted therein into a target cell (host cell) and express it in the cell. An expression vector usually contains a promoter sequence necessary for the expression of the inserted nucleic acid, an enhancer sequence that promotes expression, and the like. An expression vector containing a selection marker can also be used. When such an expression vector is used, the presence or absence (and the degree of introduction) of the expression vector can be confirmed using the selection marker.

[0070] Insertion of a nucleic acid into a vector, insertion of a selectable marker gene (if necessary), insertion of a promoter (if necessary), etc. can be carried out using standard recombinant DNA techniques (for example, well-known methods using restriction enzymes and DNA ligase; see Molecular Cloning, Third Edition, 1.84, Cold Spring Harbor Laboratory Press, New York).

[0071] As host cells, microorganisms such as Escherichia coli, filamentous fungi (Aspergillus oryzae), actinomycetes (Streptomyces, Streptomyces lividans, Streptomyces mobaraensis), Bacillus subtilis, and budding yeast (Saccharomyces cerevisiae) are preferred for ease of handling. However, any host cell capable of replicating recombinant DNA and expressing the gene for the present enzyme can be used. Examples of E. coli include E. coli BL21(DE3)pLysS when a T7 promoter is used, and E. coli JM109 when not. Examples of budding yeast include SHY2, AH22, and INVSc1 (Invitrogen).

[0072] Microorganisms carrying recombinant DNA (i.e., transformants) can be used, which can be obtained by transfection or transformation with the vector of the present invention. For example, calcium chloride method (J. Mol. Biol., Vol. 53, p. 159 (1970)), Hanahan method (J. Mol. Biology, Vol. 166, p. 557 (1983)), SEM method (Gene, Vol. 96, p. 23 (1990)), Chung et al.'s 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, P. L. et al., Proc. Natl. Acad. Sci. USA 81, 7161-7165 (1984)), and the like can be used. USA 84, 7413-7417 (1984)), conjugal transfer, etc. The above-mentioned microorganisms can be used to produce the present enzyme.

[0073] 3. Uses of the Enzyme Preparation The present invention further relates to uses of the enzymatic preparation of the present invention. That is, by allowing the enzymatic preparation of the present invention to act on a substrate (protein or peptide), the protein or peptide can be modified (e.g., improved water retention, oil retention, emulsification, emulsion stability, foaming, foam stability, protein solubility, moldability, etc.). Specifically, by allowing the enzymatic preparation of the present invention to act on a protein or peptide containing glutamine residues and lysine residues, the glutamine residues of the protein can be deamidated or the glutamine residues and lysine residues can be crosslinked, thereby improving the stickiness of the protein or peptide after enzymatic treatment. That is, the present invention may relate to a method for deamidating and crosslinking a protein or peptide, which comprises allowing an enzymatic preparation for protein deamidation and protein crosslinking to act on the protein or peptide. In particular, the protein or peptide is preferably contained in a protein-containing food or beverage, and the present invention preferably relates to a method for deamidating and crosslinking a protein-containing food or beverage, which comprises allowing an enzymatic preparation for protein deamidation and protein crosslinking to act on the protein-containing food or beverage.

[0074] The present invention may also relate to a method for producing a food or beverage, which comprises allowing an enzymatic agent for protein deamidation and protein cross-linking to act on a protein-containing food or beverage. For example, by allowing the enzymatic agent of the present invention to act on a food or beverage or food or beverage material (such as a protein-containing food or beverage) containing a protein or peptide, a food or beverage or food or beverage material containing a deamidated and cross-linked protein (such as a food or beverage containing a deamidated and cross-linked protein) can be produced. By allowing the enzymatic agent of the present invention to act on a food or beverage, the quality of the food or beverage can be improved. According to the present invention, a food or beverage treated with the enzymatic agent of the present invention (a food or beverage containing a deamidated and cross-linked protein) is provided. Furthermore, the enzymatic agent of the present invention can be used not only for producing a food or beverage or a food or beverage material (such as a protein-containing food or beverage), but also for producing industrial materials and pharmaceutical raw materials.

[0075] Specific examples of the improvement of the quality of protein-containing foods and beverages include, but are not limited to, applications for improving the adhesiveness of animal proteins or vegetable proteins (for example, applications for producing meat products (hamburger steaks, bound meat, pork cutlets, sausages, steaks, ham, meatballs, etc.), seafood products (seafood, fish eggs, kamaboko, tuna nuggets, etc.), processed egg products (chawanmushi, mayonnaise, etc.), grains and processed grain products (rice, wheat, buckwheat, millet, corn, pasta, udon), bread and confectionery (pancakes, cookies, etc.), dairy products (milk, cheese, yogurt, cream, ice cream, butter, etc.), plant-based milk, plant-based cheese, plant-based yogurt, animal gelatin, plant-based gel, animal-based gel, tofu, meat substitutes, dressings, etc.).

[0076] The method for producing a food or drink using the present enzyme preparation is not particularly limited, and examples thereof include a method comprising the following step (a): Furthermore, step (a) may be followed by steps (b) and / or (c): (a) a step of allowing the present enzyme preparation to act on a protein-containing food or drink or food or drink material (protein-containing food or drink), (b) a step of recovering the food or drink or food or drink material containing the deamidated and cross-linked protein, and (c) a step of inactivating or removing the present enzyme contained in the obtained enzyme-treated food or drink or enzyme-treated food or drink material.

[0077] Example 1: Preparation of an enzyme having protein deamidation activity and transglutaminase activity from Longimycelium tulfanense Longimycelium tulfanense cells (obtained from the National Institute of Technology and Evaluation (NITE)) (NBRC107726) were cultured in tryptophan soy broth medium (manufactured by Nissui Pharmaceutical Co., Ltd.) at 37°C for 4 days, and the supernatant was collected. The resulting supernatant was dialyzed against 20 mM phosphate buffer (pH 6) according to a standard method. The dialyzed sample was subjected to ion exchange chromatography using a cation exchange column (SP Sepharose FF, manufactured by GE Healthcare) equilibrated with the same buffer. The active fraction was desalted and concentrated, and then subjected to gel filtration on Superdex 200HR to obtain the enzyme sample.

[0078] The amino acid sequence and nucleotide sequence of this enzyme are shown below. Amino acid sequence (mature sequence only) (SEQ ID NO: 1) ATSLPPIAPPLPRGVQSKSWSVPDYIAAWEKQHGRPMTAEERYHLARGCIGVTVVNLDREDAPNPPLNLSFGTYQRAMEVQAALNEIVATRPSPREYAEQVRKHPALQGVQNLVRAFPTFIDPANLHAAIFSKRFYSKQDPNWTDEQAAEMYRPNPRTGQVDMSTYRYRARPGYVNFDYGWYDEQTNNWWHANHAEPGMKVYQSTLRYYSRPLLDFDEQVFTVAFARVA

[0079] Amino acid sequence (full length) *The underlined part is the mature sequence (SEQ ID NO: 2) MKKWLPRALVALFVLLGLPAGLAGTAHAAVVAHAAVATSLPPIAPPLPRGVQSKSWSVPDYIAAWEKQHGRPMTAEERYHLARGCIGVTVVNLDREDAPNPPLNLSFGTYQRAMEVQAALNEIVATRPSPREYAEQVRKHPALQGVQNLVRAFPTFIDPANLHAAIFSKRFYSKQDPNWTDEQAAEMYRPNPRTGQVDMSTYRYRARPGYVNFDYGWYDEQTNNWWHANHAEPGMKVYQSTLRYYSRPLLDFDEQVFTVAFARVA

[0080] Base sequence (mature body) (sequence number 3) gccacgtcgctgccaccgatcgcaccgccgcttccccggggcgtgcagagcaagagctggtcggtgccggactacattgccgcctgggagaagcaacacggtagccgatgacggccgaggacgggacggtaccacctcgcccggggctgcatcggctcaccgtggtcaacctcgaccgggaggacgcgccgaacccgccgctcaacctgtcgttcggcacttaccagcgggccatggaagtgcacagagatcgtcgcgacccggccgcttcgccgcgggagtacgccgagcaggcaggtgcgcaagcacccggcgctgcagggcgtgcagaagcacccggcgctgcagaacccggcgctcaacggacggctgcagaacccggcgtcagcaccggcgtcagagcaccggcgtcagagcaccggcgtcaga gcgttccccacgttcatcgacccggcgaacctgcacgccgccatcttctccaagcggttctactcgaagcaggacccgaactggaccgacgagcaggcggccgagatgtaccggccgaatccgcggaccggccaggtcgacatgagcacctaccgctaccgggcgcggccgg gttacgtgaacttcgactacggttggtacgacgagcagacgaacaactggtggcacgccaaccacgcggagccgggcatgaaggtctaccagagcacgttgcggtactactcccggccgctgctggatttcgacgagcaggttttcaccgtggcgttcgcgcgggtcgcctga

[0081] Base sequence (full length) (sequence number 4) gtgaagaagtggttgccgcgcgccctggtcgcgctgttcgtgctcctggggttacccgccgccggccctggccggaacggcacatgccgggtcgtcgcgcacgccgccgtcgccacgtcgctgccaccgatcgcaccgccgcttccccggggcgtgcagagcaagagctggtcggggccggactacattgccgcctgggagaagcaacacagtagccgatgacggccgaggcggtaccacctgcccggggctgcatcggctcaccgtggtcaacctcgaccgggaggacgcgccgaacccgctcaacctgtcgttcggcacttaccagcgggccatggaagtgcacagagatcgtcgcgaccggccgcttcgccgggagatc gccgagcaggtgcgcaagcaccccggcgctgcagggcgtgcagaacctggtgcgggcgttccccacgttcatcgacccggcgaacctgcacgccgccatcttctccaagcggttctactcgaagcaggacccgaactggaccgacgagcaggcggccgagatgtaccggccgaatccgcggaccggccaggtcgacatga gcacctaccgctaccgggcgcggccgggttacgtgaacttcgactacggttggtacgacgagcagacgaacaactggtggcacgccaaccacgcggagccgggcatgaaggtctaccagagcacgttgcggtactactcccggccgctgctggatttcgacgagcaggttttcaccgtggcgttcgcgcgggtcgcctga

[0082] Example 2: Confirmation of Protein Modification Effect. The protein modification effect (improvement of adhesiveness) was confirmed using this enzyme sample when casein solution was used as a substrate. (Method) Milk casein (Casein, Bovine Milk, Carbohydrate and Fatty Acid Free, Merck KGaA) was dissolved in water to prepare an 11.1% (w / v) casein solution. To 9 mL of the 11.1% (w / v) casein solution, enzyme solution was added at 0.25 mg and 1 mg per gram of casein protein, and the mixture was diluted to 10 mL with 50 mM sodium phosphate buffer (pH 7.0). The entire reaction solution was then poured into a 56 mm diameter plastic dish, incubated at 40°C for 2 hours, and then left overnight (12-16 hours) at 4°C to obtain enzyme-treated casein protein. For comparison, a similar experiment was performed using Streptomyces-derived transglutaminase (Amano Enzyme Inc.) instead of this enzyme. The physical properties of the enzyme-treated casein protein were evaluated using a rheometer. Specifically, a metal plunger with a diameter of 20 mm was used to push the sample into the sample at a rate of 60 mm / min, and the distance over which the stress (adhesive force) generated when the plunger was released from the sample was measured.

[0083] (Results) The results are shown in FIG.

[0084] (Discussion) From the results shown in FIG. 1 and Table 1, it was confirmed that the present enzyme, which has protein deamidation activity and transglutaminase activity, exhibited a significant increase in adhesiveness compared to existing transglutaminases.

[0085] Example 3: Evaluation of protein deamidation activity and transglutaminase activity on protein substrates (Method) 5 mg of insulin B chain (insulin B chain, oxidized form, derived from bovine pancreas, Sigma-Aldrich, sequence: Phe-Val-Asn-Gln-His-Leu-Cys(SOH)-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys(SOH)-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Ala (SEQ ID NO: 5)) was dissolved in 2 mL of ultrapure water to prepare a substrate solution. 20 μL of 1 mg / mL purified enzyme solution and 0.2 M potassium dihydrogen phosphate / disodium hydrogen phosphate buffer (pH 6.5) were then added to 90 μL of the substrate solution to a total volume of 180 μL to prepare a reaction solution. The reaction solution was heated to 40°C in an incubator and allowed to stand for 1 hour, after which 20 μL of 1N HCl was added to terminate the reaction. The reaction stop solution described above was analyzed by HPLC. Deamidation activity and transglutaminase activity were detected by the peak transition from insulin B chain to deamidated insulin B chain. As a comparative example, activity was detected in the same manner using Streptomyces-derived transglutaminase (Amano Enzyme Inc.). <Protein deamidation activity> The amount of deamidated insulin B chain produced was calculated from the peak areas of insulin B chain and deamidated insulin B chain calculated from the chromatogram, and protein deamidation activity was evaluated by the deamidation rate (amount of deamidated insulin B chain produced ÷ amount of substrate insulin B chain × 100). (Transglutaminase activity) The remaining amount of insulin B chain and the amount of deamidated insulin B chain produced were calculated from each peak area calculated from the chromatogram, and evaluated as the cross-linking rate ((100 - (remaining amount of insulin B chain ÷ amount of substrate insulin B chain × 100) - deamidation rate)). The ratio between the deamidation rate and the cross-linking rate calculated by the above method was used to calculate the ratio between protein deamidation activity and transglutaminase activity.

[0086] (Results) The results are shown in Table 2.

[0087] Example 4: Confirmation of protein modification effect (production of a dry composition containing a processed protein with modified physical properties) (Method) The protein materials shown in the table below were dissolved or suspended in water to prepare a protein solution with a final concentration of 5% by mass.

[0088] 1 mg of the enzyme prepared in Example 1 was added to each gram of each protein material listed in the table above, and the protein solution was incubated at 50°C for 16 hours. The enzyme was then inactivated by heating at 100°C for 5 minutes. The resulting enzyme-treated protein solution was freeze-dried to obtain a processed protein-containing dry composition (treated with this enzyme). A protein-containing dry composition that had not been subjected to enzyme treatment (enzyme-untreated) was also obtained separately. The resulting processed protein-containing dry composition and protein-containing dry composition (enzyme-untreated) were evaluated for their property modification effects in terms of A to G shown below. For some protein materials, processed protein-containing dry compositions were also obtained by treating them with Streptomyces-derived transglutaminase, and similar evaluations were performed.

[0089] (A: Water retention evaluation) An aqueous suspension was prepared by suspending 0.1 g of the processed protein-containing dry composition in 1 g of deionized water and vortexing for 30 seconds. After standing for 30 minutes, the aqueous suspension was centrifuged at 2,000 g for 10 minutes, and the supernatant was collected. The mass of the collected supernatant was measured, and the resulting measured value was subtracted from 1 g (the mass of deionized water used for suspension) to calculate the mass of water absorbed by the protein. Furthermore, the mass of water absorbed per 1 g of the processed protein-containing dry composition was calculated, and the resulting value was designated as "water retention." The water retention was also calculated in the same manner for enzyme-untreated protein-containing dry compositions. The relative value of the water retention of the processed protein-containing dry composition, when the water retention of the enzyme-untreated protein-containing dry composition was set to 1, was determined as the evaluation value of water retention. In addition, for some protein materials, the water retention capacity of the processed protein-containing dry composition was evaluated as a relative value of the water retention capacity when treated with Streptomyces-derived transglutaminase, assuming the water retention capacity of the processed protein-containing dry composition to be 1.

[0090] (Results) The results are shown in the table below.

[0091] Compared to untreated crops, the water retention capacity of soybeans, peas, broad beans, lentils, chickpeas, rye, corn, sorghum, cashew nuts, almonds, peanuts, and industrial hemp was improved when this enzyme was used. In particular, the water retention capacity of broad beans, lentils, chickpeas, rye, sorghum, cashew nuts, almonds, and industrial hemp was improved more effectively by this enzyme than by Streptomyces-derived transglutaminase.

[0092] (B: Oil Retention Evaluation) An oil suspension was prepared by suspending 0.1 g of the processed protein-containing dry composition in 1 g of canola oil and vortexing for 30 seconds. After standing for 30 minutes, the oil suspension was centrifuged at 2,000 g for 10 minutes, and the supernatant was collected. The mass of the collected supernatant was measured, and the resulting measured value was subtracted from 1 g (the mass of canola oil used for suspension) to calculate the mass of oil absorbed by the protein. Furthermore, the mass of oil absorbed per 1 g of the processed protein-containing dry composition was calculated, and the obtained value was designated as "oil retention." The oil retention was also calculated in the same manner for enzyme-untreated protein-containing dry compositions. The oil retention of the processed protein-containing dry composition relative to the oil retention of the enzyme-untreated protein-containing dry composition was designated as 1, and the evaluation value of oil retention was determined. In addition, for some protein materials, the oil retention property of the processed protein-containing dry composition when treated with Streptomyces-derived transglutaminase was set to 1, and the relative value of the oil retention property of the processed protein-containing dry composition was calculated as the evaluation value of the oil retention property.

[0093] (Results) The results are shown in the table below.

[0094] Compared to untreated samples, the oil retention of lentils, corn, cashew nuts, almonds, walnuts, coconuts, and industrial hemp was improved when this enzyme was used. In particular, the oil retention of lentils, corn, cashew nuts, almonds, and walnuts was improved more effectively by this enzyme than by Streptomyces-derived transglutaminase.

[0095] (C: Emulsifying Property Evaluation) The processed protein-containing dry composition was suspended in deionized water to prepare 30 mL of an aqueous suspension containing 1 wt% protein. 30 mL of the aqueous suspension was mixed with 10 mL of canola oil and homogenized at 10,000 rpm for 2 minutes to prepare an emulsion composition. 50 μL of the emulsion composition immediately after preparation was added to 5 mL of 0.1% SDS solution. The turbidity (A0) was measured at an absorbance of 500 nm, and the "emulsifying property" was calculated using the following formula.

[0096] The emulsifying ability was also calculated for enzyme-untreated protein-containing dry compositions. The emulsifying ability of the processed protein-containing dry composition was calculated as a relative value when the emulsifying ability of the enzyme-untreated protein-containing dry composition was set to 1. Furthermore, for some protein materials, the emulsifying ability of the processed protein-containing dry composition was calculated as a relative value when the emulsifying ability of the processed protein-containing dry composition treated with Streptomyces-derived transglutaminase was set to 1.

[0097] (Results) The results are shown in the table below.

[0098] Compared to untreated samples, the use of this enzyme resulted in improved emulsifying properties for peas, mung beans, broad beans, lentils, chickpeas, oats, rice, chia seeds, milk casein, and egg albumin. In particular, the emulsifying properties of peas, mung beans, broad beans, lentils, chickpeas, oats, chia seeds, milk casein, and egg albumin were more improved by this enzyme than by Streptomyces-derived transglutaminase.

[0099] (D: Emulsion Stability Evaluation) Evaluation was performed using the emulsion composition prepared in the emulsifying property evaluation. 50 μL of the emulsion composition immediately after preparation was taken and added to 5 mL of 0.1% SDS solution, and the turbidity (A0) was measured at an absorbance of 500 nm. 50 μL of the emulsion composition that had been left for 10 minutes was taken and added to 5 mL of 0.1% SDS solution, and the turbidity (A10) was measured at an absorbance of 500 nm. "Emulsion stability" was calculated using the following formula.

[0100] The emulsion stability was also calculated in the same manner for enzyme-untreated protein-containing dry compositions. The emulsion stability evaluation value was determined as the relative value of the emulsion stability of the processed protein-containing dry composition, where the emulsion stability of the enzyme-untreated protein-containing dry composition was set to 1. Furthermore, for some protein materials, the emulsion stability evaluation value was determined as the relative value of the emulsion stability of the processed protein-containing dry composition, where the emulsion stability of the processed protein-containing dry composition treated with Streptomyces-derived transglutaminase was set to 1.

[0101] (Results) The results are shown in the table below.

[0102] Compared to untreated samples, the use of this enzyme resulted in improved emulsion stability in soybeans, mung beans, broad beans, lentils, chickpeas, milk casein, pork powder, chicken powder, and egg albumin. In particular, the effect of this enzyme in improving emulsion stability in soybeans, lentils, chickpeas, milk casein, pork powder, and chicken powder was greater than that in the case of Streptomyces-derived transglutaminase.

[0103] (E: Foaming Property Evaluation) The processed protein-containing dry composition was suspended in deionized water to prepare 50 mL of a 0.5 wt% aqueous suspension. The aqueous suspension was homogenized at 18,000 rpm for 30 minutes and immediately transferred to a 100 mL measuring cylinder. The volume VF0 of the aqueous suspension including the foam was measured, and the "foaming property" was calculated using the following formula.

[0104] The foaming ability of the enzyme-untreated protein-containing dry composition was also calculated in the same manner. The foaming ability of the processed protein-containing dry composition was calculated as a relative value when the foaming ability of the enzyme-untreated protein-containing dry composition was set to 1. In addition, for some protein materials, the foaming ability of the processed protein-containing dry composition was calculated as a relative value when the foaming ability of the processed protein-containing dry composition treated with Streptomyces-derived transglutaminase was set to 1.

[0105] (Results) The results are shown in the table below.

[0106] Compared to untreated samples, the foaming properties of pea and broad bean were improved when the present enzyme was used. In particular, the foaming properties of pea were improved more effectively by the present enzyme than when Streptomyces-derived transglutaminase was used.

[0107] (F: Foam Stability Evaluation) The processed protein-containing dry composition was suspended in deionized water to prepare 50 mL of a 0.5 wt% aqueous suspension. The aqueous suspension was homogenized at 18,000 rpm for 30 minutes and immediately transferred to a 100 mL measuring cylinder, and the volume VF0 of the aqueous suspension including the foam was measured. After 30 minutes, the volume VF30 of the aqueous suspension including the foam was similarly measured, and the "foam stability" was calculated using the following formula.

[0108] The foam stability of the protein-containing dry composition not treated with enzyme was also calculated in the same manner. The foam stability of the processed protein-containing dry composition was calculated as the relative value of the foam stability of the processed protein-containing dry composition when the foam stability of the protein-containing dry composition not treated with enzyme was set to 1. In addition, for some protein materials, the foam stability of the processed protein-containing dry composition when treated with Streptomyces-derived transglutaminase was calculated as the relative value of the foam stability of the processed protein-containing dry composition when the foam stability of the processed protein-containing dry composition was set to 1.

[0109] (Results) The results are shown in the table below.

[0110] Compared to untreated cases, the use of this enzyme resulted in improved foam stability for milk casein and egg albumin. In particular, the foam stability improvement effect of this enzyme on egg albumin was greater than that achieved with Streptomyces-derived transglutaminase.

[0111] (G: Protein Solubility Evaluation) The processed protein-containing dry composition was suspended in deionized water to prepare a 5 wt % aqueous suspension. The prepared 5 wt % aqueous suspension was centrifuged at 15,000 g for 5 minutes. The centrifugal supernatant was collected, and the protein concentration (mg / mL) was quantified using Bradford reagent manufactured by BIO-RAD. The protein concentration (mg / mL) of an enzyme-untreated protein-containing dry composition was also quantified in the same manner. The protein solubility was evaluated by determining the relative protein concentration of the processed protein-containing dry composition relative to the protein concentration of the enzyme-untreated protein-containing dry composition, which was set to 1. Furthermore, for some protein materials, the protein solubility was evaluated by determining the relative protein concentration of the processed protein-containing dry composition relative to the protein concentration of the processed protein-containing dry composition treated with Streptomyces-derived transglutaminase, which was set to 1.

[0112] (Results) The results are shown in the table below.

[0113] Compared to untreated mungbean, the use of this enzyme resulted in improved protein solubility, which was even higher than when Streptomyces-derived transglutaminase was used.

[0114] Example 5: Confirmation of Protein Modification Effect (Production of Foods and Beverages with Modified Physical Properties) (1. Kamaboko) Surimi was prepared by adding four times the amount of water to All-Purpose Surimi Powder (Suzuhiro Kamaboko Co., Ltd., protein: 57.8 wt%). To 9 g of surimi, 0.5 mL of a solution containing the enzyme prepared in Example 1 at a concentration of 1.0 mg / mL and 0.5 mL of water were added and mixed. The mixture was quickly packed into a 56 mm diameter, 16 mm high Petri dish and molded. The mixture was then covered and incubated at 50°C for 5 hours to obtain kamaboko (treated with this enzyme). The resulting kamaboko was measured for stress (maximum force recorded during the first compression) using a rheometer (Sun Scientific Co., Ltd.). Additionally, kamaboko (untreated enzyme) was separately prepared and similarly measured for stress. The stress of the kamaboko that was not treated with the enzyme was taken as 1, and the relative value of the stress of the kamaboko that was treated with the enzyme was calculated and evaluated as the stress improvement rate.

[0115] (Results) The results are shown in the table below.

[0116] A significant improvement in stress was observed when this enzyme was used compared to when the enzyme was not used.

[0117] (2. Yogurt) One packet of starter culture (Osama no Yogurt Starter Culture, Ohtaisan Co., Ltd.) was added to 500 mL of milk (Meiji Co., Ltd., Oishii Gyunyu, protein: 34 mg / mL). 0.25 mL of a solution containing the enzyme prepared in Example 1 at a concentration of 1.0 mg / mL and 0.75 mL of water were added to 9 mL of the starter culture-containing milk and mixed. After mixing, the mixture was poured into a 56 mm diameter, 16 mm high Petri dish, covered, and incubated at 28°C for 24 hours to obtain yogurt (treated with the present enzyme). The stress of the obtained yogurt was measured in the same manner as in "1. Kamaboko." Yogurt without enzyme treatment (enzyme-untreated) was also prepared separately and similarly measured for stress. The stress of the yogurt treated with the present enzyme, relative to the stress of the untreated yogurt, was calculated as the stress improvement rate and evaluated.

[0118] (Results) The results are shown in the table below.

[0119] Compared to untreated samples, the use of this enzyme resulted in an improvement in stress.

[0120] (3. Cheese) 15 mL of 0.2 M phosphate buffer (pH 6.86) was added to 30 g of commercially available cheddar cheese and completely dissolved while stirring at 70°C. After dissolution, the cheese was cooled to 50°C and dispensed into 20 g aliquots in Petri dishes (56 mm diameter, 16 mm height). 1 mL of the enzyme prepared in Example 1 or a solution containing Streptomyces-derived transglutaminase at a concentration of 1.0 mg / mL was added and mixed. The lid was then closed and incubated at 50°C for 1 hour to obtain cheese (treated with this enzyme or Streptomyces-derived transglutaminase). The stress of the resulting cheese was measured in the same manner as in "1. Kamaboko." Cheese that had not been subjected to enzyme treatment (enzyme-untreated) was also obtained separately and stress was measured in the same manner. The stress of cheese not treated with the enzyme was set to 1, and the relative stress values ​​of cheese treated with this enzyme or cheese treated with Streptomyces-derived transglutaminase were calculated and evaluated as the stress improvement rate.

[0121] (Results) The results are shown in the table below.

[0122] By using this enzyme, a significantly higher stress could be obtained than when Streptomyces-derived transglutaminase was used.

[0123] (4. Hamburger) To 200 g of minced beef (100% domestic beef, protein: 17.1 wt%), salt was added to a final concentration of 1% and thoroughly mixed until sticky. 140 mL of water was then added, mixed again, and divided into 20 g portions. 1 mL of a solution containing the enzyme prepared in Example 1 at a concentration of 1.0 mg / mL was added to the divided portions, mixed, and shaped. The shaped meat was wrapped in plastic wrap and incubated at 50°C for 1 hour. After incubation, each side was grilled in a frying pan over medium heat for 2 minutes. After grilling, the hamburger (treated with this enzyme) was obtained by cooling to room temperature. The stress of the obtained hamburger was measured in the same manner as in "1. Kamaboko." In addition, a hamburger without enzyme treatment (untreated enzyme) was separately obtained, and the stress was measured in the same manner. The stress of the hamburger steak not treated with the enzyme was taken as 1, and the relative value of the stress of the hamburger steak treated with the present enzyme was calculated as the stress improvement rate and evaluated.

[0124] (Results) The results are shown in the table below.

[0125] Compared to untreated samples, the use of this enzyme resulted in an improvement in stress.

[0126] (5. Chicken Ham) 100 g of chicken breast meat (protein: 23.3 wt%) was pierced at appropriate intervals with a fork and softened. It was then rolled out to a uniform thickness using a rolling pin. 1.6 g of sugar was applied to both sides, followed by rubbing in 1.6 g of salt, for pretreatment. 25 g of pretreated chicken breast meat was placed in a resealable plastic bag, and 5 mL of water and 5 mL of the enzyme prepared in Example 1 or a solution containing Streptomyces-derived transglutaminase at a concentration of 1.0 mg / mL were added and mixed. The bag was sealed with a zipper, removing as much air as possible, and incubated at 50°C for 5 hours. After incubation, the chicken breast meat was removed from the resealable plastic bag and rolled into a ham shape using aluminum foil. After shaping, the chicken breast was boiled in boiling water. After boiling, the chicken ham was cooled to room temperature to obtain chicken ham (treated with the present enzyme or with Streptomyces-derived transglutaminase). The stress of the obtained chicken ham was measured in the same manner as in "1. Kamaboko". In addition, chicken ham that was not subjected to enzyme treatment (enzyme-untreated) was obtained separately and stress was measured in the same manner. The stress of the chicken ham that was not enzyme-treated was set to 1, and the relative values ​​of the stress of the chicken ham that was treated with the present enzyme or the chicken ham that was treated with Streptomyces-derived transglutaminase were calculated and evaluated as the stress improvement rate.

[0127] (Results) The results are shown in the table below.

[0128] By using this enzyme, a significantly higher stress could be obtained than when Streptomyces-derived transglutaminase was used.

[0129] (6. Chawanmushi) 180 mL of water was added to one beaten egg (protein: 12.2 wt%) and strained through a tea strainer. 0.5 mL of water and 1 mL of a solution containing the enzyme prepared in Example 1 at a concentration of 1.0 mg / mL were added to 9 mL of beaten egg, mixed, and incubated at 50°C for 4 hours. After incubation, the mixture was steamed in a boiling pot. It was then cooled to room temperature to obtain chawanmushi (treated with this enzyme). Chawanmushi that was not subjected to enzyme treatment (untreated enzyme) was also obtained separately. Each of the obtained chawanmushi was scooped with a spoon, and its physical properties were evaluated visually.

[0130] (Results) The chawanmushi that was not treated with the enzyme had a slightly rough cross section, whereas the chawanmushi that was treated with this enzyme had a smooth cross section.

[0131] (7. Pancakes) 100 g of Cooking Flour soft wheat flour (Nissin Flour Milling Welna Co., Ltd., protein: 8 wt%) and 4 g of Super Camellia Dry Yeast for Home Bakeries (Nissin Flour Milling Welna Co., Ltd.) were thoroughly mixed with 100 mL of water, and an appropriate amount of sugar and beaten egg were added. Each mixture was divided into 20 g portions, to which 1 mL of a solution containing the enzyme prepared in Example 1 at a concentration of 1.0 mg / mL was added. The mixture was then mixed and incubated at 50°C for 1 hour. After incubation, the pancakes were baked on both sides in a frying pan over medium heat for 2 minutes. After baking, the pancakes were cooled to room temperature to obtain pancakes (treated with this enzyme). The stress of the resulting pancakes was measured in the same manner as in "1. Kamaboko." Pancakes that were not treated with the enzyme (enzyme untreated) were also prepared separately and the stress was measured in the same manner. The stress of the pancake not treated with the enzyme was set to 1, and the relative value of the stress of the pancake treated with the enzyme was calculated and evaluated as the stress improvement rate.

[0132] (Results) The results are shown in the table below.

[0133] Compared to untreated samples, the use of this enzyme resulted in an improvement in stress.

[0134] (8. Tofu) 9 mL of soy milk (Sujata Meiraku Group Tofu-Making Organic Soy Milk, Protein: 50 mg / mL), 0.5 mL of bittern solution (magnesium chloride), and 0.5 mL of a solution containing the enzyme prepared in Example 1 at a concentration of 1.0 mg / mL were added and mixed. After mixing, the mixture was poured into a 56 mm diameter, 16 mm high Petri dish, covered, and incubated at 40°C for 20 hours to obtain tofu (treated with this enzyme). The stress of the obtained tofu was measured in the same manner as in "1. Kamaboko." In addition, tofu that was not subjected to enzyme treatment (enzyme-untreated) was separately obtained and similarly measured for stress. The stress of the tofu treated with this enzyme was calculated and evaluated as a relative value, where the stress of the untreated tofu was set to 1.

[0135] (Results) The results are shown in the table below.

[0136] Compared to untreated samples, the use of this enzyme resulted in an improvement in stress.

[0137] (9. Soy Milk Yogurt) One packet of starter culture (Otaisan Co., Ltd., King's Yogurt Starter Culture) was added to 500 mL of soy milk (Sujata Meiraku Group, Organic Soy Milk for Tofu, Protein: 50 mg / mL). To 9 mL of soy milk containing the starter culture, 0.5 mL of water and 0.5 mL of a solution containing the enzyme prepared in Example 1 at a concentration of 1.0 mg / mL were added and mixed. After mixing, the mixture was poured into a 56 mm diameter, 16 mm high Petri dish, covered, and incubated at 28°C for 24 hours to obtain soy milk yogurt (treated with the present enzyme). The stress of the obtained soy milk yogurt was measured in the same manner as in "1. Kamaboko." In addition, soy milk yogurt without enzyme treatment (enzyme-untreated) was separately obtained and similarly measured for stress. The relative stress of the soy milk yogurt treated with the present enzyme, defined as 1, was calculated and evaluated as the stress improvement rate.

[0138] (Results) The results are shown in the table below.

[0139] Compared to untreated samples, the use of this enzyme resulted in an improvement in stress.

[0140] (10. Alternative Hamburger) Granular vegetable protein (Marukome Co., Ltd., Soy Lab, Soybean Mince, dried type, protein: 45.4% by weight) was rehydrated in 6 times the amount of hot water for 10 minutes and swollen. To 25 g of the swollen granular vegetable protein, 1 g of methylcellulose (Shin-Etsu Chemical Co., Ltd., Metrose) and 2.75 g of pea protein (Roquette, Nutralys F85M) were added, and finally 20 mL of water was added and mixed. To the resulting raw patty, 1 mL each of the enzyme prepared in Example 1 or a solution containing Streptomyces-derived transglutaminase at a concentration of 1.0 mg / mL was added, mixed, and incubated at room temperature for 1 hour. After incubation, the patty was baked in an oven at 150°C for 5 minutes on each side. After baking, the hamburger steak was cooled to room temperature to obtain an alternative hamburger steak (treated with the present enzyme or with Streptomyces-derived transglutaminase). The stress of the obtained alternative hamburger steak was measured in the same manner as in "1. Kamaboko". In addition, an alternative hamburger steak that had not been subjected to enzyme treatment (enzyme-untreated) was separately obtained and the stress was measured in the same manner. The stress of the alternative hamburger steak that had not been enzyme-treated was set to 1, and the relative values ​​of the stress of the alternative hamburger steak that had been treated with the present enzyme or the alternative hamburger steak that had been treated with Streptomyces-derived transglutaminase were calculated and evaluated as the stress improvement rate.

[0141] (Results) The results are shown in the table below.

[0142] By using this enzyme, a higher stress could be obtained than when using transglutaminase derived from Streptomyces.

[0143] (11. Cheese Substitute) 18.8 g of pea protein (LYSAMINE GPS, Roquette), 10 g of tapioca starch, 2 g of canola oil, 12 g of coconut oil (pre-dissolved), 0.6 g of salt, 1.8 g of calcium phosphate, and 1 g of carrageenan were added as ingredients for the cheese substitute, and water was added to bring the total weight to 100 g and mixed. The mixture was homogenized at 1,000 rpm for 10 minutes while maintaining the temperature at 50°C. The homogenized mixture was dispensed into 56 mm diameter, 16 mm high Petri dishes, each containing 10 g of the enzyme prepared in Example 1 or 0.5 mL of a solution containing Streptomyces-derived transglutaminase at a concentration of 1.0 mg / mL, and mixed. The lid was placed on the container and incubated at 40°C for 20 hours to obtain substitute cheese (treated with the present enzyme or with Streptomyces-derived transglutaminase). The stress of the obtained substitute cheese was measured in the same manner as in "1. Kamaboko". In addition, a substitute cheese that had not been subjected to enzyme treatment (enzyme-untreated) was separately obtained and stress was measured in the same manner. The stress of the substitute cheese treated with the present enzyme or the substitute cheese treated with Streptomyces-derived transglutaminase was calculated as a stress improvement rate and evaluated relative to the stress of the non-enzyme-treated substitute cheese, which was set to 1.

[0144] (Results) The results are shown in the table below.

[0145] By using this enzyme, a significantly higher stress could be obtained than when Streptomyces-derived transglutaminase was used.

[0146] (12. Cow Bone Gelatin, Pig Skin Gelatin, Fish Gelatin) Each gelatin shown in the table below was dissolved in 50 mM phosphate buffer to a final concentration of 5% by mass to prepare a gelatin solution.

[0147] To 9 mL of each gelatin solution, 0.5 mL of water and 0.5 mL of the solution containing the enzyme prepared in Example 1 at a concentration of 1.0 mg / mL were added and mixed. After mixing, the mixture was poured into a Petri dish 56 mm in diameter and 16 mm in height, covered, and incubated at 40°C for 20 hours to obtain coagulated gelatin (treated with this enzyme). The stress of the obtained coagulated gelatin was measured in the same manner as in "1. Kamaboko." Note that gelatin did not coagulate when enzyme treatment was not performed.

[0148] (Results) The results are shown in the table below.

[0149] Coagulation was observed in all gelatins when this enzyme was used.

[0150] (13. Milk Gel / Soy Milk Gel) 10 mL of milk (Meiji Co., Ltd., Oishii Gyunyu) or 10 mL of soy milk (Sujata Meiraku Group, Organic Soy Milk That Can Also Be Used to Make Tofu) was mixed with 0.5 mL of either the enzyme prepared in Example 1 or a solution containing Streptomyces-derived transglutaminase at a concentration of 1.0 mg / mL. After mixing, the mixture was poured into a 56 mm diameter, 16 mm high Petri dish, covered, and incubated at 40°C for 20 hours to obtain a milk gel or a soy milk gel (treated with the enzyme or Streptomyces-derived transglutaminase). The stress of the resulting gel was measured in the same manner as in "1. Kamaboko." Furthermore, without enzyme treatment, neither the milk nor the soy milk solidified.

[0151] (Results) The results are shown in the table below.

[0152] By using this enzyme, a higher stress could be obtained than when using transglutaminase derived from Streptomyces.

[0153] (14. Egg White Gel) Egg white (protein: 10.1 wt%) was extracted from a chicken egg, 180 mL of water was added, and the mixture was strained through a tea strainer. To 9 mL of the egg white solution, 0.5 mL of water and 1 mL of either the enzyme prepared in Example 1 or a solution containing Streptomyces-derived transglutaminase at a concentration of 1.0 mg / mL were added and mixed. After mixing, the mixture was poured into a 56 mm diameter, 16 mm high Petri dish, covered, and incubated at 50°C for 4 hours. After incubation, the mixture was steamed in a boiling pot. The mixture was cooled to room temperature to obtain egg white gels (treated with this enzyme or Streptomyces-derived transglutaminase). The stress of the obtained egg white gels was measured in the same manner as in "1. Kamaboko." Additionally, egg white gels without enzyme treatment (enzyme-untreated) were separately obtained, and the stress was measured in the same manner. The stress of the egg white gel not treated with the enzyme was set to 1, and the relative stress values ​​of the egg white gel treated with the present enzyme or the egg white gel treated with Streptomyces-derived transglutaminase were calculated and evaluated as the stress improvement rate.

[0154] (Results) The results are shown in the table below.

[0155] By using this enzyme, a higher stress could be obtained than when using transglutaminase derived from Streptomyces.

[0156] (15. Chicken Egg Yolk Gel) 9 mL of egg yolk (protein: 16.5 wt%) separated from a chicken egg was mixed with 0.5 mL of water and 0.5 mL of a solution containing the enzyme prepared in Example 1 at a concentration of 1.0 mg / mL. After mixing, the mixture was poured into a Petri dish 56 mm in diameter and 16 mm in height, covered, and incubated at 50°C for 5 hours to obtain a chicken egg yolk gel (treated with the present enzyme). The stress of the obtained chicken egg yolk gel was measured in the same manner as in "1. Kamaboko." In addition, a chicken egg yolk gel that was not subjected to enzyme treatment (enzyme-untreated) was separately obtained and similarly measured for stress. The stress of the chicken egg yolk gel treated with the present enzyme, relative to the stress of the enzyme-untreated chicken egg yolk gel, was calculated and evaluated as a stress improvement rate.

[0157] (Results) The results are shown in the table below.

[0158] Compared to untreated samples, the use of this enzyme resulted in an improvement in stress.

[0159] (16. Casein Thermoreversible Gel) Milk casein was suspended in 50 mM phosphate buffer to a final concentration of 11.1% by mass, and the pH was adjusted to 7 with sodium hydroxide. The milk casein suspension was then heated in boiling water to completely dissolve it. 0.75 mL of water and 0.25 mL of the enzyme prepared in Example 1 or a solution containing Streptomyces-derived transglutaminase at a concentration of 1.0 mg / mL were added to 9 mL of casein solution and mixed. After thorough mixing, the mixture was incubated at 40°C for 20 hours to gel it. The resulting gel was autoclaved (120°C, 20 minutes) and cooled to room temperature.

[0160] (Results) Gels formed by Streptomyces-derived transglutaminase maintained their gel shape even after autoclaving. On the other hand, gels formed by this enzyme became liquid after autoclaving and regelled when cooled to room temperature. This thermoreversible gel formation was observed even after repeating heat treatment (autoclaving) and cooling at least three times.

[0161] (17. Protein Cross-Linking in Oat Milk) When the present enzyme was allowed to act on oat milk, cross-linking of oat proteins was confirmed.

[0162] (18. Tuna Nuggets) 70 g of tuna flakes (Inaba Light Tuna Flakes (Inaba Foods Co., Ltd.), protein: 11.8 wt%) containing seasoning liquid were mixed with 37.5 g of wheat flour and a small amount of salt. The resulting meat mixture was divided into 20 g portions, and 1 mL of the enzyme prepared in Example 1 or a solution containing Streptomyces-derived transglutaminase at a concentration of 1.0 mg / mL was added and further mixed. The meat mixture was packed into a petri dish with a diameter of 56 mm and a height of 16 mm, shaped, covered, and incubated at 50°C for 2 hours. After incubation, the meat mixture was removed from the petri dish and deep-fried on both sides in salad oil heated to 170°C for 2 minutes per side. Excess oil was drained using a draining net, and the tuna nuggets were cooled to room temperature to obtain tuna nuggets (treated with the present enzyme or with Streptomyces-derived transglutaminase). The stress of the obtained tuna nuggets was measured in the same manner as in "1. Kamaboko". In addition, the same procedure was repeated except that the enzyme treatment was not performed to obtain separate tuna nuggets (untreated with enzyme) and measure the stress. The stress of the tuna nuggets untreated with enzyme was set to 1, and the relative value of the stress of the tuna nuggets treated with the present enzyme or the tuna nuggets treated with Streptomyces-derived transglutaminase was calculated and evaluated as the stress improvement rate.

[0163] (Results) The results are shown in the table below.

[0164] By using this enzyme, a higher stress could be obtained than when using transglutaminase derived from Streptomyces.

[0165] (19. Butter) 2.25 mL of 0.2 M phosphate buffer solution at pH 6.86 was added to 15 g of salted butter (Snow Brand Hokkaido Butter (Megmilk Snow Brand Co., Ltd.), protein: 0.6 wt%) and stirred at 50°C for 15 minutes. 0.75 mL of either the enzyme prepared in Example 1 or a solution containing Streptomyces-derived transglutaminase at a concentration of 1.0 mg / mL was added thereto and further mixed. After mixing, the mixture was incubated at 50°C for 1 hour. After incubation, the butter was poured into a Petri dish with a diameter of 56 mm and a height of 16 mm, covered, and chilled in a refrigerator overnight to harden. After cooling overnight, the mixture was left to cool to room temperature to obtain butter (treated with this enzyme or Streptomyces-derived transglutaminase). The stress of the obtained butter was measured in the same manner as in "1. Kamaboko". In addition, the same procedure was repeated except that the enzyme treatment was not performed, and the stress was measured for separate (enzyme-untreated) butter. The stress of the enzyme-untreated butter was set to 1, and the relative stress values ​​of the butter treated with the present enzyme and the butter treated with Streptomyces-derived transglutaminase were calculated and evaluated as the stress improvement rate.

[0166] (Results) The results are shown in the table below.

[0167] By using this enzyme, a significantly higher stress could be obtained than when Streptomyces-derived transglutaminase was used.

[0168] (20. Pork Cutlets) 35 g of pork thigh meat (protein: 20.5 wt%) was pierced at appropriate intervals with a fork and softened. It was then wrapped in plastic wrap and uniformly adjusted to a uniform thickness using a rolling pin. A small amount of salt was applied to both sides of the uniformly adjusted pork thigh meat. The pork thigh meat was placed in a resealable plastic bag, and 5 mL of a solution containing the enzyme prepared in Example 1 at a concentration of 1.0 mg / mL and 5 mL of water were added, followed by further mixing. The bag was resealed after removing as much air as possible, and then incubated at 40°C for 3 hours. After incubation, the pork thigh meat was removed from the resealable plastic bag and lightly coated with flour. It was then dipped in beaten egg and coated with breadcrumbs. It was deep-fried in salad oil heated to 180°C for 2 minutes and 30 seconds, and excess oil was drained using a drainer. The pork thigh meat was then cooled to room temperature to obtain pork cutlets (treated with the present enzyme). The stress of the resulting pork cutlets was measured in the same manner as in "1. Kamaboko." Additionally, a separate pork cutlet (untreated with enzyme) was obtained and measured for stress using the same procedure, except that enzyme treatment was not performed. The stress of the untreated pork cutlet was set at 1, and the relative value of the stress of the pork cutlet treated with this enzyme was calculated and evaluated as the stress improvement rate.

[0169] (Results) The results are shown in the table below.

[0170] A significant improvement in stress was observed when this enzyme was used compared to when the enzyme was not used.

[0171] (21. Sausage) 50 g of ground pork (protein: 17.7 wt%) was mixed with 1.25 g of salt, 1.875 g of sugar, 1.25 mL of water, and 5 mL of a solution containing the enzyme prepared in Example 1 at a concentration of 1.0 mg / mL. Separately, 20 cm long sheep intestines were prepared and soaked in water for 15 minutes. The seasoned meat filling was stuffed into the intestines using a sausage stuffer, wrapped in plastic wrap, and incubated at 50°C for 3.5 hours. Each sausage contained 50 g of meat filling and had a total length of 6 cm. After incubation, the sausages were boiled in water at approximately 80°C for 20 minutes, and then grilled in a frying pan over medium heat for 5 minutes. After grilling, the sausages were cooled to room temperature to obtain sausages (treated with the present enzyme). The resulting sausages were subjected to stress measurements in the same manner as in "1. Kamaboko." In addition, the same procedure was repeated except that the enzyme treatment was not performed, and the stress of the sausage (untreated) was measured. The stress of the untreated sausage was set to 1, and the relative value of the stress of the sausage treated with the present enzyme was calculated and evaluated as the stress improvement rate.

[0172] (Results) The results are shown in the table below.

[0173] Compared to untreated samples, the use of this enzyme resulted in an improvement in stress.

[0174] (22. Chicken Meatballs) An appropriate amount of chicken thigh meat (protein: 16.6% by weight) was placed in a food processor and minced. 1.25 g of salt and 5 g of sugar were added to 300 g of the resulting minced chicken thigh meat and kneaded thoroughly until sticky. A beaten egg (the equivalent of one egg) and 15 g of flour were then added and mixed until the mixture was well combined. The meatballs were divided into 25 g portions, and 1 mL of a solution containing the enzyme prepared in Example 1 at a concentration of 1.0 mg / mL was added and mixed. The meatballs were packed into a 56 mm diameter, 16 mm high Petri dish, shaped, covered, and incubated at 50°C for 1 hour. After incubation, the mixture was boiled in boiling water for 5 minutes. After boiling, the mixture was cooled to room temperature to obtain chicken meatballs (treated with the enzyme). The stress of the resulting chicken meatballs was measured in the same manner as in "1. Kamaboko." Separately, chicken meatballs (untreated with enzyme) were obtained in the same manner, except that the enzyme treatment was not performed, and their stress was measured. The stress of the untreated chicken meatball was set at 1, and the relative value of the stress of the chicken meatball treated with the enzyme was calculated and evaluated as the stress improvement rate.

[0175] (Results) The results are shown in the table below.

[0176] Compared to untreated samples, the use of this enzyme resulted in an improvement in stress.

[0177] (23. Beef Steak) 40 g of beef tenderloin (protein: 20.5 wt%) was pierced with a fork at appropriate intervals, wrapped in plastic wrap, and the thickness was uniformed using a rolling pin. A small amount of salt was applied to both sides of the beef tenderloin. The beef tenderloin was placed in a zippered plastic bag, and 5 mL of the enzyme prepared in Example 1 or a solution containing Streptomyces-derived transglutaminase at a concentration of 1.0 mg / mL, and 5 mL of water were added, followed by further mixing. The bag was sealed after removing as much air as possible and incubated at 40°C for 4 hours. After incubation, the beef tenderloin was removed from the zippered plastic bag and grilled on both sides in a frying pan over medium heat for 2 minutes. After grilling, the beef steak (treated with this enzyme or Streptomyces-derived transglutaminase) was obtained by cooling to room temperature. The stress of the obtained beef steak was measured in the same manner as in "1. Kamaboko". In addition, a separate beef steak (untreated with enzyme) was obtained by the same procedure, except that the enzyme treatment was not performed, and the stress was measured. The stress of the beef steak untreated with enzyme was set to 1, and the relative value of the stress of the beef steak treated with this enzyme or the beef steak treated with Streptomyces-derived transglutaminase was calculated and evaluated as the stress improvement rate.

[0178] (Results) The results are shown in the table below.

[0179] By using this enzyme, a higher stress could be obtained than when using transglutaminase derived from Streptomyces.

[0180] (24. Cookies) 50 g of strong wheat flour (Nissin Camellia Strong Wheat Flour (Nissin Flour Milling Welna Co., Ltd.), protein: 12.6 wt%), 25 g of salted butter (Snow Brand Hokkaido Butter (Snow Brand Megmilk Co., Ltd.)), 25 g of sugar, 30 g of beaten egg, 0.4 g of baking powder, and a small amount of salt were added to a bowl and mixed thoroughly. The mixed cookie dough was divided into 15 g portions, and 1 mL of the enzyme prepared in Example 1 or a solution containing Streptomyces-derived transglutaminase at a concentration of 1.0 mg / mL was added and mixed. The bowl containing the cookie dough was covered with plastic wrap and incubated at 50°C for 4 hours. The dough was then rested in a refrigerator for 1 hour and shaped using a rolling pin. The front and back sides were baked for 10 minutes each in an oven preheated to 160°C (baking temperature: 160°C). After baking, the cookies were cooled to room temperature to obtain cookies (treated with the present enzyme or with Streptomyces-derived transglutaminase). The stress of the obtained cookies was measured in the same manner as in "1. Kamaboko". The measurement was terminated when the cookie broke during the measurement. In addition, the same procedure was repeated except that no enzyme treatment was performed to obtain separate cookies (untreated with enzyme) and measure their stress. The stress of the untreated cookie was set to 1, and the relative values ​​of the stress of the cookies treated with the present enzyme or the cookies treated with Streptomyces-derived transglutaminase were calculated and evaluated as the stress improvement rate.

[0181] (Results) The results are shown in the table below.

[0182] By using this enzyme, a higher stress could be obtained than when using transglutaminase derived from Streptomyces.

[0183] (25. Tuna Fillet) 20 g of raw tuna fillet (protein: 25.4 wt%) was placed in a resealable plastic bag and mixed with 2 mL of water. 2 mL of either the enzyme prepared in Example 1 or a solution containing Streptomyces-derived transglutaminase at a concentration of 1.0 mg / mL was added and further mixed. The bag was sealed with a zipper, removing as much air as possible, and incubated at 40°C for 4 hours. After incubation, the tuna fillet was removed from the resealable plastic bag and rinsed with running water. The water was drained and cooled to room temperature to obtain enzyme-treated tuna fillet (treated with the present enzyme or Streptomyces-derived transglutaminase). The resulting tuna fillet was subjected to stress measurement in the same manner as in "1. Kamaboko." Separately, a tuna fillet (untreated with enzyme) was obtained and subjected to stress measurement using the same procedure, except that the enzyme treatment was omitted. The stress of the tuna fillet treated with this enzyme or the tuna fillet treated with Streptomyces-derived transglutaminase was calculated as a relative value to the stress of the tuna fillet not treated with the enzyme, which was set to 1, and evaluated as a stress improvement rate.

[0184] (Results) The results are shown in the table below.

[0185] By using this enzyme, a higher stress could be obtained than when using transglutaminase derived from Streptomyces.

[0186] (26. Squid Fillets) 20 g of raw squid fillets (protein: 18.6 wt%) and 2 mL of water were placed in a resealable plastic bag and mixed together. 2 mL of a solution containing the enzyme prepared in Example 1 at a concentration of 1.0 mg / mL was added and further mixed together. The bag was sealed after removing as much air as possible and incubated at 40°C for 4 hours. After incubation, the squid fillets were removed from the resealable plastic bag and rinsed with running water. The water was drained and cooled to room temperature to obtain enzyme-treated squid fillets (treated with this enzyme). The stress of the obtained squid fillets was measured in the same manner as in "1. Kamaboko." Separately, squid fillets (untreated with enzyme) were obtained and measured for stress using the same procedure, except that no enzyme treatment was performed. The stress of the squid fillets treated with this enzyme, relative to the stress of the untreated squid fillets (defined as 1), was calculated and evaluated as a stress improvement rate.

[0187] (Results) The results are shown in the table below.

[0188] Compared to untreated samples, the use of this enzyme resulted in an improvement in stress.

[0189] (27. Scallop Adductor Muscles) 160 g of raw scallop adductor muscle (protein: 16.9 wt%) and 2 mL of water were placed in a resealable plastic bag and mixed together. 2 mL of a solution containing the enzyme prepared in Example 1 at a concentration of 1.0 mg / mL was added and further mixed together. The bag was sealed after removing as much air as possible and incubated at 40°C for 4 hours. After incubation, the scallop adductor muscle was removed from the resealable plastic bag and rinsed with running water. The scallop adductor muscle was drained and cooled to room temperature to obtain enzyme-treated scallop adductor muscle (treated with this enzyme). The stress of the obtained scallop adductor muscle was measured in the same manner as in "1. Kamaboko." Separately, the same procedure was repeated except that the enzyme treatment was omitted, and stress was measured on scallop adductor muscle (untreated with enzyme). The stress of the scallop adductor muscle treated with the present enzyme was calculated as a relative value to the stress of the scallop adductor muscle not treated with the enzyme, which was taken as 1, and evaluated as a stress improvement rate.

[0190] (Results) The results are shown in the table below.

[0191] Compared to untreated samples, the use of this enzyme resulted in an improvement in stress.

[0192] (28. Cod Roe) The skin was removed from cod roe (protein: 24.0 wt%), and 5 g was weighed and placed in a beaker. 0.5 mL of water and 0.5 mL of either the enzyme prepared in Example 1 or a solution containing Streptomyces-derived transglutaminase at a concentration of 1.0 mg / mL were added and mixed. The cod roe was placed in a 56 mm diameter, 16 mm high petri dish, covered, and incubated at 40°C for 4 hours. After incubation, the dish was cooled to room temperature to obtain enzyme-treated cod roe (treated with the present enzyme or Streptomyces-derived transglutaminase). The stress of the obtained cod roe was measured in the same manner as in "1. Kamaboko." Separately, a cod roe (untreated with enzyme) was obtained and its stress was measured using the same procedure except that the enzyme treatment was omitted. The stress of the cod roe not treated with the enzyme was set to 1, and the relative value of the stress of the cod roe treated with the present enzyme or the cod roe treated with Streptomyces-derived transglutaminase was calculated and evaluated as the stress improvement rate.

[0193] (Results) The results are shown in the table below.

[0194] By using this enzyme, a higher stress could be obtained than when using transglutaminase derived from Streptomyces.

[0195] (29. Ice Cream) 250 mL of milk (Meiji Co., Ltd., Oishii Gyunyu, protein: 34 mg / mL), 50 g of sugar, and 30 g of beaten eggs (protein: 12.2 wt%) were placed in a pot, mixed well, and heated over low heat until thickened to prepare a milk solution. After cooling, 3 mL of the enzyme prepared in Example 1 or a solution containing Streptomyces-derived transglutaminase at a concentration of 1.0 mg / mL was added to 60 g of the milk solution, mixed, and incubated at 40°C for 3 hours. After incubation, 20 g of the enzyme-treated milk solution was divided into petri dishes, covered, and frozen overnight in a -25°C freezer. After freezing overnight, enzyme-treated ice cream (treated with the present enzyme or Streptomyces-derived transglutaminase) was obtained. The resulting ice cream was subjected to stress measurement in the same manner as in "1. Kamaboko." In addition, ice cream (untreated) was obtained separately using the same procedure, except that the enzyme treatment was not performed, and the stress was measured. The stress of the untreated ice cream was set at 1, and the relative value of the stress of the ice cream treated with this enzyme or the ice cream treated with Streptomyces-derived transglutaminase was calculated and evaluated as the stress improvement rate.

[0196] (Results) The results are shown in the table below.

[0197] By using this enzyme, a higher stress could be obtained than when using transglutaminase derived from Streptomyces.

[0198] (30. White Rice / Brown Rice) 8.65 g of washed white rice (protein: 6.1 wt%) or brown rice (protein: 6.8 wt%) was mixed with 8 mL of water and 2 mL of a solution containing the enzyme prepared in Example 1 at a concentration of 1.0 mg / mL. The mixture was incubated at 40°C for 4 hours and then cooked. After cooling, the mixture was shaped to obtain enzyme-treated white rice or brown rice (treated with the present enzyme). The stress of the obtained white rice or brown rice was measured in the same manner as in "1. Kamaboko." Furthermore, the same procedure was repeated except that no enzyme treatment was performed to obtain separate white rice or brown rice (untreated with the enzyme) and measure its stress. The stress of the white rice or brown rice treated with the present enzyme, relative to the stress of the untreated white rice or brown rice, was set at 1, and the relative value was calculated and evaluated as the stress improvement rate.

[0199] (Results) The results are shown in the table below.

[0200] Compared to untreated samples, the use of this enzyme resulted in an improvement in stress.

[0201] (31. Pasta) 115 g of durum semolina flour (protein: 13.3 wt%), 50 g of beaten egg (protein: 12.2 wt%), and 5 g of salt were mixed and kneaded for 10 minutes until the dough came together. 10 mL of a solution containing the enzyme prepared in Example 1 at a concentration of 1.0 mg / mL was added to the dough, and the dough was kneaded for an additional 10 minutes. The dough was wrapped in plastic wrap and allowed to rest in the refrigerator for 30 minutes. The dough was then kneaded again to a uniform consistency and rolled out to a thickness of 1 mm using a rolling pin. The rolled out dough was cut into pieces measuring 5 cm in length and width. The cut pieces were arranged on a tray, wrapped in plastic wrap, and incubated at 40°C for 2 hours. After incubation, the dough was boiled in boiling water for 2 minutes and then immersed in cold water. The resulting mixture was cooled to room temperature to obtain enzyme-treated pasta (treated with the present enzyme). In addition, pasta (untreated) was obtained separately by the same procedure, except that the enzyme treatment was not performed. Two sensory panelists evaluated the texture of the dough after boiling for the untreated pasta and the pasta treated with the present enzyme.

[0202] (Results) Compared to untreated dough, the dough that was boiled using this enzyme felt chewier to the touch.

[0203] Example 6: Preparation of an enzyme having protein deamidation activity and transglutaminase activity derived from Crossiella cryophila (Enzyme 2) and an enzyme having protein deamidation activity and transglutaminase activity derived from Krasilnikova cinnamomea (Enzyme 3) 1. Preparation of transformants For Streptomyces microorganisms, the target sequence was incorporated into an expression vector. PCR amplification was performed, and transformants (heterologous expression strains derived from C. cryophila and K. cinnamomea) were obtained using standard methods. The obtained transformants were subjected to sequence analysis to confirm that the target sequence had been incorporated.

[0204] 2. C. cryophila-derived heterologous expression strain, K. cinnamomea-derived heterologous expression strain The obtained transformants were cultured in tryptophan broth medium (manufactured by Nissui Pharmaceutical Co., Ltd.) at 32 ° C for 4 days, and the supernatant was collected. The obtained supernatant was dialyzed against 20 mM phosphate buffer (pH 6) according to standard methods. The dialyzed sample was subjected to ion exchange chromatography using a cation exchange column (SP Sepharose FF, manufactured by GE Healthcare) equilibrated with the same buffer. Each enzyme sample was then further purified using a His tag protein purification column (His trap HP, manufactured by Cytiva) to obtain each enzyme sample.

[0205] The amino acid sequence and nucleotide sequence of the enzyme having protein deamidation activity and transglutaminase activity derived from C. cryophila are shown below. Amino acid sequence (mature sequence only) (SEQ ID NO: 6) GLPIAPPLRPGVESKTWKVADYIKAWETYHGRPMTADERENLARGCIGVTVVNLERGDVGNPPLGLSFGTFGKAREVQAALNEIIKSKPSAAQYAAAVRSHPLLSKLENVQRALPADLNTGELTAAIFSKRFYSKQNPNWTDEFAEKMYRANARGQVDMNSYRYVARPGYVNFDYGWYDENTRNWWHANHAEPGMKVYQSTFDYYSRDLLDFDRQVFTVAFAKKV

[0206] Amino acid sequence (full length) *The underlined part is the mature sequence (SEQ ID NO: 7) MRNSVLIRTLTTAALGLGLLIPGAAAATAAPAPSTAAAVSIVGLPIAPPLRPGVESKTWKVADYIKAWETYHGRPMTADERENLARGCIGVTVVNLERGDVGNPPLGLSFGTFGKAREVQAALNEIIKSKPSAAQYAAAVRSHPLLSKLENVQRALPADLNTGELTAAIFSKRFYSKQNPNWTDEFAEKMYRANARGQVDMNSYRYVARPGYVNFDYGWYDENTRNWWHANHAEPGMKVYQSTFDYYSRDLLDFDRQVFTVAFAKKV

[0207] Base sequence (mature body) (sequence number 8) ggcctccccatcgcccccccgctgcgcccggcgtcgagtcgaagacgtggaaggtcgcggactacatcaaggcgtggggagacgtaccatggccgccggacgaacgggaacctcgcgcggggctgcatcggggtcaccgtggtcaacctcgagcggggcgacgtggggaaatccgccgctcggccctctcgttcggcacgttcggcaaagcgcggggaggtccaagcggcgctcaacgagatcatcaagtcgaagccgtcggcgcgcagtacgcggccgggtccgctcgcaccccctgctcgaagctcgaagctcgagaacgtgcagcggg ccctcccggcggatctcaacgggcgagctcacggcggcgatcttctcgaagcggttctactcgaagcagaacccgaactggacggacgagttcgcggagaagatgtaccgggcgaacgcgcggggccaagtcgacatgaactcgtaccggtacgtcgcgcggccggg ctacgtcaacttcgactacgggctggtacgacgagaacacgcggaactggtggcacgcgaatcacgcggagccgggcatgaaggtctatcagtcgacgttcgactactactcgcgggatctcctcgacttcgaccggcaagtcttcacggtcgcgttcgcgaagaaaggtc

[0208] Base sequence (full length) (sequence number 9) atgcggaactccgtgctcatccgcacgctgaccaccgccgccctcggccctgggcctcctgatccccggggcggccgcgacggccccccggccctccaccgcggcggccgtctccattgtcggcctcccatcgcccccccgctgcgcccggcgtcgagtcgaagacgtggaaggtcgcggactacatcaaggcgtggagacgtaccatggccgcagatgaccgcggacgaacgggaacctcgcgcggggctgcatcggggtcaccgtggtcaacctcgagcggggcgacgtggggatccgccgcggccctctcgttcggcacgttcggcaaagcgcgggaggtccaagcggcgctcaacgagatcatcaagtcgaagccgtcgggggg cgcagtacgcggccgcggtccgctcgcaccccctgctctcgaagctcgagaacgtgcagcgggccctcccggcggatctcaacgggcgagctcacggcggcgatcttctcgaagcggttctactcgaagcagaacccgaactggacggacgagttcgcggagaagatgtaccgggcgaacgcgcggggccaagtcgac atgaactcgtaccggtacgtcgcgcggccgggctacgtcaacttgactacgggctggtacgacgagaacacgcggaactggtggcacgcgaatcacgcggagccgggcatgaaggtctatcagtcgacgttcgactactactcgcgggatctcctcgacttcgaccggcaagtcttcacggtcgcgttcgcgaagaaggtc

[0209] The amino acid sequence and nucleotide sequence of the enzyme having protein deamidation activity and transglutaminase activity derived from K. cinnamomea are shown below: Amino acid sequence (mature sequence only) (SEQ ID NO: 10) TESDPRSPTPARTATQAPIAEVRPAERSLANANAATATELTPLAPALPPGVSTRTWSVEDFVELFERKYGRPMTQDERNALARGCIGVTTVNLERGNINPPLGMSFGTFATSRDVQNAINDILATNPSRTQFVAAVAQHPLLSRIDNVTDSLPGGPTSQWTAVIFSKRFYSKQDPSWTDEQADQAFRPDPATGQVDMTDYRYRAKPGYVNFDYGWLDEGSGNWWHANHAEPGMKVYQSTLRHYSRPLLDFDRQVFSVTFGRVHP

[0210] Amino acid sequence (full length) *The underlined part is the mature sequence (SEQ ID NO: 11) MPKRLLRLFVIFVAGLSISTMVAAPALATESDPRSPTPARTATQAPIAEVRPAERSLANANAATATELTPLAPALPPGVSTRTWSVEDFVELFERKYGRPMTQDERNALARGCIGVTTVNLERGNINPPLGMSFGTFATSRDVQNAINDILATNPSRTQFVAAVAQHPLLSRIDNVTDSLPGGPTSQWTAVIFSKRFYSKQDPSWTDEQADQAFRPDPATGQVDMTDYRYRAKPGYVNFDYGWLDEGSGNWWHANHAEPGMKVYQSTLRHYSRPLLDFDRQVFSVTFGRVHP

[0211] Base sequence (mature body) (sequence number 12) accgaatccgacccgcggtcccccacccccgcgcgcacggccacgcaagcgccgatcgcggaagtgcggcggcgcggaacgctcctggcgaacgccaatgccgccaccgaccgaactgacccctcgccccgccctccccggggtgtcgacgcggacgtggtcggtcgaggacttcgtcgagctcttcgagcggaagtacggcggccgatgacccaagacgaacgcaatgccctcgcgcggggctgcatcggctcacgacgggtcaacctcgagcggggcaacatcaacccgccgcggcatgtcgttcggcacgagttcgcgacgtcgcgcggaggtgcacagaacgcgatcaacgacatccccaatccctccgcaccagcaatccctccgcaaccattt gtggcggccgtggcgcagcacccgctcctctcgcggatcgacaacgtcacggactcgctcccgggcggcccgacgtcgcagtggacggcggtcatcttctcgaagcggttctactcgaagcaagacccgtcgtggacggacgagcaagcggatcaagcgttccgccccgatccggcgacgggccaagtcgacatgacg gactaccggtaccgggcgaagccgggctacgtcaacttcgactacgggctggctcgacgagggctcgggcaactggtggcacgcgaaccacgcggagccggggatgaaggtgtatcagtcgacgctccggcactactcgcggccgctcctcgacttcgaccggcaagtcttctcggtcacgttcggccgggtccacccg

[0212] Base sequence (full length) (sequence number 13) atgccgaagcggctcctccggctcttcgtcatcttcgtcgcgggcctctcgatttcgaccatggtggccgccggccctcgcgaccgaatccgacccgggtcccccacccccgcgcgcgcacggccacgcaagcgccgatcgcggaagtgcggcggcggaacgctcgctggcgaacgccaatgccgccaccgccaccgaactgacccctcgccccgccctcccccgccggctgtcgagcgcagtggtcggtcgagacttcgtcgagctcttcgagcggaagtacggcggccgatgacccaagacgaacgcaatgccctcgcgcggggctgcatcggctcacgacggtcaacctcgagcggcaacatcaacccgccgcggcatgtcgttcggcacgttcgcagttcgcgacgtcgcggacgtgcagaacgc atcaacgacatcctcgcgaccaatccctcccggacccaatttgtggcggccgtggcgcagcacccgctcctctcgcggatcgacaacgtcacggactcgctcccgggcggcccgacgtcgcagtggacggcggtcatcttctcgaagcggttctactcgaagcaagacccgtcgtggacggacgagcaagcggatcaagcgttccgccccgatccggcg acgggccaagtcgacatgacggactaccggtaccgggcgaagccgggctacgtcaacttcgactacggctggctcgacgaggctcgggcaactggtggcacggaaccacgcggagccggggatgaaggtgtatcagtcgacgctccggcactactcgcggccgctcctcgacttcgaccggcaagtcttctcggtcacgttcggccgggtccacccg

[0213] Example 7 Evaluation of protein deamidation activity and transglutaminase activity on protein substrates (Method) 5 mg of insulin B chain (insulin B chain, oxidized form, derived from bovine pancreas, Sigma-Aldrich, sequence: Phe-Val-Asn-Gln-His-Leu-Cys(SOH)-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys(SOH)-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Ala (SEQ ID NO: 5)) was dissolved in 2 mL of ultrapure water to prepare a substrate solution. Next, 10 μL of each 0.2 mg / mL purified enzyme solution, 25 μL of 0.1 M sodium phosphate buffer (pH 6.5), and 0.9 μL of protease inhibitor (ProteoGuard EDTA-Free Protease Inhibitor Cocktail (TaKaRa)) were added to 22.5 μL of substrate solution for a total volume of 58.4 μL to form a reaction solution. The reaction solution was heated to 40°C in an incubator, left overnight, and then heated at 100°C for 10 minutes to terminate the reaction. The reaction-stopped solution was analyzed using HPLC. Deamidation activity and transglutaminase activity were detected by the peak transition from insulin B chain to deamidated insulin B chain. <Protein deamidation activity> The amount of deamidated insulin B chain produced was calculated from the peak areas of insulin B chain and deamidated insulin B chain calculated from the chromatogram, and protein deamidation activity was evaluated in terms of the deamidation rate (amount of deamidated insulin B chain produced ÷ amount of substrate insulin B chain × 100). (Transglutaminase activity) The amount of remaining insulin B chain and the amount of deamidated insulin B chain produced were calculated from the areas of each peak calculated from the chromatogram, and protein deamidation activity was evaluated in terms of the cross-linking rate ((100 - (amount of remaining insulin B chain ÷ amount of substrate insulin B chain × 100) - deamidation rate). The ratio of protein deamidation activity to transglutaminase activity was calculated from the ratio of the deamidation rate to the cross-linking rate calculated by the above method.

[0214] (Results) The results are shown in the table below.

[0215] (Discussion) The results shown in the table above demonstrate that the present enzymes 2 and 3 of the present invention have protein deamidation activity and transglutaminase activity. Furthermore, the adhesiveness of the present enzyme 2 was evaluated according to the method of Example 2, and an increase in adhesiveness was confirmed. It is presumed that both the present enzyme 2 (C. cryophila) and the present enzyme 3 (K. cinnamomea) have the same effect as the present enzyme (L. tulufanense).

[0216] Example 8: Preparation of an enzyme having protein deamidation activity and transglutaminase activity (Enzyme 4) derived from Longispora fulva Longispora fulva cells were cultured in tryptosoy broth medium (manufactured by Nissui Pharmaceutical Co., Ltd.) at 28°C for 6 days, and the supernatant was collected. The resulting supernatant was dialyzed against 20 mM phosphate buffer (pH 6) according to a standard method. The dialyzed sample was subjected to ion exchange chromatography using a cation exchange column (SP Sepharose FF, manufactured by GE Healthcare) equilibrated with the same buffer, and the active fraction was desalted and concentrated to obtain a crude enzyme sample.

[0217] The amino acid sequence and nucleotide sequence of the enzyme having protein deamidation activity and transglutaminase activity derived from Longispora fulva are shown below: Amino acid sequence (mature sequence only) (SEQ ID NO: 14) APPTSADRSGTPVAGLRVPSSLSAVRGAARFDAVAPEATPLPVGQKTRTWTIARYAAAWRTANGRAMTDRERDALARGCIGVTTVNIERGNINPPLGLSFGTFEKATAVRDALDAVLAGAAGRTDYAARVGADPLLSTLDNVTESLPAGDPDQWAAVVFSKRFHSGQDSTWTAAETDRAFRPDARGQVDMSAYHYRAKPGYVNFDYGWYDPATGSWWHANHAEPGMEIYQSTLKYYSRPLLDFDRQVYSVTFARKAAPAWRSSTN

[0218] Amino acid sequence (full length) *The underlined part is the mature sequence (SEQ ID NO: 15) MRNRLLRITMAAVLGVGATVGLAATTGYAAPPTSADRSGTPVAGLRVPSSLSAVRGAARFDAVAPEATPLPVGQKTRTWTIARYAAAWRTANGRAMTDRERDALARGCIGVTTVNIERGNINPPLGLSFGTFEKATAVRDALDAVLAGAAGRTDYAARVGADPLLSTLDNVTESLPAGDPDQWAAVVFSKRFHSGQDSTWTAAETDRAFRPDARGQVDMSAYHYRAKPGYVNFDYGWYDPATGSWWHANHAEPGMEIYQSTLKYYSRPLLDFDRQVYSVTFARKAAPAWRSSTN

[0219] Base sequence (mature body) (sequence number 16) gccccgccgacgtccgccgaccgctcggggacccccgtggcggggctgcgggtccctctccgccgtgcggcgcgggggccgccgggttgacgccgtggcgccggaagccaccccgctccggtcgggcgcaaaagaccgcacgcacgtggacgattgcccgctacgccgcggcctggcggaccgcgaacggccgcgcatgaccgatcgggagcgcgatgccctcgccgggggctgcatcggcgtcacgacggtcaacatcgagcggggcaacatcaacccgccgctcggctctccttcggcacgtttgaaaaagcaccgaccgggtccgcgacgcgctggacgccgtcctcgccggggccgcaccgactacgccgcgcgcgtcggggcgcggcgcgtcggcgcgctgccgccgactacgccgcgcgcgtcggcgcgcgctcggcgccgcgactacgccgcgcgcgtcggcgcgcgcgctcggcgcgcgctcggcgcgctcggcgccgcgactacgccgcgcgcgctcgg atccgctcctctccacgctcgataatgtcacggaatcgctcccggcgggggaccccggatcagtgggcggcggtcgtcttctcgaagcgctttcattccgggcaagattccacctggaccgccgggggagaccgaccgggcgtttcggcccgatgcgcgcggccaagtcgacatgtcggcgtaccactaccgggcgaagcc gggctacgtcaacttcgactacgggctggtacgacccggcgacgggctcgtggtggcacgcgaaccacgcggagccgggcatggagatctatcagtcgacgctcaagtactactcgcggccgctcctcgacttcgaccggcaagtctactcggtcacgttcgcgcggaaggcggcgccggcgtggcggtcgtcgacgaac

[0220] Base sequence (full length) (sequence number 17) atgcggaaccgcctgctccgggatcacgatggccgcggtcctgggggtgggcgccaccgtggggctgccggccaccgtggggctcgccggaccacgggctatgcggccccgcacgtccgccgaccgctcggggacccccgtggcggggctgcggggcccgtccctctccgccgtgcgcggcgccgcccggttgacgccgtggcgccggaagccaccccgctccggtcgggcgcaaaagacccgcacgtggacgattgcccgctacgccgcggcctggcggaccgcaacggccgcgcatgaccgatcgggagcgcgatgccctcgcccggggctgcatcggcgtcacgacggtcaacatcgagcggggcaacatcaacccgctcggcctctccttcggcacgtttgaaaaagcgaccgcggtccgcgacgcgctggacgccgtccgcgctggacgccgtccgcgcagctggacgccgtccgcgcagcag ggggccgcgggccgcaccgactacgccgcgcgcgtcggcgcggatccgctcctctccacgctcgataatgtcacggaatcgctcccggcgggggacccggatcagtgggcggcggtcgtcttctcgaagcgctttcattccgggcaagattccacctggaccgccggggagaccgaccgggcgtttcggcccgatgcgcgcggccaagtcgacatgtcgg cgtaccactaccgggcgaagccgggctacgtcaacttcgactacgggctggtacgacccggcgacgggctcgtggtggcacggaaccacgcggagccgggcatggagatctatcagtcgacgctcaagtactactcgcggccgctcctcgacttcgaccggcaagtctactcggtcacgttcgcgcggaaggcggcgcgggcgtggcggtcgtcgacgaac

[0221] Example 9: Evaluation of protein deamidation activity and transglutaminase activity on protein substrates (Method) 5 mg of insulin B chain (insulin B chain, oxidized form, derived from bovine pancreas, Sigma-Aldrich, sequence: Phe-Val-Asn-Gln-His-Leu-Cys(SOH)-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys(SOH)-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Ala (SEQ ID NO: 5)) was dissolved in 2 mL of ultrapure water to prepare a substrate solution. To 90 μL of the substrate solution, 40 μL of a 1 mg / mL purified enzyme solution and 0.1 M potassium dihydrogen phosphate / disodium hydrogen phosphate buffer (pH 6.5) were added to prepare a reaction solution in a total volume of 180 μL. The reaction solution was heated to 40°C in an incubator, allowed to stand for 5 hours, and then heated at 100°C for 10 minutes to terminate the reaction. The reaction-terminated solution was analyzed by HPLC. Deamidation activity and transglutaminase activity were detected by the peak transition from insulin B chain to deamidated insulin B chain.

[0222] (Results) As a result of analysis using HPLC, a peak due to a deamidation reaction was confirmed when Enzyme 4 was used. It was also estimated that Enzyme 4 had transglutaminase activity.

[0223] The enzyme preparation of the present invention contains as an active ingredient an enzyme having protein deamidation activity and transglutaminase activity, and is therefore suitable for use in the fields of food and medical applications, and has great industrial utility value.

[0224] The present invention is not limited to the above-described embodiments and examples. Various modifications within the scope of the claims and within the scope that can be easily conceived by a person skilled in the art are also included in the present invention. The contents of papers, published patent applications, patent publications, and other publications explicitly stated in this specification are incorporated herein by reference in their entirety.

Claims

1. An enzyme preparation for protein deamidation and protein cross-linking, which comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 and contains as an active ingredient an enzyme having protein deamidation activity and transglutaminase activity.

2. The enzymatic preparation for protein deamidation and protein cross-linking according to claim 1, wherein the enzyme is derived from the genus Longimycelium.

3. A method for deamidating and cross-linking a protein or peptide, which comprises allowing the enzyme preparation for protein deamidation and protein cross-linking according to claim 1 or 2 to act on the protein or peptide.

4. A method for deamidating and cross-linking protein-containing foods and beverages, which comprises allowing the enzyme preparation for protein deamidation and protein cross-linking described in claim 1 or 2 to act on the protein-containing foods and beverages.

5. A method for producing a food or drink, which comprises allowing the protein deamidation and protein cross-linking enzyme preparation according to claim 1 or 2 to act on a protein-containing food or drink.

6. A food or drink treated with the enzyme preparation for protein deamidation and protein cross-linking according to claim 1 or 2.

7. A method for producing a protein deamidating and protein cross-linking enzyme 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.

8. An enzyme preparation for protein deamidation and protein cross-linking, which comprises an amino acid sequence having 65% or more sequence identity with the amino acid sequence of SEQ ID NO: 6 and contains as an active ingredient an enzyme having protein deamidation activity and transglutaminase activity.

9. The enzymatic preparation for protein deamidation and protein cross-linking according to claim 8, wherein the enzyme is derived from the genus Crossiella.

10. A method for deamidating and cross-linking a protein or peptide, which comprises allowing the enzyme agent for protein deamidation and protein cross-linking according to claim 8 or 9 to act on the protein or peptide.

11. A method for deamidating and cross-linking a protein-containing food or drink, which comprises allowing the protein deamidating and protein cross-linking enzyme preparation according to claim 8 or 9 to act on the protein-containing food or drink.

12. A method for producing a food or drink, which comprises allowing the protein deamidating and protein cross-linking enzyme preparation according to claim 8 or 9 to act on a protein-containing food or drink.

13. A food or drink treated with the enzyme preparation for protein deamidation and protein cross-linking according to claim 8 or 9.

14. A method for producing a protein deamidating and protein cross-linking enzyme comprising an amino acid sequence having 65% or more sequence identity with the amino acid sequence of SEQ ID NO: 6, comprising the step of culturing a bacterium of the genus Crossiella.

15. An enzyme preparation for protein deamidation and protein cross-linking, comprising an amino acid sequence having 65% or more sequence identity with the amino acid sequence of SEQ ID NO: 10 and containing as an active ingredient an enzyme having protein deamidation activity and transglutaminase activity.

16. The enzymatic preparation for protein deamidation and protein cross-linking according to claim 15, wherein the enzyme is derived from the genus Krasilnikovia.

17. A method for deamidating and cross-linking a protein or peptide, which comprises allowing the enzyme agent for protein deamidation and protein cross-linking according to claim 15 or 16 to act on the protein or peptide.

18. A method for deamidating and cross-linking a protein-containing food or drink, comprising allowing the protein deamidating and protein cross-linking enzyme preparation according to claim 15 or 16 to act on the protein-containing food or drink.

19. A method for producing a food or drink, comprising allowing the protein deamidating and protein cross-linking enzyme preparation according to claim 15 or 16 to act on a protein-containing food or drink.

20. A food or drink treated with the enzyme preparation for protein deamidation and protein cross-linking according to claim 15 or 16.

21. A method for producing a protein deamidating and protein cross-linking enzyme comprising an amino acid sequence having 65% or more sequence identity with the amino acid sequence of SEQ ID NO: 10, comprising the step of culturing a bacterium of the genus Krasilnikovia.

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