Modified transglutaminase

Abstract

The present invention aims to discover new mutations that are effective in improving transglutaminase and to provide highly useful modified transglutaminase. A highly useful modified transglutaminase is disclosed that has undergone amino acid substitutions that result in improved high-temperature reactivity or decreased pH stability in the weakly acidic range.

Description

[Technical Field]

[0001] The present invention relates to a transglutaminase, and more particularly to a modified transglutaminase with altered or improved properties and uses thereof. [Background technology]

[0002] Transglutaminase 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. Therefore, the activity of transglutaminase can be utilized to modify proteins or peptides. Streptomyces-derived transglutaminase (see, for example, Patent Document 1) has been used to bind meat and produce sausages, tofu, bread, and noodles. Furthermore, the use of transglutaminase is being explored not only in the food industry but also in the textile, medical, and cosmetic fields. Along with this expansion of use, attempts have been made to improve the properties of transglutaminase (e.g., thermostability, specific activity, substrate specificity, stability, etc.) (see, for example, Patent Documents 2-4 and Non-Patent Documents 1-4). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Publication No. 4-108381 [Patent Document 2] Japanese Patent Application Laid-Open No. 2002-253272 [Patent Document 3] Japanese Patent Application Laid-Open No. 2008-194004 [Patent Document 4] International Publication No. 2019 / 107288 Brochure [Non-patent literature]

[0004] [Non-Patent Document 1] Marx CK et al., J. Biotechnol. 2008 Sep 10;136(3-4):156-62. [Non-patent document 2] Tagami U et al., Protein Eng. Des. Sel. 2009 Dec; 22(12): 747-752. [Non-patent document 3] Yokoyama K et al., Appl. Microbiol. Biotechnol. (2010) 87:2087-2096. [Non-patent document 4] Buettner K et al., Amino Acids. 2012 Feb;42(2-3):987-96. Summary of the Invention [Problem to be solved by the invention]

[0005] As described above, attempts have been made to improve transglutaminase in order to improve its heat resistance, specific activity, etc., but there remains a strong need for improved transglutaminase due to its high usefulness and further expansion of applications, etc. Therefore, an objective of the present invention is to find new mutations that are effective in improving transglutaminase, and to provide highly useful modified transglutaminase (mutants) and uses thereof. [Means for solving the problem]

[0006] To solve the above problems, the present inventors attempted to improve transglutaminase derived from Streptomyces mobaraensis by amino acid substitution (converting specific amino acid residues to other amino acids). After much trial and error, they succeeded in identifying several mutations (combinations of amino acid residues and substituted amino acids) that were effective in changing its properties (improving high-temperature reactivity and decreasing pH stability in the weakly acidic range).

[0007] In light of the common technical knowledge that enzymes of the same type have high similarities in structure (primary structure, three-dimensional structure) and that similar mutations are likely to produce similar effects, if useful mutations found in transglutaminase derived from Streptomyces mobaraensis having the amino acid sequence of SEQ ID NO: 1 are applied to other transglutaminase that is highly structurally similar to the transglutaminase in question, it is highly likely that similar effects will be achieved, and a person skilled in the art would recognize that such application is effective.

[0008] The following invention is based on the above results and considerations. [1] A modified transglutaminase having an amino acid sequence of SEQ ID NO: 1 containing any one of the following amino acid substitutions (1) to (11), or an amino acid sequence showing 80% or more identity to said amino acid sequence (with the proviso that the amino acid sequence differs in a portion other than the position of said amino acid substitution), and exhibiting a change in properties corresponding to said amino acid substitution: (1) The mutation sites are Y34 and F305, the amino acid after substitution of Y34 is W, and the amino acid after substitution of F305 is W. The property change due to the amino acid substitution is improved high-temperature reactivity; (2) The mutation site is M288, the amino acid after substitution is L, F, or Y, and the change in properties due to the amino acid substitution is a decrease in pH stability in the weakly acidic range; (3) The mutation site is D3, the amino acid after substitution is W or K, and the property change due to the amino acid substitution is improved high-temperature reactivity; (4) The mutation sites are D3 and F305, the amino acid after substitution at D3 is G, K, N, P, or W, and the amino acid after substitution at F305 is W, and the property change due to the amino acid substitution is improved high-temperature reactivity; (5) The mutation sites are V65 and F305, the amino acid after substitution at mutation site V65 is I, and the amino acid after substitution at mutation site F305 is W. The property change due to the amino acid substitution is an improvement in high-temperature reactivity; (6) The mutation sites are S303 and F305, the amino acid after the substitution at S303 is R or K, and the amino acid after the substitution at F305 is W. The property change due to the amino acid substitution is an improvement in high-temperature reactivity; (7) The mutation site is R5, the substituted amino acid is H, and the change in properties due to the amino acid substitution is a decrease in pH stability in the weakly acidic range; (8) The mutation site is V6, the amino acid after substitution is D, and the change in properties due to the amino acid substitution is a decrease in pH stability in the weakly acidic range; (9) The mutation site is W59, the amino acid after substitution is T, and the change in properties due to the amino acid substitution is a decrease in pH stability in the weakly acidic range; (10) The mutation site is S61, the amino acid after substitution is G or R, and the change in properties due to the amino acid substitution is a decrease in pH stability in the weakly acidic range; (11) The mutation site is V290, the amino acid after substitution is I, and the change in properties due to the amino acid substitution is a decrease in pH stability in the weakly acidic range; [2] The modified transglutaminase according to [1], wherein the identity is 82% or more. [3] The modified transglutaminase according to [1], wherein the identity is 85% or more. [4] The modified transglutaminase according to [1], wherein the identity is 90% or more. [5] The modified transglutaminase according to [1], which consists of any one of the amino acid sequences of SEQ ID NOs: 2 to 22. [6] A gene encoding the modified transglutaminase according to any one of [1] to [5]. [7] The gene according to [6], which contains any one of the base sequences of SEQ ID NOs: 23 to 43. [8] A recombinant DNA containing the gene described in [6] or [7]. [9] A microorganism carrying the recombinant DNA described in [8].

[10] An enzyme preparation comprising the modified transglutaminase according to any one of [1] to [5].

[11] A method for preparing a modified transglutaminase, comprising the following steps (I) to (III): (I) preparing a nucleic acid encoding the amino acid sequence of the modified transglutaminase according to any one of [1] to [5]; (II) expressing the nucleic acid; and (III) recovering the expression product.

[12] The method according to

[11] , wherein the amino acid sequence is any one of the amino acid sequences of SEQ ID NOs: 2 to 22.

[13] The method according to

[12] , wherein the nucleic acid comprises any one of the base sequences of SEQ ID NOs: 23 to 43. DETAILED DESCRIPTION OF THE INVENTION

[0009] For ease of explanation, some of the terms used in connection with the present invention are defined below. (term) The term "modified transglutaminase" refers to an enzyme obtained by modifying or mutating a reference transglutaminase (hereinafter referred to as "reference transglutaminase"). The reference transglutaminase is typically a transglutaminase derived from Streptomyces mobaraensis having the amino acid sequence of SEQ ID NO: 1.

[0010] The term "transglutaminase derived from Streptomyces mobaraensis" refers to transglutaminase whose origin is Streptomyces mobaraensis, and includes transglutaminase produced by Streptomyces mobaraensis and transglutaminase expressed in other microorganisms using the genetic information of the transglutaminase.

[0011] In the present invention, the modification or mutation is "amino acid substitution." Therefore, when the modified transglutaminase is compared with the reference transglutaminase, differences are observed in some amino acid residues. In this specification, the modified transglutaminase is also referred to as a modified enzyme or a mutant.

[0012] In accordance with the convention used herein, each amino acid is represented by a single letter as follows: Methionine: M, Serine: S, Alanine: A, Threonine: T, Valine: V, Tyrosine: Y, Leucine: L, Asparagine: N, Isoleucine: I, Glutamine: Q, Proline: P, Aspartic Acid: D, Phenylalanine: F, Glutamic Acid: E, Tryptophan: W, Lysine: K, Cysteine: C, Arginine: R, Glycine: G, Histidine: H

[0013] In this specification, the positions of the mutation points are identified by numbers assigned from the N-terminus to the C-terminus, starting with the N-terminal amino acid residue of the mature transglutaminase as the first amino acid residue.

[0014] Conventionally, the amino acid residue where the amino acid substitution occurs, i.e., the "mutation point," is expressed as a combination of a letter representing the type of amino acid and a number representing the amino acid position. Furthermore, mutations due to amino acid substitution are expressed by adding a letter representing the type of amino acid after substitution to the right of the mutation point. For example, if the mutation point is aspartic acid at position 3, it is expressed as "D3," and if aspartic acid at position 3 is substituted with glycine, it is expressed as "D3G."

[0015] In this specification, the term "weakly acidic range" refers to a range of pH 4 or higher and pH 5 or lower, and the term "neutral range" refers to a range of pH higher than 5 and lower than pH 8.

[0016] 1. Modified transglutaminase A first aspect of the present invention relates to a modified transglutaminase (hereinafter referred to as "modified enzyme"). The modified enzyme of the present invention typically has an amino acid sequence containing one or more specific amino acid substitutions (mutations) in the amino acid sequence of SEQ ID NO: 1. Due to these characteristics, the modified enzyme exhibits changes in properties such as "improved high-temperature reactivity" or "decreased pH stability in the weakly acidic range" compared to transglutaminase consisting of the amino acid sequence of SEQ ID NO: 1. Modified enzymes with improved high-temperature reactivity exhibit high activity even at high temperatures and are advantageously applied to foods and food ingredients produced under high-temperature conditions (e.g., 60°C to 80°C). On the other hand, modified enzymes with reduced pH stability in the weakly acidic range can be inactivated as the pH of the food to which they are applied decreases, making them highly useful in the production of lactic acid-fermented foods (e.g., yogurt). More specifically, when a modified enzyme with reduced pH stability in the weakly acidic range is used to produce a lactic acid-fermented food, the modified enzyme may be allowed to act on the fermentation raw material before lactic acid fermentation, or the modified enzyme may be allowed to act on the fermentation raw material while lactic acid fermentation is proceeding simultaneously. The amino acid sequence of SEQ ID NO: 1 is the sequence of transglutaminase derived from Streptomyces mobaraensis.

[0017] As used herein, "containing an amino acid substitution" means that the mutation point (i.e., the position of the amino acid residue where a specific amino acid substitution occurs) is the substituted amino acid. Therefore, when an amino acid sequence containing an amino acid substitution (mutated amino acid sequence) is compared with the amino acid sequence of SEQ ID NO: 1 that does not contain an amino acid substitution (reference amino acid sequence), a difference in the amino acid residue at the position of the amino acid substitution will be found.

[0018] High-temperature reactivity can be evaluated, for example, based on activity calculated by the activity measurement method described in the Examples below (except that the reaction temperature is changed from 37°C to 60°C). A modified enzyme with improved high-temperature reactivity has higher activity at 60°C than a reference transglutaminase. The activity of the modified enzyme at 60°C is, for example, 180% or more, preferably 200% or more, more preferably 250% or more, and particularly preferably 270% or more of the activity of the reference transglutaminase.

[0019] The decrease in pH stability in the weakly acidic range can be evaluated, for example, based on the residual activity after treatment at pH 4.0 to 5.0 at 30°C for 60 minutes (details on the measurement and evaluation of residual activity are provided in the Examples below). For modified enzymes with decreased pH stability in the weakly acidic range, the decrease in activity with decreasing pH is significant in the weakly acidic range (specifically, in the pH range of 4.0 to 5.0). Therefore, a more rapid decrease in residual activity is observed than in the reference transglutaminase. The residual activity of modified enzymes with decreased pH stability in the weakly acidic range is reduced to 0% by treatment at pH 4.0, and preferably also to 0% by treatment at pH 4.5.

[0020] Although modified enzymes with reduced pH stability in the weakly acidic range are more useful than reference transglutaminase in terms of their altered properties, it is preferable that they have high activity in the neutral range (e.g., pH 6) where activity is expected, for example, to enable a reduction in the amount (enzyme amount) used. For example, the activity at pH 6 of a modified enzyme with reduced pH stability in the weakly acidic range is preferably 30% or more of the activity of the reference transglutaminase, more preferably 50% or more, even more preferably 80% or more, and even more preferably 100% or more (i.e., exhibiting activity equal to or greater than that of the reference transglutaminase).

[0021] The amino acid substitutions (mutation points and amino acids after substitution) that result in the above-mentioned changes in properties are listed below. <Amino acid substitution effective in improving high-temperature reactivity> (1) The mutation sites are Y34 and F305, the amino acid after substitution of Y34 is W, and the amino acid after substitution of F305 is W. (3) The mutation point is D3, and the amino acid after substitution is W or K. (4) The mutation points are D3 and F305, the amino acid after substitution at mutation point D3 is G, K, N, P, or W, and the amino acid after substitution at mutation point F305 is W. (5) The mutation sites are V65 and F305, the amino acid after the substitution of the mutation site V65 is I, and the amino acid after the substitution of the mutation site F305 is W. (6) The mutation sites are S303 and F305, the amino acid after the substitution at the mutation site S303 is R or K, and the amino acid after the substitution at the mutation site F305 is W.

[0022] Among the above amino acid substitutions, the following amino acid substitutions have a large degree of improvement in high-temperature reactivity and are more preferred amino acid substitutions. D3P and F305W double mutation D3W and F305W double mutation Y34W and F305W double mutation

[0023] <Amino acid substitution effective in reducing pH stability in the weakly acidic range> (2) The mutation site is M288, and the amino acid after substitution is L, F, or Y. (7) The mutation point is R5, and the amino acid after substitution is H. (8) The mutation point is V6, and the amino acid after substitution is D. (9) The mutation site is W59, and the amino acid after substitution is T. (10) The mutation site is S61, and the amino acid after substitution is G or R. (11) The mutation site is V290 and the amino acid after substitution is I.

[0024] Among the above amino acid substitutions, the following amino acid substitutions significantly reduce pH stability in the weakly acidic range and are therefore more preferred. M288L M288Y

[0025] S61G, S61R, M288F, M288L, and V290I are highly useful in that they exhibit high activity in the neutral range (pH 6) in addition to reduced pH stability in the weakly acidic range.

[0026] Specific examples of modified enzymes of the present invention include transglutaminases consisting of any of the amino acid sequences of SEQ ID NOs: 2 to 22 (which correspond to, in order, D3G mutant, D3K mutant, D3W mutant, D3G / F305W mutant, D3K / F305W mutant, D3N / F305W mutant, D3P / F305W mutant, D3W / F305W mutant, Y34W / F305W mutant, V65I / F305W mutant, S303K / F305W mutant, S303R / F305W mutant, R5H mutant, V6D mutant, W59T mutant, S61G mutant, S61R mutant, M288F mutant, M288L mutant, M288Y mutant, and V290I mutant).

[0027] Generally, when a portion of the amino acid sequence of a protein is mutated, the resulting protein may have the same function as the unmutated protein. That is, the mutation in the amino acid sequence may not substantially affect the function of the protein, and the protein's function may be maintained before and after the mutation. On the other hand, when the amino acid sequences of two proteins are highly identical, the two proteins are likely to exhibit similar properties. Taking these technical common sense into consideration, an enzyme that has a high degree of identity with the amino acid sequence of the modified enzyme, i.e., "the amino acid sequence of SEQ ID NO: 1 containing any of the amino acid substitutions (1) to (11) above (specific examples of such amino acid sequences are the amino acid sequences of SEQ ID NOs: 2 to 22)," even if it is not completely identical (i.e., 100% identical) to the modified enzyme, and exhibits the desired change in properties, can be considered to be substantially identical to the modified enzyme (substantially identical transglutaminase). Here, identity refers to 70% or more, 80% or more, 82% or more, 85% or more, 90% or more, 93% or more, 95% or more, 98%, or 99% or more. The higher the identity, the more preferable. Thus, in the most preferred embodiment, the identity will be 99% or greater.

[0028] When comparing the modified enzyme with the substantially identical transglutaminase, slight differences in amino acid sequence will be observed, provided that the differences occur at positions other than the positions where the amino acid substitutions have been made. Therefore, for example, if the identity standard is the amino acid sequence of SEQ ID NO: 2, a position other than G at position 3; if the identity standard is the amino acid sequence of SEQ ID NO: 3, a position other than K at position 3; if the identity standard is the amino acid sequence of SEQ ID NO: 4, a position other than W at position 3; if the identity standard is the amino acid sequence of SEQ ID NO: 5, a position other than G at position 3 and W at position 305; if the identity standard is the amino acid sequence of SEQ ID NO: 6, a position other than K at position 3 and W at position 305; if the identity standard is the amino acid sequence of SEQ ID NO: 7, a position other than N at position 3 and W at position 305; if the identity standard is the amino acid sequence of SEQ ID NO: 8, a position other than P at position 3 and W at position 305; if the identity standard is the amino acid sequence of SEQ ID NO: 9, a position other than W at position 3 and W at position 305; if the identity standard is the amino acid sequence of SEQ ID NO: 10, a position other than W at position 34 and W at position 305; if the identity standard is the amino acid sequence of SEQ ID NO: 11, a position other than I at position 65 and W at position 305; if the identity standard is the amino acid sequence of SEQ ID NO: 13, differences in the amino acid sequence will occur at positions other than position 303 K and position 305 W; if the identity standard is the amino acid sequence of SEQ ID NO: 14, differences will occur at positions other than position 5 H; if the identity standard is the amino acid sequence of SEQ ID NO: 15, differences will occur at positions other than position 6 D; if the identity standard is the amino acid sequence of SEQ ID NO: 16, differences will occur at positions other than position 59 T; if the identity standard is the amino acid sequence of SEQ ID NO: 17, differences will occur at positions other than position 61 G; if the identity standard is the amino acid sequence of SEQ ID NO: 18, differences will occur at positions other than position 61 R; if the identity standard is the amino acid sequence of SEQ ID NO: 19, differences will occur at positions other than position 288 F; if the identity standard is the amino acid sequence of SEQ ID NO: 20, differences will occur at positions other than position 288 L; if the identity standard is the amino acid sequence of SEQ ID NO: 21, differences will occur at positions other than position 288 Y; In other words, in an amino acid sequence that exhibits the above-mentioned identity (70% or more, 80% or more, 82% or more, 85% or more, 90% or more, 93% or more, 95% or more, 98%, or 99% or more) with the amino acid sequence of SEQ ID NO: 2, the amino acid G at position 3 isSimilarly, in the amino acid sequence showing the above-mentioned identity with the amino acid sequence of SEQ ID NO: 3, the amino acid at position 3 is K; in the amino acid sequence showing the above-mentioned identity with the amino acid sequence of SEQ ID NO: 4, the amino acid at position 3 is W; in the amino acid sequence showing the above-mentioned identity with the amino acid sequence of SEQ ID NO: 5, the amino acid at position 3 is G and the amino acid at position 305 is W; in the amino acid sequence showing the above-mentioned identity with the amino acid sequence of SEQ ID NO: 6, the amino acid at position 3 is K and the amino acid at position 305 is W; and in the amino acid sequence showing the above-mentioned identity with the amino acid sequence of SEQ ID NO: 7, the amino acid at position 3 is N and the amino acid at position 305 is W. In the amino acid sequence showing the above-mentioned identity with the amino acid sequence of SEQ ID NO: 8, the amino acid at position 3 is P and the amino acid at position 305 is W; in the amino acid sequence showing the above-mentioned identity with the amino acid sequence of SEQ ID NO: 9, the amino acid at position 3 is W and the amino acid at position 305 is W; in the amino acid sequence showing the above-mentioned identity with the amino acid sequence of SEQ ID NO: 10, the amino acid at position 34 is W and the amino acid at position 305 is W; in the amino acid sequence showing the above-mentioned identity with the amino acid sequence of SEQ ID NO: 11, the amino acid at position 65 is I and the amino acid at position 305 is W; and in the amino acid sequence showing the above-mentioned identity with the amino acid sequence of SEQ ID NO: 12, the amino acid at position 34 is W and the amino acid at position 305 is W; In the amino acid sequence, the amino acid at position 303 is K and the amino acid at position 305 is W; in the amino acid sequence showing the above-mentioned identity with the amino acid sequence of SEQ ID NO: 13, the amino acid at position 303 is R and the amino acid at position 305 is W; in the amino acid sequence showing the above-mentioned identity with the amino acid sequence of SEQ ID NO: 14, the amino acid at position 5 is H; in the amino acid sequence showing the above-mentioned identity with the amino acid sequence of SEQ ID NO: 15, the amino acid at position 6 is D; in the amino acid sequence showing the above-mentioned identity with the amino acid sequence of SEQ ID NO: 16, the amino acid at position 59 is T; In the amino acid sequence, the amino acid at position 61 is G; in the amino acid sequence that shows the above-mentioned identity to the amino acid sequence of SEQ ID NO: 18, the amino acid at position 61 is R; in the amino acid sequence that shows the above-mentioned identity to the amino acid sequence of SEQ ID NO: 19, the amino acid at position 288 is F; in the amino acid sequence that shows the above-mentioned identity to the amino acid sequence of SEQ ID NO: 20, the amino acid at position 288 is L; in the amino acid sequence that shows the above-mentioned identity to the amino acid sequence of SEQ ID NO: 21, the amino acid at position 288 is Y; and in the amino acid sequence that shows the above-mentioned identity to the amino acid sequence of SEQ ID NO: 22, the amino acid at position 290 is I.

[0029] Here, "slight differences in amino acid sequence" are caused by amino acid deletion, substitution, addition, insertion, or a combination thereof. Typically, this refers to a mutation (change) in the amino acid sequence caused by deletion or substitution of one to several (upper limit, for example, 3, 5, 7, or 10) amino acids constituting the amino acid sequence, or addition or insertion of one to several (upper limit, for example, 3, 5, 7, or 10) amino acids, or a combination thereof. "Slight differences in amino acid sequence" are preferably caused by conservative amino acid substitution. Here, "conservative amino acid substitution" refers to the substitution of a certain 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: 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 made between amino acid residues within the same family. Since the active residue of transglutaminase derived from Streptomyces mobaraensis (SEQ ID NO: 1) is known to be cysteine ​​64, mutations should be made without affecting this acid residue.

[0030] The percent identity of two amino acid sequences or two nucleotide sequences (hereinafter, the term "two sequences" is used to include both) 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 or nucleotide) 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 can be said to be identical. The identity of two sequences is a function of the number of identical positions shared by the two sequences (i.e., identity (%) = number of identical positions / total number of positions × 100), and preferably, the number and size of gaps required for optimal alignment are also taken into consideration.

[0031] Comparison of two sequences and determination of identity can be achieved using a mathematical algorithm. An example of a mathematical algorithm that can be used for sequence comparison is the algorithm described in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the NBLAST program and XBLAST program (version 2.0) described in Altschul et al. (1990) J. Mol. Biol. 215:403-10. To obtain equivalent nucleotide sequences, for example, a BLAST nucleotide search can be performed with the NBLAST program using a score of 100 and a word length of 12. To obtain equivalent amino acid sequences, for example, a BLAST polypeptide search can be performed with the XBLAST program using a score of 50 and a word length of 3. To obtain gapped alignments for comparison, Gapped BLAST, as described in Altschul et al. (1997) Amino Acids Research 25(17):3389-3402, can be used. When using BLAST and Gapped BLAST, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used. For details, see http: / / www.ncbi.nlm.nih.gov. An example of another mathematical algorithm that can be used for sequence comparison is the algorithm described by Myers and Miller (1988) Comput Appl Biosci. 4:11-17. Such an algorithm is incorporated into the ALIGN program, available, for example, on the GENESTREAM network server (IGH Montpellier, France) or the ISREC server. When using the ALIGN program for comparing amino acid sequences, for example, the PAM120 residue mass table can be used, with a gap length penalty of 12 and a gap penalty of 4.

[0032] The identity of two amino acid sequences can be determined using the GAP program in the EMBOSS package, using a Blosum 62 matrix with a gap weight of 10 and a gap length weight of 2. The identity of two nucleotide sequences can be determined using the GAP program in the EMBOSS package (available at http: / / emboss.open-bio.org / ) with a gap weight of 50 and a gap length weight of 3.

[0033] Typically, a transglutaminase having the amino acid sequence of SEQ ID NO: 1, i.e., a transglutaminase derived from Streptomyces mobaraensis, is mutated (by any of the amino acid substitutions (1) to (11) above) to produce the modified enzyme of the present invention. The substantially identical transglutaminase can be obtained by further mutating a transglutaminase having the amino acid sequence of SEQ ID NO: 1 mutated (by any of the amino acid substitutions (1) to (11) above), by introducing an equivalent mutation into a transglutaminase having an amino acid sequence highly identical to the amino acid sequence of SEQ ID NO: 1, such as a transglutaminase from the same species or genus as the Streptomyces mobaraensis strain that produces the transglutaminase having the amino acid sequence of SEQ ID NO: 1, or by further mutating a transglutaminase obtained by such a mutation. Here, the "equivalent mutation" refers to a substitution of an amino acid residue in an amino acid sequence highly identical to the amino acid sequence of SEQ ID NO: 1 that corresponds to the amino acid residue at the mutation site in the present invention (the mutation site in any of the amino acid substitutions (1) to (11) above).Examples of transglutaminase having an amino acid sequence highly identical to the amino acid sequence of SEQ ID NO: 1 include a transglutaminase derived from Streptomyces mobaraensis (previously classified as Streptoverticillium ladakanum) and having the amino acid sequence of SEQ ID NO: 44 (amino acid sequence identity: 93%), a transglutaminase derived from Streptomyces albireticuli and having the amino acid sequence of SEQ ID NO: 45 (amino acid sequence identity: 82%), a transglutaminase derived from Streptomyces luteireticuli and having the amino acid sequence of SEQ ID NO: 46 (amino acid sequence identity: 82%), a transglutaminase derived from Streptomyces cinnamoneus and having the amino acid sequence of SEQ ID NO: 47 (amino acid sequence identity: 87%), a transglutaminase derived from Streptomyces cinnamoneus and having the amino acid sequence of SEQ ID NO: 48 (amino acid sequence identity: 88%), a transglutaminase derived from Streptomyces cinnamoneus and having the amino acid sequence of SEQ ID NO: 49 (amino acid sequence identity: 89%), a transglutaminase derived from Streptomyces cinnamoneus and having the amino acid sequence of SEQ ID NO: 50 (amino acid sequence identity: 89%), a transglutaminase derived from Streptomyces cinnamoneus and having the amino acid sequence of SEQ ID NO: 51 (amino acid sequence identity: 89%), a transglutaminase derived from Streptomyces cinnamoneus and having the amino acid sequence of SEQ ID NO: 52 (amino acid sequence identity: 89%), a transglutaminase derived from Streptomyces cinnamoneus and having the amino acid sequence of SEQ ID NO: 53 (amino acid sequence identity: 89%), a transglutam Examples of such transglutaminase include a transglutaminase derived from Streptomyces cinnamoneus and having the amino acid sequence of SEQ ID NO: 47 (amino acid sequence identity: 81%), a transglutaminase derived from Streptomyces platensis and having the amino acid sequence of SEQ ID NO: 48 (amino acid sequence identity: 80%), and a transglutaminase derived from Streptomyces hygroscopicus and having the amino acid sequence of SEQ ID NO: 49 (amino acid sequence identity: 80%).

[0034] As used herein, the term "corresponding" when used with respect to amino acid residues means that the amino acid contributes equally to the function of the proteins (enzymes) being compared. For example, when the amino acid sequence of a reference transglutaminase (the amino acid sequence of SEQ ID NO: 1) is aligned to optimize the comparison while taking into account the partial homology of the primary structure (amino acid sequence) (gaps may be introduced as necessary to optimize the alignment), the amino acid at a position corresponding to a specific amino acid in the reference amino acid sequence can be identified as the "corresponding amino acid." Instead of or in addition to comparing primary structures, "corresponding amino acids" can also be identified by comparing tertiary structures (three-dimensional structures). Using tertiary structure information can provide reliable comparison results. In this case, a method can be used in which atomic coordinates of the tertiary structures of multiple enzymes are compared and aligned. The tertiary structure information of the target enzyme to be mutated can be obtained, for example, from the Protein Data Bank (http: / / www.pdbj.org / index#j.html).

[0035] An example of a method for determining the three-dimensional structure of a protein by X-ray crystallography is shown below. (1) Crystallization of proteins. Crystallization is essential for determining three-dimensional structures, but it is also useful industrially as a method for purifying proteins to high purity and for preserving them at high density and in a stable manner. In this case, it is recommended to crystallize proteins bound to a substrate or its analogue as a ligand. (2) The prepared crystals are irradiated with X-rays to collect diffraction data. However, protein crystals are often damaged by X-ray irradiation, resulting in a deterioration of their diffraction ability. In such cases, a low-temperature measurement technique has recently become popular, in which the crystals are rapidly cooled to approximately -173°C and diffraction data are collected in this state. Finally, highly brilliant synchrotron radiation is used to collect high-resolution data for structure determination. (3) Crystal structure analysis requires phase information in addition to diffraction data. If the crystal structure of a protein related to the target protein is unknown, molecular replacement is not possible, and the phase problem must be solved using heavy atom isomorphous replacement. Heavy atom isomorphous replacement involves introducing metal atoms with high atomic numbers, such as mercury or platinum, into the crystal and utilizing the metal atoms' large X-ray scattering ability to contribute to the X-ray diffraction data to obtain phase information. The determined phase can be improved by smoothing the electron density in the solvent region of the crystal. Water molecules in the solvent region exhibit very little electron density due to their large fluctuations. Therefore, approximating the electron density in this region to zero can approximate the true electron density, thereby improving the phase. Furthermore, if the asymmetric unit contains multiple molecules, averaging the electron densities of these molecules can further improve the phase. A protein model is then fitted to the electron density map calculated using this improved phase. This process is performed on a computer graphics system using a program such as QUANTA from MSI (USA). After this, the structure is refined using a program such as MSI's X-PLOR, and the structural analysis is completed. If the crystal structure of a protein related to the target protein is known, the atomic coordinates of the known protein can be used to determine the structure using molecular replacement. Molecular replacement and structural refinement can be performed using a program such as CNS#SOLVE ver.11.

[0036] 2. Nucleic acids encoding modified transglutaminase A second aspect of the present invention provides nucleic acids related to the modified enzymes of the present invention, i.e., genes encoding the modified enzymes, nucleic acids that can be used as probes for identifying nucleic acids encoding the modified enzymes, and nucleic acids that can be used as primers for amplifying or mutating nucleic acids encoding the modified enzymes.

[0037] Genes encoding modified enzymes are typically used to prepare modified enzymes. Genetic engineering preparation methods using genes encoding modified enzymes make it possible to obtain modified enzymes in a more homogeneous state. Furthermore, these methods are also suitable for preparing large quantities of modified enzymes. The use of genes encoding modified enzymes is not limited to the preparation of modified enzymes. For example, the nucleic acids can also be used as experimental tools for elucidating the mechanism of action of modified enzymes, or as tools for designing or producing further modified enzymes.

[0038] As used herein, the term "gene encoding a modified enzyme" refers to a nucleic acid that, when expressed, yields the modified enzyme, and includes not only a nucleic acid having a base sequence corresponding to the amino acid sequence of the modified enzyme, but also a nucleic acid in which a sequence that does not encode an amino acid sequence has been added to such a nucleic acid. Codon degeneracy is also taken into consideration.

[0039] Examples of gene sequences (nucleotide sequences) encoding the modified enzymes are shown in SEQ ID NOs: 23 to 43. These sequences encode the mutants shown in the Examples below. SEQ ID NO: 23: D3G mutant SEQ ID NO: 24: D3K mutant SEQ ID NO: 25: D3W mutant SEQ ID NO: 26: D3G / F305W mutant SEQ ID NO: 27: D3K / F305W mutant SEQ ID NO: 28: D3N / F305W mutant SEQ ID NO: 29: D3P / F305W mutant SEQ ID NO: 30: D3W / F305W mutant SEQ ID NO: 31: Y34W / F305W mutant SEQ ID NO: 32: V65I / F305W mutant SEQ ID NO: 33: S303K / F305W mutant SEQ ID NO: 34: S303R / F305W mutant SEQ ID NO: 35: R5H mutant SEQ ID NO: 36: V6D mutant SEQ ID NO: 37: W59T mutant SEQ ID NO: 38: S61G mutant SEQ ID NO: 39: S61R mutant SEQ ID NO: 40: M288F mutant SEQ ID NO: 41: M288L mutant SEQ ID NO: 42: M288Y mutant SEQ ID NO: 43: V290I mutant

[0040] When expressing the gene of the present invention in a host, a gene construct in which a sequence encoding a propeptide (pro-sequence) has been added to the 5'-end of the above sequence (e.g., any of SEQ ID NOS: 23 to 43) is typically introduced into the host to stabilize the structure of the modified enzyme, which is the expression product. Furthermore, when expressing as a secreted protein, a gene construct in which a sequence encoding a pre-sequence (signal sequence) has been added to the 5'-end of the sequence encoding the pro-sequence is also prepared. When using this gene construct, a prepro-type transglutaminase in which the pre-sequence and pro-sequence are linked is expressed, and then the mature transglutaminase is obtained through cleavage of the pre-sequence (conversion to pro-type transglutaminase) and the pro-sequence. The sequences encoding the pre-sequence and the pro-sequence should preferably be the original sequences, i.e., the sequences of the reference transglutaminase (transglutaminase before modification). A specific example of a presequence is shown in SEQ ID NO: 51 (presequence of transglutaminase derived from Streptomyces mobaraensis), a specific example of a prosequence is shown in SEQ ID NO: 52 (prosequence of transglutaminase derived from Streptomyces mobaraensis), a specific example of a sequence encoding a presequence is shown in SEQ ID NO: 53 (sequence encoding the presequence of transglutaminase derived from Streptomyces mobaraensis), and a specific example of a sequence encoding a prosequence is shown in SEQ ID NO: 54 (sequence encoding the prosequence of transglutaminase derived from Streptomyces mobaraensis).

[0041] The nucleic acids of the present invention can be prepared in an isolated state by using standard genetic engineering techniques, molecular biological techniques, biochemical techniques, chemical synthesis, etc., with reference to the sequence information disclosed in this specification or the attached sequence listing.

[0042] In another aspect of the present invention, there is provided a nucleic acid (hereinafter also referred to as a "homologous nucleic acid"; a nucleotide sequence specifying a homologous nucleic acid is also referred to as a "homologous nucleotide sequence") that, when compared with the nucleotide sequence of a gene encoding the modified enzyme of the present invention, encodes a protein that is functionally equivalent but has a different nucleotide sequence in part. Examples of homologous nucleic acids include DNA that contains a nucleotide sequence containing one or more nucleotide substitutions, deletions, insertions, additions, or inversions relative to the nucleotide sequence of a nucleic acid encoding the modified enzyme of the present invention, and encodes a protein that has enzymatic activity characteristic of the modified enzyme (i.e., transglutaminase activity). Base substitutions or deletions may occur at multiple sites. Here, "multiple" refers to, for example, 2 to 40 nucleotides, preferably 2 to 20 nucleotides, and more preferably 2 to 10 nucleotides, 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.

[0043] A homologous nucleic acid has, for example, 60% or more, preferably 70% or more, more preferably 80% or more, even more preferably 85% or more, even more preferably about 90% or more, even more preferably 95% or more, and most preferably 99% or more identity to a reference base sequence.

[0044] Such homologous 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). Homologous nucleic acids can also be obtained by other methods such as ultraviolet irradiation.

[0045] Another aspect of the present invention relates to a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of a gene encoding the modified enzyme of the present invention. Yet another aspect of the present invention provides a nucleic acid having a nucleotide sequence at least about 60%, 70%, 80%, 90%, 95%, 99%, or 99.9% identical to the nucleotide sequence of a gene encoding the modified enzyme of the present invention or to a nucleotide sequence complementary thereto.

[0046] Another aspect of the present invention relates to a nucleic acid having a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence complementary to the nucleotide sequence of a gene encoding the modified enzyme of the present invention or a homologous nucleotide sequence thereof. Here, "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). Specific examples of stringent conditions include incubation at approximately 42°C to 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 to approximately 70°C using 0.1x SSC and 0.1% SDS.

[0047] Yet another aspect of the present invention provides a nucleic acid (nucleic acid fragment) having a portion of the nucleotide sequence of a gene encoding a modified enzyme of the present invention, or a portion of a nucleotide sequence complementary thereto. Such a nucleic acid fragment can be used to detect, identify, and / or amplify a nucleic acid having the nucleotide sequence of a gene encoding a modified enzyme of the present invention. For example, the nucleic acid fragment is designed to contain at least a portion that hybridizes to a continuous nucleotide portion (e.g., about 10 to about 100 bases in length, preferably about 20 to about 100 bases in length, and more preferably about 30 to about 100 bases in length) in the nucleotide sequence of a gene encoding a modified enzyme of the present invention. When used as a probe, the nucleic acid fragment can be labeled. For example, a fluorescent substance, an enzyme, or a radioisotope can be used for labeling.

[0048] Yet another aspect of the present invention relates to a recombinant DNA containing the gene of the present invention (a gene encoding a modified enzyme). The recombinant DNA of the present invention is provided, for example, in the form of a vector. As used herein, the term "vector" refers to a nucleic acid molecule that can transport a nucleic acid inserted therein into a target such as a cell.

[0049] 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, pBR322 or its modified forms (pB325, pAT153, pUC8, etc.), pET21, etc.; examples of vectors using yeast as a host include pYepSec1, pMFa, pYES2, pPIC3.5K, etc.; examples of vectors using insect cells as a host include pAc and pVL, etc.; and examples of vectors using mammalian cells as a host include pCDM8 and pMT2PC.

[0050] The vector of the present invention 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. Expression vectors 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.

[0051] Insertion of the nucleic acid of the present invention 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, see Molecular Cloning, Third Edition, 1.84, Cold Spring Harbor Laboratory Press, New York; well-known methods using restriction enzymes and DNA ligases).

[0052] As host cells, microorganisms such as koji mold (e.g., Aspergillus oryzae), Bacillus bacteria (e.g., Bacillus subtilis, Bacillus licheniformis, and Bacillus aminoliquefaciens), Brevibacillus bacteria (e.g., Brevibacillus choshinensis), Escherichia coli, and Saccharomyces cerevisiae can be used for ease of handling. However, any host cell capable of replicating recombinant DNA and expressing the gene for the modified enzyme can be used. Escherichia coli and Saccharomyces cerevisiae are preferred. Microorganisms of the Streptomyces genus (e.g., Streptomyces morabaensis) can also be used as hosts. Examples of Escherichia coli include E. coli BL21(DE3) when a T7 promoter is used, and E. coli JM109 when a T7 promoter is not used. Examples of budding yeast include budding yeast SHY2, budding yeast AH22, and budding yeast INVSc1 (Invitrogen).

[0053] Another aspect of the present invention relates to a microorganism (i.e., a transformant) harboring the recombinant DNA of the present invention. The microorganism of the present invention can be obtained by transfection or transformation using the above-mentioned 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 84, 7413-7417 (1984)) etc. The microorganism of the present invention can be used to produce the modified enzyme of the present invention.

[0054] 3. Enzyme preparation containing modified transglutaminase The modified enzyme of the present invention may be provided in the form of, for example, an enzyme preparation. In addition to the active ingredient (the modified enzyme of the present invention), the enzyme preparation may contain 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, and the like. Examples of excipients that can be used include starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, sucrose, and glycerol. Examples of buffers that can be used include phosphates, citrates, and acetates. Examples of stabilizers that can be used include propylene glycol and ascorbic acid. Examples of preservatives that can be used include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, and methylparaben. 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.

[0055] 4. Method for preparing modified transglutaminase Another aspect of the present invention relates to a method for preparing a modified enzyme. In one embodiment of the preparation method of the present invention, the modified enzyme of the present invention is prepared by genetic engineering techniques. In this embodiment, a nucleic acid encoding the amino acid sequence of the modified enzyme of the present invention (e.g., any of SEQ ID NOS: 2 to 22) is prepared (Step (I)). Here, a "nucleic acid encoding a specific amino acid sequence" refers to a nucleic acid that, when expressed, results in a polypeptide having the amino acid sequence. This nucleic acid may not only consist of a nucleotide sequence corresponding to the amino acid sequence, but may also contain an additional sequence (which may or may not encode an amino acid sequence) added to such a nucleic acid. Codon degeneracy is also taken into consideration. A "nucleic acid encoding the amino acid sequence of the modified enzyme of the present invention" can be prepared in an isolated state by standard genetic engineering, molecular biological, or biochemical techniques, with reference to the sequence information disclosed in this specification or the attached sequence listing. Here, the amino acid sequence of the modified enzyme of the present invention is a mutation of the amino acid sequence of a reference transglutaminase. Therefore, a "nucleic acid encoding the amino acid sequence of the modified enzyme of the present invention" can also be obtained by introducing necessary mutations into a gene encoding the reference transglutaminase. Numerous methods for site-specific base sequence substitution are known in the art (see, for example, Molecular Cloning, Third Edition, Cold Spring Harbor Laboratory Press, New York), and an appropriate method can be selected and used. Site-specific amino acid saturation mutagenesis can be used as a site-specific mutagenesis method. This is a "semi-rational, semi-random" method in which positions involved in the desired function are predicted based on the three-dimensional structure of the protein and amino acid saturation mutations are introduced (J. Mol. Biol. 331, 585-592 (2003)). For example, site-specific amino acid saturation mutations can be introduced using a kit such as Quick Change (Stratagene) or overlap extension PCR (Nucleic Acid Res. 16, 7351-7367 (1988)).The DNA polymerase used in PCR may be Taq polymerase, etc. However, it is preferable to use a highly accurate DNA polymerase such as KOD-PLUS- (Toyobo Co., Ltd.) or Pfu turbo (Stratagene Co., Ltd.).

[0056] Following step (I), the prepared nucleic acid is expressed (step (II)). For example, an expression vector into which the nucleic acid is inserted is first prepared, and then a host cell is transformed with this.

[0057] Next, the transformant is cultured under conditions that allow the production of the modified enzyme, which is the expression product. The culture of the transformant can be performed according to conventional methods. The carbon source used in the medium can be any assimilable carbon compound, such as glucose, sucrose, lactose, maltose, molasses, or pyruvic acid. The nitrogen source can be any usable nitrogen compound, such as peptone, meat extract, yeast extract, casein hydrolysate, or alkaline extract of soybean meal. Other salts, such as phosphate, carbonate, sulfate, magnesium, calcium, potassium, iron, manganese, or zinc, as well as specific amino acids and specific vitamins, can also be used as needed.

[0058] The culture temperature can be set within the range of 30°C to 40°C (preferably around 33 to 37°C). The culture time can be set taking into consideration the growth characteristics of the transformant to be cultured and the production characteristics of the modified enzyme. The pH of the medium is adjusted within a range that allows the transformant to grow and the enzyme to be produced. The pH of the medium is preferably around 6.0 to 9.0 (preferably around pH 7.0).

[0059] Next, the expression product (modified enzyme) is recovered (step (III)). The culture medium containing the bacterial cells after cultivation can be used as an enzyme solution directly or after concentration, removal of impurities, etc., but the expression product is generally first recovered from the culture medium or bacterial cells. If the expression product is a secretory protein, it can be recovered from the culture medium; otherwise, it can be recovered from the bacterial cells. When recovering from the culture medium, for example, the culture supernatant is filtered and centrifuged to remove insoluble matter, followed by separation and purification using a combination of techniques such as vacuum concentration, membrane concentration, salting out using ammonium sulfate or sodium sulfate, fractional precipitation using methanol, ethanol, or acetone, dialysis, heat treatment, isoelectric focusing, gel filtration, adsorption chromatography, ion exchange chromatography, and affinity chromatography (e.g., gel filtration using Sephadex gel (GE Healthcare Biosciences) or the like, DEAE Sepharose CL-6B (GE Healthcare Biosciences), Octyl Sepharose CL-6B (GE Healthcare Biosciences), and CM Sepharose CL-6B (GE Healthcare Biosciences)). On the other hand, when recovering from bacterial cells, the bacterial cells are harvested by filtration, centrifugation, etc. of the culture medium, and then disrupted by mechanical methods such as pressurization, sonication, or physical disruption, or by enzymatic methods using lysozyme or the like. The purified modified enzyme can then be separated and purified in the same manner as above.

[0060] 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 phosphate buffer, triethanolamine buffer, Tris-HCl buffer, or Good's buffer. Preferably, phosphate buffer or triethanolamine buffer is used. Examples of Good's buffer include PIPES, MES, and MOPS.

[0061] Typically, gene expression and recovery of the expression product (modified enzyme) are performed using an appropriate host-vector system as described above. However, cell-free synthesis systems can 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 generally 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. When synthesizing proteins, various amino acids, energy sources such as ATP and GTP, and other substances necessary for protein synthesis, such as creatine phosphate, are added to the cell extract. Of course, separately prepared ribosomes, various factors, and / or various enzymes may be supplemented as needed during protein synthesis.

[0062] 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 Escherichia 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.

[0063] 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. Examples of template RNA that can be used include total RNA, mRNA, and in vitro transcription products. In contrast, in vitro transcription / translation systems use DNA 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. [Example]

[0064] <Searching for mutations that are effective in improving properties> To obtain a highly useful engineered transglutaminase, we attempted to improve the enzymatic function of Streptomyces mobaraensis transglutaminase (wild-type enzyme, SEQ ID NO: 1) by protein engineering (improving thermoreactivity and decreasing pH stability in the weakly acidic range). First, we introduced mutations by amino acid substitution. We selected candidate mutation sites using a CASTing library (see, e.g., Angew Chem Int Ed Engl. 2006 Feb 13;45(8):1236-41) and alanine scanning (Ala scanning) (see, e.g., J Mol. Biol. 1995 Feb 17;246(2):317-30). Next, we prepared mutant enzymes by introducing mutations into each site using the following method.

[0065] 1. Preparation of Mutant Enzymes 1-1. Introduction of mutations (1) PCR primers for mutagenesis were designed. (2) Using a primer set for each mutation point, PCR was performed using the plasmid pET20b containing the transglutaminase gene (sequence number 50) as a template (reaction at 98°C for 1 minute, followed by 15 cycles of reaction at 98°C for 10 seconds, 60°C for 15 seconds, and 68°C for 2 minutes, followed by reaction at 68°C for 5 minutes, and then left at 4°C). (3) DpnI (1.5 μL / tube) was added to the PCR reaction solution (25 μL / tube) and treated (37°C, 3 hours or more). (4) Ligation was performed using T4 kinase (16°C, overnight). (5) E. coli BL21(DE3) was transformed with the ligation reaction mixture (11 μL / tube) and cultured in ampicillin-containing LB medium (37° C., overnight).

[0066] 1-2. Obtaining enzyme extract (1) The strain into which the mutation was introduced (mutant strain) was inoculated into ampicillin-containing TB medium and cultured for 48 hours at 33° C. IPTG (final concentration 0.1 mM) was added 24 hours after the start of culture. (2) The culture medium was centrifuged (3,000 g × 10 minutes), and the supernatant was removed to collect the bacterial cells. (3) A lysing agent is added to lyse the cells. (4) The lysate was centrifuged (3,000 g × 10 minutes), and the supernatant was collected to obtain the enzyme extract.

[0067] 1-3. Maturation (removal of the propeptide sequence) (1) Equal amounts of the enzyme extract and 2 mg / mL protease (dispase) solution were mixed and reacted (30°C, 2 hours or more). (2) The mixture was centrifuged (3,000 g × 10 minutes), and the supernatant was collected to obtain the mature enzyme.

[0068] 2. Enzyme Purification Each matured enzyme was purified using TALON Spin columns (Takara Bio) and HisTALON Buffer Set (Takara Bio) according to the attached protocol. The purified matured enzyme was used for characterization of the mutant enzymes.

[0069] 3. Characterization of Mutant Enzymes The properties of each mutant enzyme prepared were evaluated using the following activity measurement method. <Activity measurement method> The mature enzyme was diluted to an appropriate concentration with 200 mM Tris-HCl, pH 6.0 (sample solution). 100 μL of substrate solution (R-1) was added to 10 μL of sample solution, mixed, and then reacted at 37°C for 10 minutes. 100 μL of color-developing solution (R-2) was added to terminate the reaction and form an Fe complex, after which the absorbance at 525 nm was measured. As a control, a similar reaction was performed using a heat-inactivated enzyme solution, and the absorbance was measured. The difference in absorbance with the sample solution was calculated. Separately, a calibration curve was prepared using L-glutamic acid-γ-monohydroxamic acid instead of the enzyme solution, and the amount of hydroxamic acid produced was calculated from the absorbance difference. The enzyme activity required to produce 1 μmol of hydroxamic acid per minute was defined as 1 unit (1 U). (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). (Substrate solution (R-2)) 30 mL of 3M hydrochloric acid solution, 30 mL of 12% trichloroacetic acid solution, and 30 mL of 5% iron(III) chloride solution were mixed.

[0070] 3-1. Evaluation of high-temperature reactivity The matured enzyme was diluted 5-fold with 200 mM Tris-HCl pH 6.0 (sample solution), and the activity was measured at a reaction temperature of 60°C.

[0071] By comparing the activity at a reaction temperature of 60°C with that of the wild type, we identified amino acid substitutions that were effective in improving high-temperature reactivity.

[0072] The evaluation results of high-temperature reactivity are shown in Table 1. All amino acid substitutions improved high-temperature reactivity and were deemed to be effective amino acid substitutions. In particular, the D3P / F305W substitution, D3W / F305W substitution, and Y34W / F305W substitution improved activity by more than 250% compared to the wild-type, and were deemed to be particularly effective amino acid substitutions.

[0073] [Table 1]

[0074] 3-2. Evaluation of pH stability For the pH-treated samples, the matured enzyme was diluted two-fold with 200 mM Britton-Robinson buffer (pH 4, 4.5, 5, 5.5, 6) of each pH and incubated at 30°C for 60 minutes. For the untreated sample, the matured enzyme was diluted two-fold with 200 mM Tris-HCl pH 6.0. The untreated and pH-treated samples were diluted two-fold with 500 mM Tris-HCl pH 6.0 and their activities were measured. The activity of the untreated wild-type enzyme sample was set at 100, and the relative activity was evaluated.

[0075] Amino acid substitutions that resulted in a relative activity of 0 at pH 4.0 or pH 4.5 were identified.

[0076] The evaluation results of the decrease in pH stability in the weakly acidic range are shown in Table 2. All amino acid substitutions resulted in a relative activity of 0 at pH 4.0, and were therefore determined to be effective amino acid substitutions for decreasing pH stability in the weakly acidic range. The M288L and M288Y substitutions also resulted in a relative activity of 0 at pH 4.5, and were therefore determined to be particularly effective amino acid substitutions. The M288L substitution is also highly useful in that it exhibits high activity in the neutral range (pH 6). In terms of activity in the neutral range (pH 6), the S61G, S61R, M288F, and V290I substitutions can also be said to be highly useful.

[0077] [Table 2] [Industrial Applicability]

[0078] The modified transglutaminase of the present invention has improved practically important properties and is of great industrial value, and is therefore expected to be used in not only existing applications but also new applications.

[0079] 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. [Sequence List Free Text]

[0080] SEQ ID NO: 2: Description of the artificial sequence: D3G mutant SEQ ID NO: 3: Description of the artificial sequence: D3K mutant SEQ ID NO: 4: Description of the artificial sequence: D3W mutant SEQ ID NO: 5: Description of the artificial sequence: D3G / F305W mutant SEQ ID NO: 6: Description of the artificial sequence: D3K / F305W mutant SEQ ID NO: 7: Description of the artificial sequence: D3N / F305W mutant SEQ ID NO: 8: Description of the artificial sequence: D3P / F305W mutant SEQ ID NO: 9: Description of the artificial sequence: D3W / F305W mutant SEQ ID NO: 10: Description of the artificial sequence: Y34W / F305W mutant SEQ ID NO: 11: Description of the artificial sequence: V65I / F305W mutant SEQ ID NO: 12: Description of the artificial sequence: S303K / F305W mutant SEQ ID NO: 13: Description of the artificial sequence: S303R / F305W mutant SEQ ID NO: 14: Description of the artificial sequence: R5H mutant SEQ ID NO: 15: Description of the artificial sequence: V6D mutant SEQ ID NO: 16: Description of the artificial sequence: W59T mutant SEQ ID NO: 17: Description of the artificial sequence: S61G mutant SEQ ID NO: 18: Description of the artificial sequence: S61R mutant SEQ ID NO: 19: Description of the artificial sequence: M288F mutant SEQ ID NO: 20: Description of the artificial sequence: M288L mutant SEQ ID NO: 21: Description of the artificial sequence: M288Y mutant SEQ ID NO: 22: Description of the artificial sequence: V290I mutant SEQ ID NO: 23: Description of artificial sequence: D3G mutant SEQ ID NO: 24: Description of artificial sequence: D3K mutant SEQ ID NO: 25: Description of the artificial sequence: D3W mutant SEQ ID NO: 26: Description of the artificial sequence: D3G / F305W mutant SEQ ID NO: 27: Description of the artificial sequence: D3K / F305W mutant SEQ ID NO: 28: Description of the artificial sequence: D3N / F305W mutant SEQ ID NO: 29: Description of the artificial sequence: D3P / F305W mutant SEQ ID NO: 30: Description of the artificial sequence: D3W / F305W mutant SEQ ID NO: 31: Description of the artificial sequence: Y34W / F305W mutant SEQ ID NO: 32: Description of the artificial sequence: V65I / F305W mutant SEQ ID NO: 33: Description of the artificial sequence: S303K / F305W mutant SEQ ID NO: 34: Description of the artificial sequence: S303R / F305W mutant SEQ ID NO: 35: Description of artificial sequence: R5H mutant SEQ ID NO: 36: Description of the artificial sequence: V6D mutant SEQ ID NO: 37: Description of the artificial sequence: W59T mutant SEQ ID NO: 38: Description of the artificial sequence: S61G mutant SEQ ID NO: 39: Description of the artificial sequence: S61R mutant SEQ ID NO: 40: Description of the artificial sequence: M288F mutant SEQ ID NO: 41: Description of the artificial sequence: M288L mutant SEQ ID NO: 42: Description of the artificial sequence: M288Y mutant SEQ ID NO: 43: Description of the artificial sequence: V290I mutant

Claims

1. A modified transglutaminase having an amino acid sequence of SEQ ID NO: 1 containing any one of the following amino acid substitutions (1) to (6), or an amino acid sequence showing 90% or more identity to said amino acid sequence (with the proviso that the amino acid sequence differs in a portion other than the position of said amino acid substitution), and exhibiting a change in properties corresponding to said amino acid substitution: (1) The mutation site is M288, the amino acid after substitution is L, F, or Y, and the change in properties due to the amino acid substitution is a decrease in pH stability in the weakly acidic range; (2) The mutation site is R5, the amino acid after substitution is H, and the change in properties due to the amino acid substitution is a decrease in pH stability in the weakly acidic range; (3) The mutation site is V6, the amino acid after substitution is D, and the change in properties due to the amino acid substitution is a decrease in pH stability in the weakly acidic range; (4) The mutation site is W59, the amino acid after substitution is T, and the change in properties due to the amino acid substitution is a decrease in pH stability in the weakly acidic range; (5) The mutation site is S61, the amino acid after substitution is G or R, and the change in properties due to the amino acid substitution is a decrease in pH stability in the weakly acidic range; (6) The mutation site is V290, the amino acid after substitution is I, and the change in properties due to the amino acid substitution is a decrease in pH stability in the weakly acidic range;

2. The modified transglutaminase according to claim 1, wherein the identity is 93% or more.

3. The modified transglutaminase according to claim 1, wherein the identity is 95% or more.

4. The modified transglutaminase according to claim 1, wherein the identity is 98% or more.

5. The modified transglutaminase according to claim 1, consisting of an amino acid sequence of any one of SEQ ID NOs: 14 to 22.

6. A gene encoding the modified transglutaminase according to any one of claims 1 to 5.

7. The gene according to claim 6, comprising any one of the base sequences of SEQ ID NOs: 35 to 43.

8. A recombinant DNA comprising the gene according to claim 6 or 7.

9. A microorganism carrying the recombinant DNA of claim 8.

10. An enzyme preparation comprising the modified transglutaminase according to any one of claims 1 to 5.

11. A method for preparing a modified transglutaminase, comprising the following steps (I) to (III): (I) providing a nucleic acid encoding the amino acid sequence of the modified transglutaminase of any one of claims 1 to 5; (II) expressing the nucleic acid; and (III) recovering the expression product.

12. The method according to claim 11, wherein the amino acid sequence is any one of the amino acid sequences of SEQ ID NOs: 14 to 22.

13. The method according to claim 12, wherein the nucleic acid comprises a base sequence of any one of SEQ ID NOs: 35 to 43.

Images