Modified transglutaminase

Abstract

To find a new mutation effective for improving transglutaminase, and to provide a highly useful modified transglutaminase.SOLUTION: Disclosed is a highly useful modified transglutaminase having an amino acid substitution that results in a reduction in temperature stability, an improvement in heat resistance, an improvement in oxidation resistance, an improvement in reactivity, or conversion into deamidase.SELECTED DRAWING: None

Description

Technical Field

[0004] , ,

[0001] The present invention relates to transglutaminase. Specifically, it relates to a modified transglutaminase with unchanged or improved characteristics and its uses, etc. This application claims priority based on Japanese Patent Application No. 2017-231192 filed on November 30, 2017, and the entire contents of the patent application are incorporated by reference.

Background Art

[0002] Transglutaminase is an enzyme that catalyzes the acyl transfer reaction of the γ-carboxylamide group of glutamine residues in a peptide chain. When the ε-amino group of a lysine residue in a protein acts as an acyl acceptor, an ε-(γ-Gln)-Lys cross-linking bond is formed intramolecularly or intermolecularly in the protein molecule. Therefore, since the action of transglutaminase can be used to modify proteins or peptides, transglutaminase derived from the genus Streptomyces (see, for example, Patent Document 1) is used in the production of meat binding, sausages, tofu, bread, and noodles. In addition, not only in the food field, but also the use of transglutaminase in the fiber field, medical field, cosmetic field, etc. has been studied. Along with such an expansion of use, attempts have been made to improve the characteristics (heat resistance, specific activity, substrate specificity, stability, etc.) of transglutaminase (see, for example, Patent Documents 2, 3, and Non-Patent Documents 1 to 4).

Prior Art Documents

Patent Documents

[0003] Patent Document 1: Japanese Unexamined Patent Application Publication No. 4-108381 Patent Document 2: Japanese Unexamined Patent Application Publication No. 2002-253272 Patent Document 3: Japanese Unexamined Patent Application Publication No. 2008-194004 Non-Patent Documents

[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. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] As described above, attempts have been made to improve transglutaminases for purposes such as increasing heat resistance and specific activity. However, due to their high utility and the need for further expansion of their applications, the need for improved transglutaminases remains high. Therefore, the objective of this invention is to find new mutations that are effective in improving transglutaminases and to provide highly useful modified transglutaminases (mutants) and their applications. [Means for solving the problem]

[0006] To address the above challenges, the 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 (decreased temperature stability, improved heat resistance, improved antioxidant activity, improved reactivity, and deamidation). Notably, mutations that resulted in changes in two or more properties were also found. Furthermore, among the mutations that achieved such complex improvements, there were some that resulted in seemingly contradictory changes in properties, such as decreased temperature stability and improved antioxidant activity. This fact was extremely unexpected and exceeded expectations, and is worthy of special mention. On the other hand, these results are also important in that they provide information and means for designing and obtaining modified transglutaminases that can achieve the objective of improving transglutaminase.

[0007] Incidentally, it is often observed that combining two effective mutations is highly likely to produce additive or synergistic effects. Furthermore, given the common technical knowledge that enzymes of the same type have a high degree of structural similarity (primary structure, three-dimensional structure), and that similar mutations are highly likely to produce similar effects, it is highly probable that applying a useful mutation found in the transglutaminase derived from Streptomyces mobaraensis, which has the amino acid sequence of SEQ ID NO: 1, to other transglutaminases with a high degree of structural similarity to the said transglutaminase will produce equivalent effects, and those skilled in the art will 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 in the amino acid sequence of Sequence ID No. 1 that includes one of the following amino acid substitutions (1) to (134), or an amino acid sequence that is 80% or more identical to said amino acid sequence (provided that there are differences in the amino acid sequence in parts other than the position of said amino acid substitution), and exhibiting characteristic changes corresponding to said amino acid substitution: (1) The mutation site is V6, the substituted amino acid is Q, I, M, S, C, K, L, H, F, G, N, P, R, W, or Y, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (2) The mutation site is R26, the substituted amino acid is K, Q, M, H, Y, D, G, N, P, or S, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (3) The mutation site is E28, the substituted amino acid is V, Q, W, R, K, M, N, F, G, L, P, or Y, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (4) The mutation site is V30, the substituted amino acid is P, C, A, E, F, G, H, K, N, Q, R, W, Y, or L, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (5) The mutation site is Y34, the substituted amino acid is A, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (6) The mutation site is Y42, the substituted amino acid is F, A, C, D, E, G, I, L, M, Q, S, T, V, or W, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (7) The mutation site is E58, the substituted amino acid is Y, M, A, F, I, V, R, K, N, L, S, Q, G, or H, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (8) The mutation site is W59, the substituted amino acid is R, N, Y, A, S, I, V, D, G, or P, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (9) The mutation site is L60, the substituted amino acid is I, M, V, A, C, E, F, Q, S, T, W, or Y, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (10) The mutation site is Y62, the substituted amino acid is C, R, G, K, or S, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (11) The mutation site is V65, the substituted amino acid is N, L, M, F, W, or Y, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (12) The mutation site is V67, the substituted amino acid is L, N, A, C, M, Q, or S, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (13) The mutation site is T68, the substituted amino acid is C, L, A, S, M, F, N, Q, or Y, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (14) The mutation site is W69, the substituted amino acid is H, M, I, C, E, F, G, K, L, N, Q, R, S, T, or V, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (15) The mutation site is Q74, the substituted amino acid is W, D, G, or K, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (16) The mutation site is Y75, the substituted amino acid is R, Q, T, or G, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (17) The mutation site is T77, the substituted amino acid is M, H, E, C, or G, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (18) The mutation site is F85, the substituted amino acid is M, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (19) The mutation site is F90, the substituted amino acid is C, M, H, L, or V, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (20) The mutation site is F108, the substituted amino acid is R, L, T, A, I, K, N, or V, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (21) The mutation site is F117, the substituted amino acid is M or L, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (22) The mutation site is S199, the substituted amino acid is G, M, N, K, or V, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (23) The mutation site is F202, the substituted amino acid is L or W, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (24) The mutation site is W203, the substituted amino acid is F, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (25) The mutation site is F254, the substituted amino acid is Y or M, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (26) The mutation site is T273, the substituted amino acid is I, V, M, C, S, L, R, G, A, E, F, Y, D, K, W, or H, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (27) The mutation site is N276, the substituted amino acid is C, E, K, L, S, T, or V, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (28) The mutation site is Y278, the substituted amino acid is M, L, H, I, K, R, W, C, G, N, Q, S, T, or V, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (29) The mutation site is S284, and the resulting amino acid substitution is W, Y, M, F, or N. The change in properties due to amino acid substitution is a decrease in temperature stability; (30) The mutation site is Y291, the substituted amino acid is I, W, L, A, C, K, N, Q, R, S, or V, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (31) The mutation site is S299, the substituted amino acid is I, Y, V, K, M, Q, A, F, G, or E, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (32) The mutation site is S303, the substituted amino acid is N, G, C, V, P, or Y, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (33) The mutation site is Y310, the substituted amino acid is C, M, or I, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (34) The mutation site is D3, the substituted amino acid is Q, P, E, S, or Y, and the change in properties due to the amino acid substitution is improved heat resistance; (35) The mutation site is R26, the substituted amino acid is V, and the property change due to the amino acid substitution is improved heat resistance; (36) The mutation site is Y34, the substituted amino acid is W, and the change in properties due to the amino acid substitution is improved heat resistance; (37) The mutation site is V67, the substituted amino acid is H, and the property change due to the amino acid substitution is improved heat resistance; (38) The mutation site is T68, the substituted amino acid is V or I, and the property change due to the amino acid substitution is improved heat resistance; (39) The mutation site is Q74, the substituted amino acid is F, and the property change due to the amino acid substitution is improved heat resistance; (40) The mutation site is T77, the substituted amino acid is Q, and the property change due to the amino acid substitution is improved heat resistance; (41) The mutation site is S199, the substituted amino acid is C or Q, and the property change due to the amino acid substitution is improved heat resistance; (42) The mutation site is T273, the substituted amino acid is Q, and the property change due to the amino acid substitution is improved heat resistance; (43) The mutation site is S284, the amino acid after substitution is L, H, K, P or R, and the characteristic change due to amino acid substitution is an improvement in heat resistance; (44) The mutation site is S299, the amino acid after substitution is N, and the characteristic change due to amino acid substitution is an improvement in heat resistance; (45) The mutation site is S303, the amino acid after substitution is K, and the characteristic change due to amino acid substitution is an improvement in heat resistance; <000009o>(46) The mutation site is D3, the amino acid after substitution is E, Q, S or Y, and the characteristic change due to amino acid substitution is an improvement in antioxidant property; (47) The mutation site is V6, the amino acid after substitution is P, and the characteristic change due to amino acid substitution is an improvement in antioxidant property; (48) The mutation site is R26, the amino acid after substitution is M, K, W, C, Q, G, Y, E, T, N, D, I, S, P or V, and the characteristic change due to amino acid substitution is an improvement in antioxidant property; (49) The mutation site is E28, the amino acid after substitution is L, M, K, C, V, R, W, G, N, F, Y or H, and the characteristic change due to amino acid substitution is an improvement in antioxidant property; (50) The mutation site is Y42, the amino acid after substitution is L, N or F, and the characteristic change due to amino acid substitution is an improvement in antioxidant property; (51) The mutation site is E58, the amino acid after substitution is Q, A, I, V, L, T, M, K, Y, W, F or R, and the characteristic change due to amino acid substitution is an improvement in antioxidant property; (52) The mutation site is V67, the amino acid after substitution is T, S or A, and the characteristic change due to amino acid substitution is an improvement in antioxidant property; (53) The mutation site is T68, the amino acid after substitution is L, I, V or M, and the characteristic change due to amino acid substitution is an improvement in antioxidant property; (54) The mutation site is Q74, the amino acid after substitution is V, and the characteristic change due to amino acid substitution is an improvement in antioxidant property; (55) The mutation site is T77, the amino acid after substitution is D, and the characteristic change due to amino acid substitution is an improvement in antioxidant property; (56) The mutation site is S199, the amino acid after substitution is C, and the characteristic change due to amino acid substitution is an improvement in antioxidant property; (57) The mutation site is T273, the amino acid after substitution is E, and the characteristic change due to amino acid substitution is an improvement in antioxidant property; It should be noted that there was a misspelling in the original text where " " was miswritten as "<000009o>" in the translation, which has been corrected in the translation above.(58) The mutation site is S284, the substituted amino acid is R or H, and the change in properties due to the amino acid substitution is improved antioxidant activity; (59) The mutation site is Y291, the substituted amino acid is I, S, F, L, C, N, V, or M, and the change in properties due to the amino acid substitution is improved antioxidant activity; (60) The mutation site is S284, the substituted amino acid is M, K, or V, and the property change due to the amino acid substitution is improved reactivity; (61) The mutation site is F251, the substituted amino acid is Y, and the change in properties due to the amino acid substitution is improved reactivity; (62) The mutation sites are V6 and Y75, the amino acid substituted at mutation site V6 is E, the amino acid substituted at mutation site Y75 is F, and the property change due to the amino acid substitution is improved reactivity; (63) The mutation sites are D3 and T77, the amino acid substituted at mutation site D3 is P, the amino acid substituted at mutation site T77 is Q, and the property change due to the amino acid substitution is improved reactivity; (64) The mutation site is D3, the substituted amino acid is Q, S, or Y, and the property change due to the amino acid substitution is improved heat resistance and improved antioxidant properties; (65) The mutation site is D3, the substituted amino acid is P, and the property changes due to the amino acid substitution are improved heat resistance and improved reactivity; (66) The mutation site is D3, the substituted amino acid is E, and the property changes due to the amino acid substitution are improved heat resistance, improved antioxidant properties, and improved reactivity; (67) The mutation site is V6, the substituted amino acid is P, and the property changes due to the amino acid substitution are decreased temperature stability and improved antioxidant properties; (68) The mutation site is R26, the substituted amino acid is K, Q, M, Y, D, G, N, P, or S, and the property change due to the amino acid substitution is a decrease in temperature stability and an increase in antioxidant activity; (69) The mutation site is R26, the substituted amino acid is V, and the property changes due to the amino acid substitution are improved heat resistance and improved antioxidant properties; (70) The mutation site is R26, the substituted amino acid is H, and the property changes due to the amino acid substitution are decreased temperature stability, improved antioxidant activity, and improved reactivity; (71) The mutation site is E28, the substituted amino acid is V, R, K, M, N, F, G, L, or Y, and the property change due to the amino acid substitution is decreased temperature stability and improved antioxidant properties; (72) The mutation site is V30, the substituted amino acid is P, G, N, R, or Y, and the property change due to the amino acid substitution is a decrease in temperature stability and an increase in reactivity; (73) The mutation site is Y42, the substituted amino acid is F, L, or M, and the property change due to the amino acid substitution is decreased temperature stability and improved antioxidant properties; (74) The mutation site is E58, the substituted amino acid is Y, M, A, F, I, V, R, K, L, or Q, and the property change due to the amino acid substitution is decreased temperature stability and improved antioxidant properties; (75) The mutation site is L60, the substituted amino acid is I, and the property changes due to the amino acid substitution are decreased temperature stability and improved reactivity; (76) The mutation site is V67, the substituted amino acid is A or S, and the property change due to the amino acid substitution is decreased temperature stability and improved antioxidant properties; (77) The mutation site is T68, the substituted amino acid is C, L, or M, and the property change due to the amino acid substitution is decreased temperature stability and improved antioxidant properties; (78) The mutation site is T68, the substituted amino acid is V or I, and the property change due to the amino acid substitution is improved heat resistance and improved antioxidant properties; (79) The mutation site is Q74, the substituted amino acid is V, and the property changes due to the amino acid substitution are improved antioxidant activity and improved reactivity; (80) The mutation site is T77, the substituted amino acid is C, and the property changes due to the amino acid substitution are decreased temperature stability and improved antioxidant properties; (81) The mutation site is T77, the substituted amino acid is E, and the property changes due to the amino acid substitution are decreased temperature stability and improved reactivity; (82) The mutation site is T77, the substituted amino acid is D, and the property changes due to the amino acid substitution are improved antioxidant activity and improved reactivity; (83) The mutation site is S199, the substituted amino acid is G, and the property changes due to the amino acid substitution are decreased temperature stability and improved reactivity; (84) The mutation site is S199, the substituted amino acid is C, and the property changes due to the amino acid substitution are improved heat resistance, improved antioxidant properties, and improved reactivity; (85) The mutation site is T273, the substituted amino acid is E, and the property changes due to the amino acid substitution are decreased temperature stability and improved antioxidant properties; (86) The mutation site is S284, the substituted amino acid is M or F, and the property change due to the amino acid substitution is decreased temperature stability and improved reactivity; (87) The mutation site is S284, the substituted amino acid is L or K, and the property change due to the amino acid substitution is improved heat resistance and improved reactivity; (88) The mutation site is S284, the substituted amino acid is H or R, and the property change due to the amino acid substitution is improved heat resistance and improved antioxidant properties; (89) The mutation site is Y291, the substituted amino acid is I, L, C, N, S, or V, and the property change due to the amino acid substitution is decreased temperature stability and improved antioxidant properties; (90) The mutation site is S299, the substituted amino acid is V, K, M, or A, and the property change due to the amino acid substitution is a decrease in temperature stability and an increase in reactivity; (91) The mutation site is S303, the substituted amino acid is K, and the property changes due to the amino acid substitution are improved heat resistance and improved reactivity; (92) The mutation site is R5, the substituted amino acid is Q, P, C, M, I, V, Y, F, S, N, T, or G, and the change in properties due to the amino acid substitution is deamidation; (93) The mutation site is Y34, the substituted amino acid is H or M, and the change in properties due to the amino acid substitution is deamidation; (94) The mutation site is W59, the substituted amino acid is K or E, and the characteristic change due to the amino acid substitution is deamidation; (95) The mutation site is S61, the substituted amino acid is T, V, Y, D, I, L, N, E, M, F, W, Q, P, H, or K, and the change in properties due to the amino acid substitution is deamidation; (96) The mutation site is Y62, the substituted amino acid is D or P, and the characteristic change due to the amino acid substitution is deamidation; (97) The mutation site is G63, the substituted amino acid is D, Q, Y, R, K, H, P, M, N, C, F, L, W, T, V, or S, and the change in properties due to the amino acid substitution is deamidation; (98) The mutation site is V65, the substituted amino acid is C, T, or K, and the change in properties due to the amino acid substitution is deamidation; (99) The mutation site is G66, the substituted amino acid is W or K, and the change in properties due to the amino acid substitution is deamidation; (100) The mutation site is T68, the substituted amino acid is P or H, and the characteristic change due to the amino acid substitution is deamidation; (101) The mutation site is W69, the substituted amino acid is Y, D, or P, and the characteristic change due to the amino acid substitution is deamidation; (102) The mutation site is Q74, the substituted amino acid is R or P, and the characteristic change due to the amino acid substitution is deamidation; (103) The mutation site is F85, the substituted amino acid is Y, W, L, I, Q, C, T, A, V, S, R, H, or N, and the characteristic change due to the amino acid substitution is deamidation; (104) The mutation site is F108, the substituted amino acid is Q, C, E, S, or N, and the characteristic change due to the amino acid substitution is deamidation; (105) The mutation site is F117, the substituted amino acid is C, I, W, V, A, G, T, or N, and the characteristic change due to the amino acid substitution is deamidation; (106) The mutation site is Y198, the substituted amino acid is F, H, L, S, M or I, and the characteristic change due to the amino acid substitution is deamidation; (107) The mutation site is S199, the substituted amino acid is Q or P, and the characteristic change due to the amino acid substitution is deamidation; (108) The mutation site is K200, the substituted amino acid is Y, M, I, R, L, or V, and the characteristic change due to the amino acid substitution is deamidation; (109) The mutation site is H201, the substituted amino acid is K, R, M, L, or Q, and the characteristic change due to the amino acid substitution is deamidation; (110) The mutation site is F202, the substituted amino acid is Y, M, H, V, or C, and the characteristic change due to the amino acid substitution is deamidation; (111) The mutation site is W203, the substituted amino acid is Y, and the change in properties due to the amino acid substitution is deamidation; (112) The mutation site is F223, the substituted amino acid is Y, M, L, W, V, H, G, or A, and the characteristic change due to the amino acid substitution is deamidation; (113) The mutation site is R238, the substituted amino acid is A, D, V, G, H, or S, and the characteristic change due to the amino acid substitution is deamidation; (114) The mutation site is F251, the substituted amino acid is H, N, C, M, R, S, A, T, L, P, G, V, or Q, and the characteristic change due to the amino acid substitution is deamidation; (115) The mutation site is V252, the substituted amino acid is M, L, T, or C, and the characteristic change due to the amino acid substitution is deamidation; (116) The mutation site is N253, the substituted amino acid is T, V, K, C, or R, and the characteristic change due to the amino acid substitution is deamidation; (117) The mutation site is Y256, the substituted amino acid is L, V, or I, and the characteristic change due to the amino acid substitution is deamidation; (118) The mutation site is G257, the substituted amino acid is A or C, and the characteristic change due to the amino acid substitution is deamidation; (119) The mutation site is W258, the substituted amino acid is Y, F, or N, and the characteristic change due to the amino acid substitution is deamidation; (120) The mutation site is T273, the substituted amino acid is N, and the change in properties due to the amino acid substitution is deamidation; (121) The mutation site is G275, the substituted amino acid is A, C, S, V, K, I, F, H, R, L, P, Q, Y, N, M, or T, and the characteristic change due to the amino acid substitution is deamidation; (122) The mutation site is N276, the substituted amino acid is G, M, Q, or H, and the characteristic change due to the amino acid substitution is deamidation; (123) The mutation site is H277, the substituted amino acid is N, S, Q, C, E, K, D, I, R, M, P, L, W, V, Y, G, F, or T, and the change in properties due to the amino acid substitution is deamidation; (124) The mutation site is Y278, the substituted amino acid is P or D, and the characteristic change due to the amino acid substitution is deamidation; (125) The mutation site is H279, the substituted amino acid is N, and the change in properties due to the amino acid substitution is deamidation; (126) The mutation site is S284, the substituted amino acid is C, and the change in properties due to the amino acid substitution is deamidation; (127) The mutation site is M288, the substituted amino acid is V, Q, or T, and the characteristic change due to the amino acid substitution is deamidation; (128) The mutation site is V290, the substituted amino acid is T, C, L, M, A, S, or N, and the characteristic change due to the amino acid substitution is deamidation; (129) The mutation site is Y291, the substituted amino acid is H, T, E, or G, and the characteristic change due to the amino acid substitution is deamidation; (130) The mutation site is W298, the substituted amino acid is F, Y, I, L, or M, and the characteristic change due to the amino acid substitution is deamidation; (131) The mutation site is S299, the substituted amino acid is N or P, and the characteristic change due to the amino acid substitution is deamidation; (132) The mutation site is Y302, the substituted amino acid is L, H, M, V, C, T, P, S, or W, and the characteristic change due to the amino acid substitution is deamidation; (133) The mutation site is S303, the substituted amino acid is I, Q, D, H, or E, and the characteristic change due to the amino acid substitution is deamidation; (134) The mutation site is F305, the substituted amino acid is W, H, or Y, and the change in properties due to the amino acid substitution is deamide enzyme formation. [2] A modified transglutaminase according to [1], wherein the identity is 82% or more. [3] A modified transglutaminase according to [1], wherein the identity is 85% or more. [4] A modified transglutaminase according to [1], wherein the identity described above is 90% or more. [5] A modified transglutaminase described in [1], comprising any of the amino acid sequences of sequence numbers 2 to 10. A gene encoding a modified transglutaminase as described in any one of the items [6][1] to [5]. [7] The gene described in [6], which contains any of the base sequences of sequence numbers 18-26. Recombinant DNA containing the gene described in [8], [6], or [7]. Microorganisms possessing recombinant DNA as described in [9][8]. An enzyme preparation containing a modified transglutaminase as described in any one of items

[10] [1] to [5].

[11] A method for preparing modified transglutaminase, comprising the following steps (I) to (III): (I) A step of preparing a nucleic acid that encodes the amino acid sequence of any one of the modified transglutaminases described in [1] to [5]; (II) The step of expressing the nucleic acid, (III) A step of recovering the expression product.

[12] The preparation method according to

[11] , wherein the amino acid sequence is any of the amino acid sequences of Sequence ID No. 2 to 10.

[13] The preparation method according to

[12] , wherein the nucleic acid comprises any of the base sequences of sequence numbers 18 to 26. [Brief explanation of the drawing]

[0009] [Figure 1-1] Mutation sites effective in reducing temperature stability were identified. By substituting each mutation site with various amino acids and comparing the remaining activity rate of the substituted enzyme (mutant) with that of the wild-type enzyme, effective amino acid substitutions were determined. From the activity measurements, the TG (transglutaminase) activity per 1 mL of culture medium of the mutant strain was calculated and used for evaluation. [Figure 1-2] Continuation of Figure 1. [Figure 1-3] Continuation of Figure 1. [Figure 1-4] Continuation of Figure 1. [Figure 1-5] Continuation of Figure 1. [Figure 1-6] Continuation of Figure 1. [Figure 1-7] Continuation of Figure 1. [Figure 2-1] Effective mutation sites for improving heat resistance were identified. By substituting each mutation site with various amino acids and comparing the activity retention rate of the substituted enzyme (mutant) with that of the wild-type enzyme, effective amino acid substitutions were determined. From the activity measurements, the TG (transglutaminase) activity per 1 mL of culture medium of the mutant strain was calculated and used for evaluation. [Figure 2-2] Continuation of Figure 2. [Figure 3-1] Effective mutation sites for improving antioxidant activity were identified. By substituting each mutation site with various amino acids and comparing the activity retention rate of the substituted enzyme (mutant) with that of the wild-type enzyme, effective amino acid substitutions were determined. From the activity measurements, the TG (transglutaminase) activity per 1 mL of culture medium of the mutant strain was calculated and used for evaluation. [Figure 3-2] Continuation of Figure 3. [Figure 3-3] Continuation of Figure 3. [Figure 4] Effective mutation sites for improving reactivity. By substituting various amino acids at each mutation site and comparing the activity of the resulting enzyme (mutant) with that of the wild-type enzyme, effective amino acid substitutions were identified. From the activity measurements, the TG (transglutaminase) activity per 1 mL of culture medium of the mutant strain was calculated and used for evaluation. V6E / Y75F and D3P / T77Q are double mutants with amino acid substitutions at two sites. [Figure 5-1] Mutation points effective for changing two or more properties. Mutations (amino acid substitutions) effective for complex improvements are shown for each mutation point. [Figure 5-2] Continuation of Figure 5. [Figure 5-3] Continuation of Figure 5. [Figure 6-1] Mutation sites effective for deamidation enzyme formation were identified. Each mutation site was substituted with various amino acids, and the deamidation activity and transglutaminase activity of the resulting enzyme (mutant) were calculated as relative values ​​to the wild-type enzyme. The deamidation activity (ratio to wild-type) / transglutaminase activity (ratio to wild-type) ratio was then determined and used for evaluation. [Figure 6-2] Continuation of Figure 6. [Figure 6-3] Continuation of Figure 6. [Figure 6-4] Continuation of Figure 6. [Figure 6-5] Continuation of Figure 6. [Figure 6-6] Continuation of Figure 6. [Figure 6-7] Continuation of Figure 6. [Figure 6-8] Continuation of Figure 6. [Figure 6-9] Continuation of Figure 6. [Modes for carrying out the invention]

[0010] For the sake of clarity, some of the terms used in relation to this 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.

[0011] The term "transglutaminase derived from Streptomyces mobaraensis" refers to transglutaminase whose origin is Streptomyces mobaraensis, and includes transglutaminase produced by Streptomyces mobaraensis or transglutaminase expressed in other microorganisms using the genetic information of said transglutaminase.

[0012] In this invention, "amino acid substitution" is performed as a modification or mutation. Therefore, when comparing the modified transglutaminase with the reference transglutaminase, differences in some amino acid residues can be observed. In this specification, the modified transglutaminase is also referred to as the modified enzyme or mutant.

[0013] In accordance with convention, 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

[0014] In this specification, the location of mutation sites is identified by numbering the amino acid residue at the N-terminus of mature transglutaminase, starting from the N-terminus as number 1 and moving toward the C-terminus.

[0015] Following convention, the amino acid residue to which an amino acid substitution is performed, i.e., the "mutation site," is represented by a combination of a single letter indicating the type of amino acid and a number indicating the position of the amino acid. Furthermore, the mutation caused by amino acid substitution is represented by adding a single letter indicating the type of amino acid to the right of the mutation site notation. Therefore, for example, if the mutation site is valine at position 6, it is represented as "V6," and if the mutation involves valine at position 6 being replaced with glutamine, it is represented as "V6Q."

[0016] 1. Modified transglutaminase The 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 this characteristic, compared to the transglutaminase consisting of the amino acid sequence of SEQ ID NO: 1, changes in properties such as decreased temperature stability, improved heat resistance, improved antioxidant properties, improved reactivity, deamidation (improvement in the ratio of deamidation activity to transglutaminase activity), or two or more of these properties can be observed. Modified enzymes with decreased temperature stability have the practical advantage of preventing food denaturation during the enzyme's thermal deactivation process, and are highly valuable for applications such as the production of yogurt and cheese. On the other hand, modified enzymes with improved heat resistance exhibit high activity even at high temperatures and are particularly suitable for applications such as the synthesis of peptide compounds. Furthermore, modified enzymes with improved antioxidant properties have advantages such as high storage stability, and are expected to prevent deactivation during the production, storage, and use of the enzyme. Modified enzymes with improved reactivity offer advantages such as higher reaction efficiency and reduced enzyme usage, making them highly valuable for various applications. Deamidation alters substrate specificity, particularly leading to the expansion or creation of new applications (e.g., protein solubilization, emulsification, and improved foaming properties). Transglutaminase is an enzyme that catalyzes the acyl transfer reaction between the γ-carboxylamide group of a glutamine residue in a peptide chain and a primary amine. In the absence of a primary amine, water acts as an acyl acceptor, catalyzing a deamidation reaction that converts the glutamine residue to a glutamic acid residue. Modified enzymes that have undergone deamidation exhibit high deamidation catalytic activity, making them more preferable as enzymes for deamidation. The amino acid sequence of Sequence ID No. 1 is that of transglutaminase derived from Streptomyces mobaraensis.

[0017] In this specification, "containing an amino acid substitution" means that the site of mutation (i.e., the position of the amino acid residue where a specific amino acid substitution occurs) is the substituted amino acid. Therefore, if an amino acid sequence containing an amino acid substitution (mutant amino acid sequence) is compared with the amino acid sequence of Sequence ID No. 1 without an amino acid substitution (reference amino acid sequence), a difference in the amino acid residue will be observed at the position of the amino acid substitution.

[0018] Temperature stability can be evaluated, for example, based on the residual activity after treatment at 50°C for 30 minutes. Modified enzymes with reduced temperature stability will have a lower residual activity rate than standard transglutaminase. The residual activity rate of the modified enzyme is, for example, 90% or less, preferably 30% or less, and more preferably 10% or less, of the residual activity rate of standard transglutaminase. The residual activity rate is calculated as follows. Remaining activity (%) = (Enzyme activity after heat treatment) / (Enzyme activity before heat treatment) × 100

[0019] Heat resistance, like temperature stability, can be evaluated based on the remaining activity after treatment at 50°C for 30 minutes, for example. Modified enzymes with improved heat resistance exhibit a higher remaining activity rate than standard transglutaminase. The remaining activity rate of the modified enzyme is, for example, 110% or more, preferably 120% or more, and more preferably 130% or more, than that of standard transglutaminase.

[0020] Antioxidant activity can be evaluated based on the residual activity after treatment with hydrogen peroxide, an oxidizing agent. A detailed evaluation method will be described later. Modified enzymes with improved antioxidant activity have a higher residual activity rate than standard transglutaminase. The residual activity rate of the modified enzyme is, for example, 110% or more, preferably 120% or more, and more preferably 130% or more, of the residual activity rate of standard transglutaminase. The residual activity rate is calculated as follows. Remaining activity (%) = (Enzyme activity after hydrogen peroxide treatment) / (Enzyme activity before treatment) × 100

[0021] Modified enzymes with altered properties (temperature stability, heat resistance, antioxidant properties) are more useful than standard transglutaminase in terms of these altered properties. However, higher reactivity is preferable, as it allows for a reduction in the amount of enzyme used. For example, the reactivity of the modified enzyme is preferably 50% or more of that of standard transglutaminase.

[0022] Reactivity can be evaluated, for example, based on the activity calculated by the measurement method shown in the examples described later. The modified enzyme with improved reactivity exhibits higher activity than the reference transglutaminase. The reactivity (activity) of the modified enzyme is 110% or more, preferably 120% or more, and more preferably 130% or more, of the reactivity (activity) of the reference transglutaminase.

[0023] Deamidation can be evaluated based on the ratio of deamidation activity (relative to standard transglutaminase ratio) to transglutaminase activity (relative to standard transglutaminase ratio) (deamidation activity / transglutaminase activity). Detailed evaluation methods will be described later. In modified enzymes that have undergone deamidation, the ratio of deamidation activity (relative to standard transglutaminase ratio) / transglutaminase activity (relative to standard transglutaminase ratio) becomes greater than 1, preferably 1.2 or higher, more preferably 2 or higher, and even more preferably 10 or higher.

[0024] The amino acid substitutions (mutation site and substituted amino acids) that result in each of the above-mentioned characteristic changes are listed below. <Amino acid substitutions effective in reducing temperature stability> (1) The mutation site is V6, and the substituted amino acid is Q, I, M, S, C, K, L, H, F, G, N, P, R, W, or Y (2) The mutation site is R26, and the substituted amino acid is K, Q, M, H, Y, D, G, N, P, or S (3) The mutation site is E28, and the substituted amino acid is V, Q, W, R, K, M, N, F, G, L, P, or Y (4) The mutation site is V30, and the substituted amino acid is P, C, A, E, F, G, H, K, N, Q, R, W, Y, or L (5) The mutation site is Y34, and the substituted amino acid is A (6) The mutation site is Y42, and the substituted amino acid is F, A, C, D, E, G, I, L, M, Q, S, T, V, or W (7) The mutation site is E58, and the substituted amino acid is Y, M, A, F, I, V, R, K, N, L, S, Q, G, or H (8) The mutation site is W59, and the substituted amino acid is R, N, Y, A, S, I, V, D, G, or P (9) The mutation site is L60, and the substituted amino acid is I, M, V, A, C, E, F, Q, S, T, W, or Y (10) The mutation site is Y62, and the substituted amino acid is C, R, G, K, or S (11) The mutation site is V65, and the substituted amino acid is N, L, M, F, W, or Y (12) The mutation site is V67, and the substituted amino acid is L, N, A, C, M, Q, or S (13) The mutation site is T68, and the substituted amino acid is C, L, A, S, M, F, N, Q, or Y (14) The mutation site is W69, and the substituted amino acid is H, M, I, C, E, F, G, K, L, N, Q, R, S, T or is V (15) The mutation site is Q74, and the substituted amino acid is W, D, G, or K (16) The mutation site is Y75, and the substituted amino acid is R, Q, T, or G (17) The mutation site is T77, and the substituted amino acid is M, H, E, C, or G (18) The mutation site is F85, and the substituted amino acid is M (19) The mutation site is F90, and the substituted amino acid is C, M, H, L, or V (20) The mutation site is F108, and the substituted amino acid is R, L, T, A, I, K, N, or V (21) The mutation site is F117, and the substituted amino acid is M or L (22) The mutation site is S199, and the substituted amino acid is G, M, N, K, or V (23) The mutation site is F202, and the substituted amino acid is L or W (24) The mutation site is W203, and the substituted amino acid is F (25) The mutation site is F254, and the substituted amino acid is Y or M (26) The mutation site is T273, and the substituted amino acid is I, V, M, C, S, L, R, G, A, E, F, Y, D, K, W, or H (27) The mutation site is N276, and the substituted amino acid is C, E, K, L, S, T, or V (28) The mutation site is Y278, and the substituted amino acid is M, L, H, I, K, R, W, C, G, N, Q, S, T, or V (29) The mutation site is S284, and the substituted amino acid is W, Y, M, F, or N (30) The mutation site is Y291, and the substituted amino acid is I, W, L, A, C, K, N, Q, R, S, or V (31) The mutation site is S299, and the substituted amino acid is I, Y, V, K, M, Q, A, F, G, or E (32) The mutation site is S303, and the substituted amino acid is N, G, C, V, P, or Y (33) The mutation site is Y310, and the substituted amino acid is C, M, or I

[0025] Among the amino acid substitutions listed above, the following amino acid substitutions are more preferable because they significantly reduce temperature stability. V6S, V6C, V6K, V6L, V6H, V6F, V6G, V6N, V6P, V6R, V6W, V6Y R26M, R26H, R26Y, R26D, R26G, R26N, R26P, R26S E28N, E28F, E28G, E28L, E28P, E28Y V30P, V30C, V30A, V30E, V30F, V30G, V30H, V30K, V30N, V30Q, V30R, V30W, V30Y, V30L Y34A Y42A, Y42C, Y42D, Y42E, Y42G, Y42I, Y42L, Y42M, Y42Q, Y42S, Y42T, Y42V, Y42W E58G, E58H W59S, W59I, W59V, W59D, W59G, W59P L60M, L60V, L60A, L60C, L60E, L60F, L60Q, L60S, L60T, L60W, L60Y Y62C, Y62R, Y62G, Y62K, Y62S V65N, V65L, V65M, V65F, V65W, V65Y V67L, V67N, V67A, V67C, V67M, V67Q, V67S T68M, T68F, T68N, T68Q, T68Y W69M, W69I, W69C, W69E, W69F, W69G, W69K, W69L, W69N, W69Q, W69R, W69S, W69T, W69V Y75T, Y75G F85M F90V F108T, F108A, F108I, F108K, F108N, F108V F117L S199K, S199V F202W T273L, T273R, T273G, T273A, T273E, T273F, T273Y, T273D, T273K, T273W, T273H N276C, N276E, N276K, N276L, N276T, N276V, N276S Y278L, Y278H, Y278I, Y278K, Y278R, Y278W, Y278C, Y278G, Y278N, Y278Q, Y278S, Y278T, Y278V Y291W, Y291L, Y291A, Y291C, Y291K, Y291N, Y291Q, Y291R, Y291S, Y291V S299E Y310I

[0026] The following amino acid substitutions significantly reduce temperature stability and are therefore particularly preferred. V6H, V6F, V6G, V6N, V6P, V6R, V6W, V6Y R26H, R26Y, R26D, R26G, R26N, R26P, R26S E28F, E28G, E28L, E28P, E28Y V30C, V30A, V30E, V30F, V30G, V30H, V30K, V30N, V30Q, V30R, V30W, V30Y, V30L Y34A Y42A, Y42C, Y42D, Y42E, Y42G, Y42I, Y42L, Y42M, Y42Q, Y42S, Y42T, Y42V, Y42W W59I, W59V, W59D, W59G, W59P L60V, L60A, L60C, L60E, L60F, L60Q, L60S, L60T, L60W, L60Y Y62C, Y62R, Y62G, Y62K, Y62S V65M, V65F, V65W, V65Y V67A, V67C, V67M, V67Q, V67S T68F, T68N, T68Q, T68Y W69M, W69I, W69C, W69E, W69F, W69G, W69K, W69L, W69N, W69Q, W69R, W69S, W69T, W69V Y75T, Y75G F108A, F108I, F108K, F108N, F108V F117L F202W T273F, T273Y, T273D, T273K, T273W, T273H N276C, N276E, N276K, N276L, N276S, N276T, N276V Y278K, Y278R, Y278W, Y278C, Y278G, Y278N, Y278Q, Y278S, Y278T, Y278V Y291L, Y291A, Y291C, Y291K, Y291N, Y291Q, Y291R, Y291S, Y291V S299E

[0027] The following amino acid substitutions are extremely desirable because they significantly reduce temperature stability and also exhibit high reactivity. V6H, V6G, V6N, V6W R26H, R26Y, R26N, R26S E28F, E28G, E28L, E28Y V30C, V30A, V30E, V30F, V30G, V30H, V30K, V30N, V30Q, V30R, V30W, V30Y, V30L Y42C, Y42L, Y42M, Y42Q, Y42S, Y42W W59I, W59V, W59G L60V, L60C, L60F Y62C, Y62R V67C, V67M W69M, W69I, W69C, W69F, W69L, W69T, W69V Y75T T273F, T273Y, T273H Y278W Y291L, Y291K, Y291R

[0028] <Amino acid substitutions effective for improving heat resistance> (34) The mutation site is D3, and the substituted amino acid is Q, P, E, S, or Y (35) The mutation site is R26, and the substituted amino acid is V (36) The mutation site is Y34, and the substituted amino acid is W (37) The mutation site is V67, and the substituted amino acid is H (38) The mutation site is T68, and the substituted amino acid is V or I (39) The mutation site is Q74, and the substituted amino acid is F (40) The mutation site is T77, and the substituted amino acid is Q (41) The mutation site is S199, and the substituted amino acid is C or Q (42) The mutation site is T273, and the substituted amino acid is Q (43) The mutation site is S284, and the substituted amino acid is L, H, K, P, or R (44) The mutation site is S299, and the substituted amino acid is N (45) The mutation site is S303, and the substituted amino acid is K

[0029] Among the amino acid substitutions listed above, the following amino acid substitutions offer a greater improvement in heat resistance and are therefore more preferable. D3P, D3E, D3S, D3Y R26V Y34W V67H T77Q S199C, S199Q T273Q S284H, S284K, S284P, S284R S303K

[0030] The following amino acid substitutions provide a particularly significant improvement in heat resistance and are therefore highly preferred. D3E, D3S, D3Y R26V Y34W V67H S199Q T273Q S284H, S284K, S284P, S284R

[0031] The following amino acid substitutions are extremely desirable because they offer a particularly significant improvement in heat resistance and also exhibit high reactivity. D3E, D3S, D3Y R26V Y34W T273Q S284H, S284K, S284P, S284R

[0032] <Amino acid substitutions effective for improving antioxidant properties> (46) The mutation site is D3, and the substituted amino acid is E, Q, S, or Y (47) The mutation site is V6, and the substituted amino acid is P (48) The mutation site is R26, and the substituted amino acid is M, K, W, C, Q, G, Y, E, T, N, D, I, S, P, or V (49) The mutation site is E28, and the substituted amino acid is L, M, K, C, V, R, W, G, N, F, Y, or H (50) The mutation site is Y42, and the substituted amino acid is L, N, or F (51) The mutation site is E58, and the substituted amino acid is Q, A, I, V, L, T, M, K, Y, W, F or R (52) The mutation site is V67, and the substituted amino acid is T, S, or A (53) The mutation site is T68, and the substituted amino acid is L, I, V, or M (54) The mutation site is Q74, and the substituted amino acid is V (55) The mutation site is T77, and the substituted amino acid is D (56) The mutation site is S199, and the substituted amino acid is C (57) The mutation site is T273, and the substituted amino acid is E (58) The mutation site is S284, and the substituted amino acid is R or H (59) The mutation site is Y291, and the substituted amino acid is I, S, F, L, C, N, V, or M

[0033] Among the amino acid substitutions listed above, the following amino acid substitutions are more preferable because they significantly improve antioxidant activity. D3S, D3Y V6P R26W, R26C, R26Q, R26G, R26Y, R26E, R26T, R26N, R26D, R26I, R26S, R26P, R26V E28L, E28M, E28K, E28C, E28V, E28R, E28W, E28G, E28N, E28F, E28Y, E28H E58A, E58I, E58V, E58L, E58T, E58M, E58K, E58Y, E58W, E58F, E58R V67S, V67A T68I, T68V, T68M T273E S284R, S284H Y291C, Y291N, Y291V, Y291M

[0034] The following amino acid substitutions are particularly favored because they significantly improve antioxidant activity. V6P R26C, R26Q, R26G, R26Y, R26E, R26T, R26N, R26D, R26I, R26S, R26P, R26V E28V, E28R, E28W, E28G, E28N, E28F, E28Y, E28H Y42F E58I, E58V, E58L, E58T, E58M, E58K, E58Y, E58W, E58F, E58R T68I, T68V, T68M S284H

[0035] The following amino acid substitutions are extremely desirable because they offer a particularly significant improvement in antioxidant activity and high reactivity. R26C, R26Q, R26Y, R26E, R26T, R26N, R26I, R26S, R26V E28V, E28R, E28W, E28G, E28N, E28F, E28Y Y42F E58I, E58V, E58L, E58T, E58M, E58K, E58Y, E58W, E58F, E58R S284H

[0036] <Amino acid substitutions effective for improving reactivity> (60) The mutation site is S284, and the substituted amino acid is M, K, or V (61) The mutation site is F251, and the substituted amino acid is Y (62) The mutation sites are V6 and Y75, the amino acid substituted at mutation site V6 is E, and the amino acid substituted at mutation site Y75 is F (63) The mutation sites are D3 and T77, the amino acid substituted at mutation site D3 is P, and the amino acid substituted at mutation site T77 is Q.

[0037] Among the amino acid substitutions listed above, the following amino acid substitutions are more preferable due to the significant improvement in reactivity. S284V F251Y Double mutation of V6E and Y75F Double mutation of D3P and T77Q

[0038] The following amino acid substitutions are particularly favored because they significantly improve reactivity. Double mutation of V6E and Y75F Double mutation of D3P and T77Q

[0039] As shown in the examples described below, some amino acid substitutions resulted in two or more property changes. In other words, amino acid substitutions effective for complex improvements were identified. The amino acid substitutions that were successfully identified (mutation site and substituted amino acid) are listed below along with the corresponding property changes. (64) The mutation site is D3, the substituted amino acid is Q, S, or Y, and the property change due to the amino acid substitution is improved heat resistance and improved antioxidant properties. (65) The mutation site is D3, the substituted amino acid is P, and the property changes due to the amino acid substitution are improved heat resistance and improved reactivity. (66) The mutation site is D3, the substituted amino acid is E, and the property changes due to the amino acid substitution are improved heat resistance, improved antioxidant properties, and improved reactivity. (67) The mutation site is V6, the substituted amino acid is P, and the property changes due to the amino acid substitution are decreased temperature stability and improved antioxidant properties. (68) The mutation site is R26, the substituted amino acid is K, Q, M, Y, D, G, N, P, or S, and the change in properties due to the amino acid substitution is a decrease in temperature stability and an improvement in antioxidant properties. (69) The mutation site is R26, the substituted amino acid is V, and the property changes due to the amino acid substitution are improved heat resistance and improved antioxidant properties. (70) The mutation site is R26, the substituted amino acid is H, and the property changes due to the amino acid substitution are decreased temperature stability, improved antioxidant properties, and improved reactivity. (71) The mutation site is E28, the substituted amino acid is V, R, K, M, N, F, G, L, or Y, and the property change due to the amino acid substitution is decreased temperature stability and improved antioxidant properties. (72) The mutation site is V30, the substituted amino acid is P, G, N, R or Y, and the property change due to the amino acid substitution is decreased temperature stability and improved reactivity. (73) The mutation site is Y42, the substituted amino acid is F, L, or M, and the change in properties due to the amino acid substitution is a decrease in temperature stability and an improvement in antioxidant properties. (74) The mutation site is E58, the substituted amino acid is Y, M, A, F, I, V, R, K, L, or Q, and the property change due to the amino acid substitution is decreased temperature stability and improved antioxidant properties. (75) The mutation site is L60, the substituted amino acid is I, and the property changes due to the amino acid substitution are decreased temperature stability and improved reactivity. (76) The mutation site is V67, the substituted amino acid is A or S, and the property change due to the amino acid substitution is decreased temperature stability and improved antioxidant properties. (77) The mutation site is T68, the substituted amino acid is C, L, or M, and the property change due to the amino acid substitution is decreased temperature stability and improved antioxidant properties. (78) The mutation site is T68, the substituted amino acid is V or I, and the property change due to the amino acid substitution is improved heat resistance and improved antioxidant properties. (79) The mutation site is Q74, the substituted amino acid is V, and the property changes due to the amino acid substitution are improved antioxidant activity and improved reactivity. (80) The mutation site is T77, the substituted amino acid is C, and the property changes due to the amino acid substitution are decreased temperature stability and improved antioxidant properties. (81) The mutation site is T77, the substituted amino acid is E, and the property changes due to the amino acid substitution are decreased temperature stability and improved reactivity. (82) The mutation site is T77, the substituted amino acid is D, and the property changes due to the amino acid substitution are improved antioxidant activity and improved reactivity. (83) The mutation site is S199, the substituted amino acid is G, and the property changes due to the amino acid substitution are decreased temperature stability and improved reactivity. (84) The mutation site is S199, the substituted amino acid is C, and the property changes due to the amino acid substitution are improved heat resistance, improved antioxidant properties, and improved reactivity. (85) The mutation site is T273, the substituted amino acid is E, and the property changes due to the amino acid substitution are decreased temperature stability and improved antioxidant properties. (86) The mutation site is S284, the substituted amino acid is M or F, and the property change due to the amino acid substitution is decreased temperature stability and improved reactivity. (87) The mutation site is S284, the substituted amino acid is L or K, and the property change due to the amino acid substitution is improved heat resistance and improved reactivity. (88) The mutation site is S284, the substituted amino acid is H or R, and the property change due to the amino acid substitution is improved heat resistance and improved antioxidant properties. (89) The mutation site is Y291, the substituted amino acid is I, L, C, N, S, or V, and the change in properties due to the amino acid substitution is a decrease in temperature stability and an improvement in antioxidant properties. (90) The mutation site is S299, the substituted amino acid is V, K, M, or A, and the property change due to the amino acid substitution is decreased temperature stability and improved reactivity. (91) The mutation site is S303, the substituted amino acid is K, and the property changes due to the amino acid substitution are improved heat resistance and improved reactivity.

[0040] Among the amino acid substitutions listed above, the following amino acid substitutions result in two seemingly contradictory changes in properties: a decrease in temperature stability and an improvement in antioxidant activity. V6P R26K, R26Q, R26M, R26Y, R26D, R26G, R26N, R26P, R26S E28V, E28R, E28K, E28M, E28N, E28F, E28G, E28L, E28Y Y42F, Y42L, Y42M E58Y, E58M, E58A, E58F, E58I, E58V, E58R, E58K, E58L, E58Q V67A, V67S T68C, T68L, T68M T77C T273E Y291I, Y291L, Y291C, Y291N, Y291S, Y291V

[0041] <Amino acid substitutions effective for deamidation enzyme formation> (92) The mutation site is R5, and the substituted amino acid is Q, P, C, M, I, V, Y, F, S, N, T, or G (93) The mutation site is Y34, and the substituted amino acid is H or M (94) The mutation site is W59, and the substituted amino acid is K or E (95) The mutation site is S61, and the substituted amino acid is T, V, Y, D, I, L, N, E, M, F, W, Q, P, H, or K (96) The mutation site is Y62, and the substituted amino acid is D or P (97) The mutation site is G63, and the substituted amino acid is D, Q, Y, R, K, H, P, M, N, C, F, L, W, T, V, or S (98) The mutation site is V65, and the substituted amino acid is C, T, or K (99) The mutation site is G66, and the substituted amino acid is W or K (100) The mutation site is T68, and the substituted amino acid is P or H (101) The mutation site is W69, and the substituted amino acid is Y, D, or P (102) The mutation site is Q74, and the substituted amino acid is R or P (103) The mutation site is F85, and the substituted amino acid is Y, W, L, I, Q, C, T, A, V, S, R, H or N (104) The mutation site is F108, and the substituted amino acid is Q, C, E, S, or N (105) The mutation site is F117, and the substituted amino acid is C, I, W, V, A, G, T, or N (106) The mutation site is Y198, and the substituted amino acid is F, H, L, S, M or I (107) The mutation site is S199, and the substituted amino acid is Q or P (108) The mutation site is K200, and the substituted amino acid is Y, M, I, R, L, or V (109) The mutation site is H201, and the substituted amino acid is K, R, M, L, or Q (110) The mutation site is F202, and the substituted amino acid is Y, M, H, V, or C (111) The mutation site is W203, and the substituted amino acid is Y (112) The mutation site is F223, and the substituted amino acid is Y, M, L, W, V, H, G, or A (113) The mutation site is R238, and the substituted amino acid is A, D, V, G, H, or S (114) The mutation site is F251, and the substituted amino acid is H, N, C, M, R, S, A, T, L, P, G, V, or Q (115) The mutation site is V252, and the substituted amino acid is M, L, T, or C (116) The mutation site is N253, and the substituted amino acid is T, V, K, C, or R (117) The mutation site is Y256, and the substituted amino acid is L, V, or I (118) The mutation site is G257, and the substituted amino acid is A or C (119) The mutation site is W258, and the substituted amino acid is Y, F, or N (120) The mutation site is T273, and the substituted amino acid is N (121) The mutation site is G275, and the substituted amino acid is A, C, S, V, K, I, F, H, R, L, P, Q, Y, N, M or T (122) The mutation site is N276, and the substituted amino acid is G, M, Q, or H (123) The mutation site is H277, and the substituted amino acid is N, S, Q, C, E, K, D, I, R, M, P, L, W, V, Y, G, F, or T (124) The mutation site is Y278, and the substituted amino acid is P or D (125) The mutation site is H279, and the substituted amino acid is N (126) The mutation site is S284, and the substituted amino acid is C (127) The mutation site is M288, and the substituted amino acid is V, Q, or T (128) The mutation site is V290, and the substituted amino acid is T, C, L, M, A, S, or N (129) The mutation site is Y291, and the substituted amino acid is H, T, E, or G (130) The mutation site is W298, and the substituted amino acid is F, Y, I, L, or M (131) The mutation site is S299, and the substituted amino acid is N or P (132) The mutation site is Y302, and the substituted amino acid is L, H, M, V, C, T, P, S, or W (133) The mutation site is S303, and the substituted amino acid is I, Q, D, H, or E (134) The mutation site is F305, and the substituted amino acid is W, H, or Y

[0042] Among the amino acid substitutions listed above, the following amino acid substitutions are more preferable because they involve a greater degree of deamidation. R5Q, R5P, R5C, R5M, R5I, R5V, R5Y, R5F, R5S, R5N, R5T, R5G Y34H, Y34M W59K, W59E S61T, S61V, S61Y, S61D, S61I, S61L, S61N, S61E, S61M, S61F, S61W, S61Q, S61P, S61H, S61K Y62D, Y62P G63D, G63Q, G63Y, G63R, G63K, G63H, G63P, G63M, G63N, G63C, G63F, G63L, G63W, G63T, G63V, G63S V65C、V65T、V65K G66W、G66K T68P、T68H W69Y、W69D、W69P Q74R、Q74P F85Y、F85W、F85L、F85I、F85Q、F85C、F85T、F85A、F85V、F85S、F85R、F85H、F85N F108Q、F108C、F108E、F108S、F108N F117C、F117I、F117W、F117V、F117A、F117G、F117T、F117N Y198F、Y198H、Y198L、Y198S、Y198M、Y198I S199Q、S199P K200Y、K200M、K200I、K200R、K200L、K200V H201K、H201R、H201M、H201L、H201Q F202Y、F202M、F202H、F202V、F202C W203Y F223Y、F223M、F223L、F223W、F223V、F223H、F223G、F223A R238A、R238D、R238V、R238G、R238H、R238S F251H、F251N、F251C、F251M、F251R、F251S、F251A、F251T、F251L、F251P、F251G、F251V、F251Q V252M、V252L、V252T、V252C N253T、N253V、N253K、N253C、N253R Y256L、Y256V、Y256I G257A、G257C W258Y、W258F、W258N T273N G275A、G275C、G275S、G275V、G275K、G275I、G275F、G275H、G275R、G275L、G275P、G275Q、G275Y、G275N、G275M、G275T N276G, N276M, N276Q, N276H H277N, H277S, H277Q, H277C, H277E, H277K, H277D, H277I, H277R, H277M, H277P, H277L, H277W, H277V, H277Y, H277G, H277F, H277T Y278P, Y278D H279N S284C M288V, M288Q, M288T V290T, V290C, V290L, V290M, V290A, V290S, V290N Y291H, Y291T, Y291E, Y291G W298F, W298Y, W298I, W298L, W298M S299N, S299P Y302L, Y302H, Y302M, Y302V, Y302C, Y302T, Y302P, Y302S, Y302W S303I, S303Q, S303D, S303H, S303E F305W, F305H, F305Y

[0043] The following amino acid substitutions result in a greater degree of deamidation and are more preferable amino acid substitutions. It is a replacement. R5Y, R5F, R5S, R5N, R5T, R5G Y34M S61T, S61V, S61Y, S61D, S61I, S61L, S61N, S61E, S61M, S61F, S61W, S61Q, S61P, S61H, S61K Y62P G63D, G63Q, G63Y, G63R, G63K, G63H, G63P, G63M, G63N, G63C, G63F, G63L, G63W, G63T, G63V, G63S V65C, V65T, V65K G66K T68P, T68H W69D, W69P F85L、F85I、F85Q、F85C、F85T、F85A、F85V、F85S、F85R、F85H、F85N F108C, F108E, F108S, F108N F117C、F117I、F117W、F117V、F117A、F117G、F117T、F117N Y198L, Y198S, Y198M, Y198I S199Q、S199P K200Y、K200M、K200I、K200R、K200L、K200V H201K、H201R、H201M、H201L、H201Q F202M, F202H, F202V, F202C W203Y F223M, F223L, F223W, F223V, F223H, F223G, F223A R238H, R238S F251N、F251C、F251M、F251R、F251S、F251A、F251T、F251L、F251P、F251G、F251V、F251Q V252L, V252T, V252C N253T, N253V, N253K, N253C, N253R Y256L, Y256V, Y256I G257A, G257C W258Y, W258F, W258N T273N G275C, G275S, G275V, G275K, G275I, G275F, G275H, G275R, G275L, G275P, G275Q, G275Y, G275N, G275M, G275T N276G, N276M, N276Q, N276H H277S, H277Q, H277C, H277E, H277K, H277D, H277I, H277R, H277M, H277P, H277L, H277W, H277V, H277Y, H277G, H277F, H277T Y278P、Y278D M288Q, M288T V290M, V290A, V290S, V290N Y291T, Y291E, Y291G W298F, W298Y, W298I, W298L, W298M S299P Y302L, Y302H, Y302M, Y302V, Y302C, Y302T, Y302P, Y302S, Y302W F305H, F305Y

[0044] The following amino acid substitutions exhibit a particularly large degree of deamidation and are therefore particularly preferred. S61Y, S61D, S61I, S61L, S61N, S61E, S61M, S61F, S61W, S61Q, S61P, S61H, S61K Y62P G63D, G63Q, G63Y, G63R, G63K, G63H, G63P, G63M, G63N, G63C, G63F, G63L, G63W, G63T, G63V, G63S V65T, V65K G66K T68P, T68H W69D, W69P F85H, F85N F117G, F117T, F117N Y198S, Y198M, Y198I S199P K200M, K200I, K200R, K200L, K200V H201M, H201L, H201Q F202V, F202C W203Y F223W, F223V, F223H, F223G, F223A F251L, F251P, F251G, F251V, F251Q N253T, N253V, N253K, N253C, N253R Y256L, Y256V, Y256I G257C W258N G275V, G275K, G275I, G275F, G275H, G275R, G275L, G275P, G275Q, G275Y, G275N, G275M, G275T N276G, N276M, N276Q, N276H H277S, H277Q, H277C, H277E, H277K, H277D, H277I, H277R, H277M, H277P, H277L, H277W, H277V, H277Y, H277G, H277F, H277T Y278P, Y278D M288T V290S, V290N Y291G W298Y, W298I, W298L, W298M F305Y

[0045] Specific examples of the modified enzymes of the present invention include transglutaminases consisting of any of the amino acid sequences of SEQ ID NOs: 2 to 10 (corresponding to R26N, R26Y, R26S, E28N, E28G, E28F, E28Y, E58R, and E58L mutants, respectively). As shown in the examples described below, these mutants have been confirmed to have reduced temperature stability and improved antioxidant properties.

[0046] Generally, when a portion of the amino acid sequence of a protein is mutated, the mutated protein may have the same function as the original protein. That is, the amino acid sequence mutation may not substantially affect the function of the protein, and the function of the protein may be maintained before and after the mutation. On the other hand, if the amino acid sequences of two proteins are highly identical, there is a high probability that they will exhibit equivalent characteristics. Considering these common technical facts, if an amino acid sequence is not completely identical (i.e., 100% identical) to the above-mentioned modified enzyme, i.e., "the amino acid sequence of SEQ ID NO: 1, which includes any of the amino acid substitutions (1) to (134) above (specific examples of such amino acid sequences are the amino acid sequences of SEQ ID NOs: 2 to 10)," but exhibits a high degree of identity, and shows the desired change in characteristics, then it can be considered to be substantially the same enzyme (substantially identical transglutaminase) as the above-mentioned modified enzyme. Identity here 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. A higher degree of identity is preferable. Therefore, in the most preferable embodiment, the degree of identity is 99% or higher.

[0047] When comparing the above-mentioned modified enzyme with a transglutaminase that is essentially identical, slight differences in the amino acid sequence will be observed. However, these differences in the amino acid sequence will occur at positions other than those where the above-mentioned amino acid substitutions are made. Therefore, for example, if the amino acid sequence criterion for identity is the amino acid sequence of SEQ ID NO: 2, the difference will occur at positions other than N at position 26; if the amino acid sequence criterion for identity is the amino acid sequence of SEQ ID NO: 3, the difference will occur at positions other than Y at position 26; if the amino acid sequence criterion for identity is the amino acid sequence of SEQ ID NO: 4, the difference will occur at positions other than S at position 26; if the amino acid sequence criterion for identity is the amino acid sequence of SEQ ID NO: 5, the difference will occur at positions other than N at position 28; if the amino acid sequence criterion for identity is the amino acid sequence of SEQ ID NO: 6, the difference will occur at positions other than G at position 28; if the amino acid sequence criterion for identity is the amino acid sequence of SEQ ID NO: 7, the difference will occur at positions other than F at position 28; if the amino acid sequence criterion for identity is the amino acid sequence of SEQ ID NO: 8, the difference will occur at positions other than Y at position 28; if the amino acid sequence criterion for identity is the amino acid sequence of SEQ ID NO: 9, the difference will occur at positions other than R at position 58; and if the amino acid sequence criterion for identity is the amino acid sequence of SEQ ID NO: 10, the difference will occur at positions other than L at position 58. In other words, in amino acid sequences that exhibit 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 at position 26 is N. Similarly, in amino acid sequences that exhibit the above-mentioned identity with the amino acid sequence of SEQ ID NO: 3, the amino acid at position 26 is Y; in amino acid sequences that exhibit the above-mentioned identity with the amino acid sequence of SEQ ID NO: 4, the amino acid at position 26 is S; in amino acid sequences that exhibit the above-mentioned identity with the amino acid sequence of SEQ ID NO: 5, the amino acid at position 28 is N; in amino acid sequences that exhibit the above-mentioned identity with the amino acid sequence of SEQ ID NO: 6, the amino acid at position 28 is G; in amino acid sequences that exhibit the above-mentioned identity with the amino acid sequence of SEQ ID NO: 7, the amino acid at position 28 is F; in amino acid sequences that exhibit the above-mentioned identity with the amino acid sequence of SEQ ID NO: 8, the amino acid at position 28 is Y; in amino acid sequences that exhibit the above-mentioned identity with the amino acid sequence of SEQ ID NO: 9, the amino acid at position 58 is R; and in amino acid sequences that exhibit the above-mentioned identity with the amino acid sequence of SEQ ID NO: 10, the amino acid at position 58 is L.

[0048] Here, "slight differences in amino acid sequence" refers to the result of amino acid deletions, substitutions, additions, insertions, or combinations thereof. Typically, this refers to a mutation (change) in the amino acid sequence resulting from the deletion or substitution of one to several amino acids (up to, for example, 3, 5, 7, or 10) or the addition or insertion of one to several amino acids (up to, for example, 3, 5, 7, or 10) or a combination thereof. "Slight differences in amino acid sequence" are preferably caused by conservative amino acid substitutions. Here, "conservative amino acid substitution" refers to the substitution of a certain amino acid residue with an amino acid residue having a side chain of similar properties. Amino acid residues are classified into several families based on their side chains: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Conservative amino acid substitutions are preferably substitutions between amino acid residues within the same family. Since the active residue of transglutaminase from Streptomyces mobaraensis (SEQ ID NO: 1) is known to be cysteine ​​at position 64, it is advisable to avoid affecting this acidic residue when introducing mutations.

[0049] Incidentally, the identity (%) of two amino acid sequences or two base sequences (hereinafter, the term "two sequences" will be used to include these) can be determined, for example, by the following procedure. First, the two sequences are aligned in a way that allows for optimal comparison (for example, a gap may be introduced in the first sequence to optimize the alignment with the second sequence). When a molecule (amino acid residue or nucleotide) at a specific position in the first sequence is the same as the molecule at the corresponding position in the second sequence, the molecules at that position are said to be identical. The identity of the two sequences is a function of the number of identical positions common to the two sequences (i.e., identity (%) = number of identical positions / total number of positions × 100), and preferably, the number and size of gaps required for alignment optimization are also taken into consideration.

[0050] The comparison and determination of identity between two sequences can be achieved using mathematical algorithms. A concrete example of a mathematical algorithm usable for sequence comparison is the algorithm described in Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, which was modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77, but is not limited to this. Such algorithms are incorporated into the NBLAST and XBLAST programs (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 in the NBLAST program with score = 100 and wordlength = 12. To obtain equivalent amino acid sequences, for example, a BLAST polypeptide search can be performed using the XBLAST program with score = 50 and wordlength = 3. For obtaining gap alignment for comparison, Gapped BLAST, described in Altschul et al. (1997) Amino Acids Research 25(17):3389-3402, is available. When using BLAST and Gapped BLAST, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used. For more information, please refer to http: / / www.ncbi.nlm.nih.gov. Examples of other mathematical algorithms available for sequence comparison include those described in Myers and Miller (1988) Comput Appl Biosci. 4:11-17. Such algorithms are 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 to compare amino acid sequences, for example, you can use the PAM120 residue mass table and set the gap length penalty to 12 and the gap penalty to 4.

[0051] The identity of two amino acid sequences can be determined using the GAP program in the GCG software package, with the Blossom 62 matrix or PAM250 matrix, using gap weights of 12, 10, 8, 6, or 4 and gap length weights of 2, 3, or 4. The identity of two nucleotide sequences can also be determined using the GAP program in the GCG software package (available at http: / / www.gcg.com), with a gap weight of 50 and a gap length weight of 3.

[0052] Typically, a transglutaminase consisting of the amino acid sequence of SEQ ID NO: 1, i.e., a transglutaminase derived from Streptomyces mobaraensis, becomes the modified enzyme of the present invention when it undergoes a mutation (any amino acid substitution of (1) to (134) above). The substantially identical transglutaminase described above can be obtained by further mutation in a transglutaminase consisting of the amino acid sequence of SEQ ID NO: 1 that has undergone a mutation (any amino acid substitution of (1) to (134) above), by applying an equivalent mutation to a transglutaminase consisting of an amino acid sequence highly identical to the amino acid sequence of SEQ ID NO: 1, such as a transglutaminase from the same species and genus as the Streptomyces mobaraensis strain that produces the transglutaminase consisting of the amino acid sequence of SEQ ID NO: 1, or by further mutation in the result obtained by such a mutation. In this case, "equivalent mutation" means that in an amino acid sequence highly identical to the amino acid sequence of SEQ ID NO: 1, an amino acid residue corresponding to the amino acid residue at the mutation site in the present invention (the mutation site in any amino acid substitution of (1) to (134) above) is substituted.Furthermore, examples of transglutaminases with amino acid sequences highly identical to the amino acid sequence of SEQ ID NO: 1 include: a transglutaminase derived from Streptomyces mobaraensis (formerly classified as Strepoverticillium ladakanum) with the amino acid sequence of SEQ ID NO: 11 (93% amino acid sequence identity); a transglutaminase derived from Streptomyces albireticuli with the amino acid sequence of SEQ ID NO: 12 (82% amino acid sequence identity); a transglutaminase derived from Streptomyces luteireticuli with the amino acid sequence of SEQ ID NO: 13 (82% amino acid sequence identity); and Streptomyces cinnamoneus (Streptomyces Transglutaminase derived from *Streptomyces cinnamoneus*, possessing the amino acid sequence of SEQ ID NO: 14 (81% amino acid sequence identity), transglutaminase derived from *Streptomyces platensis*, possessing the amino acid sequence of SEQ ID NO: 15 (80% amino acid sequence identity), and *Streptomyces hygroscopix*. One example is a transglutaminase derived from *Hygroscopicus* that has the amino acid sequence of SEQ ID NO: 16 (80% amino acid sequence identity).

[0053] In this specification, the term "corresponding" in reference to amino acid residues means that they make equivalent contributions to the function of the proteins (enzymes) being compared. For example, when the amino acid sequence of a comparison target is arranged in a way that allows for optimal comparison with the amino acid sequence of a reference transglutaminase (amino acid sequence of SEQ ID NO: 1), while considering the partial homology of the primary structure (amino acid sequence) (gaps may be introduced as needed to optimize the alignment), the amino acid at the position corresponding to a specific amino acid in the reference amino acid sequence can be identified as the "corresponding amino acid." Instead of comparing primary structures, or in addition to comparing three-dimensional structures, "corresponding amino acids" can also be identified by comparing three-dimensional structures. By using three-dimensional structural information, highly reliable comparison results can be obtained. In this case, a method can be employed in which the atomic coordinates of the three-dimensional structures of multiple enzymes are compared while performing alignment. Three-dimensional structural information of the target enzyme can be obtained, for example, from the Protein Data Bank (http: / / www.pdbj.org / index#j.html).

[0054] An example of a method for determining the three-dimensional structure of a protein using X-ray crystallography is shown below. (1) Crystallize the protein. Crystallization is essential for determining the three-dimensional structure, but it also has industrial utility as a method for high-purity purification and high-density, stable storage of proteins. In this case, it is best to crystallize a protein to which a substrate or its analog compound has been bound as a ligand. (2) The fabricated crystals are irradiated with X-rays to collect diffraction data. However, protein crystals are often damaged by X-ray irradiation, and their diffraction ability deteriorates. In such cases, a low-temperature measurement technique has recently become popular in which the crystals are rapidly cooled to around -173°C and diffraction data is collected in that state. Finally, high-brightness synchrotron radiation is used to collect high-resolution data for structural determination. (3) In order to perform crystal structure analysis, phase information is required in addition to diffraction data. If the crystal structure of a related protein is unknown for the target protein, it is impossible to determine the structure by molecular substitution, and the phase problem must be solved by heavy atom isomorphic substitution. Heavy atom isomorphic substitution is a method that introduces metal atoms with large atomic numbers, such as mercury or platinum, into the crystal and obtains phase information by utilizing the contribution of the large X-ray scattering ability of the metal atoms to the X-ray diffraction data. The determined phase can be improved by smoothing the electron density of the solvent region in the crystal. Because water molecules in the solvent region fluctuate greatly, the electron density is hardly observed. By approximating the electron density in this region to 0, we can approach the true electron density, thereby improving the phase. Furthermore, if the asymmetric unit contains multiple molecules, averaging the electron densities of these molecules further significantly improves the phase. The protein model is then fitted to the electron density map calculated using this improved phase. This process is performed using computer graphics programs such as QUANTA from MSI (USA). After this, structural refinement is performed using programs such as X-PLOR from MSI, and the structural analysis is completed. If the crystal structure of a related protein is known for the target protein, it can be determined by molecular substitution using the atomic coordinates of the known protein. Molecular substitution and structural refinement can be performed using programs such as CNS#SOLVE ver.11.

[0055] 2. Nucleic acids encoding modified transglutaminase, etc. A second aspect of the present invention provides nucleic acids related to the modified enzyme of the present invention. Specifically, the invention provides a gene encoding the modified enzyme, a nucleic acid that can be used as a probe for identifying the nucleic acid encoding the modified enzyme, and a nucleic acid that can be used as a primer for amplifying or mutating the nucleic acid encoding the modified enzyme.

[0056] Genes encoding modified enzymes are typically used for the preparation of modified enzymes. Genetic engineering methods using genes encoding modified enzymes allow for the acquisition of more homogeneous modified enzymes. Furthermore, this method is suitable for preparing large quantities of modified enzymes. However, the uses of genes encoding modified enzymes are not limited to the preparation of modified enzymes. For example, these nucleic acids can be used as experimental tools for elucidating the mechanism of action of modified enzymes, or as tools for designing or creating further modified enzymes.

[0057] In this specification, "gene encoding a modified enzyme" refers to a nucleic acid from which the modified enzyme can be obtained when the gene is expressed. This includes not only nucleic acids having a base sequence corresponding to the amino acid sequence of the modified enzyme, but also nucleic acids obtained by adding a sequence that does not encode an amino acid sequence to such a nucleic acid. Codon degeneracy is also taken into consideration.

[0058] Examples of gene sequences (nucleotide sequences) encoding the modified enzymes are shown in Sequence IDs 18-26. These sequences encode the mutants shown in the examples described below. Sequence ID 18: R26N variant Sequence ID 19: R26Y variant Sequence ID 20: R26S variant Sequence ID 21: E28N variant Sequence ID 22: E28G variant Sequence ID 23: E28F mutant Sequence ID 24: E28Y variant Sequence ID 25: E58R variant Sequence ID 26: E58L mutant

[0059] When expressing the gene of the present invention in a host, a gene construct is usually introduced into the host in which a sequence encoding a propeptide (pro sequence) is added to the 5' end of the above sequence (for example, any of sequence numbers 18 to 26) in order to stabilize the structure of the modified enzyme, which is the expression product. Furthermore, when expressing it as a secreted protein, a gene construct is prepared in which a sequence encoding a pre sequence (signal sequence) is added to the 5' end of the sequence encoding the pro sequence. When this gene construct is used, it is expressed as a prepro transglutaminase in which the pre sequence and pro sequence are linked, and then mature transglutaminase is obtained through cleavage of the pre sequence (conversion to pro transglutaminase) and cleavage of 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 pre-sequence is shown in SEQ ID NO. 27 (pre-sequence of transglutaminase from Streptomyces mobaraensis), a specific example of a pro-sequence is shown in SEQ ID NO. 28 (pro-sequence of transglutaminase from Streptomyces mobaraensis), a specific example of a sequence encoding a pre-sequence is shown in SEQ ID NO. 29 (sequence encoding the pre-sequence of transglutaminase from Streptomyces mobaraensis), and a specific example of a sequence encoding a pro-sequence is shown in SEQ ID NO. 30 (sequence encoding the pro-sequence of transglutaminase from Streptomyces mobaraensis).

[0060] 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 herein or in the attached sequence listings.

[0061] In another aspect of the present invention, a nucleic acid is provided in which the function of the protein encoded by it is equivalent to that of the gene encoding the modified enzyme of the present invention, but in some respects the base sequence differs (hereinafter also referred to as "homologous nucleic acid"; the base sequence defining the homologous nucleic acid is also referred to as "homologous base sequence"). An example of a homologous nucleic acid is a DNA that encodes a protein having characteristic enzyme activity (i.e., transglutaminase activity) of the modified enzyme, consisting of a base sequence that includes one or more base substitutions, deletions, insertions, additions, or inversions based on the base sequence of the nucleic acid encoding the modified enzyme of the present invention. Base substitutions and deletions may occur at multiple sites. The term "multiple" here varies depending on the position and type of amino acid residues in the three-dimensional structure of the protein encoded by the nucleic acid, but is for example 2 to 40 bases, preferably 2 to 20 bases, and more preferably 2 to 10 bases.

[0062] Homologous nucleic acids have identity with respect to a reference base sequence of, 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.

[0063] Homologous nucleic acids like those described above can be obtained, for example, by restriction enzyme treatment, treatment with exonucleases or DNA ligases, or by introducing mutations using site-directed mutagenesis (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York) or random mutagenesis (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York). Homologous nucleic acids can also be obtained by other methods such as ultraviolet irradiation.

[0064] Another aspect of the present invention relates to nucleic acids 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 nucleic acids having a nucleotide sequence that is at least about 60%, 70%, 80%, 90%, 95%, 99%, and 99.9% identical to the nucleotide sequence of a gene encoding the modified enzyme of the present invention, or a nucleotide sequence complementary thereto.

[0065] A further aspect of the present invention relates to nucleic acids having a base sequence that hybridizes under stringent conditions to a base sequence complementary to the base sequence of a gene encoding the modified enzyme of the present invention or to a base sequence complementary to its homologous base sequence. Here, "stringent conditions" refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Such stringent conditions are known to those skilled in the art and can be established, for example, by referring to Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York) or Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987). As stringent conditions, for example, one can incubate the samples at approximately 42°C to 50°C using a hybridization solution (50% formamide, 10×SSC (0.15M NaCl, 15mM sodium citrate, pH 7.0), 5×Denhardt solution, 1% SDS, 10% dextran sulfate, 10 μg / ml denatured salmon sperm DNA, 50mM phosphate buffer (pH 7.5)), followed by washing at approximately 65°C to 70°C with 0.1×SSC and 0.1% SDS. Even more preferred stringent conditions include, for example, using a hybridization solution consisting of 50% formamide, 5×SSC (0.15M NaCl, 15mM sodium citrate, pH 7.0), 1×Denhardt solution, 1% SDS, 10% dextran sulfate, 10 μg / ml denatured salmon sperm DNA, and 50mM phosphate buffer (pH 7.5).

[0066] A further aspect of the present invention provides nucleic acids (nucleic acid fragments) having the nucleotide sequence of a gene encoding the modified enzyme of the present invention, or a portion of a nucleotide sequence complementary thereto. Such nucleic acid fragments can be used to detect, identify, and / or amplify nucleic acids having the nucleotide sequence of a gene encoding the modified enzyme of the present invention. The nucleic acid fragments are designed to include, for example, at least a portion that hybridizes to a continuous nucleotide portion (e.g., about 10 to about 100 nucleotides long, preferably about 20 to about 100 nucleotides long, and more preferably about 30 to about 100 nucleotides long) in the nucleotide sequence of the gene encoding the modified enzyme of the present invention. When used as a probe, the nucleic acid fragments can be labeled. For example, fluorescent substances, enzymes, and radioisotopes can be used for labeling.

[0067] A further aspect of the present invention relates to 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. In this specification, the term "vector" means a nucleic acid molecule that can transport the nucleic acid inserted therein into a target such as a cell.

[0068] An appropriate vector is selected depending on the intended use (cloning, protein expression) and the type of host cell. Examples of vectors that use E. coli as a host include M13 phage or its variants, λ phage or its variants, pBR322 or its variants (pB325, pAT153, pUC8, etc.), pET21, etc. Examples of vectors that use yeast as a host include pYepSec1, pMFa, pYES2, pPIC3.5K, etc. Examples of vectors that use insect cells as a host include pAc, pVL, etc. Examples of vectors that use mammalian cells as a host include pCDM8, pMT2PC, etc.

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

[0070] The insertion of nucleic acids into the vector, insertion of selection marker genes (if necessary), insertion of promoters (if necessary), etc., of the present invention can be carried out using standard recombinant DNA techniques (for example, well-known methods using restriction enzymes and DNA ligases, as can be seen in Molecular Cloning, Third Edition, 1.84, Cold Spring Harbor Laboratory Press, New York).

[0071] As host cells, due to their ease of handling, microorganisms such as Aspergillus oryzae (e.g., Aspergillus oryzae), Bacillus bacteria (e.g., Bacillus satillus, Bacillus licheniformis, Bacillus aminolichuaciens), Brevibacillus bacteria (e.g., Brevibacillus chosinensis), Escherichia coli, and Saccharomyces cerevisiae can be used. However, any host cell capable of replicating recombinant DNA and expressing the modified enzyme gene is acceptable. Preferably, Escherichia coli and Saccharomyces cerevisiae can be used. Streptomyces microorganisms (e.g., Streptomyces morabaensis) can also be used as hosts. As an example of Escherichia coli, E. coli BL21 (DE3) can be used when using the T7 promoter, and E. coli JM109 can be used otherwise. Furthermore, examples of budding yeasts include budding yeast SHY2, budding yeast AH22, and budding yeast INVSc1 (Invitrogen).

[0072] Another aspect of the present invention relates to microorganisms (i.e., transformants) possessing the recombinant DNA of the present invention. The microorganisms of the present invention can be obtained by transfection or transformation using the vector of the present invention described above. For example, the calcium chloride method (Journal of Molecular Biology (J.Mol. Biol.), Vol. 53, p. 159 (1970)), the Hanahan method (Journal of Molecular Biology, Vol. 166, p. 557 (1983)), the SEM method (Gene, Vol. 96, p. 23 (1990)), the Chung et al. method (Proceedings of the National Academy of Sciences of the USA, Vol. 86, p. 2172 (1989)), calcium phosphate coprecipitation method, electroporation (Potter, H. et al., Proc. Natl. Acad. Sci. USA 81, 7161-7165 (1984)), lipofection (Felgner, PL et al., Proc. Natl. This can be carried out according to Acad. Sci. USA 84,7413-7417 (1984), etc. Furthermore, the microorganism of the present invention can be used to produce the modified enzyme of the present invention.

[0073] 3. Enzyme preparations containing modified transglutaminase The modified enzyme of the present invention is provided, for example, in the form of 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 decomposition products, various extracts, various salts, various antioxidants, cysteine, glutathione, monosodium glutamate, monosodium inosinate, monosodium guanylate, calcined seashell calcium, silicon dioxide, etc. As excipients, starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, sucrose, glycerol, etc. As buffers, phosphates, citrates, acetates, etc. As stabilizers, propylene glycol, ascorbic acid, etc. As preservatives, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, etc. As preservatives, ethanol, benzalkonium chloride, parahydroxybenzoic acid, chlorobutanol, etc., can be used. Examples of proteins include soy protein, wheat protein, corn protein, milk protein, and animal-derived protein. Examples of extracts include meat extract, plant extract, and yeast extract. Examples of salts include chlorides, phosphates, polyphosphates, pyrophosphates, citrates, lactates, and carbonates. Examples of antioxidants include L-ascorbate and sodium bisulfite. The form of the enzyme preparation of the present invention is not particularly limited and may be, for example, powder, granules, liquid, or capsule.

[0074] 4. Method for preparing modified transglutaminase A further 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. In this embodiment, a nucleic acid encoding the amino acid sequence of the modified enzyme of the present invention (for example, any of sequence numbers 2 to 10) is prepared (step (I)). Here, the "nucleic acid encoding a specific amino acid sequence" is a nucleic acid that, when expressed, yields a polypeptide having the amino acid sequence, and may consist of a nucleic acid with a base sequence corresponding to the amino acid sequence, as well as a nucleic acid to which extra sequences (which may be sequences encoding an amino acid sequence or sequences that do not encode an amino acid sequence) may be added. Codon degeneracy is also taken into consideration. The "nucleic acid encoding the amino acid sequence of the modified enzyme of the present invention" can be prepared in an isolated state by using standard genetic engineering, molecular biological, or biochemical methods, 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, the "nucleic acid encoding the amino acid sequence of the modified enzyme of the present invention" can also be obtained by adding the necessary mutations to the gene encoding the reference transglutaminase. Numerous methods for site-directed base sequence substitution are known in the field (see, for example, Molecular Cloning, Third Edition, Cold Spring Harbor Laboratory Press, New York), and an appropriate method can be selected and used from among them. As a site-directed mutagenesis method, site-directed amino acid saturated mutagenesis can be employed. Site-directed amino acid saturated mutagenesis is a "semi-rational, semi-random" method that estimates the position involved in the desired function based on the three-dimensional structure of the protein and introduces amino acid saturated mutations (J.Mol.Biol.331,585-592(2003)). For example, site-directed amino acid saturated mutations can be introduced using kits such as Quick Change (Stratagene) or Overlap Extension PCR (Nucleic Acid Res. 16,7351-7367(1988)).For PCR, DNA polymerases such as Taq polymerase can be used. However, it is preferable to use high-precision DNA polymerases such as KOD-PLUS- (Toyobo Co., Ltd.) or Pfu turbo (Stratagene Corporation).

[0075] Following step (I), the prepared nucleic acid is expressed (step (II)). For example, first, an expression vector into which the above nucleic acid is inserted is prepared, and this is used to transform host cells.

[0076] Next, the transformants are cultured under conditions that produce the modified enzyme, which is the expression product. The culture of the transformants can be carried out according to standard methods. Any assimilated carbon compound can be used as the carbon source in the culture medium, such as glucose, sucrose, lactose, maltose, molasses, and pyruvate. Any available nitrogen compound can be used as the nitrogen source, such as peptone, meat extract, yeast extract, casein hydrolysate, and soybean meal alkali extract. In addition, phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese, zinc, and other salts, as well as specific amino acids and specific vitamins, may be used as needed.

[0077] On the other hand, the culture temperature can be set within the range of 30°C to 40°C (preferably around 33°C to 37°C). The culture time can be set considering the growth characteristics of the transformant being cultured and the production characteristics of the modified enzyme. The pH of the culture medium is adjusted to a range in which the transformant grows and the enzyme is produced. Preferably, the pH of the culture medium is set to about 6.0 to 9.0 (preferably around pH 7.0).

[0078] Next, the expression product (modified enzyme) is recovered (Step (III)). The culture medium containing the bacterial cells after culturing can be used as is, or after concentration, removal of impurities, etc., as an enzyme solution, but generally the expression product is recovered from the culture medium or bacterial cells. If the expression product is a secreted protein, it can be recovered from the culture medium; otherwise, it can be recovered from the bacterial cells. When recovering from a culture medium, for example, after removing insoluble matter by filtering and centrifuging the culture supernatant, a purified modified enzyme can be obtained by separating and purifying it using a combination of methods such as vacuum concentration, membrane concentration, salting out using ammonium sulfate or sodium sulfate, fractional precipitation with methanol, ethanol or acetone, dialysis, heat treatment, isoelectric focusing, gel filtration, adsorption chromatography, ion exchange chromatography, affinity chromatography, etc. (for example, gel filtration using Sephadex gel (GE Healthcare Biosciences), DEAE Sepharose CL-6B (GE Healthcare Biosciences), Octyl Sepharose CL-6B (GE Healthcare Biosciences), CM Sepharose CL-6B (GE Healthcare Biosciences)). On the other hand, when recovering from within bacterial cells, bacterial cells are collected by filtering and centrifuging the culture medium, and then the bacterial cells are destroyed by mechanical methods such as pressurization, sonication, or physical disruption, or by enzymatic methods such as lysozyme, before the purified modified enzyme can be obtained by separating and purifying it in the same manner as described above.

[0079] The purified enzyme obtained as described above can also be provided in powder form by methods such as freeze-drying, vacuum drying, or spray drying. In this case, the purified enzyme may be pre-dissolved in phosphate buffer, triethanolamine buffer, Tris-HCl buffer, or GOOD's buffer. Preferably, phosphate buffer or triethanolamine buffer can be used. Examples of GOOD's buffers include PIPES, MES, or MOPS.

[0080] Normally, gene expression and the recovery of expression products (modified enzymes) are carried out using a suitable host-vector system as described above, but a cell-free synthesis system may also be used. Here, a "cell-free synthesis system (cell-free transcription system, cell-free transcription / translation system)" refers to the in vitro synthesis of mRNA and proteins encoded by nucleic acids (DNA or mRNA) from a template, using ribosomes and transcription / translation factors derived from living cells (or obtained by genetic engineering techniques), rather than using living cells. In a cell-free synthesis system, a cell extract obtained by purifying a cell lysate as needed is generally used. The cell extract generally contains ribosomes, various factors such as initiation factors, and various enzymes such as tRNA, which are necessary for protein synthesis. When synthesizing proteins, various amino acids, energy sources such as ATP and GTP, and other substances necessary for protein synthesis, such as creatine phosphate, are added to this cell extract. Of course, ribosomes, various factors, and / or various enzymes prepared separately may be supplemented as needed during protein synthesis.

[0081] The development of transcription / translation systems that reconstitute each molecule (factor) necessary for protein synthesis has also been reported (Shimizu, Y. et al.: Nature Biotech., 19, 751-755, 2001). In this synthesis system, genes for 31 factors, consisting of three initiation factors, three elongation factors, four factors involved in termination, 20 aminoacyl-tRNA synthetases that bind each amino acid to tRNA, and methionyl-tRNA formyltransferase, which constitute the bacterial protein synthesis system, are amplified from the E. coli genome, and the protein synthesis system is reconstituted in vitro using these. The present invention may also utilize such a reconstituted synthesis system.

[0082] The term "cell-free transcription / translation system" is used interchangeably with "cell-free protein synthesis system," "in vitro translation system," or "in vitro transcription / translation system." In an in vitro translation system, RNA is used as a template to synthesize proteins. Template RNA can include total RNA, mRNA, or in vitro transcripts. In contrast, in vitro transcription / translation systems use DNA as a template. The template DNA should contain a ribosome-binding region and preferably a suitable terminator sequence. In an in vitro transcription / translation system, conditions are set up with the necessary factors added to ensure that the transcription and translation reactions proceed sequentially. [Examples]

[0083] <Searching for mutation points effective in improving characteristics> Aiming to obtain highly useful modified transglutaminases, we attempted to improve the enzyme function of transglutaminase (wild-type enzyme, SEQ ID NO: 1) derived from Streptomyces mobaraensis using protein engineering (reduced temperature stability, improved heat resistance, improved antioxidant activity, improved reactivity, and deamidation). First, we decided to introduce mutations by amino acid substitution, and selected candidate mutation sites using a CASTing library (see, for example, Angew Chem Int Ed Engl. 2006 Feb 13;45(8):1236-41.) and alanine scanning (see, for example, J Mol Biol. 1995 Feb 17;246(2):317-30.). Next, mutations were introduced at each mutation site using the following method, and mutant enzymes were prepared.

[0084] 1. Preparation of mutant enzymes 1-1. Introduction of mutations (1) Design PCR primers for introducing mutations. (2) Using primer sets for each mutation site, PCR was performed using plasmid pET20b incorporating the transglutaminase gene (SEQ ID NO: 17) as a template (after a 1 minute reaction at 98°C, followed by 15 cycles of 10 seconds at 98°C, 15 seconds at 60°C, and 2 minutes at 68°C, followed by a 5 minute reaction at 68°C, and then left to stand at 4°C). (3) Add DpnI (1.5 μL / tube) to the PCR reaction mixture (25 μL / tube) and process (at 37°C for 3 hours or more). (4) Ligation treatment with T4 kinase (16°C, overnight). (5) Transform E. coli BL21 (DE3) with ligation reaction solution (11 μL / tube) and culture in ampicillin-containing LB medium (37°C, overnight).

[0085] 1-2. Obtaining the enzyme extract (1) Inoculate the mutant strain into ampicillin-containing TB medium and culture at 33°C for 48 hours. Add IPTG (final concentration 0.1 mM) 24 hours after the start of culture. (2) After centrifuging the culture medium (3,000 g x 10 minutes), remove the supernatant and collect the bacterial cells. (3) Add a lysing agent to lyse the bacterial cells. (4) After centrifuging the lysate (3,000 g x 10 minutes), collect the supernatant to obtain the enzyme extract.

[0086] 1-3. Maturation (removal of propeptide sequence) (1) Mix equal volumes of enzyme extract and 2 mg / mL protease (dispase) solution and allow to react (at 30°C for 2 hours or more). (2) The mixture is centrifuged (3,000 g x 10 minutes), and the supernatant is collected to obtain the mature enzyme.

[0087] 2. Characterization of mutant enzymes The following activity measurement methods were used to evaluate the characteristics of each prepared mutant enzyme. <Activity measurement method> Dilute the mature enzyme to an appropriate concentration with 200 mM Tris-HCl pH 6.0 (sample solution). Add 100 μL of substrate solution (R-1) to 10 μL of the sample solution, mix, and then react at 37 °C for 10 minutes. Add 100 μL of the chromogenic solution (R-2) to stop the reaction and form an Fe complex, and then measure the absorbance at 525 nm. As a control, measure the absorbance of a reaction carried out in the same manner using a pre-heat-inactivated enzyme solution, and determine the absorbance difference from the sample solution. Separately, prepare a calibration curve using L-glutamic acid-γ-monohydroxamic acid instead of the enzyme solution, and determine the amount of hydroxamic acid produced from the absorbance difference. Define the enzyme activity that produces 1 μmol of hydroxamic acid per minute as 1 unit (1 U). (Substrate solution (R-1)) Dissolve 2.42 g of 2-amino-2-hydroxymethyl-1,3-propanediol, 0.70 g of hydroxylammonium hydrochloride, 0.31 g of reduced glutathione, and 1.01 g of Z-Gln-Gly (benzyloxycarbonyl-L-glutaminylglycine) in distilled water to a total volume of 100 mL (pH 6.0). (Substrate solution (R-2)) Mix 30 mL of 3M hydrochloric acid solution, 30 mL of 12% trichloroacetic acid solution, and 30 mL of 5% iron(III) chloride solution.

[0088] 2-1. Evaluation of temperature stability / heat resistance Dilute the mature enzyme two-fold with 200 mM Tris-HCl pH 6.0 and treat at a predetermined temperature (20 °C, 30 °C, 40 °C, 50 °C, 60 °C) for 30 minutes. Measure the activity after the treatment and calculate the residual activity rate by comparing with the activity before the treatment.

[0089] When the residual activity rate after treatment at 50 °C for 30 minutes and the relative value to the wild type were calculated using the following formula, amino acid substitutions effective for reducing temperature stability and amino acid substitutions effective for improving heat resistance were identified. Residual activity rate (%) = (enzyme activity after heat treatment) / (enzyme activity before heat treatment) × 100 Relative to wild type (%) = (residual activity rate of mutant enzyme) / (residual activity rate of wild type enzyme) × 100

[0090] Figure 1 shows the results of the temperature stability evaluation. The following amino acid substitutions (1) to (33) were considered effective if the percentage relative to the wild type (%) was 90% or less. (1) The mutation site is V6, and the substituted amino acid is Q, I, M, S, C, K, L, H, F, G, N, P, R, W, or Y (2) The mutation site is R26, and the substituted amino acid is K, Q, M, H, Y, D, G, N, P, or S (3) The mutation site is E28, and the substituted amino acid is V, Q, W, R, K, M, N, F, G, L, P, or Y (4) The mutation site is V30, and the substituted amino acid is P, C, A, E, F, G, H, K, N, Q, R, W, Y, or L (5) The mutation site is Y34, and the substituted amino acid is A (6) The mutation site is Y42, and the substituted amino acid is F, A, C, D, E, G, I, L, M, Q, S, T, V, or W (7) The mutation site is E58, and the substituted amino acid is Y, M, A, F, I, V, R, K, N, L, S, Q, G, or H (8) The mutation site is W59, and the substituted amino acid is R, N, Y, A, S, I, V, D, G, or P (9) The mutation site is L60, and the substituted amino acid is I, M, V, A, C, E, F, Q, S, T, W, or Y (10) The mutation site is Y62, and the substituted amino acid is C, R, G, K, or S (11) The mutation site is V65, and the substituted amino acid is N, L, M, F, W, or Y (12) The mutation site is V67, and the substituted amino acid is L, N, A, C, M, Q, or S (13) The mutation site is T68, and the substituted amino acid is C, L, A, S, M, F, N, Q, or Y (14) The mutation site is W69, and the substituted amino acid is H, M, I, C, E, F, G, K, L, N, Q, R, S, T, or V (15) The mutation site is Q74, and the substituted amino acid is W, D, G, or K (16) The mutation site is Y75, and the substituted amino acid is R, Q, T, or G (17) The mutation site is T77, and the substituted amino acid is M, H, E, C, or G (18) The mutation site is F85, and the substituted amino acid is M (19) The mutation site is F90, and the substituted amino acid is C, M, H, L, or V (20) The mutation site is F108, and the substituted amino acid is R, L, T, A, I, K, N, or V (21) The mutation site is F117, and the substituted amino acid is M or L (22) The mutation site is S199, and the substituted amino acid is G, M, N, K, or V (23) The mutation site is F202, and the substituted amino acid is L or W (24) The mutation site is W203, and the substituted amino acid is F (25) The mutation site is F254, and the substituted amino acid is Y or M (26) The mutation site is T273, and the substituted amino acid is I, V, M, C, S, L, R, G, A, E, F, Y, D, K, W, or H (27) The mutation site is N276, and the substituted amino acid is C, E, K, L, S, T, or V (28) The mutation site is Y278, and the substituted amino acid is M, L, H, I, K, R, W, C, G, N, Q, S, T, or V (29) The mutation site is S284, and the substituted amino acid is W, Y, M, F, or N (30) The mutation site is Y291, and the substituted amino acid is I, W, L, A, C, K, N, Q, R, S, or V (31) The mutation site is S299, and the substituted amino acid is I, Y, V, K, M, Q, A, F, G, or E (32) The mutation site is S303, and the substituted amino acid is N, G, C, V, P, or Y (33) The mutation site is Y310, and the substituted amino acid is C, M, or I

[0091] As more preferable amino acid substitutions, the following substitutions with a percentage difference from the wild type (%) of 30% or less were identified. V6S, V6C, V6K, V6L, V6H, V6F, V6G, V6N, V6P, V6R, V6W, V6Y R26M, R26H, R26Y, R26D, R26G, R26N, R26P, R26S E28N, E28F, E28G, E28L, E28P, E28Y V30P、V30C、V30A、V30E、V30F、V30G、V30H、V30K、V30N、V30Q、V30R、V30W、V30Y、V30L Y34A Y42A、Y42C、Y42D、Y42E、Y42G、Y42I、Y42L、Y42M、Y42Q、Y42S、Y42T、Y42V、Y42W E58G, E58H W59S、W59I、W59V、W59D、W59G、W59P L60M、L60V、L60A、L60C、L60E、L60F、L60Q、L60S、L60T、L60W、L60Y Y62C、Y62R、Y62G、Y62K、Y62S V65N、V65L、V65M、V65F、V65W、V65Y V67L、V67N、V67A、V67C、V67M、V67Q、V67S T68M、T68F、T68N、T68Q、T68Y W69M, W69I, W69C, W69E, W69F, W69G, W69K, W69L, W69N, W69Q, W69R, W69S, W69T, W69V Y75T, Y75G F85M F90V F108T、F108A、F108I、F108K、F108N、F108V F117L S199K, S199V F202W T273L, T273R, T273G, T273A, T273E, T273F, T273Y, T273D, T273K, T273W, T273H N276C, N276E, N276K, N276L, N276T, N276V, N276S Y278L, Y278H, Y278I, Y278K, Y278R, Y278W, Y278C, Y278G, Y278N, Y278Q, Y278S, Y278T, Y278V Y291W, Y291L, Y291A, Y291C, Y291K, Y291N, Y291Q, Y291R, Y291S, Y291V S299E Y310I

[0092] As particularly preferred amino acid substitutions, we identified the following substitutions that have a percentage difference from the wild type of 10% or less. V6H, V6F, V6G, V6N, V6P, V6R, V6W, V6Y R26H, R26Y, R26D, R26G, R26N, R26P, R26S E28F, E28G, E28L, E28P, E28Y V30C, V30A, V30E, V30F, V30G, V30H, V30K, V30N, V30Q, V30R, V30W, V30Y, V30L Y34A Y42A, Y42C, Y42D, Y42E, Y42G, Y42I, Y42L, Y42M, Y42Q, Y42S, Y42T, Y42V, Y42W W59I, W59V, W59D, W59G, W59P L60V, L60A, L60C, L60E, L60F, L60Q, L60S, L60T, L60W, L60Y Y62C, Y62R, Y62G, Y62K, Y62S V65M, V65F, V65W, V65Y V67A, V67C, V67M, V67Q, V67S T68F, T68N, T68Q, T68Y W69M, W69I, W69C, W69E, W69F, W69G, W69K, W69L, W69N, W69Q, W69R, W69S, W69T, W69V Y75T, Y75G F108A, F108I, F108K, F108N, F108V F117L F202W T273F, T273Y, T273D, T273K, T273W, T273H N276C, N276E, N276K, N276L, N276S, N276T, N276V Y278K, Y278R, Y278W, Y278C, Y278G, Y278N, Y278Q, Y278S, Y278T, Y278V Y291L, Y291A, Y291C, Y291K, Y291N, Y291Q, Y291R, Y291S, Y291V S299E

[0093] The results of the heat resistance evaluation are shown in Figure 2. The following amino acid substitutions (34) to (45), which show a percentage of 110% or more compared to the wild type, were considered effective. (34) The mutation site is D3, and the substituted amino acid is Q, P, E, S, or Y (35) The mutation site is R26, and the substituted amino acid is V (36) The mutation site is Y34, and the substituted amino acid is W (37) The mutation site is V67, and the substituted amino acid is H (38) The mutation site is T68, and the substituted amino acid is V or I (39) The mutation site is Q74, and the substituted amino acid is F (40) The mutation site is T77, and the substituted amino acid is Q (41) The mutation site is S199, and the substituted amino acid is C or Q (42) The mutation site is T273, and the substituted amino acid is Q (43) The mutation site is S284, and the substituted amino acid is L, H, K, P, or R (44) The mutation site is S299, and the substituted amino acid is N (45) The mutation site is S303, and the substituted amino acid is K

[0094] As more preferable amino acid substitutions, the following amino acid substitutions with a percentage of 120% or more compared to the wild type were identified. D3P, D3E, D3S, D3Y R26V Y34W V67H T77Q S199C, S199Q T273Q S284H, S284K, S284P, S284R S303K

[0095] As particularly preferred amino acid substitutions, we identified the following amino acid substitutions that represent a percentage of 130% or more compared to the wild type. D3E, D3S, D3Y R26V Y34W V67H S199Q T273Q S284H, S284K, S284P, S284R

[0096] 2-2. Evaluation of antioxidant properties Dilute hydrogen peroxide (30-35%) (Wako, reagent grade) with 200 mM Tris-HCl pH 6.0 to prepare a hydrogen peroxide solution (0.03%) as an oxidizing agent. Dilute the mature enzyme twice with the hydrogen peroxide solution and react at 30°C for 1 hour. Measure the reactivity and compare it to the activity before treatment to calculate the remaining activity.

[0097] The residual activity rate after hydrogen peroxide treatment and its relative value to the wild type were calculated using the following formula, and amino acid substitutions effective in improving antioxidant activity were identified. Remaining activity (%) = (Enzyme activity after hydrogen peroxide treatment) / (Enzyme activity before treatment) × 100 Vs. wild-type (%) = (Percentage of activity remaining in mutant enzyme) / (Percentage of activity remaining in wild-type enzyme) × 100

[0098] The results of the antioxidant evaluation are shown in Figure 3. The following amino acid substitutions (46) to (59), which show a percentage of 110% or more compared to the wild type, were considered effective. (46) The mutation site is D3, and the substituted amino acid is E, Q, S, or Y (47) The mutation site is V6, and the substituted amino acid is P (48) The mutation site is R26, and the substituted amino acid is M, K, W, C, Q, G, Y, E, T, N, D, I, S, P, or V (49) The mutation site is E28, and the substituted amino acid is L, M, K, C, V, R, W, G, N, F, Y, or H (50) The mutation site is Y42, and the substituted amino acid is L, N, or F (51) The mutation site is E58, and the substituted amino acid is Q, A, I, V, L, T, M, K, Y, W, F or R (52) The mutation site is V67, and the substituted amino acid is T, S, or A (53) The mutation site is T68, and the substituted amino acid is L, I, V, or M (54) The mutation site is Q74, and the substituted amino acid is V (55) The mutation site is T77, and the substituted amino acid is D (56) The mutation site is S199, and the substituted amino acid is C (57) The mutation site is T273, and the substituted amino acid is E (58) The mutation site is S284, and the substituted amino acid is R or H (59) The mutation site is Y291, and the substituted amino acid is I, S, F, L, C, N, V, or M

[0099] As more preferable amino acid substitutions, the following amino acid substitutions with a percentage of 120% or more compared to the wild type were identified. D3S, D3Y V6P R26W, R26C, R26Q, R26G, R26Y, R26E, R26T, R26N, R26D, R26I, R26S, R26P, R26V E28L, E28M, E28K, E28C, E28V, E28R, E28W, E28G, E28N, E28F, E28Y, E28H E58A, E58I, E58V, E58L, E58T, E58M, E58K, E58Y, E58W, E58F, E58R V67S, V67A T68I, T68V, T68M T273E S284R, S284H Y291C, Y291N, Y291V, Y291M

[0100] As particularly preferred amino acid substitutions, we identified the following amino acid substitutions that represent a percentage of 130% or more compared to the wild type. V6P R26C, R26Q, R26G, R26Y, R26E, R26T, R26N, R26D, R26I, R26S, R26P, R26V E28V, E28R, E28W, E28G, E28N, E28F, E28Y, E28H Y42F E58I, E58V, E58L, E58T, E58M, E58K, E58Y, E58W, E58F, E58R T68I, T68V, T68M S284H

[0101] 2-3. Evaluation of Reactivity The mature-forming enzyme was diluted to an appropriate concentration with 200 mM Tris-HCl pH 6.0 (sample solution), and its activity was measured. The relative value to the wild type was calculated using the following formula, and amino acid substitutions effective in improving reactivity were identified. Vs. wild-type (%) = (Activity of mutant enzyme) / (Activity of wild-type enzyme) × 100

[0102] Furthermore, to determine the specific activity, the absorbance of the sample solution at 280 nm is measured to calculate the protein concentration, and the specific activity is calculated from the activity measurement value and the protein concentration.

[0103] The results of the reactivity evaluation are shown in Figure 4. The following amino acid substitutions (60) to (63), which showed a reactivity rate of 110% or more compared to the wild type, were considered effective. (60) The mutation site is S284, and the substituted amino acid is M, K, or V (61) The mutation site is F251, and the substituted amino acid is Y (62) The mutation sites are V6 and Y75, the amino acid substituted at mutation site V6 is E, and the amino acid substituted at mutation site Y75 is F (63) The mutation sites are D3 and T77, the amino acid substituted at mutation site D3 is P, and the amino acid substituted at mutation site T77 is Q.

[0104] As more preferable amino acid substitutions, the following amino acid substitutions with a percentage of 120% or more compared to the wild type were identified. S284V F251Y Double mutation of V6E and Y75F Double mutation of D3P and T77Q

[0105] As particularly preferred amino acid substitutions, we identified the following amino acid substitutions that represent a percentage of 130% or more compared to the wild type. Double mutation of V6E and Y75F Double mutation of D3P and T77Q

[0106] As shown in Figure 5, (64) to (91) below were amino acid substitutions that resulted in two or more property changes and were effective for complex improvements. (64) The mutation site is D3, the substituted amino acid is Q, S, or Y, and the property change due to the amino acid substitution is improved heat resistance and improved antioxidant properties. (65) The mutation site is D3, the substituted amino acid is P, and the property changes due to the amino acid substitution are improved heat resistance and improved reactivity. (66) The mutation site is D3, the substituted amino acid is E, and the property changes due to the amino acid substitution are improved heat resistance, improved antioxidant properties, and improved reactivity. (67) The mutation site is V6, the substituted amino acid is P, and the property changes due to the amino acid substitution are decreased temperature stability and improved antioxidant properties. (68) The mutation site is R26, the substituted amino acid is K, Q, M, Y, D, G, N, P, or S, and the change in properties due to the amino acid substitution is a decrease in temperature stability and an improvement in antioxidant properties. (69) The mutation site is R26, the substituted amino acid is V, and the property changes due to the amino acid substitution are improved heat resistance and improved antioxidant properties. (70) The mutation site is R26, the substituted amino acid is H, and the property changes due to the amino acid substitution are decreased temperature stability, improved antioxidant properties, and improved reactivity. (71) The mutation site is E28, the substituted amino acid is V, S, R, K, M, N, F, G, L, or Y, and the property change due to the amino acid substitution is decreased temperature stability and improved antioxidant properties. (72) The mutation site is V30, the substituted amino acid is P, G, N, R or Y, and the property change due to the amino acid substitution is decreased temperature stability and improved reactivity. (73) The mutation site is Y42, the substituted amino acid is F, L, or M, and the change in properties due to the amino acid substitution is a decrease in temperature stability and an improvement in antioxidant properties. (74) The mutation site is E58, the substituted amino acid is Y, M, A, F, I, V, R, K, L, or Q, and the property change due to the amino acid substitution is decreased temperature stability and improved antioxidant properties. (75) The mutation site is L60, the substituted amino acid is I, and the property changes due to the amino acid substitution are decreased temperature stability and improved reactivity. (76) The mutation site is V67, the substituted amino acid is A or S, and the property change due to the amino acid substitution is decreased temperature stability and improved antioxidant properties. (77) The mutation site is T68, the substituted amino acid is C, L, or M, and the property change due to the amino acid substitution is decreased temperature stability and improved antioxidant properties. (78) The mutation site is T68, the substituted amino acid is V or I, and the property change due to the amino acid substitution is improved heat resistance and improved antioxidant properties. (79) The mutation site is Q74, the substituted amino acid is V, and the property changes due to the amino acid substitution are improved antioxidant activity and improved reactivity. (80) The mutation site is T77, the substituted amino acid is C, and the property changes due to the amino acid substitution are decreased temperature stability and improved antioxidant properties. (81) The mutation site is T77, the substituted amino acid is E, and the property changes due to the amino acid substitution are decreased temperature stability and improved reactivity. (82) The mutation site is T77, the substituted amino acid is D, and the property changes due to the amino acid substitution are improved antioxidant activity and improved reactivity. (83) The mutation site is S199, the substituted amino acid is G, and the property changes due to the amino acid substitution are decreased temperature stability and improved reactivity. (84) The mutation site is S199, the substituted amino acid is C, and the property changes due to the amino acid substitution are improved heat resistance, improved antioxidant properties, and improved reactivity. (85) The mutation site is T273, the substituted amino acid is E, and the property changes due to the amino acid substitution are decreased temperature stability and improved antioxidant properties. (86) The mutation site is S284, the substituted amino acid is M or F, and the property change due to the amino acid substitution is decreased temperature stability and improved reactivity. (87) The mutation site is S284, the substituted amino acid is L or K, and the property change due to the amino acid substitution is improved heat resistance and improved reactivity. (88) The mutation site is S284, the substituted amino acid is H or R, and the property change due to the amino acid substitution is improved heat resistance and improved antioxidant properties. (89) The mutation site is Y291, the substituted amino acid is I, L, C, N, S, or V, and the change in properties due to the amino acid substitution is a decrease in temperature stability and an improvement in antioxidant properties. (90) The mutation site is S299, the substituted amino acid is V, K, M, or A, and the property change due to the amino acid substitution is decreased temperature stability and improved reactivity. (91) The mutation site is S303, the substituted amino acid is K, and the property changes due to the amino acid substitution are improved heat resistance and improved reactivity.

[0107] Among the amino acid substitutions described above, the following substitutions result in two seemingly contradictory changes in properties: decreased temperature stability and improved antioxidant activity. The identification of such amino acid substitutions is an unexpected achievement. Modified enzymes with these distinctive amino acid substitutions are expected to be useful in food applications where enzyme inactivation at low temperatures is required. R26K, R26Q, R26M, R26Y, R26D, R26G, R26N, R26P, R26S E28V, E28R, E28K, E28M, E28N, E28F, E28G, E28L, E28Y Y42F, Y42L, Y42M E58Y, E58M, E58A, E58F, E58I, E58V, E58R, E58K, E58L, E58Q V67A, V67S T68C, T68L, T68M T77C T273E Y291I, Y291L, Y291C, Y291N, Y291S, Y291V

[0108] 2-4. Evaluation of deamidation enzyme activity The deamide activity and transglutaminase activity of the mature enzyme were calculated relative to the wild-type enzyme using the following formula. Then, the degree of change in substrate specificity (i.e., deamide formation) was evaluated based on the ratio of deamide activity (ratio to wild-type) to transglutaminase activity (ratio to wild-type), and amino acid substitutions effective for deamide formation were identified. Deamide activity was measured by the following method, and transglutaminase activity was measured using the above activity measurement method with the substrate solution (R-1). Deamide activity (ratio to wild-type) (%) = (Deamide activity of mutant enzyme) / (Ratio of wild-type enzyme) Deamide activity) × 100 Transglutaminase activity (ratio to wild-type) (%) = (transglutaminase activity of mutant enzyme) / (transglutaminase activity of wild-type enzyme) × 100

[0109] <Method for measuring deamidation activity> Dilute the mature-forming enzyme to an appropriate concentration with 200 mM potassium phosphate buffer (pH 7.0) (sample solution). Add 80 μL of substrate solution (1% Z-Gln-Gly, 200 mM potassium phosphate buffer, pH 7.0) to 20 μL of sample solution and mix, then react at 37°C for 24 hours. Using an ammonia tester (WAKO), add 40 μL of chromogenic reagent A, 20 μL of chromogenic reagent B, and 40 μL of chromogenic reagent C to 20 μL of the reaction sample and react at 37°C for 10 minutes. Mix 50 μL of the colored sample with 50 μL of deproteinized solution, centrifuge, collect the supernatant, and measure the absorbance at 630 nm.

[0110] The evaluation results for deamidation are shown in Figure 6. A value greater than 1 for deamidation activity / transglutaminase activity indicates that deamidation has occurred. The following (92) to (134) were considered effective amino acid substitutions. (92) The mutation site is R5, and the substituted amino acid is Q, P, C, M, I, V, Y, F, S, N, T, or G (93) The mutation site is Y34, and the substituted amino acid is H or M (94) The mutation site is W59, and the substituted amino acid is K or E (95) The mutation site is S61, and the substituted amino acid is T, V, Y, D, I, L, N, E, M, F, W, Q, P, H, or K (96) The mutation site is Y62, and the substituted amino acid is D or P (97) The mutation site is G63, and the substituted amino acid is D, Q, Y, R, K, H, P, M, N, C, F, L, W, T, V, or S (98) The mutation site is V65, and the substituted amino acid is C, T, or K (99) The mutation site is G66, and the substituted amino acid is W or K (100) The mutation site is T68, and the substituted amino acid is P or H (101) The mutation site is W69, and the substituted amino acid is Y, D, or P (102) The mutation site is Q74, and the substituted amino acid is R or P (103) The mutation site is F85, and the substituted amino acid is Y, W, L, I, Q, C, T, A, V, S, R, H or N (104) The mutation site is F108, and the substituted amino acid is Q, C, E, S, or N (105) The mutation site is F117, and the substituted amino acid is C, I, W, V, A, G, T, or N (106) The mutation site is Y198, and the substituted amino acid is F, H, L, S, M or I (107) The mutation site is S199, and the substituted amino acid is Q or P (108) The mutation site is K200, and the substituted amino acid is Y, M, I, R, L, or V (109) The mutation site is H201, and the substituted amino acid is K, R, M, L, or Q (110) The mutation site is F202, and the substituted amino acid is Y, M, H, V, or C (111) The mutation site is W203, and the substituted amino acid is Y (112) The mutation site is F223, and the substituted amino acid is Y, M, L, W, V, H, G, or A (113) The mutation site is R238, and the substituted amino acid is A, D, V, G, H, or S (114) The mutation site is F251, and the substituted amino acid is H, N, C, M, R, S, A, T, L, P, G, V, or Q (115) The mutation site is V252, and the substituted amino acid is M, L, T, or C (116) The mutation site is N253, and the substituted amino acid is T, V, K, C, or R (117) The mutation site is Y256, and the substituted amino acid is L, V, or I (118) The mutation site is G257, and the substituted amino acid is A or C (119) The mutation site is W258, and the substituted amino acid is Y, F, or N (120) The mutation site is T273, and the substituted amino acid is N (121) The mutation site is G275, and the substituted amino acid is A, C, S, V, K, I, F, H, R, L, P, Q, Y, N, M or T (122) The mutation site is N276, and the substituted amino acid is G, M, Q, or H (123) The mutation site is H277, and the substituted amino acid is N, S, Q, C, E, K, D, I, R, M, P, L, W, V, Y, G, F, or T (124) The mutation site is Y278, and the substituted amino acid is P or D (125) The mutation site is H279, and the substituted amino acid is N (126) The mutation site is S284, and the substituted amino acid is C (127) The mutation site is M288, and the substituted amino acid is V, Q, or T (128) The mutation site is V290, and the substituted amino acid is T, C, L, M, A, S, or N (129) The mutation site is Y291, and the substituted amino acid is H, T, E, or G (130) The mutation site is W298, and the substituted amino acid is F, Y, I, L, or M (131) The mutation site is S299, and the substituted amino acid is N or P (132) The mutation site is Y302, and the substituted amino acid is L, H, M, V, C, T, P, S, or W (133) The mutation site is S303, and the substituted amino acid is I, Q, D, H, or E (134) The mutation site is F305, and the substituted amino acid is W, H, or Y

[0111] As more preferable amino acid substitutions, the following amino acid substitutions were identified that result in a deamide activity / transglutaminase activity value of 1.2 or higher. R5Q, R5P, R5C, R5M, R5I, R5V, R5Y, R5F, R5S, R5N, R5T, R5G Y34H, Y34M W59K, W59E S61T、S61V、S61Y、S61D、S61I、S61L、S61N、S61E、S61M、S61F、S61W、S61Q、S61P、S61H、S61K Y62D, Y62P G63D、G63Q、G63Y、G63R、G63K、G63H、G63P、G63M、G63N、G63C、G63F、G63L、G63W、G63T、G63V、G63S V65C, V65T, V65K G66W, G66K T68P、T68H W69Y, W69D, W69P Q74R、Q74P F85Y、F85W、F85L、F85I、F85Q、F85C、F85T、F85A、F85V、F85S、F85R、F85H、F85N F108Q、F108C、F108E、F108S、F108N F117C、F117I、F117W、F117V、F117A、F117G、F117T、F117N Y198F、Y198H、Y198L、Y198S、Y198M、Y198I S199Q、S199P K200Y、K200M、K200I、K200R、K200L、K200V H201K、H201R、H201M、H201L、H201Q F202Y、F202M、F202H、F202V、F202C W203Y F223Y, F223M, F223L, F223W, F223V, F223H, F223G, F223A R238A、R238D、R238V、R238G、R238H、R238S F251H、F251N、F251C、F251M、F251R、F251S、F251A、F251T、F251L、F251P、F251G、F251V、F251Q V252M, V252L, V252T, V252C N253T, N253V, N253K, N253C, N253R Y256L, Y256V, Y256I G257A, G257C W258Y, W258F, W258N T273N G275A, G275C, G275S, G275V, G275K, G275I, G275F, G275H, G275R, G275L, G275P, G275Q, G275Y, G275N, G275M, G275T N276G, N276M, N276Q, N276H H277N, H277S, H277Q, H277C, H277E, H277K, H277D, H277I, H277R, H277M, H277P, H277L, H277W, H277V, H277Y, H277G, H277F, H277T Y278P、Y278D H279N S284C M288V, M288Q, M288T V290T、V290C、V290L、V290M、V290A、V290S、V290N Y291H, Y291T, Y291E, Y291G W298F, W298Y, W298I, W298L, W298M S299N, S299P Y302L, Y302H, Y302M, Y302V, Y302C, Y302T, Y302P, Y302S, Y302W S303I, S303Q, S303D, S303H, S303E F305W, F305H, F305Y

[0112] Furthermore, the following amino acid substitutions were identified as more preferable, having a deamide activity / transglutaminase activity value of 2 or higher. R5Y, R5F, R5S, R5N, R5T, R5G Y34M S61T, S61V, S61Y, S61D, S61I, S61L, S61N, S61E, S61M, S61F, S61W, S61Q, S61P, S61H, S61K Y62P G63D, G63Q, G63Y, G63R, G63K, G63H, G63P, G63M, G63N, G63C, G63F, G63L, G63W, G63T, G63V, G63S V65C, V65T, V65K G66K T68P, T68H W69D, W69P F85L, F85I, F85Q, F85C, F85T, F85A, F85V, F85S, F85R, F85H, F85N F108C, F108E, F108S, F108N F117C, F117I, F117W, F117V, F117A, F117G, F117T, F117N Y198L, Y198S, Y198M, Y198I S199Q, S199P K200Y, K200M, K200I, K200R, K200L, K200V H201K, H201R, H201M, H201L, H201Q F202M, F202H, F202V, F202C W203Y F223M, F223L, F223W, F223V, F223H, F223G, F223A R238H, R238S F251N, F251C, F251M, F251R, F251S, F251A, F251T, F251L, F251P, F251G, F251V, F251Q V252L, V252T, V252C N253T, N253V, N253K, N253C, N253R Y256L, Y256V, Y256I G257A, G257C W258Y, W258F, W258N T273N G275C, G275S, G275V, G275K, G275I, G275F, G275H, G275R, G275L, G275P, G275Q, G275Y, G275N, G275M, G275T N276G, N276M, N276Q, N276H H277S, H277Q, H277C, H277E, H277K, H277D, H277I, H277R, H277M, H277P, H277L, H277W, H277V, H277Y, H277G, H277F, H277T Y278P, Y278D M288Q, M288T V290M, V290A, V290S, V290N Y291T, Y291E, Y291G W298F, W298Y, W298I, W298L, W298M S299P Y302L, Y302H, Y302M, Y302V, Y302C, Y302T, Y302P, Y302S, Y302W F305H, F305Y

[0113] As particularly preferred amino acid substitutions, we identified the following amino acid substitutions that result in deamide activity / transglutaminase activity values ​​of 10 or higher. S61Y, S61D, S61I, S61L, S61N, S61E, S61M, S61F, S61W, S61Q, S61P, S61H, S61K Y62P G63D, G63Q, G63Y, G63R, G63K, G63H, G63P, G63M, G63N, G63C, G63F, G63L, G63W, G63T, G63V, G63S V65T, V65K G66K T68P, T68H W69D, W69P F85H, F85N F117G, F117T, F117N Y198S, Y198M, Y198I S199P K200M, K200I, K200R, K200L, K200V H201M, H201L, H201Q F202V, F202C W203Y F223W, F223V, F223H, F223G, F223A F251L, F251P, F251G, F251V, F251Q N253T, N253V, N253K, N253C, N253R Y256L, Y256V, Y256I G257C W258N G275V, G275K, G275I, G275F, G275H, G275R, G275L, G275P, G275Q, G275Y, G275N, G275M, G275T N276G, N276M, N276Q, N276H H277S, H277Q, H277C, H277E, H277K, H277D, H277I, H277R, H277M, H277P, H277L, H277W, H277V, H277Y, H277G, H277F, H277T Y278P, Y278D M288T V290S, V290N Y291G W298Y, W298I, W298L, W298M F305Y [Industrial applicability]

[0114] The modified transglutaminase of the present invention exhibits improved practically important properties and has high industrial value. Therefore, it is expected to be used not only in existing applications but also in new applications.

[0115] This invention is not limited in any way to the descriptions of embodiments and examples of the invention described above. Various modifications are also included in this invention, provided that they do not depart from the scope of the claims and are easily conceivable by those skilled in the art. The contents of papers, published patent gazettes, and other documents explicitly mentioned herein shall be cited in their entirety.

[0116] Sequence ID 2: Description of artificial sequence: R26N variant Sequence ID 3: Description of artificial sequence: R26Y variant Sequence ID 4: Description of artificial sequence: R26S variant Sequence ID 5: Description of artificial sequence: E28N variant Sequence ID 6: Description of artificial sequence: E28G variant Sequence ID 7: Description of artificial sequence: E28F variant Sequence ID 8: Description of artificial sequence: E28Y variant Sequence ID 9: Description of artificial sequence: E58R variant Sequence ID 10: Description of artificial sequence: E58L variant Sequence ID 18: Description of artificial sequence: R26N variant Sequence ID 19: Description of artificial sequence: R26Y variant Sequence ID 20: Description of artificial sequence: R26S variant Sequence ID 21: Description of artificial sequence: E28N variant Sequence ID 22: Description of artificial sequence: E28G variant Sequence ID 23: Description of artificial sequence: E28F variant Sequence ID 24: Description of artificial sequence: E28Y variant Sequence ID 25: Description of artificial sequence: E58R variant Sequence ID 26: Description of artificial sequence: E58L variant

Claims

1. A modified transglutaminase having an amino acid sequence in the amino acid sequence of Sequence ID No. 1 that includes one of the following amino acid substitutions (1) to (5), or an amino acid sequence that is 90% or more identical to said amino acid sequence (provided that there are differences in the amino acid sequence in parts other than the position of the amino acid substitution), and exhibiting characteristic changes corresponding to said amino acid substitution: (1) The mutation site is V6, the substituted amino acid is Q, I, M, S, K, L, H, or G, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (2) The mutation site is L60, the substituted amino acid is I, V, C, or F, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (3) The mutation site is V67, the substituted amino acid is L, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (4) The mutation site is T273, the substituted amino acid is V, M, C, L, F, or Y, and the change in properties due to the amino acid substitution is a decrease in temperature stability; (5) The mutation site is L60, the substituted amino acid is I, and the property changes due to the amino acid substitution are decreased temperature stability and improved reactivity;

2. A gene encoding a modified transglutaminase as described in claim 1.

3. Recombinant DNA comprising the gene described in claim 2.

4. A microorganism possessing recombinant DNA as described in claim 3.

5. An enzyme preparation comprising the modified transglutaminase described in claim 1.

6. A method for preparing modified transglutaminase, including the following steps (I) to (III): (i) A step of preparing a nucleic acid encoding the amino acid sequence of the modified transglutaminase described in claim 1; (II) The step of expressing the nucleic acid, (III) A step of recovering the expression product.

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