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]

[0001] The present invention relates to a transglutaminase. More specifically, the present invention relates to a modified transglutaminase having altered or improved properties and uses thereof. This application claims priority based on Japanese Patent Application No. 2017-231192, filed on November 30, 2017, the entire contents of which are incorporated by reference. [Background technology]

[0002] Transglutaminase is an enzyme that catalyzes the acyl transfer reaction of the γ-carboxylamide group of glutamine residues in peptide chains. When the ε-amino group of a lysine residue in a protein acts as an acyl acceptor, it forms an ε-(γ-Gln)-Lys crosslink within or between protein molecules. Therefore, the action of transglutaminase can be utilized to modify proteins or peptides, and transglutaminase derived from the genus Streptomyces (see, for example, Patent Document 1) is used for binding meat and producing sausages, tofu, bread, and noodles. In addition to the food field, the use of transglutaminase is also being considered in the fields of fiber, medicine, and cosmetics. Along with such expansion of use, attempts have been made to improve the properties of transglutaminase (such as heat resistance, specific activity, substrate specificity, and stability) (see, for example, Patent Documents 2 and 3, and Non-Patent Documents 1 to 4). [Prior art documents] [Patent documents]

[0003] Patent Document 1: Japanese Patent Application Laid-Open No. 4-108381 Patent Document 2: JP 2002-253272 A Patent Document 3: JP 2008-194004 A Non-patent literature

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

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

[0006] In order to solve the above problems, the present inventors attempted to improve transglutaminase derived from Streptomyces mobaraensis by amino acid substitution (converting a specific amino acid residue into another amino acid). After much trial and error, they succeeded in identifying multiple mutations (combinations of amino acid residues and substituted amino acids) that were effective in changing the properties (reducing temperature stability, improving thermostability, improving antioxidant properties, improving reactivity, and deamidating). Of note, some mutations brought about changes in two or more properties. Furthermore, among the mutations that achieved such combined improvements, there were some that caused changes in properties that could be said to be contradictory, namely, reducing temperature stability and improving antioxidant properties. This fact was extremely unexpected and exceeded expectations, and is worthy of special mention. On the other hand, the above results are also important in that they provide information and means for designing and obtaining modified transglutaminase that can achieve the purpose of improving transglutaminase.

[0007] However, it is often found that the combination of two effective mutations is highly likely to produce additive or synergistic effects. In addition, in view of the common general knowledge that enzymes of the same kind have high similarity in structure (primary structure, three-dimensional structure) and similar mutations are highly likely to produce similar effects, if useful mutations found in transglutaminase derived from Streptomyces mobaraensis having the amino acid sequence of SEQ ID NO: 1 are applied to other transglutaminase that has high structural similarity with the transglutaminase, it is highly likely that similar effects will be achieved, and a person skilled in the art would recognize that such application is effective.

[0008] The following invention is based on the above results and considerations. [1] A modified transglutaminase having an amino acid sequence of SEQ ID NO: 1 containing any one of the following amino acid substitutions (1) to (134), or an amino acid sequence showing 80% or more identity to said amino acid sequence (with the proviso that the amino acid sequence differs in a portion other than the position of the amino acid substitution), and having a change in characteristics corresponding to the amino acid substitution: (1) The mutation site is V6, the amino acid after substitution is Q, I, M, S, C, K, L, H, F, G, N, P, R, W or Y, and the characteristic change 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 characteristics 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 characteristics due to the amino acid substitution is a decrease in temperature stability; (4) The mutation site is V30, the amino acid after substitution is P, C, A, E, F, G, H, K, N, Q, R, W, Y or L, and the characteristic change 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 characteristics due to the amino acid substitution is a decrease in temperature stability; (6) The mutation site is Y42, the amino acid after substitution is F, A, C, D, E, G, I, L, M, Q, S, T, V or W, and the change in characteristics 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 characteristics 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 characteristics 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 characteristics 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 characteristics due to the amino acid substitution is decreased temperature stability; (11) The mutation site is V65, the substituted amino acid is N, L, M, F, W or Y, and the change in characteristics 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 characteristics due to the amino acid substitution is decreased 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 characteristics due to the amino acid substitution is decreased 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 characteristics due to the amino acid substitution is decreased temperature stability; (15) The mutation site is Q74, the substituted amino acid is W, D, G or K, and the change in characteristics due to the amino acid substitution is decreased temperature stability; (16) The mutation site is Y75, the substituted amino acid is R, Q, T or G, and the change in characteristics due to the amino acid substitution is decreased temperature stability; (17) The mutation site is T77, the substituted amino acid is M, H, E, C or G, and the change in characteristics due to the amino acid substitution is decreased temperature stability; (18) The mutation site is F85, the substituted amino acid is M, and the change in characteristics due to the amino acid substitution is decreased temperature stability; (19) The mutation site is F90, the substituted amino acid is C, M, H, L or V, and the change in characteristics due to the amino acid substitution is decreased 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 characteristics due to the amino acid substitution is decreased temperature stability; (21) The mutation site is F117, the substituted amino acid is M or L, and the change in characteristics due to the amino acid substitution is decreased temperature stability; (22) The mutation site is S199, the substituted amino acid is G, M, N, K or V, and the change in characteristics due to the amino acid substitution is decreased 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 decreased temperature stability; (24) The mutation site is W203, the substituted amino acid is F, and the change in characteristics due to the amino acid substitution is decreased temperature stability; (25) The mutation site is F254, the substituted amino acid is Y or M, and the change in characteristics due to the amino acid substitution is decreased 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 characteristics due to the amino acid substitution is decreased 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 characteristics due to the amino acid substitution is decreased 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 characteristics due to the amino acid substitution is decreased temperature stability; (29) The mutation site is S284, and the amino acid after substitution is W, Y, M, F, or N. The change in characteristics due to amino acid substitution is decreased 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 characteristics due to the amino acid substitution is decreased 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 characteristics due to the amino acid substitution is decreased temperature stability; (32) The mutation site is S303, the substituted amino acid is N, G, C, V, P or Y, and the change in characteristics due to the amino acid substitution is decreased temperature stability; (33) The mutation site is Y310, the substituted amino acid is C, M or I, and the change in characteristics due to the amino acid substitution is decreased 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 change in properties 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 change in properties 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 change in properties due to the amino acid substitution is improved heat resistance; (39) The mutation site is Q74, the substituted amino acid is F, and the change in properties due to the amino acid substitution is improved heat resistance; (40) The mutation site is T77, the substituted amino acid is Q, and the change in properties 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 change in properties due to the amino acid substitution is improved heat resistance; (42) The mutation site is T273, the substituted amino acid is Q, and the change in properties due to the amino acid substitution is improved heat resistance; (43) The mutation site is S284, the substituted amino acid is L, H, K, P or R, and the change in properties due to the amino acid substitution is improved heat resistance; (44) The mutation site is S299, the substituted amino acid is N, and the change in properties due to the amino acid substitution is improved heat resistance; (45) The mutation site is S303, the substituted amino acid is K, and the change in properties due to the amino acid substitution is improved heat resistance; (46) The mutation site is D3, the substituted amino acid is E, Q, S or Y, and the change in properties due to the amino acid substitution is an improvement in antioxidant properties; (47) The mutation site is V6, the substituted amino acid is P, and the change in properties due to the amino acid substitution is improved antioxidant activity; (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 change in properties due to the amino acid substitution is improved antioxidant activity; (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 change in properties due to the amino acid substitution is improved antioxidant activity; (50) The mutation site is Y42, the substituted amino acid is L, N or F, and the change in properties due to the amino acid substitution is improved antioxidant activity; (51) The mutation site is E58, the substituted amino acid is Q, A, I, V, L, T, M, K, Y, W, F or R, and the change in properties due to the amino acid substitution is improved antioxidant activity; (52) The mutation site is V67, the substituted amino acid is T, S or A, and the change in properties due to the amino acid substitution is improved antioxidant activity; (53) The mutation site is T68, the substituted amino acid is L, I, V or M, and the change in properties due to the amino acid substitution is improved antioxidant activity; (54) The mutation site is Q74, the substituted amino acid is V, and the change in properties due to the amino acid substitution is improved antioxidant properties; (55) The mutation site is T77, the amino acid after substitution is D, and the change in properties due to the amino acid substitution is improved antioxidant properties; (56) The mutation site is S199, the amino acid after substitution is C, and the change in properties due to the amino acid substitution is improved antioxidant properties; (57) The mutation site is T273, the amino acid after substitution is E, and the change in properties due to the amino acid substitution is improved antioxidant properties; (58) The mutation site is S284, the amino acid after substitution 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 amino acid after substitution 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 change in properties 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 after the substitution at mutation site V6 is E, and the amino acid after the substitution 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 after the substitution at mutation site D3 is P, and the amino acid after the substitution at mutation site T77 is Q, and the change in properties 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 changes due to the amino acid substitution are improved heat resistance and antioxidant properties; (65) The mutation site is D3, the substituted amino acid is P, and the changes in properties due to the amino acid substitution are improved heat resistance and reactivity; (66) The mutation site is D3, the amino acid after substitution is E, and the property changes due to the amino acid substitution are improved heat resistance, antioxidant properties, and reactivity; (67) The mutation site is V6, the substituted amino acid is P, and the changes in properties due to the amino acid substitution are decreased temperature stability and increased 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 changes due to the amino acid substitution are decreased temperature stability and increased antioxidant properties; (69) The mutation site is R26, the amino acid after substitution is V, and the property changes due to the amino acid substitution are improved heat resistance and antioxidant properties; (70) The mutation site is R26, the amino acid after substitution 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 changes due to the amino acid substitution are decreased temperature stability and increased antioxidant properties; (72) The mutation site is V30, the substituted amino acid is P, G, N, R or Y, and the characteristic changes due to the amino acid substitution are decreased temperature stability and increased reactivity; (73) The mutation site is Y42, the substituted amino acid is F, L or M, and the property changes due to the amino acid substitution are decreased temperature stability and increased antioxidant properties; (74) The mutation site is E58, and the substituted amino acid is Y, M, A, F, I, V, R, K, L or Q. The property changes due to the amino acid substitution are decreased temperature stability and increased antioxidant properties; (75) The mutation site is L60, the amino acid after substitution is I, and the changes in properties due to the amino acid substitution are decreased temperature stability and increased reactivity; (76) The mutation site is V67, the amino acid after substitution is A or S, and the changes in properties due to the amino acid substitution are decreased temperature stability and increased antioxidant properties; (77) The mutation site is T68, the substituted amino acid is C, L or M, and the property changes due to the amino acid substitution are decreased temperature stability and increased antioxidant properties; (78) The mutation site is T68, the amino acid after substitution is V or I, and the property changes due to the amino acid substitution are improved heat resistance and antioxidant properties; (79) The mutation site is Q74, the amino acid after substitution is V, and the property changes due to the amino acid substitution are improved antioxidant properties and 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 increased antioxidant properties; (81) The mutation site is T77, the amino acid after substitution is E, and the changes in properties due to the amino acid substitution are decreased temperature stability and increased reactivity; (82) The mutation site is T77, the amino acid after substitution is D, and the property changes due to the amino acid substitution are improved antioxidant properties and reactivity; (83) The mutation site is S199, the amino acid after substitution is G, and the changes in properties due to the amino acid substitution are decreased temperature stability and increased reactivity; (84) The mutation site is S199, the amino acid after substitution is C, and the changes in properties due to the amino acid substitution are improved heat resistance, antioxidant properties, and reactivity; (85) The mutation site is T273, the amino acid after substitution is E, and the changes in properties due to the amino acid substitution are decreased temperature stability and increased antioxidant properties; (86) The mutation site is S284, the substituted amino acid is M or F, and the changes in properties due to the amino acid substitution are decreased temperature stability and increased reactivity; (87) The mutation site is S284, the substituted amino acid is L or K, and the changes in properties due to the amino acid substitution are improved heat resistance and reactivity; (88) The mutation site is S284, the amino acid after substitution is H or R, and the property changes due to the amino acid substitution are improved heat resistance and antioxidant properties; (89) The mutation site is Y291, the substituted amino acid is I, L, C, N, S or V, and the property changes due to the amino acid substitution are decreased temperature stability and increased antioxidant properties; (90) The mutation site is S299, the substituted amino acid is V, K, M or A, and the characteristic changes due to the amino acid substitution are decreased temperature stability and increased reactivity; (91) The mutation site is S303, the amino acid after substitution is K, and the changes in properties due to the amino acid substitution are improved heat resistance and 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 property 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 property due to the amino acid substitution is deamidation; (94) The mutation site is W59, the substituted amino acid is K or E, and the change in property 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 property due to the amino acid substitution is deamidation; (96) The mutation site is Y62, the substituted amino acid is D or P, and the change in properties 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 property 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 change in properties 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 change in properties due to the amino acid substitution is deamidation; (102) The mutation site is Q74, the substituted amino acid is R or P, and the change in properties 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 change in properties 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 change in properties 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 change in properties 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 change in properties due to the amino acid substitution is deamidation; (107) The mutation site is S199, the substituted amino acid is Q or P, and the change in properties 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 change in properties 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 change in properties 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 change in properties 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 change in properties 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 change in properties 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 change in properties 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 change in properties 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 change in property 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 change in property due to the amino acid substitution is deamidation; (118) The mutation site is G257, the substituted amino acid is A or C, and the change in properties 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 change in property 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 change in properties 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 change in properties 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 property due to the amino acid substitution is deamidation; (124) The mutation site is Y278, the substituted amino acid is P or D, and the change in properties 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 change in property 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 change in properties 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 change in property 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 change in property due to the amino acid substitution is deamidation; (131) The mutation site is S299, the substituted amino acid is N or P, and the change in properties 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 change in properties 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 change in property 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 deamidation. [2] The modified transglutaminase according to [1], wherein the identity is 82% or more. [3] The modified transglutaminase according to [1], wherein the identity is 85% or more. [4] The modified transglutaminase according to [1], wherein the identity is 90% or more. [5] The modified transglutaminase according to [1], which consists of any one of the amino acid sequences of SEQ ID NOs: 2 to 10. [6] A gene encoding the modified transglutaminase according to any one of [1] to [5]. [7] The gene according to [6], comprising any one of the base sequences of SEQ ID NOs: 18 to 26. [8] Recombinant DNA containing the gene described in [6] or [7]. [9][8] A microorganism carrying the recombinant DNA described in the above.

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

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

[12] The preparation method described in

[11] , wherein the amino acid sequence is any one of the amino acid sequences of SEQ ID NOs: 2 to 10.

[13] The preparation method described in

[12] , wherein the nucleic acid comprises any one of the base sequences of SEQ ID NOs: 18 to 26. [Brief description of the drawings]

[0009] [Figure 1-1] Mutations that are effective in reducing temperature stability. Each mutation was replaced with various amino acids, and the residual activity of the enzyme (mutant) after replacement was compared with that of the wild-type enzyme to identify effective amino acid substitutions. From the activity measurements, the TG (transglutaminase) activity per mL of the mutant strain culture medium was calculated and used for evaluation. [Figure 1-2] Continued from Figure 1. [Figure 1-3] Continued from Figure 1. [Figure 1-4] Continued from Figure 1. [Figure 1-5] Continued from Figure 1. [Figure 1-6] Continued from Figure 1. [Figure 1-7] Continued from Figure 1. [Figure 2-1] Mutations that are effective in improving heat resistance. Each mutation was replaced with various amino acids, and the residual activity of the enzyme (mutant) after replacement was compared with that of the wild-type enzyme to identify effective amino acid substitutions. From the activity measurements, the TG (transglutaminase) activity per mL of the mutant strain culture medium was calculated and used for evaluation. [Figure 2-2] Continued from Figure 2. [Figure 3-1] Mutations that are effective in improving antioxidant properties. Each mutation was replaced with various amino acids, and the residual activity of the enzyme (mutant) after replacement was compared with that of the wild-type enzyme to identify effective amino acid substitutions. From the activity measurements, the TG (transglutaminase) activity per mL of the mutant strain culture medium was calculated and used for evaluation. [Figure 3-2] Continued from Figure 3. [Figure 3-3] Continued from Figure 3. [Figure 4] Mutations that are effective in improving reactivity. Each mutation was replaced with various amino acids, and the activity of the enzyme (mutant) after replacement was compared with that of the wild-type enzyme to identify effective amino acid substitutions. From the activity measurements, the TG (transglutaminase) activity per 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] Mutations that are effective in changing two or more characteristics. Mutations (amino acid substitutions) that are effective for multiple improvements are shown for each mutation. [Figure 5-2] Continued from Figure 5. [Figure 5-3] Continued from Figure 5. [Figure 6-1] Mutations effective for deamidation. Each mutation was replaced with a different amino acid, and the deamidation activity and transglutaminase activity of the substituted enzyme (mutant) were calculated as relative values ​​to the wild-type enzyme. The value of deamidation activity (ratio to wild-type) / transglutaminase activity (ratio to wild-type) was then calculated and used for evaluation. [Figure 6-2] Continued from Figure 6. [Figure 6-3] Continued from Figure 6. [Figure 6-4] Continued from Figure 6. [Figure 6-5] Continued from Figure 6. [Figure 6-6] Continued from Figure 6. [Figure 6-7] Continued from Figure 6. [Figure 6-8] Continued from Figure 6. [Figure 6-9] Continued from Figure 6. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] For ease of explanation, some of the terms used in connection with the present invention are defined below. (term) The term "modified transglutaminase" refers to an enzyme obtained by modifying or mutating a reference transglutaminase (hereinafter referred to as "reference transglutaminase"). The reference transglutaminase is typically a transglutaminase derived from Streptomyces mobaraensis having the amino acid sequence of SEQ ID NO: 1.

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

[0012] In the present invention, the modification or mutation is "amino acid substitution". Therefore, when the modified transglutaminase is compared with the reference transglutaminase, differences are found in some amino acid residues. In this specification, the modified transglutaminase is also called a modified enzyme or a mutant.

[0013] In accordance with the convention used herein, each amino acid is represented by a single letter as follows: Methionine: M, Serine: S, Alanine: A, Threonine: T, Valine: V, Tyrosine: Y, Leucine: L, Asparagine: N, Isoleucine: I, Glutamine: Q, Proline: P, Aspartic Acid: D, Phenylalanine: F, Glutamic Acid: E, Tryptophan: W, Lysine: K, Cysteine: C, Arginine: R, Glycine: G, Histidine: H

[0014] In the present specification, the position of the mutation site is identified by numbers starting from the N-terminus of mature transglutaminase toward the C-terminus, with the N-terminus amino acid residue being the first.

[0015] Conventionally, the amino acid residue where the amino acid substitution is made, i.e. the "mutation point", is expressed as a combination of one letter representing the type of amino acid and a number representing the position of the amino acid. In addition, a mutation due to an amino acid substitution is expressed by adding one letter representing the type of the amino acid after substitution to the right of the mutation point. Therefore, for example, if the mutation point is valine at position 6, it is expressed as "V6", and if the mutation replaces valine at position 6 with glutamine, it is expressed 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 a transglutaminase consisting of the amino acid sequence of SEQ ID NO: 1, a decrease in temperature stability, an increase in heat resistance, an increase in antioxidant activity, an increase in reactivity, deamidation (an increase in the ratio of deamidation activity to transglutaminase activity), or two or more of these characteristic changes are observed. A modified enzyme with a decreased temperature stability has a practical advantage of preventing denaturation of food in the thermal inactivation process of the enzyme, and is highly useful in applications such as the production of yogurt and cheese. On the other hand, a modified enzyme with improved heat resistance exhibits high activity even at high temperatures, and is particularly suitable for use in applications such as the synthesis of peptide compounds. In addition, a modified enzyme with improved antioxidant activity has an advantage of having high storage stability, and is expected to be effective in preventing inactivation during the production, storage, and use of the enzyme. The modified enzymes with improved reactivity have the advantages of high reaction efficiency and reduced enzyme usage, making them highly useful regardless of the application. Deamidation modifies substrate specificity, and in particular leads to the expansion or creation of applications (e.g., use in applications such as protein solubilization, emulsification, and improved foaming properties). Transglutaminase is an enzyme that catalyzes the acyl transfer reaction between the γ-carboxylamide group of glutamine residues in peptide chains and primary amines. In the absence of primary amines, water acts as an acyl acceptor, catalyzing the deamidation reaction that converts glutamine residues to glutamic acid residues. The modified enzymes that have been deamidated exhibit high deamidation catalytic ability and are more preferable as enzymes to be used for deamidation. The amino acid sequence of SEQ ID NO: 1 is the sequence of transglutaminase derived from Streptomyces mobaraensis.

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

[0018] Temperature stability can be evaluated, for example, based on the residual activity when treated at 50°C for 30 minutes. A modified enzyme with reduced temperature stability has a lower residual activity than the reference transglutaminase. The residual activity of the modified enzyme is, for example, 90% or less, preferably 30% or less, and more preferably 10% or less of the residual activity of the reference transglutaminase. The residual activity is calculated as follows: Residual activity (%) = (enzyme activity after heat treatment) / (enzyme activity before heat treatment) × 100

[0019] Like temperature stability, heat resistance can be evaluated based on the residual activity after treatment at 50°C for 30 minutes. A modified enzyme with improved heat resistance has a higher residual activity than a reference transglutaminase. The residual activity of the modified enzyme is, for example, 110% or more, preferably 120% or more, and more preferably 130% or more of the residual activity of the reference transglutaminase.

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

[0021] The modified enzymes having the above-mentioned properties (temperature stability, thermostability, antioxidant properties) are more useful than the reference transglutaminase in terms of the changed properties, but their reactivity is preferably high, since this allows for a reduction in the amount (enzyme amount) used. For example, the reactivity of the modified enzyme is preferably 50% or more of the reactivity of the reference transglutaminase.

[0022] The reactivity can be evaluated, for example, based on the activity calculated by the measurement method shown in the Examples below. A 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 reference transglutaminase ratio) to transglutaminase activity (relative to reference transglutaminase ratio) (deamidation activity / transglutaminase activity). A detailed evaluation method will be described later. In a modified enzyme in which deamidation has occurred, the deamidation activity (relative to reference transglutaminase ratio) / transglutaminase activity (relative to reference transglutaminase ratio) exceeds 1, and is preferably 1.2 or more, more preferably 2 or more, and even more preferably 10 or more.

[0024] The amino acid substitutions (mutation sites and amino acids after substitution) that result in the above-mentioned changes in characteristics are listed below. <Amino acid substitutions effective in reducing temperature stability> (1) The mutation site is V6, and the amino acid after substitution 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 amino acid after substitution is K, Q, M, H, Y, D, G, N, P, or S. (3) The mutation site is E28, and the amino acid after substitution is V, Q, W, R, K, M, N, F, G, L, P, or Y. (4) The mutation site is V30, and the amino acid after substitution is P, C, A, E, F, G, H, K, N, Q, R, W, Y, or L. (5) The mutation site is Y34, and the amino acid after substitution is A. (6) The mutation site is Y42, and the amino acid after substitution 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 amino acid after substitution is R, N, Y, A, S, I, V, D, G, or P. (9) The mutation site is L60, and the amino acid after substitution is I, M, V, A, C, E, F, Q, S, T, W, or Y. (10) The mutation site is Y62, and the amino acid after substitution is C, R, G, K, or S. (11) The mutation site is V65, and the amino acid after substitution is N, L, M, F, W, or Y. (12) The mutation site is V67, and the amino acid after substitution 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 amino acid after substitution 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 amino acid after substitution is W, D, G, or K. (16) The mutation site is Y75, and the amino acid after substitution 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 amino acid after substitution is M (19) The mutation site is F90, and the amino acid after substitution is C, M, H, L, or V. (20) The mutation site is F108, and the amino acid after substitution is R, L, T, A, I, K, N, or V. (21) The mutation site is F117, and the amino acid after substitution is M or L. (22) The mutation site is S199, and the amino acid after substitution is G, M, N, K, or V. (23) The mutation site is F202, and the amino acid after substitution 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 amino acid after substitution 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 amino acid after substitution 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 amino acid after substitution 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 amino acid after substitution 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 amino acid after substitution is C, M, or I.

[0025] Among the above amino acid substitutions, the following amino acid substitutions reduce temperature stability to a greater extent and are therefore more preferred. 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 reduce temperature stability to a particularly large extent and are particularly preferred amino acid substitutions. 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 preferable because they reduce temperature stability to a particularly large extent and are also highly reactive. 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 amino acid after substitution is Q, P, E, S, or Y. (35) The mutation site is R26, and the amino acid after substitution is V. (36) The mutation site is Y34 and the amino acid after substitution is W. (37) The mutation site is V67 and the substituted amino acid is H. (38) The mutation site is T68, and the amino acid after substitution 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 amino acid after substitution 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 amino acid after substitution is N. (45) The mutation site is S303 and the substituted amino acid is K.

[0029] Among the above amino acid substitutions, the following amino acid substitutions have a large degree of improvement in heat resistance and are more preferred. D3P, D3E, D3S, D3Y R26V Y34W V67H T77Q S199C, S199Q T273Q S284H, S284K, S284P, S284R S303K

[0030] The following amino acid substitutions improve heat resistance to a particularly large extent and are particularly preferred amino acid substitutions. D3E, D3S, D3Y R26V Y34W V67H S199Q T273Q S284H, S284K, S284P, S284R

[0031] The following amino acid substitutions are extremely preferable because they significantly improve heat resistance and also have high reactivity. D3E, D3S, D3Y R26V Y34W T273Q S284H, S284K, S284P, S284R

[0032] <Amino acid substitutions effective in improving antioxidant properties> (46) The mutation site is D3, and the amino acid after substitution 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 amino acid after substitution 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 amino acid after substitution 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 amino acid after substitution 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 amino acid after substitution 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 above amino acid substitutions, the following amino acid substitutions have a large degree of improvement in antioxidant properties and are more preferred. 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 have a particularly large degree of improvement in antioxidant properties and are particularly preferred amino acid substitutions. 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 preferable because they have a particularly large improvement in antioxidant properties and are also highly reactive. 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 following the substitution at mutation site V6 is E, and the amino acid following the substitution at mutation site Y75 is F. (63) The mutation sites are D3 and T77. The amino acid substituted at D3 is P, and the amino acid substituted at T77 is Q.

[0037] Among the above amino acid substitutions, the following amino acid substitutions have a large degree of improvement in reactivity and are more preferred. S284V F251Y V6E and Y75F double mutation D3P and T77Q double mutation

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

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

[0040] Among the above amino acid substitutions, the following amino acid substitutions are accompanied by two seemingly contradictory property changes: a decrease in temperature stability and an increase in antioxidant properties. 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> (92) The mutation site is R5, and the amino acid after substitution is Q, P, C, M, I, V, Y, F, S, N, T, or G. (93) The mutation site is Y34, and the amino acid after substitution is H or M. (94) The mutation site is W59, and the amino acid after substitution 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 amino acid after substitution 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 amino acid after substitution is C, T, or K. (99) The mutation site is G66, and the amino acid after substitution 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 amino acid after substitution is Y, D, or P. (102) The mutation site is Q74, and the amino acid after substitution 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 amino acid after substitution 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 amino acid after substitution is Q or P. (108) The mutation site is K200, and the amino acid after substitution 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 amino acid after substitution 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 amino acid after substitution is L, V, or I. (118) The mutation site is G257, and the amino acid after substitution is A or C. (119) The mutation site is W258, and the amino acid after substitution is Y, F, or N. (120) The mutation site is T273 and the amino acid after substitution 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 amino acid after substitution is P or D. (125) The mutation site is H279, and the amino acid after substitution is N. (126) The mutation site is S284, and the substituted amino acid is C. (127) The mutation site is M288, and the amino acid after substitution 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 amino acid after substitution 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 above amino acid substitutions, the following amino acid substitutions have a high degree of deamidation and are more preferred. 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 are more favorable because they have a higher degree of deamidation. It is exchange. 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 <h2 style=";text-align:left;direction:ltr">F85L、F85I、F85Q、F85C、F85T、F85A、F85V、F85S、F85R、F85H、F85N<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> F108C, F108E, F108S, F108N<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> F117C、F117I、F117W、F117V、F117A、F117G、F117T、F117N<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Y198L, Y198S, Y198M, Y198I<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> S199Q, S199P<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> K200Y、K200M、K200I、K200R、K200L、K200V<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> H201K、H201R、H201M、H201L、H201Q<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> F202M, F202H, F202V, F202C<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> W203Y<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> F223M, F223L, F223W, F223V, F223H, F223G, F223A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> R238H, R238S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> F251N、F251C、F251M、F251R、F251S、F251A、F251T、F251L、F251P、F251G、F251V、F251Q<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> V252L, V252T, V252C<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> N253T, N253V, N253K, N253C, N253R<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Y256L, Y256V, Y256I<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> G257A, G257C<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> W258Y, W258F, W258N<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> T273N<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> G275C, G275S, G275V, G275K, G275I, G275F, G275H, G275R, G275L, G275P, G275Q, G275Y, G275N, G275M, G275T<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> N276G, N276M, N276Q, N276H<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> H277S, H277Q, H277C, H277E, H277K, H277D, H277I, H277R, H277M, H277P, H277L, H277W, H277V, H277Y, H277G, H277F, H277T<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Y278P、Y278D<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> M288Q, M288T<h2 style=";text-align:left;direction:ltr"> 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 are particularly preferred because they have a particularly high degree of deamidation. 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 enzyme of the present invention include transglutaminases consisting of any one of the amino acid sequences of SEQ ID NOs: 2 to 10 (corresponding to R26N mutant, R26Y mutant, R26S mutant, E28N mutant, E28G mutant, E28F mutant, E28Y mutant, E58R mutant, and E58L mutant, respectively). As shown in the Examples below, it has been confirmed that these mutants have reduced temperature stability and improved antioxidant properties.

[0046] In general, when a part of the amino acid sequence of a protein is mutated, the mutated protein may have the same function as the protein before the mutation. That is, the mutation of the amino acid sequence 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, when the identity of the amino acid sequences of two proteins is high, the probability that both proteins will show the same characteristics is high. Considering these technical common sense, if an amino acid sequence that is not completely identical (i.e., 100% identical) to the amino acid sequence of the above modified enzyme, that is, the amino acid sequence of "the amino acid sequence of SEQ ID NO: 1 containing any of the amino acid substitutions (1) to (134) (specific examples of the amino acid sequence are the amino acid sequences of SEQ ID NOs: 2 to 10)" but has an amino acid sequence that shows high identity and exhibits a desired change in characteristics, it can be considered to be an enzyme that is substantially the same as the above modified enzyme (substantially the same transglutaminase). The identity here is 70% or more, 80% or more, 82% or more, 85% or more, 90% or more, 93% or more, 95% or more, 98% or more, or 99% or more. The higher the identity, the more preferred, thus in the most preferred embodiment the identity is 99% or greater.

[0047] When the above modified enzyme is compared with the substantially identical transglutaminase, slight differences in the amino acid sequence are observed. However, the differences in the amino acid sequence occur at positions other than the positions where the above amino acid substitutions have been made. Thus, for example, when the identity standard is the amino acid sequence of SEQ ID NO:2, the difference in the amino acid sequence occurs at a position other than N at position 26; when the identity standard is the amino acid sequence of SEQ ID NO:3, the difference in the amino acid sequence occurs at a position other than S at position 26; when the identity standard is the amino acid sequence of SEQ ID NO:4, the difference in the amino acid sequence occurs at a position other than N at position 28; when the identity standard is the amino acid sequence of SEQ ID NO:5, the difference in the amino acid sequence occurs at a position other than G at position 28; when the identity standard is the amino acid sequence of SEQ ID NO:6, the difference in the amino acid sequence occurs at a position other than F at position 28; when the identity standard is the amino acid sequence of SEQ ID NO:7, the difference in the amino acid sequence occurs at a position other than Y at position 28; when the identity standard is the amino acid sequence of SEQ ID NO:8, the difference in the amino acid sequence occurs at a position other than R at position 58; and when the identity standard is the amino acid sequence of SEQ ID NO:10, the difference in the amino acid sequence occurs at a position other than L at position 58. In other words, in the amino acid sequence showing 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) to the amino acid sequence of SEQ ID NO:2, the amino acid at position 26 is N. Similarly, in the amino acid sequence showing the above-mentioned identity to the amino acid sequence of SEQ ID NO:3, the amino acid at position 26 is Y, in the amino acid sequence showing the above-mentioned identity to the amino acid sequence of SEQ ID NO:4, the amino acid at position 28 is N, in the amino acid sequence showing the above-mentioned identity to the amino acid sequence of SEQ ID NO:5, the amino acid at position 28 is G, in the amino acid sequence showing the above-mentioned identity to the amino acid sequence of SEQ ID NO:7, the amino acid at position 28 is F, in the amino acid sequence showing the above-mentioned identity to the amino acid sequence of SEQ ID NO:8, the amino acid at position 28 is Y, in the amino acid sequence showing the above-mentioned identity to the amino acid sequence of SEQ ID NO:9, the amino acid at position 58 is R, and in the amino acid sequence showing the above-mentioned identity to the amino acid sequence of SEQ ID NO:10, the amino acid at position 58 is L.

[0048] The "slight difference in amino acid sequence" here is caused by deletion, substitution, addition, insertion, or a combination thereof of amino acids. Typically, it refers to a mutation (change) in the amino acid sequence caused by deletion or substitution of one to several (upper limit, for example, 3, 5, 7, 10) amino acids constituting the amino acid sequence, or addition or insertion of one to several (upper limit, for example, 3, 5, 7, 10) amino acids, or a combination thereof. The "slight difference in amino acid sequence" is preferably caused by conservative amino acid substitution. The "conservative amino acid substitution" here refers to the substitution of a certain amino acid residue with an amino acid residue having a side chain with similar properties. Amino acid residues are classified into several families according to their side chains, such as basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Conservative amino acid substitutions are preferably substitutions between amino acid residues within the same family. It is known that the active residue of transglutaminase derived from Streptomyces mobaraensis (SEQ ID NO: 1) is cysteine ​​at position 64, so that the acid residue is not affected when making a mutation.

[0049] Incidentally, the identity (%) of two amino acid sequences or two base sequences (hereinafter, the term "two sequences" is used to include both) can be determined, for example, by the following procedure. First, the two sequences are aligned to enable optimal comparison (for example, gaps may be introduced into the first sequence to optimize 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 a molecule at the corresponding position in the second sequence, the molecule at that position can be said to be identical. The identity of two sequences is a function of the number of identical positions shared by the two sequences (i.e., identity (%)=number of identical positions / total number of positions×100), and preferably, the number and size of gaps required for optimization of the alignment are also taken into account.

[0050] Comparison of two sequences and determination of identity can be achieved using a mathematical algorithm. A non-limiting example of a mathematical algorithm that can be used for sequence comparison is the algorithm described in Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68 and modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the NBLAST program and XBLAST program (version 2.0) described in Altschul et al. (1990) J. Mol. Biol. 215:403-10. To obtain equivalent nucleotide sequences, for example, BLAST nucleotide searches can be performed with the NBLAST program with score = 100 and wordlength = 12. Equivalent amino acid sequences can be obtained by, for example, performing a BLAST polypeptide search with the XBLAST program with score = 50 and wordlength = 3. To obtain gapped alignments for comparison, Gapped BLAST, as described in Altschul et al. (1997) Amino Acids Research 25(17):3389-3402, can be used. When using BLAST and Gapped BLAST, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used. For details, see http: / / www.ncbi.nlm.nih.gov. Examples of other mathematical algorithms that can be used for comparing sequences include the algorithm described in Myers and Miller (1988) Comput Appl Biosci. 4:11-17. Such algorithms are incorporated, for example, in the ALIGN program available on the GENESTREAM network server (IGH Montpellier, France) or the ISREC server. When using the ALIGN program to compare amino acid sequences, for example, the PAM120 residue mass table can be used, with gap length penalty=12 and gap penalty=4.

[0051] The identity of two amino acid sequences can be determined using the GAP program in the GCG software package, using a Blossom 62 matrix or a PAM250 matrix with a gap weight of 12, 10, 8, 6, or 4, and a gap length weight 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 having the amino acid sequence of SEQ ID NO: 1, i.e., a transglutaminase derived from Streptomyces mobaraensis, is mutated (by any of the amino acid substitutions (1) to (134) above) to obtain the modified enzyme of the present invention. The substantially identical transglutaminase can be obtained by further mutating a transglutaminase having the amino acid sequence of SEQ ID NO: 1 mutated (by any of the amino acid substitutions (1) to (134) above), by adding an equivalent mutation to a transglutaminase having an amino acid sequence highly identical to the amino acid sequence of SEQ ID NO: 1, such as a transglutaminase derived from the same species or genus as the Streptomyces mobaraensis strain that produces the transglutaminase having the amino acid sequence of SEQ ID NO: 1, or by further mutating a transglutaminase obtained by the above mutation. Here, the "equivalent mutation" refers to a substitution of an amino acid residue corresponding to the amino acid residue at the mutation site in the present invention (the mutation site in the amino acid substitution in any of the amino acid substitutions (1) to (134) above) in an amino acid sequence highly identical to the amino acid sequence of SEQ ID NO: 1.Examples of transglutaminase having an amino acid sequence highly identical to the amino acid sequence of SEQ ID NO:1 include transglutaminase derived from Streptomyces mobaraensis (previously classified as Streptoverticillium ladakanum) and having the amino acid sequence of SEQ ID NO:11 (93% identity of amino acid sequence), transglutaminase derived from Streptomyces albireticuli and having the amino acid sequence of SEQ ID NO:12 (82% identity of amino acid sequence), transglutaminase derived from Streptomyces luteireticuli and having the amino acid sequence of SEQ ID NO:13 (82% identity of amino acid sequence), transglutaminase derived from Streptomyces cinnamoneus and having the amino acid sequence of SEQ ID NO:14 (82% identity of amino acid sequence), transglutaminase derived from Streptomyces oxidase B ... cinnamoneus and having the amino acid sequence of SEQ ID NO: 14 (amino acid sequence identity 81%), transglutaminase derived from Streptomyces platensis and having the amino acid sequence of SEQ ID NO: 15 (amino acid sequence identity 80%), and transglutaminase derived from Streptomyces hygroscopicus and having the amino acid sequence of SEQ ID NO: 16 (amino acid sequence identity 81%). hygroscopicus) and having the amino acid sequence of SEQ ID NO: 16 (amino acid sequence identity: 80%).

[0053] The term "corresponding" when used herein with respect to amino acid residues means that the amino acid residues make an equivalent contribution to the exertion of the function of the proteins (enzymes) being compared. For example, when the amino acid sequence of the reference transglutaminase (the amino acid sequence of SEQ ID NO: 1) is aligned to allow optimal comparison while taking into account partial homology of the primary structure (amino acid sequence) (at this time, gaps may be introduced as necessary 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 or in addition to the comparison of the primary structures, the "corresponding amino acid" can also be identified by comparing the three-dimensional structures (three-dimensional structures). By using the three-dimensional structure information, a highly reliable comparison result can be obtained. In this case, a method of performing alignment while comparing the atomic coordinates of the three-dimensional structures of multiple enzymes can be adopted. The three-dimensional structure information of the target enzyme to be mutated can be obtained, for example, from the Protein Data Bank (http: / / www.pdbj.org / index#j.html).

[0054] An example of a method for determining the three-dimensional structure of a protein by X-ray crystallography is shown below. (1) Crystallize the protein. Crystallization is essential for determining the three-dimensional structure of the protein, but it is also useful industrially as a method for purifying the protein to a high degree of purity and for preserving the protein in a stable manner at a high density. In this case, it is recommended to crystallize the protein bound to a substrate or its analog compound as a ligand. (2) The prepared crystals are irradiated with X-rays to collect diffraction data. In many cases, protein crystals are damaged by X-ray irradiation, resulting in a deterioration of their diffraction ability. In such cases, a low-temperature measurement technique has recently become widespread, in which the crystals are rapidly cooled to around -173°C and diffraction data is collected in this state. Finally, highly brilliant synchrotron radiation is used to collect high-resolution data for structure determination. (3) In order to perform crystal structure analysis, phase information is required in addition to diffraction data. When the crystal structure of a protein related to the target protein is unknown, it is impossible to determine the structure by molecular replacement, and the phase problem must be solved by heavy atom isomorphous replacement. Heavy atom isomorphous replacement is a method for obtaining phase information by introducing metal atoms with large atomic numbers, such as mercury or platinum, into a crystal and utilizing the contribution of the metal atoms' large X-ray scattering ability to the X-ray diffraction data. The determined phase can be improved by smoothing the electron density in the solvent region in the crystal. Water molecules in the solvent region fluctuate greatly, so the electron density is hardly observed, By approximating the electron density in this region to 0, it is possible to approach the true electron density, and thus improve the phase. Furthermore, if the asymmetric unit contains multiple molecules, the phase can be improved even further by averaging the electron densities of these molecules. The protein model is fitted to the electron density map calculated using the improved phase in this way. This process is carried out on computer graphics using a program such as QUANTA from MSI (USA). After this, the structure is refined using a program such as X-PLOR from MSI, and the structure analysis is completed. If the crystal structure of a protein related to the target protein is known, the atomic coordinates of the known protein can be used to determine the structure by molecular replacement. Molecular replacement and structural refinement can be carried out using a program such as CNS#SOLVE ver.11.

[0055] 2. Nucleic acid encoding modified transglutaminase A second aspect of the present invention provides nucleic acids related to the modified enzymes of the present invention, i.e., genes encoding the modified enzymes, nucleic acids that can be used as probes for identifying the nucleic acids encoding the modified enzymes, and nucleic acids that can be used as primers for amplifying or mutating the nucleic acids encoding the modified enzymes.

[0056] A gene encoding a modified enzyme is typically used for preparing the modified enzyme. A genetic engineering preparation method using a gene encoding the modified enzyme makes it possible to obtain a modified enzyme in a more homogeneous state. This method is also suitable for preparing a large amount of modified enzyme. The use of the gene encoding the modified enzyme is not limited to the preparation of modified enzymes. For example, the nucleic acid can be used as an experimental tool for elucidating the mechanism of action of the modified enzyme, or as a tool for designing or producing further modified forms of the enzyme.

[0057] As used herein, the term "gene encoding a modified enzyme" refers to a nucleic acid that, when expressed, gives the modified enzyme, and includes not only a nucleic acid having a base sequence corresponding to the amino acid sequence of the modified enzyme, but also a nucleic acid in which a sequence that does not code for an amino acid sequence is added to such a nucleic acid. Codon degeneracy is also taken into consideration.

[0058] Examples of gene sequences (base sequences) encoding the modified enzymes are shown in SEQ ID NOs: 18 to 26. These sequences encode the mutants shown in the Examples below. SEQ ID NO: 18: R26N mutant SEQ ID NO: 19: R26Y mutant SEQ ID NO: 20: R26S mutant SEQ ID NO: 21: E28N mutant SEQ ID NO: 22: E28G mutant SEQ ID NO: 23: E28F mutant SEQ ID NO: 24: E28Y mutant SEQ ID NO: 25: E58R mutant SEQ ID NO: 26: E58L mutant

[0059] When expressing the gene of the present invention in a host, a gene construct in which a sequence encoding a propeptide (pro-sequence) is added to the 5'-end of the above sequence (for example, any one of SEQ ID NOs: 18 to 26) is introduced into the host in order to stabilize the structure of the modified enzyme, which is the expression product. When expressing as a secretory protein, a gene construct in which a sequence encoding a pre-sequence (signal sequence) is added to the 5'-end of the sequence encoding the pro-sequence is further prepared. When using this gene construct, the gene is expressed as a prepro-type transglutaminase in which the pre-sequence and the pro-sequence are linked, and then the mature transglutaminase is obtained through cleavage of the pre-sequence (conversion to pro-type transglutaminase) and cleavage of the pro-sequence. For the sequence encoding the pre-sequence and the sequence encoding the pro-sequence, it is preferable to use the original sequence, i.e., the sequence of the reference transglutaminase (transglutaminase before modification). A specific example of a presequence is shown in SEQ ID NO: 27 (presequence of transglutaminase derived from Streptomyces mobaraensis), a specific example of a prosequence is shown in SEQ ID NO: 28 (prosequence of transglutaminase derived from Streptomyces mobaraensis), a specific example of a sequence encoding a presequence is shown in SEQ ID NO: 29 (sequence encoding a presequence of transglutaminase derived from Streptomyces mobaraensis), and a specific example of a sequence encoding a prosequence is shown in SEQ ID NO: 30 (sequence encoding a prosequence of transglutaminase derived 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., based on the sequence information disclosed in this specification or the attached sequence listing.

[0061] In another aspect of the present invention, there is provided a nucleic acid (hereinafter also referred to as "homologous nucleic acid"; a base sequence defining a homologous nucleic acid is also referred to as "homologous base sequence") that has a part of a different base sequence, although the function of the protein it encodes is equivalent when compared with the base sequence of a gene encoding the modified enzyme of the present invention. An example of a homologous nucleic acid is a DNA that has a base sequence containing one or more base substitutions, deletions, insertions, additions, or inversions based on the base sequence of a nucleic acid encoding the modified enzyme of the present invention, and encodes a protein having an enzyme activity characteristic of the modified enzyme (i.e., transglutaminase activity). Base substitutions or deletions may occur at multiple sites. Here, "multiple" refers to, for example, 2 to 40 bases, preferably 2 to 20 bases, and more preferably 2 to 10 bases, although this varies depending on the position and type of amino acid residues in the three-dimensional structure of the protein encoded by the nucleic acid.

[0062] A homologous nucleic acid has, for example, 60% or more, preferably 70% or more, more preferably 80% or more, even more preferably 85% or more, even more preferably about 90% or more, even more preferably 95% or more, and most preferably 99% or more identity to a reference base sequence.

[0063] Such homologous nucleic acids can be obtained, for example, by restriction enzyme treatment, treatment with exonuclease or DNA ligase, or introduction of mutations by 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 a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of a gene encoding the modified enzyme of the present invention. Yet another aspect of the present invention provides a nucleic acid having a nucleotide sequence at least about 60%, 70%, 80%, 90%, 95%, 99%, or 99.9% identical to the nucleotide sequence of a gene encoding the modified enzyme of the present invention or a complementary nucleotide sequence thereto.

[0065] Yet another aspect of the present invention relates to a nucleic acid having a base sequence that hybridizes under stringent conditions to a base sequence of a gene encoding the modified enzyme of the present invention or a base sequence complementary to the homologous base sequence. Here, "stringent conditions" refers to conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed. Such stringent conditions are known to those skilled in the art and can be set with reference to, for example, Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York) or Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987). An example of stringent conditions includes incubation at about 42°C to about 50°C using a hybridization solution (50% formamide, 10xSSC (0.15M NaCl, 15mM sodium citrate, pH 7.0), 5xDenhardt's solution, 1% SDS, 10% dextran sulfate, 10µg / ml denatured salmon sperm DNA, 50mM phosphate buffer (pH 7.5)), followed by washing at about 65°C to about 70°C using 0.1xSSC, 0.1% SDS. More preferred stringent conditions include, for example, conditions using a hybridization solution containing 50% formamide, 5xSSC (0.15M NaCl, 15mM sodium citrate, pH 7.0), 1xDenhardt's solution, 1% SDS, 10% dextran sulfate, 10μg / ml denatured salmon sperm DNA, and 50mM phosphate buffer (pH 7.5).

[0066] Yet another aspect of the present invention provides a nucleic acid (nucleic acid fragment) having a part of the base sequence of a gene encoding the modified enzyme of the present invention, or a complementary base sequence thereto. Such a nucleic acid fragment can be used for detecting, identifying, and / or amplifying a nucleic acid having the base sequence of a gene encoding the modified enzyme of the present invention. For example, the nucleic acid fragment is designed to include at least a portion that hybridizes to a continuous nucleotide portion (e.g., about 10 to about 100 bases long, preferably about 20 to about 100 bases long, more preferably about 30 to about 100 bases long) in the base sequence of a gene encoding the modified enzyme of the present invention. When used as a probe, the nucleic acid fragment can be labeled. For example, a fluorescent substance, an enzyme, or a radioisotope can be used for labeling.

[0067] Yet another aspect of the present invention relates to a recombinant DNA containing the gene of the present invention (a gene encoding a modified enzyme). The recombinant DNA of the present invention is provided, for example, in the form of a vector. As used herein, the term "vector" refers to a nucleic acid molecule that can transport a nucleic acid inserted therein into a target such as a cell.

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

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

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

[0071] As host cells, microorganisms such as koji mold (e.g., Aspergillus oryzae), Bacillus bacteria (e.g., Bacillus subtilis, Bacillus licheniformis, Bacillus aminoliquefaciens), Brevibacillus bacteria (e.g., Brevibacillus choshinensis), Escherichia coli, and Saccharomyces cerevisiae can be used from the viewpoint of ease of handling, but any host cell that can replicate recombinant DNA and express the gene for the modified enzyme can be used. Preferably, Escherichia coli and Saccharomyces cerevisiae can be used. Streptomyces microorganisms (e.g., Streptomyces morabaensis) can also be used as a host. Examples of Escherichia coli include E. coli BL21(DE3) when a T7 promoter is used, and E. coli JM109 when not. Examples of budding yeast include budding yeast SHY2, budding yeast AH22, and budding yeast INVSc1 (Invitrogen).

[0072] Another aspect of the present invention relates to a microorganism carrying the recombinant DNA of the present invention (i.e., a transformant). The microorganism of the present invention can be obtained by transfection or transformation using the above-mentioned vector of the present invention. For example, the calcium chloride method (J. Mol. Biol., vol. 53, p. 159 (1970)), the Hanahan method (J. Mol. Biol., vol. 166, p. 557 (1983)), the SEM method (Gene, vol. 96, p. 23 (1990)), the method of Chung et al. (Proceedings of the National Academy of Sciences of the USA, vol. 86, p. 2172 (1989)), the 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. Acad. Sci. USA 84, 7413-7417 (1984)) etc. The microorganism of the present invention can be used to produce the modified enzyme of the present invention.

[0073] 3. Enzyme preparation containing modified transglutaminase The modified enzyme of the present invention is provided in the form of, for example, an enzyme preparation. The enzyme preparation may contain, in addition to the active ingredient (the modified enzyme of the present invention), excipients, buffers, suspending agents, stabilizers, preservatives, antiseptics, physiological saline, various proteins, various protein decomposition products, various extracts, various salts, various antioxidants, cysteine, glutathione, sodium glutamate, sodium inosinate, sodium guanylate, calcined shell calcium, silicon dioxide, and the like. Examples of excipients that can be used include starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, white sugar, glycerol, and the like. Examples of buffers that can be used include phosphates, citrates, acetates, and the like. Examples of stabilizers that can be used include propylene glycol, ascorbic acid, and the like. Examples of preservatives that can be used include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like. As the preservative, ethanol, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol, etc. can be used. Examples of proteins include soybean protein, wheat protein, corn protein, milk protein, animal-derived protein, etc. Examples of extracts include meat extract, plant extract, yeast extract, etc. Examples of salts include chloride salt, phosphate salt, polyphosphate salt, pyrophosphate salt, citrate salt, lactate salt, carbonate salt, etc. Examples of antioxidants include L-ascorbate salt, sodium bisulfite, etc. The form of the enzyme preparation of the present invention is not particularly limited, and may be, for example, powder, granule, liquid, capsule, etc.

[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 techniques. In this embodiment, a nucleic acid encoding the amino acid sequence of the modified enzyme of the present invention (for example, any one of SEQ ID NOs: 2 to 10) is prepared (step (I)). Here, the "nucleic acid encoding a specific amino acid sequence" is a nucleic acid that, when expressed, gives a polypeptide having the amino acid sequence, and may be a nucleic acid consisting of a base sequence corresponding to the amino acid sequence, or may have an extra sequence (which may be a sequence encoding an amino acid sequence or a sequence not encoding an amino acid sequence) added to such a nucleic acid. 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 techniques, molecular biological techniques, biochemical techniques, etc., 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 necessary mutations to a gene encoding a reference transglutaminase. Many methods for site-specific base sequence substitution are known in the art (see, for example, Molecular Cloning, Third Edition, Cold Spring Harbor Laboratory Press, New York), and an appropriate method can be selected and used. Site-specific amino acid saturation mutagenesis can be used as a site-specific mutagenesis method. Site-specific amino acid saturation mutagenesis is a "semi-rational, semi-random" method in which a position involved in a desired function is predicted based on the three-dimensional structure of a protein, and an amino acid saturation mutation is introduced (J. Mol. Biol. 331, 585-592 (2003)). For example, a kit such as Quick Change (Stratagene) or overlap extension PCR (Nucleic Acid Res. 16, 7351-7367 (1988)) can be used to introduce site-specific amino acid saturation mutations.The DNA polymerase used in PCR may be Taq polymerase, etc. However, it is preferable to use a highly accurate DNA polymerase such as KOD-PLUS- (Toyobo Co., Ltd.) or Pfu turbo (Stratagene Co., Ltd.).

[0075] Following step (I), the prepared nucleic acid is expressed (step (II)). For example, an expression vector into which the nucleic acid is inserted is first prepared, and then a host cell is transformed with the vector.

[0076] Next, the transformant is cultured under conditions in which the modified enzyme, which is the expression product, is produced. The transformant may be cultured according to a conventional method. The carbon source used in the medium may be any assimilable carbon compound, such as glucose, sucrose, lactose, maltose, molasses, or pyruvic acid. The nitrogen source may be any usable nitrogen compound, such as peptone, meat extract, yeast extract, casein hydrolysate, or soybean meal alkaline extract. In addition, salts such as phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids, and specific vitamins may be used as necessary.

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

[0078] Next, the expression product (modified enzyme) is recovered (step (III)). The culture solution containing the cells after the culture can be used as an enzyme solution as is or after concentration, removal of impurities, etc., but the expression product is generally first recovered from the culture solution or cells. If the expression product is a secretory protein, it can be recovered from the culture solution, and if not, it can be recovered from the cells. When recovering from the culture medium, for example, the culture supernatant is filtered and centrifuged to remove insoluble matter, and then vacuum concentration, membrane concentration, salting out using ammonium sulfate or sodium sulfate, fractional precipitation using methanol, ethanol, acetone, or the like, dialysis, heat treatment, isoelectric focusing, gel filtration, adsorption chromatography, ion exchange chromatography, affinity chromatography, and other types of chromatography (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)), and the like are combined to obtain a purified product of the modified enzyme. On the other hand, when recovering from within the cells, the cells are collected by filtering, centrifuging, or the like, and then the cells are destroyed by mechanical methods such as pressurization, ultrasonication, and physical disruption, or by enzymatic methods using lysozyme, and then the modified enzyme is separated and purified in the same manner as described above to obtain a purified product.

[0079] The purified enzyme obtained as described above can be provided in a powder form, for example, by freeze-drying, vacuum drying, or spray drying. In this case, the purified enzyme may be dissolved in advance in a phosphate buffer, triethanolamine buffer, Tris-HCl buffer, or Good's buffer. Preferably, a phosphate buffer or triethanolamine buffer can be used. Examples of Good's buffer include PIPES, MES, and MOPS.

[0080] Usually, gene expression and expression products (modified enzymes) are collected using an appropriate host-vector system as described above, but a cell-free synthesis system may also be used. Here, the term "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 template nucleic acids (DNA and mRNA) using ribosomes and transcription / translation factors derived from living cells (or obtained by genetic engineering techniques), rather than using living cells. In cell-free synthesis systems, cell extracts obtained by purifying cell lysates as necessary are generally used. Cell extracts generally contain 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 the cell extract. Of course, during protein synthesis, ribosomes, various factors, and / or various enzymes prepared separately may be supplemented as necessary.

[0081] The development of a transcription / translation system in which each molecule (factor) required for protein synthesis is reconstituted has also been reported (Shimizu, Y. et al.: Nature Biotech., 19, 751-755, 2001). In this synthesis system, the genes of 31 types of factors, consisting of three types of initiation factors, three types of elongation factors, four types of factors involved in termination, 20 types of 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. Such a reconstituted synthesis system may be used in the present invention.

[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. Total RNA, mRNA, in vitro transcription products, etc. are used as template RNA. On the other hand, in an in vitro transcription / translation system, DNA is used as a template. The template DNA should contain a ribosome binding region, and preferably contains an appropriate terminator sequence. In an in vitro transcription / translation system, conditions are set in which factors required for each reaction are added so that the transcription reaction and the translation reaction proceed consecutively. EXAMPLES

[0083] <Searching for mutations that are effective in improving properties> In order to obtain a highly useful modified transglutaminase, we attempted to improve the enzyme function (reduction of temperature stability, improvement of thermostability, improvement of antioxidant activity, improvement of reactivity, and deamidation) of transglutaminase derived from Streptomyces mobaraensis (wild-type enzyme, SEQ ID NO: 1) by protein engineering. First, we decided to introduce mutations by amino acid substitution, and selected candidates for mutation sites using the CASTing library (see, for example, Angew Chem Int Ed Engl. 2006 Feb 13;45(8):1236-41.) and alanine scanning (Ala scanning) (see, for example, J Mol Biol. 1995 Feb 17;246(2):317-30.). Next, mutations were introduced into each mutation site by 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 a primer set for each mutation site and the plasmid pET20b incorporating the transglutaminase gene (sequence number 17) as a template, PCR was performed (reacted at 98°C for 1 minute, followed by 15 cycles of reactions at 98°C for 10 seconds, 60°C for 15 seconds, and 68°C for 2 minutes, reacted at 68°C for 5 minutes, and then left at 4°C). (3) Add DpnI (1.5 μL / tube) to the PCR reaction solution (25 μL / tube) and treat (37°C, 3 hours or more). (4) Ligation is performed using T4 kinase (16°C, overnight). (5) Transform E. coli BL21(DE3) with the ligation reaction mixture (11 μL / tube) and culture in ampicillin-containing LB medium (37°C, overnight).

[0085] 1-2. Obtaining enzyme extract (1) The strain into which the mutation has been introduced (mutant strain) is inoculated into ampicillin-containing TB medium and cultured for 48 hours at 33° C. IPTG (final concentration 0.1 mM) is added 24 hours after the start of culture. (2) After centrifuging the culture solution (3,000 g × 10 minutes), the supernatant is removed and the bacterial cells are collected. (3) A lysing agent is added to lyse the bacteria. (4) After centrifuging the lysate (3,000 g × 10 minutes), collect the supernatant and use it as the enzyme extract.

[0086] 1-3. Maturation (removal of the propeptide sequence) (1) Mix equal amounts of the enzyme extract and 2 mg / mL protease (dispase) solution and allow to react (30°C, 2 hours or more). (2) After centrifuging the mixture (3,000 g × 10 minutes), the supernatant is collected and used as the mature enzyme.

[0087] 2. Characterization of Mutant Enzymes The properties of each mutant enzyme prepared were evaluated using the following activity measurement method. <Activity measurement method> The mature enzyme is diluted to an appropriate concentration with 200 mM Tris-HCl pH 6.0 (sample solution). 100 μL of substrate solution (R-1) is added to 10 μL of sample solution, mixed, and reacted at 37°C for 10 minutes. 100 μL of color-developing solution (R-2) is added to stop the reaction and form an Fe complex, and the absorbance at 525 nm is measured. As a control, the absorbance of a similar reaction is measured using an enzyme solution that has been heat-inactivated in advance, and the absorbance difference with the sample solution is calculated. Separately, a calibration curve is created using L-glutamic acid-γ-monohydroxamic acid instead of the enzyme solution, and the amount of hydroxamic acid produced is calculated from the absorbance difference. The enzyme activity that produces 1 μmole of hydroxamic acid per minute is defined as 1 unit (1U). (Substrate solution (R-1)) Dissolve 2.42 g of 2-amino-2-hydroxymethyl-1,3-propanediol, 0.70 g of hydroxyammonium 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. Temperature stability / heat resistance evaluation The mature enzyme is diluted 2-fold with 200 mM Tris-HCl pH 6.0 and treated for 30 minutes at a given temperature (20°C, 30°C, 40°C, 50°C, 60°C). After treatment, the activity is measured and compared with the activity before treatment to calculate the remaining activity.

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

[0090] The evaluation results of temperature stability are shown in Figure 1. The following (1) to (33), which have a temperature stability (%) of 90% or less relative to the wild type, were determined to be effective amino acid substitutions. (1) The mutation site is V6, and the amino acid after substitution 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 amino acid after substitution is K, Q, M, H, Y, D, G, N, P, or S. (3) The mutation site is E28, and the amino acid after substitution is V, Q, W, R, K, M, N, F, G, L, P, or Y. (4) The mutation site is V30, and the amino acid after substitution is P, C, A, E, F, G, H, K, N, Q, R, W, Y, or L. (5) The mutation site is Y34, and the amino acid after substitution is A. (6) The mutation site is Y42, and the amino acid after substitution 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 amino acid after substitution is R, N, Y, A, S, I, V, D, G, or P. (9) The mutation site is L60, and the amino acid after substitution is I, M, V, A, C, E, F, Q, S, T, W, or Y. (10) The mutation site is Y62, and the amino acid after substitution is C, R, G, K, or S. (11) The mutation site is V65, and the amino acid after substitution is N, L, M, F, W, or Y. (12) The mutation site is V67, and the amino acid after substitution 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 amino acid after substitution is W, D, G, or K. (16) The mutation site is Y75, and the amino acid after substitution 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 amino acid after substitution is M (19) The mutation site is F90, and the amino acid after substitution is C, M, H, L, or V. (20) The mutation site is F108, and the amino acid after substitution is R, L, T, A, I, K, N, or V. (21) The mutation site is F117, and the amino acid after substitution is M or L. (22) The mutation site is S199, and the amino acid after substitution is G, M, N, K, or V. (23) The mutation site is F202, and the amino acid after substitution 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 amino acid after substitution 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 amino acid after substitution 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 amino acid after substitution is C, M, or I.

[0091] As more preferable amino acid substitutions, the following amino acid substitutions, which have a ratio (%) relative to 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 <h2 style=";text-align:left;direction:ltr">V30P、V30C、V30A、V30E、V30F、V30G、V30H、V30K、V30N、V30Q、V30R、V30W、V30Y、V30L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Y34A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Y42A、Y42C、Y42D、Y42E、Y42G、Y42I、Y42L、Y42M、Y42Q、Y42S、Y42T、Y42V、Y42W<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> E58G, E58H<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> W59S、W59I、W59V、W59D、W59G、W59P<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> L60M、L60V、L60A、L60C、L60E、L60F、L60Q、L60S、L60T、L60W、L60Y<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Y62C、Y62R、Y62G、Y62K、Y62S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> V65N、V65L、V65M、V65F、V65W、V65Y<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> V67L、V67N、V67A、V67C、V67M、V67Q、V67S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> T68M、T68F、T68N、T68Q、T68Y<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> W69M, W69I, W69C, W69E, W69F, W69G, W69K, W69L, W69N, W69Q, W69R, W69S, W69T, W69V<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Y75T、Y75G<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> F85M<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> F90V<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> F108T、F108A、F108I、F108K、F108N、F108V<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> F117L<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> S199K, S199V<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> F202W<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> T273L, T273R, T273G, T273A, T273E, T273F, T273Y, T273D, T273K, T273W, T273H<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> N276C, N276E, N276K, N276L, N276T, N276V, N276S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Y278L, Y278H, Y278I, Y278K, Y278R, Y278W, Y278C, Y278G, Y278N, Y278Q, Y278S, Y278T, Y278V<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Y291W, Y291L, Y291A, Y291C, Y291K, Y291N, Y291Q, Y291R, Y291S, Y291V S299E Y310I

[0092] As particularly preferred amino acid substitutions, the following amino acid substitutions, which have a ratio (%) relative to the wild type of 10% or less, were identified. 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 evaluation of heat resistance are shown in Figure 2. The following (34) to (45), which have a heat resistance of 110% or more relative to the wild type (%), were determined to be effective amino acid substitutions. (34) The mutation site is D3, and the amino acid after substitution is Q, P, E, S, or Y. (35) The mutation site is R26, and the amino acid after substitution is V. (36) The mutation site is Y34 and the amino acid after substitution is W. (37) The mutation site is V67 and the substituted amino acid is H. (38) The mutation site is T68, and the amino acid after substitution 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 amino acid after substitution 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 amino acid after substitution 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, which have a reactivity (%) relative to the wild type of 120% or more, 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, the following amino acid substitutions, which have a reciprocal ratio (%) of 130% or more relative to the wild type, were identified. D3E, D3S, D3Y R26V Y34W V67H S199Q T273Q S284H, S284K, S284P, S284R

[0096] 2-2. Evaluation of antioxidant properties Hydrogen peroxide (30-35%) (Wako, special grade reagent) is diluted with 200 mM Tris-HCl pH 6.0 to prepare a hydrogen peroxide solution (0.03%) as an oxidizing agent. The mature enzyme is diluted two-fold with the hydrogen peroxide solution and reacted at 30°C for 1 hour. The activity is measured at the reaction level and compared with the activity before treatment to calculate the remaining activity.

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

[0098] The evaluation results of antioxidant activity are shown in Figure 3. The following (46) to (59), which have a value of 110% or more relative to the wild type (%), were determined to be effective amino acid substitutions. (46) The mutation site is D3, and the amino acid after substitution 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 amino acid after substitution 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 amino acid after substitution 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 amino acid after substitution 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 amino acid after substitution 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, which have a reactivity (%) relative to the wild type of 120% or more, 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, the following amino acid substitutions, which have a reciprocal ratio (%) of 130% or more relative to the wild type, were identified. 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 enzyme was diluted to an appropriate concentration with 200 mM Tris-HCl pH 6.0 (sample solution) and activity was measured. The relative activity to the wild type was calculated using the following formula to identify amino acid substitutions that were effective in improving reactivity. Percentage of wild type = (activity of mutant enzyme) / (activity of wild type enzyme) × 100

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

[0103] The evaluation results of the reactivity are shown in Figure 4. The following (60) to (63), which have a reactivity ratio (%) of 110% or more relative to the wild type, were determined to be effective amino acid substitutions. (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 following the substitution at mutation site V6 is E, and the amino acid following the substitution at mutation site Y75 is F. (63) The mutation sites are D3 and T77. The amino acid substituted at D3 is P, and the amino acid substituted at T77 is Q.

[0104] As more preferable amino acid substitutions, the following amino acid substitutions, which have a reactivity (%) relative to the wild type of 120% or more, were identified. S284V F251Y V6E and Y75F double mutation D3P and T77Q double mutation

[0105] As particularly preferred amino acid substitutions, the following amino acid substitutions, which have a reciprocal ratio (%) of 130% or more relative to the wild type, were identified. V6E and Y75F double mutation D3P and T77Q double mutation

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

[0107] Among the above amino acid substitutions, the following amino acid substitutions are accompanied by two seemingly contradictory changes in properties: decreased temperature stability and increased antioxidant activity. The identification of such amino acid substitutions was an unexpected result. Modified enzymes with such characteristic amino acid substitutions are expected to be used in food applications where enzymes must be inactivated at low temperatures. 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 The deamidation activity and transglutaminase activity of the mature enzyme were each calculated as a relative value to the wild-type enzyme using the following formula, and the degree of change in substrate specificity (i.e., deamidation) was evaluated based on the value of deamidation activity (ratio to wild-type) / transglutaminase activity (ratio to wild-type), and amino acid substitutions effective for deamidation were identified. Note that the deamidation activity was measured by the following method, and the transglutaminase activity was measured by the above-mentioned activity measurement method using the substrate solution (R-1). Deamidation activity (ratio to wild type) (%) = (deamidation activity of mutant enzyme) / ( Deamidation activity) x 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> The mature enzyme is diluted to an appropriate concentration with 200 mM potassium phosphate buffer (pH 7.0) (sample solution). 80 μL of substrate solution (1% Z-Gln-Gly, 200 mM potassium phosphate buffer, pH 7.0) is added to 20 μL of sample solution, mixed, and reacted at 37°C for 24 hours. Using an Ammonia Test Wako (WAKO), 40 μL of color reagent A, 20 μL of color reagent B, and 40 μL of color reagent C are added to 20 μL of reaction sample, and reacted at 37°C for 10 minutes. 50 μL of color sample is mixed with 50 μL of deproteinized solution, centrifuged, and the supernatant is collected and the absorbance at 630 nm is measured.

[0110] The evaluation results of deamidation are shown in Figure 6. If the value of deamidation activity / transglutaminase activity exceeds 1, deamidation has occurred. The following (92) to (134) were determined to be effective amino acid substitutions. (92) The mutation site is R5, and the amino acid after substitution is Q, P, C, M, I, V, Y, F, S, N, T, or G. (93) The mutation site is Y34, and the amino acid after substitution is H or M. (94) The mutation site is W59, and the amino acid after substitution 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 amino acid after substitution 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 amino acid after substitution is C, T, or K. (99) The mutation site is G66, and the amino acid after substitution 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 amino acid after substitution is Y, D, or P. (102) The mutation site is Q74, and the amino acid after substitution 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 amino acid after substitution 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 amino acid after substitution is Q or P. (108) The mutation site is K200, and the amino acid after substitution 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 amino acid after substitution 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 amino acid after substitution is L, V, or I. (118) The mutation site is G257, and the amino acid after substitution is A or C. (119) The mutation site is W258, and the amino acid after substitution is Y, F, or N. (120) The mutation site is T273 and the amino acid after substitution 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 amino acid after substitution is P or D. (125) The mutation site is H279, and the amino acid after substitution is N. (126) The mutation site is S284, and the substituted amino acid is C. (127) The mutation site is M288, and the amino acid after substitution 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 amino acid after substitution 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 preferred amino acid substitutions, the following amino acid substitutions, which have a deamidation activity / transglutaminase activity value of 1.2 or more, were identified. <h2 style=";text-align:left;direction:ltr">R5Q, R5P, R5C, R5M, R5I, R5V, R5Y, R5F, R5S, R5N, R5T, R5G<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Y34H、Y34M<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> W59K, W59E<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> S61T、S61V、S61Y、S61D、S61I、S61L、S61N、S61E、S61M、S61F、S61W、S61Q、S61P、S61H、S61K<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Y62D、Y62P<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> G63D、G63Q、G63Y、G63R、G63K、G63H、G63P、G63M、G63N、G63C、G63F、G63L、G63W、G63T、G63V、G63S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> V65C, V65T, V65K<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> G66W、G66K<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> T68P, T68H<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> W69Y, W69D, W69P<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Q74R, Q74P<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> F85Y、F85W、F85L、F85I、F85Q、F85C、F85T、F85A、F85V、F85S、F85R、F85H、F85N<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> F108Q、F108C、F108E、F108S、F108N<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> F117C、F117I、F117W、F117V、F117A、F117G、F117T、F117N<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Y198F、Y198H、Y198L、Y198S、Y198M、Y198I<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> S199Q, S199P<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> K200Y、K200M、K200I、K200R、K200L、K200V<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> H201K、H201R、H201M、H201L、H201Q<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> F202Y、F202M、F202H、F202V、F202C<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> W203Y<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> F223Y, F223M, F223L, F223W, F223V, F223H, F223G, F223A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> R238A、R238D、R238V、R238G、R238H、R238S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">F251H、F251N、F251C、F251M、F251R、F251S、F251A、F251T、F251L、F251P、F251G、F251V、F251Q<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> V252M, V252L, V252T, V252C<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> N253T, N253V, N253K, N253C, N253R<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Y256L, Y256V, Y256I<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> G257A, G257C<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> W258Y, W258F, W258N<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> T273N<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> G275A, G275C, G275S, G275V, G275K, G275I, G275F, G275H, G275R, G275L, G275P, G275Q, G275Y, G275N, G275M, G275T<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> N276G, N276M, N276Q, N276H<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> H277N, H277S, H277Q, H277C, H277E, H277K, H277D, H277I, H277R, H277M, H277P, H277L, H277W, H277V, H277Y, H277G, H277F, H277T<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Y278P、Y278D<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> H279N<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> S284C<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> M288V, M288Q, M288T<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> V290T、V290C、V290L、V290M、V290A、V290S、V290N<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Y291H, Y291T, Y291E, Y291G<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> W298F, W298Y, W298I, W298L, W298M<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> S299N, S299P<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Y302L, Y302H, Y302M, Y302V, Y302C, Y302T, Y302P, Y302S, Y302W<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> S303I, S303Q, S303D, S303H, S303E<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> F305W, F305H, F305Y<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0112] <h2 style=";text-align:left;direction:ltr"> As more preferred amino acid substitutions, the following amino acid substitutions, which have a deamidation activity / transglutaminase activity value of 2 or more, were identified. 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, the following amino acid substitutions, which have a deamidation activity / transglutaminase activity value of 10 or more, were identified. 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 has improved practically important properties and is of high industrial value, and is therefore expected to be used in new applications as well as in existing applications.

[0115] The present invention is not limited to the above-mentioned embodiment and examples. Various modifications within the scope of the claims and within the scope that can be easily conceived by a person skilled in the art are also included in the present invention. The contents of papers, published patent publications, patent publications, etc. specified in this specification are all incorporated by reference.

[0116] SEQ ID NO: 2: Description of the artificial sequence: R26N mutant SEQ ID NO: 3: Description of the artificial sequence: R26Y mutant SEQ ID NO: 4: Description of the artificial sequence: R26S mutant SEQ ID NO: 5: Description of the artificial sequence: E28N mutant SEQ ID NO: 6: Description of the artificial sequence: E28G mutant SEQ ID NO: 7: Description of the artificial sequence: E28F mutant SEQ ID NO: 8: Description of the artificial sequence: E28Y mutant SEQ ID NO: 9: Description of the artificial sequence: E58R mutant SEQ ID NO: 10: Description of the artificial sequence: E58L mutant SEQ ID NO: 18: Description of the artificial sequence: R26N mutant SEQ ID NO: 19: Description of the artificial sequence: R26Y mutant SEQ ID NO: 20: Description of the artificial sequence: R26S mutant SEQ ID NO: 21: Description of the artificial sequence: E28N mutant SEQ ID NO: 22: Description of the artificial sequence: E28G mutant SEQ ID NO: 23: Description of the artificial sequence: E28F mutant SEQ ID NO: 24: Description of the artificial sequence: E28Y mutant SEQ ID NO: 25: Description of the artificial sequence: E58R mutant SEQ ID NO: 26: Description of the artificial sequence: E58L mutant

Claims

1. A modified transglutaminase having an amino acid sequence of SEQ ID NO: 1 containing any one of the following amino acid substitutions (1) to (12), or an amino acid sequence showing 90% or more identity with said amino acid sequence (with the proviso that the amino acid sequence differs in a portion other than the position of the amino acid substitution), and having a change in properties corresponding to the amino acid substitution: (1) The mutation site is V6, the amino acid after substitution is Q, I, M, S, C, K, L, H, F, G, N, R, W or Y, and the change in characteristics due to the amino acid substitution is a decrease in temperature stability; (2) 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 characteristics due to the amino acid substitution is a decrease in temperature stability; (3) The mutation site is V67, the amino acid after substitution is L, N, C, M, or Q, and the change in characteristics due to the amino acid substitution is a decrease in temperature stability; (4) The mutation site is F85, the substituted amino acid is M, and the change in characteristics due to the amino acid substitution is a decrease in temperature stability; (5) the mutation site is F90, the substituted amino acid is C, M, H, L or V, and the change in characteristics due to the amino acid substitution is a decrease in temperature stability; (6) the mutation site is F108, the substituted amino acid is R, L, T, A, I, K, N or V, and the change in characteristics due to the amino acid substitution is a decrease in temperature stability; (7) The mutation site is F117, the substituted amino acid is M or L, and the change in characteristics due to the amino acid substitution is decreased temperature stability; (8) The mutation site is W203, the amino acid after substitution is F, and the characteristic change due to the amino acid substitution is a decrease in temperature stability; (9) The mutation site is T273, the substituted amino acid is I, V, M, C, S, L, R, G, A, F, Y, D, K, W or H, and the change in characteristics due to the amino acid substitution is decreased temperature stability; (10) the mutation site is N276, the substituted amino acid is C, E, K, L, S, T or V, and the characteristic change due to the amino acid substitution is a decrease in temperature stability; (11) The mutation site is Y310, the substituted amino acid is C, M or I, and the change in characteristics due to the amino acid substitution is decreased temperature stability; (12) The mutation site is L60, the amino acid after substitution is I, and the characteristic changes due to the amino acid substitution are decreased temperature stability and increased reactivity;

2. A gene encoding the modified transglutaminase according to claim 1.

3. A recombinant DNA comprising the gene according to claim 2.

4. A microorganism carrying the recombinant DNA of claim 3.

5. An enzyme preparation comprising the modified transglutaminase according to claim 1.

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

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