Mutant transglutaminase

Abstract

Provided is a mutant transglutaminase (mutant TG) having high heat tolerance and / or high pH stability. This mutant TG has a mutation at an amino acid residue such as D1, Y24, R48, S101, G102, N139, D142, L147, K152, G157, R167, N176, K181, E182, H188, D189, R208, T245, S246, G250, G275, S284, H289, G301 or K327 and / or a mutation that introduces a disulfide bond.

Description

Technical Field

[0001] The present invention relates to a mutant transglutaminase (mutant TG) having high heat resistance and / or pH stability and its use.

Background Art

[0002] Transglutaminase (TG) is known as an enzyme that catalyzes protein cross-linking and is used to improve the physical properties of foods.

[0003] Also, various mutant TGs with modified functions are known, such as mutant TGs with improved heat resistance and mutant TGs with improved pH stability. As mutant TGs with improved heat resistance, for example, the mutant TGs described in Patent Documents 1 to 4 are known. As mutant TGs with improved pH stability, for example, the mutant TGs described in Patent Document 4 are known.

[0004] Also, Patent Document 1 discloses mutations such as G275A as mutations that deamidate TG.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Summary of the Invention

Problems to be Solved by the Invention

[0006] An object of the present invention is to provide a mutant TG having high heat resistance and / or pH stability. [Means for solving the problem]

[0007] The inventors of this invention have discovered a mutation that improves the heat resistance and / or pH stability of TG, thereby completing the present invention.

[0008] In other words, the present invention can be illustrated as follows. [1] A method for manufacturing food, The process includes treating food ingredients with a variant transglutaminase under heating, acidic, or alkaline conditions. The mutant transglutaminase is a protein that has a specific mutation in the amino acid sequence of wild-type transglutaminase and possesses transglutaminase activity. A method wherein the specific mutation is a mutation that improves heat resistance and / or pH stability. [2] The method wherein the specific mutation includes the following mutations (A) and / or (B): Mutation (A): A mutation in one or more amino acid residues selected from the following: G275, D1, Y24, R48, S101, G102, N139, D142, L147, K152, G157, R167, N176 , K181, E182, H188, D189, R208, T245, S246, G250, S284, H289, G301, K327; Mutation (B): A mutation that introduces a disulfide bond. [3] The method wherein the mutation (A) comprises a mutation in one or more amino acid residues selected from the following: G275, S101, G157, R208, G250. [4] The method wherein the mutation (A) includes one or more mutations selected from the following: G275A, D1F, Y24(G, N), R48(I, K), S101(G, A, V, I, P, F, N, Q, Y, K, R, E), G102N, N139S, D142A, L147W, K152T, G157(A, V, I, S, N, K, R, H, D, E), R167G, N176D, K181R, E182D, H188Y, D189I, R208(L, A, E), T245A, S246(K, N, R), G250(A, V, L, M, P, F, W, S, T, N, Q, Y, K, R, H, D), S284P, H289I, G301W, K327F. [5] The method wherein the mutation (A) includes one or more mutations selected from the following: G275A, S101P, G157(A, R, S), R208E, and G250(N, R, S). [6] The method wherein the mutation (A) includes any of the following mutations: S101P / G157(A, R, S) / R208E / G250(N, R, S) / G275A, S101P / G157(A, R, S) / G250(N, R, S), S101P / G157(A, R, S) / R208E / G250(N, R, S). [7] The method wherein the mutation (A) includes any of the following mutations: S101P / G157R / R208E / G250S / G275A, S101P / G157S / R208E / G250R / G275A, S101P / G157S / R208E / G250S, S101P / G157A / R208E / G250S, S101P / G157R / R208E / G250S, S101P / G157R / R208E / G250N. [8] The method wherein the mutation (A) includes any of the following mutations: S101P / G157R / R208E / G250S / G275A, S101P / G157S / R208E / G250R / G275A. [9] The method wherein the mutation (B) includes one or more mutations selected from the following: D3C / G283C, A81C / V311C, E93C / V112C, A106C / D213C, E107C / Y217C, A160C / G228C, S2C / N282C, S2C / G283C, T7C / E58C, P17C / W330C, D46C / S318C, S84C / K121C, R79C / P169C, A113C / P220C, E119C / S299C, and R89C / S116C.

[10] The method wherein the mutation (B) is a mutation that introduces two or more disulfide bonds.

[11] The method wherein the mutation (B) is a mutation that introduces three or more disulfide bonds.

[12] The method wherein the mutation (B) includes D3C / G283C.

[13] The method wherein the mutation (B) includes a combination of D3C / G283C and one or more mutations selected from E93C / V112C, A81C / V311C, A106C / D213C, E107C / Y217C, A160C / G228C, S84C / K121C, R79C / P169C, A113C / P220C, E119C / S299C, and R89C / S116C.

[14] The method wherein the mutation (B) includes any of the following mutations: D3C / G283C / E93C / V112C, D3C / G283C / A81C / V311C, D3C / G283C / A106C / D213C, D3C / G283C / E107C / Y217C, D3C / G283C / A160C / G228C, D3C / G283C / S84C / K121C, D3C / G283C / R79C / P169C, D3C / G283C / A113C / P220C, D3C / G283C / E119C / S299C, D3C / G283C / R89C / S116C, D3C / G283C / E93C / V112C / E107C / Y217C, D3C / G283C / E93C / V112C / A113C / P220C, D3C / G283C / E93C / V112C / E119C / S299C, D3C / G283C / E107C / Y217C / S84C / K121C, D3C / G283C / S84C / K121C / A113C / P220C.

[15] The method, wherein the specific mutation comprises the mutations (A) and (B).

[16] The method, wherein the residual activity of the mutant transglutaminase after heat treatment at 65°C and pH 6.0 for 10 minutes is 15% or more.

[17] The method, wherein the residual activity of the mutant transglutaminase after heat treatment at 75°C and pH 6.0 for 10 minutes is 10% or more.

[18] The method, wherein the residual activity of the mutant transglutaminase after treatment at pH 4.0 and 37°C for 4 hours is 60% or more as a relative value with respect to 100% of the residual activity of the mutant TG after treatment at pH 6.0 and 37°C for 4 hours.

[19] The method, wherein the wild-type transglutaminase is a protein comprising the amino acid sequence of the mature transglutaminase of a bacterium belonging to the genus Streptomyces.

[20] The method, wherein the bacterium belonging to the genus Streptomyces is Streptomyces mobaraensis.

[21] The method wherein the wild-type transglutaminase is one of the following proteins: (a) Proteins containing the amino acid sequence shown in Sequence ID No. 2; (b) Proteins containing an amino acid sequence in which 1 to 10 amino acid residues are substituted, deleted, inserted, and / or added in the amino acid sequence shown in Sequence ID No. 2; (c) A protein containing an amino acid sequence that has 90% or more identity with the amino acid sequence shown in Sequence ID No. 2. [twenty two] The method described above, which satisfies any of the following (1) to (3): (1) The process is carried out under heating conditions, and the food is a dairy product, an egg product, a plant product, a meat product, a seafood product, or a gelatin product; (2) The process is carried out under acidic conditions, and the food is a grain, potato, bean, nut, meat, seafood, dairy product, oil and fat, seasoning, beverage, fruit juice, or processed product thereof; (3) The process is carried out under alkaline conditions, and the food is a plant-based beverage, a seaweed extract, or a processed product thereof. [twenty three] The method described above, which satisfies any of the following (1) to (3): (1) The process is carried out under heating conditions, and the food is processed cheese, pre-incubated yogurt, omelet, steamed egg custard, egg tofu, tofu, yuba (tofu skin), noodles, bread, ham, sausage, hamburger, ice cream, wheat dough, jelly, gummy candy, margarine, or processed food products; (2) The process is carried out under acidic conditions, and the food is white rice, brown rice, vinegared rice, oatmeal, rice flour, wheat flour, buckwheat flour, rice bran, mochi, kneaded rice, bread, noodles, gluten, buckwheat, potatoes, soy milk, chocolate, fried tofu, pea protein, meat, gelatin, shrimp, crab, scallop, herring roe, yogurt, butter, cheese, ice cream, mayonnaise, ketchup, mustard, soy sauce, miso, coffee-based beverages, energy drinks, fruit juices, cocoa, beer, sake lees, fruit gummies, or processed products thereof; (3) The above process is carried out under alkaline conditions, and the food is soy milk, kelp broth, or a processed product thereof. [twenty four] The method wherein the heating temperature in the step is 60°C or higher. [twenty five] A composition for the manufacture of food products, It contains mutant transglutaminase, The aforementioned food is a food that is heated during manufacturing or a food with a low or high pH. The mutant transglutaminase is a protein that has a specific mutation in the amino acid sequence of wild-type transglutaminase and possesses transglutaminase activity. A composition in which the specific mutation is a mutation that improves heat resistance and / or pH stability.

[26] The composition wherein the specific mutation comprises the following mutations (A) and / or (B): Mutation (A): A mutation in one or more amino acid residues selected from the following: G275, D1, Y24, R48, S101, G102, N139, D142, L147, K152, G157, R167, N176 , K181, E182, H188, D189, R208, T245, S246, G250, S284, H289, G301, K327; Mutation (B): A mutation that introduces a disulfide bond.

[27] The composition wherein the mutation (A) comprises a mutation in one or more amino acid residues selected from the following: G275, S101, G157, R208, G250.

[28] The composition wherein the mutation (A) comprises one or more mutations selected from the following: G275A, D1F, Y24(G, N), R48(I, K), S101(G, A, V, I, P, F, N, Q, Y, K, R, E), G102N, N139S, D142A, L147W, K152T, G157(A, V, I, S, N, K, R, H, D, E), R167G, N176D, K181R, E182D, H188Y, D189I, R208(L, A, E), T245A, S246(K, N, R), G250(A, V, L, M, P, F, W, S, T, N, Q, Y, K, R, H, D), S284P, H289I, G301W, K327F.

[29] The composition wherein the mutation (A) comprises one or more mutations selected from the following: G275A, S101P, G157(A, R, S), R208E, and G250(N, R, S).

[30] The composition wherein the mutation (A) includes any of the following mutations: S101P / G157(A, R, S) / R208E / G250(N, R, S) / G275A, S101P / G157(A, R, S) / G250(N, R, S), S101P / G157(A, R, S) / R208E / G250(N, R, S).

[31] The composition wherein the mutation (A) includes any of the following mutations: S101P / G157R / R208E / G250S / G275A, S101P / G157S / R208E / G250R / G275A, S101P / G157S / R208E / G250S, S101P / G157A / R208E / G250S, S101P / G157R / R208E / G250S, S101P / G157R / R208E / G250N.

[32] The composition wherein the mutation (A) includes any of the following mutations: S101P / G157R / R208E / G250S / G275A, S101P / G157S / R208E / G250R / G275A.

[33] The composition wherein the mutation (B) comprises one or more mutations selected from the following: D3C / G283C, A81C / V311C, E93C / V112C, A106C / D213C, E107C / Y217C, A160C / G228C, S2C / N282C, S2C / G283C, T7C / E58C, P17C / W330C, D46C / S318C, S84C / K121C, R79C / P169C, A113C / P220C, E119C / S299C, and R89C / S116C.

[34] The composition wherein the mutation (B) is a mutation that introduces two or more disulfide bonds.

[35] The composition wherein the mutation (B) is a mutation that introduces three or more disulfide bonds.

[36] The composition wherein the mutation (B) comprises D3C / G283C.

[37] The composition wherein the mutation (B) comprises a combination of D3C / G283C and one or more mutations selected from E93C / V112C, A81C / V311C, A106C / D213C, E107C / Y217C, A160C / G228C, S84C / K121C, R79C / P169C, A113C / P220C, E119C / S299C, and R89C / S116C.

[38] The composition wherein the mutation (B) comprises any of the following mutations: D3C / G283C / E93C / V112C, D3C / G283C / A81C / V311C, D3C / G283C / A106C / D213C, D3C / G283C / E107C / Y217C, D3C / G283C / A160C / G228C, D3C / G283C / S84C / K121C, D3C / G283C / R79C / P169C, D3C / G283C / A113C / P220C, D3C / G283C / E119C / S2 99C, D3C / G283C / R89C / S116C, D3C / G283C / E93C / V112C / E107C / Y217C, D3C / G283C / E93C / V112C / A113C / P220C, D3C / G283C / E93C / V112C / E119C / S299C, D3C / G283C / E107C / Y217C / S84C / K121C, D3C / G283C / S84C / K121C / A113C / P220C.

[39] The composition wherein the specific mutation comprises the mutations (A) and (B).

[40] The composition wherein the residual activity of the mutant transglutaminase after heat treatment at 65°C and pH 6.0 for 10 minutes is 15% or more.

[41] The composition wherein the residual activity of the mutant transglutaminase after heat treatment at 75°C and pH 6.0 for 10 minutes is 10% or more.

[42] The composition wherein the residual activity of the mutant transglutaminase after treatment at pH 4.0 and 37°C for 4 hours is 60% or more, relative to the residual activity of the mutant TG after treatment at pH 6.0 and 37°C for 4 hours, which is set to 100%.

[43] The composition wherein the wild-type transglutaminase is a protein containing the amino acid sequence of mature transglutaminase of a bacterium of the genus Streptomyces.

[44] The composition wherein the Streptomyces bacterium is Streptomyces mobaraensis.

[45] The composition wherein the wild-type transglutaminase is one of the following proteins: (a) Proteins containing the amino acid sequence shown in Sequence ID No. 2; (b) Proteins containing an amino acid sequence in which 1 to 10 amino acid residues are substituted, deleted, inserted, and / or added in the amino acid sequence shown in Sequence ID No. 2; (c) A protein containing an amino acid sequence that has 90% or more identity with the amino acid sequence shown in Sequence ID No. 2.

[46] The composition that satisfies any of the following (1) to (3): (1) The process is carried out under heating conditions, and the food is a dairy product, an egg product, a plant product, a meat product, a seafood product, or a gelatin product; (2) The process is carried out under acidic conditions, and the food is a grain, potato, bean, nut, meat, seafood, dairy product, oil and fat, seasoning, beverage, fruit juice, or processed product thereof; (3) The process is carried out under alkaline conditions, and the food is a plant-based beverage, a seaweed extract, or a processed product thereof.

[47] The composition that satisfies any of the following (1) to (3): (1) The process is carried out under heating conditions, and the food is processed cheese, pre-incubated yogurt, omelet, steamed egg custard, egg tofu, tofu, yuba (tofu skin), noodles, bread, ham, sausage, hamburger, ice cream, wheat dough, jelly, gummy candy, margarine, or processed food products; (2) The process is carried out under acidic conditions, and the food is white rice, brown rice, vinegared rice, oatmeal, rice flour, wheat flour, buckwheat flour, rice bran, mochi, kneaded rice, bread, noodles, gluten, buckwheat, potatoes, soy milk, chocolate, fried tofu, pea protein, meat, gelatin, shrimp, crab, scallop, herring roe, yogurt, butter, cheese, ice cream, mayonnaise, ketchup, mustard, soy sauce, miso, coffee-based beverages, energy drinks, fruit juices, cocoa, beer, sake lees, fruit gummies, or processed products thereof; (3) The above process is carried out under alkaline conditions, and the food is soy milk, kelp broth, or a processed product thereof.

[48] The composition wherein the heating temperature in the manufacturing process is 60°C or higher.

[49] A mutant transglutaminase, It has a specific mutation in the amino acid sequence of wild-type transglutaminase and possesses transglutaminase activity. The aforementioned specific mutation is one that improves heat resistance and / or pH stability. The aforementioned specific mutations are the following mutations (A) and / or (B): Mutation (A): A mutation in one or more amino acid residues selected from the following: G275, D1, Y24, R48, S101, G102, N139, D142, L147, K152, G157, R167, N176 , K181, E182, H188, D189, R208, T245, S246, G250, S284, H289, G301, K327; Mutation (B): A mutation that introduces a disulfide bond. Includes, Mutant transglutaminases that satisfy the following (B1) and / or (A1): (A1) The specific mutation comprises the mutation (A), and the mutation (A) comprises a mutation in one or more amino acid residues selected from the following: D1, R48, G102, N139, D142, L147, K152, R167, N176, K181, E182, D189, S246, H289, G301, K327; However, if the mutation at R48 is selected alone, the mutation at R48 is R48I. However, if the mutation at S246 is selected alone, the mutation at S246 is S246N; (B1) The particular mutation comprises the mutation (B), wherein the mutation (B) comprises a combination of D3C / G283C and one or more mutations selected from E93C / V112C, A81C / V311C, A106C / D213C, E107C / Y217C, A160C / G228C, S84C / K121C, R79C / P169C, A113C / P220C, E119C / S299C, and R89C / S116C.

[50] The mutant transglutaminase wherein the mutation (A) comprises a mutation in one or more amino acid residues selected from the following: G275, S101, G157, R208, G250.

[51] The mutant transglutaminase wherein the mutation (A) comprises one or more mutations selected from the following: G275A, D1F, Y24(G, N), R48(I, K), S101(G, A, V, I, P, F, N, Q, Y, K, R, E), G102N, N139S, D142A, L147W, K152T, G157(A, V, I, S, N, K, R, H, D, E), R167G, N176D, K181R, E182D, H188Y, D189I, R208(L, A, E), T245A, S246(K, N, R), G250(A, V, L, M, P, F, W, S, T, N, Q, Y, K, R, H, D), S284P, H289I, G301W, K327F.

[52] The mutant transglutaminase wherein the mutation (A) comprises one or more mutations selected from the following: G275A, S101P, G157(A, R, S), R208E, and G250(N, R, S).

[53] The mutant transglutaminase in which the mutation (A) includes any of the following mutations: S101P / G157(A, R, S) / R208E / G250(N, R, S) / G275A, S101P / G157(A, R, S) / G250(N, R, S), S101P / G157(A, R, S) / R208E / G250(N, R, S).

[54] The mutant transglutaminase in which the mutation (A) includes any of the following mutations: S101P / G157R / R208E / G250S / G275A, S101P / G157S / R208E / G250R / G275A, S101P / G157S / R208E / G250S, S101P / G157A / R208E / G250S, S101P / G157R / R208E / G250S, S101P / G157R / R208E / G250N.

[55] The mutant transglutaminase in which the mutation (A) includes any of the following mutations: S101P / G157R / R208E / G250S / G275A, S101P / G157S / R208E / G250R / G275A.

[56] The mutant transglutaminase wherein the mutation (B) includes one or more mutations selected from the following: D3C / G283C, A81C / V311C, E93C / V112C, A106C / D213C, E107C / Y217C, A160C / G228C, S2C / N282C, S2C / G283C, T7C / E58C, P17C / W330C, D46C / S318C, S84C / K121C, R79C / P169C, A113C / P220C, E119C / S299C, and R89C / S116C.

[57] The mutant transglutaminase wherein the mutation (B) is three or more mutations that introduce disulfide bonds.

[58] The mutant transglutaminase in which the mutation (B) includes any of the following mutations: D3C / G283C / E93C / V112C, D3C / G283C / A81C / V311C, D3C / G283C / A106C / D213C, D3C / G283C / E107C / Y217C, D3C / G283C / A160C / G228C, D3C / G283C / S84C / K121C, D3C / G283C / R79C / P169C, D3C / G283C / A113C / P220C, D3C / G283C / E119C / S2 99C, D3C / G283C / R89C / S116C, D3C / G283C / E93C / V112C / E107C / Y217C, D3C / G283C / E93C / V112C / A113C / P220C, D3C / G283C / E93C / V112C / E119C / S299C, D3C / G283C / E107C / Y217C / S84C / K121C, D3C / G283C / S84C / K121C / A113C / P220C.

[59] The mutant transglutaminase wherein the specific mutation comprises the mutations (A) and (B).

[60] The mutant transglutaminase, wherein the residual activity after heat treatment at 65°C and pH 6.0 for 10 minutes is 15% or more.

[61] The mutant transglutaminase, wherein the residual activity after heat treatment at 75°C and pH 6.0 for 10 minutes is 10% or more.

[62] The mutant transglutaminase, wherein the residual activity after treatment of the mutant transglutaminase at pH 4.0 and 37°C for 4 hours is 60% or more, relative to the residual activity after treatment of the mutant TG at pH 6.0 and 37°C for 4 hours, which is set to 100%.

[63] The mutant transglutaminase is a protein in which the wild-type transglutaminase contains the amino acid sequence of mature transglutaminase from a bacterium of the genus Streptomyces.

[64] The mutant transglutaminase wherein the bacterium of the genus Streptomyces is Streptomyces mobaraensis.

[65] The mutant transglutaminase is one of the following proteins: (a) Proteins containing the amino acid sequence shown in Sequence ID No. 2; (b) Proteins containing an amino acid sequence in which 1 to 10 amino acid residues are substituted, deleted, inserted, and / or added in the amino acid sequence shown in Sequence ID No. 2; (c) A protein containing an amino acid sequence that has 90% or more identity with the amino acid sequence shown in Sequence ID No. 2.

[66] The gene encoding the aforementioned mutant transglutaminase.

[67] A vector containing the aforementioned gene.

[68] A microorganism possessing the aforementioned gene.

[69] The microorganism, which is a bacterium or yeast.

[70] The aforementioned microorganism is a Corynebacterium or a bacterium of the Enterobacteriaceae family.

[71] The microorganism is a bacterium of the genus Corynebacterium or Escherichia.

[72] The microorganism is Corynebacterium glutamicum or Escherichia coli. [Brief explanation of the drawing]

[0009] [Figure 1] This diagram shows the compressive stress of processed cheese produced without TG (triglyceride), with wild-type TG, or with each mutant TG (HT-03, HT-10, or HT-14) added. [Figure 2] A diagram (photograph) showing the shape retention during heating of processed cheese produced without TG additives, or with the addition of wild-type TG or mutant TG HT-10. [Figure 3] A diagram (photograph) showing the compressive stress and heat retention of processed cheese produced without TG, or with the addition of wild-type TG or mutant TG HT-16. [Figure 4] This figure (photograph) shows the results of shape retention and sensory evaluation of ice cream produced without TG or with the addition of mutant TG HT-16. [Figure 5] A diagram (photograph) showing the compressive stress and heat retention of gummy candies manufactured without TG or with the addition of mutant TG HT-16. [Figure 6] This figure shows the results of a compression test of jelly (Formulation 1) manufactured with wild-type TG or mutant TG HT-16 added. [Figure 7] This figure shows the results of a compression test of jelly (formulation 2) prepared with wild-type TG or mutant TG HT-16 added. [Figure 8] This figure shows the results of a compression test of jelly (formulation 3) manufactured with wild-type TG or mutant TG HT-16 added. [Modes for carrying out the invention]

[0010] <1> Mutant transglutaminase (mutant TG) The present invention provides transglutaminase (TG) having the “specific mutations” described herein.

[0011] "Transglutaminase (TG)" may refer to a protein that has the activity to catalyze an acyl group transfer reaction between the amide group of a glutamine residue in a protein and a primary amine (EC 2.3.2.13, etc.). This activity is also called "TG activity." The gene that codes for TG is also called the "TG gene." Examples of primary amines include lysine residues in proteins. In other words, "TG activity" may specifically refer to the activity that catalyzes a crosslinking reaction between glutamine residues and lysine residues in a protein. The crosslinking reaction may result in intramolecular crosslinking and / or intermolecular crosslinking. Typically, the crosslinking reaction may result in at least intermolecular crosslinking.

[0012] TG activity can be measured, for example, by hydroxamate (Lorand, L., et al.: Anal. Biochem., 44, 221-213 (1971)) or fluorescence (Takagi, J., et al.: Anal. Biochem., 153, 296-298 (1986)). Unless otherwise specified, "TG activity" may refer to TG activity measured by hydroxamate.

[0013] The procedure for measuring TG activity by the hydroxamate method is as follows: TG activity can be measured by incubating the enzyme with substrates (i.e., benzyloxycarbonyl-L-glutaminylglycine and hydroxylamine) at 37°C and pH 6.0, and measuring the enzyme and substrate-dependent production of hydroxamic acid. Hydroxamic acid production can be measured by forming an iron complex of hydroxamic acid in the presence of trichloroacetic acid, with the increase in absorbance at 525 nm serving as an indicator. Under the above conditions, the amount of enzyme that catalyzes the production of 1 μmol of hydroxamic acid per minute is defined as 1 U (unit).

[0014] The procedure for measuring TG activity by fluorescence is as follows: TG activity can be measured by incubating the enzyme with substrates (i.e., dimethylated casein and monodansylcadaverine (MDC)) at 37°C and pH 7.5, and measuring the enzyme and substrate-dependent incorporation of MDC into dimethylated casein. MDC incorporation can be measured by an increase in fluorescence (excitation wavelength 350 nm, emission wavelength 480 nm).

[0015] A triglyceridyl transgenic (TG) gene possessing a specific mutation is also called a "mutant TG gene." Furthermore, the gene encoding a mutant TG gene is also called a "mutant TG gene."

[0016] TG that does not possess a "specific mutation" is also called "wild-type TG." The gene that codes for wild-type TG is also called the "wild-type TG gene." Here, "wild-type" is a convenient designation to distinguish "wild-type" TG from "mutant" TG, and is not limited to naturally occurring TG as long as it does not possess a "specific mutation." "TG does not possess a 'specific mutation'" can be interpreted as TG not possessing the mutation selected as a "specific mutation." Wild-type TG may or may not possess mutations that were not selected as "specific mutations," as long as it does not possess the mutation selected as a "specific mutation."

[0017] When a certain wild-type TG and a certain mutant TG are identical except for the presence or absence of a "specific mutation," the wild-type TG is also referred to as the "wild-type TG corresponding to a certain mutant TG," and the mutant TG is also referred to as the "mutant TG corresponding to a certain wild-type TG."

[0018] The following explains the wild type TG.

[0019] Wild-type TG may or may not have TG activity, as long as its corresponding mutant TG has TG activity. Wild-type TG usually has TG activity.

[0020] Examples of wild-type triglycerides (TGs) include those of actinomycetes (Appl. Environ. Microbiol., 2003, 69(1), 358-366). Examples of actinomycetes include bacteria of the genus Streptomyces. Examples of Streptomyces bacteria include Streptomyces mobaraensis, Streptomyces cinnamoneus, and Streptomyces griseocarneus. Note that the genus Streptomyces also includes bacteria that were formerly classified under the genus Streptoverticillium. For example, "Streptomyces mobaraensis" includes bacteria that were formerly classified as Streptoverticillium mobaraense or Streptoverticillium ladakanum. Similarly, "Streptomyces cinnamoneus" includes bacteria that were formerly classified as Streptoverticillium cinnamoneum. Furthermore, "Streptomyces griseocarneus" includes bacteria that were formerly classified as Streptoverticillium griseocarneum. TG can be expressed in a form including the pro-structure, and the pro-structure can be removed to become a mature protein. The mature protein of TG is also called "mature TG." Specifically, the mature TG of the organisms exemplified above can be cited as an example of TG from those organisms. Sequence ID 1 shows the nucleotide sequence of the portion of the TG gene in Streptoverticillium mobaraense that codes for mature TG, and Sequence ID 2 shows the amino acid sequence of the mature TG encoded by the same gene.In other words, the wild-type TG gene may be, for example, a gene having the nucleotide sequence of the TG gene of the organism exemplified above (for example, the nucleotide sequence shown in Sequence ID No. 1). Also, the wild-type TG may be, for example, a protein having the amino acid sequence of the TG of the organism exemplified above (for example, the amino acid sequence shown in Sequence ID No. 2). Unless otherwise specified, the expression "a gene or protein has a nucleotide sequence or amino acid sequence" may mean that the gene or protein contains the said nucleotide sequence or amino acid sequence, and may also include cases where the gene or protein consists of the said nucleotide sequence or amino acid sequence.

[0021] A wild-type TG gene may be a variant of the wild-type TG gene exemplified above (for example, a gene with the nucleotide sequence shown in SEQ ID NO: 1), as long as the encoding TG does not have a "specific mutation." Similarly, a wild-type TG may be a variant of the wild-type TG gene exemplified above (for example, a protein with the amino acid sequence shown in SEQ ID NO: 2), as long as it does not have a "specific mutation." In other words, the term "wild-type TG gene" may encompass not only the wild-type TG gene exemplified above (for example, a gene with the nucleotide sequence shown in SEQ ID NO: 1) but also its variants. Similarly, the term "wild-type TG" may encompass not only the wild-type TG gene exemplified above (for example, a protein with the amino acid sequence shown in SEQ ID NO: 2) but also its variants. Note that the gene identified in the species of origin is not limited to the gene itself found in that species, but also includes genes having the nucleotide sequence of the gene found in that species and their variants. Furthermore, the protein identified in the species of origin is not limited to the protein itself found in that species, but also includes proteins having the amino acid sequence of the protein found in that species and their variants. These variants may or may not be found in the species in question. That is, for example, "TG of Streptoverticillium bacteria" is not limited to TG itself found in Streptoverticillium bacteria, but includes proteins having the amino acid sequence of TG found in Streptoverticillium bacteria and their variants. Examples of variants include homologs and artificially modified versions of the genes and proteins exemplified above.

[0022] Homologs of the wild-type TG gene or homologs of wild-type TG can be easily determined from public databases, for example, by BLAST or FASTA searches using the nucleotide sequence of the wild-type TG gene or the amino acid sequence of the wild-type TG gene exemplified above as query sequences. Alternatively, homologs of the wild-type TG gene can be obtained, for example, by PCR using the chromosomes of various organisms as templates and oligonucleotides prepared based on the nucleotide sequences of the wild-type TG gene exemplified above as primers.

[0023] A wild-type TG gene may encode a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added at one or several positions in the above amino acid sequence (for example, the amino acid sequence shown in SEQ ID NO: 2), unless the encoding TG has a "specific mutation." For example, the encoded protein may have an elongated or shortened N-terminus and / or C-terminus. The above "one or several" will vary depending on the position and type of amino acid residue in the three-dimensional structure of the protein, but specifically, it may be, for example, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 5, or 1 to 3.

[0024] The above substitutions, deletions, insertions, or additions of one or more amino acids are conservative mutations that maintain the original function of the protein. A typical example of a conservative mutation is a conservative substitution. A conservative substitution is a mutation in which the substitution site is between Phe, Trp, and Tyr if the substitution site is an aromatic amino acid; between Leu, Ile, and Val if the substitution site is a hydrophobic amino acid; between Gln and Asn if the substitution site is a polar amino acid; between Lys, Arg, and His if the substitution site is a basic amino acid; between Asp and Glu if the substitution site is an acidic amino acid; and between Ser and Thr if the amino acid has a hydroxyl group. Substitutions considered conservative include, specifically, substitutions from Ala to Ser or Thr, from Arg to Gln, His or Lys, from Asn to Glu, Gln, Lys, His or Asp, from Asp to Asn, Glu or Gln, from Cys to Ser or Ala, from Gln to Asn, Glu, Lys, His, Asp or Arg, from Glu to Gly, Asn, Gln, Lys or Asp, from Gly to Pro, from His to Asn, Lys, Gln, Arg or Tyr, and Il Examples of substitutions include e to Leu, Met, Val, or Phe; Leu to Ile, Met, Val, or Phe; Lys to Asn, Glu, Gln, His, or Arg; Met to Ile, Leu, Val, or Phe; Phe to Trp, Tyr, Met, Ile, or Leu; Ser to Thr or Ala; Thr to Ser or Ala; Trp to Phe or Tyr; Tyr to His, Phe, or Trp; and Val to Met, Ile, or Leu. Furthermore, such amino acid substitutions, deletions, insertions, or additions may also result from naturally occurring mutations (mutants or variants) based on individual differences or species differences in the organisms from which the genes originate.

[0025] Furthermore, the wild-type TG gene may also be a gene that encodes a protein having an amino acid sequence that is identical to, for example, 50% or more, 65% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more of the entire amino acid sequence, as long as the encoding TG does not have a "specific mutation".

[0026] Furthermore, the wild-type TG gene may also be a gene, such as DNA, that hybridizes under stringent conditions with a probe, such as a complementary sequence to all or part of the above base sequence, which can be prepared from the above base sequence (for example, the base sequence shown in Sequence ID No. 1), as long as the encoding TG does not have a "specific mutation." "Stringent conditions" may mean conditions under which so-called specific hybrids are formed and nonspecific hybrids are not formed. For example, conditions can be described as those in which DNA with high identity, such as 50% or more, 65% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more, hybridize with each other, while DNA with lower identity does not hybridize. Alternatively, conditions can be described as washing once, preferably two to three times, at a salt concentration and temperature equivalent to the washing conditions for normal Southern hybridization: 60°C, 1×SSC, 0.1% SDS, preferably 60°C, 0.1×SSC, 0.1% SDS, more preferably 68°C, 0.1×SSC, 0.1% SDS.

[0027] As described above, the probe used in the hybridization may be a part of the complementary sequence of the gene. Such probes can be prepared by PCR using oligonucleotides prepared based on known gene sequences as primers and a DNA fragment containing the gene as a template. For example, a DNA fragment approximately 300 bp in length can be used as a probe. When using a DNA fragment approximately 300 bp in length as a probe, the conditions for hybridization washing may be 50°C, 2×SSC, and 0.1% SDS.

[0028] Furthermore, since codon degeneracy differs depending on the host, the wild-type TG gene may be one in which any codon is replaced with an equivalent codon. In other words, the wild-type TG gene may be a variant of the wild-type TG gene exemplified above due to the degeneracy of the genetic code. For example, the wild-type TG gene may be modified to have the optimal codons depending on the codon usage frequency of the host being used.

[0029] Note that "identity" between amino acid sequences refers to the identity between amino acid sequences calculated using the default Scoring Parameters (Matrix: BLOSUM62; Gap Costs: Existence=11, Extension=1; Compositional Adjustments: Conditional compositional score matrix adjustment) by blastp. Furthermore, "identity" between nucleotide sequences refers to the identity between nucleotide sequences calculated using the default Scoring Parameters (Match / Mismatch Scores=1,-2; Gap Costs=Linear) by blastn.

[0030] The following describes mutant TG.

[0031] Mutant TG has TG activity. The degree of TG activity of mutant TG is not particularly limited, as long as mutant TG can be used for the desired application. The TG activity of mutant TG may be, for example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 120% or more, 150% or more, or 200% or more of the TG activity of the wild-type TG corresponding to the mutant TG and / or the TG activity of the wild-type TG consisting of the amino acid sequence shown in Sequence ID No. 2, or 10000% or less, 1000% or less, 200% or less, 150% or less, 120% or less, or 100% or less, or any non-inconsistent combination thereof.

[0032] Mutant TG has "specific mutations" in the amino acid sequence of wild-type TG.

[0033] In other words, mutant TG may be a protein having an amino acid sequence that has a "specific mutation" in the amino acid sequence shown in Sequence ID No. 2, for example. Alternatively, mutant TG may be a protein having an amino acid sequence that has a "specific mutation" in the amino acid sequence shown in Sequence ID No. 2, and also includes one or more additional amino acid substitutions, deletions, insertions, and / or additions at locations other than the "specific mutation", and possessing TG activity.

[0034] In other words, mutant TG may be a protein having the same amino acid sequence as wild-type TG except for having a "specific mutation." That is, mutant TG may be a protein having the amino acid sequence shown in Sequence ID No. 2 except for having a "specific mutation." Also, mutant TG may be a protein having an amino acid sequence that includes one or more amino acid substitutions, deletions, insertions, and / or additions in the amino acid sequence shown in Sequence ID No. 2, except for having a "specific mutation," and possessing TG activity. Furthermore, mutant TG may be a protein having an amino acid sequence that has 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 97% or more, identity with the amino acid sequence shown in Sequence ID No. 2, except for having a "specific mutation," and possessing TG activity.

[0035] Mutant TG may contain other amino acid sequences in addition to the mutant TG amino acid sequence exemplified above. That is, mutant TG may be a fusion protein with other amino acid sequences. Furthermore, mutant TG may be expressed in a form containing other amino acid sequences (i.e., as a fusion protein with other amino acid sequences) and may ultimately lose part or all of those other amino acid sequences. "Mutant TG contains other amino acid sequences" means, unless otherwise specified, that the ultimately obtained mutant TG contains other amino acid sequences. On the other hand, "Mutant TG is expressed in a form containing other amino acid sequences" means, unless otherwise specified, that mutant TG contains other amino acid sequences at least during expression, and does not necessarily mean that the ultimately obtained mutant TG contains other amino acid sequences. The same applies to wild-type TG. "Other amino acid sequences" are not particularly limited as long as mutant TG has TG activity. "Other amino acid sequences" can be appropriately selected according to various conditions such as the intended use. Examples of "other amino acid sequences" include peptide tags, signal peptides (also called signal sequences), pro-structures, and protease recognition sequences. The "other amino acid sequence" may be, for example, ligated to the N-terminus, C-terminus, or both of the mutant TG. The "other amino acid sequence" may be a single amino acid sequence, or a combination of two or more amino acid sequences.

[0036] Peptide tags include His tags, FLAG tags, GST tags, Myc tags, MBP (maltose binding protein), CBP (cellulose binding protein), TRX (thioredoxin), GFP (green fluorescent protein), HRP (horseradish peroxidase), ALP (alkaline phosphate), and the Fc region of antibodies. A 6xHis tag is an example of a His tag. Peptide tags can be used, for example, to detect and purify expressed mutant TG.

[0037] The signal peptide is not particularly limited as long as it functions in a host expressing the mutant TG. Examples of signal peptides include those recognized by the Sec secretory pathway and those recognized by the Tat secretory pathway. Specifically, examples of signal peptides recognized by the Sec secretory pathway include signal peptides of cell surface proteins of Corynebacteria. Specifically, examples of signal peptides of cell surface proteins of Corynebacteria include the PS1 signal sequence and PS2 (CspB) signal sequence of C. glutamicum (Japanese Patent Publication No. 6-502548) and the SlpA (CspA) signal sequence of C. stationis (Japanese Patent Publication No. 10-108675). Specifically, signal peptides recognized by the Tat secretory pathway include the TorA signal sequence of E. coli, the SufI signal sequence of E. coli, the PhoD signal sequence of Bacillus subtilis, the LipA signal sequence of Bacillus subtilis, and the IMD signal sequence of Arthrobacter globiformis (WO2013 / 118544). Signal peptides can be used, for example, in the secretory production of mutant TG. When mutant TG is secreted using signal peptides, the signal peptide is cleaved during secretion, and mutant TG without the signal peptide may be secreted outside the bacterial cell. In other words, typically, the mutant TG that is ultimately obtained does not need to have the signal peptide.

[0038] Specifically, examples of pro-structures include the pro-structures of each wild-type TG as illustrated above. Sequence ID 3 shows the nucleotide sequence of the Streptoverticillium mobaraense TG gene containing the portion encoding the pro-structure, and Sequence ID 4 shows the amino acid sequence of TG containing the pro-structure encoded by the same gene. In Sequence ID 3, positions 1 to 135 correspond to the portion encoding the pro-structure, and position 136 onwards corresponds to the portion encoding mature TG (i.e., Sequence ID 1). In Sequence ID 4, positions 1 to 45 correspond to the pro-structure, and position 46 onwards corresponds to mature TG (i.e., Sequence ID 2). Mutant TG may be expressed in a form containing the pro-structure, and the pro-structure may be removed to form a mature protein. When mutant TG is expressed in a form containing the pro-structure, the removal of the pro-structure can activate the mutant TG. Therefore, typically, the final mutant TG does not need to have a pro-structure. Removal of the pro-structure can be carried out, for example, using a processing enzyme. Examples of processing enzymes include proteases such as SAM-P45 (Appl. Environ. Microbiol., 2003, 69(1), 358-366) and alcalase. Furthermore, as will be described later, when expressing mutant TG by inserting a protease recognition sequence into the junction between the pro structure and the mutant TG, the pro structure can be removed using the corresponding protease.

[0039] Specific examples of protease recognition sequences include those for Factor Xa protease and proTEV protease. Protease recognition sequences can be used, for example, to cleave expressed mutant TG. Specifically, for example, when expressing mutant TG as a fusion protein with other amino acid sequences such as peptide tags or pro-structures, inserting a protease recognition sequence into the linkage between the mutant TG and the other amino acid sequence allows the expressed mutant TG to use the corresponding protease to cleave the other amino acid sequence, thereby obtaining a mutant TG that does not contain the other amino acid sequence.

[0040] The mutant TG gene is not particularly limited as long as it encodes the mutant TG described above. In this invention, the term "gene" is not limited to DNA, but may include any polynucleotide as long as it encodes the target protein. That is, "mutant TG gene" may mean any polynucleotide that encodes mutant TG. The mutant TG gene may be DNA, RNA, or a combination thereof. The mutant TG gene may be single-stranded or double-stranded. The mutant TG gene may be single-stranded DNA or single-stranded RNA. The mutant TG gene may be double-stranded DNA, double-stranded RNA, or a hybrid strand consisting of a DNA strand and an RNA strand. The mutant TG gene may contain both DNA residues and RNA residues in a single polynucleotide chain. When the mutant TG gene contains RNA, the descriptions of DNA such as the exemplified base sequences above may be appropriately read in accordance with RNA. The form of the mutant TG gene can be appropriately selected according to various conditions such as its intended use.

[0041] The following describes "specific mutations."

[0042] A specific mutation is one that improves heat resistance and / or pH stability. That is, the mutant TG has high heat resistance and / or pH stability. The mutant TG may have higher heat resistance and / or pH stability than the wild-type TG. For example, the mutant TG may have higher heat resistance and / or pH stability than the wild-type TG corresponding to the mutant TG and / or the wild-type TG consisting of the amino acid sequence shown in Sequence ID No. 2. A specific mutation may, at the very least, be a mutation that improves heat resistance.

[0043] "Heat resistance" can mean resistance to deactivation by heat. In other words, "mutant TG has high heat resistance" can mean that the degree of deactivation caused by heat treatment of mutant TG is small, or to put it another way, that the residual activity of mutant TG after heat treatment is large. Also, "mutant TG has higher heat resistance than wild-type TG" can mean that the degree of deactivation caused by heat treatment of mutant TG is smaller than the degree of deactivation caused by heat treatment of wild-type TG under the same conditions, or to put it another way, that the residual activity of mutant TG after heat treatment is greater than the residual activity of wild-type TG after heat treatment under the same conditions.

[0044] "The mutant TG has high heat resistance" may mean, for example, that the residual activity of the mutant TG after heat treatment is 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more. "The mutant TG has high heat resistance" may also mean, for example, that the residual activity of the mutant TG after heat treatment is 1.1 times or more, 1.3 times or more, 1.5 times or more, 2 times or more, 2.5 times or more, 3 times or more, 5 times or more, 10 times or more, 15 times or more, or 20 times or more, compared to the residual activity of wild-type TG (for example, wild-type TG corresponding to the mutant TG or wild-type TG consisting of the amino acid sequence shown in Sequence ID No. 2) after heat treatment under the same conditions.

[0045] Heat treatment options include treatment at 60°C, 65°C, 70°C, 72°C, 75°C, 77°C, or 80°C at pH 6.0 for 10 minutes. "Residual activity after heat treatment of TG" refers to the ratio of the TG activity after heat treatment to the TG activity before heat treatment. "Heat treatment of TG" means placing the TG under the heat treatment conditions described above.

[0046] For example, the residual activity after heat treatment of mutant TG at 60°C, 65°C, 70°C, or 72°C at pH 6.0 for 10 minutes may be 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more. Also, for example, the residual activity after heat treatment of mutant TG at 75°C at pH 6.0 for 10 minutes may be 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, or 75% or more. Furthermore, for example, the residual activity after heat treatment of mutant TG at 77°C and pH 6.0 for 10 minutes may be 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, or 45% or more. Furthermore, for example, the residual activity after heat treatment of mutant TG at 80°C and pH 6.0 for 10 minutes may be 5% or more.

[0047] "pH stability" may mean resistance to deactivation under acidic and / or alkaline conditions. Resistance to deactivation under acidic conditions is also called "acid resistance." Resistance to deactivation under alkaline conditions is also called "alkali resistance." In other words, "mutant TG has high pH stability" may mean that mutant TG has high acid and / or alkali resistance. "Mutant TG has high acid or alkali resistance" may mean that the degree of deactivation caused by treatment of mutant TG under acidic or alkaline conditions is small, or in other words, that the residual activity of mutant TG after treatment under acidic or alkaline conditions is high. Furthermore, "mutant TG has higher acid or alkali resistance than wild-type TG" may mean that the degree of inactivation of mutant TG by treating it under acidic or alkaline conditions is less than the degree of inactivation of wild-type TG by treating it under the same conditions; in other words, it may mean that the residual activity of mutant TG after treating it under acidic or alkaline conditions is greater than the residual activity of wild-type TG after treating it under the same conditions.

[0048] "The variant TG has high acid or alkali resistance" may mean, for example, that the residual activity after treating the variant TG under acidic or alkaline conditions is 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more. "The variant TG has high acid or alkali resistance" may mean, for example, that the residual activity of the variant TG after treatment under acidic or alkaline conditions is 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more, relative to the residual activity of the variant TG after treatment under control conditions, which is set to 100%. "The mutant TG has high acid or alkali resistance" may mean, for example, that the residual activity of the mutant TG after treatment under acidic or alkaline conditions is 1.1 times or more, 1.3 times or more, 1.5 times or more, 2 times or more, 2.5 times or more, 3 times or more, 5 times or more, 10 times or more, 15 times or more, or 20 times or more than the residual activity of the wild-type TG (for example, the wild-type TG corresponding to the mutant TG or the wild-type TG consisting of the amino acid sequence shown in Sequence ID No. 2) after treatment under the same conditions.

[0049] Examples of acidic treatments include treatment at pH 5.0, pH 4.5, pH 4.0, pH 3.5, pH 3.0, or pH 2.5 at 37°C for 4 hours. Examples of alkaline treatments include treatment at pH 7.5, pH 8.0, pH 8.5, pH 9.0, pH 9.5, or pH 10.0 at 37°C for 4 hours. An example of a control treatment is treatment at pH 6.0 at 37°C for 4 hours. "Residual activity after treatment of TG under certain conditions" refers to the ratio of the TG activity after treatment under those conditions to the TG activity before treatment under those conditions. "Treatment of TG under certain conditions" means placing TG under those conditions.

[0050] For example, the residual activity of mutant TG after treatment at pH 4.0 at 37°C for 4 hours may be 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more, relative to the residual activity of mutant TG after treatment at pH 6.0 at 37°C for 4 hours, which is set to 100%.

[0051] Examples of "specific mutations" include mutations (A) and (B) described below, as well as other known mutations that improve heat resistance and / or pH stability. Known mutations that improve heat resistance and / or pH stability include those described in WO2010 / 101256, WO2008 / 099898, and WO2019094301.

[0052] A “specific mutation” may be a mutation in a single amino acid residue, or a combination of mutations in two or more amino acid residues. A “specific mutation” may include, for example, mutation (A) and / or (B). A “specific mutation” may be, for example, mutation (A), mutation (B), or a combination of mutations (A) and (B).

[0053] Mutation (A) is a mutation in the following amino acid residue: D1, Y24, R48, S101, G102, N139, D142, L147, K152, G157, R167, N176, K181 , E182, H188, D189, R208, T245, S246, G250, G275, S284, H289, G301, K327.

[0054] Mutation (A) may be a mutation in a single amino acid residue, or a combination of mutations in two or more amino acid residues. That is, mutation (A) may include, for example, a mutation in one or more amino acid residues selected from these amino acid residues. Mutation (A) may be, for example, a mutation in a single amino acid residue selected from these amino acid residues, or a combination of mutations in two or more amino acid residues selected from these amino acid residues.

[0055] Any mutation at any of the amino acid residues may be selected individually or not.

[0056] In one embodiment, mutation (A) is particularly limited to mutations at S101, G157, R208, G250, and G275. That is, mutation (A) may include mutations at one or more amino acid residues selected from S101, G157, R208, G250, and G275. Mutation (A) may be, for example, a mutation at one or more amino acid residues selected from S101, G157, R208, G250, and G275, or a combination of mutations at one or more amino acid residues selected from S101, G157, R208, G250, and G275 and mutations at one or more other amino acid residues.

[0057] In another embodiment, mutation (A) may include mutations in D1, R48, G102, N139, D142, L147, K152, R167, N176, K181, E182, D189, S246, H289, G301, and K327. That is, mutation (A) may include mutations in one or more amino acid residues selected from, for example, D1, R48, G102, N139, D142, L147, K152, R167, N176, K181, E182, D189, S246, H289, G301, and K327. Mutation (A) may be, for example, a mutation in one or more amino acid residues selected from D1, R48, G102, N139, D142, L147, K152, R167, N176, K181, E182, D189, S246, H289, G301, and K327, or a combination of a mutation in one or more amino acid residues selected from D1, R48, G102, N139, D142, L147, K152, R167, N176, K181, E182, D189, S246, H289, G301, and K327 and a mutation in one or more other amino acid residues.

[0058] In another embodiment, mutation (A) may more particularly include mutations in D1, G102, N139, D142, L147, K152, R167, N176, K181, E182, D189, H289, G301, and K327. That is, mutation (A) may include mutations in one or more amino acid residues selected from, for example, D1, G102, N139, D142, L147, K152, R167, N176, K181, E182, D189, H289, G301, and K327. Mutation (A) may be, for example, a mutation in one or more amino acid residues selected from D1, G102, N139, D142, L147, K152, R167, N176, K181, E182, D189, H289, G301, and K327, or a combination of a mutation in one or more amino acid residues selected from D1, G102, N139, D142, L147, K152, R167, N176, K181, E182, D189, H289, G301, and K327 and a mutation in one or more other amino acid residues.

[0059] In the above notation used to identify amino acid residues, the number indicates the position in the amino acid sequence shown in Sequence ID No. 2, and the letter to the left of the number indicates the amino acid residue at each position in the amino acid sequence shown in Sequence ID No. 2 (i.e., the amino acid residue at each position before modification). For example, "G275" indicates the G (Gly) residue at position 275 in the amino acid sequence shown in Sequence ID No. 2.

[0060] In any wild-type TG, these amino acid residues each represent "the amino acid residue corresponding to the amino acid residue shown in the amino acid sequence shown in Sequence ID No. 2." That is, for example, "G275" in any wild-type TG represents the amino acid residue corresponding to the G (Gly) residue at position 275 in the amino acid sequence shown in Sequence ID No. 2.

[0061] Each of the above mutations may be a substitution of an amino acid residue. In each of the above mutations, the modified amino acid residue may be any amino acid residue other than the original amino acid residue, as long as the heat resistance and / or pH stability of TG is improved. In other words, the modified amino acid residue should be one that improves the heat resistance and / or pH stability of TG. Specifically, examples of modified amino acid residues include amino acid residues selected from K (Lys), R (Arg), H (His), A (Ala), V (Val), L (Leu), I (Ile), G (Gly), S (Ser), T (Thr), P (Pro), F (Phe), W (Trp), Y (Tyr), C (Cys), M (Met), D (Asp), E (Glu), N (Asn), and Q (Gln), other than the original amino acid residue.

[0062] Specifically, the following mutations can be considered as mutation (A): D1F, Y24(G, N), R48(I, K), S101(G, A, V, I, P, F, N, Q, Y, K, R, E), G102N, N139S, D142A, L147W, K152T, G157(A, V, I, S, N, K, R, H, D, E), R167G, N176D, K181R, E182D, H188Y, D189I, R208(L, A, E), T245A, S246(K, N, R), G250(A, V, L, M, P, F, W, S, T, N, Q, Y, K, R, H, D), G275A, S284P, H289I, G301W, K327F.

[0063] That is, mutation (A) may include, for example, one or more mutations selected from these mutations. Mutation (A) may be, for example, one mutation selected from these mutations, or a combination of two or more mutations selected from these mutations. Mutation (A) may also be, for example, a combination of one or more mutations selected from these mutations and one or more other mutations (for example, mutations in one or more amino acid residues selected from other D1, Y24, R48, S101, G102, N139, D142, L147, K152, G157, R167, N176, K181, E182, H188, D189, R208, T245, S246, G250, G275, S284, H289, G301, and K327).

[0064] In other words, the mutations in D1, Y24, R48, S101, G102, N139, D142, L147, K152, G157, R167, N176, K181, E182, H188, D189, R208, T245, S246, G250, G275, S284, H289, G301, and K327 are, respectively, D1F, Y24(G, N), R48(I, K), S101(G, A, V, I, P, F, N, Q, Y, K, R, E), G102N, N139S, D142A, L147W, K152T, G157(A, V, I, S, N, K, R, H, D, Examples include E), R167G, N176D, K181R, E182D, H188Y, D189I, R208(L, A, E), T245A, S246(K, N, R), G250(A, V, L, M, P, F, W, S, T, N, Q, Y, K, R, H, D), G275A, S284P, H289I, G301W, and K327F.

[0065] In particular, S101P is a notable mutation in S101.

[0066] In particular, G157(A, K, R, S) is a notable variant in G157. Even more specifically, G157(A, R, S) is a notable variant in G157. Even more specifically, G157(R, S) is a notable variant in G157.

[0067] In particular, R208E is a notable mutation in R208.

[0068] In particular, G250(A, F, S, N, R) is a notable mutation in G250. Even more specifically, G250(N, R, S) is a notable mutation in G250. Even more specifically, G250(R, S) is a notable mutation in G250.

[0069] In particular, R48I is a notable mutation in R48. For example, when a mutation in R48 is selected alone, the mutation in R48 may be R48I.

[0070] In particular, S246N is a notable mutation at S246. For example, when a mutation at S246 is selected alone, the mutation at S246 may be S246N.

[0071] In one embodiment, examples of mutations (A) include S101(G, A, V, I, P, F, N, Q, Y, K, R, E), G157(A, V, I, S, N, K, R, H, D, E), R208(L, A, E), G250(A, V, L, M, P, F, W, S, T, N, Q, Y, K, R, H, D), and G275A. That is, mutation (A) may include one or more mutations selected from, for example, S101(G, A, V, I, P, F, N, Q, Y, K, R, E), G157(A, V, I, S, N, K, R, H, D, E), R208(L, A, E), G250(A, V, L, M, P, F, W, S, T, N, Q, Y, K, R, H, D), and G275A. Mutation (A) may be, for example, one mutation selected from S101(G, A, V, I, P, F, N, Q, Y, K, R, E), G157(A, V, I, S, N, K, R, H, D, E), R208(L, A, E), G250(A, V, L, M, P, F, W, S, T, N, Q, Y, K, R, H, D), and G275A. D), and a combination of two or more mutations selected from G275A.Furthermore, mutations (A) include, for example, S101 (G, A, V, I, P, F, N, Q, Y, K, R, E), G157 (A, V, I, S, N, K, R, H, D, E), R208 (L, A, E), G250 (A, V, L, M, P, F, W, S, T, N, Q, Y, K, R, H, D), and G275A may be combined with one or more other mutations (for example, mutations in one or more amino acid residues selected from D1, Y24, R48, S101, G102, N139, D142, L147, K152, G157, R167, N176, K181, E182, H188, D189, R208, T245, S246, G250, G275, S284, H289, G301, and K327).

[0072] In one embodiment, examples of mutation (A) include S101P, G157(A, K, R, S), R208E, G250(A, F, S, N, R), and G275A. In one embodiment, examples of mutation (A) include S101P, G157(A, R, S), R208E, G250(N, R, S), and G275A. In one embodiment, examples of mutation (A) include S101P, G157(R, S), R208E, G250(R, S), and G275A. That is, mutation (A) may include one or more mutations selected from, for example, S101P, G157(R, S), R208E, G250(R, S), and G275A. Mutation (A) may be, for example, a single mutation selected from S101P, G157(R, S), R208E, G250(R, S), and G275A, or a combination of two or more mutations selected from S101P, G157(R, S), R208E, G250(R, S), and G275A. Furthermore, mutation (A) may be a combination of one or more mutations selected from, for example, S101P, G157(R, S), R208E, G250(R, S), and G275A, and one or more other mutations (for example, mutations in amino acid residues selected from other D1, Y24, R48, S101, G102, N139, D142, L147, K152, G157, R167, N176, K181, E182, H188, D189, R208, T245, S246, G250, G275, S284, H289, G301, and K327).

[0073] In the above notation for identifying mutations, the meaning of the numbers and the letters to their left is the same as described above. In the above notation for identifying mutations, the letters to the right of the numbers indicate the modified amino acid residue at each position. For example, "G275A" indicates a mutation in which the G (Gly) residue at position 275 in the amino acid sequence shown in Sequence ID No. 2 is replaced with an A (Ala) residue. Also, "G157(R, S)" indicates a mutation in which the G (Gly) residue at position 157 in the amino acid sequence shown in Sequence ID No. 2 is replaced with an R (Arg) residue or an S (Ser) residue.

[0074] In any wild-type TG, these mutations each represent "a mutation corresponding to the mutation in the amino acid sequence shown in SEQ ID NO: 2". In any wild-type TG, "a mutation corresponding to a mutation in which the amino acid residue at position X in the amino acid sequence shown in SEQ ID NO: 2 is replaced with a certain amino acid residue" should be read as "a mutation in which the amino acid residue corresponding to the amino acid residue at position X in the amino acid sequence shown in SEQ ID NO: 2 is replaced with a certain amino acid residue". That is, for example, in any wild-type TG, "G275A" represents a mutation in which the amino acid residue corresponding to the G (Gly) residue at position 275 in the amino acid sequence shown in SEQ ID NO: 2 is replaced with an A (Ala) residue.

[0075] There are no particular restrictions on the combinations of mutations for mutation (A). The following are examples of possible combinations of mutations for mutation (A): S101P / G157(A, R, S) / G250(N, R, S), S101P / G157(A, R, S) / R208E / G250(N, R, S), S101P / G157(A, R, S) / R208E / G250(N, R, S) / G275A.

[0076] The following are specific examples of mutation combinations for mutation (A): S101P / G157S / G250R, S101P / G157S / R208E / G250S, S101P / G157A / R208E / G250S, S101P / G157R / R208E / G25 0S, S101P / G157R / R208E / G250N, S101P / G157R / R208E / G250S / G275A, S101P / G157S / R208E / G250R / G275A.

[0077] Regarding mutation (A), the following mutation combinations are particularly noteworthy: S101P / G157S / R208E / G250S, S101P / G157A / R208E / G250S, S101P / G157R / R208E / G250S, S101P / G157R / R208E / G250N, S101P / G157R / R208E / G250S / G275A, and S101P / G157S / R208E / G250R / G275A.

[0078] More specifically, the following are possible mutation combinations for mutation (A): S101P / G157R / R208E / G250S / G275A and S101P / G157S / R208E / G250R / G275A.

[0079] That is, mutation (A) may include, for example, any combination of these. Mutation (A) may also be, for example, any combination of these and one or more other mutations (for example, mutations in one or more amino acid residues selected from other D1, Y24, R48, S101, G102, N139, D142, L147, K152, G157, R167, N176, K181, E182, H188, D189, R208, T245, S246, G250, G275, S284, H289, G301, and K327).

[0080] In the above notation for identifying combinations, the meaning of the numbers and the letters to their left and right is the same as described above. In the above notation for identifying combinations, the listing of two or more mutations separated by " / " indicates a double mutation or multiple mutation. For example, "S101P / G157S / G250R" indicates a triple mutation of S101P, G157S, and G250R.

[0081] Mutation (B) is a mutation that introduces a disulfide bond.

[0082] Examples of mutations (B) include the following: D3C / G283C, A81C / V311C, E93C / V112C, A106C / D213C, E107C / Y217C, A160C / G228C, S2C / N282C, S2C / G283C, T7C / E58C, P17C / W330C, D46C / S318C, S84C / K121C, R79C / P169C, A113C / P220C, E119C / S299C, R89C / S116C.

[0083] In one embodiment, mutation (B) includes the following mutations: D3C / G283C, A81C / V311C, E93C / V112C, A106C / D213C, E107C / Y217C, A16 0C / G228C, S2C / N282C, S2C / G283C, T7C / E58C, P17C / W330C, D46C / S318C.

[0084] In particular, the following mutations are examples of mutation (B): D3C / G283C, A81C / V311C, E93C / V112C, A106C / D213C, E107C / Y217C, A160C / G228C, S84C / K121C, R79C / P169C, A113C / P220C, E119C / S299C, R89C / S116C.

[0085] In one embodiment, mutation (B) includes, in particular, the following mutations: D3C / G283C, A81C / V311C, E93C / V112C, A106C / D213C, E107C / Y217C, A160C / G228C.

[0086] The notation for mutations is the same as that for mutation (A). For the sake of clarity, in the explanation of mutation (B), a double mutation introducing one disulfide bond will be considered as a single mutation.

[0087] Mutation (B) may be a single mutation, or a combination of two or more mutations. Mutation (B) may be, for example, a combination of two or more, three or more, or four or more mutations. Specifically, Mutation (B) may be, for example, a combination of one, two, three, or four mutations. That is, Mutation (B) may include, for example, one or more mutations selected from these mutations. Mutation (B) may be, for example, a single mutation selected from these mutations, or a combination of two or more mutations selected from these mutations. Also, Mutation (B) may be, for example, a combination of one or more mutations selected from these mutations and one or more other mutations that introduce a disulfide bond.

[0088] There are no particular restrictions on the combination of mutations for mutation (B).

[0089] Mutation (B) may, for example, include at least D3C / G283C. That is, a combination of mutations for mutation (B) is a combination of D3C / G283C and one or more other mutations that introduce a disulfide bond. Specifically, a combination of mutations for mutation (B) is a combination of D3C / G283C and one or more mutations selected from A81C / V311C, E93C / V112C, A106C / D213C, E107C / Y217C, A160C / G228C, S84C / K121C, R79C / P169C, A113C / P220C, E119C / S299C, and R89C / S116C. In one embodiment, a specific example of a mutation combination for mutation (B) is a combination of D3C / G283C and one or more mutations selected from A81C / V311C, E93C / V112C, A106C / D213C, E107C / Y217C, and A160C / G228C.

[0090] Regarding mutation (B), the following combinations are particularly noteworthy: D3C / G283C / A81C / V311C, D3C / G283C / E93C / V112C, D3C / G283C / A106C / D213C, D3C / G283C / E107C / Y217C, D3C / G283C / A160C / G228C, D3C / G283C / S84C / K121C, D3C / G283C / R79C / P169C, D3C / G283C / A113C / P220C, D3C / G283C / E119C / S2 99C, D3C / G283C / R89C / S116C, D3C / G283C / E93C / V112C / E107C / Y217C, D3C / G283C / E93C / V112C / A113C / P220C, D3C / G283C / E93C / V112C / E119C / S299C, D3C / G283C / E107C / Y217C / S84C / K121C, D3C / G283C / S84C / K121C / A113C / P220C.

[0091] In one embodiment, the following combinations of mutations for mutation (B) are particularly noteworthy: D3C / G283C / A81C / V311C, D3C / G283C / E93C / V112C, D3C / G283C / A106C / D213C, D3C / G283C / E107C / Y217C, D3C / G283C / A160C / G228C.

[0092] In other words, mutation (B) may include, for example, any combination of these. Mutation (B) may also be, for example, any combination of these. Furthermore, mutation (B) may be, for example, a combination of any combination of these with one or more other mutations that introduce a disulfide bond.

[0093] There are no particular restrictions on the combination of mutations (A) and (B). The mutations (A) and (B) constituting the combination may each be a single mutation, or a combination of two or more mutations. For example, the mutations (A) and (B) constituting the combination can be selected from those exemplified above.

[0094] The following are possible combinations of mutations (A) and (B): Mutation (A): S101P / G157(A, R, S) / G250(N, R, S), S101P / G157(A, R, S) / R208E / G250(N, R, S), or S101P / G157(A, R, S) / R208E / G250(N, R, S) / G275A; Mutation (B): One or more mutations selected from D3C / G283C, A81C / V311C, E93C / V112C, A106C / D213C, E107C / Y217C, A160C / G228C, S84C / K121C, R79C / P169C, A113C / P220C, E119C / S299C, and R89C / S116C.

[0095] In one embodiment, the following combinations of mutations (A) and (B) are given: Mutation (A): S101P / G157(A, R, S) / G250(N, R, S), S101P / G157(A, R, S) / R208E / G250(N, R, S), or S101P / G157(A, R, S) / R208E / G250(N, R, S) / G275A; Mutation (B): One or more mutations selected from D3C / G283C, A81C / V311C, E93C / V112C, A106C / D213C, E107C / Y217C, and A160C / G228C.

[0096] In particular, the following combinations of mutations (A) and (B) are examples of mutations (A) and (B): Mutation (A): S101P / G157S / R208E / G250S, S101P / G157A / R208E / G250S, S101P / G157R / R208E / G250S, S101P / G157R / R208E / G250N, S101P / G157R / R208E / G250S / G275A, or S101P / G157S / R208E / G250R / G275A; Mutation (B): A combination of D3C / G283C and one or more mutations selected from A81C / V311C, E93C / V112C, A106C / D213C, E107C / Y217C, A160C / G228C, S84C / K121C, R79C / P169C, A113C / P220C, E119C / S299C, and R89C / S116C.

[0097] In one embodiment, the following combinations of mutations (A) and (B) are particularly noteworthy: Mutation (A): S101P / G157S / R208E / G250S, S101P / G157A / R208E / G250S, S101P / G157R / R208E / G250S, S101P / G157R / R208E / G250N, S101P / G157R / R208E / G250S / G275A, or S101P / G157S / R208E / G250R / G275A; Mutation (B): A combination of D3C / G283C and one or more mutations selected from A81C / V311C, E93C / V112C, A106C / D213C, E107C / Y217C, and A160C / G228C.

[0098] More specifically, the following combinations of mutations (A) and (B) are possible: Mutation (A): S101P / G157R / R208E / G250S / G275A or S101P / G157S / R208E / G250R / G275A; Mutation (B): D3C / G283C, D3C / G283C / A81C / V311C, D3C / G283C / E93C / V112C, D3C / G283C / A106C / D213C, D3C / G283C / E107C / Y21 7C, D3C / G283C / A160C / G228C, D3C / G283C / S84C / K121C, D3C / G283C / R79C / P169C, D3C / G283C / A113C / P220C, D3C / G283C / E 119C / S299C, D3C / G283C / R89C / S116C, D3C / G283C / E93C / V112C / E107C / Y217C, D3C / G283C / E93C / V112C / A113C / P220C, D3C / G283C / E93C / V112C / E119C / S299C, D3C / G283C / E107C / Y217C / S84C / K121C, or D3C / G283C / S84C / K121C / A113C / P220C.

[0099] In one embodiment, the following combinations of mutations (A) and (B) are particularly relevant: Mutation (A): S101P / G157R / R208E / G250S / G275A or S101P / G157S / R208E / G250R / G275A; Mutation (B): D3C / G283C, D3C / G283C / A81C / V311C, D3C / G283C / E93C / V112C, D3C / G 283C / A106C / D213C, D3C / G283C / E107C / Y217C, or D3C / G283C / A160C / G228C.

[0100] In other words, a “specific mutation” may include, for example, any combination of these. A “specific mutation” may also be, for example, any combination of these and one or more other mutations (for example, one or more other mutations that introduce a disulfide bond).

[0101] The positions of the amino acid residues mentioned in each of the above mutations are for convenience in identifying the amino acid residues being modified and do not need to indicate their absolute positions in wild-type TG. In other words, the positions of the amino acid residues in each of the above mutations indicate relative positions based on the amino acid sequence shown in SEQ ID NO: 2, and their absolute positions may change due to deletions, insertions, or additions of amino acid residues. For example, if one amino acid residue is deleted or inserted at a position closer to the N-terminus than position X in the amino acid sequence shown in SEQ ID NO: 2, the original amino acid residue at position X becomes the (X-1) or (X+1) amino acid residue from the N-terminus, respectively, but is considered to be "the amino acid residue corresponding to the amino acid residue at position X in the amino acid sequence shown in SEQ ID NO: 2". Furthermore, the pre-modification amino acid residues mentioned in each of the above mutations are for convenience in identifying the amino acid residues being modified and do not need to be conserved in wild-type TG. In other words, if wild-type TG does not have the amino acid sequence shown in SEQ ID NO: 2, the pre-modification amino acid residues mentioned in each of the above mutations may not be conserved. In other words, each of the above mutations may include mutations in which the amino acid residue before modification mentioned in each mutation is replaced with another amino acid residue (for example, the modified amino acid residue mentioned in each mutation) if that amino acid residue is not conserved. For example, "mutation at G275" is not limited to mutations in which the amino acid residue corresponding to G275 is replaced with another amino acid residue when that amino acid residue is conserved (i.e., it is a G(Gly) residue), but may also include mutations in which the amino acid residue corresponding to G275 is replaced with another amino acid residue when that amino acid residue is not conserved (i.e., it is not a G(Gly) residue). The amino acid residues before and after modification are selected so as not to be identical to each other. Furthermore, for any mutant TG, the corresponding wild-type TG may be one in which the amino acid residue before modification mentioned in each of the above mutations is conserved.

[0102] The amino acid residue that corresponds to the amino acid residue at position X in the amino acid sequence shown in Sequence ID No. 2 in any given TG amino acid sequence can be determined by aligning the amino acid sequence of the given TG with the amino acid sequence shown in Sequence ID No. 2. Alignment can be performed, for example, using known gene analysis software. Specific examples of such software include DNASIS from Hitachi Solutions and GENETYX from Genetics (Elizabeth C. Tyler et al., Computers and Biomedical Research, 24(1), 72-96, 1991; Barton GJ et al., Journal of molecular biology, 198(2), 327-37, 1987).

[0103] <2> Manufacturing of mutant TG Mutant TG can be produced, for example, by expressing the mutant TG gene in a host that possesses the mutant TG gene.

[0104] Furthermore, mutant TG can also be produced, for example, by expressing the mutant TG gene in a cell-free protein synthesis system.

[0105] The following details the production of mutant TG using a host with a mutant TG gene.

[0106] <2-1>Host A host possessing a mutant TG gene can be obtained by introducing the mutant TG gene into a suitable host. "Introducing a mutant TG gene into a host" may also include modifying the host's existing TG gene, such as the wild-type TG gene, to encode the mutant TG gene. "Possessing a mutant TG gene" is also referred to as "having mutant TG."

[0107] The host is not particularly restricted as long as it can express a functional mutant TG. Examples of hosts include microorganisms, plant cells, insect cells, and animal cells. Microorganisms are particularly noteworthy as hosts. Examples of microorganisms include bacteria and yeast. Bacteria are particularly noteworthy as microorganisms.

[0108] Examples of bacteria include those belonging to the family Enterobacteriaceae, Corynebacteria, and Bacillus.

[0109] Bacteria belonging to the Enterobacteriaceae family include those belonging to genera such as Escherichia, Enterobacter, Pantoea, Klebsiella, Serratia, Erwinia, Photorhabdus, Providencia, Salmonella, and Morganella. Specifically, bacteria classified under the Enterobacteriaceae family according to the classification method used in the NCBI (National Center for Biotechnology Information) database (http: / / www.ncbi.nlm.nih.gov / Taxonomy / Browser / wwwtax.cgi?id=91347) can be used. While there are no particular restrictions on Escherichia bacteria, those classified under the Escherichia genus according to classifications known to microbiology experts are generally considered. Examples of Escherichia bacteria include those described in the book by Neidhardt et al. (Backmann, BJ 1996. Derivations and Genotypes of some mutant derivatives of Escherichia coli K-12, p. 2460-2488. Table 1. In FD Neidhardt (ed.), Escherichia coli and Salmonella Cellular and Molecular Biology / Second Edition, American Society for Microbiology Press, Washington, DC). An example of Escherichia bacteria is Escherichia coli. Examples of Escherichia coli include Escherichia coli K-12 strains such as strain W3110 (ATCC 27325) and strain MG1655 (ATCC 47076); Escherichia coli K5 strain (ATCC 23506); Escherichia coli B strains such as strain BL21 (DE3); and their derivative strains.Examples of Enterobacter bacteria include Enterobacter agglomerans and Enterobacter aerogenes. Examples of Pantoea bacteria include Pantoea ananatis, Pantoea stewartii, Pantoea agglomerans, and Pantoea citrea. Examples of Erwinia bacteria include Erwinia amylovora and Erwinia carotovora. Examples of Klebsiella bacteria include Klebsiella planticola. It should be noted that bacteria belonging to the Enterobacteriaceae family have recently been reclassified into multiple families through comprehensive comparative genomic analysis (Adelou M. et al., Genome-based phylogeny and taxonomy of the 'Enterobacteriales': proposal for Enterobacterales ord. nov. divided into the families Enterobacteriaceae, Erwiniaceae fam. nov., Pectobacteriaceae fam. nov., Yersiniaceae fam. nov., Hafniaceae fam. nov., Morganellaceae fam. nov., and Budviciaceae fam. nov., Int. J. Syst. Evol. Microbiol., 2016, 66:5575-5599). However, in this invention, bacteria that were conventionally classified under the Enterobacteriaceae family will be treated as bacteria belonging to the Enterobacteriaceae family.

[0110] Examples of coryne-type bacteria include those belonging to genera such as Corynebacterium, Brevibacterium, and Microbacterium.

[0111] Examples of Corynebacteria include the following species: Corynebacterium acetoacidophilum Corynebacterium acetoglutamicum Corynebacterium alkanolyticum Corynebacterium callunae Corynebacterium crenatum Corynebacterium glutamicum Corynebacterium lilium Corynebacterium melassecola Corynebacterium thermoaminogenes (Corynebacterium efficiens) Corynebacterium herculis Brevibacterium divaricatum (Corynebacterium glutamicum) Brevibacterium flavum (Corynebacterium glutamicum) Brevibacterium immariophilum Brevibacterium lactofermentum (Corynebacterium glutamicum) Brevibacterium roseum Brevibacterium saccharolyticum Brevibacterium thiogenitalis Corynebacterium ammoniagenes (Corynebacterium stationis) Brevibacterium album Brevibacterium cerinum Microbacterium ammoniaphilum

[0112] Examples of Corynebacterium strains include the following: Corynebacterium acetoacidophilum ATCC 13870 Corynebacterium acetoglutamicum ATCC 15806 Corynebacterium alkanolyticum ATCC 21511 Corynebacterium callunae ATCC 15991 Corynebacterium crenatum AS1.542 Corynebacterium glutamicum ATCC 13020, ATCC 13032, ATCC 13060, ATCC 13869, FERM BP-734 Corynebacterium lilium ATCC 15990 Corynebacterium molassecola ATCC 17965 Corynebacterium efficiens (Corynebacterium thermoaminogenes) AJ12340 (FERM BP-1539) Corynebacterium herculis ATCC 13868 Brevibacterium divaricatum (Corynebacterium glutamicum) ATCC 14020 Brevibacterium flavum (Corynebacterium glutamicum) ATCC 13826, ATCC 14067, AJ12418 (FERM BP-2205) Brevibacterium immariophilum ATCC 14068 Brevibacterium lactofermentum (Corynebacterium glutamicum) ATCC 13869 Brevibacterium roseum ATCC 13825 Brevibacterium saccharolyticum ATCC 14066 Brevibacterium thiogenitalis ATCC 19240 Corynebacterium ammoniagenes (Corynebacterium stationis) ATCC 6871, ATCC 6872 Brevibacterium album ATCC 15111 Brevibacterium cerinum ATCC 15112 Microbacterium ammoniaphilum ATCC 15354

[0113] Furthermore, the genus Corynebacterium includes bacteria that were previously classified under the genus Brevibacterium but have now been integrated into the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 255 (1991)). Additionally, the genus Corynebacterium statyonis includes bacteria that were previously classified under Corynebacterium ammoniagenes but have been reclassified as Corynebacterium statyonis based on 16S rRNA sequencing analysis (Int. J. Syst. Evol. Microbiol., 60, 874-879 (2010)).

[0114] Examples of bacteria belonging to the genus Bacillus include Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus polymixa, and Bacillus stearothermophilus. Specific examples of Bacillus subtilis include Bacillus subtilis strain 168 Marburg (ATCC 6051) and Bacillus subtilis strain PY79 (Plasmid, 1984, 12, 1-9). Examples of Bacillus amyloricephasiens include Bacillus amyloricephasiens strain T (ATCC 23842) and Bacillus amyloricephasiens strain N (ATCC 23845).

[0115] Examples of yeasts include those belonging to genera such as Saccharomyces cerevisiae, Candida utilis, Pichia, Pichia pastoris, Hansenula, Hansenula polymorpha, and Schizosaccharomyces pombe.

[0116] These strains can be obtained, for example, from the American Type Culture Collection (address: 12301 Parklawn Drive, Rockville, Maryland 20852 PO Box 1549, Manassas, VA 20108, United States of America). Each strain is assigned a registration number, which can be used to obtain them (see http: / / www.atcc.org / ). The registration numbers for each strain are listed in the American Type Culture Collection catalog. These strains can also be obtained, for example, from the depositary institutions where each strain is deposited.

[0117] A mutant TG gene can be obtained, for example, by modifying a wild-type TG gene so that the encoded TG has a "specific mutation." The wild-type TG gene used as the basis for modification can be obtained, for example, by cloning from an organism that possesses the wild-type TG gene, or by chemical synthesis. Alternatively, a mutant TG gene can be obtained without going through the wild-type TG gene. The mutant TG gene may be obtained directly, for example, by chemical synthesis. The obtained mutant TG gene may be used as is or after further modification. For example, a mutant TG gene of one form may be obtained by modifying a mutant TG gene of another form.

[0118] Gene modification can be performed using known methods. For example, site-directed mutagenesis (SMU) can be used to introduce a desired mutation at a target site in DNA. That is, for example, SMU can be used to modify the coding region of a gene so that the encoded protein includes substitution, deletion, insertion, and / or addition of amino acid residues at a specific site. Examples of SMU methods include methods using PCR (Higuchi, R., 61, in PCR technology, Erlich, HA Eds., Stockton press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)) and methods using phages (Kramer, W. and Frits, HJ, Meth. in Enzymol., 154, 350 (1987); Kunkel, TA et al., Meth. in Enzymol., 154, 367 (1987)).

[0119] The method for introducing the mutant TG gene into a host is not particularly limited. The mutant TG gene only needs to be held in the host in an expressible state. That is, in the host, the mutant TG gene only needs to be held in an expressible state under the control of a promoter that functions in that host. In the host, the mutant TG gene may be present on a vector that autonomously replicates outside the chromosome, such as a plasmid, or it may be introduced onto a chromosome. The host may have only one copy of the mutant TG gene, or it may have two or more copies. The host may have only one type of mutant TG gene, or it may have two or more types of mutant TG genes.

[0120] The promoter used to express the mutant TG gene is not particularly limited as long as it functions in the host. A "promoter that functions in the host" means a promoter that has promoter activity in the host. The promoter may be a promoter of host origin or a promoter of heterologous origin. The promoter may be the intrinsic promoter of the TG gene or a promoter of another gene. The promoter may be a more potent promoter than the intrinsic promoter of the TG gene. Examples of potent promoters that function in Enterobacteriaceae bacteria such as Escherichia coli include the T7 promoter, trp promoter, trc promoter, lac promoter, tac promoter, tet promoter, araBAD promoter, rpoH promoter, msrA promoter, and the Bifidobacterium-derived Pm1 promoter, PR promoter, and PL promoter. Furthermore, potent promoters that function in Corynebacteria include the artificially modified P54-6 promoter (Appl. Microbiol. Biotechnolo., 53, 674-679 (2000)), the pta, aceA, aceB, adh, and amyE promoters that can be induced in Corynebacteria with acetic acid, ethanol, pyruvate, etc., and the cspB, SOD, and tuf((EF-Tu)) promoters, which are potent promoters with high expression levels in Corynebacteria (Journal of Biotechnology 104 (2003) 311-323, Appl Environ Microbiol. 2005). Examples include the P2 promoter (WO2018 / 079684), P3 promoter (WO2018 / 079684), lac promoter, tac promoter, trc promoter, and F1 promoter (WO2018 / 179834). Furthermore, as a strong promoter, highly active versions of conventional promoters may be obtained and used by using various reporter genes. For example, promoter activity can be increased by bringing the -35 and -10 regions within the promoter region closer to the consensus sequence (International Publication No. 00 / 18935).Examples of highly active promoters include various tac-like promoters (Katashkina JI et al. Russian Federation Patent application 2006134574) and the pnlp8 promoter (WO2010 / 027045). Methods for evaluating promoter strength and examples of strong promoters are described in Goldstein et al.'s paper (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995)), among others.

[0121] Furthermore, a terminator for transcription termination can be placed downstream of the mutant TG gene. The terminator is not particularly limited as long as it functions in the host. The terminator may be of host origin or of heterologous origin. The terminator may be a terminator specific to the TG gene or a terminator of another gene. Specific examples of terminators include, for example, the T7 terminator, T4 terminator, fd phage terminator, tet terminator, and trpA terminator.

[0122] A mutant TG gene can be introduced into a host, for example, using a vector containing the gene. A vector containing a mutant TG gene is also called a mutant TG gene expression vector or recombinant vector. A mutant TG gene expression vector can be constructed, for example, by ligating a DNA fragment containing the mutant TG gene with a vector that functions in the host. By transforming the host with a mutant TG gene expression vector, a transformant into which the vector has been introduced can be obtained, that is, the gene can be introduced into the host. As the vector, a vector capable of autonomous replication within the host cell can be used. The vector is preferably a multicopy vector. Furthermore, the vector is preferably equipped with a marker such as an antibiotic resistance gene for selecting transformants. The vector may also be equipped with a promoter or terminator for expressing the inserted gene. The vector may be, for example, a bacterial plasmid-derived vector, a yeast plasmid-derived vector, a bacteriophage-derived vector, a cosmid, or a phagemid. Examples of vectors capable of autonomous replication in Enterobacteriaceae bacteria such as Escherichia coli include pUC19, pUC18, pHSG299, pHSG399, pHSG398, pBR322, pSTV29 (all available from Takara Bio), pACYC184, pMW219 (Nippon Gene), pTrc99A (Pharmacia), pPROK vector (Clontech), pKK233-2 (Clontech), pET vector (Novagen), pQE vector (Qiagen), pCold TF DNA (Takara Bio), pACYC vector, and the broad-host-range vector RSF1010.Specifically, examples of vectors capable of autonomous replication in Corynebacteria include pHM1519 (Agric. Biol. Chem., 48, 2901-2903 (1984)); pAM330 (Agric. Biol. Chem., 48, 2901-2903 (1984)); plasmids containing improved drug resistance genes; pCRY30 (JP-A-3-210184); pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE, and pCRY3KX (JP-A-2-72876, U.S. Patent No. 5,185,262); pCRY2 and pCRY Examples include 3 (JP-A-1-191686); pAJ655, pAJ611, and pAJ1844 (JP-A-58-192900); pCG1 (JP-A-57-134500); pCG2 (JP-A-58-35197); pCG4 and pCG11 (JP-A-57-183799); pPK4 (US Patent No. 6,090,597); pVK4 (JP-A-9-322774); pVK7 (JP-A-10-215883); pVK9 (WO2007 / 046389); pVS7 (WO2013 / 069634); and pVC7 (JP-A-9-070291). Furthermore, specific examples of vectors capable of autonomous replication in Corynebacteria include variants of pVC7, such as pVC7H2 (WO2018 / 179834). When constructing an expression vector, for example, the mutant TG gene containing a unique promoter region may be directly incorporated into the vector, the coding region of the mutant TG may be bound downstream of the promoter as described above before being incorporated into the vector, or the coding region of the mutant TG may be incorporated downstream of the promoter originally present on the vector.

[0123] Vectors, promoters, and terminators usable in various microorganisms are described in detail in, for example, "Basic Microbiology Course 8: Genetic Engineering," Kyoritsu Shuppan, 1987, and these can be utilized.

[0124] Furthermore, mutant TG genes can be introduced, for example, onto host chromosomes. Gene introduction into chromosomes can be carried out, for example, using homologous recombination (Miller I, JH Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). Examples of gene introduction methods using homologous recombination include methods using linear DNA such as Red-driven integration (Datsenko, K. A, and Wanner, BL Proc. Natl. Acad. Sci. US A. 97:6640-6645 (2000)), methods using plasmids containing temperature-sensitive origins of replication, methods using conjugate-transferable plasmids, methods using suicide vectors that do not have origins of replication that function in the host, and phage-based transduction methods. Only one copy of the gene may be introduced, or two or more copies may be introduced. For example, multiple copies of the gene can be introduced into the chromosome by performing homologous recombination targeting sequences that have many copies on the chromosome. Sequences with multiple copies on a chromosome include repetitive DNA sequences and inverted repeats located at both ends of transposons. Homologous recombination may also be performed targeting appropriate sequences on the chromosome, such as genes that are not necessary for carrying out the present invention. Genes can also be randomly introduced onto the chromosome using transposons or Mini-Mu (Japanese Patent Publication No. 2-109985, US5,882,888, EP805867B1). When introducing a gene into a chromosome, for example, a mutant TG gene containing a unique promoter region may be directly incorporated into the chromosome, the coding region of the mutant TG may be bound downstream of the promoter as described above before being incorporated into the chromosome, or the coding region of the mutant TG may be incorporated downstream of a promoter that is originally present on the chromosome.

[0125] The introduction of a gene onto a chromosome can be confirmed, for example, by Southern hybridization using a probe with a nucleotide sequence complementary to all or part of the gene, or by PCR using primers created based on the nucleotide sequence of the gene.

[0126] The transformation method is not particularly limited, and conventionally known methods can be used. Examples of transformation methods include the method of treating receptor bacterial cells with calcium chloride to increase DNA permeability, as reported for Escherichia coli K-12 (Mandel, M. and Higa, A.,J. Mol. Biol. 1970, 53, 159-162), and the method of preparing competent cells from cells in the growth stage and introducing DNA, as reported for Bacillus subtilis (Duncan, CH, Wilson, GA and Young, FE., 1977. Gene 1: 153-167). Furthermore, as a transformation method, a method in which the cells of DNA-receiving bacteria are made into protoplasts or spheroplasts that readily incorporate recombinant DNA, as is known for Bacillus subtilis, actinomycetes, and yeasts, and recombinant DNA is introduced into the DNA-receiving bacteria (Chang, S. and Choen, SN, 1979. Mol. Gen. Genet. 168: 111-115; Bibb, MJ, Ward, JM and Hopwood, OA 1978. Nature 274: 398-400; Hinnen, A., Hicks, JB and Fink, GR 1978. Proc. Natl. Acad. Sci. USA 75: 1929-1933) can also be applied. Additionally, as a transformation method, the electropulse method (Japanese Patent Publication No. Hei 2-207791), as reported for Corynebacteria, can also be used.

[0127] Furthermore, if a host already possesses a TG gene, such as a wild-type TG gene, on its chromosomes, the host can be modified to possess a mutant TG gene by altering the TG gene to encode a mutant TG gene. "Introduction of a mutant TG gene" may also include altering the host's existing TG gene to a mutant TG gene. Modification of TG genes present on chromosomes can be carried out, for example, by spontaneous mutation, mutation treatment, or genetic engineering.

[0128] The host may or may not have the wild-type TG gene. In particular, the host does not need to have the wild-type TG gene.

[0129] The host may have any properties as long as it can produce mutant TG.

[0130] <2-2> Host culture By culturing a host containing a mutant TG gene, mutant TG can be expressed.

[0131] The culture medium used is not particularly limited, as long as it allows the host to grow and express a functional mutant TG. For example, a standard culture medium used for culturing microorganisms such as bacteria and yeast can be used. The medium may contain, as needed, a carbon source, nitrogen source, phosphate source, sulfur source, and other various organic and inorganic components. The types and concentrations of the culture medium components may be appropriately set according to various conditions, such as the type of host.

[0132] Specific carbon sources include, for example, sugars such as glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, molasses, hydrolyzed starch, and hydrolyzed biomass; organic acids such as acetic acid, citric acid, succinic acid, and gluconic acid; alcohols such as ethanol, glycerol, and crude glycerol; and fatty acids. Plant-derived raw materials are preferably used as carbon sources. Examples of plants include corn, rice, wheat, soybeans, sugarcane, beets, and cotton. Examples of plant-derived raw materials include organs such as roots, stems, trunks, branches, leaves, flowers, and seeds; plant bodies containing these organs; and decomposition products of these plant organs. The form of use of plant-derived raw materials is not particularly limited and can be used in any form, such as unprocessed products, juices, pulverized products, or refined products. In addition, pentoses such as xylose, hexoses such as glucose, or mixtures thereof can be obtained and used, for example, from plant biomass. Specifically, these sugars can be obtained by subjecting plant biomass to treatments such as steam treatment, concentrated acid hydrolysis, dilute acid hydrolysis, hydrolysis by enzymes such as cellulase, and alkaline treatment. Since hemicellulose is generally more easily hydrolyzed than cellulose, hemicellulose in plant biomass may be hydrolyzed beforehand to release pentoses, and then cellulose may be hydrolyzed to produce hexoses. Xylose may also be supplied by converting hexoses, such as glucose, into xylose, for example, by providing the host with a conversion pathway from hexoses. As a carbon source, one type of carbon source may be used, or two or more types of carbon sources may be used in combination.

[0133] The concentration of the carbon source in the culture medium is not particularly limited, as long as the host can grow and functional mutant TG is expressed. The concentration of the carbon source in the culture medium may be as high as possible, for example, without inhibiting the production of mutant TG. The initial concentration of the carbon source in the culture medium may be, for example, usually 5-30 w / v%, preferably 10-20 w / v%. In addition, the carbon source may be supplied to the culture medium as appropriate. For example, the carbon source may be supplied to the culture medium in response to the decrease or depletion of the carbon source as the culture progresses. The carbon source may be temporarily depleted as long as mutant TG is eventually produced, but it is preferable in some cases to carry out the culture in a way that prevents the carbon source from being depleted or to prevent a state of carbon source depletion from continuing.

[0134] Specific examples of nitrogen sources include ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate; peptone; organic nitrogen sources such as yeast extract, meat extract, and soy protein hydrolysate; ammonia; and urea. Ammonia gas or ammonia water used for pH adjustment may also be used as a nitrogen source. One nitrogen source may be used, or two or more nitrogen sources may be used in combination.

[0135] Examples of phosphate sources include phosphates such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphate polymers such as pyrophosphate. One phosphate source may be used, or two or more phosphate sources may be used in combination.

[0136] Examples of sulfur sources include inorganic sulfur compounds such as sulfates, thiosulfates, and sulfites, and sulfur-containing amino acids such as cysteine, cystine, and glutathione. A single sulfur source may be used, or a combination of two or more sulfur sources may be used.

[0137] Other various organic and inorganic components include, specifically, inorganic salts such as sodium chloride and potassium chloride; trace metals such as iron, manganese, magnesium, and calcium; vitamins such as vitamin B1, vitamin B2, vitamin B6, nicotinic acid, nicotinamide, and vitamin B12; amino acids; nucleic acids; and organic components containing these, such as peptone, casamino acid, yeast extract, and soy protein hydrolysate. These other organic and inorganic components may be used individually, or in combination of two or more components.

[0138] Furthermore, when using a nutrient-dependent mutant strain that requires nutrients such as amino acids for growth, it is preferable to supplement the culture medium with such required nutrients.

[0139] The culture conditions are not particularly limited, as long as the host can grow and a functional mutant TG is expressed. Culturing can be carried out under the usual conditions used for culturing microorganisms such as bacteria and yeast. The culture conditions may be set appropriately depending on various factors, such as the type of host. Furthermore, the expression of the mutant TG gene can be induced as needed.

[0140] Culturing can be carried out using a liquid medium. During culturing, for example, the host may be cultured on a solid medium such as agar medium and then directly inoculated into the liquid medium, or the host may be seed cultured on a liquid medium and then inoculated into the liquid medium for the main culture. In other words, culturing may be carried out separately as seed culture and main culture. In this case, the culture conditions for seed culture and main culture may be the same or different. Mutant TG should be expressed at least in the main culture. The amount of host contained in the medium at the start of culturing is not particularly limited. For example, a seed culture solution with OD660 = 4 to 100 may be added to the medium for the main culture at the start of culturing in an amount of 0.1% to 100% by mass, preferably 1% to 50% by mass.

[0141] Culture can be carried out by batch culture, fed-batch culture, continuous culture, or a combination thereof. The culture medium used at the start of culture is also called the "initial medium." The culture medium supplied to the culture system (e.g., a fermenter) in fed-batch or continuous culture is also called the "fed-batch medium." The act of supplying fed-batch medium to the culture system in fed-batch or continuous culture is also called "fed-batch." When culture is carried out separately as a seed culture and a main culture, the culture forms of the seed culture and the main culture may or may not be the same. For example, both the seed culture and the main culture may be carried out as batch cultures, or the seed culture may be carried out as a batch culture and the main culture as a fed-batch or continuous culture.

[0142] In the present invention, various components such as carbon sources may be contained in the initial culture medium, the fed-batch medium, or both. That is, during the culture process, various components such as carbon sources may be supplied to the medium individually or in any combination. These components may be supplied once, multiple times, or continuously. The types of components contained in the initial culture medium may be the same as, or different from, the types of components contained in the fed-batch medium. Furthermore, the concentration of each component contained in the initial culture medium may be the same as, or different from, the concentration of each component contained in the fed-batch medium. In addition, two or more types of fed-batch media with different types and / or concentrations of components may be used. For example, if multiple feedings are performed intermittently, the types and / or concentrations of components contained in each fed-batch medium may be the same or different.

[0143] Culturing can be carried out, for example, under aerobic conditions. "Aerobic conditions" may mean that the dissolved oxygen concentration in the culture medium is 0.33 ppm or higher, preferably 1.5 ppm or higher. Specifically, the oxygen concentration may be controlled to, for example, 1 to 50% of the saturated oxygen concentration, preferably about 5%. Culturing can be carried out, for example, by aeration culture or shaking culture. The pH of the culture medium may be, for example, pH 3 to 10, preferably pH 4.0 to 9.5. During cultivation, the pH of the culture medium can be adjusted as needed. The pH of the culture medium can be adjusted using various alkaline or acidic substances such as ammonia gas, ammonia water, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, and magnesium hydroxide. The culture temperature may be, for example, 20 to 45°C, preferably 25 to 37°C. The culture period may be, for example, 10 to 120 hours. The culture may be continued, for example, until the carbon source in the culture medium is consumed or until the host becomes inactive.

[0144] By culturing the host in this manner, a culture containing mutant TG is obtained. The mutant TG may accumulate, for example, within the host's cells. "Bacterial cells" may be appropriately replaced with "cells" depending on the type of host. Depending on the type of host and / or the design of the mutant TG gene, the mutant TG may accumulate, for example, in the periplasm, or be secreted outside the cell.

[0145] Mutant TG may be used as is contained in the culture (specifically, the culture medium or bacterial cells), or it may be used after purification from the culture (specifically, the culture medium or bacterial cells). Purification can be carried out to any desired degree. That is, mutant TG may be purified mutant TG or a fraction containing mutant TG. In other words, mutant TG may be used in the form of a purified enzyme, in the form of such a fraction (i.e., in the form contained in such a fraction), or in a combination thereof. Such fractions are not particularly limited as long as they contain mutant TG so that it can act on their substrate. Such fractions may be cultures of hosts having the mutant TG gene (i.e., hosts having mutant TG), bacterial cells recovered from the same culture, culture supernatant recovered from the same culture, processed products thereof (e.g., processed products of bacterial cells such as cell lysates, cell lysates, cell extracts, and immobilized cells), partially purified products thereof (i.e., crude products), or combinations thereof. Furthermore, "purified mutant TG" may include crude products. These fractions may be used individually or in combination with purified mutant TG. Mutant TG may be used, for example, in a form not contained within bacterial cells. Mutant TG may also be used, for example, in the form of the compositions of the present invention described later.

[0146] If mutant TG accumulates in the culture medium, the culture supernatant can be obtained by, for example, centrifugation, and the mutant TG can be purified from the culture supernatant. Alternatively, if mutant TG accumulates within the host bacterial cells, the bacterial cells can be subjected to processes such as disruption, lysis, or extraction, and the mutant TG can be purified from the treated material. Purification of mutant TG can be carried out, for example, by known methods used for protein purification. Such methods include ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration chromatography, and isoelectric point precipitation. These methods can be used individually or in appropriate combinations.

[0147] The uses of mutant TG are not particularly limited.

[0148] Mutant TG may be used, for example, to modify food. Furthermore, mutant TG may be used, for example, in the manufacture of food. Food manufactured using mutant TG may be modified food. In other words, mutant TG may be specifically used, for example, in the manufacture of modified food. To put it another way, modified food may be obtained through food modification.

[0149] Examples of foods include foods that are heated during manufacturing. Specifically, examples of foods include protein-containing foods that are heated during manufacturing. Examples of foods that are heated during manufacturing (for example, protein-containing foods that are heated during manufacturing) include processed dairy products, processed egg products, processed plant products, processed meat products, processed seafood products, and processed gelatin products. "Processed dairy products," "processed egg products," "processed plant products," "processed meat products," "processed seafood products," and "processed gelatin products" mean foods manufactured by processing milk, eggs, plants, meat, seafood, and gelatin, respectively. In other words, "processed dairy products," "processed egg products," "processed plant products," "processed meat products," "processed seafood products," and "processed gelatin products" may mean processed foods manufactured using at least milk, eggs, plants, meat, seafood, and gelatin as ingredients, respectively.

[0150] Milk includes milk from mammals such as cow's milk, goat's milk, sheep's milk, buffalo milk, reindeer's milk, donkey's milk, and camel's milk. Eggs include chicken eggs and other bird eggs. Plants include grains such as beans and wheat. Beans include soybeans, peas, broad beans, chickpeas, almonds, peanuts, and lupin beans. Wheat includes wheat, barley, and rye. "Meat" refers to meat for consumption. Meat includes animal meat and bird meat. Animals include livestock such as cows, pigs, horses, sheep, goats, and rabbits; wild animals such as wild boars, deer, and bears; and marine mammals such as whales, dolphins, and sea lions. Birds include chickens, turkeys, ducks, geese, guinea fowl, quail, and ostriches. Seafood includes fish such as horse mackerel, salmon, cod, pufferfish, sillago, conger eel, hoki, and hake; crustaceans such as shrimp and crab; shellfish such as scallops and oysters; and other marine products such as squid and octopus. Gelatin can be obtained from the hides or bones of animals such as cows and pigs.

[0151] Examples of dairy products include processed cheese, pre-incubation yogurt, ice cream, and margarine. Examples of egg products include tamagoyaki (Japanese rolled omelet), chawanmushi (steamed egg custard), and egg tofu. Examples of plant-based products include tofu, yuba (tofu skin), noodles, bread, wheat dough, and veggie meat. "Veggie meat" may refer to ingredients that have been processed to resemble meat using raw materials containing plant protein, or foods manufactured using such ingredients. Examples of plant protein include the plant proteins exemplified above. Examples of plant protein include legume protein. Examples of plant protein include soy protein. In other words, veggie meat specifically refers to products manufactured using raw materials containing soy protein (also called "soy meat"). Examples of plant-based products, meat products, or seafood products include ham, sausages, hamburgers, meatballs, and processed fish products. Meatballs may include, for example, those used as fillings for wrapped noodle products such as shumai, or those used as fillings for cabbage rolls. For example, sausages and hamburgers include those made from processed vegetarian meats such as soy meat, sausages and hamburgers made from processed meats such as beef and pork, and sausages and hamburgers made from processed seafood such as fish. Processed fish products include kamaboko (fish cake), kanikamaboko (crab fish cake), and fried kamaboko. Gelatin-based foods include jelly and gummy candies.

[0152] Examples of foods include those with low or high pH. Specifically, examples of foods include protein-containing foods with low or high pH. Foods with low or high pH may be, for example, foods manufactured using raw materials with low or high pH, ​​or foods that experience a decrease or increase in pH during manufacturing. pH may decrease, for example, through fermentation. That is, examples of foods that experience a decrease in pH during manufacturing include foods manufactured by fermentation. "Low pH" may be, for example, pH 5.0 or less, pH 4.5 or less, pH 4.0 or less, pH 3.5 or less, pH 3.0 or less, or pH 2.5 or less, or pH 1.5 or higher, pH 2.0 or higher, pH 2.5 or higher, pH 3.0 or higher, pH 3.5 or higher, or pH 4.0 or higher, or any non-contradictory combination thereof. "High pH" means, for example, pH 7.5 or higher, pH 8.0 or higher, pH 8.5 or higher, pH 9.0 or higher, pH 9.5 or higher, or pH 10.0 or higher, or pH 11.0 or lower, pH 10.5 or lower, pH 10.0 or lower, pH 9.5 or lower, pH 9.0 or lower, or pH 8.5 or lower, and any non-contradictory combination thereof is also acceptable.

[0153] Foods with a low pH include grains, potatoes, beans, nuts, meat, seafood, gelatin, dairy products, oils and fats, seasonings, beverages, fruit juices, and processed products thereof. Examples of grains and their processed products include white rice, brown rice, vinegared rice, oatmeal, rice flour, wheat flour, buckwheat flour, rice bran, and processed products thereof. Examples of rice flour processed products include mochi and gyuhi (a type of mochi). Examples of wheat flour processed products include bread, noodles (pasta, etc.), and fu (wheat gluten). Examples of buckwheat flour processed products include soba noodles. Examples of potatoes and their processed products include potatoes and their processed products. Examples of beans and their processed products include soy milk, chocolate, fried tofu, pea protein, and processed products thereof. Examples of meat and its processed products include the meats and their processed products mentioned above. Examples of seafood and its processed products include shrimp, crab, scallops, herring roe, and processed products thereof. Examples of dairy products include yogurt, butter, cheese, and ice cream. Examples of ice cream include those containing yogurt and / or fruit. Examples of fats and oils include margarine. Examples of seasonings include mayonnaise, ketchup, mustard, soy sauce, and miso. Examples of beverages include coffee-based beverages, energy drinks, fruit juices, cocoa, beer, and sake lees. Examples of processed fruit juice products include fruit juice gummies. Beverages may also be provided as high-viscosity beverages such as jelly drinks or smoothies. In addition, low-pH foods include foods that are heated during manufacturing as exemplified above and have a low pH (for example, those adjusted to a low pH).

[0154] Examples of high-pH foods include plant-based beverages such as soy milk, seaweed extracts such as kelp broth, and processed products thereof. Examples of processed soy milk products include tofu and yuba (tofu skin). Examples of processed seaweed extract products include beverages containing seaweed extract and foods containing seaweed extract, such as jelly foods. In addition, high-pH foods can also include foods that are heated during manufacturing, as exemplified above, and have a high pH (for example, those that have been adjusted to a high pH).

[0155] Furthermore, food products manufactured using mutant TG may be used as raw materials to manufacture other food products. For the sake of explanation, food products manufactured using mutant TG may also be called "intermediate products," and other food products manufactured using intermediate products as raw materials may be called "final products." In other words, such final products can also be considered as food products. Examples of final products include foods containing the examples given above (for example, processed cheese). The final product may also be modified by manufacturing the final product using the intermediate product as a raw material. The type of modification in the final product may be the same as the type of modification in the intermediate product, or it may not be. "Using mutant TG in the modification or manufacture of food products" may also include cases where mutant TG is indirectly used in the modification or manufacture of the final product by manufacturing the final product using the intermediate product as a raw material.

[0156] Food modification can include improvements in physical properties and flavor. Improvements in physical properties include increased fracture strength, increased compressive strength (i.e., compressive stress), increased shape retention (e.g., shape retention during heating and shape retention at room temperature), prevention of syneresis, imparting a smooth texture, and increased viscosity. Improvements in physical properties can be particularly seen in increased fracture strength, increased compressive strength, and increased shape retention during heating. Improvements in physical properties can be measured, for example, using physical property measuring instruments such as a texture analyzer. Improvements in physical properties can also be measured, for example, by sensory evaluation by a panel of experts. For example, improvements in physical properties such as increased fracture strength or increased compressive strength may be measured by sensory evaluation as an increase in hardness, flexibility, or elongation. Improvements in flavor can be measured, for example, by sensory evaluation by a panel of experts. Improvements in flavor can include increased richness or increased fattiness. Increased richness or increased fattiness can be obtained, for example, in dairy processed foods such as ice cream. In other words, for example, by using mutant TG, it may be possible to obtain improved physical properties and / or improved flavor in processed cheese and other foods produced in the examples compared to when mutant TG is not used. Specifically, for example, by manufacturing processed cheese and other foods produced in the examples using mutant TG, it may be possible to obtain processed cheese and other foods produced in the examples with improved physical properties, such as increased fracture strength, increased compressive strength, increased shape retention during heating, prevention of syneresis, and / or a smoother texture, compared to when processed cheese and other foods produced in the examples are manufactured without using mutant TG.

[0157] <3> Composition of the present invention The composition of the present invention is a composition containing mutant TG.

[0158] The composition of the present invention may be, for example, a composition used for the applications of mutant TG as illustrated above. That is, the composition of the present invention may be, for example, a composition for modifying food products such as processed cheese. The composition of the present invention may also be, for example, a composition for manufacturing food products such as processed cheese. More specifically, the composition of the present invention may be, for example, a composition for manufacturing modified food products such as modified processed cheese.

[0159] The composition of the present invention may consist of mutant TG, or it may contain components other than mutant TG.

[0160] Other components besides mutant TG are not particularly limited, as long as they do not impair the function of mutant TG. Other components can be those acceptable depending on the intended use of the composition of the present invention. Examples of other components include those incorporated into foods or pharmaceuticals.

[0161] As a composition of the present invention, for example, a variant TG in the embodiment exemplified above may be used as is or after being appropriately formulated. In formulation, appropriate additives may be used depending on the intended use of the composition of the present invention. Examples of additives include excipients, binders, disintegrants, lubricants, stabilizers, flavoring and odor-correcting agents, diluents, surfactants, and solvents. Additives can be appropriately selected, for example, depending on various conditions such as the shape of the composition of the present invention.

[0162] The form of the composition of the present invention is not particularly limited. The composition of the present invention may be provided in any form, such as powder, flakes, tablets, paste, or liquid.

[0163] <4> Method of the present invention The method of the present invention utilizes mutant TG.

[0164] In the method of the present invention, mutant TG may be used for applications of mutant TG as illustrated above. That is, the method of the present invention may be a method for modifying food products such as processed cheese. The method of the present invention may also be a method for producing food products such as processed cheese. More specifically, the method of the present invention may be a method for producing modified food products such as modified processed cheese.

[0165] In the modification or manufacture of food, mutant TG can be used to treat food ingredients. That is, one use of mutant TG is to treat food ingredients with mutant TG. In other words, the method of the present invention may be a method for modifying food, for example, which includes a step of treating food ingredients with mutant TG. The method of the present invention may also be a method for manufacturing food, for example, which includes a step of treating food ingredients with mutant TG. Furthermore, the method of the present invention may specifically be a method for manufacturing modified food, for example, which includes a step of treating food ingredients with mutant TG. Note that "treating food ingredients with mutant TG" is also called "applying mutant TG to food ingredients." The step of "treating food ingredients with mutant TG" is also called a "processing step." In other words, the method of the present invention may include a processing step.

[0166] Food ingredients can be appropriately selected according to various conditions such as the type of food. For example, if the food is a processed dairy product, an egg product, a plant product, a meat product, a seafood product, or a gelatin product, the food ingredients could be milk, eggs, plants, meat, seafood, and gelatin, respectively. These food ingredients may or may not be processed. For example, if the food is processed cheese, the milk may be used as a food ingredient in a state where it has already been processed into cheese such as natural cheese. That is, for example, if the food is processed cheese, the food ingredients could be cheese such as natural cheese.

[0167] Mutant TG may be used in the treatment of food ingredients in any manner that allows it to act on the food ingredients. Mutant TG may be used in the treatment of food ingredients in the form exemplified above, for example. Specifically, mutant TG may be used in the treatment of food ingredients in the form of the composition of the present invention, for example. That is, "treating food ingredients with mutant TG" also includes treating food ingredients with the composition of the present invention. Mutant TG can act on food ingredients by being used as is, or by being prepared in a desired form such as a solution and coexisting with the food ingredients. For example, mutant TG may be added to food ingredients, or a treatment solution containing mutant TG may be mixed with food ingredients. Such operations of coexisting mutant TG with food ingredients are collectively referred to as "addition" of mutant TG.

[0168] The modification or manufacture of food may be carried out using the same food ingredients and under the same manufacturing conditions as ordinary food, except for the use of mutant TG. Furthermore, the food ingredients and manufacturing conditions may be modified as appropriate for use in the modification or manufacture of food. The method of the present invention may include a step of manufacturing food from food ingredients. This step is also called the "food manufacturing step." The processing step may also be a step of manufacturing food by treating food ingredients with mutant TG.

[0169] Mutant TG may be used under heating conditions. For example, if mutant TG has high heat resistance, mutant TG may be used under heating conditions. Also, for example, if the food is heated during manufacturing, mutant TG may be used under heating conditions. In other words, heating may be performed in the processing step. That is, the processing step may be a step of treating food raw materials with mutant TG under heating conditions, or a step of applying mutant TG to food raw materials under heating conditions. In other words, the processing step may be a step of adding mutant TG to food raw materials and heating them. It should also be noted that when heating is performed at a certain heating temperature in the processing step, it is also said that "mutant TG is used at a certain temperature."

[0170] Mutant TG may be used under acidic or alkaline conditions. For example, if mutant TG has high acid or alkali resistance, it may be used under acidic or alkaline conditions. Also, for example, if the food is a low-pH or high-pH food, mutant TG may be used under acidic or alkaline conditions. In other words, the processing step may be a step of treating food raw materials with mutant TG under acidic or alkaline conditions, or a step of reacting food raw materials with mutant TG under acidic or alkaline conditions. It should also be noted that when processing is carried out at a certain pH in the processing step, it is also said that "mutant TG is used at a certain pH."

[0171] Mutant TG may be used under acidic or alkaline heating conditions.

[0172] Mutant TG can be used under desired conditions (e.g., heating conditions, acidic conditions, or alkaline conditions) and applied to food ingredients at any stage of the food manufacturing process, as long as the desired effect (e.g., food modification effect) is obtained. In other words, the treatment process can be carried out at any stage of the food manufacturing process, as long as the desired effect (e.g., food modification effect) is obtained.

[0173] The conditions for carrying out the processing steps (e.g., heating conditions) are not particularly limited as long as the desired effect (e.g., food modification effect) is obtained. In other words, the processing steps (e.g., heating) should be carried out in such a way that the mutant TG can act sufficiently on the food ingredients. Furthermore, the mutant TG may or may not be deactivated during the processing steps (e.g., heating), as long as it can act sufficiently on the food ingredients. The conditions for carrying out the processing steps (e.g., heating conditions) can be appropriately set according to various conditions such as the properties and amount of mutant TG added and the type of food.

[0174] When the processing step is carried out under heating conditions, the heating conditions (e.g., heating temperature, heating time, heating method, pH during heating) may be the same as, or modified as appropriate, the heating conditions used in the production of ordinary foods. The heating temperature may be, for example, 60°C or higher, 65°C or higher, 70°C or higher, 72°C or higher, or 75°C or higher, or 100°C or lower, 95°C or lower, 90°C or lower, 85°C or lower, 80°C or lower, 77°C or lower, 75°C or lower, 72°C or lower, or 70°C or lower, or any non-contradictory combination thereof. Specifically, the heating temperature may be, for example, 60°C to 100°C, 60°C to 90°C, 65°C to 80°C, or 65°C to 75°C. For example, if the food is processed cheese, the heating temperature may be 65°C to 80°C or 65°C to 75°C. The heating time may be, for example, 5 minutes or more, 10 minutes or more, 15 minutes or more, 30 minutes or more, or 1 hour or more, or 6 hours or less, 3 hours or less, 1 hour or less, 30 minutes or less, 15 minutes or less, or 10 minutes or less, or any non-contradictory combination thereof. Specifically, the heating time may be, for example, 5 minutes to 6 hours, 5 minutes to 3 hours, 5 minutes to 1 hour, 5 minutes to 30 minutes, 5 minutes to 15 minutes, 30 minutes to 6 hours, 30 minutes to 3 hours, 30 minutes to 1 hour, 1 hour to 6 hours, or 1 hour to 3 hours. For example, if the food is processed cheese, the heating time may be 5 minutes to 30 minutes or 5 minutes to 15 minutes. Heating methods include incubating, baking, steaming, boiling, and frying. The pH during heating may be, for example, acidic, neutral, or alkaline. Regarding the pH during heating, for example, the description of the treatment pH when the processing step is carried out under acidic or alkaline conditions may be applied mutatis mutandis.

[0175] When the processing step is carried out under acidic or alkaline conditions, the conditions for carrying out the processing step (e.g., processing pH, processing temperature, processing time) may be the same as, or with appropriate modifications, the conditions for processing using TG in the production of ordinary food products. When the processing step is carried out under acidic conditions, the processing pH may be, for example, pH 5.0 or less, pH 4.5 or less, pH 4.0 or less, pH 3.5 or less, pH 3.0 or less, or pH 2.5 or less, or pH 1.5 or higher, pH 2.0 or higher, pH 2.5 or higher, pH 3.0 or higher, pH 3.5 or higher, or pH 4.0 or higher, or any non-contradictory combination thereof. When the processing step is carried out under alkaline conditions, the processing pH may be, for example, pH 7.5 or higher, pH 8.0 or higher, pH 8.5 or higher, pH 9.0 or higher, pH 9.5 or higher, or pH 10.0 or higher, or pH 11.0 or lower, pH 10.5 or lower, pH 10.0 or lower, pH 9.5 or lower, pH 9.0 or lower, or pH 8.5 or lower, and any non-contradictory combination thereof may be used. The processing temperature may be, for example, 10°C or higher, 20°C or higher, 30°C or higher, 40°C or higher, 50°C or higher, 60°C or higher, 65°C or higher, 70°C or higher, 72°C or higher, or 75°C or higher, or 100°C or lower, 95°C or lower, 90°C or lower, 85°C or lower, 80°C or lower, 75°C or lower, 72°C or lower, 70°C or lower, 65°C or lower, 60°C or lower, 50°C or lower, 40°C or lower, or 30°C or lower, and any non-contradictory combination thereof may be used. Regarding the processing temperature, for example, the description of the heating temperature when the processing step is carried out under heating conditions may be applied mutatis mutandis. The processing time may be, for example, 5 minutes or more, 10 minutes or more, 15 minutes or more, 30 minutes or more, or 1 hour or more, or 6 hours or less, 3 hours or less, 1 hour or less, 30 minutes or less, 15 minutes or less, or 10 minutes or less, or any non-contradictory combination thereof. Specifically, the processing time may be, for example, 5 minutes to 6 hours, 5 minutes to 3 hours, 5 minutes to 1 hour, 5 minutes to 30 minutes, 5 minutes to 15 minutes, 30 minutes to 6 hours, 30 minutes to 3 hours, 30 minutes to 1 hour, 1 hour to 6 hours, or 1 hour to 3 hours.

[0176] The processing steps may be carried out in conjunction with the food manufacturing process, or they may be carried out separately from the food manufacturing process. Alternatively, a portion of the processing steps may be carried out in conjunction with the food manufacturing process, while the remainder is carried out separately from the food manufacturing process.

[0177] In the method of the present invention, ingredients other than those exemplified above (e.g., food ingredients and mutant TG) may be used, as long as the desired effect (e.g., food modification effect) is obtained. Such ingredients are also called "additional ingredients." Additional ingredients include ingredients other than those exemplified above that are commonly used in the manufacture of food. Additional ingredients may be mixed with the food ingredients beforehand, or they may be added to the food ingredients during the manufacture of the food as appropriate. For example, if the food is processed cheese, additional ingredients may include emulsifiers and water.

[0178] The amount and ratio of each component added in the method of the present invention are not particularly limited, as long as the desired effect (e.g., food modification effect) is obtained. The amount and ratio of each component used in the method of the present invention can be appropriately set according to various conditions such as the type of each component.

[0179] The amount of mutant TG added may be, for example, 0.00033U or more, 0.001U or more, 0.0033U or more, 0.01U or more, 0.033U or more, 0.1U or more, 0.33U or more, or 1U or more per gram of protein contained in the food raw material, or 100U or less, 33U or less, 10U or less, 3.3U or less, or 1U or less, and any non-inconsistent combination thereof may be used. Specifically, the amount of mutant TG added may be, for example, 0.00033U to 33U, or 0.0033U to 3.3U per gram of protein contained in the food raw material. [Examples]

[0180] The present invention will be described in more detail below with reference to non-limiting embodiments.

[0181] Example 1: Construction of mutant transglutaminase (mutant TG) and analysis of its heat resistance In this example, mutant TG was constructed and its heat resistance was analyzed.

[0182] <1> Experimental methods In this embodiment, the quantification of TG and the measurement of TG activity were performed according to the following procedure unless otherwise specified.

[0183] <1-1>Quantitative determination of TG TG was quantified by HPLC analysis using BSA as the standard. The analytical conditions are as follows. Mobile phase A: 0.1% trifluoroacetic acid (TFA) Mobile phase B: 0.1% TFA, 80% acetonitrile Flow rate: 1.0 ml / min Column temperature: 40℃ Detection: UV 280 nm Column: Proteonavi, 4.6 × 150 mm, 5 μm (manufactured by Osaka Soda Co., Ltd.) Gradient: 0 min (B: 30%), 0-20 min (B: 30-50%), 20-25 min (B: 50-100%), 25-26 min (B: 100%), 26-27 min (B: 100-30%), 27-30 min (B: 30%)

[0184] <1-2> Measurement of TG activity TG activity was measured by the hydroxamate method. 500 μL of solution A (50 mM MES, 100 mM NH2OH, 10 mM reduced glutathione, 30 mM CBZ-Gln-Gly (Z-QG), adjusted to pH 6.0 with NaOH) was added to a 1.5 mL tube and incubated at 37°C for 5 minutes. 50 μL of enzyme solution was added, and after reacting at 37°C for 10 minutes, 500 μL of solution B (1N HCl, 4% TCA, 1.67% FeCl3·6H2O) was added to stop the reaction. 200 μL of the stop solution was added to a 96-well plate, and the absorbance at 525 nm was measured using a plate reader and taken as the absorbance of the enzyme sample. As a control, the absorbance of a sample reacted similarly using 20 mM MES pH 6.0 was measured and taken as the absorbance of the control sample. The absorbance difference between the control sample and the enzyme sample was determined. Separately, a calibration curve was created using L-glutamic acid-γ-monohydroxamic acid instead of the enzyme solution, and the amount of hydroxamic acid produced was determined from the absorbance difference. An enzyme activity that produces 1 μmol of hydroxamic acid per minute was defined as 1 U.

[0185] <2> Mutation introduction into TG In the example in WO2010 / 101256, mutations effective in improving heat resistance were comprehensively analyzed, and it was revealed that G157S / A / R, R208A / L / E, and G250R / F / S / N contribute to improved heat resistance. Therefore, in order to confirm the synergistic effect of these mutation combinations, mutations were introduced into the known heat-resistant mutant TG "MSS2βB" (D3C / G283C / S101P / G157S / G250R; hereinafter referred to as "HT00"; WO2010 / 101256) to construct mutant TG HT01 (D3C / G283C / S101P / G157S / R208E / G250S), HT02 (D3C / G283C / S101P / G157A / R208E / G250S), HT03 (D3C / G283C / S101P / G157R / R208E / G250S), and HT04 (D3C / G283C / S101P / G157R / R208E / G250N). Note that the variant TG is sometimes referred to as "HT-X" instead of "HTX" (where X is a positive integer). The procedure is as follows.

[0186] Using the expression plasmid of mutant TG HT00 (i.e., the expression plasmid of MSS2βB; WO2010 / 101256) as a template, PCR was performed using PrimeSTAR(R) Max DNA Polymerase (manufactured by Takara Bio Inc.) and the primers shown in Table 1 to prepare two kinds of PCR fragments. By ligating the two kinds of PCR fragments using the In-Fusion(R) HD Cloning Kit (manufactured by Takara Bio Inc.), a TG expression plasmid with the desired mutation introduced was obtained. The expression plasmid of the double mutant was constructed by introducing additional mutations in the same procedure using the constructed expression plasmid of the single mutant as a template. The expression plasmid of the triple or more mutants was constructed by introducing additional mutations in the same procedure using the constructed expression plasmid of the double or more mutants as a template.

[0187]

Table 1

[0188] <3> Construction of mutant TG-expressing strains the above <2> The mutant TG expression plasmid constructed using the method described above was introduced into Corynebacterium glutamicum YDK010 (WO 2002 / 081694 A1) by electroporation. YDK010 is a deficient strain of the cell surface protein PS2 from C. glutamicum AJ12036 (FERM BP-734). AJ12036 was originally deposited as an international deposit on March 26, 1984, at the National Institute of Microbial Science, Agency of Industrial Science and Technology (now the Patent Organism Depositary Center, National Institute of Technology and Evaluation, postal code: 292-0818, address: Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan), and was assigned accession number FERM BP-734. The bacterial cells after electroporation were cultured at 30°C on a CM-Dex plate containing 25 mg / L kanamycin (glucose 5 g / L, polypeptone 10 g / L, yeast extract 10 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.4 g / L, urea 3 g / L, FeSO4·7H2O 0.01 g / L, MnSO4·5H2O 0.01 g / L, biotin 10 μg / L, soy hydrochloride hydrolysate (as total nitrogen) 1.2 g / L, pH adjusted to 7.0 with KOH, agar 20 g / L). The obtained colonies were purified on a CM-Dex plate containing 25 mg / L kanamycin to obtain mutant TG-expressing strains. The obtained strains were cultured in 3 mL of CM-Dex liquid medium containing 25 mg / L kanamycin at 30°C for approximately 16 hours. 0.6 mL of the culture solution was mixed with 0.6 mL of 40% glycerol and stored as a glycerol stock at -80°C.

[0189] <4> Culture of mutant TG-expressing strains the above <3> A small amount of glycerol stock of the mutant TG-expressing strain obtained was scraped off and spread onto a CM-Dex plate containing 25 mg / L kanamycin, and incubated at 20°C for 3 days. The cells grown on the plate were inoculated into 10 mL of CM2G medium containing 25 mg / L kanamycin (5 g / L glucose, 10 g / L polypeptone, 10 g / L yeast extract, 5 g / L NaCl, 0.2 g / L DL-methionine, pH adjusted to 7.0 with KOH), and incubated with shaking at 30°C for 24 hours using a large test tube. 2.5 mL of the obtained culture medium was inoculated into 50 mL of TG production medium containing 25 mg / L kanamycin and 50 g / L CaCO3 (glucose 60 g / L, MgSO4·7H2O 1 g / L, FeSO4·7H2O 0.01 g / L, MnSO4·5H2O 0.01 g / L, (NH4)2SO4 30 g / L, KH2PO4 1.5 g / L, DL-methionine 0.15 g / L, thiamine hydrochloride 0.45 mg / L, Biotin 0.45 mg / L, pH adjusted to 7.5 with KOH), and cultured at 30°C for 48 hours using a Sakaguchi flask. For the Cys mutant-introduced TG-expressing strain, DTT was added at 5 hours after the start of culture to a final concentration of 3 mM. After the end of culture, the culture medium was collected in a plastic bottle and stored at -80°C.

[0190] <5> Purification of mutant TG the above <4> After thawing the culture medium obtained, the sterilized solution was obtained by centrifugation (8000 rpm, 4°C, 20 min) and filtration with a 0.45 μm filter. The sterilized solution was replaced with 20 mM MES buffer (pH 5.5) at room temperature using Sephadex G25(M) (GE Healthcare). After adjusting the pH to 7.0 with NaOH, alcalase (Sigma-Aldrich, P4860-50ML) was added to a weight ratio of 0.5% relative to mutant TG, and the reaction was carried out at 30°C for 17 hours to activate mutant TG. After the reaction was complete, the pH was adjusted to 5.5 with 10% acetic acid, and the entire volume was applied to a cation exchange column (Resource S 6 mL, GE Healthcare) that had been fully equilibrated with 20 mM MES buffer (pH 5.5). After re-equilibriumization with the same buffer, the protein fraction that eluted with a linear concentration gradient from 0 to 0.5 M NaCl, with NaCl eluting around 200 mM, was fractionated using UV absorption at a wavelength of 280 nm as an indicator. The TG activity and TG amount of each fraction were measured using the method described in Example 1 above, and fractions with low specific activity were removed, and fractions near the peak top with nearly equivalent specific activity were collected. The collected fractions were desalted with 20 mM phosphate buffer (pH 6.0) using HiPrep 26 / 10 Desalting (GE Healthcare), and the mutant TG was used in the following experiments. Both cation exchange chromatography and buffer substitution were performed at 4°C.

[0191] <6> Evaluation of the heat resistance of mutant TG (evaluation of mutant point optimized plants) the above <5> The purified enzyme of the mutant TG obtained was diluted in 20 mM MES buffer (pH 6.0) to 0.05 mg / mL (BSA equivalent) and heat-treated at 65°C or 70°C for 10 minutes using a thermal cycler. The enzyme reaction was carried out using 50 μL of the heat-treated sample according to the procedure in <1-2> above, and the absorbance difference (i.e., the absorbance difference between the control sample and the enzyme sample) was calculated. Similarly, the enzyme reaction was carried out on the untreated sample, and the absorbance difference was calculated. The absorbance difference in the heat-treated sample at each temperature was calculated as a relative value with the absorbance difference in the untreated sample set to 100%. This relative value of absorbance difference was considered as the residual TG activity after heat treatment at each temperature. In addition, as a control for non-heat-stable TG, the heat resistance of wild-type TG (prepared from Activa(R)) was analyzed using the same procedure. Furthermore, as a control for heat-stable TG, mutant TG HT00 (expression plasmid of mutant TG HT00 (WO2010 / 101256) was used in the same procedure as described above. <3> ~ <5> The heat resistance of the product (prepared according to the procedure) was analyzed.

[0192] The results are shown in Table 2. Compared to wild-type TG and mutant TG HT00, mutant TG HT01 to HT04 all showed high residual TG activity. Therefore, it was revealed that mutant TG HT01 to HT04 have improved heat resistance.

[0193] [Table 2]

[0194] <7> Evaluation of the heat resistance of mutant TG (Identification of mutation sites that contribute to improved heat resistance of HT03) the above <6> We investigated the mutation sites that contribute to the improved heat resistance of HT03, which showed the highest thermal stability. Using the mutant TG HT00 expression plasmid (WO2010 / 101256) as a template and the primers listed in Table 1, the above <2> Expression plasmids of mutant TG were constructed by introducing the single mutations G157R, R208E, and G250S into HT00 according to the procedure described above, and the purified enzymes of these mutant TG were used as described above. <3> ~ <5> The preparation was carried out according to the following procedure. The purified enzyme of the obtained mutant TG was diluted with 20 mM MES buffer (pH 6.0) to 0.05 mg / mL (BSA equivalent) and heated in a thermal cycler at 65°C or 70°C for 10 minutes. The enzyme reaction was carried out using 50 μL of the heat-treated sample according to the procedure in <1-2> above, and the absorbance difference (i.e., the absorbance difference between the control sample and the enzyme sample) was calculated. Similarly, the enzyme reaction was carried out on the untreated sample, and the absorbance difference was calculated. The absorbance difference in the heat-treated sample at each temperature was calculated as a relative value with the absorbance difference in the untreated sample set to 100%. This relative value of absorbance difference was considered as the residual TG activity after heat treatment at each temperature. In addition, as a control for non-heat-stable TG, the heat resistance of wild-type TG (prepared from Activa(R)) was analyzed using the same procedure. Furthermore, the heat resistance of HT00 and HT03 was analyzed as a control for the heat-resistant TG.

[0195] The results are shown in Table 3. The introduction of R208E improved the heat resistance of HT00 to the same level as HT03, indicating that R208E contributes to the improvement in the heat resistance of HT03.

[0196] [Table 3]

[0197] <8> Evaluation of heat tolerance of mutant TG (introduction of a single mutation into wild-type TG) Using the wild-type TG expression plasmid pPSPTG1 (Appl. Environ. Microbiol., 2003, 69(1), 358-366) as a template, and using the primers listed in Table 4, the above <2> Following the procedure, an expression plasmid for mutant TG was constructed by introducing the single mutations listed in Table 4 into wild-type TG, and the purified enzyme of these mutant TG was used as described above. <3> ~ <5> The following procedure was followed for preparation. pPSPTG1 is a secretory expression plasmid of the pro-structure-attached TG of the wild-type TG, Streptoverticillium mobaraense. The purified enzyme of the obtained mutant TG was diluted with 20 mM MES buffer (pH 6.0) to 0.05 mg / mL (BSA equivalent) and heat-treated at 60°C for 10 minutes using a thermal cycler. The enzymatic reaction was carried out using 50 μL of the heat-treated sample according to the procedure in <1-2> above, and the absorbance difference (i.e., the absorbance difference between the control sample and the enzyme sample) was calculated. Similarly, the enzymatic reaction was carried out with the untreated sample, and the absorbance difference was calculated. The absorbance difference in the heat-treated sample was calculated as a relative value with the absorbance difference in the untreated sample set to 100%. This relative absorbance difference was considered as the residual TG activity after heat treatment. Furthermore, as a control for non-heat-resistant TG, the heat resistance of wild-type TG (prepared from Activa(R)) was analyzed using the same procedure.

[0198] The results are shown in Table 4. Improved heat resistance was observed in all variant TGs. In particular, G275A was shown to contribute to the improved heat resistance.

[0199] [Table 4]

[0200] <9> Evaluation of heat resistance of mutant TG (introduction of the G275A mutation into heat-resistant TG) For HT03 and HT00 + R208E, which showed high heat resistance in <6> and <7> above, the heat resistance of the mutant TG with the introduced G275A found in <8> above was evaluated. Using the expression plasmids of mutant TG HT03 and HT00 + R208E as templates, and using the primers described in Table 1, the expression plasmids of mutant TG with G275A introduced into HT03 and HT00 + R208E were constructed according to the procedure in <2> above, and the purified enzymes of these mutant TGs were prepared according to the procedures in <3> to <5> above. The purified enzyme of the obtained mutant TG was diluted with 20 mM MES buffer (pH 6.0) to 0.05 mg / mL (in terms of BSA), and heat-treated at 70 °C or 72 °C for 10 minutes using a thermal cycler. Using 50 μL of the heat-treated sample, an enzyme reaction was carried out according to the procedure in <1-2> above, and the absorbance difference (that is, the absorbance difference between the control sample and the enzyme sample) was calculated. Similarly, an enzyme reaction was carried out on the untreated sample, and the absorbance difference was calculated. The absorbance difference in the sample after heat treatment at each temperature was calculated as a relative value with the absorbance difference in the untreated sample as 100%. The relative value of the absorbance difference was regarded as the residual TG activity after heat treatment at each temperature. Also, as a control, the heat resistance of wild-type TG (prepared from Activa (R)) was analyzed by the same procedure.

[0201] The results are shown in Table 5. The heat resistance of both HT00 + R208E and HT03 was improved by the introduction of G275A, indicating that G275A contributes to the improvement of heat resistance. The mutant TG (D3C / G283C / S101P / G157R / R208E / G250S / G275A) with G275A introduced into HT03 was named "HT10", and its expression plasmid was named "pPSPTG1-HT10". The mutant TG (D3C / G283C / S101P / G157S / R208E / G250R / G275A) with R208E and G275A introduced into HT00 was named "HT14", and its expression plasmid was named "pPSPTG1-HT14".

[0202]

Table 5

[0203] <10> Evaluation of the heat resistance of mutant TG - Introduction of SS bonds into heat-resistant TG - (1) the above <9> HT10 (D3C / G283C / S101P / G157R / R208E / G250S / G275A), which showed high thermal stability, was given SS bonds, and the heat resistance of the resulting mutant TG was evaluated.

[0204] <10-1>Introduction of SS bonds to HT10 the above <9> Using the TG expression plasmid pPSPTG1-HT10 constructed in [preparation method] as a template, PCR was performed using PrimeSTAR(R) Max DNA Polymerase (Takara Bio Inc.) and the primers listed in Table 6 to prepare two PCR fragments. By ligating the two PCR fragments using the In-Fusion(R) HD Cloning Kit (Takara Bio Inc.), a TG expression plasmid with the desired mutation was obtained.

[0205] [Table 6]

[0206] <10-2> Heat Resistance Evaluation The purified enzyme of each mutant TG is as described above. <3> ~ <5> The preparation was carried out according to the following procedure. The purified enzyme of the obtained mutant TG was diluted with 20 mM MES buffer (pH 6.0) to 0.05 mg / mL (BSA equivalent) and heated in a thermal cycler at 65-80°C for 10 minutes. The enzymatic reaction was carried out using 50 μL of the heat-treated sample according to the procedure in <1-2> above, and the absorbance difference (i.e., the absorbance difference between the control sample and the enzyme sample) was calculated. Similarly, the enzymatic reaction was carried out on the untreated sample, and the absorbance difference was calculated. The absorbance difference in the heat-treated sample at each temperature was calculated as a relative value with the absorbance difference in the untreated sample set to 100%. This relative value of absorbance difference was considered as the residual TG activity after heat treatment at each temperature. In addition, the heat resistance of wild-type TG (prepared from Activa(R)), HT00, HT03, and HT10 was analyzed using the same procedure.

[0207] The results are shown in Table 7. It was demonstrated that the introduction of SS bonds significantly improved heat resistance. HT10+E93C / V112C(D3C / G283C / S101P / G157R / R208E / G250S / G275A+E93C / V112C) was named "HT-16".

[0208] [Table 7]

[0209] <11> Evaluation of the heat resistance of mutant TG - Introduction of SS bonds into heat-resistant TG - (2) the above <9> HT10 (D3C / G283C / S101P / G157R / R208E / G250S / G275A), which showed high thermal stability, was given SS bonds, and the heat resistance of the resulting mutant TG was evaluated.

[0210] <11-1> Introduction of SS bonds to HT10 the above <9> Using the TG expression plasmid pPSPTG1-HT10 constructed as a template, two site-direct mutagenesis procedures were sequentially performed by PCR using PrimeSTAR(R) Max DNA Polymerase (Takara Bio Inc.) and the primers listed in Table 8 to obtain TG expression plasmids into which the target mutations were introduced.

[0211] [Table 8]

[0212] <11-2> Culture of mutant TG expression strains For each mutant TG constructed above <11-1>, <3> Glycerol stocks of mutant TG-expressing strains were obtained according to the following procedure. A small amount of the obtained mutant TG-expressing strain glycerol stock was scraped off and spread onto a CM-Dex plate containing 25 mg / L kanamycin, and incubated at 30°C for 2 days. The cells grown on the plate were inoculated into 800 μL of CM2G medium containing 25 mg / L kanamycin (5 g / L glucose, 10 g / L polypeptone, 10 g / L yeast extract, 5 g / L NaCl, 0.2 g / L DL-methionine, pH adjusted to 7.0 with KOH), and incubated with shaking at 30°C for 24 hours using a 96-well plate. 40 μL of the obtained culture medium was inoculated into 760 μL of TG production medium containing 25 mg / L kanamycin and 50 g / L CaCO3 (glucose 60 g / L, MgSO4·7H2O 1 g / L, FeSO4·7H2O 0.01 g / L, MnSO4·5H2O 0.01 g / L, (NH4)2SO4 30 g / L, KH2PO4 1.5 g / L, DL-methionine 0.15 g / L, thiamine hydrochloride 0.45 mg / L, Biotin 0.45 mg / L, pH adjusted to 7.5 with KOH), and cultured at 30°C for 48 hours using a 96-well plate. For the Cys mutant-introduced TG-expressing strain, DTT was added at 5 hours after the start of culture to a final concentration of 3 mM. After the end of culture, the culture medium was stored at -80°C.

[0213] Furthermore, for each mutant TG constructed in <10-1> above, <3> ~ <4> The culture medium of the mutant TG-expressing strain was obtained according to the procedure described above and stored at -80°C.

[0214] <11-3> Preparation of mutant TG solution After thawing the culture medium obtained in <11-2> above, the culture supernatant was obtained by centrifugation (5000 rpm, 20°C, 10 min). The protein concentration of the culture supernatant was quantified and diluted to 0.2 mg / mL. Protein concentration quantification was performed using a ThermoFisher Scientific kit and the Bradford method with BSA as the standard. 10 μL of 0.4% alcalase (Sigma-Aldrich, P4860-50ML) was added to 1000 μL of this diluted solution and reacted at 30°C for 17 hours to activate mutant TG. After the reaction was complete, 10 μL of 100 mM PFSF was added and allowed to stand at room temperature for 1 hour to obtain the mutant TG solution.

[0215] <11-4> Heat Resistance Evaluation The obtained mutant TG solution was diluted with 20 mM MES buffer (pH 6.0) to 0.05 mg / mL (BSA equivalent) and heated in a thermal cycler at 65-80°C for 10 minutes. The enzymatic reaction was carried out using 50 μL of the heat-treated sample according to the procedure in <1-2> above, and the absorbance difference (i.e., the absorbance difference between the control sample and the enzyme sample) was calculated. Similarly, the enzymatic reaction was carried out on the untreated sample, and the absorbance difference was calculated. The absorbance difference in the heat-treated sample at each temperature was calculated as a relative value with the absorbance difference in the untreated sample set to 100%. This relative absorbance difference was considered as the residual TG activity after heat treatment at each temperature. The heat resistance of wild-type TG (prepared from Activa(R)), HT00, and HT10 was also analyzed using the same procedure.

[0216] The results are shown in Table 9. It was demonstrated that the introduction of SS bonds significantly improved heat resistance.

[0217] [Table 9]

[0218] Example 2: Preparation and analysis of processed cheese using mutant transglutaminase (mutant TG). In this example, the effect of mutant TG on the physical properties of processed cheese was evaluated.

[0219] <1> Evaluation of mutant TG HT-03, HT-10, and HT-14 <1-1> Making Processed Cheese Processed cheese was prepared using an RVA (Rapid Visco Analyzer, Perten Instruments). Red cheddar cheese (Ito-Yokado Co., Ltd., USA), trisodium polyphosphate (Taihei Chemical Industry), and tap water were weighed in portions of 21g, 0.6g, and 8.4g respectively into RVA-specific aluminum containers. Wild-type TG (prepared from a pPSPTG1 expressing strain) or mutant TG (HT-03, HT-10, or HT-14; obtained in Example 1) were prepared as aqueous solutions in advance and added to the cheese so that the TG activity per 1g of protein was 2 U. For samples to which enzyme solution was added, the amount of tap water added was reduced by the amount of enzyme solution added. After enzyme addition, the aluminum containers were quickly placed in the RVA apparatus and heated and stirred at 70°C for 11 minutes. The stirring intensity was set to 200 rpm. Prior to the stirring step, a 30-second step of raising the temperature to 70°C without stirring was included. After heating and stirring, the temperature was raised to 85°C in 1 minute, and the enzyme was deactivated by heating and stirring for 5 minutes. Then, the aluminum container was removed, and the contents were poured into approximately 30 mL plastic cups, each containing about 10 g, and stored at 5°C overnight.

[0220] <1-2> Measurement of compressive stress value It is known that covalent bonds are formed between lysine residues or glutamine residues by the reaction of TG, improving the compressive stress of protein-containing foods. Therefore, as an index of the reactivity of each TG, the compressive stress of the prepared process cheese was measured using a Texture analyzer. The measurement conditions are shown below. Two sub-samples were used per sample, and the measurement was carried out with n = 2.

[0221] <Measurement conditions by Texture analyzer> Plunger used: Cylindrical (diameter 1 cm) Contact Force: 0.5 g Test Mode: Compression Test speed: 0.5 mm / sec Target mode: Strain Strain: 90% Trigger type: Auto Trigger force: 0.5 g

[0222] The results are shown in Figure 1. There was no significant difference in the compressive stress values between the wild-type TG-added product and the TG-free product (Control), and the wild-type TG did not contribute to the improvement of the compressive stress value. On the other hand, all mutant TGs contributed to the improvement of the compressive stress value. In particular, HT-10 showed the highest effect of improving the compressive stress, and the compressive stress value of the HT-10-added product was about twice that of the TG-free product (Control).

[0223] <1-3>Measurement of shape retention during heating Improving the shape retention of process cheese during heating is an issue when using process cheese in processed products where a heating process is carried out, and it has been found to be an added value. For the HT-10-added product with the highest compressive stress value in <2> above, the shape retention during heating was compared with the wild-type TG-added product and the TG-free product. Each process cheese was heated at 150 °C for 1 minute using an electric oven (SHARP, Helsio), and the melting behavior was observed.

[0224] The results (photos after 1-minute heating) are shown in Fig. 2. The wild-type TG-added product and the TG-free product were almost completely melted, but the wild-type TG-added product showed slightly higher shape retention than the TG-free product. Also, the HT-10-added product generally maintained its shape before heating, while the wild-type TG-added product and the TG-free product were almost completely melted. Therefore, it was shown that HT-10 contributes to the improvement of shape retention during heating.

[0225] <2>Evaluation of mutant TG HT-16 <2-1>Production of process cheese Using wild-type TG (prepared from the expression strain of pPSPTG1) or mutant TG HT-16 (obtained in Example 1) as TG, process cheese was produced according to the procedure of <1-1> above.

[0226] <2-2>Measurement of compression stress value The compression stress of the produced process cheese was measured with a Texture analyzer. The measurement conditions are shown below. Two small sub-samples were used per sample, and the measurement was carried out with n = 2.

[0227] <Measurement conditions by Texture analyzer> Plunger used: cylindrical (diameter 12.7 mm) Contact Force: 0.5 g Test Mode: Compression Test speed: 1.0 mm / sec Target mode: Distance Distance: 5 mm Trigger type: Auto Trigger force: 0.5 g

[0228] The results are shown in panel (A) of Fig. 3. The effect of improving compression stress was recognized by the addition of TG. Also, the addition of mutant TG HT-16 showed a higher effect of improving compression stress compared to the case where wild-type TG was added.

[0229] <2-3> Measurement of shape retention during heating The heat retention of the prepared processed cheese was measured according to the procedure described in <1-3> above.

[0230] The results (photographs after 1 minute of heating) are shown in panel (B) of Figure 3. The wild-type TG-added and TG-free samples melted almost completely. On the other hand, the HT-16-added sample largely retained its shape from before heating. Therefore, it was shown that HT-16 contributes to improved shape retention during heating.

[0231] Example 3: Analysis of acid resistance of mutant transglutaminase (mutant TG) In this example, the acid resistance of mutant TG was analyzed.

[0232] The purified enzyme of the mutant TG shown in Table 10 (obtained in Example 1) was diluted with 20 mM sodium acetate buffer (pH 4.0 or 5.0) or 20 mM MES buffer (pH 6.0) to a concentration of 0.56 U / mL and treated at 37°C for 4 hours. The residual TG activity after treatment at each pH was measured by hydroxamate. 500 μL of solution A (50 mM MES, 100 mM NH2OH, 10 mM reduced glutathione, 30 mM CBZ-Gln-Gly (Z-QG), adjusted to pH 6.0 with NaOH) was added to a 1.5 mL tube and heated at 37°C for 5 minutes. 150 μL of the enzyme solution after treatment at each pH was added, and the reaction was stopped at 37°C for 30 minutes. Then, 500 μL of solution B (1N HCl, 4% TCA, 1.67% FeCl3·6H2O) was added to stop the reaction. 200 μL of reaction stop solution was added to a 96-well plate, and the absorbance at 525 nm was measured using a plate reader to determine the absorbance of the enzyme sample. As a control, the absorbance of a sample reacted similarly with 20 mM MES pH 6.0 was measured and determined to be the absorbance of the control sample. The difference in absorbance between the control sample and the enzyme sample was calculated. The difference in absorbance in the samples after treatment at each pH was calculated as a relative value, with the difference in absorbance in the sample after treatment at pH 6.0 set to 100%. This relative value of absorbance difference was considered as the relative value of the remaining TG activity after treatment at each pH, ​​with the remaining TG activity after treatment at pH 6.0 set to 100%. The acid resistance of wild-type TG (prepared from Activa(R)) was also analyzed using the same procedure.

[0233] The results are shown in Table 10. While a decrease in residual TG activity was observed in wild-type TG after acid treatment, no decrease in residual TG activity was observed in mutant TG. Therefore, it was clear that mutant TG exhibits improved acid resistance.

[0234] [Table 10]

[0235] Example 4: Construction of mutant transglutaminase (mutant TG) and analysis of its heat resistance <1> Introducing SS binding to wild-type TG and mutant TG (D3C / G283C) A mutant TG expression plasmid was created by total synthesis by introducing a mutation into the TG gene of the TG expression plasmid pPSPTG1. The mutant name and mutation site are shown in Table 11.

[0236] [Table 11]

[0237] <2> Heat resistance evaluation of SS-binding-introduced mutants The heat resistance of the obtained mutant TG was evaluated. Based on the results of WO2008 / 099898, WT1 to WT5 are expected to show heat resistance equivalent to or less than that of wild-type TG (WT). On the other hand, based on the results of Example 1, SS1 to SS5 are expected to show improved heat resistance compared to SS. The purified enzyme of each mutant TG was used in Example 1. <3> ~ <5> The preparation was carried out according to the following procedure. The purified enzyme of the obtained mutant TG was diluted with 20 mM MES buffer (pH 6.0) to 0.05 mg / mL (BSA equivalent) and heat-treated at 60°C to 70°C for 10 minutes using a thermal cycler. The enzymatic reaction was carried out using 50 μL of the heat-treated sample according to the procedure of Example 1<1-2>, and the absorbance difference (i.e., the absorbance difference between the control sample and the enzyme sample) was calculated. Similarly, the enzymatic reaction was carried out on the untreated sample, and the absorbance difference was calculated. The absorbance difference in the samples after heat treatment at each temperature was calculated as a relative value with the absorbance difference in the untreated sample set to 100%. This relative value of absorbance difference was considered as the residual TG activity after heat treatment at each temperature. As a control, the heat resistance of wild-type TG (prepared from Activa(R)) was analyzed using the same procedure.

[0238] The results are shown in Table 12. WT1 to WT5 tended to exhibit heat resistance equivalent to or lower than that of wild-type TG(WT). SS1 to SS5 showed improved heat resistance compared to SS.

[0239] [Table 12]

[0240] Example 5: Analysis of alkali resistance of mutant transglutaminase (mutant TG) In this example, the alkali resistance of mutant TG was analyzed.

[0241] The purified enzyme of the mutant TG shown in Table 13 (obtained in Example 1) was diluted to 0.56 U / mL with 20 mM MES buffer (pH 6.0), 20 mM Tris-HCl buffer (pH 8.0), and 20 mM CAPS buffer (pH 10.0), and treated at 37°C for 4 hours. The residual TG activity after treatment at each pH was measured by the hydroxamate method. 500 μL of Solution A (50 mM MES, 100 mM NH2OH, 10 mM reduced glutathione, 30 mM CBZ-Gln-Gly (Z-QG), adjusted to pH 6.0 with NaOH) was added to a 1.5 mL tube and incubated at 37°C for 5 minutes. 150 μL of the enzyme solution treated at each pH was added, and the reaction was incubated at 37°C for 30 minutes. Then, 500 μL of solution B (1N HCl, 4% TCA, 1.67% FeCl3·6H2O) was added to stop the reaction. 200 μL of the stop solution was added to a 96-well plate, and the absorbance at 525 nm was measured using a plate reader and recorded as the absorbance of the enzyme sample. As a control, the absorbance of a sample reacted similarly using 20 mM MES pH 6.0 was measured and recorded as the absorbance of the control sample. The absorbance difference between the control sample and the enzyme sample was calculated. The absorbance difference in the samples treated at each pH was calculated as a relative value, with the absorbance difference in the sample treated at pH 6.0 set to 100%. This relative absorbance difference was considered as the relative value of the residual TG activity after treatment at each pH, ​​with the residual TG activity after treatment at pH 6.0 set to 100%. Furthermore, the alkali resistance of wild-type TG (prepared from Activa(R)) was analyzed using a similar procedure.

[0242] The results are shown in Table 13. Wild-type TG showed a decrease in residual TG activity upon alkaline treatment, while mutant TG showed a smaller decrease in residual TG activity upon alkaline treatment compared to wild-type TG. Therefore, it was revealed that mutant TG has improved alkali tolerance.

[0243] [Table 13]

[0244] Example 6: Preparation and analysis of ice cream using mutant transglutaminase (mutant TG). In this example, the effects of mutant TG on the physical properties and sensory quality of ice cream were evaluated.

[0245] Ice cream was produced using the formulations shown in Table 14 according to the following procedure. Mutant TG HT-16 (obtained in Example 1) was used as the triglyceride (TG). Ice cream produced without the addition of TG was used as the control.

[0246] [Table 14]

[0247] <Manufacturing Procedure> Liquid raw material mixing; Powdered raw material mixing (at this stage, 0.1% of TG HT-16 (100 U / g) is added); Heating and dissolution (75°C, 1 hour); Emulsification and homogenization (homogenizer, 70°C); Sterilization (88℃, 33 seconds); cooling; Aging (5°C for 2 hours); Freezing (-4℃); Filling (overrun 50%).

[0248] A shape retention test (50 mL, room temperature, 75 minutes standing) was conducted on the manufactured ice cream. In addition, a sensory evaluation was conducted on the manufactured ice cream by a panel of experts. In the sensory evaluation, the intensity of "richness and fattiness" of the control was set to 3 points, and the intensity of "richness and fattiness" of the TG-added product was evaluated on a scale of 1 point (weak) to 5 points (strong) in 0.5 point increments.

[0249] The results are shown in Figure 4. The addition of mutant TG HT-16 improved the shape retention and sensory quality (specifically, richness and fattiness) of the ice cream.

[0250] Example 7: Preparation and analysis of gummy candies using mutant transglutaminase (mutant TG). In this example, the effect of mutant TG on the physical properties of gummy candy was evaluated.

[0251] Gummy candies were manufactured using the formulations shown in Table 15 according to the following procedure. Mutant TG HT-16 (obtained in Example 1) was used as the triglyceride (TG). Gummy candies manufactured without the addition of TG were used as the control. [Table 15]

[0252] <Manufacturing Procedure> Add water for swelling to the gelatin and let it swell for at least 20 minutes (at room temperature); Heat and dissolve the corn syrup and granulated sugar at 90°C; Mix pre-swollen gelatin (a mixture of gelatin and swelling water) with water and dissolve in a 60°C water bath; Combine the sugar solution at 90°C and the gelatin solution at 60°C, and keep warm at 75°C. Add 50% citric acid solution, adjust pH to 3.5, and adjust Brix to 75. Add 0.3% TG HT-16 (100 U / g) and react at 60°C for 10 minutes, then at 75°C. Molding.

[0253] For the manufactured gummy candies, the 80% compression stress was measured using a Texture analyzer under the following conditions. Also, a shape retention test (40 °C, 2 hours) was conducted on the manufactured gummy candies.

[0254] <Measurement conditions by Texture analyzer> Plunger used: Wedge type (diameter 20.0 mm) Contact Force: 0.5 g Test Mode: Compression Test speed: 1.0 mm / sec Target mode: Strain Strain: 80% Trigger type: Auto Trigger force: 0.5 g

[0255] The results are shown in Fig. 5. The addition of the mutant TG HT-16 improved the compression stress and shape retention during heating of the gummy candies.

[0256] Example 8: Preparation and analysis of meatballs using mutant transglutaminase (mutant TG) In this example, the effect of mutant TG on the physical properties of meatballs was evaluated.

[0257] Three types of meatballs with different pH values were manufactured according to the following procedure with the formulations shown in Table 16. As TG, wild-type TG (prepared from the expression strain of pPSPTG1) or mutant TG HT-16 (obtained in Example 1) was used. The meatballs manufactured without adding TG were used as the Control.

[0258]

Table 16

[0259] <Manufacturing procedure> Raw material mixing (mix half the amount of only water; here, add 0.38% of TG (wild-type TG or HT-16; 100 U / g)); Stir (kitchen mixer, 10 sec); Add the remaining water; Stir (kitchen mixer, 10 sec); Mold (length 40 mm, width 90 mm, thickness 20 mm); Heat (80 °C, 30 min); Inactivate (90 °C, 30 min).

[0260] For the prepared meatballs, the breaking strength was measured using a Texture analyzer under the following conditions.

[0261] <Measurement conditions by Texture analyzer> Plunger used: Disk type (diameter 7 mm) Contact Force: 0.5 g Test Mode: Compression Test speed: 1.0 mm / sec Target mode: Distance Distance: 15 mm Trigger type: Auto Trigger force: 0.5 g

[0262] The results are shown in Table 17. At any pH condition, the addition of TG improved the breaking strength of the meatballs. Also, at any pH condition, the addition of the mutant TG HT-16 improved the breaking strength of the meatballs compared to the case where wild-type TG was added.

[0263]

Table 17

[0264] Example 9: Preparation and analysis of chawanmushi using mutant transglutaminase (mutant TG) In this example, the effect of mutant TG on the physical properties of chawanmushi was evaluated.

[0265] Two types of steamed eggs with different pH values were produced according to the following procedure with the formulations shown in Table 18. As the TG, wild-type TG (prepared from the expression strain of pPSPTG1) or mutant TG HT-16 (obtained in Example 1) was used.

[0266]

Table 18

[0267] <Manufacturing procedure> Mix liquid raw materials; Stir; Dispense (here, add TG (wild-type TG or HT-16; 100 U / g) at 0.49%); Dispense 20 mL each; Heat (75 °C, 20 minutes); Inactivate (90 °C, 20 minutes); Cool (room temperature, 7 hours).

[0268] For the produced steamed eggs, a 15-mm compression test (n = 5) was carried out using a Texture analyzer under the following conditions.

[0269] <Measurement conditions by Texture analyzer> Plunger used: Cylindrical (diameter 10 mm) Contact Force: 0.5 g Test Mode: Compression Test speed: 1.0 mm / sec Target mode: Distance Distance: 15 mm Trigger type: Auto Trigger force: 0.5 g

[0270] The results are shown in Table 19. At any pH condition, the compression strength of the steamed eggs was improved by the addition of TG. Also, at any pH condition, the addition of mutant TG HT-16 improved the compression strength of the steamed eggs as compared with the case where wild-type TG was added.

[0271]

Table 19

[0272] Example 10: Preparation and Analysis of Jelly Using Mutant Transglutaminase (Mutant TG) In this example, the effect of mutant TG on the physical properties of jelly (i.e., gelled gelatin) was evaluated.

[0273] Three types of jelly were produced according to the following procedure with the formulations shown in Table 20. As TG, wild-type TG (prepared from the expression strain of pPSPTG1) or mutant TG HT-16 (obtained in Example 1) was used.

[0274]

Table 20

[0275] <Manufacturing Procedure> Add 0.3% of TG (wild-type TG or HT-16; 100 U / g) to 15 g of the raw material mixture; Enzyme reaction (standing, 70 °C, 20 minutes); Enzyme inactivation (standing, 90 °C, 20 minutes); Cool at room temperature for 60 minutes; Cool overnight at 5 °C.

[0276] For the produced jelly, a compression test was performed using a Texture analyzer under the following conditions to measure the breaking strength and compressive strength.

[0277] <Measurement Conditions by Texture Analyzer> Plunger used: Cylindrical (diameter 12.7 mm) Contact Force: 0.5 g Test Mode: Compression Test speed: 1.0 mm / sec Target mode: Force Force: 1019.7 g Trigger type: Auto Trigger force: 0.5g

[0278] The results for the jellies of formulations 1 to 3 are shown in Figures 6 to 8, respectively. In all compositions, the addition of mutant TG HT-16 increased gel strength and improved the jelly's fracture strength and compressive strength (i.e., compressive stress) compared to the case with wild-type TG.

[0279] Example 11: Preparation and analysis of crab paste using mutant transglutaminase (mutant TG) In this example, the effects of mutant TG on the physical properties and sensory quality of crab paste were evaluated.

[0280] Crab fish paste was produced using the formulation shown in Table 21 and the following procedure. Mutant TG HT-16 (obtained in Example 1) was used as the triglyceride (TG). Crab fish paste produced without the addition of TG was used as the control.

[0281] [Table 21]

[0282] <Manufacturing Procedure> Mixing of raw materials excluding salt (at this stage, 0.2% of TG HT-16 (100 U / g) is added); Rough grinding; Adding salt; Salt rubbing; Drum heating (94℃); molding; Sterilization (90℃, 20 minutes).

[0283] The fracture strength of the manufactured crab fish cakes was measured using a texture analyzer. In addition, the manufactured crab fish cakes were subjected to sensory evaluation by a panel of experts. In the sensory evaluation, the strength of "hardness" and "flexibility and elongation" of the control sample was set to 3 points, while the strength of "hardness" and "flexibility and elongation" of the TG-added sample was evaluated on a scale from 1 point (weak) to 5 points (strong) in increments of 0.5 points.

[0284] The results are shown in Table 22. The addition of mutant TG HT-16 improved the fracture strength and sensory quality (specifically, "hardness" and "flexibility and elongation") of the crab fish cake.

[0285] [Table 22]

[0286] Example 12: Preparation and analysis of fried fish cake using mutant transglutaminase (mutant TG). In this example, the effect of mutant TG on the physical properties of fried fish cake was evaluated.

[0287] Fried fish cakes were manufactured using the formulation shown in Table 23 according to the following procedure. Mutant TG HT-16 (obtained in Example 1) was used as the triglyceride (TG). Fried fish cakes manufactured without the addition of TG were used as the control.

[0288] [Table 23]

[0289] <Manufacturing Procedure> Mixing of raw materials excluding salt (at this stage, 0.2% of TG HT-16 (100 U / g) is added); Rough grinding; Adding salt; Salt rubbing; Forming (10 mm thick satsuma-age); Oil condition (1) (135℃, 120 seconds); Oil condition (2) (165℃, 90 seconds); Cooling.

[0290] The fracture strength of the manufactured fried fish cake was measured using a texture analyzer.

[0291] The results are shown in Table 24. The addition of mutant TG HT-16 improved the breaking strength of the fried fish cake.

[0292] [Table 24]

[0293] <Explanation of Sequence Listings> Sequence ID 1: Base sequence of the portion of the TG gene in Streptoverticillium mobaraense that codes for mature TG. Sequence ID 2: Amino acid sequence of mature TG from Streptoverticillium mobaraense Sequence ID 3: Base sequence of the TG gene in Streptoverticillium mobaraense Sequence ID 4: Amino acid sequence of TG containing the pro-structural component of Streptoverticillium mobaraense Sequence IDs 5-114: Primers [Industrial applicability]

[0294] According to the present invention, a variant TG with high heat resistance and / or pH stability can be provided. This variant TG with high heat resistance and / or pH stability is useful, for example, in the modification or manufacture of food products such as processed cheese.

Claims

1. A method for manufacturing food, The process includes treating food ingredients with a variant transglutaminase under heating, acidic, or alkaline conditions. The mutant transglutaminase is a protein that has a specific mutation in the amino acid sequence of wild-type transglutaminase and possesses transglutaminase activity. The wild-type transglutaminase is a protein containing the amino acid sequence shown in Sequence ID No. 2, The mutant transglutaminase is a protein containing an amino acid sequence that has 90% or more identity with the amino acid sequence shown in Sequence ID No.

2. The aforementioned specific mutation is one that improves heat resistance and / or pH stability. The aforementioned specific mutation includes the following mutations (B) and / or (A): Mutation (B): A mutation that introduces a disulfide bond; Mutation (A): A mutation in one or more amino acid residues selected from the following: G275A, D1F, Y24(G, N), R48(I, K), S101(G, A, V, I, P, F, N, Q, Y, K, R, E), G102N, N139S, D142A, L147W, K152T, G157(A, V, I, S, N, K, R, H, D, E), R167G, N176D, K181R, E182D, H188Y, D189I, R208(L, A, E), T245A, S246(K, N, R), G250(A, V, L, M, P, F, W, S, T, N, Q, Y, K, R, H, D), S284P, H289I, G301W, K327F; The mutant transglutaminase is a mutant transglutaminase that satisfies (B1) and / or (A1) below, Method: (B1) The particular mutation includes the mutation (B), and the mutation (B) includes at least a combination of D3C / G283C and one or more mutations selected from E93C / V112C, A81C / V311C, A106C / D213C, E107C / Y217C, A160C / G228C, S84C / K121C, R79C / P169C, A113C / P220C, E119C / S299C, and R89C / S116C; (A1) The particular mutation includes mutation (A), and mutation (A) includes at least the following mutations: G275A.

2. The method according to claim 1, wherein the mutation (A) includes the following mutation: S101P / G157(A, R, S) / R208E / G250(N, R, S) / G275A.

3. The method according to claim 1 or 2, wherein the mutation (A) includes any of the following mutations: S101P / G157R / R208E / G250S / G275A, S101P / G157S / R208E / G250R / G275A.

4. The method according to any one of claims 1 to 3, wherein the mutation (B) includes a combination of D3C / G283C and E93C / V112C.

5. The method according to any one of claims 1 to 4, wherein the specific mutation includes the mutations (A) and (B).

6. The method according to any one of claims 1 to 5, wherein the residual activity of the mutant transglutaminase after heat treatment at 65°C and pH 6.0 for 10 minutes is 15% or more.

7. The method according to any one of claims 1 to 6, wherein the residual activity of the mutant transglutaminase after heat treatment at 75°C and pH 6.0 for 10 minutes is 10% or more.

8. The method according to any one of claims 1 to 7, wherein the residual activity of the mutant transglutaminase after treatment at pH 4.0 and 37°C for 4 hours is 60% or more, relative to the residual activity of the mutant transglutaminase after treatment at pH 6.0 and 37°C for 4 hours, which is set to 100%.

9. The method according to any one of claims 1 to 8, wherein the wild-type transglutaminase is a protein containing the amino acid sequence of mature transglutaminase of a bacterium of the genus Streptomyces.

10. The method according to claim 9, wherein the Streptomyces bacterium is Streptomyces mobaraensis.

11. The method according to any one of claims 1 to 10, satisfying any of (1) to (3) below: (1) The process is carried out under heating conditions, and the food is a dairy product, an egg product, a plant product, a meat product, a seafood product, or a gelatin product; (2) The process is carried out under acidic conditions, and the food is a grain, potato, bean, nut, meat, seafood, dairy product, oil and fat, seasoning, beverage, fruit juice, or processed product thereof; (3) The above process is carried out under alkaline conditions, and the food is a plant-based beverage, a seaweed extract, or a processed product thereof.

12. The method according to any one of claims 1 to 11, satisfying any of (1) to (3) below: (1) The process is carried out under heating conditions, and the food is processed cheese, pre-incubated yogurt, omelet, steamed egg custard, egg tofu, tofu, yuba (tofu skin), noodles, bread, ham, sausage, hamburger, ice cream, wheat dough, jelly, gummy candy, margarine, or processed food products; (2) The above process is carried out under acidic conditions, and the food is white rice, brown rice, vinegared rice, oatmeal, rice flour, wheat flour, buckwheat flour, rice bran, mochi, kneaded rice, bread, noodles, gluten, buckwheat, potatoes, soy milk, chocolate, fried tofu, pea protein, meat, gelatin, shrimp, crab, scallops, herring roe, yogurt, butter, cheese, ice cream, mayonnaise, ketchup, mustard, soy sauce, miso, coffee-based beverages, energy drinks, fruit juices, cocoa, beer, or alcohol. It is pulp, fruit juice gummies, or processed products thereof; (3) The above process is carried out under alkaline conditions, and the food is soy milk, kelp broth, or a processed product thereof.

13. The method according to any one of claims 1 to 12, wherein the heating temperature in the above step is 60°C or higher.

14. A composition for the manufacture of food products, It contains mutant transglutaminase, The aforementioned food is a food that is heated during manufacturing or a food with a low or high pH. The mutant transglutaminase is a protein that has a specific mutation in the amino acid sequence of wild-type transglutaminase and possesses transglutaminase activity. The wild-type transglutaminase is a protein containing the amino acid sequence shown in Sequence ID No. 2, The mutant transglutaminase is a protein containing an amino acid sequence that has 90% or more identity with the amino acid sequence shown in Sequence ID No.

2. The aforementioned specific mutation is one that improves heat resistance and / or pH stability. The aforementioned specific mutation includes the following mutations (B) and / or (A): Mutation (B): A mutation that introduces a disulfide bond; Mutation (A): A mutation in one or more amino acid residues selected from the following: G275A, D1F, Y24(G, N), R48(I, K), S101(G, A, V, I, P, F, N, Q, Y, K, R, E), G102N, N139S, D142A, L147W, K152T, G157(A, V, I, S, N, K, R, H, D, E), R167G, N176D, K181R, E182D, H188Y, D189I, R208(L, A, E), T245A, S246(K, N, R), G250(A, V, L, M, P, F, W, S, T, N, Q, Y, K, R, H, D), S284P, H289I, G301W, K327F; The composition wherein the mutant transglutaminase is a mutant transglutaminase that satisfies (B1) and / or (A1) below: (B1) The particular mutation comprises the mutation (B), and the mutation (B) comprises a combination of D3C / G283C and one or more mutations selected from E93C / V112C, A81C / V311C, A106C / D213C, E107C / Y217C, A160C / G228C, S84C / K121C, R79C / P169C, A113C / P220C, E119C / S299C, and R89C / S116C; (A1) The particular mutation includes mutation (A), and mutation (A) includes at least the following mutations: G275A.

15. The composition according to claim 14, wherein the mutation (A) includes the following mutation: S101P / G157(A, R, S) / R208E / G250(N, R, S) / G275A.

16. The mutation (A) includes any one of the following mutations, according to claim 14 or 15. The compositions described in the section: S101P / G157R / R208E / G250S / G275A, S101P / G157S / R208E / G250R / G275A.

17. The composition according to any one of claims 14 to 16, wherein the mutation (B) includes a combination of D3C / G283C and E93C / V112C.

18. The composition according to any one of claims 14 to 17, wherein the specific mutation comprises the mutations (A) and (B).

19. After heating the mutant transglutaminase at 65°C and pH 6.0 for 10 minutes The composition according to any one of claims 14 to 18, wherein the residual activity is 15% or more.

20. The composition according to any one of claims 14 to 19, wherein the residual activity of the mutant transglutaminase after heat treatment at 75°C and pH 6.0 for 10 minutes is 10% or more.

21. The composition according to any one of claims 14 to 20, wherein the residual activity of the mutant transglutaminase after treatment at pH 4.0 and 37°C for 4 hours is 60% or more, relative to the residual activity of the mutant transglutaminase after treatment at pH 6.0 and 37°C for 4 hours, which is set to 100%.

22. The composition according to any one of claims 14 to 21, wherein the wild-type transglutaminase is a protein containing the amino acid sequence of mature transglutaminase of a bacterium of the genus Streptomyces.

23. The composition according to claim 22, wherein the Streptomyces bacterium is Streptomyces mobaraensis.

24. A composition according to any one of claims 14 to 23, satisfying any of the following (1) to (3): (1) The process is carried out under heating conditions, and the food is a dairy product, an egg product, a plant product, a meat product, a seafood product, or a gelatin product; (2) The process is carried out under acidic conditions, and the food is a grain, potato, bean, nut, meat, seafood, dairy product, oil and fat, seasoning, beverage, fruit juice, or processed product thereof; (3) The above process is carried out under alkaline conditions, and the food is a plant-based beverage, a seaweed extract, or a processed product thereof.

25. A composition according to any one of claims 14 to 24, satisfying any of the following (1) to (3): (1) The process is carried out under heating conditions, and the food is processed cheese, pre-incubated yogurt, omelet, steamed egg custard, egg tofu, tofu, yuba (tofu skin), noodles, bread, ham, sausage, hamburger, ice cream, wheat dough, jelly, gummy candy, margarine, or processed food products; (2) The process is carried out under acidic conditions, and the food is white rice, brown rice, vinegared rice, oatmeal, rice flour, wheat flour, buckwheat flour, rice bran, mochi, kneaded rice, bread, noodles, gluten, buckwheat, potatoes, soy milk, chocolate, fried tofu, pea protein, meat, gelatin, shrimp, crab, scallop, herring roe, yogurt, butter, cheese, ice cream, mayonnaise, ketchup, mustard, soy sauce, miso, coffee-based beverages, energy drinks, fruit juices, cocoa, beer, sake lees, fruit gummies, or processed products thereof; (3) The above process is carried out under alkaline conditions, and the food is soy milk, kelp broth, or a processed product thereof.

26. The composition according to any one of claims 14 to 25, wherein the heating temperature in the manufacturing process is 60°C or higher.

27. A mutant transglutaminase, It has a specific mutation in the amino acid sequence of wild-type transglutaminase and possesses transglutaminase activity. The wild-type transglutaminase is a protein containing the amino acid sequence shown in Sequence ID No. 2, The mutant transglutaminase is 90% of the amino acid sequence shown in SEQ ID NO 2. This is a protein containing an amino acid sequence having the above-mentioned identity, The aforementioned specific mutation is one that improves heat resistance and / or pH stability. The aforementioned specific mutation is the following mutation (B) and / or (A): Mutation (B): A mutation that introduces a disulfide bond; Mutation (A): A mutation in one or more amino acid residues selected from the following: G275A, D1F, Y24(G, N), R48(I, K), S101(G, A, V, I, P, F, N, Q, Y, K, R, E), G102N, N139S, D142A, L147W, K152T, G157(A, V, I, S, N, K, R, H, D, E), R167G, N176D, K181R, E182D, H188Y, D189I, R208(L, A, E), T245A, S246(K, N, R), G250(A, V, L, M, P, F, W, S, T, N, Q, Y, K, R, H, D), S284P, H289I, G301W, K327F; Includes, Mutant transglutaminases that satisfy the following (B1) and / or (A1): (B1) The particular mutation includes the mutation (B), and the mutation (B) includes at least a combination of D3C / G283C and one or more mutations selected from E93C / V112C, A81C / V311C, A106C / D213C, E107C / Y217C, A160C / G228C, S84C / K121C, R79C / P169C, A113C / P220C, E119C / S299C, and R89C / S116C; (A1) The particular mutation includes mutation (A), and mutation (A) includes at least the following mutations: G275A.

28. The mutant transglutamin according to claim 27, wherein the mutation (A) includes the following mutation. -Ze: S101P / G157(A, R, S) / R208E / G250(N, R, S) / G275A.

29. The mutation (A) includes any one of the following mutations, according to claim 27 or 28. Mutant transglutaminases described in the section: S101P / G157R / R208E / G250S / G275A, S101P / G157S / R208E / G250R / G275A.

30. The mutant transglutaminase according to any one of claims 27 to 29, wherein the mutation (B) includes a combination of D3C / G283C and E93C / V112C.

31. The mutant transglutaminase according to any one of claims 27 to 30, wherein the specific mutation comprises the mutations (A) and (B).

32. The mutant transglutaminase according to any one of claims 27 to 31, wherein the residual activity after heating the mutant transglutaminase at 65°C and pH 6.0 for 10 minutes is 15% or more.

33. The mutant transglutaminase according to any one of claims 27 to 32, wherein the residual activity after heating the mutant transglutaminase at 75°C and pH 6.0 for 10 minutes is 10% or more.

34. The mutant transglutaminase according to any one of claims 27 to 33, wherein the residual activity after treating the mutant transglutaminase at pH 4.0 and 37°C for 4 hours is 60% or more, relative to the residual activity after treating the mutant transglutaminase at pH 6.0 and 37°C for 4 hours, which is set to 100%.

35. The mutant transglutaminase according to any one of claims 27 to 34, wherein the wild-type transglutaminase is a protein containing the amino acid sequence of mature transglutaminase of a bacterium of the genus Streptomyces.

36. The mutant transglutaminase according to claim 35, wherein the Streptomyces bacterium is Streptomyces mobaraensis.

37. A gene encoding a mutant transglutaminase according to any one of claims 27 to 36.

38. A vector carrying the gene described in claim 37.

39. A microorganism having the gene described in claim 37.

40. The microorganism according to claim 39, which is a bacterium or yeast.

41. The microorganism according to claim 39 or 40, which is a Corynebacterium or a bacterium of the Enterobacteriaceae family.

42. The microorganism according to any one of claims 39 to 41, which is a bacterium of the genus Corynebacterium or Escherichia.

43. The microorganism according to any one of claims 39 to 42, which is Corynebacterium glutamicum or Escherichia coli.

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