MODIFIED TRANSGLUTAMINASE

Abstract

The present invention addresses the issues of finding a novel mutation effective for the improvement of transglutaminase and providing a highly useful modified transglutaminase. Disclosed is a highly useful modified transglutaminase having an amino acid substitution that results in an increase of high temperature reactivity or a lowering in pH stability in a weakly acidic region.

Claims

1. A modified transglutaminase having an amino acid sequence containing any one of following amino acid substitutions (1) to (11) in an amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having 80% or more of identity to the amino acid sequence (provided that a difference in the amino acid sequence occurs at a position other than the position of the amino acid substitution), wherein a change in properties corresponding to the amino acid substitution(s) is observed: (1) amino acid substitutions at mutation points Y34 and F305, wherein an amino acid after the substitution at the mutation point Y34 is W, an amino acid after the substitution at the mutation point F305 is W, and a change in properties due to the amino acid substitutions is an improvement of high-temperature reactivity; (2) an amino acid substitution at a mutation point of M288, wherein an amino acid after the substitution is L, F, or Y, and a change in properties due to the amino acid substitution is a decrease in the pH stability in the range of weak acidity; (3) an amino acid substitution at a mutation point of D3, wherein an amino acid after the substitution is W or K, and a change in properties due to the amino acid substitution is an improvement of high-temperature reactivity; (4) amino acid substitutions at mutation points of D3 and F305, wherein an amino acid after the substitution at the mutation point D3 is G, K, N, P, or W, an amino acid after the substitution at the mutation point F305 is W, and a change in properties due to the amino acid substitutions is an improvement of high-temperature reactivity; (5) amino acid substitutions at mutation points V65 and F305, wherein an amino acid after the substitution at the mutation point V65 is I, an amino acid after the substitution at the mutation point F305 is W, and a change in properties due to the amino acid substitutions is an improvement of high-temperature reactivity; (6) amino acid substitutions at mutation points S303 and F305, wherein an amino acid after the substitution at the mutation point S303 is R or K, an amino acid after the substitution at the mutation point F305 is W, and a change in properties due to the amino acid substitutions is an improvement of high-temperature reactivity; (7) an amino acid substitution at a mutation point of R5, wherein an amino acid after the substitution is H, and a change in properties due to the amino acid substitution is a decrease in the pH stability in the range of weak acidity; (8) an amino acid substitution at a mutation point of V6, wherein an amino acid after the substitution is D, and a change in properties due to the amino acid substitution is a decrease in the pH stability in the range of weak acidity; (9) an amino acid substitution at a mutation point of W59, wherein an amino acid after the substitution is T, and a change in properties due to the amino acid substitution is a decrease in the pH stability in the range of weak acidity; (10) an amino acid substitution at a mutation point of S61, wherein an amino acid after the substitution is G or R, and a change in properties due to the amino acid substitution is a decrease in the pH stability in the range of weak acidity; (11) an amino acid substitution at a mutation point of V290, wherein an amino acid after the substitution is I, and a change in properties due to the amino acid substitution is a decrease in the pH stability in the range of weak acidity;

2. The modified transglutaminase according to claim 1, wherein the identity is 82% or more.

3. The modified transglutaminase according to claim 1, wherein the identity is 85% or more.

4. The modified transglutaminase according to claim 1, wherein the identity is 90% or more.

5. The modified transglutaminase according to claim 1, consisting of an amino acid sequence of any of SEQ ID NOs: 2 to 22.

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

7. The gene according to claim 6, including a nucleotide sequence of any of SEQ ID NOs: 23 to 43.

8. A recombinant DNA including the gene according to claim 6.

9. A microorganism having the recombinant DNA according to claim 8.

10. An enzyme preparation containing the modified transglutaminase according to claim 1.

11. A method for preparing a modified transglutaminase, including the following steps (I) to (III): (I) a step of providing a nucleic acid encoding the amino acid sequence of the modified transglutaminase of claim 1; (II) a step of expressing the nucleic acid; and (III) a step of recovering an expressed product.

12. The preparing method according to claim 11, wherein the amino acid sequence is an amino acid sequence of any of SEQ ID NOs: 2 to 22.

13. The preparing method according to claim 12, wherein the nucleic acid includes a nucleotide sequence of any of SEQ ID NOs: 23 to 43.

Description

EXAMPLES

<Search for Effective Mutation Points to Improve Properties>

[0138] For obtaining highly useful modified transglutaminases, an attempt was made to improve the enzyme function (improvement of high-temperature reactivity, a decrease in pH stability in the range of weak acidity) of a transglutaminase derived from Streptomyces mobaraensis (wild type enzyme; SEQ ID NO. 1) by using protein engineering. First, for introducing a mutation due to an amino acid substitution, a CASTing library (for example, see Angew Chem Int Ed Engl. 2006 February 13; 45(8): 123641) and alanine scanning (Ala scanning) (for example, J Mol Biol 1995 February 17; 246(2): 317-30) were utilized to select candidate mutation points. Next, a mutation was introduced into each mutation point by the following method to prepare a mutated enzyme.

1. Preparation of Mutated Enzyme

1-1. Introduction of Mutation

[0139] (1) A PCR primer was designed for introduction of a mutation.

[0140] (2) With use of a primer set for each mutation point, PCR was performed by using, as a template, plasmid pET20b in which transglutaminase gene (SEQ ID NO. 50) was incorporated (15 cycles of reactions at 98° C. for 1 minute, at 98° C. for 10 seconds, at 60° C. for 15 seconds and at 68° C. for 2 minutes were performed, and the resultant product was reacted at 68° C. for 5 minutes, and then allowed to stand at 4° C.).

[0141] (3) DpnI (1.5 μL/tube) was added to the PCR reaction solution (25 μL/tube) for treatment (37° C. 3 hours or longer).

[0142] (4) Ligation treatment (16° C., overnight) was performed using T4 kinase.

[0143] (5) E. coli BL21 (DE3) was transformed with the ligation reaction solution (11 μL/tube), and cultured in an LB medium containing ampicillin (37° C., overnight).

1-2. Obtainment of Enzyme Extract

[0144] (1) A strain into which a mutation was introduced (mutated strain) is inoculated into a TB medium containing ampicillin, and culture at 33° C. for 48 hours. IPTG (final concentration: 0.1 mM) was added 24 hours after the start of the culture.

[0145] (2) After centrifugation of the culture solution (3,000 g 10 minutes), the supernatant was removed, and the bacterial cells are recovered.

[0146] (3) A bacteriolytic agent is added to lyse the bacterial cells.

[0147] (4) After centrifugation of the bacterial lysate (3,000 g×10 minutes), the supernatant was recovered and used as an enzyme extract.

1-3. Maturation (Removal of Propeptide Sequence)

[0148] (1) Equal amounts of the enzyme extract and a 2 mg/mL protease (Dispase) solution were mixed to cause a reaction (30° C., 2 hours or longer).

[0149] (2) After centrifugation of the mixed solution (3,000 g×10 minutes), the supernatant was recovered and used as a maturated enzyme.

2. Purification of Enzyme

[0150] Each maturated enzyme was purified by using TALON Spin columns (Takara Bio Inc.) and HisTALON Buffer Set (Takara Bio Inc.), according to the attached protocol. The purified maturated enzymes were used for evaluating properties of the mutated enzymes.

3. Evaluation of Property of Mutated Enzyme

[0151] The following activity measurement methods were used to evaluate the properties of each prepared mutated enzyme.

<Activity Measurement Method>

[0152] The maturated enzyme is diluted to an appropriate concentration with 200 mM Tris-HCl pH 6.0 (sample solution). To 10 μL of the sample solution, 100 μL of the substrate solution (R-1) was added, and the solution mixture is mixed and caused to react at 37° C. for 10 minutes. A coloring solution (R-2) (100 μL) was added to stop the reaction and form an Fe complex, and then the absorbance at 525 nm was measured. As a control, the absorbance of a previously heat-inactivated enzyme solution subjected to a similar reaction was measured, and the absorbance difference thereof from the sample solution was determined. Separately, a calibration curve was prepared using L-glutamic acid-γ-monohydroxamic acid instead of the enzyme liquid, and the amount of hydroxamic acid generated from the absorbance difference was determined. The enzyme activity that produced 1 μmol of hydroxamic acid per minute was defined as one unit (1 U).

(Substrate Solution (R-1))

[0153] 2.42 g of 2-Amino-2-hydroxymethyl-1.3-propanediol, 0.70 g of hydroxyammonium hydrochloride, 0.31 g of reduced glutathione, and 1.01 g of Z-Gln-Gly (benzyloxycarbonyl-L-glutaminylglycine) were dissolved in distilled water to attain a total volume of 100 mL (pH 6.0).

(Substrate Solution (R-2))

[0154] Mixed were 30 mL of 3M hydrochloric acid solution, 30 mL of 12% trichloroacetic acid solution, and 30 mL of 5% iron (III) chloride solution.

3-1. Evaluation of High-Temperature Reactivity

[0155] The maturated enzyme was diluted 5 times with 200 mM Tris-HCl pH 6.0 (sample solution), and the activity was measured at a reaction temperature of 60° C.

[0156] The activity at the reaction temperature of 60° C. was compared with that of a wild type, and amino acid substitutions effective for improving the high-temperature reactivity were identified.

[0157] FIG. 1 shows the evaluation results regarding the high-temperature reactivity. All of the amino acid substitutions, resulting in an improvement of the high-temperature reactivity, were determined as effective amino acid substitutions. Particularly in the cases of D3P/F305W substitution, D3W/F305W substitution, and Y34W/F305W substitution, the activity was improved to 250% or more of that of the wild type, and these substitutions were determined to be particularly effective amino acid substitutions.

TABLE-US-00001 TABLE 1 Mutation Activity at 60° C. (U/g) No mutation (wild type) 11 D3G F305W 24 D3K — 25 D3K F305W 21 D3N F305W 26 D3P F305W 34 D3W — 26 D3W F305W 31 Y34W F305W 30 V65I F305W 20 S303K F305W 24 S303R F305W 21

3-2. Evaluation of pH Stability

[0158] The maturated enzyme was diluted 2 times with 200 mM Britton-Robinson Buffer at each pH (pH 4, 4.5, 5, 5.5, 6), and treated at 30° C. for 60 minutes, to obtain pH-treated samples. The maturated enzyme was diluted 2 times with 200 mM Tris-HCl pH 6.0 to obtain untreated samples. The untreated samples and the pH-treated samples were diluted 2 times with 500 mM Tris-HCl pH 6.0, then activities were measured, and evaluation was made on relative activities thereof with respect to the activity of the untreated sample of the wild-type enzyme, which was assumed to be 100.

[0159] Amino acid substitutions resulting in relative activities of 0 at pH 4.0 or pH 4.5 were identified.

[0160] Table 2 shows the evaluation results regarding a decrease in pH stability in the range of weak acidity. All of the amino acid substitutions, resulting in relative activities at pH 4.0 of 0, were determined to be amino acid substitutions effective for decreasing pH stability in the range of weak acidity. In the case of the M288L substitution and the M288Y substitution, the relative activity at pH 4.5 was also 0, and thus it was determined to be a particularly effective amino acid substitution. The M288L substitution is highly useful also in that it shows high activity in the neutral range (pH 6). From the viewpoint of activity in the neutral range (pH 6), it can be said that the S61G substitution, the S61R substitution, the M288F substitution, and the V290I substitution are also highly useful.

TABLE-US-00002 TABLE 2 Relative activity to activity of wild-type enzyme w/o pH treatment assumed to be 100 Mutation Untreated pH 4 pH 4.5 pH 5 pH 5.5 pH 6 No mutation 100 73 103 109 107 120 (wild type) R5H 42 0 35 43 39 67 V6D 37 0 38 28 36 52 W59T 60 0 47 64 85 82 S61G 114 0 53 98 131 120 S61R 166 0 127 182 121 145 M288F 122 0 36 95 133 139 M288L 116 0 0 58 103 126 M288Y 83 0 0 51 75 70 V290I 97 0 84 125 116 119

INDUSTRIAL APPLICABILITY

[0161] The modified transglutaminase of the present invention has improved practically important properties, and has high industrial value. Therefore, the modified transglutaminase of the present invention is expected to be used in new applications as well as in existing applications.

[0162] The present invention is not limited to the description of the embodiments and examples of the invention. Various modifications that can be easily conceived by those skilled in the art without departing from the scope of the claims are also included in the present invention. The entire contents of the articles, published patent publications, patent publications, and the like specified in the present specification are incorporated herein by reference.

SEQUENCE LISTING FREE TEXT

[0163] SEQ ID NO: 2: Explanation of artificial sequence: D3G variant

[0164] SEQ ID NO: 3: Explanation of artificial sequence: D3K variant

[0165] SEQ ID NO: 4: Explanation of artificial sequence: D3W variant

[0166] SEQ ID NO: 5: Explanation of artificial sequence: D3G/F305W variant

[0167] SEQ ID NO: 6: Explanation of artificial sequence: D3K/F305W variant

[0168] SEQ ID NO: 7: Explanation of artificial sequence: D3N/F305W variant

[0169] SEQ ID NO: 8: Explanation of artificial sequence: D3P/F305W variant

[0170] SEQ ID NO: 9: Explanation of artificial sequence: D3W/F305W variant

[0171] SEQ ID NO: 10: Explanation of artificial sequence: Y34W/F305W variant

[0172] SEQ ID NO: I1: Explanation of artificial sequence: V65I/F305W variant

[0173] SEQ ID NO: 12: Explanation of artificial sequence: S303K/F305W variant

[0174] SEQ ID NO: 13: Explanation of artificial sequence: S303R/F305W variant

[0175] SEQ ID NO: 14: Explanation of artificial sequence: R5H variant

[0176] SEQ ID NO: 15: Explanation of artificial sequence: V6D variant

[0177] SEQ ID NO: 16: Explanation of artificial sequence: W59T variant

[0178] SEQ ID NO: 17: Explanation of artificial sequence: S61G variant

[0179] SEQ ID NO: 18: Explanation of artificial sequence: S61R variant

[0180] SEQ ID NO: 19: Explanation of artificial sequence: M288F variant

[0181] SEQ ID NO: 20: Explanation of artificial sequence: M288L variant

[0182] SEQ ID NO: 21: Explanation of artificial sequence: M288Y variant

[0183] SEQ ID NO: 22: Explanation of artificial sequence: V290I variant

[0184] SEQ ID NO: 23: Explanation of artificial sequence: D3G variant

[0185] SEQ ID NO: 24: Explanation of artificial sequence: D3K variant

[0186] SEQ ID NO: 25: Explanation of artificial sequence: D3W variant

[0187] SEQ ID NO: 26: Explanation of artificial sequence: D3G/F305W variant

[0188] SEQ ID NO: 27: Explanation of artificial sequence: D3K/F305W variant

[0189] SEQ ID NO: 28: Explanation of artificial sequence: D3N/F305W variant

[0190] SEQ ID NO: 29: Explanation of artificial sequence: D3P/F305W variant

[0191] SEQ ID NO: 30: Explanation of artificial sequence: D3W/F305W variant

[0192] SEQ ID NO: 31: Explanation of artificial sequence: Y34W/F305W variant

[0193] SEQ ID NO: 32: Explanation of artificial sequence: V65I/F305W variant

[0194] SEQ ID NO: 33: Explanation of artificial sequence: S303K/F305W variant

[0195] SEQ ID NO: 34: Explanation of artificial sequence: S303R/F305W variant

[0196] SEQ ID NO: 35: Explanation of artificial sequence: R5H variant

[0197] SEQ ID NO: 36: Explanation of artificial sequence: V6D variant

[0198] SEQ ID NO: 37: Explanation of artificial sequence: W59T variant

[0199] SEQ ID NO: 38: Explanation of artificial sequence: S61G variant

[0200] SEQ ID NO: 39: Explanation of artificial sequence: S61R variant

[0201] SEQ ID NO: 40: Explanation of artificial sequence: M288F variant

[0202] SEQ ID NO: 41: Explanation of artificial sequence: M288L variant

[0203] SEQ ID NO: 42: Explanation of artificial sequence: M288Y variant

[0204] SEQ ID NO: 43: Explanation of artificial sequence: V290I variant