JP2008194004A - Mutant transglutaminase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a transglutaminase mutant protein improved in its activity for a polymer substrate. <P>SOLUTION: This transglutaminase mutant protein improved in the activity for the polymer substrate (especially, the protein substrate materials such as casein, gelatin, collagen, keratin, etc.) is obtained by introducing a suitable mutation into a WT (wild type) transglutaminase. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a mutant protein of actinomycete-derived transglutaminase. Transglutaminase (also simply referred to as TG) is widely used in food processing and the like because it forms a gel-like substance by forming a crosslink between proteins. Mutant TGs with improved transglutaminase activity and thermal stability contribute to the reduction of the required amount, and mutant TGs with altered substrate specificity and optimum pH make it possible to apply this enzyme to new fields.

Transglutaminase is an enzyme that catalyzes an acyl transfer reaction of a γ-carboxamide group in a peptide chain of a protein. When this enzyme is allowed to act on a protein, ε- (γ-Glu) -Lys cross-linking reaction and substitution reaction to Glu by deamidation of Gln can occur. As transglutaminase, an animal-derived one and a microorganism-derived one have been known so far. The former is a Ca ²⁺ -dependent enzyme and is distributed in animal organs, skin, blood and the like. For example, there are guinea pig liver transglutaminase (Non-patent document 1), human epidermal keratinocyte transglutaminase (Non-patent document 2), human blood coagulation factor XIII (Non-patent document 3), and the like. About the latter, the thing of Ca2 ⁺ independent was discovered from the microbe of Streptomyces genus. Examples include Streptomyces griseocarneus NBRC 12776, Streptomyces cinnamoneus NBRC 12852, Streptomyces mobaraensis NBRC 13819, and the like. Among these, the transglutaminase found in the culture supernatant of Streptomyces mobaraensis variant is called MTG (Microbial Transglutaminase). Furthermore, Ca ²⁺ -independent transglutaminase has also been discovered from Streptomyces lydicus NRRL B-3446 (Patent Document 1). As a result of peptide mapping and gene structure analysis, it has been found that the primary structure of transglutaminase produced by these microorganisms has no homology with those derived from animals (Patent Document 2).

MTG is a monomeric protein consisting of 331 amino acids and having a molecular weight of about 38,000 (Non-patent Document 4). Since MTG is produced from a culture such as the above fungus through a purification operation, there are problems in terms of supply amount, efficiency, and the like. Attempts have also been made to produce transglutaminase by genetic engineering techniques. Escherichia coli (E. coli), a method by secretory expression by yeast or the like (Patent Document 3), or after expressing MTG as a protein inclusion body in Escherichia coli, the inclusion body is solubilized with a protein denaturant, A method for producing active MTG by regenerating through a denaturant treatment (Patent Document 4) and a method for secreting and expressing MTG in Corynebacterium glutamicum (Patent Document 5) have been reported. . Unlike animal-derived transglutaminase, MTG and other microorganism-derived transglutaminases are Ca ²⁺ -independent. Therefore, production of gelled foods such as jelly, yogurt, cheese, or gel-like cosmetics and meat (Patent Document 6). In addition, it is an enzyme with high industrial applicability, such as being used for the production of heat-stable microcapsule materials and immobilized enzyme carriers. As for the enzyme reaction conditions, for example, in the case of gelled food, if the enzyme reaction time is short, it does not harden. Conversely, if the reaction time is too long, it becomes too hard and does not become a commercial product. Therefore, when MTG is used for the production of gelled foods such as jelly, yogurt, cheese, or gel cosmetics, and for improving meat quality, etc., by adjusting the concentration of substrate and enzyme, reaction temperature, and reaction time, The object is being made. However, as foods and reagents using MTG diversify, it may not be possible to produce the target product by simply adjusting the concentration, temperature, time, etc., and the necessity of modifying the enzyme activity of MTG arises. ing.
Patent Document 7 discloses the three-dimensional structure of MTG, which discloses that the active center is the Cys residue at the 64th position, but improves the enzyme activity by mutating an amino acid near the active center in the three-dimensional structure. It does not specifically show that

  So far, MTG has been mainly used in the food field. Although the possibility of application in various applications such as fibers, chemical products (photographic film, leather tannery), feed, cosmetics, and medicine has been suggested, there are few that have been put to practical use in these fields. The reason for this is that MTG has low reactivity when used industrially in this field. MTG reacts with glutamine residues in proteins, but does not react with all glutamine residues in proteins, but reacts with glutamine residues that react with the primary sequence and conformation of amino acids around glutamine residues. It is known that there are not glutamine residues. In addition, it is known that the reactivity to a reactive glutamine residue and an individual glutamine residue differs depending on the type of transglutaminase (Patent Document 8). When this mutant transglutaminase with altered reactivity to glutamine residues is allowed to act on the protein, the degree of cross-linking of the protein, ie gelation, is greater than when the original (ie, wild type) transglutaminase is allowed to act. The degree of will also change.

  In the textile field, modification of wool with transglutaminase is known. That is, it is known that by treating wool with transglutaminase, it is possible to impart shrinkage resistance, anti-pilling property and hydrophobicity while maintaining the texture (Patent Document 9). However, when transglutaminase is used in the fiber field, its price is high, so if the amount of transglutaminase used can be reduced by improving the activity of wool against keratin, transglutaminase can be used to modify wool. It will be possible to do this.

  It is known that photographic film, photographic paper and the like are coated with gelatin crosslinked by a chemical crosslinking agent. Gelatin is also a food material and is known to be cross-linked with transglutaminase. It is also known that a photographic film using transglutaminase as an alternative to a chemical crosslinking agent is useful (Patent Document 10). However, when transglutaminase is used for photographic film, its price is high, so if the amount of transglutaminase used can be reduced by improving the activity against gelatin, transglutaminase should be used for the production of photographic film. Will be possible.

  Skin tanning is the process of making hide / skin an animal skin (hide / skin) that is processed and processed in multiple steps to make the leather durable and flexible. This is done by crosslinking the collagen of the skin with hexavalent chromium. Hexavalent chromium is harmful and undesirable for release into the environment, so there is a strong demand for the development of alternative methods. Regarding the use of transglutaminase for tanning, Patent Document 11 discloses that microbial-derived transglutaminase can be used for tanning, but there is no example that actually acts on skin, and it has not yet been put into practical use. . Jelenska et al. Reported that when animal-derived transglutaminase is used, the activity against natural collagen is low (Non-patent Document 5). Microorganism-derived transglutaminase is also considered to have not been put into practical use because of its low activity against natural collagen, and when transglutaminase is used for tanning, a mutant transglutaminase with high activity against collagen is considered necessary. Although collagen is insoluble, gelatin, which is a heat-denatured product thereof, is soluble, and its activity is easily evaluated as a substrate for transglutaminase. A mutant transglutaminase having high activity against gelatin is expected to have high activity against collagen and is considered to be a mutant transglutaminase applicable to tanning.

  In finishing leather products, there is a process called casein finishing, and it is known that leather is provided with a casein finish obtained by curing with a chemical crosslinking agent. Also in use for casein finishing, in practical use, it is considered that a reduction in the amount of use by improving activity against casein is required.

  As described above, when TG is used in a general-purpose area (fiber processing, photographic film production, leather tanning, etc.), it is necessary to improve the enzyme activity of TG and reduce the amount used. Improvement of enzyme activity is inevitable.

In order to modify the enzyme activity of MTG, it is necessary to prepare a mutant of MTG, evaluate its activity, etc., and find a superior mutant. In order to produce a mutant, it is necessary to manipulate a wild-type gene, and therefore it is necessary that a recombinant protein can be prepared. In the case of MTG, a secretory expression system using Corynebacterium glutamicum has been established (Patent Document 5). Excellent mutants are mutants with improved activity against polymer substrates such as casein, gelatin, collagen and keratin.
Japanese National Patent Publication No. 10-504721 European Patent Publication 0 481 504 A1 Japanese Patent Laid-Open No. 5-199883 JP-A-6-30771 International Publication No. 2002/081694 Pamphlet Japanese Unexamined Patent Publication No. 64-27471 International Publication No. 2002/014518 Pamphlet JP 2001-321192 A JP-A-3-213574 JP 2004-29383 A US Patent 6,849,095 K. Ikura et al., Biochemistry, 27, 2898, 1988 MA Phillips et al., Proc. Natl. Acad. Sci. USA, 87, 9333, 1990 A. Ichinose et al., Biochemistry, 25, 6900, 1990 T. Kanaji et al., Journal of Biological Chemistry. 268, 11565, 1993 MM Jelenska et al., Biochimica et Biophysica Acta, 616, 167-178, 1980

  An object of the present invention is to provide a transglutaminase mutant protein having improved activity against a macromolecular substrate (particularly a protein substrate) as compared to wild-type (hereinafter also simply referred to as WT) transglutaminase. In particular, by providing a transglutaminase mutant protein with improved activity of cross-linking casein, gelatin, collagen, and keratin, the amount of enzyme used is reduced, thereby modifying wool, producing photographic films, tanning, etc. Makes it possible to apply transglutaminase.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have introduced transmutaminase mutations with improved activity against protein substrates such as casein, gelatin, collagen and keratin by introducing appropriate mutations into WT transglutaminase. It has been found that body proteins can be produced, and the present invention has been completed.

The present invention is outlined as follows.
[1] A protein having transglutaminase activity and having the following amino acid sequence of A, B, C, or D:
(A) In SEQ ID NO: 2, 3, 5, 10, 12, 14, 16, 26, 28, 30, 32, 34, 38, 42, 49, 58, 59, 65, 74, 75, 77, 199, An amino acid sequence having a mutation at at least one position selected from positions 238, 241, 248, 284, 289, 299, 303, 312 and 320;
(B) In the arrangement of (A), 3, 5, 10, 12, 14, 16, 26, 28, 30, 32, 34, 38, 42, 49, 58, 59, 65, 74, 75, 77, Amino acid sequences having 1 to several residue substitutions, deletions, additions and / or insertions at positions other than positions 199, 238, 241, 248, 284, 289, 299, 303, 312 or 320;
(C) An amino acid sequence selected from SEQ ID NOs: 4, 6, 8, 10 and 12 or an amino acid sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10 and 12 has a homology of 70% or more 3, 5, 10, 12, 14, 16, 26, 28, 30, 32, 34, 38, 42, 49, 58, 59, SEQ ID NO: 2 in the amino acid sequence of the protein having transglutaminase activity. An amino acid sequence having a mutation at at least one position selected from positions corresponding to positions 65, 74, 75, 77, 199, 238, 241, 248, 284, 289, 299, 303, 312 and 320; or (D ) In the sequence of (C), 3, 5, 10, 12, 14, 16, 26, 28, 30, 32, 34, 38, 42, 49, 58, 59, 65, 74, 75 of SEQ ID NO: 2. Amino acids having 1 to several residue substitutions, deletions, additions and / or insertions at positions other than positions corresponding to positions 7, 199, 238, 241, 248, 284, 289, 299, 303, 312 or 320 An array.
[2] The position of the mutation is selected from positions 10, 12, 14, 16, 30, 32, 34, 38, 42, 49, 65, 77, 199, 299, 312 and 320 [1] The protein according to 1.
[3] The protein according to [1] or [2] above, wherein the mutation is selected from the following mutations:
1) 3rd position: substitution with phenylalanine, valine, leucine, isoleucine or asparagine;
2) 5-position: substitution with lysine;
3) 10th position: substitution with serine;
4) 12th position: substitution with serine;
5) 14th position: substitution with asparagine;
6) 16th position: substitution with threonine, serine, leucine, lysine or asparagine;
7) 26th position: substitution with phenylalanine;
8) 28th position: substitution with aspartic acid;
9) Position 30: substitution to aspartic acid;
10) Position 32: substitution to aspartic acid;
11) 34th position: substitution with phenylalanine;
12) 38th position: substitution with phenylalanine;
13) Position 42: substitution to histidine;
14) 49th position: substitution with arginine;
15) 58th position: substitution with aspartic acid;
16) 59th position: substitution with leucine or phenylalanine;
17) 65th position: substitution with isoleucine;
18) 74th position: substitution with asparagine, alanine or serine;
19) 75th position: substitution with phenylalanine, alanine, histidine, isoleucine, tryptophan, valine or proline;
20) Position 77: substitution with serine, alanine, phenylalanine, leucine, valine, asparagine, tryptophan, tyrosine or proline;
21) Position 199: substitution with alanine, leucine or threonine;
22) Position 238: substitution to phenylalanine or leucine;
23) Position 241: substitution with alanine;
24) Position 248: substitution with serine;
25) position 284: substitution to glycine or threonine;
26) Position 289: substitution with phenylalanine or tyrosine;
27) Position 299: substitution with leucine;
28) 303 position: substitution with phenylalanine;
29) 312 position: substitution with valine; and
30) Position 320: substitution with aspartic acid.
[4] A polynucleotide encoding the protein according to any one of [1] to [3] above.
[5] A recombinant vector comprising the polynucleotide according to [4] above.
[6] A host cell transformed with the recombinant vector according to [5] above.
[7] The production of the protein according to any one of [1] to [3] above, wherein the host cell according to [6] is cultured and a protein having transglutaminase activity is collected. Method.
[8] The protein according to any one of [1] to [3] above, the protein produced by the method according to [7] above, or the host cell according to [6] above as a polymer substrate A method for processing the polymer substrate, comprising a step of acting.
[9] The method according to [8] above, wherein the polymer substrate is a substrate protein.
[10] The method according to [9] above, wherein the substrate protein is collagen, gelatin, casein or keratin.
[11] The method according to any one of [8] to [10], wherein the method is for leather tanning, fiber processing, or photographic film production.

  ADVANTAGE OF THE INVENTION According to this invention, the transglutaminase which improved the activity with respect to a polymeric substrate can be provided by modification | change of WT transglutaminase. Furthermore, a new product and a new technique can be provided by using transglutaminase having improved activity against a polymer substrate.

  Hereinafter, the present invention will be specifically described. Transglutaminase is widely used for the production of foods such as gelatin, cheese, yogurt, tofu, salmon, ham, sausage, noodles and the like, and for improving meat quality (Japanese Patent Laid-Open No. 64-27471). In addition, it is an enzyme used for various industrial purposes, such as being used in the production of heat-stable microcapsule materials and immobilized enzyme carriers. Transglutaminase catalyzes the acyl transfer reaction of the γ-carboxyamide group of a glutamine residue in the peptide chain of a protein molecule. When the ε-amino group of the lysine residue in the protein molecule acts as an acyl acceptor, an ε- (γ-Glu) -Lys bond is formed in the protein molecule and between the molecules.

Transglutaminase is broadly divided into those of the Ca ²⁺ -independent and microbial origin that of Ca ²⁺ dependence of animal origin. As TG derived from microorganisms, those derived from actinomycetes are known. Representative examples of nucleotide sequences and amino acid sequences of TGs derived from various actinomycetes are shown in the following table.

Even when a transglutaminase homolog other than these is used, a mutant protein having improved transglutaminase activity (particularly, activity against a macromolecular substrate such as a protein substrate) can be obtained according to the method described herein. That is, since transglutaminase may have a slightly different sequence depending on the microorganism species and strain, the amino acid sequence of transglutaminase that can be modified to obtain the mutant protein of the present invention is the above amino acid sequence 2, 4, 6 , 8, 10 and 12 as long as the protein has transglutaminase activity, SEQ ID NO: 2, 4, 6, 8, 10 or 12, respectively 70%, preferably 80%, more preferably 90%, Particularly preferably, if the protein has a homology of 95% or more, most preferably 98% or more, alignment is performed in the same manner as described later, and the corresponding amino acid residue is identified, whereby the amino acid residue is identified. Mutations can be introduced into the mutants of the present invention. Here, “homology” refers to identity.
The homology analysis can be calculated using, for example, default parameters of “Genetyx ver. 7 (Genetyx Corporation)”.
Further, a polynucleotide that hybridizes under stringent conditions with a sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, or 11 or a probe that can be prepared from these sequences. A polynucleotide encoding a protein having transglutaminase activity can also be used for the production of the mutant protein of the present invention.
Here, “stringent conditions” means, for example, nucleotide sequences having high homology, for example, nucleotide sequences having homology of 80, 90, 95, 97 or 99% or more hybridize to each other, and homology is more than that. Under conditions where nucleotide sequences with low specificity do not hybridize, specifically, washing conditions for normal Southern hybridization, 60 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, more preferably 68 The conditions include washing once, more preferably 2-3 times at a salt concentration and temperature corresponding to ° C., 0.1 × SSC, and 0.1% SDS.
As a probe, a partial sequence of the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9 or 11 can also be used. Such a probe can be prepared by PCR using an oligonucleotide prepared based on the nucleotide sequence as a primer and a DNA fragment containing the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9 or 11 as a template. it can. For example, when a DNA fragment having a length of about 300 bp is used as a probe, hybridization washing conditions include 50 ° C., 2 × SSC, and 0.1% SDS.

  “Transglutaminase activity” means the activity of forming a bridge between a glutamine residue and a lysine residue in a protein as described above. In the present specification, it particularly refers to the activity of forming a cross-link between substrate proteins (eg, casein, gelatin, collagen, keratin, etc.), that is, gelling the substrate protein. That is, the activity with respect to protein refers to the activity of crosslinking (gelling) the protein.

  “WT (wild type) transglutaminase” means a naturally occurring transglutaminase into which no amino acid sequence mutation has been introduced. When the activity of a transglutaminase variant is “improved” compared to this WT transglutaminase activity, this variant is a preferred variant of the present invention.

  The “transglutaminase mutant protein (referred to as the transglutaminase mutant of the present invention or simply the mutant of the present invention)” is represented by (A) 3, 5, 10, 12, 14, 16, 26, 28, 30, 32, 34, 38, 42, 49, 58, 59, 65, 74, 75, 77, 199, 238, 241, 248, 284, 289, 299, 303, 312 and 320 A protein having an amino acid sequence having a mutation at at least one position; or (B) an amino acid sequence having substitution, deletion, addition and / or insertion of 1 to several residues at positions other than these positions. And (C) an amino acid sequence having a mutation at at least one position selected from positions corresponding to the above positions in an amino acid sequence selected from SEQ ID NOs: 4, 6, 8, 10 and 12; or (D) Proteins having amino acid sequences having one to several residue substitutions, deletions, additions and / or insertions at positions other than such corresponding positions are also within the scope of the present invention.

  The positions of the mutations are positions 10, 12, 14, 16, 30, 32, 34, 38, 42, 49, 65, 77, 199, 299, 312 and 320 (in the sequences (C) and (D) above. (Including the corresponding position). Mutations at these positions are preferable from the viewpoint of activity.

  In the present specification, in the amino acid sequence selected from SEQ ID NOs: 4, 6, 8, 10 and 12, the position “corresponding” to the above position is obtained by aligning these sequences with the amino acid sequence of SEQ ID NO: 2. It is determined. It is also possible to align the amino acid sequence of a transglutaminase homolog other than those shown in this specification with the amino acid sequence of SEQ ID NO: 2 to determine the “corresponding” position, and to introduce a mutation at that position. is there. For example, if gaps are introduced when aligning SEQ ID NO: 2 with other sequences, it should be noted that the positions referred to above may shift back and forth. See, for example, FIG. 3 for the corresponding positions.

  Algorithms used for alignment of amino acid sequences are described in NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool), Karlin et al., Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993). [The algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) (Altschul et al., Nucleic Acids Res., 25: 3389-3402 (1997))], Needleman et al., J. Mol. Biol., 48 The algorithm described in 444-453 (1970) [the algorithm is incorporated into the GAP program in the GCG software package], the algorithm described in Myers and Miller, CABIOS, 4: 11-17 (1988) [the algorithm Is incorporated into the ALIGN program (version 2.0) which is part of the CGC sequence alignment software package], an algorithm described in Pearson et al., Proc. Natl. Acad. Sci. USA, 85: 2444-2448 (1988) [ The Algorithm is incorporated into FASTA program in the GCG software package] and the like.

  In addition to Streptomyces mobaraensis-derived transglutaminase (MTG), an enzyme having transglutaminase activity in which homology between this enzyme and the amino acid sequence is recognized, or a trans that is expected to have a three-dimensional structure similar to this enzyme. It is sufficiently possible to modify an enzyme having glutaminase activity based on the three-dimensional structure of MTG. In MTG, amino acid substitution that is effective for modification of substrate specificity is also effective for related enzymes derived from microorganisms such as Streptomyces cinnamonius and Streptomyces rigix (Japanese translation of PCT publication No. 10-504721). It is predicted.

  The mutation at each of the above positions is preferably a substitution of an amino acid residue. As long as the resulting transglutaminase mutant protein has transglutaminase activity, particularly as long as it has higher activity than WT transglutaminase. Any substitution can be used.

Such mutations are preferably selected from the following mutations:
1) 3rd position: substitution with phenylalanine, valine, leucine, isoleucine or asparagine;
2) 5-position: substitution with lysine;
3) 10th position: substitution with serine;
4) 12th position: substitution with serine;
5) 14th position: substitution with asparagine;
6) 16th position: substitution with threonine, serine, leucine, lysine or asparagine;
7) 26th position: substitution with phenylalanine;
8) 28th position: substitution with aspartic acid;
9) Position 30: substitution to aspartic acid;
10) Position 32: substitution to aspartic acid;
11) 34th position: substitution with phenylalanine;
12) 38th position: substitution with phenylalanine;
13) Position 42: substitution to histidine;
14) 49th position: substitution with arginine;
15) 58th position: substitution with aspartic acid;
16) 59th position: substitution with leucine or phenylalanine;
17) 65th position: substitution with isoleucine;
18) 74th position: substitution with asparagine, alanine or serine;
19) 75th position: substitution with phenylalanine, alanine, histidine, isoleucine, tryptophan, valine or proline;
20) Position 77: substitution with serine, alanine, phenylalanine, leucine, valine, asparagine, tryptophan, tyrosine or proline;
21) Position 199: substitution with alanine, leucine or threonine;
22) Position 238: substitution to phenylalanine or leucine;
23) Position 241: substitution with alanine;
24) Position 248: substitution with serine;
25) position 284: substitution to glycine or threonine;
26) Position 289: substitution with phenylalanine or tyrosine;
27) Position 299: substitution with leucine;
28) 303 position: substitution with phenylalanine;
29) 312 position: substitution with valine; and
30) Position 320: substitution with aspartic acid.
These positions are also “corresponding positions” in the sequences (C) and (D). The mutant of the present invention may have only one or a combination of these amino acid mutations. Such a mutant protein can be prepared by using site-directed mutagenesis methods known in the art. For example, the mutation is introduced using Stratagene's QuikChange II Site-Directed Mutagenesis Kit, etc., and the mutation is introduced into a TG expression plasmid (eg, pPSPTG11), and the plasmid containing the desired mutation is transformed into various host cells (eg, E by transporation into a .coli or coryneform bacterium), a transglutaminase mutant protein production strain (hereinafter also referred to as a mutant TG production strain) can be obtained. Transglutaminase mutant protein can be obtained by culturing the obtained mutant TG production strain and producing / acquiring the protein.

  Substitutions, deletions, additions and / or insertions at positions other than the above positions are as long as the resulting transglutaminase mutant protein has transglutaminase activity, and particularly has higher activity than WT transglutaminase. There is no particular limitation. The number of substitutions, deletions, additions and / or insertions of such a mutant protein is 1 to several residues (1 to 30, preferably 1 to 15, more preferably 1 to 5, 4, 3 or 2). Residue), but as long as the mutant protein has transglutaminase activity, in particular as long as it has higher activity than WT transglutaminase, any number of amino acid substitutions, insertions, Additions and deletions may be included. For example, when this mutation is a substitution, substitution with a similar amino acid (ie, conservative amino acid substitution) is expected to hardly affect the function of the protein, and therefore substitution with a similar amino acid is preferable. Here, “similar amino acids” mean amino acids that are similar in physicochemical properties, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids ( Gln, Asn), basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids with hydroxyl groups (Ser, Thr), amino acids with small side chains (Gly, Ala, Ser, Thr, Met) Amino acids classified into the same group. Examples of conservative amino acid substitutions are well known in the art.

  The present invention also provides a polynucleotide encoding the transglutaminase mutant protein of the present invention (referred to as the polynucleotide of the present invention). Such polynucleotides can be obtained using methods known in the art (for example, the methods described herein). Using an appropriate restriction enzyme, this polynucleotide can be inserted into a vector to obtain a recombinant vector.

  The vector backbone used in the recombinant vector of the present invention is appropriately selected according to its purpose (for example, cloning, protein expression) or a host cell into which this vector is to be introduced, preferably suitable for a microorganism, Can be used. Where the recombinant vector is an expression vector, the expression vector comprises a polynucleotide of the invention operably linked to a suitable promoter, preferably a transcription termination signal, ie a terminator, downstream of the polynucleotide of the invention. Includes area. Furthermore, a selection marker gene (drug resistance gene, gene complementing an auxotrophic mutation, etc.) for selecting a transformant can be further included. Further, it may contain a sequence encoding a tag sequence useful for separation and purification of the expressed protein. In addition, the vector may be one that is integrated into the genome of the target host cell.

  Vectors are known transformation techniques such as competent cell method, protoplast method, calcium phosphate coprecipitation method, polyethylene glycol method, lithium method, electroporation method, microinjection method, liposome fusion method, particle gun method, etc. Can be introduced into the subject host cell.

  The present invention also provides a host cell transformed with such a recombinant vector (hereinafter also referred to as the transformant of the present invention). Examples of host cells include prokaryotic cells such as E. coli and actinomycetes, and eukaryotic cells such as yeast. If it is desired to express the protein, the host cell is a host cell that is suitable for the production of the protein and is capable of expressing the mutant protein of the invention in a functional form. The transformant of the present invention can be obtained using the above transformation technique. Any microorganism can be used as a host cell as long as it is a developed recombinant vector system, and preferred host cells include E. coli, Corynebacterium glutamicum, yeast, Examples include, but are not limited to, Bacillus bacteria and various actinomycetes.

  The transformant of the present invention can be cultured under conditions usually used in the art. For example, the culture temperature, time, medium composition, pH, stirring conditions and the like can be appropriately selected depending on the transformant to be cultured.

  The transglutaminase mutant protein of the present invention is expressed by the transformant of the present invention. The transglutaminase mutant protein may be used by being secreted from the transformant or separated and purified from the transformant, or may be used as it is as a transformant containing the mutant protein. . Separation and purification of the protein can be performed according to a method known per se.

  Further, the transglutaminase mutant protein of the present invention may be expressed as a pro form and then treated with an appropriate protease (eg, subtilisin) to form a mature form, or the pro form and protease may be simultaneously used in the target host cell. It may be expressed to obtain a mature body.

The obtained transformant can be cultured and the transglutaminase mutant protein can be separated and purified as follows, for example. Dispense CM2G medium containing kanamycin into test tubes. The transformant is inoculated and pre-cultured at 30 ° C. for 24 hours. Dispense 50 ml each of MM medium containing kanamycin and CaCO ₃ into the Sakaguchi flask, subculture the preculture, and culture. After centrifugation of the culture solution, the supernatant is filtered and the solvent is replaced with 20 mM phosphate buffer, pH 7.0 using Sephadex G25 (M). Subtilisin is added in an amount of 1/100 of transglutaminase and reacted at 30 ° C. for 16 hours to activate transglutaminase. The activated solution is exchanged for cation exchange chromatography equilibration buffer using Sephadex G25 (M). Next, the entire amount is loaded onto a cation exchange column (Resource S 6 ml; manufactured by GE Healthcare Bioscience) that has been sufficiently equilibrated with the same buffer. After re-equilibration with the same buffer, fractionate the protein fraction that is eluted with a linear concentration gradient from 0 to 0.5 M NaCl, with the UV absorption at a wavelength of 280 nm as the index, and the NaCl concentration eluting around 200 mM. . The activity of the fraction and the amount of protein are measured, and the fraction near the peak top, which has almost the same specific activity except for the fraction with low specific activity, is collected. The collected fraction is replaced with 20 mM phosphate buffer, pH 6.0 using Sephadex G25 (M). The resulting sample is diluted to about 1 mg / ml with 20 mM phosphate buffer, pH 6.0, and stored at -80 ° C until use.

  The present invention also provides a method for processing a macromolecular substrate (particularly a substrate protein) using the transglutaminase mutant protein of the present invention or a transformant expressing the protein. Examples of the protein that can serve as a substrate for transglutaminase include proteins having glutamine and / or lysine residues accessible to transglutaminase. In particular, preferred substrate proteins of the present invention are collagen, gelatin, casein and keratin. This method can be applied to various uses including a protein crosslinking step (for example, tanning, production of photographic film, processing of fibers (eg, wool)), and is particularly suitable for tanning.

  Based on the above findings obtained by the present inventors, a polymer substrate (for example, a substrate protein such as casein, gelatin, collagen, keratin, etc.) can be obtained by selectively introducing mutations into the above-mentioned residues of transglutaminase. A transglutaminase mutant protein with improved cross-linking (ie gelling) activity is obtained.

  The transglutaminase mutant protein of the present invention may be obtained by introducing a random mutation or a site-specific mutation and conducting screening to obtain a mutant transglutaminase having the same activity or higher activity.

  For example, introduction of a random mutation into the MTG gene and primary screening of the MTG mutant can be performed as follows. The sequence of S. mobaraense-derived transglutaminase gene (ie, MTG gene) has already been determined (Eur. J. Biochem., 257, 570-576, 1998). With reference to this sequence, the mutation is introduced into the mature body region (about 1,000 bp) encoding the MTG active body so that the mutation is 2 to 3 sites at the base sequence level. The MTG with the mutation introduced into the MTG gene portion of pPSPTG1 by treating the PCR product with the mutation and the MTG expression vector pPSPTG1 (App. Env. Micro., 2003, 69, 358-366) with restriction enzyme and ligation. It can be easily replaced with a gene. E. coli is transformed with the plasmid prepared by ligation, and the resulting colony is mixed culture and plasmid extraction is performed. By electroporation, Corynebacterium glutamicum AJ12036 (FERM BP-734) (WO 2002/081694) is transformed with the obtained plasmid and the pro-cleaving enzyme expression plasmid pVSSI.

  Alternatively, a specific amino acid residue may be selected, and a mutant may be created in which each selected residue is substituted with several different amino acids. In this case, a mutation may be introduced near the 64th Cys residue of the active center. Mutation can be introduced by introducing a mutation into a pro-TG expression plasmid (eg, pPSPTG11) using various known site-specific mutation introduction methods (eg, QuikChange II Site-Directed Mutagenesis Kit from Stratagene). Good. A plasmid containing the desired mutation is introduced into Corynebacterium by electroporation.

  In addition, mutation points can be analyzed by confirming the base sequence of the strain obtained by screening. The specific activity can be calculated by measuring the activity and the amount of protein with respect to the confirmed mutation point. The activity and protein amount can be measured by the methods described in the literature (Prot. Exp. Puri., 2002, 26, 329-335).

  After identifying the highly active mutation site by confirming the base sequence, if there is a mutant with multiple mutations, create a mutant with only one mutation point, The effect on the activity of the mutation point can also be accurately evaluated. Mutation can be easily introduced by using Stratagene's QuikChange II Site-Directed Mutagenesis Kit.

  Transformants can be assayed in a plate assay (described in Example 1) and highly active variants can be subjected to further screening. It can also be assayed by the usual hydroxamate method (J. Biol. Chem., 241, 5518-5525, 1966). The assay may use 96 wells or test tubes. If a strain into which no mutation has been introduced is used as a control, one having an absorbance equal to or higher than that of the control can be selected.

  The activity unit of transglutaminase in the hydroxamate method is defined as follows. That is, the reaction is carried out using benzyloxycarbonyl-L-glutaminylglycine and hydroxylamine as substrates, and the resulting hydroxamic acid is converted to an iron complex in the presence of trichloroacetic acid, and then the amount is measured by absorbance at 525 nm. A calibration curve is prepared from the amount of hydroxamic acid in this way, and the amount of enzyme that produces 1 μmol of hydroxamate per minute is defined as one unit of transglutaminase activity. The details of this measurement method are as already reported (for example, see Japanese Patent Application Laid-Open No. 64-27471).

  As specifically described in Example 6, when the substrate is a polymer such as casein or gelatin, the TG activity measured by the hydroxamate method using low molecules (benzyloxycarbonyl-L-glutaminylglycine and hydroxylamine) as substrates is The TG activity measured by gelation of such a polymer does not always coincide. Therefore, TG activity is preferably measured directly by gelation of a macromolecular substrate (substrate protein).

  The activity of the separated and purified mutant protein can be evaluated by casein gelation or gelatin gelation. The casein gelation time is measured as follows. Dispense 1 ml of 10% sodium caseinate solution (pH 7.5) into a test tube and preincubate at 37 ° C. Add 100 μl of 0.2 mg / ml TG solution, incubate at 37 ° C., and set the gelation time when the gel stops flowing for 5 seconds even if it falls. The gelatin gelation time can be measured as follows. Dispense 1 ml of 10% gelatin solution (pH 5.0) into a test tube and preincubate at 37 ° C. Add 100 μl of 0.2 mg / ml TG solution, incubate at 37 ° C., and set the gelation time when the gel stops flowing for 5 seconds even if it falls. Since the gelation time varies depending on the substrate quality and dissolution state, the gelation ability can be determined by comparing the gelation time of the purified MTG standard (control) measured with the same lot of substrate at the same time. That is, the gelation ability of the mutant = the gelation time of the purified MTG preparation / the gelation time of the mutant. If this value is greater than 1, the mutant will have a faster gel time than the purified MTG preparation.

  Collagen is an insoluble protein, but is known to be cross-linked by transglutaminase to improve storage elastic modulus (Tissue Eng. 12 (6), 1467-74, 2006). The activity against collagen can be evaluated by comparing the storage elastic modulus of collagen cross-linked with each mutant and natural transglutaminase.

  Keratin is also an insoluble protein, but it is known that the tensile strength is increased by treating wool (keratin is a major component) with transglutaminase (J. Biotechnol. 6, 116 (4), 379-86. , 2005). The activity against keratin can be evaluated by comparing the tensile strength of the wool crosslinked with the mutant and the natural transglutaminase, respectively.

  The selected strain can be evaluated by casein gelation, gelatin gelation, or the like as it is by adding a serine protease inhibitor after culturing in test tube or subtilisin. Alternatively, the purified mutant can be evaluated by culturing in a Sakaguchi flask and purifying by cation exchange chromatography.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, this invention is not limited to these Examples.

  In the present specification and drawings, when amino acids and the like are indicated by abbreviations, each indication is based on an abbreviation by IUPAC-IUB Commission on Biochemical Nomenclature or a common abbreviation in the field.

Example 1 Establishment of Plate Assay Method When a random mutation is introduced into the MTG gene, it is necessary to efficiently assay a highly active mutant. Therefore, it was decided to construct a plate assay method using a nitrocellulose membrane. The enzyme activity of MTG can be measured by the hydroxamate method (J. Biol. Chem., 241, 5518-5525, 1966). In the hydroxamate method, the enzymatic activity of MTG is determined by performing an MTG enzymatic reaction using benzyloxycarbonyl-L-glutamylglycine and hydroxylammonium chloride as substrates, and quantifying the resulting complex of hydroxamic acid and iron (showing a red color). taking measurement. In the plate assay method, the hydroxamate method is applied, the strain is grown on the nitrocellulose membrane (secreting MTG), the substrate is sprayed, and the strain showing a red color around the colony by the TG reaction is activated MTG secretion. It was decided to visually judge the stock. Corynebacterium (App. Env. Micro., 2003, 69, 358-366), which holds TG expression plasmid pPSPTG1 and pro-cleaving enzyme expression plasmid pVSS1 and can secrete and express TG, and TG expression plasmid Corynebacterium not retained (retained vectors pPK4 and pVSS1, control) CM2G plate containing 25 μg / ml kanamycin and 5 μg / ml chloramphenicol (Glucose 5 g, Polypepton 10 g, Yeast extract 10 g, NaCl 5 g, DL-Met 0.2 g, Agar 1.5 g, pH 7.2 / 1 L), cultured at 30 ° C. for 24 hours, and transferred to a nitrocellulose membrane. MM secretion plate containing the same concentration of kanamycin and chloramphenicol (Glucose 60g, MgSO ₄ · 7H ₂ O 1g, FeSO ₄ · 7H ₂ O 0.01g, MnSO ₄ · 5H ₂ O 0.01g) , (NH ₄ ) ₂ SO ₄ 30 g, KH ₂ PO ₄ 1.5 g, VB1-HCl 0.45 mg, Biotin 0.45 mg, DL-Met 0.15 g, pH 7.5 / 1 L), and cultured at 30 ° C. for 24 hours. After incubation, the nitrocellulose membrane is peeled off from the plate and sprayed with reagent A (0.2 M MES, 0.4 M NH ₂ OH, 0.04 M Glutathion, 0.12 M CBZ-Gln-Gly, pH 6.0) in a glass sprayer. After reaction at 45 ° C. for 45 minutes, reagent B (1N HCl, 4% TCA, 1.67% FeCl ₃ .6H ₂ O) containing a reaction terminator and a color former was sprayed. As a result, those with enzyme activity developed a red color around the colonies, and those without enzyme activity remained yellow (corynebacterium had a yellow color) (FIG. 1).

Example 2 Introduction of random mutation into MTG gene and primary screening of MTG mutant The MTG gene consists of three regions: a pre-region, a pro region, and a mature region. Mutations were introduced into the mature region (about 1000 bp) encoding the MTG active body by error-prone PCR. The sequence of the transglutaminase gene from the S. mobaraense DSMZ strain has already been determined (Eur. J. Biochem., 257, 570-576, 1998). With reference to this sequence, forward primer and reverse primer were synthesized upstream and downstream of the mature body region, respectively, and error-prone PCR was performed under the following reaction solution composition and conditions. The reaction solution composition was set so that mutations were introduced at 2 to 3 mutations at the base sequence level. In order to ligate the obtained PCR product and vector pPSPTG1, it was treated with BamHI (37 ° C., 2 hours) and then with BstEII (60 ° C., 3 hours). The vector was cut out and dissolved in 60 μl of ddH ₂ O. The PCR product was purified by phenol chloroform and ethanol precipitation. After mixing so that it might become a vector: PCR product = 1: 5, it was made to ligate by making it react at 16 degreeC and 5min by 2 * ligation mix (Nippon Gene). E. coli DH5α was transformed with the plasmid prepared by ligation, and 8,000 colonies obtained were mixed and cultured in units of 100, and plasmid extraction was performed. By electroporation, Corynebacterium glutamicum AJ12036 (FERM BP-734) (WO 2002/081694) was transformed with the 80 plasmids obtained and the pro-cleaving enzyme expression plasmid pVSSI, and 300 plasmids per type. After being cultured in CM2G medium (30 ° C., 24 hours), the transformant was transferred to a nitrocellulose membrane and placed on an MM secretion plate (30 ° C., 24 hours). Electroporation was performed by the method described in the literature (FEMS Microbiol. Lett., 53, 299-303, 1989). About 24,000 strains were assayed by a plate assay, and about 5,000 active mutants were selected and subjected to secondary screening.
・ PCR reaction solution composition
Template DNA (1 ng / μl) 10 μl
100 mM Tris-HCl (pH8.8) 10 μl
500 mM KCl 10 μl
1 mg / ml BSA 10 μl
50 mM MgCl ₂ 10 μl
2.5 mM dNTPmix 10 μl
Forward primer (1 pmol / l) 2.5μl
Reverce primer (1 pmol / l) 2.5μl
DMSO 10μl
rTaq polymerase (TAKARA) 0.5μl
ddH ₂ O 24.5 μl
100 μl
PCR conditions (1) 94 ℃ 3 min
(2) 94 ℃ 2 min
58 ℃ 30 sec
72 ℃ 3 min x 25 cycles (3) 4 ℃

Example 3 Secondary and tertiary screening of mutants
CM2G medium (Glucose 5g, Polypepton 10g, Yeast extract 10g, NaCl 5g, DL-Met 0.2g, pH7.2 / 1L) containing 25μg / ml kanamycin, 5μg / ml chloramphenicol in 96-well deep wells Dispensed. The mutant strain (about 5000 strains) selected in Example 2 was inoculated and pre-cultured at 1,500 rpm at 30 ° C. for 24 hours. MM medium containing 25 µg / ml kanamycin, 5 µg / ml chloramphenicol and 50 g / L CaCO ₃ (Glucose 60 g, MgSO ₄ · 7H ₂ O 1 g, FeSO ₄ · 7H ₂ O 0.01 g, MnSO ₄ · 5H ₂ O 0.01g, (NH ₄ ) ₂ SO ₄ 30g, KH ₂ PO ₄ 1.5g, VB1-HCl 0.45mg, Biotin 0.45mg, DL-Met 0.15g, pH 7.5 / 1L) Then, 50 μl of the preculture was subcultured. The cells were cultured at 1,500 rpm at 30 ° C. for 48 hours. The culture was diluted 10-fold with 20 mM phosphate buffer, pH 6, and secondary screening was performed by measuring the activity by the hydroxamate method. In the activity measurement, 30 μl of 10-fold diluted culture solution was dispensed into a 96-well and pre-warmed at 37 ° C. for 5 minutes. 50 μl of A solution (0.05M MES, 0.1M NH ₂ OH, 0.01M Glutathion, 0.03M CBZ-Gln-Gly, pH 6.0) pre-warmed at 37 ° C. for 5 minutes was dispensed into each well at 37 ° C. After reaction for 20 minutes, 50 μl of B solution (1N HCl, 4% TCA, 1.67% FeCl ₃ · 6H ₂ O) was dispensed into each well to stop the reaction, and the absorbance at 525 nm was measured with a plate reader. It was measured. About 200 strains having an absorbance equal to or higher than that of the control were selected with the strain having no mutation introduced as a control. Next, 3 ml of CM2G medium containing 25 μg / ml kanamycin and 5 μg / ml chloramphenicol was dispensed into test tubes. Mutant strains (about 200 strains) selected by the secondary screening were inoculated and pre-cultured at 30 ° C. for 24 hours. Dispense 4 ml of MM medium containing 25 μg / ml kanamycin, 5 μg / ml chloramphenicol and 50 g / L CaCO ₃ into each test tube, subculture 50 μl of the preculture, and incubate at 30 ° C. for 48 hours. . Tertiary screening was performed by appropriately diluting the culture medium and measuring the activity by the hydroxamate method. The activity was measured by dispensing 100 μl of diluted culture solution in a test tube and preheating at 37 ° C. for 5 minutes. Dispense 1 ml each of solution A (0.05M MES, 0.1M NH ₂ OH, 0.01M Glutathion, 0.03M CBZ-Gln-Gly, pH 6.0) preheated at 37 ° C for 5 minutes, and react at 37 ° C for 10 minutes. Thereafter, liquid B (1N HCl, 4% TCA, 1.67% FeCl ₃ .6H ₂ O) was dispensed in 1 ml portions to stop the reaction, and the absorbance at 525 nm was measured. 80 strains having an absorbance equal to or higher than that of the control were selected with the strain having no mutation introduced as a control.

Example 4 Mutation point analysis of tertiary screening strain Mutation points were analyzed by confirming the base sequence of the strain obtained in the tertiary screening. The strain obtained by the third screening was cultured in CM2G medium containing 25 μg / ml kanamycin, 5 μg / ml chloramphenicol at 30 ° C. for 24 hours, and then the plasmid was extracted with QIAGEN's QIAprepSpin Miniprep Kit. It was transformed into JM109 strain. The transformed E. coli JM109 strain was cultured in LB medium at 37 ° C. for 16 hours, and then the plasmid was extracted in the same manner. Using ABI PRISM Cycle Sequencing Kit, the nucleotide sequence was analyzed according to the method recommended by the supplier. As a result, the presence of mutation points was confirmed in 34 strains (Table 2). For those with confirmed mutation points, the activity and protein amount were measured, and the specific activity was calculated. The activity and protein amount were measured by methods described in the literature (Prot. Exp. Puri., 2002, 26, 329-335). Here, the hydroxamate specific activity of the control was 22 to 24 (U / mg), and the mutation points of the mutants showing a specific activity of 25 or more were analyzed.

Example 5 Construction of a single mutation-introduced strain and purification of the mutant enzyme In Example 4, a highly active mutation site was identified. However, the presence of mutants containing multiple mutations, TG that does not occur and remains in a pro form (considered to be caused by the weak activity of the pro-sequence cleaving enzyme), and includes sequences other than the natural mature N-terminal sequence (N-terminal) (By amino acid sequence analysis). Therefore, a mutant in which only one mutation point was introduced was prepared, and after culture, pro-sequence cleavage activation, purification was performed, and the effect on the mutation point activity was accurately evaluated. To introduce mutations, use Stratagene's QuikChange II Site-Directed Mutagenesis Kit, and follow the method recommended by the manufacturer to mutate the proTG expression plasmid pPSPTG11 (App. Env. Micro., 2003, 69, 3011-3014). Introduced. Whether or not a mutation was introduced was confirmed by analyzing the base sequence in the same manner as described above. Table 3 shows the list of mutants prepared. The original mutation of the 16th methionine was threonine, but a mutant in which another amino acid was substituted was also created. Similarly, although the original mutation of the 199th serine is alanine, a mutant in which another amino acid is substituted was also created. The plasmid containing the desired mutation was introduced into the aforementioned Corynebacterium by electroporation. Note that the pro-sequence cleavage enzyme co-expressed with Corynebacterium had a weak activity, and the pro-form cleavage enzyme expression plasmid was not transformed at the same time.
CM2G medium containing 25 μg / ml kanamycin was dispensed into 3 ml test tubes. The mutant strain was inoculated and pre-cultured at 30 ° C. for 24 hours. 50 ml each of MM medium containing 25 μg / ml kanamycin and 50 g / L CaCO ₃ was dispensed into the Sakaguchi flask, subcultured 2.5 ml of the preculture, and cultured at 30 ° C. for 48 hours. The culture solution was centrifuged (10,000 rpm, 10 minutes), the supernatant was filtered, and the solvent was replaced with 20 mM phosphate buffer, pH 7.0 using Sephadex G25 (M). Subtilisin was added in an amount of 1/100 of MTG and reacted at 30 ° C. for 16 hours to activate MTG. The activated solution was exchanged into a cation exchange chromatography equilibration buffer (20 mM sodium acetate buffer, pH 5.5) using Sephadex G25 (M). Next, the entire amount was loaded onto a cation exchange column (Resource S 6 ml; manufactured by GE Healthcare Bioscience) that was sufficiently equilibrated with the same buffer. After re-equilibration with the same buffer, the fraction of protein eluted with a linear concentration gradient from 0 to 0.5 M NaCl, with the UV absorption at a wavelength of 280 nm as the index, was eluted around 200 mM NaCl. . By measuring the activity of the fraction and the amount of protein by the method described above, the fraction near the peak top having almost the same specific activity was collected except for the fraction having a low specific activity. The collected fraction was replaced with 20 mM phosphate buffer, pH 6.0 using Sephadex G25 (M). All chromatography was performed at room temperature. The obtained sample was diluted to a concentration of about 1 mg / ml with 20 mM phosphate buffer, pH 6.0, and stored at −80 ° C. until use.

Example 6 Casein Gelling Ability, Gelatin Gelling Ability Evaluation Casein gelation time was measured as follows. Dissolve sodium caseinate in 0.1M Tris-HCl buffer, pH 7.5 to 10% (W / W) (stir overnight). A test tube was dispensed with 1 ml of 10% sodium caseinate solution and preincubated at 37 ° C. 100 μl of 0.2 mg / ml TG solution was added. The gelation time was defined as the point at which the gel stopped flowing for 5 seconds after incubating at 37 ° C. When purified MTG preparation is used, gelation takes about 85 minutes.
The gelatin gelation time was measured as follows. Gelatin derived from cowhide was dissolved in 50 mM sodium acetate buffer, pH 5.0 so as to be 10% (W / W) (dissolved by heating at 50 to 55 ° C.). 1 ml of 10% gelatin solution was dispensed into a test tube and preincubated at 37 ° C. 100 μl of 0.2 mg / ml TG solution was added. The gelation time was defined as the point at which the gel stopped flowing for 5 seconds after incubating at 37 ° C. When purified MTG preparation is used, gelation takes about 85 minutes.
Since the gelation time varies depending on the substrate quality and the dissolution state, the gelation ability was determined by comparing the gelation time of the purified MTG preparation measured with the same lot of substrate at the same time. That is, the gelation ability of the mutant = the gelation time of the purified MTG preparation / the gelation time of the mutant. If this value is greater than 1, the mutant will have a faster gel time than the purified MTG preparation.
The results of gelation ability evaluation are summarized in Table 4. However, mutants with high specific activity by hydroxamate method (for example, 199S → A mutant) do not necessarily have high gelation ability. The importance of performing the assay on a polymer substrate was confirmed. The enzyme activity of TG is usually measured by the hydroxamate method (see Patent Document 6, etc.), but since it uses low molecules (benzyloxycarbonyl-L-glutaminylglycine and hydroxylamine) as substrates, hydroxamate The increase in activity by the method is not necessarily consistent with the increase in activity on the polymer substrate. In fact, in this example, it was found that the improvement in specific activity measured by the hydroxamate method did not coincide with the improvement in casein gelation and gelatin gelation ability. Moreover, it was found that excellent mutations that improve gelation ability are concentrated near the N-terminus.

Example 7 Preparation of mutants by modification near the active center and primary and secondary evaluation In the three-dimensional structure, 43 residues that are within 15 mm from the 64th Cys of the active center and have high solvent exposure were extracted. Mutants were created in which each extracted residue was substituted with several different amino acids. As in Example 5, the mutation was introduced into the pro-TG expression plasmid pPSPTG11 using the QuikChange II Site-Directed Mutagenesis Kit from Stratagene. Whether or not a mutation was introduced was confirmed by analyzing the base sequence in the same manner as described above. A plasmid containing the desired mutation was introduced into Corynebacterium by electroporation.
1 ml of CM2G medium containing 25 μg / ml kanamycin was dispensed into 96 wells. Mutant strains (about 220 strains) were inoculated and pre-cultured at 1,500 rpm at 30 ° C. for 24 hours. 1 ml each of MM medium containing 25 μg / ml kanamycin and 50 g / L CaCO ₃ was dispensed into a 96-well deep well, and 50 μl of the preculture was subcultured. The cells were cultured at 1,500 rpm at 30 ° C. for 48 hours. The culture was diluted 10-fold with 20 mM phosphate buffer (pH 7), 1/100 volume of MTG was added, and reacted at 30 ° C. for 16 hours to activate MTG. As in Example 3, primary evaluation was performed by measuring activity by hydroxamate method using a 96-well. About 100 strains having an absorbance equal to or higher than that of the control were selected with the strain having no mutation introduced as a control.
Next, 3 ml of CM2G medium containing 25 μg / ml kanamycin was dispensed into test tubes. The mutant strain selected in the primary evaluation was inoculated and pre-cultured at 30 ° C. for 24 hours. 4 ml each of MM medium containing 25 μg / ml kanamycin and 50 g / L CaCO ₃ was dispensed into a test tube, and 50 μl of the preculture was subcultured and cultured at 30 ° C. for 48 hours. The culture was diluted 10-fold with 20 mM phosphate buffer, pH 7, and subtilisin was added to 1/100 of MTG and reacted at 30 ° C. for 16 hours to activate MTG. The culture solution was appropriately diluted, and the tertiary evaluation was performed by measuring the activity by the hydroxamate method. 80 strains having an absorbance equal to or higher than that of the control were selected with the strain having no mutation introduced as a control.

Example 8 Casein gelling ability and gelatin gelling ability evaluation As in Example 6, the mutants selected in Example 7 were evaluated for casein gelling ability and gelatin gelling ability. Evaluation was performed as follows. 3 ml of CM2G medium containing 25 μg / ml kanamycin was dispensed into test tubes. The mutant strain selected in Example 7 was inoculated and pre-cultured at 30 ° C. for 24 hours. 4 ml each of MM medium containing 25 μg / ml kanamycin and 50 g / L CaCO ₃ was dispensed into a test tube, and 50 μl of the preculture was subcultured and cultured at 30 ° C. for 48 hours. The culture solution was replaced with 20 mM phosphate buffer (pH 7) using Sephadex G25 (M). Subtilisin was added in an amount of 1/100 of MTG and reacted at 30 ° C. for 16 hours to activate MTG. A serine protease inhibitor PMSF was added to the activated MTG to a final concentration of 5 mM, and the casein gelation ability and gelatin gelation ability were measured by the same method as in Example 6. As a result of the measurement, a mutant that was considered to have a gelling ability equivalent to or higher than that of MTG was prepared by a method using cation exchange chromatography described in Example 4, and the same method as in Example 6, Casein gelation ability and gelatin gelation ability were measured. The results obtained are shown in Table 6.

  The present invention can provide a transglutaminase mutant protein having improved activity against a high molecular weight substrate (particularly protein substrates such as casein, gelatin, collagen, keratin) as compared with WT protein. The amount of this enzyme used in various applications including quality, photographic film production, tannery, etc. can be reduced.

FIG. 3 shows exemplary results of the plate assay shown in Example 1. Strains that retain the TG expression plasmid develop color (shows red color), and strains that do not retain the TG expression plasmid do not develop color. It is a figure which shows the three-dimensional structure (left) and amino acid sequence (right) of MTG. It is a figure showing the alignment of the amino acid sequence of MTG and TG derived from various actinomycetes. Conserved amino acid residues are indicated by *.

Claims (11)

A protein having transglutaminase activity and having the following amino acid sequence of A, B, C or D:
(A) In SEQ ID NO: 2, 3, 5, 10, 12, 14, 16, 26, 28, 30, 32, 34, 38, 42, 49, 58, 59, 65, 74, 75, 77, 199, An amino acid sequence having a mutation at at least one position selected from positions 238, 241, 248, 284, 289, 299, 303, 312 and 320;
(B) In the arrangement of (A), 3, 5, 10, 12, 14, 16, 26, 28, 30, 32, 34, 38, 42, 49, 58, 59, 65, 74, 75, 77, Amino acid sequences having 1 to several residue substitutions, deletions, additions and / or insertions at positions other than positions 199, 238, 241, 248, 284, 289, 299, 303, 312 or 320;
(C) An amino acid sequence selected from SEQ ID NOs: 4, 6, 8, 10 and 12 or an amino acid sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10 and 12 has a homology of 70% or more 3, 5, 10, 12, 14, 16, 26, 28, 30, 32, 34, 38, 42, 49, 58, 59, SEQ ID NO: 2 in the amino acid sequence of the protein having transglutaminase activity. An amino acid sequence having a mutation at at least one position selected from positions corresponding to positions 65, 74, 75, 77, 199, 238, 241, 248, 284, 289, 299, 303, 312 and 320; or (D ) In the sequence of (C), 3, 5, 10, 12, 14, 16, 26, 28, 30, 32, 34, 38, 42, 49, 58, 59, 65, 74, 75 of SEQ ID NO: 2. Amino acids having 1 to several residue substitutions, deletions, additions and / or insertions at positions other than positions corresponding to positions 7, 199, 238, 241, 248, 284, 289, 299, 303, 312 or 320 An array.   The protein according to claim 1, wherein the position of the mutation is selected from positions 10, 12, 14, 16, 30, 32, 34, 38, 42, 49, 65, 77, 199, 299, 312 and 320. . The protein according to claim 1 or 2, wherein the mutation is selected from the following mutations:
1) 3rd position: substitution with phenylalanine, valine, leucine, isoleucine or asparagine;
2) 5-position: substitution with lysine;
3) 10th position: substitution with serine;
4) 12th position: substitution with serine;
5) 14th position: substitution with asparagine;
6) 16th position: substitution with threonine, serine, leucine, lysine or asparagine;
7) 26th position: substitution with phenylalanine;
8) 28th position: substitution with aspartic acid;
9) Position 30: substitution to aspartic acid;
10) Position 32: substitution to aspartic acid;
11) 34th position: substitution with phenylalanine;
12) 38th position: substitution with phenylalanine;
13) Position 42: substitution to histidine;
14) 49th position: substitution with arginine;
15) 58th position: substitution with aspartic acid;
16) 59th position: substitution with leucine or phenylalanine;
17) 65th position: substitution with isoleucine;
18) 74th position: substitution with asparagine, alanine or serine;
19) 75th position: substitution with phenylalanine, alanine, histidine, isoleucine, tryptophan, valine or proline;
20) Position 77: substitution with serine, alanine, phenylalanine, leucine, valine, asparagine, tryptophan, tyrosine or proline;
21) Position 199: substitution with alanine, leucine or threonine;
22) Position 238: substitution to phenylalanine or leucine;
23) Position 241: substitution with alanine;
24) Position 248: substitution with serine;
25) position 284: substitution to glycine or threonine;
26) Position 289: substitution with phenylalanine or tyrosine;
27) Position 299: substitution with leucine;
28) 303 position: substitution with phenylalanine;
29) 312 position: substitution with valine; and
30) Position 320: substitution with aspartic acid.   The polynucleotide which codes the protein of any one of Claims 1-3.   A recombinant vector comprising the polynucleotide according to claim 4.   A host cell transformed with the recombinant vector according to claim 5.   The method for producing a protein according to any one of claims 1 to 3, wherein the host cell according to claim 6 is cultured, and a protein having transglutaminase activity is collected.   The protein according to any one of claims 1 to 3, the protein produced by the method according to claim 7, or the step of causing the host cell according to claim 6 to act on a macromolecular substrate. A method for processing molecular substrates.   The method of claim 8, wherein the macromolecular substrate is a substrate protein.   The method according to claim 9, wherein the substrate protein is collagen, gelatin, casein or keratin.   The method according to any one of claims 8 to 10, which is used for tanning, fiber processing, or photographic film production.