MODIFIED PROTEIN GLUTAMINASE

Abstract

The purpose of the present invention is to provide a protein glutaminase with improved oxidative stability. A protein glutaminase comprising a polypeptide containing an amino acid sequence in which at least one substitution of (A) a substitution of a valine or alanine residue for the amino acid residue at position 121 and (B) a substitution of a cysteine ​​or aspartic acid residue for the amino acid residue at position 142 has been introduced into an amino acid sequence shown in SEQ ID NO: 1, exhibits improved oxidative stability compared to that of a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1.

Description

Technical field

[0001] The present invention relates to a modified protein glutaminase. In particular, the present invention relates to a protein glutaminase that is modified to improve its oxidative stability. State of the art

[0002] Protein glutaminase is an enzyme that acts on a protein, which is a polymer, and catalyzes a reaction in which an amide-containing side chain is broken down (i.e., deamidated) without cleaving peptide bonds and cross-linking the protein. Protein glutaminase deamidates glutamine residues in a protein, generating negatively charged carboxyl groups, thus causing the protein to undergo various characteristic changes. For example, an increase in hydration strength and an increase in electrostatic repulsion due to a decrease in the protein's isoelectric point lead to a reduction in inter-protein interactions (i.e., a deterioration of association properties) and consequently increase the protein's solubility and water dispersibility.Furthermore, the exposure of internal hydrophobic regions due to a change in the higher-order structure of the protein imparts interfacial activity to the protein, consequently improving its emulsifying capacity, emulsion stability, foaming properties, and foam stability. Protein glutaminase can significantly alter the properties of a protein, as described above, and has therefore greatly expanded its applications. For this reason, protein glutaminase is very useful and has garnered considerable attention in the field.

[0003] Protein glutaminase was first discovered in Chryseobacterium proteolyticum in 2000 (non-patent document 1). Since then, protein glutaminase derived from C. proteolyticum has been used industrially and commercially for a long time as the sole active ingredient in protein glutaminase enzyme preparations. Document list - Non-patent documents

[0004] NPL 1: A novel protein-deamidating enzyme from Chryseobacterium proteolyticum sp. nov., a newly isolated bacterium from soil. Applied and Environmental Microbiology 2000; 66(8): 3337-43 Summary of the invention: Technical problem

[0005] Protein glutaminase derived from C. proteolyticum exhibits poor oxidative stability and is difficult to preserve for extended periods. Therefore, handling protein glutaminase derived from C. proteolyticum in this context requires its use or storage in an environment where oxidation is unlikely to occur.

[0006] On the other hand, in view of the possibility of further expanding the applications of protein glutaminase, which is expected due to its great usefulness, it is desirable that protein glutaminase can be stored for a long period of time, and for this purpose an improvement in the oxidative stability of protein glutaminase is desirable.

[0007] Accordingly, it is an object of the present invention to provide a protein glutaminase that exhibits improved oxidative stability. Solution to the problem

[0008] The present inventors have conducted extensive investigations and, as a result, discovered a new mutation that can improve the oxidative stability of protein glutaminase (in particular, it improves the residual activity of protein glutaminase after hydrogen peroxide treatment compared to the residual activity of wild-type protein glutaminase). The present invention was developed based on these findings. That is to say, the present invention provides objects of aspects as specified below.

[0009] Item 1. Modified protein glutaminase comprising one of the following polypeptides (I) to (III): (I) a polypeptide consisting of an amino acid sequence modified by the introduction of at least one of: (A) a substitution of the amino acid residue at position 121 with a valine residue or an alanine residue, (B) a substitution of the amino acid residue at position 142 with a cysteine ​​residue or an aspartic acid residue, (C) a substitution of the amino acid residue at position 22 with a tyrosine residue, (D) a substitution of the amino acid residue at position 26 with a tyrosine residue or a cysteine ​​residue, (E) a substitution of the amino acid residue at position 30 with a threonine residue, (F) a substitution of the amino acid residue at position 33 with a valine residue, (G) a substitution of the amino acid residue at position 35 with a serine residue, (H) a substitution of the amino acid residue at position 36 with an isoleucine residue, (I) a substitution of the amino acid residue at position 43 with a phenylalanine residue,(J) a substitution of the amino acid residue at position 73 with an isoleucine residue, (K) a substitution of the amino acid residue at position 113 with a lysine residue, (L) a substitution of the amino acid residue at position 156 with a proline residue, (M) a substitution of the amino acid residue at position 157 with a phenylalanine residue, (N) a substitution of the amino acid residue at position 169 with a phenylalanine residue, (O) a substitution of the amino acid residue at position 170 with a threonine residue, (P) a substitution of the amino acid residue at position 182 with an arginine residue, and (Q) a substitution of the amino acid residue at position 183 with a glycine residue, into the amino acid sequence that is specified as SEQ ID NO: 1; (II) a polypeptide in which one or more amino acid residue(s), which is / are different from the substituted amino acid residue(s), is / are substituted, added, inserted or deleted in the amino acid sequence in which at least one of the substitutions (A) to (Q) is introduced, and the residual activity of protein glutaminase after hydrogen peroxide treatment is improved compared with the residual activity of the polypeptide consisting of the amino acid sequence specified as SEQ ID NO: 1; and (III) a polypeptide in which the sequence identity of regions not including the substituted amino acid sequences is 70% or more in the amino acid sequence in which at least one of the substitutions (A) to (Q) is introduced, and the residual activity of protein glutaminase after hydrogen peroxide treatment is improved compared with the residual activity of the polypeptide consisting of the amino acid sequence specified as SEQ ID NO: 1.

[0010] Item 2. DNA encoding the modified protein glutaminase according to Item 1.

[0011] Item 3. Expression cassette or recombinant vector comprising the DNA according to Item 2.

[0012] Item 4. Transformant obtained by transforming a host using the expression cassette or the recombinant vector according to Item 3.

[0013] Item 5. Method for the production of a modified protein glutaminase, comprising the step of cultivating the transformant according to Item 4.

[0014] Item 6. Enzyme agent comprising the modified protein glutaminase according to Item 1.

[0015] Item 7. Modifying agent for a protein material comprising the modified protein glutaminase according to Item 1.

[0016] Item 8. Method for producing a modified protein material comprising the step of applying the modified protein glutaminase according to Item 1 to a protein material. Advantageous effect of the invention

[0017] According to the invention, a modified protein glutaminase with improved oxidative stability is provided. Description of embodiments

[0018] The present invention is described in detail below. The 20 types of amino acid residues in the amino acid sequence can be represented by a letter abbreviation. In particular, glycine (Gly) is G, alanine (Ala) is A, valine (Val) is V, leucine (Leu) is L, isoleucine (Ile) is I, phenylalanine (Phe) is F, tyrosine (Tyr) is Y, tryptophan (Trp) is W, serine (Ser) is S, threonine (Thr) is T, cysteine ​​(Cys) is C, methionine (Met) is M, aspartic acid (Asp) is D, glutamic acid (Glu) is E, asparagine (Asn) is N, glutamine (Gln) is Q, lysine (Lys) is K, arginine (Arg) is R, histidine (His) is H, and proline (Pro) is P.

[0019] In the amino acid sequence described here, the left end is an N-terminus and the right end is a C-terminus.

[0020] The term "nonpolar amino acid," as used here, includes glycine, alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan. The term "uncharged amino acids," as used here, includes glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. An "acidic amino acid" includes aspartic acid and glutamic acid. A "basic amino acid" includes lysine, arginine, and histidine.

[0021] The term "substitution," as used here, encompasses not only cases where an amino acid residue substitution is artificially introduced, but also cases where an amino acid residue substitution occurs naturally, i.e., cases where the amino acid residues are intrinsically different. The substitution of an amino acid residue, as used here, can be either artificial or natural, but is preferably artificial. 1. Modified protein glutaminase

[0022] The modified protein glutaminase of the present invention comprises one of the following polypeptides (I) to (III).

[0023] (I) A polypeptide consisting of an amino acid sequence formed by the introduction of at least one of (A) a substitution of the amino acid residue at position 121 with a valine residue or an alanine residue, (B) a substitution of the amino acid residue at position 142 with a cysteine ​​residue or an aspartic acid residue, (C) a substitution of the amino acid residue at position 22 with a tyrosine residue, (D) a substitution of the amino acid residue at position 26 with a tyrosine residue or a cysteine ​​residue, (E) a substitution of the amino acid residue at position 30 with a threonine residue, (F) a substitution of the amino acid residue at position 33 with a valine residue, (G) a substitution of the amino acid residue at position 35 with a serine residue, (H) a substitution of the amino acid residue at position 36 with an isoleucine residue, (I) a substitution of the amino acid residue at position 43 with a phenylalanine residue, (J) a substitution of the amino acid residue at position 73 with an isoleucine residue, (K) a substitution of the amino acid residue at position 113 with a lysine residue, (L) a substitution of the amino acid residue at position 156 with a proline residue, (M) a substitution of the amino acid residue at position 157 with a phenylalanine residue, (N) a substitution of the amino acid residue at position 169 with a phenylalanine residue, (O) a substitution of the amino acid residue at position 170 with a threonine residue, (P) a substitution of the amino acid residue at position 182 with an arginine residue, and (Q) a substitution of the amino acid residue at position 183 with a glycine residue, is obtained in the amino acid sequence specified as SEQ ID NO: 1.

[0024] (II) A polypeptide in which one or more amino acid residue(s) different from the substituted amino acid residue(s) is / are substituted, added, inserted or deleted in the amino acid sequence in which at least one of the substitutions (A) to (Q) is introduced, and the residual activity of protein glutaminase after hydrogen peroxide treatment is improved compared with the residual activity of the polypeptide consisting of the amino acid sequence specified as SEQ ID NO: 1.

[0025] (III) A polypeptide in which the sequence identity of regions not including the substituted amino acid sequences is 70% or more in the amino acid sequence in which at least one of the substitutions (A) to (Q) is introduced, and the residual activity of protein glutaminase after hydrogen peroxide treatment is improved compared with the residual activity of the polypeptide consisting of the amino acid sequence specified as SEQ ID NO: 1.

[0026] The amino acid sequence, which is specified as SEQ ID NO: 1, is a mature sequence of the full-length sequence (the sequence which includes the signal sequence and the pro sequence) of protein glutaminase derived from Chryseobacterium proteolyticum, which is specified as SEQ ID NO: 2.

[0027] The polypeptides (I) to (III) can be polypeptides containing the substitutions (A) to (Q) individually (single-substituted forms of protein glutaminase derived from C. proteolyticum), or they can be polypeptides containing the substitutions (A) to (Q) in a combination of two or more of them (multiple-substituted forms of protein glutaminase derived from C. proteolyticum). Examples of preferred polypeptides from polypeptides (I) to (III) include: polypeptides containing at least one of the substitutions (A), (B), (D), (F), (G), (H), (J), (L), (M), (N), and (P) from substitutions (A) to (Q); polypeptides containing substitutions (E) and (K) from substitutions (A) to (Q); Polypeptides containing substitutions (D), (I) and (Q) from substitutions (A) to (Q); and polypeptides containing substitutions (C) and (O) from substitutions (A) to (Q).

[0028] As a specific example of polypeptide (I), the specific amino acid sequences of the polypeptides exhibiting substitution (A) with a valine residue and a substitution with an alanine residue are given as SEQ ID NO: 3 and SEQ ID NO: 4 respectively, and the specific amino acid sequences of the polypeptides exhibiting substitution (B) with a cysteine ​​residue and an aspartic acid residue are given as SEQ ID NO: 5 and SEQ ID NO: 6 respectively.

[0029] In the polypeptide (II), the introduced amino acid modification can comprise only one type of modification (for example, only substitution) or two or more types of modification (for example, substitution and insertion), as well as substitution, addition, insertion, and deletion. In the polypeptide (II), the number of amino acid differences at any given difference site can be 1 or more and is, for example, 1 to 18, preferably 1 to 10, more preferably 1 to 8, 1 to 7, 1 to 6, 1 to 5, or 1 to 4, even more preferably 1 to 3, and particularly preferably 1 or 2 or 1.

[0030] In the polypeptide (III), the sequence identity with the amino acid sequence specified as SEQ ID NO: 1 can be 70% or more, and is preferably 80% or more or 85% or more, more preferably 90% or more or 93% or more, even more preferably 95% or more, even more preferably 98% or more, even more preferably 98.5% or more or 99% or more, and particularly preferably 99.3% or more or 99.5% or more.

[0031] In polypeptide (III), the sequence identity with each amino acid sequence specified as SEQ ID NO: 1 is the sequence identity calculated by comparison with the amino acid sequence specified as SEQ ID NO: 1. The "sequence identity" indicates the identity value of an amino acid sequence obtained by the bI2seq program (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol. Lett., Vol. 174, pp. 247-250, 1999) of BLASTPACKAGE [sgi32 bit edition, version 2.0.12; available from the National Center for Biotechnology Information (NCBI)]. The parameters can be set to "Gap insertion Cost value": 11 and "Gap extension Cost value": 1.

[0032] In the polypeptides of (II) and (III), the amino acid residues corresponding to position 42 (cysteine ​​residue), position 83 (histidine residue) and position 103 (aspartic acid residue) in the amino acid sequence specified as SEQ ID NO: 1 can be active catalyst residues, and therefore it is preferred not to introduce any substitutions or deletions at these positions.

[0033] When an amino acid substitution is introduced into polypeptides (II) and (III) in SEQ ID NO: 1, a preferred type of amino acid substitution introduced is a conservative substitution. That is, for example, the substitution in polypeptides (II) and (III) is such that if the amino acid prior to substitution is a nonpolar amino acid, a substitution with another nonpolar amino acid is introduced; or if the amino acid prior to substitution is an uncharged amino acid, a substitution with another uncharged amino acid is introduced; or if the amino acid prior to substitution is an acidic amino acid, a substitution with another acidic-polar amino acid is introduced; or if the amino acid prior to substitution is a basic amino acid, a substitution with another basic amino acid is introduced.

[0034] Specific examples of polypeptides (II) and (III) include polypeptides obtained by introducing the substitution into a protein glutaminase analogous to the protein glutaminase derived from C. proteolyticum. Examples of the analogous protein glutaminase into which the substitution is introduced include a protein glutaminase derived from Chryseobacterium sp., in particular an amino acid sequence designated as SEQ ID NO: 7 or SEQ ID NO: 8, which is a mature sequence of protein glutaminase derived from Chryseobacterium sp., or an amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8 in which the amino acid residue at position 115 is substituted with another amino acid residue (for example, a serine residue). SEQ ID NO: 7 has a sequence identity of 86.9% with SEQ ID NO: 1 and SEQ ID NO: 8 has a sequence identity of 87.4% with SEQ ID NO: 1.The full-length sequence of SEQ ID NO: 7 is the amino acid sequence specified as SEQ ID NO: 9, and the full-length sequence of SEQ ID NO: 8 is the amino acid sequence specified as SEQ ID NO: 10.

[0035] The aforementioned polypeptides (I) to (III) exhibit protein glutaminase activity and the property of improved oxidative stability. The expression "property of improved oxidative stability" means that the residual activity of protein glutaminase after hydrogen peroxide treatment is improved compared to the residual activity of a polypeptide consisting of the amino acid sequence specified as SEQ ID NO: 1 (wild-type protein glutaminase derived from Chryseobacterium proteolyticum), noting in particular that the residual activity is improved when 0.1 mg / mL of an aqueous polypeptide solution is treated with hydrogen peroxide at a final concentration of 0.001% at 37°C for 1 hour (hereinafter also referred to as "residual activity after oxidation treatment A"). 0.001% “ denoted), 2 times or more of the residual activity after an oxidation treatment A 0.001 %of wild-type protein glutaminase. As a preferred example of the oxidation stability of the aforementioned polypeptides (I) to (III), the residual activity after oxidation treatment is A 0,001% 3 times or more, more preferably 4 times or more, even more preferably 5 times or more and even more preferably 5.5 times or more of the residual activity after an oxidation treatment A 0,001% of wild-type protein glutaminase. The upper limit of residual activity after oxidation treatment A 0,001% The activity of polypeptides (I) to (III) is not limited and is, for example, 20 times or less or 10 times or less of the residual activity after oxidation treatment A 0,001%of wild-type protein glutaminase. Regarding the residual activity when 0.1 mg / mL of the aqueous polypeptide solution is treated with hydrogen peroxide at a final concentration of 0.0025% at 37°C for 1 hour (hereinafter also referred to as "residual activity after oxidation treatment B"). 0,0025% “ denoted), the property is that the oxidation stability in the polypeptides (I) to (III) is improved, for example by 5 times or more, preferably by 7 times or more, more preferably by 10 times or more and more preferably by 20 times or more of the residual activity after an oxidation treatment B 0,0025% of wild-type protein glutaminase. The upper limit of residual activity after oxidation treatment B 0,025% The activity of polypeptides (I) to (III) is not limited and is, for example, 60 times or less or 50 times or less of the residual activity after oxidation treatment B. 0,0025%of wild-type protein glutaminase.

[0036] The specific activity of polypeptides (I) to (III) is not limited, and the specific activity of polypeptides (I) to (III) at 37 °C is, for example, 20% or more, 40% or more, or 60% or more, if the specific activity of the polypeptide consisting of the amino acid sequence specified as SEQ ID NO: 1 (wild-type protein glutaminase derived from Chryseobacterium proteolyticum) is set to 100% at 37 °C. The specific activity of polypeptides (I) to (III) is preferably equivalent to the specific activity of the polypeptide consisting of the amino acid sequence specified as SEQ ID NO: 1 (wild-type protein glutaminase derived from Chryseobacterium proteolyticum).The equivalent specific activity, in particular the specific activity of polypeptides (I) to (III) at 37 °C, where the specific activity at 37 °C of the polypeptide consisting of the amino acid sequence specified as SEQ ID NO: 1 is set as 100%, is 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 100% or more and more preferably 105% or more.

[0037] The amount of an enzyme that produces 1 µmol of ammonia per minute using Z-Gln-Gly (benzyloxycarbonyl-L-glutaminylglycine) as a substrate is defined as one unit (1 U) of protein glutaminase activity.

[0038] The modified protein glutaminase of the present invention can be used as the active ingredient of an enzyme agent described later, or can be used to form part of a larger protein that is integrated in the form of a fusion protein or the like with peptides or proteins consisting of other amino acid sequences (hereinafter also referred to as "other proteins, etc."). Examples of the other proteins, etc., include peptides used for protein purification, such as a polyhistidine residue, and those derived from an additional sequence to ensure mRNA stability during recombinant production. 2. DNA

[0039] The DNA of the present invention is a DNA encoding the modified protein glutaminase described above in “1. Modified Protein Glutaminase”.

[0040] The DNA of the present invention is not limited as long as it has a nucleotide sequence encoding a modified protein glutaminase, including any of the polypeptides (I) to (III) described above in “1. Modified Protein Glutaminase”. Examples of the nucleotide sequence of DNA encoding the amino acid sequence specified as SEQ ID NO: 1 (protein glutaminase derived from Chryseobacterium proteolyticum), which is a reference sequence for the polypeptides (I) to (III), include SEQ ID NO: 11. Therefore, a person skilled in the art can design the DNA of the present invention using SEQ ID NO: 11 as a reference sequence in a suitable manner.

[0041] Examples of the DNA of the present invention include the following DNAs [i] to [iii].

[0042] [i] A DNA consisting of a nucleotide sequence formed by the introduction of at least one of (a) a substitution at positions 361 to 363 with a nucleotide sequence encoding a valine residue or an alanine residue, (b) a substitution at positions 424 to 426 with a nucleotide sequence encoding a cysteine ​​residue or an aspartic acid residue, (c) a substitution at positions 64 to 66 with a nucleotide sequence encoding a tyrosine residue, (d) a substitution at positions 76 to 78 with a nucleotide sequence encoding a tyrosine residue or a cysteine ​​residue, (e) a substitution at positions 88 to 90 with a nucleotide sequence encoding a threonine residue, (f) a substitution at positions 97 to 99 with a nucleotide sequence encoding a valine residue, (g) a substitution at positions 103 to 105 with a nucleotide sequence encoding a serine residue, (h) a substitution at positions 106 to 108 with a nucleotide sequence encoding an isoleucine residue, (i) a substitution at positions 127 to 129 with a nucleotide sequence encoding a phenylalanine residue, (j) a substitution at positions 217 to 219 with a nucleotide sequence encoding an isoleucine residue, (k) a substitution at positions 337 to 339 with a nucleotide sequence encoding a lysine residue, (l) a substitution at positions 466 to 468 with a nucleotide sequence encoding a proline residue, (m) a substitution at positions 469 to 471 with a nucleotide sequence encoding a phenylalanine residue, (n) a substitution at positions 505 to 507 with a nucleotide sequence encoding a phenylalanine residue, (o) a substitution at positions 508 to 510 with a nucleotide sequence encoding a threonine residue, (p) a substitution at positions 544 to 546 with a nucleotide sequence encoding an arginine residue, and (q) a substitution at positions 547 to 549 with a nucleotide sequence encoding a glycine residue, is obtained in the nucleotide sequence specified as SEQ ID NO: 11.

[0043] [ii] A DNA encoding a polypeptide in which the residual activity of protein glutaminase after hydrogen peroxide treatment is enhanced compared with the residual activity of the polypeptide consisting of the amino acid sequence specified as SEQ ID NO: 1, wherein the DNA is hybridized under stringent conditions with a DNA consisting of a nucleotide sequence complementary to the DNA [i].

[0044] [iii] A DNA encoding a polypeptide in which the residual activity of protein glutaminase after hydrogen peroxide treatment is enhanced compared with the residual activity of the polypeptide consisting of the amino acid sequence specified as SEQ ID NO: 1, wherein the DNA has a homology of 70% or more with the DNA [i].

[0045] As a specific example for DNA [i], the specific nucleotide sequences of DNAs that have substitution [a] a substitution with a nucleotide sequence encoding a valine residue and a substitution with a nucleotide sequence encoding an alanine residue are given as SEQ ID NO: 12 and SEQ ID NO: 13 respectively, and the specific nucleotide sequences of DNAs that have substitution [b] a substitution with a nucleotide sequence encoding a cysteine ​​residue and a substitution with a nucleotide sequence encoding an aspartic acid residue are given as SEQ ID NO: 14 and SEQ ID NO: 15 respectively.

[0046] For DNA [ii], the phrase “under stringent conditions” refers to conditions in which DNA is kept at 50 °C to 65 °C for 4 hours to one night in 6 × SSC (1 × SSC consists of 0.15 M NaCl and 0.015 M sodium citrate at a pH of 7.0) containing 0.5% SDS, 5 × Denhartz’s [0.1% bovine serum albumin (BSA), 0.1% polyvinylpyrrolidone, 0.1% Ficoll 400] and 100 µg / ml salmon sperm DNA.

[0047] Hybridization under stringent conditions is carried out in particular by the following procedure. That is, a nylon membrane on which a DNA library or a cDNA library is immobilized is prepared, and the nylon membrane is blocked at 65 °C in a pre-hybridization solution containing 6 × SSC, 0.5% SDS, 5 × Denhartz's, and 100 µg / ml salmon sperm DNA. Subsequently, each probe containing 32P-labeled, is added to the nylon membrane, which is kept at 65 °C overnight. The nylon membrane is washed at room temperature for 10 minutes in 6 × SSC, at room temperature for 10 minutes in 2 × SSC containing 0.1% SDS, and at 45 °C for 30 minutes in 0.2 × SSC containing 0.1% SDS, and then subjected to autoradiography, which allows the detection of DNA specifically hybridized to the probe.

[0048] For the DNA [iii], the homology can be 70% or more, and it is preferably 80% or more or 85% or more, more preferably 90% or more or 93% or more, even more preferably 95% or more, even more preferably 98% or more, even more preferably 98.5% or more or 99% or more and particularly preferably 99.3% or more or 99.5% or more.

[0049] The "homology" of DNA is calculated using published or commercially available software with an algorithm that performs a comparison using a reference sequence as the query sequence. Specifically, BLAST, FASTA, GENETYX (manufactured by GENETYX KK), or similar software can be used, and they can be configured with default parameters.

[0050] The DNA of the present invention can be obtained, for example, by introducing at least one of the substitutions (a) to (q) into DNA encoding a polypeptide consisting of the amino acid sequence specified as SEQ ID NO: 1 (i.e., a protein glutaminase derived from C. proteolyticum) or its analogous protein glutaminase (as described above; specific examples include Chryseobacterium sp., in particular a polypeptide consisting of the amino acid sequence specified as SEQ ID NO: 7 or SEQ ID NO: 8, or an amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8 in which the amino acid residue at position 115 is substituted with another amino acid residue (for example, a serine residue)). Furthermore, the DNA of the present invention can be artificially synthesized by a total gene synthesis process.

[0051] In the case of DNA encoding a polypeptide consisting of the amino acid sequence specified as SEQ ID NO: 1, from a nucleic acid construct, such as a plasmid, incorporating the nucleotide sequence specified as SEQ ID NO: 11, the nucleotide sequence can be obtained by a conventional PCR-based procedure. Along with the nucleotide sequence specified as SEQ ID NO: 11, a sequence obtained by adding a signal sequence and a pro sequence to the 5' end of the nucleotide sequence can be incorporated into the nucleic acid construct. Examples of sequences to which such a signal sequence and pro sequence are added include the nucleotide sequence specified as SEQ ID NO: 16, which encodes the full-length sequence of protein glutaminase produced by C.proteolyticum, which is specified as SEQ ID NO: 2, and the nucleotide sequence, which is specified as SEQ ID NO: 17 or SEQ ID NO: 18, which encodes the full-length sequence of protein glutaminase, which is derived from Chryseobacterium sp., analogous to a protein glutaminase, which is specified as SEQ ID NO: 9 or 10.

[0052] The method for artificially modifying an amino acid sequence by introducing a mutation into a gene can include known methods such as the Kunkel method and the "gapped duplex" method, as well as a mutation introduction kit using a site-directed mutagenesis method, such as the QuikChange (brand) site-directed mutagenesis kit (Stratagene Company), the GeneTailor (brand) site-directed mutagenesis system (Invitrogen Company), the TaKaRa site-directed mutagenesis system (Mutan-K, Mutan-Super Express Km, etc.: Takara Bio Inc.) and the like.

[0053] The DNA of the present invention comprises various types of DNA derived from codon degeneration. The artificial production of different types of DNA encoding the same amino acid sequence can be easily accomplished using a known genetic engineering technique. For example, in the genetic engineering of a protein, the expression level of a protein of interest may be low if the frequency of use of a codon in an original gene encoding the protein is low in the host. In this case, strong expression of the protein of interest can be achieved by optimizing the frequency of codon use for the host without altering the encoded amino acid sequence.

[0054] As an index for codon usage frequency, the optimal overall frequency of codon usage for the host can be used for each codon. The optimal codon is defined as the codon whose usage frequency is highest among codons corresponding to the same amino acid. Codon usage frequency is not limited as long as it is optimized for the host, and examples of optimal codons in E. coli include the following. F: phenylalanine (ttt), L: leucine (ctg), I: isoleucine (att), M: methionine (atg), V: valine (gtg), Y: tyrosine (tat), stop codon (taa), H: histidine (cat), Q: glutamine (cag), N: asparagine (aat), K: lysine (aaa), D: aspartic acid (gat), E: glutamic acid (gaa), S: serine (agc), P: proline (ccg), T: threonine (acc), A: alanine (gcg), C: cysteine ​​(tgc), W: tryptophan (tgg), R: arginine (cgc), G: glycine (ggc).

[0055] The nucleotide sequence of DNA in which a mutation has been introduced in a base sequence can be confirmed by sequencing using a conventional method. Specific examples of sequencing methods include a dideoxynucleotide chain termination method (Sanger et al. (1977), Proc. Natl. Acad. Sci. USA 74: 5463) and a sequence analysis method using a suitable DNA sequencer. Examples of methods for confirming whether DNA encodes a polypeptide of interest include a method in which a sequenced nucleotide sequence is compared to an unsubstituted nucleotide sequence, such as the nucleotide sequence designated as SEQ ID NO: 11, and a method in which an amino acid sequence determined from the sequenced nucleotide sequence is compared to an unsubstituted amino acid sequence, such as the amino acid sequence designated as SEQ ID NO: 1. 3. Expression cassette or recombinant vector

[0056] The expression cassette or recombinant vector of the present invention contains the DNA of the present invention, which is described above in “2. DNA”. The expression cassette or recombinant vector of the present invention can be obtained by combining a promoter and a terminator with the DNA of the present invention or by inserting the expression cassette of the present invention or the DNA of the present invention into the expression vector.

[0057] The expression cassette or recombinant vector of the present invention may optionally include, in addition to the promoter and terminator, transcriptional elements such as an enhancer, a CCAAT box, a TATA box, or an SPI site as control factors. These control factors may be functionally linked to the DNA of the present invention. The term "functionally linked" means that the DNA of the present invention and various control factors that regulate the DNA of the present invention are linked in a state in which they are functional in host cells.

[0058] Regarding the recombinant vector of the present invention, a suitable expression vector for genetic recombination is one constructed from a phage, plasmid, or virus capable of autonomous growth within a host. Such an expression vector is known, and examples include commercially available expression vectors such as pQE-based vectors (Qiagen Corporation), pDR540 and pRIT2T (GE Healthcare Bio-Sciences AB), and pET-based vectors (Merck KGaA). A suitable combination with host cells can be selected and used for the expression vector. For example, when E. coli is used as the host cell, a combination of a pET-based vector and a DH5α E. coli strain, a combination of a pET-based vector and a BL21 (DE3) E. coli strain, or a combination of a pDR540 vector and a JM109 E. coli strain can be used. coli strain can be used. 4. Transformant

[0059] The transformant of the present invention is obtained by transforming a host with the expression cassette or recombinant vector of the present invention, which is / are described above in “3. Expression cassette or recombinant vector”.

[0060] The host used to produce the transformant of the present invention is not limited, provided that a gene can be introduced into it, the stability of the expression cassette or recombinant vector is ensured, and the host can autonomously grow and express the features of a gene containing the DNA of the present invention. Preferred examples include bacteria belonging to the genus Escherichia, such as Escherichia coli, the genus Bacillus, such as Bacillus subtilis, the genus Pseudomonas, such as Pseudomonasputida, and the genus Chryseobacterium proteolyticum, such as Chryseobacterium proteolyticum; and yeast; and may also include animal cells, insect cells, plant cells, and the like.

[0061] The transformant of the present invention can be obtained by introducing the expression cassette or recombinant vector of the present invention into a host. The site at which the DNA of the present invention is introduced is not restricted, as long as a gene of interest can be expressed, and the DNA can be introduced into a plasmid or a genome. Examples of the specific method for introducing the expression cassette or recombinant vector of the present invention include a recombinant vector method and a genome editing method. Conditions for introducing the expression cassette or recombinant vector into the host can be adjusted appropriately according to the nature of the host and the like.If the host is bacteria, methods such as using competent cells with calcium ion treatment and electroporation are applicable. If the host is yeast, methods such as electroporation, spheroplast treatment, and lithium acetate treatment are applicable. If the host is animal cells, methods such as electroporation, calcium phosphate treatment, and lipofection treatment are applicable. If the host is insect cells, methods such as calcium phosphate treatment, lipofection treatment, and electroporation treatment are applicable. If the host is plant cells, methods such as electroporation, Agrobacterium treatment, particle gun treatment, and PEG treatment are applicable.

[0062] Whether the expression cassette of the present invention or the recombinant vector of the present invention has been introduced into a host can be confirmed by a PCR method, a Southern hybridization method, a Northern hybridization method or the like.

[0063] If it has been confirmed by a PCR method whether the expression cassette of the present invention or the recombinant vector of the present invention has been included in a host, for example genomic DNA, the expression cassette or the recombinant vector can be separated and purified from a transformant.

[0064] For example, if the host is bacteria, the expression cassette or recombinant vector is separated and purified with a lysate obtained by lyseing bacteria. The lysis method might involve treatment with a lytic enzyme, such as lysozyme, and optionally a combination of a protease, other enzymes, and a surfactant, such as sodium lauryl sulfate (SDS).

[0065] Physical comminution methods, such as freeze-thaw and French press treatment, can also be combined. The DNA can be separated and purified from the lysate by a suitable combination of, for example, a deproteinization treatment based on phenol and protease treatment, a ribonuclease treatment, an alcohol precipitation treatment, and a commercially available kit.

[0066] DNA can be cleaved using a conventional method, for example, by restriction enzyme treatment. A type II restriction enzyme, which acts on a specific nucleotide sequence, is used as the restriction enzyme. The DNA and the expression cassette or expression vector are then bound together, for example, using a DNA ligase.

[0067] Subsequently, a primer specific to the DNA of the present invention is designed using the separated and purified DNA as a template, and PCR is performed. The amplification product obtained by PCR is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, or the like, and stained with ethidium bromide, SYBR green solution, or the like. This allows the amplification product to be detected as a band, thus confirming that the transformation has taken place.

[0068] It is also possible to detect the amplification product by performing PCR using a primer that has been pre-labeled with a fluorescent dye or the like. Alternatively, a method can be used in which the amplification product is bound to a solid phase, such as a microplate, and its presence is confirmed by fluorescence detection, an enzymatic reaction, or the like. 5. Method for the production of a modified protein glutaminase

[0069] The method for producing a modified protein glutaminase according to the present invention is a method for producing an enzyme described in the preceding “1. Modified Protein Glutaminase”, wherein the method comprises the step of cultivating the transformant of the present invention. If at least one of the substitutions (A) to (Q) contained in the modified protein glutaminase is introduced naturally, the modified protein glutaminase can be obtained by a manufacturing process that includes the step of cultivating microorganisms that produce the modified protein glutaminase.

[0070] The culture conditions can be adjusted according to the nutritional and physiological properties of the transformant or the microorganisms, and a liquid culture is preferred. In the case of industrial or commercial production, an aerated and stirred culture is preferred. Nutrient sources for the medium can be those that are suitable for the growth of the transformant or the microorganisms. The carbon source can be any consumable carbon compound, and examples include glucose, sucrose, lactose, maltose, molasses, and pyruvic acid. The nitrogen source can be any consumable nitrogen compound, and examples include peptone, meat extract, yeast extract, casein hydrolysate, and alkaline soybean press cake extract.In addition to the carbon source and the nitrogen source, phosphates, carbonates, sulfates, salts of magnesium, calcium, potassium, iron, manganese, zinc and the like, specific amino acids, specific vitamins and the like may be used if necessary.

[0071] The culture temperature can be adjusted appropriately as long as the transformant of the present invention or the microorganisms can grow and produce a modified protein glutaminase. The culture temperature is preferably about 15 to 37 °C. The culture can be completed in a suitable time, which is judged to be the time when the modified protein glutaminase reaches its highest yield, and the culture time is typically about 12 to 48 hours.

[0072] After culturing the transformant or microorganisms, a process such as centrifugation is applied to the culture solution to isolate the culture supernatant and / or bacterial cells. A mechanical process, such as ultrasonic treatment or French pressing, or treatment with a lytic enzyme, such as lysozyme, is applied to the bacterial cells, and optionally, an enzyme, such as protease, or a surfactant, such as sodium lauryl sulfate (SDS), is used to solubilize the bacterial cells, yielding a water-soluble fraction containing a predefined modified protein glutaminase. It is also possible to secrete the expressed modified protein glutaminase into the culture solution by selecting a suitable expression cassette or expression vector and host.

[0073] The resulting water-soluble fraction containing the modified protein glutaminase can be subjected to purification treatment directly or after the modified protein glutaminase has been concentrated in the water-soluble fraction. Concentration can be achieved, for example, by reduced-pressure concentration, membrane concentration, salting-out treatment, or fractional precipitation with hydrophilic organic solvents (e.g., methanol, ethanol, and acetone).

[0074] The purification of the modified protein glutaminase can be carried out, for example, by appropriately combining methods such as gel filtration, adsorption chromatography, ion exchange chromatography, and affinity chromatography. The purified modified protein glutaminase can then be pulverized, if necessary, by lyophilization, vacuum drying, spray drying, or similar methods. 6. Enzyme agents

[0075] The modified protein glutaminase can be provided in the form of an enzyme agent. Accordingly, the present invention also provides an enzyme agent containing the modified protein glutaminase described above in "1. Modified Protein Glutaminase" as an active ingredient.

[0076] The content of the modified protein glutaminase in the enzyme composition of the present invention is not limited, and the lower limit of the content is, for example, 1 U / g or more, preferably 10 U / g or more, more preferably 50 U / g or more, even more preferably 100 U / g or more, and particularly preferably 200 U / g or more. The upper limit of the content is, for example, 10,000 U / g or less, preferably 5,000 U / g or less, more preferably 2,000 U / g or less, even more preferably 1,000 U / g or less, and particularly preferably 800 U / g or less.

[0077] It may or may not be necessary for the enzyme composition of the present invention to contain, in addition to the modified protein glutaminase, further components to such an extent that the effect of the present invention is not impaired. Examples of such further components include other enzymes different from the modified protein glutaminase, additives, and culture residues produced by the manufacturing process described above.

[0078] Examples of the additional enzyme include amylase (α-amylase, β-amylase, glucoamylase), glucosidase (α-glucosidase, β-glucosidase), galactosidase (α-galactosidase, β-galactosidase), protease (acidic protease, neutral protease, alkaline protease), peptidase (leucine peptidase, aminopeptidase), lipase, esterase, cellulase, phosphatase (acidic phosphatase, alkaline phosphatase), nuclease, deaminase, oxidase, dehydrogenase, glutaminase, pectinase, catalase, dextranase, transglutaminase, protein deamidase (excluding the modified protein glutaminase described above), and pullulanase. The additional enzymes may be present alone or in combination with two or more of them.

[0079] Examples of additives include an excipient, a buffer, a suspending agent, a stabilizer, a preservative, an antiseptic, and physiological saline. Examples of excipients include starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, sucrose, and glycerol. Examples of buffers include phosphate, citrate, and acetate. Examples of stabilizers include propylene glycol and ascorbic acid. Examples of preservatives include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, and methylparaben. Examples of antiseptics include ethanol, benzalkonium chloride, paraoxybenzoic acid, and chlorobutanol. These additives may be present alone or in a combination of two or more.

[0080] Examples of culture residue include a component originating from a culture medium, a contaminating protein, and a bacterial cell component.

[0081] The form of the enzyme agent of the present invention is not specifically limited, and examples include a liquid form and a solid form (a powder, granules, and the like). The enzyme agent in the form described above can be prepared by a generally known process. 7. Modifying agents for the protein material

[0082] The modified protein glutaminase can be used for known applications of protein glutaminase. For example, the modified protein glutaminase can be used for the purpose of modifying a protein material. Accordingly, the present invention also provides a modifier for a protein material containing a modified protein glutaminase.

[0083] The specific aspect of modifying a protein material is not limited, as long as it involves a change in the protein's properties caused by the generation of a carboxyl group due to the deamidation of the γ-amide and β-amide groups of the glutamine and asparagine residues of the protein. In particular, examples of protein material modification include increasing the protein's solubility, increasing its water dispersibility, and improving its emulsifying properties and emulsion stability. A specific procedure for using the modifying agent on a protein material is as described below in "8. Procedure for Preparing a Modified Protein Material." 8. Method for producing a modified protein material

[0084] As described above, the modified protein glutaminase can be used for the purpose of modifying a protein material. Accordingly, the present invention also provides a method for producing a modified protein material, comprising the step of applying the modified protein glutaminase to a protein material.

[0085] In the manufacturing process of the present invention, a mixture containing a protein material and a modified protein glutaminase is exposed to conditions for applying the modified protein glutaminase to cause a protein modification reaction to take place.

[0086] The protein material is not specifically restricted as long as it contains protein. The protein material can be either for edible or non-edible use. Edible protein material can be used as a food / beverage or as a material for manufacturing a food / beverage. Non-edible protein material can be used for protein experiments, as a medical material, a fiber material, a cosmetic material, or the like.

[0087] Specific examples of protein material include a protein source itself and a preparation obtained from a protein source by performing a treatment to increase the protein content using a known method selected appropriately by a person skilled in the art. Examples of edible protein material include a preparation obtained from a food product containing plant protein as the plant protein material; and a preparation made from a food product containing animal protein as the animal protein material. Examples of edible plant protein include bean protein, such as soybean protein, broad bean protein, pea protein, chickpea protein, green bean protein, and lupin protein; cereal protein, such as...Wheat protein, rye protein, oat protein, and corn protein; and seed protein from canary grass, flaxseed, almonds, cashews, hazelnuts, pecans, macadamia nuts, pistachios, walnuts, Brazil nuts, peanuts, coconuts, hemp seeds (industrial hemp), pili, chestnuts, sesame, pine nuts, and the like. Examples of edible animal protein include protein from livestock meat, fish, eggs, and milk. Examples of non-food protein material include albumin and globulin, obtained from a biological sample, such as egg white or serum; and silk and wool.

[0088] The protein content in the protein material is not limited and is, for example, 30 wt% or more, 40 wt% or more, or 50 wt% or more, preferably 60 wt% or more, more preferably 70 wt% or more, and even more preferably 80 wt% or more. The upper limit of the protein content in the protein material is not limited and is, for example, 95 wt% or less, 90 wt% or less, 85 wt% or less, 80 wt% or less, 70 wt% or less, or 60 wt% or less.

[0089] Examples of the protein content in the mixture include an amount such that the concentration of protein contained in the protein material in the mixture is, for example, 0.1 wt% or more, or 0.3 wt% or more, preferably 0.7 wt% or more, and more preferably 1.4 wt% or more. The upper limit of the concentration of protein contained in the protein material in the mixture is not limited and is, for example, 80 wt% or less, 60 wt% or less, 40 wt% or less, 30 wt% or less, 20 wt% or less, 15 wt% or less, 10 wt% or less, 8 wt% or less, 5 wt% or less, 3 wt% or less, or 2 wt% or less.

[0090] The amount of modified protein glutaminase used is not limited, and the amount of modified protein glutaminase used per gram of protein contained in the protein material is, for example, 0.1 U or more, preferably 0.5 U or more, more preferably 1 U or more, even more preferably 2 U or more, even more preferably 3.5 U or more, and even more preferably 4.5 U or more. The upper limit of the amount of modified protein glutaminase used per gram of protein contained in the protein material is not limited and is, for example, 45 U or less, 35 U or less, 20 U or less, 10 U or less, 8 U or less, or 5.5 U or less.

[0091] The amount of modified protein glutaminase used per gram of protein material is, for example, 0.01 U or more, 0.05 U or more, or 0.1 U or more, preferably 0.5 U or more, more preferably 1 U or more, even more preferably 2 U or more, even more preferably 3 U or more, and even more preferably 4 U or more. The upper limit of the amount of modified protein glutaminase used per gram of protein material is not limited and is, for example, 40 U or less, 30 U or less, 20 U or less, 10 U or less, 7 U or less, or 5 U or less.

[0092] The conditions for the application of the modified protein glutaminase are determined in a suitable manner based on the optimal temperature and pH value for the modified protein glutaminase used.

[0093] The temperature condition for the application of the modified protein glutaminase can be appropriately determined by a person skilled in the art according to the optimal temperature for the modified protein glutaminase and the like. For example, a specific temperature condition may be 40 to 70 °C, preferably 48 to 67 °C, more preferably 53 to 65 °C, and even more preferably 57 to 63 °C.

[0094] The pH conditions for the application of the modified protein glutaminase can be appropriately determined by a person skilled in the art according to the optimal pH value for the modified protein glutaminase and the like. For example, a specific pH value of 2 to 12, preferably 3 to 10, and more preferably 4 to 9, may be required.

[0095] The duration of application of the modified protein glutaminase is not limited and can be determined in a suitable manner according to the production scale or the like, and is, for example, 1 hour or more, preferably 8 hours or more, more preferably 16 hours or more, and even more preferably 20 hours or more. The upper limit of the time range is not limited and is, for example, 40 hours or less, 30 hours or less, or 25 hours or less.

[0096] After the reaction is complete, an enzyme deactivation treatment is performed, followed by cooling and, if necessary, post-treatment, resulting in a modified protein material. Examples

[0097] The present invention is described in detail below by means of examples which should not be construed as limiting the present invention. Test example 1(1) Production of a modified protein glutaminase

[0098] A sequence (SEQ ID NO: 11) encoding the amino acid sequence (SEQ ID NO: 1) of protein glutaminase derived from wild-type C. proteolyticum and introduced into the pET21 vector was used as a template into which various mutations were introduced to produce the following modified protein glutaminases. [Table 1] Example 1 C121V mutant of SEQ ID NO: 1 (SEQ ID NO: 3) Example 2 C121A mutant of SEQ ID NO: 1 (SEQ ID NO: 4) Example 3 Y142C mutant of SEQ ID NO: 1 (SEQ ID NO: 5) Example 4 Y142D mutant of SEQ ID NO: 1 (SEQ ID NO: 6)

[0099] In particular, PCR was performed using the following primers and the PrimeSTAR mutagenesis basal kit (Takara) with a conventional method for introducing mutations. (Primer for the C121V mutation) Rv: 5'-ATCTGTTACAGGACCGCTTGAAAATAGTGAAGGATC-3' (SEQ ID NO: 19) Fw: 5'-ACAGCATGGAGAAACGCTgttGTTAACACCTCTTGCGGATCTGCATCC-3' (SEQ ID NO: 20) (Primer for the C121A mutation) Rv: 5'-ATCTGTTACAGGACCGCTTGAAAATAGTGAAGGATC-3' (SEQ ID NO: 21) Fw: 5'-ACAGCATGGAGAAACGCTgcgGTTAACACCTCTTGCGGATCTGCATCC-3' (SEQ ID NO: 22) (Primer for the Y142C mutation) Rv: 5'-CTCTTGCGGATCTGCATCCGTTTCCTCTTATGCT-3' (SEQ ID NO: 23) Fw: 5'-AATACTGCAGGAAATGTTtgtTACAGAAGTCCTAGTAATTCTTACCTGTATGACAACAATC-3' (SEQ ID NO: 24) (Primer for the Y142D mutation) Rv: 5'-CTCTTGCGGATCTGCATCCGTTTCCTCTTATGCT-3' (SEQ ID NO: 25) Fw: 5'-AATACTGCAGGAAATGTTgatTACAGAAGTCCTAGTAATTCTTACCTGTATGACAACAATC-3' (SEQ ID NO: 26)

[0100] Each of the obtained mutated gene products was transformed into E. coli BL21 (DE3) using a conventional method to obtain gene expression vectors. The protein glutaminase sequence introduced into the gene expression vector was validated. The transformant was cultured in LB medium at 37 °C for 16 hours with shaking. The transformant was then transferred to Terrific Broth medium and cultured at 37 °C for 4 hours, and IPTG (final concentration: 0.5 mM) was added. The culture was then shaken at 33 °C for 20 hours. Bacterial cells were isolated from the culture solution and lysed using the bacterial cell lysis reagent B-PER (brand name) (manufactured by Thermo Scientific) using a conventional method and centrifuged at 15,000 rpm for 10 minutes.The centrifuged supernatant was isolated as a crude enzyme solution, trypsin was added to the solution at a final concentration of 50 µg / mL, and the mixture was allowed to stand at 37 °C for 1 hour and then purified by a conventional procedure using TALON (registered trademark) spin columns (manufactured by Takara Bio Inc.), yielding an enzyme sample. (2) Methods for measuring protein glutaminase activity

[0101] N-Benzyloxycarbonyl-L-glutaminylglycine (Z-Gln-Gly; Peptide Institute, Inc.) was dissolved in a 0.2 mol / L phosphate buffer (pH 6.5) at 30 mmol / L, and the resulting solution was used as the substrate solution. 0.1 mL of an enzyme solution whose activity was to be measured was added to a test tube and incubated in a water bath at constant temperature (37 ± 0.5 °C) for 1 minute. Then, 1 mL of a substrate solution that had been previously incubated at 37 ± 0.5 °C for 10 minutes was added, and the mixture was immediately mixed. This solution was allowed to stand for 10 minutes to allow an enzymatic reaction, and 1 mL of a 0.4 mol / L trichloroacetic acid solution was added to stop the enzymatic reaction.A blank sample was prepared by adding 0.1 mL of an enzyme solution to a test tube, followed by 1 mL of a 0.4 mol / L trichloroacetic acid solution and 1 mL of a substrate solution, in that order. A color development reaction using the Wako ammonia test (FUJIFILM Wako Pure Chemical Corporation) was performed, and ammonia released by an enzyme reaction over 10 minutes was quantified by the absorbance value at a wavelength of 630 nm. The ammonia concentration in the reaction solution was determined from a calibration curve showing the relationship between ammonia concentration and absorbance (630 nm), which was prepared using a standard ammonia solution (ammonium chloride). The enzymatic activity of protein glutaminase was calculated from the following equation, where the amount of enzyme producing 1 µmol of ammonia per minute is defined as one unit (1U).In the equation, the amount of reaction solution is 2.1, the amount of enzyme solution is 0.1, and Df is a dilution factor of the enzyme solution. The value "17.03" represents the molecular weight of ammonia. [Chemical Formula 1] Enzymatic activity (U / mL) = Ammonia concentration in the reaction solution (mg / L) × (1 / 17.03) × (Amount of reaction solution / Amount of enzyme solution) × (1 / 10) × Df (3) Confirmation of the specific activity

[0102] The specific activity at 37 °C was measured using the obtained samples of the modified protein glutaminases from Examples 1 to 4. The results showed that, when the specific activity of Chryseobacterium proteolyticum-derived protein glutaminase (wild type) is set as 100%, the specific activity was 84% ​​for the enzyme sample of the modified protein glutaminase from Example 1, 93% for the enzyme sample of the modified protein glutaminase from Example 2, 137% for the enzyme sample of the modified protein glutaminase from Example 3, and 90% for the enzyme sample of the modified protein glutaminase from Example 4. It was confirmed that the activity of all samples is equivalent to or better than that of the wild type. (4) Confirmation of oxidation stability

[0103] Hydrogen peroxide was added to a solution in which the protein content of the purified enzyme solution had been adjusted to 0.1 mg / mL, at a final concentration of 0.001 wt%. The protein glutaminase activity after treatment at 37 °C for 1 hour was measured, and the residual activity (residual activity after oxidation treatment A) was determined. 0.001 % ) was calculated from the ratio of protein glutaminase activity after hydrogen peroxide treatment to protein glutaminase activity without hydrogen peroxide treatment. The ratio of residual activity after oxidation treatment A 0,001% of the modified protein glutaminase to the residual activity after an oxidation treatment A 0,001% The ratio of wild-type protein glutaminase to wild type was calculated. Furthermore, the residual activity after oxidation treatment B was determined. 0,0025%and the ratio to the wild type was calculated accordingly, except that the amount of added hydrogen peroxide was adjusted to a final concentration of 0.0025 wt.%. The results are shown in the table below. [Table 2] Specific activity (ratio to the activity of the wild type, which is set as 100%) 0.001% hydrogen peroxide treatment 0.0025% hydrogen peroxide treatment Activity after oxidation treatment A 0,001% Relationship to wild type Activity after oxidation treatment B 0,0025% Relationship to wild type Reference example 1 Wild type 100 % 9% 1 1 % 1 Example 1 C121V mutant 84 % 56 % 5,9 23 % 26,1 Example 2 C121A mutant 93 % 41 % 4,4 7% 7,9 Example 3 Y142C mutant 137 % 58 % 6,2 9% 10,6 Example 4 Y142D mutant 90 % 47 % 5 20 % 22,8

[0104] As shown in the table above, the residual activity after hydrogen peroxide treatment in the modified protein glutaminases of Examples 1 to 4 was considerably improved. Test example 2

[0105] Using a statistical mutation introduction kit (Gene Morph II Statistical Mutagenesis Kit, Diversify PCR Statistical Mutagenesis Kit, manufactured by Agilent Technologies, Inc.), a statistical mutation was introduced into the sequence (SEQ ID NO: 11) encoding the amino acid sequence of a protein glutaminase derived from C. proteolyticum (SEQ ID NO: 1), according to the instructions for the kit.

[0106] The gene was inserted into a pET21a vector after mutation introduction using the In-Fusion (registered trademark) HD ​​cloning kit (manufactured by Clontech Laboratories, Inc.). E. coli BL21 (DE3) was transformed using a conventional method, and the resulting transformant was cultured with shaking in LB medium at 37 °C for 16 hours. The transformant was then transferred to Terrific Broth and cultured at 37 °C for 4 hours, and IPTG (final concentration: 0.5 mM) was added, followed by a shake culture at 33 °C for 20 hours. Bacterial cells were isolated from the culture solution and lysed using B-PER (trademark) bacterial cell lysis reagent (manufactured by Thermo Scientific) using a conventional method and centrifuged at 15,000 rpm for 10 minutes.The centrifuged supernatant was isolated as a crude enzyme solution, trypsin was added to the solution at a final concentration of 50 µg / mL, and the mixture was left to stand at 37 °C for 1 hour and then purified using a conventional method with TALON (registered trademark) spin columns (manufactured by Takara Bio Inc.), yielding an enzyme sample.

[0107] The specific activity and oxidative stability of the obtained enzyme sample were evaluated using the procedure from Test Example 1. For enzyme samples with improved oxidative stability, the gene sequence was confirmed and the mutation identified. The results are shown in the table below. Table 3] Specific activity (ratio to the activity of the wild type, which is set as 100%) 0.0025% hydrogen peroxide treatment Activity after oxidation treatment B 0,0025% Relationship to wild type Reference example 1 Wild type 100 % 2% 1 Example 5 R36I mutant 75 % 52 % 25 Example 6 S30T / T113K mutant 74 % 23 % 11 Example 7 I157F mutant 74 % 25 % 12 Example 8 L156P mutant 63 % 52 % 26 Example 9 S26C / Y43F / C183G mutant 28 % 79 % 38 Example 10 I33V mutant 69 % 66 % 32 Example 11 L169F mutant 69 % 32 % 15 Example 12 S182R mutant 76 % 56 % 28 Example 13 T73I mutant 31 % 49 % 24 Example 14 S26Y mutant 63 % 87 % 42 Example 15 F35S mutant 81 % 38 % 18 Example 16 S22Y / S170T-Mutant 111 % 88 % 43

[0108] As shown in the table above, the residual activity after hydrogen peroxide treatment in the modified protein glutaminases of Examples 5 to 16 was considerably improved. Sequence protocol, free text

[0109] SEQ ID NOS: 19 to 26 correspond to primers. QUOTES INCLUDED IN THE DESCRIPTION

[0000] This list of documents cited by the applicant was automatically generated and is included solely for the reader's convenience. The list is not part of the German patent or utility model application. The DPMA accepts no liability for any errors or omissions. Cited non-patent literature

[0000] A novel protein-deamidating enzyme from Chryseobacterium proteolyticum sp. nov., a newly isolated bacterium from soil. Applied and Environmental Microbiology 2000; 66(8): 3337-43

[0004] Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol. Lett., Volume 174, Pages 247-250, 1999

[0031] Sanger et al. (1977), Proc. Natl. Acad. Sci. USA 74:5463

[0055]

Claims

[1] Modified protein glutaminase comprising one of the following polypeptides (I) to (III): (I) a polypeptide consisting of an amino acid sequence obtained by introducing at least one of (A) a substitution of the amino acid residue at position 121 with a valine residue or an alanine residue, (B) a substitution of the amino acid residue at position 142 with a cysteine ​​residue or an aspartic acid residue, (C) a substitution of the amino acid residue at position 22 with a tyrosine residue, (D) a substitution of the amino acid residue at position 26 with a tyrosine residue or a cysteine ​​residue, (E) a substitution of the amino acid residue at position 30 with a threonine residue, (F) a substitution of the amino acid residue at position 33 with a valine residue, (G) a substitution of the amino acid residue at position 35 with a serine residue, (H) a substitution of the amino acid residue at position 36 with an isoleucine residue, (I) a substitution of the amino acid residue at position 43 with a phenylalanine residue, (J) a substitution of the amino acid residue at position 73 with an isoleucine residue, (K) a substitution of the amino acid residue at position 113 with a lysine residue, (L) a substitution of the amino acid residue at position 156 with a proline residue, (M) a substitution of the amino acid residue at position 157 with a phenylalanine residue, (N) a substitution of the amino acid residue at position 169 with a phenylalanine residue, (O) a substitution of the amino acid residue at position 170 with a threonine residue, (P) a substitution of the amino acid residue at position 182 with an arginine residue, and (Q) a substitution of the amino acid residue at position 183 with a glycine residue, into the amino acid sequence given as SEQ ID NO: 1; (II) a polypeptide in which one or more amino acid residues other than the substituted amino acid residues are substituted, added, inserted or deleted in the amino acid sequence in which at least one of the substitutions (A) to (Q) is introduced, and the residual activity of protein glutaminase after hydrogen peroxide treatment is improved compared to the residual activity of the polypeptide consisting of the amino acid sequence given as SEQ ID NO: 1; and (III) a polypeptide in which the sequence identity of regions not comprising the substituted amino acid sequences is 70% or more in the amino acid sequence in which at least one of the substitutions (A) to (Q) is introduced, and the residual activity of protein glutaminase after hydrogen peroxide treatment is improved compared with the residual activity of the polypeptide consisting of the amino acid sequence given as SEQ ID NO:

1. [2] DNA encoding the modified protein glutaminase according to claim 1. [3] An expression cassette or recombinant vector comprising the DNA of claim 2. [4] A transformant obtained by transforming a host using the expression cassette or recombinant vector according to claim 3. [5] A process for producing a modified protein glutaminase, which comprises the step of culturing the transformant according to claim 4. [6] An enzyme agent comprising the modified protein glutaminase according to claim 1. [7] A modifier for a protein material comprising the modified protein glutaminase according to claim 1. [8] A process for producing a modified protein material comprising the step of applying the modified protein glutaminase according to claim 1 to a protein material.