Protein, method for producing protein, and method for producing protein having asparagine residue deamidated

Abstract

To provide a novel protein deamidation enzyme (protein asparaginase) that catalyzes a reaction of deamidation of an asparagine residue contained in a protein.SOLUTION: A protein characterized by having activity to catalyze a reaction in which an asparagine residue in a protein is deamidated, and by having one β-barrel domain at the C-terminus or having no β-barrel domain at the C-terminus.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a novel protein deamidase, a modified protein deamidase, a polynucleotide encoding the enzyme or the modified protein deamidase, a recombinant vector comprising the polynucleotide, a transformant into which the vector has been introduced, a method for producing the enzyme or the modified protein deamidase, and a method for deamidating a protein using the enzyme or the modified protein deamidase. [Background technology]

[0002] Protein deamidation improves various functional properties of proteins (Non-Patent Document 1). Therefore, protein deamidation is expected to expand the applications of proteins. Among the amino acids that make up proteins, glutamine and asparagine have amide groups. These are converted to glutamic acid and aspartic acid, respectively, by deamidation. Deamidation increases the negative charge of proteins and lowers their isoelectric point, significantly improving the solubility and water dispersibility of proteins. Furthermore, as electrostatic repulsion increases, protein-protein interactions, i.e., association, decrease. Furthermore, the tertiary structure of proteins unravels, the higher-order structure changes, and hydrophobic regions buried inside the molecules become exposed on the molecular surface. This gives deamidated proteins amphiphilic properties, improving the emulsifying power, emulsion stability, foaming ability, and foam stability of proteins.

[0003] Protein deamidation methods can be divided into chemical methods and enzymatic methods. Many chemical deamidation methods using mild acid or alkaline treatment have been reported. However, all of these methods have problems, such as being non-specific reactions, cleavage of peptide bonds under acid or alkaline conditions, and the generation of unexpected by-products. Furthermore, they require equipment for using chemical substances, which places a heavy burden on the environment. Enzymatic methods can overcome these problems associated with chemical methods. Examples of enzymatic deamidation methods that have been reported include methods using protein glutaminase (Patent Documents 1-2, Non-Patent Document 2), methods using proteases (Non-Patent Documents 3-4), methods using transglutaminase (Non-Patent Document 5), and methods using peptidoglutaminase (Non-Patent Documents 6-7).

[0004] Among these, only protein glutaminase can catalyze the deamidation of high-molecular-weight proteins without side reactions. Proteases and transglutaminases primarily catalyze the cleavage of peptide bonds and the formation of isopeptide bonds between glutamine and lysine, respectively, through cross-linking. Peptidoglutaminases, on the other hand, exclusively catalyze the deamidation of low-molecular-weight peptides, and therefore both have their drawbacks. Protein glutaminase is considered highly practical as an enzyme with high deamidation ability for high-molecular-weight proteins. There are already findings regarding the use of protein glutaminase to improve the functionality of wheat proteins, milk proteins (casein and whey proteins), and soybean proteins (Non-Patent Documents 8-11). Patent findings regarding the use of protein glutaminase to improve the quality of foods such as yogurt, ice cream, coffee whitener, noodles, and meat products have also been reported (Patent Documents 3-7). However, it has been clearly stated that this protein glutaminase uses glutamine residues in proteins as a substrate and has no effect at all on asparagine residues (Non-Patent Document 2, Non-Patent Document 12), and the treatment effect is limited. In fact, the amino acids that make up plant and animal proteins contain a large amount of asparagine, and in order to obtain a further functional modification effect by deamidation, it is preferable to deamidate not only glutamine but also asparagine.

[0005] Asparaginase (EC number: 3.5.1.1) hydrolyzes asparagine to aspartic acid. Asparaginase is widely known as an enzyme that catalyzes the deamidation of asparagine residues in peptides or proteins with large molecular weights. However, asparaginase is an enzyme that acts specifically on free asparagine and cannot deamidate asparagine residues in peptides or proteins with large molecular weights. In addition, an enzyme that catalyzes the deamidation of N-terminal asparagine residues that have a free α-amino group is known (Non-Patent Document 13). However, this enzyme cannot deamidate asparagine residues in proteins other than the N-terminal asparagine residue.

[0006] The present inventors have identified novel enzymes that deamidate asparagine residues in proteins (other than N-terminal asparagine residues with a free α-amino group) from Luteimicrobium album, Agromyces sp., and Microbacterium diffusum. reported a protein deamidase derived from Microbacterium testaceum, Leifsonia xyli, or Leifsonia aquatica (Patent Document 8). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Laid-Open No. 2001-218590 [Patent Document 2] Japanese Patent Application Laid-Open No. 2005-052158 [Patent Document 3] International Publication No. 2011 / 024994 [Patent Document 4] International Publication No. 2011 / 125826 [Patent Document 5] International Publication No. 2011 / 108633 [Patent Document 6] Japanese Patent Application Laid-Open No. 2009-219419 [Patent Document 7] International Publication No. 2006 / 075771 [Patent Document 8] International Publication No. 2015 / 133590 [Non-patent literature]

[0008] [Non-Patent Document 1] Hamada JS 1994. Critical Reviews in Food Science and Nutrition, 34, 283. [Non-patent document 2] Yamaguchi S, Jeenes DJ and Archer DB. 2001. Eur. J. Biochem. 268(5), 1410.

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Non-patent document 10

[0009] An objective of the present invention is to provide a novel protein deamidase (protein asparaginase) that catalyzes the deamidation of asparagine residues in proteins. [Means for solving the problem]

[0010] As a result of extensive research to solve the above problems, the present inventors discovered that a part of a protein having a certain level of homology to a partial sequence of protein deamidase previously reported (WO 2015 / 133590) catalyzes a reaction that specifically deamidates asparagine residues, thereby completing the present invention.

[0011] That is, the present invention can be exemplified as follows. [1] A compound having an activity of catalyzing the deamidation of an asparagine residue in a protein, and A protein characterized by having one β-barrel domain at the C-terminus or no β-barrel domain at the C-terminus. [2] The protein according to [1], which is derived from Amycolatopsis deserti, Microbispora corallina, or Motilibacter peucedani. [3] The protein according to [1] or [2], which is a protein according to the following (A), (B), or (C): (A) a protein comprising the amino acid sequence shown in SEQ ID NO: 1, 3, or 5, the amino acid sequence of positions 129 to 785 of SEQ ID NO: 1, positions 130 to 808 of SEQ ID NO: 3, or positions 143 to 818 of SEQ ID NO: 5; (B) A protein comprising an amino acid sequence containing a substitution, deletion, insertion, or addition of one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 1, 3, or 5, or the amino acid sequence of positions 129 to 785 of SEQ ID NO: 1, positions 130 to 808 of SEQ ID NO: 3, or positions 143 to 818 of SEQ ID NO: 5, and having the activity of catalyzing a reaction of deamidating an asparagine residue in a protein. (C) A protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 1, 3, or 5, or the amino acid sequence of positions 129 to 785 of SEQ ID NO: 1, positions 130 to 808 of SEQ ID NO: 3, or positions 143 to 818 of SEQ ID NO: 5, and having the activity of catalyzing a reaction to deamidate an asparagine residue in a protein. [4] A protein selected from the group consisting of (A'), (B'), and (C') below: (A') a protein having the amino acid sequence of positions 1 to 1074, 132 to 1074, 240 to 1074, 1 to 1166, 132 to 1166, or 240 to 1166 of SEQ ID NO: 9; positions 1 to 894, 67 to 894, 181 to 894, 1 to 1007, 67 to 1007, or 181 to 1007 of SEQ ID NO: 11; positions 1 to 932, 70 to 932, 193 to 932, 1 to 1018, 70 to 1018, or 193 to 1018 of SEQ ID NO: 13; or positions 1 to 831, 81 to 831, 1 to 939, or 81 to 939 of SEQ ID NO: 14; (B') amino acids at positions 1 to 1074, 132 to 1074, 240 to 1074, 1 to 1166, 132 to 1166, or 240 to 1166 of SEQ ID NO: 9; amino acids at positions 1 to 894, 67 to 894, 181 to 894, 1 to 1007, 67 to 1007, or 181 to 1007 of SEQ ID NO: 11; amino acids at positions 1 to 932, 70 to 932, 193 to 932, 1 to 1018, 70 to 1018, or 193 to 1018 of SEQ ID NO: 13; or amino acids at positions 1 to 831, 81 to 831, 1 to 939, or 81 to 939 of SEQ ID NO: 14 a protein having an amino acid sequence containing substitution, deletion, insertion, or addition of one or several amino acid residues, and having the activity of catalyzing the reaction of deamidating asparagine residues in proteins; (C') positions 1 to 1074, 132 to 1074, 240 to 1074, 1 to 1166, 132 to 1166, or 240 to 1166 of SEQ ID NO: 9; positions 1 to 894, 67 to 894, 181 to 894, 1 to 1007, 67 to 1007, or 181 to 1007 of SEQ ID NO: 11; positions 1 to 932, 70 to 932, 193 a protein having an amino acid sequence that is 90% or more identical to the amino acid sequence of positions 1 to 932, 1 to 1018, 70 to 1018, or 193 to 1018 of SEQ ID NO: 14, or positions 1 to 831, 81 to 831, 1 to 939, or 81 to 939 of SEQ ID NO: 14, and having the activity of catalyzing a reaction to deamidate an asparagine residue in a protein. [5] A polynucleotide encoding the protein according to any one of [1] to [4]. [6] A recombinant vector comprising the polynucleotide according to [5]. [7] A transformant into which the recombinant vector described in [6] has been introduced. [8] A method for producing a protein having an activity to catalyze a reaction to deamidate an asparagine residue in a protein, the method comprising culturing the transformant according to [7] in a medium to produce the protein according to any one of [1] to [4], and recovering the protein from the culture. [9] A method for producing a protein having the activity of catalyzing a reaction to deamidate an asparagine residue in a protein, the method comprising culturing an organism capable of producing the protein according to any one of [1] to [4] in a medium to produce the protein, and recovering the protein from the culture.

[10] The method according to [8] or [9], which comprises treating the protein with a processing enzyme.

[11] The method according to

[10] , wherein the processing enzyme is a protease.

[12] A method for producing a protein variant having improved activity based on the amino acid sequence of a protein that has an activity to catalyze a reaction to deamidate an asparagine residue in a protein and is characterized by having two or more β-barrel domains at the C-terminus, comprising: A method comprising designing a polynucleotide encoding a protein variant having an amino acid sequence obtained by removing at least one amino acid sequence corresponding to the C-terminal β-barrel domain from the amino acid sequence of the protein.

[13] The method according to

[12] , wherein the protein is a protein selected from the group consisting of (a), (b), and (c): (a) a protein comprising the amino acid sequence set forth in SEQ ID NO: 9, 11, 13, or 14, the amino acid sequence of positions 240 to 1355 of SEQ ID NO: 9, the amino acid sequence of positions 181 to 1180 or positions 67 to 1180 of SEQ ID NO: 11, the amino acid sequence of positions 193 to 1172 or positions 70 to 1172 of SEQ ID NO: 13, or the amino acid sequence of positions 81 to 1119 of SEQ ID NO: 14; (b) an amino acid sequence containing one or several substitutions, deletions, insertions, or additions of amino acid residues in the amino acid sequence set forth in SEQ ID NO: 9, 11, 13, or 14, the amino acid sequence of positions 240 to 1355 of SEQ ID NO: 9, the amino acid sequence of positions 181 to 1180 or positions 67 to 1180 of SEQ ID NO: 11, the amino acid sequence of positions 193 to 1172 or positions 70 to 1172 of SEQ ID NO: 13, or the amino acid sequence of positions 81 to 1119 of SEQ ID NO: 14, and having activity of catalyzing a reaction of deamidating an asparagine residue in a protein; (c) a protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 9, 11, 13 or 14, the amino acid sequence of positions 240 to 1355 of SEQ ID NO: 9, the amino acid sequence of positions 181 to 1180 or positions 67 to 1180 of SEQ ID NO: 11, the amino acid sequence of positions 193 to 1172 or positions 70 to 1172 of SEQ ID NO: 13, or the amino acid sequence of positions 81 to 1119 of SEQ ID NO: 14, and having the activity of catalyzing a reaction of deamidating an asparagine residue in a protein.

[14] The amino acid sequence corresponding to the C-terminal β-barrel domain is the following (a'), ( The method according to

[12] or

[13] , wherein the amino acid sequence is the amino acid sequence of (b') or (c'): (a') the amino acid sequence of positions 1075 to 1166, 1167 to 1257, or 1258 to 1355 of SEQ ID NO: 9, positions 895 to 1007 or 1008 to 1180 of SEQ ID NO: 11, positions 933 to 1018 or 1019 to 1172 of SEQ ID NO: 13, or positions 832 to 939 or 940 to 1119 of SEQ ID NO: 14; (b') an amino acid sequence comprising substitution, deletion, insertion, or addition of one or several amino acid residues in the amino acid sequence of positions 1075 to 1166, 1167 to 1257, or 1258 to 1355 of SEQ ID NO: 9, positions 895 to 1007 or 1008 to 1180 of SEQ ID NO: 11, positions 933 to 1018 or 1019 to 1172 of SEQ ID NO: 13, or positions 832 to 939 or 940 to 1119 of SEQ ID NO: 14; (c') An amino acid sequence having 90% or more identity to the amino acid sequence of positions 1075 to 1166, 1167 to 1257, or 1258 to 1355 of SEQ ID NO: 9, positions 895 to 1007 or 1008 to 1180 of SEQ ID NO: 11, positions 933 to 1018 or 1019 to 1172 of SEQ ID NO: 13, or positions 832 to 939 or 940 to 1119 of SEQ ID NO: 14.

[15] The method according to any one of

[12] to

[14] , which comprises introducing a recombinant vector containing the polynucleotide into a host to obtain a transformant.

[16] The method according to

[15] , comprising culturing the transformant in a medium to produce the protein variant, and recovering the protein variant from the culture obtained by the culturing.

[17] The method according to

[16] , which comprises treating the protein variant with a processing enzyme.

[18] The method according to

[17] , wherein the processing enzyme is a protease.

[19] A method for producing a protein in which an asparagine residue is deamidated, comprising allowing the protein according to any one of [1] to [4] to act on a protein containing an asparagine residue.

[20] The method according to

[19] , wherein the protein containing the asparagine residue is contained in a food or beverage or an ingredient thereof.

[21] The method according to

[19] or

[20] , wherein the food or drink is selected from mayonnaise, dressing, cream, yogurt, meat products, and bread.

[22] The method according to any one of

[19] to

[21] , wherein 0.001 to 2000 U of the protein according to any one of [1] to [4] is used per 1 g of the protein containing an asparagine residue.

[23] A method for modifying a food or drink or a raw material thereof containing a protein having an asparagine residue, the method comprising allowing the protein according to any one of [1] to [4] to act on the food or drink or a raw material thereof.

[24] The method according to

[23] , wherein the food or beverage is selected from mayonnaise, dressing, cream, yogurt, meat products, and bread.

[25] The method according to

[23] or

[24] , wherein 0.001 to 2000 U of the protein according to any one of [1] to [4] is used per 1 g of the protein containing an asparagine residue.

[26] A method for producing a modified food or beverage product or a raw material thereof, comprising allowing the protein according to any one of [1] to [4] to act on a food or beverage product or a raw material thereof containing a protein having an asparagine residue.

[27] The method according to

[26] , wherein the food or beverage is selected from mayonnaise, dressing, cream, yogurt, meat products, and bread.

[28] The method according to

[26] or

[27] , wherein 0.001 to 2000 U of the protein according to claim 1 or 4 is used per 1 g of the protein containing the asparagine residue. [Effects of the Invention]

[0012] The present invention provides a novel protein deamidase (protein asparaginase) that catalyzes the deamidation of asparagine residues in proteins. [Brief explanation of the drawings]

[0013] [Figure 1] Predicted three-dimensional structure of protein deamidase from Amycolatopsis deserti (photo instead of drawing). [Figure 2] Predicted three-dimensional structure of the protein deamidase derived from Luteimicrobium album (photo instead of drawing). [Figure 3] Predicted three-dimensional structure of the protein deamidase from Microbacterium testaceum (photo instead of drawing). [Figure 4] Predicted three-dimensional structure of protein deamidase from Microbispora corallina (photo instead of drawing). [Figure 5] Predicted three-dimensional structure of protein deamidase from Motilibacter peucedani (photo instead of drawing). [Figure 6] The three-dimensional structure of the protein deamidase derived from Amycolatopsis deserti obtained by X-ray crystal structure analysis (photograph instead of drawing). [Figure 7] A diagram (photograph) showing the predicted 3D structure of the protein deamidase derived from Amycolatopsis deserti superimposed on the 3D structure obtained by X-ray crystal structure analysis. Gray: predicted 3D structure, black: 3D structure obtained by X-ray crystal structure analysis. DETAILED DESCRIPTION OF THE INVENTION

[0014] <1> protein deamidase A first embodiment of the present invention is a protein deamidase, Specifically, the following: having an activity of catalyzing a reaction of deamidating an asparagine residue in a protein, and A protein characterized by having one β-barrel domain at the C-terminus or no β-barrel domain at the C-terminus.

[0015] In the present invention, "protein deamidase" refers to a protein having an activity to catalyze a reaction to deamidate asparagine residues in a protein. This activity is also referred to as "protein asparaginase activity." In the present invention, protein deamidase is also referred to as "protein asparaginase."

[0016] The term "asparagine residue in a protein" refers to an asparagine residue present in a protein other than an N-terminal asparagine residue having a free α-amino group. Protein deamidase may or may not have the activity to catalyze the deamidation of an N-terminal asparagine residue having a free α-amino group, as long as it has the activity to catalyze the deamidation of such an "asparagine residue in a protein." Protein deamidase may or may not have the activity to catalyze the deamidation of a monomeric asparagine.

[0017] In the present invention, a protein to be deamidated by protein deamidase is also referred to as a "substrate protein." The length of the substrate protein is not particularly limited as long as it is two residues (dipeptide) or more. The length of the substrate protein may be, for example, two residues (dipeptide) or more, three residues (tripeptide) or more, 10 residues or more, 50 residues or more, or 100 residues or more. In other words, unless otherwise specified, the substrate protein also encompasses embodiments called peptides, such as oligopeptides and polypeptides.

[0018] Asparagine residues are hydrolyzed to aspartic acid residues and ammonia by deamidation. Therefore, protein asparaginase activity can be measured, for example, by utilizing the generation of ammonia accompanying the deamidation of asparagine residues. For example, protein asparaginase activity can be measured by using a peptide of two or more residues (e.g., CBZ-Asn-Gly) that contains an asparagine residue but does not contain a glutamine residue as a substrate. Protein asparaginase activity can be calculated based on the amount of ammonia liberated by allowing protein deamidase to act on the asparaginase. Specifically, protein asparaginase activity can be calculated, for example, under the conditions described in the Examples. That is, protein asparaginase activity can be measured by adding 10 μL of an enzyme solution of an appropriate concentration to 190 μL of 200 mM phosphate buffer (pH 6.5) containing 30 mM CBZ-Asn-Gly, incubating at 37°C for 10 minutes, and then adding 12% trichloroethylene. The reaction was stopped by adding 200 μL of isopropyl alcohol solution, and the product (CBZ-Asp-Gly or ammonia) in the supernatant was collected. In the present invention, the enzyme activity that produces 1 μmol of ammonia per minute under these conditions is defined as 1 unit (U) of protein asparaginase activity. Let's say.

[0019] It is preferable that protein deamidase does not substantially have the activity of catalyzing the reaction of deamidating glutamine residues in proteins. This activity is also referred to as "protein glutaminase activity." Glutamine residues are hydrolyzed into glutamic acid residues and ammonia by deamidation. Therefore, protein glutaminase activity can be measured, for example, based on the production of ammonia accompanying the deamidation of glutamine residues. For example, protein deamidase is allowed to act on a peptide (e.g., CBZ-Gln-Gly) of two or more residues containing glutamine residues but not asparagine residues as a substrate, and the released ammonia is measured. Protein glutaminase activity can be calculated based on the amount of ammonia. Specifically, for example, protein glutaminase activity can be calculated by performing an enzyme reaction under the conditions for measuring protein asparaginase activity described in the Examples, but substituting CBZ-Gln-Gly for CBZ-Asn-Gly. That is, protein glutaminase activity can be measured by adding 10 μL of an enzyme solution of an appropriate concentration to 190 μL of 200 mM phosphate buffer (pH 6.5) containing 30 mM CBZ-Gln-Gly, incubating at 37° C. for 10 minutes, and then adding 200 μL of 12% trichloroacetic acid solution to stop the reaction. and measuring the concentration of the product (CBZ-Glu-Gly or ammonia) in the supernatant. In the present invention, the enzyme activity required to produce 1 μmol of ammonia per minute under these conditions is is defined as 1 unit (U) of protein glutaminase activity. The phrase "protein deamidase has substantially no protein glutaminase activity" may mean, for example, that the ratio of protein glutaminase activity to protein asparaginase activity in protein deamidase (i.e., specific activity of protein glutaminase activity / specific activity of protein asparaginase activity) is 1 / 100 or less, 1 / 1000 or less, 1 / 10,000 or less, or 0 (zero). The specific activity ratio can be calculated by measuring the protein asparaginase activity and the protein glutaminase activity, respectively.

[0020] It is preferable that protein deamidase does not substantially have the activity of catalyzing the reaction of hydrolyzing peptide bonds in proteins. This activity is also referred to as "protease activity." Protease activity can be measured, for example, by known techniques. Specifically, protease activity can be calculated using azocasein as a substrate under the following conditions: That is, an appropriate concentration of azocasein is added to 1.0 mL of 50 mM Tris-HCl buffer (pH 8.0) containing 1% azocasein. Add 0.5 mL of enzyme solution and incubate at 37°C for 30 minutes. Then, add 2.0 mL of 12% trichloroacetic acid solution to stop the reaction. After centrifugation (15,000 rpm, 4°C, 5 minutes), the supernatant A 405 As controls, a test section was prepared by adding 0.5 mL of water instead of the enzyme solution, and a test section was prepared by adding 1.0 mL of 50 mM Tris-HCl buffer (pH 8.0) without azocasein instead of 50 mM Tris-HCl buffer (pH 8.0) containing azocasein in order to subtract the effect of the color of components that may be contained in the enzyme solution. 405 This is measured. et al. A 405 Based on the measured values ​​of A 405 In the present invention, under these conditions, the increase in A per minute is calculated. 405 The enzyme activity that increases the amount of protease by 0.01 is defined as 1 unit (U) of protease activity. The phrase "protein deamidase has substantially no protease activity" means, for example, It may be that when a protein is treated with protein deamidase so that deamidation of asparagine residues occurs to a desired extent, no significant degradation of the protein by cleavage of peptide bonds is observed before or after the treatment. Furthermore, "protein deamidase has substantially no protease activity" may mean, for example, that the ratio of the protease activity of protein deamidase to the protein asparaginase activity (i.e., specific activity of protease activity / specific activity of protein asparaginase activity) is 1 / 100 or less, 1 / 1000 or less, 1 / 10,000 or less, or 0 (zero). The specific activity ratio can be calculated by measuring the protein asparaginase activity and the protease activity, respectively.

[0021] It is preferable that protein deamidase does not substantially have the activity of catalyzing a protein cross-linking reaction. This activity is also referred to as "protein cross-linking activity." Protein cross-linking activity can be measured, for example, by known techniques. "Protein deamidase does not substantially have protein cross-linking activity" may mean, for example, that when a protein is treated with protein deamidase so that deamidation of asparagine residues occurs to a desired extent, no significant polymerization of the protein due to cross-linking is observed before or after the treatment. Specifically, "protein deamidase does not substantially have protein cross-linking activity" means, for example, that when 25 μL of an enzyme solution adjusted to a protein asparaginase activity of 2.7 U / mL is added to 500 μL of 20 mM sodium phosphate buffer (pH 7.0) containing 2% w / v sodium caseinate, the reaction is allowed to proceed at 37°C for 1 hour, and the enzyme is inactivated by treatment at 100°C for 5 minutes, the amount of uncross-linked casein is 1 / 2 times that of a control (a sample in which 25 μL of water was added instead of the enzyme solution and the reaction was carried out in the same manner). The amount of uncrosslinked casein may be 90% or more, or 95% or more of the amount of uncrosslinked casein in the sample. The "amount of uncrosslinked casein" can be measured and compared by known techniques. Measurement and comparison of the "amount of uncrosslinked casein" may be carried out, for example, by measuring the molecular weight distribution by gel filtration chromatography, or by confirming the position and concentration of a target band by SDS-PAGE.

[0022] The protein deamidase of this embodiment has one β-barrel domain at the C-terminus or does not have any β-barrel domain at the C-terminus. A β-barrel domain is one of the tertiary structures of a protein, and refers to a structure in which both ends of a β-sheet are connected by hydrogen bonds. Having one β-barrel domain at the C-terminus means that, among the regular secondary and tertiary structural motifs possessed by the protein, the motif located closest to the C-terminus is a single β-barrel domain. In other words, this means that the protein does not have a regular secondary structure, such as an α-helix or β-sheet, C-terminally of the β-barrel domain, but this does not preclude the presence of structures that are considered to be irregular, such as loops. On the other hand, having two or more β-barrel domains at the C-terminus means that the two or more motifs located closest to the C-terminus are all β-barrel domains.

[0023] Examples of protein deamidases having one β-barrel domain at the C-terminus or no β-barrel domain at the C-terminus include protein deamidases derived from Amycolatopsis deserti, Microbispora corallina, or Motilibacter peucedani. .

[0024] For example, the protein deamidase from Amycolatopsis deserti is represented by SEQ ID NO: 1 and and 2, respectively, and the nucleotide sequence of the gene encoding them, and is thought to have the tertiary structure shown in Figure 1. In Figure 1, the domain described as "Main β barrel" is present at the C-terminus of the protein, and no other β barrel domains are present further C-terminally.

[0025] On the other hand, proteins derived from Luteimicrobium album Deamidase is thought to have the tertiary structure shown in Figure 2. As shown in Figure 2, the protein deamidase from Luteimicrobium album is thought to have a short α-helix on the C-terminal side of a domain called the Main β-barrel, and four further β-barrel domains on the C-terminal side.

[0026] The amino acid sequence of protein deamidase derived from Microbispora corallina and the nucleotide sequence of the gene encoding it are shown in SEQ ID NOs: 3 and 4, respectively. The amino acid sequence of protein deamidase derived from Motilibacter peucedani and the nucleotide sequence of the gene encoding it are shown in SEQ ID NOs: 5 and 6, respectively. That is, protein deamidase may be, for example, a protein having the amino acid sequence shown in SEQ ID NO: 1, 3, or 5. Furthermore, protein deamidase may be, for example, a protein encoded by a gene having the nucleotide sequence shown in SEQ ID NO: 2, 4, or 6. The expression "having an (amino acid or nucleotide) sequence" encompasses the cases where the phrase "comprises the (amino acid or nucleotide) sequence" and the case where the phrase "consists of the (amino acid or nucleotide) sequence."

[0027] Luteimicrobium album AJ111072 (NITE P-01650) was deposited on July 5, 2013, at the Patent Microorganisms Depositary Center, National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu, Chiba Prefecture, Japan, 292-0818) under the accession number NITE P-01650.

[0028] The amino acid sequence shown in SEQ ID NO: 1, 3 or 5 may contain a pro-sequence. A protein containing a pro-sequence is also called a "pro-protein." A protein not containing a pro-sequence is also called a "mature protein." Protein deamidase may be, for example, a protein having the amino acid sequence of a portion of the amino acid sequence shown in SEQ ID NO: 1, 3 or 5 excluding the pro-sequence (i.e., the amino acid sequence of the mature protein). The amino acid sequence of the mature protein of protein deamidase derived from Amycolatopsis deserti is SEQ ID NO: 1 It corresponds to positions 129 to 785 of the protein deamidation product derived from Amycolatopsis deserti. The amino acid sequence of the proprotein of the enzyme corresponds to the amino acid sequence shown in SEQ ID NO: 1. The amino acid sequence of the mature protein of protein deamidase derived from Microbispora corallina corresponds to positions 130 to 808 of SEQ ID NO: 3. The amino acid sequence of the proprotein of protein deamidase derived from Microbispora corallina corresponds to the amino acid sequence shown in SEQ ID NO: 3. The amino acid sequence of the mature protein of protein deamidase derived from Motilibacter peucedani corresponds to positions 143 to 818 of SEQ ID NO: 5. The amino acid sequence of the proprotein of protein deamidase derived from Motilibacter peucedani corresponds to the amino acid sequence shown in SEQ ID NO: 5. The position of the N-terminal residue of the mature protein may vary by, for example, several residues depending on various conditions such as the type of processing enzyme. The "several residues" referred to here may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues. That is, for example, in the case of protein deamidase derived from Amycolatopsis deserti, The amino acid sequence of the mature protein may correspond to residues 129+ / - through 785 of SEQ ID NO:1.

[0029] Protein deamidase may be a variant of the above-exemplified protein deamidases (for example, proteins having the amino acid sequence shown in SEQ ID NO: 1, 3, or 5, or a part of these amino acid sequences), so long as the original function is maintained. Similarly, a gene encoding protein deamidase (also referred to as a "protein deamidase gene") may be a variant of the above-exemplified protein deamidase genes (for example, genes having the nucleotide sequence shown in SEQ ID NO: 2, 4, or 6, or a part of these nucleotide sequences), so long as the original function is maintained. Such variants that maintain the original function are sometimes referred to as "conservative variants." Examples of conservative variants include homologs and artificially modified forms of the above-exemplified protein deamidases or genes encoding them.

[0030] "Maintaining the original function" means that a gene or protein variant has a function (activity or property) corresponding to the function (activity or property) of the original gene or protein. That is, in the case of protein deamidase, "maintaining the original function" means that the protein variant has protein asparaginase activity. Furthermore, in the case of protein deamidase genes, "maintaining the original function" means that the gene variant encodes a protein that maintains the original function (i.e., a protein that has protein asparaginase activity).

[0031] As long as the original function of protein deamidase is maintained, the protein may be a protein having an amino acid sequence in which one or several amino acids have been substituted, deleted, inserted or added at one or several positions in the above amino acid sequence (for example, the amino acid sequence shown in SEQ ID NO: 1, 3 or 5, or a part thereof (such as the mature protein portion or proprotein portion)). Note that the term "one or several" varies depending on the position or type of amino acid residue in the three-dimensional structure of the protein, and specifically means, for example, 1 to 50, 1 to 40, 1 to 30, preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and particularly preferably 1 to 3.

[0032] The above-mentioned substitution, deletion, insertion, or addition of one or several amino acids is a conservative mutation that maintains the normal function of the protein. A typical conservative mutation is a conservative substitution. When the substitution site is an aromatic amino acid, a conservative substitution is a substitution between Phe, Trp, and Tyr. If the substitution site is a hydrophobic amino acid, it is between Leu, Ile, and Val. If the substitution site is a polar amino acid, it is between Leu, Ile, and Val. In the case of Gln and Asn, basic amino acids are present, and in the case of Lys, Arg, and His, acidic amino acids are present. If it is an acid, it is between Asp and Glu, and if it is an amino acid with a hydroxyl group, it is Ser. Conservative substitutions are substituted for each other between Thr and Thr. These include substitutions of Ala to Ser or Thr, Arg to Gln, His or Lys, Asn to Glu, Gln, Lys, His or Asp, Asp to Asn, Glu or Gln, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp or Arg, Glu to Gly, Asn, Gln, Lys or Asp, Gly to Pro, His to Asn, Lys, Gln, Arg or Tyr, Ile to Leu, Met , substitution of Lys with Ile, Leu, Val, or Phe, substitution of Lys with Asn, Glu, Gln, His, or Arg, substitution of Met with Ile, Leu, Val, or Phe, substitution of Phe with Trp, Tyr, Met, Ile, or Leu, substitution of Ser with Thr or Ala, substitution of Thr with Ser or Ala, substitution of Trp with Phe or Tyr, substitution of Tyr with His, Phe, or Trp, and substitution of Val with Met, Ile, or Leu. Furthermore, the above-mentioned amino acid substitutions, deletions, and insertions can also be included. The additions, inversions, and the like also include those that arise due to naturally occurring mutations (mutants or variants) based on individual differences or differences in species of the organism from which the protein is derived.

[0033] Furthermore, protein deamidase may be a protein having an amino acid sequence that is 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more homologous to the entire amino acid sequence described above, so long as the original function is maintained. In this specification, "homology" may also refer to "identity."

[0034] Furthermore, protein deamidase may be a protein encoded by DNA that hybridizes under stringent conditions with a probe that can be prepared from the above-mentioned nucleotide sequence (for example, the nucleotide sequence shown in SEQ ID NO: 2, 4, or 6, or a part thereof (a part encoding a mature protein or a proprotein)), for example, a sequence complementary to the entire or part of the above-mentioned nucleotide sequence, so long as the original function is maintained. Such a probe can be, for example, The DNA fragment containing the above-mentioned base sequence can be prepared by PCR using oligonucleotides prepared based on the above-mentioned base sequence as primers and a DNA fragment containing the above-mentioned base sequence as a template. The "conditions" refer to conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed. For example, DNAs with high homology, for example, 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more, are hybridized. The DNA fragments are washed once, preferably 2 to 3 times, under conditions where DNA fragments having the above homology hybridize with each other but DNA fragments having lower homology do not hybridize with each other, or under the conditions of washing in ordinary Southern hybridization, which are 60°C, 1 x SSC, 0.1% SDS, preferably 60°C, 0.1 x SSC, 0.1% SDS, more preferably 68°C, 0.1 x SSC, 0.1% SDS. For example, a probe containing DNA of about 300 bp in length can be used. When fragments are used, washing conditions for hybridization include 50°C, 2 x SSC, and 0.1% SDS.

[0035] The protein deamidase may be any of the above-mentioned protein deamidases excluding those whose amino acid sequences or nucleotide sequences of the genes encoding them are publicly known at the time of filing of the present invention.

[0036] Protein deamidase may be a fusion protein with another amino acid sequence. The "other amino acid sequence" can be appropriately selected depending on various conditions such as the intended use. Examples of the "other amino acid sequence" include a peptide tag, a signal sequence (pre-sequence), a pro-sequence, and a protease recognition sequence. The "other amino acid sequence" may be linked to, for example, the N-terminus or C-terminus of protein deamidase, or both. As the "other amino acid sequence," one type of amino acid sequence may be used, or two or more types of amino acid sequences may be used in combination.

[0037] Peptide tags can be used, for example, for detection and purification of expressed protein deamidase. Specific examples of peptide tags include His tags, FLAG tags, GST tags, Myc tags, maltose binding protein (MBP), cellulose binding protein (CBP), thioredoxin (TRX), green fluorescent protein (GFP), horseradish peroxidase (HRP), alkaline phosphatase (ALP), and antibody Fc regions. An example of a His tag is a 6xHis tag.

[0038] The signal sequence can be used, for example, for secretory production of protein deamidase. Examples of the signal sequence include a signal sequence recognized by the Sec secretory pathway and a signal sequence recognized by the Tat secretory pathway. Specific examples of the signal sequence recognized by the Sec secretory pathway include: Examples of signal sequences recognized by the Tat secretion pathway include the signal sequences of cell surface proteins of coryneform bacteria. Examples of cell surface proteins of coryneform bacteria include PS1 (CspA) and PS2 (CspB) of C. glutamicum (JP Patent Publication No. 6-502548 A) and SlpA (CspA) of C. ammoniagenes (C. stationis) (JP Patent Publication No. 10-108675 A). Specific examples of signal sequences recognized by the Tat secretion pathway include the TorA signal sequence of E. coli, the SufI signal sequence of E. coli, the PhoD signal sequence of Bacillus subtilis, the LipA signal sequence of Bacillus subtilis, and the IMD signal sequence of Arthrobacter globiformis (WO2013 / 118544). Furthermore, examples of signal sequences include the signal sequences recognized by the protein deaminase inhibitor (PDE) and the signal sequences recognized by the Tat secretion pathway. A signal sequence of a protein deamidase may be used. The signal sequence can be added, for example, to the N-terminus of a protein to be produced. Specifically, the signal sequence can be added, for example, to the N-terminus of a proprotein or mature protein of protein deamidase. A signal sequence is generally cleaved by a signal peptidase when the translation product is secreted outside the bacterial cell. Therefore, when protein deamidase is secreted and produced using a signal sequence, protein deamidase without a signal sequence can be secreted outside the bacterial cell.

[0039] Specific examples of pro-sequences include the pro-sequence of protein deamidase. Examples of pro-sequences of protein deamidase include the sequence from positions 1 to 128 of SEQ ID NO: 1. The pro-sequence can be used, for example, by adding it to the N-terminus of the protein to be produced. Specifically, a pro-sequence can be used by, for example, adding it to the N-terminus of the mature protein of protein deamidase. Furthermore, when protein deamidase is secreted and produced, for example, protein deamidase may be expressed so as to contain, in order from the N-terminus, a signal sequence, a pro-sequence, and the sequence of the mature protein. Expressing protein deamidase in a form having a pro-sequence may contribute to stabilizing the structure of protein deamidase. On the other hand, from the viewpoint of protein asparaginase activity, it is preferable that the finally obtained protein deamidase does not have a pro-sequence.

[0040] The protease recognition sequence can be used, for example, for cleavage of expressed protein deamidase. The protease recognition sequence is preferably a recognition sequence of a protease with high substrate specificity. Specific examples of the recognition sequence of a protease with high substrate specificity include Factor Xa protease. The recognition sequences of Factor Xa protease and proTEV protease are examples. The proTEV protease recognizes the amino acid sequence Ile-Glu-Gly-Arg (=IEGR) (SEQ ID NO: 7) in a protein, and the proTEV protease recognizes the amino acid sequence Glu-Asn-Leu-Tyr-Phe-Gln (=ENLYFQ) (SEQ ID NO: 8) in a protein, and specifically cleaves the C-terminal side of each sequence. For example, when protein deamidase is expressed as a fusion protein with another amino acid sequence such as a peptide tag or a prosequence, by further introducing a protease recognition sequence between the protein deamidase and the other amino acid sequence, the other amino acid sequence can be removed from the expressed protein deamidase using the protease, thereby obtaining protein deamidase that does not have the other amino acid sequence.

[0041] The protein deamidase gene may be one in which any codon in the nucleotide sequence of the above-exemplified protein deamidase gene or a conservative variant thereof is substituted with an equivalent codon. For example, the protein deamidase gene may be modified to have optimal codons depending on the codon usage frequency of the host to be used.

[0042] In the present invention, the term "gene" is not limited to DNA and may include any polynucleotide as long as it encodes a protein of interest. The term "amidase gene" may refer to any polynucleotide encoding a protein deamidase. The protein deamidase gene may be DNA, RNA, or a combination thereof. The protein deamidase gene may be single-stranded or double-stranded. The protein deamidase gene may be single-stranded DNA, but The protein deamidase gene may be double-stranded DNA, double-stranded RNA, or a hybrid chain consisting of a DNA strand and an RNA strand. The protein deamidase gene may contain both DNA residues and RNA residues in a single polynucleotide chain. When the protein deamidase gene contains RNA, The descriptions relating to DNA such as the nucleotide sequence exemplified above may be appropriately interpreted in accordance with RNA. The form of the protein deamidase gene can be appropriately selected depending on various conditions such as its mode of use.

[0043] <2> Production of protein deamidase Protein deamidase can be produced by utilizing an organism capable of producing protein deamidase. That is, a second embodiment of the present invention is a method for producing protein deamidase, comprising culturing an organism capable of producing protein deamidase in a medium to produce protein deamidase, and recovering the protein deamidase from the culture. This method is also referred to as the "method for producing protein deamidase of the present invention." Protein deamidase can also be produced by expressing a protein deamidase gene in a cell-free protein synthesis system.

[0044] The organism having the ability to produce protein deamidase may be an organism that inherently has the ability to produce protein deamidase, or an organism that has the ability to produce protein deamidase. It may be modified to have a force.

[0045] Examples of organisms capable of producing protein deamidase include the above-mentioned Amycolatopsis deserti, Microbispora corallina, and Motilibacter peucedani.

[0046] Organisms capable of producing protein deamidase also include hosts into which a protein deamidase gene has been introduced.

[0047] The host into which the protein deamidase gene is introduced is not particularly limited as long as it is capable of expressing a functional protein deamidase. Examples of the host include bacteria, actinomycetes, yeast, fungi, plant cells, insect cells, and animal cells. Preferred hosts include microorganisms such as bacteria and yeast. More preferred hosts include bacteria. Examples of bacteria include gram-negative bacteria and gram-positive bacteria. Examples of gram-negative bacteria include bacteria of the genus Escherichia and bacteria of the genus Enterobacter. Bacteria belonging to the Enterobacteriaceae family, such as Pantoea, Examples of Gram-positive bacteria include coryneform bacteria such as bacteria of the genus Bacillus and bacteria of the genus Corynebacterium. Escherichia coli can be preferably used. When protein deamidase is produced and secreted outside the bacterial cell, coryneform bacteria such as Corynebacterium glutamicum and Corynebacterium stationis can be particularly preferably used as the host (WO2013 / 065869, WO2013 / 065772, WO2013 / 118544, WO2013 / 062029).

[0048] The protein deamidase gene can be obtained by cloning from an organism that has the protein deamidase gene. Cloning involves the use of genomic DNA, cDNA, etc. containing the gene. Protein deamidase genes can also be obtained by chemical synthesis (Gene, 60(1), 115-127 (1987)).

[0049] Specifically, the protein deamidase gene can be cloned from an organism having the ability to produce the above-mentioned protein deamidase by the method described below.

[0050] First, protein deamidase is suitably isolated and purified from an organism capable of producing protein deamidase, and information on its partial amino acid sequence is obtained. In determining the partial amino acid sequence, for example, the purified protein deamidase is directly subjected to the Edman degradation method according to a conventional method [Journal of Biological Chemistry, Vol. 256, pp. 7990-7997]. (1981)] for amino acid sequence analysis (such as using a protein sequencer, Shimadzu Corporation PPSQ-21A), or by using a protease to perform limited hydrolysis, separating and purifying the resulting peptide fragments, and then analyzing the amino acid sequence of the purified peptide fragments. Subsequently, the base sequence of the genomic DNA extracted from the microorganism can be analyzed using a next-generation sequencer ( After decoding by a DNA sequencer (Illumina, Miseq, etc.), the partial amino acid sequence obtained by the above method was That is, the base sequence of the obtained genomic DNA is searched using CLC Genomics Workbench (Chemical Laboratory, Inc.). Contig sequences were created using the CLC Bio Japan Co., Ltd., and the proteins previously obtained were Based on the partial amino acid sequence of protein deamidase, the nucleotide sequence of the gene encoding the enzyme can be determined. Based on the determined nucleotide sequence, the protein can be isolated by a general PCR method. The enzyme deamidase gene can be cloned.

[0051] When using PCR, the following method can be used. The genomic DNA of an organism capable of producing amido-enzyme is used as a template to obtain information on the partial amino acid sequence. PCR was performed using synthetic oligonucleotide primers designed based on the information obtained. The PCR method uses PCR technology to obtain a DNA fragment containing a part of the protein deamidase gene. PCR is carried out according to the method described in PCR Technology, edited by Erlich HA, Stockton Press, 1989. The amplified DNA fragments are then subjected to the usual PCR amplification. When the nucleotide sequence is determined by the method used, for example, the dideoxy chain terminator method, a sequence corresponding to a partial amino acid sequence of protein deamidase is found in the determined sequence in addition to the sequence of the synthetic oligonucleotide primer, i.e., a partial nucleotide sequence of the target protein deamidase gene can be determined. Furthermore, by performing a hybridization method or the like using the obtained gene fragment as a probe, the full-length protein deamidase gene can be cloned.

[0052] Furthermore, the protein deamidase gene obtained as described above can be appropriately modified to obtain its variants. Gene modification can be performed by known techniques. For example, a desired mutation can be introduced into a target site in the gene by site-directed mutagenesis. That is, for example, site-directed mutagenesis can be used to modify the coding region of the gene so that an amino acid residue at a specific site in the encoded protein contains a substitution, deletion, insertion, or addition. Examples of site-directed mutagenesis include methods using PCR (Higuchi, R., 61, in PCR technology, Erlich, H.A. Eds., Stockton Press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)) and methods using phage (Kramer, W. and Frits, H.J., Meth. in Enzymol., 154, 350 (1987); Kunkel, T.A. et al., Meth. in Enzymol., 154, 367 (1987)). Variants of the protein deamidase gene can also be obtained by, for example, mutagenesis. Examples of mutagenesis include in vitro treatment of the gene itself with hydroxylamine or the like, or treatment of a microorganism carrying the protein deamidase gene, such as a bacterium belonging to the class Actinobacteria, with X-rays, ultraviolet light, or N-methyl-N'-nitro-N-nitrosoguanidine (NTG), ethyl methanesulfonate (EMS), methyl methanesulfonate (MS), or the like. Examples of such methods include treatment with a mutagen such as methyl methyl methacrylate (MMS), Erasprone PCR (Cadwell, RC PCR Meth. Appl. 2, 28 (1992)), DNA shuffling (Stemmer, WP Nature 370, 389 (1994)), and StEP-PCR (Zhao, H. Nature Biotechnol. 16, 258 (1998)).

[0053] The method for introducing the protein deamidase gene into a host is not particularly limited. In the host, the protein deamidase gene may be maintained in an expressible manner under the control of a promoter that functions in the host. In the host, the protein deamidase gene may be present on an extrachromosomally autonomously replicating vector such as a plasmid, or may be introduced into the chromosome. The host may have only one copy of the protein deamidase gene, or may have two or more copies. The host may have only one type of protein deamidase gene, or may have two or more types of protein deamidase genes.

[0054] The promoter for expressing the protein deamidase gene is not particularly limited as long as it functions in the host. A "promoter that functions in the host" refers to a promoter that has promoter activity in the host. The promoter may be a promoter derived from the host or a heterologous promoter. The promoter may be a promoter specific to the protein deamidase gene or a promoter of another gene. The promoter may be a strong promoter that can achieve a high expression level. Specific examples of strong promoters that function in Enterobacteriaceae bacteria such as Escherichia coli include the T7 promoter, trp promoter, trc promoter, lac promoter, tac promoter, tet promoter, and araBAD promoter. Examples of strong promoters that function in coryneform bacteria include the artificially engineered P54-6 promoter. Promoter (Appl. Microbiol. Biotechnolo., 53, 674-679(2000)), pta, aceA, aceB, adh, amyE promoters that can be induced in coryneform bacteria by acetic acid, ethanol, pyruvate, etc. , cspB, SOD, tuf (EF-Tu), which are strong promoters highly expressed in coryneform bacteria. ) promoter (Journal of Biotechnology 104 (2003) 311-323, Appl Environ Microbiol. 2005 Dec;71(12):8587-96.), lac promoter, tac promoter, trc promoter. Furthermore, highly active promoters of conventional promoters may be obtained and used by using various reporter genes. For example, promoter activity can be increased by adjusting the -35 and -10 regions of the promoter region to a consensus sequence (International Publication No. 00 / 18935). Highly active promoters include various tac-like promoters (Katashkina JI et al., Russian Federation Patent Application 2006134574) and the pnlp8 promoter (WO2010 / 027045). Methods for evaluating promoter strength and examples of strong promoters are described in a paper by Goldstein et al. (Prokaryotic Promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995)).

[0055] Furthermore, a terminator for terminating transcription can be placed downstream of the protein deamidase gene. The terminator is not particularly limited as long as it functions in the host. The terminator may be a terminator derived from the host or a heterologous terminator. The terminator may be a terminator inherent to the protein deamidase gene or a terminator of another gene. Specific examples of terminators include the T7 terminator, T4 terminator, fd phage terminator, tet terminator, and trpA terminator.

[0056] The protein deamidase gene can be introduced into a host using, for example, a vector containing the gene. A vector containing the protein deamidase gene is also called an expression vector or recombinant vector for the protein deamidase gene. The expression vector for the protein deamidase gene is, for example, a vector that expresses a DNA fragment containing the protein deamidase gene in a host. The protein deamidase gene can be constructed by linking it to a functional vector. By transforming a host with an expression vector for the protein deamidase gene, a transformant into which the vector has been introduced can be obtained, that is, the gene can be introduced into the host. A vector capable of autonomous replication in host cells can be used. The vector is preferably a multicopy vector. Furthermore, the vector preferably has a marker such as an antibiotic resistance gene to select transformants. Furthermore, the vector may have a promoter or terminator for expressing the inserted gene. The vector may be, for example, a bacterial plasmid-derived vector, a yeast plasmid-derived vector, a bacteriophage-derived vector, a cosmid, or a phagemid. Specific examples of vectors capable of autonomous replication in Enterobacteriaceae bacteria such as Escherichia coli include pUC19, pUC18, pHSG299, pHSG399, pHSG398, pBR322, and pSTV29 (all of which are transcription factors). (available from Calabio), pACYC184, pMW219 (Nippon Gene), pTrc99A (Far pPROK series vectors (Clontech), pKK233-2 (Clontech), pET series vectors (Novagen), pQE series vectors (Qiagen), pACYC, broad-host-range vectors Specific examples of vectors capable of autonomous replication in coryneform bacteria include: For example, pHM1519 (Agric. Biol. Chem., 48, 2901-2903 (1984)); pAM330 (Agric. Biol. Chem., 48, 2901-2903 (1984)); and plasmids having drug resistance genes improved from these; Plasmid pCRY30 described in Japanese Patent Application Laid-Open No. 3-210184; plasmids pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE, and the like described in Japanese Patent Application Laid-Open No. 2-72876 and U.S. Pat. No. 5,185,262; and pCRY3KX; plasmids pCRY2 and pCRY3 described in Japanese Patent Laid-Open No. 1-191686; pAJ655, pAJ611, and pAJ1844 described in Japanese Patent Laid-Open No. 58-192900; pCG1 described in Japanese Patent Laid-Open No. 57-134500; pCG2 described in Japanese Patent Laid-Open No. 58-35197; and pCG4 and pCG11 described in Japanese Patent Laid-Open No. 57-183799. When constructing an expression vector, for example, a protein deamidase gene containing an intrinsic promoter region may be directly incorporated into the vector, or the coding region of protein deamidase may be ligated downstream of such a promoter and then incorporated into the vector. Alternatively, the coding region for protein deamidase may be inserted downstream of a promoter that is originally present on the vector.

[0057] Vectors, promoters, and terminators that can be used in various organisms are described in detail in, for example, "Basic Microbiology Lectures 8: Genetic Engineering, Kyoritsu Shuppan, 1987," and they can be used.

[0058] Furthermore, the protein deamidase gene can be introduced, for example, into the chromosome of the host. Introduction of the gene into the chromosome can be carried out, for example, by utilizing homologous recombination. Examples of gene introduction methods that utilize homologous recombination include Red-driven integration (RED-RI). method (WO2005 / 010175), transduction using phages such as P1 phage, a method using a conjugative transfer vector, a method using a strain of phage that does not have a replication origin that functions in the host, Examples of methods using vectors include those using vectors. Only one copy of a gene may be introduced, or two or more copies may be introduced. For example, multiple copies of a gene can be introduced into a chromosome by performing homologous recombination targeting a sequence that exists in multiple copies in a chromosome. Examples of sequences that exist in multiple copies in a chromosome include repetitive DNA sequences and inverted repeats at both ends of a transposon. In addition, for example, by using transposons or Mini-Mu, genes can be inserted onto chromosomes. It can also be introduced randomly (Japanese Patent Laid-Open Publication No. 2-109985, US Pat. No. 5,882,888, EP805867B1). When introducing a gene into a chromosome, for example, the protein deamidase gene including an intrinsic promoter region may be integrated directly into the chromosome, the protein deamidase coding region may be linked downstream of the above-mentioned promoter and then integrated into the chromosome, or the protein deamidase coding region may be integrated downstream of a promoter that is originally present on the chromosome.

[0059] The introduction of a gene into a chromosome can be confirmed, for example, by Southern hybridization using a probe having a nucleotide sequence complementary to all or part of the gene, or by PCR using primers prepared based on the nucleotide sequence of the gene.

[0060] The transformation method is not particularly limited, and a conventionally known method can be used. For example, the transformation method is a method for transforming recipient cells, such as that reported for Escherichia coli K-12. treatment with calcium chloride to increase DNA permeability (Mandel, M. and Higa, A., J. Mol. Biol. 1970, 53, 159-162), as reported for Bacillus subtilis. Examples include a method in which competent cells are prepared from cells in the growth stage and DNA is introduced into them (Duncan, CH, Wilson, GA and Young, FE, 1997. Gene 1: 153-167). As a transformation method, a method known for Bacillus subtilis, actinomycetes, and yeasts, in which the cells of the DNA recipient are transformed into protoplasts or spheroplasts that can easily incorporate recombinant DNA, and then the recombinant DNA is introduced into the DNA recipient bacteria (Chang, S. and Choen, SN, 1979. Mol. Gen. Genet. 168: 111-115; Bibb, MJ, Ward, JM, and Hopwood, OA 1978. Nature 274: 398-400; Hinnen, A., Hicks, JB, and Fink, GR 1978. Proc. Natl. Acad. Sci. USA 75: 1929-1933) can also be applied. It is also possible to use an electric pulse method (Japanese Patent Laid-Open Publication No. 2-207791) as reported for type bacteria.

[0061] Alternatively, a host that inherently has a protein deamidase gene may be modified so that expression of the protein deamidase gene is increased. Techniques for increasing expression of the protein deamidase gene include increasing the copy number of the protein deamidase gene and improving the transcription efficiency of the protein deamidase gene. The copy number of the protein deamidase gene can be increased by introducing the protein deamidase gene into the host. Introduction of the protein deamidase gene can be carried out as described above. The protein deamidase gene to be introduced may be derived from the same species or from a different species. The transcription efficiency of the protein deamidase gene can be improved by replacing the promoter of the protein deamidase gene with a stronger promoter. Examples of stronger promoters include the strong promoters described above.

[0062] Protein deamidase can be expressed by culturing an organism capable of producing the above-mentioned protein deamidase in a medium. In this case, gene expression may be induced, if necessary. The culture conditions for the organism and the conditions for inducing gene expression may be appropriately selected depending on various conditions, such as the type of marker, the type of promoter, and the type of the organism. The medium used for culturing is not particularly limited as long as it allows the organism to grow and allows protein deamidase to be expressed. As the medium, for example, a conventional medium containing a carbon source, a nitrogen source, a sulfur source, inorganic ions, and, if necessary, other organic components, can be used.

[0063] Examples of carbon sources include sugars such as glucose, fructose, sucrose, molasses, and starch hydrolysates; alcohols such as glycerol and ethanol; and organic acids such as fumaric acid, citric acid, and succinic acid.

[0064] Examples of nitrogen sources include inorganic ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, organic nitrogen such as that from soybean hydrolysate, ammonia gas, and aqueous ammonia.

[0065] Examples of sulfur sources include inorganic sulfur compounds such as sulfates, sulfites, sulfides, hyposulfites, and thiosulfates.

[0066] Examples of inorganic ions include calcium ions, magnesium ions, manganese ions, potassium ions, iron ions, and phosphate ions.

[0067] Other organic components include organic trace nutrient sources, such as required substances such as vitamin B1 and yeast extracts containing these.

[0068] The culture method may be liquid culture or solid culture, but liquid culture is preferred. The culture is preferably carried out aerobically. Examples of aerobic culture methods include shaking culture and aerobic submerged culture using a jar fermenter. The oxygen concentration during this process may be adjusted to, for example, 5 to 50% of the saturation concentration, preferably about 10%. The culture temperature may be, for example, The culture temperature may be 10 to 50°C, preferably 20 to 45°C, and more preferably 25 to 40°C. The pH of the medium may be adjusted to, for example, 3 to 9, and preferably 5 to 8. An inorganic or organic acidic or alkaline substance, such as calcium carbonate, ammonia gas, or aqueous ammonia, can be used to adjust the pH. The culture period may be, for example, 12 hours to 20 days, and preferably 1 to 7 days.

[0069] Cultivation under the above conditions allows for the production of a culture containing protein deamidase. Protein deamidase accumulates, for example, within the host bacterial cells and / or in the medium. The term "bacterial cells" may be appropriately interpreted as "cells" depending on the type of host. Depending on the host used and the design of the protein deamidase gene, it is also possible to cause protein deamidase to accumulate in the periplasm or to cause protein deamidase to be secreted and produced outside the bacterial cells.

[0070] The protein deamidase may be used as it is contained in the culture, or may be extracted from the culture and used as known Alternatively, the enzyme may be separated and purified from the bacterial cells or the like and used as a crude enzyme fraction or a purified enzyme.

[0071] That is, for example, when protein deamidase accumulates in the host cells, the cells can be disrupted, lysed, extracted, or the like as appropriate to recover protein deamidase. The cells can be recovered from the culture by centrifugation or the like. Disruption, lysis, extraction, or the like of cells can be carried out by known methods. Examples of such methods include ultrasonic disruption, Dynomill method, bead disruption, French press disruption, and lysozyme treatment. One of these methods may be used alone, or two or more of them may be used in appropriate combination. Furthermore, for example, when protein deamidase accumulates in the medium, the culture supernatant can be obtained by centrifugation or the like, and protein deamidase can be recovered from the culture supernatant.

[0072] Protein deamidase can be purified by a known method used for enzyme purification. Examples of such methods include ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration chromatography, and isoelectric precipitation. These methods may be used alone or in appropriate combination of two or more. Protein deamidase can be purified to a desired degree.

[0073] The purified protein deamidase can be used for deamidating proteins as a “protein deamidase.” Protein deamidase may be used in a free state or in the form of an immobilized enzyme immobilized on a solid phase such as a resin.

[0074] Furthermore, the protein deamidase is not limited to purified protein deamidase, and any fraction containing protein deamidase may be used as the "protein deamidase" for protein deamidation. The fraction containing protein deamidase is not particularly limited as long as it is contained in a manner that allows protein deamidase to act on a substrate protein. Examples of such fractions include a culture of an organism capable of producing protein deamidase, bacterial cells recovered from the culture (cultured bacterial cells), a disrupted product of the bacterial cells, a lysate of the bacterial cells, an extract (cell-free extract) of the bacterial cells, a processed bacterial cell product such as immobilized bacterial cells obtained by immobilizing the bacterial cells on a carrier such as acrylamide or carrageenan, a culture supernatant recovered from the culture, a partially purified product (crude product) thereof, and a combination thereof. Any of these fractions may be used alone or together with purified protein deamidase.

[0075] The recovered protein deamidase may be formulated as appropriate. The dosage form is not particularly limited and can be appropriately determined depending on various conditions such as the intended use of the protein deamidase. Examples of dosage forms include liquids, suspensions, powders, tablets, pills, and capsules. When formulating the protein deamidase, pharmacologically acceptable additives such as excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, odorants, fragrances, diluents, and surfactants can be used.

[0076] When protein deamidase has a pro-sequence, removal of the pro-sequence can improve protein asparaginase activity. Therefore, the method for producing protein deamidase of the present invention may further comprise removing the pro-sequence from protein deamidase having a pro-sequence. Removal of the pro-sequence can be carried out, for example, by treating protein deamidase with a processing enzyme. Examples of processing enzymes include proteases. Specific examples of proteases include erythrocyte proteases such as subtilisin, chymotrypsin, trypsin, and alcalase. Serine proteases; cysteine ​​proteases such as papain, bromelain, caspase, and calpain protease; acid protease such as pepsin and cathepsin; metalloprotease such as thermolysin. In addition, specific proteases are inserted between the pro-sequence and the mature protein sequence. When a recognition sequence for the above is inserted to express protein deamidase, the pro-sequence can be specifically removed using the specific protease. The origin of the protease is not particularly limited, and proteases of any origin, such as microorganisms, animals, or plants, may be used. Furthermore, the protease may be a homologue of a known protease or an artificially modified protease. The protease may be in any form, such as a culture of a microorganism that produces the protease, a culture supernatant separated from the culture, bacterial cells separated from the culture, a processed product of the bacterial cells, agricultural or aquatic livestock products containing the protease, a processed product of the agricultural or aquatic livestock product, a protease separated therefrom, or a commercially available protease preparation. The protease may be purified to a desired degree. Examples of microorganisms that produce the protease include bacteria of the genus Bacillus and fungi of the genus Aspergillus.

[0077] <3> Production of modified protein deamidase with improved activity A third embodiment of the present invention is a method for producing a protein variant having improved activity, based on the amino acid sequence of a protein that has an activity to catalyze a reaction of deamidating an asparagine residue in a protein and is characterized by having two or more β-barrel domains at the C-terminus, comprising: The method comprises designing a polynucleotide encoding a protein variant having an amino acid sequence obtained by removing at least one amino acid sequence corresponding to the C-terminal β-barrel domain from the amino acid sequence of the protein. That is, the third embodiment of the present invention is a method for producing a modified protein deamidase with improved activity based on the amino acid sequence of protein deamidase having two or more β-barrel domains at the C-terminus.

[0078] Protein deamidase of the present embodiment has two or more β-barrel domains at the C-terminus. Having two or more β-barrel domains at the C-terminus means that the two or more motifs located closest to the C-terminus are all β-barrel domains. For specific details, see the description of the first embodiment.

[0079] The modified protein deamidase has an amino acid sequence from which at least one amino acid sequence corresponding to the C-terminal β-barrel domain has been removed. The removed β-barrel domain may be the one located closest to the C-terminus among two or more β-barrel domains present at the C-terminus, or may be one of the β-barrel domains located at the second or subsequent positions counting from the C-terminus. Furthermore, when there are three or more β-barrel domains present at the C-terminus, it is preferable to remove all of these β-barrel domains except for the one located closest to the N-terminus. When a modified protein deamidase has an amino acid sequence from which at least one amino acid sequence corresponding to the C-terminal β-barrel domain has been deleted, the enzyme activity is improved. Although the reason for this is unclear, it is thought that this may be due to an improvement in the expression level of the protein.

[0080] Examples of protein deamidases having two or more β-barrel domains at the C-terminus include protein deamidases derived from Luteimicrobium album, Agromyces sp. AJ111073 (NITE BP-01782), Microbacterium testaceum, or Leifsonia aquatica.

[0081] The amino acid sequence of the protein deamidase from Luteimicrobium album AJ111072 (NITE P-01650) and the nucleotide sequence of the gene encoding it are shown in SEQ ID NOs: 9 and 10, respectively. The amino acid sequence of the original protein deamidase and the nucleotide sequence of the gene encoding it are shown in SEQ ID NOs: 11 and 12, respectively. The amino acid sequence of protein deamidase from Microbacterium testaceum is shown in SEQ ID NO: 13. The amino acid sequence of protein deamidase from Leifsonia aquatica is shown in SEQ ID NO: 14. That is, a protein deamidase having two or more β-barrel domains at its C-terminus may be, for example, a protein having the amino acid sequence shown in SEQ ID NO: 9, 11, 13, or 14. Furthermore, protein deamidase may be, for example, a protein encoded by a gene having the nucleotide sequence shown in SEQ ID NO: 10 or 12. The expression "having an (amino acid or nucleotide) sequence" encompasses the cases of "comprising the (amino acid or nucleotide) sequence" and "consisting of the (amino acid or nucleotide) sequence."

[0082] A species of the genus Agromyces, AJ111073 (NITE BP-01782), was approved by the Independent Administrative Agency on December 11, 2013. The original deposit was made at the Patent Microorganisms Depositary Center, National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu, Chiba Prefecture, Japan, 292-0818) under the accession number NITE P-01782, and was transferred to an international deposit under the Budapest Treaty on March 4, 2015, under the accession number NITE BP-01782 (accession number It has been granted the NITE ABP-01782 certification.

[0083] The amino acid sequence shown in SEQ ID NO: 9, 11, 13, or 14 may contain a prepro region (a pre sequence and a pro sequence). A protein containing a prepro region is also referred to as a "prepro protein." A protein containing a pro sequence but not a pre sequence is also referred to as a "pro protein." A protein not containing a prepro region is also referred to as a "mature protein." Protein deamidase may be, for example, a protein having the amino acid sequence of the portion of the amino acid sequence shown in SEQ ID NO: 9, 11, 13, or 14 excluding the prepro region (i.e., the amino acid sequence of the mature protein), or a protein having the amino acid sequence of the portion excluding the pre sequence (i.e., the amino acid sequence of the pro protein). The amino acid sequence of the mature protein of protein deamidase from Luteimicrobium album corresponds to positions 240 to 1355 of SEQ ID NO: 9. The amino acid sequence of the mature protein of protein deamidase from a species of Agromyces corresponds to positions 181 to 1180 of SEQ ID NO: 11. The amino acid sequence of the mature protein of protein deamidase from Microbacterium testaceum corresponds to positions 193 to 1172 of SEQ ID NO: 13. The amino acid sequence of the proprotein of protein deamidase from a species of Agromyces corresponds to positions 67 to 1180 of SEQ ID NO: 11. The amino acid sequence of the proprotein of protein deamidase from Microbacterium testaceum corresponds to positions 70 to 1172 of SEQ ID NO: 13. The amino acid sequence of the mature protein of protein deamidase from Leifsonia aquatica corresponds to positions 81 to 1119 of SEQ ID NO: 14. Furthermore, protein deamidase may be, for example, a protein encoded by a gene having a nucleotide sequence encoding the amino acid sequence of a portion of the nucleotide sequence shown in SEQ ID NO: 10 or 12 excluding the prepro region (i.e., a nucleotide sequence encoding the amino acid sequence of the mature protein), or a protein encoded by a gene having a nucleotide sequence encoding the amino acid sequence of a portion excluding the presequence (i.e., a nucleotide sequence encoding the amino acid sequence of the proprotein). The nucleotide sequence encoding the amino acid sequence of the mature protein of protein deamidase from Luteimicrobium album corresponds to positions 718 to 4068 of SEQ ID NO:10.The nucleotide sequence encoding the amino acid sequence of the mature protein of protein deamidase of Agromyces sp. corresponds to positions 541 to 3543 of SEQ ID NO: 12. The nucleotide sequence encoding the amino acid sequence of the proprotein of protein deamidase of Agromyces sp. corresponds to positions 199 to 3543 of SEQ ID NO: 12. The position of the N-terminal residue of the mature protein may vary, for example, by several residues, depending on various conditions such as the type of processing enzyme. The term "several residues" used here may mean, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues. That is, for example, the amino acid sequence of the mature protein of protein deamidase of Agromyces sp. may correspond to positions 181 to 1180 of SEQ ID NO: 11. In one embodiment, The amino acid sequence of the mature protein of protein deamidase of a species of the genus Agromyces may correspond to positions 177 to 1180 of SEQ ID NO:11.

[0084] Protein deamidase may be a variant of the above-exemplified protein deamidases (for example, proteins having the amino acid sequence shown in SEQ ID NO: 9, 11, 13, or 14, or a part of these amino acid sequences), so long as the original function is maintained. Similarly, a gene encoding protein deamidase (also referred to as a "protein deamidase gene") may be a variant of the above-exemplified protein deamidase genes (for example, genes having the nucleotide sequence shown in SEQ ID NO: 10 or 12, or a part of these nucleotide sequences), so long as the original function is maintained.

[0085] As long as the original function of protein deamidase is maintained, the protein may be a protein having the above-mentioned amino acid sequence (for example, the amino acid sequence shown in SEQ ID NO: 9, 11, 13, or 14, or a part thereof (such as the mature protein portion or proprotein portion)) in which one or several amino acids have been substituted, deleted, inserted, or added at one or several positions. Note that the term "one or several" varies depending on the position or type of amino acid residue in the three-dimensional structure of the protein, and specifically means, for example, 1 to 50, 1 to 40, 1 to 30, preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and particularly preferably 1 to 3.

[0086] The substitution, deletion, insertion, or addition of one or several amino acids is a conservative mutation that maintains normal protein function. Representative conservative mutations are the same as those in the first embodiment.

[0087] Furthermore, protein deamidase may be a protein having an amino acid sequence that is 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more homologous to the entire amino acid sequence described above, so long as the original function is maintained.

[0088] Furthermore, protein deamidase may be a protein encoded by DNA that hybridizes under stringent conditions with a probe that can be prepared from the above-mentioned nucleotide sequence (for example, the nucleotide sequence shown in SEQ ID NO: 10 or 12, or a part thereof (a part encoding a mature protein or a proprotein)), for example, a sequence complementary to the entire or part of the above-mentioned nucleotide sequence, so long as the original function is maintained. Such a probe can be, for example, The oligonucleotides prepared based on the above-mentioned base sequence can be used as primers and PCR can be performed using a DNA fragment containing the above-mentioned base sequence as a template. The "stringent conditions" are the same as those in the first embodiment.

[0089] The protein deamidase may be any of the above-mentioned protein deamidases excluding those whose amino acid sequences or nucleotide sequences of the genes encoding them are publicly known at the time of filing of the present invention.

[0090] Protein deamidase may be a fusion protein with another amino acid sequence. The "other amino acid sequence" is the same as in the first embodiment.

[0091] The protein deamidase gene may be one in which any codon in the nucleotide sequence of the above-mentioned protein deamidase gene or a conservative variant thereof is substituted with an equivalent codon. For example, the protein deamidase gene may be modified to have optimal codons depending on the codon usage frequency of the host to be used.

[0092] As shown in the Examples below, a modified protein deamidase having improved protein asparaginase activity can be obtained by deleting at least one amino acid sequence corresponding to the C-terminal β-barrel domain from the amino acid sequence of the protein deamidase. That is, a modified protein having improved protein asparaginase activity can be produced by designing a polynucleotide encoding a modified protein having an amino acid sequence obtained by deleting at least one amino acid sequence corresponding to the C-terminal β-barrel domain from the amino acid sequence of the protein.

[0093] The amino acid sequence corresponding to the C-terminal β-barrel domain of protein deamidase from Luteimicrobium album corresponds to positions 1075 to 1166, 1167 to 1257, or 1258 to 1355 of SEQ ID NO: 9. The amino acid sequence corresponding to the C-terminal β-barrel domain of protein deamidase from Agromyces sp. AJ111073 (NITE BP-01782) corresponds to positions 1075 to 1166, 1167 to 1257, or 1258 to 1355 of SEQ ID NO: 9. The amino acid sequence corresponding to the C-terminal β-barrel domain of protein deamidase from Microbacterium testaceum corresponds to positions 895 to 1007 or positions 1008 to 1180 of SEQ ID NO: 11. The amino acid sequence corresponding to the C-terminal β-barrel domain of protein deamidase from Microbacterium testaceum corresponds to positions 933 to 1018 or positions 1019 to 1172 of SEQ ID NO: 13. The amino acid sequence corresponding to the C-terminal β-barrel domain of protein deamidase from Leifsonia aquatica corresponds to positions 832 to 939 or positions 940 to 1119 of SEQ ID NO: 14. Furthermore, the amino acid sequence corresponding to the C-terminal β-barrel domain of protein deamidase may be, for example, an amino acid sequence encoded by a nucleotide sequence encoding an amino acid sequence corresponding to the C-terminal β-barrel domain of protein deamidase from the nucleotide sequence shown in SEQ ID NO: 10 or 12. A nucleotide sequence encoding an amino acid sequence corresponding to the C-terminal β-barrel domain of protein deamidase from Luteimicrobium album corresponds to positions 3223 to 3500, 3551 to 3771, or 3772 to 4065 of SEQ ID NO: 10. A nucleotide sequence encoding an amino acid sequence corresponding to the C-terminal β-barrel domain of protein deamidase from a species of Agromyces corresponds to positions 2683 to 3021 or 3022 to 3540 of SEQ ID NO: 12.

[0094] The C-terminal β-barrel domain of protein deamidase may be a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, or added at one or several positions in the above amino acid sequence (e.g., the portion corresponding to the C-terminal β-barrel domain of the amino acid sequence shown in SEQ ID NO: 9, 11, 13, or 14), so long as the β-barrel domain motif is maintained. Note that the term "one or several" varies depending on the position or type of amino acid residue in the three-dimensional structure of the protein, and specifically means, for example, 1 to 10, preferably 1 to 5, and more preferably 1 to 3.

[0095] The substitution, deletion, insertion, or addition of one or several amino acids is a conservative mutation that maintains normal protein function. Representative conservative mutations are the same as those in the first embodiment.

[0096] Furthermore, the C-terminal β-barrel domain of protein deamidase may be a protein having an amino acid sequence that is 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more homologous to the entire amino acid sequence, as long as the β-barrel domain motif is maintained.

[0097] Furthermore, the C-terminal β-barrel domain of protein deamidase may be a protein encoded by DNA that hybridizes under stringent conditions with a probe that can be prepared from the above-mentioned nucleotide sequence (e.g., a portion encoding the C-terminal β-barrel domain of the nucleotide sequence shown in SEQ ID NO: 10 or 12), for example, a sequence complementary to all or part of the above-mentioned nucleotide sequence, as long as the β-barrel domain motif is maintained. Such a probe can be, for example, For example, an oligonucleotide prepared based on the above base sequence is used as a primer, and the above base sequence is The nucleic acid sequence can be prepared by PCR using a DNA fragment containing the sequence as a template. The "stringent conditions" are the same as those in the first embodiment.

[0098] The method of this embodiment may comprise obtaining a transformant by introducing into a host a recombinant vector containing a polynucleotide encoding a protein deamidase variant having an amino acid sequence obtained by removing at least one amino acid sequence corresponding to the C-terminal β-barrel domain from the amino acid sequence of a protein deamidase having two or more C-terminal β-barrel domains. Hereinafter, a polynucleotide encoding the protein deamidase variant may be referred to as a "protein deamidase variant gene."

[0099] A modified protein deamidase gene can be obtained by modifying a protein deamidase gene having two or more β-barrel domains at the C-terminus. Alternatively, the protein deamidase gene can be obtained by cloning from an organism having the protein deamidase gene. Nucleic acids such as genomic DNA and cDNA containing the gene can be used for cloning. Specifically, cloning can be performed by the method described in the second embodiment. Here, preferred organisms capable of producing protein deamidase are Luteimicrobium album, a species of the genus Agromyces, Microbacterium testaceum, or Leifsonia aquatica. Genetic modification can be carried out by known techniques. Alternatively, a modified protein deamidase gene can be obtained by chemical synthesis (Gene, 60(1), 115-127 (1987)).

[0100] The recombinant vector may be, for example, a DNA fragment containing a modified protein deamidase gene. The protein deamidase variant can be constructed by ligating the vector to a vector that functions in a host. By transforming a host with an expression vector for the protein deamidase variant gene, a transformant into which the vector has been introduced can be obtained, i.e., the gene can be introduced into the host.

[0101] The host, the method for introducing the gene into the host, the vector, and the transformation method may be the same as those described in the second embodiment. The protein deamidase variant gene may be introduced into the chromosome of the host by using homologous recombination or the like.

[0102] Furthermore, the method of the present embodiment may include culturing the transformant in a medium to produce the protein deamidase variant, and recovering the protein deamidase variant from the culture obtained by the culturing. The culture conditions for the transformant, the conditions for inducing gene expression, and the method for recovering the modified protein deamidase may be the same as those described in the second embodiment.

[0103] Furthermore, the method of this embodiment may include treating the modified protein deamidase with a processing enzyme. When the modified protein deamidase has a pro-sequence, removal of the pro-sequence can further improve the protein asparaginase activity. Examples of the processing enzyme include those similar to those listed in the second embodiment.

[0104] <4> Modified protein deamidase According to the third embodiment of the present invention, a modified protein deamidase with improved activity can be produced. Specific examples include modified protein deamidases having an amino acid sequence obtained by removing at least one amino acid sequence corresponding to the C-terminal β-barrel domain of protein deamidase derived from Luteimicrobium album, a species of the genus Agromyces, Microbacterium testaceum, or Leifsonia aquatica.

[0105] The modified protein deamidase derived from Luteimicrobium album, Agromyces sp., Microbacterium testaceum, or Leifsonia aquatica may be any of a preproprotein, proprotein, or mature protein. That is, the amino acid sequence excluding at least one amino acid sequence corresponding to the C-terminal β-barrel domain of protein deamidase derived from Luteimicrobium album corresponds to positions 1 to 1074, 132 to 1074, 240 to 1074, 1 to 1166, 132 to 1166, 240 to 1166, 1 to 1257, 132 to 1257, or 240 to 1257 of SEQ ID NO: 9. Preferably, the amino acid sequence is positions 1 to 1074, 132 to 1074, 240 to 1074, 1 to 1166, 132 to 1166, or 240 to 1166 of SEQ ID NO: 9. The amino acid sequence from which at least one amino acid sequence corresponding to the C-terminal β-barrel domain of protein deamidase derived from a species of the genus Agromyces is removed corresponds to positions 1 to 894, 67 to 894, 181 to 894, 1 to 1007, 67 to 1007, or 181 to 1007 of SEQ ID NO: 11. The amino acid sequence from which at least one amino acid sequence corresponding to the C-terminal β-barrel domain of protein deamidase derived from Microbacterium testaceum is removed corresponds to positions 1 to 932, 70 to 932, 193 to 932, 1 to 1018, 70 to 1018, or 193 to 1018 of SEQ ID NO: 13. Preferably, it is positions 1 to 932, 70 to 932, or 193 to 932 of SEQ ID NO: 13. The amino acid sequence from which at least one amino acid sequence corresponding to the C-terminal β-barrel domain of protein deamidase derived from Leifsonia aquatica is deleted corresponds to positions 1 to 831, 81 to 831, 1 to 939, or 81 to 939 of SEQ ID NO: 14. Furthermore, the modified protein deamidase may be, for example, a protein encoded by a gene having a nucleotide sequence encoding an amino acid sequence from which at least one amino acid sequence corresponding to the C-terminal β-barrel domain of protein deamidase is deleted, within the nucleotide sequence shown in SEQ ID NO: 10 or 12.The nucleotide sequence encoding the amino acid sequence of the modified protein deamidase derived from Luteimicrobium album corresponds to positions 1 to 3222, 394 to 3222, 718 to 3222, 1 to 3498, 394 to 3498, 718 to 3771, 1 to 3771, 394 to 3771, or 718 to 3771 of SEQ ID NO: 10. Preferably, it corresponds to positions 1 to 3222, 394 to 3222, 718 to 3222, 1 to 3498, or 394 to 3498 of SEQ ID NO: 10. The nucleotide sequence encoding the amino acid sequence of the modified protein deamidase derived from a species of the genus Agromyces corresponds to positions 1 to 2682, 199 to 2682, 541 to 2682, 1 to 3021, 199 to 3021, or 541 to 3021 of SEQ ID NO: 12. The position of the C-terminal residue may vary by, for example, several residues depending on various conditions. The "several residues" referred to here may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues. That is, for example, the amino acid sequence of a modified protein deamidase from Luteimicrobium album may correspond to positions 1 to 1074 of SEQ ID NO: 9 plus or minus several residues.

[0106] As long as the original function is maintained, the modified protein deamidase may be a variant of the above-exemplified modified protein deamidase (for example, a protein having a part of the amino acid sequence shown in SEQ ID NO: 9, 11, 13, or 14). Similarly, as long as the original function is maintained, the modified protein deamidase gene may be a variant of the above-exemplified modified protein deamidase gene (for example, a gene having a part of the nucleotide sequence shown in SEQ ID NO: 10 or 12).

[0107] As long as the original function is maintained, the modified protein deamidase may be a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added at one or several positions in the above amino acid sequence (e.g., a part of the amino acid sequence shown in SEQ ID NO: 9, 11, 13 or 14). The term "one or several" varies depending on the position and type of amino acid residue in the three-dimensional structure of the protein, and specifically, for example, 1 to 50, 1 to 40, 1 to 30, preferably 1 to 20. More preferably, it means 1 to 10, even more preferably 1 to 5, and particularly preferably 1 to 3.

[0108] The substitution, deletion, insertion, or addition of one or several amino acids is a conservative mutation that maintains normal protein function. Representative conservative mutations are the same as those in the first embodiment.

[0109] Furthermore, the modified protein deamidase may be a protein having an amino acid sequence that is 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more homologous to the entire amino acid sequence described above, so long as the original function is maintained.

[0110] Furthermore, as long as the original function is maintained, the modified protein deamidase may be a protein encoded by DNA that hybridizes under stringent conditions with a probe that can be prepared from the above-mentioned nucleotide sequence (e.g., the nucleotide sequence shown in SEQ ID NO: 10 or 12, or a part thereof (a part encoding a mature protein or a proprotein)), for example, a sequence complementary to the entire or part of the above-mentioned nucleotide sequence. Such a probe can be, for example, For example, it can be prepared by PCR using oligonucleotides prepared based on the above-mentioned base sequence as primers and a DNA fragment containing the above-mentioned base sequence as a template. The "stringent conditions" are the same as those in the first embodiment.

[0111] The modified protein deamidase may be any of the above-mentioned protein deamidases excluding those whose amino acid sequences or nucleotide sequences of the genes encoding them are publicly known at the time of filing of the present invention.

[0112] The modified protein deamidase may be a fusion protein with another amino acid sequence. The "other amino acid sequence" is the same as that in the first embodiment.

[0113] The modified protein deamidase gene may be one in which any codon in the nucleotide sequence of the above-mentioned modified protein deamidase gene or a conservative variant thereof is substituted with an equivalent codon. For example, the modified protein deamidase gene may be modified to have optimal codons depending on the codon usage frequency of the host to be used.

[0114] <5> Use of protein deamidase In the present invention, asparagine residues in proteins can be deamidated using protein deamidase or a modified protein deamidase (hereinafter, both may be collectively referred to as "protein deamidase, etc."). That is, the present invention provides a method for deamidating asparagine residues in proteins, which comprises allowing protein deamidase, etc. to act on the protein. This method is also referred to as the "deamidation method of the present invention." One embodiment of this method is a method for producing a protein in which an asparagine residue has been deamidated, which comprises allowing protein deamidase, etc. to act on the protein.

[0115] The protein (substrate protein) to be deamidated by protein deamidase or the like is not particularly limited as long as it contains an asparagine residue. As mentioned above, the length of the substrate protein is not particularly limited as long as it is two residues (dipeptide) or more, and unless otherwise specified, the substrate protein also includes forms called peptides, such as oligopeptides and polypeptides. In other words, the term "protein (substrate protein)" may specifically mean a protein and / or a peptide. The substrate protein may be a natural product or an artificial product.

[0116] The substrate protein may be a protein itself or a material containing a protein. In other words, the substrate protein may be subjected to the deamidation reaction alone (i.e., in an isolated state) or may be subjected to the deamidation reaction in a state where it is contained in any material. Examples of substrate proteins include protein-containing agricultural, aquatic, and livestock products, processed products thereof, and proteins separated therefrom. Examples of materials containing plant proteins include grains such as soybeans, wheat, barley, corn, and rice, and processed products thereof. Examples of materials containing animal proteins include meats such as beef, pork, and chicken, fish meat, milk, eggs, and processed products thereof. Examples of plant proteins include soybean proteins such as glycinin, wheat proteins such as gluten, glutenin, and gliadin, and corn proteins such as corn gluten meal. Examples of animal proteins include milk proteins such as casein, lactalbumin, and β-lactoglobulin, egg proteins such as ovalbumin, meat proteins such as myosin and actin, blood proteins such as serum albumin, and tendon proteins such as gelatin and collagen. Further, examples of substrate proteins include proteins partially decomposed chemically with acids or alkalis or enzymatically with proteases, proteins chemically modified with various reagents, recombinant proteins produced in appropriate hosts, and synthetic peptides. The substrate protein may be one that has been subjected to appropriate treatments such as heating, steaming, pulverization, freezing, thawing, and drying. The protein-containing material may contain one type of protein, or two or more types of proteins. As the substrate protein, one type of protein, or two or more types of proteins may be used.

[0117] The substrate protein may be, for example, a food or beverage containing protein, or a raw material for a food or beverage containing protein. In other words, the substrate protein may be subjected to the deamidation reaction while contained in, for example, a food or beverage or a raw material thereof. The type and form of the food or beverage or raw material thereof are not particularly limited, as long as the food or beverage or raw material thereof contains protein. For example, the above-mentioned protein-containing agricultural, aquatic, and livestock products, processed products thereof, and proteins separated therefrom may all be used alone as a food or beverage or raw material thereof, or two or more of them may be appropriately combined and used as a food or beverage or raw material thereof, or one or more of them may be appropriately combined and used as a food or beverage or raw material thereof with other components. By deamidating a protein-containing food or beverage or raw material thereof with protein deamidase, a food or beverage or raw material thereof containing a protein in which an asparagine residue has been deamidated can be obtained. Furthermore, a food or beverage containing a protein in which an asparagine residue has been deamidated can be produced using a raw material for a food or beverage containing a protein in which an asparagine residue has been deamidated.

[0118] That is, one embodiment of the deamidation method of the present invention may be a method for producing a food or drink containing a protein at which an asparagine residue has been deamidated, or a raw material thereof, which comprises allowing protein deamidase to act on a food or drink containing a protein, or a raw material thereof. A food or drink containing a protein at which an asparagine residue has been deamidated, obtained by one embodiment of the deamidation method of the present invention, is also referred to as the "food or drink of the present invention." The food or drink of the present invention can be produced using the same raw materials and by the same method as ordinary foods and drinks, except for the treatment with protein deamidase. Foods and drinks also include seasonings. Specific examples of foods and drinks include mayonnaise, dressing, cream, yogurt, meat products, and bread.

[0119] The substrate protein may be subjected to the deamidation reaction in the form of, for example, a solution, suspension, slurry, or paste. The concentration of the substrate protein in the solution is not particularly limited as long as deamidation is achieved to the desired degree. The concentration of the substrate protein in the solution can be appropriately set depending on various conditions such as the type and properties of the substrate protein and the desired deamidation rate. The solution containing the substrate protein is not limited to an aqueous solution, but may also be an emulsion with oil or fat. The solution containing the substrate protein may consist of the substrate protein and a solvent. The composition may contain other ingredients, such as salts, sugars, proteins, fragrances, moisturizers, and coloring agents.

[0120] The reaction conditions (enzyme amount, reaction time, reaction temperature, reaction pH, etc.) are not particularly limited as long as deamidation is achieved to the desired degree. The reaction conditions can be appropriately set depending on various conditions, such as the type and purity of protein deamidase, the type and purity of protein, and the desired degree of deamidation. The amount of enzyme may be, for example, 0.001 to 2000 U, preferably 0.01 to 500 U, and more preferably 0.1 to 100 U per 1 g of substrate protein. The reaction temperature may be, for example, 5 to 80°C, preferably 5 to 40°C. The pH of the reaction solution may be, for example, 2 to 10, preferably 4 to 8. The reaction time may be, for example, 10 seconds to 48 hours, preferably 10 minutes to 24 hours.

[0121] Deamidation can be carried out to a desired degree. The deamidation rate (number of deamidated asparagine residues in the substrate protein / number of asparagine residues before deamidation in the substrate protein) may be, for example, 0.1% or more, 1% or more, 5% or more, 10% or more, or 20% or more, or 100% or less, 70% or less, or 50% or less.

[0122] Protein deamidation increases the negative charge of proteins. This increase in negative charge can result in effects such as a lower isoelectric point (pI), increased hydration potential, and increased electrostatic repulsion. Protein deamidation also changes the higher-order structure of proteins, resulting in effects such as increased surface hydrophobicity. These effects can improve protein functionality, such as improved solubility and dispersibility, foaming ability and foam stability, and emulsifying ability and emulsion stability. Proteins with improved functionality can greatly expand their applications, for example, in the food industry. For example, many plant proteins have poor solubility, dispersibility, and emulsifying properties, especially under weakly acidic conditions, which correspond to the pH range of typical foods. This has limited their use in many foods, such as coffee whitener, acidic beverages such as fruit juice, dressings, mayonnaise, and creams. However, if proteins can be deamidated with protein deamidating enzymes to improve their solubility, dispersibility, and emulsifying properties, they can be used in a wide range of foods.

[0123] Furthermore, deamidating a protein with protein deamidating enzymes can reduce the mineral sensitivity of the protein and increase the soluble mineral content in a solution containing the protein and minerals. It is generally known that the absorbability of calcium in foods to the human body can be improved by solubilizing calcium with an organic acid or casein phosphopeptide. Therefore, if the soluble mineral content in foods and beverages is increased by deamidating a protein with protein deamidating enzymes or the like, the absorbability of minerals such as calcium to the human body can be improved. That is, protein deamidating enzymes or the like can also be used, for example, as an active ingredient in a calcium absorption enhancer.

[0124] Furthermore, in the production of seasonings made from proteins as raw materials, such as hydrolyzed animal protein (HAP), hydrolyzed vegetable protein (HVP), miso, soy sauce, etc., deamidation of proteins with protein deamidating enzymes can reduce bitterness and improve the rate of proteolysis by proteases. Hydrophobic peptides are generally known to cause bitterness, but deamidation can increase the hydrophilicity of peptides and reduce bitterness. Deamidation can also change the higher-order structure of proteins, increasing their susceptibility to proteases. For example, deamidation can improve the low rate of proteolysis, which is one of the problems encountered in the enzymatic production of HAP and HVP.

[0125] In this way, deamidation can modify the physical properties and functions of proteins or materials containing such proteins. These modifications of physical properties and functions are collectively called "modification." In other words, the deamidation method of the present invention may be a method for modifying a protein, which comprises allowing protein deamidase or the like to act on a protein containing an asparagine residue, or a method for producing a modified protein, which comprises allowing protein deamidase or the like to act on a protein containing an asparagine residue. Furthermore, one aspect of the method may be, for example, a method for modifying a food or drink or a raw material thereof, which comprises allowing protein deamidase or the like to act on a food or drink containing a protein containing an asparagine residue, or a method for producing a modified food or drink or a raw material thereof, which comprises allowing protein deamidase or the like to act on a food or drink containing a protein containing an asparagine residue, or a raw material thereof.

[0126] Furthermore, protein deamidase and the like may be used in combination with protein glutaminase. Protein glutaminase is an enzyme that catalyzes the deamidation of glutamine residues in proteins. The combined use of protein deamidase and protein glutaminase makes it possible to deamidate both asparagine residues and glutamine residues in proteins without causing the proteins to become smaller in molecular weight. In other words, the combined use of these enzymes further increases the protein deamidation rate and shifts the isoelectric point (pI) of the protein to a more acidic side, thereby improving the solubility of proteins at pH levels more acidic than those in which proteins are normally prone to insolubilization, thereby further improving the functionality of the protein.

[0127] Furthermore, protein deamidase and the like may be used in combination with transglutaminase. Transglutaminase is an enzyme that catalyzes a reaction in which glutamine and lysine residues in a protein are bonded to crosslink the protein. Crosslinking can cause protein gelation and improve the functionality of the protein. For this reason, transglutaminase is widely used as a protein modifier in industrial applications, including the food industry. Here, when both protein crosslinking and deamidation are performed, deamidation of the protein with protein glutaminase converts glutamine residues, which are the substrate of transglutaminase, to glutamic acid residues, thereby inhibiting the crosslinking reaction by transglutaminase. On the other hand, since the substrate of protein deamidase and the like is asparagine residue, they do not compete with transglutaminase for the substrate, and therefore both protein crosslinking and deamidation can be efficiently performed by using them in combination with transglutaminase.

[0128] The type of enzyme used in combination with protein deamidase, etc. can be selected appropriately depending on various conditions such as the properties of protein deamidase, etc. When protein deamidase, etc. is used in combination with another enzyme, there are no particular limitations on the timing or order of adding each enzyme. Both enzymes may be added simultaneously or at different times. Furthermore, when protein deamidase, etc. is used in combination with another enzyme, there are no particular limitations on the reaction conditions (enzyme amount, reaction time, reaction temperature, reaction pH, etc.) as long as the desired effect is obtained. The reaction conditions can be set appropriately depending on various conditions such as the type of other enzyme. For example, when protein deamidase, etc. is used in combination with protein glutaminase, the amount of protein glutaminase used per 1 g of substrate protein is The amount of transglutaminase used may be preferably 0.001 to 100 U per 1 g of substrate protein. When protein deamidase or the like is used in combination with transglutaminase, the amount of transglutaminase used may be preferably 0.001 to 100 U per 1 g of substrate protein.

[0129] Protein deamidases and the like can also be used as protein engineering reagents for modifying the function of proteins. When the substrate protein is an enzyme, the enzymatic and physicochemical properties of the enzyme can be modified. For example, by deamidating an enzyme protein with protein deamidase and the like, the isoelectric point of the enzyme protein can be lowered, thereby modifying its pH stability. Furthermore, by changing the structure and electrical environment of the active site of the enzyme protein, the properties of the enzyme protein, such as substrate affinity, substrate specificity, reaction rate, pH dependency, temperature dependency, and temperature stability, can be modified.

[0130] Furthermore, protein deamidase and the like can also be used as reagents for protein analysis and research, such as reagents for quantifying the amide content of proteins and reagents for solubilizing proteins.

[0131] Furthermore, protein deamidating enzymes and the like can be used to improve the extraction efficiency and concentration efficiency of proteins from grains and pulses. Generally, many proteins from grains and pulses, such as wheat and soybeans, are insoluble in water, and it is not easy to extract these proteins. However, for example, by treating a suspension of wheat flour or soybean flour with protein deamidating enzymes and the like to increase the solubility of the proteins, the proteins can be easily extracted and a high-content protein isolate can be obtained.

[0132] Protein deamidating enzymes and the like can also be used to improve the efficiency of animal protein extraction. For example, gelatin is industrially produced primarily from cattle bones, cattle hides, and pigskins. To efficiently extract high-quality gelatin, pretreatment with inorganic acids such as hydrochloric acid or sulfuric acid (acid treatment) or pretreatment with lime (alkali treatment) is performed. However, both of these methods have a high environmental impact and require a long time for processing. On the other hand, protein deamidating enzymes and the like can easily extract proteins, and because they are enzymatic methods, they reduce the environmental impact.

[0133] The protein with improved functionality obtained in this way shows excellent effects when used in various foods such as meat and fish products and noodles, and may enable the production of foods with new textures and functions. [Example]

[0134] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0135] The experimental procedures used in the examples are as follows.

[0136] <Quantitative determination of protein deamidase> The Quick Start™ Bradford Protein Assay Kit (Bio-Rad) was used. The procedure was carried out according to the protocol provided with the kit. Bovine serum albumin (BSA) of known concentration was used as the standard titer. It was used as a protein.

[0137] <Protein asparaginase activity measurement> Protein asparaginase activity is measured using CBZ-Asn-Gly (hereinafter also referred to as "Z-NG") as a substrate. The enzyme reaction was carried out using the enzyme, and the enzyme was determined from the quantitative value of the produced CBZ-Asp-Gly (hereinafter also referred to as "Z-DG"). The elementary activity was calculated. Specifically, 190 μL of substrate solution (30 mM Z-NG, 200 mM phosphate buffer, pH 6.5) was added to a 1.5 mL tube. The mixture was added to a tube and heated at 37°C for 10 minutes. 10 μL of enzyme solution was added and the mixture was incubated at 37°C for 10 minutes. Then, 200 μL of a stop solution (12% trichloroacetic acid solution) was added to stop the reaction. The enzyme activity required to produce 1 μmol of Z-DG per minute was defined as 1 U. The substrate (Z-NG) and product (Z-DG) were quantified by UHPLC under the following analytical conditions. Mobile phase A: 0.1% trifluoroacetic acid Mobile phase B: 0.1% trifluoroacetic acid, 80% acetonitrile Flow rate: 0.6 mL / min Column temperature: 40℃ Detection: UV 215 nm Column: ZORBAX RRHD Eclipse Plus C18 3.0 x 100 mm (Agilent Technologies) Gradient conditions: 0-5 min (B: 22.5%), 5-5.1 min (B: 22.5-100%), 5.1-6 min (B: 100%) 6-6.1 min(B: 100-22.5%), 6.1-8 min(B: 22.5%)

[0138] Experiment 1: Analysis of modified protein deamidases <Structure prediction> Protein deamidase derived from Luteimicrobium album (hereinafter referred to as "LalPA") Sometimes referred to as) and the protein deamidase derived from Microbacterium testaceum (hereinafter sometimes referred to as "MtePA"). The three-dimensional structures of these were predicted using AlphaFold2 (Jumper, J., Evans, R., Pritzel, A. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583-589 (2021).) and are shown in Figures 2 and 3 respectively. These enzymes each possess 4 and 2 β-barrel domains at their C-termini, and the numbers are not conserved in both organisms. From the above, this sequence was predicted to be a sequence unnecessary for protein asparaginase activity.

[0139] <Construction of E. coli expression plasmid> To investigate whether the C-terminal β-barrel domain is a sequence necessary for protein asparaginase activity, a plasmid was constructed to express a protein with the C-terminus of the protein deamidase derived from Rutimicrobium album truncated. Five types of mutants described in Table 1 were designed. After codon optimization for E. coli, each DNA sequence was synthesized by artificial gene synthesis and cloned into the NdeI-XhoI site of pET29b(+) (Novagen) to obtain the target expression plasmid.

Table 1

[0140] <Expression in E. coli> The obtained plasmid was introduced into E. coli BL21(DE3) (Novagen) by transformation. The resulting The transformant was inoculated into 3 mL of LB medium containing 50 μg / mL of kanamycin and cultured at 37°C for 16 to 18 hours to obtain a preculture solution. 2.5 mL of the preculture solution was inoculated into 50 mL of Overnight Express™ Instant TB medium (Novagen) containing 50 μg / mL of kanamycin and cultured at 20°C for 24 hours. The culture medium was centrifuged to collect the bacterial cells, which were then frozen and stored at -80°C.

[0141] <Activity measurement using bacterial cell lysate> The collected frozen cells were thawed, and then 2 / 5 the volume of BugBuster (registered trademark) Master Mix (Merck) was added to disrupt the cells. The supernatant was collected by centrifugation to prepare a disrupted cell solution. A 100 mg / mL trypsin solution (derived from porcine pancreas, Sigma-Aldrich T4799-5G, dissolved in 20 mM sodium phosphate buffer (pH 6.5)) was added in an amount of 1 / 10 of the cell lysate, and the mixture was treated at 37°C for 2 hours for maturation. Protein asparaginase activity was measured using the cell lysate after maturation. No activity was observed in βx0, whereas protein asparaginase activity was observed in βx1, βx2, βx3, and βx4. This suggests that the activity of protein deamidase derived from Luteimicrobium album may require at least one β-barrel domain (Table 2). Furthermore, the fewer the number of β-barrel domains, the lower the activity per culture medium. These results suggest that the C-terminal β-barrel domain is not essential for protein asparaginase activity. [Table 2]

[0142] Experiment 2: Search for novel protein deamidating enzymes <Screening for novel protein deamidases using the amino acid sequence of protein deamidase from Luteimicrobium album> The C-terminal β-barrel domain is considered not to be essential for protein asparaginase activity, and since the activity per culture medium improved as the number of β-barrel domains decreased, among the amino acid sequences of the protein deamidase derived from Lutimicrobium album, a protein having a sequence highly homologous to the partial sequence not containing the C-terminal β-barrel domain was searched for.

[0143] <Construction of E. coli expression plasmid> As a result, proteins derived from Amycolatopsis deserti, Microbispora corallina, and Motilibacter peucedani were identified. Based on the amino acid sequence information of these proteins (SEQ ID NO: 1, 3, or 5), after optimizing the codons for E. coli, the DNA sequences shown in SEQ ID NO: 2, 4, and 6 were synthesized by the artificial gene synthesis method. These DNA sequences were cloned into the NdeI-XhoI site of pET29b(+) (Novagen) to obtain the target expression plasmid.

[0144] <Expression in E. coli> The obtained plasmid was introduced into E. coli BL21(DE3) (Novagen) by transformation. The resulting transformant was inoculated into 3 mL of LB medium containing 50 μg / mL of kanamycin and cultured at 37 °C for 16 to 18 hours to obtain a preculture solution. 2.5 mL of the preculture solution was inoculated into 50 mL of Overnight Express (trademark) Instant TB medium (Novagen) containing 50 μg / mL of kanamycin and cultured at 20 °C for 24 hours. The culture solution was centrifuged to collect the cells, which were stored frozen at -80 °C.

[0145] <Activity measurement in cell lysate> (A) Measurement of activity after trypsin digestion The collected frozen cells were thawed, and then 2 / 5 the volume of BugBuster (registered trademark) Master Mix (Merck) was added to disrupt the cells. The supernatant was collected by centrifugation to prepare a disrupted cell solution. A 100 mg / mL trypsin solution (derived from porcine pancreas, Sigma-Aldrich T4799-5G, dissolved in 20 mM sodium phosphate buffer (pH 6.5)) was added in an amount of 1 / 10 of the bacterial cell lysate, and the mixture was treated at 37°C for 2 hours for maturation. Protein asparaginase activity was measured using the maturated bacterial cell lysate, and the results are shown in Table 3. Hereinafter, the proteins derived from Amycolatopsis deserti, Microbispora corallina, and Motilibacter peucedani may be referred to as AdePA, McoPA, and MpePA, respectively. [Table 3]

[0146] (a) Measurement of activity after digestion with Alcalase A cell lysate of a transformant expressing McoPA or MpePA was prepared in the same manner as in (A) above. Then, Alcalase (Sigma-Aldrich P4860-50ML) was added to a final concentration of 1%, and the mixture was treated at 30°C for 1 hour for maturation. Protein asparagus was then prepared using the cell lysate after maturation. The ginase activity was measured, and the results are shown in Table 4. [Table 4]

[0147] <Structure prediction> The three-dimensional structures of AdePA, McoPA, and MpePA were predicted using AlphaFold2. Figures 1, 4, and 5 show the structures of AdePA, McoPA, and MpePA, respectively. is shown. When comparing these protein deaminases with the predicted three-dimensional structures (Figures 2 and 3) of protein deamidases derived from Lutimicrobium album and Microbacterium testaceum, it can be seen that the three-dimensional structures of the pro-sequence and the part excluding the β-barrel domain located on the C-terminal side rather than the Mainβ-barrel (hereinafter sometimes referred to as the "active domain") are conserved among species. On the other hand, LalPA and MtePA have a β-barrel domain on the C-terminal side rather than the Mainβ-barrel, while AdePA, McoPA, and MpePA do not have a β-barrel domain at the C-terminus. That is, it was shown that the β-barrel domain on the C-terminal side rather than the active domain containing the Mainβ-barrel is not essential for protein asparaginase activity.

[0148] <Preparation of purified AdePA enzyme> A cell disruption solution of the transformant expressing AdePA was prepared by the above method. A 1 / 10 volume of a 100 mg / mL trypsin solution (derived from porcine pancreas, Sigma-Aldrich T4799-5G, dissolved in 20 mM sodium phosphate buffer (pH 6.5)) was added to the cell disruption solution and treated at 37°C for 2 hours for maturation. After adjusting the pH of the matured cell disruption solution to 7.5 with NaOH, the centrifuged supernatant was collected and filter-filtered to obtain a clear solution. The entire amount of the above solution was added to a 5 mL HisTALON (trademark) Superflow Cartridge (Takara Bio Inc.) sufficiently equilibrated with Buffer A (50 mM Tris-HCl, 300 mM NaCl (pH 7.5)), and washed with 3.3% of 50 mL of Buffer B (50 mM Tris-HCl, 300 mM NaCl, 150 mM imid azole (pH 7.5)), and then the protein bound to the column was eluted using 100% of Buffer B. The elution fraction with the peak UV absorption was selected, and the solvent of the elution fraction was replaced with 20 mM sodium phosphate buffer (pH 6.5) using HiPrep 26 / 10 Desalting (Cytiva) ​Subsequently, the purified AdePA enzyme was obtained.

[0149] <Evaluation of thermal stability> After diluting the purified AdePA enzyme to 3 U / mL, 15 μL was added to a PCR tube and heated at 30, 40, or 50 °C for 10 minutes. The residual activity after heating was calculated by comparing with the activity before heating (Table 6). LalPA and MtePA obtained in a previous report (International Publication No. 2015 / 133590) were also evaluated simultaneously as controls. When heated at 40 °C for 10 minutes, AdePA showed a residual activity of 25%, while LalPA and MtePA had residual activities of 3% and 1% respectively, indicating that AdePA has high thermal stability. It was shown.

Table 5

[0150] <Evaluation of pH stability> After diluting the purified AdePA enzyme to 5 U / mL, it was further diluted 9-fold with buffers of pH 4 - 9 (pH 4, 5: 20 mM sodium acetate, pH 6, 6.5: 20 mM sodium phosphate, pH 7, 8, 9: 20 mM Tris-HCl) and treated at 25 °C for 1 hour. 30 μL of the purified enzyme treated at each pH was added to 190 μL of the substrate solution, reacted at 37 °C for 10 minutes, and then 200 μL of the stop solution (12% trichloroacetic acid solution) was added to stop the reaction. The enzyme activity was calculated from the amount of the product, and the relative activity with the maximum activity = 100% was calculated. The results are shown in Table 6. AdePA was shown to have higher stability at pH 4 - 6.5 compared with LalPA and MtePA, and higher stability in the weakly acidic region. It was shown to have high stability.

Table 6

[0151] <Evaluation of substrate reactivity> Edamame protein (50's Co., Ltd.), soybean protein (Cargill), casein sodium (Sigma), BSA (Sigma), fish-derived gelatin (Sigma), or egg white albumin (Sigma) in 200 μL of a 2% aqueous solution (200 mM sodium phosphate buffer, pH 6.5), 20 μL of purified enzyme diluted to 5 U / mL was added, and after reacting at 37 °C for 60 minutes, 220 μL of a 12% trichloroacetic acid solution was added to stop the reaction. The amount of free ammonia in the reaction stop solution was quantified using a LabAssay (trademark) ammonia kit (Fuji Film Wako Shibayagi Co., Ltd.). The results are shown in Table 7. AdePA was shown to have equivalent reactivity to fish-derived gelatin compared to LalPA and MtePA. reactivity.

Table 7

[0152] Experiment 3: Stereostructure analysis of AdePA <Large-scale purification of AdePA> The transformant expressing AdePA obtained in Experiment 2 was inoculated into 50 mL of LB medium containing 50 μg / mL of kanamycin, and cultured at 37 °C for 16 - 18 hours to obtain a preculture solution. 10 mL of the preculture solution was inoculated into 1 L of LB medium (Nacalai) containing 50 μg / mL of kanamycin, and cultured at 37 °C with rotary shaking until the OD value reached about 0.6. After standing at 15 °C for 30 minutes, isopropyl-β-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM, and cultured with rotary shaking at 15 °C overnight, followed by cell collection. To the obtained cells, 40 mL of disruption buffer (50 mM HEPES pH 7.5, 100 mM NaCl, 25 mM Imidazole) was added, and disrupted using an ultrasonic disruptor ISONATER (KUBOTA) 600 at 150 W for 20 minutes. After centrifuging the disrupted solution at 15000×g, 4 °C for 30 minutes, the supernatant was collected and passed through a 0.2 μm filter. ​​

[0153] Next, the supernatant of the crushed solution was purified by affinity chromatography using AKTA pure (Cytiva). A HisTrap FF crude 5 mL column (Cytiva) was equilibrated with the crushing buffer. The supernatant of the crushed solution was applied to the column, washed with the crushing buffer, and then eluted with a gradient of HisTrap elution buffer -(50 mM HEPES pH 7.5, 100 mM NaCl, 500 mM Imidazole). The obtained HisTrap elution fraction was diluted 10-fold with 50 mM HEPES, pH 7.5.

[0154] Next, it was purified by anion exchange chromatography. After equilibrating a Resource Q column 6 mL (Cytiva) with Resource Q equilibration buffer (50 mM HEPES, pH 7.5), the diluted HisTrap elution fraction was applied and eluted with a gradient of Resource Q elution buffer (50 mM HEPES, 1 M NaCl, pH 7.5). Next, the elution fraction obtained by anion exchange chromatography was concentrated using Amicon Ultra-15 50

[0155] kDa (Merck) and purified by gel filtration chromatography. A Superdex 200 Increase 10 / 300 GL column (Cytiva) was used, and 10 mM HEPES, 100 mM NaCl, pH 7.5 was used as the gel filtration buffer. The elution fraction obtained by gel filtration chromatography was collected, the protein concentration was quantified by the Bradford method, and it was stored at 4°C to a concentration of 10 mg / mL. All purification was carried out at 4°C. <Crystallization of AdePA>

[0156] <AdePA's crystallization> From the obtained elution fraction, AdePA was crystallized using the Hanging drop method. 500 μL of a reservoir solution (25% PEG3350, 0.1 M Bis-Tris pH 6.4) was added to each well of a 24-well plate (Funakoshi), and 1.5 μL of a crystallization sample solution containing protein (10 mg / mL pro-AdePA, 10 mM HEPES pH 7.5, 100 mM NaCl) and 1 μL of the reservoir solution were dropped onto a cover slide, mixed, and a drop was made. The cover slide was turned over so that the drop faced downward, covered the well, sealed with grease, and incubated at 20 °C.

[0157] <Obtaining and analyzing X-ray diffraction data> To obtain X-ray diffraction data, the beamline AR-NW12A of the Photon Factory, a synchrotron radiation research facility of the High Energy Accelerator Research Organization (KEK), a large-scale synchrotron radiation facility, was used. As an antifreeze agent, 27% PEG3350, 0.1 M Bis-Tris pH 6.4 was used. Using the diffraction data analysis program XDS, the obtained X-ray diffraction intensity data was converted into reflection data. Next, an initial molecular model was constructed using the molecular replacement program Molrep included in the protein structure analysis software package CCP4. The structure refinement calculation using the structure refinement program Refmac5 included in the CCP4 software and the auto-fitting or manual fitting using the analysis program COOT for model building were alternately repeated to perform model building that matches the electron density and densify the crystal structure. The obtained three-dimensional structure is shown as a cartoon model in Figure 6. In Figure 6, the model of the observed water molecules is indicated by dots.

[0158] <Comparison with the predicted structure> The three-dimensional structure of AdePA obtained by X-ray crystal structure analysis was superimposed on the predicted structure of AlphaFold2 using the software pymol. At the same time, the root mean square deviation (RMSD) between the corresponding Cα atoms of the main chain was calculated to be 0.47 Å, which was in excellent agreement. The results are shown in Figure 7 as a cartoon model (black: X-ray crystal structure analysis, gray: predicted structure). [Industrial Applicability]

[0159] The present invention provides a novel protein deamidase (protein asparaginase) that catalyzes the deamidation of asparagine residues in proteins. In one aspect, the enzyme can be used to deamidate asparagine residues in proteins.

Claims

1. having an activity of catalyzing a reaction of deamidating an asparagine residue in a protein, and A protein characterized by having one β-barrel domain at the C-terminus or no β-barrel domain at the C-terminus.

2. Derived from Amycolatopsis deserti, Microbispora corallina, or Motilibacter peucedani. The protein of claim 1.

3. The protein according to claim 1 or 2, which is a protein according to the following (A), (B), or (C): (A) a protein comprising the amino acid sequence shown in SEQ ID NO: 1, 3, or 5, the amino acid sequence of positions 129 to 785 of SEQ ID NO: 1, positions 130 to 808 of SEQ ID NO: 3, or positions 143 to 818 of SEQ ID NO: 5; (B) A protein comprising an amino acid sequence containing a substitution, deletion, insertion, or addition of one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 1, 3, or 5, or the amino acid sequence of positions 129 to 785 of SEQ ID NO: 1, positions 130 to 808 of SEQ ID NO: 3, or positions 143 to 818 of SEQ ID NO: 5, and having the activity of catalyzing a reaction of deamidating an asparagine residue in a protein. (C) A protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 1, 3, or 5, or the amino acid sequence of positions 129 to 785 of SEQ ID NO: 1, positions 130 to 808 of SEQ ID NO: 3, or positions 143 to 818 of SEQ ID NO: 5, and having the activity of catalyzing a reaction of deamidating an asparagine residue in a protein.

4. A protein described in the following (A'), (B', or (C'): (A') a protein having the amino acid sequence of positions 1 to 1074, 132 to 1074, 240 to 1074, 1 to 1166, 132 to 1166, or 240 to 1166 of SEQ ID NO: 9; positions 1 to 894, 67 to 894, 181 to 894, 1 to 1007, 67 to 1007, or 181 to 1007 of SEQ ID NO: 11; positions 1 to 932, 70 to 932, 193 to 932, 1 to 1018, 70 to 1018, or 193 to 1018 of SEQ ID NO: 13; or positions 1 to 831, 81 to 831, 1 to 939, or 81 to 939 of SEQ ID NO: 14; (B') positions 1 to 1074, 132 to 1074, 240 to 1074, 1 to 1166, 132 to 1166, or 240 to 1166 of SEQ ID NO: 9; positions 1 to 894, 67 to 894, 181 to 894, 1 to 1007, 67 to 1007, or 181 to 1007 of SEQ ID NO: 11; positions 1 to 932, 70 to 932, 193 to 932, 1 to 10 of SEQ ID NO: 13 a protein having an amino acid sequence containing substitution, deletion, insertion, or addition of one or several amino acid residues in the amino acid sequence of positions 18, 70 to 1018, or 193 to 1018, or positions 1 to 831, 81 to 831, 1 to 939, or 81 to 939 of SEQ ID NO: 14, and having an activity to catalyze a reaction of deamidating an asparagine residue in a protein; (C') positions 1 to 1074, 132 to 1074, 240 to 1074, 1 to 1166, 132 to 1166, or 240 to 1166 of SEQ ID NO: 9; positions 1 to 894, 67 to 894, 181 to 894, 1 to 1007, 67 to 1007, or 181 to 1007 of SEQ ID NO: 11; positions 1 to 932, 70 to 932, 193 a protein having an amino acid sequence that is 90% or more identical to the amino acid sequence of positions 1 to 932, 1 to 1018, 70 to 1018, or 193 to 1018 of SEQ ID NO: 14, or positions 1 to 831, 81 to 831, 1 to 939, or 81 to 939 of SEQ ID NO: 14, and having an activity to catalyze a reaction of deamidating an asparagine residue in a protein.

5. A polynucleotide encoding the protein of claim 1 or 4.

6. A recombinant vector comprising the polynucleotide of claim 5.

7. A transformant into which the recombinant vector according to claim 6 has been introduced.

8. A method for producing a protein having an activity to catalyze a reaction to deamidate an asparagine residue in a protein, the method comprising culturing the transformant described in claim 7 in a medium to produce the protein described in claim 1 or 4, and recovering the protein from the culture.

9. 10. A method for producing a protein having an activity to catalyze a reaction to deamidate an asparagine residue in a protein, the method comprising culturing an organism capable of producing the protein according to claim 1 or 4 in a medium to produce the protein, and recovering the protein from the culture.

10. 9. The method of claim 8, comprising treating the protein with a processing enzyme.

11. The method of claim 10, wherein the processing enzyme is a protease.

12. A method for producing a protein variant having improved activity, based on the amino acid sequence of a protein having an activity to catalyze a reaction of deamidating an asparagine residue in a protein and having two or more β-barrel domains at the C-terminus, comprising: The method comprises designing a polynucleotide encoding a protein variant having an amino acid sequence obtained by removing at least one amino acid sequence corresponding to the C-terminal β-barrel domain from the amino acid sequence of the protein.

13. The method according to claim 12, wherein the protein is a protein described in (a), (b), or (c) below: (a) a protein comprising the amino acid sequence set forth in SEQ ID NO: 9, 11, 13, or 14, the amino acid sequence of positions 240 to 1355 of SEQ ID NO: 9, the amino acid sequence of positions 181 to 1180 or positions 67 to 1180 of SEQ ID NO: 11, the amino acid sequence of positions 193 to 1172 or positions 70 to 1172 of SEQ ID NO: 13, or the amino acid sequence of positions 81 to 1119 of SEQ ID NO: 14; (b) an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 9, 11, 13, or 14, the amino acid sequence of positions 240 to 1355 of SEQ ID NO: 9, the amino acid sequence of positions 181 to 1180 or positions 67 to 1180 of SEQ ID NO: 11, the amino acid sequence of positions 193 to 1172 or positions 70 to 1172 of SEQ ID NO: 13, or the amino acid sequence of positions 81 to 1119 of SEQ ID NO: 14, but containing a substitution, deletion, insertion, or addition of one or several amino acid residues, and having an activity of catalyzing a reaction of deamidating an asparagine residue in a protein. (c) a protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:9, 11, 13 or 14, the amino acid sequence of positions 240 to 1355 of SEQ ID NO:9, the amino acid sequence of positions 181 to 1180 or positions 67 to 1180 of SEQ ID NO:11, the amino acid sequence of positions 193 to 1172 or positions 70 to 1172 of SEQ ID NO:13, or the amino acid sequence of positions 81 to 1119 of SEQ ID NO:14, and having the activity of catalyzing a reaction of deamidating an asparagine residue in a protein.

14. The method according to claim 12 or 13, wherein the amino acid sequence corresponding to the C-terminal β-barrel domain is the amino acid sequence set forth in the following (a'), (b'), or (c'): (a') positions 1075 to 1166, 1167 to 1257, or 1258 to 1355 of SEQ ID NO: 9, positions 895 to 1007 or 1008 to 1180 of SEQ ID NO: 11, positions 933 to 1018 or 1019 to 1172 of SEQ ID NO: 13, or positions 1075 to 1166, 1167 to 1257, or 1258 to 1355 of SEQ ID NO: 14 the amino acid sequence of positions 832 to 939 or 940 to 1119; (b') an amino acid sequence comprising substitution, deletion, insertion, or addition of one or several amino acid residues in the amino acid sequence of positions 1075 to 1166, 1167 to 1257, or 1258 to 1355 of SEQ ID NO: 9, positions 895 to 1007 or 1008 to 1180 of SEQ ID NO: 11, positions 933 to 1018 or 1019 to 1172 of SEQ ID NO: 13, or positions 832 to 939 or 940 to 1119 of SEQ ID NO: 14; (c') an amino acid sequence having 90% or more identity to the amino acid sequence of positions 1075 to 1166, 1167 to 1257, or 1258 to 1355 of SEQ ID NO: 9, positions 895 to 1007 or 1008 to 1180 of SEQ ID NO: 11, positions 933 to 1018 or 1019 to 1172 of SEQ ID NO: 13, or positions 832 to 939 or 940 to 1119 of SEQ ID NO:

14.

15. The method according to claim 12 or 13, comprising introducing a recombinant vector containing the polynucleotide into a host to obtain a transformant.

16. The method according to claim 15, comprising culturing the transformant in a medium to produce the protein variant, and recovering the protein variant from the culture obtained by the culturing.

17. The method of claim 16, comprising treating the protein variant with a processing enzyme.

18. 18. The method of claim 17, wherein the processing enzyme is a protease.

19. A method for producing a protein in which an asparagine residue has been deamidated, comprising allowing the protein according to claim 1 or 4 to act on a protein containing an asparagine residue.

20. The method according to claim 19, wherein the protein containing the asparagine residue is contained in a food or drink or an ingredient thereof.

21. 20. The method of claim 19, wherein the food or beverage is selected from mayonnaise, dressing, cream, yogurt, meat products, and bread.

22. The method according to claim 19, wherein 0.001 to 2000 U of the protein according to claim 1 or 4 is used per gram of the protein containing an asparagine residue.

23. A method for modifying a food or drink or a raw material thereof containing a protein having an asparagine residue, the method comprising allowing the protein according to claim 1 or 4 to act on the food or drink or a raw material thereof.

24. 24. The method of claim 23, wherein the food or beverage is selected from mayonnaise, dressing, cream, yogurt, meat products, and bread.

25. The method according to claim 23, wherein 0.001 to 2000 U of the protein according to claim 1 or 4 is used per gram of the protein containing an asparagine residue.

26. A method for producing a modified food or drink or a raw material thereof, which comprises allowing the protein according to claim 1 or 4 to act on a food or drink or a raw material thereof containing a protein having an asparagine residue.

27. 27. The method of claim 26, wherein the food or beverage is selected from mayonnaise, dressing, cream, yogurt, meat products, and bread.

28. The method according to claim 26, wherein 0.001 to 2000 U of the protein according to claim 1 or 4 is used per gram of the protein containing an asparagine residue.

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