Enzyme agent for deamidating protein, and use thereof

Abstract

The present invention addresses the problem of providing: an enzyme agent for deamidating a protein, the enzyme agent containing a protein deamidase; and a use of the enzyme agent. According to the present invention, there is provided an enzyme agent for deamidating a protein, the enzyme agent containing, as an active ingredient, a protein deamidase comprising an amino acid sequence having 90% or higher sequence identity with the amino acid sequence represented by SEQ ID NO: 1.

Description

Protein deamidation enzyme preparation and its use

[0001] The present invention relates to an enzyme preparation containing protein deamidating enzyme as an active ingredient (enzyme preparation for protein deamidation) and uses thereof.

[0002] Protein deamidase is an enzyme that acts on macromolecular proteins to catalyze deamidation reactions. Protein deamidase generates negatively charged carboxyl groups by deamidating glutamine residues in proteins, thereby causing various changes in protein properties. For example, an increase in hydration force and electrostatic repulsion due to a decrease in the isoelectric point of a protein reduces protein-protein interactions (i.e., reduces association), thereby increasing the solubility and water dispersibility of the protein. Furthermore, the exposure of internal hydrophobic regions due to changes in the higher-order structure of a protein confers surface activity to the protein, thereby improving the emulsifying power, emulsion stability, foaming ability, and foam stability of the protein. Because protein deamidase can significantly change the properties of proteins, it has dramatically expanded the applications of proteins. For this reason, protein deamidase is highly useful and has attracted great interest in the technical field.

[0003] The first protein deamidase discovered was protein glutaminase, which was found in Chryseobacterium proteolyticum in 2000 (Non-Patent Document 1). However, despite its high usefulness and the great interest it has attracted, protein glutaminase is also an extremely unique enzyme, and no new ones have been discovered for the long period of about 20 years since then. Approximately 20 years later, protein glutaminase was discovered in Bacteroides helcogenes (Non-Patent Document 2), but as of February 2024, the only protein glutaminase in industrial use is that derived from C. proteolyticum.

[0004] A novel protein-deamidating enzyme from Chryseobacterium proteolyticum sp. nov., a newly isolated bacterium from soil. Applied and Environmental Microbiology 2000; 66(8): 3337-43A novel protein glutaminase from Bacteroides helcogenes-characterization and comparison, Applied Microbiology and Biotechnology (2020) 104:187-199

[0005] The problem to be solved by the present invention is to provide a novel enzymatic agent having protein deamidation activity.

[0006] The present inventors have now newly discovered that a polypeptide derived from Longimycelium tulfanense has protein deamidation activity. The present invention was completed based on the above findings.

[0007] According to the present invention, the following inventions are provided: <1> A protein deamidating enzyme preparation comprising, as an active ingredient, a protein deamidating enzyme having an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1. <2> The protein deamidating enzyme preparation according to <1>, wherein the protein deamidating enzyme is derived from the genus Longimycelium. <3> A method for deamidating a protein or peptide, comprising allowing the protein deamidating enzyme preparation according to <1> or <2> to act on the protein or peptide. <4> A method for deamidating a protein-containing food or drink, comprising allowing the protein deamidating enzyme preparation according to <1> or <2> to act on the protein-containing food or drink. <5> A method for producing a food or drink, comprising allowing the protein deamidating enzyme preparation according to <1> or <2> to act on the protein-containing food or drink. <6> A food or drink treated with the protein deamidating enzyme preparation according to <1> or <2>. <7> A method for producing a protein deamidating enzyme having an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, comprising the step of culturing a bacterium of the genus Longimycelium. <8> An enzyme preparation for protein deamidation, comprising, as an active ingredient, a protein deamidating enzyme consisting of an amino acid sequence having 60% or more sequence identity with the amino acid sequence of SEQ ID NO: 1. <9> The enzyme preparation for protein deamidation according to <8>, wherein the protein deamidating enzyme is derived from the genus Longimycelium, Crossiella, or Krasilnikovia. <10> A method for deamidating a protein or peptide, comprising allowing the protein deamidating enzyme preparation according to <8> or <9> to act on the protein or peptide. <11> A method for deamidating a protein-containing food or drink, comprising allowing the protein deamidating enzyme preparation according to <8> or <9> to act on the protein-containing food or drink. <12> A method for producing a food or drink, comprising allowing the protein deamidating enzyme preparation according to <8> or <9> to act on the protein-containing food or drink. <13> A food or drink treated with the protein deamidating enzyme preparation according to <8> or <9>.<14> A method for producing a protein deamidase comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, the method comprising the step of culturing bacteria of the genus Longimycelium, Crossiella, or Krasilnikovia. <15> An enzymatic agent for protein deamidation, comprising, as an active ingredient, a protein deamidating enzyme comprising an amino acid sequence having 50% or more sequence identity with the amino acid sequence of SEQ ID NO: 1. <16> The enzymatic agent for protein deamidation according to <15>, wherein the protein deamidating enzyme is derived from the genus Longimycelium, Crossiella, Krasilnikovia, or Longispora. <17> A method for deamidating a protein or peptide, the method comprising allowing the protein deamidating enzyme agent according to <15> or <16> to act on the protein or peptide. <18> A method for deamidating a protein-containing food or drink, the method comprising allowing the protein deamidating enzyme agent according to <15> or <16> to act on the protein-containing food or drink. <19> A method for producing a food or drink, comprising allowing the protein deamidating enzyme preparation according to <15> or <16> to act on a protein-containing food or drink. <20> A food or drink treated with the protein deamidating enzyme preparation according to <15> or <16>. <21> A method for producing a protein deamidating enzyme comprising an amino acid sequence having 50% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, comprising a step of culturing bacteria of the genus Longimycelium, Crossiella, Krasilnikovia or Longispora.

[0008] Use of an enzymatic agent for protein deamidation, the enzymatic agent comprising, as an active ingredient, a protein deamidase consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1.

[0009] An enzymatic agent for protein deamidation containing the protein deamidating enzyme according to the present invention is suitable for use in the fields of food and medical applications.

[0010] 1. Enzymatic Preparation for Protein Deamidation The enzymatic preparation for protein deamidation of the present invention comprises, as an active ingredient, a protein deamidase consisting of the amino acid sequence of SEQ ID NO: 1, 6, 10, or 14, or an amino acid sequence equivalent to said amino acid sequence. The protein deamidase of the present invention is an enzyme that exhibits the action of degrading amide group-containing side chains of proteins, and may further have the action of degrading amide group-containing side chains of proteins, which involves cleavage of peptide bonds and protein crosslinking, so long as it has the above-mentioned action. Specifically, the protein deamidase of the present invention is an enzyme (protein glutaminase) that deamidates glutamine residues in proteins, converting them to glutamic acid.

[0011] An "equivalent amino acid sequence" refers to an amino acid sequence that is partially different from a reference amino acid sequence (the amino acid sequence of SEQ ID NO: 1, 6, 10, or 14), but the difference does not substantially affect the function of the protein (here, protein deamidation activity). Thus, an enzyme having an equivalent amino acid sequence catalyzes the enzymatic reaction of protein deamidase. The level of activity is not particularly limited as long as it can exhibit the function of protein deamidation. However, it is preferable that the activity is the same as or higher than that of an enzyme consisting of the reference amino acid sequence (having the amino acid sequence of SEQ ID NO: 1, 6, 10, or 14).

[0012] The protein deamidase is preferably derived from the genus Longimycelium, Crossiella, Krasilnikova, or Longispora, and more preferably derived from Longimycelium tulfanense, Crossiella cryophila, Krasilnikova cinnamomea, or Longispora fulva. The amino acid sequence of SEQ ID NO: 1 is the amino acid sequence (mature form) of protein deamidase derived from Longimycelium tulfanense. The full-length amino acid sequence of protein deamidase derived from Longimycelium tulfanense is shown in SEQ ID NO: 2. The amino acid sequence of SEQ ID NO: 6 is the amino acid sequence (mature form) of protein deamidase derived from Crossiella cryophila. The full-length amino acid sequence of protein deamidase derived from Crossiella cryophila is shown in SEQ ID NO: 7. The amino acid sequence of SEQ ID NO: 10 is the amino acid sequence (mature form) of protein deamidase derived from Krasilnikova cinnamomea. The full-length amino acid sequence of protein deamidase derived from Krasilnikova cinnamomea is shown in SEQ ID NO: 11. The amino acid sequence of SEQ ID NO: 14 is the amino acid sequence (mature form) of protein deamidase derived from Longispora fulva. The full-length amino acid sequence of protein deamidase derived from Longispora fulva is shown in SEQ ID NO: 15.

[0013] A "partial difference in the amino acid sequence" occurs, for example, by deletion or substitution of one or more amino acids among the amino acids constituting the amino acid sequence, addition or insertion of one or more amino acids to the amino acid sequence, or any combination thereof. A partial difference in the amino acid sequence is permissible as long as protein deamidation activity is maintained (although the activity may vary to some extent). As long as this condition is satisfied, the position at which the amino acid sequence differs is not particularly limited. Furthermore, differences in the amino acid sequence may occur at multiple positions (locations).

[0014] The number of amino acids that result in a partial difference in the amino acid sequence is, for example, a number corresponding to less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% of all amino acids that make up the amino acid sequence, preferably a number corresponding to less than about 8%, more preferably a number corresponding to less than about 6%, even more preferably a number corresponding to less than about 4%, even more preferably a number corresponding to less than about 2%, and most preferably a number corresponding to less than about 1%. Thus, an equivalent protein has, for example, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 81% or more, about 82% or more, about 83% or more, about 84% or more, about 85% or more, about 86% or more, about 87% or more, about 88% or more, about 89% or more, about 90% or more, about 91% or more, about 92% or more, preferably about 93% or more, more preferably about 94% or more, even more preferably about 95% or more, even more preferably about 96% or more, even more preferably about 97% or more, even more preferably about 98% or more, and most preferably about 99% or more identity to a reference amino acid sequence.

[0015] A typical example of a "partial difference in the amino acid sequence" is a mutation (change) in the amino acid sequence due to deletion or substitution of 1 to 40 (preferably 1 to 30, more preferably 1 to 10, even more preferably 1 to 7, even more preferably 1 to 5, and even more preferably 1 to 3) amino acids among the amino acids constituting the amino acid sequence, addition or insertion of 1 to 40 (preferably 1 to 30, more preferably 1 to 10, even more preferably 1 to 7, even more preferably 1 to 5, and even more preferably 1 to 3) amino acids to the amino acid sequence, or a combination thereof.

[0016] Preferably, an equivalent amino acid sequence is obtained by conservative amino acid substitution at an amino acid residue that is not essential for protein deamidation activity. Here, "conservative amino acid substitution" refers to the substitution of an amino acid residue with an amino acid residue having a side chain with similar properties. Amino acid residues are classified into several families based on their side chains, such as basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Conservative amino acid substitutions are preferably substitutions between amino acid residues within the same family.

[0017] The percent identity of two amino acid sequences can be determined, for example, by the following procedure. First, the two sequences are aligned to enable optimal comparison (for example, gaps may be introduced into the first sequence to optimize alignment with the second sequence). When a molecule (amino acid residue) at a specific position in the first sequence is the same as a molecule at the corresponding position in the second sequence, the molecules at that position are considered to be identical. The identity of two sequences is a function of the number of identical positions shared by the two sequences (i.e., percent identity = number of identical positions / total number of positions × 100), and preferably takes into account the number and size of gaps required for optimal alignment.

[0018] Comparison of two sequences and determination of identity can be achieved using a mathematical algorithm. A specific example of a mathematical algorithm that can be used for sequence comparison is the algorithm described in Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68 and modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77, but is not limited to this. Amino acid sequence identity can be obtained, for example, using blastp (protein-protein BLAST) from the National Center for Biotechnology Information (NCBI). Default parameters can be used, for example, using the BLOSUM62 matrix and setting Gap Costs to Existence: 11 and Extension: 1.

[0019] Protein deamidase, which is an active ingredient of the enzymatic preparation of the present invention, may be a part of a larger protein (e.g., a fusion protein). Examples of sequences added to the fusion protein include sequences that are useful for purification, such as multiple histidine residues, and additional sequences that ensure stability during recombinant production.

[0020] The protein deamidase of the present invention (hereinafter also referred to as the present enzyme) can be obtained by culturing a microorganism that produces the protein deamidase (a protein deamidase-producing strain), for example, a bacterium of the genus Longimycelium, a bacterium of the genus Crossiella, a bacterium of the genus Krasilnikova, or a bacterium of the genus Longispora. As an example, the protein deamidase can be obtained by culturing Longimycelium tulufanense, a bacterium of the genus Crossiella, a bacterium of the genus Krasilnikova, or a bacterium of the genus Longispora. That is, the present invention may relate to a method for producing a protein deamidase comprising an amino acid sequence having 50% or more (preferably 60% or more, 70% or more, 80% or more, or 90% or more) sequence identity with the amino acid sequence of SEQ ID NO: 1, the method comprising the step of culturing bacteria belonging to the genus Longimycelium, Crossiella, Krasilnikova, or Longispora. The protein deamidase-producing strain may be a wild-type strain or a mutant strain (e.g., a mutant strain obtained by ultraviolet irradiation). A specific example of a protein deamidase-producing strain is Longimycelium tulfanense (NBRC107726). Longimycelium tulfanense (NBRC107726) is a strain stored at NBRC (National Institute of Technology and Evaluation, Biotechnology Center), and can be purchased by following the prescribed procedures.

[0021] The present enzyme can be prepared from the culture medium and / or cells of a microorganism that produces the present enzyme. The culture conditions and method are not particularly limited as long as the present enzyme is produced. That is, a method and conditions suitable for culturing the microorganism to be used can be appropriately set, provided that the present enzyme is produced. The culture method may be either liquid culture or solid culture, but liquid culture is preferably used. The culture conditions will be explained using liquid culture as an example.

[0022] The medium is not particularly limited as long as it allows the growth of the microorganisms used. For example, a medium containing a carbon source such as glucose, sucrose, gentiobiose, soluble starch, glycerin, dextrin, molasses, or organic acid, a nitrogen source such as ammonium sulfate, ammonium carbonate, ammonium phosphate, or ammonium acetate, or gelatin, peptone, yeast extract, corn steep liquor, casein hydrolysate, bran, or meat extract, or an inorganic salt such as potassium salt, magnesium salt, sodium salt, phosphate, manganese salt, iron salt, or zinc salt, may be used. Vitamins, amino acids, and the like may be added to the medium to promote the growth of the microorganisms used. The pH of the medium is adjusted to, for example, about 3 to 8, preferably about 4 to 7, and the culture temperature is usually about 20 to 40°C, preferably about 25 to 35°C, under aerobic conditions for 1 to 20 days, preferably about 3 to 10 days. Examples of culture methods that can be used include shaking culture and aerobic submerged culture using a jar fermenter.

[0023] After culturing under the above conditions, the target enzyme is recovered from the culture medium or bacterial cells. When recovering from the culture medium, for example, the culture supernatant is filtered, centrifuged, or the like to remove insoluble matter, and then the enzyme can be obtained by separating and purifying the enzyme using an appropriate combination of methods such as concentration using an ultrafiltration membrane, salting out using ammonium sulfate precipitation, dialysis, and various types of chromatography using ion exchange resins. On the other hand, when recovering from bacterial cells, the enzyme can be obtained by disrupting the bacterial cells, for example, by pressure treatment or ultrasonic treatment, and then separating and purifying the enzyme in the same manner as above. Note that the bacterial cells may be recovered from the culture medium in advance by filtration, centrifugation, or the like, and then the above series of steps (disruption, separation, and purification of the bacterial cells) may be carried out.

[0024] The present enzyme can also be easily prepared by genetic engineering techniques. For example, it can be prepared by transforming a suitable host cell (e.g., Escherichia coli) with DNA encoding the present enzyme and recovering the protein expressed in the transformant. The recovered protein is then purified as appropriate depending on the purpose. Obtaining the desired enzyme as a recombinant protein in this way allows for various modifications. For example, by inserting the DNA encoding the present enzyme and other appropriate DNA into the same vector and producing a recombinant protein using the vector, it is possible to obtain the present enzyme as a recombinant protein to which any peptide or protein is linked. Furthermore, modifications such as the addition of sugar chains and / or lipids, or modifications that cause N- or C-terminal processing, may also be performed. These modifications can simplify the extraction and purification of the recombinant protein, or impart biological functions to the protein.

[0025] Typically, gene expression and recovery of the expression product (the present enzyme) are carried out using an appropriate host-vector system as described above. However, cell-free synthesis systems may also be used. Here, "cell-free synthesis systems (cell-free transcription systems, cell-free transcription / translation systems)" refer to in vitro synthesis of mRNA and proteins encoded by template nucleic acids (DNA and mRNA) using ribosomes and transcription / translation factors derived from living cells (or obtained by genetic engineering techniques), rather than using living cells. Cell-free synthesis systems typically use cell extracts obtained by purifying cell lysates as needed. Cell extracts generally contain ribosomes, various factors such as initiation factors, and various enzymes such as tRNA, all of which are necessary for protein synthesis. During protein synthesis, various amino acids, energy sources such as ATP and GTP, and other substances necessary for protein synthesis, such as creatine phosphate, are added to the cell extract. Of course, separately prepared ribosomes, various factors, and / or various enzymes may be supplemented as needed during protein synthesis.

[0026] The development of a transcription / translation system in which each molecule (factor) required for protein synthesis is reconstituted has also been reported (Shimizu, Y. et al.: Nature Biotech., 19, 751-755, 2001). In this synthesis system, the genes for 31 factors that make up the bacterial protein synthesis system, including three initiation factors, three elongation factors, four factors involved in termination, 20 aminoacyl-tRNA synthetases that bind each amino acid to tRNA, and methionyl-tRNA formyltransferase, were amplified from the E. coli genome, and the protein synthesis system was reconstituted in vitro using these genes. Such a reconstituted synthesis system may be used in the present invention.

[0027] The term "cell-free transcription / translation system" is used interchangeably with cell-free protein synthesis system, in vitro translation system, or in vitro transcription / translation system. In an in vitro translation system, RNA is used as a template to synthesize proteins. The template RNA can be total RNA, mRNA, or an in vitro transcription product. On the other hand, in an in vitro transcription / translation system, DNA is used as a template. The template DNA should contain a ribosome binding domain and preferably contains an appropriate terminator sequence. In an in vitro transcription / translation system, conditions are established in which factors required for each reaction are added so that the transcription and translation reactions proceed sequentially.

[0028] The purified enzyme obtained as described above can be provided as a powder by, for example, freeze-drying, vacuum drying, or spray-drying. In this case, the purified enzyme may be dissolved in advance in acetate buffer, phosphate buffer, triethanolamine buffer, Tris-HCl buffer, or Good's buffer. Preferably, acetate buffer, phosphate buffer, or triethanolamine buffer can be used. Examples of Good's buffer include PIPES, MES, and MOPS.

[0029] The degree of purification of the enzyme is not particularly limited, and the final form may be liquid or solid (including powder).

[0030] The present enzyme deamidates the γ-amide group of glutamine residues in proteins. The content of the active ingredient (the present enzyme) in the enzyme preparation of the present invention is not particularly limited, but the content of the active ingredient can be set or adjusted so that the protein deamidation activity per gram of the present enzyme preparation is 0.01 U to 5000 U, preferably 0.1 U to 2000 U, and more preferably 1 U to 1000 U.

[0031] The protein deamidation activity can be measured using the following method using a synthetic substrate. 0.1 mL of a sample solution containing protein deamidase is added to 1 mL of 0.2 M phosphate buffer (pH 6.5) containing 30 mM Z-Gln-Gly, and the mixture is left to stand at 37°C for 10 minutes. Then, 1 mL of 0.4 M TCA (trichloroacetic acid) solution is added to terminate the reaction. As a blank, 1 mL of 0.4 M TCA solution is added to 1 mL of 0.2 M phosphate buffer (pH 6.5) containing 30 mM Z-Gln-Gly, and 0.1 mL of the sample solution containing protein deamidase is further added, followed by standing at 37°C for 10 minutes.

[0032] The amount of ammonia generated in the reaction solution obtained above is measured using an Ammonia Test Wako (Fujifilm Wako Pure Chemical Industries). The ammonia concentration in the reaction solution is determined from a calibration curve showing the relationship between ammonia concentration and absorbance (630 nm) prepared using an ammonia standard solution (ammonium chloride).

[0033] The activity of protein deamidase is calculated from the following formula, where 1 unit (1 U) is the amount of enzyme that produces 1 μmol of ammonia per minute. In the formula, the volume of the reaction solution is 2.1, the volume of the enzyme solution is 0.1, Df is the dilution factor of the enzyme solution, and 17.03 is the molecular weight of ammonia.

[0034]

[0035] On the other hand, the protein deamidation activity for a protein substrate can be calculated from the amount of deamidated insulin B chain produced when insulin B chain is used as a substrate. Details of the measurement conditions, measurement procedures, etc. are described in the Examples below.

[0036] The protein deamidation activity of the enzyme preparation of the present invention is not particularly limited. For example, the lower limit of the deamidation rate in an evaluation method using insulin B chain is 4% or more, 5% or more, 7% or more, preferably 10% or more, and more preferably 15% or more, and the upper limit of the deamidation rate is 99% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, preferably 40% or less, more preferably 30% or less, and even more preferably 20% or less.

[0037] Furthermore, the protein deamidation activity of the enzymatic preparation of the present invention can also be evaluated by detecting a peak transition by analyzing the reaction stopped solution after the reaction with a synthetic substrate using HPLC, as described above. Details of the measurement conditions, measurement procedures, etc. will be described later in the Examples.

[0038] The enzyme preparation of the present invention may contain, in addition to the active ingredient (protein deamidase of the present invention), excipients, buffers, suspending agents, stabilizers, preservatives, antiseptics, physiological saline, various proteins, various protein hydrolysates, various extracts, various salts, various antioxidants, cysteine, glutathione, sodium glutamate, sodium inosinate, sodium guanylate, calcined shell calcium, silicon dioxide, and the like. Examples of excipients that can be used include starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, sucrose, and glycerol. Examples of buffers that can be used include phosphates, citrates, and acetates. Examples of stabilizers that can be used include propylene glycol and ascorbic acid. Examples of preservatives that can be used include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, and methylparaben. Examples of preservatives that can be used include ethanol, benzalkonium chloride, parahydroxybenzoic acid, and chlorobutanol. Examples of proteins include soybean protein, wheat protein, corn protein, milk protein, and animal-derived protein. Examples of extracts include meat extract, plant extract, and yeast extract. Examples of salts include chloride, phosphate, polyphosphate, pyrophosphate, citrate, lactate, and carbonate. Examples of antioxidants include L-ascorbate and sodium bisulfite. The form of the enzyme preparation of the present invention is not particularly limited and may be, for example, powder, granules, liquid, or capsule.

[0039] 2. Gene The gene encoding the present enzyme consists of DNA encoding a protein containing the amino acid sequence of SEQ ID NO: 1, 6, 10, or 14. Specific examples of this embodiment include DNA consisting of the nucleotide sequence shown in SEQ ID NO: 3, 8, 12, or 16, and DNA consisting of the nucleotide sequence shown in SEQ ID NO: 4, 9, 13, or 17. The DNA of SEQ ID NO: 3 encodes only the mature amino acid sequence (SEQ ID NO: 1), while the DNA of SEQ ID NO: 4 encodes the mature amino acid sequence (SEQ ID NO: 1) plus a signal peptide and pro-sequence. The DNA of SEQ ID NO: 8 encodes only the mature amino acid sequence (SEQ ID NO: 6), while the DNA of SEQ ID NO: 9 encodes the mature amino acid sequence (SEQ ID NO: 6) plus a signal peptide and pro-sequence. The DNA of SEQ ID NO: 12 encodes only the mature amino acid sequence (SEQ ID NO: 10), while the DNA of SEQ ID NO: 13 encodes the mature amino acid sequence (SEQ ID NO: 10) plus a signal peptide and pro-sequence. The DNA of SEQ ID NO: 16 encodes only the mature amino acid sequence (SEQ ID NO: 14), while the DNA of SEQ ID NO: 17 encodes the mature amino acid sequence (SEQ ID NO: 14) as well as a signal peptide and a pro-sequence.

[0040] The gene encoding the present enzyme is typically used to prepare the present enzyme. A genetic engineering preparation method using the gene encoding the present enzyme makes it possible to obtain the present enzyme in a more homogeneous state. This method is also suitable for preparing large quantities of the present enzyme. The use of the gene encoding the present enzyme is not limited to the preparation of the present enzyme. For example, the nucleic acid can also be used as an experimental tool for elucidating the mechanism of action of the present enzyme, or as a tool for designing or creating mutants (modified forms) of the present enzyme.

[0041] As used herein, the term "gene encoding the present enzyme" refers to a nucleic acid that, when expressed, gives the present enzyme, and includes not only a nucleic acid having a base sequence corresponding to the amino acid sequence of the present enzyme, but also a nucleic acid in which a sequence that does not encode an amino acid sequence is added to such a nucleic acid. Codon degeneracy is also taken into consideration.

[0042] Nucleic acids can be prepared in an isolated state by standard genetic engineering techniques, molecular biological techniques, biochemical techniques, chemical synthesis, PCR (e.g., overlap PCR), or a combination thereof, using the sequence information disclosed in this specification or the attached sequence listing as a reference.

[0043] Nucleic acids that, when compared with the nucleotide sequence of the gene encoding the present enzyme, have the same function as the protein they encode but differ in part from the nucleotide sequence (hereinafter also referred to as "equivalent nucleic acids"; a nucleotide sequence specifying an equivalent nucleic acid is also referred to as "equivalent nucleotide sequence"). Examples of equivalent nucleic acids include DNAs that have a nucleotide sequence containing one or more nucleotide substitutions, deletions, insertions, additions, or inversions based on the nucleotide sequence of the nucleic acid encoding the present enzyme, and that encode a protein that has the enzymatic activity characteristic of the present enzyme (i.e., protein deamidation activity). Base substitutions or deletions may occur at multiple sites. Here, "multiple" refers to, for example, 2 to 40 bases, preferably 2 to 20 bases, and more preferably 2 to 10 bases, although this varies depending on the position and type of amino acid residues in the three-dimensional structure of the protein encoded by the nucleic acid. Equivalent nucleic acids have, for example, 50% or more, 60% or more, 70% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, preferably 93% or more, more preferably 94% or more, even more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably about 98% or more, and most preferably 99% or more sequence identity to the reference base sequence (SEQ ID NO: 3, 4, 8, 9, 12, 13, 16, or 17).

[0044] Such equivalent nucleic acids can be obtained, for example, by restriction enzyme treatment, treatment with exonuclease or DNA ligase, or by introducing mutations using site-directed mutagenesis (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York) or random mutagenesis (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York). Equivalent nucleic acids can also be obtained by other methods, such as ultraviolet irradiation.

[0045] A nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of the gene encoding the present enzyme may be used, or a nucleic acid having a nucleotide sequence that is at least about 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of the gene encoding the present enzyme or a nucleotide sequence complementary thereto.

[0046] Nucleic acids having a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the gene encoding the present enzyme or a nucleotide sequence complementary to the equivalent nucleotide sequence may also be used. "Stringent conditions" refer to conditions under which so-called specific hybrids are formed and nonspecific hybrids are not formed. Such stringent conditions are well known to those skilled in the art and can be determined with reference to, for example, Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York) or Current Protocols in Molecular Biology (edited by Frederick M. Ausubel et al., 1987). An example of stringent conditions includes incubation at approximately 50°C using a hybridization solution (50% formamide, 10x SSC (0.15 M NaCl, 15 mM sodium citrate, pH 7.0), 5x Denhardt's solution, 1% SDS, 10% dextran sulfate, 10 μg / ml denatured salmon sperm DNA, 50 mM phosphate buffer (pH 7.5)), followed by washing at approximately 65°C using 0.1x SSC and 0.1% SDS. More preferred stringent conditions include, for example, a hybridization solution containing 50% formamide, 5x SSC (0.15 M NaCl, 15 mM sodium citrate, pH 7.0), 1x Denhardt's solution, 1% SDS, 10% dextran sulfate, 10 μg / ml denatured salmon sperm DNA, and 50 mM phosphate buffer (pH 7.5).

[0047] Recombinant DNA containing a gene encoding the present enzyme may be used. The recombinant DNA may be provided, for example, in the form of a vector. As used herein, the term "vector" refers to a nucleic acid molecule that can transport an inserted nucleic acid into a target such as a cell.

[0048] An appropriate vector is selected depending on the intended use (cloning, protein expression) and the type of host cell. Examples of vectors using E. coli as a host include M13 phage or its modified forms, λ phage or its modified forms, and pBR322 or its modified forms (pB325, pAT153, pUC8, etc.), vectors using yeast as a host include pYepSec1, pMFa, pYES2, etc., vectors using insect cells as a host include pAc and pVL, and vectors using mammalian cells as a host include pCDM8 and pMT2PC.

[0049] The vector is preferably an expression vector. An "expression vector" refers to a vector that can introduce a nucleic acid inserted therein into a target cell (host cell) and express it in the cell. An expression vector usually contains a promoter sequence necessary for the expression of the inserted nucleic acid, an enhancer sequence that promotes expression, and the like. An expression vector containing a selection marker can also be used. When such an expression vector is used, the presence or absence (and the degree of introduction) of the expression vector can be confirmed using the selection marker.

[0050] Insertion of a nucleic acid into a vector, insertion of a selectable marker gene (if necessary), insertion of a promoter (if necessary), etc. can be carried out using standard recombinant DNA techniques (for example, well-known methods using restriction enzymes and DNA ligase; see Molecular Cloning, Third Edition, 1.84, Cold Spring Harbor Laboratory Press, New York).

[0051] As host cells, microorganisms such as Escherichia coli, filamentous fungi (Aspergillus oryzae), actinomycetes (Streptomyces, Streptomyces lividans, Streptomyces mobaraensis), Bacillus subtilis, and budding yeast (Saccharomyces cerevisiae) are preferred for ease of handling. However, any host cell capable of replicating recombinant DNA and expressing the gene for the present enzyme can be used. Examples of E. coli include E. coli BL21(DE3)pLysS when a T7 promoter is used, and E. coli JM109 when not. Examples of budding yeast include SHY2, AH22, and INVSc1 (Invitrogen).

[0052] Microorganisms carrying recombinant DNA (i.e., transformants) can be used, which can be obtained by transfection or transformation with the vector of the present invention. For example, calcium chloride method (J. Mol. Biol., Vol. 53, p. 159 (1970)), Hanahan method (J. Mol. Biology, Vol. 166, p. 557 (1983)), SEM method (Gene, Vol. 96, p. 23 (1990)), Chung et al.'s method (Proceedings of the National Academy of Sciences of the USA, Vol. 86, p. 2172 (1989)), calcium phosphate coprecipitation method, electroporation (Potter, H. et al., Proc. Natl. Acad. Sci. USA 81, 7161-7165 (1984)), lipofection (Felgner, P. L. et al., Proc. Natl. Acad. Sci. USA 81, 7161-7165 (1984)), and the like can be used. USA 84, 7413-7417 (1984)), conjugal transfer, etc. The above-mentioned microorganisms can be used to produce the present enzyme.

[0053] 3. Uses of the Enzyme Preparation The present invention further relates to uses of the enzymatic preparation of the present invention. That is, by allowing the enzymatic preparation of the present invention to act on a substrate (protein or peptide), the protein or peptide can be modified. Specifically, by allowing the enzymatic preparation of the present invention to act on a protein or peptide containing a glutamine residue, the glutamine residue of the protein can be deamidated. That is, the present invention may also relate to a method for deamidating a protein or peptide, comprising allowing the enzymatic preparation for protein deamidation to act on the protein or peptide. Furthermore, the enzymatic preparation of the present invention can deamidate the glutamine residue of a protein even when acting on a protein or peptide containing both glutamine and lysine residues. It is known that conventional general transglutaminases hardly catalyze deamidation reactions when acting on a protein or peptide containing both glutamine and lysine residues (when acting in the presence of an acyl acceptor). However, the high-performance material of the present invention can deamidate the glutamine residue of a protein even when a lysine residue is present as an acyl acceptor.

[0054] The protein or peptide is preferably contained in a protein-containing food or drink, and the present invention preferably relates to a method for deamidating a protein-containing food or drink, which comprises allowing a protein deamidating enzyme agent to act on the protein-containing food or drink.

[0055] The present invention may also relate to a method for producing a food or beverage, which comprises allowing a protein-containing food or beverage to undergo the action of an enzymatic preparation for protein deamidation. For example, by allowing the enzymatic preparation of the present invention to act on a food or beverage or food or beverage material (such as a protein-containing food or beverage) containing a protein or peptide, a food or beverage or food or beverage material (such as a deamidated protein-containing food or beverage) containing a deamidated protein can be produced. By allowing the enzymatic preparation of the present invention to act on a protein-containing food or beverage, the quality of the protein-containing food or beverage can be improved. According to the present invention, a food or beverage treated with the enzymatic preparation of the present invention (a deamidated protein-containing food or beverage) is provided. Furthermore, the enzymatic preparation of the present invention can be used not only for producing a food or beverage or food or beverage material (such as a protein-containing food or beverage), but also for producing industrial materials and pharmaceutical raw materials.

[0056] Specific examples of the improvement of the quality of protein-containing foods and beverages include, but are not limited to, applications for improving the solubility, dispersibility, emulsifying properties, etc. of animal proteins and vegetable proteins (for example, applications for producing dairy products (milk, yogurt, etc.), vegetable milk, vegetable yogurt, coffee whitener, beverages such as juice, dressings, mayonnaise, and cream); increasing the solubility and dispersibility of hardly soluble vegetable proteins (for example, applications for producing tempura flour using wheat gluten); applications for modifying dough in bread and confectionery (for example, applications for producing crackers, biscuits, cookies, pizza or pie crusts); applications for improving allergenic proteins in foods and beverages (for example, applications for producing crackers, biscuits, cookies, pizza or pie crusts); Examples of uses include removing or reducing allergens in foods (e.g., in the production of foods for patients with wheat allergies); reducing the mineral sensitivity of proteins, increasing the soluble mineral content in liquids containing proteins and minerals, and increasing the absorbability of minerals into the human body (e.g., in the production of mineral-rich (e.g., calcium) beverages and mineral (e.g., calcium) absorption enhancers); reducing bitterness, improving the proteolysis rate of proteases, and / or increasing the glutamic acid content (e.g., in the production of amino acid-based seasonings (hydrolyzed animal protein (HAP), hydrolyzed vegetable protein (HVP)), miso and soy sauce).

[0057] The method for producing a food or drink using the present enzyme preparation is not particularly limited, and examples thereof include a method comprising the following step (a): Furthermore, step (a) may be followed by steps (b) and / or (c): (a) a step of allowing the present enzyme preparation to act on a protein-containing food or food material (protein-containing food or drink), (b) a step of recovering a food or food material containing a deamidated protein, and (c) a step of inactivating or removing the present enzyme contained in the obtained enzyme-treated food or enzyme-treated food material.

[0058] Example 1: Preparation of protein deamidase derived from Longimycelium tulfanense Longimycelium tulfanense cells (obtained from the National Institute of Technology and Evaluation (NITE)) (NBRC107726) were cultured in tryptophan soy broth medium (manufactured by Nissui Pharmaceutical Co., Ltd.) at 37°C for 4 days, and the supernatant was collected. The resulting supernatant was dialyzed against 20 mM phosphate buffer (pH 6) according to a standard method. The dialyzed sample was subjected to ion exchange chromatography using a cation exchange column (SP Sepharose FF, manufactured by GE Healthcare) equilibrated with the same buffer. The active fraction was desalted and concentrated, and then subjected to gel filtration on Superdex 200HR to obtain the enzyme sample.

[0059] The amino acid sequence and nucleotide sequence of this enzyme are shown below. Amino acid sequence (mature sequence only) (SEQ ID NO: 1) ATSLPPIAPPLPRGVQSKSWSVPDYIAAWEKQHGRPMTAEERYHLARGCIGVTVVNLDREDAPNPPLNLSFGTYQRAMEVQAALNEIVATRPSPREYAEQVRKHPALQGVQNLVRAFPTFIDPANLHAAIFSKRFYSKQDPNWTDEQAAEMYRPNPRTGQVDMSTYRYRARPGYVNFDYGWYDEQTNNWWHANHAEPGMKVYQSTLRYYSRPLLDFDEQVFTVAFARVA

[0060] Amino acid sequence (full length) *The underlined part is the mature sequence (SEQ ID NO: 2) MKKWLPRALVALFVLLGLPAGLAGTAHAAVVAHAAVATSLPPIAPPLPRGVQSKSWSVPDYIAAWEKQHGRPMTAEERYHLARGCIGVTVVNLDREDAPNPPLNLSFGTYQRAMEVQAALNEIVATRPSPREYAEQVRKHPALQGVQNLVRAFPTFIDPANLHAAIFSKRFYSKQDPNWTDEQAAEMYRPNPRTGQVDMSTYRYRARPGYVNFDYGWYDEQTNNWWHANHAEPGMKVYQSTLRYYSRPLLDFDEQVFTVAFARVA

[0061] Base sequence (mature body) (sequence number 3) gccacgtcgctgccaccgatcgcaccgccgcttccccggggcgtgcagagcaagagctggtcggtgccggactacattgccgcctgggagaagcaacacggtagccgatgacggccgaggacgggacggtaccacctcgcccggggctgcatcggctcaccgtggtcaacctcgaccgggaggacgcgccgaacccgccgctcaacctgtcgttcggcacttaccagcgggccatggaagtgcacagagatcgtcgcgacccggccgcttcgccgcgggagtacgccgagcaggcaggtgcgcaagcacccggcgctgcagggcgtgcagaagcacccggcgctgcagaacccggcgctcaacggacggctgcagaacccggcgtcagcaccggcgtcagagcaccggcgtcagagcaccggcgtcaga gcgttccccacgttcatcgacccggcgaacctgcacgccgccatcttctccaagcggttctactcgaagcaggacccgaactggaccgacgagcaggcggccgagatgtaccggccgaatccgcggaccggccaggtcgacatgagcacctaccgctaccgggcgcggccgg gttacgtgaacttcgactacggttggtacgacgagcagacgaacaactggtggcacgccaaccacgcggagccgggcatgaaggtctaccagagcacgttgcggtactactcccggccgctgctggatttcgacgagcaggttttcaccgtggcgttcgcgcgggtcgcctga

[0062] Base sequence (full length) (sequence number 4) gtgaagaagtggttgccgcgcgccctggtcgcgctgttcgtgctcctggggttacccgccgccggccctggccggaacggcacatgccgggtcgtcgcgcacgccgccgtcgccacgtcgctgccaccgatcgcaccgccgcttccccggggcgtgcagagcaagagctggtcggggccggactacattgccgcctgggagaagcaacacagtagccgatgacggccgaggcggtaccacctgcccggggctgcatcggctcaccgtggtcaacctcgaccgggaggacgcgccgaacccgctcaacctgtcgttcggcacttaccagcgggccatggaagtgcacagagatcgtcgcgaccggccgcttcgccgggagatc gccgagcaggtgcgcaagcaccccggcgctgcagggcgtgcagaacctggtgcgggcgttccccacgttcatcgacccggcgaacctgcacgccgccatcttctccaagcggttctactcgaagcaggacccgaactggaccgacgagcaggcggccgagatgtaccggccgaatccgcggaccggccaggtcgacatga gcacctaccgctaccgggcgcggccgggttacgtgaacttcgactacggttggtacgacgagcagacgaacaactggtggcacgccaaccacgcggagccgggcatgaaggtctaccagagcacgttgcggtactactcccggccgctgctggatttcgacgagcaggttttcaccgtggcgttcgcgcgggtcgcctga

[0063] Example 2: Confirmation of protein deamidation activity The protein deamidation activity of this enzyme sample was confirmed by the production of deamidated insulin B chain when insulin B chain (a peptide containing glutamine and lysine residues) was used as a substrate. (Method) 5 mg of insulin B chain (insulin B chain, oxidized form, derived from bovine pancreas, Sigma-Aldrich, sequence: Phe-Val-Asn-Gln-His-Leu-Cys(SOH)-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys(SOH)-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Ala (SEQ ID NO: 5)) was dissolved in 2 mL of ultrapure water to prepare a substrate solution. Subsequently, 10 μL or 20 μL of 1 mg / mL purified enzyme solution and 0.2 M potassium dihydrogen phosphate / disodium hydrogen phosphate buffer (pH 6.5) were added to 90 μL of substrate solution to a total volume of 180 μL to prepare a reaction solution. The reaction solution was heated to 40°C in an incubator and allowed to stand for 1 hour, after which 20 μL of 1 N HCl was added to terminate the reaction. The reaction-stop solution described above was analyzed by HPLC. Deamidation activity was detected by the peak transition from insulin B chain to deamidated insulin B chain. As a comparative example, deamidation activity was similarly detected using Streptomyces-derived transglutaminase (Amano Enzyme Inc.). <Deamidation Evaluation> The amount of deamidated insulin B chain produced was calculated from the peak areas of insulin B chain and deamidated insulin B chain calculated from the chromatogram, and evaluated as the deamidation rate (amount of deamidated insulin B chain produced ÷ amount of substrate insulin B chain × 100).

[0064] (Results) The results are shown in Table 1.

[0065] (Discussion) In the results shown in Table 1, the transglutaminase of the comparative example produced only a small amount of deamidated insulin B chain, and the amount produced did not change even when the amount of enzyme added was increased. This confirmed that transglutaminase does not catalyze the deamidation reaction of peptides containing glutamine and lysine residues. In contrast, the protein deamidase of the present invention produced deamidated insulin B chain, and the amount produced increased with increasing the amount of enzyme added, indicating that it catalyzes the deamidation reaction.

[0066] Example 3: Preparation of Crossiella cryophila-derived protein deamidase and Krasilnikova cinnamomea-derived protein deamidase 1. Preparation of transformants A target sequence was incorporated into an expression vector for Streptomyces microorganisms. The sequence was amplified by PCR, and transformants (C. cryophila-derived protein deamidase heterologous expression strain and K. cinnamomea-derived protein deamidase heterologous expression strain) were obtained using standard methods. The obtained transformants were subjected to sequence analysis to confirm whether the target sequence had been incorporated.

[0067] 2. C. cryophila-derived protein deamidase heterologous expression strain, K. cinnamomea-derived protein deamidase heterologous expression strain The obtained transformants were cultured in tryptophan broth medium (manufactured by Nissui Pharmaceutical Co., Ltd.) at 32 ° C for 4 days, and the supernatant was collected. The obtained supernatant was dialyzed against 20 mM phosphate buffer (pH 6) according to a standard method. The dialyzed sample was subjected to ion exchange chromatography using a cation exchange column (SP Sepharose FF, manufactured by GE Healthcare) equilibrated with the same buffer. Each enzyme sample was then further purified using a His tag protein purification column (His trap HP, manufactured by Cytiva) to obtain each enzyme sample.

[0068] The amino acid sequence and nucleotide sequence of protein deamidase derived from C. cryophila are shown below. Amino acid sequence (mature sequence only) (SEQ ID NO: 6) GLPIAPPLRPGVESKTWKVADYIKAWETYHGRPMTADERENLARGCIGVTVVNLERGDVGNPPLGLSFGTFGKAREVQAALNEIIKSKPSAAQYAAAVRSHPLLSKLENVQRALPADLNTGELTAAIFSKRFYSKQNPNWTDEFAEKMYRANARGQVDMNSYRYVARPGYVNFDYGWYDENTRNWWHANHAEPGMKVYQSTFDYYSRDLLDFDRQVFTVAFAKKV

[0069] Amino acid sequence (full length) *The underlined part is the mature sequence (SEQ ID NO: 7) MRNSVLIRTLTTAALGLGLLIPGAAAATAAPAPSTAAAVSIVGLPIAPPLRPGVESKTWKVADYIKAWETYHGRPMTADERENLARGCIGVTVVNLERGDVGNPPLGLSFGTFGKAREVQAALNEIIKSKPSAAQYAAAVRSHPLLSKLENVQRALPADLNTGELTAAIFSKRFYSKQNPNWTDEFAEKMYRANARGQVDMNSYRYVARPGYVNFDYGWYDENTRNWWHANHAEPGMKVYQSTFDYYSRDLLDFDRQVFTVAFAKKV

[0070] Base sequence (mature body) (sequence number 8) ggcctccccatcgcccccccgctgcgcccggcgtcgagtcgaagacgtggaaggtcgcggactacatcaaggcgtggggagacgtaccatggccgccggacgaacgggaacctcgcgcggggctgcatcggggtcaccgtggtcaacctcgagcggggcgacgtggggaaatccgccgctcggccctctcgttcggcacgttcggcaaagcgcggggaggtccaagcggcgctcaacgagatcatcaagtcgaagccgtcggcgcgcagtacgcggccgggtccgctcgcaccccctgctcgaagctcgaagctcgagaacgtgcagcggg ccctcccggcggatctcaacgggcgagctcacggcggcgatcttctcgaagcggttctactcgaagcagaacccgaactggacggacgagttcgcggagaagatgtaccgggcgaacgcgcggggccaagtcgacatgaactcgtaccggtacgtcgcgcggccggg ctacgtcaacttcgactacgggctggtacgacgagaacacgcggaactggtggcacgcgaatcacgcggagccgggcatgaaggtctatcagtcgacgttcgactactactcgcgggatctcctcgacttcgaccggcaagtcttcacggtcgcgttcgcgaagaaaggtc

[0071] Base sequence (full length) (sequence number 9) atgcggaactccgtgctcatccgcacgctgaccaccgccgccctcggccctgggcctcctgatccccggggcggccgcgacggccccccggccctccaccgcggcggccgtctccattgtcggcctcccatcgcccccccgctgcgcccggcgtcgagtcgaagacgtggaaggtcgcggactacatcaaggcgtggagacgtaccatggccgcagatgaccgcggacgaacgggaacctcgcgcggggctgcatcggggtcaccgtggtcaacctcgagcggggcgacgtggggatccgccgcggccctctcgttcggcacgttcggcaaagcgcgggaggtccaagcggcgctcaacgagatcatcaagtcgaagccgtcgggggg cgcagtacgcggccgcggtccgctcgcaccccctgctctcgaagctcgagaacgtgcagcgggccctcccggcggatctcaacgggcgagctcacggcggcgatcttctcgaagcggttctactcgaagcagaacccgaactggacggacgagttcgcggagaagatgtaccgggcgaacgcgcggggccaagtcgac atgaactcgtaccggtacgtcgcgcggccgggctacgtcaacttgactacgggctggtacgacgagaacacgcggaactggtggcacgcgaatcacgcggagccgggcatgaaggtctatcagtcgacgttcgactactactcgcgggatctcctcgacttcgaccggcaagtcttcacggtcgcgttcgcgaagaaggtc

[0072] The amino acid sequence and nucleotide sequence of protein deamidase derived from K. cinnamomea are shown below. Amino acid sequence (mature sequence only) (SEQ ID NO: 10) TESDPRSPTPARTATQAPIAEVRPAERSLANANAATATELTPLAPALPPGVSTRTWSVEDFVELFERKYGRPMTQDERNALARGCIGVTTVNLERGNINPPLGMSFGTFATSRDVQNAINDILATNPSRTQFVAAVAQHPLLSRIDNVTDSLPGGPTSQWTAVIFSKRFYSKQDPSWTDEQADQAFRPDPATGQVDMTDYRYRAKPGYVNFDYGWLDEGSGNWWHANHAEPGMKVYQSTLRHYSRPLLDFDRQVFSVTFGRVHP

[0073] Amino acid sequence (full length) *The underlined part is the mature sequence (SEQ ID NO: 11) MPKRLLRLFVIFVAGLSISTMVAAPALATESDPRSPTPARTATQAPIAEVRPAERSLANANAATATELTPLAPALPPGVSTRTWSVEDFVELFERKYGRPMTQDERNALARGCIGVTTVNLERGNINPPLGMSFGTFATSRDVQNAINDILATNPSRTQFVAAVAQHPLLSRIDNVTDSLPGGPTSQWTAVIFSKRFYSKQDPSWTDEQADQAFRPDPATGQVDMTDYRYRAKPGYVNFDYGWLDEGSGNWWHANHAEPGMKVYQSTLRHYSRPLLDFDRQVFSVTFGRVHP

[0074] Base sequence (mature body) (sequence number 12) accgaatccgacccgcggtcccccacccccgcgcgcacggccacgcaagcgccgatcgcggaagtgcggcggcgcggaacgctcctggcgaacgccaatgccgccaccgaccgaactgacccctcgccccgccctccccggggtgtcgacgcggacgtggtcggtcgaggacttcgtcgagctcttcgagcggaagtacggcggccgatgacccaagacgaacgcaatgccctcgcgcggggctgcatcggctcacgacgggtcaacctcgagcggggcaacatcaacccgccgcggcatgtcgttcggcacgagttcgcgacgtcgcgcggaggtgcacagaacgcgatcaacgacatccccaatccctccgcaccagcaatccctccgcaaccattt gtggcggccgtggcgcagcacccgctcctctcgcggatcgacaacgtcacggactcgctcccgggcggcccgacgtcgcagtggacggcggtcatcttctcgaagcggttctactcgaagcaagacccgtcgtggacggacgagcaagcggatcaagcgttccgccccgatccggcgacgggccaagtcgacatgacg gactaccggtaccgggcgaagccgggctacgtcaacttcgactacgggctggctcgacgagggctcgggcaactggtggcacggaaccacgcggagccggggatgaaggtgtatcagtcgacgctccggcactactcgcggccgctcctcgacttcgaccggcaagtcttctcggtcacgttcggccgggtccacccg

[0075] Base sequence (full length) (sequence number 13) atgccgaagcggctcctccggctcttcgtcatcttcgtcgcgggcctctcgatttcgaccatggtggccgccggccctcgcgaccgaatccgacccgggtcccccacccccgcgcgcgcacggccacgcaagcgccgatcgcggaagtgcggcggcggaacgctcgctggcgaacgccaatgccgccaccgccaccgaactgacccctcgccccgccctcccccgccggctgtcgagcgcagtggtcggtcgagacttcgtcgagctcttcgagcggaagtacggcggccgatgacccaagacgaacgcaatgccctcgcgcggggctgcatcggctcacgacggtcaacctcgagcggcaacatcaacccgccgcggcatgtcgttcggcacgttcgcagttcgcgacgtcgcggacgtgcagaacgc atcaacgacatcctcgcgaccaatccctcccggacccaatttgtggcggccgtggcgcagcacccgctcctctcgcggatcgacaacgtcacggactcgctcccgggcggcccgacgtcgcagtggacggcggtcatcttctcgaagcggttctactcgaagcaagacccgtcgtggacggacgagcaagcggatcaagcgttccgccccgatccggcg acgggccaagtcgacatgacggactaccggtaccgggcgaagccgggctacgtcaacttcgactacggctggctcgacgaggctcgggcaactggtggcacggaaccacgcggagccggggatgaaggtgtatcagtcgacgctccggcactactcgcggccgctcctcgacttcgaccggcaagtcttctcggtcacgttcggccgggtccacccg

[0076] Example 4 Confirmation of Protein Deamidation Activity (Method) 5 mg of insulin B chain (insulin B chain, oxidized form, derived from bovine pancreas, Sigma-Aldrich, sequence: Phe-Val-Asn-Gln-His-Leu-Cys(SOH)-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys(SOH)-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Ala (SEQ ID NO: 5)) was dissolved in 2 mL of ultrapure water to prepare a substrate solution. Subsequently, 10 μL of each 0.2 mg / mL purified enzyme solution, 25 μL of 0.1 M sodium phosphate buffer (pH 6.5), and 0.9 μL of protease inhibitor (ProteoGuard EDTA-Free Protease Inhibitor Cocktail (TaKaRa)) were added to 22.5 μL of substrate solution to a total volume of 58.4 μL to form a reaction solution. The reaction solution was heated to 40°C in an incubator, left overnight, and then heated at 100°C for 10 minutes to terminate the reaction. The reaction-stop solution was analyzed using HPLC. Deamidation activity was detected by the peak transition from insulin B chain to deamidated insulin B chain. <Deamidation Evaluation> The amount of deamidated insulin B chain produced was calculated from the peak areas of insulin B chain and deamidated insulin B chain calculated from the chromatogram, and evaluated as the deamidation rate (amount of deamidated insulin B chain produced ÷ amount of substrate insulin B chain × 100).

[0077] (Results) The results are shown in Table 2. (Discussion) The results shown in Table 2 indicate that the protein deamidase derived from K. cinnamomea and the protein deamidase derived from C. cryophila of the present invention catalyze a deamidation reaction, since they produced deamidated insulin B chain.

[0078] Example 5: Confirmation of protein deamidation activity Preparation of protein deamidating enzyme derived from Longispora fulva Longispora fulva cells were cultured in tryptosoy broth medium (manufactured by Nissui Pharmaceutical Co., Ltd.) at 28°C for 6 days, and the supernatant was collected. The obtained supernatant was dialyzed against 20 mM phosphate buffer (pH 6) according to a standard method. The dialyzed sample was subjected to ion exchange chromatography using a cation exchange column (SP Sepharose FF, manufactured by GE Healthcare) equilibrated with the same buffer, and the active fraction was desalted and concentrated to obtain a crude enzyme sample.

[0079] The amino acid sequence and nucleotide sequence of protein deamidase derived from Longispora fulva are shown below. Amino acid sequence (mature sequence only) (SEQ ID NO: 14) APPTSADRSGTPVAGLRVPSSLSAVRGAARFDAVAPEATPLPVGQKTRTWTIARYAAAWRTANGRAMTDRERDALARGCIGVTTVNIERGNINPPLGLSFGTFEKATAVRDALDAVLAGAAGRTDYAARVGADPLLSTLDNVTESLPAGDPDQWAAVVFSKRFHSGQDSTWTAAETDRAFRPDARGQVDMSAYHYRAKPGYVNFDYGWYDPATGSWWHANHAEPGMEIYQSTLKYYSRPLLDFDRQVYSVTFARKAAPAWRSSTN

[0080] Amino acid sequence (full length) *The underlined part is the mature sequence (SEQ ID NO: 15) MRNRLLRITMAAVLGVGATVGLAATTGYAAPPTSADRSGTPVAGLRVPSSLSAVRGAARFDAVAPEATPLPVGQKTRTWTIARYAAAWRTANGRAMTDRERDALARGCIGVTTVNIERGNINPPLGLSFGTFEKATAVRDALDAVLAGAAGRTDYAARVGADPLLSTLDNVTESLPAGDPDQWAAVVFSKRFHSGQDSTWTAAETDRAFRPDARGQVDMSAYHYRAKPGYVNFDYGWYDPATGSWWHANHAEPGMEIYQSTLKYYSRPLLDFDRQVYSVTFARKAAPAWRSSTN

[0081] Base sequence (mature body) (sequence number 16) gccccgccgacgtccgccgaccgctcggggacccccgtggcggggctgcgggtccctctccgccgtgcggcgcgggggccgccgggttgacgccgtggcgccggaagccaccccgctccggtcgggcgcaaaagaccgcacgcacgtggacgattgcccgctacgccgcggcctggcggaccgcgaacggccgcgcatgaccgatcgggagcgcgatgccctcgccgggggctgcatcggcgtcacgacggtcaacatcgagcggggcaacatcaacccgccgctcggctctccttcggcacgtttgaaaaagcaccgaccgggtccgcgacgcgctggacgccgtcctcgccggggccgcaccgactacgccgcgcgcgtcggggcgcggcgcgtcggcgcgctgccgccgactacgccgcgcgcgtcggcgcgcgctcggcgccgcgactacgccgcgcgcgtcggcgcgcgcgctcggcgcgcgctcggcgcgctcggcgccgcgactacgccgcgcgcgctcgg atccgctcctctccacgctcgataatgtcacggaatcgctcccggcgggggaccccggatcagtgggcggcggtcgtcttctcgaagcgctttcattccgggcaagattccacctggaccgccgggggagaccgaccgggcgtttcggcccgatgcgcgcggccaagtcgacatgtcggcgtaccactaccgggcgaagcc gggctacgtcaacttcgactacgggctggtacgacccggcgacgggctcgtggtggcacgcgaaccacgcggagccgggcatggagatctatcagtcgacgctcaagtactactcgcggccgctcctcgacttcgaccggcaagtctactcggtcacgttcgcgcggaaggcggcgccggcgtggcggtcgtcgacgaac

[0082] Base sequence (full length) (sequence number 17) atgcggaaccgcctgctccgggatcacgatggccgcggtcctgggggtgggcgccaccgtggggctgccggccaccgtggggctcgccggaccacgggctatgcggccccgcacgtccgccgaccgctcggggacccccgtggcggggctgcggggcccgtccctctccgccgtgcgcggcgccgcccggttgacgccgtggcgccggaagccaccccgctccggtcgggcgcaaaagacccgcacgtggacgattgcccgctacgccgcggcctggcggaccgcaacggccgcgcatgaccgatcgggagcgcgatgccctcgcccggggctgcatcggcgtcacgacggtcaacatcgagcggggcaacatcaacccgctcggcctctccttcggcacgtttgaaaaagcgaccgcggtccgcgacgcgctggacgccgtccgcgctggacgccgtccgcgcagctggacgccgtccgcgcagcag ggggccgcgggccgcaccgactacgccgcgcgcgtcggcgcggatccgctcctctccacgctcgataatgtcacggaatcgctcccggcgggggacccggatcagtgggcggcggtcgtcttctcgaagcgctttcattccgggcaagattccacctggaccgccggggagaccgaccgggcgtttcggcccgatgcgcgcggccaagtcgacatgtcgg cgtaccactaccgggcgaagccgggctacgtcaacttcgactacgggctggtacgacccggcgacgggctcgtggtggcacggaaccacgcggagccgggcatggagatctatcagtcgacgctcaagtactactcgcggccgctcctcgacttcgaccggcaagtctactcggtcacgttcgcgcggaaggcggcgcgggcgtggcggtcgtcgacgaac

[0083] Example 6: Evaluation of protein deamidation activity against protein substrate (Method) 5 mg of insulin B chain (insulin B chain, oxidized form, derived from bovine pancreas, Sigma-Aldrich, sequence: Phe-Val-Asn-Gln-His-Leu-Cys(SOH)-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys(SOH)-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Ala (SEQ ID NO: 5)) was dissolved in 2 mL of ultrapure water to prepare a substrate solution. To 90 μL of the substrate solution, 40 μL of a 1 mg / mL purified enzyme solution and 0.1 M potassium dihydrogen phosphate / disodium hydrogen phosphate buffer (pH 6.5) were added to prepare a reaction solution in a total volume of 180 μL. The reaction solution was heated to 40°C in an incubator, allowed to stand for 5 hours, and then heated at 100°C for 10 minutes to terminate the reaction. The reaction-terminated solution was analyzed by HPLC. Deamidation activity was detected by the peak transition from insulin B chain to deamidated insulin B chain.

[0084] (Results) As a result of the analysis using HPLC, when protein deamidase derived from Longispora fulva was used, a peak due to the deamidation reaction was confirmed.

[0085] The enzymatic preparation of the present invention contains protein deamidase as an active ingredient. Therefore, it is suitable for use in the fields of food and medical applications and has high industrial utility value. The present invention is not limited in any way to the above-described embodiments and examples of the invention. Various modifications within the scope of the claims and within the scope that can be easily conceived by a person skilled in the art are also included in the present invention. The contents of papers, published patent applications, patent publications, etc. specified in this specification are hereby incorporated by reference in their entirety.

Claims

1. An enzyme preparation for protein deamidation, comprising as an active ingredient a protein deamidating enzyme consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO:

1.

2. The protein deamidating enzyme preparation according to claim 1, wherein the protein deamidating enzyme is derived from the genus Longimycelium.

3. A method for deamidating a protein or peptide, which comprises allowing the protein deamidating enzyme preparation according to claim 1 or 2 to act on the protein or peptide.

4. A method for deamidating a protein-containing food or drink, which comprises allowing the protein deamidating enzyme preparation according to claim 1 or 2 to act on the protein-containing food or drink.

5. A method for producing a food or drink, which comprises allowing the protein deamidating enzyme preparation according to claim 1 or 2 to act on a protein-containing food or drink.

6. A food or drink treated with the protein deamidating enzyme preparation according to claim 1 or 2.

7. A method for producing protein deamidase consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, comprising the step of culturing a bacterium of the genus Longimycelium.

8. An enzyme preparation for protein deamidation, comprising as an active ingredient a protein deamidating enzyme consisting of an amino acid sequence having 60% or more sequence identity with the amino acid sequence of SEQ ID NO:

1.

9. The protein deamidating enzyme preparation according to claim 8, wherein the protein deamidating enzyme is derived from the genus Longimycelium, Crossiella or Krasilnikovia.

10. A method for deamidating a protein or peptide, which comprises allowing the protein deamidating enzyme preparation according to claim 8 or 9 to act on the protein or peptide.

11. A method for deamidating a protein-containing food or drink, which comprises allowing the protein deamidating enzyme preparation according to claim 8 or 9 to act on the protein-containing food or drink.

12. A method for producing a food or drink, which comprises allowing the protein deamidating enzyme preparation according to claim 8 or 9 to act on a protein-containing food or drink.

13. A food or drink treated with the protein deamidating enzyme preparation according to claim 8 or 9.

14. A method for producing protein deamidase consisting of an amino acid sequence having 60% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, comprising the step of culturing bacteria of the genus Longimycelium, Crossiella, or Krasilnikovia.

15. An enzyme preparation for protein deamidation, comprising as an active ingredient a protein deamidating enzyme consisting of an amino acid sequence having 50% or more sequence identity with the amino acid sequence of SEQ ID NO:

1.

16. The protein deamidating enzyme preparation according to claim 15, wherein the protein deamidating enzyme is derived from the genus Longimycelium, Crossiella, Krasilnikovia, or Longispora.

17. A method for deamidating a protein or peptide, which comprises allowing the protein deamidating enzyme preparation according to claim 15 or 16 to act on the protein or peptide.

18. A method for deamidating a protein-containing food or drink, comprising allowing the protein deamidating enzyme preparation according to claim 15 or 16 to act on the protein-containing food or drink.

19. A method for producing a food or drink, which comprises allowing the protein deamidating enzyme preparation according to claim 15 or 16 to act on a protein-containing food or drink.

20. A food or drink treated with the protein deamidating enzyme preparation according to claim 15 or 16.

21. A method for producing protein deamidase consisting of an amino acid sequence having 50% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, comprising the step of culturing a bacterium of the genus Longimycelium, Crossiella, Krasilnikovia or Longispora.

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