Glutamic acid decarboxylase and application thereof

Abstract

The present invention addresses the problem of providing a novel glutamic acid decarboxylase for the production of GABA by an enzymatic method. The present invention relates to a glutamic acid decarboxylase comprising any of the polypeptides (1) to (3): (1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1; (2) a polypeptide comprising an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO: 1 by substitution, addition, insertion, or deletion of one or several amino acid residues, and having glutamic acid decarboxylase activity; and (3) a polypeptide having at least 75% sequence identity to the amino acid sequence represented by SEQ ID NO: 1 and having glutamic acid decarboxylase activity.

Description

Glutamic acid decarboxylase and its applications

[0001] The present invention relates to glutamic acid decarboxylase, DNA encoding glutamic acid decarboxylase, an expression cassette containing the DNA, a recombinant vector containing the DNA, a transformant, a method for producing glutamic acid decarboxylase, a method for producing γ-aminobutyric acid, a method for reducing the taste of foods and beverages, and a γ-aminobutyric acid-producing enzyme agent.

[0002] γ-aminobutyric acid (GABA) is an amino acid that acts as a neurotransmitter and is biosynthesized from glutamic acid by glutamic acid decarboxylase (GAD) in vivo. Glutamic acid decarboxylase is present in a wide range of living organisms, from plants and microorganisms to higher animals.

[0003] γ-aminobutyric acid has been reported to have physiological effects such as lowering blood pressure and stabilizing the mind (anti-stress effect), and is attracting attention as it is expected to prevent arteriosclerosis, heart disease, and stroke caused by high blood pressure. For this reason, many foods containing γ-aminobutyric acid have become commercially available in recent years. For example, γ-aminobutyric acid is found in familiar beverages and foods such as tomato juice, chocolate, pickles, and fermented foods such as miso, and its easy availability has led to growing consumer interest.

[0004] Glutamic acid decarboxylase (GAD) catalyzes the reaction of glutamic acid as a substrate to produce γ-aminobutyric acid (GABA). When used as a food additive, GABA is industrially produced by chemical synthesis or fermentation. Enzymatic production methods using microbial glutamic acid decarboxylase are also known.

[0005] For example, Patent Document 1 discloses a fermentation method for producing GABA using Lactobacillus hilgardii K-3 strain. Patent Document 2 discloses an enzymatic method for producing GABA using bacteria of the genus Saccharomyces. Patent Document 3 discloses an enzymatic method for producing GABA using recombinant glutamic acid decarboxylase derived from the genus Lactiplantibacillus. Furthermore, Non-Patent Document 1 investigates the GABA production ability by fermentation when a recombinant glutamic acid decarboxylase derived from Saccharomyces cerevisiae is expressed in Escherichia coli.

[0006] JP 2003-70462 A JP 2013-150585 A JP 2024-73190 A

[0007] Qiang Xiong et al.,Appl.Biochem.Biotechnol.,(2017), 183:1390-1400

[0008] As mentioned above, various methods for producing GABA are known, but currently, production by fermentation using lactic acid bacteria is the mainstream, and the influence of components derived from the culture medium on the final product is a concern. Therefore, an object of the present invention is to provide a novel glutamic acid decarboxylase for enzymatic production of GABA.

[0009] Examples of specific embodiments of the present invention are given below.

[0010] [1] A glutamic acid decarboxylase comprising a polypeptide shown in any one of (1) to (3) below: (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; (2) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 with one or several amino acid residues substituted, added, inserted or deleted, and having glutamic acid decarboxylase activity; (3) a polypeptide having 75% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1 and having glutamic acid decarboxylase activity. [2] The glutamic acid decarboxylase according to [1], wherein the polypeptide is a polypeptide derived from Sporobolomyces pararoseus. [3] The glutamic acid decarboxylase according to [1] or [2], wherein the TM-score between the three-dimensional structure of a protein having the amino acid sequence shown in SEQ ID NO: 1 and the three-dimensional structure of the polypeptide shown in any one of (1) to (3) is 0.6 or more. [4] A glutamic acid decarboxylase comprising a polypeptide having glutamic acid decarboxylase activity and having a TM-score of 0.6 or more with respect to the three-dimensional structure of a protein having the amino acid sequence shown in SEQ ID NO: 1. [5] A DNA comprising a polynucleotide shown in any of the following (1) to (3): (1) a polynucleotide encoding the polypeptide of claim 1; (2) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3; (3) a polynucleotide comprising a sequence equivalent to the nucleotide sequence shown in SEQ ID NO: 3 and encoding a polypeptide having glutamic acid decarboxylase activity. [6] An expression cassette comprising the DNA according to [5]. [7] A recombinant vector comprising the DNA according to [5]. [8] A transformant comprising the DNA according to [5], the expression cassette according to [6], or the recombinant vector according to [7]. [9] A method for producing glutamic acid decarboxylase, comprising a step of culturing the transformant according to [8].

[10] A method for producing glutamic acid decarboxylase, comprising a step of culturing Sporobolomyces pararoseus.

[11] A method for producing γ-aminobutyric acid, comprising the step of allowing the glutamic acid decarboxylase according to any one of [1] to [4] to act on a raw material containing glutamic acid or a salt thereof.

[12] A method for reducing the taste of a food or drink, comprising the step of allowing the glutamic acid decarboxylase according to any one of [1] to [4] to act on a raw material containing glutamic acid or a salt thereof.

[13] A γ-aminobutyric acid-producing enzyme preparation, comprising the glutamic acid decarboxylase according to any one of [1] to [4].

[0011] According to the present invention, a novel glutamic acid decarboxylase for enzymatic production of GABA can be provided.

[0012] Fig. 1 is a graph showing the amount of GABA produced when the glutamic acid decarboxylase of the present invention was allowed to act under various temperature conditions. Fig. 2 shows the three-dimensional structure of the amino acid sequence of SEQ ID NO: 1 predicted using AlphaFold version 2.3.0.

[0013] The present invention will be described in detail below. The following description of the constituent elements may be based on representative embodiments or specific examples, but the present invention is not limited to such embodiments. In this specification, a numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits.

[0014] The present invention will be described in detail below. Except for the sequence listing, the 20 types of amino acid residues in the amino acid sequence may be expressed by single-letter abbreviations. That is, glycine (Gly) is G, alanine (Ala) is A, valine (Val) is V, leucine (Leu) is L, isoleucine (Ile) is I, phenylalanine (Phe) is F, tyrosine (Tyr) is Y, tryptophan (Trp) is W, serine (Ser) is S, threonine (Thr) is T, cysteine ​​(Cys) is C, methionine (Met) is M, aspartic acid (Asp) is D, glutamic acid (Glu) is E, asparagine (Asn) is N, glutamine (Gln) is Q, lysine (Lys) is K, arginine (Arg) is R, histidine (His) is H, and proline (Pro) is P. In the present specification, the left end of the amino acid sequence is the N-terminus, and the right end is the C-terminus.

[0015] As used herein, "nonpolar amino acids" include alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan. "Uncharged amino acids" include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. "Acidic amino acids" include aspartic acid and glutamic acid. "Basic amino acids" include lysine, arginine, and histidine.

[0016] As used herein, the term "substitution" refers not only to cases where an amino acid residue substitution is artificially introduced, but also to cases where an amino acid residue substitution is naturally introduced, i.e., cases where the amino acid residue is originally different. As used herein, the amino acid residue substitution may be either an artificial substitution or a natural substitution, with artificial substitution being preferred.

[0017] (Glutamic acid decarboxylase) This embodiment relates to a glutamic acid decarboxylase comprising a polypeptide shown in any one of the following (1) to (3): (1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1. (2) A polypeptide having glutamic acid decarboxylase activity, in which one or several amino acid residues have been substituted, added, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 1. (3) A polypeptide having 75% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1 and having glutamic acid decarboxylase activity.

[0018] Glutamic acid decarboxylase is an enzyme that decarboxylates the carboxy group of glutamic acid to produce γ-aminobutyric acid. The glutamic acid decarboxylase of this embodiment is preferably a glutamic acid decarboxylase consisting of any one of the polypeptides (1) to (3) above.

[0019] In this embodiment, γ-aminobutyric acid (GABA) can be produced from a glutamic acid-containing raw material by allowing the polypeptide to act on glutamic acid. Furthermore, in this embodiment, glutamic acid can be converted to γ-aminobutyric acid (GABA) with high efficiency. For example, the conversion rate from glutamic acid to γ-aminobutyric acid (GABA), calculated using the following formula, is preferably 1% or higher, more preferably 2% or higher, even more preferably 5% or higher, and particularly preferably 8% or higher. The upper limit of the conversion rate from glutamic acid to γ-aminobutyric acid (GABA) is not particularly limited and may be 100%.

[0020] The conversion rate of glutamic acid to γ-aminobutyric acid (GABA) can be calculated using the following formula:

[0021] In the above formula, [GABA] is the concentration of γ-aminobutyric acid produced, [GABA] ｍａｘ is the maximum production concentration of γ-aminobutyric acid, [GABA] ｃｏｎｔ． is the concentration of γ-aminobutyric acid in the solution without the addition of glutamic acid decarboxylase, [Glu] ｃｏｎｔ． represents the concentration of glutamic acid in a solution without the addition of glutamic acid decarboxylase, 103.1 represents the molecular weight of γ-aminobutyric acid, and 147.1 represents the molecular weight of glutamic acid. The concentrations of γ-aminobutyric acid and glutamic acid can be quantified by HPLC (high performance liquid chromatography) or the like.

[0022] The type and origin of the polypeptide described in (1) above are not particularly limited. Examples of polypeptides exhibiting glutamic acid decarboxylase activity include glutamic acid decarboxylases derived from lactic acid bacteria, yeast, and koji. These glutamic acid decarboxylases may be used alone or in combination.

[0023] Among these, the polypeptide shown in (1) above is preferably a polypeptide derived from the genus Sporobolomyces, and particularly preferably a polypeptide derived from Sporobolomyces pararoseus. That is, the polypeptide shown in (1) above is preferably glutamic acid decarboxylase derived from Sporobolomyces pararoseus. Note that in this embodiment, γ-aminobutyric acid can also be produced by fermentation using Sporobolomyces pararoseus.

[0024] The polypeptides (2) and (3) above are variants of the polypeptide (1) above. The polypeptides (1) to (3) above include not only polypeptides obtained by artificial substitution, but also polypeptides that originally have such amino acid sequences.

[0025] The polypeptide (2) above is a polypeptide having glutamic acid decarboxylase activity, which comprises the amino acid sequence shown in SEQ ID NO: 1, with one or several amino acid residues substituted, added, inserted, or deleted. In the polypeptide (2), the introduced amino acid modification may include only one type of modification (e.g., substitution only) from the group consisting of substitution, addition, insertion, and deletion, or may include two or more types of modifications (e.g., substitution and insertion). In the polypeptide (2), the number of amino acid differences at any difference site may be one or several, for example, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, preferably 1 to 3, more preferably 1 or 2, and particularly preferably 1.

[0026] The polypeptide of (3) above is a polypeptide having a sequence identity of 75% or more to the amino acid sequence shown in SEQ ID NO: 1 and having glutamic acid decarboxylase activity. In the polypeptide of (3) above, the sequence identity to the amino acid sequence shown in SEQ ID NO: 1 may be 75% or more, but is preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, and particularly preferably 99% or more. Here, in the polypeptide of (3) above, the sequence identity to the amino acid sequence shown in SEQ ID NO: 1 refers to the sequence identity calculated by comparing the amino acid sequence shown in SEQ ID NO: 1. Furthermore, "sequence identity" refers to the amino acid sequence identity value obtained by the bl2seq program (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol. Lett., Vol. 174, pp. 247-250, 1999) of BLASTPACKAGE [sgi32 bit edition, Version 2.0.12; available from the National Center for Biotechnology Information (NCBI)]. The parameters are calculated by setting Gap insertion cost value: 11, Gap extension cost value: 1.

[0027] In the polypeptides of (2) and (3) above, when an amino acid substitution is introduced into SEQ ID NO: 1, a preferred embodiment of the amino acid substitution to be introduced is a conservative substitution. Examples of the amino acid substitution in the polypeptides of (2) and (3) above include substitution with another nonpolar amino acid if the amino acid before substitution is a nonpolar amino acid, substitution with another uncharged amino acid if the amino acid before substitution is an uncharged amino acid, substitution with another acidic amino acid if the amino acid before substitution is an acidic amino acid, and substitution with another basic amino acid if the amino acid before substitution is a basic amino acid.

[0028] In the polypeptides (2) and (3) above, the amino acid residues corresponding to positions 244 (threonine residue) and 275 (aspartic acid residue) in the amino acid sequence shown in SEQ ID NO: 1 are considered to be active center residues. Furthermore, in the polypeptides (2) and (3) above, the amino acid residue corresponding to position 308 (lysine residue) in the amino acid sequence shown in SEQ ID NO: 1 is considered to be a PLP-binding residue. Therefore, it is desirable not to introduce substitutions or deletions into these sites.

[0029] The polypeptides (2) and (3) above are polypeptides having glutamic acid decarboxylase activity. For example, when the conversion rate from glutamic acid to γ-aminobutyric acid (GABA) is equal to or higher when the polypeptides (2) and (3) above are used compared to when the polypeptide (1) above is used, the polypeptides can be determined to have glutamic acid decarboxylase activity. More specifically, compared to when the polypeptides (1) above are used, the conversion rate from glutamic acid to γ-aminobutyric acid (GABA) when the polypeptides (2) and (3) above are used is preferably 0.8 to 1.2, more preferably 0.9 to 1.1, and even more preferably 0.95 to 1.05.

[0030] The polypeptides (1) to (3) above may be part of a larger protein (e.g., a fusion protein). Additional sequences in the fusion protein include sequences that aid in purification, such as multiple histidine residues, and additional sequences that ensure stability during recombinant production.

[0031] The glutamic acid decarboxylase of this embodiment can exhibit glutamic acid decarboxylase activity even in the absence of the coenzyme pyridoxal-5-phosphate (PLP). In a preferred embodiment, the glutamic acid decarboxylase of this embodiment can exhibit glutamic acid decarboxylase activity in the absence of PLP equivalent to that in the presence of PLP. In this case, the glutamic acid decarboxylase of this embodiment is preferably a PLP-binding glutamic acid decarboxylase.

[0032] The glutamic acid decarboxylase of this embodiment may have an activity ratio calculated by the following formula of 60% or more, 70% or more, or 80% or more: Activity ratio: (GABA production amount without PLP addition / GABA production amount with PLP addition) × 100. The GABA production amount without PLP addition is the amount of GABA produced when a predetermined concentration of glutamic acid decarboxylase is added to a substrate solution with a glutamic acid concentration of 5.0 mM and PLP 0.0 mM, and the GABA production amount with PLP addition is the amount of GABA produced when a predetermined concentration of glutamic acid decarboxylase is added to a substrate solution with a glutamic acid concentration of 5.0 mM and PLP 0.05 mM.

[0033] The TM-score between the three-dimensional structure of the protein having the amino acid sequence shown in SEQ ID NO: 1 and the three-dimensional structure of the polypeptide shown in any one of (1) to (3) above is preferably 0.6 or more, more preferably 0.61 or more, 0.62 or more, 0.63 or more, 0.64 or more, 0.65 or more, 0.66 or more, 0.67 or more, 0.68 or more, 0.69 or more, 0.71 or more, 0.72 or more, 0.73 or more, 0.74 or more, 0.75 or more, 0.76 or more, 0.77 or more, 0.78 or more, 0.79 or more, 0.80 or more, 0.81 or more, 0.82 or more, 0.83 or more, 0.84 or more, 0.85 or more, 0.86 or more, 0.87 or more, 0.88 or more, 0.89 or more, 0.90 or more, 0.91 or more, 0.92 or more, 0.93 or more, 0.94 or more, 0.95 or more, 0.96 or more, 0.97 or more, 0.98 or more, 0.99 or more, 0.99 or more, 10 ... 0.74 or more, 0.75 or more, 0.76 or more, 0.77 or more, 0.78 or more, 0.79 or more, 0.8 or more, 0.81 or more, 0.82 or more, 0.83 or more, 0.84 or more, 0.85 or more, 0.86 or more, 0.87 or more, 0.88 or more, 0.89 or more, 0.9 or more, 0.91 or more, 0.92 or more, 0.93 or more, 0.94 or more, 0.95 or more, 0.96 or more, 0.97 or more, 0.98 or more, 0.99 or more.

[0034] The second embodiment of the present invention may relate to a glutamic acid decarboxylase comprising a polypeptide having glutamic acid decarboxylase activity and having a TM-score of 0.6 or more with respect to the three-dimensional structure of a protein having the amino acid sequence shown in SEQ ID NO: 1. That is, the present invention relates to a glutamic acid decarboxylase comprising a polypeptide shown in any of the following (1) to (4): (1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1. (2) A polypeptide having glutamic acid decarboxylase activity, in which one or more amino acid residues have been substituted, added, inserted, or deleted in the amino acid sequence shown in SEQ ID NO: 1. (3) A polypeptide having 75% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1 and having glutamic acid decarboxylase activity. (4) A polypeptide having TM-score of 0.6 or more with respect to the three-dimensional structure of a protein having the amino acid sequence shown in SEQ ID NO: 1 and having glutamic acid decarboxylase activity.

[0035] In the above (4), the TM-score is preferably 0.61 or more, more preferably 0.62 or more, 0.63 or more, 0.64 or more, 0.65 or more, 0.66 or more, 0.67 or more, 0.68 or more, 0.69 or more, 0.71 or more, 0.72 or more, 0.73 or more, 0.74 or more, 0.75 or more, 0.76 or more, 0.77 or more, 0.78 or more, 0.79 or more, 0.8 or more, 0.81 or more, 0.82 or more, 0.83 or more, 0.84 or more, 0.85 or more, 0.86 or more, 0.87 or more, 0.88 or more, 0.89 or more, 0.9 or more, 0.91 or more, 0.92 or more, 0.93 or more, 0.94 or more, 0.95 or more, 0.96 or more, 0.97 or more, 0.98 or more, 0.99 or more.

[0036] The three-dimensional structure of the protein having the amino acid sequence set forth in SEQ ID NO: 1 and the three-dimensional structure of the polypeptide of the present invention are determined using AlphaFold, a computational method for predicting the three-dimensional structure of a polypeptide from its amino acid sequence (Jumper et al., Highly accurate protein structure prediction with AlphaFold. Nature, 2021). The predicted structures of millions of polypeptides registered in the UniProt database are registered in the AlphaFold Protein Structure Database using the AlphaFold Monomer v2.0 model (Varadi et al., AlphaFold Protein Structure Database: Massively Expanding the Structural Coverage of Protein-Sequence Space with High-Accuracy Models. Nucleic Acids Research, 2021). In the AlphaFold Protein Structure Database, the three-dimensional structure of a polypeptide can be obtained by searching the UniProt accession number of the polypeptide.

[0037] In addition to the many three-dimensional structures already published, code for reproducing and predicting the structure of new polypeptides is available in source code repositories such as deepmind / alphafold / under Github.com using AlphaFold v2.3.0 or later. Furthermore, code for reproducing and predicting the structure of new polypeptides is also available in sokrypton / ColabFold under Github.com. For technical details, see Jumper et al., supra. It is preferable to use the latest version of AlphaFold.

[0038] AlphaFold generates a per-residue confidence estimate on a scale of 0 to 100. This confidence measure, called pLDDT, corresponds to the model's predicted score on the IDDT-Cα index. It is stored in the B-factor field of the downloadable mmCIF and PDB files (however, unlike the B-factor, a higher pLDDT is better). Regions with a pLDDT score above 90 are expected to be modeled with high accuracy. They are considered suitable for any application that benefits from high accuracy (e.g., binding site characterization). Regions with pLDDT scores between 70 and 90 generally correspond to good backbone predictions and are expected to have well-modeled three-dimensional structures.

[0039] Traditionally, comparisons of relatedness between amino acid sequences have been determined solely by the parameter of "sequence identity." However, the functional relationships between polypeptides can be better assessed if the three-dimensional structures of the polypeptides can be compared.

[0040] The structural similarity between three-dimensional structures determined using AlphaFold can be determined by the TM-score calculated using the following formula (Zhang & Skolnick, Proteins 57:702-710, 2004). The closer the TM-score value is to 1.0, the higher the structural similarity. A TM-score value of 0.6 or higher indicates approximately an 80% probability that the proteins belong to the same topology family and share a common functional mechanism. A TM-score value of 0.7 or higher indicates approximately a 90% probability that the proteins belong to the same fold family and are highly likely to have a high degree of functional similarity. A TM-score value of 0.8 or higher indicates a very high probability that the proteins have similar enzymatic activity if their structures are nearly identical and the active site is conserved. A TM-score value of 0.9 or higher indicates a very high probability that the proteins are nearly identical and have the same function. A TM-score value of 1.0 (theoretical maximum value) indicates a completely identical structure.

[0041]

[0042] Here, L Ｎindicates the length (number of residues) of the reference protein, and L Ｔ indicates the length (number of residues) of the protein to be compared, and d ｉ represents the distance between the i-th pair of aligned residues (the interatomic distance between the three-dimensional coordinates of the Cα atoms of the corresponding amino acid residues), and d ０ is a scale parameter for normalizing the degree of match, and Max denotes the maximum value (maximum score) after optimal spatial registration.

[0043] In addition, L Ｎ is always the length of the reference protein. To prevent artificially large TM-scores from arising from the sequence of certain substructures such as α-helices, motifs, and domain structures, L Ｎ Alternatively, a fixed reference length L may be used.

[0044]

[0045] Before calculating the TM-score, structural alignment of the three-dimensional structures of the two polypeptides may be performed. For example, when the amino acid sequences of the two polypeptides to be compared are different, structural alignment of the three-dimensional structures is preferable. Structural alignment is achieved by an algorithm that optimizes structural overlap, and several methods are available, such as CEAlign (Shindyalov and Bourne, Protein Eng., 11, 739-747, 1998), DALI (Holm and Sander, Trends Biochem. Sci., 20, 478-480, 1995), or TMAlign (Nucleic Acids Res., 33:2302-2309, 2005).

[0046] In this embodiment, TMAlign is preferably applied. TM-score is integrated into the TMAlign software, which can be accessed at the following website (https: / / zhanggroup.org / TM-align / ). The version of TMAlign is preferably updated after August 22, 2019, and the TM-score between a reference protein and a comparison protein can be determined by, for example, uploading two PDB files to the website and running a command. The website calculates the TM-score and reports it in the output along with several other parameters from the alignment.

[0047] (γ-aminobutyric acid-producing enzyme agent) This embodiment may relate to a γ-aminobutyric acid-producing enzyme agent containing the above-mentioned glutamic acid decarboxylase. The γ-aminobutyric acid-producing enzyme agent contains the above-mentioned polypeptide as an active ingredient. The enzyme agent may consist of the above-mentioned polypeptide.

[0048] The content of the polypeptide in the enzyme preparation of this embodiment is not particularly limited, and can be appropriately set within a range in which glutamic acid decarboxylase activity is exerted.

[0049] The γ-aminobutyric acid-producing enzyme preparation of this embodiment may contain optional components other than the above-described polypeptide to the extent that the effects of the present invention are not affected. Examples of optional components include coenzymes. Examples of coenzymes that may be contained include pyridoxal-5-phosphate, pyridoxamine-5-phosphate, pyridoxine-5-phosphate, vitamin B6 (pyridoxine, pyridoxal, pyridoxamine), etc. However, in this embodiment, an embodiment that does not contain pyridoxal-5-phosphate, etc. is also preferred, and the γ-aminobutyric acid-producing enzyme preparation of this embodiment can produce γ-aminobutyric acid with high efficiency even without containing pyridoxal-5-phosphate, etc.

[0050] When pyridoxal-5-phosphate or the like is contained in the γ-aminobutyric acid-producing enzyme agent of this embodiment, the content of pyridoxal-5-phosphate or the like is preferably 0.01 μM or more, more preferably 0.05 μM or more, and even more preferably 0.1 μM or more. Furthermore, the content of pyridoxal-5-phosphate is preferably 5000 μM or less, more preferably 1000 μM or less, and even more preferably 500 μM or less. The content of pyridoxal-5-phosphate or the like is the total content of pyridoxal-5-phosphate, pyridoxine, pyridoxal, pyridoxamine, pyridoxine-5-phosphate, and pyridoxamine-5-phosphate.

[0051] The γ-aminobutyric acid-producing enzyme preparation of this embodiment may contain other enzymes as optional components. Examples of other enzymes include amylases (α-amylase, β-amylase, glucoamylase), glucosidases (α-glucosidase, β-glucosidase), galactosidases (α-galactosidase, β-galactosidase), proteases (acid proteases, neutral proteases, alkaline proteases), peptidases (leucine peptidase, aminopeptidase), lipase, esterase, cellulase, phosphatase (acid phosphatase, alkaline phosphatase), nuclease, deaminase, oxidase, dehydrogenase, glutaminase, pectinase, catalase, dextranase, transglutaminase, protein deamidating enzyme, pullulanase, etc. These other enzymes may be contained alone or in combination.

[0052] The γ-aminobutyric acid-producing enzyme preparation of this embodiment may contain additives as optional components. Examples of additives include excipients, buffers, suspending agents, stabilizers, preservatives, antiseptics, pH adjusters, and physiological saline. Examples of excipients include starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, sucrose, and glycerol. Examples of buffers include phosphates, citrates, and acetates. Examples of stabilizers include propylene glycol and ascorbic acid. Examples of preservatives include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, and methylparaben. Examples of preservatives include ethanol, benzalkonium chloride, parahydroxybenzoic acid, and chlorobutanol. Examples of pH adjusters include acetate buffer, phosphate buffer, MES buffer, HEPES buffer, PIPES buffer, Bis-tris buffer, and MOPS buffer. These additives may be contained alone or in combination of two or more.

[0053] The γ-aminobutyric acid-producing enzyme preparation of this embodiment may also contain culture residues generated in the production process of glutamic acid decarboxylase. Examples of culture residues include components derived from the culture medium, contaminating proteins, and bacterial components.

[0054] The γ-aminobutyric acid-producing enzyme agent of this embodiment may be in any form, such as powder, solid (powder, granules, etc.), gel, or liquid.

[0055] (DNA) This embodiment relates to a DNA comprising a polynucleotide shown in any one of (1) to (3) below. (1) A polynucleotide encoding the above-described polypeptide. (2) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3. (3) A polynucleotide comprising a sequence equivalent to the nucleotide sequence shown in SEQ ID NO: 3 and encoding a polypeptide having glutamic acid decarboxylase activity.

[0056] The DNA of this embodiment is preferably a DNA consisting of a polynucleotide described in any one of (1) to (3) above.

[0057] The DNA of this embodiment may contain a polypeptide consisting of the nucleotide sequence shown in SEQ ID NO: 2, or may consist of a polypeptide consisting of the nucleotide sequence shown in SEQ ID NO: 2. The nucleotide sequence shown in SEQ ID NO: 2 is the genomic sequence of a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, and the nucleotide sequence shown in SEQ ID NO: 3 is the cDNA sequence of a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1.

[0058] The polynucleotide (3) above is a polynucleotide that contains a sequence equivalent to the nucleotide sequence shown in SEQ ID NO: 3 and encodes a polypeptide having glutamic acid decarboxylase activity. The homology of the polynucleotide (3) above to the nucleotide sequence shown in SEQ ID NO: 3 is preferably 75% or more, more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, and particularly preferably 99% or more. Even if a polynucleotide has a sequence different from the nucleotide sequence shown in SEQ ID NO: 3, it can be said to be equivalent to the nucleotide sequence shown in SEQ ID NO: 3 as long as it encodes a polypeptide having glutamic acid decarboxylase activity, and is included in the DNA of the present invention.

[0059] DNA "homology" is calculated using publicly available or commercially available software with an algorithm for comparing a reference sequence with a query sequence. Specifically, BLAST, FASTA, or GENETYX (Software Development Co., Ltd.) can be used, and these may be used with default parameters.

[0060] Furthermore, a base sequence encoding a polypeptide in which one or more amino acid residues have been substituted, added, inserted or deleted at any difference site in the amino acid sequence is also included in the DNA of the present invention, as long as it encodes a polypeptide having glutamic acid decarboxylase activity.

[0061] Furthermore, DNA that hybridizes under stringent conditions with DNA consisting of a nucleotide sequence complementary to the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 3 is also included in the DNA of the present invention, as long as it encodes a polypeptide having glutamic acid decarboxylase activity. Here, "under stringent conditions" refers to incubation for 4 hours to overnight at 50°C to 65°C in 6xSSC (1xSSC is 0.15M NaCl, 0.015M sodium citrate, pH 7.0) containing 0.5% SDS, 5x Denhartz's [Denhartz's, 0.1% bovine serum albumin (BSA), 0.1% polyvinylpyrrolidone, 0.1% Ficoll 400], and 100 μg / ml salmon sperm DNA. Hybridization under stringent conditions is specifically carried out by the following method. Specifically, a nylon membrane immobilized with a DNA or cDNA library is prepared, and the nylon membrane is blocked at 65°C in a prehybridization solution containing 6x SSC, 0.5% SDS, 5x Denhardt's buffer, and 100 μg / ml salmon sperm DNA. Each 32P-labeled probe is then added, and the membrane is incubated overnight at 65°C. The nylon membrane is then washed in 6x SSC at room temperature for 10 minutes, in 2x SSC containing 0.1% SDS at room temperature for 10 minutes, and in 0.2x SSC containing 0.1% SDS at 45°C for 30 minutes, after which autoradiography is performed to detect DNA that specifically hybridizes with the probe.

[0062] The DNA of this embodiment can be isolated from a microorganism that produces the predetermined polypeptide. For example, the target DNA can be isolated from the genome of Sporobolomyces pararoseus by PCR or hybridization using the genomic DNA of Sporobolomyces pararoseus as a template and primers or probes designed from known amino acid sequence information taking gene degeneracy into consideration, or primers or probes designed based on known nucleotide sequence information.

[0063] The DNA of this embodiment encompasses various types of DNA resulting from codon degeneracy. Various types of DNA encoding the same amino acid sequence can be artificially produced easily using known genetic engineering techniques. For example, in the production of a protein by genetic engineering, if the codons used in the original gene encoding the target protein are infrequently used in the host, the protein expression level may be low. In such cases, high expression of the target protein can be achieved by optimizing the codon usage frequency for the host without changing the encoded amino acid sequence. The sum of the host optimal codon usage frequencies for each codon can be used as an index of codon usage. An optimal codon is defined as the codon with the highest usage frequency among codons corresponding to the same amino acid. The codon usage frequency is not particularly limited as long as it is optimized for the host. For example, the following is an example of an optimal codon for Escherichia coli: F: phenylalanine (ttt), L: leucine (ctg), I: isoleucine (att), M: methionine (atg), V: valine (gtg), Y: tyrosine (tat), stop codon (taa), H: histidine (cat), Q: glutamine (cag), N: asparagine (aat), K: lysine (aaa), D: aspartic acid (gat), E: glutamic acid (gaa), S: serine (agc), P: proline (ccg), T: threonine (acc), A: alanine (gcg), C: cysteine ​​(tgc), W: tryptophan (tgg), R: arginine (cgc), G: glycine (ggc).

[0064] Methods for introducing a mutation into a gene and artificially modifying the amino acid sequence include known techniques such as the Kunkel method and the Gapped duplex method, and mutagenesis kits using site-directed mutagenesis, such as QuikChange™ Site-Directed Mutagenesis Kit (Stratagene), GeneTailor™ Site-Directed Mutagenesis System (Invitrogen), and TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, etc.: Takara Bio Inc.).

[0065] The DNA base sequence can be confirmed by conventional sequencing, such as the dideoxynucleotide chain termination method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74:5463). Alternatively, the sequence can be analyzed using an appropriate DNA sequencer.

[0066] Whether the obtained DNA encodes the polypeptide of interest can be confirmed by comparing the determined nucleotide sequence with the nucleotide sequence set forth in SEQ ID NO: 2 or 3. Alternatively, the amino acid sequence deduced from the determined nucleotide sequence can be compared with the amino acid sequence set forth in SEQ ID NO: 1.

[0067] (Expression Cassette / Recombinant Vector) This embodiment may relate to an expression cassette containing the above-described DNA, or a recombinant vector containing the above-described DNA. The expression cassette or recombinant vector of this embodiment can be obtained by linking a promoter and a terminator to the above-described DNA. Furthermore, a recombinant vector can be obtained by inserting the expression cassette of this embodiment or the DNA of this embodiment into an expression vector.

[0068] The expression cassette of this embodiment or the recombinant vector of this embodiment may contain, as control elements, transcription elements such as an enhancer, a CCAAT box, a TATA box, or an SPI site, as necessary, in addition to a promoter and a terminator. These control elements may be operably linked to the DNA of this embodiment. "Operably linked" means that the DNA of this embodiment is linked to various control elements that regulate the DNA of this embodiment in a state that allows it to operate in a host cell.

[0069] The expression vector used to construct the recombinant vector of this embodiment is preferably one constructed for genetic recombination from a phage, plasmid, or virus capable of autonomously replicating in a host. Such expression vectors are known, and commercially available expression vectors include pQE-based vectors (Qiagen, Inc.), pDR540, pRIT2T (GE Healthcare Biosciences, Inc.), and pET-based vectors (Merck & Co., Inc.). The expression vector may be used in an appropriate combination with the host cell. For example, when Escherichia coli is used as the host cell, examples include a combination of a pET-based vector and a DH5α E. coli strain, a combination of a pET-based vector and a BL21(DE3) E. coli strain, or a combination of a pDR540 vector and a JM109 E. coli strain.

[0070] (Transformant) This embodiment may relate to a transformant having the above-described DNA, the above-described expression cassette, or the above-described recombinant vector. That is, the transformant of this embodiment is a transformant obtained by transforming a host with an expression cassette containing the above-described DNA or a recombinant vector containing the above-described DNA.

[0071] The host used for producing the transformant is not particularly limited as long as it can express the traits of the gene containing the DNA of this embodiment, but it is preferably one into which the gene can be introduced, which can stably retain the expression cassette or recombinant vector, and which is capable of autonomous replication. Suitable examples of the host include bacteria belonging to the genus Escherichia, such as Escherichia coli, the genus Bacillus, such as Bacillus subtilis, and the genus Pseudomonas, such as Pseudomonas putida; yeast; and the like. However, other host cells, such as animal cells, insect cells, and plants may also be used.

[0072] The transformant of this embodiment can be obtained by introducing an expression cassette containing the above-described DNA or a recombinant vector containing the above-described DNA into a host. The location of the DNA introduction is not particularly limited as long as the target gene is expressed, and may be on a plasmid or on the genome. Specific methods for introducing the expression cassette or recombinant vector into a host include, for example, recombinant vector methods and genome editing methods. Conditions for introducing the expression cassette or recombinant vector into a host may be appropriately determined depending on the type of host, etc. When the host is a bacterium, examples of the methods include a method using competent cells treated with calcium ions and electroporation. When the host is a yeast, examples of the methods include electroporation, spheroplast method, and lithium acetate method. When the host is an animal cell, examples of the methods include electroporation, calcium phosphate method, and lipofection method. When the host is an insect cell, examples of the methods include calcium phosphate method, lipofection method, and electroporation method. When the host is a plant cell, examples of the method include electroporation, Agrobacterium method, particle gun method, PEG method, etc.

[0073] Whether or not the expression cassette or recombinant vector has been incorporated into the host can be confirmed by PCR, Southern hybridization, Northern hybridization, or the like.

[0074] When PCR is used to confirm whether an expression cassette or recombinant vector has been incorporated into a host, for example, genomic DNA, expression cassette, or recombinant vector can be isolated and purified from a transformant. For example, when the host is a bacterium, isolation and purification of genomic DNA, expression cassette, or recombinant vector can be achieved by lysing the bacterium to obtain a lysate. Lysis can be achieved by treatment with a lytic enzyme such as lysozyme, optionally in combination with protease, other enzymes, and surfactants such as sodium lauryl sulfate (SDS). Lysis can also be achieved by combining physical disruption methods such as freeze-thawing and French press treatment. DNA can be isolated and purified from the lysate by, for example, deproteinization using phenol or protease treatment, ribonuclease treatment, alcohol precipitation, and an appropriate combination of commercially available kits.

[0075] Using the separated and purified DNA as a template, primers specific to the DNA of this embodiment are designed and PCR is performed. The amplified product obtained by PCR is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., and then stained with ethidium bromide and SYBR Green solution, etc., and the amplified product is detected as a band, thereby confirming transformation. DNA cleavage can be performed according to standard methods, for example, using restriction enzyme treatment. As the restriction enzyme, for example, a type II restriction enzyme that acts on a specific nucleotide sequence is used. DNA is linked to an expression cassette or expression vector using, for example, DNA ligase.

[0076] Furthermore, when PCR is used to confirm whether an expression cassette or a recombinant vector has been incorporated into a host, PCR can be performed using primers pre-labeled with a fluorescent dye or the like to detect the amplified product. Furthermore, a method may be employed in which the amplified product is bound to a solid phase such as a microplate and the amplified product is confirmed by fluorescence, enzymatic reaction, or the like.

[0077] (Method for Producing Glutamic Acid Decarboxylase) This embodiment relates to a method for producing glutamic acid decarboxylase, which includes a step of culturing the above-mentioned transformant. This embodiment also relates to a method for producing glutamic acid decarboxylase, which includes a step of culturing a microorganism (preferably Sporobolomyces pararoseus).

[0078] The culture conditions may be appropriately determined taking into consideration the nutritional and physiological properties of the transformant or microorganism, and are preferably liquid culture. For industrial production, aeration and agitation culture is preferred.

[0079] The nutrient sources used in the medium may be those required for the growth of the transformant or the microorganism. The carbon source may be any assimilable carbon compound, such as glucose, sucrose, lactose, maltose, molasses, or pyruvic acid. The nitrogen source may be any assimilable nitrogen compound, such as peptone, meat extract, yeast extract, casein hydrolysate, or alkaline extract of soybean meal. In addition to the carbon and nitrogen sources, salts such as phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese, and zinc, as well as specific amino acids and specific vitamins, may also be used as needed.

[0080] The culture temperature can be appropriately set within a range in which the transformant or the microorganism can grow and the transformant or the microorganism can produce the specified laccase, and is preferably about 15 to 37° C. The culture may be completed at an appropriate time when the specified glutamic acid decarboxylase reaches its maximum yield, and the culture time is usually about 12 to 48 hours.

[0081] When a glutamic acid decarboxylase-secreting microorganism is used, the enzyme can be separated and / or purified after bacterial cells are recovered from the transformant or the culture medium of the microorganism by filtration, centrifugation, etc., as necessary. In this case, by selecting an appropriate expression cassette or expression vector and host, the expressed glutamic acid decarboxylase can be secreted into the culture medium.

[0082] When a glutamic acid decarboxylase-non-secreting microorganism is used, the bacterial cells may be recovered from the culture medium in advance, if necessary, and then disrupted by pressure treatment, ultrasonic treatment, or the like to expose the enzyme, after which the enzyme can be separated and / or purified. For example, after culturing the transformant or the microorganism, the culture medium is centrifuged or the bacterial cells are recovered, and the bacterial cells are treated mechanically, by ultrasonication or a French press, or with a lytic enzyme such as lysozyme, and then solubilized, if necessary, with an enzyme such as protease or a surfactant such as sodium lauryl sulfate (SDS), to obtain a water-soluble fraction containing the predetermined glutamic acid decarboxylase.

[0083] The water-soluble fraction containing the desired glutamic acid decarboxylase obtained as described above may be subjected to purification treatment as is, or the desired glutamic acid decarboxylase in the water-soluble fraction may be concentrated and then subjected to purification treatment. Concentration can be performed, for example, by vacuum concentration, membrane concentration, salting out, fractional precipitation using a hydrophilic organic solvent (e.g., methanol, ethanol, and acetone), etc. Purification can also be performed by an appropriate combination of methods such as gel filtration, adsorption chromatography, ion exchange chromatography, and affinity chromatography. The separated and / or purified enzyme can also be prepared into a composition by adding appropriate additives and sterilizing by filtration. The separated and / or purified desired glutamic acid decarboxylase can also be powdered, if necessary, by freeze-drying, vacuum drying, spray-drying, etc.

[0084] (Method for Producing γ-Aminobutyric Acid) This embodiment may relate to a method for producing γ-aminobutyric acid, which includes a step of allowing the glutamic acid decarboxylase to act on a raw material containing glutamic acid or a salt thereof. Examples of glutamic acid or a salt thereof include sodium glutamate, potassium glutamate, calcium glutamate, and magnesium glutamate.

[0085] Examples of raw materials containing glutamic acid or its salts include glutamic acid-containing materials. Examples of glutamic acid-containing materials include seasonings and protein-containing foods containing free glutamic acid or its salts. Examples of such foods include natural seasonings such as kelp extract and its powder, and chicken extract and its powder. Furthermore, raw materials containing glutamic acid or its salts may also be those to which glutamic acid or its salts have been separately added.

[0086] The raw material containing glutamic acid or a salt thereof is preferably a protein-containing raw material. That is, this embodiment relates to a method for producing γ-aminobutyric acid, which includes a step of treating a protein-containing raw material with the above-described composition for generating γ-aminobutyric acid. Examples of protein-containing raw materials include plant protein-containing foods such as beans, wheat, nuts, fungi, and vegetables (spinach, carrots, onions, cabbage, and radishes), and animal protein-containing foods such as beef, chicken, pork, fish, and insects. Furthermore, amino acid or peptide mixtures obtained by acid hydrolysis or enzymatic degradation of plant protein-containing foods or animal protein-containing foods can also be used. Furthermore, fermented products of plant protein-containing foods or animal protein-containing foods can also be used.

[0087] Among these, specific examples of raw materials containing glutamic acid or a salt thereof preferably include tomato paste, tomato juice, pickles, miso, yogurt, spinach, carrots, onions, cabbage, radishes, fungi, etc. The above-mentioned composition for generating γ-aminobutyric acid may be used for adding to the above-mentioned foods, or may be a glutamic acid-containing food additive.

[0088] The content of glutamic acid or its salts contained in the raw material containing glutamic acid or its salts is preferably 0.05 mM or more, more preferably 0.1 mM or more, even more preferably 0.5 mM or more, and particularly preferably 1.0 mM or more. Furthermore, the content of glutamic acid or its salts contained in the raw material containing glutamic acid or its salts is preferably 1 M or less, more preferably 0.8 M or less, even more preferably 0.5 M or less, even more preferably 0.2 M or less, and even more preferably 0.1 M or less. By ensuring that the content of glutamic acid or its salts contained in the raw material containing glutamic acid or its salts falls within the above range, it becomes easier to convert glutamic acid to γ-aminobutyric acid (GABA) with high efficiency.

[0089] The form of the raw material containing glutamic acid or a salt thereof is not particularly limited, and may be liquid, powder, solid, slurry, etc. Among these, the raw material containing glutamic acid or a salt thereof is preferably in liquid or slurry form.

[0090] The amount of glutamic acid decarboxylase used in the production of γ-aminobutyric acid is not particularly limited, but is, for example, 0.00001 U or more, preferably 0.0001 U or more, more preferably 0.0005 U or more, even more preferably 0.001 U or more, and even more preferably 0.002 U or more, per gram of raw material containing glutamic acid or a salt thereof. Regarding the activity of glutamic acid decarboxylase, one unit (1 U) of the enzyme is defined as the amount of enzyme that liberates 1 μmol of GABA per minute when treated at 30° C. using a 5 mmol / L final concentration of sodium glutamate solution as the substrate, a 0.05 mM final concentration of pyridoxal phosphate (PLP) solution as the coenzyme, and a 30 mM final concentration of acetate buffer (pH 4.5).

[0091] In the step of allowing the above-described composition for generating γ-aminobutyric acid to act on a raw material containing glutamic acid or a salt thereof, pyridoxal-5-phosphate may be added. An embodiment in which pyridoxal-5-phosphate is not added is also preferred. In this embodiment, even in the step of allowing the above-described composition for generating γ-aminobutyric acid to act on a raw material containing glutamic acid or a salt thereof, γ-aminobutyric acid can be produced with high efficiency even in the absence of pyridoxal-5-phosphate.

[0092] In the step of treating a raw material containing glutamic acid or a salt thereof with the above-described composition for generating γ-aminobutyric acid, when pyridoxal-5-phosphate is present, the content (amount added) of pyridoxal-5-phosphate is preferably more than 0 parts by mass, more preferably 0.001 parts by mass or more, even more preferably 0.01 parts by mass or more, even more preferably 0.1 parts by mass or more, and even more preferably 0.5 parts by mass or more, relative to 100 parts by mass of the raw material containing glutamic acid or a salt thereof. Furthermore, when pyridoxal-5-phosphate is present, the content (amount added) of pyridoxal-5-phosphate is preferably 150 parts by mass or less, more preferably 100 parts by mass or less, even more preferably 50 parts by mass or less, even more preferably 20 parts by mass or less, even more preferably 10 parts by mass or less, and even more preferably 5 parts by mass or less, relative to 100 parts by mass of the raw material containing glutamic acid or a salt thereof.

[0093] In the step of allowing the above-described composition for generating γ-aminobutyric acid to act on a raw material containing glutamic acid or a salt thereof, if pyridoxal-5-phosphate is present, the method may include a step of mixing the raw material (substrate) containing glutamic acid or a salt thereof with pyridoxal-5-phosphate (coenzyme) to prepare a substrate solution, and a step of mixing the above-described composition for generating γ-aminobutyric acid with the substrate solution. Furthermore, in the step of allowing the above-described composition for generating γ-aminobutyric acid to act on a raw material (substrate) containing glutamic acid or a salt thereof, the above-described composition for generating γ-aminobutyric acid and pyridoxal-5-phosphate (coenzyme) may be simultaneously added to and mixed with the raw material (substrate) containing glutamic acid or a salt thereof.

[0094] The reaction time, temperature, and pH of the reaction solution for reacting a raw material containing glutamic acid or a salt thereof with a composition for producing γ-aminobutyric acid are not particularly limited. The reaction temperature is preferably, for example, 10 to 80°C, more preferably 15 to 60°C, and even more preferably 20 to 40°C. The pH of the reaction solution is, for example, 2 to 9, preferably 3 to 8, and more preferably 4 to 7. The reaction time is, for example, 30 seconds to 48 hours, preferably 1 minute to 24 hours, and more preferably 30 minutes to 12 hours. By adopting the above reaction conditions, it becomes easy to convert glutamic acid to γ-aminobutyric acid (GABA) with high efficiency. The optimal reaction conditions can be determined through preliminary experiments, etc.

[0095] The method for producing γ-aminobutyric acid according to this embodiment preferably includes the following steps (1) and (2), and may further include steps (3) and / or (4): (1) a step of preparing a raw material containing glutamic acid or a salt thereof, (2) a step of treating the raw material with the glutamic acid decarboxylase described above, (3) a step of inactivating the enzyme, and (4) a step of recovering γ-aminobutyric acid.

[0096] Step (3) may include a step of inactivating glutamic acid decarboxylase, for example, by heating the raw material treated with glutamic acid decarboxylase to 80°C or higher.

[0097] In step (4), the reaction product can be recovered as is and used as a food or beverage. Alternatively, the reaction product may be subjected to centrifugation, diatomaceous earth filtration, membrane filtration, or ultrafiltration to desalt and remove impurities, and then concentrated and recovered. The recovered γ-aminobutyric acid may be further processed into a liquid, powder, tablet, or the like.

[0098] (Method for reducing the taste of food and drink) This embodiment may relate to a method for reducing the taste of food and drink, which includes a step of allowing the glutamic acid decarboxylase described above to act on a raw material containing glutamic acid or a salt thereof. For example, by allowing the glutamic acid decarboxylase described above to act on a raw material containing glutamic acid or a salt thereof, the umami of the food and drink can be reduced, resulting in a refreshing taste. In other words, the method for reducing the taste of this embodiment may be a method for reducing umami.

[0099] Furthermore, the present embodiment may also relate to a γ-aminobutyric acid-containing substance or a γ-aminobutyric acid-containing food obtained through the above-described production method. Such a γ-aminobutyric acid-containing substance or a γ-aminobutyric acid-containing food may contain a high concentration of γ-aminobutyric acid.

[0100] The features of the present invention will be explained in more detail below with reference to examples and comparative examples. The materials, amounts used, ratios, treatment contents, treatment procedures, etc. shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed as being limited by the specific examples shown below.

[0101] <Obtaining and Identifying the Enzyme> From the applicant's strain library, Sporobolomyces pararoseus NBRC1036 strain was selected based on its glutamic acid decarboxylase activity and its independence from the addition of PLP.

[0102] <Preparation of glutamic acid decarboxylase> The components in Table 1 were dissolved in water to the indicated concentrations, and the mixture was autoclaved at 121°C for 20 minutes to prepare a medium. The selected strain was inoculated into the medium and cultured with shaking at 30°C for 24 hours. The culture solution was then inoculated into a new medium and cultured with shaking at 30°C for 4 days. After culture, the medium was centrifuged and the supernatant was collected. The supernatant was filtered through a 0.45 μm membrane filter to remove the bacterial cells. The enzyme was then purified and concentrated by conventional methods such as ultrafiltration, column adsorption, and chromatography to obtain a purified enzyme solution.

[0103]

[0104] <Sequence Confirmation> SDS-PAGE was performed on the purified enzyme solution, and the 62 kDa band corresponding to glutamic acid decarboxylase was excised and subjected to PMF analysis. The sequence coverage with Hypothetical Protein JCM5350 000455 was confirmed for the analysis. As a result, the amino acid sequence of glutamic acid decarboxylase derived from Sporobolomyces pararoseus was predicted. Next, the predicted genomic region was amplified using PrimeSTAR GXL DNA Polymerase (manufactured by Takara Bio Inc.). The amplified fragment was recovered using NucleoSpin Gel and PCR Clean-up (manufactured by Takara Bio Inc.), and sequence analysis was performed using degenerate primers. RNA was also extracted from the cultured cells using standard methods. cDNA was synthesized from the RNA and the amino acid sequence was confirmed by sequence analysis using the following primers.

[0105]

[0106] <Confirmation of 3D structure> Using AlphaFold version 2.3.0, the 3D structure of the amino acid sequence of SEQ ID NO: 1 was predicted and a PDB file was created. The 3D structure of the PDB file was illustrated using PyMOL (Figure 2).

[0107] <Activity Measurement Method> A 50 mM solution of sodium glutamate monohydrate was prepared as a substrate. A 0.5 mM solution of pyridoxal phosphate (PLP) was also prepared as a coenzyme. A substrate solution was prepared by mixing 100 μL of the 50 mM solution of sodium glutamate monohydrate, 100 μL of a 0.5 mM solution of pyridoxal phosphate (PLP), and 300 μL of 100 mM acetate buffer (pH 4.5). 500 μL of this substrate solution and 500 μL of purified enzyme solution were added to a 1.5 mL microtube, mixed, and reacted at 30°C. After the reaction, the mixture was boiled to terminate the reaction. GABA was then detected by HPLC (reverse phase) analysis. The analysis was performed using an HPH-C18 (2.7 μm, 3.0 × 100 mm; manufactured by Poroshell) at a flow rate of 0.65 mL / min, with 20 mM dibasic sodium phosphate (pH 8.2) as the mobile phase for solution A and methanol:acetonitrile:water = 45:45:10 as the mobile phase for solution B. The analysis program was as shown in Table 3. Using glutamic acid as a substrate, the enzyme activity catalyzing the production of 1 μmol of GABA per minute was defined as 1 unit (U), and activity was measured.

[0108]

[0109] Test Example 1: 10 μL of a 50 mM solution of sodium glutamate monohydrate, 10 μL of a solution of PLP (pyridoxal phosphate) at an arbitrary concentration, and 30 μL of 100 mM acetate buffer (pH 4.5) were mixed to prepare a substrate solution with the PLP concentration listed in Table 4. 50 μL of this substrate solution and 50 μL of glutamic acid decarboxylase (to give a final concentration of 0.0034 U / g-glutamic acid) were added to a microplate, mixed, and incubated at 30°C for 20 hours. GABA was then detected by HPLC (reverse-phase). For the analysis, an HPH-C18 column (2.7 μm, 3.0 × 100 mm; manufactured by Poroshell) was used at a flow rate of 0.65 mL / min. The mobile phase used was 20 mM dibasic sodium phosphate (pH 8.2) for Solution A and 45:45:10 methanol:acetonitrile:water for Solution B. The analysis program was as shown in Table 3.

[0110]

[0111] As shown in the results in the above table, it was confirmed that the glutamic acid decarboxylase of the present invention produces GABA regardless of the concentration of PLP added, and it was found that the glutamic acid decarboxylase of the present invention is a PLP-binding glutamic acid decarboxylase.

[0112] Test Example 2: Glutamic acid decarboxylase was added to commercially available tomato juice (Del Monte Lycopene Rich) so that the concentration was 0.002 U per 1 g of glutamic acid contained in the tomato juice, and the mixture was allowed to react for 1 hour at 30° C. GABA was detected in the same manner as in Test Example 1, and the amount of GABA produced increased by approximately 142%.

[0113] Test Example 3: 10 mL of substrate solution was prepared by mixing 8 mL of 100 mM acetate buffer (pH 4.5), 1 mL of 50 mM sodium glutamate monohydrate, and 1 mL of 0.5 mM pyridoxal phosphate (PLP). 50 μL of glutamic acid decarboxylase was added to 450 μL of substrate solution to make a total volume of 500 μL. The resulting reaction solution was incubated for 60 minutes at temperatures of 20°C, 30°C, 40°C, 50°C, 60°C, and 70°C with stirring at 800 rpm. The optimal temperature for this enzyme was determined by measuring the amount of GABA produced under each condition. As shown in Figure 1, the optimal temperature was around 40-50°C.

[0114] SEQ ID NO: 1 (amino acid sequence of glutamic acid decarboxylase derived from Sporobolomyces pararoseus) MSLSRHVNPDRIIEESREHAKKHGRTSIHDLMGRKEPGFMSRDVHNNNQDDGTEPIPKYTFPRHGIPGREAYETVTNELSLDGNPLLNLASFVHTHMDEYGTKIAIENMSKNLIDSDEYPATTLLHSRCVSMLAELWHADGQKESATGTATTGSSEAIALGGLAMKKRWQAKRKAEGKSIHEPGPNIVMGANAQVALEKFARYFDVEARMVPVDKSTNYCMDPKRAIELVDENTIGVFVIWGSTYTGHYENVQEMSDLLDEYEKKTGISVPIHVDGAS GAMFSPFATPSVKWDFQIKRVVSINTSGHKWGKAYVGVGWVVFRDKEHLPKELVFELHYLGSVEYSYSINFSRPAAPILAQYYNFLKLGGFDGYRKISLHDAKNARLLARALENSKYYDVVSDIHRPKSEESLTEKAKHS IGLADDIDHYKPALPVVAFKFSESFKKEFPRIKQSAIQHGLRQNNWIVPNYELPPNAQNEEVLRVVMRETFNEDMVERLVVDIIQTTESLMEEHKNDVQNPGSLLASHGKEKNKSHKSSSERRAANHGEGVRPTGHDSVC

[0115]

[0116]

Claims

1. A glutamic acid decarboxylase comprising a polypeptide shown in any one of (1) to (3) below: (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; (2) a polypeptide in which one or more amino acid residues have been substituted, added, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 1, and which has glutamic acid decarboxylase activity; (3) a polypeptide which has a sequence identity of 75% or more to the amino acid sequence shown in SEQ ID NO: 1, and which has glutamic acid decarboxylase activity.

2. The glutamic acid decarboxylase according to claim 1, wherein the polypeptide is derived from Sporobolomyces pararoseus.

3. The glutamic acid decarboxylase according to claim 1, wherein the TM-score between the three-dimensional structure of a protein having the amino acid sequence set forth in SEQ ID NO: 1 and the three-dimensional structure of a polypeptide set forth in any one of (1) to (3) is 0.6 or more.

4. A glutamic acid decarboxylase comprising a polypeptide having a TM-score of 0.6 or more with respect to the three-dimensional structure of a protein having the amino acid sequence shown in SEQ ID NO: 1 and having glutamic acid decarboxylase activity.

5. DNA comprising a polynucleotide shown in any one of (1) to (3) below: (1) a polynucleotide encoding the polypeptide described in claim 1; (2) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 3; (3) a polynucleotide comprising a sequence equivalent to the base sequence shown in SEQ ID NO: 3 and encoding a polypeptide having glutamic acid decarboxylase activity.

6. An expression cassette comprising the DNA of claim 5.

7. A recombinant vector containing the DNA of claim 5.

8. A transformant having the DNA of claim 5, the expression cassette of claim 4, or the recombinant vector of claim 5.

9. A method for producing glutamic acid decarboxylase, which comprises culturing the transformant according to claim 8.

10. A method for producing glutamic acid decarboxylase, comprising the step of culturing Sporobolomyces pararoseus.

11. A method for producing γ-aminobutyric acid, comprising the step of allowing the glutamic acid decarboxylase according to any one of claims 1 to 4 to act on a raw material containing glutamic acid or a salt thereof.

12. A method for reducing the taste of food or drink, comprising the step of allowing the glutamic acid decarboxylase according to any one of claims 1 to 4 to act on a raw material containing glutamic acid or a salt thereof.

13. A γ-aminobutyric acid-producing enzyme preparation comprising the glutamic acid decarboxylase according to any one of claims 1 to 4.

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