JP7754409B2 - Engineered glutamic acid decarboxylase - Google Patents

Description

The present invention relates to a modified polypeptide having glutamic acid decarboxylase activity, an enzyme preparation for producing gamma-aminobutyric acid (GABA) containing this polypeptide, and a method for producing GABA that includes using this enzyme preparation for producing GABA. The present invention further relates to a polynucleotide encoding the polypeptide, a vector containing this polynucleotide, a host cell transformed with this vector, and a method for producing GABA that includes using this host cell.

Gamma-aminobutyric acid (GABA) acts as an inhibitory neurotransmitter in the body and is known to have physiological effects such as lowering blood pressure and diuretic effects. It is also known to be found in large amounts in foods such as germinated brown rice, miso, and pickles (such as kimchi) (Patent Document 1). GABA is also known as a monomer that makes up nylon 4. Nylon 4 has biodegradability not found in conventional nylon 6 or nylon 66, and also has superior strength and heat resistance compared to conventional biodegradable plastic materials (Patent Document 1).

Traditionally, GABA has been produced by chemical synthesis, fermentation, and enzymatic methods. The enzymatic method involves the action of glutamate decarboxylase (GAD; EC 4.1.1.15) on glutamic acid. GAD is endogenous to microorganisms and plants, and since GABA is often used in food applications, it is produced using GAD endogenous to lactic acid bacteria and molds used in fermented food production (Patent Documents 2 and 3). For example, Patent Documents 2 and 3 disclose methods for producing γ-aminobutyric acid, which involve culturing lactic acid bacteria and koji mold, respectively.

Examples of GABA production using genetic recombination technology have also been reported. For example, Non-Patent Document 1 describes the expression of GAD derived from the Lactobacillus plantarum WCFS1 strain in Escherichia coli, the preparation of GAD, and the production of GABA using a purified enzyme. Furthermore, Patent Document 1 discloses a method for producing gamma-aminobutyric acid using a thermostable GAD isolated from a thermophilic archaea.

Patent No. 4243685 Japanese Patent Application Laid-Open No. 2007-135416 Japanese Patent Application Laid-Open No. 2007-028998

Microbiol. Biotechnol. Lett., 2015, 43, 300-305

In order to produce GABA industrially, there is a need for the development of GAD with excellent productivity and stability at high temperatures. GABA production using microorganisms containing GAD, such as lactic acid bacteria and koji mold (see, for example, Patent Documents 2 and 3), has the problem of low bioconversion rates from glutamic acid or glutamate, the GAD substrate, to GABA. Conventional GAD also has the disadvantage of low stability at high temperatures. Non-Patent Document 1 improves the thermal stability of purified GAD by immobilizing it on beads, but does not alter the properties of the enzyme itself. While a thermostable GAD derived from thermophilic archaea has been discovered (Patent Document 1), the enzyme must be extracted in a high-salt environment, and a process for removing the salt carried over was required before it could be used to produce GABA.

The present invention therefore aims to provide a GAD that has higher GABA productivity and properties suitable for industrial-scale production (enzymatic thermostability, pH, productivity), as well as a GABA production method that includes using this GAD, and a GABA production method that includes using host cells into which this GAD gene has been introduced.

The present inventors aimed to create an engineered glutamic acid decarboxylase and designed numerous artificial GABA synthases, screening them through functional analysis. Among these, they discovered that at least two types of artificial GABA synthesized using glutamic acid decarboxylase derived from Lactobacillus brevis as a template possess higher GABA productivity and properties suitable for industrial-scale production, leading to the completion of the present invention.

According to the present invention, the following inventions are provided.
[1] A polypeptide comprising an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 1 or 2 and having glutamic acid decarboxylase activity.
[2] The polypeptide according to [1], comprising the amino acid sequence set forth in SEQ ID NO: 1 or 2.
[3] An enzyme preparation for producing γ-aminobutyric acid (GABA), comprising the polypeptide according to [1] or [2].
[4] A method for producing GABA, comprising using the enzyme preparation for producing GABA described in [3].
[5] The method for producing GABA according to [4], wherein the substrate is glutamic acid and/or glutamate.

[6] A polynucleotide encoding the polypeptide according to [1] or [2].
[7] A vector comprising the polynucleotide according to [6].
[8] A host cell transformed with the vector according to [7].
[9] The host cell according to [8], which is a cell of Escherichia coli (E. coli), Bacillus, Lactobacillus, Lactococcus, Corynebacterium, Aspergillus, or yeast.
[10] A method for producing GABA, comprising treating the host cell according to [8] or [9].
[11] A method for producing glutamic acid decarboxylase, comprising culturing the host cell according to [8] or [9] and recovering glutamic acid decarboxylase from the culture.
[12] A method for producing a food, feed, cosmetic, or pharmaceutical, comprising a step of obtaining a food, feed, cosmetic, or pharmaceutical using GABA obtained by carrying out the method according to any one of [4], [5], and [10].
[13] A method for producing a GABA-containing food, comprising contacting the polypeptide described in [1] or [2], the enzyme preparation described in [3], or the host cell described in [8] or [9] with a food material containing glutamic acid and/or glutamate.

The present invention provides a novel modified polypeptide having glutamic acid decarboxylase activity.

FIG. 1A shows the amino acid sequences of wild-type GAD (GAD-Wild_GU987102.1) and modified GADs (GAD-NSK01, GAD-NSK02) derived from Lactobacillus brevis CGMCC 1306. FIG. 1B shows the nucleotide sequences of wild-type GAD (GAD-Wild) and modified GAD (GAD-NSK01, GAD-NSK02) contained in the vectors used for recombination. FIG. 1C is a continuation of FIG. 1B and shows the nucleotide sequences of wild-type GAD (GAD-Wild) and modified GAD (GAD-NSK01, GAD-NSK02) contained in the vectors used for recombination. 2 is an SDS-PAGE showing purified GAD-Wild, GAD-NSK01, and GAD-NSK02. M: molecular weight marker; lanes 1 to 4: GAD-NSK01; lanes 5 to 8: GAD-NSK02; lanes 9 to 12: GAD-Wild; lanes 1, 5, and 9: inelutable fraction; lanes 2, 6, and 10: eluate (5 mM); lanes 3, 7, and 11: eluate (50 mM); and lanes 4, 8, and 12: eluate (250 mM). FIG. 3 is a graph showing the relative activities of purified GAD-Wild, GAD-NSK01, and GAD-NSK02 at each reaction pH. 4A is a graph showing the relative activity of purified GAD-Wild, GAD-NSK01, and GAD-NSK02 when the enzyme reaction was carried out at each reaction temperature for 10 minutes. FIG. 4B is a graph showing the relative residual activity of purified GAD-Wild, GAD-NSK01, and GAD-NSK02 when they were maintained at each temperature for 10 minutes. The change in GABA concentration over time during the reaction is shown in Figure 5. The reaction was carried out by adding the GAD-NSK02-expressing bacteria to a mixture of glutamic acid and water at 40°C or 50°C with vortex stirring.

The following description of the present invention may be based on representative embodiments and specific examples, but the present invention is not limited to such embodiments. In this specification, when a numerical value is accompanied by the word "about," it is intended to include a range of ±10% of that value. Furthermore, a numerical range expressed using "to" means a range that includes the numerical values before and after "to" as the lower and upper limits.

(Polypeptide)
The present invention relates to a polypeptide (hereinafter sometimes referred to as a modified polypeptide) comprising an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 1 or 2 and having glutamic acid decarboxylase activity. In one embodiment, the polypeptide of the present invention may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 1 or 2.

"Glutamic acid decarboxylase activity" refers to the activity of catalyzing the decarboxylation of glutamic acid (L-glutamic acid), which converts glutamic acid to GABA (gamma-aminobutyric acid).

The modified polypeptides of the present invention were designed by inserting amino acid mutations into GAD derived from Lactobacillus brevis CGMCC 1306 (DDBJ/EMBL/GenBank Accession number: GU987102.1, SEQ ID NO: 3, hereinafter referred to as GAD-Wild). Lactobacillus brevis CGMCC 1306 was isolated from milk and has been reported to have high GAD activity (Chinese J Chem Eng, 15, 157-161 (2007)). The present inventors have discovered that two types of modified polypeptides, which are artificially designed GADs and have the amino acid sequences set forth in SEQ ID NOs: 1 and 2, respectively, have higher GAD productivity than the wild-type (GAD-Wild), excellent enzymatic thermostability, an optimum pH suitable for GABA production, and high GABA productivity.

The theoretical molecular weight of the modified polypeptide of the present invention (without a histidine tag) is 54 kDa. The theoretical molecular weight of the modified polypeptide of the present invention with a histidine tag is 56 kDa, and the molecular weight measured by SDS-PAGE is approximately 56 kDa.

The modified polypeptides of the present invention can have improved expression efficiency in host cells. As described in Example 2 below, wild-type GAD and modified GAD were inserted into the same type of vector, and the expression efficiency of each GAD in the transformant was compared. Compared to the wild-type, the modified polypeptides GAD-NSK01 and GAD-NSK02 showed a more than 2-fold increase in expression, and more than 20-fold increase in expression, respectively. Fan et al. (Ann Microbiol (2012) 62:689-69) described the cloning of Lactobacillus brevis CGMCC 1306 and the analysis of the properties of the recombinant enzyme. While Fan et al. reported that the enzyme production per 1 L of culture was 13 mg, the modified polypeptide GAD-NSK02 of the present invention produced 253 mg/L, resulting in an approximately 19.5-fold increase in expression. Improving GAD productivity in host cells leads to improved GABA production efficiency and purity, whether using an enzymatic method or a fermentation method, and also reduces the amount of contaminants in the produced GABA, thereby contributing to a reduction in the purification burden during GABA production.

The glutamate decarboxylase activity of a modified polypeptide is evaluated by reacting the modified polypeptide with glutamate as a substrate and detecting the resulting reaction product (GABA). The reaction product can be detected using high-performance liquid chromatography (HPLC) with a fluorescence detector, RI detector, or charged aerosol detector. Detection can also be performed using thin-layer chromatography (TLC) and high-performance ion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD).

When measured at 40°C using glutamic acid as a substrate, the modified polypeptides of the present invention exhibited maximum activity at pH 3.5, and the pH ranges at which they exhibited 80% or more of the maximum activity were 3.0 to 3.5 (GAD-NSK01) and 3.0 to 4.0 (GAD-NSK02).On the other hand, wild-type GAD exhibited maximum activity at pH 4.5.
When measured at pH 3.5 using glutamic acid as a substrate, the modified polypeptides of the present invention exhibit maximum activity at 70°C, with the temperature ranges at which they exhibit 80% or more of the maximum activity being 50°C to 70°C (GAD-NSK01) and 70°C to 80°C (GAD-NSK02). Furthermore, in a test in which the modified polypeptides were maintained at pH 7.0 and temperatures of 30°C to 90°C for 10 minutes, they exhibited a residual activity of nearly 100% at 70°C or below (GAD-NSK01) or approximately 90% or more at 65°C or below (GAD-NSK02), demonstrating stability. On the other hand, when measured at pH 4.5, wild-type GAD exhibited maximum activity at 30°C, with a residual activity of 15% or less at 70°C. The temperature at which maximum activity was exhibited and the temperature stability were determined by measuring the amount of glutamic acid produced under the conditions shown in Example 3.

The modified polypeptide of the present invention has an optimum pH in the acidic range. This is advantageous because the aqueous solution of glutamic acid used as a substrate for GAD is acidic (pH 2.9-3.9 at 0.7%), eliminating the need for pH adjustment of the culture medium. Maintaining a low reaction pH also has the advantage of preventing the growth of contaminating microorganisms in the reaction solution. The modified polypeptide of the present invention has an optimum temperature of 70°C, is stable at least below 65°C, and exhibits high activity at a much higher temperature range than wild-type GAD, making it suitable for the industrial production of GABA.

In one embodiment, the modified polypeptide of the present invention may be a polypeptide comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, or at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or 2, and having glutamic acid decarboxylase activity.

"Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with those in the reference polypeptide after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished by a variety of methods within the skill of one in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

Furthermore, the modified polypeptide of the present invention may be a polypeptide having glutamic acid decarboxylase activity, and consisting of a protein consisting of an amino acid sequence in which one or several amino acids have been substituted, inserted, deleted, and/or added in the amino acid sequence set forth in SEQ ID NO: 1 or 2. As used herein, the range of "one or several" in the "amino acid sequence in which one or several amino acids have been substituted, inserted, deleted, and/or added" is not particularly limited, but means, for example, about 1 to 20, preferably 1 to 10, more preferably 1 to 7, even more preferably 1 to 5, and particularly preferably 1 to 3.
For the method for producing the modified polypeptide of the present invention, see <Production of Modified Polypeptides> below.

(enzyme preparation)
The present invention relates to an enzyme preparation for producing γ-aminobutyric acid (GABA), which comprises a polypeptide having glutamic acid decarboxylase activity and an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 1 or 2. The enzyme preparation for producing GABA of the present invention may comprise a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 1 or 2. The enzyme preparation of the present invention can be used to produce GABA using glutamic acid or glutamate as a substrate. The enzyme preparation of the present invention may be an isolated and purified modified polypeptide. Furthermore, the enzyme preparation of the present invention may be an enzyme composition containing the isolated and purified modified polypeptide as an active ingredient. The enzyme composition may contain additional components in addition to the modified polypeptide, as long as they do not inhibit the glutamic acid decarboxylase reaction. These may be components commonly used in enzyme compositions, such as buffers, stabilizers, and excipients. Such additional components are known in the prior art and are well known to those skilled in the art. The enzyme preparation of the present invention may also be in any form, including solid (e.g., powder) or liquid. The enzyme preparation of the present invention can be used, for example, by adding the solid or liquid form to food materials.

The enzyme preparation of the present invention can be provided, for example, as an enzyme preparation in which a polypeptide having glutamic acid decarboxylase activity and containing an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 1 or 2 is immobilized on an immobilization carrier. By using an immobilized enzyme preparation, for example, it is possible to produce a reaction product at a high substrate concentration and at a high temperature, and it is also possible to produce a reaction product using a bioreactor system.

The immobilization carrier is not particularly limited, and any carrier can be used as long as it can adsorb or crosslink a polypeptide having glutamic acid decarboxylase activity and retain the activity of the modified polypeptide of the present invention. Examples of the immobilization carrier include anion exchange carriers, cation exchange carriers, and hydrophobic carriers. Specific examples of the immobilization carrier include ion exchange gels. Examples of the ion exchange gel include the Diaion (registered trademark) series (manufactured by Mitsubishi Chemical Corporation), such as Diaion (registered trademark) SK1B, Diaion (registered trademark) PK212, Diaion (registered trademark) HPA25, Diaion (registered trademark) UBK550, and UBK555; the Sepabeads (registered trademark) series (manufactured by Mitsubishi Chemical Corporation), such as Sepabeads (registered trademark) SP-207 and Sepabeads (registered trademark) SP-850; and the Duolite (manufactured by Sumika Chemtex Co., Ltd.), such as Duolite A568, Duolite PWA7, and Duolite XAD761. The shape of the immobilization carrier is not particularly limited and may be, for example, a membrane, beads, or plate. The method for immobilizing the modified polypeptide to the immobilization carrier is not particularly limited and can be carried out, for example, by adding the modified polypeptide of the present invention and the immobilization carrier to a solvent such as a buffer solution and shaking the mixture.

(GABA manufacturing method)
The present invention relates to a method for producing GABA, which comprises using the above-mentioned enzymatic preparation for producing GABA. The substrate for the method for producing GABA of the present invention is glutamic acid and/or glutamate, particularly free glutamic acid or glutamate, and examples of glutamate include sodium glutamate, potassium glutamate, calcium glutamate, magnesium glutamate, etc. That is, the present invention provides a method for producing GABA, which comprises the step of allowing the enzymatic preparation for producing GABA of the present invention to act on glutamic acid and/or glutamate to obtain GABA.

The enzymatic preparation of the present invention can be allowed to act on glutamic acid and/or glutamate by, for example, preparing an aqueous solution of glutamic acid and/or glutamate, adjusting the pH as necessary, and then adding the enzymatic preparation of the present invention to the aqueous solution. Furthermore, the enzymatic preparation can be prepared by immobilizing a polypeptide containing an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 1 or 2 of the present invention and having glutamic acid decarboxylase activity on a support, and then contacting the enzymatic preparation with an aqueous solution of glutamic acid and/or glutamate. This is because the glutamic acid decarboxylase reaction using the enzymatic preparation of the present invention can be carried out in a reaction solution.

Glutamic acid and/or glutamate may be used in pure form, or other materials or ingredients (such as food ingredients) containing glutamic acid and/or glutamate may be used. Specific examples of the glutamic acid or glutamate-containing food materials include seasonings, proteinaceous materials, and other food ingredients, processed foods, and food residues. Examples of such glutamic acid or glutamate-containing food materials include natural seasonings extracted from natural ingredients, such as kelp extract and chicken extract; food residues (food processing residues and cooking residues) such as used tea leaves, defatted soybeans, and okara; amino acid and peptide mixtures obtained by acid hydrolysis or enzymatic degradation of vegetable proteins such as soybeans and wheat, or animal proteins such as pork and chicken; beans such as soybeans, black beans, adzuki beans, edamame, kidney beans, and peas; wheat and grains such as barley, wheat, oats, and job's barley; nuts and seeds such as sesame, peanuts, almonds, and walnuts; and cereals such as rice, corn, buckwheat, foxtail millet, millet, barnyard millet, and amaranth; and fermented grains and other cereals in which free glutamic acid is produced by microbial fermentation of glutamic acid-containing raw materials or extracts thereof. Furthermore, microorganisms that produce glutamic acid (such as mold, lactic acid bacteria, bifidobacteria, natto bacteria, acetic acid bacteria, yeast, and actinomycetes) can also be used. The present invention includes a method for producing a GABA-containing food product, which comprises contacting the modified polypeptide, the enzyme preparation, or the host cell with a food material containing glutamic acid and/or glutamate. For example, GABA can be easily ingested in daily meals by adding a dressing containing the modified polypeptide to a tomato salad.

The reaction temperature when the polypeptide contained in the enzyme preparation of the present invention is reacted with glutamic acid and/or glutamate is not particularly limited, as long as it is within the temperature range in which enzymatic activity is expressed. As described in the Examples, the polypeptide contained in the enzyme preparation of the present invention exhibits thermostability up to at least 65°C when reacted at pH 7.0, and has an optimum temperature of 70°C when reacted at pH 3.5. Some conventional glutamic acid decarboxylases have limitations on their use on an industrial scale due to their thermostability. For example, the optimum temperature of wild-type GAD derived from Lactobacillus brevis CGMCC 1306 is 30°C, and after 10 minutes of treatment at 70°C, the remaining activity is 15% or less. However, the polypeptide contained in the enzyme preparation of the present invention exhibits high thermostability and has thermostability suitable for use on an industrial scale. Therefore, the reaction temperature (liquid temperature of the reaction solution) when the enzyme preparation of the present invention is reacted can be set over a wide temperature range, such as 35-65°C. Accurate temperature control can be difficult in industrial-scale reaction systems, but the modified polypeptides of the present invention have the advantage of being stable over a wide temperature range.

The reaction temperature can be set in the range of 35 to 65°C, but in the case of pH 3.5, it is preferably set in the range of 50 to 65°C. A reaction temperature of 50°C or higher makes it difficult for contaminating bacteria to grow, while a temperature of 65°C or lower stabilizes the polypeptide of the present invention.

In the GABA production method, as described below, the enzyme preparation of the present invention can be used in combination with other enzymes. When other enzymes are used in combination, the reaction temperature need only be within a temperature range in which the protein contained in the enzyme preparation of the present invention can act stably. As described above, the protein contained in the enzyme preparation of the present invention is stable over a wide temperature range, so it is often possible to set the temperature within a range in which the activity of all enzymes used in the reaction system can be fully utilized, taking into account the temperature at which the other enzymes used in combination are stably active.

The reaction temperature does not need to be constant throughout the entire reaction time. If it is desired to increase the activity of another enzyme used in combination in the early stages of the reaction time, the temperature at the beginning of the reaction can be set to a temperature range where the activity of that enzyme is high, and the temperature at the middle and late stages of the reaction can be set to a temperature range where the activity of the protein contained in the enzyme preparation of the present invention is high.

The pH of the reaction solution when the polypeptide contained in the enzyme preparation of the present invention is reacted with glutamic acid and/or glutamate is not particularly limited as long as it is within the pH range in which the enzyme activity is expressed, but can be set in the range of pH 2.5 to 5.0. This is because, as described in the Examples, the polypeptide contained in the enzyme preparation of the present invention can react at least in the pH range of 2.5 to 5.0. To efficiently obtain GABA, it is preferable to set the pH of the reaction solution when the polypeptide contained in the enzyme preparation of the present invention is reacted in the range of pH 3.0 to 4.0. In particular, GAD-NSK02 has not only higher GAD productivity but also higher specific activity than the wild-type, making it usable over a wide range of pH conditions. Specific activity refers to the amount of enzyme (units/mg) contained per mg of enzyme sample protein. The pH of the reaction solution can be adjusted by adding acid or alkali as needed.

The reaction time between the substrate and the polypeptide contained in the enzyme preparation of the present invention in the GABA production method can be determined appropriately taking into consideration the reaction temperature, substrate concentration, and, if other enzymes are used in combination, the characteristics of the enzymes used. Furthermore, an appropriate reaction time for efficient GABA production can be determined appropriately based on conventional technology in this field. Specific examples of reaction times include, but are not limited to, 5 minutes to 72 hours. The enzyme preparation of the present invention can be added appropriately during the reaction.

The concentration of glutamic acid and/or glutamate in the reaction solution is not particularly limited. For example, within the range in which the enzymatic reaction can proceed, a higher concentration of glutamic acid and/or glutamate is more economically advantageous. When glutamic acid is used as a substrate, the glutamic acid concentration is in the range of 0.1 g to 100 g per 100 g of solvent. The solvent for the reaction solution is not particularly limited, and examples include water, buffer solutions, etc. Note that if the selected substrate has low solubility in the solvent, it may not be completely dissolved at the desired concentration. When performing an enzymatic reaction in a batch system, for example, the substrate does not need to be completely dissolved at the beginning of the reaction; a substrate concentration that is dissolved at the end of the enzymatic reaction may be selected. On the other hand, when using a column packed with an immobilized enzyme agent for the enzymatic reaction, it is preferable that the substrate be completely dissolved to avoid pressure loss due to column clogging. When using a column packed with an immobilized enzyme agent for the enzymatic reaction, it is preferable, for example, to reflux the substrate solution through the column and continuously pass it through.

As a specific example of GABA production using the enzyme preparation of the present invention, when glutamic acid is used as a substrate, an aqueous solution of glutamic acid at approximately 0.1 g to 100 g per 100 g of water is prepared, and the pH of the aqueous solution is adjusted to approximately 2.5 to 4.5, preferably approximately 3.0 to 4.0, using an acid or alkali as needed. The enzyme preparation of the present invention is added to the aqueous solution, and the solution is then maintained at a temperature in the range of 35 to 65°C for approximately 5 minutes to 72 hours, thereby producing GABA.

Furthermore, in the GABA production method, the enzyme preparation of the present invention can be used in combination with other enzymes. The other enzymes may be, but are not limited to, those that produce glutamic acid and/or glutamate, which can serve as substrates for the enzymes of the present invention that have the activity of catalyzing the glutamic acid decarboxylase reaction. Examples of other enzymes that can be used include, but are not limited to, enzymes involved in glutamic acid synthesis (e.g., glutamate synthase, glutamate dehydrogenase, aminotransferase), proteases, peptidases, etc. By using the enzyme preparation of the present invention (i.e., an enzyme preparation for catalyzing the glutamic acid decarboxylase reaction) in combination with an enzyme involved in glutamic acid synthesis as the other enzyme, it is possible to produce glutamic acid while simultaneously producing GABA from glutamic acid via the glutamic acid decarboxylase reaction. By using the enzyme preparation of the present invention (i.e., an enzyme preparation for catalyzing the glutamic acid decarboxylase reaction) in combination with other enzymes such as protease or peptidase, proteins contained in food, food ingredients, food waste, and food residues (e.g., used tea leaves, defatted soybeans, and okara) can be hydrolyzed to obtain glutamic acid, while GABA can be produced from glutamic acid through the glutamic acid decarboxylase reaction.

The above manufacturing method produces an aqueous solution containing GABA. This GABA-containing aqueous solution can be subjected to standard processes such as decolorization, desalination, and purification, as needed. Furthermore, unreacted substrates can be removed and the purity of GABA increased by using resin fractionation, precipitation with organic solvents such as ethanol, chromatographic fractionation, or ultrafiltration membrane treatment. GABA can be purified more efficiently by using a single purification process or a combination of several processes.

The present invention provides a method for producing food, feed, cosmetic, or pharmaceutical, which includes a step of obtaining food, feed, cosmetic, or pharmaceutical using GABA obtained by carrying out the above-mentioned GABA production method. In the step of obtaining food, feed, cosmetic, or pharmaceutical using GABA obtained by carrying out the GABA production method, GABA can be used as one of the raw materials to produce food, feed, cosmetic, or pharmaceutical, or GABA itself can be prepared in an appropriate form (powder, liquid, etc.) and provided as food, feed, cosmetic, or pharmaceutical.

Examples of foods that can be produced using the method of the present invention include, but are not limited to, various carbohydrates (bread, noodles, cooked rice, mochi), various Japanese sweets (rice crackers, arare, okoshi, gyuhi, mochi, manju, dorayaki, uriwara, bean paste, yokan, mizu yokan, kingyoku, castella, candy), various Western sweets (bread, biscuits, crackers, cookies, pies, donuts, steamed cakes, puddings, jellies, mousses, bavarois, custard cream, cream puffs, waffles, sponge cakes, chocolates, chewing gum, caramel, nougat, candies, syrups), various frozen desserts (ice cream, sorbet, gelato, shaved ice), various paste-like foods (flour paste, peanut paste, margarine, fruit paste), various beverages (fruit juice-containing beverages, fruit juice, vegetable juice, cider, ginger ale, isotonic drinks, amino acid drinks, jelly drinks, coffee drinks, green tea, black tea, oolong tea, barley tea, milk drinks, lactic acid bacteria drinks, cocoa, beer, happoshu, third beer, non-alcoholic drinks, beer-flavored drinks, liqueurs, chuhai, sake, fruit wine, distilled alcohol, energy drinks, health drinks, powdered drinks), processed fruit and vegetable products (jam, marmalade, preserved fruit in syrup, candied fruit, pickles), various dairy products (cheese, yogurt, butter, condensed milk, powdered milk), powdered foods (powdered soup, powdered mousse, powdered jelly, powdered sweeteners), nutritional foods, diet foods, nutritional foods for sports, liquid foods, semi-solid liquid foods, nursing care foods, foods for dysphagia, etc.

Examples of feeds and baits produced using the method of the present invention include, but are not limited to, feeds and baits for livestock, poultry, seafood, and insects (honeybees, silkworms, etc.). These feeds and baits may be in the form of powder, pellets, tablets, paste, capsules, etc.

Examples of cosmetics produced using the method of the present invention include, but are not limited to, moisturizers and beauty products. These may be in the form of lotions, creams, emulsions, etc.

Examples of pharmaceuticals produced by the method of the present invention include, but are not limited to, brain function improvers, blood pressure inhibitors, sleep improvers, fatigue reducers, and stress relievers, and may be in the form of tablets, powders, liquids, capsules, etc.

<Production of modified polypeptide>
The method for obtaining the modified polypeptide contained in the enzyme preparation of the present invention is not particularly limited, and the modified polypeptide may be a protein synthesized by chemical synthesis or a recombinant modified polypeptide produced by genetic recombination technology. The production of a recombinant polypeptide will be described below.

A polypeptide having the amino acid sequence set forth in SEQ ID NO: 1 or 2, or an amino acid sequence having 85% or more amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or 2, can be prepared by genetic engineering techniques. For example, a gene encoding the amino acid sequence set forth in SEQ ID NO: 1 or 2 can be replicated within a host cell, or incorporated into a chromosome and contained in an expressible DNA molecule, particularly inserted into an expression vector, by transforming the host cell and culturing the host cell to produce the protein. This DNA molecule can be obtained by incorporating a DNA fragment encoding the amino acid sequence set forth in SEQ ID NO: 1 or 2, or an amino acid sequence having 85% or more amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or 2, into a vector molecule. In a preferred embodiment of the present invention, the vector is a plasmid. The DNA molecule of the present invention can be produced according to the method described in Molecular Cloning: A Laboratory Manual.

The present invention relates to a polynucleotide that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 1 or 2 and encodes a polypeptide having glutamic acid decarboxylase activity. In one embodiment of the present invention, the polynucleotide encoding the modified polypeptide of the present invention comprises the nucleotide sequence set forth in SEQ ID NO: 4 or 5, or its complementary strand sequence. The present invention also includes polynucleotides that have at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% nucleotide sequence identity to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 4 or 5 or its complementary strand sequence, and that encode a polypeptide having the activity of catalyzing a glutamic acid decarboxylase reaction. Sequence identity is defined as the percentage of bases that are identical between two sequences after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Sequence identity can be determined using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software.

The present invention further relates to a vector comprising the above-described polynucleotide. Vectors that can be used in the present invention can be appropriately selected from viruses, plasmids, cosmid vectors, etc., taking into consideration the type of host cell used. For example, when the host cell is Bacillus subtilis, pHT-series plasmids are used; when the host cell is Escherichia coli, λ-phage-series bacteriophages, pET-series, pUC-series, pCold-series, and pGEX-series plasmids are used; and when the host cell is yeast, YEp-series, YCp-series, and YIp-series vectors, or pLeu4-series, pPPLeu4-series, pJPLeu4-series, etc., are used. This plasmid may contain a marker for selecting transformants; the selection marker can be, but is not limited to, a drug resistance marker or an auxotrophic marker gene.

Furthermore, expression vectors usable in the present invention can contain DNA sequences necessary for the expression of enzyme genes, such as promoters, terminators, ribosome binding sites, transcriptional regulatory signals such as transcription termination signals, and translational regulatory signals. Examples of promoters that can be used include, but are not limited to, promoters such as subtilisin and SPAC in Bacillus subtilis, and promoters such as alcohol dehydrogenase (ADH), acid phosphatase (PHO), galactose gene (GAL), and glyceraldehyde-3-phosphate dehydrogenase gene (GAP) in yeast. Adding a signal peptide is preferred because it allows the target enzyme to be secreted into the culture supernatant, facilitating purification. Signal peptides can also be replaced with those derived from Bacillus subtilis or yeast (e.g., invertase signal, acid phosphatase signal, λ-factor signal, etc.). In addition to commonly used lac and T7 promoters, in E. coli, more efficient expression can be achieved by simultaneously expressing molecular chaperones using promoters such as the cspA promoter.

The present invention relates to a method for producing glutamic acid decarboxylase, which comprises culturing host cells transformed with the above vector and recovering glutamic acid decarboxylase from the culture. The transformed host cells can be cultured using a method commonly used for the host cells used. Typically, the enzyme is produced and accumulated in the intracellular or extracellular culture after approximately 1 to 4 days of culture. Culture conditions (medium, pH, temperature, etc.) are typically 25 to 37°C for bacteria, 25 to 30°C for yeast, and approximately 37°C for eukaryotic cells. For information on culture conditions, see the Gene Expression Experiment Manual (Kodansha), etc.

Host cells that can be used include bacteria such as Escherichia coli, Bacillus, Lactobacillus, Lactococcus, Corynebacterium, and Aspergillus; yeasts such as Candida utilis, Saccaromyces cerevisiae, and Pichia pastoris; Rhizopus niveus, Rhizopus delemar, and higher eukaryotes (e.g., CHO cells). For the purpose of producing the modified polypeptides of the present invention, yeast, filamentous fungi, or bacteria are preferred as host cells, with bacteria being more preferred, and Escherichia coli and Bacillus subtilis being particularly preferred. Furthermore, the modified polypeptides of the present invention can be produced using cell-free protein expression systems using rabbit reticulocyte lysate (RRL), wheat germ extract, Escherichia coli lysate, insect cell lysate (such as SF9 or SF21), and the like.

Polypeptides produced by transformants can be isolated and purified by an appropriate combination of known separation and purification methods. These separation and purification methods include, for example, methods that exploit differences in solubility, such as salt precipitation and solvent precipitation; methods that exploit differences in molecular weight, such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis; methods that exploit differences in charge, such as ion exchange chromatography; methods that exploit differences in hydrophobicity, such as hydrophobic chromatography and reversed-phase chromatography; and methods that exploit differences in isoelectric point, such as isoelectric focusing. Furthermore, affinity chromatography can also be used. In addition to the purification methods described in the examples, general separation and purification methods can be found in, for example, Basic Experimental Methods for Proteins and Enzymes (Nankodo).

(Fermentation method)
The present invention relates to a method for producing GABA, which comprises using a host cell transformed with the vector. Specifically, using a host cell can be carried out, for example, by preparing an aqueous solution of glutamic acid and/or a glutamate, adjusting the pH as necessary, and then adding the host cell of the present invention to the aqueous solution. Examples of host cells that can be used include lactic acid bacteria (Lactobacillus, Lactococcus, e.g., Lactococcus), actinomycetes (Corynebacterium), Aspergillus, Escherichia coli, Bacillus, or yeast. In one embodiment, Corynebacterium glutamicum is preferably used as the host cell. C. glutamicum is used for industrial fermentation production of monosodium glutamate. Transforming C. glutamicum with a modified glutamate decarboxylase gene may enable GABA production using glutamate produced by C. glutamicum itself, without the need for added glutamate and/or glutamate. Protein expression in the host cells can be induced before use. When E. coli is used as the host cell, isopropyl-β-thiogalactopyranoside (IPTG) can be used as an inducer. The concentration of glutamate and/or glutamate in the aqueous solution can range from 0.01 to 3 M. The aqueous solution of glutamate and/or glutamate containing the host cells can be incubated for a period of time that is not particularly limited, but can range from 5 minutes to 72 hours. Incubation can be performed at room temperature, with shaking or agitation, or at a temperature suitable for protein expression in the host cells, for example, at a temperature between 30 and 55°C, with shaking or agitation. The pH of the aqueous solution of glutamic acid and/or glutamate can be set to a value suitable for the host cell.

GABA production methods can be broadly divided into two: fermentation methods using microbial fermentation and enzymatic methods using enzymes. Generally, fermentation methods have the drawback of requiring longer production times and lower substrate conversion efficiency. On the other hand, enzymatic methods shorten reaction times, but require labor and cost for the addition of the coenzyme pyridoxal phosphate (PLP) and the purification of the enzyme itself. Compared to the above methods, the modified glutamic acid decarboxylase of the present invention is superior to conventional enzymes in that it has a faster reaction rate due to its improved specific activity and thermostability, resulting in high substrate conversion efficiency and the production of high-concentration GABA in a short period of time. Furthermore, the modified glutamic acid decarboxylase of the present invention has high enzyme productivity and a high enzyme amount per cell weight. Therefore, by using cells expressing the modified glutamic acid decarboxylase of the present invention, GABA can be produced with reduced costs for coenzyme addition and enzyme purification. In Example 4 described below, 2.5 M GABA was produced in 50 minutes, with a conversion rate of glutamic acid to GABA of over 90%.

The present invention will be explained in more detail based on the following examples, but the present invention is not limited to these examples. In this specification, unless otherwise specified, "%" and other percentages are based on mass, and numerical ranges are stated as including their endpoints.

Example 1: Design of modified glutamate decarboxylases
Using GAD derived from Lactobacillus brevis CGMCC 1306 (hereinafter referred to as GAD-Wild, DDBJ/EMBL/GenBank Accession number: GU987102.1, SEQ ID NO: 3) as a template, 10,000 similar sequences were obtained from Blastp. The analysis conditions were an Expectec threshold value of 1.0E-4 and Max target sequences of 10,000. The obtained similar sequences were analyzed, and sequences similar to the template sequence were removed. Then, the resulting sequences were combined with the corresponding sequences from Reference A (Nakano, S., Motoyama, T., Miyashita, Y., Ishizuka, Y., Matsuo, N., Tokiwa, H., Shinoda, S., Asano, Y., and Ito, S. (2018) Benchmark Analysis of Native and Artificial NAD ⁺ -Dependent Enzymes Generated by a Sequence-Based Design Method with or without Phylogenetic Data, Biochemistry 57, 3722-3732.) and Reference B (Nakano, S., Niwa, M., Asano, Y., and Ito, S. (2019) Following the Evolutionary Track of a Highly Specific l-Arginine Oxidase by Reconstruction and Biochemical Analysis of Ancestral and Native Enzymes, Appl Environ Microbiol 85, Using the techniques used in [e00459-00419], a total of eight modified glutamate decarboxylase sequences were designed. Genes encoding these eight sequences were artificially synthesized, and the feasibility of enzyme production in recombinant E. coli was confirmed. Five of the sequences were confirmed to produce glutamate decarboxylase. These five sequences were purified using His tags, and those with improved activity compared to the wild-type were selected, yielding two modified glutamate decarboxylases designated GAD-NSK01 and GAD-NSK02. GAD-NSK01 and GAD-NSK02 have the amino acid sequences set forth in SEQ ID NOs: 1 and 2, respectively (Figure 1A).

Example 2: Preparation of enzymes GAD-Wild, GAD-NSK01 and GAD-NSK02 were prepared as follows.
An artificially synthesized DNA (GeneScript) was inserted into the NdeI/BamHI restriction enzyme sites of pET15b (Merck) as a vector (FIG. 1B). pET15b contains a histidine tag (MGSSHHHHHHSSGLVPAGSH, SEQ ID NO: 7).

Next, Escherichia coli BL21(DE3) (Nippon Gene Co., Ltd.) was transformed with the above cloning plasmid and cultured in LB medium at 37°C. When the turbidity (OD600) reached 0.4-0.6, induction with isopropyl-β-thiogalactopyranoside (IPTG) was performed, and the culture was cultured overnight at 25°C. After culture, the cells were collected and suspended in cell disruption buffer (10 mM phosphate buffer, 50 mM NaCl, pH 7.0). This cell suspension was subjected to ultrasonic disruption and centrifugation, and the centrifuged supernatant was used as a crude enzyme solution. The crude enzyme solution was subjected to affinity chromatography using a Ni column (GE Healthcare Japan Co., Ltd.), and SDS-PAGE confirmed that purified GAD-Wild, GAD-NSK01, and GAD-NSK02 were obtained (Figure 2). Polyacrylamide gel (12.5%) e-Pagel (ATTO Corporation) was used for SDS-PAGE. 10 μg of protein was applied for measurement, and electrophoresis was performed at a constant current of 20 mA. WideView ^™ Prestained Protein Size Marker III (Fujifilm Wako Pure Chemical Industries, Ltd.) was used as a molecular weight marker. The concentration of the prepared GAD was measured by absorbance at 280 nm, and the productivity of each GAD was calculated from the solution volume and concentration. The results are shown in Table 1. Specifically, the GAD productivity (mg/L) in Table 1 was calculated by dividing the weight of GAD obtained from the volume and concentration of the purified GAD solution by the initial medium volume.

When wild-type GAD and modified GAD were inserted into the same vector and the expression efficiency of each GAD in the transformant was compared, GAD-NSK01 showed a more than 2-fold increase in expression compared to GAD-Wild, and GAD-NSK02 showed a more than 20-fold increase in expression compared to GAD-Wild. Furthermore, compared to the GAD used in the comparative example (Fan et al., 2012, supra), GAD-NSK02 showed an approximately 19.5-fold increase in expression.
It is believed that the increase in GAD productivity in E. coli into which modified GAD has been introduced will lead to improved GABA production efficiency and purity, whether GABA is produced using purified enzymes or by culturing E. coli into which modified GAD has been introduced.

Example 3: Analysis of Enzyme Characteristics a) Activity Measurement To 26 μL of a mixed solution consisting of 5 to 30 mM glutamic acid, 0.2 mM pyridoxal phosphate, and 100 mM phosphate-citrate buffer (pH 3.5), 4 μL of GAD-NSK01 or GAD-NSK02 (100 to 500 μg/mL) prepared in Example 1 was added, and the mixture was kept at 40° C. for 10 minutes and then kept at 95° C. for 10 minutes to terminate the reaction. Similarly, for GAD-Wild, 4 μL of GAD-Wild (100-500 μg/mL) prepared in Example 1 was added to 26 μL of a mixture consisting of 5-30 mM glutamic acid, 0.2 mM pyridoxal phosphate, and 100 mM phosphate-citrate buffer (pH 4.5). The mixture was then incubated at 40°C for 10 minutes, followed by 95°C for 10 minutes to terminate the reaction. Next, 100 μL of R1 enzyme reagent solution and 100 μL of R2 enzyme reagent solution from the L-glutamic acid measurement kit "Yamasa" NEO (Yamasa Shoyu Co., Ltd.) were added. After incubation at 30°C for 20 minutes, the absorbance (A555) was measured, and the reaction rate was calculated from the amount of glutamic acid consumed. Glutamic acid (0-30 mM) was used to generate a calibration curve. One unit of enzyme activity was defined as the amount of enzyme that consumes 1 μmol of glutamate per minute of reaction under the above conditions. Next, using OriginPro2021 (Lightstone Co., Ltd.), nonlinear approximation of the reaction rate at each concentration was performed, and the reaction rate constant was calculated by applying the Michaelis-Menten equation. The results are shown in Table 2.

b) Optimal pH
The optimum pH was determined by measuring enzyme activity at pH 2.5 to 6.0. Specifically, 4 μL of 200 μg/mL GAD-Wild, GAD-NSK01, or GAD-NSK02 was added to 26 μL of a mixture consisting of 10 mM glutamic acid, 0.2 mM pyridoxal phosphate, and 100 mM phosphate-citrate buffer (pH 2.5 to 6.0). The enzyme reaction was allowed to proceed at 40°C for 10 minutes, and then stopped at 95°C for 10 minutes. Next, 100 μL of R1 enzyme reagent solution and 100 μL of R2 enzyme reagent solution from the L-glutamic acid measurement kit "Yamasa" NEO (Yamasa Shoyu Co., Ltd.) were added, and the mixture was incubated at 30°C for 20 minutes, after which the absorbance (A555) was measured. Enzyme activity was calculated based on the amount of glutamic acid consumed according to the method described above in a). The relative activity, with the enzyme activity showing maximum activity taken as 100%, is shown in Figure 3. Analysis revealed that the optimum pH for GAD-Wild was 4.5, while the optimum pH for GAD-NSK01 and GAD-NSK02 was 3.5. Specifically, GAD-NSK01 and GAD-NSK02 exhibited activities of 6.9 U/mg and 17.2 U/mg, respectively, at pH 3.5. On the other hand, GAD-Wild exhibited activities of 2.7 U/mg at pH 3.5 and 5.4 U/mg at pH 4.5. GAD-NSK01 exhibited superior activity compared to GAD-Wild in the acidic range, while GAD-NSK02 exhibited superior activity over a wider pH range.

Since the aqueous solution of glutamic acid used as a substrate for GAD is acidic (pH 2.9-3.9 at 0.7%), the optimal pH for GAD-NSK01 and GAD-NSK02 is in the acidic range, which is advantageous as it eliminates the need for pH adjustment in the medium. Another advantage is that maintaining a low reaction pH can prevent the growth of contaminating microorganisms in the reaction solution.

c) Optimum Temperature and Temperature Stability The optimum temperature was determined by measuring the enzyme activity at 10 to 90° C. Specifically, 4 μL of 200 μg/mL GAD-NSK01 or GAD-NSK02 was added to 26 μL of a mixture consisting of 10 mM glutamic acid, 0.2 mM pyridoxal phosphate, and 100 mM phosphate-citrate buffer (pH 3.5), and the enzyme reaction was allowed to proceed by maintaining the mixture at 10 to 90° C. for 10 minutes, and the reaction was terminated by maintaining the mixture at 95° C. for 10 minutes. Similarly, for GAD-Wild, 4 μL of 200 μg/mL GAD-Wild was added to 26 μL of a mixture consisting of 10 mM glutamic acid, 0.2 mM pyridoxal phosphate, and 100 mM phosphate-citrate buffer (pH 4.5). The enzyme reaction was allowed to proceed at 10-90°C for 10 minutes, and then stopped at 95°C for 10 minutes. Next, 100 μL of R1 enzyme reagent solution and 100 μL of R2 enzyme reagent solution from the L-glutamic acid measurement kit "Yamasa" NEO (Yamasa Shoyu Co., Ltd.) were added, and the mixture was incubated at 30°C for 20 minutes, after which the absorbance (A555) was measured. Enzyme activity was calculated based on the amount of glutamic acid consumed according to the method described above in a). Figure 4A shows the relative activity, with the maximum enzyme activity taken as 100%.
Temperature stability was determined by incubating 500 μg/mL of GAD-NSK01 or GAD-NSK02 in 10 mM phosphate buffer, 50 mM NaCl, pH 7.0 at 30 to 90°C for 10 minutes, then cooling to 4°C and measuring the enzyme activity. The enzyme activity was measured according to the method described in a) above. The relative activity (residual activity) is shown in Figure 4B, where the enzyme activity showing the maximum residual activity is defined as 100%.
Analysis showed that GAD-Wild had an optimum temperature of 30°C, exhibited a residual activity of 87.3% or more at 50°C or below, and was stable at 50°C or below. The residual activity at 70°C was 15% or less. GAD-NSK01 had an optimum temperature of 70°C and was stable at 70°C or below. GAD-NSK02 had an optimum temperature of 70°C, exhibited a residual activity of 89.6% or more at 65°C or below, and was stable at 65°C or below. Specifically, GAD-NSK01 and GAD-NSK02 exhibited activities of 10.5 U/mg and 34.3 U/mg, respectively, at 70°C. On the other hand, GAD-Wild exhibited an activity of 1.2 U/mg at 70°C and 5.4 U/mg at 30°C. The activity of GAD-NSK02 was superior to that of GAD-Wild in all temperature ranges, and GAD-NSK01 showed superior activity to GAD-Wild especially at temperatures of 40°C or higher.
Both GAD-NSK01 and GAD-NSK02 have an optimum temperature of 70° C. and are stable at temperatures below 70° C. and below 65° C., respectively, and can be said to have properties suitable for the industrial production of GABA.

Example 4: Production of GABA 3 g (wet weight) of GAD-NSK02-expressing bacteria was added to a mixture of 26.4 g of glutamic acid and 60 ml of ultrapure water in a 500 ml baffled Erlenmeyer flask, and GABA was produced by vortex stirring at 40°C or 50°C. Sampling was performed every 10 minutes, and the mixture was diluted 10-fold with ultrapure water and boiled for 10 minutes to stop the reaction. The amount of GABA produced was then confirmed by HPLC analysis using an AccQ Tag Derivatization Chemistry Kit (Waters) under the following conditions.
<HPLC analysis conditions>
Column: AccQ-Taq Column (150 mm x 3.9 mm, manufactured by Waters)
Column temperature: 50°C
Eluents: (A) AccQ Taq, (B) acetonitrile, (C) degassed ultrapure water Gradient: shown in Table 3.
Flow rate: 1.0 mL/min. Detector: Fluorescence detector (excitation wavelength 250 nm, fluorescence wavelength 395 nm)
Injection volume: 10 μL
Analysis time: 20 minutes

After 60 minutes of reaction, the reaction solution was collected and centrifuged to remove the bacterial cells, yielding a reaction solution. This reaction solution was dried under reduced pressure to obtain GABA powder. The powder was dissolved in ultrapure water and subjected to HPLC analysis under the above conditions to determine the purity of GABA.
The analytical results, which show the change in GABA concentration over time during the reaction, are shown in Figure 5. After 50 minutes of reaction at 50°C, the GABA concentration reached approximately 2.5 M. The powder obtained by drying under reduced pressure had a GABA content of over 98%, and the conversion rate from glutamic acid to GABA was over 90%.

SEQ ID NO: 1: Amino acid sequence of modified glutamic acid decarboxylase GAD-NSK01 SEQ ID NO: 2: Amino acid sequence of modified glutamic acid decarboxylase GAD-NSK02 SEQ ID NO: 3: Amino acid sequence of glutamic acid decarboxylase derived from Lactobacillus brevis CGMCC 1306 SEQ ID NO: 4: Nucleotide sequence encoding modified glutamic acid decarboxylase GAD-NSK01 SEQ ID NO: 5: Nucleotide sequence encoding modified glutamic acid decarboxylase GAD-NSK02 SEQ ID NO: 6: Nucleotide sequence encoding glutamic acid decarboxylase derived from Lactobacillus brevis CGMCC 1306 SEQ ID NO: 7: Histidine tag sequence

Claims (13)

A polypeptide comprising an amino acid sequence that is at least 90 % identical to the amino acid sequence set forth in SEQ ID NO: 1 or 2, and having glutamic acid decarboxylase activity. The polypeptide of claim 1, comprising the amino acid sequence set forth in SEQ ID NO: 1 or 2. An enzyme preparation for producing gamma-aminobutyric acid (GABA), comprising the polypeptide of claim 1 or 2. A method for producing GABA, comprising using the enzyme preparation for producing GABA described in claim 3. The GABA production method according to claim 4, wherein the substrate is glutamic acid and/or glutamate. A polynucleotide encoding the polypeptide of claim 1 or 2. A vector comprising the polynucleotide of claim 6. A host cell transformed with the vector of claim 7. The host cell of claim 8, which is a cell of Escherichia coli (E. coli), Bacillus, Lactobacillus, Lactococcus, Corynebacterium, Aspergillus, or yeast. A method for producing GABA, comprising inducing the host cell of claim 8 or 9 to act. A method for producing glutamic acid decarboxylase, comprising culturing the host cell of claim 8 or 9 and recovering glutamic acid decarboxylase from the culture. A method for producing a GABA- containing feed, a GABA-containing cosmetic, or a GABA-containing pharmaceutical, comprising contacting the polypeptide according to claim 1 or 2, the enzyme preparation according to claim 3, or the host cell according to claim 8 or 9 with a material containing glutamic acid and/ or glutamate . A method for producing a GABA-containing food product, comprising contacting the polypeptide of claim 1 or 2, the enzyme preparation of claim 3, or the host cell of claim 8 or 9 with a food material containing glutamic acid and/or glutamate.