JP7181712B2 - Glutathione manufacturing method - Google Patents

Description

The present invention relates to a method for producing glutathione.

Glutathione (γ-glutamyl-L-cysteinylglycine) is a tripeptide consisting of L-cysteine, L-glutamic acid, and glycine. It is an important compound for living organisms such as oxidation.

As a method for producing glutathione, a method of producing glutathione in yeast by fermentation using yeast (Patent Document 1), γ-glutamylcysteine produced by a microorganism having the ability to produce glutathione, L-cysteine, L-glutamic acid, and glycine. An enzymatic method for synthesis using synthase or glutathione synthase is known (Patent Document 2).

There is also a method of producing a target organic compound by using a microorganism as a reaction system for a synthetic reaction and utilizing the metabolic pathway of the microorganism. In order to synthesize organic compounds more efficiently with this method, instead of modifying the metabolic pathways of living microorganisms, multiple metabolic enzymes are modularized in advance and combined arbitrarily to specialize in substance production. There is also known an artificial metabolic system that artificially constructs a synthetic pathway that has been developed (Patent Document 3). Since this artificial metabolic system uses multiple enzymes, the stability of the enzymes is important. Therefore, a thermostable enzyme is used. In addition, since a plurality of enzymes are used, there is a problem that purification of the enzyme is costly, but the purification cost can be reduced by using a thermostable enzyme.

As thermostable γ-glutamylcysteine synthetase and glutathione synthetase, enzymes having activity by reaction at 45° C. are known (Non-Patent Document 1). In addition, it was reported that oxidized glutathione was detected in the cell extract of the hyperthermophilic archaea Sulfolobus solfataricus (Non-Patent Document 2). Glutathione was not detected in the cell extracts of S. achidolacdarius and the thermophilic bacterium Thermus thermophilus in excess of the amount brought in from the medium components.

JP-A-8-70884 JP-A-60-27396 WO2016/136620

Zhang et al., 2017, J. Biotech Heinemann J et al., 2014, Biochim Biophys Acta.

By using an artificial metabolic system, it may be possible to produce organic compounds that have been difficult to produce in organic synthesis. , can efficiently produce glutathione. Accordingly, an object of the present application is to find a thermostable γ-glutamylcysteine synthase or glutathione synthase and to provide a novel method for producing glutathione.

The present inventors provide novel thermostable γ-glutamylcysteine synthase and glutathione synthase, and provide a method for producing glutathione using these enzymes.

Glutathione synthase was identified from Thermosynechococcus elongatus, a thermophilic cyanobacterium. Furthermore, a gene with γ-glutamylcysteine synthase activity was also identified from genes of unknown function.

The present invention is the following invention.
(1) A polypeptide having γ-glutamylcysteine synthase activity consisting of the amino acid sequence of SEQ ID NO: 1 in L-glutamic acid and L-cysteine, or having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1 A method for producing γ-glutamylcysteine, comprising the step of producing γ-glutamylcysteine by allowing a polypeptide having γ-glutamylcysteine synthase activity to act,
(2) A polypeptide having γ-glutamylcysteine synthase activity consisting of the amino acid sequence of SEQ ID NO: 1 in L-glutamic acid and L-cysteine, or having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1 A step of reacting a polypeptide having γ-glutamylcysteine synthase activity to produce γ-glutamylcysteine, then synthesizing glutathione consisting of the amino acid sequence of SEQ ID NO: 2 or 3 with glycine and γ-glutamylcysteine A step of producing glutathione by reacting a polypeptide having enzymatic activity or a polypeptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 2 or 3 and having glutathione synthetase activity , a method for the production of glutathione.
(3) A polypeptide having glutathione synthetase activity consisting of glycine and γ-glutamylcysteine, consisting of the amino acid sequence of SEQ ID NO: 2 or 3, or sequence identity of at least 90% or more to the amino acid sequence of SEQ ID NO: 2 or 3 A method for producing glutathione, comprising the step of producing glutathione by allowing a polypeptide having a glutathione synthetase activity to act.

γ-GC production from L-glutamic acid and L-cysteine, glutathione production from glycine and γ-GC, and furthermore L-glutamic acid, L-cysteine and glycine using a thermostable enzyme with an optimum temperature of 65°C. It is possible to perform glutathione production from E. coli at a much higher temperature than has been the case in the past (45°C).

Results of calculating specific activity based on the amount of γ-GC produced (temperature) Results of calculating specific activity based on the amount of γ-GC produced (pH) Amount of γ-GC and residual L-cysteine produced Result of calculating specific activity based on the amount of glutathione produced (temperature) Results of calculating specific activity based on the amount of glutathione produced (pH) Glutathione and γ-GC produced, amount of residual L-cysteine

The present invention relates to a method for producing glutathione, wherein L-glutamic acid, L-cysteine and glycine are used as substrates and glutathione is produced by thermophile-derived γ-glutamylcysteine synthase and thermophile-derived glutathione synthase.

In the present application, γ-glutamylcysteine synthetase may be abbreviated as γ-GCL, glutathione synthase as GSHS, glutathione as GSH, and oxidized glutathione as GSSG.

γ-GCL of the present invention catalyzes a reaction in which L-cysteine is used as a substrate in the presence of adenosine triphosphate (ATP) and is bound to L-glutamic acid to produce γ-glutamylcysteine (γ-Glu-Cys). It is an enzyme that has the activity to

GSHS of the present invention uses γ-glutamylcysteine as a substrate in the presence of adenosine triphosphate (ATP), binds to glycine, and produces γ-glutamyl-L-cysteinylglycine (γ-Glu-Cys-Gly: glutathione). is an enzyme that has the activity to catalyze the reaction that produces

The thermophilic bacterium of the present invention is a bacterium that expresses an enzyme that can maintain its activity without denaturation at 50°C or higher, particularly at 60 to 110°C. γ-GCL and GSHS in the present invention may be derived from thermophilic bacteria, but may not be derived from thermophilic bacteria as long as the enzymes are not denatured at 50° C. or higher.

γ-GCL of the present invention has a polypeptide having the amino acid sequence of SEQ ID NO: 1 and an amino acid sequence having at least 90% or more, or at least 95% or more sequence identity with SEQ ID NO: 1, It is an active polypeptide.

The GSHS of the present invention has a polypeptide having the amino acid sequence of SEQ ID NO: 2 or 3 and an amino acid sequence having at least 90% or more, or at least 95% or more sequence identity to SEQ ID NO: 2 or 3, It is an active polypeptide.

The γ-GCL and GSHS of the present invention can use those having the amino acid sequences up to the preceding paragraph and further derived from Thermosynechococcus elongatus, which is a thermophilic cyanobacterium.

The method for obtaining γ-GCL and GSHS used in the present invention is not particularly limited. For example, when it is obtained using genetic engineering techniques, a gene encoding the desired enzyme is inserted into an appropriate vector to construct a recombinant vector. An enzyme can be expressed and produced by transforming the recombinant vector into a host cell capable of enzyme production. In the present invention, since multiple enzymes are used, Escherichia coli such as DH5α and MG1655 strains, Gram-negative bacteria such as Pseudomonas, and Gram-positive bacteria such as Corynebacterium, Bacillus, and Rhodococcus, which can be easily transformed, are suitable. there is Specifically, for γ-GCL and GSHS, the gene amplified by PCR from the genomic DNA of Thermosynechococcus elongatus or the like is ligated to, for example, pET21a and expressed under the control of the T7 promoter. Genomic DNA of Thermosynechococcus elongatus etc. can be obtained from RIKEN BioResource Center, National Institute for Environmental Studies, National Institute of Technology and Evaluation Biotechnology Center (NBRC), Kazusa DNA Research Institute, etc. be. DNA synthesized from the amino acid sequence information of SEQ ID NOS: 1 to 3 can also be used as the DNA to be used, and the synthetic DNA is a DNA sequence whose amino acid sequence is designed to match the codon usage frequency of E. coli. From these DNAs, E. coli, such as BL21 (DE3) pLysS manufactured by Novagen, is transformed with an expression vector containing the gene in question as described above. Alternatively, a DNA fragment may be inserted by homologous recombination or transposon. A general method may be used for the transformation method.

Enzymes required in the present invention are selected from Escherichia coli such as DH5α and MG1655 strains, Gram-negative bacteria such as the genus Pseudomonas, Gram-positive bacteria such as the genus Corynebacterium, the genus Bacillus, and the genus Rhodococcus, and γ-GCL and GSHS. It can be obtained by expression. γ-GCL and GSHS may be obtained simultaneously. The fungi used in this paragraph need not be thermophilic, as they readily remove host-derived proteins, as described below. Although all necessary enzymes may be expressed simultaneously, γ-GCL and GSHS can be obtained by dividing into a plurality of Escherichia coli or the like from the viewpoint of expression efficiency and the like, as described in the Examples. In the present invention, the thermophile-derived enzyme is obtained by expressing it in Escherichia coli or the like, so proteins derived from the host such as Escherichia coli can be easily removed. For example, the protein derived from E. coli is denatured by heat treatment at a high temperature of 60° C. to 80° C. after expression in E. coli, but the target enzyme is not denatured by heat because it is derived from thermophilic bacteria. Thus, the necessary crude enzyme solution can be easily obtained by removing proteins denatured by heat treatment. The heat treatment may be performed by directly heat-treating the cultured cells. Alternatively, the cell extract may be heat-treated. An extraction method can be selected without particular limitation. After crushing the cells by ultrasonic crushing or the like, the extracted liquid may be heat-treated. Specifically, for example, wet cells of recombinant Escherichia coli are suspended in 50 mM HEPES-NaOH (pH 8.0) so as to have a concentration of 200 mg wet cells/ml, and the suspension is subjected to ultrasonication treatment. The body is crushed to obtain a cell-free extract. The cell-free extract is heat-treated at 70° C. for 30 minutes to denature the host-derived protein. By removing cell debris and denatured protein by centrifugation, the supernatant can be used as a crude enzyme solution for the production of glutathione.

The method for producing glutathione according to the present invention includes a step of reacting L-cysteine and L-glutamic acid with the aforementioned γ-GCL in the presence of ATP to produce γ-glutamylcysteine; It includes the step of producing glutathione in the presence of ATP by the aforementioned GSHS.

An enzymatic reaction with γ-GCL and GSHS can be carried out in a general reaction solution containing a solvent adjusted to an appropriate pH. The substrate concentration (total concentration of L-cysteine, L-glutamic acid, and glycine) can react at a general concentration. The quantitative ratio of L-cysteine, L-glutamic acid and glycine can be selected without any particular limitation. Generally, equal amounts of L-cysteine, L-glutamic acid, and glycine are sufficient. Buffers such as HEPES, TAPS, and CHES can be used as solvents. A particularly preferred buffer uses TAPS buffer. The pH of the reaction is pH 6-10, more preferably pH 8-9. The γ-GCL reaction temperature is 60° C. to 75° C., preferably 65° C. or higher. The reaction time can be adjusted as appropriate, and can be from 10 minutes to 120 minutes.

Glutathione of the present invention may be produced by γ-GCL to produce γ-glutamylcysteine (γ-GC), and then by GSHS to produce glutathione by γ-GC and glycine. GC production and glutathione production may be performed simultaneously. When performed separately, the reaction of γ-GCL is performed under the conditions of Paragraph No. 0024. The GSHS reaction can also be performed under the conditions of paragraph 0024, but by setting the temperature to 55 to 65° C., glutathione can be produced more efficiently.

The concentration of each enzyme varies depending on the preparation of the crude enzyme solution, and may be appropriately adjusted by a general method. The concentration ratio of γ-GCL and GSHS can also be adjusted as appropriate and is not limited.

In addition, ATP, magnesium chloride and the like are required for the reaction and can be used under general conditions.

Although the present invention will be described in more detail below, the present invention is not limited to the following methods.

(Adjustment of crude enzyme solution)
Wet cells of the recombinant E. coli were suspended in 50 mM HEPES-NaOH (pH 8.0) at 200 mg wet cells/ml. By subjecting the suspension to ultrasonication, the cells were crushed to obtain a cell-free extract. The cell-free extract was heat-treated at 60° C. for 30 minutes to denature the host-derived protein. The supernatant obtained by removing cell debris and denatured protein by centrifugation was used as a crude enzyme solution for activity measurement.

・Used enzyme gene, strain, culture
Thermosynechococcus elongatus-derived γ-glutamylcystein ligase (γ-GCL) and glutathione synthetase (GSHS) were expressed under the control of the T7 promoter by ligating synthetic DNAs corresponding to SEQ ID NOs: 1 and 2 to pET21a. All of these gene expression vectors were introduced into Novagen's Rosetta 2 (DE3) pLysS. For Rosetta2 (DE3) pLysS, 100 mg/l ampicillin and 34 mg/l chloramphenicol were added to Luria-Bertani medium and cultured aerobically at 37°C. At the late logarithmic growth stage, 0.2 mM IPTG was added to the culture to induce the target enzyme gene.

The concentrations of γ-GC and glutathione were measured according to the method reported by Steele et al. (Steele et al., 2012, Analytical Biochemistry). Specifically, it is as follows.

・Add 1 mM TCEP (Tris(2-carboxyethyl)phosphine hydrochloride) to each 160 μl of sample and standard (mixture of 100 μM each of L-cysteine, γ-GC, and GSH) after derivatization enzyme reaction, and heat at 35°C. Warmed for 5 minutes. Subsequently, 200 μl of Borate buffer (0.1 M Borate buffer (pH 9.3) with 1 mM EDTA) and 60 μl of ABD-F (1 mg/ml 4-Fluoro-7-aminosulfonylbenzofurazan in Borate buffer) were added. C. for 10 minutes. After adding 100 μl of 2 M HCl to stop the reaction, the mixture was centrifuged, and the centrifugal supernatant obtained was used for the measurement of glutathione.

・High-performance liquid chromatography (HPLC) measurement conditions Eluent: A solution of acetic acid diluted to 0.1 M with ultrapure water, adjusted to pH 4.0 with sodium hydroxide, and mixed with methanol at a volume ratio of 86:14. : 0.8ml/min
Column: COSMOSIL Packed Column 5C18-AR-II 4.6ID x 250mm
Column temperature: 35°C
Measurement wavelength: UV 390 nm (excitation wavelength: 390 nm, fluorescence wavelength: 510 nm)

(Enzyme activity evaluation of γ-GCL)
- Examination of Optimal Temperature An enzyme solution was prepared with the composition shown in Table 1, and used to prepare a reaction solution with the composition shown in Table 2 excluding ATP. After incubating for 2 minutes at each temperature of 40, 45, 50, 55, 60, 65, 70 and 75°C, the reaction was initiated by adding ATP. After reacting for 10 minutes, an equal amount of methanol was added to stop the reaction, and the γ-GC produced was quantified by HPLC. FIG. 1 shows the result of calculating the specific activity based on the amount of γ-GC produced. From this result, it became clear that the optimum temperature for γ-GCL is 65°C.

Examination of optimal pH An enzyme solution was prepared with the composition shown in Table 1, and used to prepare a reaction solution with the composition shown in Table 3, excluding ATP. As shown in FIG. 1, the optimum temperature for γ-GCL was 65° C., so after incubating at 65° C. for 2 minutes, ATP was added to initiate the reaction. After reacting for 10 minutes, an equal volume of methanol was added to stop the reaction, and the γ-GC produced was quantified by HPLC. FIG. 2 shows the result of calculating the specific activity based on the amount of γ-GC produced. This result revealed that the optimum pH of γ-GCL was 9.0, and that the activity was particularly high when TAPS was used as a buffer.

(Generation of γ-GC)
Enzyme solutions were prepared with the compositions shown in Table 1, and reaction solutions were prepared with the compositions shown in Table 4, excluding ATP. After incubating at 65° C. for 2 minutes, the reaction was initiated by adding ATP. After 1, 3, 5, 10, 20 and 30 minutes, samples were taken, and an equal amount of methanol was added to each to stop the reaction, and the γ-GC produced was quantified by HPLC. As a result, 1.21 mM γ-GC could be confirmed 30 minutes after the start of the reaction. The amounts of γ-GC produced and residual L-cysteine are shown in FIG.

(Enzyme activity evaluation of GSHS)
· Examination of optimal temperature An enzyme solution was prepared with the composition shown in Table 5, and used to prepare a reaction solution with the composition shown in Table 6, excluding ATP. After incubating for 2 minutes at each temperature of 40, 45, 50, 55, 60, 65, 70 and 75°C, the reaction was initiated by adding ATP. After reacting for 10 minutes, an equal amount of methanol was added to stop the reaction, and the produced glutathione was quantified by HPLC. FIG. 4 shows the result of calculating the specific activity based on the amount of glutathione produced. From this result, it was clarified that although the optimum temperature of GSHS is 55°C, almost the same activity is exhibited up to 55 to 65°C.

Examination of optimal pH An enzyme solution was prepared with the composition shown in Table 5, and used to prepare a reaction solution with the composition shown in Table 7, excluding ATP. As shown in FIG. 4, since the activity of GSHS was almost the same from the optimum temperature of 55° C. to 65° C., the reaction was initiated by adding ATP after incubating at 65° C. for 2 minutes. After reacting for 10 minutes, an equal amount of methanol was added to stop the reaction, and the produced glutathione was quantified by HPLC. FIG. 5 shows the results of calculating the specific activity based on the amount of glutathione produced. From this result, it was clarified that the optimum pH of GSHS is 9.0, and the activity is particularly high when TAPS is used as a buffer.

(Generation of glutathione (GSH))
Enzyme solutions were prepared with the compositions shown in Tables 1 and 5, and used to prepare reaction solutions with the compositions shown in Table 8 excluding ATP. After incubating at 65° C. for 2 minutes, the reaction was initiated by adding ATP. After 1, 3, 5, 10, 20 and 30 minutes, samples were taken, an equal amount of methanol was added to each to stop the reaction, and the produced glutathione was quantified by HPLC. As a result, 0.54 mM glutathione was confirmed 30 minutes after the start of the reaction. FIG. 6 shows the amount of glutathione produced, γ-GC, and residual L-cysteine. According to this, the concentration of γ-GC reached a maximum 10 minutes after the start of the reaction and decreased thereafter. This is probably because the production rate of γ-GC by γ-GCL exceeded the consumption rate of γ-GC by GSHS until 10 minutes after the start of the reaction. In fact, the amount of glutathione produced uniformly increased, suggesting that glutathione production by GSHS was the rate-limiting step.

Based on the above, in the present invention, a thermostable enzyme having an optimum temperature in the high temperature range of 65° C. is used to produce γ-GC from L-glutamic acid and L-cysteine, to produce glutathione from glycine and γ-GC, and further to produce L- Glutathione production from glutamic acid, L-cysteine and glycine can be carried out at much higher temperatures than in the past (45° C.).

Claims (3)

A polypeptide having γ-glutamylcysteine synthase activity consisting of the amino acid sequence of SEQ ID NO: 1 for non-thermophilic bacteria , or γ-glutamylcysteine having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1 a step of expressing a polypeptide having synthase activity; a step of heat-treating a culture solution containing the polypeptide at 60 to 80° C. to obtain a crude enzyme solution; A method for producing γ-glutamylcysteine, comprising the step of reacting at 60 to 75°C to produce γ-glutamylcysteine. A polypeptide having γ-glutamylcysteine synthase activity consisting of the amino acid sequence of SEQ ID NO: 1 for non-thermophilic bacteria , or γ-glutamylcysteine having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1 A step of expressing a polypeptide having synthase activity, a step of heat-treating a culture solution containing the polypeptide at 60 to 80°C to obtain a crude enzyme solution, and a step of converting the enzyme solution into L-glutamic acid and L-cysteine. A step of acting at 60 to 75° C. to produce γ-glutamylcysteine, and then a polypeptide having glutathione synthetase activity consisting of the amino acid sequence of SEQ ID NO: 2 or 3, or SEQ ID NO: 2 or 3, on non-thermophilic bacteria A step of expressing a polypeptide having at least 90% sequence identity with the amino acid sequence of and having glutathione synthase activity, and heat-treating a culture medium containing the polypeptide at 60 to 80 ° C. to obtain a crude enzyme A method for producing glutathione, comprising the steps of: obtaining a liquid; and allowing the enzyme liquid to act on glycine and γ-glutamylcysteine at 55 to 65°C to produce glutathione. A polypeptide having glutathione synthetase activity consisting of the amino acid sequence of SEQ ID NO: 2 or 3 in non-thermophilic bacteria , or a glutathione synthase having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 2 or 3 a step of expressing a polypeptide having activity; a step of heat-treating a culture solution containing the polypeptide at 60 to 80°C to obtain a crude enzyme solution; A method for producing glutathione, comprising the step of producing glutathione by acting at 65°C .

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