METHOD FOR PRODUCING GAMMA-GLUTAMYLCYSTEINE AND GLUTATHIONE

Abstract

The technical problem to be solved by the present invention is to provide a method for producing glutathione and a precursor thereof, -glutamylcysteine, at a high yield. As a means for solving the problem, the method for producing glutathione according to the present invention includes step A of reacting L-cysteine and L-glutamic acid under a low-oxygen atmosphere to produce -glutamylcysteine and step B of reacting -glutamylcysteine and glycine under a low-oxygen atmosphere to produce glutathione.

Claims

1. A method for producing -glutamylcysteine, comprising: reacting L-cysteine and L-glutamic acid under an atmosphere having a lower oxygen concentration than atmospheric air to produce -glutamylcysteine.

2. The method according to claim 1, wherein the reacting of L-cysteine and L-glutamic acid is carried out by the action of at least one enzyme selected from the group consisting of -glutamylcysteine synthetase and a bifunctional glutathione synthetase in the presence of adenosine triphosphate (ATP).

3. The method according to claim 2, wherein the reacting of L-cysteine and L-glutamic acid is carried out in conjugation with an ATP regeneration reaction for regenerating adenosine diphosphate (ADP) into adenosine triphosphate (ATP).

4. The method according to claim 2, wherein the -glutamylcysteine synthetase is derived from Escherichia coli.

5. The method according to claim 2, wherein the bifunctional glutathione synthetase is derived from Streptococcus agalactiae.

6. A method for producing glutathione, comprising: reacting -glutamylcysteine and glycine under an atmosphere having a lower oxygen concentration than atmospheric air to produce glutathione.

7. The method according to claim 6, wherein the reacting of -glutamylcysteine and glycine is carried out by the action of at least one enzyme selected from the group consisting of glutathione synthetase and a bifunctional glutathione synthetase in the presence of adenosine triphosphate (ATP).

8. The method according to claim 7, wherein the reacting of -glutamylcysteine and glycine is carried out in conjugation with an ATP regeneration reaction for regenerating adenosine diphosphate (ADP) into adenosine triphosphate (ATP).

9. The method according to claim 7, wherein the glutathione synthetase is derived from Escherichia coli.

10. The method according to claim 7, wherein the bifunctional glutathione synthetase is derived from Streptococcus agalactiae.

11. The method according to claim 6, further comprising, prior to the reacting of -glutamylcysteine and glycine: reacting L-cysteine and L-glutamic acid under an atmosphere having a lower oxygen concentration than atmospheric air to produce the -glutamylcysteine.

12. The method according to claim 11, wherein the reacting of L-cysteine and L-glutamic acid is carried out by the action of at least one enzyme selected from the group consisting of -glutamylcysteine synthetase and a bifunctional glutathione synthetase in the presence of adenosine triphosphate (ATP).

13. The method according to claim 12, wherein the reacting of L-cysteine and L-glutamic acid is carried out in conjugation with an ATP regeneration reaction for regenerating adenosine diphosphate (ADP) into adenosine triphosphate (ATP).

14. The method according to claim 12, wherein the -glutamylcysteine synthetase is derived from Escherichia coli.

15. The method according to claim 12, wherein the bifunctional glutathione synthetase is derived from Streptococcus agalactiae.

16. The method according to claim 2, wherein the -glutamylcysteine synthetase has an amino acid sequence having 95% or more identity to the amino acid sequence of SEQ ID NO: 9, and the bifunctional glutathione synthetase has an amino acid sequence having 95% or more identity to the amino acid sequence of SEQ ID NO: 12.

17. The method according to claim 7, wherein the glutathione synthetase has an amino acid sequence having 95% or more identity to the amino acid sequence of SEQ ID NO: 10.

18. The method according to claim 12, wherein the reacting of -glutamylcysteine and glycine is carried out by the action of at least one enzyme selected from the group consisting of glutathione synthetase and a bifunctional glutathione synthetase in the presence of adenosine triphosphate (ATP), the -glutamylcysteine synthetase has an amino acid sequence having 95% or more identity to the amino acid sequence of SEQ ID NO: 9, the bifunctional glutathione synthetase has an amino acid sequence having 95% or more identity to the amino acid sequence of SEQ ID NO: 12, and the glutathione synthetase has an amino acid sequence having 95% or more identity to the amino acid sequence of SEQ ID NO: 10.

Description

EXAMPLES

<Experiment 1>

[0138] Preparation of -glutamylcysteine Synthetase (GSH I) Derived from Escherichia coli K12 Strain

[0139] A DNA primer (Primer-1: SEQ ID NO: 2) having a restriction enzyme Sad cleavage site and an SD sequence bound to the nucleotide sequence corresponding to the N terminal portion of the GSH I gene (SEQ ID NO: 1); and a DNA primer (Primer-2: SEQ ID NO: 3) having a restriction enzyme KpnI cleavage site bound to the nucleotide sequence corresponding to the C terminal portion of the GSH I gene (SEQ ID NO: 1) were prepared. Using these DNA primers, DNA between these sequences was amplified by PCR to obtain a DNA fragment containing a full length of the GSH I gene. The template used in the PCR amplification was the genomic DNA of Escherichia coli K12 strain. The result of analysis of the nucleotide sequence of the obtained DNA fragment showed that the full length of GSH I gene (SEQ ID NO: 1) was contained. The obtained DNA fragment was inserted between a Sad recognition site and a KpnI recognition site present at the downstream of a lac promoter of plasmid pUC18 (GenBank Accession No. L09136, manufactured by Takara Bio Inc.) to construct a recombinant vector, pUCGSHI. E. coli HB101 competent cell (manufactured by Takara Bio Inc.) was transformed with the recombinant vector pUCGSHI to obtain E. coli HB101 (pUCGSHI). The obtained transformant was inoculated on 50 ml of 2YT culture medium (tryptone 1.6%, yeast extract 1.0%, NaCl 0.5%, pH7.0) containing 200 g/ml ampicillin and subjected to shaking culture at 37 C. for 24 hours. Then, enzyme activity was measured. As a result, GSH I activity was 5 U/ml and the activity of ADK derived from Escherichia coli used as a host cell was 90 U/ml. Subsequently, bacterial cells were centrifugally collected, suspended in 2.5 ml of a 100 mM phosphate buffer (pH7.0) and sonicated to obtain an enzyme solution.

<Experiment 2>

[0140] Preparation of Glutathione Synthetase (GSH II) Derived from Escherichia coli K12 Strain

[0141] A DNA primer (Primer-3: SEQ ID NO: 5) having a restriction enzyme NdeI cleavage site bound to the nucleotide sequence corresponding to the N terminal portion of a GSH II gene (SEQ ID NO: 4); and a DNA primer (Primer-4: SEQ ID NO: 6) having a restriction enzyme EcoRI cleavage site bound to the nucleotide sequence corresponding to the C terminal portion of GSH II gene (SEQ ID NO: 4) were prepared. Using these DNA primers, DNA between these sequences was amplified by PCR to obtain a DNA fragment containing a full length of the GSH II gene. The template used in the PCR amplification was the genomic DNA of Escherichia coli K12 strain. The result of analysis of the nucleotide sequence of the obtained DNA fragment showed that the full length of GSH II gene (SEQ ID NO: 4) was contained. The obtained DNA fragment was inserted between a NdeI recognition site and an EcoRI recognition site present at the downstream of a lac promoter of plasmid pUCN18 to construct a recombinant vector, pNGSHII. The plasmid pUCN18 was a plasmid obtained by substituting T at the 185th positions of pUC18 (GenBank Accession No. L09136 manufactured by Takara Bio Inc.) with A to destruct an NdeI site and further substituting GC at the 471-472nd positions with TG to newly introduce a NdeI site. E. coli HB101 competent cell (manufactured by Takara Bio Inc.) was transformed with the recombinant vector pNGSHII to obtain E. coli HB 101 (pNGSHII). The obtained transformant was inoculated on 50 ml of 2YT culture medium (tryptone 1.6%, yeast extract 1.0%, NaCl 0.5%, pH7.0) containing 200 g/ml ampicillin and subjected to shaking culture at 37 C. for 24 hours. Then, enzyme activity was measured. As a result, GSH II activity was 5 U/ml and the activity of ADK derived from Escherichia coli used as a host cell was 90 U/ml. Subsequently, bacterial cells were centrifugally collected, suspended in 2.5 ml of a 100 mM phosphate buffer (pH7.0) and sonicated to obtain an enzyme solution.

[0142] <Experiment 3>

[0143] Preparation of Bifunctional Glutathione Synthetase (GSH F) Derived from Streptococcus agalactiae

[0144] A GSH F gene fragment (SEQ ID NO: 7) derived from Streptococcus agalactiae was manufactured in accordance with a gene synthesis method (by Eurogentec). In this GSH F gene fragment, the codons were optimized for expression in Escherichia coli; and a restriction enzyme NdeI cleavage site was bound to the nucleotide sequence corresponding to the N terminal portion; and a restriction enzyme EcoRI cleavage site was bound to the nucleotide sequence corresponding to the C terminal portion. The obtained DNA fragment was inserted between a NdeI recognition site and an EcoRI recognition site present at the downstream of a lac promoter of plasmid pUCN18 to construct a recombinant vector, pNGSHF. The plasmid pUCN18 was a plasmid obtained by substituting T at the 185th position of pUC18 (GenBank Accession No. L09136 manufactured by Takara Bio Inc.) with A to destruct an NdeI site and further substituting GC at the 471-472nd positions with TG to newly introduce a NdeI site. E. coli HB101 competent cell (manufactured by Takara Bio Inc.) was transformed with the recombinant vector pNGSHF to obtain E. coli HB101 (pNGSHF). The obtained transformant was inoculated on 50 ml of 2YT culture medium (tryptone 1.6%, yeast extract 1.0%, NaCl 0.5%, pH7.0) containing 200 g/ml ampicillin and subjected to shaking culture at 37 C. for 24 hours. Then, enzyme activity was measured. As a result, GSH F activity was 3 U/ml and the activity of ADK derived from Escherichia coli used as a host cell was 90 U/ml. Subsequently, bacterial cells were centrifugally collected, suspended in 2.5 ml of a 100 mM phosphate buffer (pH7.0) and sonicated to obtain an enzyme solution.

<Experiment 4>

[0145] Preparation of AMP phosphotransferase (PAP) derived from Acinetobacter johnsonii A PAP gene fragment (SEQ ID NO: 8) was manufactured in accordance with a gene synthesis method (by Eurogentec). In this PAP gene fragment, the codons were optimized for expression in Escherichia coli; and a restriction enzyme NdeI cleavage site was bound to the nucleotide sequence corresponding to the N terminal portion; and a restriction enzyme EcoRI cleavage site was bound to the nucleotide sequence corresponding to the C terminal portion. The obtained DNA fragment was inserted between a NdeI recognition site and an EcoRI recognition site present at the downstream of a lac promoter of plasmid pUCN18 to construct a recombinant vector, pNPAP. The plasmid pUCN18 was a plasmid obtained by substituting T at the 185th position of pUC18 (GenBank Accession No. L09136 manufactured by Takara Bio Inc.) with A to destruct an NdeI site and further substituting GC at the 471-472nd positions with TG to newly introduce a NdeI site. E. coli HB101 competent cell (manufactured by Takara Bio Inc.) was transformed with the recombinant vector pNPAP to obtain E. coli HB101 (pNPAP). The obtained transformant was inoculated on 50 ml of 2YT culture medium (tryptone 1.6%, yeast extract 1.0%, NaCl 0.5%, pH7.0) containing, 200 g/m1 ampicillin and subjected to shaking culture at 37 C. for 24 hours. Then, enzyme activity was measured. As a result, PAP activity was 40 U/ml and the activity of ADK derived from Escherichia coli used as a host cell was 90 U/ml. Subsequently, bacterial cells were centrifugally collected, suspended in 2.5 ml of a 100 mM phosphate buffer (pH7.0) and sonicated to obtain an enzyme solution.

<Calculation of Yield>

[0146] A method of calculating the yield of each of the compounds obtained in the Experiments herein was as follows.

[0147] A reaction product was quantified based on analysis by high-performance liquid chromatography and then the yield thereof was determined by the following expression:

Yield: the production amount (mol) of compound/starting L-cysteine (mol)100

[0148] The conditions of the above high-performance liquid chromatography are as follows. In the elution conditions, glutathione (GSH), -glutamylcysteine (-GC), oxidized -GC, oxidized glutathione (GSSG) sequentially elute in this order.

[Analysis of Yield]

[0149] Column: ODS-HG-3 (4.6 mm150 mm, manufactured by Nomura Chemical Co., Ltd.);

[0150] Eluent: Solution prepared by dissolving potassium dihydrogenphosphate (12.2 g) and sodium heptane sulfonate (3.6 g) in distilled water (1.8 L), controlling pH of the solution to be pH 2.8 with phosphoric acid and further adding methanol (186 ml);

[0151] Flow rate: 1.0 ml/minute;

[0152] Column temperature: 40 C.;

[0153] Measurement wavelength: 210 nm.

Example 1

Reaction in System Containing an Equivalent Amount of ATP Under a Nitrogen Atmosphere

[0154] The reaction of Example 1 described below was carried out under a nitrogen atmosphere.

[0155] (Production of -glutamylcysteine)

##STR00002##

[0156] Monosodium L-glutamate monohydrate (0.3668 g (2.17 mmol)), L-cysteine hydrochloride monohydrate (0.3636 g (2.07 mmol)), magnesium sulfate hepta hydrate (1.02 g), ATP (1.19 g (2.16 mmol)), and distilled water (15 g) were mixed. The pH of the mixture was controlled to be 7.5 with a 15 wt % aqueous sodium hydroxide solution (1.4 g). To the mixture, the -glutamylcysteine synthetase (GSH I) solution (1 g) prepared in Experiment 1 was added and a reaction was initiated. The reaction was carried out in a reaction vessel provided with a nitrogen port and an exhaust port in the conditions where the oxygen concentration of the gaseous phase was maintained at 0 vol % as much as possible by supplying nitrogen at a rate of 10 ml/min through the nitrogen port to purge the air (gaseous phase) within the reaction vessel. The temperature during the reaction was set at 30 C. The reaction continuously proceeded and produced -glutamylcysteine and oxidized glutamylcysteine. Six hours after initiation of the reaction, L-cysteine disappeared. The yields of -glutamylcysteine, and oxidized glutamylcysteine six hours after initiation of the reaction relative to the starting L-cysteine were 97 mol % and 1 mol %, respectively.

[0157] (Production of Glutathione)

##STR00003##

[0158] After the 6-hour reaction, to the reaction solution, glycine (0.192 g (2.56 mmol)), magnesium sulfate hepta hydrate (1.02 g) and ATP (1.19 g (2.16 mmol)) were mixed and the pH of the mixture was controlled to be 7.5 with a 15 wt % aqueous sodium hydroxide solution (1.4 g). To the reaction mixture, the glutathione synthetase (GSH II) solution (1 g) prepared in Experiment 2 was added and a reaction was initiated. The reaction was carried out under a nitrogen atmosphere in the same manner as in the step of producing -glutamylcysteine. The temperature during the reaction was set at 30 C. The reaction continuously proceeded. Four hours after initiation of the reaction, -glutamylcysteine and oxidized glutamylcysteine disappeared and glutathione and oxidized glutathione were produced. The yields of glutathione and oxidized glutathione four hours after initiation of the reaction relative to the starting L-cysteine were 94 mol % and 2 mol %, respectively.

Example 2

Reaction with ATP Regenerating System Under a Nitrogen Atmosphere

[0159] The reaction of Example 2 described below was carried out under a nitrogen atmosphere.

[0160] (Production of -glutamylcysteine)

##STR00004##

[0161] Monosodium L-glutamate monohydrate (0.389 g (2.30 mmol)), L-cysteine hydrochloride monohydrate (0.3714 g (2.11 mmol)), magnesium sulfate hepta hydrate (0.7068 g), ATP (0.0588 g (0.11 mmol)), sodium metaphosphate (0.8 g) and distilled water (14 g) were mixed and the pH of the mixture was controlled to be 7.5 with a 15 wt % aqueous sodium hydroxide solution (0.8 g). To the mixture, the -glutamylcysteine synthetase (GSH I) solution (1 g) prepared in Experiment 1 and the PAP solution (1 g) prepared in Experiment 4 were added and a reaction was initiated. The reaction was carried out in a reaction vessel provided with a nitrogen port and an exhaust port in the conditions where the oxygen concentration of the gaseous phase was maintained at 0 vol % as much as possible by supplying nitrogen at a rate of 10 ml/min through the nitrogen port to purge the air (gaseous phase) within the reaction vessel. The temperature during the reaction was set at 30 C. The reaction continuously proceeded and produced -glutamylcysteine and oxidized glutamylcysteine. Six hours after initiation of the reaction, L-cysteine disappeared. The yields of -glutamylcysteine and oxidized glutamylcysteine six hours after initiation of the reaction relative to the starting L-cysteine were 95 mol % and 1 mol %, respectively.

[0162] (Production of Glutathione)

##STR00005##

[0163] After the 6-hour reaction, to the reaction solution, glycine (0.19 g (2.53 mmol)) was added and the pH of the mixture was controlled to be 7.5 with a 15 wt % aqueous sodium hydroxide solution (0.2 g). To the reaction mixture, the glutathione synthetase (GSH II) solution (1 g) prepared in Experiment 2 and the PAP solution (1 g) prepared in Experiment 4 were added and a reaction was initiated. The reaction was carried out under a nitrogen atmosphere in the same manner as in the step of producing -glutamylcysteine. The temperature during the reaction was set at 30 C. The reaction continuously proceeded and produced glutathione and oxidized glutathione. Six hours after initiation of the reaction, -glutamylcysteine and oxidized glutamylcysteine disappeared. The yields of glutathione and oxidized glutathione six hours after initiation of the reaction relative to the starting L-cysteine were 82 mol % and 2 mol %, respectively.

[0164] Since the GSH I solution prepared in Experiment 1, GSH II solution prepared in Experiment 2 and PAP solution prepared in Experiment 4 had ADK activity derived from Escherichia coli used as a host cell, no separate preparation of an ADK solution was required in the aforementioned two steps.

Example 3

Reaction with ATP Regenerating System Under a Nitrogen Atmosphere

[0165] The reaction of the Example described below was carried out under a nitrogen atmosphere.

[0166] (Production of Glutathione)

##STR00006##

[0167] Monosodium L-glutamate monohydrate (0.185 g (1.09 mmol)), L-cysteine hydrochloride monohydrate (0.175 g (1.00 mmol)), glycine (1.09 mmol), magnesium sulfate hepta hydrate (0.35 g), ATP (0.055 g (0.10 mmol)), sodium metaphosphate (0.4 g) and distilled water (16 g) were mixed. The pH of the mixture was controlled to be 7.5 with a 15 wt % aqueous sodium hydroxide solution (0.44 g). To the mixture, the bifunctional glutathione synthetase (GSH F) solution (1 g) prepared in Experiment 3 and the PAP solution (1 g) prepared in Experiment 4 were added and a reaction was initiated. The reaction was carried out in a reaction vessel provided with a nitrogen port and an exhaust port in the conditions where the oxygen concentration of the gaseous phase was maintained at 0 vol % as much as possible by supplying nitrogen at a rate of 10 ml/min through the nitrogen port to purge the air (gaseous phase) within the reaction vessel. The temperature during the reaction was set at 30 C. The reaction continuously proceeded and produced glutathione and oxidized glutathione. Six hours after initiation of the reaction, L-cysteine disappeared. The yields of glutathione and oxidized glutathione six hours after initiation of the reaction relative to the starting L-cysteine were 90 mol % and 1 mol %, respectively.

[0168] Since the GSH F solution prepared in Experiment 3 and the PAP solution prepared in Experiment 4 had ADK activity derived from Escherichia coli used as a host cell, no separate preparation of an ADK solution was required in the step mentioned above.

Comparative Example 1

Reaction in System Containing an Equivalent Amount of ATP Under an Air Atmosphere>

[0169] The reaction of Comparative Example 1 described below was carried out under an air atmosphere.

[0170] (Production of -glutamylcysteine)

##STR00007##

[0171] Monosodium L-glutamate monohydrate (0.3668 g (2.17 mmol)), L-cysteine hydrochloride monohydrate (0.3636 g (2.07 mmol)), magnesium sulfate hepta hydrate (1.02 g), ATP (1.19 g (2.16 mmol)), and distilled water (15 g) were mixed. The pH of the mixture was controlled to be 7.5 with a 15 wt % aqueous sodium hydroxide solution (1.4 g). To the mixture, the -glutamylcysteine synthetase (GSH I) solution (1 g) prepared in Experiment 1 was added and a reaction was initiated. The reaction was carried out in the conditions where the reaction solution was in contact with the air within a reaction vessel without replacement with nitrogen. The temperature during the reaction was set at 30 C. The reaction continuously proceeded and produced -glutamylcysteine and oxidized glutamylcysteine. Six hours after initiation of the reaction, L-cysteine disappeared. The yields of -glutamylcysteine and oxidized glutamylcysteine six hours after initiation of the reaction relative to the starting L-cysteine were 78 mol % and 10 mol %, respectively.

[0172] (Production of Glutathione)

##STR00008##

[0173] After the 6-hour reaction, to the reaction solution, glycine (0.192 g (2.56 mmol)), magnesium sulfate hepta hydrate (1.02 g) and ATP (1.19 g (2.16 mmol)) were added and the pH of the mixture to be 7.5 with a 15 wt % aqueous sodium hydroxide solution (1.4 g). To the reaction mixture, the glutathione synthetase (GSH II) solution (1 g) prepared in Experiment 2 was added and a reaction was initiated. The reaction was carried out in the conditions where the reaction solution was in contact with the air within a reaction vessel without replacement with nitrogen. The temperature during the reaction was set at 30 C. The reaction continuously proceeded. Four hours after initiation of the reaction, -glutamylcysteine and oxidized glutamylcysteine disappeared and glutathione and oxidized glutathione were produced. The yields of glutathione and oxidized glutathione four hours after initiation of the reaction relative to the starting L-cysteine were 58 mol % and 20 mol %, respectively.

Comparative Example 2

Reaction with ATP Regenerating System Under an Air Atmosphere

[0174] The reaction of Comparative Example 2 described below was carried out under an air atmosphere.

[0175] (Production of -glutamylcysteine)

##STR00009##

[0176] Monosodium L-glutamate monohydrate (0.389 g (2.30 mmol)), L-cysteine hydrochloride monohydrate (0.3714 g (2.11 mmol)), magnesium sulfate hepta hydrate (0.7068 g), ATP (0.0588 g (0.11 mmol)), sodium metaphosphate (0.8 g) and distilled water (14 g) were mixed. The pH of the mixture was controlled to be 7.5 with a 15 wt % aqueous sodium hydroxide solution (0.8 g). To the mixture, the -glutamylcysteine synthetase (GSH I) solution (1 g) prepared in Experiment 1 and the PAP solution (1 g) prepared in Experiment 4 were added and a reaction was initiated. The reaction was carried out in the conditions where the reaction solution was in contact with the air within a reaction vessel without replacement with nitrogen. The temperature during the reaction was set at 30 C. The reaction continuously proceeded and produced -glutamylcysteine and oxidized glutamylcysteine. Six hours after initiation of the reaction, L-cysteine disappeared. The yields of -glutamylcysteine and oxidized glutamylcysteine six hours after initiation of the reaction relative to the starting L-cysteine were 72 mol % and 13 mol %, respectively.

[0177] (Production of Glutathione)

##STR00010##

[0178] After the 6-hour reaction, to the reaction solution, glycine (0.19 g (2.53 mmol)) was added and the pH of the mixture was controlled to be 7.5 with a 15 wt % aqueous sodium hydroxide solution (0.2 g). To the reaction mixture, the glutathione synthetase (GSH II) solution (1 g) prepared in Experiment 2 and the PAP solution (1 g) prepared in Experiment 4 were added and a reaction was initiated. The reaction was carried out in the conditions where the reaction solution was in contact with the air within a reaction vessel without replacement with nitrogen. The temperature during the reaction was set at 30 C. The reaction continuously proceeded and produced glutathione and oxidized glutathione. Six hours after initiation of the reaction, -glutamylcysteine and oxidized -glutamylcysteine disappeared. The yields of glutathione and oxidized glutathione six hours after initiation of the reaction relative to the starting L-cysteine were 51 mol % and 22 mol %, respectively.

[0179] Since the GSH I solution prepared in Experiment 1, GSH II solution prepared in Experiment 2 and PAP solution prepared in Experiment 4 had ADK activity derived from Escherichia coli used as a host cell, no separate preparation of an ADK solution was required in the aforementioned two steps.

Comparative Example 3

Reaction with ATP Regenerating System Under an Air Atmosphere

[0180] The reaction of the Comparative Example described below was carried out under an air atmosphere.

[0181] (Production of Glutathione)

##STR00011##

[0182] Monosodium L-glutamate monohydrate (0.185 g (1.09 mmol)), L-cysteine hydrochloride monohydrate (0.175 g (1.00 mmol)), glycine (1.09 mmol), magnesium sulfate hepta hydrate (0.35 g), ATP (0.055 g (0.10 mmol)), sodium metaphosphate (0.4 g) and distilled water (16 g) were mixed. The pH of the mixture was controlled to be 7.5 with a 15 wt % aqueous sodium hydroxide solution (0.44 g). To the mixture, the bifunctional glutathione synthetase (GSH F) solution (1 g) prepared in Experiment 3 and the PAP solution (1 g) prepared in Experiment 4 were added and a reaction was initiated. The reaction was carried out in the conditions where the reaction solution was in contact with the air within a reaction vessel without replacement with nitrogen. The temperature during the reaction was set at 30 C. The reaction continuously proceeded and produced glutathione and oxidized glutathione. Six hours after initiation of the reaction, L-cysteine disappeared. The yields of glutathione and oxidized glutathione six hours after initiation of the reaction relative to the starting L-cysteine were 36 mol % and 14 mol %, respectively. Note that oxidized -glutamylcysteine was produced at a yield of 6 mol %.

[0183] Since the GSH F solution prepared in Experiment 3 and the PAP solution prepared in Experiment 4 have ADK activity derived from Escherichia coli used as a host cell, no separate preparation of an ADK solution was required in the step mentioned above.

Example 4

Reaction with ATP Regenerating System Using Unground Cells Under a Nitrogen Atmosphere

[0184] The reaction described below was carried out under a nitrogen atmosphere.

[0185] (Production of Glutathione)

##STR00012##

[0186] Monosodium L-glutamate monohydrate (0.185 g (1.09 mmol)), L-cysteine hydrochloride monohydrate (0.175 g (1.00 mmol)), glycine (1.09 mmol), magnesium sulfate hepta hydrate (0.35 g), ATP (0.055 g (0.10 mmol)), sodium metaphosphate (0.4 g) and distilled water (16 g) were mixed. The pH of the mixture was controlled to be 7.5 with a 15 wt % aqueous sodium hydroxide solution (0.44 g). To the mixture, a suspension (1 g) containing unground cells of a recombinant Escherichia coli expressing GSH F and a suspension (1 g) containing unground cells of a recombinant Escherichia coli expressing PAP were added and a reaction was initiated. The reaction was carried out in a reaction vessel provided with a nitrogen port and an exhaust port in the conditions where the oxygen concentration of the gaseous phase was maintained at 0 vol % as much as possible by supplying nitrogen at a rate of 10 ml/min through the nitrogen port to purge the air (gaseous phase) within the reaction vessel. The temperature during the reaction was set at 30 C. One hour after initiation of the reaction, the reaction mixture was analyzed. As a result, production of glutathione and oxidized glutathione was found. The conversion rates of them relative to the starting L-cysteine were 7.3 mol % and 0.6 mol %, respectively. Thereafter, the reaction proceeded and virtually stopped seven hours after initiation of the reaction. The conversion rates of them relative to the starting L-cysteine were 10.8 mol % and 1.2 mol %, respectively. ATP, ADP and AMP almost disappeared. Decomposed products of AMP, i.e., adenine, adenosine and hypoxanthine, were found.

[0187] The suspension containing unground cells of a recombinant Escherichia coli expressing GSH F was prepared in the same manner and using the same materials as in Example 3 except that an operation described at the end of the description of Experiment 3 by the phrase: Subsequently, bacterial cells were centrifugally collected, suspended in 2.5 ml of a 100 mM phosphate buffer (pH7.0) and sonicated to obtain an enzyme solution was replaced by an operation described by the phrase: Subsequently, bacterial cells were centrifugally collected, suspended in 2.5 ml of a 100 mM phosphate buffer (pH7.0) to obtain a suspension containing unground cells of a recombinant Escherichia coli expressing GSH F.

[0188] The suspension containing unground cells of a recombinant Escherichia coli expressing PAP was prepared in the same manner and using the same materials as in Example 4 except that an operation described at the end of the description of Experiment 4 by the phrase: Subsequently, bacterial cells were centrifugally collected, suspended in 2.5 ml of a 100 mM phosphate buffer (pH7.0) and sonicated to obtain an enzyme solution was replaced by an operation described by the phrase: Subsequently, bacterial cells were centrifugally collected, suspended in 2.5 ml of a 100 mM phosphate buffer (pH7.0) to obtain a suspension containing unground cells of a recombinant Escherichia coli expressing PAP.

[0189] Since the suspension containing unground cells of a recombinant Escherichia coli expressing GSH F and the suspension containing unground cells of a recombinant Escherichia coli expressing PAP prepared by the aforementioned methods had ADK activity derived from Escherichia coli used as a host cell, no separate preparation of an ADK solution was required in the step mentioned above.

Sequence Listing Free Text

[0190] SEQ ID NO: 2: primer

[0191] SEQ ID NO: 3: primer

[0192] SEQ ID NO: 5: primer

[0193] SEQ ID NO: 6: primer

[0194] All publications including Patents and Patent Applications cited in the specification are incorporated in the specification by reference in their entirety.