Method of continuously producing glutathione using photosynthetic membrane vesicles

Abstract

The present invention relates to a method of producing glutathione, wherein photosynthetic membrane vesicles and enzymes catalyzing glutathione synthesis are combined and glutamate, cysteine and glycine are used as reaction substrates. As enzymes catalyzing glutathione synthesis, γ-glutamylcysteine synthetase and glutathione synthetase may be used together, or bifunctional glutathione synthetase may be used alone. According to the conventional methods, there is a problem in that expensive adenosine triphosphate should be continuously supplied when glutathione is produced. However, according to the present invention, since photosynthetic membrane vesicles are used as a source to regenerate adenosine triphosphate, it is possible to continuously produce glutathione without additionally adding adenosine triphosphate, thereby reducing production costs of glutathione.

1 . A method of producing glutathione, comprising:
 a) a step of generating adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and an inorganic phosphate by irradiating photosynthetic membrane vesicles with light; and   b) a step of synthesizing glutathione by enzymes that catalyze glutathione synthesis using ATP generated in step a), and forming ADP and an inorganic phosphate.   
     
     
         2 . The method according to  claim 1 , further comprising c) a step of reusing ADP and an inorganic phosphate forming in step b) to generate ATP in photosynthetic membrane vesicles. 
     
     
         3 . The method according to  claim 1 , wherein the photosynthetic membrane vesicles are selected from the group consisting of chromatophore membrane vesicles isolated from purple non-sulfur bacteria and thylakoid membrane vesicles isolated from cyanobacteria or algae. 
     
     
         4 . The method according to  claim 1 , wherein the enzymes that catalyze glutathione synthesis are one or more selected from the group consisting of γ-glutamylcysteine synthetase (GSH-I), glutathione synthetase (GSH-II) and bifunctional glutathione synthetase (bifunctional γ-glutamylcysteine synthetase/glutathione synthetase, GshF). 
     
     
         5 . The method according to  claim 1 , further comprising a step of adding glutamate, cysteine and glycine as substrates for producing glutathione. 
     
     
         6 . The method according to  claim 1 , wherein step b) comprises a step of synthesizing γ-glutamylcysteine from glutamate and cysteine by γ-glutamylcysteine synthetase while converting adenosine triphosphate generated in step a) into adenosine diphosphate and an inorganic phosphate; and a step of synthesizing glutathione from the synthesized γ-glutamylcysteine and glycine by glutathione synthetase while converting adenosine triphosphate generated in step a) into adenosine diphosphate and an inorganic phosphate. 
     
     
         7 . The method according to  claim 1 , wherein step b) comprises a step of synthesizing glutathione from glutamate, cysteine and glycine by bifunctional glutathione synthetase while converting adenosine triphosphate generated in step a) into adenosine diphosphate and an inorganic phosphate. 
     
     
         8 . The method according to  claim 6 , wherein a relative activity ratio of γ-glutamylcysteine synthetase to glutathione synthetase is 4:1 to 20:1. 
     
     
         9 . The method according to  claim 6 , wherein a relative activity ratio of the photosynthetic membrane vesicles to γ-glutamylcysteine synthetase to glutathione synthetase is 1:12:1 to 50:12:1. 
     
     
         10 . The method according to  claim 7 , wherein a relative activity ratio of photosynthetic membrane vesicles to bifunctional glutathione synthetase is 10:1 to 500:1. 
     
     
         11 . A composition for producing glutathione, comprising: i) photosynthetic membrane vesicles; and ii) enzymes that catalyze glutathione synthesis, wherein the enzymes are selected from the group consisting of γ-glutamylcysteine synthetase (GSH-I), glutathione synthetase (GSH-II) and bifunctional glutathione synthetase (bifunctional γ-glutamylcysteine synthetase/glutathione synthetase, GshF). 
     
     
         12 . A system for continuously producing glutathione, comprising: i) photosynthetic membrane vesicles as a means for supplying adenosine triphosphate; ii) enzymes that catalyze glutathione synthesis, wherein the enzymes are selected from the group consisting of γ-glutamylcysteine synthetase (GSH-I), glutathione synthetase (GSH-II) and bifunctional glutathione synthetase (bifunctional γ-glutamylcysteine synthetase/glutathione synthetase, GshF), as a means for catalyzing glutathione synthesis 
     
     
         13 . A method of continuously producing glutathione, the method comprising a step of adding glutamate, cysteine and glycine as substrates for producing glutathione to the system according to  claim 12 .