Abstract
In the present study, permeated yeast cells were used as the catalyst to synthesize glutathione. When waste cells of brewer’s yeast were incubated with the three precursor amino acids and glucose for 36 h, 899 mg/L of glutathione were produced. To release the feedback inhibition of γ-glutamylcysteine synthetase caused by glutathione, two-stage reaction was adopted. In the first stage, glycine was omitted from the reaction mixture and only γ-glutamylcysteine was formed. Glycine was then added in the second stage, and 1,569 mg/L of glutathione were produced. The conditions of the two-stage reaction were optimized using Plackett–Burman design and response surface methodology. Under the optimized condition, commercially available baker’s yeast produced 3,440 mg/L of glutathione in 30 h, and most of the produced glutathione was in the medium. The two-stage reaction could effectively reduce the feedback inhibition caused by glutathione, but degradation of glutathione was significant.
Similar content being viewed by others
References
Meister A, Anderson ME (1983) Glutathione. Annu Rev Biochem 52:711–760
Penninckx MJ, Elskens M (1993) Metabolism and functions of glutathione in micro-organisms. Adv Microb Physiol 34:239–301
Carmel-Harel O, Storz GR (2000) Roles of the glutathione and thioredoxin-dependent reduction systems in the Escherichia coli and Saccharomyces cerevisiae responses to oxidative stress. Annu Rev Microbiol 54:439–501
Richman PG, Meister A (1975) Regulation of γ-glutamycysteine synthetase by nonallosteric feedback inhibition by glutathione. J Biol Chem 250:1422–1426
Tate S, Meister A (1981) γ-Glutamyltranspeptidase: catalytical, structural and functional aspects. Mol Cell Biochem 39:357–368
Harington CR, Mead TH (1935) Synthesis of glutathione. Biochem J 29:1602–1611
Liang GB, Du GC, Chen J (2008) A novel strategy of enhanced glutathione production in high cell density cultivation of Candida utilis—cysteine addition combined with dissolved oxygen controlling. Enzyme Microb Technol 42:284–289
Tong Q, Wang G, Du GC, Chen J (2003) Effect of feeding method on production of glutathione by Saccharomyces cerevisiae. J Ind Microbiol 33:19–22
Alfafara CG, Kanda A, Shioi T, Shimizu H, Shioya S, Suga K, Suzuki K (1993) Fuzzy control of ethanol concentration and its application to maximum glutathione production in yeast fed-batch culture. Biotechnol Bioeng 41:493–501
Alfafara CG, Kanda A, Shioi T, Shimizu H, Shioya S, Suga K (1992) Effect of amino acids on glutathione production by Saccharomyces cerevisiae. Appl Microbiol Biotechnol 36:538–540
Alfafara CG, Miura K, Shimizu H, Shioya S, Suga K (1992) Cysteine addition strategy for maximum glutathione production in fed-batch culture of Saccharomyces cerevisiae. Appl Microbiol Biotechnol 37:141–146
Liang G, Du GC, Chen J (2008) Enhanced glutathione production by using low-pH stress coupled with cysteine addition in the treatment of high cell density culture of Candida utilis. Lett Appl Microbiol 46:507–512
Gushima H, Miya T, Murata K, Kimura A (1983) Construction of glutathione-producing strains of Escherichia coli B by recombinant DNA techniques. J Appl Biochem 5:43–52
Ohtake Y, Watanabe K, Tezuka H, Ogata T, Yabuuchi S, Murata K, Kimura A (1988) The expression of γ-glutamylcysteine synthetase gene of Escherichia coli in Saccharomyces cerevisiae. Agric Biol Chem 52:2753–2762
Ohtake Y, Watanabe K, Tezuka H, Ogata T, Yabuuchi S, Murata K, Kimura A (1989) Expression of the glutathione synthetase gene of Escherichia coli B in Saccharomyces cerevisiae. J Ferment Bioeng 68:390–394
Anderson ME (1998) Glutathione: an overview of biosynthesis and modulation. Chem Biol Interact 111–112:1–14
Tietze F (1969) Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem 27:502–522
Plackett RL, Burman JP (1946) The Design of optimum multifactorial experiments. Biometrika 33:305–325
Deming SN, Morgan SL (1987) Experimental design: a chemomatric approach. Elsevier, Oxford
Murata K, Tan K, Kato J, Chibata I (1981) Glutathione production by immobilized Saccharomyces cerevisiae cells containing an ATP regeneration system. Appl Microbiol Biotechnol 11:72–77
Larsson C, Nilsson A, Blomberg A, Gustafsson L (1997) Glycolytic flux is conditionally correlated with ATP concentration in Saccharomyces cerevisiae: a chemostat study under carbon- or nitrogen-limiting conditions. J Bacteriol 179:7243–7250
Penninckx MJ (2002) An overview on glutathione in Saccharomyces versus non-conventional yeasts. FEMS Yeast Res 2:295–305
Acknowledgment
We thank Asia-Pacific Brewery Co., Shangahi, China, for providing the waste brewer’s yeast, and Chyuan Chemical Co., Hubei, China, for providing l-cysteine. This work was partly supported by the project of Shanghai Leading Academic Disciplines No. B505.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Li, W., Li, Z. & Ye, Q. Enzymatic synthesis of glutathione using yeast cells in two-stage reaction. Bioprocess Biosyst Eng 33, 675–682 (2010). https://doi.org/10.1007/s00449-009-0361-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00449-009-0361-6