Bioprocess optimization of glutathione production by Saccharomyces boulardii: biochemical characterization of glutathione peroxidase.

Autor: Badr H; Department of Industrial Biotechnology, Genetic Engineering and Biotechnology Research Institute, University of Sadat City, Sadat City, 22857/79, Egypt., El-Baz A; Department of Industrial Biotechnology, Genetic Engineering and Biotechnology Research Institute, University of Sadat City, Sadat City, 22857/79, Egypt. ashrafhawase@gmail.com., Mohamed I; Department of Microbial Molecular Biology, Agricultural Genetic Engineering Research Institute (AGERI), Agricultural Research Center (ARC), Giza, Egypt., Shetaia Y; Department of Microbiology, Faculty of Science, Ain Shams University, Cairo, Egypt. you_shetaia@hotmail.com., El-Sayed ASA; Botany and Microbiology Department, Faculty of Science, Zagazig University, Zagazig, 44519, Egypt., Sorour N; Department of Industrial Biotechnology, Genetic Engineering and Biotechnology Research Institute, University of Sadat City, Sadat City, 22857/79, Egypt.
Jazyk: angličtina
Zdroj: Archives of microbiology [Arch Microbiol] 2021 Dec; Vol. 203 (10), pp. 6183-6196. Date of Electronic Publication: 2021 Sep 27.
DOI: 10.1007/s00203-021-02584-0
Abstrakt: The well-known probiotic GRAS Saccharomyces boulardii (CNCM I-745) was used for the first time to produce glutathione (GSH). The culture conditions affecting GSH biosynthesis were screened using a Plackett-Burman design (PBD). Analyzing the regression coefficients for 12 tested variables, yeast extract, glucose, peptone, cysteine, temperature and agitation rate had a positive significant effect on GSH production with a maximum yeild 192 mg/L. The impact of kinetics of adding cysteine was investigated in 19 experiments during the growth time course (0-36 h), and the maximum yield of glutathione (235 mg/L) was obtained by addition of cysteine after 8 h post-inoculation. The most significant variables were further explored at five levels using central composite rotatable design (CCRD), giving a maximum production of GSH (552 mg/L). Using baffled flasks, the yield of GSH was increased to 730 mg/L, i.e., 1.32-fold increment. The two rate-limiting genes of GSH biosynthesis "γ-glutamyl cysteine synthetase (GSH1) and GSH-synthetase (GSH2)" were amplified and sequenced to validate the GSH biosynthetic potency of S. boulardii. The sequences of genes showed 99% similarity with GSH1 and GSH2 genes of S. cerevisiae. Glutathione peroxidase was purified and characterized from S. boulardii with molecular mass and subunit structure of 80 kDa and 35 kDa as revealed from native and SDS-PAGE, ensuring its homodimeric identity. The activity of GPx was reduced by 2.5-fold upon demetallization confirming its metalloproteinic identity. The GPx was strongly inhibited by hydroxylamine and DTNB, ensuring the implication of surface lysine and cysteine residues on the enzyme active site domains.
(© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)
Databáze: MEDLINE
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