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

Hossam Badr,Yousseria Shetaia,Noha Sorour,Ismail Mohamed,Ashraf El-Baz,Ashraf S A El-Sayed

doi:10.1007/s00203-021-02584-0

Abstract

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 192mg/L. The impact of kinetics of adding cysteine was investigated in 19 experiments during the growth time course (0-36h), and the maximum yield of glutathione (235mg/L) was obtained by addition of cysteine after 8h post-inoculation. The most significant variables were further explored at five levels using central composite rotatable design (CCRD), giving a maximum production of GSH (552mg/L). Using baffled flasks, the yield of GSH was increased to 730mg/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 80kDa and 35kDa 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.

Highlights

During the last decades, biomaterials, fine chemicals, nutraceuticals, nanomaterials, and pharmaceutically relevant compounds have become increasingly biosynthesized by different microorganisms (El-Baz et al, 2011, 2016; 2018; Cui et al, 2019; Hassan et al, 2019)
S. boulardii is a variety of S. cerevisiae, it differs from S. cerevisiae in its taxonomic, metabolic, and genetic properties (Mitterdorfer et al, 2002; Edwards-Ingram et al, 2007; Lei et al, 2016)
Molecular study was used to assure the genetic relatedness of S. boulardii isolate to the Saccharomyces spp. and the GSH enzyme production

Summary

Introduction

Biomaterials, fine chemicals, nutraceuticals, nanomaterials, and pharmaceutically relevant compounds have become increasingly biosynthesized by different microorganisms (El-Baz et al, 2011, 2016; 2018; Cui et al, 2019; Hassan et al, 2019). The organisms of choice for efficient production of GSH are the yeasts, especially S. cerevisiae and Candida utilis (Wei et al, 2003; Santos et al, 2007; Xiong et al, 2009; Liang et al, 2010; Wang et al, 2012; Lorenz et al, 2016; Prima et al, 2017). Yeasts provide advantage such as, their ability for fast growing to high cell densities in low-price media (Bachhawat et al, 2009). Cha et al (2004) reported that by the optimization of concentrations of glucose, yeast extract, K2HPO4, and cysteine in the medium; the productivity of GSH was increased 2.27-fold as compared with the basal medium

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