d(GC) - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and Pharmacy
Vol. 6 (2) 241-254 April 2012, ISSN 0973-8916 (Print), 2230-7303 (Online)
Enhanced Production of Glutathione from
Saccharomyces Cerevisiae using Metabolic Precursor
and Purification with New Approach
Parbatsingh Rajpurohit, Ashwini Tilay, Shrikant Survase and Uday Annapure*
Food Engineering and Technology Department, Institute of Chemical Technology,
Matunga, Mumbai 400 019, India
*For Correspondence - us.annapure@ictmumbai.edu.in
Abstract
This paper reports on the enhanced
fermentative production of glutathione by
Saccharomyces cerevisiae NCIM 3454 using a
statistical approach and its successive
purification by alternative technique. In the first
step, one factor at-a-time method was used to
examine the effect of carbon sources, nitrogen
sources and pH on glutathione (GSH) production.
Subsequently, statistical mathematical model
was used to identify the optimum concentrations
of the key nutrients for higher GSH production.
Glutathione production increased significantly
from 55.28 to 148.45 mg/L when Saccharomyces
cerevisiae NCIM 3454 was cultivated using
optimized medium, as compared to basal
medium. Further, glutathione production was
considerably increased to 163.12 mg/L by using
cysteine amino acid as one of the metabolic
precursor. In this study aqueous two phase
system (ATPS) was found to be most useful
technique to abolish contaminating proteins in
glutathione purification. Further enhanced
purification was carried out by adsorption
chromatography (ion exchange) using a variety
of Amberlite resins.
Keywords: Glutathione, Saccharomyces
cerevisiae, fermentation, precursor,
chromatography.
Introduction
Glutathione (α-glutamyl-L-cysteinylglycine,
GSH) is the most abundant water soluble non-
Enhanced Production of Glutathione
241
protein consisting of thiol group which is widely
distributed in living organisms and predominantly,
in eukaryotic ells (1). It functions in many cellular
processes including the protection of cells against
xenobiotics, carcinogens, radiation and reactive
oxygen species (2,3) hence it has medicinally
important value in areas like health care,
functional foods, cosmetics and its commercial
demand is intensifying (4). Other functions of
GSH include storage and transport of cysteine,
regulation of cell proliferation, synthesis of
deoxyribonucleotides, and regulation of
leukotriene and prostaglandin metabolism (5). It
also works as a neurotransmitter,
neuromodulator and regulator in cell proliferation
and apoptosis (6). An imbalance of GSH is
observed in a wide range of pathologies
including, cancer, neurodegenerative disorders,
cystic fibrosis, HIV and aging.
Normally, most of the glutathione is present
in the reduced form GSH while several additional
forms of glutathione are present in (microbial)
cells, tissues, and plasmas. Oxidized form of
glutathione (glutathione disulfide, GSSG) upon
oxidation of GSH, can in turn be reduced to GSH
by glutathione reductase at the expense of
NADPH (7). It is less easily oxidized than its
precursors, cysteine and ã-glutamyl cysteine (8).
It can be produced by using chemical synthesis
(9) enzymatic methods (10) or by direct
fermentative methods (11, 12). Although
production of GSH by enzymatic method gives
maximum concentration (up to 9 g/L) but