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)
broth) in a 250 mL Erlenmeyer flask and
incubated at 30±2 °C and 180 rpm for 18 h.
Media optimization by one factor at-a-time
method
Effect of initial pH: In order to investigate the
effect of initial pH on GSH production,
fermentation runs were carried out by adjusting
initial pH (before autoclaving) of the medium in
the pH range of 4 to 8. The pH was adjusted
using 1 N HCl and/or 1 N NaOH. Each media
was inoculated with 3 % of inoculum suspension
and placed on a shaker for 24 h at 180 rpm at
30±2 ºC.
Effect of carbon sources: Glucose in the media
was replaced with different carbon sources viz
galactose, glycerol, sucrose, soluble starch,
maltose, lactose, fructose at a concentration of
5.2 % to determine their effect on growth and
GSH production. Each media was inoculated with
3 % of inoculum suspension and placed on a
shaker for 24 h at 180 rpm at 30±2 ºC.
Effect of nitrogen sources: The effect of
various organic nitrogen sources on the formation
of GSH by S. cerevisiae NCIM 3454 was
investigated. Peptone in the production media
was substituted with different organic nitrogen
sources such as yeast extract, malt extract, beef
extract, casein peptone at 4.84 %. The 50 mL of
autoclaved medium was inoculated with 3 % of
mycelium suspension of and incubated for 24 h
at 180 rpm at 30±2 ºC. Simultaneously, the effects
different inorganic nitrogen sources such as
sodium nitrate, ammonium sulphate, ammonium
chloride at 4.84 % were also studied.
Media optimization by Response Surface
method (RSM): A central composite rotatable
design (CCRD) for three independent variables
(glucose, yeast extract and magnesium sulphate)
was used to obtain the combination of values that
optimizes the response within the region of three
dimensional observation spaces, which allows
Enhanced Production of Glutathione
243
one to design a minimal no. of experiments. The
experiments were designed using the software,
Design Expert Version 6.0.10 trial version
(StatEase, Minneapolis, MN). The medium
components (independent variables) selected for
the optimization were glucose, yeast extract, and
magnesium sulphate. Regression analysis was
performed on the data obtained from the design
experiments. Coding of the variables was done
according to the following Eq. 1
xi = (Xi - Xcp) / ΔXi, i =1, 2, 3, . . . , k (1)
Where xi, dimensionless value of an
independent variable; Xi, real value of an
independent variable; Xcp, real value of an
independent variable at the centre point; and ÄXi,
step change of real value of the variable i
corresponding to a variation of a unit for the
dimensionless value of the variable i. The
experiments were carried out at least in triplicate,
which was necessary to estimate the variability
of measurements, i.e. the repeatability of the
phenomenon. Replicates at the centre of the
domain in three blocks permit the checking of
the absence of bias between several sets of
experiments. The relationship of the independent
variables and the response was calculated by
the second order polynomial Eq. 2:
k k
Y = β +Σ βi X +Σ β X X +Σi Σj β X X (2)
0 i ii i j ij i j
i=1 i=1 i< j
Where Y is the predicted response; β a 0
constant; β the linear coefficient; β the squared
i ii
coefficient; and β the cross-product coefficient,
ij
k is no. of factors. The second order polynomial
coefficients were calculated using the software
package Design Expert Version 6.0.10 to
estimate the responses of the dependent
variable. Response surface plots were also
obtained using Design Expert Version 6.0.10.
Effect of amino acids as a stimulator for GSH
production: Effect of cysteine, glycine, glutamic
acid, methionine, serine, tyrosine and lysine on
GSH production was studied at different
concentrations. To 50 mL of autoclaved medium