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)
was added and were kept for 2 h on rocker
shaker for equilibration. The resin was allowed
to settle and the supernatant was used to quantify
for GSH content. The resin was washed twice
with the equilibration buffer. Adsorbed GSH was
calculated by subtracting amt obtained in
supernatant from the initial sample of the same
pH. Thus, bound percentage was calculated
using the following formula.
Total GSH content of bound
% BOUND = X 100 (4)
Total GSH content in loaded
The pH with maximum binding was binding pH,
and that with least binding can be used as elution
pH. The resins were regenerated with 0.5 M
NaOH and reused.
Determination of static binding capacity and
further purification by column
chromatography: Different dilutions of the
filtered sample containing different
concentrations of GSH (0.5 to 3.5 mg/mL) were
prepared using phosphate buffer of pH 7. These
dilutions were loaded to 0.5 mL of Amberlite IR
120 H previously equilibrated with binding buffer
at 25±2 °C) and kept for equilibration. After
equilibration it was allowed to settle; the
supernatant was removed and quantified for
GSH. The isotherms obtained by plotting
concentrations of GSH adsorbed (q*) as (µg/mL
of resin) vs corresponding equilibrium
concentration of GSH in the supernatant (C*) (i.e.
unadsorbed concentration; µg/mL), this signify
the nature of adsorption. The amt of GSH bound
to the adsorbent q * 1
*
q
k d 1 1
= × + *
q max C q max
was calculated as the
difference between the total amt of GSH loaded
and that present in the supernatant after 2 h of
equilibration. Maximum adsorption capacity qmax was determined from the plot of q* vs C*
(isotherm) and the type of isotherm was found
by plotting a graph of 1/q* vs 1/C*.
Adsorption isotherm equation:
Enhanced Production of Glutathione
Where k d = Langmuir isotherm constant.
Rearranging Eq. (5), we get,
(5)
(6)
245
Value of k d can be determined from straightline
plot of 1/q* against 1/C*. The intercept of
such plots on the 1/q* axis is at 1/q max and slope
is k d /q max .
A batch study was carried out on strong
cation exchange resin Amberlite IR 120H packed
in column of diameter 1.1 cm, with bed height
5.7 mL. The headspace of the column was filled
with buffer completely to avoid any air gap and
then equilibrated with acetate buffer (pH 4.0).
Supernatant obtained after ammonium sulphate
precipitation was mixed with buffer and loaded
on the column until exhaustion point.
Concentration of GSH to be loaded was
calculated according to static binding capacity
of the resin. The pH of the sample was kept at 4
to ensure binding of the GSH to the matrix.
Volumetric flow rate of 0.5 mL /min was
maintained by peristaltic pump. Washing with
same equilibrating buffer to remove the unbound
or weakly bound was done.
Three different elution strategies were
employed to elute the adsorbed GSH from
Amberlite IR 120H resin, as elution with 1.5 M
NaCl, elution by changing the buffer pH and
elution with 1 %. Each time volumetric flow rate
was maintained at 0.5 mL/min and 3 mL fractions
were collected at a time and analyzed for GSH
concentration by alloxan method.
Results and Discussion
Media optimization by one factor at-a-time
method
Effects of pH: At an initial pH 6.0, maximum
production of GSH, 62.18 mg/L (biomass, 5.55