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)
indicating that GSH was partitioned in both
phases. Also percent protein in top phase was
38.23 % indicating that remaining proteins were
in bottom phase with PEG 8000. Total percent
protein in top phase was found to be (53.48 %)
with PEG 8000 which was undesirable. Results
suggested that with use of PEG 6000 around
61.87 % unwanted proteins could be separated
from GSH cell extract in a single step. Hence,
PEG 6000 was selected for further studies.
The effect of concentration of PEG on
partitioning of GSH and protein was further taken
in to consideration. As partitioning of GSH in top
phase increased as concentration of PEG was
increased. Maximum Kd (3.55) was achieved at
PEG 6000 concentration of 20 % which
decreased to 3.1 with further increase in PEG
concentration. This could be due to increased
viscosity of the system at high PEG concentration
which in turn causes resistance to mass transfer.
The percent protein in top phase was found
minimum (38.36 %). The effect of ammonium
sulphate concentration on partitioning of GSH
and protein was also studied. At 12 % ammonium
sulphate concentration maximum Kd (3.7) was
achieved. The percent proteins in top phase were
38.5 % indicating that remaining were separated
from GSH cell extract. At low salt concentrations
percent proteins in top phase were less which
may be due to solubilization of proteins at low
salt concentration (salting in effect). At high salt
concentration percent protein in top phase were
found to be increased which may be because of
the fact that proteins tend to precipitate and are
retained in upper phase.
Ion exchange chromatography
Selection of optimal binding pH for GSH on
different resins: The percentages of GSH
bound on various matrices at different pH are
shown in Fig. 7. The adsorption of GSH was
found to be maximum on Amberlite IR 120H (at
pH 4.0, 74.39 %), Indion CAM (at pH 3.0, 65.34
%), Amberlite XAD-16 (at pH 3.0, 52.14 %) and
Indion 830 (at pH 5.0, 45.60 %). Since maximum
binding was on Amberlite IR 120H resin at pH
Enhanced Production of Glutathione
253
4.0 it was selected for further studies.
Determination of static binding capacity and
further purification by column chromatography:
Equilibrium adsorption isotherm was
studied to find out adsorption isotherm pattern
for purification of GSH using Amberlite IR 120H
resin. The adsorption isotherm data was used to
determine the adsorption capacity of the matrix.
The GSH adsorption followed a typical Langmuir
type of isotherm as shown in Fig. 8 for Amberlite
IR 120H resin. The maximum capacity of the
matrix (q ) for GSH was found to be 3.081 mg/
max
mL. A plot of 1/q* Vs 1/C* (Fig. 9) gave a linear
correlation confirming the adsorption to be of the
Langmuir type. Lower elution efficiency with 1.5
M NaCl (8.64 %) and 100 mM phosphate buffer
of pH 8 (5.91 %) was observed (Table 5). Elution
with 1 % H SO showed the best elution pattern.
2 4
Percent elution was very high (89.19 %) as
compared to salt and pH elution.
Conclusions
Optimization of the fermentation medium
could increase the GSH production by S.
cerevisiae NCIM 3454 from 55.28 mg/L to 148.45
mg/L. L-cysteine acted as stimulator in production
of glutathione (163.12 mg/L). Aqueous two phase
system was found to be good alternative during
purification of GSH prior to adsorption
chromatography. System containing PEG 20 %,
and ammonium sulphate 12 % gave maximum
protein recovery of GSH in top phase and
separating maximum proteins in bottom phase.
Amberlite IR 120H showed maximum GSH
adsorption (74.39 %) at pH 4 and maximum
elution was achieved with 1 % H SO 2 4.
Acknowledgement
Authors are thankful to the Department of
Biotechnology, Ministry of Science and
Technology, India, for providing financial
assistance during the course of this research.
References
1 Anderson, M.E. and Meister, A. (1983).
Glutathione. Annu Rev Biochem. 52: 711-
760.