d(GC) - Association of Biotechnology and Pharmacy
d(GC) - Association of Biotechnology and Pharmacy
d(GC) - Association of Biotechnology and Pharmacy
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Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong><br />
Vol. 6 (2) 241-254 April 2012, ISSN 0973-8916 (Print), 2230-7303 (Online)<br />
indicating that GSH was partitioned in both<br />
phases. Also percent protein in top phase was<br />
38.23 % indicating that remaining proteins were<br />
in bottom phase with PEG 8000. Total percent<br />
protein in top phase was found to be (53.48 %)<br />
with PEG 8000 which was undesirable. Results<br />
suggested that with use <strong>of</strong> PEG 6000 around<br />
61.87 % unwanted proteins could be separated<br />
from GSH cell extract in a single step. Hence,<br />
PEG 6000 was selected for further studies.<br />
The effect <strong>of</strong> concentration <strong>of</strong> PEG on<br />
partitioning <strong>of</strong> GSH <strong>and</strong> protein was further taken<br />
in to consideration. As partitioning <strong>of</strong> GSH in top<br />
phase increased as concentration <strong>of</strong> PEG was<br />
increased. Maximum Kd (3.55) was achieved at<br />
PEG 6000 concentration <strong>of</strong> 20 % which<br />
decreased to 3.1 with further increase in PEG<br />
concentration. This could be due to increased<br />
viscosity <strong>of</strong> the system at high PEG concentration<br />
which in turn causes resistance to mass transfer.<br />
The percent protein in top phase was found<br />
minimum (38.36 %). The effect <strong>of</strong> ammonium<br />
sulphate concentration on partitioning <strong>of</strong> GSH<br />
<strong>and</strong> protein was also studied. At 12 % ammonium<br />
sulphate concentration maximum Kd (3.7) was<br />
achieved. The percent proteins in top phase were<br />
38.5 % indicating that remaining were separated<br />
from GSH cell extract. At low salt concentrations<br />
percent proteins in top phase were less which<br />
may be due to solubilization <strong>of</strong> proteins at low<br />
salt concentration (salting in effect). At high salt<br />
concentration percent protein in top phase were<br />
found to be increased which may be because <strong>of</strong><br />
the fact that proteins tend to precipitate <strong>and</strong> are<br />
retained in upper phase.<br />
Ion exchange chromatography<br />
Selection <strong>of</strong> optimal binding pH for GSH on<br />
different resins: The percentages <strong>of</strong> GSH<br />
bound on various matrices at different pH are<br />
shown in Fig. 7. The adsorption <strong>of</strong> GSH was<br />
found to be maximum on Amberlite IR 120H (at<br />
pH 4.0, 74.39 %), Indion CAM (at pH 3.0, 65.34<br />
%), Amberlite XAD-16 (at pH 3.0, 52.14 %) <strong>and</strong><br />
Indion 830 (at pH 5.0, 45.60 %). Since maximum<br />
binding was on Amberlite IR 120H resin at pH<br />
Enhanced Production <strong>of</strong> Glutathione<br />
253<br />
4.0 it was selected for further studies.<br />
Determination <strong>of</strong> static binding capacity <strong>and</strong><br />
further purification by column chromatography:<br />
Equilibrium adsorption isotherm was<br />
studied to find out adsorption isotherm pattern<br />
for purification <strong>of</strong> GSH using Amberlite IR 120H<br />
resin. The adsorption isotherm data was used to<br />
determine the adsorption capacity <strong>of</strong> the matrix.<br />
The GSH adsorption followed a typical Langmuir<br />
type <strong>of</strong> isotherm as shown in Fig. 8 for Amberlite<br />
IR 120H resin. The maximum capacity <strong>of</strong> the<br />
matrix (q ) for GSH was found to be 3.081 mg/<br />
max<br />
mL. A plot <strong>of</strong> 1/q* Vs 1/C* (Fig. 9) gave a linear<br />
correlation confirming the adsorption to be <strong>of</strong> the<br />
Langmuir type. Lower elution efficiency with 1.5<br />
M NaCl (8.64 %) <strong>and</strong> 100 mM phosphate buffer<br />
<strong>of</strong> pH 8 (5.91 %) was observed (Table 5). Elution<br />
with 1 % H SO showed the best elution pattern.<br />
2 4<br />
Percent elution was very high (89.19 %) as<br />
compared to salt <strong>and</strong> pH elution.<br />
Conclusions<br />
Optimization <strong>of</strong> the fermentation medium<br />
could increase the GSH production by S.<br />
cerevisiae NCIM 3454 from 55.28 mg/L to 148.45<br />
mg/L. L-cysteine acted as stimulator in production<br />
<strong>of</strong> glutathione (163.12 mg/L). Aqueous two phase<br />
system was found to be good alternative during<br />
purification <strong>of</strong> GSH prior to adsorption<br />
chromatography. System containing PEG 20 %,<br />
<strong>and</strong> ammonium sulphate 12 % gave maximum<br />
protein recovery <strong>of</strong> GSH in top phase <strong>and</strong><br />
separating maximum proteins in bottom phase.<br />
Amberlite IR 120H showed maximum GSH<br />
adsorption (74.39 %) at pH 4 <strong>and</strong> maximum<br />
elution was achieved with 1 % H SO 2 4.<br />
Acknowledgement<br />
Authors are thankful to the Department <strong>of</strong><br />
<strong>Biotechnology</strong>, Ministry <strong>of</strong> Science <strong>and</strong><br />
Technology, India, for providing financial<br />
assistance during the course <strong>of</strong> this research.<br />
References<br />
1 Anderson, M.E. <strong>and</strong> Meister, A. (1983).<br />
Glutathione. Annu Rev Biochem. 52: 711-<br />
760.