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 />
was inoculated with 3 % <strong>of</strong> mycelium suspension<br />
<strong>of</strong> <strong>and</strong> incubated for 24 h at 180 rpm at 30±2 ºC.<br />
Analytical determinations<br />
Determination <strong>of</strong> dry cell weight (DCW):<br />
Fermentation broth was centrifuged at 8000 rpm<br />
for 10 min <strong>and</strong> biomass was separated.<br />
Separated biomass was suspended in 25 mL,<br />
40 % ethanol <strong>and</strong> kept for extraction for 2 h on a<br />
rotary shaker at 30±2 °C. The extract was again<br />
centrifuged at 8000 rpm for 15 min <strong>and</strong> GSH<br />
concentration in the supernatant was determined<br />
by alloxan method. Biomass obtained after<br />
centrifugation was dried on a preweighed filter<br />
paper at 100 °C to constant weight for<br />
determination <strong>of</strong> dry cell weight.<br />
Estimation <strong>of</strong> GSH : GSH forms colored<br />
compound upon reaction with alloxan so the<br />
concentration <strong>of</strong> GSH can be determined by UV<br />
spectrophotometer, at 305 nm. One gram per liter<br />
alloxan was prepared in 0.1 M HCI solution.<br />
Glycine (0.1 M) <strong>and</strong> 0.24 M NaHPO 4 –NaH 2 PO 4<br />
buffer (pH 7.6) were prepared in deionized<br />
water. The st<strong>and</strong>ard curve was prepared<br />
accordingly using st<strong>and</strong>ard GSH. Each <strong>of</strong> the<br />
st<strong>and</strong>ards was added to a cuvette containing 3.5<br />
mL 0.24 M NaHPO 4 –NaH 2 PO 4 buffer (pH 7.6)<br />
<strong>and</strong> 0.5 mL 0.1 M glycine. The reaction was<br />
started by addition <strong>of</strong> 1 mL alloxan solutions <strong>and</strong><br />
the durations <strong>of</strong> the reactions were 20 min.<br />
Similar reaction was carried out for extract <strong>of</strong><br />
GSH from fermentation broth, replacing st<strong>and</strong>ard<br />
GSH (17).<br />
Purification <strong>of</strong> GSH<br />
Aqueous two phase system (ATPS): Effect <strong>of</strong><br />
PEG molecular weight, PEG concentration <strong>and</strong><br />
ammonium sulphate concentration: Four different<br />
ATPS’s systems were studied to find out effect<br />
<strong>of</strong> molecular weight <strong>of</strong> PEG (1500, 4000, 6000,<br />
<strong>and</strong> 8000), 20 %; ammonium sulphate, 10 %;<br />
cell extract, 10 % <strong>and</strong> deionized water, 60 %.<br />
Similarly, to study the effect <strong>of</strong> PEG 6000<br />
concentration on partitioning <strong>of</strong> GSH <strong>and</strong> proteins<br />
Parbatsingh Rajpurohit et al<br />
244<br />
five different systems were prepared each<br />
containing PEG 6000 at different concentration<br />
(5 %, 10 %, 15 %, 20 %, <strong>and</strong> 25 %), ammonium<br />
sulphate 10 %, cell extract 10 % <strong>and</strong><br />
concentration <strong>of</strong> deionized water was adjusted<br />
to make final volume to 100 %. To find out the<br />
effect <strong>of</strong> ammonium sulphate concentration, four<br />
different systems were prepared each containing<br />
ammonium sulphate at different concentration<br />
(10 %, 12 %, 15 %, 20 %), PEG 6000 20 %, cell<br />
extract 10 % <strong>and</strong> concentration <strong>of</strong> deionized<br />
water was adjusted to make the final volume to<br />
100 %.<br />
In all above mention cases, entire<br />
system was mixed <strong>and</strong> phases dispersed by<br />
vortex mixer for 1 min at 30±2 °C. Phases were<br />
allowed to separate at 30±2 °C for 12 h. Visual<br />
estimates <strong>of</strong> the volumes <strong>of</strong> top <strong>and</strong> bottom<br />
phases were made. Samples were carefully<br />
extracted from the phases <strong>and</strong> analyzed for GSH<br />
content by alloxan method <strong>and</strong> for protein content<br />
by Bradford method. Kd was calculated for each<br />
system by the formula given below:<br />
Concentration <strong>of</strong> GSH in top phase<br />
Kd = (3)<br />
Concentration <strong>of</strong> GSH in lower phase<br />
Ion exchange chromatography<br />
Selection <strong>of</strong> optimal binding pH for GSH<br />
using different resins: The optimum binding<br />
pH for GSH on different selected resins like<br />
Indion CAM-I, Amberlite XAD-16, Indion 830,<br />
Amberlite IR 120H was determined. Based on<br />
this, a suitable ion exchange resin was selected<br />
for further study. The resin (0.5 mL) was added<br />
to each <strong>of</strong> the tubes, <strong>and</strong> equilibrated to different<br />
pH viz., 3, 4, 5, 6, 7 <strong>and</strong> 8 by washing twice with<br />
equilibration buffer (5 mL <strong>of</strong> equilibration buffer<br />
was added to each tube, <strong>and</strong> kept for 2 h on<br />
rocker shaker). Glycine-HCl buffer (100 mM) was<br />
used for pH 3, acetate buffer (100 mM) was used<br />
for pH 4 <strong>and</strong> 5, <strong>and</strong> phosphate buffer (100 mM)<br />
was used for pH 6. In each tube, 2 mL <strong>of</strong> sample