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) 145-165 April 2012, ISSN 0973-8916 (Print), 2230-7303 (Online)<br />
intracellular in the SmF <strong>and</strong> LSF processes<br />
during the first 48 h <strong>of</strong> fermentation.<br />
Subsequently, a larger portion <strong>of</strong> the enzyme is<br />
excreted in the SmF <strong>and</strong> LSF processes <strong>and</strong> the<br />
ratio <strong>of</strong> intracellular to extracellular tannase is<br />
about 1:6 in SmF <strong>and</strong> 1: 1 in LSF at the peak<br />
enzyme titre levels at 144 h (1).<br />
The results obtained in three different<br />
fermentation conditions for extracellular tannase<br />
production by P. Variotii shows a significant<br />
difference in tannase yield between fermentation<br />
processes. Maximum (167 ± 3.6 U/ml) tannase<br />
production was obtained by SSF at a relatively<br />
short incubation time (60 h) followed by SmF (123<br />
± 3.6 U/ml) at 72 h <strong>and</strong> LSF (102 ± 4.2 U/ml) at<br />
96 h, respectively. In another report <strong>of</strong> Rana <strong>and</strong><br />
Bhat, (88) tannase-producing efficiency <strong>of</strong> LSF<br />
<strong>and</strong> SSF vis-à-vis SmF was investigated in a<br />
strain <strong>of</strong> Aspergillus niger, besides finding out if<br />
there was a change in the activity pattern <strong>of</strong><br />
tannase in these fermentation processes. The<br />
studies on the physicochemical properties were<br />
confined to intracellular tannase as only this form<br />
<strong>of</strong> enzyme was produced by A. niger in all three<br />
fermentation processes. In LSF <strong>and</strong> SmF, the<br />
maximum production <strong>of</strong> tannase was observed<br />
by 120 h, whereas in SSF its activity peaked at<br />
96 h <strong>of</strong> growth (88).<br />
Purification <strong>and</strong> Immobilization Techniques<br />
Purification : Battestin et al. (60) reported<br />
fractional precipitation <strong>of</strong> tannase with 80%<br />
ammonium sulphate saturation that removed<br />
some <strong>of</strong> the non-enzymatic proteins at lower<br />
concentration with about 34% recovery. Further<br />
the elution pr<strong>of</strong>ile <strong>of</strong> the tannase obtained from<br />
the diethylaminoethyl (DEAE)-sepharose column<br />
showed five protein peaks, but tannase activity<br />
was reported only in two peaks with 10-fold<br />
purification <strong>and</strong> 3% yield. In another approach,<br />
Bhardwaj et al. (95) reported a two-step<br />
purification procedure for a fungal tannase. In<br />
the first step, contaminating proteins precipitated<br />
from broth supernatants by ammonium sulphate<br />
at 60% (w/v) saturation, were pelleted by<br />
Dinesh Prasad et al<br />
152<br />
centrifugation, discarded <strong>and</strong> tannase<br />
precipitated from the supernatant at 80%<br />
saturation. While in the second, it was purified<br />
by column chromatography using a DEAEcellulose<br />
column to homogeneity. Mahapatra et<br />
al. (96) partially purified the tannase by acetone<br />
precipitation <strong>and</strong> further by gel filtration<br />
chromatography (GFC) using Sephadex G-100<br />
column. High performance liquid<br />
chromatography (HPLC using GF-250 column)<br />
analysis showed a single major peak with the<br />
elution time <strong>of</strong> 6.8 min. Aqueous two phase<br />
separation (ATPS) is yet another useful technique<br />
for purification <strong>of</strong> enzymes (97). Tannase from<br />
A. heteromorphus was partially purified using<br />
ultrafiltration (30 kDa membrane) <strong>and</strong> ATPS but<br />
the recovery was not very significant in case <strong>of</strong><br />
ATPS (98).<br />
Two extracellular tannin acyl hydrolases<br />
(TAH I <strong>and</strong> TAH II) produced by Verticillium sp.<br />
were purified to homogeneity (7.9 <strong>and</strong> 10.5 fold<br />
with a yield <strong>of</strong> 1.6 <strong>and</strong> 0.9%, respectively) by<br />
Kasieczka-Burnecka et al. (65). Tannase from<br />
P. variable IARI 2031 was purified by a two-step<br />
purification strategy comprising <strong>of</strong> ultrafiltration<br />
using 100 kDa molecular weight cut-<strong>of</strong>f<br />
membrane <strong>and</strong> gel-filtration using sephadex G-<br />
200. Also HPLC analysis <strong>of</strong> the purified tannase<br />
showed that the enzyme eluted as a single peak<br />
with retention time at 6.31 min (99). A similar<br />
strategy <strong>of</strong> ultrafiltration for partial purification <strong>and</strong><br />
concentration <strong>of</strong> A. niger LCF 8 tannase was<br />
reported by using a 200 kDa cut <strong>of</strong>f membrane.<br />
Permeate obtained was again filtered through<br />
100 kDa cut <strong>of</strong>f membrane to eliminate impurities<br />
<strong>of</strong> lower molecular masse that resulted in 80%<br />
recovery with a 14.9 purification fold (85).<br />
Mahendran et al. (23) attempted to purify<br />
tannase from Paecilomyces variotii. The dark<br />
brown extracellular extract was treated with 1%<br />
(w/v) activated carbon that removed more than<br />
50% <strong>of</strong> the coloured impurities. Further fractional<br />
precipitation with 50% saturation <strong>of</strong> ammonium<br />
sulphate removed some <strong>of</strong> the non-enzymatic