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Purification of a protease inhibitor from <i>Dolichos biflorus</i> using immobilized metal affinity chromatography

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Title Purification of a protease inhibitor from Dolichos biflorus using immobilized metal affinity chromatography
 
Creator Kuhar, Kalika
Mittal, Anuradha
Kansal, Rekha
Gupta, Vijay Kumar
 
Subject Trypsin
Chymotrypsin
Dolichos biflorus
Immobilized metal affinity chromatography
Protease inhibitor
Purification
 
Description 66-74
Plant protease inhibitors (PIs) are
generally small proteins which play key roles in regulation of endogenous
proteases and may exhibit antifeedant, antifungal, antitumor and cytokine
inducing activities. Dolichos biflorus
(horse gram) is an unexploited
legume, which is rich in nutrients and also has therapeutic importance. It
contains a double-headed PI, which is an anti-nutritional factor. As there is
no report available on its simultaneous removal and purification in single
step, in this study, a double-headed PI active against both trypsin and
chymotrypsin was purified from Dolichos
biflorus to ~14-fold with ~84% recovery using an immobilized metal affinity
chromatography (IMAC) medium consisting of Zn-alginate
beads. The method was single-step, fast, simple, reliable and economical. The
purified inhibitor showed a single band on
SDS-PAGE corresponding to molecular mass of 16 kDa and was stable over a pH
range of 2.0-12.0 and up to a temperature of 100°C for 20 min. The optimum
temperature for trypsin and chymotrypsin inhibitor was observed to be 50°C and
37°C, respectively and pH optimum was pH 7.0 and 8.0, respectively. Thus, IMAC
using Zn-alginate beads was useful in simultaneous purification and removal of
an anti-nutritional factor from horse gram flour in single step. This procedure
may also be employed for purification of other plant PIs in one step.
 
Date 2014-03-03T17:46:08Z
2014-03-03T17:46:08Z
2014-02
 
Type Article
 
Identifier 0975-0959 (Online); 0301-1208 (Print)
http://hdl.handle.net/123456789/27289
 
Language en_US
 
Rights CC Attribution-Noncommercial-No Derivative Works 2.5 India
 
Publisher NISCAIR-CSIR, India
 
Source IJBB Vol.51(1) [February 2014]