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Title: Purification of a protease inhibitor from Dolichos biflorus using immobilized metal affinity chromatography
Authors: Kuhar, Kalika
Mittal, Anuradha
Kansal, Rekha
Gupta, Vijay Kumar
Keywords: Trypsin;Chymotrypsin;Dolichos biflorus;Immobilized metal affinity chromatography;Protease inhibitor;Purification
Issue Date: Feb-2014
Publisher: NISCAIR-CSIR, India
Abstract: 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.
Page(s): 66-74
ISSN: 0975-0959 (Online); 0301-1208 (Print)
Appears in Collections:IJBB Vol.51(1) [February 2014]

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