Purification of a protease inhibitor from Dolichos biflorus using immobilized metal affinity chromatography.

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.

Isolation, purification and characterization of alkali and thermo stable Xylanase from bacillus sp. Ks09.

Nine bacterial strains were isolated using xylan rich media. The bacterial strain KS09 was selected on the basis of qualitative and quantitative test. It was identified as Bacillus sp. via physiological, morphological and biochemical characterization. The xylanase was purified to homogeneity from crude extract of Bacillus sp. KS09 using ammonium sulphate fractioning and CM-Sephadex C-50. The final purification fold was 10.20 with a recovery of 36.18%. The enzyme was optimally active at 50°C, pH 7.0 and stable over a broad pH range of 6.0-11.0. The residual activity at 6.0-11.0 pH was 100% even upto 3 h of incubation. The enzyme showed 75, 70 and 60% thermal stability at 50, 55 and 60°C, respectively after 1 h of incubation. The kinetic parameters (Km 22.59 mM; Vmax 76.93 IU/mL) were estimated using Lineweaver-Burk plot for purified xylanase. The xylanase activity was inhibited by all the metal ions applied. The characteristic studies revealed that xylanase including its cellulase free nature, broad pH stability and temperature stability are particularly suited its industrial applications.