Abstract
Present study was undertaken to develop cross-linked enzyme aggregate (CLEA)of alkaline serine proteases (sp) from Pythium myriotylum (Pm), a necrotrophic oomycete reported to considerably secrete serine proteases. Among various precipitants screened for spPm1-CLEA preparation, ammonium sulfate at 80% saturation (w/v) yielded 100% activity recovery and retention of spherical morphology as observed by SEM analysis. Addition of glutaraldehyde as cross-linker at 1% (v/v) concentration with optimized ammonium sulfate concentration for 1 hour at 100 rpm yielded 100% activity recovery of spPm1-CLEA from 8-day old P. myriotylum culture filtrate. Addition of BSA (10 mg/ml) to CLEA cross-linking reaction mix reduced CLEA size from the range of 1.82–1.19 µm to 394–647 nm. spPm1-CLEA preparations retained 100% activity at temperature of 80 °C and pH 12.0 signifying their potential commercial applications. In terms of kinetic parameters, present process enhanced kinetic parameters as revealed by 1.67 U.mg−1 specific activity, Km of 0.062 mM and Vmax of 0.145 µmol.min−1.mg−1 for the spPm1-CLEA compared to 0.288 U.mg−1 specific activity, Km of 0.060 mM and Vmax of 0.20 µmol.min−1.mg−1 determined for the free spPm1 enzyme. Study has successfully demonstrated the concept of CLEA in enhancing spPm1 stability and the results so generated can be translated in future towards development of robust biocatalysts.
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