Studies of proteases from biological sources

Abstract

An extracellular protease was purified from Pseudomonas thermaerum GW1 a new strain identified by morphological, biochemical and 16S rDNA sequencing. It was isolated from soil of Poultry waste site at Ghazipur near Ghaziabad, Delhi. This strain produces extra cellular protease in the culture media that was maintained at 37°C at 140 rpm after 48 hrs of incubation. Purification steps involved ammonium sulphate precipitation, DEAE-cellulose chromatography followed by casein zymography studies. Enzyme remained stable between at 60°C at pH 8.0. Interestingly Mn2+ strongly activated enzyme activity by 5 fold, where as Zn2+, Fe2+ and Hg2+ inhibited enzyme activity. The protease was stable in presence of 50 % (v/v) ethylacetate and acetone whereas isopropanol, methanol and benzene increased protease activity by 2.7, 1.3 and 1.1 fold respectively suggesting its potential industrial application. Additionally protease from senesced leaves of the weed Lantana camara was purified by a two-step procedure involving ammonium sulphate precipitation and Sephadex G 250 gel permeation chromatography which makes it a cheaper enzyme source for detergent industry. Enzyme showed 28.31 fold purification with a yield of 6.19%. It is a low molecular weight cysteine alkaline protease of 43 kDa as seen by SDS-PAGE. It is strongly activated by metal ions such as Cu2+, Zn2+, Mg2, Co2+ and Mn2+and remained active at 60°C, pH 10.5 even after one hour of incubation and remained compatible with detergents and 60% enzyme activity retaining.