Abstract

In the present study, the isolation, purification and partial characterization of thermostable serine alkaline protease produced from Bacillus thuringinsis SH-II-1A was reported. The culture was isolated from soil of slaughter house waste and identified further from ribosomal sequence. The crude enzyme was purified by ammonium sulfate precipitation, dialysis and Sephadex G-200 gel permeation chromatography up to 17.04 fold with recovery of 8.47%. Relative molecular weight (67 kDa) of purified enzyme was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Maximum production of enzyme and cell biomass was observed for 48 h of incubation period at 45°C. Strong activity of enzyme was observed at pH 10 to 11; also stability of up to 2 and 20 h incubation at the same pH range confirms alkaline protease. Optimum temperature recorded for protease activity was 45°C, and 100% thermal stability up to 350 min of incubation was recorded. Among different natural substrates tried, casein was found as ideal substrate. Enzyme activity was strongly enhanced by metal ions like Ca²⁺, Mg²⁺ and Mn²⁺ whereas, 100% enzyme activity was inhibited by phenylmethylsulphonyl fluoride (PMSF), and up to 92% inhibition by diisopropyl fluorophosphates (DFP) confirmed serine protease. Detergent compatibility of the enzyme was studied in the presence of 10 mM CaCl₂ and 1 M glycine at 45°C. This indicates 80 to 100% stability for a period of 0.5 to 2.5 h incubation. Improved washing performance and removal of blood stains from the cotton cloth was observed when detergent Surf excel was used with enzyme. Overall, the observed properties of isolated protease conclude its commercial application in detergent and leather industries.

Key words: Thermostable serine alkaline protease, Bacillus thuringinsis, purification, partial characterization.

Abbreviation

NCCS, National Centre for Cell Sciences; DEAE, di ethyl amino ethyl; CBB, co-ommassie brilliant blue; SDS-PAGE, sodium dodecyl sulphate poly-acrylamide gel electrophoresis; TCA, tri-chloro acetic acid; UV, ultra violet; BSA, bovine serum albumin; p-CMB, para chloro mercuric benzoate; PMSF, phenyl methyl sulfonyl fluoride; β-ME, β-mercapto ethanol; DFP, di isopropyl fluoro phosphate.