Study about Isolation, Purification and Partial Characterization of Thermostable Serine Alkaline Protease from a Newly Isolated Bacillus thuringinsis
The isolation, purification, and partial characterisation of thermostable serine alkaline protease generated by Bacillus thuringiensis were described in this study. Using ribosomal sequence analysis, the culture was isolated from slaughterhouse waste soil and identified. Using ammonium sulphate precipitation, dialysis, and Sephadex G-200 gel permeation chromatography, the crude enzyme was purified up to 17.04 fold with an 8.47 percent recovery. The relative molecular weight of the isolated enzyme was determined using SDS-PAGE (67 kDa). After 48 hours of incubation at 45°C, the maximal enzyme and cell biomass output was reported. At pH 10 to 11, enzyme activity was high, and alkaline protease was confirmed after incubation for up to 2 and 20 hours at the same pH range. The best temperature for protease activity was 45°C, with thermal stability of 100 percent evaluated after 350 minutes of incubation. Among the natural substrates studied, casein was shown to be the best. Metal ions such as Ca+2, Mg+2, and Mn+2 significantly increased enzyme activity, but phenylmethylsulphonyl fluoride (PMSF) reduced enzyme activity completely, and diisopropyl fluorophosphates (DFP) decreased enzyme activity up to 92 percent, confirming serine protease. The detergent compatibility of the enzyme was tested at 45°C with 10 mM CaCl2 and 1 M glycine. This indicates 80 to 100 percent stability after 0.5 to 2.5 hours of incubation. The washing performance and removal of blood spots from the cotton material improved after using the detergent Surf excel with enzyme. Overall, the characteristics of the isolated protease support its commercial applicability in the detergent and leather industries.
Sunil L. Harer
Department of Pharmaceutical Chemistry, Dattakala College of Pharmacy, Swami Chincholi, Daund, Pune 413130 (MS), India
Priya S. Harer
Department of Pharmaceutics, PES’s, Modern College of Pharmacy, Yamunanagar, Nigdi, Pune 411044 (MS), India.
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