Study about Isolation, Purification and Partial Characterization of Thermostable Serine Alkaline Protease from a Newly Isolated Bacillus thuringinsis

Abstract

In the present study, the author reported the isolation, purification and partial characterization of thermostable serine alkaline protease produced from Bacillus thuringinsis. The culture was isolated from slaughterhouse waste soil and identified using ribosomal sequence analysis. The crude enzyme was purified up to 17.04 fold with an 8.47 percent recovery using ammonium sulphate precipitation, dialysis, and Sephadex G-200 gel permeation chromatography. SDS-PAGE revealed the isolated enzyme's relative molecular weight (67 kDa). At 45°C, maximum enzyme and cell biomass production was recorded after 48 hours of incubation. At pH 10 to 11, enzyme activity was high, and stability of up to 2 and 20 hours incubation at the same pH range confirmed alkaline protease. The optimal temperature for protease activity was 45°C, with 100 percent thermal stability measured up to 350 minutes of incubation. Casein was discovered to be the best natural substrate among those tested.Enzyme activity was strongly enhanced by metal ions like Ca+2, Mg+2 and Mn+2 whereas, 100% enzyme activity was inhibited by phenylmethylsulphonyl fluoride (PMSF), and up to 92% inhibition by diisopropyl fluorophosphates (DFP) confirmed serine protease. The enzyme's detergent compatibility was investigated at 45°C in the presence of 10 mM CaCl2 and 1 M glycine. For a period of 0.5 to 2.5 hours incubation, this suggests 80 to 100 percent stability. When the detergent Surf excel with enzyme was applied, the washing performance and elimination of blood stains from the cotton cloth improved. Overall, the isolated protease's capabilities support its commercial application in the detergent and leather sectors