Isolation and screening of alkaline protease producing bacteria and physio-chemical characterization of the enzyme

Abstract

Soil samples from different habitats including tanneries, soap industries, garden soil and soil compost were screened for the presence of alkalophilic Bacillus isolates capable of producing alkaline protease in large quantities. One hundred and eighteen (118) isolates were found having proteolytic activity on skim milk agar plates. Isolates forming larger zones, as a result of casein hydrolysis were further studied for quantitative production of extracellular alkaline protease activity in the shake flask studies. Isolate CEMB10370 gave maximum activity. Time course studies indicated that strain CEMB10370 had the highest protease activity (380 APU/mL) after 48 h of fermentation. The wild type enzyme was biochemically characterized. The enzyme exhibits optimal activity at 50°C and pH 11.5. The protease enzyme was completely inhibited by phenylmethylsulfonyl (PMSF, serine protease inhibitor) and its isoelectric point was ~9.5. The enzyme was purified by ion-exchange chromatography using CMSepharose column as a ~29 Kilo Dalton (kDa) protein.

Key words: Alkaline protease, alkalophilic ,Bacillus subtilis