Abstract
In this study, extracellular alkaline protease producing bacterial isolates EN-2 and EN-3 from the agricultural soil of C.R.C. Pantnagar were identified as Bacillus megaterium strains EN-2 and Bacillus subtilis strain EN-3 on the basis of 16S rDNA gene sequencing. During kinetic characterization, optimum pH for EN-2 and EN-3 protease activity was 10 and 9, respectively. While optimum temperature, for maximum protease activity in both isolates, was 50°C. The crude extracellular alkaline protease from isolates EN-2 and EN-3 were partially purified using ammonium sulphate fractionation and dialysis to 1.50 and 1.42 fold with 53.77% and 42% recovery respectively. The observed values of Vmax and Km for protease from isolate EN-2 were found to be 11.57 U/ml and 17.442 mg/ml, while for EN-3 protease these were 42 U/ml and 10.62 mg/ml, respectively. The partially purified enzyme from both bacterial strains was then immobilized in sodium alginate beads with maximum immobilization efficiency at 3% (w/w) and some change in their kinetic properties. The immobilized alkaline protease from EN-2 and EN-3 showed their maximum protease activity at pH 9 and 10, and temperature 60 and 50°C, respectively. Due to these properties, isolated extracellular alkaline proteases from the two strains are ideal choice for application in detergent formulation, leather and food industries. Key words: Bacillus megaterium, Bacillus subtilis, alkaline protease, immobilization.
Highlights
Proteases (EC 3.2.21.24) are the single class of enzyme which occupies a pivotal position with respect to their applications in both physiological and commercial fields
Luria Broth (LB) media, and Nutrient Agar media were procured from HiMedia Laboratories, India
Bacterial strains EN-2 and EN-3 showed positive results for the production of protease by forming the zone of lysis around their colonies in skim milk agar plate. It was confirmed by protease assay in production media in which EN-2 and EN-3 showed the protease activity (90.60 and 80.66 U/ml, respectively)
Summary
Proteases (EC 3.2.21.24) are the single class of enzyme which occupies a pivotal position with respect to their applications in both physiological and commercial fields. These are degradative enzymes which catalyze the total hydrolysis of proteins by the cleavage of peptide bonds. Bacterial proteases are the most significant when compared with those of animals and plants (Gupta et al, 2002). This enzyme accounts for nearly 60% of the total worldwide enzyme sales (Adinarayana et al, 2003; Beg et al, 2003). Alkaline protease of microbial origin possess considerable industrial potential due to their biochemical diversity and wide applications in
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