Gurvinder Singh, KocherInderjeet Kaur2017-06-152017-06-152011http://krishikosh.egranth.ac.in/handle/1/5810020520The present study was conducted to characterize alkaline protease of Bacillus circulans MTCC 7906. Cotton deoiled meal (2.1%) supported optimum enzyme production in 144 hours of incubation. Among the agricultural byproducts used to improve alkaline protease production, the combination of Soybean meal and Glucose supported maximum enzyme production in 96 hours of incubation which was comparable with the control Reese medium (Casein + Glucose) in terms of alkaline protease activity as well as incubation time required for enzyme production. The alkaline protease was purified (16.2 fold) to homogeneity from the culture supernatant by the combination of ammonium sulphate precipitation (30-60%) and DEAE-cellulose anion exchange chromatography. The biochemical characterization of partially purified alkaline protease displayed maximum activity at a pH of 9.0, temperature of 60°C, an enzyme concentration of 0.1 ml/3.0 ml and substrate concentration of 12 mg/ml. The Km and Vmax of partially purified alkaline protease were found to be 4.5 mg/ml and 5555 nmole tyrosine min-1 ml-1, respectively. The enzyme activity was activated by divalent ions whereas a metal chelator EDTA and ammonium hydroxide reduced the activity. The molecular weight of the enzyme was estimated to be 46 kDa on SDS-PAGE. For, molecular analysis, full length alkaline protease gene (BcAP, 1329 bp) was PCR amplified from B. circulans DNA and cloned into pTrcHisA vector. Sequencing and alignment of nucleotide and predicted amino acid sequences of alkaline protease region identified a number of substitutions (mutation) which established that alkaline protease of B. circulans MTCC 7906 is a novel gene. The multiple alignment of nucleotide and amino acid sequences of alkaline protease gene of B. circulans established major matches with three closely related subspecies of B. subtilis (B. subtilis subspecies subtilis strain 168, B. subtilis BSn5 and B. subtilis subspecies spizizenii strain W23). Molecular evolution of B. circulans strain was determined by phylogenetic analysis of alkaline protease gene and predicted amino acid sequences. This analysis also suggested that alkaline protease gene is novel gene, which has been accessioned in GenBank with accession number JN645176.1. Recombinant expression of alkaline protease gene studied by inducible expression and analysis by SDS-PAGE, established that the protein with estimated molecular size of 46 kDa as observed earlier.ennullThesis