Purification, molecular cloning, expression and biochemical characterization of subtilisin JB1 from a newly isolated Bacillus subtilis JB1

Abstract

An extracellular gelatinolytic enzyme (subtilisin JB1) obtained from a newly isolated Bacillus subtilis JB1, which was a thermophilic microorganism relevant to the aerobic biodegradation process of fish-meal waste (FMW), was purified via ammonium sulfate flocculation, Sephadex-200 Gel filtration chromatography, and 1-DE separation, and subsequently identified via PMF and CAF MALDI-TOF-MS. The subtilisin JB1 gene was cloned and sequenced. Its recombinant protein was expressed in E. coli DH5α MCR using the pGEX-4T-1 vector and characterized. The soluble prosubtilisin JB1 was purified into 62 kDa fusion proteins, and its protease activity was digested not only with gelatin but also with BSA, azocazein, fibrinogen, and the fluorogenic peptide substrate AAF-AMC. The serine protease inhibitors PMSF and chymostatin completely inhibited its enzyme activity at the optimal pH of 7.5. Thus, our results show that subtilisin JB1 may serve as a potential source material for use in industrial applications including fishery waste degradation and fish by-product processing of proteolytic enzymes.