Organic solvent stable protease isolation and characterization from organic solvent tolerant strain of Lysinibacillus sphaericus PAP02

In this study, a bacterium producing solvent-stable protease was isolated from salt-enriched soil after incubation in benzene and toluene enriched-media and identified as Lysinibacillus sphaericus PAP02 by 16S rRNA sequencing. Further, the enzyme was purified by 3.64-fold with yield of 12.07%. The purified protease molecular mass was determined to be 33 kDa by SDS-PAGE and the substrate activity was confirmed by the presence of a protease band. Further, the purified protease was characterized and found to be active at pH 10 and a temperature of 40°C. In addition, metal ions in the form of CoCl2 and ZnSO4 slightly upregulated the enzyme activity. The activity was reduced in EDTA, indicating that it was a metallo-type protease. The significant levels of protease were retained after incubation (14 days) in the presence of organic solvents at 25 and 50% concentrations. Activity was induced by hexane at both concentrations, but toluene only induced at 25%. The isolated organic solvent stable protease will be useful as a biocatalyst for many industrial applications, including peptide synthesis in non-aqueous media.