Cloning, Expression, Purification and Biochemical Characterization of Novel Thermostable Subtilisin-like Serine...

The persistent rise in demand for biocatalysts in industries has prompted the exploration of novel enzymes. Proteases are very important class of enzymes with many industrial applications. However, majority of the reported proteases are from mesophiles and exhibit low stability under harsh conditions limiting their potential applications. This study presents the expression and characterization of a novel thermostable recombinant subtilisin-like serine protease derived from Thermococcus onnurineus NA1. The subtilisin serine protease gene was successfully cloned and expressed in E. coli BL21. The recombinant protease was purified by affinity chromatography and has an estimated molecular mass of 29 kDa following SDS-PAGE analysis. It exhibited an optimal activity at a pH of 10 and temperature of 90 °C. The recombinant subtilisin-like serine protease maintained 96% of its activity at 100 °C. Additionally, Na⁺, Mg²⁺, Cu²⁺, and Mn²⁺ had minimal effect on the recombinant protease activity whereas the presence of Ca²⁺ significantly enhanced the enzyme activity. Interestingly, the recombinant protease was stable in the presence of different surfactants and inhibitors. These properties illustrate the potential of recombinant subtilisin-like serine protease for industrial applications.