Purification and Characterization of Recombinant Bacillus stearothermophilus Subtilisin J

Abstract

Subtilisin J produced in Bacillus subtilis DB104/pZS101 containing the gene encoding subtilisin J of Bacillus stearothermophilus NCIMB10278 (Jang et al., 1992) was purified to study the kinetic properties of the enzyme. Subtilisin J was purified to homogeneity from a culture medium using CM-cellulose ion exchange chromatography. The molecular weight of the enzyme was estimated to be approximately 27,500 kDa. The NH2-terminal sequence of subtilisin J showed a high degree of homology with the same sequence of other subtilisins. The optimum pH for the proteolytic activity of subtilisin J was 9.0. Ca2+ stabilized the enzyme upon heat treatment and maximum proteolytic activity was obtained at 60℃. The enzyme retains about 50% of its activity even after treatment at60℃ for 30 min in the presence of 2 mM calcium chloride.