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To develop a large-scale production of the protease of Bacillus subtilis strain D-2, the full-length gene apr-D2 (1,149 bp) encoding the alkaline protease was cloned into plasmid pET-32a and expressed as a secretory protein in Escherichia coli. Sequence analysis of the deduced amino acid sequence revealed high homology with the catalytic domains of the subtilisin serine proteases. From SDS-PAGE analysis, the recombinant protein had a molecular mass of 60.4 kDa. The expressed protease was secreted into the culture medium in a functional active form. The purified recombinant protease showed a pH optimum of 10.5 and temperature optimum of 55 °C, and it was stable in the pH range from 5.0 to 13.0. The enzyme activity was slightly enhanced by Ca2+, Mg2+, Ba2+, and SBT1. However, it was highly inhibited by Ag+ and PMSF. A theoretical structural model of mature protein was constructed by comparative modeling, which showed a putative catalytic triad (Asp-32, His-64 and Ser-221) with high similarity to the template. The structural characteristics that confer enzymatic specificity of the protease were analyzed. Taken together, the data suggested that the secretory expression system with pET-32a in E. coli was successfully constructed. Additionally, enzymatic specificity analysis of the alkaline protease indicated that it was suitable for various processing industries.

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