Protein engineering on subtilisin E.

Protein engineering was carried out by site-directed and random mutagenesis on subtilisin E gene. Four mutants were obtained. They are M222A; M222A, N118S; M222A, N118S, Q103R; and M222A, N118S, Q103R, D60N. The mutant genes were recombined in pBE-2, an E. coli-B. subtilis shuttle vector, and transformed into B. subtilis DB104, an alkaline and neutral proteinase deficient strain. The subtilisin E mutations obtained from their gene expressions were purified. The properties of these mutants showed that the M222A mutation made the enzyme resistant to oxidation, N118S mutation increased the thermal stability, while Q103R and D60N mutations enhanced the specific activity of the enzyme but decreased the thermal stability and, in particular, D60N mutation caused the enzyme to be very unstable. The IEF-PAGE showed that the wild type and M222A mutant had the same pI of 8.92, while those of double mutant, triple mutant, and quadruple mutant were 8.88, 9.10, and 9.17, respectively. The optimum pH range was 7.5-9.5 for suc-AAPF-pNA substrate and was 10-12 for casein substrate.