Improved autoprocessing efficiency of mutant subtilisins E with altered specificity by engineering of the pro-region.

Modification of substrate specificity of an autoprocessing enzyme is accompanied by a risk of significant failure of self-cleavage of the pro-region essential for activation. Therefore, to enhance processing, we engineered the pro-region of mutant subtilisins E of Bacillus subtilis with altered substrate specificity. A high-activity mutant subtilisin E with Ile31Leu replacement (I31L) as well as the wild-type enzyme show poor recognition of acid residues as the P1 substrate. To increase the P1 substrate preference for acid residues, Glu156Gln and Gly166Lys/Arg substitutions were introduced into the I31L gene based upon a report on subtilisin BPN' [Wells et al. (1987) Proc. Natl. Acad. Sci. USA 84, 1219-1223]. The apparent P1 specificity of four mutants (E156Q/G166K, E156Q/G166R, G166K, and G166R) was extended to acid residues, but the halo-forming activity of Escherichia coli expressing the mutant genes on skim milk-containing plates was significantly decreased due to the lower autoprocessing efficiency. A marked increase in active enzyme production occurred when Tyr(-1) in the pro-region of these mutants was then replaced by Asp or Glu. Five mutants with Glu(-2)Ala/Val/Gly or Tyr(-1)Cys/Ser substitution showing enhanced halo-forming activity were further isolated by PCR random mutagenesis in the pro-region of the E156Q/G166K mutant. These results indicated that introduction of an optimum arrangement at the cleavage site in the pro-region is an effective method for obtaining a higher yield of active enzymes.