Construction of novel subtilisin E with high specificity, activity and productivity through multiple amino acid substitutions

Through three cumulative amino acid substitutions, we constructed novelmutant subtilisins E of Bacillus subtilis, all with high specificity,activity and productivity. The substitution of conserved Gly127,constituting P1 substrate-binding pocket, with Ala and Val showed a markedpreference for the small P1 substrate. Leu was then substituted for Ile31next to the catalytic Asp32 to enhance the catalytic activity. Both doublemutants (I31L/G127A and I31L/G127V) showed a 3-5- fold increase incatalytic efficiency due to a large kcat, without any change in thespecificity of the mutants at position 127. Molecular modeling suggeststhat large P1 residues were unable to access the pocket because of sterichindrance. A third mutation was introduced by replacing Tyr(-1) with Ala inthe propeptide essential for autoprocessing to active mature subtilisin invivo. A prominent 7-20- fold increase in active enzyme production occurredin the triple mutants (Y-1A/I31L/G127A and Y-1A/I31L/G127V).