237 this single substitution causes a shift in catalytic activity toward ester substrates verses anilide substrates of almost 30 fold as compared to naturally occurring subtilisin it also results in a partial elimination of the kink so that the a helix extends from residue 221 237 modifications of this position with other amino acids are also expected to produce mutant enzymes demonstrating a shift in catalytic activity for different substrates and similar structural consequences thus other amino acids other than alanine may be used to replace the proline at position 225 such amino acids preferably include leucine methionine glutamine valine and serine most preferably serine and leucine these amino acids are expected to extend the a helix removing the discontinuity or kink in a manner analogous to the alanine substitution mutant enzymes containing such substituted amino acids at position 225 are also expected to demonstrate a shift in catalytic activity towards ester substrates as compared to anilide or amide substrates the procedure used to substitute alanine for proline at position 225 in the bacillus amyloliquefaciens subtilisin gene is illustrated generally in fig4 primer extension mutagenesis on a single stranded m13 subclone using the mutagenic oligonucleotide in fig4 was employed plasmid ppro225a was used to transform e coli mm294 clones from this transformation were used to transform b subtilis and transformants were plated on luria agar containing skim milk in order to detect protease secretion one protease secreting transformant was selected for enzyme purification and characterization and for sequence analysis to ensure.