Site-specific mutagenesis of subtilisin coding gene

Abstract

Subtilisin is a alkaline protease secreted by Bacilli. It``s catalytic triad is composed of Asp 32, His 64 and Ser 221. Also, His 67 is located nearby His 64 in a-helix segment. To analyze the functional role of subtilisin, His 64 and His 67 were changed to Gly 64 and Gly 67, by oligonucleotide-directed site-specific mutagenesis. Furthermore, double mutant was obtained from Gly 64 and Gly 67 mutant by simple gene manipulation. Then, we were examined for streptokinase activity of each mutant. This experiment was based upon the fact that SK has evolved from a serine protease by gene duplication and fusion. In addition, catalytic site of SK is composed of Gly 24 ASP 66 and Ser 157. When transformed into B. subtilkis DB104, each mutant had not shown any detectable protease activity compared with wild-type and had not shown SK activity. Interstingly, the effect of double mutant for protease production was almost same as Gly 64 mutant. from these results, we suggest that His 67 is also necessary for the catalytic activity of subtilisin as well as His 64.