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 $\underline{B}$. $\underline{subtilis}$ DB104, each mutant had not shown any detectable protease activity compared with wild-type and had not shown SK activity. Interestingly, 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.
pZAll8 은 pKWZ 보다 B. subtilis DB 104 에서 발현이 더 잘 되었다. 부위 특이 돌연변이를 이용하여 서브틸리신의 His 64 와 His 67 을 각각 Gly 64 와 Gly 67로 치환시켰으며 변이 확인을 용이하게 하기 위하여 합성한 primer 에 새로운 KpnI site 를 도입하여 돌연변이를 시킨후 pZAll8ml 과 pZAll8m2 를 KpnI 으로 잘라 봄으로써 확인하였다. 또한, 이 두 변이 플라스미드를 이용하여 double mutant 즉 pZAll8m3 를 얻게 되었는데 이 변이체는 그의 염기 배열에 5개의 mismatched bases 를 갖고 있었다. pZAll8m1, pZAll8m2 and pZAll8m3 를 고초균에 형질전환시켜 성장 속도를 조사했더니 변이 플라스미드가 들어있는 고초균에서의 성장속도가 높게 나타났다. 그러나 LB-skim milk plate 에 streaking 시 거의 protease 활성이 나타나지 않았으며, 천연기질인 azocasein 을 이용시 변이체들은 야생형에 비해 protease 활성이 없어졌다. 또한, 변이체들의 SK 활성도 거의 나타나지 않았다. 이들 결과로부터 His 67 도 His 64 와 더불어 소크틸리신의 활성에 필수적 임을 알 수 있었다.
