枯草芽胞杆菌突变株ZC-7中性蛋白酶催化区域氨基酸突变导致其活力提高

Amino Acids Mutation in Catalytic Region of Neutral Protease in Bacillus subtilis Mutant ZC-7 Led to its High Activity

目的:探讨枯草芽胞杆菌突变株ZC-7高产中性蛋白酶的原因。方法:用PCR方法分别扩增突变株ZC-7与出发菌株枯草芽胞杆菌AS1.398产中性蛋白酶的编码基因,测序比较二者基因的不同;在CPHmodels Server网站进行氨基酸序列分析,模拟突变前后中性蛋白酶的二级结构。结果:对比结果显示成熟肽中有5个氨基酸位点发生突变,其中3个位于酶的催化区域内;从预测的二级结构模型上可以看到突变位点所处区域的折叠结构发生细微变化。结论:先前研究中发现枯草芽胞杆菌AS1.398和突变株ZC-7发酵液中的酶蛋白含量基本相同,因此推测高产的原因不是酶量的增加,而是突变的氨基酸使酶与底物结合的部位更加适合催化水解反应,从而提高其比活力。

Objective： To investigate the reasotas for high-yield of Bacillus subtilis mutant ZC-7 obtained by implantation with 30 keV N^＋ ions beam to B.subtilis AS1.398. Methods： The DNA fragments of the neutral protease genes in the original strain and the mutant were cloned and sequenced, respectively. Then, they were compared to analyze the gene difference between ZC-7 and AS1.398 neutral protease. Subsequently, the two protein secondary structures were simulated on CPHmodels Server net. Results： The alignment results showed that five amino acids of the mature peptide changed and three of them were in the key region. The protein secondary structures prediction results indicated that some conformation changed in the fixed range because of the mutation sites in the central region occurred. Conclusion： In previous research, the neutral protease productions of the two strains were almost the same. Therefore, these mutant amino acids located in the key domain of substrate binding led to the conformation change of ZC-7 neutral protease, and it was responsible for more appropriate for substrate binding, in another word, higher activity.