通过定点突变提高乳链菌肽对热及pH的稳定性

Improving heat and pH stability of nisin by site-directed mutagenesis

摘要:

摘要：【目的】通过定点突变技术改变乳链菌肽（nisin）特定位置氨基酸，获得性质改善的nisin突变体，为扩大其应用范围提供依据。【方法】在抑菌谱扩大的nisin单突变体M21K nisinZ的基础上，对M21K nisZ基因第29位丝氨酸密码子进行定点突变；将其克隆至乳酸菌表达载体pMG36e，并在Lactococcus lactis NZ9800中进行表达；双突变体M21K/S29K nisinZ经分离纯化后检测其在抑菌活性、抑菌谱和稳定性等方面的变化。【结果】与单突变体M21K nisinZ及野生型nisinZ（wild-type，WT）相比，双突变体M21K/S29K nisinZ对指示菌的抑菌活性虽有所下降，但其对温度及pH值的稳定性有显著提高。同时其抑菌谱与M21K nisinZ相同，可抑制革兰氏阴性菌，扩大了WT的抑菌谱。【结论】通过改变nisin分子特定位置的氨基酸可以改善nisin分子的理化性质，有可能得到应用范围更广的nisin品种。

关键词:关键词：乳链菌肽;稳定性;温度;pH;定点突变

Abstract:

Abstract: [Objective] The aim of this study was to optimize the property of nisin through altering its specific amino acid by site-directed mutagenesis method. [Methods] On the basis of M21K nisinZ, a former reported nisinZ mutant that exhibited antimicrobial activity against Gram-negative bacteria, the 29th amino acid of it was mutated from serine to lysine. The mutant M21K/S29K nisZ gene was cloned into vector pMG36e and the recombinant plasmid was introduced into Lacotococcus lactis NZ9800. The resulting M21K/S29K nisinZ was then isolated and purified, and its antibacterial activity, antibacterial spectrum and stability were analyzed and compared to those of M21K nisinZ and nisinZ. [Results] Compared with wild-type nisinZ and M21K nisinZ, the M21K/S29K nisinZ displayed reduced antimicrobial activity, but showed significantly increased stabilities to heat and pH stress. Moreover, M21K/S29K nisinZ also exhibited antimicrobial activity against Gram-negative bacteria as M21K nisinZ did. [Conclusion] By changing the 29th amino acid of nisin, we can optimize the property of nisin, especially its stability to heat and pH stress.

Key words:Keywords：nisin; stability; heat; pH; site-directed mutagenesis