定点突变改造提高纺锤形赖氨酸芽孢杆菌氨基甲酸乙酯水解酶稳定性

Stability enhancement of urethanase from Lysinibacillus fusiformis by site-directed mutagenesis

摘要:

氨基甲酸乙酯 (ethyl carbamate，EC) 是一种存在于发酵食品和酿造酒精饮料中的潜在致癌物质。利用生物酶法去除食品饮料中的EC是一种较为安全有效的方法。本研究以来源于赖氨酸芽孢杆菌Lysinibacillus fusiformis SC02的氨基甲酸乙酯水解酶为研究对象，采用计算机辅助设计突变位点，构建了其不稳定区域Q328位点的饱和突变体。通过酶学性质分析发现，突变体Q328C和Q328V在40 ℃下的半衰期分别提高了7.46和1.99倍，Q328R在高温下也有比原酶更好的耐受性。此外，突变体Q328C对乙醇的耐受性和酸耐受性也有所提高。对氨基甲酸乙酯水解酶分子改造的结果表明，通过改造其不稳定区域Q328位点，可以提高酶的热稳定性及对酸和乙醇的耐受性。

关键词:定点突变，氨基甲酸乙酯水解酶，氨基甲酸乙酯，酶稳定性

Abstract:

Ethyl carbamate as a potential carcinogen commonly exists in traditional fermented foods and beverages. Enzymatic removal of ethyl carbamate from fermented foods and beverages is an efficient and safe method. In this study, we mutated urethanase from Lysinibacillus fusiformis SC02 on the Q328 site through computer aided design approaches. The half-life of resulting mutants Q328C and Q328V was detected to be 7.46 and 1.96 folds higher than that of the original enzyme, and Q328R presented better thermal-tolerance than the original urethanase when incubated at high temperature. The tolerance of Q328C to ethanol and acid also increased when compared with that of the original enzyme. The stability and tolerance to acid and ethanol of urethanase could be improved by modification on its Q328 site.

Key words:site-directed mutagenesis, urethanase, ethyl carbamate, enzyme stability