定点突变提高枯草芽孢杆菌L-天冬酰胺酶的活力及稳定性

Improving the Activity and Stability of L-Asparaginase from Bacillus subtilis by Site-Directed Mutagenesis

DOI：10.3969/j.issn.1673-1689.2015.11.002

中文关键词: L-天冬酰胺酶 定点突变 热稳定性 pH稳定性

英文关键词: L-asparaginase,site-directed mutagenesis,thermal stability,pH stability

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中文摘要:

L-天冬酰胺酶可以催化L-天冬酰胺转化为L-天冬氨酸和氨。它可以通过降解食品原料中的L-天冬酰胺而降低高温烹制食品中丙烯酰胺的含量。由于食品预处理环境的复杂性，只有具有高酶活且稳定的酶才能满足食品生产中的应用。作者通过点突变提高来源于Bacillus subtilis B11-06的L-天冬酰胺酶（BsAII）的酶活和热稳定性。通过序列比对和同源模拟选择5个点进行突变，构建6个突变菌株。酶活测定结果表明，突变体酶S299Nansz和P348Aansz的酶活较BsAII分别提高28%和32%。其中P348Aansz的热稳定性和pH稳定性较BsAII均有明显提高。本研究表明，第299和348位氨基酸残基对酶的催化作用有较大影响，对该酶的催化机理的研究提供了一定的基础，并提高了该酶的工业应用潜力。

英文摘要:

L-Asparaginase catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonia，which can hydrolyze free L-Asn to aspartic acid and decrease acrylamide formation during food processingd. In order to meet the high requirements and complicated procedures of food processing，the activity and stability of L-Asparaginase need to beimproved. In this study，we successfully improved the enzyme activity and stability of L-asparaginase （BsAII） from Bacillus subtilis B11-06 by site-directed mutagenesis. Five residues of BsAII were selected and used to construct the mutants by sequence alignment and homology modeling. The enzyme activity assayshowed that the enzyme activity of S299Nansz and P348Aaansz were increased by 28% and 32%，respectively，as compared to the BsAII. The thermal stability and pH stability of P348Aansz were significantly improved compared to that of BsAII. This study showed that residues 299 and 348 in amino acid sequence of BsAII had a great influence on the catalytic action，which provided a basis for the study of the catalytic mechanism of the enzyme，and extended the application in food industry.

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