Acid stabilization of Bacillus licheniformis alpha amylase through introduction of mutations
Acid stabilization of Bacillus licheniformis alpha amylase through introduction of mutations
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DOI：
10.1007/s00253-008-1580-5
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发表时间：
2008-10-01
影响因子：
5
通讯作者：
Gao, Chen
中科院分区：
文献类型：
--
作者：
Liu, Yi-han;Lu, Fu-ping;Gao, Chen
本文进一步了解了地衣芽孢杆菌α-淀粉酶(BLA)两个关键位点Leu134和Ser320的突变导致不同突变体的结构特征与耐酸性的关系。为了研究这两个位置对酸稳定性的影响，我们对野生型和单突变体L134R和S320A以及双突变体L134R/S320A进行了详细的鉴定。L134R/S320A的k(Cat)/Km最高，pH为4.5，约为野生型的14倍。L134R和S320A的k(Cat)/Km介于野生型和L134R/S320A之间。此外，与活性下降较快的野生型相比，L134R/S320A在低pH条件下仍能保持较强的活性。同时测定了L134R和S320A在酸性条件下的耐受性低于L134R/S320A。令人惊讶的是，通过定向进化，L134R/S320A的耐酸能力显著增强。这些结果与三维结构分析相结合，表明静电效应在决定BLA在134和320这两个关键位置的稳定性方面起着重要作用。
This paper provided further understanding of the relationships between acid resistance and structural features of different mutants in Bacillus licheniformis alpha amylase (BLA) due to the changes of two crucial positions Leu134 and Ser320. In order to investigate effect of the two positions on the acid stability, we described the detailed characterization of wild-type and the single mutants L134R and S320A as well as the double mutant L134R/S320A. The highest k (cat) /Km with pH 4.5, approximately 14 times that of wild type, was observed in L134R/S320A. The k (cat) /Km corresponding to L134R and S320A were at an intermediate values between those for wild type and L134R/S320A. In addition, compared with wild type, which had a rapid decline of the activity, L134R/S320A could maintain its activity strongly in low pH. Meanwhile, lower tolerance of L134R and S320A in acidic conditions than that of L134R/S320A was determined. Surprisingly, the acid-resistant capability of L134R/S320A was significantly enhanced by directed evolution. These results, combined with three-dimensional structure analysis, show that the electrostatic effects play a significant role in determining the stability of BLA at two crucial positions, 134 and 320.