Combination of computational prescreening and experimental library construction can accelerate enzyme optimization by directed evolution
Combination of computational prescreening and experimental library construction can accelerate enzyme optimization by directed evolution
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DOI：
10.1093/protein/gzi062
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发表时间：
2005-11-01
影响因子：
2.4
通讯作者：
Thiel, W
中科院分区：
文献类型：
--
作者：
Funke, SA;Otte, N;Thiel, W
使用生物催化剂可以有效地生产手性化合物。然而，野生型酶通常不能满足生产过程的要求，因此需要通过合理设计或定向进化进行优化。在这里，我们从理论上和实验上研究了模型底物 1-(2-萘基)乙酸乙酯的脂肪酶催化水解。我们发现，基于 QM/MM 方法的丙氨酸扫描诱变的计算等效可用于识别对酶活性重要的氨基酸位置。理论结果与使用完全饱和诱变和高通量筛选目标生物催化剂（来自枯草芽孢杆菌的脂肪酶）的伴随实验工作一致。基于 QM/MM 的计算和分子生物学实验都将组氨酸 76 识别为强烈影响催化活性的残基。实验证明了它对对映选择性的重要影响。
Chiral compounds can be produced efficiently by using biocatalysts. However, wild-type enzymes often do not meet the requirements of a production process, making optimization by rational design or directed evolution necessary. Here, we studied the lipase-catalyzed hydrolysis of the model substrate 1-(2-naphthyl)ethyl acetate both theoretically and experimentally. We found that a computational equivalent of alanine scanning mutagenesis based on QM/MM methodology can be applied to identify amino acid positions important for the activity of the enzyme. The theoretical results are consistent with concomitant experimental work using complete saturation mutagenesis and high-throughput screening of the target biocatalyst, a lipase from Bacillus subtilis. Both QM/MM-based calculations and molecular biology experiments identify histidine 76 as a residue that strongly affects the catalytic activity. The experiments demonstrate its important influence on enantioselectivity.