Max-Planck-Institut für Kohlenforschung, Department of Synthetic Organic Chemistry, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr, Germany.
Chemphyschem. 2011 Jun 6;12(8):1550-7. doi: 10.1002/cphc.201100031. Epub 2011 Apr 5.
The most thoroughly studied enzyme in directed evolution is the lipase from Pseudomonas aeruginosa (PAL) as a catalyst in the hydrolytic kinetic resolution of 2-methyldecanoic acid p-nitrophenyl ester. Seminal studies utilized epPCR, saturation mutagenesis and DNA shuffling or combinations thereof. With current emphasis on efficacy in laboratory evolution, however, we recently applied our previously developed method, iterative saturation mutagenesis (ISM), to the same catalytic system, discovering that this approach is much more efficient than the original strategies. Herein, we consider PAL once more, this time testing ISM as a means to broaden the substrate scope of this lipase by studying bulky substrates of the type 2-phenylalkanoic acid esters as substrates that are not accepted by the WT. Highly active and enantioselective (E up to 436) mutants were evolved, a process that required only small mutant libraries and thus a minimum of screening effort. A theoretical investigation using molecular dynamics simulations and docking experiments revealed the source of enhanced activity and stereoselectivity.
在定向进化中研究得最彻底的酶是假单胞菌属脂酶（PAL），它可以作为催化剂用于 2-甲基癸酸对硝基苯酯的水解动力学拆分。开创性的研究利用易错 PCR、饱和突变和 DNA 重排或它们的组合。然而，由于目前强调实验室进化的效果，我们最近将我们之前开发的方法，即迭代饱和突变（ISM），应用于相同的催化体系，发现这种方法比原始策略效率更高。在这里，我们再次考虑 PAL，这次通过研究 WT 不接受的类型 2-苯基链烷酸酯作为底物，来测试 ISM 作为拓宽这种脂肪酶的底物范围的一种手段。进化出了高活性和对映选择性（高达 436 的 E）的突变体，这个过程只需要小型突变文库，因此筛选工作量最小。使用分子动力学模拟和对接实验的理论研究揭示了增强活性和立体选择性的来源。
