Saturation mutagenesis constitutes a powerful method in the directed evolution of enzymes. Traditional protocols of whole plasmid amplification such as Stratagene's QuikChange™ sometimes fail when the templates are difficult to amplify. In order to overcome such restrictions, we have devised a simple two-primer, two-stage polymerase chain reaction (PCR) method which constitutes an improvement over existing protocols. In the first stage of the PCR, both the mutagenic primer and the antiprimer that are not complementary anneal to the template. In the second stage, the amplified sequence is used as a megaprimer. Sites composed of one or more residues can be randomized in a single PCR reaction, irrespective of their location in the gene sequence.The method has been applied to several enzymes successfully, including P450-BM3 from Bacillus megaterium, the lipases from Pseudomonas aeruginosa and Candida antarctica and the epoxide hydrolase from Aspergillus niger. Here, we show that megaprimer size as well as the direction and design of the antiprimer are determining factors in the amplification of the plasmid. Comparison of the results with the performances of previous protocols reveals the efficiency of the improved method.
Candida antarctica lipase B (CALB) is a robust and easily expressed enzyme used widely in academic and industrial laboratories with many different kinds of applications. In fine chemicals production, examples include acylating kinetic resolution of racemic secondary alcohols and amines as well as desymmetrization of prochiral diols (or the reverse hydrolytic reactions). However, in the case of hydrolytic kinetic resolution of esters or esterifying kinetic resolution of acids in which chirality resides in the carboxylic acid part of the substrate, rate and stereoselectivity are generally poor. In the present study, directed evolution based on iterative saturation mutagenesis was applied to solve the latter problem. Mutants with highly improved activity and enantioselectivity relative to wild-type CALB were evolved for the hydrolytic kinetic resolution of p-nitrophenyl 2-phenylpropanoate, with the selectivity factor increasing from E = 1.2 (S) to E = 72 (S) or reverting to E = 42 (R) on an optional basis. Surprisingly, point mutations both in the acyl and alcohol pockets of CALB proved to be necessary. Some of the evolved CALB mutants are also efficient biocatalysts in the kinetic resolution of other chiral esters without performing new mutagenesis experiments. Another noteworthy result concerns the finding that enantiocomplementary CALB mutants for α-substituted carboxylic acid esters also show stereocomplementarity in the hydrolytic kinetic resolution of esters derived from chiral secondary alcohols. Insight into the source of stereoselectivity was gained by molecular dynamics simulations and docking experiments.
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