Biotransformation and Biocatalysis, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, the Netherlands.
Chembiochem. 2012 Jan 2;13(1):137-48. doi: 10.1002/cbic.201100579. Epub 2011 Nov 23.
We used directed evolution to obtain enantiocomplementary haloalkane dehalogenase variants that convert the toxic waste compound 1,2,3-trichloropropane (TCP) into highly enantioenriched (R)- or (S)-2,3-dichloropropan-1-ol, which can easily be converted into optically active epichlorohydrins-attractive intermediates for the synthesis of enantiopure fine chemicals. A dehalogenase with improved catalytic activity but very low enantioselectivity was used as the starting point. A strategy that made optimal use of the limited capacity of the screening assay, which was based on chiral gas chromatography, was developed. We used pair-wise site-saturation mutagenesis (SSM) of all 16 noncatalytic active-site residues during the initial two rounds of evolution. The resulting best R- and S-enantioselective variants were further improved in two rounds of site-restricted mutagenesis (SRM), with incorporation of carefully selected sets of amino acids at a larger number of positions, including sites that are more distant from the active site. Finally, the most promising mutations and positions were promoted to a combinatorial library by using a multi-site mutagenesis protocol with restricted codon sets. To guide the design of partly undefined (ambiguous) codon sets for these restricted libraries we employed structural information, the results of multiple sequence alignments, and knowledge from earlier rounds. After five rounds of evolution with screening of only 5500 clones, we obtained two strongly diverged haloalkane dehalogenase variants that give access to (R)-epichlorohydrin with 90 % ee and to (S)-epichlorohydrin with 97 % ee, containing 13 and 17 mutations, respectively, around their active sites.
我们采用定向进化的方法获得了对映体互补的卤代烷烃脱卤酶变体，可将有毒废物化合物 1,2,3-三氯丙烷（TCP）转化为高对映体过量（R）-或（S）-2,3-二氯-1-丙醇，该化合物可轻松转化为光学活性的表氯醇-合成手性精细化学品的有吸引力的中间体。我们以催化活性提高但对映选择性非常低的脱卤酶为起点。开发了一种充分利用基于手性气相色谱的筛选测定法的有限容量的策略。在最初的两轮进化中，我们对所有 16 个非催化活性位点残基进行了两两定点饱和突变（SSM）。所得最佳 R-和 S-对映选择性变体在两轮定点限制突变（SRM）中进一步得到改善，在更大数量的位置包括离活性位点更远的位置，掺入了精心选择的氨基酸集。最后，通过使用具有受限密码子集的多部位诱变方案，将最有前途的突变和位置提升到组合文库中。为了指导这些受限文库部分未定义（模糊）密码子集的设计，我们采用了结构信息、多重序列比对的结果以及早期轮次的知识。经过五轮仅筛选 5500 个克隆的进化，我们获得了两种强烈分化的卤代烷烃脱卤酶变体，可分别获得 90%ee 的（R）-表氯醇和 97%ee 的（S）-表氯醇，其活性位点周围分别包含 13 和 17 个突变。
