无色杆菌JA81基因组中多个假定烯烃还原酶的性能及其在贫电子烯烃不对称还原中的应用

Enoate reductases catalyze the reduction of alkenes with the creation of up to two chiral centers, and its application in asymmetric synthesis is a recently-developed research area in the field of bioreduction. We have previously identified several enoate reductase-producing strains, and one of them, Achromobacter sp. JA81, is the first strain of this genus that is reported to catalyze the asymmetric reduction of C=C double bond. The present project will focus on the systematic investigation of the multiple (at least 12) putative enoate reductases mined from the whole genome sequencing of the strain. Those putative enzymes will be cloned and heterologously expressed in Escherichia coli. Their enzymatic properties, and transcriptional and translational status in JA81, as well as their substrate spectra and the corresponding structure-functional relationships will be investigated.Key amino acid residues related to important catalytical characteristics will be identified.This project is expected to yield multiple efficient novel enoate reductases that can catalyze activated alkenes with excellent selectivity and activity with proprietary intellectual property rights, which will enrich the tool-box for asymmetric reduction of alkenes, and provide valuable information for future database mining. The use of recombinant enzymes increases the availability and facilitates the handling of biocatalysts, which would contribute substantially to the application of enoate reductases in organic synthesis.

烯烃还原酶催化的贫电子烯烃不对称还原是生物还原领域近年来发展的一个新的研究方向，是构建碳手性中心的高效手段之一。我们前期研究中筛选到的多个产烯烃还原酶菌株，其中无色杆菌JA81是该属中首次发现的具有不对称C=C还原能力的菌株。本项目将利用该菌株全基因组测序信息，系统性发掘其中多个(至少12个)假定的烯烃还原酶，研究其在JA81中的转录翻译情况；研究重组表达的酶蛋白的酶学性质及在若干类型贫电子烯烃不对称还原中的底物谱和催化底物的结构功能关系；识别影响重组蛋白催化性能的关键氨基酸残基，从而获得多个新型的具有自主知识产权的、高效高选择性的重组烯烃还原酶，丰富生物催化烯烃还原反应的工具酶库，为今后的基因组数据挖掘工作提供重要参考。重组酶可获得性高，使用简洁便利，将有望大力推动烯烃生物还原在不对称有机合成中的应用。