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OALib Journal期刊
ISSN: 2333-9721
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烯酮还原酶基因的克隆与优化表达
DOI: 10.3724/SP.J.1145.2011.00087, PP. 87-90
Keywords: 烯酮还原酶,优化表达,不对称还原,c=c双键,丙酮丁醇梭菌
Abstract:
烯酮/酯还原酶可以专一性地还原烯酮/酯中的碳碳双键，并引入两个手性中心，是手性化合物生物催化合成的重要酶类之一；在使用烯酮/酯还原酶进行全细胞手性分子生物催化合成中，其他还原酶的存在常导致副反应的产生.为研究烯酮/酯还原酶的理化性质、底物的专一性、产物的手性特点等，需要经过纯化的烯酮/酯还原酶.为此，根据烯酮还原酶（enoatereductase，ers，ec1.3.1.31）的基因序列设计引物，以丙酮丁醇梭菌（clostridiumacetobutylicum）atcc824基因组dna为模板，通过pcr扩增技术得到了目标酶的基因，目的片段全长为1995bp，共编码664个氨基酸，相对分子质量为73×103.将烯酮还原酶基因连接到pet-32a(+)上，构建表达质粒pet-32a（+）-ers，并成功在escherichiacolirosetta（de3）plyss中表达.在摇床上优化的表达条件为：诱导温度为18℃，诱导起始时菌体d600nm为0.6~0.8，转速为100r/min，诱导剂iptg浓度为0.1mmol/l，诱导培养时间为12h，表达的烯酮还原酶大部分以可溶性形式存在于菌体内.图5参16
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