【作者】 张敏；

【导师】 吕早生；

【作者基本信息】 武汉科技大学 ， 化学工艺， 2008， 硕士

【摘要】 具有特定功能基团的手性醇是合成手性药物的重要中间体,从羰基化合物不对称还原合成手性醇,已成为手性合成的一个重要部分。在羰基的不对称催化还原反应研究中,生物催化以其温和的反应条件、对环境的压力较小和较高的光学选择性,在羰基不对称还原合成中占有很重要的地位。选择合适的生物催化剂对于生物催化至关重要。2006年Bali等报道了短链脱氢酶家族(short-chain dehydrogenase superfamily, SDR)成员之一的糖多孢红霉菌聚酮合成酶中的酮还原酶对很多的底物,都有比较好的生物选择性还原能力,特别是对结构中含有环己酮的底物。本实验以糖多孢红霉菌基因组作为DNA供体,并根据2005年Alexandros的报道设计特异性引物,扩增野生型的糖多孢红霉菌聚酮合成酶中的酮还原酶域基因(eryKR1)。再根据Genbank中报道的eryKR1基因和放线菌素聚酮合成酶中的酮还原酶基因(ActKR),设计了用于定点突变的特异性引物,利用重叠PCR技术将KR1中决定其底物特异性的位点定点突变为ActKR中决定其底物专一性的那段基因位点,得到突变的KR1片段命为eryKR1M。将其克隆到表达载体pET-28a上,从中挑选阳性克隆菌株pET-eryKR1和pET-eryKR1M进行测序。用同样的方法构建重组质粒pET-GDH作为辅酶再生。将重组质粒pET-GDH,pET-eryKR1和pET-eryKR1M转化到大肠杆菌BL21中进行表达。加定量的IPTG诱导6小时后,通过SDS-PAGE检测重组蛋白的表达。最后通过发酵检验野生型和突变后的酮还原酶对四种底物(4-氯乙酰乙酸乙酯,苯乙酮,2-辛酮和环己酮)还原作用。结果表明,扩增eryKR1,eryKR1M基因的最佳退火温度为68℃,GDH基因的最佳退火温度为54℃。扩增的eryKR1,eryKR1M和GDH基因所对应的蛋白序列与报道的同源性高达98%。pET-eryKR1M中的目标基因与Genbank中报道的KR1基因仅在控制底物专一性的位点处不同,被放线菌素聚酮合成酶中的酮还原酶基因(ActKR)上的这段位点所取代,这也与我们设想一致。另外,SDS-PAGE检测出eryKR1,eryKR1M,GDH目标蛋白带。最后,发酵实验结果表明野生型的eryKR1对环己酮有很好的还原作用,而eryKR1M对环己酮的作用降低。这为今后该工程菌应用于手性醇的生物催化奠定了一定的基础。更多还原

【Abstract】 Chiral alcohols with special functional groups are important building blocks for synthesizing chiral drug, and synthesizing chiral alcohols by the asymmetric reduction of carbonyl compounds has become an important part of chiral synthesis. Biocatalysis plays a very important role in carbonyl asymmetric reduction because of its mild reaction conditions, less stressful environment and efficient stereoselectivity. Screening the most suitable biocatalyst is critical. In 2006, Bali reported that ketoreductase of polyketides synthase in Saccharopolyspora erythraea as one of the members of short-chain dehydrogenase superfamily existed optimistic ability of biological selective reduction, especially to the substrates whose structures contain cyclohexanone.In this work, DNA of S.erythraea was extracted.The primer sequences were designed according to the result Alexandros reported.We cloned the mild eryKR1 gene. Based on the nucleotide sequences of the ketoreductase from the first extension module of the erythromycin polyketide synthase, and the nucleotide sequences of ketoreductase of polyketide synthase in Actinorhodin (ActKR) in the Genbank, gene specific primers were designed. Through the overlapping PCR manner, the gene sites determining its substrate specificity in the ketoreductase (KR1) domain is replaced by that determining its specificity in ActKR, and we get the mutated KR1 domain DNA fragment eryKR1M. We cloned eryKR1 and eryKR1M into vector pET-28a, built the plasmid pET-eryKR1M and introduced the plasmid pET-eryKR1M and pET-eryKR1 into Escherichia coli BL21.Then we built the plasmid pET-GDH with the same method as the coenzyme regeneration. After 6 hours of inducing by IPTG, we detected the expression of the protein eryKR1M ,eryKR1,GDH by SDS-PAGE. At last, we examined its effect on four kinds of different substrates (ethyl 4-chloro-3-oxobutanoate, acetophenone, 2-octanone and cyclohexanone) using the fermentation technology.The results showed that the best annealing temperature was 68℃.The protein sequences of the cloned genes had the high homology compared with the reported protein sequences(reached 98%). The target gene of pET-eryKR1M differented from the gene of the ketoreductase from the first extension module of the erythromycin polyketide synthase only in the sites determing the specificity of the substrates, and was replaced by the sites of ketoreductase of polyketide synthase in Actinorhodin(ActKR), which is accordant with our initial presumption. In addition, through SDS-PAGE, all the target protein eryKR1M,eryKR1,GDH were detected. At last, the results of fermentation showed the wild gene existed optimic effects on Cyclohexanone ,which suggests that the engineering bacteria would be used for the biocatalysis of chiral alcohols the in the future.更多还原