马克斯克鲁维酵母羰基还原酶基因的克隆与表达

摘要

[Objective] The gene encoding carbonyl reductase was cloned and expressed for the synthesis of the chiral drug intermediates.[Methods] Based on the N-terminal amino acid sequence of carbonyl reductase from GenBank,cmcr was cloned from Kluyveromyce marxianus CGMCC 2.1977 and sequenced,an expression vector,pET28a-cMCR containing the full length of cmcr was constructed and introduced into Escherichia coli BL21(DE3) and Rosetta(DE3) to express the enzyme separately.[Results] This gene showing 100％ similarity to reported mer contains an open reading frame of 1 038 bp encoding 345 amino acid residues.CMCR was overexpressed in Rosetta(DE3) with a protein molecular weight of 42 kD.The optimal temperature was 40 ℃ and pH 8.The enzyme has low thermal and pH stability.Ca2+ activates the enzyme activity,especially when its concentration is 0.5 mmol/L.The asymmetric reduction of 4-chloro-3-oxobutanoate ethyl ester to (S)-4-chloro-3-hydroxyl butanoate ethyl ester (CHBE) with Rosetta(pET28a-cMCR) cells,in which cmcr was expressed,as a catalyst was investigated (100％ e.e.,81.0％ yield).The application of the cells to the asymmetric reduction of N,N-dimethyl-3-keto-3-(2-thienyl)-1-prapanamine(DKTP) to (S)-N,N-dimethyl-3-hydroxy-(2-thienyl)-1-propanamine [(S)-DHTP] was also attempted.[Conclusion] The gene encoding carbonyl reductase was cloned and expressed successfully in E.coli form Kluyveromyce marxianus CGMCC 2.1977 and can be applied to asymmetric reduction.%[目的]研究羰基还原酶基因的克隆、表达及其在不对称生物催化中的应用.[方法]对羰基还原酶氨基酸序列进行BLAST推导出核苷酸序列,设计引物,以马克斯克鲁维酵母(Kluyveromyce marxianus) CGMCC 2.1977全基因组为模板,通过PCR扩增目的片段,与载体pET-28a连接,转化大肠杆菌获得重组菌BL21 (DE3)-(pET28a-cMCR)和Rosetta(DE3)-(pET28a-cMCR).[结果]扩增的序列与已报道的mer序列有100％同源性,全长1 038 bp,共编码345个氨基酸.目的蛋白在Rosetta(DE3)-(pET28a-cMCR)得到了高效表达,大小为42 kD.该酶最适反应温度为40℃,最适反应pH是8,热稳定性与pH稳定性较差.Ca2+对酶活具有明显的激活作用,且浓度为0.5 mmol/L时效果最好.重组菌可还原4-氯乙酰乙酸乙酯(COBE)为(S)-4-氯-3-羟基丁酸乙酯[(S)-CHBE],光学纯度为100％,转化率为81.0％.重组菌在制备度洛西汀关键中间体(S)-氮,氮-二甲基-3-羟基-(2-噻吩)-1-丙胺[(S)-DHTP]中也得到初步应用.[结论]从菌株马克斯克鲁维酵母(Kluyveromyce marxianus) CGMCC 2.1977中克隆获得了羰基还原酶基因,在大肠杆菌中成功表达,并可应用于不对称还原.