马克斯克鲁维酵母羰基还原酶基因的克隆与表达

【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°C,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)-l-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。

Cloning and expression of Kluyveromyce marxianus gene encoding carbonyl reductase in Escherichia coli

[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 °C 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.