一种新型醛酮还原酶的克隆表达、性质研究以及在不对称合成(R)-CHBE中的应用

Overexpression,characterization of a novel aldo-keto reductase for asymmetric synthesis of (R)-CHBE

为开发催化4-氯乙酰乙酸乙酯(COBE)制备(R)-4-氯-3-羟基丁酸乙酯((R)-CHBE)的新型催化剂,挖掘到了来自白色念珠菌SC5314中的一种NADPH辅酶依赖型醛酮还原酶CAK基因(cak),并将该基因在大肠杆菌中表达。将重组酶进行纯化后,测定其酶学性质,并构建了以葡萄糖为辅底物的双酶偶联辅酶再生系统,考察其不对称转化制备(R)-CHBE的能力。结果表明:CAK对多种醛酮类化合物有催化活性,其催化COBE的最适反应温度为40℃,最适p H为5。CAK在40℃下以及酸性条件中能保持较好的稳定性。Mg2+、Na+、K+对酶活有一定的激活作用,而Cu2+存在条件下酶会彻底失活。乙酸乙酯、邻苯二甲酸二丁酯对酶活的抑制作用较小。利用双酶偶联辅酶再生系统不对称转化制备(R)-CHBE。在合适的条件下,转化600 mmol/L的底物,产率达80.6%,产物对映体过量值(e.e.值)>99%。

An NADPH?dependent aldo?keto reductase ( CAK) gene from Candida albicans SC5314 was discovered for reducing 4?chloroacetoacetate ethyl ester ( COBE ) to ethyl ( R )?4?chloro?3?hydroxybutanoate ( ( R )?CHBE ) . The CAK gene was cloned and overexpressed in Escherichia coli Rosetta. The catalytic properties of purified CAK were studied. Furthermore,an enzyme?coupled coenzyme regeneration system with co?substrate glucose was constructed, and its capability of reducing COBE to ( R)?CHBE was studied. The results showed that CAK had catalytic activity for a variety of aldehydes and ketones. CAK had a maximum activity at 40℃ and pH 5. The enzyme was stable below 40℃ and under acidic conditions. Mg2+, Na+ and K+ could enhance the activity of CAK. When Cu2+ was added, no enzyme activity was detected. The inhibit effect of ethyl acetate and dibutyl?O?phthalate was relatively light. When the coenzyme regeneration system for conversion reaction was applied,the molar conversion yield reached 80?6% with 600 mmol/L COBE,and the e?e? value was over 99%.