羰基还原酶突变体高选择性催化不对称还原大位阻羰基化合物

Highly Stereoselective Reduction of Bulky Carbonyl Compounds by Carbonyl Reductase Variant

以立体选择性羰基还原酶(RCR)的双突变酶RCR-F285A/W286A为对象,考察了该突变酶对酮酯类和杂环酮类底物的催化性能(包括酶活力和动力学参数)及对映体选择性,发现其对2-氧代-4-苯基丁酸乙酯(OPBE)、苯甲酰基乙酸乙酯(EBA)和1-苄基-3-吡咯烷酮均具有酶活性,并且以高光学纯度生成相应手性醇(e.e.>99%).考察了反应条件对产率最高的OPBE的不对称转化反应的影响,结果表明,当pH=7. 0,反应温度30℃,3. 5%(体积分数)的异丙醇作助溶剂,加入1 g/L底物时,产率达到80. 3%.进一步通过分批补料减弱底物抑制作用,在10 g/L的底物浓度下,时空产率为0. 062 g·L^(-1)·h^(-1),为酶法不对称合成(R)-2-羟基-4-苯基丁酸乙酯[(R)-HPBE]的工业应用提供了实验基础.

RCR-F285A/W286A,the variant of the(R)-specific carbonyl reductase(RCR),was used as the biocatalyst for asymmetric reduction of carbonyl compounds with bulky groups.In this study,ketoesters and heterocyclic ketone,including ethyl 2-oxo-4-phenylbutyrate(OPBE),ethyl benzoylacetate(EBA)and 1-benzyl-3-pyrrolidone,were employed.Catalytic property(including enzyme activity and kinetic parameters)and enantioselectivity of RCR-F285A/W286A towards tested bulky substrates were measured.RCR-F285A/W286A exhibited enzymatic activity towards all of the tested substrates.The corresponding chiral alcohols of the tested substrates acting as important pharmaceutical intermediates were prepared with high optical purity(e.e.>99%).Moreover,the effects of reaction conditions,including pH,temperature,substrate concentration,cosolvents and ratio of the optimum cosolvent,on the asymmetric reduction of OPBE with the highest yield were investigated.The results showed that the yield of 80.3%was obtained at pH=7.0,30℃,1 g/L substrate and isopropanol with volume fraction of 3.5%.Furthermore,fed-batch strategy was adopted to weaken substrate inhibition with the space-time yield 0.062 g·L-1·h-1 at 10 g/L OPBE.This result would provide the experimental basis for the industrial application of the enzyme-mediated asymmetric synthesis of ethyl(R)-2-hydroxy-4-phenylbutanoate.