不对称还原大位阻二芳香基甲酮的羰基还原酶的基因克隆及性质分析

Cloning and characterization of a novel carbonyl reductase for asymmetric reduction of bulky diaryl ketones

摘要:

由于二芳香基甲酮的位阻大及羰基两侧的取代基差异性较小，对其进行不对称还原是生物催化中具有挑战性的难题之一。文中通过对毕赤酵母GS115基因组序列的分析，发现了一个潜在羰基还原酶基因pascr。将该基因克隆、表达在大肠杆菌Rosetta2 (DE3) 中，通过Ni-NTA对重组蛋白进行了分离纯化，并对酶的性质进行了研究。PasCR专一性利用NADPH作为辅酶，其最适反应pH为6.5；最适反应温度为35 ℃；凝胶层析实验结合SDS-PAGE分析表明PasCR在溶液中以二聚体形式存在。PasCR能够不对称还原位阻较大的二芳香基甲酮类化合物，如4-甲基二苯甲酮、4-氯二苯甲酮、2-氯二苯甲酮等，对4-甲基二苯甲酮的还原产物的ee值达到了85％。

Abstract:

Asymmetric reduction of bulky diaryl ketones is still one of the challenging tasks in biocatalysis. By genomic data mining, a putative carbonyl reductase gene pascr was found in Pichia pastoris GS115. pascr was cloned and over-expressed in Escherichia coli Rosseta2 (DE3). The recombinant enzyme was purified to homogeneity by Ni-NTA column and its catalytic properties were studied. PasCR strictly used NADPH as cofactor, gel filtration and SDS-PAGE analysis suggested that the native form of PasCR was a dimmer. PasCR exhibited the highest activity at 35 °C in phosphate buffer at pH 6.5. The enzyme catalyzed the reduction of some bulky diaryl ketones, such as 4-methylbenzophenone, 2-methylbenzophenone and 4-chlorobenzophenone, especially for 4-methylbenzophenone, the product S - alcohol was obtained with 85% ee.