高通量筛选具有催化活性和立体选择性羰基还原酶

High throughput screening of active and stereoselective carbonyl reductases

摘要:

根据羰基还原酶催化可逆氧化还原反应的原理，利用与偶氮还原酶催化偶氮染料还原反应耦合的颜色变化，建立了一种新的羰基还原酶筛选方法。由于羰基还原酶在催化醇底物氧化反应时会产生NAD(P)H，当在反应体系中加入偶氮还原酶AzoB和偶氮染料金橙Ⅰ的时候，偶氮还原酶可以利用NAD(P)H作为电子的供体与底物金橙Ⅰ发生反应，导致反应体系颜色的变化，这样就能够根据明显的颜色变化推断出该羰基还原酶是否对所选底物表现出特定的活性，进而可以筛选出有活性的羰基还原酶。同时，使用不同构型的手性醇作为底物时，根据体系的颜色变化，可以实现羰基还原酶的活性和立体选择性的同时筛选。

Abstract:

In this study, a fast carbonyl reductases colorimetric screening method for discovering stereoselective carbonyl reductases was established by combining the reverse alcohol oxidation with the azoreductase-catalyzed reduction of azo dye. When azo dye (OrangeⅠ, 4-(4-hydroxy-1-naphthylazo) benzenesulfonic acid) and azoreductase (AzoB) were added into the reaction system of alcohol oxidation catalyzed by carbonyl reductase, the produced NAD(P)H served as electron donor for the azoreductase to reduce the azo dye, resulting the color fade. Hence, the carbonyl reductases can be screened by the obvious color change. When chiral alcohol was used as the substrate, the activity and stereoselectivity of carbonyl reductases can be screened at the same time.