Gene mining-based identification of aldo-keto reductases for highly stereoselective reduction of bulky ketones
Gene mining-based identification of aldo-keto reductases for highly stereoselective reduction of bulky ketones
复制标题
基于基因挖掘的醛酮还原酶鉴定，用于高度立体选择性还原大体积酮
DOI：
10.1186/s40643-018-0220-x
复制
发表时间：
2018-01-01
影响因子：
4.6
通讯作者：
Xu, Yan
中科院分区：
文献类型：
--
作者：
Liang, Chen;Nie, Yao;Xu, Yan
背景：醛酮还原酶(AKR)或乙醇脱氢酶(ADH)介导的前手性羰基化合物的立体选择性还原是制备单一手性醇类对映体的有效方法。然而，底物的空间位阻会影响酶的催化性能。本研究旨在发现和鉴定能够催化空间位阻酮的高度立体选择性还原的AKRs/ADHS。结果：通过基因挖掘鉴定了来自不同微生物(CACR、SCCR、KmCR、CPR-C1和CPR-C2)的5个AKRs，并在重组大肠杆菌BL21(DE3)中高效表达。进一步评价了AKRs的比活性和立体选择性对各种酮酯类和杂环酮类化合物的影响，这些化合物体积大，在工业上有应用价值。每个纯化的酶对一个或多个测试底物表现出催化活性。在这些酶中，SCCR表现出比其他酶更宽的底物光谱。关于与底物结合相关的K-m值，我们还提供了对某些酶的特异性和偏好的见解。因此，对映体(R)-扁桃酸甲酯、(R)-扁桃酸乙酯、(R)-2-羟基-4-苯基丁酸乙酯和(S)-N-苄基-3-吡咯烷醇(>；99%e.E.)结论：通过基因挖掘获得了立体特异性AKRs，在重要光学活性醇类化合物的制备中具有潜在的应用前景。
Background: Aldo-keto reductase (AKR) or alcohol dehydrogenases (ADH)-mediated stereoselective reduction of prochiral carbonyl compounds is an efficient way of preparing single enantiomers of chiral alcohols. However, steric hindrance of substrate affects the catalytic performance of enzymes. The present study aims to discover and identify AKRs/ADHs capable of catalyzing highly stereoselective reduction of sterically hindered ketones.Results: Five AKRs from different microorganisms (CaCR, ScCR, KmCR, CPR-C1, and CPR-C2) were identified through gene mining, and overexpressed in recombinant Escherichia coli BL21(DE3). The specific activity and stereoselectivity of the AKRs were further evaluated towards various ketoesters and heterocyclic ketones, which are sterically bulky and are valuable in industrial applications. Each purified enzyme exhibited catalytic activity to one or more of the tested substrates. Among the enzymes, ScCR showed a broader substrate spectrum compared to the others. Regarding K-m values related to substrate association, we also provided insights into the specificity and preference of certain enzymes. Consequently, enantiopure (R)-methyl mandelate, ethyl (R)-mandelate, ethyl (R)-2-hydroxy-4-phenylbutyrate, and (S)-N-benzyl-3-pyrrolidinol (>99%e.e.) were obtained through the identified AKRs.Conclusion: The stereospecific AKRs were obtained through gene mining, which possesses the potential for application in the preparation of important optically active alcohols.