Abstract：
Catalytic activity is undoubtedly a key focus in enzyme engineering. The complicated reaction conditions hinder some enzymes from industrialization even though they have relatively promising activity. This has occurred to some dehydrogenases. Hydroxysteroid dehydrogenases (HSDHs) specifically catalyze the conversion between hydroxyl and keto groups, and hold immense potential in the synthesis of steroid medicines. We underscored the importance of 7α-HSDH activity, and analyzed the overall robustness and underlying mechanisms. Employing a high-throughput screening approach, we comprehensively assessed a mutation library, and obtained a mutant with enhanced enzymatic activity and overall stability/tolerance. The superior mutant (I201M) was identified to harbor improved thermal stability, substrate susceptibility, cofactor affinity, as well as the yield. This mutant displayed a 1.88-fold increase in enzymatic activity, a 1.37-fold improvement in substrate tolerance, and a 1.45-fold increase in thermal stability when compared with the wild type (WT) enzyme. The I201M mutant showed a 2.25-fold increase in the kcat/KM ratio (indicative of a stronger binding affinity for the cofactor). This mutant did not exhibit the highest enzyme activity compared with all the tested mutants, but these improved characteristics contributed synergistically to the highest yield. When a substrate at 100 mM was present, the 24 h yield by I201M reached 89.7%, significantly higher than the 61.2% yield elicited by the WT enzyme. This is the first report revealing enhancement of the catalytic efficiency, cofactor affinity, substrate tolerance, and thermal stability of NAD(H)-dependent 7α-HSDH through a single-point mutation. The mutated enzyme reached the highest enzymatic activity of 7α-HSDH ever reported. High enzymatic activity is undoubtedly crucial for enabling the industrialization of an enzyme. Our findings demonstrated that, when compared with other mutants boasting even higher enzymatic activity, mutants with excellent overall robustness were superior for industrial applications. This principle was exemplified by highly active enzymes such as 7α-HSDH.
摘要：
催化活性无疑是酶工程中的一个重点。复杂的反应条件阻碍了一些酶的工业化，尽管它们具有相对有希望的活性。这已经发生在一些脱氢酶上。羟基类固醇脱氢酶（HSDHs）专门催化羟基和酮基之间的转化，并在合成类固醇药物方面具有巨大潜力。我们强调了7α-HSDH活性的重要性，并分析了整体稳健性和潜在机制。采用高通量筛选方法，我们全面评估了一个突变库，并获得了具有增强的酶活性和总体稳定性/耐受性的突变体。鉴定出优越的突变体(I201M)具有改善的热稳定性，基底磁化率，辅因子亲和力，以及产量。该突变体的酶活性增加了1.88倍，基材耐受性提高1.37倍，与野生型(WT)酶相比,热稳定性提高1.45倍。I201M突变体显示kcat/KM比率增加2.25倍(指示对辅因子的更强结合亲和力)。与所有测试的突变体相比，该突变体没有表现出最高的酶活性，但是这些改善的特性协同作用于最高的产量。当存在100mM的底物时，I201M的24小时产量达到89.7%，显著高于WT酶引起的61.2%的产量。这是第一份揭示催化效率提高的报告，辅因子亲和力，基材公差,和通过单点突变的NAD（H）依赖性7α-HSDH的热稳定性。突变的酶达到了7α-HSDH的最高酶活性。高酶活性对于实现酶的工业化无疑是至关重要的。我们的研究结果表明,与其他具有更高酶活性的突变体相比，具有优异的整体鲁棒性的突变体对于工业应用是优越的。该原理由高活性酶如7α-HSDH举例说明。
