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 k_cat/K_M 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.

中文翻译：

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不仅是活性，而且是最佳点：脱氢酶点突变可协同提高活性、底物耐受性、热稳定性和产量

催化活性无疑是酶工程的一个重点。一些酶虽然具有较好的活性，但复杂的反应条件阻碍了其工业化。某些脱氢酶已发生这种情况。羟基类固醇脱氢酶（HSDH）特异性催化羟基和酮基之间的转化，在类固醇药物的合成中具有巨大的潜力。我们强调了 7α-HSDH 活性的重要性，并分析了其整体稳健性和潜在机制。我们采用高通量筛选方法，对突变库进行了全面评估，获得了具有增强的酶活性和整体稳定性/耐受性的突变体。优良突变体 (I201M) 经鉴定具有改善的热稳定性、底物敏感性、辅因子亲和力以及产量。与野生型 (WT) 酶相比，该突变体的酶活性提高了 1.88 倍，底物耐受性提高了 1.37 倍，热稳定性提高了 1.45 倍。 I201M 突变体的k _cat / K _M比率增加了 2.25 倍（表明对辅因子有更强的结合亲和力）。与所有测试的突变体相比，该突变体并未表现出最高的酶活性，但这些改进的特性协同促进了最高产量。当存在 100 mM 底物时，I201M 24 小时产率达到 89.7%，显着高于 WT 酶产生的 61.2% 产率。这是第一份揭示通过单点突变增强 NAD(H) 依赖性 7α-HSDH 的催化效率、辅因子亲和力、底物耐受性和热稳定性的报告。突变酶达到了迄今为​​止报道的 7α-HSDH 的最高酶活性。高酶活性无疑对于酶的工业化至关重要。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，具有优异整体稳健性的突变体更适合工业应用。 7α-HSDH 等高活性酶就是这一原理的例证。