Alcohol dehydrogenases (ADHs) are used in reductive biotransformations for the production of valuable chiral alcohols. In this study, we used a high-throughput screening approach based on the NADPH biosensor pSenSox and fluorescence-activated cell sorting (FACS) to search for variants of the NADPH-dependent ADH of Lactobacillus brevis (LbADH) with improved activity for the reduction of 2,5-hexanedione to (2R,5R)-hexanediol. In a library of approx. 1.4 × 106 clones created by random mutagenesis we identified the variant LbADHK71E. Kinetic analysis of the purified enzyme revealed that LbADHK71E had a ~ 16% lowered KM value and a 17% higher Vmax for 2,5-hexanedione compared to the wild-type LbADH. Higher activities were also observed for the alternative substrates acetophenone, acetylpyridine, 2-hexanone, 4-hydroxy-2-butanone, and methyl acetoacetate. K71 is solvent-exposed on the surface of LbADH and not located within or close to the active site. Therefore, K71 is not an obvious target for rational protein engineering. The study demonstrates that high-throughput screening using the NADPH biosensor pSenSox represents a powerful method to find unexpected beneficial mutations in NADPH-dependent alcohol dehydrogenases that can be favorable in industrial biotransformations.

中文翻译：

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基于NADPH生物传感器的酒精脱氢酶变异体的鉴定，该变异体具有改善的催化特性，这是由于蛋白质表面的一次电荷反转引起的。

醇脱氢酶（ADH）用于还原性生物转化，以生产有价值的手性醇。在这项研究中，我们使用了基于NADPH生物传感器pSenSox和荧光激活细胞分选（FACS）的高通量筛选方法，搜索了短乳杆菌（LbADH）的NADPH依赖性ADH变体，其活性降低了。 2，5-己二酮转化为（2R，5R）-己二醇。在大约一个库中。通过随机诱变创建的1.4×106个克隆，我们鉴定出LbADHK71E变异体。纯化酶的动力学分析表明，与野生型LbADH相比，LbADHK71E的2,5-己二酮的KM值降低了〜16％，Vmax升高了17％。还观察到替代底物苯乙酮，乙酰吡啶，2-己酮，4-羟基-2-丁酮和乙酰乙酸甲酯的活性较高。K71溶剂暴露在LbADH的表面上，而不位于活性位点内或附近。因此，K71不是合理的蛋白质工程的明显目标。这项研究表明，使用NADPH生物传感器pSenSox进行的高通量筛选代表了一种强大的方法，可以发现NADPH依赖性酒精脱氢酶中意想不到的有益突变，这种突变对工业生物转化非常有利。