Imine reductases (IREDs) have been identified as an important class of biocatalysts to synthesize chiral amines with substantial promise for industrial application. Here, we report the promiscuous imine reductase activity of a glucose dehydrogenase (GDH). Starting from enzyme GDH Rd1bb, a commercial glucose dehydrogenase variant typically used for NAD(P)H regeneration, eight rounds of directed evolution were used to convert this enzyme into a highly active IRED for the chemo- and stereoselective manufacture of a chiral neprilysin inhibitor precursor, improved NADH cofactor specificity, and superior thermal stability. The evolved variant GDH Rd6bb achieved high productivity, with 99% conversion over 3 h at 50 g/L keto substrate concentration and 10% enzyme loading with respect to the keto substrate. Early process development studies at multigram scale provided the product in 94% yield with >99% purity as a single stereoisomer with an er of >99.9:0.1 and a dr of >99.9:0.1.

中文翻译：

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增强混杂葡萄糖脱氢酶的亚胺还原酶活性，用于大规模生产手性脑啡肽酶抑制剂前体

亚胺还原酶（IRED）已被确定为一类重要的生物催化剂，用于合成手性胺，具有工业应用的巨大前景。在这里，我们报告了葡萄糖脱氢酶（GDH）的混杂亚胺还原酶活性。从酶 GDH Rd1bb（一种通常用于 NAD(P)H 再生的商业葡萄糖脱氢酶变体）开始，使用八轮定向进化将该酶转化为高活性 IRED，用于化学和立体选择性制造手性脑啡肽酶抑制剂前体、改进的 NADH 辅因子特异性和卓越的热稳定性。进化后的变体 GDH Rd6bb 实现了高生产率，在 50 g/L 酮底物浓度和相对于酮底物的酶负载量为 10% 的情况下，3 小时内转化率为 99%。多克规模的早期工艺开发研究提供了该产品的收率 94%，单一立体异构体纯度 >99%，er >99.9:0.1，dr >99.9:0.1。