Photometric Characterization of the Reductive Amination Scope of the Imine Reductases from Streptomyces tsukubaensis and Streptomyces ipomoeae
Photometric Characterization of the Reductive Amination Scope of the Imine Reductases from Streptomyces tsukubaensis and Streptomyces ipomoeae
复制标题
DOI：
10.1002/cbic.201700257
复制
发表时间：
2017-10-18
期刊：
影响因子：
3.2
通讯作者：
Hoehne, Matthias
中科院分区：
文献类型：
--
作者：
Matzel, Philipp;Krautschick, Lukas;Hoehne, Matthias
亚胺还原酶(IREDS)是一种很有前途的酶，可用于从羰基底物开始不对称合成仲胺和叔胺。筛选还原胺化反应的底物特异性通常是通过耗时的GC分析来完成的。我们在我们的酶集合中发现了两个高活性的IRED，来自Tsukubaensis的IR-20和来自Streptomyces ipomoeae的IR-sip，它们允许用光度NADPH法进行全面的底物筛选。我们筛选出39种羰基底物和17种胺类化合物作为亲核剂。来自663个组合的活性数据提供了关于底物特异性和这些酶的还原胺化能力的清晰图景。除脂肪醛外，红外区还可接受各种环(C-4-C-8)和无环酮，优先与甲胺结合。IR-sip还接受了一系列伯胺和仲胺作为亲核剂。在生物催化反应中，(R)-3-甲基环己酮与二甲胺或吡咯烷具有很高的非对映选择性(>；94-96%De)。亲核受体光谱取决于所使用的羰基底物。如果使用粗酶解物作为酶的来源，也可以检测到被接受的底物的转化。
Imine reductases (IREDs) have emerged as promising enzymes for the asymmetric synthesis of secondary and tertiary amines starting from carbonyl substrates. Screening the substrate specificity of the reductive amination reaction is usually performed by time-consuming GC analytics. We found two highly active IREDs in our enzyme collection, IR-20 from Streptomyces tsukubaensis and IR-Sip from Streptomyces ipomoeae, that allowed a comprehensive substrate screening with a photometric NADPH assay. We screened 39 carbonyl substrates combined with 17 amines as nucleophiles. Activity data from 663 combinations provided a clear picture about substrate specificity and capabilities in the reductive amination of these enzymes. Besides aliphatic aldehydes, the IREDs accepted various cyclic (C-4-C-8) and acyclic ketones, preferentially with methylamine. IR-Sip also accepted a range of primary and secondary amines as nucleophiles. In biocatalytic reactions, IR-Sip converted (R)-3-methylcyclohexanone with dimethylamine or pyrrolidine with high diastereoselectivity (>94-96% de). The nucleophile acceptor spectrum depended on the carbonyl substrate employed. The conversion of well-accepted substrates could also be detected if crude lysates were employed as the enzyme source.