浑球红细菌Rhodobacter sphaeroides LHS-305腈水解酶基因的克隆及表达

以筛选的产腈水解酶浑球红细菌Rhodobacter sphaeroides LHS-305为出发菌株,根据已知腈水解酶基因保守区域设计简并引物,从总DNA中成功扩增得到腈水解酶基因的部分片段(402 bp)。通过染色体步移扩增片段的上下游序列,经拼接,得到的腈水解酶基因全长为969 bp(在Genebank数据库中的登录号为JN635494)。该基因与已知腈水解酶序列的最高相似性为82%。构建pET28a-nit表达载体,转化大肠杆菌Rosetta(DE3),得到重组菌。SDS-PAGE电泳结果表明该基因在大肠杆菌中成功表达。重组酶对底物3-氰基吡啶催化结果显示了较高的酶活力,具有跟原始菌相同的特性。

Reductive amination of ketones with ammonium catalyzed by a newly identified Brevibacterium epidermidis strain for the synthesis of(S)-chiral amines

The asymmetric reductive amination of achiral ketones with ammonia is a particularly attractive reaction for the synthesis of chiral amines.Although several engineered amine dehydrogenases have been developed by protein engineering for the asymmetric reductive amination of ketones,they all display(R)‐stereoselectivity.To date,there is no report of an(S)‐stereoselective biocatalyst for this reaction.Herein,a microorganism named Brevibacterium epidermidis ECU1015 that catalyzes the(S)‐selective reductive amination of ketones with ammonium has been successfully isolated from soil.Using B.epidermidis ECU1015 as the catalyst,the asymmetric reductive amination of a set of phenylacetone derivatives was successfully carried out,yielding the corresponding(S)‐chiral amines with moderate conversion and>99%enantiomeric excess.