Saini Priya, Kumar Naveen, Wani Shadil Ibrahim, Sharma Shilpi, Chimni Swapandeep Singh, Sareen Dipti
Department of Biochemistry, Panjab University, Sector 25, Chandigarh, 160 014, India.
Department of Chemistry, Guru Nanak Dev University, Amritsar, Punjab, 143 005, India.
World J Microbiol Biotechnol. 2017 May;33(5):82. doi: 10.1007/s11274-017-2248-z. Epub 2017 Apr 4.
In order to produce enantiomerically pure epoxides for the synthesis of value-added chemicals, a novel putative epoxide hydrolase (EH) sgeh was cloned and overexpressed in pET28a/Escherichia coli BL21(DE3). The 1047 bp sgeh gene was mined from Streptomyces griseus NBRC 13350 genome sequence. The recombinant hexahistidyl-tagged SGEH was purified (16.6-fold) by immobilized metal-affinity chromatography, with 90% yield as a homodimer of 100 kDa. The recombinant E. coli whole cells overexpressing SGEH could kinetically resolve racemic phenyl glycidyl ether (PGE) into (R)-PGE with 98% ee, 40% yield, and enantiomeric ratio (E) of 20. This was achieved under the optimized reaction conditions i.e. cell/substrate ratio of 20:1 (w/w) at pH 7.5 and 20 °C in 10% (v/v) dimethylformamide (DMF) in a 10 h reaction. 99% enantiopure (R)-PGE was obtained when the reaction time was prolonged to 12 h with a yield of 34%. In conclusion, an economically viable and environment friendly green process for the production of enantiopure (R)-PGE was developed by using wet cells of E. coli expressing recombinant SGEH.
为了生产对映体纯的环氧化物用于合成高附加值化学品，克隆了一种新型假定的环氧化物水解酶（EH）sgeh，并在pET28a/大肠杆菌BL21(DE3)中进行了过表达。从灰色链霉菌NBRC 13350基因组序列中挖掘出1047 bp的sgeh基因。重组的带有六聚组氨酸标签的SGEH通过固定化金属亲和色谱法进行纯化（纯化倍数为16.6倍），以100 kDa的同型二聚体形式获得，产率为90%。过表达SGEH的重组大肠杆菌全细胞能够将外消旋苯基缩水甘油醚（PGE）动力学拆分得到ee值为98%的(R)-PGE，产率为40%，对映体比例（E）为20。这是在优化的反应条件下实现的，即在pH 7.5、20℃、10%（v/v）二甲基甲酰胺（DMF）中，细胞/底物比例为20:1（w/w），反应10小时。当反应时间延长至12小时，产率为34%时，可获得ee值为99%的对映体纯(R)-PGE。总之，利用表达重组SGEH的大肠杆菌湿细胞开发了一种经济可行且环境友好的绿色工艺来生产对映体纯的(R)-PGE。
