Screening Of A Regioselective Nitrile Hydratase And Its Application In The Biosynthesis Of 1-cyanocyclohexaneacetamide

Gabapentin is a novel orally active antiepileptic drug.Except for its ideal pharmacokinetic properties,this medicine also has advantages of less side effect,good curative effect,high safety and favorable tolerance.1-cyanocyclohexaneacetamide?1-CCHAM?is a key intermediate for the synthesisofgabapentin.Regioselectivehydrationof1-cyanocyclohexaneacetonitrile?1-CCHAN?by Nitrile hydratase?NHase?provides a green access to 1-CCHAM due to the high yield,mild conditions and environment-friendly nature.This paper focused on the bioconversion of 1-CCHAN to 1-CCHAM and investigated in aspects of regioselective NHase screening,conversion process conditions optimization,as well as separation and purification of product.The strain ZJB-1208 exhibiting high hydration activity and strong tolerance towards 1-CCHAN was isolated from soil samples through enrichment culture.It was identified as Rhodococcus aetherivorans based on its morphological and physiological features along with Biolog identification result and 16S rDNA sequencing method.The effects of reaction conditions and media on NHase activity were also evaluated.It was indicated that the NHase exhibited a specfic activity of 1442.3 U g^-1 DCW at 35?C,4.735 g DCW L^-1 cell concentration and 0.5mol L^-11 substrate concentration,applying distilled water as reaction medium.In order to overcome the limitation that caused by low solubility of substrate to further increase the accumulation of 1-CCHAM,the reaction was then carried out in fed-batch mode.Total 6 M substrate was added into the reaction mixture through five feedings,and 966.7 g L^-1 1-CCHAM was produced after 73 h of incubation with a conversion of 97%and catalyst yield(g_product/g_cat)of 204.2.Finally,a novel process for separation and purification of 1-CCHAM was developed.Purified product was obtained through threes phases including pretreatment,crude extraction and refinement.The total yield of this product separation and purification process was 86.2%with a purity of99.5%.The structure of the refined product was further confirmed by IR,MS and NMR spectra.