Characteristic Of The Nitrile Hydratase By R.boritolerans FW815 And Its Application

Nitrile hydratases(NHases),a group of metalloenzymes,can catalyze nitriles hydration to the corresponding amides,and have been widely used in organic synthesis,and environmental management.The most successful application of NHase is the industrial production of acrylamide.The goals of this dissertation are to purify and characterize the NHase from R.boritolerans FW815,optimize the hydration conditions of 2-(2-oxopyrrolidin-l-yl)butyronitrile with R.boritolerans FW815,and develop 2,2-dimethylcyclopropanecarbonitrile(DMCPCN)hydration in aqueous-organic two-phase system.The purification process of NHase from R.boritolerans FW815 consisted of four steps:cell-free extract preparation,High Q column chromatography,t-butyl HIC chromatography,and UNO Q column chromatography.The owerall yield of NHase was 14%and the maximal sepecific activity was 918.4 U/mg.According to the results of SDS-PAGE,the NHase was composed of two subunits,whose molecular weights were 24 kDal and 22 kDal.Analytical size exclusion chromatography analysis revealed that this NHase has a molecular weight of 40.4 kDal.Hence,it was concluded that R.boritolerans FW815 NHase is a heterodimernWe further investigated the enzymatic property of the purified R.boritolerans FW815 NHase.This NHase exhibited maximum activity at 37 ?,pH7.0,with half lives of 1h at 30?40 ?,respectively.Cu2+,Zn2+,Ni2+ and EDTA displayed inhibitory effects towards R.boritolerans FW815 nitrile hydratase.Besides,no resurrection phenomenon of this NHase,pre-treated with EDTA and then exposed to varying concentrations of Fe2+,Fe3+,Co2+ and Cu2+ and Zn2+,was observed.Kinetic constants of R.boritolerans FW815 nitrile hydratase including Km and Vmax,were regressed to be 0.293 mmol/L and 209.6 ?mol/(min,mL)when DMCPCN was used as the substrate.In addition to DMCPCN and 2-amino-2,3-dimethylbutyronitrile,it can efficiently hydrate 2-(2-oxopyrrolidin-1-yl)butyronitrile,acrylonitrile and 2-amino-4-methylthio-l-butyronitrile,in the order of DMCPCN>acrylonitrile>2-amino-4-methylthio-1-butyronitrile>2-(2-oxopyrrolidin-1-yl)butyronitrile.Influences of reaction conditions on R.boritolerans FW815 activity for 2-(2-oxopyrrolidin-1-yl)butyronitrile were further evaluated.It was indicated that R.boritolerans FW815 exhibited maximum activity 35?,in pH8.0,20.0 mmol/L Na2HPO4-NaH2PO4 bufferring system.R.boritolerans FW815 had poor thermostability and was almost inactivated after 2 h incubation at 35?.It fitted the Arrhenius equation between 25? and 35 ?,with apparent activation energy Ea of 139.4 kJ/mol.Kinetic studies of the NHase catalyzed reaction showed that the kinetic constants were as follows:Km= 240.8 mmol/L and Vmax=0.176 mmol·L-1·min-1.DMCPCN hydration to 2,2-dimethyl-cyclopropanecarboxamide in aqueous-organic two phase system was studied with R.boritolerans FW815.The organic solvent,aqueous/organic solvent ratio,conversion temperature,conversion pH and ionic strength were evaluated,respectively.Results demonstrated that aqueous-hexane two-phase system was preferential.The optimal conditions for 2,2-dimethyl-cyclopropane carbonitrile hydration in aqueous-hexane two-phase system were as follows:the ratio of organic solvent/water of 3:7,conversion temperature 40 ?,pH7.0,ionic strength 10 mmol/L.Compared with the aqueous monophase system,aqueous-hexane two-phase system eliminated the substrate inhibition to somewhat extent.