Study On Catalytic Characteristics And Enatioselectivity Of Recombinant Delftia Tsur Uhatensis Amid Ase

Amidase(EC 3.5.1.4)from Delftia tsuruhatensis,as an important member of Nitril converting enzyme,has broad application prospects in the preparation of chiral intermediates of drugs and agrochemicals due to its broad substrate spectrum and loyal stereoselectivity.The stereoselectivity is mainly produced in the amide hydrolysis reaction in the nitrile hydratase/amidase enzyme system.Our lab has kinetic resolution of cilastatin key intermediates(S-2,2-dimethylCyclopropane carboxamide)by Delftia tsuruhatensis ZJB-05174 amidase through selectively hydrolyzed R-2,2-dimethyl cyclopropanecarboxamide.The regulation of amidase's stereoselectivity may build a nitrile hydratase/Optical amidase base of biocatalysis preparation of chiral drugs and agrochemicals intermediates technology platform.This essay firstly clone the amidase gene from Delftia tsuruhatensis ZJB-05174 and highly expressed by E.coli,then study the catalytic characteristics and stereoselectivity of amidase so that provides a theoretical basis for its industrial application.D.turuhatensis ZJB-05174 whole genome as a template,the size of 1401 bp of amidase gene was gain by PCR.Connected the gene to expression vector pET28b and transformed into E.coli BL21(DE3),then engineered bacteria E.coli BL21(DE3)/pET28b-amidase was constructed successfully.Recombinant bacteria which induced by IPTG could highly express amidase,SDS-PAGE analysis of recombinant lactamase molecular weight was approximately 50 kDa.After the separation and purification,the 2,2-dimethylcyclopropane carboxamide specific enzyme activity was 52.4 U/mg by enzyme activity assay.Km and Vmax of different amides hydrolysised by E.coli BL21(DE3)/pET-28b-amidase have constructed the specificity of amidase.It showed that the Km of aliphatic amide was lower than aromatic and heterocyclic.Activity of amidase decreased or without vitality due to steric hindrance caused by H on the benzene or heterocyclic ring substituted by larger group.Thermal inactivation process of E.coli BL21(DE3)/pET28b-amidase was not in line with the one level inactivation mechanics,rendered as a non-linear relationship.Ping-pong kinetic constants of amidase catalytic nicotinamide acyl transfer reaction determined by the colorimetric method was Kamide=16.5 mM,KNH2OH?20.9mM,it show that amide binds to amidase more easily than hydro xylamine.Selecting 2,2-dimethylcyclopropcarboxamide as substrate,eep was reduced from 95.1%to-71.4%as the temperature increased from 30?to 60 ?;while mandelic amide as substrate,eep declined from 90.8%to 57.5%as the temperature increased from 30? to 55 ?;when substrate is the 2-phenylbutanamide,eep decreased from 99.2%to-11.5%.The eep of 2,2-cyclopropylcarboxamide acyl transfer reaction catalysis by E.coli BL21(DE3)/pET28b-amidase was 90%(untreated)down to 17%(55?for 7 min)and-4%(55 ? for 8 min).The results of temperature effect on the E.coli BL21(DE3)/pET28b-amidase catalysised three chiral amides showed:the stereoselective of amidase catalytic process is regulated by temperature(when the temperature increases,the stereoselectivity of the enzyme is reduced;the stereoselectivity of the enzyme increased when the temperature decreased).Furthermore,the selectivity of the E.coli BL21(DE3)/pET28b-amidase can be reversed by temperature in the process of stereoselective catalytic 2,2-cyclopropylcarboxamide and 2-phenylbutanamide by amidase:from the R-type selectivity to S-type selectivity,however,for mandelamide,this change was not the same("reversal" enantioselectivity was not a general rule).