Screening For Enantioselective Amidase And Its Substrate Specificities

With wide substrate spectrum and strict stereospecificity,amidases turned out to be an attractive tool in industries for the synthesis of wide variety of carboxylic acids,hydroxamic acids and hydrazide.Relative to the nitrile hydratase and nitrilase,the industrial application of amidase far not been developed.In-depth studies on the amidase can extend the industrial chain of the nitrile hydratase,conducive to building a technology platform for biological catalysis based on nitrile hydratase/enantioselectivity amidase for preparation of chiral drugs and agrochemicals intermediates.This paper focused on screening,identification,substrate specificity and optimization of transformation conditions of the biocatalysts.With an enantioselective amidase screening method which employed acyl transfer activity of amidase in the presence of hydroxylamine,leading to the formation of hydroxamic acids,followed by spectrophotometric quantification of hydroxamic acid/iron(III)complex,we obtained 13 microorganism strains with enantioselective amidase from a variety of soil samples.The extracellular amidase from ZJB-09214 is the first report.Strain ZJB-09214 was identified as Galactomyces geotrichum.This is the first report on strains in these species with enantioselective amidase activity.At the same time,the extracellular amidase from ZJB-09214 is the first report.The optimized medium composition was as follows(g/L):glucose 10,yeast extraction 5,peptone 2,KH2PO4 1,K2HPO4 1,MgS04.7H20 0.2,FeSO4.7H2O 0.01,acetamide 1,NaCl 1,pH 7.5.Simultaneously,Influences of reaction conditions on activity and enantioselectivity of extracellular amidase from G.geotrichum ZJB-09214 were also investigated.Results indicated that optimal working pH of the extracellular amidase with acrylamide as substrate was 7.5,the amidase showed highest activity at 35℃.Substrate specificity of G.geotrichum ZJB-09214 indicated that G.geotrichum ZJB-09214 amidase can hydrolyse wide variety of amides,especially aliphatic amides.The enzymic activity of G.geotrichum ZJB-09214 with butylamide、isobutyramide and acetamide as substrate were 448.7,298.5 and 250.4 U/L.The G.geotrichum amidase exhibited R-enantioselectivity for(R,S)-2,2-Dimethylcyclopropanecarboxamide and(R,S)-etiracetam.The ee for the two subsrate were 91.2%and 31.5%,respectively,E value were 23.9 and 1.9,respectively.The G.geotrichum amidase exhibited L-enantioselectivity for(D,L)-methioninamide and(D,L)-tryptophanamide.The ee for the two subsrate were 90.8%and 99.9%,respectively,E value were>200 and 28,respectively.It couldnot catalyzed aromatic amides.Strain ZJB-09215 was identified as Ochrobactrum anthropi.The hydrolysis response catalyzed by O.anthropi ZJB-09215 amidase with tryptophanamide as substrate was studied.It was indicated that the amidase exhibited maximal activity in Tris-HCl buffer(pH 8.0)at 45 ℃.Substrate inhibition could occur when substrate concentration reach 90mM or above.The kinetic constants were as follows:Vm and Km were 2.66×10-3 mol/Lmin and 6.18×10-2 mol/L,respectively.The O.anthropi ZJB-09215 amidase can hydrolyse wide variety of amides,especially acrylamide、acetamide and tryptophanamide.The enzymic activities were 1784.1,632.5 and 261.7 U/L.It exhibited strict enantioselectivity for(R,S)-2,2-dimethylcyclopropane carboxamide and(D,L)-methioninamide and(D,L)-tryptophanamide.The ee value of the three were>98%.Based on morphology,physiological tests,ATB system and the 16S/18S rDNA sequence,the other five strains were identified.They are Galactomyces geotrichum ZJB-09214,Ochrobactrum anthropi ZJB-09215,Delftia acidovorans ZJB-09212,Enterobacter cloacae ZJB-09213,Enterobacter oryzae ZJB-09219,Klebsiella pneumoniae ZJB-09217 and Candida tropicalis ZJB-09216,respectively.E.cloacae ZJB-09213 and E.oryzae ZJB-09219 were the first reports on strains in these species with enantioselective amidase activity.Substrate specificity indicated that K.pneumoniae ZJB-09217 amidase can hydrolyse aliphatic amides and a few heterocyclic amides.The amidase showed higher activity with acetamide as subsrate,the enzymic acitivity was 1120.4 U/L.It exhibited strict L-enantioselectivity for(D,L)-methioninamide and(D,L)-tryptophanamide and R-enantioselectivity for(R,S)-2,2-dimethylcyclopropane carboxamide.The ee values of the three were 99.9%,99.9%and 97%.The E value were>200,>200 and 130.5.