Biocatalysis Of L-glufosinate By Nitrilase-producing Strain

Enzymatic hydrolysis of nitriles has been widely applied to synthesis of chemicals intermediates such as the optical unnatural amino acids.Due to the mild conditions,high yields and ecofriendly nature,enzymatic hydrolysis of nitriles meets the demands of green chemistry.Glufosinate is the second largest transgenic crop resistant herbicide worldwide.Only the L-enantiomer has herbicidal activity.The biological activity of the natural L-enantiomer is twice as strong as that of the racemic mixture.The objective of the present study was to screen for a strain which could be used as a biocatalyst in the resolution of 2-amino-4-(hydroxy-methyl-phosphory)-butyronitrile to L-glufosinate.The activity of wild strain was then be increased through optimization of cultivation and bioconversion conditions,and then study the recovery of L-glufosinate,thus lay a theoretical foundation for the industry use.Firstly,a chiral pre-column derivatization method for optical purity of L-Glufosinate has been established by RP-HPLC through common C18 column.Then stain ZJB-09280,which with high stereselectivity and activity toward 2-amino-4-(hydroxy-methyl-phosphory)-butyronitrile was isolated from soil samples.Based on morphology,physiological tests and the 16S rRNA sequence,ZJB-09280 was identified as Bacillus cereus.The medium components and the culture conditions of Bacillus cereus ZJB-09280 were optimized.By single-factor and orthogonal method,the medium components was optimized as follows(g/L):carbamide 2.5,glucose 12.0,yeast extract 10,KH2PO4 0.75,K2HPO4 0.75,MnCl2 0.01.The optimum conditions for cell growth and enzyme production were as follows:initial pH 7.5,inoculum volume 4%(v/v).Under these conditions,a specific activity of 55.65 U/L was achieved after cultivation for 50 h,which was 2.76 times higher than that obtained under initial conditions.The influences of reaction conditions on nitrilase activity were also evaluated.Results indicated that the optimal pH was 8.5 in Tris-HCl buffer.The optimal wet cell concentration was 0.2 g wet cells in 10ml reaction system.The optimal temperature was 45 ℃,It fitted the Arrhenius equation between 25-45 ℃,the apparent activation energy Ea was 26.15 kJ/mol.The nitrilase of Bacillus cereus ZJB-09280 had good thermostability with half-life of 76.49 h at 35 ℃.The nitrilase had poor thermostability with half-life of 29.15 h at 45 ℃.The suitable concentration of the substrate was 50 mM and the initial rate reached 132.5 μmolmin-1g.After 4 reuse cycles,24%of the activity was remained.The kinetic studies of the nitrilase mediated reaction indicated that the kinetic constants were as follows:Km=5.01 mM and Vm=0.1294 mM/min.The recovery of L-glufosinate using ion-exchange process was developed.In ion exchange column experiments,the breakthrough curves of L-glufosinate from the solution on resin 201 were determined at different flow rates and L-glufosinate was eluted with different concentrations of NaCl.The favorable breakthrough curve and optimal eluant concentration were obtained.The results were used for the separation of L-glufosinate biosynthesized from 2-amino-4-(hydroxy-methyl-phosphory)-butyronitrile with nitrilase.At last,the product L-glufosinate with purity of 89%,e.e.of 90%was obtained by rotary evaporation and anhydrous ethanol elution process.