The Screening Of Amino Nitrilases And The Application Of The Industrial In L-glufosinate

Glufosinate is a kind of new and effective herbicide,which draws a wide attention for excellent herbicidal effects and applications in transgenic plants.In recent years,many researchers reported many chemical and biological synthesis methods of glufosinate,but only a few methods could be established viable industrialial processes.The synthesis of glufosinate,which stereoselective hydrolysis 2-amino-4-?phosphono-methyl-ethyl?–butyronitrile,solved the most critical step in the process of the synthesis reaction,with a high conversion rate and mild conditions.In this work,we separated the aminonitrile from the mixture and establish a complete separation process.Meanwhile,two high-throughput screening methods of nitrilases were established.At last,we optimized the catalytic conditions of nitrilase-producing microorganisms and set up a biological path for L-glufosinate.Then using an ion-exchange?IEX?process investigated the isolation of aminonitrile from the mixture.Resin 001×7 has the largest adsorption capacity among nine anion exchange resins.The best pH of the sample solution is 2.0.In the fixed bed experiments,the sample run at a flow of1.0 mL/min,0.4 M ammonia solution was worked as eluent,flow rate of eluent was 0.3 mL/min.Finally,the purity of aminonitrile was over 90%,which could be directly used to the next step of the synthesis of L-glufosinate and the screening of nitrilases.We established two feasible high-throughput assay strategies for the screening of nitrilases and optimized the phenol-hypochlorite method,the solution components was optimized as follows?g/L?:NaOH 4,NaClO0.2,sodium nitroprusside 0.075,phenol 10.These methods proved to be reliable after comparison of the results were assayed by high-throughput screening methods and HPLC,so these methods could be widely used in the screening of nitrilase-producing microorganisms.Then,2-amino-4-?phosphono-methyl-ethyl?-butyronitrile were used as the sole source of nitrogen to screen nitrilase-producing microorganism,twenty-four candidates were selected by using the high-throught screening methods.Enterobacter sp.ZJB-15078 had the highest conversion rate.The medium components and the culture conditions of Enterobacter sp.ZJB-15078 were optimized by single-factor method,the components medium was optimized as follows?g/L?:lactose 15,beef extract 10,caprolactam 1.5,ZnCl₂ 0.01,KH₂PO₄ 1.0,K₂HPO₄ 1.0.The optimum conditions for cell growth and enzyme production were as follows:initial pH 6.5,inoculum volume 3%?v/v?.Under these conditions,a specific activity of 20.57 U/L was achieved after cultivation for 36 h,which was1.3 times higher than that obtained under initial conditions.But the activity of our nitrilases were still low so that a further screening would be needed for a better one.