The Study On Microbial Production Of ?-aminobutyramide

?-aminobutyramide is a critical intermediate of Levetiracetam which is a kind of antiepileptic drug.The purpose of this study is to establish a method of biotransformation process of?-aminobutyramide from?-aminobutyronitrile through nitrile hydratase.Several works were involved,that was the establishment of the detection method of?-aminobutyramide,the screening of microorganisms which produced nitrile hydratase,and the optimization research on cultiviation conditions and catalytic condition.The thin layer chromatography was established to separate the?-aminobutyronitrile and?-aminobutyramide.The developing agent was80%methanol and the chromogenic agent was 0.2%ninhydrin.For the precise assay,a high performance liquid chromatography?HPLC?was established based on 2,4-dinitrofluorobenzene?DNFB?derivatization of?-aminobutyramide.The adding of DNFB was 0.7 mL and its concentration was 89.23 mM.The process was completed under shade environment at 70^oC and 50 min.The Nocardia.globerula with a high nitrile hydratase activity was screened from several strains preservatized in the lab through thin layer chromatography and the high performance liquid chromatography.The nitrile hydratase activity was 0.681 U/mg.Optimization of cultiviation conditions for nitrile hydratase production by Nocardia globerula was conducted,including carbon sources and its concentration,nitrogen source and its concentration,inducers and its concentration,initial media pH values,medium volume and inoculation quantity.Finally,the optimal fermentation medium was determined and its composition was:glucose 12 g/L,peptone 6 g/L,?-caprolactam 2 g/L,KH₂PO₄ 1 g/L,K₂HPO₄?12H₂O 1 g/L,MgSO₄·7H₂O0.2 g/L,NaCl 1 g/L,FeSO₄·7H₂O 0.01 g/L,pH 7.0.The medium volum value was 45 mL/250 mL,the inculation quantity was 3%.The maximum nitrile hydratase reached 0.914 U/mg,which was increased by 48.9%as compared to the fermentation using initial medium.Finally,various reaction conditions for the efficient production of?-aminobutyramide were investigated.Results indicated the nitrile hydratase showed good activity at 10 mL phosphate buffer?pH 7.0?and30 ^oC.The 96.98 mM of?-aminobutyramide could be got at 100 mM?-aminobutyronitrile and 12 g/L wet cell weight.183.45 mM?-aminobutyramide could be got through the fed-batch of?-aminobutyronitrile and the wet cell to 500 mM,13.2 g/L.