Study On Microbial Production Of 2-Amino-2,3-Dimethylbutyramide

2-amino-2,3-dimethylbutyramide is an important precursor of broad spectrum herbicides imidazolones.Biosynthesis of 2-amino-2,3-dimethylbutyramide has great potential for industrialization because its traditional chemical methods consumed large quantity of raw materials and caused severe environmental pollution. 2-amino-2,3-dimethylbutyronitrile,anα-aminonitrile,is prone to decompose in aqueous solution to the corresponding carbonyl compound and cyanide,which is a strong inhibitor of nitrile hydratase.Thus, obtaining of cyanide-tolerance nitrile hydratase is the key step of the whole enzymatic process.In the dissertation,a biotechnological process for production of 2-amino-2,3-dimethylbutyramide was established after isolation of a novel nitrile hydratases and optimization of its cultivation and reaction conditions.Firstly,strain ZJB-09141 capable of hydration of 2-amino-2,3- dimethylbutyronitrile to 2-amino-2,3-dimethyl-butyramide,was isolated from the soil samples by enrichment culture technique.CN^- inhibition test showed that the nitrile hydratase had good cyanide resistance activity. Based on morphology,physiological tests and 16S rRNA sequence,the new isolate was identified as Nocardia globerula.Secondly,the cultivation conditions of N.globerula ZJB-09141 for production of NHase were optimized.After single factor experiments,9 factors were selected for the evaluation,by using a Plackett-Burmam design,of the effects on the NHase production.The results showed that the concentration of yeast extract,peptone andε-caprolactam had significant effects on the NHase activity production.A Box-Behnken design was applied to determine the optimal concentration of those three significant variables.The optimum fermentation medium were yeast extract 5.46g 1^-1,peptone 2.39 g 1^-1,ε-caprolactam 3.77 g 1^-1,allowing the NHase activity to increase from 7.62 U/ml to 9.36 U/ml in flask-shaking batch fermentation.Finally,various reaction conditions for the efficient production of 2-amino-2,3-dimethyl-butyramide were investigated with resting cells of N.globerula ZJB-09141.Results indicated the NHase exhibited maximal activity at pH value 7.2 in the temperature range of 30 and 35℃.Results of thermal stability of the NHase demonstrated that the enzyme is stable at 30℃,while decrease sharply above 40℃.With the addition of 5 mM KCN,half of the NHase activity was lost.Reaction kinetics studies showed that the Michaelis-Menten constants K_m and V_max of the NHase were 74.42 mM and 6.734μmol min^-1 mg·dcw^-1,respectively.