Study On The Production Of Methionine By Nitrilase Hydrolysis

Methionine is an important amino acid which is mainly used in feed industry.The domestic production capacity of Methionine is far from able to meet the demand of it.So most of the demands for Methionine mainly depend on imports.Compared with traditional chemical synthesis,the biocatalysis of 2-amino-4-methylthio butylnitrile to product Methionine attracts many people's attention because its reaction conditions are mild and the product is easily to be separated.In this paper,recombinant E.coli BL21(DE3)/pET28b-NIT harbouring nirtilase was studied to convert 2-amino-4-methylthio butanenitrile to methionine,and the catalytic conditions for methionine synthesis were optimized.The optimal temperature and pH for methionine synthesis was 40 ? and 7.5(Tris-HCl),respectively.When the concentration of substrate was 150 to 250 mM,the specific activity reached to maximum and the maximum was 2800 U/gDCW.With the increase of substrate concentration,the activity of nitril ase had been a certain degree of inhibition.And the reusability of the resting cell was bad,and only 50%of activity was remained in reaction after one batch.Through comparing several different immobilization methods,it showed that the sodium alginate(SA)-polyethylene glycol(PEG)was the most appropriate method.And the method was optimized by single factor and orthogonal method.The optimal immobilized conditions as follows:2.5%SA,2%CaCl2,immobilization time 6 h,60 mg/ml wet cell,100 mg/ml PEG6000.The transformation conditions of the immobilized cells was studied and compared with free cells.It found that the optimal pH and the temperature of the immobilized cells was the same as the free cells indicating that the immobilized cells had good permeability.Thermal stability of immobilized cells had significantly improved compared with free cells.The halflife of the immobilized cells at 40 ? and 50 ? was 1.29 and 1.45 times than the free cells.After reusing nine times the immobilized cells could still maintain more than 75%of the activity.Whereas the activity of the free cells after using two times had dropped below 50%and almost lost all activity when the reuse cycle was 9.Finally the process of continuous production of methionine by immobilized cells was studied in packed bed reactor whose specification was ? 35×400.The optimum operating conditions as follows:the substrate(40 mM)flowed from the top of the reactor at the rate of 0.4 ml/min,the product solution flowed from the bottom of the reactor after 5 h at the same speed.After 100 h continuous reaction,the conversion rate was maintained at more than 85%.The production of methionine reached to 1.29 g/(h-gDCW)which was 14.3 times than reported.And the algebraic dynamics equationin of the packed bed reactor was L=0.18/?[32lncs/cs0+(cs-cs0)].The equation was related to Km rmax,conversion rate,porosity and intraparticle diffusion efficiency factor.Through calculation,the empirical equation was L =-0.58×[32lncs/cs0+(cs-cs0)]when substrate concentration was 40 mM.The sample was analyzed by HPLC and NMR which proved that the sample was methionine,and the purity reached to more than 99%.