Study On Methionine Production By Biological Method

Nitrilase has important applications in organic synthesis,nitrilase is broadly applied to synthesis of a wide range of bulk chemicals,pharmaceutical and agrochemical intermediates from nitriles or amides and environmental protection.Methionine is used extensively in pharma-ceutical,health and fitness products,the global market demand to methionine gradually increasing at rate of 4 to 5 percent every year.The production methods of methionine divided into chemical and biological.This paper studies the biocatalytic 2-amino-4-methylthio butylnitrile to product methionine,this paper focus on the construction of genetic engineering bacterium,culture medium and culture conditions,and the optimization of converting conditions optimization.Firstly,through the technique of genetic engineering,we structured a gene engineering bacteria with nitrilase activity.Recombin ant expression was induced by IPTG and analyzed by SDS-PAGE.The result indicated that the molecular mass is about 43KDa,and the activity is 960U/mg.Studied on natrilse’s properties and found that:the optimum pH and temperature of the purified enzyme are 7.5(Tris-HCl buffer)and 40℃;the natrilase has good thermostability with half-life of 5.52h at 40℃.Studied of the nitrilase catalyzed reaction showed that the kinetic constants are as follows:Km= 0.096mol/L and Vmax= 0.0197mol/(L-min).The substrate specificity research shows E.coli J10-4 can hydrolysis aliphatic acrylic,but it can’t hydrolysis aromatic nitriles and heterocyclic nitriles.Secondly,the medium composition and cultivation conditions was optimized by single factors.The optimized medium and medium composition are as follow:Tryptone 12.0g/L,Yeast extract 8.0g/L,NaCl 8.0g/L.The optimal concentration of IPTG is 0.2mM,the induction temperature is 28 ℃,the optimal induction time is 12h.Initial pH value is 7.0,medium volumetric ratio is 20%(v/v),seed culture time is 12h,inoclulum volume is 3%.Influences of reaction conditions on natrilase activity were also evaluated.It indicated that the enzymes exhibited maximum activity in Tris-HC1 buffer(pH=8.0),the natrilase has good pH-stability.It fitted the Arrhenius equation between 25-40℃,the apparent activation energy Ea is 44.56 kJ/mol.The natrilase has good thermostability with half-life of 18.23 and 7.8h at 30℃ and 40 ℃.Substrate concentration in the 150 mM to 250 mM,the natrilase activity achieved maximum;the maximum activity is 2800DCW U/g;along with the increase of substrate concentration,natrilase activity is crimped.At last,studied on the fed-culture conditions of E.coli J10-4 in the 5L fermenter.Finally the optimization of the medium and medium composition as follow:Glucose 5.0g/L,Tryptone 8.0g/L,Yeast extract 5.4g/L,NaCl 8.0g/L;initial pH value is 7.0,and control fermentation pH at 7.0;at fermentation 6h,add the induction of IPTG,the optimal concentration of TPTG is 0.4mM,induce temperature is 28℃.In the fermentation,detecting the glucose concentration,when the glucose concentration reduce to 2g/L,begining to add glucose(60 g/L),maintain the glucose concentration between 2g/L and 3g/L;from fermentation 6h,interval 2h to add 50ml tryptone(60g/L)and yeast extract(40g/L),add 4 times;the fermentation process last for 18-20h.At last,cell dry weight was achieved to be 11.5g/L,enzyme activity achieved to be 31000U/L.