Optimization Of Culture Conditions Of Recombinant Escherichia Coil Nitrilase And Study On The Catalytic Properties Of Cell

R-Mandelic acid is used as an important pharmaceutical intermediate and chiral resolving agent,Biotransformation of mandelonitrile to R-mandelic acid with nitrilase is one of the most attractive method because of its high specificity,high yields,mild reaction condition,ecofriendly nature and good selectivity.The culture conditions of Recombinant Escherichia coil nitrilase was optimized firstly.The optimum medium compositions were as follows:coRN paste powder 25.00g·L-1,yeast extract paste 5.00g·L-1,NaCl10.00g·L-1,the iron(Fe3+)content 0.5mmol·L-1,initial pH 7.5.Lactose was used as the inducer and added to the culture at 0.5g·L-1 after 6h inoculation.The highest nitrilase production reached 6161.46U·L-1 after 28h culture at 30?.Then the fermentation of Escherichia coil nitrilase was scaled up in 5L bioreactor,the glycerol was demonstrated as the optimal fed-batch culture medium.The nitrilase production could be significantly enhanced by adding 20%glycerol with 20ml·h-1 for 25 h.After 42h culture from addition of inducer,the nitrilase production and biomass reached 20057.03U·L-1 and 17.00 g dcw·L-1,respectively.The bioconversion of mandelonitrile by the Recombinant nitrilase was carried out.The optimum pH and temperature were 7.0 and 45?,respectively.under this condictions,the molar yield of R-(-)-mandelic acid against 30 mmol·L-1 substrate reach above 98.37%with high e.e.value(>99%)in 30min.Substrate feeding experiment showed that the mandeloitrile inputs to reach a total of 400mmol·L-1 after 320min in 500ml reaction kettle,with a conversion rate of 69%,and e.e.value maintain in 99%.The substrate specificity of E.coil nitrilase was also investigated.The K,n values of mandelonitrile,2-chloromandelonitrile,3-chloromandelonitrile and 4-chloromandelonitrile were 1.77,2.01,4.22 and 4.31mmol·L-1,respectively.Recombinant bacteria catalyzes the conversion of four kinds of substrates by the substrate inhibition strength from strong to weak in turn are 4-chloromandelonitrile,mandelonitrile,3-chloromandelonitrile and 2-chloromandelonitrile,Ks values were 6.76,26.45,33.38 and 91.70mmol·L-1.Foused on the reaction of mandelonitrile translate to mandelic acid catalyzed by resting cells,substrate inhibition concentration is 6.84mmol·L-1.Reaction of the other three chlorinated derivative substrates(2-chloromandelonitrile,3-chloromandelonitrile and 4-chloromandelonitrile)to generate the corresponding R-chlorinated derivative acid catalyzed by resting cells gave the conversion rate 80.06%,75.16%and 99.95%,e.e.values were about 61%,75%and 78%,respectively.