Biosynthesis Of Mandelic Acid And Its Derivatives By Recombination Nitrilase

Mandelic acid and its derivatives are an important pharmaceutical intermediate.In this work,it is objective to hydrolyze mandelic nitrile and its derivatives using nitrilase as catalyst.In addition,metal chelated carrier with agar as the matrix was prepared and employed for recombinant nitrilase immobilization.and it is of practical and theoretical values.Firstly,Nitrilase used as catalyst to hydrolysize mandelic nitrile and its derivatives.The enantioselectivity(E)of nitrilase hydrolysis to four substrates with the optimal reaction of pH,temperature,substrates concentration was represented as follows:the E of mandelonitrile was more than 200,the E of 2-chloromandelonitrile was 3.16,the E of 3-chloromandelonitrile was 9.07,and the E of 4-chloromandelonitrile was 19.58.Effects of patterns of Cl substitution group on the reaction conditions was studied,and the resistance result was presented as follows:ortho-position>meta-position>para-position.Further more,this observation was explained by enzyme-substrate docking studies.Therefore,2-chloromandelonitrile showed big steric effect.Secondly,the conditions to immobilize nitrilase were optimizied by single factor method and the characters of immobilized nitrilase were studied.Mandelonitrile was adopted as a typical substrate for researching the properties of immonilized nitrilase.Results were showed as follows:Zn2+ was selected as the most suitable chealtor;the concentration of ionic had no effect on immogilization of nitrilase;when the concentration of Zn2+was 0.3 mol/L and the amount of loaded enzyme was 15.6 mg/g,the metal chelated carrier showed a obviously high adsorption capacity of recombinant nitrilase at pH 8.0 and 40 °C,after 1 h.The maximum conversion of mandelonitrile to(R)-(-)-mandelic acid by this immobilized nitrilase was obtained at pH 7.0 and temperature 50 ?.The surface of carrier was also investigated.The pH and temperature were 7.0 and 50 ?respectively for immobilized nitrilase,while 7.5 and 40 ? for free nitrilase.The stabilities of immobilized ntrilase on temperature,operation,pH and storage were improved.Some metal ions had inhibitory effects on immobilized nitrilase and free nitrilase.The Km and Vmax of free nitrilase were 4.74 mmol/L and 15.85 ?mol/min-mg,respectively,while 81.39 mmol/L and 2.15 ?mol/min-mg for immobilized nitrilase.Finally,the reaction conditions to the hydrolysis of mandelonitrile and its derivatives by immobilized nitrilase were studied.The results showed that the optimal reaction conditions of mandelonitrile were represented as follows:pH was 7.5,the concentration of immobilized nitrilase was 0.05 g/mL,temperature was 35 ?,substrate concentration was 10 mM;The optimal reaction conditions of 2-chloromandelonitrile were represented as follows:pH was 7.5,the concentration of immobilized nitrilase was 0.05 g/mL,temperature was 50 °C,substrate concentration was 10 mM;the optimal reaction conditions of 3-chloromandelonitrile were represented as follows:pH was 7.0,the concentration of immobilized nitrilase was 0.05 g/mL,temperature was 45 ?,substrate concentration was 10 mM;the optimal reaction conditions of 4-chloromandelonitrile were represented as follows:pH was 7.0,the concentration of immobilized nitrilase was 0.05 g/mL,temperature was 40 ?,substrate concentration was 10 mM.