Biosynthesis Of Optically Pure Mandelic Acid Derivatives By Stereoselective Nitrilase

Mandelic acid is an important pharmaceutical intermediate,it has many different synthesis methods,one of the mainstream way is nitrilase enzymatic synthesis.The mandelic acid derivatives,which is an important pharmaceutical intermediates and chemical reagents too.For example,chloromandelic acid is an important intermediate to synthsis anti-platelet aggregation drugs.The stereoselective nitrilase can synthesis some intermediates which is hard to synthesis in chemical way.In our study,we use chiral nitrilase as catalyst to hydrolysis of mandelic nitrile derivatives,and make optically pure mandelic acid derivatives.The strains were screened and cultured at the conditions which is the optimal transformation conditions and cultivate conditions,and also studied the theoretical basis for industrial applications.Firstly,we screened strain in the laboratory and get a genetically stable strains which is Alcaligenes faecalis ZJUTB10,using this strain we catalytic conversion reaction.Explorated detect method of three derivatives by HPLC,then analytical methods were established to analysis products conversion,and catalytic esterification and OD-H chiral column were used to establishing chiral analytical methods of derivatives,separation of two peaks were reached 1.5 or more.By single factor,nitrilase medium components and component concentrations were optimizided and the types of inducers we take chloromandelic as a typical representative,more appropriate medium composition were determined(g/1):ammonium citrate 10,yeast extract 10,is nitrile 3.0,potassium hydrogen phosphate 5,sodium chloride 1,magnesium sulfate 0.2.After optimizide the growth components of the conditions for enzyme production,the culture conditions optimization were also obtained:temperature 30 ?,medium volume 100ml/500ml,inoculation of 6%(v/v),in this condition cultured 26h,the enzyme activity Chloromandelic nitrile maximum 150.2U/g,biomass 4g/l.Resting cells was investigated racemic mandelic nitrile derivatives to catalyzing the process of transformation conditions on the enzyme activities.The results show the appropriate conversion of 2-chloromandelonitrile conversion conditions as follow:pH7.5-8.0 phosphate buffer,transition temperature of 40 ?,cell concentration 0.75mg/ml,substrate concentration 20mM,the conversion rate of 86.4%after 8h,ee value reached 60.0%,the concentration of the substrate in 40mM produce substrate represent inhibition.The appropriate conversion of 3-chloromandelonitrile conversion conditions as follow:pH8.5 phosphate buffer,transition temperature of 30 ?,cell concentration 1.0mg/ml,substrate concentration 20mM,conversion rate after 3.5h is 92.0%,ee value reached 98.7%,40mM substrate concentration produced substrate was represented inhibition.The appropriate conversion of 4-chloromandelonitrile conversion conditions as follow:pH8.0 phosphate buffer,transition temperature of 40 ?,cell concentration 0.75mg/ml,substrate concentration 20mM,after 3h reaction conditions,the conversion rate 93.2%,ee value reached 99%,50mM substrate concentration in the produced substrate was represented inhibition.On the kinetics of enzymatic reaction of 2-chloromandelic nitrile maximum reaction rate Vmax = 10.59mM/min,Michaelis constant Km =9.47mmol/l;3-chloromandelic nitrile maximum reaction rate Vmax = 8.69mM/min,Michaelis constant Km = 6.79mmol/l;4-chloromandelic nitrile maximum rate Vmax =6.33mM/min,Michaelis constant Km = 4.06mmol/l.In the two-phase system of 2-chloromandelic nitrile,The optimal reaction conditions were represented as follows:water:organic phase(70:30,v/v),temperature 30 ?,phosphate buffer pH8.0,different octanes were used as the organic solvent,the reaction activity reach 151.44U/g,S configuration ee value was reached 99%.As for the 3-chloromandelonitrile and 4-chloromandelonitrile,organic solvents have little effect or even detrimental to the acquisition of R configuration products.Compared mandelic acid with its derivatives show that the potential effects and steric effects impact its catalytic efficiency and selectivity is significant,its different substrate affinity can also indirectly verify this effect,a resistance results:2-chloromandelonitrile>3-chloromandelonitrile>4-chloromandelonitrile>mandelonitrile.In particular 2-chloromandelonitrile shows big steric effect,which is the cause of relatively low catalytic activity.