R-(-)-Mandelic Acid Produced From Racemic Mendelonitrile By Enantioselective Nitrilase

Hydrolysis of nitrile compounds has been widely used in the organic synthesis of amides, carboxylic acids and their derivatives. Enzymatic hydrolysis of nitriles offers alternatives to conventional nitrile hydrolysis due to the mild conditions, high yields and ecofriendly nature and meets the demands of "green chemistry". Owning to its chemo-, region-, or enantio-selectrvity, many organic chemicals can be obtained which can not be achieved by traditional ways. R-（-）-mandelic acid, the desired product of this research, is a valuable intermediate in pharmaceutical industry which has been primary prepared through the separation of racemate. Enantioselective nitrilase-mediated pathway offers a straightforward method to produce optically pure R-（-）-mandelic from mamdelonitrile. The objective of the present study was to screen for a strain with high yield and specific nitrilase activity which could be used as a biocatalyst in the resolution of racemic mandelonitrile to R-（-）-mandelic acid. The activity of wild strain was then be increased via mutagenesis and through optimization of culture and bioconversion conditions, thus lay a theoretical foundation for the industry use.A high throughput screening model was established employing phenylacetonitrile as carbon and nitrogen source through conventional enrichment cultivation and then colorimetric assay in the rapid detection of aid producing. According to this method plus HPLC, strain WT10 bearing nitrilase with high activity and selectivity was selected for further study. Based on morphology, physiological tests and 16S rDNA sequence, strain WT10 was identified as Alcaligenes faecalis WT10. A. faecalis WT10 was then used as a starting strain, using several mutation methods. A genetically stable mutant ZJUTB10 with nitrilase activity of 260 U/g comparing with 53 U/g of WT10 was selected for further research.The medium composition was optimized by single-factor method and response surface methodology （RSM）. The optimized medium composition was as follows （g/l）: ammonium acetate 12.14, yeast extract 7.79, K₂HPO₄ 5, MgSO₄, 0.2, NaCl 1, n-butyronitrile, 3.29. The optimum conditions for cell growth and enzyme producing were as follows: 30℃, initial pH, 7.5, inoculum volume, 6 % （v/v） , medium capacity 20 %（v/v）. Under these conditions, a biomass of 2.94 g/l and a specific activity of 1197 U/l were achieved after cultivation for 20 h. A. faecalis WT10 was then cultivated in a 151 scale batch bioreactor with medium volume of 81, and the biomass and specific activity were 2.64 g/l and 997 U/l respectively after 20 h.The fermentation kinetics of A. faecalis ZJUTB10 through shake flask cultivation were studied. Logistic equation was employed in cell growth modelbuilding which was as follow: C_x =（?）.It was presumed thatnitrilase producing was correlated with cell growth, thus the enzyme producingmodel was C_e=（?）.The effect of bioconversion conditions on specific activity and reaction rate was also investigated. It showed that the optimal pH and temperature were 8.0-8.4 and 35℃separately. Under this condition, the bioconversion rate and the enantiomeric excess （e.e.） reached 95% and 99% respectively when the substrate concentration was 20mM and cell concentration was 1mg/ml. It showed no substrate inhibition while the substrate concentration was under 50 mM. However, mandelic acid inhibited the hydrolysis at concentration above 10 mM. The kinetic studies of the nitrilase mediated reaction indicated that the kinetic constants were as follows: K_m=0.342 mmol·l^-1、v_m=1.288 mmol·l^-1·min^-1 and k_ip =20.61 mmol·l^-1. Nitrilase in A.faecalis ZJUTB10 showed good thermal stability with half-life at 30℃（14d）, 38℃（lld）, 45℃（1.5d）, 53（240 min） and 60℃（55 min） . The study of pH stability demonstrated that the resting cells of A.faecalis ZJUTB10 exhibited better stability in neutral or alkaline condition than in acidic condition.