Screening For α-Oxophenylacetonitrilase And Its Application

Benzoylformic acid is an important intermediate for organic synthesisbecause it could be widely used in the synthesis of pesticides and medicine.With the development of science and technology, the application ofbenzoylformic acid continues to expand and the demand is also increasingyear by year. Many experts and scholars take more and more attention to thehighly effective synthesis. Many methods can be used for synthesizingbenzoylformic acid. The nitrilase-mediated method for biotransformingα-oxophenylacetonitrile into benzoylformic acid gains great interest undermild reaction conditions compared to chemical methods. The quality ofproduct is good and the process is in line with the requirements of thedevelopment of green chemistry, so it has a broad prospect for development.In this study, a high-throughput nitrilase screening method and the isolation ofnitrilase-producer strain, the optimization of fermentation conditions andcatalytic reaction conditions, expanding the substrate spectrum, andimmobilizing cells by the calcium-alginate immobilization method and theoptimization of some catalytic conditions of immobilized cells were described.Finally, the main product of biotransforming α-oxophenylacetonitrile byCCZU10-1was analyzed.Firstly, a high-throughput nitrilase screening method was built based on amodified ferric hydroxamate spectrophotometry. The color-generationreactions were optimized as follows:0.5mL dicyclohexylcarbodiimide(0.60mol·L^-1) and1.0mL hydroxyl-ammonium perchlorate (0.070mol·L^-1)were added into the50μL of the testing sample，and then the mixture wasincubated for15min at room temperature （25℃³5℃）, before0.7mL ofacidic ferric perchlorate solution (0.040mol·L^-1) was added into the mixture.Finally, the purple ferric hydroxamate formed was assayed at520nm by aUV–Vis spectrophotometer. The nitrilase screening method was simple, rapid,and high-throughput. Furthermore, the accuracy of this method was alsovalidated. The results indicated that the accuracy of this method for assayingcarboxylic acids was as high as the HPLC-based method.Secondly, a strain with high α-oxophenylacetonitrile-hydrolyzing activity has been isolated using3-cyanopyridine and α-oxophenylacetonitrile as solenitrogen source by the built high-throughput nitrilase screening method. It wasidentified as Rhodococcus by its morphological, physiological properties and16S rDNA sequence analysis. Finally, it was named Rhodococcus sp.CCZU10-1.Thirdly, the preferred carbon/nitrogen sources and inducer of the strainCCZU10^-1 were glucose (10g·L^-1), the combination of peptone (10g·L^-1)plus yeast extract (5g·L^-1), and caprolactam (5mmol·L^-1) which was added atfermentation for24h, respectively. Under above conditions, the nitrilaseactivity of38.34U·mL^-1was obtained. The optimum catalytic reactionconditions was as follows: temperature was30℃, initial pH was7.0, andsubstrate concentration was100mmol·L^-1, the cosolvent was tolune （2%v/v）,and metal ions was Ca2+(0.1mmol·L^-1). Under the optimum catalyticreaction conditions, the yield of benzoylformic acid was98.22%after60h.Moreover, it was found that the objective strain had a broad substratespectrum.Finally, the free cells were immobilized by the calcium-alginateimmobilization method. For the immobilized cells, the optimum reactionconditions as follows: substrate concentration was150mmol·L^-1, the reactiontemperature was35℃, the reaction pH was pH7.0. After cell immobilization,the stability of immobilized cells was better than free cells, the immobilizedcells can be reused many times. Moerover, for the catalytic reaction by theimmobilized cells, the product is easily separated and the operation is simple.Furthermore, the product from the hydrolysis of α-oxophenylacetonitrile bythe immobilized cells was obtained. The analysis of HPLC and13C-NMRresults indicated that it was benzoylformic acid.