The Selective Hydrolysis And Synthetic Application Of Cyano Compounds By Nitrilase

4-Cyanobenzoic acid serves as a key precursor for the synthesis of 4-(Aminomethyl)benzoic acid,tranps-4-aminomethylcyclohexane and liquid crystal materials.4-(Aminomethyl)benzoic acid and trans-4-aminomethylcycIohexane are outstanding antifibrinolytic agents and suitable for abnormal bleeding during surgery.In industry,4-(Aminomethyl)benzoic acid and trans-4-aminomethylcyclohexane are mainly produced from 4-cyanobenzoic acid by catalytic hydrogenation.In this paper,nitrilases genetically engineered strain free cells were achieved as catalysts,and terephthalonitrile was used as substrate to synthesize high concentration and high yield of 4-cyanobenzoic acid.In this paper,by screening our laboratory’s nitrilase strains,a strain of nitrilase named PaNIT genetic engineering strain derived from Pantoea sp.AS-PWVM4 was finally screened by gradually increasing the substrate concentration.PaNIT could catalyze 300 mmol/L of terephthalonitrile.We performed expression purification of the PaNIT which was obtained by screening.And the enzyme activity of the nitrilase PaNIT pure enzyme was 84.9 U/mg.Subsequently,we studied the optimum reaction conditions of the nitrilase PaNIT.The optimum pH of the nitrilase PaNIT was 8.0(NaHPO4-Na2HPO4,100 mmol/L),and the optimum reaction temperature was 37℃.High and stable enzyme activity could be maintained at 30℃-37℃.The enzyme had high catalytic activity in organic solvent DMSO and it had a broad substrate specificity.The substrate biotransformation experiment of the nitrilase PaNIT was carried out.The concentration of the substrate terephthalonitrile could reach 100 g/L,and it could be completely converted within 4 h.The separation yield of the target product to cyanobenzoic acid could also be achieved 91%,and the conversion of the product was 624 g·L-1·d-1.In this paper,the preliminary exploration of substrate specificity and temperature tolerance of nitrilase PyNIT was also carried out.In the substrate specificity study,we found that the nitrilase PyNIT exhibited catalytic activity for some para-substituted cyano compounds and cinnamonitrile.In the temperature tolerance study,the nitrilase PyNIT was found to be able to adapt to higher temperatures and still retain higher enzyme activity at 70℃.