Regioselective Nitrilase Hydrolysis 1-cyanocyclohexylacetonitrile For Production Of 1-cyanocyclohexaneacetic Acid

1-Cyanocyclohexaneacetic acid is an important intermediate for the synthesis of antiepileptic drug gabapentin.Although it can be prepared by regioselective nitrilase through one-step hydrolysis which is a promising pathway for its high selectivity and mild conditions,it usually hampered due to the poor stability and difficult recovery of catalyst.To solve these problems,first,the effects of seven factors on the stability of resting cell were investigated by single factor test,high concentration of product existed in the reaction system was found played main role in the stability of resting cell.The activity of free enzyme and cells in the system were investigated,we found that the activity of cells declined with the increase of product concentrations.The substrates were mainly catalyzed by the residual activity of cells.Although the free enzyme had catalytic activity under low product concentration,when the production concentration increased with the extended reaction time,the free enzyme could hardly catalyze substrate due to product inhibition.The apparent kinetics of nitrilase under different product concentrations were determined which illustrate the inhibition might be attributed to reversible competitive inhibition.Several immobilization methods and stabilizers were compared,an efficient production of 1-CHAA was developed by the addition of carbonate to improve the enzyme recovery which was based on the polyethyleneimine(PEI)/glutaraldehyde(GA)cross-linking.The effects of several parameters on the immobilization of whole cells were optimized,the enzyme recovery of potassium bicarbonate,potassium carbonate and ammonium bicarbonate added immobilized cells were 65.0%?69.8% and 64.4% respectively when 200 mM,150 mM,100 mM carbonate added respectively with 3%PEI(5%w/w)and 1% GA(25%w/w)were used as crosslinker in the process of immobilization.The optimum pH,temperature and thermostability were investigated.The specific activities of the immobilized preparation were all found to be maximal at pH 7.0.The temperature response of the ammonium bicarbonate,potassium carbonate and potassium bicarbonate added immobilized cells showed a maximum activity respectively at 50?,60? and 60? respectively.The half-lives of the traditional,ammonium bicarbonate,potassium carbonate and potassium bicarbonate added immobilized cells reached as long as 187.3,115.5,203.8,182.4 h at 40?,which were2.0-,1.23-,2.17-and 1.94-fold those of the free cells.The carbonate added immobilized cell didn't show better operational stability due to the intolerance of product under 750 mM substrate.The dosages of crosslinker were increased under the premise of identical batch time and enzyme recovery,the potassium carbonate added immobilized cells were successfully recycled for 13 cycles with more than 93% residual activity was retained while potassium bicarbonate added immobilized cells with 83% original activity in the 12 th cycle.These two immobilized cells show better operational stability when compare with traditional immobilized cells.Finally,the stability of immobilization cells was investigated.We found that the activity of immobilization cells was related to the water content in the case of air-dry.The activity of immobilization cells which either dried at room temperature or in vacuum were both around 20%.Several inorganic salts and agents reported in literatures were selected to determine the optimum preserved condition.The optimum preserve system were determined as 0.6 M sodium sulfite,pH 7.0,20?C of sodium sulfite system and 10% ammonium sulfate,pH 6.5,-20?C of ammonium sulfate preserve system.Finally,storage stability of immobilized cells under optimum conditions with the two preserve systems were compared.Results showed that both the ammonium sulfate preserve system exhibited remarkable stabilizing effect on immobilized cells with no obvious activity lost in 28 days.