Study Of L-Cyano-Cyclohexyl-Acetonitrile By Recombinant E. Coli Containing The Nitrilase

This thesis mainly concentrated on the optimization of the fermentation conditions, the reaction conditions and the immobilization method of recombinant Escherichia coli cells containing nitrilase. By using calcium alginate-glutaraldehydepolyethylenimine, the recombinant cells were immobilized and utilized in the biocatalytic hydrolysis of 1-cyanocyclohexyl acetonitrile to obtain 1-cyanocyclohexyl acid with high-yield production which is the intermediate of organic synthesis and pharmaceutical.In the second part of the thesis, the fermentation conditions of nitrilase produced by recombinant E. coli were optimized by shake flask through the way of single factor method. The effect of nutrient media components(carbon source, nitrogen source) and fermentation conditions(temperature, initial p H, inoculum volume fraction, shake bottle loading liquid volume, the time of adding IPTG, the final concentration of IPTG, enzyme production cycle) on the production of the nitrilase were investigated. Results showed that the best medium for nitrilase production were made up of 10g/L peptone, 10g/L yeast extract, 5 g/L Na Cl. The optimum fermentation conditions were culture temperature 30℃, initial p H value 7.0, inoculum volume fraction 4%, shake flask loading liquid volume 20%, the time for adding IPTG at 4h, 0.2m M IPTG, and fermentation period 10 h. The final nitrilase activitycan reach to 50.60U/g, after optimization.The third chapter of this thesis studied the effect of reaction conditions on the nitrilase activity including organic solvent, the amount of cell catalyst, reaction time, reaction temperature, reaction p H, p H stability, temperature stability, reaction volume, substrate particle size, substrate inhibition test, product inhibition test, and the time for storing. Experimental results showed that high conversion rate was obtained when reaction system consisted of 1mol/L substrate, 15g/L cell mass, 5ml reaction volume without adding any co-solvent, phosphate buffer p H 7.3, reaction temperature 30℃. In addition, our results also showed that there was no effect of substrate particle size on nitrilase activity and no substrate inhibition phenomenon appeared in the reaction.However the product inhibition was proved to be existed in the reaction.The forth chapter of the thesis presented the immobilization method for the recombinant Escherichia coli cells by using alginate--glutaraldehyde-- polyethyleneimine. The process for immobilization conditions were optimized as following: 30g/L loaded cells, 2g/100 m L sodium alginate, 12 h for hardening time, 0.1M glutaraldehyde, 0.143 m M polyethyleneimine, and 20 min for cross-linked time. Then the properties of the immobilized cells were examined and compared with free cells. Results indicated that the optimum temperature of immobilized and free cells were 70℃ and 60℃, and the optimum p H were 8.0 and 7.3 respectively, Furthermore the p H stability and temperature stability of the immobilized cells were proved to be both improved profoundly. The immobilized cells retained 59.7% activity in Tris-HCL buffer(p H=8.0) containing 30 m M calcium chloride after stored at 4 ℃ for 55 days, while the free cells retained only 26.67% initial activity under the same condition. In the final recycled experiment, it was found that the immobilized cells can be reused for 20 times without obvious decrease of conversion when 0.5M substrate and 35g/L cells were utilized in the reaction. Under the above reaction conditions, 166.19 g 1-cyanidecyclohexyl acetate/L/d, while the conversion of the free cells started to decrease after five cycles and the conversion rate of substrate was only 6% after 20 times recycle. Our experiments confirmed that immobilized cells can be recycled 20 times in cycling reaction mixture containing Ca2+ without immobilized cells crushed and no loss of enzyme activity. However it was also found that the immobilized cells were all broken after 7 times in cycling reaction mixture without Ca2+, resulting in the interrupting of the reaction. It was supposed that the addition of Ca2+ to reaction solution can maintain the structure of the nitrilase inimmobilized cells, thus increase the reaction batch of the immobilized cells, and the yield of final product.