| 1-Cyanocyclohexaneacetic acid?1-CHAA?is an important intermediate in the synthesis of gabapentin which is used in the treatment of epilepsy.Although nitrilase-mediated biocatalysis pathway is a promisingforbio-productionof1-CHAAfrom1-cyanocyclohexaneacetonitrile?1-CHAN?,it is hampered due to the high cost of biocatalyst,the poor operational stability,the difficulty of recycling product.To solve the problem,immobilized nitrilase and immobilized cells harboring nitrilase are studied in this paper.The concrete results are as following:Firstly,selecting the silicon oxide support for the immobilization of nitrilase from several carriers.The process of immobilizing the nitrilase and the catalytic property of immobilized enzyme were investigated,the results showed that 30 mM silicic acid,TETA as the inducer,a molar ratio of[Si]:[N]=1:1 and 0.15 mg/mL free enzyme concentration in the final solution,this optimized parameters gave the best immobilized effects,affording high activity recovery of 96.6%and high immobilization efficiency of 98.1%.The catalytic property of immobilized enzyme showed that the pH stability and storage stability were obviously improved compared with free enzyme.The activity of immobilized enzyme was approximately 75%-85%higher at 50 to 60°C than that of free enzyme when the thermostability was studied and after 20 cycle's reaction,the immobilized enzyme still retained 77.2%relative activity.In order to simplify the immobilization process and make the immobilization method more suitable for application in an industrial scale.The immobilized cells harboring nitrilase was investigated.Firstly,comparing different methods of immobilized cells.an efficient production of 1-CHAA was developed using polyethyleneimine?PEI?/glutaraldehyde?GA?cross-linked cells which provided obvious advantages such as cheap starting material,easy preparation,good stability.The effects of seven parameters on the immobilization of whole cells were optimized,the residual activity of immobilized cells retained 85%after shaking for24 h and the activity recovery increased from 45.3%to 57.8%when the parameters were set as 3%?v/v?of PEI,1%?v/v?of GA,100 mg/mL of cell loading,7.0 of cross-linking pH,0.6%?w/v?of diatomite,1 h of PEI cross-linking time and 30 min of GA cross-linking time.The SEM data and SDS-PAGE analyses further proved the stability of the immobilized whole cell.Finally,the biocatalysis pathway of immobilized cells was investigated,the optimum temperature of immobilized cells was 45°C and the optimum p H was 7.0.When the cell concentration was 250mg/mL,the substrate concentration was 1.0 M.914.6 mM of product concentration was achieved in 7 h,affording an extremely high productivity of 244.5 g·L-1·d-1 and 91.5%yield.The stability of immobilized cells showed that pH stability was significantly improved,the good storage stability made immobilized cells retain 84.2%of its activity after 120 h of incubation at 4°C,the good thermal stability of immobilized cells improved the half-lives at 45,50,and 55°C and were11.6-,5.0-,8.5-fold those of the free cells,the good operational stability made the immobilized cells retain more than 80%of the original activity during 30 cycles of reaction. |
PDF Full Text Request