High-density Fermentation Of Nitrialse And The Application In 1-Cyanocyclohexyl Acetate Synthesis

Nitrilases,as an important biocatalyst,can catalyze a,co-dinrtrile into?-nitrilecarboxylic acid with high activity and excellent regioselectivity.l-cyanocyclohexaneacetic acid is a useful precursor for the synthesis of gabapentin.An attractive approach to the synthesis of 1-cyanocyclohexaneacetic acid is the regioselective hydrolysis of dinitriles to cyanocarboxylic acids by nitrilase.Therefore,there is a need for the preparation of low-cost and efficient nitrilases..In this work,we investigated the high cell density cultivation of recombinant Escherichia coli to produce nitrilase and explored the biocatalytic process by this nitrilase.The effects of medium composition,pH,inducer and induction temperature on the nitrilase fermentation were firstly examined in a 5 L fermenter.The results showed that the higher nitrilase production was obtained with modified 2xYT media(g L-1 peptone 15,yeast extract 12,NaCl 10,glycerol 15,K2HPO4·3 H2O 2.28,KH2PO41.36,MgSO4·7H2O 0.375)at pH6.5,30? of induction temperature by using 12.5 g/L of lactose as inducer.Under these optimal conditions,the biomass reached 10.3 g DCW/L with specific activity of 1759 U/g DCW.The volume activity was calculated to be 18117 U/L,which was 5 times higher compared with LB media and 2.2 times higher compared with initial 2×YT media(g L-1 peptone 16,yeast extract 10,NaCl 5).Three different feeding strategies including constant feeding,exponential feeding and DO-Stat feeding were then explored for high cell density cultivation of recombinant E.coli.The feedback control stratety led to a high nitrilase production than the non-feedback control strategies.When the DO-Stat feeding strategy with dissolved oxygen at 15%was used to control the fermentation process and 34 g/L lactose added in two batches as inducer,the biomass reach 69.3 g DCW/L with the specific activity of 1479 U/g DCW.The total nitrilase was kept above 90000 U/L.The nitrilase was used for the syntheisiof 1-cyanocyclohexaneacetic acid from 1-(cyanomethyl)cyclohexanecarbonitrile.The catalytic system PBS buffer can be replaced by distilled water.The Reactor Volume amplificated to 5 L from 200 mL,the catalyst usage dropped from 100 g/L to 50 g/L(wet weight of the cell).Then,separation of the product was attempted,the purity of 1-cyanocyclohexaneacetic acid obtained 97%and the yield reached 92%.