Reaction System Improvement And Immobilization Of Recombinant E.coli Expressing Nitrilase

In this thesis, the recombinant nitrilase was researched which was the expression of Rhodobacter sp. LHS-305in E.coli. On the base of studying its catalytic characteristic, solving methods were presented to deal with the existing problems. Adding cyclodextrin(CD) to increase catalysis efficiency, e.e. value of production and substrate concentration. The stability was enhanced by immobilizing the recombinant cells to composite materials.First, substrate spectrum was analysed to confirm its regioselectivity and stereoselectivity. The temperature, pH and substrate concentration of cell reaction was tested.Second, CD was used to handle substrate to improve the catalysis efficiency. It was showed that β-CD, HP-β-CD and RM-β-CD could improve the catalysis efficiency to245%,234%and204%. The inclusion conditions were optimized to confirm that the optimum temperature was50℃, inclusion time was80min, ratio of CD and substrate was1:1. With improving, the substrate concentration reached the maximum reaction speed increased from100mM to200mM, while which only increased from100mM to150mM by two-phase system. It took3/8the time before improvement to reach50%of the conversion, side-product was inhibited to make the e.e. value improve from65%to83%.Third, with studying the advantage of the common immobilization materials, a kind of novel composite material was made with the magnetic materials as the inorganic kernel, PVA as the outside shell, and using SA as the molding assistance, the problem of PVA prilling was solved and the loss of cells and enzymes were reduced because of the magnetic materials, meanwhile, stability was enhanced because of the embedding structure.Preparation conditions were optimized to confirm the optimum concentration of PVA was7.0%, SA was1.5%, the best cell upload amount was50g/L. It made optimum temperature of reaction increase from40℃to45℃. Meanwhile, stability was enhanced because of the embedding structure. After used for10batches, residual activity of immolized cells was85%while the free cells were only40%, substrate inhibition was eliminated further.In conclusion, methods used in this paper have improved the enzyme properties, they have certain universality, which can provide the reference for related researches.