| Bacterial surface display system has been recognized as a new technology in protein applications, which has shown a promising applying prospect in many biotechnological fields. In this dissertation, the N-terminal domain of a newly-identified ice nucleation protein (InaQ) from Pseudomonas syringae, was engineered as a functional carrier protein to immobilize a bacterial laccase onto the surface of P. putida AB92019 strain through the C-terminal fusion pattern, followed by further comparative analyses of the display efficiencies of three recombinant cell-surface display systems constituting of 1,2 or 3 tandemly repeated InaQ-N anchor and the laccase. The effect on dye decolorization treating with the optimized laccase-displaying system was subsequently investigated.The mutated bacterial laccase gene wlacD was PCR-amplified from its parent plasmid pMB172, then ligated into the expression vector pMB104, pMB111 and pMB112 to yield the recombinant plasmids pMB281, pMB282 and pMB283, which carrying the recombinant inaQ-N-wlacD, (inaQ-N)2-wlacD and (inaQ-N)3-wlacD fusion genes, respectively. Three recombinant strains, MB284, MB285 and MB286, which were endowed the surface-display activities, were subsequently obtained by respectively introducing those recombinant genes into P. putida AB92019 strain by electroporation. The surface localization of the fusion proteins expressed by the recombinant strains were confirmed using SDS-PAGE and Western blot analyses of cell fractions, immunofluorescence microscopic examinations and flow cytometry analyses of intact cells. Whole-cell laccase activities of three recombinant strains were further determined, it showed that the recombinant MB285 expressing (InaQ-N)2-WlacD, exhibited the highest enzymatic activity by 6.9 U/mL, was an apparent optimum strain that displaying laccase on the surface.Two dye materials, acid green and acid red, were selected as the substrates for dye decolorization test using the intact MB285 cells with optimum laccase-displaying efficiency, it showed that MB285 cells exhibited the substantial decolorazation effect on both of two dye substrates, the decolorization rate of acid green was by 35.5%, while the decolorization rate of acid red was by 17%.The absolute dye decolorization capacity of MB285 under the optimized culture conditions was evaluated. The time course of enzymatic activity of MB285 whole cells, showed a considerable coordination with that of cell growth of this bacterium, it therefore provides an approach to enhance the absolute dye decolorization by simply increasing the cell's growth density. MB285 culture conditions in flask fermentation (culture temperature, initial pH, medium volume and inoculum) was investigated and optimized by means of the single factor test. As a result, an optimum culture condition was found by culturing MB285 with an initial pH 7.0, incubation temperature 30℃, 20% of medium load (v/v) and 4% of inoculum (v/v), while using an optimum medium that was made up of 2% glucose (w/v),3% corn steep liquor,2% ammonium sulfate,0.2% sodium chloride,0.08% MgSO4·7H2O and 0.05% K2HPO4·3H2O. The optimized cell culture can reach up to 1.1×1011 CFU/mL in cell density, which was about 2.3-fold in contrast to the original culture condition. Furthermore, by using the optimized cultures to treat two dye materials, it found that the absolute decoloration rate of dye acid green had increased from 36.5% to 45%, whereas the absolute decoloration rate of dye acid red increased from 17.9% to 28%.
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