恶臭假单胞菌表面展示细菌漆酶及对染料的脱色性能
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摘要
细菌表面展示系统是一种蛋白质应用新技术,已在多个生物技术领域显示出应用前景。本研究利用丁香假单胞菌冰晶核蛋白(Ice Nucleation Protein) InaQ的N-末端结构域作为运载蛋白,采用C-端融合的方法,将一种细菌漆酶成功展示在恶臭假单胞菌(Pseudomonas putida) AB92019菌株的表面,然后对3种不同重复单元InaQ-N、(InaQ-N)2和(InaQ-N)3作为运载蛋白表面展示细菌漆酶的性能进行了比较分析,并对优化系统进一步用于染料脱色的性能进行了研究。
从携带细菌漆酶基因的重组质粒pMB172中通过PCR反应扩增得到突变型漆酶基因wlacD,然后酶切连接至分别含有1、2和3个inaQ-N基因重复的表达载体pMB104、pMB111和pMB112,构建得到分别含有融合基因inaQ-N-wlacD、(inaQ-N)2-wlacD和(inaQ-N)3-wlacD的重组质粒pMB281、pMB282和pMB283,并通过电转化导入恶臭假单胞菌AB92019菌株,筛选得到恶臭假单胞菌表面展示重组菌MB284、MB285和MB286。经对重组菌的SDS-PAGE、Western Blot、免疫荧光显微镜镜检以及流式细胞仪分析,结果证明重组菌MB284、MB285和MB286均能成功地在细胞表面分别展示其融合蛋白InaQ-N-WlacD、(InaQ-N)2-WlacD和(InaQ-N)3-WlacD。通过对3个重组菌全细胞漆酶酶活的检测发现,表面展示(InaQ-N)2-WlacD融合蛋白的重组菌MB285酶活最高,达到6.9U/mL。因此,选择重组菌MB285进行染料的脱色研究。
选取酸性绿和酸性红2种染料进行脱色试验。实验结果发现,重组菌MB285对两种染料均具有明显的脱色性能,其中对酸性绿的脱色率达到35.5%,对酸性红的脱色率达到17%。
对重组菌在优化培养条件下对两种染料的绝对脱色性能进行了考察。测定了重组菌MB285的全细胞酶活曲线,发现该曲线与重组菌的生长曲线具有同步性。通过单因子试验探讨重组菌MB285的摇瓶发酵条件(培养温度、初始pH、装液量和接种量)对生长量的影响。确定最佳培养条件为初始pH7.0,培养温度30℃,装液量20%和接种量4%。通过单因素和正交试验确定重组菌MB285发酵培养基的最佳配比为(W/V)2%葡萄糖、3%玉米浆、2%硫酸铵、0.2%氯化钠、0.08%MgSO4·7H2O和0.05% K2HPO4·3H2O。筛选出的优化培养基活菌数为1.1×1011CFU/mL,与优化前相比提高了约2.3倍。重组菌MB285摇瓶发酵优化后绝对脱色效率明显的提高,对酸性绿的绝对脱色率由优化前的36.5%提高到优化后的45%,对酸性红的绝对脱色率由优化前的17.9%提高到优化后的28%。
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%.
从携带细菌漆酶基因的重组质粒pMB172中通过PCR反应扩增得到突变型漆酶基因wlacD,然后酶切连接至分别含有1、2和3个inaQ-N基因重复的表达载体pMB104、pMB111和pMB112,构建得到分别含有融合基因inaQ-N-wlacD、(inaQ-N)2-wlacD和(inaQ-N)3-wlacD的重组质粒pMB281、pMB282和pMB283,并通过电转化导入恶臭假单胞菌AB92019菌株,筛选得到恶臭假单胞菌表面展示重组菌MB284、MB285和MB286。经对重组菌的SDS-PAGE、Western Blot、免疫荧光显微镜镜检以及流式细胞仪分析,结果证明重组菌MB284、MB285和MB286均能成功地在细胞表面分别展示其融合蛋白InaQ-N-WlacD、(InaQ-N)2-WlacD和(InaQ-N)3-WlacD。通过对3个重组菌全细胞漆酶酶活的检测发现,表面展示(InaQ-N)2-WlacD融合蛋白的重组菌MB285酶活最高,达到6.9U/mL。因此,选择重组菌MB285进行染料的脱色研究。
选取酸性绿和酸性红2种染料进行脱色试验。实验结果发现,重组菌MB285对两种染料均具有明显的脱色性能,其中对酸性绿的脱色率达到35.5%,对酸性红的脱色率达到17%。
对重组菌在优化培养条件下对两种染料的绝对脱色性能进行了考察。测定了重组菌MB285的全细胞酶活曲线,发现该曲线与重组菌的生长曲线具有同步性。通过单因子试验探讨重组菌MB285的摇瓶发酵条件(培养温度、初始pH、装液量和接种量)对生长量的影响。确定最佳培养条件为初始pH7.0,培养温度30℃,装液量20%和接种量4%。通过单因素和正交试验确定重组菌MB285发酵培养基的最佳配比为(W/V)2%葡萄糖、3%玉米浆、2%硫酸铵、0.2%氯化钠、0.08%MgSO4·7H2O和0.05% K2HPO4·3H2O。筛选出的优化培养基活菌数为1.1×1011CFU/mL,与优化前相比提高了约2.3倍。重组菌MB285摇瓶发酵优化后绝对脱色效率明显的提高,对酸性绿的绝对脱色率由优化前的36.5%提高到优化后的45%,对酸性红的绝对脱色率由优化前的17.9%提高到优化后的28%。
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%.
引文
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