假单胞菌的快速检测及其与表面活性剂的作用机理研究
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摘要
假单胞菌是一属在环境生物技术和工业生物技术中占据着重要位置的微生物,为了实现对该菌属整细胞的控制和催化过程强化,需要对其细胞特性进行快速的定量和监测。
本论文以假单胞菌为重点研究对象,通过应用生物发光及荧光技术,在饮用水及其过滤系统内的生物膜体系中对混合菌群结构进行解析,提出了一种快速定量目标菌的新方法;在表面活性剂对恶臭假单胞菌代谢影响机理的解析中,利用LUX-CFP实现了对整细胞及其胞内酶活性的快速准确表征,为基于表面活性剂控制假单胞菌整细胞及其代谢特性提供了新的手段。
实验结果表明,饮用水过滤器系统虽然能在一定程度上简化饮用水中的菌群结构,但因为在其内部会形成生物膜,导致出水中的微生物学指标升高,而生物膜的形成与过滤器的结构和温度都有关系。另外,在饮用水中检出了恶臭假单胞菌,但经过过滤系统后,该菌没有再被检测到。
提出并成功建立了一种基于KinExA仪器的免疫检测病原菌的方法,该方法对菌体的定量检测具有操作简单、灵敏度高、准确度高、重复性高和检测时间短等优点,用该方法检测铜绿假单胞菌的结果,接近文献报道的最优值,而其灵敏度比常规ELISA的高出100倍,并具有应用于混合微生物种群解析的潜力。
通过成功构建带有生物发光和荧光蛋白标记的基因工程菌P. putida BLU-CFP,实现了用LUX-CFP双生物标记简单而全面的表征整细胞及其胞内蛋白总体特性的目的。其中,LUX反映细胞的代谢活性,CFP可用其来衡量细胞膜通透性及细胞形态完整性的变化,并可用以表征细胞内大部分蛋白的活性。
对季铵盐阳离子表面活性剂DTAB和P. putida整细胞作用过程的机理模型研究表明,按照DTAB从低浓度到高浓度的顺序,DTAB对细胞的作用可以分为五个阶段:正常适应→休止细胞→代谢活性被抑制→失去繁殖能力→细胞裂解。而胞内蛋白主要经历了三个变化阶段:正常→部分酶活被抑制,但底物或产物的传质速率增加→酶泄漏到胞外→酶失活。此外,研究发现在该作用的过程中,阳离子表面活性剂的疏水基团起到了十分关键的作用。
Pseudomonas is an important genus both for environmental biotechnology and industrial biotechnology. In order to control the bacteria of this genus and intensify the metabolism of the whole cells, rapid and accurate monitoring of cell number and cellular characteristics is indispensable.
This thesis aimed at establishing rapid and simple tools for quantification and monitoring of the whole cells and cellular activities of Pseudomonas sp., by means of fluorescence and bioluminescence techniques.
To study the rapid quantification of Pseudomonas sp., the system of drinking water with its purifier was chosen, for its bacterialogical quality is closely related to the human health. The water purifier was found to be capable of simplifying the structure of bacterial community in drinking water by API kits identification. While, the biofilm formed inside the purifier would significantly increase the total number of bacteria (HPC) and the formation was closely related to the configuration and temperature of the purifier. P. putida was detected in the drinking water, but became non-detectable either in the filtered water or the biofilm formed inside the purifier.
A novel immunoassay-based bacterial quantification method was developed by using the KinExA instrument, showing the superior performances with high sensitivity, high accuracy, high reproducibility, simple manipulation and short detection time, etc. By this method, the sensitivity and dynamice range for P. aeruginosa detection were close to or even better than those of the best immunological detection method reported so far, and the sensitivity was 100 times higher than that of ELISA.
To analyze the whole cell characteristics and intracellular metabolic activities under the presence of cationic surfactants, a genetically engineered strain, P. putida BLU-CFP, was constructed by employing the LUX-CFP dual bioremarkers. Results showed that LUX reflected the metabolic activity of the cells, while CFP could estimate the membrane permeability, the shape integrity of the cells and also the activities of most intracellular proteins. By using this dual bioremarkers of LUX-CFP, the monitoring of whole cell catalysis would be much simpler.
The action process of DTAB (one of the cationic surfactants) on the whole cell of P. putida could be divided into five stages according to the changes of the cells by the treatment of DTAB from low to high concentrations: normalcells resting metabolic activity inhibited cell viability lost lysed. And according to the changes of intracellular enzymes, the DTAB effect was described as normal partly inhibited, while mass transfer speed enhanced leaked outside the cell completely denatured. In addition, the hydrophobic group of quarternary ammonium compounds was found playing a key role in the action of cationic surfactant on the whole cells and its intracellular enzymes.
本论文以假单胞菌为重点研究对象,通过应用生物发光及荧光技术,在饮用水及其过滤系统内的生物膜体系中对混合菌群结构进行解析,提出了一种快速定量目标菌的新方法;在表面活性剂对恶臭假单胞菌代谢影响机理的解析中,利用LUX-CFP实现了对整细胞及其胞内酶活性的快速准确表征,为基于表面活性剂控制假单胞菌整细胞及其代谢特性提供了新的手段。
实验结果表明,饮用水过滤器系统虽然能在一定程度上简化饮用水中的菌群结构,但因为在其内部会形成生物膜,导致出水中的微生物学指标升高,而生物膜的形成与过滤器的结构和温度都有关系。另外,在饮用水中检出了恶臭假单胞菌,但经过过滤系统后,该菌没有再被检测到。
提出并成功建立了一种基于KinExA仪器的免疫检测病原菌的方法,该方法对菌体的定量检测具有操作简单、灵敏度高、准确度高、重复性高和检测时间短等优点,用该方法检测铜绿假单胞菌的结果,接近文献报道的最优值,而其灵敏度比常规ELISA的高出100倍,并具有应用于混合微生物种群解析的潜力。
通过成功构建带有生物发光和荧光蛋白标记的基因工程菌P. putida BLU-CFP,实现了用LUX-CFP双生物标记简单而全面的表征整细胞及其胞内蛋白总体特性的目的。其中,LUX反映细胞的代谢活性,CFP可用其来衡量细胞膜通透性及细胞形态完整性的变化,并可用以表征细胞内大部分蛋白的活性。
对季铵盐阳离子表面活性剂DTAB和P. putida整细胞作用过程的机理模型研究表明,按照DTAB从低浓度到高浓度的顺序,DTAB对细胞的作用可以分为五个阶段:正常适应→休止细胞→代谢活性被抑制→失去繁殖能力→细胞裂解。而胞内蛋白主要经历了三个变化阶段:正常→部分酶活被抑制,但底物或产物的传质速率增加→酶泄漏到胞外→酶失活。此外,研究发现在该作用的过程中,阳离子表面活性剂的疏水基团起到了十分关键的作用。
Pseudomonas is an important genus both for environmental biotechnology and industrial biotechnology. In order to control the bacteria of this genus and intensify the metabolism of the whole cells, rapid and accurate monitoring of cell number and cellular characteristics is indispensable.
This thesis aimed at establishing rapid and simple tools for quantification and monitoring of the whole cells and cellular activities of Pseudomonas sp., by means of fluorescence and bioluminescence techniques.
To study the rapid quantification of Pseudomonas sp., the system of drinking water with its purifier was chosen, for its bacterialogical quality is closely related to the human health. The water purifier was found to be capable of simplifying the structure of bacterial community in drinking water by API kits identification. While, the biofilm formed inside the purifier would significantly increase the total number of bacteria (HPC) and the formation was closely related to the configuration and temperature of the purifier. P. putida was detected in the drinking water, but became non-detectable either in the filtered water or the biofilm formed inside the purifier.
A novel immunoassay-based bacterial quantification method was developed by using the KinExA instrument, showing the superior performances with high sensitivity, high accuracy, high reproducibility, simple manipulation and short detection time, etc. By this method, the sensitivity and dynamice range for P. aeruginosa detection were close to or even better than those of the best immunological detection method reported so far, and the sensitivity was 100 times higher than that of ELISA.
To analyze the whole cell characteristics and intracellular metabolic activities under the presence of cationic surfactants, a genetically engineered strain, P. putida BLU-CFP, was constructed by employing the LUX-CFP dual bioremarkers. Results showed that LUX reflected the metabolic activity of the cells, while CFP could estimate the membrane permeability, the shape integrity of the cells and also the activities of most intracellular proteins. By using this dual bioremarkers of LUX-CFP, the monitoring of whole cell catalysis would be much simpler.
The action process of DTAB (one of the cationic surfactants) on the whole cell of P. putida could be divided into five stages according to the changes of the cells by the treatment of DTAB from low to high concentrations: normalcells resting metabolic activity inhibited cell viability lost lysed. And according to the changes of intracellular enzymes, the DTAB effect was described as normal partly inhibited, while mass transfer speed enhanced leaked outside the cell completely denatured. In addition, the hydrophobic group of quarternary ammonium compounds was found playing a key role in the action of cationic surfactant on the whole cells and its intracellular enzymes.
引文
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