Construction Of A Genetically Engineered Bacterium For Efficiently Degrading Azo Dyes

Biodegradation of azo dyes has been studied for years, and many microorganism degrading azo dyes were screened. Some azoreductase genes were cloned and expressed in E. coli efficiently, while enzyme application in waste water treatment is limited by its high cost. Then microorganism itself is asked to be more efficient to promote practical treatment.The purpose of this dissertation is to construct a genetically engineered bacterium with higher azoreductase activity. In order to get this goal, the azoreductase gene from the donor (Rhodobacter sphaeroides AS 1.1737) is inserted in a Rhodopseudomonas palustris/E. coli shuttle vector, which is transformed into the acceptor (Rhodopseudomonas palustris AS 1.2352). And the feasibility of this process is proved by investigating the azo dye decolorization by donor and acceptor strains, and the knowledge on plasmids in acceptor cells.The decolorization of azo dyes by cells and crude enzyme of Rhodobacter sphaeroides AS1.173 7 was investigated, and several kinds of azo dyes can be decolorized over 90% in 24 h. While azo dyes can not be the sole carbon source for growth of this strain. Besides, the optimal conditions of decolorization are 35 - 40℃ and pH 7-8. The crude enzyme extracted from this strain has azoreductase activity, but can not be well purified by Red Sepharose CL-6B. Making methyl orange as model dye, the optimal decolorization conditions are 50℃ and pH 8.0, and the catalysis corresponded to ping-pong mechanism, where the Michaelis-Menten constants for Methyl Red and NADH are 0.45 mM and 1.25 mM, respectively.The azoreductase activities of the cells and intracellular crude enzyme of Rhodopseudomonas palustris AS1.2352 were reported, and LAS (Sodium linear alkylbenzene sulphonate) and SDS (Sodium dodecyl sulfate) were added in cultures to simulate practical dyeing wastewater. It is found that this strain can endure surfactant and degrade them aerobically, and inhibitions of LAS on growth and decolorization are higher than SDS. Besides, the optimal temperature of decolorization with SDS (30℃) is much lower than that without SDS, which shows that SDS made the decolorization of this strain sensitive to higher temperatures.The plasmid extraction of Rhodopseudomonas palustris was optimized, so that the native plasmids of this strain could be obtained with higher quantity and quality for further operations. And four kinds of plasmids were found in this strain, one of which was identified...