Cloning And Expression Of Comamonas Testosteroni7aipha-hydroxysteroid Dehydrogenase And Regulation Analysis

With the development of industrialization, more and more environmentalpollutants are discharged into the nature, including organic pollutants. Due to thesimilar structure with human and animal hormone, the increasing concentration ofsteroidal compounds affects the growth and development of human beings andanimals,which causes the dysplasia of animals and the metabolic diseases of humanbeings. The traditional technology in dealing with the pollutants has the short-comings of low efficiency, high-cost, and easily causing the second pollution by thetoxic and hazardous intermediates. Nowadays the bioremediation degradation ofsteroidal compounds is a quite hot research topic in solving the environmentalpollution.Comamonas testosteroni is a bacterium that can use steroidal compounds assole carbon source for growth and metabolism of Gram-negative bacteria.Comamonas testosterone bacteria metabolic pathways of degradation steroidalcompounds are complex, about dozens of kinds of enzymes involved. Among themany enzymes7α-hydroxysteroid dehydrogenase capable of specifically acting onthe steroid nucleus7carbon atoms, hydroxyl group α to oxidation. Comamonastestosterone ATCC11996sequencing work has been completed, found in itsgenome does exist another7α-hydroxy steroid dehydrogenase possible sequences.Get the PCR production of7α-hydroxysteroid dehydrogenase gene and itsneighboring LysR, and to analyze the conserved sequences, presumably its basiccharacters. After7α-hydroxysteroid dehydrogenase gene and its neighboring LysR get purified protein expression vector. And get7α-hydroxysteroid dehydrogenasepolyclonal antibodies, established ELISA methods. ELISA method proved7α-hydroxysteroid dehydrogenase expression can be changed by vary steroidhormone changes.The7α-hydroxysteroid dehydrogenase gene and its300bp regulatorysequences upstream made plasmids were transformed into E. coli genes with LysRprove LysR gene is indeed involved in7α-hydroxysteroid dehydrogenase gene isregulated, when the presence of hormones7α-hydroxysteroid dehydrogenaseexpression was significantly increased. To further prove7α-hydroxysteroiddehydrogenase in the degradation of steroid compounds play an important role inthis study, suicide plasmid approach would make7α-hydroxysteroid dehydrogenaseknockout, and found that the mutation greatly reduced capacity in plant growth, anddegradation of hormones. Due to microbial degradation of pollutants is by variousenzymes, so the mode of enzymes and the regulation of its own way has a veryimportant significance. This research attempts7α-hydroxysteroid dehydrogenaseresearch, in order to further understand Comamonas testosterone mechanism ofaction of bacteria lay a foundation.