Functional Analysis And Application Of Steroidal Hormone-degrading Enzyme In Comamonas Testosteroni

In recent years, hormone pollution problems are becoming more and more serious, the pollutant with food chain gradually transferred to the organism, which causes serious damage to a large number of organisms. Steroid hormone is a kind of important pollutants existing in today’s environment, functional analysis of biological degradation and degradation genes of steroid hormone increasingly attention by people and attention.Comamonas testosteroni strain is a steroid hormone use as a sole source of carbon and energy Gram-negative bacteria. In this paper, gene cloning, gene knockout technology electricity conversion, enzyme-linked immune adsorption experiments, HPLC techniques for cloning and induced testosterone comamonas bacteria SDR short-chain dehydrogenase expression, functional analysis, and specific gene knockout were studied. SDR gene was cloned by PCR, the gene was constructed prokaryotic expression system pET-15b-SDR/E.coli BL21(DE3), IPTG induction of recombinant protein expression and purification using His-Binding-resin purification column, immune BALB/c mouse, obtained using the murine polyclonal antibodies with high titer established ELISA methods SDR (ELISA). The molecular weight of the final protein expression30.6kDa, the concentration of purified8.7mg/mL, murine antibody titer of5×104. The concentration of17β-estradiol,estriol, cholesterol and testosterone cultured comamonas testosterone bacteria were added in different culture medium, ELISA test results showed SDR induced by steroid hormones, especially for SDR-induced cholesterol effect was significantly higher than the expression of non-steroidal hormones added.Using gene knockout technology construction SDR gene deletion mutant strain, using HPLC detecting the wild-type gene deletion mutant strain induced by steroid hormone, found:SDR cholesterol, testosterone plays an important role in the use, SDR gene deletion mutant strain of cholesterol degradation of loss of function, efficiency in the use of testosterone reduced by more than90%;The above findings provide large number of theoretical foundations for applications of comamonas testosteroni in environmental bioremediation, conducive to rational development and utilization of comamonas testosteroni’s genetically engineered bacteria.