The Construction And Application Of A System For Flourescence Steroid Determination

3a-Hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni is a key enzyme in the degradation of steroids in the environment. In this work the hsdA gene was replaced by the green fluorescent protein (GFP) gene, which was homologous integrated into the chromosomal DNA, the C. testosteroni mutant strain CT-GFP5-1 was generated. In the mutant strain GFP gene could be expressed when some steroid inducers were added in the culture medium. The Fluorescence intensity is correlated to the quantity of the steroids. We have discussed the relation between thefluorescence intensity and quantity of the steroids. Result:the fluorescence intensity is correlated to the quantity of the steroids.The cell-free system is more sensitive than cell-based system. And the determination of estradiol is more sensitive than testosterone. It is a rapid sensitive biosensor system for flourescence steroid determination. Compared to the results of HPLC, the identical results were got with our biosensor method. And the bioassay is more rapidity, simplity, stability and sensitivity. It is viable to speared and used to determinate the steroid in the environment as a new biosensor system.