Cloning, Expression And Enzymology Properties Research Of A 17 Beta Hydroxy Steroid Dehydrogenase In Comamonas Testosteroni

Steroid hormones as the only carbon source and energy in the environment to provide the Comonas testosterone survival. A series of enzymes in the Comonas testosterone can degrade the steroid hormones into the H2 O and CO2.The short chain dehydrogenase(SDRs)which in the Comonas testosterone are a kind of REDOX enzyme which dependent on NAD(P)H.17 beta hydroxy steroid dehydrogenase is a member of the SDR suoerfamiLy. Its main function is CataLytic steroid hydroxyL/ketone group of oxidation reduction. We have cloned a 17 beta hydroxy steroid dehydrogenase(17?- HSDX) gene from Comamonas testosteroni(C. testosterone) ATCC11996, Constructed the recombinant expression vector pET-15b-17?-HSDX. To send the recombinant expression vector into e. coLi BL21. 17 beta HSDX protein was successfully expressed and purified by the method of Ni coLumn affinity chromatography.The 17 beta hydroxy steroid dehydrogenase(17?-HSDX) gene contains 750 bp and translates into a protein of 249 amino acid residues. Using the purified protein immune rabbits, obtained using the rabbit polyclonal antibodies with high titer(1:16000) established the standard curve of 17 beta HSDX Determination.Induction of the Comonas testosterone with T,ES,E2 and E3.The The expression of 17 beta HSDX protein was measured by Elsia. Fuond that The expression quantity of 17 beta HSDX protein was higher when Induction of the Comonas testosterone with T and ES.17 beta hydroxy steroid dehydrogenase was knocked out by using the method of homoLogous recombination. Mutant strain was obtained successfuly without 17 beta HSDX gene. The residual quantity of induction of hormone in culture medium which has mutant and wild type strain was detected by high performance liquid chromatography mehhod, and we have found that 17?- HSDX plays an important role in degrading testosterone and oestrone.