[Isolation and identification of Comamonas testosteroni: cloning and overexpression of 3alpha-hydroxysteroid dehydrogenase in E.coli].

OBJECTIVE: To isolate and identify 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) producing Comamonas testosteroni from soil, and to clone and overexpress 3alpha-HSD in E.coli. METHODS: Samples of pond mud were inoculated into cultural medium with androsterone as sole carbon source. The primary identification was performed according to the morphological observation, biochemical reaction and cultural characterization. To further identify the bacteria, a couple of primers were designed according to the 3alpha-HSD gene of Comamonas testosteroni. An 800 bp fragment containing 3alpha-HSD gene was obtained by PCR amplification. Then the PCR products were inserted into plasmids pET-15b to construct recombinant plasmids pET-15b. Afterwards the host bacteria containing recombinant plasmids pET-15b with proper orientation grew with isopropyl-beta-D-thioglactopyranoside (IPTG) induction. RESULTS: The isolated bacteria which could use androsterone as the sole carbon source had 85% consistency with Comamonas testosteroni. After 5 hours of IPTG induction, a recombinant protein about 29 x10(3) with enzyme activity was overexpressed in the host bacteria E.coli. BL21(DE3) pLysS. This protein could catalyze the dehydrogenization reaction of androsterone (3alpha-hydroxysteroid). CONCLUSION: A strain of Gramjnegative 3alpha-HSD producing Comamonas testosteroni was isolated from pond mud, and recombinant 3alpha-HSD with enzyme activity was overexpressed in E.coli. This work laid good foundation for the purification of recombinant 3alpha-HSD by metal chelate chromatography, and also for the construction of an enzymatic cycling method to measure serum total bile acids with recombinant 3alpha-HSD as the tool enzyme.

An enzymatic cycling method for the determination of serum total bile acids with recombinant 3alpha-hydroxysteroid dehydrogenase.

A highly sensitive enzymatic cycling method was developed for the serum total bile acids assay. We constructed a prokaryotic expression system to prepare the recombinant 3alpha-hydroxysteroid dehydrogenase in place of the natural enzyme and for the first time used it in the total bile acids assay. The production rate of thio-NADH correlated with the bile acids concentration and was measured by the change of absorbance at 405/660 nm. The enzymatic cycling method could detect 0.22 micromol/L total bile acids in serum. Within-run and between-run imprecisions were 1.2-3.7% and 2.3-4.8%, respectively. The calibration curve for total bile acids in serum was linear between 0.5 and 180 micromol/L. This method was free from interference by bilirubin, hemoglobin, ascorbate, and lactate dehydrogenase. In conclusion, serum total bile acids could be measured by the enzymatic cycling method with recombinant 3alpha-hydroxysteroid dehydrogenase as the tool enzyme.