17β-HYDROXYSTEROID DEHYDROGENASE FROM THE FUNGUS : BIOSYNTHESIS IN ACTINOBACTERIAL CELLS AND FUNCTIONAL CHARACTERISTICS

17β-Hydroxysteroid dehydrogenase (17β-HSD) is an enzyme used in biotechnology for obtaining testosterone from phytosterol. Heterologous 17β-HSD from the fungus catalyzes the NADPH-dependent reduction of the 17-oxo group of androstenedione/androstadienedione, formed in the cells of mycolicibacteria as a result of their inherent polyenzyme process of oxidation of the aliphatic side chain of phytosterol, with the formation of testosterone/Δ-dehydrotestosterone, respectively. The object of the study was heterologous 17β-HSD from the fungus (17β-HSD) with a 6xHis tag (6×His-17β-HSD), synthesized in the cells of actinobacteria . Isolation and purification of the recombinant enzyme was carried out using affinity chromatography. The enzyme preparation 6×His-17β-HSD exhibited the highest activity towards androstenedione. The activity of 6×His-17β-HSD depended on NADPH and was manifested in the pH range from 6.0 to 9.0 with an optimum at pH 7.0. Analysis of kinetic characteristics showed that the properties of the heterologous 6×His-17β-HSD enzyme synthesized in cells are comparable to those obtained for the 17β-HSD enzyme isolated from the fungus , as well as for the recombinant 17β-HSD enzymes synthesized in and cells. The results expand our knowledge of microbial 17β-HSDs and indicate the potential for using recombinant strains expressing a codon-optimized cDNA sequence encoding 17β-HSD from the fungus for producing testosterone from phytosterol.