Background17β-estradiol (E2) residues exhibit harmful effects both for human and animals and have got global attention of the scientific community. Microbial enzymes are considered as one of the effective strategies having great potential for removal E2 residues from the environment. However, limited literature is available on the removal of E2 from wastewater using short-chain dehydrogenase.ResultsIn this study, 17β-estradiol degrading enzyme (17β-HSD-0095) was expressed and purified fromMicrobacteriumsp. MZT7. The optimal pH and temperature for reaction was 7 and 40 °C, respectively. Molecular docking studies have shown that the ARG215 residue form a hydrogen bond with oxygen atom of the substrate E2. Likewise, the point mutation results have revealed that the ARG215 residue play an important role in the E2 degradation by 17β-HSD-0095. In addition, 17β-HSD-0095 could remediate E2 contamination in synthetic livestock wastewater.ConclusionsThese findings offer some fresh perspectives on the molecular process of E2 degradation and the creation of enzyme preparations that can degrade E2.Graphical Abstract