Covalent Immobilization of Human Placental 17β-Hydroxysteroid Dehydrogenase Type 1 onto Glutaraldehyde Activated Silica Coupled with LC-TOF/MS for Anti-Cancer Drug Screening Applications

Abstract

Human 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1), a potential target in breast cancer prevention and therapy, was extracted from human placenta and immobilized on nonporous silica (∼5 μm) with a covalent method for the first time. The optimum initial enzyme concentration and immobilization time during the immobilization process were 0.42 mg mL and 12 h, repectively. The binding was confirmed by scanning electron microscope (SEM) and infrared spectroscopy (FT-IR). It could improve the pH, thermal and storage stability compared to free enzyme. Moreover, the immobilized enzyme could be reused at least four times. A screening method based on it coupled with liquid chromatography-time-of-flight mass spectrometer (LC-TOF/MS) was established, and the half-maximal inhibitory concentration (IC 50) of apigenin for the immobilized enzyme was 291 nM. Subsequently, 10 natural products were evaluated leading to inhibition of the activity of 17β-HSD1 at the concentration of 25 μM, and six of them inhibit the activity over 50%.