Expression and purification of TetX2 monooxygenase enzyme and investigating the effect of HAuCl4 on its structure and activity

Zomorodimanesh, Sadegh; Razavi, Seyed Hadi; Hosseinkhani, Saman; Emam Jomeh, Zahra

doi:10.22034/bmmj.2026.733632

Expression and purification of TetX2 monooxygenase enzyme and investigating the effect of HAuCl4 on its structure and activity

Document Type : Article

Authors

Sadegh Zomorodimanesh ¹

Seyed Hadi Razavi ²

Saman Hosseinkhani ³

Zahra Emam Jomeh ²

¹ Department of Food science and biotechnology, university of Tehran

² Department of Food Science and Engineering, university of Tehran

³ Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

10.22034/bmmj.2026.733632

Abstract

TetX2 monooxygenase is an enzyme with a molecular weight of 44 kDa. This enzyme catalyzes the selective hydroxylation of tetracycline to 11a-hydroxytetracycline due to the presence of NADPH as a cofactor and O2 as an electron acceptor. Gold is one of the metals with low toxicity, good solubility, and easy preparation. It is commonly used in the field of biological sciences, particularly in the production of diagnostic biosensors. In this study, recombinant Bacteroides thetaiotaomicron TetX2 monooxygenase was expressed and then purified based on N-terminal His-tags. The effect of HAuCl4 on the tertiary structure of TetX2 monooxygenase, as well as its kinetic parameters, size, thermal stability, and remaning activity, was investigated using a spectrophotometer, fluorescence spectrophotometer, and dynamic light scattering. The results showed that km, Vmax of the enzyme decrease in the presence of HAuCl4. Also, increasing the concentration of HAuCl4 in the presence of TetX2 monooxygenase enzyme caused a decrease in the emission intensity of tryptophan amino acid residues in the protein structure at a wavelength of 295 nm. The remaning activity, thermal stability, and size of the enzyme in the presence of HAuCl4 have also decreased compared to the native enzyme. Evidence showed that HAuCl4 affects the structure of TetX2 monooxygenase enzyme.

Keywords

Bacteroides thetaiotaomicron

TetX2 monooxygenase

tetracycline

protein tertiary structure

gold

Subjects