Expression Optimization, Purification, and Fluorescence Spectroscopic Assessment of Creatinase Enzyme in the Presence of Gold Nanoparticles

Nowadays, the use of laboratory methods based on enzymatic reactions to monitor the health of individuals, especially in the field of military forces, has become widespread. One of the enzymatic methods is the use of creatinase enzyme for clinical measurement of creatine and creatinine in body fluids, through which the health of kidneys, muscles, and thyroid gland can be understood. This study aimed to optimize the expression of the creatinase enzyme and to investigate the effect of gold nanoparticles on the stability of the creatinase enzyme.

This research was conducted in the spring and summer of 2021 in the electrochemical institute located at the University of Tehran, Iran. In this study, E. coli BL21 containing pET28a amplification vector was cultured in TB medium and then induced at 18, 22, 28, 32, and 37°C. After sonication, purification of enzymes was performed by affinity chromatography. After confirmation of expression by SDS-PAGE, the concentrations of enzymes expressed at different induction temperatures were measured and compared by the Bradford method. Then the activity of enzymes was evaluated and compared by the colorimetric method. Finally, the effect of gold nanoparticles on the structure of the creatinase enzyme was investigated by fluorescence spectroscopy.

Examination of creatinase expression at different temperatures in the TB medium showed that at 28°C the highest amount of enzyme with the highest activity was obtained. On the other hand, fluorescence spectroscopy of creatinase enzyme with the presence and absence of gold nanoparticles at excitation wavelength of 295 nm showed a decrease in fluorescence intensity in the presence of gold nanoparticles.

The induction temperature of 28°C in the TB medium provides a more active and more concentrated enzyme. Also, the presence of gold nanoparticles reduced the fluorescence intensity of the creatinase enzyme, which could indicate a change in the fluorescent amino acid microenvironment of the creatinase enzyme structure.