Expression and characterization of cholesterol oxidase with high thermal and pH stability from Janthinobacterium agaricidamnosum

Abstract

Cholesterol oxidases (COXases) have a diverse array of applications including analysis of blood cholesterol levels, synthesis of steroids, and utilization as an insecticidal protein. The COXase gene from Janthinobacterium agaricidamnosum was cloned and expressed in Escherichia coli. The purified COXase showed an optimal temperature of 60 °C and maintained about 96 and 72% of its initial activity after 30 min at 60 and 70 °C, respectively. In addition, the purified COXase exhibited a pH optimum at 7.0 and high pH stability over the broad pH range of 3.0–12.0. The pH stability of the COXase at pH 12.0 was higher than that of highly stable COXase from Chromobacterium sp. DS-1. The COXase oxidized cholesterol and β-cholestanol at higher rates than other 3β-hydroxysteroids. The Km, Vmax, and kcat values for cholesterol were 156 μM, 13.7 μmol/min/mg protein, and 14.4 s⁻¹, respectively. These results showed that this enzyme could be very useful in the clinical determination of cholesterol in serum and the production of steroidal compounds. This is the first report to characterize a COXase from the genus Janthinobacterium.

Keywords:

• Cholesterol
• cholesterol oxidase
• clinical examination
• Janthinobacterium agaricidamnosum
• stable enzyme
• vanillyl-alcohol oxidase family
• word

Acknowledgment

We would like to thank Yura Fujii, Taisei Kurihara, and Prof. Hiroaki Hasegawa for their technical support.

Ethical approval

The authors confirm that there are no ethical issues in the publication of the manuscript.

Author contributions

ND designed the experiments and contributed to paper writing. ND, YI, and TS conducted the experiment and contributed to the data analysis.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

All data generated or analyzed during this study are included in this submitted article.

Additional information

Funding

This work was supported in part by a Grant for Bioresilience Research Project at Toyo University, and by a Grant for the Programme for the Strategic Research Foundation at Private Universities S1101017, organized by the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan.