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

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 ß-cholestanol at higher rates than other 3ß-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.

Improving the organic solvent tolerance of Escherichia coli with vanillin, and the involvement of an AcrAB-TolC efflux pump in vanillin tolerance.

Vanillin is used as a flavor ingredient in a diverse range of food categories and is known to inhibit microbial fermentation. In this study, we found that vanillin improved the hydrophobic organic solvent tolerance of Escherichia coli and showed that the AcrAB-TolC efflux pump is implicated in that tolerance. The expression level of the pump was enhanced by the addition of vanillin. AcrAB-TolC efflux pump expression is known to be regulated by transcription activators such as MarA, SoxS, and Rob. Among these three transcription factors, marA transcription was significantly elevated by the addition of vanillin. We found that the AcrAB-TolC efflux pump is involved also in vanillin tolerance. The ΔacrB mutant was more sensitive to vanillin than the parent strain. A complementation test revealed that the introduction of the acrB gene recovered the vanillin tolerance of the ΔacrB mutant.