Azurin Involved in Alcohol Oxidation System in Pseudomonas putida HK5: Expression Analysis and Gene Cloning

Abstract

Expression of azurin in Pseudomonas putida HK5 was examined by immunoblot analysis. Similar amounts of azurin were found in the cells grown into the stationary phase on any carbon sources, including LB medium without alcohol, where no quinoprotein alcohol dehydrogenases appeared. In the early exponential phase, the highest amount of azurin was found in the cells grown on 1-butanol, but here was none in the case of LB medium, suggesting that expression of azurin is cooperative with that of the alcohol oxidase system, especially the system including quinohemoprotein alcohol dehydrogenase IIB. The azurin gene (azu) was cloned and sequenced. azu is monocistronic, and in its promoter region, FNR-binding consensus sequence was found. However, its relative position suggests different transcriptional regulation from that in azu of P. aeruginosa. The molecular weight of the mature protein without copper ion calculated from the amino acid sequence was consistent with the value of the purified azurin measured by mass spectrometry.