Characteristics Of Enzymes Related To Caffeine Degradation In Paraburkholderia Caffeinilytica CF1

Caffeine(1,3,7 trimethylxanthine)is a naturally occurring environmental contaminant that belongs to the class of purine alkaloids.The emerging biodegradation method is attracting widespread attention because of its advantages of environmental protection,high efficiency and convenience.Therefore,it should come as no surprise that some caffeine-degrading microorganisms have been isolated from the environment,however,little is known concerning the enzymology of caffeine degradation.In order to better study bacterial N-demethylases,the enzymes that catalyze N-demethylation have been purified and characterized in this study.A novel caffeine-degrading bacterium,Paraburkholderia caffeinilytica CF1,was isolated from the soil by an enrichment procedure using caffeine as the sole source of carbon and nitrogen.Initial results suggested that caffeine was degraded by sequential N-demethylation to form xanthine.The enzymes,Cdn A,Cdn B&Cdn C,related to caffeine-degradation belong to the class of two-component Rieske oxygenases and require an oxidoreductase,Cdn D,for efficient catalysis.In this report,cdn D and cdn A gene from the g DNA of P.caffeinilytica CF1 have been isolated and cloned in a p ET28a expression vector.Soluble Cdn A,Cdn B,Cdn C and Cdn D were seperatly over-expressed in E.coli cells and purified by Ni²⁺NTA chromatography.Monomeric molecular mass of the proteins were found to be 44,58,47 and 64 k Da,respectively.The characterization of enzymes was performed due to the determination of specific substrates,optimal conditions and sequence analysis.