Expressão e purificação de proteínas recombinantes da família GH23 de Chromobacterium violaceum ATCC 12472 com potencial antibacteriano

Abstract

In this study, Escherichia coli BL21(DE3) strains transformed with the plasmids pET32a-Cv2641 and pET32a-Cv0348 were used for the expression of proteins from Chromobacterium violaceum ATCC 12472, a Gram-negative bacterium with a fully sequenced genome. Genomic analysis of this microrganism revealed several open reading frames (ORFs) with biotechnological potential, including glycoside hydrolases (GHs; EC 3.2.1), enzymes that catalyze the modification and cleavage of carbohydrates through an acid-base catalysis mechanism. The aim of this study was to express and purify two recombinant enzymes from the GH23 family of C. violaceum ATCC 12472, encoded by the genes Cv2641 and Cv0348, and to evaluate their antibacterial activity. The recombinant proteins (rCv2641 and rCv0348) were successfully expressed in their soluble fraction and purified by affinity chromatography using a nickel-immobilized Sepharose matrix. The proteins of interest were eluted using phosphate buffer containing 250 mM imidazole. In silico analyses and previous studies suggest that GH23 enzymes exhibit lysozyme-like activity, hydrolyzing β-(1,4) glycosidic bonds between the carbohydrates N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) in bacterian peptidoglycan. Therefore, an antibacterial assay was performed in sterile 96-well microplates against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 33951 and Pseudomonas aeruginosa ATCC 27853 for both recombinant proteins. The results confirmed antibacterial activity, with a minimum inhibitory concentration (MIC) of 86.4 µg/ml for rCv2641 against all tested microrganisms and 186.8 µg/ml for rCv0348, but only for E. coli ATCC 25922 and S.aureus ATCC 33951. These findings support the potential of these enzymes for future biochemical characterization and biotechnological applications.