Produção e caracterização in silico de uma quitosanase de Chromobacterium violaceum em estirpes de Escherichia coli recombinantes com potencial antibacteriano

Abstract

Chromobacterium violaceum is a Gram-negative, saprophytic and free-living bacterium. The genome sequencing of this organism revealed several ORFs encoding proteins with biotechnological potential. Among them are various glycoside hydrolases, including chitinases and chitosanases. Chitosanases are enzymes capable of degrading chitosan, a polymer of β-D-glucosamine and the product of this degradation, the oligomers of chitosan or oligomers of GlcN, are of great industrial interest, presenting antitumor and anticholesterol activity, among others, besides being used in food e having antimicrobial activity. The present work aimed to produce a recombinant chitosanase (Cv3931) from the 46 family of glycoside hydrolases, from C. violaceum ATCC 12472. The ORF encoding Cv3931 was amplified by polymerase chain reaction (PCR) and cloned into pET303/CT-His for expression in three strains of Escherichia coli. The recombinant protein was secreted into the culture medium by the three strains, in its soluble and functional form, and it was purified by affinity chromatography. Bioinformatics analyzes were also used for a more in-depth study of the molecular interactions carried out by this protein. The chitosanase produced in the strain OrigamiB (DE3) pLysS apparently showed a higher enzymatic activity, probably due to the favoring of the disulphide bridge formation by this strain. The oligomers released by the hydrolysis of chitosan by rCv3931 showed signs of antimicrobial activity. From the generated three-dimensional model and the molecular docking with different ligands it was observed that Cv3931 shows an extremely conserved sequence with a chitosanase from Bacillus circulans. Further studies on rCv3931 and the oligomers released when this protein hydrolyzes chitosan may make this enzyme an important indrutrial tool.