Expressão em Escherichia coli e Pichia pastoris de uma quitinase antifúngica da família 19 das glicosídeo hidrolases de Chromobacterium violaceum.

Abstract

Chromobacterium violaceum is a Gram-negative, saprophytic, non-pathogenic and free living bacterium. The annotation of the genome of this species revealed many genes encoding products with potential applications in many areas. Among these products are several chitinases, which are enzymes capable of degrading chitin, a polysaccharide of N-acetyl-β-D-glucosamine. Chitin is found in the cell wall of many fungi and in the exoskeleton of insects and crustaceans. Chitinases are of great biotechnological interest because these enzymes can be used to produce useful derivatives from chitin, and also to be exploited for the control of fungi and insects that cause damages in crops. This study aimed to carry out the biochemical, biological and structural characterization of a recombinant chitinase (CV1897) belonging to family GH19 from C. violaceum ATCC 12472. The complete sequence of the ORF CV1897 (encoding CV1897SP+, with the native signal peptide) was amplified by polymerase chain reaction (PCR) and cloned into the vectors pET302/NT-His and pPICZαA, for expression in Escherichia coli and Pichia pastoris, respectively. The partial sequence of the ORF CV1897 (encoding CV1897SP-, without the native signal peptide) was expressed only in P. pastoris. In E. coli the rCV1897SP+ was mainly produced as insoluble inclusion bodies. In P. pastoris, the two versions of the protein (rCV1897PS+ and rCV1897PS-) were secreted into the culture medium, in their soluble and functional form. The enzymes were purified by affinity chromatography on immobilized nickel ions, with yields of 1.8 mg/L (rCV1897PS+) and 2.1 mg/L (rCV1897PS-), being detected by electrophoresis as two isoforms with different molecular weights. The recombinant chitinase (rCV1897PS+) showed optimal hydrolytic activity at pH 5.0 and was active after treatement at temperatures up to 40 °C for 30 min. The rCV1897 showed chitinolytic activity against colloidal chitin and glycol-chitin on SDS-PAGE. Circular dichroism spectra showed that the protein has predominantly α-helical arrangements (37%), also having 26% of β-strands and 38% disordered structure. The fluorescence spectra revealed that the protein was produced in their folded conformation. The rCV1897PS+ inhibited the conidial germination and mycelial growth of the phytopathogenic fungi Penicillium herquei and Colletotrichum lindemuthianum. Both rCV1897PS+ and rCV1897PS- in combination with fluconazole acted synergistically against different strains of the human pathogenic yeast Candida tropicalis. The biochemical, structural and biological studies of rCV1897 may be useful for biotechnological application of this enzyme.