Uma quitinase termoestável de Chromobacterium violaceum ATCC 12472 expressa em Escherichia coli e que inibe a germinação de conídeos de dois fungos fitopatogênicos isolados do abacaxi ornamental

Abstract

Chromobacterium violaceum is a Gram-negative, saprophytic, facultative anaerobic and free-living β-proteobacterium. This bacterium inhabits several ecosystems in the tropical and subtropical regions of the world, especially in samples of water or moist soil. It is also characterized by a versatile energy metabolism, using of numerous enzymes that allow the use of various energy sources. In the C. violaceum genome, several ORFs (open reading frames) were identified, coding for proteins supposedly responsible for mediating the interaction of C. violaceum with the various elements present in the environments in which it inhabits. Among these proteins are various chitinases. Chitinases are enzymes capable of hydrolyzing chitin, which is a polysaccharide composed of residues of N-acetyl-β-D-glucosamine bound by O-glycosidic bonds of the β- (1,4) type. In this work, the objective was to express a chitinase (CV1440) of C. violaceum ATCC 12472 in E. coli, to purify the recombinant protein and to characterize it in relation to biochemical and biological aspects. The sequence encoded by the CV1440 ORF was amplified by DNA polymerase chain reaction (PCR) and added to the plasmid pET303/CT-His expression cassette. The recombinant plasmid was cloned into E. coli TOP10F 'and subsequently introduced into E. coli BL21 (DE3) for expression. Recombinant protein was secreted into the culture medium via the endogenous signal peptide. The chitinase was then purified from the cell free medium by ammonium sulfate precipitation (95% saturation), chitin affinity chromatography, followed by a nickel affinity chromatography immobilized on a sepharose matrix. When subjected to electrophoresis under denaturing conditions (SDS-PAGE), the purified protein had a single band with apparent molecular mass of about 46 kDa. The identity of this band as corresponding to chitinase CV1440 was confirmed by mass spectrometry. The yield of the purified recombinant product was approximately 3 mg per liter of culture. The purified recombinant chitinase presented optimum activity at pH 5.0 and had an optimum temperature of 60 °C, was moderately stable at pH variation and highly stable at temperature variations, maintaining more than 50% of the chitinolytic activity after incubation at 100 °C for 1 hour. The activity of the enzyme was negatively affected by the Cu2+, Hg2+, La3+ and Fe2+ ions and by the chemical agents DTT, β-mercaptoethanol, and SDS, but there was no increase in the presence of other ions, suggesting that the enzyme has no cofactor. The enzyme was able to inhibit the germination of conidia of phytopathogenic fungi Fusarium oxysporum and F. proliferatum. Atomic force microscopy assays revealed pronounced morphological changes on the surface of spores incubated with CvChi47 when compared to conidia not treated with the enzyme. Together, these results indicate the possibility of future use of chitinase encoded by ORF CV1440 as a potential tool in the control of phytopathogenic fungi.