Obtenção de uma quitinase mutante recombinante e avaliação da sua interferência no crescimento do fungo R. stolonifer

Abstract

Chitin is the second most abundant biopolymer in nature and its structure is formed by N-acetyl-β-D-glucosamine units linked through β-1,4- glycosidic bonds. Different organisms have chitin such as crustaceans, insects and fungi. The enzymes that hydrolyze chitin, called chitinases, belong to the class of glycosyl hydrolases and are produced by several organisms. Biotechnological applications of chitinases include biocontrol of fungi and insects. The objective of this work was to obtain a recombinant chitinase from the leaf-cutter ant Atta sexdens (AsChtII-C4B1-1) and evaluate its interference with the growth of the fungus Rhizopus stolonifer. AsChtII-C4B1-1 shows 99.8% identity with the AsChtII-C4B1 enzyme and can therefore be considered isoforms. Both enzymes present a catalytic domain for chitinase in a chitin-binding domain (CBM). AsChtII-C4B1-1 shows greater activity at pH 6.0 and 600C. Against the colloidal chitin substrate, the AsChtII-C4B1 enzyme shows greater activity, requiring 15.8% less AsChtII-C4B1 to release 1µmol of N-Ac than AsChtII-C4B1-1. The two enzymes were able to interfere with the growth of R. stolonifer, an important fungus in agriculture. AsChtII-C4B1 and AsChtII-C4B1-1 reduced 57% and 53% growth, respectively. The 3D Model of the AsChtII-C4B1-1 enzyme suggests that the sequential differences between the enzymes may be related to the different optimal pH and temperature values found and also to the difference in biological activity.