Obtenção e aplicação biológica de quitinases recombinantes da formiga cortadeira Atta sexdens
Resumen
Chitinases catalyze the cleavage of
the β-1,4 bond of chitin, a polysaccharide produced by several organisms including
insects. From a biotechnological point of view, these enzymes have potential
application in the medical field and in biological control. Despite their importance,
insect chitinases have been little studied. The objective of this work was to obtain
and characterize three recombinant chitinases from the leaf-cutter ant Atta sexdens,
containing different catalytic domains (C) and chitin-binding sites, CBM (B). A.
sexdens does not have a known genome, as yet, but from the genome of
Acromyrmex echinatior, also a leafcutter, primers were designed and it was possible
to amplify DNA from A. sexdens cDNA that were cloned into the pPICZαA vector, to
proteins being expressed in Pichia pastoris. The enzymes, called AsChtII-C2B3,
AsChtII-C3C4 and AsChtII-C5B1, presented an optimum pH between 4-5 and
greater activities at 50 0C using a colloidal chitin as a substrate. Specific activities
evaluated showed that the presence of CBM does not interfere with the activity, the
AsChtII-C3C4, which does not contain a chitin binding site, having the highest
activity in colloidal chitin. The three enzymes were evaluated for fungicidal activity
using Candida albicans and Aspergilius fumigatus, human fungal pathogens, as
models. The enzyme AsChtII-C5B1 inhibited the growth of C.albicans by 87.6% (at
150 ug/mL) and AsChtII-C2B3 and AsChtII-C3C4 inhibited it by 61% and 54.5%,
respectively, at 50 ug/mL. AsChtII-C2B3 and AsChtII-C5B1 inhibited the growth of A.
fumigatus by 66% and 61% at 50 ug/mL. The enzyme AsChtII-C3C4, which does not
present CBM, inhibited the growth of the fungus by 60% at 25 μg/mL. Another
objective of the work was to characterize a previously obtained recombinant
chitinase, AsChtII-C4B1, against α-chitin and β-chitin, using a combination of
techniques such as 13C NMR, X-ray diffraction, viscosity analysis and SEM-FEG. α-
chitin is a constituent of the cell wall of fungi and the exoskeleton of crustaceans and
β-chitin is found in squid gladia. The results obtained showed that AsChtII-C4B1 is
capable of hydrolyzing α- and β-chitin, showing the biotechnological potential of the
enzyme, with its use as a fungicide, insecticide and in obtaining chitosan from crab
and shrimp shells being seen, for example. Thus, the interference of AsChtII-C4B1 in
the growth of the phytopathogenic fungus Lasiodiplodia theobromae was evaluated.
The delay in the development of mycelia with visual changes in the hyphae led to
analysis by scanning electron microscopy, SEM, of the same, which showed that
treatment with AsChtII -C4B1 caused important changes such as wrinkling, rounding
of the edges and holes on the surface of the hyphae. Together, the set of data
obtained from the different approaches of the present study showed that the
recombinant proteins obtained act on different types of chitin and have great
biotechnological potential.
Colecciones
El ítem tiene asociados los siguientes ficheros de licencia: