Proteases e quitinases de fungos anamórficos de insetos da Amazônia com uso potencial em biocontrole

Abstract

Hydrolytic enzymes are used in many areas of industry and biotechnology, for example of hydrolytic enzymes we can nominate the proteases and chitinases. These enzymes are secreted by fungi that cause diseases in other organisms, and they represente an important virulence factor for them. The aim of this work was to isolate fungi from lace bug (Vatiga illudens) and mealybug (Orthezia sp.), evaluating the proteolytic potential of the extracts originated from isolates, and to determine the optimal temperature and pH of the enzyme, as well as to perform partial purification of proteases and chitinases produced by the fungi. For fungi isolation, insects were placed in a moist chamber at 25 °C, followed by the inoculum in Petri dishes containing Sabouraund agar. After that, the fungi were underwent to submerged cultivation with MGYP medium. The crude extract was recovered by vacuum filtration and inoculated into cup-plate, in casein solid medium 10% (w/v) at 37 °C. The effect of temperature and pH on the proteolytic activity was determined using azocasein 1% (w/v) as substrate at different pH buffers (5-10) and temperatures (25 °C-60 °C). Five fungal isolates were selected from the obtained from V. illudens and Orthezia sp. The selected extracts were subjected to partial protein purification by ammonium sulfate fractionation in fractions 0-60% and 60-80% saturation. After dialysis, protein concentration, proteolytic and chitinolytic activity, specific activity, yield and purification factor of each of the fractions and crude extracts were determined. From V. illudens the identified fungi were: Paecilomyces sp. and P. javanicus. In Orthezia sp was found the greater diversity of fungi, represented by Fusarium decemcellulare, Penicillium aurantiogriseum, P. brevicompactum, P. fellutanum, P. glabrum, P. solitum and the ascomycetes Eupenicillium javanicum. The isolates formed halos of 10-19 mm diameter in solid medium and the crude extract expressed proteolytic activity ranging from 1.40 to 27.07 U/mL. The enzymatic activity of temperature and pH optima varied among isolates. In the F2 fraction (60-80%) were recorded the highest specific activities of proteases and chitinases. The semi-purified extracts of P. brevicompactum (C3) and P. fellutanum (C1) showed the highest specific activities of protease (18.72 U / mg) and chitinase (0.88 mg / mg), respectively