Purificação e caracterização bioquímica de uma quitinase de Parkia multijuga com ação antifúngica

Abstract

The fungi stand out because they cause great losses in the main agricultural crops and trigger diseases in animals and humans. Big investments are focused on drug development, but many fungi develop resistance to treatments. Thus, the search for new antifungal molecules has become incessant. The present work aimed to prospect proteins from Parkia multijuga seeds with antimicrobial action, which could be used as alternatives to control fungi of importance for agriculture and human health. Initially, the 0.1 M glycine buffer, pH 9.0, with 0.15 M NaCl was established as the best protein extraction buffer. Then, the protein extract was fractionated using different methods and solvents to obtain the albumin, globulin, prolamin and acid and basic glutelin fractions. The globulin fraction was active against Fusarium oxysporum, Fusarium solani, Botrytis cinerea, Candida albicans, Candida krusei, Candida parapsilosis and Candida tropicalis. In view of these results, we proceeded with the characterization of bioactive proteins present in the globulin fraction, being detected the chitinase and beta-1,3-glucanase activities, proteins toxic to fungi. As a further step, the globulin fraction was subjected to ion exchange chromatography on a DEAE-cellulose matrix. Proteins eluted with 0.1 M glycine buffer, pH 9.0, with 0.4 M NaCl, presented a protein band on SDS-PAGE and increased chitinase activity by 44-fold when compared to the activity found in the globulin fraction. The purified chitinase had an apparent molecular weight of 65 kDa, showed a pH optimum of 7.0 and optimum temperature of activity at 45 °C. Chitinase showed activity against the fungi Glomerella cingulata and Penicillium herquei, which showed increased cellular permeability. With this, Pm-Chi was shown to have biotechnological potential to be used as an alternative for agricultural pest control.