Produção e caracterização bioquímica de proteases de fungos isolados de amostras de solos do semiárido pernambucano

Abstract

Soils are habitat for a diverse range of macro and microorganisms that form a network of interrelationships capable of allowing and ensuring the maintenance of life in this place. Even with abiotic (climate, temperature, availability of nutrients and water) and biotic factors that interfere with the development of species, fungi are one of the groups of microorganisms most adaptable to environments like this, since these factors end up conferring resistance to these organisms, that use strategies such as the production of secondary metabolites to ensure their maintenance, an example of this are enzymes. Therefore, this work aimed to evaluate the ability of protease production by filamentous fungi (Aspergillus sp., Paecilomyces sp., Penicillium sp. and Cladosporium sp.), isolated from soil samples from the semi-arid region of Pernambuco, and to characterize the enzymatic extract with better relationship between enzymatic index (IE) and fungal biomass. Six isolates were obtained from the microbiological collection at LAPEEMI, and were submitted to macroscopic analysis, microscopic analysis, and their potential for proteolytic production in solid Milk-Agar medium. The production capacity was tested through three processes, being production in milk-agar medium (solid), which served as an initial screening for better peptidases producers. Only four isolates with better EI and tested under submerged production, in half fiber skimmed milk, with agitation of 150 rpm, 37°C and submerged production without agitation, in triplicate in both cultures, for 6 days, after this time the samples were centrifuged to obtain the enzymatic extract. As a result, it is observed that there is a significant difference between the cultivation method with and without agitation, for biomass production and enzymatic production respectively for Paecilomyces sp. (0.368 and 0.602g and 80.3 and 302 U/mL), Penicillium sp. (1.756 and 2.913g; 75.7 and 177 U/mL), Aspergillus sp. (2.852 and 3.759; 91 and 292.7 U/ml). The latter produced more biomass and the same enzymatic level as Paecilomyces sp. which had lower production of biomass in stationary fermentation. So this isolate was selected for the biochemical characterization tests of the enzymes. The enzymatic extract of Paecilomyces sp. was tested for peptidases classes, optimal temperature, thermostability and pH-optimal. Inhibitors for four classes of proteases were used: serine protease, aspartate protease, metalloprotease, cysteine proteases, of which it was observed that only 20.3% of the proteases were inhibited, 10% of which were serine protease, and 10.3% in aspartate protease, demonstrating that most of the enzymes present do not belong to these classes. The enzymatic activity was analyzed at different temperatures (27 to 97 °C) with an increment of 5°C at each new temperature, demonstrating greater activity at 47°C. As for thermostability, proteases from the crude extract, after being incubated at temperatures (27°C; 37°C; 47°C to 97°C), also with increments of 5°C, demonstrated greater stability in the temperature ranges between 27 °C and 47°C. The enzymatic extract showed two peaks of proteolytic activity at different pH, one at alkaline pH (pH 8.0) and the second peak, this higher one, at acidic pH (pH 6.0). In view of the results obtained, it is concluded that the fungi studied were capable of producing proteases by submerged fermentation in both methodologies using skimmed milk as substrate, with emphasis on stationary cultivation, that fiber skimmed milk medium can be considered a good alternative medium for production of these enzymes and that the extract of proteases from Paecilomyces sp. presents desired characteristics for industrial proteases.