Poultry farming is one of the sectors of great impact on the Brazilian economy. In recent years, there has been an increase in the production of broiler chicken, making this sector of the industry responsible for the generation of tons of feathers, which have polluting potential when improperly disposed of in the environment. The disposal of feathers can become an economic problem for companies, which must invest in their correct destination to avoid possible environmental impacts. Given this, there is a need for sustainable alternatives for recycling these protein rich waste. The objective of this study was to investigate the production of proteases secreted by filamentous fungus, isolated from the soil of the Amazon Forest in Roraima, using chicken feathers as the only source of energy, to characterize the strain as to the optimum temperature and pH of production, as well as the effects chemical products, in addition to evaluating the effect of different growth substrates on proteolytic activity. Initially, a screening of 40 strains of filamentous fungi obtained from soil samples from Parna Viruá (preserved in the Culture Collection of the Microbiology Laboratory-PRONAT) was carried out, in order to select an isolate with the potential for the production of extracellular protease. The identification of the selected fungus was carried out through macroscopic and microscopic observations, inoculating the isolate in specific culture media, which was identified as Aspergillus sp. The fungus was inoculated on milk agar and feather meal agar to verify the qualitative production of proteases and keratinases, respectively. The formation of halo indicated positivity for both enzyme productions. As for the quantitative assessment of proteolytic and keratinolytic activities, obtained through spectrophotometry at 420 nm, the results indicated the ability of Aspergillus sp. in producing both proteases and keratinases. The ability to secrete proteases was evaluated on different growth substrates (whole feather, feather meal, peptone, casein, chicken beak, hair and gelatin). The highest production of enzymes occurred in culture media with peptone and feather meal, indicating that Aspergillus sp. is a keratinolytic microorganism, capable of efficiently degrading keratinous materials. The protease showed optimal activity at pH 5.0 and temperature of 37 ° C. The enzymatic activity was enhanced with the addition of CaCl2, MnSO4, KCl, MgSO4 and CuSO4. The detergents Tween 20 and Triton x-100 tended to stimulate activity. The enzymes were resistant to organic solvents (methanol, acetone, butanol, acetonitrile, isoprapanol and DMSO), keeping the enzymatic activity close to the control (100%). The β-mercaptoethanol inhibitors and ethylenediaminetetraacetium acid (EDTA) did not inhibit proteolytic activity in enzymatic assays, suggesting that the enzymes present in the enzyme hydrolyzate are neither metalloproteases nor cysteine proteases. From the perspective of industrial microbiology, the results of this research suggest that the crude protease extract can potentially be used in the bioconversion of keratinous residues, considered difficult to break down in the environment, and the possibility of applying protein hydrolyzate in animal feed supplementation in use as biofertilizers.
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