“PRODUCTION OF COLLAGENOLYTIC PROTEASES FROM Aspergillus heteromorphus URM 0269 AND EXTRACTION BY AQUEOUS TWO-PHASE SYSTEMS FOR APPLICATION IN COLLAGEN HYDROLYSIS

 The increase in consumer demand regarding the ingredients and beneficial properties of foods, supplements and cosmetics has led different industrial segments to seek the inclusion of functional ingredients in their formulations, in order to make their products more attractive to the population. In this way, collagen peptides emerge as a potential agent capable of assisting in the prevention and treatment of chronic diseases such as diabetes, hypertension, cancer, among other inflammatory and autoimmune diseases. However, the steps used and the methods necessary for the production of collagen peptides make the process more expensive, which limits its application and consumption. Therefore, the search for alternatives that may make one or more stages of the production of these peptides cheaper is constantly encouraged, leading to the investigation of the use of filamentous fungi for the production of proteases, of simpler means of extraction and purification of these enzymes and of the potential of fish waste as sources of collagen. In this context, this work aimed to produce and purify collagenolytic proteases from Aspergillus heteromorphus URM 0269 using solid state fermentation (FES) for application in the production of collagen peptides. For this, the microorganism was cultivated under predetermined fermentation conditions (3g of wheat bran, 20% humidity, 30°C, 96 hours of fermentation) to obtain the crude enzymatic extract. The crude extract was then submitted to a factorial design (2³) for the purification of collagenolytic proteases using the two-phase aqueous system (ATPS), having as independent variables: PEG molar mass (M_PEG), PEG concentration (C_PEG) and of sulfate (C_sulf). Subsequently, the proteases extracted by PEG/sulfate SDFA were evaluated for their stability at different pH levels (4.0 – 11.0). The enzyme was partitioned preferentially to the PEG-rich phase whose highest purification and recovery factor (PF = 6.256 and Y= 404.432%) was obtained using M_PEG 8000 g/mol, C_PEG 30%, C_sulf 10%. The evaluation of the effect of pH on the enzymatic activity revealed that the extraction in SDFA was able to increase the pH range with high enzymatic activity (7.0 – 11.0) compared to that observed in the crude extract (6.0 – 7.0). Furthermore, the enzymatic samples of SDFA were more stable in relation to the crude extract, maintaining at least 80% of their protease activity after 20 hours of incubation for all pH levels analyzed, except for pH 11.0. These results demonstrate that SDFA, a simple, fast and economical extraction technique, benefits the activity and stability of proteases produced by Aspergillus heteromorphus URM0269, essential parameters for increasing the shelf life and feasibility of using these enzymes in the production of collagen peptides. .