Produção de colagenase por Rhizopus microsporus UCP 1296 e sua aplicação na obtenção de peptídeos de colágeno

Abstract

The search for new enzymes is a constant challenge, mainly due to the need to develop more sustainable and financially viable production conditions. With the advance of biotechnology, researches aiming the discovery of microbial enzymes have been developed mainly due to the favorable production conditions in relation to other organisms. Enzymes that have high specificity and can be used in small quantities are extremely interesting from a biotechnological point of view. Filamentous fungi have stood out in the production of enzymes of industrial interest, especially collagenases. In this context, the filamentous fungus Rhizopus microsporus (UCP1296) isolated from Caatinga soil was selected for collagenase production. In this work the submerged fermentation system (FS) was chosen to obtain the crude extract and the two aqueous phase system (SDFA) as a purification strategy. The gelatin culture medium was used for fermentation and with 120h the collagenolytic activity obtained was 550 U/mL, biomass of 0.42 g/L and specific collagenolytic activity of 808.23 U/mg. A factorial planning was applied and an increase of 32% was recorded in the enzymatic production, being equivalent to 727.50 U/mL of collagenolytic activity. The partial purification by SDFA was effective with the highest values of yield and partition coefficient obtained with PEG 8000 g/mol at 12.5% (w/w) concentration, pH= 8 and phosphate concentration at 10.0% (w/w). The parameters, optimum pH and temperature and influence of inhibitors were determined for the characterization of the purified enzyme. In this context, the optimum pH and temperature were 8.0 and 37 °C. The enzyme showed partial inhibition to ethylenediaminetetraacetic acid (EDTA) and iodoacetic acid (IAA), thus, it may have portions of metallo and cysteine proteases. The results suggest that the enzyme produced presents itself as a promising biotechnological product with applicability in several areas.