Imobilização de proteases colagenolíticas obtidas de Aspergillus sclerotiorum URM 5792 em nanopartículas magnéticas revestidas com quitosana

Abstract

Proteases are enzymes that have been used extensively in several industry segments. Despite their high interest and numerous advantages, their application still has some limitations. The immobilization process has become an alternative since it increases enzyme stability. The use of magnetic supports has been widely explored, due to its versatility in the biotechnology sector for the purification of biomolecules, and its coating with organic supports such as chitosan, facilitates the process, preventing the oxidation of magnetic nanoparticles (MNP's), giving ideal properties for immobilization. The present work aimed to select, immobilize and characterize collagenolytic proteases obtained from Aspergillus sclerotiorum URM5792 in MNP’s coated with chitosan. For production, a complete factorial planning was carried out 22; with the best production condition (7g of wheat bran and 60% humidity, submitted to 30°C for 72h of fermentation, with proteolytic activity of 56,27 U/mL and e collagenolytic of 303,00 U/mL). The immobilization process was carried out using complete factorial planning 23 aiming to evaluate the influence of independent variables: glutaraldehyde concentration, activation time and immobilization time under the enzyme immobilization yield. In the assay composed of 4% glutaraldehyde concentration, 2.5h of activation and 1.5h of immobilization yields of 86.25% for protein activity and 83.83% for collagenolytic activity were obtained. The immobilized enzyme showed more than 95% of the initial activity after 28 days of storage and retained more than 60% of the residual activity in the twelfth cycle of reuse. The influence of pH and temperature on enzyme activity in free and immobilized form were also analyzed, both had an optimal pH in the range of 9.0, as well as an optimal temperature of 30 ° C and 40 ° C, respectively. The pH and temperature stability of the free enzyme was maintained with more than 80% and 60% of residual activity within 24 hours and 180 minutes, respectively. And the stability of the immobilized enzyme was maintained with 60% and 70% of residual activity in the same times. The solid-state fermentation was effective with high enzymatic production and the immobilization of protease collagenolytic in magnetic nanoparticles coated with chitosan and activated in glutaraldehyde, proved to be efficient methods in the yield, storage and reuse of the enzyme. The immobilized enzyme showed greater affinity to the substrate in relation to the free enzyme, it was inhibited by the Cu2+ ion, SDS and PMSF indicating the presence of active serine protease sites. These results indicate that Aspergillus sclerotiorum URM 5792 is a potential source for production of collagenolytic protease with possible biotechnological applications in various sectors of the industry, in the production of detergents, in the textile and pharmaceutical industry, in the treatment and regeneration of tissues in necrosis.