Produção, purificação e caracterização bioquímica de uma amilase produzida pelo fungo termofílico Rasamsonia composticola

Abstract

Amylases or amylolytic complex enzymes are part of a class of enzymes responsible for hydrolyzing glycosidic bonds in starch, and can be classified as: α-amylase, β-amylase and glucoamylase. These enzymes are used in various industrial sectors, with emphasis on the food industry. Filamentous fungi stand out in the production of amylases, using agro-industrial substrates as a carbon source. This work aimed to optimize the production, purification and biochemical characterization of an amylase produced by the thermophilic fungus Rasamsonia composticola. For the production of amylase, cultivation was carried out in semi-solid state fermentation (FES) using low-cost agro-industrial residues. Parameters such as carbon sources, mixtures of carbon sources, nitrogen and the effect of temperature on growth, enzyme production and cultivation time were also evaluated. Amylase purification was carried out in two chromatographic steps: ion exchange (DEAE-Fractogel) followed by hydrophobic chromatography (Phenyl-Sepharose). The enzyme was characterized biochemically, regarding the effect of pH, temperature, ions and substrates. Among the waste used, the mixture of passion fruit peel and extruded orange peel in FES (1:1) was better (3.87 U/mL for the production of amylase). The best nitrogen source for amylase production was peptone (1%). The most suitable temperature and cultivation time for the production of amylases were 40ºC and 96 hours. Amylase was purified 18 times with a yield of 69%. The optimum pH and temperature were 4.6 and 55ºC, respectively. The enzyme hydrolyzed starch (100%) and xylan (59%). It was activated in the presence of CaCl2 (264%) and inhibited by HgCl2, CuSO4 and EDTA. We suggest that it is a glucoamylase (E.C. 3.2.1.3.) with the characteristics obtained. The thermophilic fungus Rasamsonia composticola produced amylase from low-cost agro-industrial waste, and purification and characterization demonstrate the enzyme's potential for future biotechnological use