Invertase Intracelular de Aspergillus Aculeatus OI4C2 induzida com farelo de soja: purificação, caracterização bioquímica e produção de Frutooligossacarídeos

Abstract

Agriculture in Brazil is one of the main bases of the country's economy, and as a consequence large amounts of waste and by-products are also generated per day, a worrying factor, as these wastes can have serious environmental impacts. However, these residues can be useful as nutrients or carbon sources for fungal growth and induce the production of enzymes applicable in biotechnology, such as invertase. The β-fructofuranosidase or invertase is an enzyme that hydrolyzes the β (1-2) bond of sucrose, producing invert sugar, at high concentrations of sucrose it can still have transfutosylation activity and produce fructooligosaccharides (FOS). And these oligosaccharides have commercial importance mainly in the food and pharmaceutical industries. In this context, this study aimed to carry out a study on invertases produced by fungi using agro-industrial residues or by-products as alternative sources of carbon, as well as to purify and biochemically characterize the intracellular invertase produced by the filamentous fungus Aspergillus aculeatus OI4C2. The cultivation of A. aculeatus was carried out under stationary liquid culture conditions for 5 days using soybean meal as a carbon source. The intracellular crude extract after dialysis was used for enzyme purification on DEAE-Sepharose and Sephadex G-75 molecular exclusion column. After all purification processes, the final enzyme yield was 26.8%, specific activity 14,191 U/mg, and estimated molecular mass of 45 kDa. The optimal pH for invertase activity was 5.5 with stability in the range of 4.5 to 7.5; the optimum temperature of 60ºC, and thermal stability at 50, 55, 60 and 65ºC for up to 120 minutes, except at 70ºC with a half-life of 25 minutes. This invertase with fructosyltransferase activity produced FOS from sucrose with predominance of kestose (48 and 49%) in 15 minutes, in 1h of reaction (37% and 55%), and nystose (102.05 g/L) in 24 hours, The enzyme was tolerant to high concentrations of glucose, without affecting the formation of FOS. Therefore, A. aculeatus OI4C2 invertase presents potential for FOS synthesis from sucrose, mainly forming kestose and nystose, a very promising characteristic for industrial application.