Resumo

Many enzymes with biotechnological interest are produced by filamentous fungi as, for example, β-fructofuranosidases (EC 3.2.1.26) that are able to hydrolyze the sucrose molecule producing an equimolar mixture of D-fructose and D-glucose known as invert sugar. Hence, our aim was to investigate the production of intracellular β-fructofuranosidase by A. parasiticus, as well as to determine some of its biochemical properties. The submerged fermentation (SbmF) was conducted in different culture media (Khanna, Adams, SR, Czapeck, M5 and M6), added with different carbon sources and maintained at 30°C and 100 rpm for different periods (24 to 120 h). The cultures were harvested under vacuum, resulting in a mycelium that was dried using filter paper, macerated, suspended in distilled water and centrifuged (12000g). The supernatant (crude extract intracellular) obtained was dialyzed against distilled water overnight at 4°C. The enzyme activity was determined using 1% sucrose as substrate in 100 mM sodium acetate buffer, pH 5.5 and the reducing sugars released were quantified using DNS, and protein using the Folin reagent. One unit of enzyme activity was defined as the quantity of enzyme that releases 1 μmol of glucose per minute under assay conditions. High enzymatic levels were obtained with Khanna medium containing sucrose (2% w/V) as carbon source for 72 h under orbital agitation at 30ºC (51.2 U/mg), but interesting results were also observed when industrial residues as sugarcane bagasse (59 U/mg) were used as carbon source for 24 h under orbital agitation at 30ºC. Addition of 0.25% (w/v) of glucose and fructose in culture medium reduced 30% the enzyme production, while the addition of 1% (w/v) sodium phosphate, potassium phosphate and ammonium phosphate increased at least 125%. The A. parasiticus β-fructofuranosidase has optimum temperature of activity at a range from 40-56ºC and optimum pH from 4.8-6.1, determined by factorial experiment (CCRD). When the enzyme was incubated at 50ºC showed a t₅₀ of 28 min and it was stable between pH 5.0 and 10.0 for 60 min. β-fructofuranosidase activity was increased Na⁺ and NH₃⁺. Addition of Ag²⁺, β-mercaptoethanol and urea, does not reduce the enzymatic activity. In conclusion, A. parasiticus intracellular β-fructofuranosidase showed interesting characteristics for future applications.