Resumo

Microbial enzymes, mainly from filamentous fungi, have attracted the industrial interest for different purposes as, for example, β-fructofuranosidases (EC 3.2.1.26), which are mainly applied in food industry. This enzyme hydrolyzes the sucrose molecule producing an equimolar mixture of D-fructose and D-glucose known as invert sugar. Our objective was to optimize the production of extracellular β-fructofuranosidase by A. parasiticus as well as their purification and biochemical characterization. The optimization was performed using factorial design with analysis of variance. A Central Composite Rotational Design (CCRD) for three independent variables (temperature, sugarcane bagasse concentration and time of culture) selected by a Plackett-Burman was performed. The cultures were harvested under vacuum, resulting in cell-free filtrate (extracellular crude extract). The enzyme was purified by two chromatographic steps, DEAE-cellulose and Sephacryl S-200. The enzyme activity was determined using 1% sucrose as substrate in 100 mM sodium cetate 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. The culture conditions optimized were temperature from 27-33°C, sugarcane bagasse concentration from 1.5-2.5%, and time of culture of 72-120 h. The extracellular β-fructofuranosidase was purified 119-fold with recuperation of 16%. The native molecular mass was 131 kDa with two subunits of 59 kDa (7% SDS-PAGE). The purified enzyme showed optimal temperature of activity from 38-56ºC and optimum of pH from 4.5 to 6.2, determined by CCRD. The enzyme incubated at 50ºC showed a t₅₀ of 8 min and was stable between pH 5.0 and 10.0 for 60 min. The β-fructofuranosidase activity was increased by addition Mg²⁺, Ba²⁺ and Na⁺. Addition of Ag²⁺, β-mercaptoethanol and urea, does not reduce the enzymatic activity. The K_m and V_max values were 10 mM and 1565 U/mg of protein for sucrose, and 19 mM and 1965 U/mg of protein for raffinose, respectively. In conclusion, A. parasiticus was a good producer of extracellular β-fructofuranosidase using an industrial residue as carbon source and the biochemical characteristics presented make the enzyme interesting for the future applications.