Isolation of a thermostable acid phytase from Aspergillus niger UFV-1 with strong proteolysis resistance

An Aspergillus niger UFV-1 phytase was characterized and made available for industrial application. The enzyme was purified via ultrafiltration followed by acid precipitation, ion exchange and gel filtration chromatography. This protein exhibited a molecular mass of 161 kDa in gel filtration and 81 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), indicating that it may be a dimer. It presented an optimum temperature of 60 °C and optimum pH of 2.0. The KM for sodium phytate hydrolysis was 30.9 mM, while the kcat and kcat/KM were 1.46 ×105 s−1 and 4.7 × 106s−1.M−1, respectively. The purified phytase exhibited broad specificity on a range of phosphorylated compounds, presenting activity on sodium phytate, p-NPP, 2- naphthylphosphate, 1- naphthylphosphate, ATP, phenyl-phosphate, glucose-6-phosphate, calcium phytate and other substrates. Enzymatic activity was slightly inhibited by Mg2+, Cd2+, K+ and Ca2+, and it was drastically inhibited by F−. The enzyme displayed high thermostability, retaining more than 90% activity at 60 °C during 120 h and displayed a t1/2 of 94.5 h and 6.2 h at 70 °C and 80 °C, respectively. The enzyme demonstrated strong resistance toward pepsin and trypsin, and it retained more than 90% residual activity for both enzymes after 1 h treatment. Additionally, the enzyme efficiently hydrolyzed phytate in livestock feed, liberating 15.3 μmol phosphate/mL after 2.5 h of treatment

Development of a method to quantify sucrose in soybean grains

The sucrose content of soybean seeds affects the final flavor of soy-derived products. The aim of this work was to develop a simple, low-cost, spectrophotometric method for sucrose quantification in soybean seeds. To achieve this goal, we combined the action of invertase, an enzyme that hydrolyses sucrose into fructose and glucose, with glucose oxidase, an enzyme widely used for glucose quantification. This system was adapted to ELISA plates, making large-scale analyses possible at low cost, with potential application in routine analyses. To validate this method, sucrose content was determined in seeds of 14 soybean cultivars by this new method, as well as by HPLC and the enzymatic method of Stitt. The correlation coefficients were high and significant between the results obtained with the new method and the HPLC method (r = 0.9766) and the Stiff method (0.9461)