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

Abstract

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 K M for so- dium phytate hydrolysis was 30.9 mM, while the k cat and k cat /K M were 1.46 x10^ 5 s^ -1 and 4.7 x 10^ 6 s^ -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- naphthyl- phosphate, ATP, phenyl-phosphate, glucose-6-phosphate, calcium phytate and other substrates. Enzymatic activity was slightly inhibited by Mg^ 2+ , Cd^ 2+ , K^ + and Ca^ 2+ , 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 t 1/2 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 live- stock feed, liberating 15.3 mmol phosphate/mL after 2.5 h of treatment.