Characterization of a HAP–phytase from a thermophilic mould Sporotrichum thermophile

Abstract

The phytase of Sporotrichum thermophile was purified to homogeneity using acetone precipitation followed by ion-exchange and gel-filtration column chromatography. The purified phytase is a homopentamer with a molecular mass of ∼456 kDa and p I of 4.9. It is a glycoprotein with about 14% carbohydrate, and optimally active at pH 5.0 and 60 °C with a T 1/2 of 16 h at 60 °C and 1.5 h at 80 °C. The activation energy of the enzyme reaction is 48.6 KJ mol −1 with a temperature quotient of 1.66, and it displayed broad substrate specificity. Mg 2+ exhibited a slight stimulatory effect on the enzyme activity, while it was markedly inhibited by 2,3-butanedione suggesting a possible role of arginine in its catalysis. The chaotropic agents such as guanidinium hydrochloride, urea and potassium iodide strongly inhibited phytase activity. Inorganic phosphate inhibited enzyme activity beyond 3 mM. The maximum hydrolysis rate ( V max) and apparent Michaelis–Menten constant ( K m) for sodium phytate were 83 nmol mg −1 s −1 and 0.156 mM, respectively. The catalytic turnover number ( K cat) and catalytic efficiency ( K cat/ K m) of phytase were 37.8 s −1 and 2.4 × 10 5 M −1 s −1, respectively. Based on the N-terminal and MALDI–LC–MS/MS identified amino acid sequences of the peptides, the enzyme did not show a significant homology with the known phytases.