Biochemical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolases): Catalytic properties

Supplementation with phytase is an effective way to increase the availabili ty of phosphorus in seed-based animal feed. The biochemical characteristics of an ideal phytase for this application are still largely unknown. To ext end the biochemical characterization of wild-type phytases, the catalytic p roperties of a series of fungal phytases, as well as Escherichia coli phyta se, were determined. The specific activities of the fungal phytases at 37 d egrees C ranged from 23 to 196 U . (mg of protein)(-1), and the pH optima r anged from 2.5 to 7.0. When excess phytase was used, all of the phytases we re able to release fire phosphate groups of phytic acid (myo-inositol hexak isphosphate), which left myo-inositol 2-monophosphate as the end produc. A combination consisting of a phytase and Aspergillus niger pH 2.5 acid phosp hatase was able to liberate all six phosphate groups. When substrate specif icity was examined, the A. niger, Aspergillus terreus, and E. coli phytases were rather specific for phytic acid. On the other hand, the Aspergillus f umigatus, Emericella nidulans, and Mycleiophthora thermophila phytases exhi bited considerable activity with a broad range of phosphate compounds, incl uding phenyl phosphate, p-nitrophenyl phosphate, sugar phosphates, alpha- a nd beta-glycerophosphates, phosphoenolpyruvate, 3-phosphoglycerate, ADP, an d ATP. Both phosphate liberation kinetics and a time course experiment in w hich high-performance liquid chromatography separation of the degradation i ntermediates was used showed that all of the myo-inositol phosphates from t he hexakisphosphate to the bisphosphate were efficiently cleaved by A. fumi gatus phytase, In contrast, phosphate liberation by A. niger or A. terreus phytase decreased with incubation time, and the myo-inositol tris- and bisp hosphates accumulated, suggesting that these compounds are worse substrates than phytic acid is. To test whether broad substrate specificity may be ad vantageous for feed application, phosphate liberation kinetics were studied in vitro by using feed suspensions supplemented with 250 or 500 U of eithe r A. fumigatus phytase or A. niger phytase (Natuphos) per kg of feed. Initi ally, phosphate liberation was linear and identical for the two phytases, b ut considerably more phosphate was liberated by the A. fumigatus phytase th an by the A. niger phytase at later stages of incubation.