Markus Wyss1, Roland Brugger1, Alexandra Kronenberger1, Roland Rémy1, Rachel Fimbel1, Gottfried Oesterhelt2, Martin Lehmann1, Adolphus P. G. M. van Loon1
1VFB Department1 and
2PRPI-S Department,2 F. Hoffmann-La Roche Ltd., 4070 Basel, Switzerland
Tóm tắt
ABSTRACT
Supplementation with phytase is an effective way to increase the availability of phosphorus in seed-based animal feed. The biochemical characteristics of an ideal phytase for this application are still largely unknown. To extend the biochemical characterization of wild-type phytases, the catalytic properties of a series of fungal phytases, as well as
Escherichia coli
phytase, were determined. The specific activities of the fungal phytases at 37°C ranged from 23 to 196 U · (mg of protein)
−1
, and the pH optima ranged from 2.5 to 7.0. When excess phytase was used, all of the phytases were able to release five phosphate groups of phytic acid (
myo
-inositol hexakisphosphate), which left
myo
-inositol 2-monophosphate as the end product. A combination consisting of a phytase and
Aspergillus niger
pH 2.5 acid phosphatase was able to liberate all six phosphate groups. When substrate specificity was examined, the
A. niger
,
Aspergillus terreus
, and
E. coli
phytases were rather specific for phytic acid. On the other hand, the
Aspergillus fumigatus
,
Emericella nidulans
, and
Myceliophthora thermophila
phytases exhibited considerable activity with a broad range of phosphate compounds, including phenyl phosphate,
p
-nitrophenyl phosphate, sugar phosphates, α- and β-glycerophosphates, phosphoenolpyruvate, 3-phosphoglycerate, ADP, and ATP. Both phosphate liberation kinetics and a time course experiment in which high-performance liquid chromatography separation of the degradation intermediates was used showed that all of the
myo
-inositol phosphates from the hexakisphosphate to the bisphosphate were efficiently cleaved by
A. fumigatus
phytase. In contrast, phosphate liberation by
A. niger
or
A. terreus
phytase decreased with incubation time, and the
myo
-inositol tris- and bisphosphates accumulated, suggesting that these compounds are worse substrates than phytic acid is. To test whether broad substrate specificity may be advantageous for feed application, phosphate liberation kinetics were studied in vitro by using feed suspensions supplemented with 250 or 500 U of either
A. fumigatus
phytase or
A. niger
phytase (Natuphos) per kg of feed. Initially, phosphate liberation was linear and identical for the two phytases, but considerably more phosphate was liberated by the
A. fumigatus
phytase than by the
A. niger
phytase at later stages of incubation.
