Expression, Gene Cloning, and Characterization of Five Novel Phytases from Four Basidiomycete Fungi: Peniophora lycii, Agrocybe pediades , a Ceriporia sp., and Trametes pubescens

Abstract

ABSTRACT Phytases catalyze the hydrolysis of phosphomonoester bonds of phytate ( myo -inositol hexakisphosphate), thereby creating lower forms of myo -inositol phosphates and inorganic phosphate. In this study, cDNA expression libraries were constructed from four basidiomycete fungi ( Peniophora lycii, Agrocybe pediades , a Ceriporia sp., and Trametes pubescens ) and screened for phytase activity in yeast. One full-length phytase-encoding cDNA was isolated from each library, except for the Ceriporia sp. library where two different phytase-encoding cDNAs were found. All five phytases were expressed in Aspergillus oryzae , purified, and characterized. The phytases revealed temperature optima between 40 and 60°C and pH optima at 5.0 to 6.0, except for the P. lycii phytase, which has a pH optimum at 4.0 to 5.0. They exhibited specific activities in the range of 400 to 1,200 U · mg, of protein −1 and were capable of hydrolyzing phytate down to myo -inositol monophosphate. Surprisingly, 1 H nuclear magnetic resonance analysis of the hydrolysis of phytate by all five basidiomycete phytases showed a preference for initial attack at the 6-phosphate group of phytic acid, a characteristic that was believed so far not to be seen with fungal phytases. Accordingly, the basidiomycete phytases described here should be grouped as 6-phytases (EC 3.1.3.26 ).