Biochemical characterization of cloned Aspergillus fumigatus phytase (phyA)

The gene for Aspergillus fumigatus phytase (phyA) was cloned and expressed in Pichia pastoris. The enzyme expressed was purified to near homogeneity u sing sequential ion-exchange chromatography and was characterized biochemic ally. Although A. fumigatus phytase shows 66.2% sequence homology with A. f icuum phytase, the most widely studied enzyme, the cloned phytase showed id entical molecular weight and temperature optima profile to the benchmark ph ytase, The pH profile of activity and kinetic parameters, however, differed from A. ficuum phytase. The cloned enzyme contains the septapeptide RHGARY P motif, which is also identical to the active site motif of A. ficuum phyt ase. Chemical probing of the active site Arg residues using both cyclohexan edione and phenylglyoxal resulted in the inactivation of phytase, The clone d A fumigatus phytase, however, was more resistant to phenylglyoxal-induced inactivation. Both cloned A. fumigatus and A. ficuum phytases were identic ally affected by cyclohexanedione. Both the thermal characterization data a nd kinetic parameters of cloned and expressed A. fumigatus phytase indicate that this biocatalyst is not superior to the benchmark enzyme. The sequenc e difference between A. fumigatus and A. ficuum phytase may explain why the former enzyme catalyzes poorly compared to the benchmark enzyme. In additi on, differential sensitivity toward the Arg modifier, phenylglyoxal, indica tes a different chemical environment at the active site for each of the phy tases. (C) 2000 Academic Press.