Submitted to: Biochemical and Biophysical Research Communications
Publication Type: Peer reviewed journal
Publication Acceptance Date: 6/20/2003
Publication Date: 6/27/2003
Citation: Ullah, A.H., Sethumadhavan, K., Mullaney, E.J., Ziegelhoffer, T., Austin-Phillips, S. 2003. Fungal phyA gene expressed in potato leaves produces active and stable phytase. Biochemical and Biophysical Research Communications. 306(2):603-609. Interpretive Summary: Phytic acid is present copiously in many legumes and cereals. It binds minerals and proteins present in the feedstuffs; thereby, it interferes with mineral availability to poultry and hogs. To breakdown phytic acid, the anti-nutrient in the feedstuffs, we have developed an enzyme-based technology. The fungally produced enzyme phytase was cloned and over-expressed. In this paper, we showed that the gene coding for phytase was cloned into the potato plant. The transformed potato plant produced stable and active enzyme in its leaf. We purified the cloned enzyme and then ran a series of experiments to characterize its properties. We showed that the cloned enzyme retained all the important biochemical properties of the original enzyme. Farmers in less developed countries now may produce a recombinant phytase by cropping the transformed potato plant. This method of producing microbial enzymes in plants is called "Bio-farming." Our research effort is ushering in this new field of "Bio-farming," which is at its infancy.
Technical Abstract: Fungal phyA gene from Aspergillus ficuum (niger) was cloned and expressed in potato leaves. The recombinant enzyme was stable and catalytically active. The expressed protein in the leaves of the dicotyledonous plant retained most of the physical and catalytic properties of the benchmark Aspergillus ficuum phytase. The expressed enzyme was however 15% less glycosylated than the native phytase. The usual bi-hump pH optima profile, which is characteristic of the fungal phytase, was altered; however, the pH optimum at 5.0 was unchanged for phytate and at 4.0 for synthetic substrate p-nitrophenyl phosphate. The temperature was, however, unchanged. The expressed phytase was found to be as sensitive as the native enzyme to the inhibitory action of pseudo substrate, myo-inositol hexasulfate while losing about 90% of the activity at 20mM inhibitor concentration. Similar to the benchmark phytase, the expressed phytase in leaves was completely inactivated by Arg modifier phenylglyoxal at 60nM. In addition, the expressed phytase in the leaves was inhibited by antibody raised against a 20-mer internal peptide, which is present on the surface of the molecule as shown by the X-ray deduced 3-D structure of fungal phytase. Taken together, the biochemical evidences indicate that fungal phytase when cloned and expressed in potato leaves produces a stable and active biocatalyst. 'Biofarming,' therefore, is an alternative way to produce functional hydrolytic enzymes as exemplified by the expression of Aspergillus ficuum (niger) phyA gene in potato leaf.