Cloned and expressed fungal phyA gene in alfalfa produces a stable phytase.
The phyA gene from Aspergillus ficuum that codes for a 441-amino-acid full-length phosphomonoesterase (phytase) was cloned and expressed in Medicago sativa (alfalfa) leaves. The expressed enzyme from alfalfa leaves was purified to homogeneity and biochemically characterized, and its catalytic properties were elucidated. The expressed phytase in alfalfa leaves retained all the biochemical properties of the benchmark A. ficuum phytase. Although the characteristic bi-hump pH optima were retained in the cloned phytase, the optimal pH shifted downward from 5.5 to 5.0. Also, the recombinant phytase was inhibited by the pseudo-substrate myo-inositol hexasulfate and also by antibody raised against a 20-mer peptide belonging to fungal phytase. The expressed phytase in alfalfa could also be modified by phenylglyoxal. Taken together, the results indicate that fungal phytase when cloned and expressed in alfalfa leaves produces stable and catalytically active phytase while retaining all the properties of the benchmark phytase. This affirms our view that "molecular biofarming" could be an alternative means of producing stable hydrolytic enzymes such as phytase.
Ullah
Abul H J
AH
Southern Regional Research Center, ARS, USDA, New Orleans, Louisiana 70124, USA.
Sethumadhavan
Kandan
K
Mullaney
Edward J
EJ
Ziegelhoffer
Thomas
T
Austin-Phillips
Sandra
S
D010832:6-Phytase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D015870:Gene Expression; D005800:Genes, Fungal; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007294:Inositol / Q000031:analogs & derivatives* / Q000494:pharmacology; D007700:Kinetics; D000455:Medicago sativa / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010658:Phenylglyoxal / Q000494:pharmacology; D030821:Plants, Genetically Modified; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
D000906:Antibodies; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; C013063:inositol hexasulfate; D007294:Inositol; D010832:6-Phytase; D010658:Phenylglyoxal