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PLANT PHYSIOLOGY , Vol 114, Issue 3 1103-1111, Copyright © 1997 by American Society of Plant Biologists
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GENE REGULATION AND MOLECULAR GENETICS |
Secretion of Active Recombinant Phytase from Soybean Cell-Suspension Cultures
J. Li, C. E. Hegeman, R. W. Hanlon, G. H. Lacy, D. M. Denbow and E. A. Grabau
Department of Plant Pathology, Physiology and Weed Science (J.L., C.E.H., R.W.H., G.H.L., E.A.G.), and Department of Animal and Poultry Sciences (D.M.D.), Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061-0346
Phytase, an enzyme that degrades the phosphorus storage compound phytate,
has the potential to enhance phosphorus availability in animal diets when
engineered into soybean (Glycine max) seeds. The phytase gene from
Aspergillus niger was inserted into soybean transformation plasmids under
control of constitutive and seed-specific promoters, with and without a
plant signal sequence. Suspension cultures were used to confirm phytase
expression in soybean cells. Phytase mRNA was observed in cultures
containing constitutively expressed constructs. Phytase activity was
detected in the culture medium from transformants that received constructs
containing the plant signal sequence, confirming expectations that the
protein would follow the default secretory pathway. Secretion also
facilitated characterization of the biochemical properties of recombinant
phytase. Soybean-synthesized phytase had a lower molecular mass than did
the fungal enzyme. However, deglycosylation of the recombinant and fungal
phytase yielded polypeptides of identical molecular mass (49 kD).
Temperature and pH optima of the recombinant phytase were indistinguishable
from the commercially available fungal phytase. Thermal inactivation
studies of the recombinant phytase suggested that the additional protein
stability would be required to withstand the elevated temperatures involved
in soybean processing.
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