Secretion of active recombinant phytase from soybean cell-suspension cultures.
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.
Li
J
J
Department of Plant Pathology, Physiology and Weed Science Virginia Polytechnic Institute and State University, Blacksburg 24061-0346, USA.
Hegeman
C E
CE
Hanlon
R W
RW
Lacy
G H
GH
Denbow
M D
MD
Grabau
E A
EA
D010832:6-Phytase / Q000096:biosynthesis*; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D002478:Cells, Cultured; D017931:DNA Primers; D005800:Genes, Fungal; D005822:Genetic Vectors; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D012639:Seeds / Q000201:enzymology; D013025:Glycine max / Q000166:cytology / Q000201:enzymology*; D014162:Transfection
D017931:DNA Primers; D011994:Recombinant Proteins; D010832:6-Phytase