SECRETION OF ACTIVE RECOMBINANT PHYTASE FROM SOYBEAN CELL-SUSPENSION CULTURES

Phytase, an enzyme that degrades the phosphorus storage compound phyta te, has the potential to enhance phosphorus availability in animal die ts when engineered into soybean (Glycine max) seeds. The phytase gene from Aspergillus niger was inserted into soybean transformation plasmi ds under control of constitutive and seed-specific promoters, with and without a plant signal sequence. Suspension cultures were used to con firm phytase expression in soybean cells. Phytase mRNA was observed in cultures containing constitutively expressed constructs. Phytase acti vity was detected in the culture medium from transformants that receiv ed constructs containing the plant signal sequence, confirming expecta tions that the protein would follow the default secretory pathway. Sec retion also facilitated characterization of the biochemical properties of recombinant phytase. Soybean-synthesized phytase had a lower molec ular mass than did the fungal enzyme. However, deglycosylation of the recombinant and fungal phytase yielded polypeptides of identical molec ular mass (49 kD). Temperature and pH optima of the recombinant phytas e were indistinguishable from the commercially available fungal phytas e. Thermal inactivation studies of the recombinant phytase suggested t hat the additional protein stability would be required to withstand th e elevated temperatures involved in soybean processing.