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Production of Two Highly Active Bacterial Phytases with Broad pH
Optima in Germinated Transgenic Rice Seeds
Transgenic Res. 2004 Feb;13(1):29-39.
Hong CY, Cheng KJ, Tseng TH, Wang CS, Liu LF, Yu SM.
Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 11529, Taiwan,
ROC.
Phytate is the main storage form of phosphorus in many plant seeds, but
phosphate bound in this form is not available to monogastric animals. Phytase,
an enzyme that hydrolyzes phosphate from phytate, has the potential to enhance
phosphorus availability in animal diets when engineered in rice seeds as a feed
additive. Two genes, derived from a ruminal bacterium Selenomonas ruminantium
(SrPf6) and Escherichia coli (appA), encoding highly active phytases were
expressed in germinated transgenic rice seeds. Phytase expression was controlled
by a germination inducible alpha-amylase gene (alphaAmy8) promoter, and
extracellular phytase secretion directed by an betaAmy8 signal peptide sequence.
The two phytases were expressed in germinated transgenic rice seeds transiently
and in a temporally controlled and tissue-specific manner. No adverse effect on
plant development or seed formation was observed. Up to 0.6 and 1.4 U of phytase
activity per mg of total extracted cellular proteins were obtained in germinated
transgenic rice seeds expressing appA and SrPf6 phytases, respectively, which
represent 46-60 times of phytase activities compared to the non-transformant.
The appA and SrPf6 phytases produced in germinated transgenic rice seeds had
high activity over broad pH ranges of 3.0-5.5 and 2.0-6.0, respectively. Phytase
levels and inheritance of transgenes in one highly expressing plant were stable
over four generations. Germinated transgenic rice seeds, which produce a
highly active recombinant phytase and are rich in hydrolytic enzymes, nutrients
and minerals, could potentially be an ideal feed additive for improving the
phytate-phosphorus digestibility in monogastric animals.
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