Expression of an Aspergillus niger phytase gene (phyA) in Saccharomyces cerevisiae.
Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals and reduces phosphorus pollution of animal waste. Our objectives were to express an Aspergillus niger phytase gene (phyA) in Saccharomyces cerevisiae and to determine the effects of glycosylation on the phytase's activity and thermostability. A 1.4-kb DNA fragment containing the coding region of the phyA gene was inserted into the expression vector pYES2 and was expressed in S. cerevisiae as an active, extracellular phytase. The yield of total extracellular phytase activity was affected by the signal peptide and the medium composition. The expressed phytase had two pH optima (2 to 2.5 and 5 to 5.5) and a temperature optimum between 55 and 60 degrees C, and it cross-reacted with a rabbit polyclonal antibody against the wild-type enzyme. Due to the heavy glycosylation, the expressed phytase had a molecular size of approximately 120 kDa and appeared to be more thermostable than the commercial enzyme. Deglycosylation of the phytase resulted in losses of 9% of its activity and 40% of its thermostability. The recombinant phytase was effective in hydrolyzing phytate phosphorus from corn or soybean meal in vitro. In conclusion, the phyA gene was expressed as an active, extracellular phytase in S. cerevisiae, and its thermostability was affected by glycosylation.
Han
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Department of Animal Science, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853, USA.
Wilson
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DB
Lei
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D010832:6-Phytase / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D000818:Animals; D000908:Antibodies, Fungal; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004795:Enzyme Stability; D015870:Gene Expression; D005800:Genes, Fungal; D006031:Glycosylation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010758:Phosphorus / Q000378:metabolism; D010833:Phytic Acid / Q000378:metabolism; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013696:Temperature
D000908:Antibodies, Fungal; D017931:DNA Primers; D010758:Phosphorus; D010833:Phytic Acid; D010832:6-Phytase