title: Heat-stable phytases in transgenic wheat (Triticum aestivum L.): deposition pattern, thermostability, and phytate hydrolysis.
abstract: The present paper addresses the question of thermotolerance of in planta synthesized heterologous enzymes using phytase as a model. Two individual transgenic wheat materials expressing an Aspergillus fumigatus phytase with a low denaturation temperature (62.5 degrees C) but a high refolding capacity, and a rationally designed consensus phytase engineered to a high denaturation temperature (89.3 degrees C), were evaluated. High levels of endosperm specific expression were ensured by the wheat high molecular weight glutenin 1DX5 promoter. Immunodetection at the light and electron microscopical level shows unequivocally that the heterologous phytase is deposited in the vacuole, albeit that the transformation constructs were designed for secretion to the apoplast. Evaluation of heat stability properties and kinetic properties unraveled that, under these deposition conditions, heat stability based on high unfolding temperature is superior to high refolding capacity and represents a realistic strategy for improving phosphate and mineral bioavailability in cereal-based feed and food.
authors.affiliation: Research Centre Flakkebjerg, Department of Genetics and Biotechnology, Danish Institute of Agricultural Sciences, DK-4200 Slagelse, Denmark. Henrik.brinchpedersen@agrsci.dk
authors.forename: Henrik
authors.initials: H
authors.lastname: Brinch-Pedersen
authors.forename: Frank
authors.initials: F
authors.lastname: Hatzack
authors.forename: Eva
authors.initials: E
authors.lastname: Stöger
authors.forename: Elsa
authors.initials: E
authors.lastname: Arcalis
authors.forename: Katrine
authors.initials: K
authors.lastname: Pontopidan
authors.forename: Preben B
authors.initials: PB
authors.lastname: Holm
mesh_terms: D010832:6-Phytase / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D001232:Aspergillus fumigatus / Q000201:enzymology / Q000235:genetics; D004795:Enzyme Stability; D015870:Gene Expression; D006358:Hot Temperature; D006868:Hydrolysis; D007295:Inositol Phosphates / Q000378:metabolism; D010833:Phytic Acid / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000201:enzymology*; D011489:Protein Denaturation; D014908:Triticum / Q000201:enzymology* / Q000235:genetics
chemical_list: D007295:Inositol Phosphates; D010833:Phytic Acid; D010832:6-Phytase