Heat-stable phytases in transgenic wheat (Triticum aestivum L.): deposition pattern, thermostability, and phytate hydrolysis.
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.
Brinch-Pedersen
Henrik
H
Research Centre Flakkebjerg, Department of Genetics and Biotechnology, Danish Institute of Agricultural Sciences, DK-4200 Slagelse, Denmark. Henrik.brinchpedersen@agrsci.dk
Hatzack
Frank
F
Stöger
Eva
E
Arcalis
Elsa
E
Pontopidan
Katrine
K
Holm
Preben B
PB
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
D007295:Inositol Phosphates; D010833:Phytic Acid; D010832:6-Phytase