Determination of the activity of acidic phytate-degrading enzymes in cereal seeds.
Five different methods were compared to elucidate the total activity of the acidic phytate-degrading enzymes present in the seeds of rye, wheat, and barley. Phytate-degrading activity was studied at pH 5.0 by quantifying the liberated phosphate. Rye showed the highest acid phytate-degrading activity among the cereals studied. Using an aqueous extract, only 30-50% of the activity was found (rye, 3443 mU g(-1) of grain; wheat, 1026 mU g(-1) of grain; barley, 1032 mU g(-1) of grain) compared to that found by the direct incubation of the dry-milled cereal grains in a buffered phytate-containing solution (rye, 6752 mU g(-1) of grain; wheat, 2931 mU g(-1) of grain; barley, 2093 mU g(-1) of grain). Extending the extraction time resulted in an increase in extractable phytate-degrading activity by, at maximum, 10-15%. Extraction of phytate-degrading activity is strongly enhanced in the presence of Triton X-100 and the protease inhibitor phenylmethylsulfonyl fluoride (rye, 6536 mU g(-1) of grain; wheat, 2873 mU g(-1) of grain; barley, 2023 mU g(-1) of grain), suggesting at least a partial association with membrane structures and a degradation by proteolytic activity during extraction. In addition, it was shown that determining phytate-degrading activity by quantification of the liberated inorganic phosphate is more robust and precise than determining phytate-degrading activity by quantification of the residual phytate.
Greiner
Ralf
R
Centre for Molecular Biology, Federal Research Centre for Nutrition, Haid-und-Neu-Strasse 9, 76131 Karlsruhe, Germany. ralf.greiner@bfe.uni-karlsruhe.de
Egli
Ines
I
D010832:6-Phytase / Q000302:isolation & purification / Q000378:metabolism*; D002523:Edible Grain / Q000201:enzymology*; D001467:Hordeum / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D017830:Octoxynol / Q000494:pharmacology; D012434:Secale / Q000201:enzymology; D012639:Seeds / Q000201:enzymology*; D014908:Triticum / Q000201:enzymology
D017830:Octoxynol; D010832:6-Phytase