Purification and characterization of phytase from cotyledons of germinating soybean seeds.
Soybean phytase (myo-inositol-hexakisphosphate phosphohydrolase; EC 3.1.3.8) was purified from 10-day-old germinating cotyledons using a four-step purification scheme. Phytase was separable from the major acid phosphatase present, and stained as a minor band of the three acid phosphatases detectable by activity staining after gel electrophoresis. The purified enzyme exhibited two closely migrating bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of approximately 59 and 60 KDa. The molar extinction coefficient of the enzyme at 280 nm was estimated to be 7.5 X 10(4) M-1 cm-1. The isoelectric point of phytase, as judged by the elution profile on chromatofocusing, was about 5.5. The enzyme was totally absorbed to a Procion Red HE3B column and eluted as a single protein component at a salt concentration of 250-300 mM. The enzyme possessed a high affinity for phytic acid (apparent Km = 48 microM), and was strongly inhibited by phosphate (apparent Ki = 18 microM), vanadate, and fluoride. Characteristic of other plant phytases, the pH and temperature optima were 4.5-4.8 and 55 degrees C, respectively.
Gibson
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DM
Southern Regional Research Center, U.S. Department of Agriculture, New Orleans, Louisiana 70179.
Ullah
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AH
D010832:6-Phytase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D000135:Acid Phosphatase / Q000302:isolation & purification / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D052179:Antigens, Plant; D002845:Chromatography; D002846:Chromatography, Affinity; D004396:Coloring Agents; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D005916:Globulins / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007118:Immunoassay; D055314:Seed Storage Proteins; D012639:Seeds / Q000201:enzymology*; D030262:Soybean Proteins; D013025:Glycine max / Q000201:enzymology*; D013053:Spectrophotometry; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors; D014227:Triazines
D000596:Amino Acids; D052179:Antigens, Plant; D004396:Coloring Agents; D005916:Globulins; D055314:Seed Storage Proteins; D030262:Soybean Proteins; D014227:Triazines; C013894:beta-conglycinin protein, Glycine max; C020795:procion red HE-3B; C011474:glycinin; D000135:Acid Phosphatase; D010832:6-Phytase