Purification and characterization of an oat fructan exohydrolase that preferentially hydrolyzes beta-2,6-fructans.
Oat (Avena sativa cv Fulghum) fructan hydrolase was purified by ammonium sulfate precipitation and anion-exchange, hydrophobic interaction, and size-exclusion chromatography. The enzyme was purified to homogeneity as determined by the presence of a single band (43 kD) on a silver-stained sodium dodecyl sulfate-polyacrylamide gel. A mixture of beta-2,6-linked fructan (neokestin) isolated from oat was used as the substrate to purify fructan hydrolase. Neokestin and small degree of polymerization fructan isomers were used to characterize the substrate specificity of the purified enzyme. The purified fructan hydrolase catalyzed hydrolysis of the terminal beta-2,6 linkage of 6G,6-kestotetraose 3.5 times more rapidly than it hydrolyzed the terminal beta-2,6 linkage of 6G-kestotriose and approximately 10 times faster than it hydrolyzed the terminal beta-2,1 linkage of chicory inulin. Sucrose and 1-kestose were not substrates. The Km for neokestin (beta-2,6-linked fructans with a degree of polymerization of 7-14) hydrolysis was 2.8% (w/v), and the Vmax was 0.041 mumol min-1 mL-1. The Km for hydrolysis of 6G,6-kestotetraose was 5.6% (w/v), and the Vmax was 0.138 mumol min-1 mL-1. Catalysis was exolytic and by multiple chain attack. Hydrolysis of neokestin was maximal at pH 4.5 to 5.0.
Henson
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CA
Agricultural Research Service, United States Department of Agriculture.
Livingston
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DP
D018554:Avena / Q000201:enzymology* / Q000254:growth & development; D001426:Bacterial Proteins; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005630:Fructans / Q000737:chemistry / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013379:Substrate Specificity
D001426:Bacterial Proteins; D005630:Fructans; D006026:Glycoside Hydrolases; C021512:levanase