Purification and characterization of p-coumaroyl-D-glucose hydroxylase of sweet potato (Ipomoea batatas) roots.
p-Coumaroyl-D-glucose hydroxylase in sweet potato (Ipomoea batatas Lam.) has been purified to apparent electrophoretic homogeneity using a combination of anion-and cation-exchange, hydrophobic and gel filtration chromatography. The purified enzyme was a monomer with a molecular weight of 33,000 and pI of 8.3. The purified enzyme showed not only hydroxylase activity but also polyphenol oxidase activity. L-Ascorbic acid was the best electron donor for the hydroxylation reaction, which had an optimum pH of 7.0. The enzyme hydroxylated p-coumaroyl-D-glucose, p-coumaric acid, and p-cresol but did not act on o-coumaric acid, m-coumaric acid, 4-hydroxy-3-methoxycinnamic acid, p-hydroxybenzoic acid or L-tyrosine. While the enzyme utilized p-coumaroyl-D-glucose and p-coumaric acid equally at pH 7.0, it hydroxylated only p-coumaroyl-D-glucose at pH 5.5. The enzyme oxidized diphenols such as D,L-(3,4-dihydroxyphenyl) alanine and caffeic acid, but exhibited no clear pH optimum in this reaction characteristic of polyphenol oxidase. Both the hydroxylase and the polyphenol oxidase activities were strongly inhibited by beta-mercaptoethanol, diethyldithiocarbamate, KCN, and p-coumaric acid (in concentrations higher than 5 mM). Ammonium sulfate and sodium chloride activated the hydroxylase activity but not the polyphenol oxidase activity of the enzyme. The enzyme activity and L-ascorbic acid contents changed in a manner suggesting their involvements in chlorogenic acid biosynthesis during incubation of sliced sweet potato root tissues.
Tanaka
M
M
Institute for Biochemical Regulation, Faculty of Agriculture, Nagoya University, Japan.
Kojima
M
M
D000645:Ammonium Sulfate / Q000494:pharmacology; D001205:Ascorbic Acid / Q000378:metabolism; D002726:Chlorogenic Acid / Q000378:metabolism; D003373:Coumaric Acids / Q000494:pharmacology; D005419:Flavonoids; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007526:Isoelectric Point; D006899:Mixed Function Oxygenases; D008970:Molecular Weight; D010636:Phenols / Q000378:metabolism; D010944:Plants / Q000201:enzymology*; D011108:Polymers / Q000378:metabolism; D059808:Polyphenols; D011422:Propionates; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity
D003373:Coumaric Acids; D005419:Flavonoids; D010636:Phenols; D011108:Polymers; D059808:Polyphenols; D011422:Propionates; D002726:Chlorogenic Acid; D012965:Sodium Chloride; D006899:Mixed Function Oxygenases; C066923:4-coumaroyl-D-glucose hydroxylase; D006867:Hydrolases; C495469:p-coumaric acid; D001205:Ascorbic Acid; D000645:Ammonium Sulfate