Purification and characterization of a novel esterase induced by growth of Aspergillus niger on sugar-beet pulp.
An inducible esterase has been isolated from a liquid culture of Aspergillus niger grown on sugar-beet pulp. The enzyme was active on methyl esters of cinnamic acids, caffeic > p-coumaric > ferulic, and is therefore termed a cinnamoyl esterase. The enzyme was not active on methyl sinapinate, a good substrate for ferulic acid esterase III, which was purified previously from A. niger [Faulds and Williamson (1994) Microbiology 140, 779-787]. With methyl caffeate as substrate the enzyme had temperature and pH optima of 50 degrees C and 6.0 respectively, and a specific activity of 96.9 units per mg of protein. The purified protein (native molecular mass 145 000 Da) gave a single heavily stained band on SDS/PAGE, suggesting the protein was a dimer, and seemed to be heavily glycosylated. Isoelectric focusing gave a single band corresponding to a pl of 4.80. The pure enzyme was free of other carbohydrase activities. The activity of the pure enzyme was inhibited by more than 99% after treatment with the serine-specific protease inhibitor aminoethylbenzenesulphonylfluoride (1 mM) for 12 h. The enzyme was capable of releasing ferulic acid from sugar beet pulp.
Kroon
P A
PA
Food Molecular Biochemistry Department, Institute of Food Research, Norwich Laboratory, Colney, U.K.
Faulds
C B
CB
Williamson
G
G
D000645:Ammonium Sulfate / Q000737:chemistry; D001234:Aspergillus niger / Q000201:enzymology* / Q000254:growth & development; D002109:Caffeic Acids / Q000737:chemistry / Q000378:metabolism; D002241:Carbohydrates; D002934:Cinnamates / Q000737:chemistry / Q000378:metabolism; D003373:Coumaric Acids / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004950:Esterases / Q000302:isolation & purification* / Q000378:metabolism; D006031:Glycosylation; D007525:Isoelectric Focusing; D008970:Molecular Weight; D011422:Propionates; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013450:Sulfones / Q000737:chemistry; D014361:Trypsin Inhibitors / Q000737:chemistry; D014462:Ultrafiltration
D002109:Caffeic Acids; D002241:Carbohydrates; D002934:Cinnamates; D003373:Coumaric Acids; D011422:Propionates; D013450:Sulfones; D014361:Trypsin Inhibitors; C029010:cinnamic acid; C002010:4-(2-aminoethyl)benzenesulfonylfluoride; C004999:ferulic acid; D004950:Esterases; C495469:p-coumaric acid; D000645:Ammonium Sulfate; C040048:caffeic acid