Purification and characterization of a peroxidase from corn steep water.
Three cationic peroxidases have been detected in early, middle, and late corn steep water, with pI values of approximately 8.9, approximately 9.5, and >10.0. The major cationic corn steep water peroxidase (CSWP), with a pI >10, was purified 36400-fold with a 12% recovery from late steep water by a combination of acetone and ammonium sulfate precipitation and sequential chromatography on CM-cellulose, phenyl-Sepharose, and Sephadex G-75. The UV-vis spectrum of purified CSWP is typical of other plant class III peroxidases. The RZ (A(403)/A(280)) of CSWP was between 2.6 and 2.9. It is not glycosylated and exhibited an M(r) of 30662 +/- 7 by MALDI-TOF MS. The pH optimum of CSWP depends on the substrate, and it is active on 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid), guaiacol, ferulic acid, o-dianisidine, o-phenylenediamine, and pyrogallol but is not active on either syringaldazine or ascorbate. At 75 degrees C and pH 4.5, the enzyme has half-lives of 22.7 min (0 mM Ca(2+)) and 248 min (1 mM Ca(2+)). The enzyme is stable at room temperature (22-25 degrees C), losing <3% of the activity at pH 4.5 and <10% at pH 6.2 over 400 h in the presence of 1 mM Ca(2+).
Gray
James S S
JS
Department of Biochemistry, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City 52245, USA. james-gray@uiowa.edu
Montgomery
Rex
R
D000596:Amino Acids / Q000032:analysis; D002241:Carbohydrates / Q000032:analysis; D002412:Cations; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D013058:Mass Spectrometry; D008970:Molecular Weight; D010544:Peroxidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D014867:Water / Q000737:chemistry*; D003313:Zea mays / Q000201:enzymology*
D000596:Amino Acids; D002241:Carbohydrates; D002412:Cations; D014867:Water; D010544:Peroxidases