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 pl values of similar to8.9, similar to9.5, and >10.0. The major cationic corn steep water peroxidase (CSWP), with a pl >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 degreesC and pH 4.5, the enzyme has half-lives of 22.7 min (0 mM Ca2+) and 248 min (1 mM Ca2+). The enzyme is stable at room temperature (22-25 degreesC), losing <3% of the activity at pH 4.5 and <10% at pH 6.2 over 400 h in the presence of 1 mM Ca2+.