Chromatographic isolation and characterization of a novel peroxidase from large lima legumes.
A novel peroxidase with antifungal activity was isolated from the large lima bean (Phaseolus limensis) seeds. The procedure entailed extraction, ammonium sulfate precipitation, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-Toyopearl, and gel filtration on Sephadex G-75. The enzyme was adsorbed on Affi-gel blue gel and SP-Toyopearl, and possessed a molecular weight of 34 kDa in SDS-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. There was an almost 110-fold increase in specific activity of the purified peroxidase compared with that of the crude extract. The enzyme exhibited a pI of 8.6 by isoelectric focusing electrophoresis, indicating that it is a basic protein. The optimum pH and the optimum temperature were 5.5 and 30 degreeC, respectively. The enzyme was stable up to 55 degreeC. It potently suppressed mycelial growth in Fusarium solani, Mycosphaerella arachidicola, and Pythium aphanidermatum with an IC50 of 76, 103, and 119 microM, respectively.
Wang
S Y
SY
College of Biological Science & Technology, Fuzhou Univ.,Fuzhou 350002, China. shywang@fzu.edu.cn
Gong
Y S
YS
Zhou
J J
JJ
D000645:Ammonium Sulfate; D002412:Cations / Q000494:pharmacology; D011232:Chemical Precipitation; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005659:Fungicides, Industrial / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008970:Molecular Weight; D025282:Mycelium / Q000187:drug effects / Q000254:growth & development; D009195:Peroxidase / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D027805:Phaseolus / Q000201:enzymology*; D012639:Seeds / Q000201:enzymology*
D002412:Cations; D005659:Fungicides, Industrial; D009195:Peroxidase; D000645:Ammonium Sulfate