Purification and characterisation of an extracellular peroxidase from white-rot fungus Pleurotus ostreatus.
A peroxidase was purified 98.3-fold from the culture filtrate of Pleurotus ostreatus with an overall yield of 12.4%. The molecular mass determined by gel filtration was found to be approx. 140 kDa. SDS-PAGE revealed that the enzyme consists of two identical subunits with a molecular mass of approx. 72 kDa. The pI value of this enzyme is approx. 4.3. The enzyme contains 41% carbohydrate by weight, and aspartic acid and asparagine (16.8%), and glutamic acid and glutamine (12.0%). The enzyme has the highest affinity toward synaptic acid and affinity towards various phenolic compounds containing methoxyl and p-hydroxyl groups, directly attached to the benzene ring. However, the enzyme does not react with veratryl alcohol and shows no affinity for nonphenolic compounds. The optimal reaction pH and temperature are 4.0 and 40 degrees C, respectively. The catalytic mechanism of the enzymic reaction is of the Ping-Pong type. The activity of the enzyme is competitively inhibited by high concentrations of H2O2 and its Ki value is 1.70 mM against H2O2. This enzyme contains approx. 1 mol of heme per mol of one subunit of the enzyme. The pyridine hemochrome spectrum of the enzyme indicates that the heme of P. ostreatus peroxidase is iron protoporphyrin IX. The EPR spectrum of the native peroxidase shows the presence of a high-spin ferric complex with g values at 6.102, 5.643 and 1.991.
Kang
S O
SO
Department of Microbiology, College of Natural Sciences, Seoul National University, South Korea.
Shin
K S
KS
Han
Y H
YH
Youn
H D
HD
Hah
Y C
YC
D000596:Amino Acids / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D008031:Lignin / Q000378:metabolism; D008970:Molecular Weight; D009195:Peroxidase / Q000737:chemistry / Q000302:isolation & purification*; D011124:Polyporaceae / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
D000596:Amino Acids; D008031:Lignin; D009195:Peroxidase