Novel bacterial peroxidase without catalase activity from Flavobacterium meningosepticum: purification and characterization

A novel bacterial peroxidase co-produced intracellularly with H2O2-forming nucleoside oxidase, was purified from the cell-free extract of Flavobacteri um meningosepticum to homogeneity with 10.3% overall recovery through simpl e purification procedures including successive DEAE-Sephacel, phenyl-Sephar ose CL-4B and Sephacryl S-300 chromatography. The relative molecular mass o f the native enzyme was 220 000 Da, and that of its subunit was 54 000 Da. In contrast to other major intercellular peroxidases of bacterial origin, t he enzyme did not show any catalase activity. The amino acid sequences of t he 92 NH2-terminal amino acids and three internal peptides showed no signif icant homology with known peroxidases. The enzyme was not sensitive to the typical peroxidase inhibitors NaCN, NaF and NaN3, while mercuric ion strong ly inhibited the enzyme activity, and some carbonyl reagents were also foun d to have inhibitory effects. The enzyme showed a small K-m value for H2O2 (9.5 mu M) compared to other peroxidases. On the basis of its visible absor ption spectrum, the enzyme contained about 1.3 mol of heme per molecule. (C ) 1999 Elsevier Science B.V. All rights reserved.