Nucleoside oxidase that was purified to an electrophretically homogeneousstate catalyzed the oxidation of hydroquinone only in the presence of nucleosides simultaneously with the oxidation of nucleosides. From the identification of the reaction products and a chemical balance study, we concluded that the oxidation of hydroquinone proceeded by the following scheme: 2 hydroquinone + O2->2p-quinone + 2H2O. The enzyme also catalyzed the oxidation of various phenolic compounds and strongly colored substances were formed from phenolic compoundswith 4-aminoantipyrine in proportion to the amountof nucleosides. As described in earlier communications,1~3) we found a new enzyme that catalyzes the oxidation of nucleosides to nucleoside-5'-carboxylic acid via nucleoside-5'-aldehyde without a requirement for an exogenous co factor. The enzymewas purified from the cell-free extract of Pseudomonas maltophilia LB-86 by a procedure involving column chromatographies on DEAE-Toyopearl and gel filtration on Sephacryl S-200. The final preparation was homogeneous on polyacrylamide gel electrophoresis. The purified enzyme oxidized various nucleosides but not did attack nucleotides, bases, or ribose. During the investigation on this enzyme, we found that the enzyme catalyzes the oxidation of hydroquinone to pquinone. This oxidation reaction was identical with that of laccase (EC 1.10.3.2), but this activity of nucleoside oxidase appeared only in the presence of nucleoside. This paper describes an identification of the reaction product and the stoichiometry of enzyme reaction using hydroquinone as a substrate. The activities against various phenolic compoundsand the coupling reaction of phenolic compounds and 4-aminoantipyrine are also presented.
A novel lysozyme inhibitor, hen egg white lysozyme inhibitor (Hewli) was isolated from the culture broth of a bacterium (strain 1-139) identified as Bacillus subtilis. Maximum lysozyme inhibitory activity was obtained when the bacterium was grown aerobically in a mediumconsisting of 0.65% glucose, 0.5 %sodium glutamate, 1 % meat extract, and 4 %Polypepton (pH 7.0), at 30°C after 30 to 35 hr. Hewli was purified 69-fold from the culture supernatant of B. subtilis 1-139 by ammoniumsulfate fractionation, DEAE-Sephadex A-50 column chromatography, Sephacryl S-200 gel chromatography, DEAE-Toyopearl650Mcolumn chromatography, affinity chromatography on a column of immobilized egg white lysozyme, and acetone extraction. The purified lysozyme inhibitor has an amino acid composition of aspartic acid, glutamic acid, valine, and leucine in a 1 : 1 : 1 : 4 molar ratio and fatty acid composition of palmitic acid and/or vaccenic acid. Hewli inhibited B. subtilis 1-139 glycanase and animal lysozymes such as the hen, turkey, or human enzymes.
The enzyme contains 1 mol of covalently bound FAD, 2g atoms of nonheme iron, 2 mol of labile sulfides, and 1 mol of heme per mol enzyme protein. The absorption spectrum of nucleoside oxidase had maxima 278 and 390nm, and shoulders at 343 and 450nm. The enzyme catalyzes the oxidation of various nucleosides, and the Kmvalue for inosine was 4.4 x 10"5 M. The enzyme was most active at pH 5-6, and was most stable between pH 5.0-6.0 and at temperatures below 60°C. The activity was strongly inhibited by /V-bromosuccinimide and potassium cyanide.
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