Non-Heme Hydroquinone Peroxidase from Azotobacter beijerinckii HM121

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In order to purify a lignin-degrading enzyme from the bacterium Azotobacter beijerinckii HM121,which rapidly decolorizes lignin, the enzyme activity was monitored by observing the hydroquinone oxidizing activity in the presence of hydrogen peroxide. The activity was bound to the membrane and was solubilized by treating disrupted cells with 0.1% Triton X-100. The enzyme was purified to homogeneity by ammonium sulfate fractionation, hydrophobic chromatography on butyl-Toyopearl 650M, and gel filtration on Toyopearl HW-55F. the relative molecular mass of the enzyme was determined to be 59,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 55,000 by gel filtration on Toyopearl HW-55F, suggesting it is a monomeric enzyme. The purified enzyme exhibited the maximal activity for hydroquinone oxidation at pH 7.0 and 50℃ and was stable up to 70℃ for 1 h. The optimum concentration of hydrogen peroxide was 6.0 mM. The enzyme was active toward hydroquinone, vanillin, catechol, caffeic acid, p-phenylenediamine, pyrogallol, methylhydroquinone, and dimethylhydroquinone. Based on these results the enzyme was named hydroquinone peroxidase. The enzyme also decolorized and degraded lignin. Spectral analysis revealed that this peroxidase did not have iron-heme. Inductively coupled plasmma analysis showed the presence of equimolar manganese. The enzyme activity was inhibited by 1 mM EDTA and by 1 mM 1,10-phenanthroline; the activity of the EDTA-inactivated enzyme was restored by the addition of manganese ions. However, the enzyme activity did not require exogenous manganese ions and was not promoted by the addition of manganese. the peroxidase oxidized Mn(II) to MN(III) in the presence of hydrogen peroxide. The activity was promoted 4 fold by the addition of 0.1 mM ZnCl_2. The enzyme formed a hydroxy radical from hydrogen peroxide as an active oxygen molecule. From the above results, the hydroquinone peroxidase was judged to be a new type of lignin-degrading enzyme.
社団法人日本生物工学会の論文
1997-07-25

Non-Heme Hydroquinone Peroxidase from Azotobacter beijerinckii HM121

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