IDENTIFICATION AND PARTIAL-PURIFICATION OF AN OXIDASE FROM LIGNIFYINGXYLEM OF LEYLAND CYPRESS (X CUPRESSOCYPARIS-LEYLANDII)

Oxidase activity was exclusively present in lignifying cells of develo ping xylem of Leyland cypress. The oxidase was enriched in 200 mM CaCl 2 extracts of crude cell walls and seems to be ionically associated wi th the cell walls. Oxidase activity was selected and concentrated usin g affinity chromatography on Concanavalin-A Sepharose which suggests t hat it is a high-mannose type glycoprotein. A subsequent purification step using gel permeation chromatography on Sephadex GF-150 partially separated the oxidase activity from peroxidase activity. An oxidase ba nd of apparent Mr 92 kD capable of oxidising N, N, N', N'-tetramethyl phenylene diamine/alpha-naphthol was identified after non-denaturing s odium dodecyl sulphate polyacrylamide gel electrophoresis. The 92 kD o xidase band was enriched in the oxidase-rich fraction and absent from the peroxidase-rich fraction from the gel permeation step. In addition , the 92 kD oxidase band could be differentiated from peroxidase bands because it was not intensified by the addition of hydrogen peroxide. The partially purified oxidase effectively oxidised and polymerised co niferyl alcohol to form insoluble material that yielded a Fourier tran sform infra-red spectrum similar to dehydrogenation polymers of conife ryl alcohol. This coniferyl alcohol oxidase appears to be specific to lignifying xylem cells and may participate in lignin deposition but fu rther studies are required to fully define this oxidase and its possib le homology with other oxidases identified in the lignifying xylem of different species of trees.