IDENTIFICATION AND PARTIAL CHARACTERIZATION OF A CONIFERYL ALCOHOL OXIDASE FROM LIGNIFYING XYLEM OF SITKA SPRUCE (PICEA-SITCHENSIS)

Oxidase activity in the developing xylem of branches of Sitka spruce [ Picea sitchensis] (Bong) Carr. was expressed in synchrony with the dep osition of lignin. The activity was closely associated with the cell w all but it could be extracted by elution with salt solutions such as I hi NaCl or CaCl2. A number of different oxidase isoforms with isoelec tric points in the range 8-5 were present in these cell wall extracts. These enzymes displayed a marked preference for the oxidation of coni feryl alcohol and efficiently initiated polymerization of coniferyl al cohol into insoluble, lignin-like polymers. They also had a substrate preference and profile of sensitivity to inhibitors that was dissimila r to those reported for classical catechol oxidase or laccase-type pol yphenol oxidases. A novel procedure that combines extraction and affin ity chromatography on Concanavalin-A to select high-mannose-type glyco proteins provided oxidase activity at higher purity and yield than pre viously used methods. A single band of oxidase activity (apparent M-r approx. 84 kDa) which was capable of oxidizing alpha-naphthol/N,N,N'N' -tetramethyl p-phenylene diamine in the absence of added hydrogen pero xide was detected in these cell wall extracts using non-denaturing sod ium dodecyl sulfate-polyacrylamide gel electrophoresis. The addition o f hydrogen peroxide did not intensify the staining of this band but it confirmed the presence of a true peroxidase band of apparent M-r appr ox. 40 kDa. The properties of this coniferyl alcohol oxidase are diffe rent from those of laccase-type polyphenol oxidases (EC 1.10.3.2) prev iously implicated in lignin deposition in tree species, and their poss ible roles in this process are discussed.