A LACCASE-TYPE POLYPHENOL OXIDASE FROM LIGNIFYING XYLEM OF TOBACCO

Lignifying xylem from tobacco (Nicotiana tabacum) expresses oxidase ac tivity capable of oxidizing a range of chromogenic substrates by a non -peroxidative mechanism. These oxidases appear to be ionically bound t o the cell wall and can be extracted using 1M NaCl. The extracted oxid ases can oxidize the monolignol coniferyl alcohol. Extracts from xylem cell walls contain a number of different oxidase isoforms with isoele ctric points in the neutral to mildly acidic range. Xylem from younger , apical tobacco stems yield a different set of oxidase isoforms than xylem from older, basal areas which suggests that oxidase isoforms may be differently expressed during xylem maturation. Non-denaturing SDS- PAGE confirms the presence of a band of oxidase activity (M, ca 100 kD a) in these extracts that has properties similar to those of the lacca se-type polyphenol oxidases (p-diphenol: O-2 oxidoreductase; EC 1.14.1 8.1) previously identified in the lignifying tissues of trees. Ion-exc hange chromatography on DEAE-Sepharose retained this 100 kDa laccase-l ike activity and resulted in a ten-fold purification and a six-fold in crease in the recovery of oxidase activity, probably as the result of the removal of inhibitors. In contrast, a subsequent hydrophobic inter action chromatography step was unsuccessful, probably as a result of t he precipitation of the laccase-like oxidase in the concentrated ammon ium sulphate buffers required for this procedure. Copyright (C) 1996 E lsevier Science Ltd