Use of a fluorescence labelled, carbohydrate-binding module from Phanerochaete chrysosporium Cel7D for studying wood cell wall ultrastructure.

Abstract

The alpha-amino group of the carbohydrate-binding module (CBM) from Phanerochaete chrysosporium cellulase Cel7D was covalently labelled with fluorescein isothiocyanate. The fluorescein-labelled CBM was characterised regarding substrate binding, showing specificity only to cellulose and not to mannan and xylan. Conjugation of fluorescein isothiocyanate to CBM did not affect its binding to cellulose. The labelled CBM was successfully used as a probe for detecting cellulose in lignocellulose material such as never dried spruce and birch wood as well as pulp fibres. Bmax, maximum binding capacity; BMCC, bacterial microcrystalline cellulose; CBH, cellobiohydrolase; CBM, carbohydrate-binding module; CBM-FITC, carbohydrate-binding module labelled with fluorescein isothiocyanate; CBM1, carbohydrate-binding module of family 1; FITC, fluorescein isothiocyanate; KD, dissociation constant; RP-HPLC, reversed phase HPLC; S1-S3, Secondary cell wall layers 1-3.