Purification and characterization of wall-localized cellulase from maize coleoptiles

Wall-localized cellulase was partially purified from freeze-dried maize coleoptiles by a combination of DEAE-Sepharose, Superdex-200 gel filtration and Hydroxyapatite column chromatography. Activity was measured by both reducing sugar assay and dot assay on agarose gel containing carboxymethylcellulose(CMC). In situ activity staining on a nondenaturing gel overlaid on agarose gel containing CMC turned out to be a quite reliable method to detect cellulase activity. The molecular mass of partially-purified cellulase was determined to be about 53 kD based on SDS-PAGE, and the N-terminal amino acid sequence of this cellulase was NH₂-AGAKGANXLGGLXRA. The enzyme hydrolyzed CMC with an optimal pH of 4.5 and optimal temperature of 40°C. It also catalyzed carboxymethylcellulose with aK _m of 2.02 mg/mL and aV _max of 160 ng/h/mL The β-1,4-glucosyl linkages of CMC, fibrous cellulose and lichenan were cleaved specifically by this enzyme. Reducing reagents such as cysteine-HCI, dithiothreitol and glutathione strongly enhanced the activity, suggesting that SH-groups of the enzyme were protected from oxidation. N-ethylmaleimide which is a sulfhydryl-reacting reagent did not seem to inhibit the activity, indicating that cysteine residues were not located near the active site of the enzyme. These results will be valuable in understanding the structure of wall-localized cellulase in maize coleoptiles and in predicting its possible function in the cell wall.