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Appl. Environ. Microbiol., Aug 1995, 2976-2980, Vol 61, No. 8
Copyright © 1995, American Society for Microbiology

Purification and Characterization of a Cellulose-Binding (beta)-Glucosidase from Cellulose-Degrading Cultures of Phanerochaete chrysosporium

ES Lymar, B Li and V Renganathan
Department of Chemistry, Biochemistry, and Molecular Biology, Oregon Graduate Institute of Science & Technology, Portland, Oregon 97291-1000

Extracellular (beta)-glucosidase from cellulose-degrading cultures of Phanerochaete chrysosporium was purified by DEAE-Sephadex chromatography, by Sephacryl S-200 chromatography, and by fast protein liquid chromatography (FPLC) using a Mono Q anion-exchange column. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic (SDS-PAGE) analysis of FPLC-purified (beta)-glucosidase indicated the presence of three enzyme forms with molecular weights of 96,000, 98,000, and 114,000. On further fractionation with a microcrystalline cellulose column, the 114,000-molecular-weight (beta)-glucosidase, which had 82% of the (beta)-glucosidase activity, was bound to cellulose. The (beta)-glucosidases with molecular weights of 96,000 and 98,000 did not bind to cellulose. The cellulose-bound (beta)-glucosidase was eluted completely from the cellulose matrix with water. Cellulose-bound (beta)-glucosidase catalyzed p-nitrophenylglucoside hydrolysis, suggesting that the catalytic site is not involved in cellulose binding. When the cellulose-binding form was incubated with papain for 20 h, no decrease in the enzyme activity was observed; however, approximately 74% of the papain-treated glucosidase did not bind to microcrystalline cellulose. SDS-PAGE analysis of the nonbinding glucosidase produced by papain indicated the presence of three bands with molecular weights in the range of 95,000 to 97,000. On the basis of these results, we propose that the low-molecular-weight (96,000 and 98,000) non-cellulose-binding (beta)-glucosidase forms are most probably formed from the higher-molecular-weight (114,000) cellulose-binding (beta)-glucosidase via extracellular proteolytic hydrolysis. Also, it appears that the extracellular (beta)-glucosidase from P. chrysosporium might be organized into two domains, a cellulose-binding domain and a catalytic domain. Kinetic characterization of the cellulose-binding form is also presented.

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