Processive endoglucanase active in crystalline cellulose hydrolysis by the brown rot basidiomycete Gloeophyllum trabeum

Abstract

Brown rot basidiomycetes have long been thought to lack the processive cellulases that release soluble sugars from crystalline cellulose. On the other hand, these fungi remove all of the cellulose, both crystalline and amorphous, from wood when they degrade it. To resolve this discrepancy, we grew Gloeophyllum trabeum on microcrystalline cellulose (Avicel) and purified the major glycosylhydrolases it produced. The most abundant extracellular enzymes in these cultures were a 42-kDa endoglucanase (Cel5A), a 39-kDa xylanase (Xyn10A), and a 28-kDa endoglucanase (Cel12A). Cel5A had significant Avicelase activity--4.5 nmol glucose equivalents released/min/mg protein. It is a processive endoglucanase, because it hydrolyzed Avicel to cellobiose as the major product while introducing only a small proportion of reducing sugars into the remaining, insoluble substrate. Therefore, since G. trabeum is already known to produce a beta-glucosidase, it is now clear that this brown rot fungus produces enzymes capable of yielding assimilable glucose from crystalline cellulose.

FIG. 1.

SDS-PAGE analysis of extracellular proteins in the G. trabeum crude extract (4 μg) and of the purified Cel5A, Xyn10A, and Cel12A samples (0.5 μg each) that were submitted for partial amino acid sequencing. Lane M contained molecular mass standards.

FIG. 2.

Peptide sequence comparisons between glycosylhydrolases of G. trabeum and other organisms. A. G. trabeum Cel5A (GtCel5A) compared with Hypocrea jecorina Cel5A (HjCel5A, National Center for Biotechnology Information [http://ncbi.nlm.nih.gov/entrez/query.fcgi?] accession no. P07982) and Macrophomina phaseolina Cel5 (MpCel5, accession no. 1085763). B. G. trabeum Xyn10A (GtXyn10A) compared with Agaricus bisporus Xyn10 (AbXyn10, accession no. CAB05886) and C. fimi Xyn10A (CfXyn10A, accession no. P07986). C. G. trabeum Cel12A (GtCel12A) compared with Aspergillus aculeatus F-50 Cel12 (AaCel12, accession no. P22669) and A. aculeatus KSM 510 Cel12 (AaCel12′, accession no. AAD02275). All sequences are internal except the first Cel5A sequence of G. trabeum, which is N-terminal.

FIG. 3.

Two-dimensional IEF/SDS-PAGE of purified G. trabeum Cel5A. The locations of Cel5A and the tropomyosin internal standard (tr) are indicated.

FIG. 4.

Thin layer chromatography showing products released from Avicel or from oligoglucosides. The leftmost lane shows standards of glucose (G1), cellobiose (G2), cellotriose (G3), cellotetraose (G4), and cellopentaose (G5). The other lanes show, from left to right, products released by G. trabeum cultures from Avicel, products released by the crude extracellular extract from Avicel, products released by purified Cel5A from Avicel, and products released by purified Cel5A from the oligoglucosides G2 through G5.