Dataset Information

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.

SUBMITTER: Cohen R

PROVIDER: S-EPMC1087581 | biostudies-literature | 2005 May

REPOSITORIES: biostudies-literature

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

Cohen Roni R Suzuki Melissa R MR Hammel Kenneth E KE

Applied and environmental microbiology 20050501 5

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 ...[more]

PMID: 15870328

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Project description:Fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-rot fungi, such as Gloeophyllum trabeum, tend to have few LPMOs, and information on these enzymes is scarce. The genome of G. trabeum encodes four auxiliary activity 9 (AA9) LPMOs (GtLPMO9s), whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants of GtLPMO9A seem to be produced, a single-domain variant, GtLPMO9A-1, and a longer variant, GtLPMO9A-2, which contains a C-terminal domain comprising approximately 55 residues without a predicted function. We have overexpressed the phylogenetically distinct GtLPMO9A-2 in Pichia pastoris and investigated its properties. Standard analyses using high-performance anion-exchange chromatography-pulsed amperometric detection (HPAEC-PAD) and mass spectrometry (MS) showed that GtLPMO9A-2 is active on cellulose, carboxymethyl cellulose, and xyloglucan. Importantly, compared to other known xyloglucan-active LPMOs, GtLPMO9A-2 has broad specificity, cleaving at any position along the β-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic viscosity measurements to compare the hemicellulolytic action of GtLPMO9A-2 to that of a well-characterized hemicellulolytic LPMO, NcLPMO9C from Neurospora crassa revealed that GtLPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurements also revealed minor activity on glucomannan that could not be detected by the analysis of soluble products by HPAEC-PAD and MS and that was lower than the activity of NcLPMO9C. Experiments with copolymeric substrates showed an inhibitory effect of hemicellulose coating on cellulolytic LPMO activity and did not reveal additional activities of GtLPMO9A-2. These results provide insight into the LPMO potential of G. trabeum and provide a novel sensitive method, a measurement of dynamic viscosity, for monitoring LPMO activity.ImportanceCurrently, there are only a few methods available to analyze end products of lytic polysaccharide monooxygenase (LPMO) activity, the most common ones being liquid chromatography and mass spectrometry. Here, we present an alternative and sensitive method based on measurement of dynamic viscosity for real-time continuous monitoring of LPMO activity in the presence of water-soluble hemicelluloses, such as xyloglucan. We have used both these novel and existing analytical methods to characterize a xyloglucan-active LPMO from a brown-rot fungus. This enzyme, GtLPMO9A-2, differs from previously characterized LPMOs in having broad substrate specificity, enabling almost random cleavage of the xyloglucan backbone. GtLPMO9A-2 acts preferentially on free xyloglucan, suggesting a preference for xyloglucan chains that tether cellulose fibers together. The xyloglucan-degrading potential of GtLPMO9A-2 suggests a role in decreasing wood strength at the initial stage of brown rot through degradation of the primary cell wall.

Dataset Information

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

Publications

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

Similar Datasets

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