Substrate specificity and mode of action of the cellulases from the thermophilic fungus <i>Thermoascus aurantiacus</i>

Shepherd, Maxwell G.; Tong, Chow Chin; Cole, A. L. J.

doi:10.1042/bj1930067

Cited by 43 publications

(17 citation statements)

References 22 publications

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“…Endoglucanases hydrolyze internal β‐1,4‐glycosidic bonds in mixed glucans, carboxymethylcellulose (CMC) and in some cases cellulose. Here we report structure determination of a β‐1,4‐endoglucanase from this fungus, which according to its abundance and characteristics [5] is probably identical or similar to endocellulases identified by others [2–4,6]. These enzymes all showed an apparent molecular weight of approximately 32–34 kDa, high temperature stability and optimum (around 70°) and low optimum pH (3–4.5).…”

Section: Introductionmentioning

confidence: 83%

The 1.62 Å structure of Thermoascus aurantiacus endoglucanase: completing the structural picture of subfamilies in glycoside hydrolase family 5

Leggio

Larsen

2002

FEBS Letters

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The crystal structure of Thermoascus aurantiacus endoglucanase (Cel5A), a family 5 glycoside hydrolase, has been determined to 1.62 A î resolution by multiple isomorphous replacement with anomalous scattering. It is the ¢rst report of a structure in the subfamily to which Cel5A belongs. Cel5A consists solely of a catalytic module with compact eight-fold L L/K K barrel architecture. The length of the tryptophan-rich substrate binding groove suggests the presence of substrate binding subsites 3 34 to +3. Structural comparison shows that two glycines are completely conserved in the family, in addition to the two catalytic glutamates and six other conserved residues previously identi¢ed. Gly 44 in particular is part of a type IV C-terminal helix capping motif, whose disruption is likely to a¡ect the position of an essential conserved arginine. One aromatic residue (Trp 170 in Cel5A), not conserved in term of sequence, is nonetheless spatially conserved in the substrate binding groove. Its role might be to force the bend that occurs in the polysaccharide chain on binding, thus favoring substrate distortion at subsite 3 31. ß 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

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Section: Introductionmentioning

confidence: 83%

The 1.62 Å structure of Thermoascus aurantiacus endoglucanase: completing the structural picture of subfamilies in glycoside hydrolase family 5

Leggio

Larsen

2002

FEBS Letters

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“…Viscosity measurements have occasionally been used to assess the activity of endoglucanases on water-soluble polysaccharides (56–58). Here, we show that dynamic viscosity measurements provide an alternative and sensitive method for assessing and comparing LPMO activity on water-soluble polysaccharides.…”

Section: Discussionmentioning

confidence: 99%

A Lytic Polysaccharide Monooxygenase with Broad Xyloglucan Specificity from the Brown-Rot Fungus Gloeophyllum trabeum and Its Action on Cellulose-Xyloglucan Complexes

Kojima

Várnai

Ishida

et al. 2016

Appl Environ Microbiol

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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.IMPORTANCE Currently, 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.

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“…However, when this endoglucanase was incubated with an exoglucanase, even of a different origin, it showed a high synergistic action on crystalline cellulose. On the other hand, cellulase components of T. aurantiacus showed no synergism (228). Why some cellulase enzymes show synergism whereas others do not is of great interest.…”

Section: Cellulasementioning

confidence: 99%

Thermophilic Fungi: Their Physiology and Enzymes

Maheshwari

Bharadwaj

Bhat

2000

Microbiol Mol Biol Rev

652

380

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SUMMARY Thermophilic fungi are a small assemblage in mycota that have a minimum temperature of growth at or above 20°C and a maximum temperature of growth extending up to 60 to 62°C. As the only representatives of eukaryotic organisms that can grow at temperatures above 45°C, the thermophilic fungi are valuable experimental systems for investigations of mechanisms that allow growth at moderately high temperature yet limit their growth beyond 60 to 62°C. Although widespread in terrestrial habitats, they have remained underexplored compared to thermophilic species of eubacteria and archaea. However, thermophilic fungi are potential sources of enzymes with scientific and commercial interests. This review, for the first time, compiles information on the physiology and enzymes of thermophilic fungi. Thermophilic fungi can be grown in minimal media with metabolic rates and growth yields comparable to those of mesophilic fungi. Studies of their growth kinetics, respiration, mixed-substrate utilization, nutrient uptake, and protein breakdown rate have provided some basic information not only on thermophilic fungi but also on filamentous fungi in general. Some species have the ability to grow at ambient temperatures if cultures are initiated with germinated spores or mycelial inoculum or if a nutritionally rich medium is used. Thermophilic fungi have a powerful ability to degrade polysaccharide constituents of biomass. The properties of their enzymes show differences not only among species but also among strains of the same species. Their extracellular enzymes display temperature optima for activity that are close to or above the optimum temperature for the growth of organism and, in general, are more heat stable than those of the mesophilic fungi. Some extracellular enzymes from thermophilic fungi are being produced commercially, and a few others have commercial prospects. Genes of thermophilic fungi encoding lipase, protease, xylanase, and cellulase have been cloned and overexpressed in heterologous fungi, and pure crystalline proteins have been obtained for elucidation of the mechanisms of their intrinsic thermostability and catalysis. By contrast, the thermal stability of the few intracellular enzymes that have been purified is comparable to or, in some cases, lower than that of enzymes from the mesophilic fungi. Although rigorous data are lacking, it appears that eukaryotic thermophily involves several mechanisms of stabilization of enzymes or optimization of their activity, with different mechanisms operating for different enzymes.

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Substrate specificity and mode of action of the cellulases from the thermophilic fungus Thermoascus aurantiacus

Cited by 43 publications

References 22 publications

The 1.62 Å structure of Thermoascus aurantiacus endoglucanase: completing the structural picture of subfamilies in glycoside hydrolase family 5

The 1.62 Å structure of Thermoascus aurantiacus endoglucanase: completing the structural picture of subfamilies in glycoside hydrolase family 5

A Lytic Polysaccharide Monooxygenase with Broad Xyloglucan Specificity from the Brown-Rot Fungus Gloeophyllum trabeum and Its Action on Cellulose-Xyloglucan Complexes

Thermophilic Fungi: Their Physiology and Enzymes

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