Curdlan

Glucans are polymers of D-glucose with differing linkages in linear or branched sequences. They are constituents of microbial and plant cell-walls and involved in important bio-recognition processes, including immunomodulation, anticancer activities, pathogen virulence, and plant cell-wall biodegradation. Translational possibilities for these activities in medicine and biotechnology are considerable. High-throughput micro-methods are needed to screen proteins for recognition of specific glucan sequences as a lead to structure–function studies and their exploitation. We describe construction of a “glucome” microarray, the first sequence-defined glycome-scale microarray, using a “designer” approach from targeted ligand-bearing glucans in conjunction with a novel high-sensitivity mass spectrometric sequencing method, as a screening tool to assign glucan recognition motifs. The glucome microarray comprises 153 oligosaccharide probes with high purity, representing major sequences in glucans. Negative-ion electrospray tandem mass spectrometry with collision-induced dissociation was used for complete linkage analysis of gluco-oligosaccharides in linear “homo” and “hetero” and branched sequences. The system is validated using antibodies and carbohydrate-binding modules known to target α- or β-glucans in different biological contexts, extending knowledge on their specificities, and applied to reveal new information on glucan recognition by two signaling molecules of the immune system against pathogens: Dectin-1 and DC-SIGN. The sequencing of the glucan oligosaccharides by the MS method and their interrogation on the microarrays provides detailed information on linkage, sequence and chain length requirements of glucan-recognizing proteins, and are a sensitive means of revealing unsuspected sequences in the polysaccharides.

The aim of the present study was to investigate whether β-glucans obtained from the lactic acid bacteria (LAB) Levilactobacillus (L.) brevis and Pediococcus (P.) claussenii exhibit similar physiological effects such as cholesterol-binding capacity (CBC) as the structurally different β-glucans from oat, barley, and yeast as well as curdlan. After in vitro fermentation, fermentation supernatants (FSs) and/or -pellets (FPs) were analyzed regarding the concentrations of short-chain fatty acids (SCFAs), ammonia, bile acids, the relative abundance of bacterial taxa and chemopreventive effects (growth inhibition, apoptosis, genotoxicity) in LT97 colon adenoma cells. Compared to other glucans, the highest CBC was determined for oat β-glucan (65.9 ± 8.8 mg g^-1, p < 0.05). Concentrations of SCFA were increased in FSs of all β-glucans (up to 2.7-fold). The lowest concentrations of ammonia (down to 0.8 ± 0.3 mmol L^-1) and bile acids (2.5-5.2 μg mL^-1) were detected in FSs of the β-glucans from oat, barley, yeast, and curdlan. The various β-glucans differentially modulated the relative abundance of bacteria families and reduced the Firmicutes/Bacteroidetes ratio. Treatment of LT97 cells with the FSs led to a significant dose-dependent growth reduction and increase in caspase-3 activity without exhibiting genotoxic effects. Though the different β-glucans show different fermentation profiles as well as cholesterol- and bile acid-reducing properties, they exhibit comparable chemopreventive effects.

Background: The discovery of lytic polysaccharide monooxygenases (LPMOs) has fundamentally changed our understanding of microbial lignocellulose degradation. Cellulomonas bacteria have a rich history of study due to their ability to degrade recalcitrant cellulose, yet little is known about the predicted LPMOs that they encode from Auxiliary Activity Family 10 (AA10). Results: Here, we present the comprehensive biochemical characterization of three AA10 LPMOs from Cellulomonas flavigena (CflaLPMO10A, CflaLPMO10B, and CflaLPMO10C) and one LPMO from Cellulomonas fimi (CfiLPMO10). We demonstrate that these four enzymes oxidize insoluble cellulose with C1 regioselectivity and show a preference for substrates with high surface area. In addition, CflaLPMO10B, CflaLPMO10C, and CfiLPMO10 exhibit limited capacity to perform mixed C1/C4 regioselective oxidative cleavage. Thermostability analysis indicates that these LPMOs can refold spontaneously following denaturation dependent on the presence of copper coordination. Scanning and transmission electron microscopy revealed substrate-specific surface and structural morphological changes following LPMO action on Avicel and phosphoric acid-swollen cellulose (PASC). Further, we demonstrate that the LPMOs encoded by Cellulomonas flavigena exhibit synergy in cellulose degradation, which is due in part to decreased autoinactivation. Conclusions: Together, these results advance understanding of the cellulose utilization machinery of historically important Cellulomonas species beyond hydrolytic enzymes to include lytic cleavage. This work also contributes to the broader mapping of enzyme activity in Auxiliary Activity Family 10 and provides new biocatalysts for potential applications in biomass modification.

β-glucans are the dietary nutrients present in oats, barley, algae, and mushrooms. The macromolecules are well known for their immune-modulatory activity; however, how the human gut bacteria digest them is vaguely understood. In this study, Bacteroides uniformis JCM 13288^T was found to grow on laminarin, pustulan, and porphyran. We sequenced the genome of the strain, which was about 5.05 megabase pairs and contained 4868 protein-coding genes. On the basis of growth patterns of the bacterium, two putative polysaccharide utilization loci for β-glucans were identified from the genome, and associated four putative genes were cloned, expressed, purified, and characterized. Three glycoside hydrolases (GHs) that were endo-acting enzymes (BuGH16, BuGH30, and BuGH158), and one which was an exo-acting (BuGH3) enzyme. The BuGH3, BuGH16, and BuGH158 can cleave linear exo/endo-β-1-3 linkages while BuGH30 can digest endo-β- 1-6 linkages. BuGH30 and BuGH158 were further explored for their roles in digesting β- glucans and generation of oligosaccharides, respectively. The BuGH30 predominately found to cleave long chain β-1-6 linked glucans, and obtained final product was gentiobiose. The BuGH158 used for producing oligosaccharides varying from degree of polymerization 2 to 7 from soluble curdlan. We demonstrated that these oligosaccharides can be utilized by gut bacteria, which either did not grow or poorly grew on laminarin. Thus, B. uniformis JCM 13288^T is not only capable of utilizing β-glucans but also shares these glycans with human gut bacteria for potentially maintaining the gut microbial homeostasis.

The present study describes the heterogeneous carboxymethylation of xylan,α-1,3-glucan, glucomannan, pullulan, curdlan, galactoglucomannan, and agarose with sodium monochloracetate (SMCA) using iso-propanol as slurry medium in the presence of caustic soda. Using heteropolysaccharides for the carboxymethylation, higher DS values are obtained compared to the DS of homopolysaccharides. The influence of the amount caustic soda in the reaction medium is studied. The characterization of the products obtained is performed by means of ¹³C-NMR spectroscopy. Carboxymethylation transforms the investigated polysaccharides into water-soluble products.

A novel β-1,3-1,4-glucanase in the glycoside hydrolase family 5 (GH5) has been identified in the secretome of Paenibacillus polymyxa KF-1. The recombinant GH5 enzyme PpBglu5A shows broad substrate specificity, with strong lichenase activity, medium β-1,3-glucanase activity, and minimal cellulase activity. Barley β-glucan, lichenan, curdlan, and carboxymethyl cellulose are hydrolyzed to varying degrees by PpBglu5A, with the highest catalytic activity being observed with barley β-glucan. Hydrolysates from barley β-glucan or lichenan are primarily glucan oligosaccharides with degrees of polymerization from 2 to 4. PpBglu5A also hydrolyzes oat bran into oligosaccharides mainly consisted of di-, tri-, and tetra- oligosaccharides that are useful in the preparation of gluco-oligosaccharides. In addition to hydrolytic activity, transglycosylation was also observed with PpBglu5A and cellotriose as substrate. An in vitro assay indicated that the recombinant PpBglu5A has antifungal activity and can inhibit the growth of Canidia albicans. These results suggest that PpBglu5A exhibits unique properties and may be useful as an antifungal agent.

An exoglucanase (Exg-D) from the glycoside hydrolase family 5 subfamily 38 (GH5_38) was heterologously expressed and structurally and biochemically characterised at a molecular level for its application in alkyl glycoside synthesis. The purified Exg-D existed in both dimeric and monomeric forms in solution, which showed highest activity on mixed-linked β-glucan (88.0 and 86.7 U/mg protein, respectively) and lichenin (24.5 and 23.7 U/mg protein, respectively). They displayed a broad optimum pH range from 5.5 to 7 and a temperature optimum from 40 to 60 °C. Kinetic studies demonstrated that Exg-D had a higher affinity towards β-glucan, with a k_m of 7.9 mg/mL and a k_cat of 117.2 s^-1, compared to lichenin which had a k_m of 21.5 mg/mL and a k_cat of 70.0 s^-1. The circular dichroism profile of Exg-D showed that its secondary structure consisted of 11% α-helices, 36% β-strands and 53% coils. Exg-D performed transglycosylation using p-nitrophenyl cellobioside as a glycosyl donor and several primary alcohols as acceptors to produce methyl-, ethyl- and propyl-cellobiosides. These products were identified and quantified via thin-layer chromatography (TLC) and liquid chromatography-mass spectrometry (LC-MS). We concluded that Exg-D is a novel and promising oligomeric glycoside hydrolase for the one-step synthesis of alkyl glycosides with more than one monosaccharide unit.

Cellulases play important roles in the dietary fibre digestion in pigs, and have multiple industrial applications. The porcine intestinal microbiota display a unique feature in rapid cellulose digestion. Herein, we have expressed a cellulase gene, p4818Cel5_2A, which singly encoded a catalytic domain belonging to glycoside hydrolase family 5 subfamily 2, and was previously identified from a metagenomic expression library constructed from porcine gut microbiome after feeding grower pigs with a cellulose-supplemented diet. The activity of purified p4818Cel5_2A was maximal at pH 6.0 and 50°C and displayed resistance to trypsin digestion. This enzyme exhibited activities towards a wide variety of plant polysaccharides, including cellulosic substrates of avicel and solka-Floc^®, and the hemicelluloses of β-(1 → 4)/(1 → 3)-glucans, xyloglucan, glucomannan and galactomannan. Viscosity, reducing sugar distribution and hydrolysis product analyses further revealed that this enzyme was a processive endo-β-(1 → 4)-glucanase capable of hydrolyzing cellulose into cellobiose and cellotriose as the primary end products. These catalytic features of p4818Cel5_2A were further explored in the context of a three-dimensional homology model. Altogether, results of this study report a microbial processive endoglucanase identified from the porcine gut microbiome, and it may be tailored as an efficient biocatalyst candidate for potential industrial applications.

Laminarinases exhibit potential in a wide range of industrial applications including the production of biofuels and pharmaceuticals. In this study, we present the genetic and biochemical characteristics of FLamA and FLamB, two laminarinases derived from a metagenomic sample from a hot spring in the Azores. Sequence comparison revealed that both genes had high similarities to genes from Fervidobacterium nodosum Rt17-B1. The two proteins showed sequence similarities of 62% to each other and belong to the glycoside hydrolase (GH) family 16. For biochemical characterization, both laminarinases were heterologously produced in Escherichia coli and purified to homogeneity. FLamA and FLamB exhibited similar properties and both showed highest activity towards laminarin at 90° C and pH 6.5. The two enzymes were thermostable but differed in their half-life at 80° C with 5 h and 1 h for FLamA and FLamB, respectively. In contrast to other laminarinases, both enzymes prefer β-1,3-glucans and mixed-linked glucans as substrates. However, FLamA and FLamB differ in their catalytic efficiency towards laminarin. Structure predictions were made and showed minor differences particularly in a kink adjacent to the active site cleft. The high specific activities and resistance to elevated temperatures and various additives make both enzymes suitable candidates for application in biomass conversion.

Among non-viral vectors, the cationic polymer chitosan has gained attention as a gene delivery system. We hypothesized that the addition of casein into the nanoparticle’s structure would facilitate a proper gene transfer. The work herein presented aimed to optimize the production method of chitosan-casein nanoparticles (ChiCas NPs) and to test their ability as a gene delivery system. ChiCas NPs formulation optimization was carried out by analyzing several characteristics such as NP size, zeta potential, and chitosan and casein incorporation efficacy. The best formulation developed presented small and homogenous particle size (around 335 nm) and positive zeta potential (≈ + 38 mV), and showed to be stable for 34 weeks both, at 4°C and 20°C. The particles were further used to entrap or to adsorb DNA and form NPs-DNA complexes. In vitro transfection studies, carried out in COS-7 cells, suggested a low transfection efficiency of the different NPs:DNA ratios tested, comparatively to the positive control. Nonetheless, we could observe that the complexes with larger sizes presented better transfection results than those with smaller diameters. To conclude, ChiCas NPs have great technological potential since the preparation process is very simple, and the DNA incorporation efficacy is very high and shows to be physically very stable. The NPs:DNA ratio still needs to be optimized with the aim of achieving better transfection results and being able to anticipate a high gene expression on DNA-based vaccination studies.

We cloned two glycoside hydrolase family 74 genes, the sav_1856 gene and the sav_2574 gene, from Streptomyces avermitilis NBRC14893 and characterized the resultant recombinant proteins. The sav_1856 gene product (SaGH74A) consisted of a catalytic domain and a family 2 carbohydrate-binding module at the C terminus, while the sav_2574 gene product (SaGH74B) consisted of only a catalytic domain. SaGH74A and SaGH74B were expressed successfully and had molecular masses of 92 and 78 kDa, respectively. Both recombinant proteins were xyloglucanases. SaGH74A had optimal activity at 60°C and pH 5.5, while SaGH74B had optimal activity at 55°C and pH 6.0. SaGH74A was stable over a broad pH range (pH 4.5 to 9.0), whereas SaGH74B was stable over a relatively narrow pH range (pH 6.0 to 6.5). Analysis of the hydrolysis products of tamarind xyloglucan and xyloglucan-derived oligosaccharides indicated that SaGH74A was endo-processive, while SaGH74B was a typical endo-enzyme. The C terminus of SaGH74A, which was annotated as a carbohydrate-binding module, bound to β-1,4-linked glucan-containing soluble polysaccharides such as hydroxyethyl cellulose, barley glucan, and xyloglucan.

Here we report the cloning of the Pa_3_10940 gene from the coprophilic fungus Podospora anserina, which encodes a C-terminal family 1 carbohydrate binding module (CBM1) linked to a domain of unknown function. The function of the gene was investigated by expression of the full-length protein and a truncated derivative without the CBM1 domain in the yeast Pichia pastoris. Using a library of polysaccharides of different origins, we demonstrated that the full-length enzyme displays activity toward a broad range of β-glucan polysaccharides, including laminarin, curdlan, pachyman, lichenan, pustulan, and cellulosic derivatives. Analysis of the products released from polysaccharides revealed that this β-glucanase is an exo-acting enzyme on β-(1,3)- and β-(1,6)-linked glucan substrates and an endo-acting enzyme on β-(1,4)-linked glucan substrates. Hydrolysis of short β-(1,3), β-(1,4), and β-(1,3)/&beta-(1,4) gluco-oligosaccharides confirmed this striking feature and revealed that the enzyme performs in an exo-type mode on the nonreducing end of gluco-oligosaccharides. Excision of the CBM1 domain resulted in an inactive enzyme on all substrates tested. To our knowledge, this is the first report of an enzyme that displays bifunctional exo-β-(1,3)/(1,6) and endo-β-(1,4) activities toward beta-glucans and therefore cannot readily be assigned to existing Enzyme Commission groups. The amino acid sequence has high sequence identity to hypothetical proteins within the fungal taxa and thus defines a new family of glycoside hydrolases, the GH131 family.

Botryosphaeria rhodina MAMB-05 produced β-1,3-glucanases and botryosphaeran when grown on glucose, while Trichoderma harzianum Rifai only produced the enzyme. A comparison of long-term cultivation (300 h) by B. rhodina demonstrated a correlation between the formation of botryosphaeran (48 h) and its consumption (after 108 h), and de-repression of β-1,3-glucanase synthesis when glucose was depleted from the nutrient medium, whereas for T. harzianum enzyme production commenced during exponential growth. Growth profiles and levels of β-1,3-glucanases produced by both fungi on botryosphaeran also differed, as well as the production of β-1,3-glucanases and β-1,6-glucanases on glucose, lactose, laminarin, botryosphaeran, lasiodiplodan, curdlan, Brewer's yeast powder and lyophilized fungal mycelium, which were dependent upon the carbon source used. A statistical mixture-design used to optimize β-1,3-glucanase production by both fungi evaluated botryosphaeran, glucose and lactose concentrations as variables. For B. rhodina, glucose and lactose promoted enzyme production at the same levels (2.30 U mL^-1), whereas botryosphaeran added to these substrates exerted a synergic effect favorable for β-glucanase production by T. harzianum (4.25 U mL^-1).

There is an increasing interest in the development of therapeutic antibodies (Ab) to improve the control of fungal pathogens, but none of these reagents is available for clinical use. We previously described a murine monoclonal antibody (mAb 2G8) targeting β-glucan, a cell wall polysaccharide common to most pathogenic fungi, which conferred significant protection against Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans in animal models. Transfer of this wide-spectrum, antifungal mAb into the clinical setting would allow the control of most frequent fungal infections in many different categories of patients. To this aim, two chimeric mouse–human Ab derivatives from mAb 2G8, in the format of complete IgG or scFv-Fc, were generated, transiently expressed in Nicotiana benthamiana plants and purified from leaves with high yields (approximately 50 mg Ab/kg of plant tissues). Both recombinant Abs fully retained the β-glucan-binding specificity and the antifungal activities of the cognate murine mAb against C. albicans. In fact, they recognized preferentially β1,3-linked glucan molecules present at the fungal cell surface and directly inhibited the growth of C. albicans and its adhesion to human epithelial cells in vitro. In addition, both the IgG and the scFv-Fc promoted C. albicans killing by isolated, human polymorphonuclear neutrophils in ex vivo assays and conferred significant antifungal protection in animal models of systemic or vulvovaginal C. albicans infection. These recombinant Abs represent valuable molecules for developing novel, plant-derived immunotherapeutics against candidiasis and, possibly, other fungal diseases.

The cell wall of the fruiting body of the mushroom Lentinula edodes is degraded after harvesting by enzymes such as β-1,3-glucanase. In this study, a novel endo-type β-1,3-glucanase, GLU1, was purified from L. edodes fruiting bodies after harvesting. The gene encoding it, glu1, was isolated by rapid amplification of cDNA ends (RACE)-PCR using primers designed from the N-terminal amino acid sequence of GLU1. The putative amino acid sequence of the mature protein contained 247 amino acid residues with a molecular mass of 26 kDa and a pI of 3.87, and recombinant GLU1 expressed in Pichia pastoris exhibited β-1,3-glucanase activity. GLU1 catalyzed depolymerization of glucans composed of β-1,3-linked main chains, and reaction product analysis by thin-layer chromatography (TLC) clearly indicated that the enzyme had an endolytic mode. However, the amino acid sequence of GLU1 showed no significant similarity to known glycoside hydrolases. GLU1 has similarity to several hypothetical proteins in fungi, and GLU1 and highly similar proteins should be classified as a novel glycoside hydrolase family (GH128).

β-glucans of bacterial and fungal origin are known immuno-modulators, but data in the literature also indicate that lichen and cereal-derived β-glucans may have immuno-modulatory functions. The aim of the current study was to test the effect of different sources of β-glucans on neutrophils in an ex-vivo whole blood stimulation assay. Whole blood samples were either treated with curdlan, a linear β-(1→3)-D-glucan from the non-pathogenic Alcaligenes faecalis, lichenan, a mixed linked β-(1→3),(1→4)-D-glucan from Islandic moss (Cetraria islandica) or zymosan, prepared from yeast cell walls and being rich in branched β–(1→3),(1→6)–D-glucan. The blood cell were either stimulated alone or in combination with lipopolysaccharide (LPS) and compared with LPS-treated and untreated control samples. Preliminary results show that Zymosan, had a different effect than lichenan and curdlan, on the surface expression of Toll-like Receptor (TLR) 2 and 4, but not significantly on the signal regulatory protein SIRPα after a stimulation either alone or in combination with LPS. Thus, branching may appear to be important for the different effect, but an effect of impurities in the Zymosan preparation cannot be ruled out.

The plant cell wall is a complex material in which the cellulose microfibrils are embedded within a mesh of other polysaccharides, some of which are loosely termed “hemicellulose.” One such hemicellulose is xyloglucan, which displays a β-1,4-linked D-glucose backbone substituted with xylose, galactose, and occasionally fucose moieties. Both xyloglucan and the enzymes responsible for its modification and degradation are finding increasing prominence, reflecting both the drive for enzymatic biomass conversion, their role in detergent applications, and the utility of modified xyloglucans for cellulose fiber modification. Here we present the enzymatic characterization and three-dimensional structures in ligand-free and xyloglucan-oligosaccharide complexed forms of two distinct xyloglucanases from glycoside hydrolase families GH5 and GH12. The enzymes, Paenibacillus pabuli XG5 and Bacillus licheniformis XG12, both display open active center grooves grafted upon their respective (β/α)₈ and β-jelly roll folds, in which the side chain decorations of xyloglucan may be accommodated. For the β-jelly roll enzyme topology of GH12, binding of xylosyl and pendant galactosyl moieties is tolerated, but the enzyme is similarly competent in the degradation of unbranched glucans. In the case of the (β/α)₈ GH5 enzyme, kinetically productive interactions are made with both xylose and galactose substituents, as reflected in both a high specific activity on xyloglucan and the kinetics of a series of aryl glycosides. The differential strategies for the accommodation of the side chains of xyloglucan presumably facilitate the action of these microbial hydrolases in milieus where diverse and differently substituted substrates may be encountered.

Dectin-1 is a C-type lectin-like receptor on leukocytes that mediates phagocytosis and inflammatory mediator production in innate immunity to fungal pathogens. Dectin-1 lacks residues involved in calcium ligation that mediates carbohydrate-binding by classical C-type lectins; nevertheless, it binds zymosan, a particulate β-glucan-rich extract of Saccharomyces cerevisiae, and binding is inhibited by polysaccharides rich in β1,3- or both β1,3- and β1,6-linked glucose. The oligosaccharide ligands on glucans recognized by Dectin-1 have not yet been delineated precisely. It is also not known whether Dectin-1 can interact with other types of carbohydrates. We have investigated this, since Dectin-1 shows glucan-independent binding to a subset of T-lymphocytes and is involved in triggering their proliferation. Here we assign oligosaccharide ligands for Dectin-1 using the neoglycolipid-based oligosaccharide microarray technology, a unique approach for constructing microarrays of lipid-linked oligosaccharide probes from desired sources. We generate “designer” microarrays from three glucan polysaccharides, a neutral soluble glucan isolated from S. cerevisiae and two bacterial glucans, curdlan from Alcaligenes faecalis and pustulan from Umbilicaria papullosa, and use these in conjunction with 187 diverse, sequence-defined, predominantly mammalian-type, oligosaccharide probes. Among these, Dectin-1 binding is detected exclusively to 1,3-linked glucose oligomers, the minimum length required for detectable binding being a 10- or 11-mer. Thus, the ligands assigned so far are exogenous rather than endogenous. We further show that Dectin-1 ligands, 11-13 gluco-oligomers, in clustered form (displayed on liposomes), mimic the macromolecular β-glucans and compete with zymosan binding and triggering of tumor necrosis factor-α secretion by a Dectin-1-expressing macrophage cell line.

Polysaccharide components in hot-water extract of Ganoderma lucidum fruiting bodies were separated and their molecular weights and distribution profiles were measured using high-performance size-exclusion chromatography with multi-angle laser light scattering detection (HPSEC-MALLS). Three polysaccharide groups were found in the extract with a weight percentage of 31, 46 and 23% respectively. The weight-average molecular weights of these groups were 2.08 x 10⁶, 2.3 x 10⁴, and 1.2 x 10⁴ g/mol, respectively. The representative fractions of these 3 groups were obtained using differential precipitation with ethanol at 20, 60 and 80% (v/v) concentrations for further property study. The first group that had the largest molecular size was branched (1→3)-β-D-glucan with single β-D glucosyl side chains at the O-6 positions, as revealed from sugar composition analysis, aniline blue assay and ¹H- and ¹³C-NMR spectroscopy. (1→3, 1→6)-β-D-glucan was the major active polysaccharide and showed significant tumor necrosis factor-α (TNF-α) releasing stimulation activity from human mononuclear cells (MNC). The glucan was slightly soluble in water at ambient temperature. The second and third groups that showed slight modulating activity consisted of D-glucose, D-galactose and D-mannose at different ratios. Aggregation of polysaccharide molecules was also revealed by HPSEC-MALLS study.

The eglA gene, encoding a thermostable endoglucanase from the hyperthermophilic archaeon Pyrococcus furiosus, was cloned and expressed in Escherichia coli. The nucleotide sequence of the gene predicts a 319-amino-acid protein with a calculated molecular mass of 35.9 kDa. The endoglucanase has a 19-amino-acid signal peptide but not cellulose-binding domain. The P. furiosus endoglucanase has significant amino acid sequence similarities, including the conserved catalytic nucleophile and proton donor, with endoglucanases from glucosyl hydrolase family 12. The purified recombinant enzyme hydrolyzed β-1,4 but not β-1,3 glucosidic linkages and had the highest specific activity on cellopentaose (degree of polymerization [DP] = 5) and cellohexaose (DP = 6) oligosaccharides. To a lesser extent, EglA also hydrolyzed shorter cellodextrins (DP < 5) as well as the amorphous portions of polysaccharides which contain only β-1,4 bonds such as carboxymethyl cellulose, microcrystalline cellulose, Whatman paper, and cotton linter. The highest specific activity toward polysaccharides occurred with mixed-linkage β-glucans such as barley β-glucan and lichenan. Kinetics studies with cellooliogsaccharides and p-nitrophenyl-cellooligosaccharides indicated that the enzyme had three glucose binding subsites (−I, −II, and −III) for the nonreducing end and two glucose binding subsites (+I and +II) for the reducing end from the scissile glycosidic linkage. The enzyme had temperature and pH optima of 100°C and 6.0, respectively; a half-life of 40 h at 95°C; and a denaturing temperature of 112°C as determined by differential scanning calorimetry. The discovery of a thermostable enzyme with this substrate specificity has implications for both the evolution of enzymes involved in polysaccharide hydrolysis and the occurrence of growth substrates in hydrothermal vent environments.