cellulase and laminaran

In this study, we characterized Gly5M, originating from a marine bacterium, as a novel β-1,3-1,6-endoglucanase in glycoside hydrolase family 5 (GH5) in the Carbohydrate-Active enZyme database. The gly5M gene encodes Gly5M, a newly characterized enzyme from GH5 subfamily 47 (GH5_47) in Saccharophagus degradans 2-40(T) The gly5M gene was cloned and overexpressed in Escherichia coli Through analysis of the enzymatic reaction products by thin-layer chromatography, high-performance liquid chromatography, and matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry, Gly5M was identified as a novel β-1,3-endoglucanase (EC 3.2.1.39) and bacterial β-1,6-glucanase (EC 3.2.1.75) in GH5. The β-1,3-endoglucanase and β-1,6-endoglucanase activities were detected by using laminarin (a β-1,3-glucan with β-1,6-glycosidic linkages derived from brown macroalgae) and pustulan (a β-1,6-glucan derived from fungal cell walls) as the substrates, respectively. This enzyme also showed transglycosylase activity toward β-1,3-oligosaccharides when laminarioligosaccharides were used as the substrates. Since laminarin is the major form of glucan storage in brown macroalgae, Gly5M could be used to produce glucose and laminarioligosaccharides, using brown macroalgae, for industrial purposes.. In this study, we have discovered a novel β-1,3-1,6-endoglucanase with a unique transglycosylase activity, namely, Gly5M, from a marine bacterium, Saccharophagus degradans 2-40(T) Gly5M was identified as the newly found β-1,3-endoglucanase and bacterial β-1,6-glucanase in GH5. Gly5M is capable of cleaving glycosidic linkages of both β-1,3-glucans and β-1,6-glucans. Gly5M also possesses a transglycosylase activity toward β-1,3-oligosacchrides. Due to the broad specificity of Gly5M, this enzyme can be used to produce glucose or high-value β-1,3- and/or β-1,6-oligosaccharides.

Topics: Cellulase; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Cloning, Molecular; Escherichia coli; Gammaproteobacteria; Gene Expression; Glucans; Hydrolysis; Polysaccharides; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Substrate Specificity

Although many endo-ß-1,4-glucanases have been isolated in invertebrates, their cellulolytic systems are not fully understood. In particular, gastropod feeding on seaweed is considered an excellent model system for production of bioethanol and renewable bioenergy from third-generation feedstocks (microalgae and seaweeds). In this study, enzymes involved in the conversion of cellulose and other polysaccharides to glucose in digestive fluids of the sea hare (Aplysia kurodai) were screened and characterized to determine how the sea hare obtains glucose from sea lettuce (Ulva pertusa). Four endo-ß-1,4-glucanases (21K, 45K, 65K, and 95K cellulase) and 2 ß-glucosidases (110K and 210K) were purified to a homogeneous state, and the synergistic action of these enzymes during cellulose digestion was analyzed. All cellulases exhibited cellulase and lichenase activities and showed distinct cleavage specificities against cellooligosaccharides and filter paper. Filter paper was digested to cellobiose, cellotriose, and cellotetraose by 21K cellulase, whereas 45K and 65K enzymes hydrolyzed the filter paper to cellobiose and glucose. 210K ß-glucosidase showed unique substrate specificity against synthetic and natural substrates, and 4-methylumbelliferyl (4MU)-ß-glucoside, 4MU-ß-galactoside, cello-oligosaccharides, laminarin, and lichenan were suitable substrates. Furthermore, 210K ß-glucosidase possesses lactase activity. Although ß-glucosidase and cellulase are necessary for efficient hydrolysis of carboxymethylcellulose to glucose, laminarin is hydrolyzed to glucose only by 210K ß-glucosidase. Kinetic analysis of the inhibition of 210K ß-glucosidase by D-glucono-1,5-lactone suggested the presence of 2 active sites similar to those of mammalian lactase-phlorizin hydrolase. Saccharification of sea lettuce was considerably stimulated by the synergistic action of 45K cellulase and 210K ß-glucosidase. Our results indicate that 45K cellulase and 210K ß-glucosidase are the core components of the sea hare digestive system for efficient production of glucose from sea lettuce. These findings contribute important new insights into the development of biofuel processing biotechnologies from seaweed.

Topics: Amino Acid Sequence; Animals; Aplysia; Aquatic Organisms; beta-Glucosidase; Cellobiose; Cellulase; Cellulose; Enzyme Assays; Gastrointestinal Tract; Glucans; Gluconates; Glucose; Glucosides; Kinetics; Lactones; Molecular Sequence Data; Polysaccharides; Substrate Specificity; Tetroses; Ulva

A series of β-(1,3)-d-glucans have been synthesized incorporating structural variations specifically on the reducing end of the oligomers. Both O- and C-glucosides derived from di- and trisaccharides have been obtained in good overall yields and with complete selectivity. Whereas the O-glycosides were obtained via a classical Koenigs-Knorr glycosylation, the corresponding C-glycosides were obtained through allylation of the anomeric carbon and further cross-metathesis reaction. Finally, the compounds were evaluated against two glycosidases and two endo-glucanases and no inhibitory activity was observed.

Topics: Carbohydrate Sequence; Cellulase; Chemistry Techniques, Synthetic; Clostridium thermocellum; Disaccharides; Enzyme Inhibitors; Glucans; Glycoside Hydrolases; Glycosides; Glycosylation; Hordeum; Hydrophobic and Hydrophilic Interactions; Molecular Sequence Data; Monosaccharides; Polysaccharides; Structure-Activity Relationship; Trisaccharides

Clostridium thermocellum, an anaerobic, thermophilic, and ethanogenic bacterium produces a large cellulase complex termed the cellulosome and many free glycosyl hydrolases. Most cellulase genes scatter around the genome. We mapped the transcripts of the six-gene cluster celC-glyR3-licA-orf4-manB-celT and determined their transcription initiation sites by primer extension. Northern blot showed that celC-glyR3-licA were co-transcribed into a polycistronic messenger with the transcription initiation site at -20 bp. Furthermore, RT-PCR mapping showed that manB and celT, two cellulosomal genes immediately downstream, were co-transcribed into a bicistronic messenger with the initiation site at -233 bp. In contrast, rf4 was transcribed alone with the two initiation sites at -130 and -138 bp, respectively. Finally, quantitative RT-PCR analysis showed that celC, glyR3, and licA were coordinately induced by growing on laminarin, a β-1,3 glucan. Gene expression peaked at the late exponential phase. Taking together with our previous report that GlyR3 binds to the celC promoter in the absence of laminaribiose, a β-1,3 glucose dimer, these results indicate that celC, glyR3, and licA form an operon repressible by GlyR3 and inducible by laminaribiose, signaling the availability of β-1,3 glucan. The celC operon is the first glycosyl hydrolase operon reported in this bacterium.

Topics: Bacterial Proteins; Base Sequence; beta-Glucosidase; Cellulase; Clostridium thermocellum; Disaccharides; Gene Expression Regulation, Bacterial; Genes, Bacterial; Glucans; Mannose-6-Phosphate Isomerase; Molecular Sequence Data; Multienzyme Complexes; Multigene Family; N-Glycosyl Hydrolases; Nucleotidyltransferases; Operon; Polysaccharides; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic

In the horseshoe crab, the recognition of beta-1,3-D-glucans by factor G triggers hemolymph coagulation. Factor G contains a domain of two tandem xylanase Z-like modules (Z1-Z2), each of which recognizes beta-1,3-D-glucans. To gain an insight into the recognition of beta-1,3-D-glucans from a structural view point, recombinants of Z1-Z2, the C-terminal module Z2, Z2 with a Cys to Ala substitution (Z2A), and its tandem repeat Z2A-Z2A were characterized. Z2 and Z1-Z2, but not Z2A and Z2A-Z2A, formed insoluble aggregates at higher concentrations more than approximately 30 and 3 microM, respectively. Z1-Z2 and Z2A-Z2A bound more strongly to an insoluble beta-1,3-D-glucan (curdlan) than Z2A. The affinity of Z2A for a soluble beta-1,3-D-glucan (laminarin) was equivalent to those of Z1-Z2, Z2A-Z2A, and native factor G, suggesting that the binding of a single xylanase Z-like module prevents the subsequent binding of another module to laminarin. Interestingly, Z2A as well as intact factor G exhibited fungal agglutinating activity, and fungi were specifically detected with fluorescently tagged Z2A by microscopy. The chemical shift perturbation of Z2A induced by the interaction with laminaripentaose was analyzed by nuclear magnetic resonance spectroscopy. The ligand-binding site of Z2A was located in a cleft on a beta-sheet in a predicted beta-sandwich structure, which was superimposed onto cleft B in a cellulose-binding module of endoglucanase 5A from the soil bacterium Cellvibrio mixtus. We conclude that the pattern recognition for beta-1,3-D-glucans by factor G is accomplished via a carbohydrate-binding cleft that is evolutionally conserved between horseshoe crab and bacteria.

Topics: Animals; beta-Glucans; Binding Sites; Cellulase; Cellvibrio; Conserved Sequence; Endo-1,4-beta Xylanases; Evolution, Molecular; Glucans; Horseshoe Crabs; Lectins; Polysaccharides; Proteoglycans

A new method for extracting soil enzymes is described and a microplate method for assaying soil beta-1,4-glucanases (cellulases) and beta-1,3-glucanases (laminarinases). Soil samples were mechanically disrupted to produce crude enzyme extracts, and diluted preps incubated in microplates containing either carboxymethyl cellulose (CMC) to determine cellulase activity or laminarin substrate to determine laminarinase activity. The resulting glucose was measured using the fluorometric Amplex Red glucose assay. The method was reproducible, could be completed in 1 day and measured twice as much enzyme activity than the standard passive soil enzyme extraction procedure. The method described herein facilitates the development of high-throughput soil multiplex enzymatic assays from several soil samples at one time, and is well suited to the study of functional microbial ecology.

Topics: Carboxymethylcellulose Sodium; Cellulase; Cellulases; Glucans; Glucose; Polysaccharides; Reproducibility of Results; Sensitivity and Specificity; Soil

Hydrolysis of plant cell wall polysaccharides, a process which is of intrinsic biological and biotechnological importance, requires the concerted action of an extensive repertoire of microbial cellulases and hemicellulases. Here, we report the identification of the gene cluster unk16A, regA and cel5B in the aerobic soil bacterium Cellvibrio mixtus, encoding a family 16 (CmUnk16A) glycoside hydrolase (GH), an AraC/XylS transcription activator (CmRegA) and a family 5 (CmCel5B) endo-glucanase, respectively. CmUnk16A is a modular enzyme comprising, in addition to the catalytic domain, two family 32 carbohydrate-binding modules (CBMs), termed CBM32-1 and CBM32-2, a CBM4 and a domain of unknown function. We show that CBM32-2 binds weakly to laminarin and pustulan. CmRegA is also a modular protein containing a highly hydrophobic N-terminal domain and a C-terminal DNA-binding domain of the AraC/XylS family. The role of the identified enzymes in the hydrolysis of cell wall polysaccharides by aerobic bacteria is discussed.

Topics: Amino Acid Sequence; Bacterial Proteins; Cell Wall; Cellulase; Cellvibrio; Cloning, Molecular; Glucans; Molecular Sequence Data; Multigene Family; Polysaccharides; Sequence Alignment

Two endoglucanases (EGs), EG A and EG B, were purified to homogeneity from Penicillium occitanis mutant Pol 6 culture medium. The molecular weights of EG A and EG B were 31,000 and 28,000 kDa, respectively. The pI was about 3 for EG A and 7.5 for EG B. Optimal activity was obtained at pH 3.5 for both endoglucanases. Optimal temperature for enzyme activity was 60 degrees C for EG A and 50 degrees C for EG B. EG A was thermostable at 60 degrees C and remained active after 1 h at 70 degrees C. EGs hydrolyzed carboxymethylcellulose, phosphoric acid swollen cellulose, and beta-glucan efficiently, whereas microcrystalline cellulose (Avicel) and laminarin were poorly hydrolyzed. Only EG B showed xylanase activity. Furthermore, these EGs were insensitive to the action of glucose and cellobiose but were inhibited by the divalent cations Hg2+, Co2+, and Mn2+.

Topics: beta-Glucans; Carboxymethylcellulose Sodium; Cations, Divalent; Cellulase; Cellulose; Chromatography, Gel; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Glucans; Hydrogen-Ion Concentration; Isoelectric Point; Kinetics; Molecular Weight; Mutation; Penicillium; Polysaccharides; Substrate Specificity; Temperature; Xylans

In this paper, we present the first detailed analysis of the modes of action of three purified, thermostable endo-beta-D-glucanases (EG V-VII) against a range of soluble beta-linked glucans. Studies indicated that EG V-VII, purified to homogeneity from a new source, the thermophilic fungus Talaromyces emersonii, are strict beta-glucanases that exhibit maximum activity against mixed-link 1,3;1,4-beta-D-glucans. Time-course hydrolysis studies of 1,4-beta-D-glucan (carboxymethylcellulose; CMC), 1,3;1,4-beta-D-glucan from barley (BBG) and lichenan confirmed the endo-acting nature of EG V-VII and verified preference for 1,3;1,4-beta-D-glucan substrates. The results suggest that EG VI and EG VII belong to EC 3.2.1.6, as both enzymes also exhibit activity against 1,3-beta-glucan (laminaran), in contrast to EG V. Although cellobiose, cellotriose and glucose were the main glucooligosaccharide products released, the range and relative amount of each product was dependent on the particular enzyme, substrate and reaction time. Kinetic constants (Km, Vmax, kcat and kcat/Km) determined for EG V-VII with BBG as substrate yielded similar Km and Vmax values for EG V and EG VI. EG VII exhibited highest affinity for BBG (Km value of 9.1 mg ml(-1)) and the highest catalytic efficiency (kcat/Km of 12.63 s(-1) mg(-1) ml).

Topics: Carbohydrate Conformation; Carboxymethylcellulose Sodium; Catalysis; Cellulase; Chromatography, Ion Exchange; Glucans; Glycoside Hydrolases; Hordeum; Kinetics; Polysaccharides; Solubility; Substrate Specificity; Talaromyces; Viscosity

The secretion of nonstarchy polysaccharide-degrading enzymes from an anaerobic human intestinal bacterium, Clostridium butyricum- beijerinckii (isolated from human feces), was investigated. Growth of the bacterium was found when laminarin, konjac glucomannan, and pectic acid were added separately to the culture media as sole carbon source. The corresponding degrading enzymes for these dietary fibers, laminarinase (endo-1,3- beta-glucanase), endo-1,4-beta-mannanase, endo- and exo-pectate lyases, and pectin methylesterase, were then purified and characterized. These extracelluar enzymes, which were secreted by the bacterium in the human large intestine, were considered to contribute to digestion of the ingested dietary fibers to their oligosaccharides, following by short-chain fatty acid fermentation by the bacterium. We have developed cell immobilization techniques of the bacterium on cellulose-foam carriers that are effective for continuous production of the oligosaccharides from the dietary fibers in a fed-batch reactor system. From 9 g of pectic acid, a total of 3.96 g of 4,5-unsaturated digalacturonic acid was produced over 40 h in four 500-ml batchcultures. In the same manner, the corresponding oligosaccharides were obtained from konjac glucomannan and laminarin with average conversion rates of around 30-40%.

Topics: Adsorption; Amino Acid Sequence; Cells, Immobilized; Cellulase; Clostridium; Dietary Fiber; Glucans; Humans; Hydrogen-Ion Concentration; Intestines; Mannans; Molecular Sequence Data; Oligosaccharides; Polysaccharides

We report here the first molecular characterization of an endo-beta-1,3-glucanase from an archaeon. Pyrococcus furiosus is a hyperthermophilic archaeon that is capable of saccharolytic growth. The isolated lamA gene encodes an extracellular enzyme that shares homology with both endo-beta-1,3- and endo-beta-1,3-1,4-glucanases of the glycosyl hydrolase family 16. After deletion of the N-terminal leader sequence, a lamA fragment encoding an active endo-beta-1,3-glucanase was overexpressed in Escherichia coli using the T7-expression system. The purified P. furiosus endoglucanase has highest hydrolytic activity on the beta-1,3-glucose polymer laminarin and has some hydrolytic activity on the beta-1,3-1,4 glucose polymers lichenan and barley beta-glucan. The enzyme is the most thermostable endo-beta-1,3-glucanase described up to now; it has optimal activity at 100-105 degrees C. In the predicted active site of glycosyl hydrolases of family 16 that show predominantly endo-beta-1,3-glucanase activity, an additional methionine residue is present. Deletion of this methionine did not change the substrate specificity of the endoglucanase, but it did cause a severe reduction in its catalytic activity, suggesting a structural role of this residue in constituting the active site. High performance liquid chromatography analysis showed in vitro hydrolysis of laminarin by the endo-beta-1,3-glucanase proceeds more efficiently in combination with an exo-beta-glycosidase from P. furiosus (CelB). This most probably reflects the physiological role of these enzymes: cooperation during growth of P. furiosus on beta-glucans.

Topics: Amino Acid Sequence; Cellulase; Cloning, Molecular; Fermentation; Glucan Endo-1,3-beta-D-Glucosidase; Glucans; Hydrogen-Ion Concentration; Molecular Sequence Data; Mutagenesis, Site-Directed; Polysaccharides; Pyrococcus; Sequence Alignment; Sequence Homology, Amino Acid; Substrate Specificity; Temperature

The deduced amino acid sequence derived from a Macrophomina phaseolina beta-1,4-endoglucanase-encoding gene revealed 48% identity (over 119 amino acids) with egl1 from the phytopathogen Pseudomonas solanacearum. Its similarity to saprophyte endoglucanases was not significant. Its minimum substrate size, unlike that of any known saprophyte endoglucanase, was cellopentaose. The unique characteristics of M. phaseolina egl1-encoded endoglucanase suggest that it is phytopathogen specific.

Topics: Amino Acid Sequence; Base Sequence; Cellulase; DNA, Complementary; Fungal Proteins; Galactans; Genes, Fungal; Glucans; Mitosporic Fungi; Molecular Sequence Data; Polysaccharides; Sequence Alignment; Sequence Homology, Amino Acid; Substrate Specificity; Xylans

Two genes encoding beta-1,3 glucanase activity were cloned from the gram-negative soil bacterium Cellvibrio mixtus. The two clones, designated cwd (cell wall degradation) and lam (laminarin degradation), had distinct endonuclease restriction patterns and encoded enzymes with distinct substrate specificities. The 3.7-kilobase cwd insert encoded an enzyme which degraded yeast cell walls as well as the soluble beta-1,3 glucan laminarin and the insoluble beta-1,3 glucans zymosan and pachyman. The 1.8-kilobase lam insert encoded an enzyme which degraded laminarin only. Both enzymes degraded laminarin in an endohydrolytic manner to yield laminarobiose, laminarotriose, and laminarotetraose as major end products. Radiolabeled translation products of the cwd and lam transcripts were identified.

Topics: Bacterial Proteins; Cellulase; Genes, Bacterial; Glucans; Gram-Negative Aerobic Bacteria; Polysaccharides; Recombinant Fusion Proteins; Substrate Specificity; Zymosan

A cel gene from Bacteroides succinogenes inserted into the vector pUC8 coded for an enzyme which exhibited high hydrolytic activity on carboxymethylcellulose, p-nitrophenylcellobioside, and lichenan and low activity on laminarin and xylan. The enzyme was not synthesized by the Escherichia coli host when cells were cultured in complex medium containing added glucose. In the absence of added glucose, the endoglucanase and cellobiosidase activities synthesized were partitioned into the periplasmic space during growth, and practically all enzyme was located in the periplasm when the stationary phase of growth was reached. The enzyme exhibited 17- and sixfold higher Km values for the hydrolysis of carboxymethylcellulose and lichenan, respectively, than did the extracellular endoglucanase complex from B. succinogenes. The Cel endoglucanase had a pH optimum similar to that of the B. succinogenes enzyme except that the range was narrower, and the Cel endoglucanase was more readily inactivated on exposure to high temperature, detergents, and certain metals. Its activity was stimulated by calcium and magnesium. Nondenaturing polyacrylamide gel electrophoresis at different acrylamide concentrations revealed the presence of three endoglucanase components, two with molecular weights of 43,000 and one with a molecular weight of 55,000.

Topics: Bacteroides; Carboxymethylcellulose Sodium; Cellulase; Cellulose; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Genes, Bacterial; Glucans; Glucosides; Hydrogen-Ion Concentration; Molecular Weight; Polysaccharides; Xylans