cellulase and cellotetraose

Exploring new multifunctional enzymes and understanding the mechanisms of catalytic promiscuity will be of enormous industrial and academic values. In the present study, we reported the discovery and characterization of a multifunctional enzyme BSGH13 from Bacillus subtilis BS-5. Remarkably, BSGH13 possessed α-amylase, endoglucanase, and xylanase activities. To our knowledge, this was the first report on an amylase from Bacillus species having additional endoglucanase and xylanase activities. Subsequently, we analyzed the effects of aromatic residues substitution at each site of the active site architecture on ligand-binding affinity and catalytic specificity of BSGH13 by a combination of virtual mutation and site-directed mutagenesis approaches. Our results indicated that the introduction of aromatic amino acids Phe or Trp at the positions L182 and L183 altered the local interaction network of BSGH13 towards different substrates, thus changing the multifunctional properties of BSGH13. Moreover, we provided an expanded perspective on studies of multifunctional enzymes.

Topics: alpha-Amylases; Amino Acid Substitution; Bacillus subtilis; Bacterial Proteins; Catalytic Domain; Cellulase; Cellulose; Cloning, Molecular; Endo-1,4-beta Xylanases; Escherichia coli; Gene Expression; Genetic Vectors; Kinetics; Maltose; Models, Molecular; Mutation; Phenylalanine; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Recombinant Proteins; Structural Homology, Protein; Structure-Activity Relationship; Substrate Specificity; Tetroses; Tryptophan; Xylans

Topics: Amino Acid Sequence; Bacillus subtilis; Bacterial Proteins; Biomass; Cellulase; Cellulose; Cloning, Molecular; Enzyme Stability; Glucans; Hydrolysis; Plant Stems; Protein Structure, Tertiary; Recombinant Proteins; Soil Microbiology; Substrate Specificity; Tetroses

Lignocellulose feedstock constitutes the most abundant carbon source in the biosphere; however, its recalcitrance remains a challenge for microbial conversion into biofuel and bioproducts. Bacillus licheniformis is a microbial mesophilic bacterium capable of secreting a large number of glycoside hydrolase (GH) enzymes, including a glycoside hydrolase from GH family 9 (BlCel9). Here, we conducted biochemical and biophysical studies of recombinant BlCel9, and its low-resolution molecular shape was retrieved from small angle X-ray scattering (SAXS) data. BlCel9 is an endoglucanase exhibiting maximum catalytic efficiency at pH 7.0 and 60 °C. Furthermore, it retains 80% of catalytic activity within a broad range of pH values (5.5-8.5) and temperatures (up to 50 °C) for extended periods of time (over 48 h). It exhibits the highest hydrolytic activity against phosphoric acid swollen cellulose (PASC), followed by bacterial cellulose (BC), filter paper (FP), and to a lesser extent carboxymethylcellulose (CMC). The HPAEC-PAD analysis of the hydrolytic products demonstrated that the end product of the enzymatic hydrolysis is primarily cellobiose, and also small amounts of glucose, cellotriose, and cellotetraose are produced. SAXS data analysis revealed that the enzyme adopts a monomeric state in solution and has a molecular mass of 65.8 kDa as estimated from SAXS data. The BlCel9 has an elongated shape composed of an N-terminal family 3 carbohydrate-binding module (CBM3c) and a C-terminal GH9 catalytic domain joined together by 20 amino acid residue long linker peptides. The domains are closely juxtaposed in an extended conformation and form a relatively rigid structure in solution, indicating that the interactions between the CBM3c and GH9 catalytic domains might play a key role in cooperative cellulose biomass recognition and hydrolysis.

Topics: Bacillus licheniformis; Catalysis; Cellobiose; Cellulase; Cellulose; Glucose; Glycoside Hydrolases; Hydrogen-Ion Concentration; Lignin; Scattering, Small Angle; Tetroses; Trioses; X-Ray Diffraction

PoCel12A, PoCel12B, and PoCel12C are genes that encode glycoside hydrolase family 12 (GH12) enzymes in Penicillium oxalicum. PoCel12A and PoCel12B are typical GH12 enzymes that belong to fungal subfamilies 12-1 and 12-2, respectively. PoCel12C contains a low-complexity region (LCR) domain, which is not found in PoCel12A or PoCel12B and independent of fungal subfamily 12-1 or 12-2. Recombinant enzymes (named rCel12A, rCel12B and rCel12C) demonstrate existing diversity in the substrate specificities. Although most members in GH family 12 are typical endoglucanases and preferentially hydrolyze β-1,4-glucan (e.g., carboxymethylcellulose), recombinant PoCel12A is a non-typical endo-(1-4)-β-glucanase; it preferentially hydrolyzes mix-linked β-glucan (barley β-glucan, β-1,3-1,4-glucan) and slightly hydrolyzes β-1,4-glucan (carboxymethylcellulose). Recombinant PoCel12B possesses a significantly high activity against xyloglucan. A specific activity of rCel12B toward xyloglucan (239 µmol/min/mg) is the second-highest value known. Recombinant PoCel12C shows low activity toward β-glucan, carboxymethylcellulose, or xyloglucan. All three enzymes can degrade phosphoric acid-swollen cellulose (PASC). However, the hydrolysis products toward PASC by enzymes are different: the main hydrolysis products are cellotriose, cellotetraose, and cellobiose for rCel12A, rCel12B, and rCel12C, correspondingly. A synergistic action toward PASC among rCel12A and rCel12B is observed, thereby suggesting a potential application for preparing enzyme cocktails used in lignocellulose hydrolysis.

Topics: beta-Glucans; Cellulase; Cellulose; Glucans; Glycoside Hydrolases; Hydrogen-Ion Concentration; Hydrolysis; Lignin; Penicillium; Phylogeny; Substrate Specificity; Tetroses; Trioses; Xylans

Optimal catalytic activity of endoglucanase Cel5D from the thermophilic anaerobic bacterium Caldicellulosiruptor bescii requires the presence of a carbohydrate-binding module of family 28, CbCBM28. The binding properties of CbСВМ28 with cello-, laminari-, xylo- and chito-oligosaccharides were studied by isothermal titration calorimetry. CbСВМ28 bound only cello-oligosaccharides comprising at least four glucose residues with binding constants of 2.5·10

Topics: Binding Sites; Calorimetry; Cellulase; Cellulose; Crystallins; Firmicutes; Glucose; Hydrogen-Ion Concentration; Oligosaccharides; Tetroses

Economic sustainability of modern biochemical technologies for plant cell wall transformations in renewable fuels, green chemicals, and sustainable materials is considerably impacted by the elevated cost of enzymes. Therefore, there is a significant drive toward discovery and characterization of novel carbohydrate-active enzymes. Here, the BlCel48 cellulase from Bacillus licheniformis, a glycoside hydrolase family 48 member (GH48), was functionally and biochemically characterized. The enzyme is catalytically stable in a broad range of temperatures and pH conditions with its enzymatic activity at pH5.0 and 60°C. BlCel48 exhibits high hydrolytic activity against phosphoric acid swollen cellulose (PASC) and bacterial cellulose (BC) and significantly lower activity against carboxymethylcellulose (CMC). BlCel48 releases predominantly cellobiose, and also small amounts of cellotriose and cellotetraose as products from PASC hydrolysis. Small-angle X-ray scattering (SAXS) data analysis revealed a globular molecular shape and monomeric state of the enzyme in solution. Its molecular mass estimated based on SAXS data is ~77.2kDa. BlCel48 has an (αα)

Topics: Bacillus licheniformis; Carboxymethylcellulose Sodium; Cellulase; Cellulose; Hydrolysis; Kinetics; Scattering, Small Angle; Substrate Specificity; Tetroses; X-Ray Diffraction

Endo-ß-1,4-glucanase AkEG21 belonging to glycosyl hydrolase family 45 (GHF45) is the most abundant cellulase in the digestive fluid of sea hare (Aplysia kurodai). The specific activity of this 21-kDa enzyme is considerably lower than those of other endo ß-1,4-glucanases in the digestive fluid of A. kurodai, therefore its role in whole cellulose hydrolysis by sea hare is still uncertain. Although AkEG21 has a catalytic domain without a cellulose binding domain, it demonstrated stable binding to cellulose fibers, similar to that of fungal cellobiohydrolase (CBH) 1 and CBH 2, which is strongly inhibited by cellohexaose, suggesting the involvement of the catalytic site in cellulose binding. Cellulose-bound AkEG21 hydrolyzed cellulose to cellobiose, cellotriose and cellotetraose, but could not digest an external substrate, azo-carboxymethyl cellulose. Cellulose hydrolysis was considerably stimulated by the synergistic action of cellulose-bound AkEG21 and AkEG45, another ß-1,4-endoglucanase present in the digestive fluid of sea hare; however no synergy in carboxymethylcellulose hydrolysis was observed. When AkEG21 was removed from the digestive fluid by immunoprecipitation, the cellulose hydrolyzing activity of the fluid was significantly reduced, indicating a critical role of AkEG21 in cellulose hydrolysis by A. kurodai. These findings suggest that AkEG21 is a processive endoglucanase functionally equivalent to the CBH, which provides a CBH-independent mechanism for the mollusk to digest seaweed cellulose to glucose.

Topics: Animals; Aplysia; Catalytic Domain; Cellobiose; Cellulase; Cellulose; Digestion; Glucose; Hydrolysis; Kinetics; Oligosaccharides; Protein Binding; Protein Domains; Tetroses

The sco0765 gene was annotated as a glycosyl hydrolase family 5 endoglucanase from the genomic sequence of Streptomyces coelicolor A3(2) and consisted of 2,241 bp encoding a polypeptide of 747 amino acids (molecular weight of 80.5 kDa) with a 29-amino acid signal peptide for secretion. The SCO0765 recombinant protein was heterogeneously over-expressed in Streptomyces lividans TK24 under the control of a strong ermE* promoter. The purified SCO0765 protein showed the expected molecular weight of the mature form (718 aa, 77.6 kDa) on sodium dodecyl sulfate-polyacryl amide gel electrophoresis. SCO0765 showed high activity toward β-glucan and carboxymethyl cellulose (CMC) and negligible activity to Avicel, xylan, and xyloglucan. The SCO0765 cellulase had a maximum activity at pH 6.0 and 40°C toward CMC and at pH 9.0 and 50-60°C toward β-glucan. Thin layer chromatography of the hydrolyzed products of CMC and β-glucan by SCO0765 gave cellotriose as the major product and cellotetraose, cellopentaose, and longer oligosaccharides as the minor products. These results clearly demonstrate that SCO0765 is an endo-β-1,4-cellulase, hydrolyzing the β-1,4 glycosidic bond of cellulose into cellotriose.

Topics: beta-Glucans; Cellulase; Cellulose; Enzyme Stability; Fungal Proteins; Hydrogen-Ion Concentration; Kinetics; Molecular Weight; Streptomyces coelicolor; Substrate Specificity; Tetroses

Ionic liquids (ILs), that is, salts with melting points <100°C, have recently attracted a lot of attention in biomass processing due to their ability to dissolve lignocellulosics. In this work, we studied how two imidazolium-based, hydrophilic, cellulose dissolving ionic liquids 1,3-dimethylimidazolium dimethylphosphate [DMIM]DMP and 1-ethyl-3-methylimidazolium acetate [EMIM]AcO affect the usually employed analytical methods for mono- and oligosaccharides, typical products from hydrolytic treatments of biomass. HPLC methods were severely hampered by the presence of ILs with loss of separation power and severe baseline problems, making their use for saccharide quantification extremely challenging. Problems in DNS photometric assay and chromatography were also encountered at high ionic liquid concentrations and many capillary electrophoresis (CE) methods did not allow an efficient analysis of saccharides in these matrices. In this paper we describe an optimized CE method with pre-column derivatization for the qualitative and quantitative analysis of mono- and oligosaccharides in sample matrices containing moderate (20-40% (v/v)) concentrations of ILs. The IL content and type in the sample matrix was found to affect both peak shape and quantification parameters. Generally, the presence of high IL concentrations (≥20% (v/v)) had a dampening effect on the detection of the analytes. IL in lower concentrations of <20% (v/v) was, however, found to improve peak shape and/or separation in some cases. The optimized CE method has good sensitivity in moderate concentrations of the ionic liquids used, with limits of detection of 5mg/L for cellooligomers up to the size of cellotetraose and 5-20mg/L for cellopentaose and cellohexaose, depending on the matrix. The method was used for analysing the action of a commercial β-glucosidase in ILs and for analysing saccharides in the IL containing hydrolysates from the hydrolysis of microcrystalline cellulose with Trichoderma reesei endoglucanase Cel5A. According to the results, [DMIM]DMP and [EMIM]AcO] showed clear differences in enzyme inactivation.

Topics: Calibration; Cellulase; Cellulose; Electrolytes; Electrophoresis, Capillary; Imidazoles; Ionic Liquids; Oligosaccharides; Photometry; Tetroses; Trichoderma

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

Cellulases, distributed in at least 15 families of glycoside hydrolases, will play a key role in biomass conversion and renewable energy challenges of the future. Cel5B from Clostridium thermocellum is a β-1,4-endoglucanase and a member of family 5 of glycoside hydrolases (GH5) and is characterized by an (α/β)(8) barrel structure. In contrast to other retaining enzymes, in which the catalytic carboxylate groups (glutamate or aspartate) are positioned ≈ 5.5 Å apart to facilitate nucleophilic attack on the anomeric carbon of the sugar substrate, these two residues in Cel5B are positioned ≈ 10 Å from each other in the unliganded wild-type structure. The structure of the enzyme solved in complex with a cleavage product (cellobiose) revealed ligand-induced conformational changes in the loop carrying Glu140 (proton donor). The reorientation of Glu140 in the complex reduces the separation of the catalytic glutamate residues to 4.3 Å. In this study, we took advantage of conventional and steered molecular dynamics (MD) simulations along with in silico and in vitro mutagenesis to investigate the ligand-induced changes of the enzyme and interactions involved in preservation of Cel5B conformations in the presence and absence of substrate. We determined that the variation in separation of catalytic glutamates in the absence and presence of substrate is due to the different protonation states of the proton donor glutamate that is largely governed by conformational changes in the β3α3 loop. In the absence of substrate, the conformation of Cel5B is preserved by an electrostatic interaction between deprotonated Glu140 and protonated His91. The ion pair is interrupted upon the binding of substrate, and the positional displacement of the β3α3 loop allows Glu140 to become oriented within the active site in a less hydrophilic microenvironment that assists in Glu140 protonation.

Topics: Amino Acid Sequence; Catalytic Domain; Cellulase; Cellulose; Glutamic Acid; Histidine; Hydrolysis; Ligands; Molecular Dynamics Simulation; Protein Conformation; Protons; Sequence Alignment; Substrate Specificity; Tetroses

Neurospora crassa colonizes burnt grasslands in the wild and metabolizes both cellulose and hemicellulose from plant cell walls. When switched from a favored carbon source such as sucrose to cellulose, N. crassa dramatically upregulates expression and secretion of a wide variety of genes encoding lignocellulolytic enzymes. However, the means by which N. crassa and other filamentous fungi sense the presence of cellulose in the environment remains unclear. Here, we show that an N. crassa mutant carrying deletions of two genes encoding extracellular β-glucosidase enzymes and one intracellular β-glucosidase lacks β-glucosidase activity, but efficiently induces cellulase gene expression in the presence of cellobiose, cellotriose, or cellotetraose as a sole carbon source. These data indicate that cellobiose, or a modified version of cellobiose, functions as an inducer of lignocellulolytic gene expression in N. crassa. Furthermore, the inclusion of a deletion of the catabolite repressor gene, cre-1, in the triple β-glucosidase mutant resulted in a strain that produces higher concentrations of secreted active cellulases on cellobiose. Thus, the ability to induce cellulase gene expression using a common and soluble carbon source simplifies enzyme production and characterization, which could be applied to other cellulolytic filamentous fungi.

Topics: Cellobiose; Cellulase; Cellulases; Cellulose; Cluster Analysis; Dextrins; Fungal Proteins; Gene Deletion; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Fungal; Lignin; Mass Spectrometry; Mutation; Neurospora crassa; Phylogeny; Reverse Transcriptase Polymerase Chain Reaction; Tetroses; Trioses

A metagenomic fosmid library was constructed using genomic DNA isolated from abalone intestine. Screening of a library of 3,840 clones revealed a 36 kb insert of a cellulase positive clone (pAMHElO). A shotgun clone library was constructed using the positive clone (pAMHElO) and further screening of 3,840 shotgun clones with an approximately 5 kb insert size using a Congo red overlay revealed only one cellulase positive clone (pAMHL9). The pAMHL9 consisted of a 5,293-bp DNA sequence and three open reading frames (ORFs). Among the three ORFs, cellulase activity was only shown in the recombinant protein (CelAMll) coded by ORF3, which showed 100% identity with outer membrane protein A from Vibrio alginolyticus 12G01, but no significant sequence homology to known cellulases. The expressed protein (CelAMll) has a molecular weight of approximately 37 kDa and the highest CMC hydrolysis activity was observed at pH 7.0 and 37°C. The carboxymethyl cellulase activity was determined by zymogram active staining and different degraded product profiles for CelAMll were obtained when cellotetraose and cellopentaose were used as the substrates, while no substrate hydrolysis was observed on oligosaccharides such as cellobiose and cellotriose.

Topics: Animals; Carboxymethylcellulose Sodium; Cellulase; Cellulose; Cloning, Molecular; Enzyme Stability; Gastrointestinal Tract; Gastropoda; Gene Library; Hydrogen-Ion Concentration; Metagenome; Molecular Sequence Data; Molecular Weight; Oligosaccharides; Open Reading Frames; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Substrate Specificity; Temperature; Tetroses; Vibrio alginolyticus

Three isozymes with both lichenase and endo-β-1,4-glucanase activity were purified and characterised from the midgut gland of the herbivorous gecarcinid land crab, Gecarcoidea natalis. The three isozymes, termed 1a, 1b and 2, had respective molecular masses of 53 ± 0 (3), 43 ± 0 (3) and 47.4 ± 0(3) kDa. All isozymes possessed similar V(max) values and thus hydrolysed both carboxy methyl cellulose and lichenan equally. Furthermore the chromatography profiles for lichenase activities mirrored that for endo-β-1,4-glucanase activities suggesting that the same enzyme possessed both activities. Given this, the endo-β-1,4-glucanase enzymes described for other animals, may, like the isozymes described in this study, may be able to hydrolyse lichenan. However this ability needs to be confirmed. The main digestive function of these isozymes may be to hydrolyse hemicelluloses such as lichenan and mixed beta-D-glucan. All three isozymes randomly hydrolysed internal glycosidic bonds within carboxy methyl cellulose and lichenan to release short oligomers of 4-5 glucose units in length. They also hydrolysed cellotetraose to either two units of cellobiose or cellotriose and glucose. Cellotriose was hydrolysed to cellobiose and glucose. All three enzymes lacked β-1,4-glucosidase activity as they could not hydrolyse cellobiose.

Topics: Adaptation, Biological; Animals; Brachyura; Carboxymethylcellulose Sodium; Cellulase; Cellulose; Glucans; Glycoside Hydrolases; Hydrogen-Ion Concentration; Isoenzymes; Substrate Specificity; Tetroses

Two genes encoding endoglucanase, designated as egl2 and egl3, were cloned from a lignocellulosic decomposing fungus Aspergillus fumigatus Z5 and were successfully expressed in Pichia pastoris X33. The deduced amino acid sequences encoded by egl2 and egl3 showed strong similarity with the sequence of glycoside hydrolase family 5. SDS-PAGE and western blot assays indicated that the recombinant enzymes were secreted into the culture medium and the zymogram analysis confirmed that both recombinant enzymes had endoglucanase activity. Several biochemical properties of the two recombinant enzymes were studied: Egl2 and Egl3 showed optimal activity at pH 5.0 and 4.0, respectively, and at 50 and 60°C, respectively. Egl2 and Egl3 showed good pH stability in the range of 4-7, and both enzymes demonstrated good thermostability ranging from 30 to 60°C. The K(m) and V(max) values using carboxymethyl cellulose (CMC, soluble cellulose, polymerized by β-1, 4-linked glucose residues) as the substrate at optimal conditions were determined. The activities of the enzymes on a variety of cello-oligosaccharide substrates were investigated, and Egl2 can hydrolyze cellotetraose and cellopentaose but not cellobiose and cellotriose, whereas Egl3 can hydrolyze all cello-oligosaccharides, except cellobiose.

Topics: Amino Acid Sequence; Aspergillus fumigatus; Blotting, Western; Cellulase; Cellulose; Chromatography, Thin Layer; Cloning, Molecular; Electrophoresis, Polyacrylamide Gel; Hydrogen-Ion Concentration; Hydrolysis; Isoenzymes; Kinetics; Lignin; Molecular Sequence Data; Oligosaccharides; Pichia; Plasmids; Recombinant Proteins; Sequence Alignment; Soil; Substrate Specificity; Temperature; Tetroses; Transformation, Genetic

The hyperthermophilic endoglucanase Cel5A from Thermotoga maritima can find applications in lignocellulosic biofuel production, because it catalyzes the hydrolysis of glucan- and mannan-based polysaccharides. Here, we report the crystal structures in apo-form and in complex with three ligands, cellotetraose, cellobiose and mannotriose, at 1.29Å to 2.40Å resolution. The open carbohydrate-binding cavity which can accommodate oligosaccharide substrates with extensively branched chains explained the dual specificity of the enzyme. Combining our structural information and the previous kinetic data, it is suggested that this enzyme prefers β-glucosyl and β-mannosyl moieties at the reducing end and uses two conserved catalytic residues, E253 (nucleophile) and E136 (general acid/base), to hydrolyze the glycosidic bonds. Moreover, our results also suggest that the wide spectrum of Tm_Cel5A substrates might be due to the lack of steric hindrance around the C2-hydroxyl group of the glucose or mannose unit from active-site residues.

Topics: Binding Sites; Catalytic Domain; Cellobiose; Cellulase; Cellulose; Crystallography, X-Ray; Macromolecular Substances; Models, Molecular; Mutagenesis, Site-Directed; Protein Binding; Protein Structure, Quaternary; Substrate Specificity; Tetroses; Thermotoga maritima; Trisaccharides

A gene encoding a glycoside hydrolase family 44 (GH44) protein from Clostridium acetobutylicum ATCC 824 was synthesized and transformed into Escherichia coli. The previously uncharacterized protein was expressed with a C-terminal His tag and purified by nickel-nitrilotriacetic acid affinity chromatography. Crystallization and X-ray diffraction to a 2.2-A resolution revealed a triose phosphate isomerase (TIM) barrel-like structure with additional Greek key and beta-sandwich folds, similar to other GH44 crystal structures. The enzyme hydrolyzes cellotetraose and larger cellooligosaccharides, yielding an unbalanced product distribution, including some glucose. It attacks carboxymethylcellulose and xylan at approximately the same rates. Its activity on carboxymethylcellulose is much higher than that of the isolated C. acetobutylicum cellulosome. It also extensively converts lichenan to oligosaccharides of intermediate size and attacks Avicel to a limited extent. The enzyme has an optimal temperature in a 10-min assay of 55 degrees C and an optimal pH of 5.0.

Topics: Carboxymethylcellulose Sodium; Cellulase; Cellulose; Clostridium acetobutylicum; Crystallization; Crystallography, X-Ray; Enzyme Stability; Escherichia coli; Gene Expression; Glucans; Glucose; Hydrogen-Ion Concentration; Kinetics; Models, Molecular; Oligosaccharides; Phylogeny; Protein Structure, Tertiary; Recombinant Proteins; Sequence Homology, Amino Acid; Substrate Specificity; Temperature; Tetroses; Transformation, Genetic; Xylans

Pseudoalteromonas sp. NO3 was isolated from the hemolymph of diseased sea squirts (Halocynthia rorentzi) with symptoms of soft tunic syndrome. The strain was found to produce an extracellular cellulase (CelY) that consisted of a 1,476 bp open reading frame encoding 491 amino acid residues with an approximate molecular mass of 52 kDa. Homologies of the deduced amino acid sequence of celY with the products of the celA, celX, celG and cel5Z genes were 92.6, 93.3, 92.6, and 59.1%, respectively. Additionally, CelY had 50-80% remnant catalytic activity at temperatures of 10-20 degrees C. Highest carboxymethyl cellulose (CMC) hydrolysis was observed at pH 8.0 and 40 degrees C. CMC activity was determined by zymogram active staining and different degraded product profiles for CelY were obtained when cellotetraose, cellopentaose, and CMC were used as substrates. This study identified a transglycosylation activity in CelY that allows the enzyme to digest G4 to G2 and G3 without the production of G1.

Topics: Animals; Carboxymethylcellulose Sodium; Cellulase; Cellulose; DNA, Bacterial; DNA, Ribosomal; Enzyme Stability; Hemolymph; Hydrogen-Ion Concentration; Molecular Sequence Data; Molecular Weight; Oligosaccharides; Open Reading Frames; Pseudoalteromonas; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Temperature; Tetroses; Urochordata

The direct conversion of plant cell wall polysaccharides into soluble sugars is one of the most important reactions on earth, and is performed by certain microorganisms such as Clostridium thermocellum (Ct). These organisms produce extracellular multi-subunit complexes (i.e. cellulosomes) comprising a consortium of enzymes, which contain noncatalytic carbohydrate-binding modules (CBM) that increase the activity of the catalytic module. In the present study, we describe a combined approach by X-ray crystallography, NMR and computational chemistry that aimed to gain further insight into the binding mode of different carbohydrates (cellobiose, cellotetraose and cellohexaose) to the binding pocket of the family 11 CBM. The crystal structure of C. thermocellum CBM11 has been resolved to 1.98 A in the apo form. Since the structure with a bound substrate could not be obtained, computational studies with cellobiose, cellotetraose and cellohexaose were carried out to determine the molecular recognition of glucose polymers by CtCBM11. These studies revealed a specificity area at the CtCBM11 binding cleft, which is lined with several aspartate residues. In addition, a cluster of aromatic residues was found to be important for guiding and packing of the polysaccharide. The binding cleft of CtCBM11 interacts more strongly with the central glucose units of cellotetraose and cellohexaose, mainly through interactions with the sugar units at positions 2 and 6. This model of binding is supported by saturation transfer difference NMR experiments and linebroadening NMR studies.

Topics: Bacterial Proteins; Binding Sites; Carbohydrate Conformation; Carbohydrate Sequence; Cellobiose; Cellulase; Cellulose; Clostridium thermocellum; Computer Simulation; Crystallography, X-Ray; Models, Molecular; Molecular Sequence Data; Molecular Structure; Multienzyme Complexes; Nuclear Magnetic Resonance, Biomolecular; Oligosaccharides; Protein Conformation; Substrate Specificity; Tetroses

Two model sodium carboxymethyl celluloses (CMC) with similar monomer composition but with significant differences in the viscoelastic properties, that could not be assigned to variations in the average molar mass or molar mass distribution, were investigated with respect to the fraction of nonsubstituted cellulose segments in the polymers. The CMCs were hydrolyzed by a purified highly selective endoglucanase. The average molar mass and molar mass distribution of the enzyme products, as measured by size-exclusion chromatography with online multi-angle light scattering and refractive index detection (SEC/MALS/RI), revealed that the enzyme-catalyzed hydrolysis was more effective on one of the CMCs. To investigate whether this was due to a higher fraction of nonsubstituted cellulose segments in the polymer, the concentrations of nonsubstituted enzyme products, e.g., cellotetraose and cellopentaose, were measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). It was concluded that the two CMCs displayed significant differences in the fraction of nonsubstituted cellulose segments. Furthermore, the CMC with the strongest attractive intermolecular interactions, according to rheometry, also contained the highest fraction of nonsubstituted cellulose segments.

Topics: Biocompatible Materials; Carboxymethylcellulose Sodium; Cellulase; Cellulose; Chemistry; Elasticity; Enzymes; Hydrolysis; Models, Chemical; Molecular Weight; Oligosaccharides; Rheology; Sodium; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tetroses; Trichoderma

Xylella fastidiosa was the first plant pathogen whose complete genome sequence was available. X. fastidiosa causes citrus variegated chlorosis, but the physiological basis of the disease in unknown. Through comparative sequence analysis, several putative plant cell wall-degrading enzymes were identified on the X. fastidiosa genome. We have cloned Xf818, a putative endoglucanase ORF, into expression vectors pET20b and pET28b, and purified a recombinant form of Xf818 containing a His(6) tag. Through biochemical assays, we have characterized the endoglucanase activity of this protein. The best conditions for hydrolysis over carboxymethyl cellulose (CMC) were on pH 5.2 at 65 degrees C. Xf818 hydrolyzed CMC, acid swollen cellulose, Avicel, birch wood, oat spels xylans, and the oligosaccharides cellotetraose and cellopentaose. Xf818 carried out transglycosylation and had a functional cellulose-binding domain.

Topics: Bacterial Proteins; Carboxymethylcellulose Sodium; Cellulase; Cellulose; Chromatography, Thin Layer; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Gene Expression; Genetic Vectors; Hydrogen-Ion Concentration; Hydrolysis; Oligosaccharides; Recombinant Proteins; Temperature; Tetroses; Transformation, Bacterial; Xylans; Xylella

Endoglucanase Cel6A from Thermobifida fusca hydrolyzes the beta-1,4 linkages in cellulose at accessible points along the polymer. The structure of the catalytic domain of Cel6A from T. fusca in complex with a nonhydrolysable substrate analogue that acts as an inhibitor, methylcellobiosyl-4-thio-beta-cellobioside (Glc(2)-S-Glc(2)), has been determined to 1.5 A resolution. The glycosyl unit in subsite -1 was sterically hindered by Tyr73 and forced into a distorted (2)S(o) conformation. In the enzyme where Tyr73 was mutated to a serine residue, the hindrance was removed and the glycosyl unit in subsite -1 had a relaxed (4)C(1) chair conformation. The relaxed conformation was seen in two complex structures of the mutated enzyme, with cellotetrose (Glc(4)) at 1.64 A and Glc(2)-S-Glc(2) at 1.04 A resolution.

Topics: Bacterial Proteins; Catalytic Domain; Cellobiose; Cellulase; Cellulose; Crystallography, X-Ray; Enzyme Inhibitors; Protein Binding; Substrate Specificity; Tetroses; Tyrosine

As part of an ongoing enzyme discovery program to investigate the properties and catalytic mechanism of glycoside hydrolase family 12 (GH 12) endoglucanases, a GH family that contains several cellulases that are of interest in industrial applications, we have solved four new crystal structures of wild-type Humicola grisea Cel12A in complexes formed by soaking with cellobiose, cellotetraose, cellopentaose, and a thio-linked cellotetraose derivative (G2SG2). These complex structures allow mapping of the non-covalent interactions between the enzyme and the glucosyl chain bound in subsites -4 to +2 of the enzyme, and shed light on the mechanism and function of GH 12 cellulases. The unhydrolysed cellopentaose and the G2SG2 cello-oligomers span the active site of the catalytically active H.grisea Cel12A enzyme, with the pyranoside bound in subsite -1 displaying a S31 skew boat conformation. After soaking in cellotetraose, the cello-oligomer that is found bound in site -4 to -1 contains a beta-1,3-linkage between the two cellobiose units in the oligomer, which is believed to have been formed by a transglycosylation reaction that has occurred during the ligand soak of the protein crystals. The close fit of this ligand and the binding sites occupied suggest a novel mixed beta-glucanase activity for this enzyme.

Topics: Ascomycota; Binding Sites; Catalysis; Cellobiose; Cellulase; Cellulose; Crystallography, X-Ray; Models, Molecular; Oligosaccharides; Protein Binding; Protein Conformation; Structure-Activity Relationship; Substrate Specificity; Tetroses

The crystal structure of the Clostridium cellulovorans carbohydrate-binding module (CBM) belonging to family 17 has been solved to 1.7 A resolution by multiple anomalous dispersion methods. CBM17 binds to non-crystalline cellulose and soluble beta-1,4-glucans, with a minimal binding requirement of cellotriose and optimal affinity for cellohexaose. The crystal structure of CBM17 complexed with cellotetraose solved at 2.0 A resolution revealed that binding occurs in a cleft on the surface of the molecule involving two tryptophan residues and several charged amino acids. Thermodynamic binding studies and alanine scanning mutagenesis in combination with the cellotetraose complex structure allowed the mapping of the CBM17 binding cleft. In contrast to the binding groove characteristic of family 4 CBMs, family 17 CBMs appear to have a very shallow binding cleft that may be more accessible to cellulose chains in non-crystalline cellulose than the deeper binding clefts of family 4 CBMs. The structural differences in these two modules may reflect non-overlapping binding niches on cellulose surfaces.

Topics: Alanine; Binding Sites; Calorimetry; Cellulase; Cellulose; Clostridium; Crystallography, X-Ray; Hydrogen Bonding; Ligands; Models, Molecular; Mutation; Oligosaccharides; Protein Binding; Protein Structure, Quaternary; Protein Structure, Secondary; Protein Structure, Tertiary; Static Electricity; Substrate Specificity; Tetroses; Thermodynamics; Titrimetry; Trioses; Tryptophan

Fifteen mutant genes in six loop residues and eight mutant genes in five conserved noncatalytic active site residues of Thermobifida fusca Cel6B were constructed, cloned and expressed in Escherichia coli or Streptomyces lividans. The mutant enzymes were assayed for catalytic activity on carboxymethyl cellulose (CMC), swollen cellulose (SC), filter paper (FP), and bacterial microcrystalline cellulose (BMCC) as well as cellotetraose, cellopentaose, and 2, 4-dinitrophenyl-beta-D-cellobioside. They were also assayed for ligand binding, enzyme processivity, thermostability, and cellobiose feedback inhibition. Two double Cys mutations that formed disulfide bonds across two tunnel forming loops were found to significantly weaken binding to ligands, lower all activities, and processivity, demonstrating that the movement of these loops is important but not essential for Cel6B function. Two single mutant enzymes, G234S and G284P, had higher activity on SC and FP, and the double mutant enzyme had threefold and twofold higher activity on these substrates, respectively. However, synergism with endocellulase T. fusca Cel5A was not increased with these mutant enzymes. All mutant enzymes with lower activity on filter paper, BMCC, and SC had lower processivity. This trend was not true for CMC, suggesting that processivity in Cel6B is a key factor in the hydrolysis of insoluble and crystalline cellulose. Three mutations (E495D, H326A and W329C) located near putative glycosyl substrate subsites -2, +1 and +2, were found to significantly increase resistance to cellobiose feedback inhibition. Both the A229V and L230C mutations specifically decreased activity on BMCC, suggesting that BMCC hydrolysis has a different rate limiting step than the other substrates. Most of the mutant enzymes had reduced thermostability although Cel6B G234S maintained wild-type thermostability. The properties of the different mutant enzymes provide insight into the catalytic mechanism of Cel6B.

Topics: Actinomycetales; Amino Acid Sequence; Bacterial Proteins; Binding Sites; Catalysis; Cellobiose; Cellulase; Cellulose; Cellulose 1,4-beta-Cellobiosidase; Escherichia coli; Genes, Bacterial; Glucosides; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligosaccharides; Polysaccharides; Protein Binding; Protein Conformation; Protein Denaturation; Recombinant Fusion Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Substrate Specificity; Tetroses

Cellulase Cel48F from Clostridium cellulolyticum was described as a processive endo-cellulase. The active site is composed of a 25 A long tunnel which is followed by an open cleft. During the processive action, the cellulose substrate has to slide through the tunnel to continuously supply the leaving group site with sugar residues after the catalytic cleavage. To study this processive action in the tunnel, the native catalytic module of Cel48F and the inactive mutant E55Q, have been cocrystallized with cellobiitol, two thio-oligosaccharide inhibitors (PIPS-IG3 and IG4) and the cello-oligosaccharides cellobiose, -tetraose and -hexaose. Seven sub-sites in the tunnel section of the active center could be identified and three of the four previously reported sub-sites in the open cleft section were reconfirmed. The sub-sites observed for the thio-oligosaccharide inhibitors and oligosaccharides, respectively, were located at two different positions in the tunnel corresponding to a shift in the chain direction of about a half sugar subunit. These two positions have different patterns of stacking interactions with aromatic residues present in the tunnel. Multiple patterns are not observed in nonprocessive endo-cellulases, where only one sugar position is favored by aromatic stacking. It is therefore proposed that the aromatic residues serve as lubricating agents to reduce the sliding barrier in the processive action.

Topics: Binding Sites; Cellobiose; Cellulase; Cellulose; Clostridium; Crystallography, X-Ray; Enzyme Inhibitors; Hydrogen Bonding; Hydrolysis; Macromolecular Substances; Models, Chemical; Models, Molecular; Mutagenesis, Site-Directed; Oligosaccharides; Substrate Specificity; Tetroses

The mechanisms of crystalline cellulose degradation by cellulases are of paramount importance for the exploitation of these enzymes in applied processes, such as biomass conversion. Cellulases have traditionally been classified into cellobiohydrolases, which are effective in the degradation of crystalline materials, and endoglucanases, which appear to act on "soluble" regions of the substrate. Humicola insolensCel6A (CBH II) is a cellobiohydrolase from glycoside hydrolase family 6 whose native structure has been determined at 1.9 A resolution [Varrot, A., Hastrup, S., Schülein, M., and Davies, G. J. (1999) Biochem. J. 337, 297-304]. Here we present the structure of the catalytic core domain of Humicola insolens cellobiohydrolase II Cel6A in complex with glucose/cellotetraose at 1.7 A resolution. Crystals of Cel6A, grown in the presence of cellobiose, reveal six binding subsites, with a single glucose moiety bound in the -2 subsite and cellotetraose in the +1 to +4 subsites. The complex structure is strongly supportive of the assignment of Asp 226 as the catalytic acid and consistent with proposals that Asp 405 acts as the catalytic base. The structure undergoes several conformational changes upon substrate binding, the most significant of which is a closing of the two active site loops (residues 174-196 and 397-435) with main-chain movements of up to 4.5 A observed. This complex not only defines the polysaccharide-enzyme interactions but also provides the first three-dimensional demonstration of conformational change in this class of enzymes.

Topics: Binding Sites; Carbohydrates; Catalysis; Cellulase; Cellulose; Cellulose 1,4-beta-Cellobiosidase; Computer Simulation; Crystallization; Crystallography, X-Ray; Fungal Proteins; Glucose; Ligands; Macromolecular Substances; Mitosporic Fungi; Models, Molecular; Oligosaccharides; Protein Conformation; Solutions; Substrate Specificity; Tetroses

Values of kcat. and Km for the hydrolysis of cellotetraose, cellotriose, beta-cellobiosyl fluoride and various beta-aryl cellobiosides by endoglucanase A (CenA) from Cellulomonas fimi indicate that specific binding interactions between the reducing-end glucose residues of cellotetraose and cellotriose and the enzyme at the transition state provide enormous stabilization, endowing glucose with the "effective leaving group ability' of 2,4-dinitrophenol. As has been seen with several other inverting glycosidases, CenA hydrolyses the "wrong' anomer of its glycosyl fluoride substrate, alpha-cellobiosyl fluoride, according to non-Michaelian kinetics. This indicates that CenA carries out this hydrolysis by a mechanism involving binding of two substrate molecules in the active site (Hehre, Brewer and Genghof (1979) J. Biol. Chem. 254, 5942-5950] in contrast with that reported for cellobiohydrolase II, another family-6 enzyme [Konstantinidis, Marsden and Sinnott (1993) Biochem. J. 291, 833-838]. The pH profiles for wild-type CenA indicate that kcat. for CenA depends on the presence of both a protonated group and a deprotonated group for full activity, consistent with the presence of an acid and a base catalyst at the active site. By contrast, the profile for the Asp252Ala mutant of CenA shows a dependence only on a base-catalytic group, thereby confirming the role of Asp-252 as an acid catalyst. These results show that hydrolysis by CenA occurs by a typical inverting mechanism involving both acid and base catalysis, as first proposed by Koshland. It also suggests that endoglucanases from family 6 may function by fundamentally different mechanisms for exoglucanases in this family.

Topics: Actinomycetales; beta-Glucosidase; Binding Sites; Carbohydrate Sequence; Cellobiose; Cellulase; Cellulose; Glucan 1,3-beta-Glucosidase; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Molecular Sequence Data; Substrate Specificity; Tetroses; Trisaccharides

The crystal structure of the catalytic domain of the thermostable endocellulase E1 from Acidothermus cellulolyticus in complex with cellotetraose has been solved by multiple isomorphous replacement and refined at 2.4 A resolution to an R-factor of 0.18 (Rfree = 0.24). E1cd is a member of the 4/7 superfamily of hydrolases, and as expected, its structure is an (alpha/beta)8 barrel, which constitutes a prototype for family 5-subfamily 1 cellulases. The cellotetraose molecule binds in a manner consistent with the expected Michaelis complex for the glycosylation half-reaction and reveals that all eight residues conserved in family 5 enzymes are involved in recognition of the glycosyl group attacked during cleavage. Whereas only three residues are conserved in the whole 4/7 superfamily (the Asn/Glu duo and the Glu from which the name is derived), structural comparisons show that all eight residues conserved in family 5 have functional equivalents in the other 4/7 superfamily members, strengthening the case that mechanistic details are conserved throughout the superfamily. On the basis of the structure, a detailed sequence of physical steps of the cleavage mechanism is proposed. A close approach of two key glutamate residues provides an elegant mechanism for the shift in the pKa of the acid/base for the glycosylation and deglycosylation half-reactions. Finally, purely structural based comparisons are used to show that significant differences exist in structural similarity scores resulting from different methods and suggest that caution should be exercised in interpreting such results in terms of implied evolutional relationships.

Topics: Amino Acid Sequence; Binding Sites; Catalysis; Cellulase; Cellulose; Crystallography, X-Ray; Enzyme Stability; Gram-Negative Aerobic Bacteria; Models, Molecular; Molecular Sequence Data; Protein Binding; Protein Conformation; Sequence Homology, Amino Acid; Tetroses

The hydrolysis of soluble cello-oligosaccharides, with a degree of polymerisation of 4-6, catalysed by cellobiohydrolase II from Trichoderma reesei was studied using 1H-NMR spectroscopy and HPLC. The experimental progress curves were analysed by fitting numerically integrated kinetic equations, which provided cleavage patterns and kinetic constants for each oligosaccharide. This analysis procedure accounts for product inhibition and avoids the initial slope approximation. No glucose was detected at the beginning of the reaction indicating that only the internal glycosidic linkages are attacked. For cellotetraose only the second glycosidic linkage was cleaved. For cellopentaose and cellohexaose the second and the third glycosidic linkage from the non-reducing end were cleaved with approximately equal probability. The degradation rates of these cello-oligosaccharides, 1-12 s-1 at 27 degrees C, are about 10-100 times faster than for the 4-methylumbelliferyl substituted analogs or for collotriose. No intermediate products larger than cellotriose were released. The degradation rate for cellotetraose were higher than its off-rate, which accounts for the processive degradation of cellohexaose. A high cellohexaose/enzyme ratio caused slow reversible inactivation of the enzyme.

Topics: Binding Sites; Cellulase; Cellulose; Cellulose 1,4-beta-Cellobiosidase; Chromatography, High Pressure Liquid; Hydrolysis; Kinetics; Magnetic Resonance Spectroscopy; Oligosaccharides; Substrate Specificity; Tetroses; Trichoderma

Molecular mechanics calculations have been used to place a cellotetraose substrate into the active site of the crystallographically determined structure of endocellulase E2 from Thermomonospora fusca. In the lowest energy model structure, the second residue of the substrate oligosaccharide is tilted away from the planar ribbon geometry of cellulose as it is in the X-ray structure of the E2cd-cellobiose co-crystal. This tilt is the result of the topology of the binding site, and results in several strong carbohydrate-protein hydrogen bonds. The tilting produces a twisting of the glycosidic linkage of the cleavage site between residues two and three. In the predicted enzyme-substrate complex both of the Asp residues believed to function in general acid and base roles in the previously proposed model for the mechanism are distant from the bond being cleaved. Molecular dynamics simulations of the complex were conducted, and while the putative catalytic Asp residues remained distant from the cleavage site, the proton of Tyr73 briefly came within van der Waals contact of the linkage oxygen.

Topics: Actinomycetales; Binding Sites; Cellulase; Cellulose; Computer Simulation; Hydrogen Bonding; Models, Molecular; Molecular Conformation; Tetroses; Thermodynamics

A cellulase gene (endA) was isolated from a library of Ruminococcus flavefaciens strain 17 DNA fragments inserted in pUC13. The endA product showed activity against acid-swollen cellulose, carboxymethyl-cellulose, lichenan, cellopentaose and cellotetraose, but showed no activity against cellotriose or binding to avicel. Nucleotide sequencing indicated an encoded product of 455 amino acids which showed significant sequence similarity (ranging from 56% to 61%) with three endoglucanases from Ruminococcus albus, and with Clostridium thermocellum endoglucanase E. Little relatedness was found with a cellodextrinase previously isolated from R. flavefaciens FD1.

Topics: Amino Acid Sequence; Base Sequence; Carboxymethylcellulose Sodium; Cellulase; Cellulose; Cloning, Molecular; Genes, Bacterial; Glucans; Molecular Sequence Data; Oligosaccharides; Restriction Mapping; Sequence Homology, Nucleic Acid; Substrate Specificity; Tetroses; Transformation, Bacterial; Trisaccharides

An endoglucanase encoded by a gene of Clostridium josui was expressed in Escherichia coli and purified. The homogeneous enzyme, with a molecular weight of 39,000, revealed maximum endoglucanase activity at pH 7.2 to 7.5 and a temperature of 65 to 70 degrees C. The enzyme was stable at a temperature lower than 45 degrees C (the growth temperature of the bacterium) in the range of pH 4.5 to 9.0. The amino acid sequence of the enzyme at the N terminus was Val-Glu-Glu-Asp-Ser-Ser-His-Leu-Ile-Thr-Asn-Gln-Ala-Lys-Lys----. The enzyme hydrolyzed cellotetraose to cellobiose and then transferred cellobiose to the residual cellotetraose. The resulting cellohexaose was cleaved to cellotriose.

Topics: Amino Acid Sequence; Cellobiose; Cellulase; Cellulose; Clostridium; Escherichia coli; Genes, Bacterial; Hydrogen-Ion Concentration; Molecular Sequence Data; Molecular Weight; Oligosaccharides; Protein Biosynthesis; Substrate Specificity; Temperature; Tetroses; Transformation, Genetic

The stereochemical courses of the hydrolyses catalysed by three glycosidases have been determined directly by 1H nmr. The anomeric configuration of the initially formed product was ascertained in each case by observation of the chemical shift and coupling constant of the anomeric proton at the new hemiacetal centre. Two of the enzymes investigated, an endo-glucanase and an exo-glucanase are components of the cellulase complex of Cellulomonas fimi. The third enzyme is the beta-glucosidase from almond emulsin. Two of these enzymes, the exo-glucanase and the almond beta-glucosidase catalysed hydrolysis with retention of anomeric configuration, in agreement with previous observations on the almond enzyme. The endo-glucanase catalysed hydrolysis with inversion of configuration, this result being confirmed by optical rotation measurements. This 1H nmr approach has several advantages over other techniques in that it is applicable to a wide variety of glycosidases and substrates and it is non-destructive, allowing recovery of the enzyme.

Topics: beta-Glucosidase; Cellobiose; Cellulase; Cellulose; Glucan 1,4-beta-Glucosidase; Glucosidases; Glycoside Hydrolases; Hydrolysis; Magnetic Resonance Spectroscopy; Oligosaccharides; Optical Rotation; Stereoisomerism; Tetroses