Components of cellulase from the higher termite,Nasutitermes walkeri

The cellulase from the termite Nasutitermes walkeri consists of two enzymes. Each has broad specificity with predominantly one activity. One enzyme is an endo-gb-1,4-glucanase (EC 3.2.1.4) which predominantly cleaves cellulose randomly to glucose, cellobiose and cellotriose. It hydrolyses cellotetraose to cellobiose but will not hydrolyse cellobiose or cellotriose. The second enzyme component is a β-1,4-glucosidase (EC 3.2.1.21) as its major activity is to hydrolyse cellobiose, cellotriose and cellotetraose to glucose; it has some exoglucosidase activity as glucose is the only product produced from cellulose. Its cellobiase activity is inhibited by glucono-δ-lactone.     

Kinetic studies of cellodextrins hydrolyses by exocellulase from Trichoderma reesei

The kinetics of the hydrolyses of cellotriose and of cellotetraose by cellobiohydrolase were studied using a convenient integral technique. Reaction mechanisms and mathematical models were postulated to describe the reactions. The end-products of the reaction were found to be inhibitory toward hydrolysis in a competitive mode. Hydrolysis of cellotetraose produces cellobiose and hydrolysis of cellotriose produces cellobiose and glucose. Both sugars inhibit the enzyme with cellobiose being a stronger inhibitor.     

Kinetic characterization of a glycoside hydrolase family 44 xyloglucanase/endoglucanase from Ruminococcus flavefaciens FD-1

Two forms of Ruminococcus flavefaciens FD-1 endoglucanase B, a member of glycoside hydrolase family 44, one with only a catalytic domain and the other with a catalytic domain and a carbohydrate binding domain (CBM), were produced. Both forms hydrolyzed cellotetraose, cellopentaose, cellohexaose, carboxymethylcellulose (CMC), birchwood and larchwood xylan, xyloglucan, lichenan, and Avicel but not cellobiose, cellotriose, mannan, or pullulan. Addition of the CBM increased catalytic efficiencies on both CMC and birchwood xylan but not on xyloglucan, and it decreased rates of cellopentaose and cellohexaose hydrolysis. Catalytic efficiencies were much higher on xyloglucan than on other polysaccharides. Hydrolysis rates increased with increasing cellooligosaccharide chain length. Cellotetraose hydrolysis yielded only cellotriose and glucose. Hydrolysis of cellopentaose gave large amounts of cellotetraose and glucose, somewhat more of the former than of the latter, and much smaller amounts of cellobiose and cellotriose. Cellohexaose hydrolysis yielded much more cellotetraose than cellobiose and small amounts of glucose and cellotriose, along with a low and transient amount of cellopentaose.     

Enzymatic studies on a cellulase system of Trichoderma viride. III. Transglycosylation properties of two cellulase components of random type.

Two highly purified cellulases [EC 3.2.1.4], II-A, and II-B, were obtained from the cellulase system of Trichoderma viride. Both cellulases split cellopentaose retaining the beta-configuration of the anomeric carbon atoms in the hydrolysis products at both pH 3.5 and 5.0. The Km values of cellulases II-A and II-B for cellotetraose were different, but their Vmax values were similar and those for cellooligosaccharides increased in parallel with chain length. Both cellulases produced predominantly cellobiose and glucose from various cellulosic substrates as well as from higher cellooligosaccharides. Cellulase II-A preferentially attacked the holoside linkage of rho-nitrophenyl beta-D-cellobioside, whereas cellulase II-B attacked mainly the aglycone linkage of this cellobioside. Both cellulases were found to catalyze the synthesis of cellotriose from rho-nitrophenyl beta-D-cellobioside by transfer of a glucosyl residue, possibly to cellobiose produced in the reaction mixture. They were also found to catalyze the rapid synthesis of cellotetraose from cellobiose, with accompanying formation of cellotriose and glucose, which seemed to be produced by secondary random hydrolysis of the cellotetraose produced. The capacity to synthesize cellotetraose from cellobiose appeared to be greater with cellulase II-B than with cellulase II-A.     

DNA sequences and expression in Streptomyces lividans of an exoglucanase gene and an endoglucanase gene from Thermomonospora fusca.

Two genes encoding cellulases E1 and E4 from Thermomonospora fusca have been cloned in Escherichia coli, and their DNA sequences have been determined. Both genes were introduced into Streptomyces lividans, and the enzymes were purified from the culture supernatants of transformants. E1 and E4 were expressed 18- and 4-fold higher, respectively, in S. lividans than in E. coli. Thin-layer chromatography of digestion products showed that E1 digests cellotriose, cellotetraose, and cellopentaose to cellobiose and a trace of glucose. E4 is poor at degrading cellotriose and cleaves cellopentaose to cellotetraose and glucose or cellotriose and cellobiose. It readily cleaves cellotetraose to cellobiose. E1 shows 59% identity to Cellulomonas fumi CenC in a 689-amino-acid overlap, and E4 shows 80% identity to the N terminus of C. fimi CenB in a 441-amino-acid overlap; all of these proteins are members of cellulase family E. Alignment of the amino acid sequences of Clostridium thermocellum celD, E1, E4, and four other members of family E demonstrates a clear relationship between their catalytic domains, although there is as little as 25% identity between some of them. Residues in celD that have been identified by site-directed mutagenesis and chemical modification to be important for catalytic activity are conserved in all seven proteins. The catalytic domains of E1 and E4 are not similar to those of T. fusca E2 or E5, but all four enzymes share similar cellulose-binding domains and have the same 14-bp inverted repeat upstream of their initiation codons. This sequence has been identified previously as the binding site for a protein that regulates induction.     

Purification, characterization, and synergistic action of endoglucanases from Fusarium lini

Five endoglucanases (1,4-beta-D-glucan-glucanohydrolase, EC 3.2.1.4) were isolated from Fusarium lini. Endo I and II were purified by preparative gel electrophoresis and Endo III, IV, and V were purified in a single-step procedure involving preparative flat-bed isoelectric focusing. All the endoglucanases were homogenous on disk gel electrophoresis and analytical isoelectric focusing in polyacrylamide gel. The pi values were between 6 and 6.6 for Endo III, IV, and V; for Endo I, the pi value was 8. The molecular weights of the enzymes were between 4 x 10(4) and 6.5 x 10(4). The K(m) values for endoglucanases using carboxymethyl cellulose (CM-cellulose) as the substrate were 2-12 mg/mL. The specificity of the enzymes was restricted to beta-1, 4-linkages. All the enzymes showed activity towards D-xylan. The endoglucanases had high viscosity reducing activity with CM-cellulose. Striking synergism was observed for the hydrolysis of CM-cellulose by endoglucanases. Endo II, IV, and V attacked cellopentaose and cellotetraose more readily than cellotriose. Endo II and V hydrolyzed cellotriose, cellotetraose, and cellopentaose, yielding a mixture of cellobiose with a trace amount of glucose; endo IV produced only cellobiose.     

Purification and properties of a novel type of exo-1,4-beta-glucanase (avicelase II) from the cellulolytic thermophile Clostridium stercorarium

Avicelase II was purified to homogeneity from culture supernatants of Clostridium stercorarium. A complete separation from the major cellulolytic enzyme activity (avicelase I) was achieved by FPLC gel filtration on Superose 12 due to selective retardation of avicelase II. The enzyme has an apparent molecular mass of 87 kDa and a pI of 3.9. Determination of the N-terminal amino acid indicates that avicelase II is not a proteolytically processed product of avicelase I. Maximal activity of avicelase II is observed between pH 5 and 6. In the presence of Ca2+, the enzyme is highly thermostable, exhibiting a temperature optimum around 75 degrees C. Hydrolysis of avicel occurs at a linear rate for three days at 70 degrees C. Avicelase II is active towards unsubstituted celluloses, cellotetraose and larger cellodextrins. It lacks activity towards carboxymethylcellulose and barley beta-glucan. Unlike other bacterial exoglucanases, avicelase II does not hydrolyze aryl-beta-D-cellobiosides. Avicel is degraded to cellobiose and cellotriose at a molar ratio of approximately 4:1. With acid-swollen avicel as substrate, cellotetraose is also formed as an intermediary product, which is further cleaved to cellobiose. The degradation patterns of reduced cellodextrins differ from that expected for a cellobiohydrolase attacking the non-reducing ends of chains; cellopentaitol is degraded to cellobiitol and cellotriose, while cellohexaitol is initially cleaved into cellobiitol and cellotetraose. These findings, taken together, indicate that avicelase II represents a novel type of exoglucanase (cellodextrinohydrolase), which, depending on the accessibility of the substrate, releases cellotetraose, cellotriose, or cellobiose from the non-reducing end of the cellulose chains.     

Differential cellulolytic activity of native-form and C-terminal tagged-form cellulase derived from Coptotermes formosanus and expressed in E. coli

An endogenous cellulase gene (CfEG3a) of Coptotermes formosanus, an economically important pest termite, was cloned and overexpressed in both native form (nCfEG) and C-terminal His-tagged form (tCfEG) in Escherichia coli. Both forms of recombinant cellulases showed hydrolytic activity on cellulosic substrates. The nCfEG was more active and stable than tCfEG even though the latter could be purified to near homogeneity with a simple procedure. The differential activities of nCfEG and tCfEG were also evidenced by hydrolytic products they produced on different substrates. On CMC, both acted as an endoglucanase, randomly hydrolyzing internal β-1,4-glycosidic bonds and resulting in a smear of polymers with different lengths, although cellobiose, cellotriose, and cellotetraose equivalents were noticeable. The hydrolytic products of tCfEG were one unit sugar less than those produced by nCfEG. Using filter paper as substrate, however, the major hydrolytic products of nCfEG were cellobiose, cellotriose and trace of glucose; those of tCfEG were cellobiose, cellotriose and trace of cellotetraose, indicating a property similar to that of cellobiohydrolase, an exoglucanase. The results presented in this report uncovered the biochemical properties of the recombinant cellulase derived from the intact gene of Formosan subterranean termites. The recombinant cellulase would be useful in designing cellulase-inhibiting termiticides and incorporating into a sugar-based biofuel production program.     

Affinity chromatography of extracellular cellulase from chaetomium cellulolyticum

Summary An endo--glucanase of C.cellulolyticum was purified by a procedure involving concanavalin A (Con A)-Sepharose chromatography and polyacrylamide gel electrophoresis (PAGE). The enzyme produced G1 and oligosaccharides from CMC. Chromatography on Procion Red HE3B-Agarose proved to be useful in the separation of cellobiase from cellobiose dehydrogenase.Abbreviations CMC carboxymethyl cellulose - CM-cellulase carboxymethyl cellulase - FP-cellulase filter paper degrading cellulase - G1 glucose - G2 cellobiose - G3 cellotriose - G4 cellotetraose - G5 cellopentaose - G6 cellohexaose - G7 celloheptaose - -MG methyl--D-glucoside - pNPG p-nitrophenyl--glucopyranoside - pNP p-nitrophenol - CBDH cellobiose dehydrogenase     

Purification and properties of an endo-1,4-beta-glucanase translated from a Clostridium josui gene in Escherichia coli

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.     

Molecular Docking Study of Beta-Glucosidase with Cellobiose,Cellotetraose and Cellotetriose

Beta-glucosidase (3.2.1.21) plays an essential role in the removal of non-reducing terminal glucosyl residues from glycosides. Recently, beta-glucosidase has been of interest for biomass conversion that acts in synergy with two other enzymes, endoglucanase and exo-glucanase. However, there is not much information available on the catalytic interactions of beta-glucosidase with its substrates. Thus, this study reports on the binding modes between beta-glucosidase from glycoside hydrolase family 1 namely BglB with cellobiose, cellotetraose and cellotetriose via molecular docking simulation. From the results, the binding affinities of BglB-cellobiose, BglB-cellotetraose, and BglB-cellotetriose complexes were reported to be -6.2kJ/mol , -5.68 kJ/mol and -5.63 kJ/mol, respectively. The detail interactions were also been investigated that revealed the key residues involved in forming hydrogen bonds (h-bond) with the substrates. These findings may provide valuable insigths in designing beta-glucosidase with higher cellobiose-hydrolyzing efficiency.     

Purification and properties of an endo-1,4-beta-glucanase translated from a Clostridium josui gene in Escherichia coli.

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.     

Characterization of a cellobiose phosphorylase from a hyperthermophilic eubacterium,Thermotoga maritima MSB8

The cepA putative gene encoding a cellobiose phosphorylase of Thermotoga maritima MSB8 was cloned, expressed in Escherichia coli BL21-codonplus-RIL and characterized in detail. The maximal enzyme activity was observed at pH 6.2 and 80 degrees C. The energy of activation was 74 kJ/mol. The enzyme was stable for 30 min at 70 degrees C in the pH range of 6-8. The enzyme phosphorolyzed cellobiose in an random-ordered bi bi mechanism with the random binding of cellobiose and phosphate followed by the ordered release of D-glucose and alpha-D-glucose-1-phosphate. The Km for cellobiose and phosphate were 0.29 and 0.15 mM respectively, and the kcat was 5.4 s(-1). In the synthetic reaction, D-glucose, D-mannose, 2-deoxy-D-glucose, D-glucosamine, D-xylose, and 6-deoxy-D-glucose were found to act as glucosyl acceptors. Methyl-beta-D-glucoside also acted as a substrate for the enzyme and is reported here for the first time as a substrate for cellobiose phosphorylases. D-Xylose had the highest (40 s(-1)) kcat followed by 6-deoxy-D-glucose (17 s(-1)) and 2-deoxy-D-glucose (16 s(-1)). The natural substrate, D-glucose with the kcat of 8.0 s(-1) had the highest (1.1 x 10(4) M(-1) s(-1)) kcat/Km compared with other glucosyl acceptors. D-Glucose, a substrate of cellobiose phosphorylase, acted as a competitive inhibitor of the other substrate, alpha-D-glucose-1-phosphate, at higher concentrations.     

Studies of the cellulolytic system of Trichoderma reesei QM 9414. Binding of small ligands to the 1,4-beta-glucan cellobiohydrolase II and influence of glucose on their affinity

Binding onto cellobiohydrolase II from Trichoderma reesei of glucose, cellobiose, cellotriose, derivatized and analogous compounds, is monitored by protein-difference-absorption spectroscopy and by titration of ligand fluorescence, either at equilibrium or by the stopped-flow technique. The data complete earlier results [van Tilbeurgh, H., Pettersson, L. G., Bhikhabhai, R., De Boeck, H. and Claeyssens, M. (1985) Eur. J. Biochem. 148, 329-334] indicating an extended active center, with putative subsites ABCD. Subsite A specifically complexes with beta-D-glucosides and D-glucose; at 25 degrees C the latter influences the concomitant binding of other ligands at neighbouring sites. For several ligands this cooperative effect for binding (at 0.33 M glucose and temperature range 4-37 degrees C) was characterized by a substantial increase of the enthalpic term (delta delta H = -35 kJ mol-1). Glucose (0.33 M) decreases the association and dissociation rate parameters of 4-methylumbelliferyl beta-D-cellobioside by one order of magnitude: k+ = (3.6 +/- 0.5) x 10(-5) M-1 s-1 versus (5.1 +/- 0.1) x 10(-6) M-1 s-1 (in the absence of glucose) and k- = (1.3 +/- 0.1) s-1 versus (14.0 +/- 0.3) s-1. As deduced from substrate-specificity studies and inhibition experiments, subsite B interacts with terminal non-reducing glucopyranosyl residues of oligomeric ligands and substrates, whereas catalytic (hydrolytic) cleavage occurs between C and D. Association constants 10-100 times higher than those for cellobiose or its glycosides were observed for D-glucopyranosyl-(1----4)-beta-D-xylopyranose and cellobionolactone derivatives, suggesting 'transition-state'-type binding for these ligands at subsite C. Although subsite D can accomodate a bulky chromophoric group (MeUmb) its preference for a glucosyl residue is reflected in the lower binding enthalpy of cellotriose (-34 kJ mol-1) as compared to cellobiose (-28.3 kJ mol-1) and MeUmb(Glc)2 (-11.6 kJ mol-1). This model indicates that oligomeric ligands (substrates) interact through cooperativity of their subunits at the extended binding site of cellobiohydrolase II.     

Proteomic and physiological experiments to test Thermotoga neapolitana constraint-based model hypotheses of carbon source utilization

Constraint-based models of biochemical reaction networks require experimental validation to test model-derived hypotheses and iteratively improve the model. Physiological and proteomic analysis of Thermotoga neapolitana growth on cellotetraose was conducted to identify gene products related to growth on cellotetraose to improve a constraint-based model of T. neapolitana central carbon metabolism with incomplete cellotetraose pathways. In physiological experiments comparing cellotetraose to cellobiose and glucose as growth substrates, product formation yields on cellotetraose, cellobiose, and glucose were similar; however cell yields per mol carbon consumed were higher on cellotetraose than on cellobiose or glucose. Proteomic analysis showed increased expression of several proteins from cells grown on cellotetraose compared with glucose cell cultures, including cellobiose phosphorylase (CTN_0783), endo-1,4-β-glucosidase (CTN_1106), and an ATP-binding protein (CTN_1296). The CTN_1296 gene product should be evaluated further for participation in cellotetraose metabolism and is included as one of two hypothetical gene-protein-reaction associations in the T. neapolitana constraint-based model to reinstate cellotetraose metabolism in model simulations.     

Utilization of individual cellodextrins by three predominant ruminal cellulolytic bacteria.

Growth of the ruminal bacteria Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD-1, and R. albus 7 followed Monod kinetics with respect to concentrations of individual pure cellodextrins (cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose). Under the conditions tested, R. flavefaciens FD-1 possesses the greatest capacity to compete for low concentrations of these cellodextrins.     

Endo-β-Glucanase from Acetobacter xylinum: Purification and Characterization

A cellulose-producing acetic acid bacterium, Acetobacter xylinum KU-1, abundantly produces an extracellular endo-β-glucanase (EC 3.2.1.4) in the culture broth. The enzyme was purified to homogeneity by DEAE- and CM- Toyopearl 650M ion-exchange chromatography, Butyl-Toyopearl 650M hydrophobic chromatography, and Toyopearl HW-50 gel filtration. The purified enzyme showed the maximum activity at pH 5 and 50°C: it was stable up to 50°C at pH 5, activated by Co²⁺, and competitively inhibited by Hg²⁺; the apparent K _i was 7 μM. The molecular weight of the enzyme was determined to be about 39,000 by sodium dodesyl sulfate/polyacrylamide gel electrophoresis, and about 41,000 by Toyopearl HW-50 gel filtration; the enzyme is monomeric. The enzyme hydrolyzed carboxymethylcellulose with an apparent K _m of 30 mg/ml and V _max of 1.2 μM/min. It hydrolyzed cellohexaose to cellobiose, cellotriose and cellotetraose, and also cellopentaose to cellobiose and cellotriose, but did not act on cellobiose, cellotriose, or cellotetraose. Received: 3 October 1996 / Accepted: 5 November 1996     

An Avicel-affinity site in an Avicel-digesting exocellulase from a Trichoderma viride mutant.

A single form of exo-type cellulase (Exo I; MW, 65,000), purified from a Trichoderma viride protease-depressed mutant, HK-75, digested Avicel to cellobiose exowise, and hydrolyzed cellotriose, cellotetraose, and cellopentaose in the strict manner of splitting off by cellobiose units. Exo I, however, hydrolyzed cellohexaose by both cellobiose and cellotriose units. Exo I was proteolyzed by papain into two fragments; GPExo (MW, 9,000) and Exo I' (MW, 56,000). The GPExo intensively adsorbed onto Avicel but did not hydrolyze it. Exo I' had nearly identical activity to that of intact Exo I toward cellooligosaccharides but was almost inert to Avicel in digestion and adsorption. Sequence analysis of N-terminal and C-terminal amino acids showed that GPExo was between Gly435 and Leu496 and Exo I' between Glu1 and Gly434 in Exo I. Exo I therefore consists of two domains, one for adsorption to Avicel, as demonstrated by the Avicel-affinity site, GPExo and the other for the cleavage of glycosidic linkages as demonstrated in Exo I'.     

酶法制备功能性纤维低聚糖的研究

研究里氏木霉(Trichoderma reesei)Rut C30纤维素酶单一组分EGI、EGII和CBHI降解纤维素的机理及纤维低聚糖酶法制备技术,进而初步研究纤维低聚糖对青春双歧杆菌的增殖作用。以内切葡聚糖酶EGII酶法制备纤维低聚糖,每克纤维素最佳酶用量1 U,最佳酶解时间90 min,制备得到的纤维低聚糖中纤维二糖、纤维三糖和纤维四糖占总糖的比例分别为43.8%、34.8%和7.9%。以纤维二糖、纤维低聚糖为C源增殖青春双歧杆菌,菌体质量浓度增殖倍数分别为2.14、2.84。     

Cloning and characterization of the glucooligosaccharide catabolic pathway beta-glucan glucohydrolase and cellobiose phosphorylase in the marine hyperthermophile Thermotoga neapolitana

Characterization in Thermotoga neapolitana of a catabolic gene cluster encoding two glycosyl hydrolases, 1,4-beta-D-glucan glucohydrolase (GghA) and cellobiose phosphorylase (CbpA), and the apparent absence of a cellobiohydrolase (Cbh) suggest a nonconventional pathway for glucan utilization in Thermotogales. GghA purified from T. neapolitana is a 52.5-kDa family 1 glycosyl hydrolase with optimal activity at pH 6.5 and 95 degrees C. GghA releases glucose from soluble glucooligomers, with a preference for longer oligomers: k(cat)/K(m) values are 155.2, 76.0, and 9.9 mM(-1) s(-1) for cellotetraose, cellotriose, and cellobiose, respectively. GghA has broad substrate specificity, with specific activities of 236 U/mg towards cellobiose and 251 U/mg towards lactose. With p-nitrophenyl-beta-glucoside as the substrate, GghA exhibits biphasic kinetic behavior, involving both substrate- and end product-directed activation. Its capacity for transglycosylation is a factor in this activation. Cloning of gghA revealed a contiguous upstream gene (cbpA) encoding a 93.5-kDa cellobiose phosphorylase. Recombinant CbpA has optimal activity at pH 5.0 and 85 degrees C. It has specific activity of 11.8 U/mg and a K(m) of 1.42 mM for cellobiose, but shows no activity towards other disaccharides or cellotriose. With its single substrate specificity and low K(m) for cellobiose (compared to GghA's K(m) of 28.6 mM), CbpA may be the primary enzyme for attacking cellobiose in Thermotoga spp. By phosphorolysis of cellobiose, CbpA releases one activated glucosyl molecule while conserving one ATP molecule per disaccharide. CbpA is the first hyperthermophilic cellobiose phosphorylase to be characterized.