Cellobiose Units Research Articles

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'.

Evidence for the lack of exo-cellobiohydrolase activity in the cellulase system of Trichoderma reesei QM 9414

A cellulolytic enzyme (pI 4.3) was isolated from culture filtrates of Trichoderma reesei QM 9414 by column chromatography and preparative flat bed isoelectric focusing. The focusing experiments were carried out using an extremely shallow pH gradient (pH 3.7–4.6), which made it possible to recover one single protein band out of numerous Trichoderma isoenzymes and/or multi-enzyme complexes in the acidic pH region. The purified hydrolase is homogeneous as assessed by analytical isoelectric focusing, SDS gel electrophoresis, and titration curve analysis under detergent/ urea conditions. It was found to have an apparent molar mass of 68 000 g mol −1 (SDS-PAGE) and showed its optimum activity towards cellotetraose at pH 4 and 60°C. It is a glycoprotein with a carbohydrate content of 27% (as glucose). The hydrolase does not act with an endo-mechanism (CMC as substrate) and splits off mainly cellobiose from microcrystalline cellulose. In the current literature the enzyme would thus be termed an exo-cellobiohydrolase [1,4-β-D-glucan cellobiohydrolase (CBH), EC 3.2.1.91]. However, when the enzyme's action pattern was further evaluated using defined soluble cellodextrins (DP 2–7) as substrates, it became obvious from the recorded concentration-time course data that the enzyme does not exclusively split off cellobiose units from the oligomers. In conclusion, we do not believe that a cellobiohydrolase analogous to β-amylase (EC 3.2.1.2) exists, at least in the cellulase system of Trichoderma reesei. The term CBH or cellobiohydrolase should thus not be used any longer in the literature.

Degradation of barley ?-glucan by endoglucanase C of Clostridium thermocellum

Endoglucanase C encoded by the celC gene of Clostridium thermocellum has been purified to homogeneity from a recombinant Escherichia coli strain. It was found that this enzyme is highly efficient in degrading glucans with alternating β-1,4- and β-1,3-linkages but lacks activity on unmodified cellulosic substrates. The properties of endoglucanase C were compared to those of Bacillus subtilis β-glucanase, an enzyme used in the brewing industry for β-glucan degradation. Both enzyme cause a rapid decrease of the viscosity of barley β-glucan as a result of internal chain cleavage. Endoglucanase C hydrolyses non-specifically β-1,3- and β-1,4-bonds adjacent to unsubstituted or 4-O′-substituted cellobiose units. Due to its lower pH optimum and increased thermostability endoglucanase C compares favourably with B. subtilis β-glucanase and seems suitable for use in the mashing process of beer brewing.

Macromolecular Organization of the Cellulolytic Enzyme Complex of Clostridium thermocellum as Revealed by Electron Microscopy

Clostridium thermocellum JW20 and YM4 both synthesize cellulolytic enzyme complexes, cellulosomes, when grown on medium containing cellulose. Electron microscopic studies showed that, in the early stages of growth of strain JW20, clusters of tightly packed cellulosomes, i.e., polycellulosomes, were located on the cell surface and were bound to cellulose. The polycellulosome was estimated to have a particle mass of 50 x 10 to 80 x 10 daltons (Da), while that of the cellulosome was estimated to be 2 x 10 to 2.5 x 10 Da and to contain about 35 polypeptides ranging from 20 to 200 kDa. The cellulosome produced by strain YM4 was found to be somewhat larger, with the estimated particle mass being 3.5 x 10 Da, and the number of polypeptides was counted to be 45 to 50, ranging from 20 to 200 kDa. In the early stages of cultivation, the cellulosomes from both species exist as tightly packed complexes (tight cellulosomes). These subsequently decompose to loosely packed complexes (loose cellulosomes) and ultimately to free polypeptides. Examination of the loose cellulosomal particles showed that they contain rows of equidistantly spaced, similarly sized polypeptide subunits, with an apparently identical orientation arranged parallel to the major axis of the cellulosome. It is postulated that on binding of a cellulose chain alongside such a row of subunits a simultaneous multicutting event occurs that leads to the release of cellooligosaccharides of four cellobiose units in length (C(4)). Rows of smaller-sized subunits with lower center-to-center distances, which are also present in the cellulosome, subsequently cleave the C(4) fragments (or cellulose) to C(2) (cellotetraose) or C(1) (cellobiose). In this way the cellulosome can catalyze the complete hydrolysis of cellulose.

Purification and properties of the endoglucanase C of Clostridium thermocellum produced in Escherichia coli

The celC gene, which codes for a new endoglucanase of Clostridium thermocellum, termed endoglucanase C, was found to be expressed when cloned in Escherichia coli. The enzyme was purified to electrophoretic homogeneneity from E. coli and its biochemical properties were studied. It differs from the previously studied endoglucanases A and B. In particular, endoglucanase C displays features common to endo- and exoglucanases, since it had a high activity on carboxymethylcellulose and on p-nitrophenyl-beta-D-cellobioside where only the agluconic bond was split. In addition, the enzyme was able to release cellobiose units from G3, G4 and G5 cellodextrins. Endoglucanase C was characterized by Western blot in a culture supernatant from C. thermocellum grown on cellulose, using an antiserum raised against the enzyme produced by E. coli.

Structures and Antitumor Activities of the Polysaccharides Isolated from Fruiting Body and the Growing Culture of Mycelium of Ganoderma lucidum

Several β-D-glucans, appertaining to the same molecular species but having different degrees of branching, were isolated from water and alkali extracts of the fruiting body of Ganoderma lucidum (Reishi). The purified glucans that were mostly water-insoluble had a backbone of (1 →3)-linked D-glucose residues, attached mainly with single D-glucosyl units at 0-6 and also with a few short (l→4)-linked glucosyl units at 0-2 positions. However, their degrees of branching appeared to differ in the range of d.b. 1/3 ~ 1/23, depending on the extracted glucan fractions. In addition to the ^-glucans, the fruiting body contained water-soluble heteropolysaccharides, comprising D-glucose, D-galactose, D-mannose, L-(or D)-arabinose, D-xylose, and L-fucose. A branched (1 →3)-β-D-glucan was also isolated from the culture filtrate of G. lucidum grown in a glucose-yeast extract medium. The extracellular β-D-glucan was less soluble in water after purification, but soluble in dilute alkali. This glucan has essentially the same structure as that of hot-water extracted polysaccharide from the fruiting body. The repeating unit of the glucan contains a backbone chain of (1 →3)-linked D-glucose residues, five out of sixteen D-glucose residues being substituted at 0-6 positions with single D-glucosyl units and one D-glucose residue at 0-2 positions probably with a cellobiose unit. The hot-water extractable fruiting body glucan and the extracellular glucan of the culture of growing mycelium showed relatively high growth-inhibition activities against Sarcoma 180 solid tumor in mice, when administered by. successive intraperitoneal injections. When the moderately branched glucans were modified to D-glucan-polyols by periodate oxidation and borohydride reduction, they exhibited higher antitumor activities, confirming the previous conclusion that the attachment of polyol groups to the (1 →3)-lmked backbone significantly enhances its host-mediated antitumor effect.

Structures and antitumor activities of the polysaccharides isolated from fruiting body and the growing culture of mycelium of Ganoderma lucidum.

Several β-D-glucans, appertaining to the same molecular species but having different degrees of branching, were isolated from water and alkali extracts of the fruiting body of Ganoderma lucidum (Reishi). The purified glucans that were mostly water-insoluble had a backbone of (1→3)-linked D-glucose residues, attached mainly with single D-glucosyl units at O-6 and also with a few short (1→4)-linked glucosyl units at O-2 positions. However, their degrees of branching appeared to differ in the range of d.b. 1/3-1/23, depending on the extracted glucan fractions. In addition to the β-glucans, the fruiting body contained water-soluble heteropolysaccharides, comprising D-glucose, D-galactose, D-mannose, L-(or o)-arabinose, D-xylose, and L-fucose. A branched (1→3)-β-D-glucan was also isolated from the culture filtrate of G. lucidum grown in a glucose-yeast extract medium. The extracellular β-D-glucan was less soluble in water after purification, but soluble in dilute alkali. This glucan has essentially the same structure as that of hot-water extracted polysaccharide from the fruiting body. The repeating unit of the glucan contains a backbone chain of (1→3)-linked D-glucose residues, five out of sixteen D-glucose residues being substituted at O-6 positions with single D-glucosyl units and one D-glucose residue at O-2 positions probably with a cellobiose unit. The hot-water extractable fruiting body glucan and the extracellular glucan of the culture of growing mycelium showed relatively high growth-inhibition activities against Sarcoma 180 solid tumor in mice, when administered by- successive intraperitoneal injections. When the moderately branched glucans were modified to D-glucan-polyols by periodate oxidation and borohydride reduction, they exhibited higher antitumor activities, confirming the previous conclusion that the attachment of polyol groups to the (1→3)-linked backbone significantly enhances its host-mediated antitumor effect.

An assay for selective determination of exo-1,4,-β-glucanases in a mixture of cellulolytic enzymes

A selective procedure using synthetic substrates for determination of exo-1,4,-β-glucanases in a mixture of exoglucanases, endoglucanases, and β-glucosidases is formulated. The heterobiosides, p-nithrophenyl-β-d-cellobioside (pNPC) or p-nitrophenyl-β-d-lactoside (pNPL), were used as selective substrates for the measurement of exoglucanase activity. The exoglucanases (especially cellobiohydrolases, which split off cellobiose units from the nonreducing end of the cellulose chain) specifically act on the agluconic bond (between p-nitrophenyl and the disaccharide moiety) and not on the holosidic bond (between the two glucose units of cellobiose). The interfering effect of β-glucosidase, which acts on both agluconic and holosidic bonds, is overcome by the addition of d-glucono-1,5-δ-lactone, a specific inhibitor of β-glucosidases. The interference of endoglucanases, which also act on both agluconic and holosidic bonds, can be compensated for by prior standardization of the assay procedure with a purified endoglucanase from the studied mixture of cellulases.

Polysaccharides ofEremurus. XV. Structure of the glucomannan ofEremurus lactiflorus

Ten oligosaccharides have been isolated from the products of the partial hydrolysis of a native acetylated glucomannan obtained fromEremurus lactiflorus O. Fedtsch. Their structures have been studied with the aid of acid hydrolysis before and after reduction with NaBH4, by the GLC method, and also by chromatography with markers. The compositions and sequence of the monomers in tetra- and heptaoligo-saccharides have been determined by the13C NMR method. The glucommannan ofE. lactiflorus differs from theEremurus glucomannans studied previously by the ratio of monosaccharides, the presence of 0-Ac groups, the degree of polymerization, and the presence of a cellobiose unit (Glcp-Glcp) in the polymer chain. The repeating unit consists of 14 hexose residues.

Isolation and characterization of a new extracellular polysaccharide from a cellulose-negative strain of Acetobacter xylinum.

An extracellular, acidic polysaccharide has been isolated from the culture medium of a spontaneous cellulose-negative strain of Acetobacter xylinum. Chemical analysis shows that the polymer is composed of glucose, mannose, rhamnose, and glucuronic acid in a molar ratio approximating 3:1:1:1. No evidence for the presence of cellobiose units as structural parts in the polysaccharide has been found.

The nature and mode of action of the cellulolytic component C1 of Trichoderma koningii on native cellulose.

1. A purified cellulolytic component C(1) was isolated free from associated activities of the cellulase complex and shown to act as a beta-1,4-glucan cellobiohydrolase on both simple and complex forms of native cellulose. 2. The enzyme releases terminal cellobiose units from cellulose, its extent of action being determined principally by the product and by the nature of the substrate. 3. Component C(x) of the cellulase system is not required for the action of component C(1) (cellobiohydrolase). The enzyme synergizes extensively with cellobiase in extending the hydrolysis of native and of less-complex forms of cellulose to at least 70% with the liberation of glucose. 4. The cellobiohydrolase is relatively unstable, with an optimum at pH5 and a K(m) of 0.05mg/ml. The enzyme is inhibited by its product, from which it is released by cellobiase. 5. Of other compounds tested against the cellobiohydrolase the metal ions Cu(2+), Zn(2+), phenylmercuric and Fe(3+) are increasingly effective inhibitors. Glucose has no action at concentrations found inhibitory with cellobiose. 6. The relationship of the enzyme to the entire cellulase complex is discussed.

Effect of aromatic groups on localization of high energy in cellulose

AbstractWhen γ‐radiation from 60Co interacted with fibrous cotton cellulose, the localization of at least part of the high energy resulted in cellulosic chain cleavage and loss in breaking strength of the irradiated fibers. The substitution of aromatic groups on the cotton cellulose molecule affected this localization of energy and decreased the radiation degradation of the fiber. The nature of the linkage of the aromatic group to the cellulose molecule was not as important as the radiation stability of the linkage. For example, if localization of energy occurred which cleaved the aromatic group from the cellulose molecule, the radioprotection of the cellulosic molecular chain by the aromatic group was not effective. If the aromatic group was so modified that the effective number of π‐electrons was decreased, the radioprotection of the cellulosic chain was also decreased. The radioprotection of the cellulosic molecular chain by benzhydryl, trityl, benzoyl, and cinnamoyl groups was effective over distances equivalent to several cellobiose units. The radioprotection of the cellulosic chain by naphthoyl groups was significant but not as effective as the listed groups. Due to the sharing of π‐electrons in the naphthoyl group, the effective number of π‐electrons was reduced, and consequently the radioprotective effect of the group was also reduced. Benzyl groups were cleaved from the cellulose molecule on irradiation and offered no radioprotection to the cellulosic chain, at least at the high radiation dosages used. The ESR spectra of the irradiated celluloses, both substituted and unsubstituted, were similar. This indicated that the presence of aromatic groups did not change the nature of the long‐lived free radicals induced in cellulose on irradiation. It was suggested that selective energy absorption by the aromatic group from the spur of high‐energy electrons produced on interaction of γ‐radiation with the cellulose molecule could account for the radioprotection of the cellulosic molecular chain.

Immunochemical studies on some immune systems involving ß(1,4) linked glucose

Rabbit antiserum to a BSA-cellobiose conjugate was examined by immunodiffusion and quantitative hapten inhibition employing the synthetic polyvalent hapten phloroglucinol triazophenyl-ß- d-cellobioside as antigen. The isomeric series of ß-linked glucobioses and mixed oligosaccharides possessing either terminal nonreducing or internal (subterminal) cellobiose were assayed for their ability to inhibit precipitation. Anticonjugate was more effectively inhibited by oligosaccharides possessing a terminal non-reducing cellobiose unit than by compounds with subterminal cellobiose groups. antibody could readily distinguish the ß-(1,4) from the ß-(1,3) and ß-(1,6) glucosidic linkages. The portion of horse anti-VIII, precipitable by BSA-Cello was examined by hapten inhibition and p- nitrophenyl-ß- d-cellobioside , cellotriose and cellotetraose found to be equipotent inhibitors of precipitation suggesting a specificity directed against the ß- d-cellobiosyl or cellotriose unit.

Intramolecular transfer of high energy in cellulose

AbstractThe intramolecular transfer of high energy in purified cotton cellulose has been demonstrated to occur over distances several times greater than the length of the b axis of a cellobiose unit. The electron spin resonance spectra of fibrous benzoylated cotton cellulose have shown that the concentrations of free radicals initiated in the cellulosic molecule by γ‐radiation from Co60 are decreased by the presence of substituted benzoyl groups spaced at least as far apart as 70–80 A. The retention of the breaking strength of fibrous cotton cellulose on exposure to γ‐radiation is increased by substitution of benzoyl groups on the cellulose molecule. The substitution of benzoyl groups, spaced at least as far apart as 10–20 A., results in about 80% retention of the initial strength of the fibrous cellulose at dosages as high as 1.3 × 1021 e.v./g. cellulose, as compared with about 20% retention of the initial strength of unsubstituted cellulose at the same dosage. Based on the breaking strengths of the irradiated fibrous celluloses, at a dosage of 1.3 × 1021 e.v./g. cellulose, the average calculated distances between molecular cleavages are increased from about 500 A. for unsubstituted cellulose to about 3000 A. for benzoylated cellulose (degree of substitution 2.0). These energy transfer effects in cellulose of high molecular weight indicate that polymeric carbohydrates can be radioprotected.

Infrared spectra of crystalline polysaccharides. I. Hydrogen bonds in native celluloses

AbstractThe infrared spectra of oriented films of valonia cellulose and of ramie and bacterial cellulose crystallites have been observed in the 3 μ region. Polarization properties of the bands have also been determined. The differences between the polarized spectra of bacterial and ramie crystallites in this region are attributed to per cent crystallinity and orientation effects. Two new bands in the CH stretching region have been observed. With this new information the CH2 symmetric and antlisymmetric stretching modes are assigned to parallel and perpendicular bands, respectively, requiring a specific orientation of the CH2OH group. From the observed polarization of the bands in the OH stretching region, a system of hydrogen bonding in the crystal structure of cellulose I is proposed. This involves a change in conformation of the cellobiose unit to permit an intramolecular hydrogen bond between the C3 hydroxyl and the ring oxygen of contiguous glucose units. Two sets of intermolecular hydrogen bonds are proposed: in the 101 plane the C6 hydroxyls of the antiparallel chains are joined to the bridge oxygens of the adjacent parallel chains; in the 101 plane the C6 hydroxyls of the parallel chains are hydrogen‐bonded to the bridge oxygens of the adjacent antiparallel chains.

The molecular and crystal structure of cellulose triacetate

AbstractAn x‐ray diffraction study has been made of cellulose triacetate II. Small films of highly crystalline triacetate with good fibrous orientation and a fair degree of biaxial orientation have been prepared by repeated rolling at successively higher temperatures between 160 and 215°C. The x‐ray diffraction patterns exhibited several new reflections which made it possible to determine the unit cell. It has been found that cellulose triacetate II has four cellobiose acetate residues in a pseudo‐orthorhombic cell with a = 24.5, b = 11.56, and c = 10.43 A. Pairs of these residues are related by twofold screw axes in the c or fiber‐axis direction. The paired arrangement seems to be determined by a fitting of the natural zigzagging shape of the cellulose backbone which is exaggerated by the presence of the secondary acetyl groups. The glucose residues of such a pair are parallel and inclined at about 45° with respect to the a axis. A neighboring chain pair points in the opposite direction and the median planes of the cellobiose units of two such pairs are approximately mutually perpendicular. To accommodate this type of packing, one half of the primary acetyl groups must be twisted sharply out of the median plane of the parent glucose residues. A second statistical model which basically is very similar is also considered. There do not appear to be any strong interactions between chains.

THE MECHANISM OF DEGRADATION OF A CELLODEXTRIN BY MYROTHECIUM CELLULASE

A cellodextrin with a number-average degree of polymerization (D.P.) of 24 was hydrolyzed by Myrothecium cellulase in the presence of C14-labelled cellobiose. The specific activities of cellotriose and cellotetraose isolated at various stages of the hydrolysis were less than 20% of that of cellobiose isolated at the same time. It is concluded that transfer reactions involving cellobiose contributed little to the formation of cellotriose and cellotetraose. The ratio moles of cellotriose formed/mole of cellobiose formed was at least 0.7 and the corresponding ratio for cellotetraose at least 0.4. The number-average D.P. of the substrate decreased slightly during the hydrolysis. The results exclude a degradation of the substrate by cleavage of cellobiose units from the ends of its chains but are consistent with degradation by random cleavage.

THE MECHANISM OF DEGRADATION OF A CELLODEXTRIN BY MYROTHECIUM CELLULASE

A cellodextrin with a number-average degree of polymerization (D.P.) of 24 was hydrolyzed by Myrothecium cellulase in the presence of C14-labelled cellobiose. The specific activities of cellotriose and cellotetraose isolated at various stages of the hydrolysis were less than 20% of that of cellobiose isolated at the same time. It is concluded that transfer reactions involving cellobiose contributed little to the formation of cellotriose and cellotetraose. The ratio moles of cellotriose formed/mole of cellobiose formed was at least 0.7 and the corresponding ratio for cellotetraose at least 0.4. The number-average D.P. of the substrate decreased slightly during the hydrolysis. The results exclude a degradation of the substrate by cleavage of cellobiose units from the ends of its chains but are consistent with degradation by random cleavage.

Solid Organic Materials

CELLULOSE is Nature's strong material. It is the chief constituent of cotton flax and wood. Wood can be turned into sugars by treatment with hydrochloric acid Bergius process) and by certain termites; horses and cows break down the cellulose in grass into sugar before digesting it. So it is not surprising that the cellulose polymer is built up of what are practically molecules of a glucose (“Barley Sugar”). Each β glucose unit is twisted about its axis through 180 deg.; the combination of two such units makes up that is called a cellulose unit which has the structure shown in Fig. 2. The cellulose polymer is a long, straight chain made up from these cellobiose units, and each chain probably contains about 70 such units.