Reducing End Groups Research Articles

Processive action of cellobiohydrolase Cel7A from Trichoderma reesei is revealed as 'burst' kinetics on fluorescent polymeric model substrates.

Reaction conditions for the reducing-end-specific derivatization of cellulose substrates with the fluorogenic compound, anthranilic acid, have been established. Hydrolysis of fluorescence-labelled celluloses by cellobiohydrolase Cel7A from Trichoderma reesei was consistent with the active-site titration kinetics (burst kinetics), which allowed the quantification of the processivity of the enzyme. The processivity values of 88+/-10, 42+/-10 and 34+/-2.0 cellobiose units were found for Cel7A acting on labelled bacterial cellulose, bacterial microcrystalline cellulose and endoglucanase-pretreated bacterial cellulose respectively. The anthranilic acid derivatization also provides an alternative means for estimating the average degree of polymerization of cellulose and, furthermore, allows the quantitative monitoring of the production of reducing end groups on solid cellulose on hydrolysis by cellulases. Hydrolysis of bacterial cellulose by cellulases from T. reesei revealed that, by contrast with endoglucanase Cel5A, neither cellobiohydrolases Cel7A nor Cel6A produced detectable amounts of new reducing end groups on residual cellulose.

LC/MS Characterization of Meningococcal Depolymerized Polysaccharide Group C Reducing Endgroup and Internal Repeating Unit

Hydrogen peroxide has been used to cleave the native Neisseria meningiditis polysaccharide (PS) from mega-Dalton molecular weight to a smaller size (approximately 20 kDa) depolymerized polysaccharide. The polysaccharide was examined after partial peroxide depolymerization to verify the presence of the carboxyl group at position 1 and the intactness of the internal sialic acid repeating units. The reducing end group of meningococcal polysaccharide type C was also examined after derivatization by L-tyrosine hydrazide. Partial peroxide depolymerization did not result in loss of the position 1 carboxyl group at the reducing end of the polysaccharide. In addition, no loss of structural integrity was noted for the internal sialic repeat units.

Structural reinvestigation of the core oligosaccharide of a mutant form of Aeromonas salmonicida lipopolysaccharide containing an O-4 phosphorylated and O-5 substituted Kdo reducing end group using electrospray QqTOF-MS/MS.

The molecular structure of the mutant form of the lipopolysaccharide of Aeromonas salmonicida was determined to contain an O-4 phosphorylated and O-5 substituted Kdo reducing group, and is proposed as the following: [molecular structure: see text] It was established that during the cleavage of this LPS with 1% acetic acid, to release the core oligosaccharide from the Lipid A portion, we obtained a degraded core oligosaccharide which eliminated its phosphate group with extreme facility. The precise molecular structure of this dephosphorylated core was deduced by electrospray mass spectrometry and is proposed as the following:[molecular structure: see text] Low energy collision ESI-QqTOF-MS/MS analysis of the dephosphorylated core oligosaccharide confirmed the presence of the O-5 glycosylated 4,8- and 4,7-anhydro derivatives of the enolizable alpha-keto-acids. The CID tandem mass spectrometric analysis of the heterogeneous mixture of the permethylated core oligosaccharide established the unreported methylation reaction on the diastereomeric 4,8- and 4,7-anhydro alpha-keto-acids and the complete permethylation and addition reaction of the O-5 glycosylated open chain reducing end terminal D-arabino-3-en-2-ulonic acid. The stereo-specific fragmentation routes obtained during the tandem mass spectrometric analysis permitted the precise sequencing of this dephosphorylated rough core oligosaccharide of the mutant LPS of A. salmonicida.

Viscometric determination of dialdehyde content in periodate oxycellulose. Part I. Methodology

Periodate oxycellulose was subjected to alkaline hydrolysis at room temperature in both homogeneous (cupriethylenediamine) and heterogeneous (sodium hydroxide) medium, and the degradation kinetics was followed for nearly three months. By comparing the obtained results with the degradation kinetics of both hydrocellulose and periodate oxycellulose reduced with tert-butylamine borane, it was demonstrated that the β-alkoxy fragmentation of oxidised sites is a very fast reaction, which reaches completeness during the preparation of samples for viscometric analyses. The subsequent degradation is due to other mechanisms, such as autoxidation and peeling. A comparison between the degrees of polymerisation of periodate oxycellulose before and after its reduction allows the quantitative determination of dialdehyde groups, without the interference of reducing end groups. Although this technique might not be valid for other kinds of oxycellulose, it supplies a simple and fast method for the analysis of mildly oxidised cellulose.

A novel method for the determination of carbonyl groups in cellulosics by fluorescence labeling. 3. Monitoring oxidative processes.

The fluorescence-based CCOA method for determination of carbonyl group profiles in cellulosic substrates was employed to study the mechanisms of various oxidative and degradation processes involving celluloses in greater detail. The approach comprises labeling with the marker carbazole-9-carboxylic acid [2-(2-aminooxyethoxy)ethoxy]amide (CCOA), followed by gel permeation chromatography in DMAc/LiCl with fluorescence, multiangle laser light scattering, and refractive index detection. At first, the CCOA method was applied to study solutions of pulp in N-methylmorpholine-N-oxide monohydrate (NMMO), as occurring in the production of Lyocell-type fibers. NMMO is a rather strong oxidant that on one hand converts reducing end groups to carboxyl structures, thus lowering the overall carbonyl content, but generates new keto structures along the chain by nonselective oxidation on the other hand. The CCOA method allowed for the first time to distinguish the carbonyl course in different molecular weight ranges. Second, alkalization and aging of pulp, which are used in the industrial preparation of cellulose derivatives, e.g., as an element of the preripening process in viscose rayon production, were investigated. The CCOA method shows a clear reduction of the molecular weight, accompanied by a fast loss of carbonyls in the first phase, which is due to removal of low-molecular weight material by dissolution, and a slow decrease in the second phase, which is caused by further oxidation of carbonyl groups. Also here, differences in the carbonyl course in different molecular weight regions were monitored. Third, electron beaming, proposed as a means of pulp activation, was shown to decrease and narrow the molecular weight distribution, under generation of comparatively low amounts of carbonyls, which, however, are also introduced into high molecular weight, crystalline domains, as shown by a comparison of homogeneous and heterogeneous CCOA labeling approach. Finally, as the fourth application, thermal treatment of cellulose at temperatures between 105 and 165 degrees C was shown to bring about a small reduction of the molecular weight, which only at higher drying temperatures is accompanied by an introduction of carbonyls over the whole molecular weight range.

Synthesis and Characterization of Oligomaltose-Grafted Lipids with Application to Liposomes

Novel glycolipids, which contain 2 and 15 oligomaltose units and a phosphatidylethanolamine, were synthesized and characterized by FTIR and 1H-NMR spectroscopies. The well-defined linear structure of the glycolipids was assured by an end-point conjugation strategy using selective oxidation of the reducing end groups of maltose oligosaccharides, followed by aminolysis with distearoylphosphatidylethanolamine. The intermediate acids react selectively with amines to form amide linkages, catalyzed by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide. Conformations of the glycolipids at the air–water interface were proposed based on the film balance measurements. The unique conformations of glycolipids at interfaces may offer advantages over traditional PEO-derived lipids in regard to their applications for sterically stabilizing liposomes. The glycolipids demonstrated the ability for sterically stabilizing liposome dispersions, as determined by turbidity measurements.

Hydrolytic Processes and Condensation Reactions in the Cellulose Solvent System N,N-Dimethylacetamide/Lithium Chloride. Part 1.

Summary Refluxing of cellulose samples in N,N-dimethylacetamide (DMAc, 1), or in DMAc containing low concentrations of LiCl, is a common protocol to facilitate dissolution of the cellulose for GPC measurements. The formation of chromophoric condensation products, whose structures have been elucidated for the first time, and of secondary chromophores by reaction with carbohydrate structures has been demonstrated. DMAc containing traces of water is slowly hydrolyzed to dimethylammonium acetate (2) under reflux conditions. The reaction is accelerated in the presence of lithium chloride. In contrast, absolute DMAc produces five different condensation products upon thermal treatment, which were isolated by non-traditional column chromatography approach. The primary condensation product N,N-dimethylacetoacetamide (3) is formed as the major component and acts as the precursor to the other chromophore products, namely dehydroacetic acid and isodehydroacetic acid derivatives. Compound 3 is able to form furan derivatives by reaction with reducing end groups or carbonyls in carbohydrate structures as demonstrated by reactions with model compounds. As a consequence, it must be concluded that heating/refluxing in DMAc or DMAc/LiCl generates a number of potent chromophores from the solvent and, furthermore, can chemically alter pulp samples by introducing reactive structures.

A xylosyltransferase that synthesizes beta-(1-->4)-xylans in wheat (Triticum aestivum L.) seedlings.

A particulate preparation from 6-day-old seedlings of wheat (Triticum aestivum L.) was found to contain a xylosyltransferase (XylTase) which incorporated xylose (Xyl) from UDP-xylose into exogenous beta-(1-->4)-xylooligosaccharides with 2-aminopyridine-derivatized reducing end groups. High-performance liquid chromatographic analysis showed that the chain elongation of pyridylaminated beta-(1-->4)-xylotriose (Xyl3-PA) occurred by attachment of a series of one, two, or three xylosyl residues, depending on substrate concentrations and reaction times. Methylation analysis and beta-xylosidase digestion of the newly synthesized Xyl4-PA confirmed that the xylosyl residues were incorporated through beta-(1-->4)-linkages. The enzyme was maximally active at pH 6.8 and 20 degrees C, and required Triton X-100, which enhanced activity 5-fold at a concentration of 0.05-2%. Divalent ions, including Mn2+ and Mg2+, did not affect activity. Enzyme activity increased with increasing polymerization of xylosyl residues of the acceptor substrates: for instance, Xyl5-PA was almost 7 times as efficient as Xyl2-PA. The apparent Michaelis constants of the enzyme for Xyl3-PA and UDP-xylose were 13.5 and 7.9 mM, respectively. The enzyme also catalyzed incorporation of radioactive sugars (Xyl together with a small portion of L-arabinose) from UDP-[14C]xylose into higher beta-(1-->4)-xylooligosaccharides (degree of polymerization > 7) with or without (4-O-methyl-)glucuronosyl side chains at activities comparable to those observed for pyridylaminated xylooligosaccharides, and into several heteroxylans but with much lower efficiency. Enzymatic hydrolysis of the product with a beta-xylanase degraded it into mainly xylobiose, providing further evidence that the xylosyl residues are incorporated through beta-(1-->4)-linkages.

Structure Properties of Dextran. 2. Dilute Solution

Dilute solution properties of 13 dextrans with different molar masses in water and 0.5 M NaOH were investigated. Seven of these samples were products of Sigma prepared by bacteria from Leuconostoc mesenteroides, one was obtained by fractionation of the dextran with the highest molar mass, and five samples were obtained by degradation via controlled acid hydrolysis. Static and dynamic light scattering, viscometry and analysis of the reducing end group were applied. The molar mass dependencies of the radius of gyration Rg, the hydrodynamic radius Rh, the second virial coefficient A2 and the intrinsic viscosity [η] for the nonfractionated samples were determined. The results are compared with data from the literature. Size exclusion chromatography in on-line combination with multiple-angle laser light scattering and viscosity detection revealed different calibration curves Mi vs Ve for the studied samples. The molar mass dependencies of the radii and intrinsic viscosities could be measured. The data of the r...

Stability studies of chondroitin sulfate

The stability of chondroitin sulfate (CS) was studied under acidic, neutral and basic conditions at 30 and 60 °C. CS is remarkably stable under neutral conditions at low temperature, while it degrades at 60 °C producing low-molecular-mass fragments and desulfated products. This decomposition process begins at ca. 500–600 h and is consistent with an acid-catalyzed hydrolysis of glycosidic linkages caused by a drop in pH resulting from acidic products. Under basic conditions, a breakdown of glycosidic linkages causes a decrease in molecular mass due to the β-elimination reaction, confirmed by a strong increase of absorbance at 232 nm and 1H NMR. Virtually no loss of O-sulfate groups can be detected in the base-treated CS. Under acidic conditions, the molecular mass decreases probably through hydrolysis of polysaccharidic linkages resulting in an increased number of reducing end groups. Little or no β-elimination occurs. A loss of O-sulfate groups was detected, producing desulfated derivatives.

Degradation of Cellulosic Materials Under the Alkaline Conditions of a Cementitious Repository for Low- and Intermediate-Level Radioactive Waste. II. Degradation Kinetics

The degradation of cellulosic materials, differing mainly in the degree of polymerization and the number of reducing end groups, was studied under the alkaline conditions similar to those existing in a cementitious repository for low- and intermediate-level radioactive waste (pH 13.3, T = 25°C). The kinetics of alkaline degradation (peeling-off reaction) were studied and the data analyzed by the model of Haas et al. [13]. The observed kinetic parameters for the propagation reaction and overall stopping reaction were compared with literature data. Although measured under different experimental conditions, literature data and data from this study show a consistent picture. Differences in the extent of degradation observed for the different cellulosic materials could be satisfactorily explained by differences in reducing end group content and, consequently, by differences in the degrees of polymerization. Besides the number of reducing end groups, the degree of amorphousness also plays an important role. The main degradation products formed under the experimental conditions used are α- and β-(gluco)isosaccharinic acid. This is in agreement with many other studies on alkaline degradation of cellulose. The two isomers are formed in roughly equal amounts.

Grafting and initiation reactions

AbstractGrafting of acrylic monomers onto cellulose substrates in acid aqueous medium using complex of Mn3+ ions as a redox initiator is described. Assuming that the /Mn3+/ ions react with aldehydes and vicinal diols, the rate of these reactions has been measured spectroscopically. (ESR and visible light) using model compounds. It is concluded that the /Mn3+/ grafting onto cellulose is a radical reaction initiated mainly by oxidation of aldehydes at reducing end groups and C‐C bond scission of vicinal diols at the end groups and along the cellulose chains. The initiation rates of the Ce4+, /Mn3+/ and VO22+ redox ions are related to their oxidation potential.

Cellulase Hydrolysis of Acetylcelluloses Differing in Dissociation.

The cellulase-hydrolysis of acetylcelluloses with acetyl substitution (DS) from 0.20 to 2.20 was assessed based on glucose and reducing end group production and changes in viscosity of substrate solution following enzymatic hydrolysis. The hydrolysis of water-soluble acetylcellulose by cellulase may be explained as due to DS, while that of water-insoluble acetylcellulose, not only DS but also the dissociation of substrate molecules brought about through solvation, swelling and dissolution of the substrate.Acetylcellulose with only DS=1.56 was found to undergo hydrolysis in aqueous cellulase solution subsequent to pre-swelling with 75 vol% aqueous ethanol. Acetylcellulose with higher DS (2.07) was hydrolyzed significantly in 80 vol% aqueous acetone by encapsulate cellulase. Acetylcelluloses with relatively high DS may thus be concluded to interact well with cellulase molecules and be hydrolyzed to varying degrees through the dissociation of solid substrates in appropriate organic solvents and cellulase can be used in such a way as to prevent it from coming into contact with the organic solvent.

Self Assembled Oligosaccharide Surfactant Visualized by Atomic Force Microscopy

The physisorption of oligosaccharide surfactants is being developed as a method for modifying biomaterials to prevent protein adsorption. In this study, we visualize the surface adsorption and ordering of oligosaccharide surfactant using the atomic force microscope (AFM). Contact mode imaging is used to provide topographical images of the surface. Tapping mode imaging also provides topographical images, but reduces the amount of the lateral forces applied to the surface by driving the cantilever and contacting the surface once per oscillation. A phase image, which is the measure of phase shift between the cantilever response and the driving signal in tapping mode, gives information on variations in the viscoelastic response of the sample surface.The maltose diblock surfactant was prepared by the selective oxidation and lactonization of the maltose reducing end group, followed by aminolysis with dodecylamine. The chemical structure is Maltose--CONH-(CH2)11CH3.The solutions used for adsorption were prepared by diluting a stock solution of the surfactant at the critical micelle concentration (CMC) to the desired concentrations.

Methylglyoxal determination from different carbohydrates during heat processing

Heat induced formation of methylglyoxal (MG) was examined in the caramelisation and Maillard reaction. Mono-, oligo- and polysaccharides (i.e. D-glucose, dextrin 15, starch) were caramelised using three different conditions. MG concentration was analysed with HPLC as a methylquinoxaline derivate. The concentration of MG formed from dextrin 15 was higher than that of starch because of the presence of more reducing end groups. The effect of Maillard reaction on the formation of MG was examined. The different carbohydrates were heated in the presence of o-phenylenediamine (o-PD) and glycine. In the formation of MG there was a catalytic effect of the amino group, so the MG concentration was higher than in caramelisation. When carbohydrates were heated with glycine, the MG concentration was not as high because of the base strength of the amino acid. Both in caramelisation and Maillard reaction the concentration of MG was higher in the presence of water.

Review of the kinetics of alkaline degradation of cellulose in view of its relevance for safety assessment of radioactive waste repositories

The degradation of cellulose (a substantial component of low- and intermediate-level radioactive waste) under alkaline conditions occurs via two main processes: a peeling-off reaction and a basecatalyzed cleavage of glycosidic bonds (hydrolysis). Both processes show pseudo-first-order kinetics. At ambient temperature, the peeling-off process is the dominant degradation mechanism, resulting in the formation of mainly isosaccharinic acid. The degradation depends strongly on the degree of polymerization (DP) and on the number of reducing end groups present in cellulose. Beyond pH 12.5, the OH- concentration has only a minor effect on the degradation rate. It was estimated that under repository conditions (alkaline environment, pH 13.3-12.5) about 10% of the cellulosic materials (average DP = 1000-2000) will degrade in the first stage (up to 105 years) by the peeling-off reaction and will cause an ingrowth of isosaccharinic acid in the interstitial cement pore water. In the second stage (105-106 years), alkaline hydrolysis will control the further degradation of the cellulose. The potential role of microorganisms in the degradation of cellulose under alkaline conditions could not be evaluated. Proper assessment of the effect of cellulose degradation on the mobilization of radionuclides basically requires knowing the concentration of isosaccharinic acid in the pore water. This concentration, however, depends on several factors such as the stability of ISA under alkaline conditions, sorption of ISA on cement, formation of sparingly soluble ISA-salts, etc. A discussion of all the relevant processes involved, however, is far beyond the scope of the presented overview.

Assay for cellobiose dehydrogenase in the presence of laccase

The currently used assay for cellobiose dehydrogenase (CDH), an enzyme produced by many wood degrading fungi, lacks specificity and can give false results. The presence of laccase interferes with the standard assay. We have developed an assay for CDH that is insensitive to the presence of both laccase and other phenoloxidases. The method is based on the decrease of reducing end groups in lactose determined by the DNS method. Ferricyanide is present as electron acceptor. Advantages and drawbacks of CDH assay methods are discussed

Effects of Agitation and Endoglucanase Pretreatment on the Hydrolysis of Cotton Fabrics by a Total Cellulase

We have attempted to clarify the effects of low and high levels of mechanical agitation on the cellulolytic activity of a pure endoglucanase ( EG ) and a total cellulase mixture (TC) on scoured and bleached cotton fabric, along with the effect of EG pretreatment on subsequent treatments with TC. Methods used to follow the progress of fabric treatments include weight loss, breaking load loss, bending hysteresis, and the pro duction of soluble reducing sugars and reducing end groups in the fibers. The results show that agitation rate affects the mechanism of cellulolytic attack, and that this has implications for delivering desired finishing effects.

The analysis of fluorophore-labeled carbohydrates by polyacrylamide gel electrophoresis.

The glycans of glycoconjugates mediate numerous important biological processes. Their separation and structural determination present considerable difficulties because of the small quantities that are available from biological sources and the inherent difficulty of analyzing the wide variety of complex structures that exist. A method for the analysis of reducing saccharides by PAGE that uses specific fluorophore labeling and is simple, rapid, sensitive, and readily available to biological researchers, has been developed. This method is known acronimically either as PAGEFS (PAGE of Fluorophore-labeled Saccharides) or in one commercial format as FACE (Fluorophore-Assisted Carbohydrate Electrophoresis). In the PAGEFS method, saccharides having an aldehydic reducing end group are labeled quantitatively with a fluorophore and then separated with high resolution by PAGE. Two fluorophores, 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) and 2-aminoacridone (AMAC), have been used to enable the separation of a variety of saccharide positional isomers, anomers, and epimers. Subpicomolar quantities of individual saccharides can be detected using a sensitive imaging system. Mixtures of oligosaccharides obtained by enzymatic cleavage from glycoproteins can be labeled and electrophoresed to yield an oligosaccharide profile of each protein. AMAC can be used to distinguish unequivocally between acidic and neutral oligosaccharides. Methods of obtaining saccharide sequence information from purified oligosaccharides have been developed using enzymatic degradation. Other applications and the potential of the system are described.

Two Chains of Rhamnogalacturonan II Are Cross-Linked by Borate-Diol Ester Bonds in Higher Plant Cell Walls.

Polysaccharide moiety of the boron-polysaccharide complex (T. Matoh, K. Ishigaki, K. Ohno, J. Azuma [1993] Plant Cell Physiol 34: 639-642) isolated from radish (Raphanus sativus) roots has been shown to be rhamnogalacturonan II by glycosyl-linkage analysis and the presence of diagnostic monosaccharides, including apiose, aceric acid, 2-O-methylfucose, and 3-deoxy-D-manno-2-octulosonic acid. Removal of boron from the complex reduced the molecular weight by one-half without causing a significant increase in the number of reducing end groups, indicating that boron, as boric acid, links two rhamnogalacturonan II chains together to form the boron-polysaccharide complex.