Characterization of water-soluble hemicelluloses from spruce and aspen employing SEC/MALDI mass spectroscopy

Partly depolymerized hemicelluloses isolated from wood chips of spruce and aspen employing microwave treatment were resolved using size-exclusion chromatography (SEC) into oligo- and polysaccharide fractions containing components with a narrow range of sizes, as determined by MALDI mass spectroscopy. The degree of substitution with acetyl moieties (DS) was also calculated on the basis of the MALDI-MS spectra obtained prior to and following deacetylation. For spruce hemicelluloses, the low molecular mass fraction contained small arabino-4-O-methylglucuronoxylan oligosaccharides, with DP values ranging from 4 to approximately 20, separated primarily on the basis of their charge density. The fraction eluted last consisted of an O-acetyl-(galacto)glucomannan polysaccharide of peak-average DP value (DP(p)) 14. The degree of substitution with acetyl groups (DS) decreased with decreasing DP, a value DS of 0.39 being obtained for the fraction with DP(p) 12. For the aspen hemicelluloses, the SEC fractions eluted first contained an acidic O-acetyl-4-O-methylglucuronoxylan polysaccharide with DP ranging from 10 to approximately 28 and an average DS of approximately 0.75. The fractions eluted last consisted of oligosaccharide mixtures composed primarily of small neutral O-acetyl-xylooligosaccharides (DP(p) 6, DS 0.41), together with minor quantities of an O-acetyl-glucomannan.     

Characterization of O-acetyl-(4-O-methylglucurono)xylan isolated from birch and beech

The structures of water-soluble birch and beech xylans, extracted from holocellulose using dimethyl sulfoxide, were determined employing 1H and 13C NMR spectroscopy together with chemical analysis. These polysaccharides were found to be O-acetyl-(4-O-methylglucurono)xylans containing one 4-O-methylglucuronic acid substituent for approximately every 15 D-xylose residues. The average degree of acetylation of the xylose residues in these polymers was 0.4. The presence of the structural element -->4)[4-O-Me-alpha-D-GlcpA-(1-->2)][3-O-Ac]-beta-D-Xylp-(1--> was demonstrated. Additional acetyl groups were present as substituents at C-2 and/or C-3 of the xylopyranosyl residues. Utilizing size-exclusion chromatography in combination with mass spectroscopy, the weight-average molar masses (and polydispersities) were shown to be 8000 (1.09) and 11,100 (1.08) for birch and beech xylan, respectively.     

Characterization of acetylated 4-O-methylglucuronoxylan isolated from aspen employing 1H and 13C NMR spectroscopy

Water-soluble hemicelluloses were extracted from milled aspen wood (Populus tremula) employing microwave oven treatment at 180 degrees C for 10 min. The final pH of this extract was 3.5. From this extract oligo- and polysaccharides were isolated and subsequently fractionated by size-exclusion chromatography. The structures of the saccharides in three of the fractions obtained were determined by 1H and 13C NMR spectroscopy, using homonuclear and heteronuclear two-dimensional techniques. The polysaccharides present in the two fractions eluted first were O-acetyl-(4-O-methylglucurono)xylans. The average degree of acetylation of the xylose residues in these compounds was 0.6. The structural element -->4)[4-O-Me-alpha-D-GlcpA-(1-->2)][3-O-Ac]-beta-D-Xylp-(1 --> could also be identified. On the average, these two xylans were composed of the following (1-->4)-linked beta-D-xylopyranosyl structural elements: unsubstituted (50 mol%), 2-O-acetylated (13 mol%), 3-O-acetylated (21 mol%), 2,3-di-O-acetylated (6 mol%) and [MeGlcA alpha-(1-->2)][3-O-acetylated] (10 mol%). Most of the 4-O-methylglucuronyl and acetyl substituents in the isolated polysaccharides survived the microwave oven treatment. The third fraction, eluted last, contained acetylated xylo-oligosaccharides, with minor contamination by an acetylated mannan. In the case of these xylo-oligosaccharides, the average degree of acetylation was 0.3.     

Size-exclusion chromatography of enzymatically treated cellulose and related polysaccharides: a review

Size-exclusion chromatography (SEC) of wood pulp polysaccharides (cellulose and hemicelluloses) has become widely used for the analysis of these polymers, including monitoring of transformations occurring under various chemical and biological processes. The present review provides information on the different procedures and methodologies developed for the characterization of molecular mass distribution (MMD) and chemical composition of cellulose and hemicelluloses by SEC. This paper also includes the significant results obtained in the characterization of enzymatically treated wood polysaccharides. Conventional SEC with double detection (refractometric and UV), "hyphenated" with a fractional dissolution, was demonstrated to be an efficient method for simultaneous determination of MMD and chemical heterogeneity.     

Comparative study of the degradation of flax shive by strains of Pleurotus

Summary Among the four strains of Pleurotus tested, hybrid strain A was found to degrade flax shive faster than the other three strains. Strain A produced more polygalacturonase, pectinlyase, cellulase and laccase enzymes than the other three strains of Pleurotus. Maximum activities of laccase and polysaccharide degrading enzymes were correlated with high weight loss, reduction in the yield of lignin and holocellulose and the degree of polymerisation of holocellulose. The addition of crude extract of flax shive on unextracted-shive increased the production of primordia of all the four strains, but it did not increase the degradation of flax shive. Flavonoid type compounds were detected in the crude extract of flax shive and they may be responsible for the increase in the production of mushroom primordia.     

Preparation and characterization of hemicellulosic derivatives containing carbamoylethyl and carboxyethyl groups

A series of novel water-soluble hemicellulosic derivatives, containing carbamoylethyl and carboxyethyl groups, were heterogeneously synthesized from wheat-straw hemicelluloses with acrylamide (AA) under alkaline conditions. The factors such as reaction temperature, reaction time, molar ratio of catalysis to xylose unit in hemicelluloses and molar ratio of acrylamide to xylose unit in hemicelluloses, were investigated. The average degree of substitution (DS) was calculated by ¹H NMR spectroscopy. DS values up to 0.23 in a one-step synthesis of hemicelluloses derivatives were obtained. Under optimum conditions (60 °C, NaOH to xylose unit in hemicelluloses molar ratio of 0.8, AA to xylose unit in hemicelluloses molar ratio of 8.0, reaction time of 1 h) an expected ratio of carbamoylethyl group to carboxyethyl group of 4.8 in the hemicellulosic derivatives was obtained. The structural features of the hemicellulosic derivatives were characterized by FTIR, NMR spectroscopy, and by elemental analysis. The current work provides a facile method for the synthesis of hemicellulose derivatives with bifunctional groups, which could be used as wet-end additives in the papermaking industry.     

Separation and quantification of neoagaro- and agaro-oligosaccharide products generated from agarose digestion by beta-agarase and HCl in liquid chromatography systems

A series of neoagaro-oligosaccharides (NAOS) were separated and isolated by beta-agarase digestion and agaro-oligosaccharides (AOS) by HCl hydrolysis from agarose with defined quantity and degree of polymerization (DP). Profiles of the oligomer length in the crude product mixtures were monitored by two high-performance liquid chromatography (HPLC) systems: size-exclusion chromatography (SEC) and NH2-column chromatography (NH2-HPLC), coupled with an evaporative light-scattering detector (ELSD). Calibration curves were established separately to identify the DP and quantify the amount of the oligomer products analyzed in the two systems. Each system was optimized to generate a spectrum of saccharide oligomers with various DP, where the reaction yield for NAOS was 52.7% by 4U/mg beta-agarase and for AOS was 45.6% by 0.4M HCl. SEC resolved the product in size ranges consisting of DP 1-22 for NAOS and DP 1-14 for AOS. NH2-HPLC clearly resolved both distinct saccharide product sizes within DP 12. The optimized system was connected with a fraction collector to isolate and quantify these individually separated products. The total product yields of the recovered NAOS of DP 1-22 and AOS of DP 1-14 by the SEC system were 84.7% and 82.9%, respectively. NH2-HPLC recovered NAOS and AOS, both with a DP of 1-10 with total product yields of 48.9% and 90.0%, respectively. Isolated NAOS and AOS product fractions were inspected by (1)H NMR spectroscopy and ESIMS spectrometry to confirm structure, molecular mass, and purity. This study established feasible systems for the preparation and qualitative and quantitative measurements, as well as for the isolation of various sizes of oligomers generated from agarose.     

Fractionation and characterization of a Polysaccharide from the sclerotia of Pleurotus tuber-regium by preparative size-exclusion chromatography

A kind of regenerated cellulose gel (RCG) particles were treated by toluene to obtain particles with smaller mean pore size, then was mixed with the cellulose gel with pore size of 370 and 525 nm. A preparative size-exclusion chromatography (SEC) column (700 x 20 mm) packed with three gel particles was used to fractionate water-soluble polysaccharide (WEP) extracted from the sclerotia of Pleurotus tuber-regium by aqueous solution. The exclusion limit and fractionation range of the stationary phase of the preparative SEC were molecular mass 8 x 10(5) and 5 x 10(3) to 8 x 10(5), respectively. The calibration curve of the preparative SEC was represented as: log M=13.96-0.53 Ve. The WEP sample (weight-average molecular mass M(w)=2.2 x 10(4), polydispersity=2.4) was divided into three fractions with M(w) ranging from 1.4 x 10(4) to 3.4 x 10(4) by the preparative SEC column, and the fractions were characterized by gas chromatography GC, SEC combined with laser light scattering (SEC-LLS) and viscometry. The unfractionated WEP exhibited triple peaks due to different molecular mass, but each fraction exhibited single peak with the polydispersity of 1.1-1.8 in the SEC patterns. The results indicated that the preparative SEC was efficient for fractionation of polysaccharides having low molecular weight and for determination of their molecular mass.     

Mechanistic investigation of a starch-branching enzyme using hydrodynamic volume SEC analysis

Two linear alpha-(1,4)-D-glucans substrates, of degrees of polymerization DP approximately 150 and 6000, were exposed to maize starch-branching enzyme IIa (mSBEIIa) in vitro. The resulting branched alpha-glucans and their constituent chains (obtained by debranching) were analyzed by nuclear magnetic resonance (NMR) and size-exclusion chromatography (SEC). SEC data for the debranched species are presented as chain-length distributions, while those for branched species are presented as hydrodynamic volume distributions (HVDs), which is the most meaningful way to present such data (because SEC separates by size, not molar mass, and a sample of branched polymers with the same size can have a range of molar masses). A rigorous interpretation of the HVDs of the substrate and its branched product show that at least part of the branching is an interchain transfer mechanism in both the short- and long-chain substrate cases. A bimodal HVD of the in vitro branched alpha-glucan derived from the short-chain substrate was observed, and it is postulated that the divergence of the two populations is due to very small chains being unable to undergo branching. In the case of the in vitro branching of the long-chain substrate, the formation of maltohexaose during the reaction and the presence of a monomodal HVD were observed, suggesting a distinct mode of action of mSBEIIa on this substrate. Quantification of the branching level by NMR showed the branched glucans from both substrates had substantial amounts of branching (2.1-4.5%), ascribed to the intrinsic nature of the action of mSBEIIa on the two substrates. It is postulated that differences in the degrees of substrate association affect the pattern of branching catalyzed by the enzyme, and a putative active site structure is proposed based on the appearance of maltohexaose. The molar mass distribution of the constituent chains of the in vitro branched alpha-glucans obtained by isoamylase treatment reveals the transfer of chains of specific size and supports the supposition given in the literature that mSBEIIa is responsible for short-chain branching in amylopectin. It is suggested that hydrodynamic volume SEC analysis should be used as a tool for the mechanistic investigation of SBEs, allowing SEC data of in vitro branched alpha-glucans to be both comparable and quantitative.     

A reverse-phase liquid chromatography/mass spectrometry method for the analysis of high-molecular-weight fructooligosaccharides

Many important crop and forage plants accumulate polymeric water-soluble carbohydrates as fructooligosaccharides (or fructans). We have developed an improved method for the analysis of the full fructan complement in plant extracts based on porous graphitized carbon chromatography coupled to negative electrospray ionization mass spectrometry. By the use of profile data collection and multiple charge state ions, the effective mass range of the ion trap was extended to allow for the analysis of very high-molecular-weight oligosaccharides. This method allows the separation and quantification of isomeric fructan oligomers ranging from degree of polymerization (DP) 3 to DP 49.     

Characterization of an acetylated heteroxylan from Eucalyptus globulus Labill

A heteroxylan was isolated from Eucalyptus globulus wood by extraction of peracetic acid delignified holocellulose with dimethyl sulfoxide. Besides (1-->4)-linked beta-D-xylopyranosyl units of the backbone and short side chains of terminal (1-->2)-linked 4-O-methyl-alpha-D-glucuronosyl residues (MeGlcA) in a 1:10 molar ratio, this hemicellulose contained galactosyl and glucosyl units attached at O-2 of MeGlcA originating from rhamnoarabinogalactan and glucan backbones, respectively. About 30% of MeGlcA units were branched at O-2. The O-acetyl-(4-O-methylglucurono)xylan showed an acetylation degree of 0.61, as determined by 1H NMR spectroscopy, and a weight-average molecular weight (M(w)) of about 36 kDa (P=1.05) as revealed from size-exclusion chromatography (SEC) analysis. About half of the beta-D-xylopyranosyl units of the backbone were found as acetylated moieties at O-3 (34 mol%), O-2 (15 mol%) or O-2,3 (6 mol%). Practically, all beta-D-xylopyranosyl units linked at O-2 with MeGlcA residues were 3-O-acetylated (10 mol%).     

Characterization of gelatine and acid soluble collagen by size exclusion chromatography coupled with multi angle light scattering (SEC-MALS)

Acid soluble collagen (ASC) and a cattle hide gelatine were analyzed by size exclusion chromatography (SEC) coupled with multi angle light scattering (MALS). The SEC system was calibrated with ASC and its cyan bromide cleavage products. The accuracy of calibration was confirmed by MALS by measuring the mass-average molar masses (Mw). ASC acted as a mixture of two polymer standards of Mw = 90 and 180 kg/mol, respectively. The elution behavior of the gelatine in SEC-MALS was similar to that of ASC. Therefore, the determination of the molar mass distribution of this gelatine was possible either by SEC, using a calibration curve, or by MALS by direct measurement of Mw. According to the scaling law (1/2) = KMalpha, alpha = 0.78 was determined for the gelatine. This alpha could reflect a structure in solution, which is more similar to an ellipsoid than to a random coil.     

Solution properties of viilian, the exopolysaccharide from Lactococcus lactis subsp. cremoris SBT 0495

The exopolysaccharide (EPS) "viilian" was isolated from a large-batch fermentation of Lactococcus lactis subsp. cremoris SBT 0495. After applying a newly developed purification procedure, pure viilian with a weight-averaged molar mass of 2.64 x 10(3) kg/mol was obtained in a yield of 0.6 g/L culture broth. The native EPS, as well as lower molar mass fractions obtained by sonication of the native polymer, were studied by capillary viscometry and size-exclusion chromatography (SEC) coupled to multiangle laser light scattering detection (MALLS). From the viscosity data at various ionic strengths, we extracted a Mark-Houwink-Kuhn-Sakurada exponent a = 0.79, and a Smidsrod B value of 0.03. By application of the Hearst, Bohdanecky, and Odijk models for stiff polymer coils, in connection to the experimental viscosity data, we established the characteristic ratio to be C(infinity) = 44 and the intrinsic persistence length q(0) = 11.5 nm. The rms radii of gyration predicted from each of the models were in good agreement with the experimental radii (e.g., (1/2)(w) = 162 nm for native viilian in 0.2M NaNO(3)), as determined by SEC-MALLS. In addition, the Odijk model predicts correct ionic strength-linear charge density dependence of the rms radius of gyration. From the combined viscosity and SEC-MALLS experiments we concluded that, in dilute aqueous solutions, viilian behaves as an intermediately stiff, random coil polyelectrolyte system.Copyright 2000 John Wiley & Sons, Inc.     

Biochemical and catalytic properties of an endoxylanase purified from the culture filtrate of Sporotrichum thermophile

An endo-beta-1,4-xylanase (1,4-beta-D-xylan xylanoxydrolase, EC 3.2.1.8) present in culture filtrates of Sporotrichum thermophile ATCC 34628 was purified to homogeneity by Q-Sepharose and Sephacryl S-200 column chromatographies. The enzyme has a molecular mass of 25,000 Da, an isoelectric point of 6.7, and is optimally active at pH 5 and at 70 degrees C. Thin-layer chromatography (TLC) analysis showed that endo-xylanase liberates mainly xylose (Xyl) and xylobiose (Xyl2) from beechwood 4-O-methyl-D-glucuronoxylan, O-acetyl-4-O-methylglucuronoxylan and rhodymenan (a beta-(1-->4)-beta(1-->3)-xylan). Also, the enzyme releases an acidic xylo-oligosaccharide from 4-O-methyl-D-glucuronoxylan, and an isomeric xylotetraose and an isomeric xylopentaose from rhodymenan. Analysis of reaction mixtures by high performance liquid chromatography (HPLC) revealed that the enzyme cleaves preferentially the internal glycosidic bonds of xylooligosaccharides, [1-3H]-xylooligosaccharides and xylan. The enzyme also hydrolyses the 4-methylumbelliferyl glycosides of beta-xylobiose and beta-xylotriose at the second glycosidic bond adjacent to the aglycon. The endoxylanase is not active on pNPX and pNPC. The enzyme mediates a decrease in the viscosity of xylan associated with a release of only small amounts of reducing sugar. The enzyme is irreversibly inhibited by series of omega-epoxyalkyl glycosides of D-xylopyranose. The results suggest that the endoxylanase from S. thermophile has catalytic properties similar to the enzymes belonging to family 11.     

Rapid esterification of wheat straw hemicelluloses induced by microwave irradiation

Esterification of wheat straw hemicelluloses with acetyl chloride, propionyl chloride, n-octanoyl chloride, lauroyl chloride, palmitoyl chloride, stearoyl chloride, and oleoyl chloride, respectively, using N-bromosuccinimide (NBS) as a catalyst was achieved in DMF/LICl medium by microwave irradiation. The effects of various acyl chlorides and the molar ratios of xylose units in hemicelluloses/acyl chloride on the degree of substitution (DS) were investigated and DS reached up to 1.34 by a few minutes. ¹³C NMR studies showed that the esterification occurred preferentially at the C-3 and C-2 positions. On the other hand, microwave irradiation brought a partial degradation of the polymer, and therefore resulted in a slight decrease in thermal stability of the hemicellulosic derivatives in comparison with conventional heating technique.     

Enzymatic degradation of carboxymethyl cellulose hydrolyzed by the endoglucanases Cel5A, Cel7B, and Cel45A from Humicola insolens and Cel7B, Cel12A and Cel45Acore from Trichoderma reesei.

Enzymatic hydrolysis of carboxymethyl cellulose (CMC) has been studied with purified endoglucanases Hi Cel5A (EG II), Hi Cel7B (EG I), and Hi Cel45A (EG V) from Humicola insolens, and Tr Cel7B (EG I), Tr Cel12A (EG III), and Tr Cel45Acore (EG V) from Trichoderma reesei. The CMC, with a degree of substitution (DS) of 0.7, was hydrolyzed with a single enzyme until no further hydrolysis was observed. The hydrolysates were analyzed for production of substituted and non-substituted oligosaccharides with size exclusion chromatography (SEC) and with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS). Production of reducing ends and of nonsubstituted oligosaccharides was determined as well. The two most effective endoglucanases for CMC hydrolysis were Hi Cel5A and Tr Cel7B. These enzymes degraded CMC to lower molar mass fragments compared with the other endoglucanases. The products had the highest DS determined by MALDI-TOF-MS. Thus, Hi Cel5A and Tr Cel7B were less inhibited by the substituents than the other endoglucanases. The endoglucanase with clearly the lowest activity on CMC was Tr Cel45Acore. It produced less than half of the amount of reducing ends compared to Tr Cel7B; furthermore, the products had significantly lower DS. By MALDI-TOF-MS, oligosaccharides with different degree of polymerization (DP) and with different number of substituents could be separated and identified. The average oligosaccharide DS as function of DP could be measured for each enzyme after hydrolysis. The combination of techniques for analysis of product formation gave information on average length of unsubstituted blocks of CMC.     

Structural changes in microcrystalline cellulose in subcritical water treatment

Subcritical water is a high potential green chemical for the hydrolysis of cellulose. In this study microcrystalline cellulose was treated in subcritical water to study structural changes of the cellulose residues. The alterations in particle size and appearance were studied by scanning electron microscopy (SEM) and those in the degree of polymerization (DP) and molar mass distributions by gel permeation chromatography (GPC). Further, changes in crystallinity and crystallite dimensions were quantified by wide-angle X-ray scattering and (13)C solid-state NMR. The results showed that the crystallinity remained practically unchanged throughout the treatment, whereas the size of the remaining cellulose crystallites increased. Microcrystalline cellulose underwent significant depolymerization in subcritical water. However, depolymerization leveled off at a relatively high degree of polymerization. The molar mass distributions of the residues showed a bimodal form. We infer that cellulose gets dissolved in subcritical water only after extensive depolymerization.     

A comparison of molecular mass determination of hyaluronic acid using SEC/MALLS and sedimentation equilibrium

Hyaluronic acid (HA) is a natural polysaccharide with importance in the pharmaceutical, medical and cosmetic industries. Determining factors in its final applications are its physicochemical properties, particularly molecular mass. A high molecular mass HA was degraded using five different hydroxyl free-radical starting concentrations chemically produced from ascorbic acid and hydrogen peroxide. The aims of the study were to investigate the effect of different hydroxyl free-radical concentrations on the chain length of HA and compare the molecular masses obtained from analytical ultracentrifugation using sedimentation equilibrium experiments and size exclusion chromatography/multi-angle laser light scattering (SEC/MALLS). The results indicated that their molecular masses varied, depending on the degree of hydroxyl free-radical starting concentration. Molecular mass values obtained from sedimentation equilibrium experiments for each sample showed the same trend as those obtained from the SEC/MALLS in the range of molecular masses studied. The molecular masses obtained from sedimentation equilibrium for high molecular mass samples from reciprocal plots of apparent weight average molecular mass against concentration gave values similar to those obtained by SEC/MALLS. In contrast, the molecular mass from conventional plots for high molecular mass samples were much lower than those from SEC/MALLS, even when high ionic strength buffers were used.     

Characterization of a polysaccharide-protein complex from Ganoderma tsugae mycelium by size-exclusion chromatography combined with laser light scattering

A water-soluble polysaccharide-protein complex (GM3) extracted from the mycelium of Ganoderma tsugae was characterized using size-exclusion chromatography combined with laser light scattering (SEC-LLS). Two peaks coded as fractions I and II appeared in the SEC pattern of GM3 in 0.5 M NaCl aqueous solution, corresponding to the weight-average molecular mass (M(w)) of 355 x 10(4) and 6.3 x 10(4), respectively. The relationship between the radius of gyration ((z)(1/2)) and M(w) showed that molecules of fraction I exhibited more compact coil conformation than that of fraction II in 0.5 M NaCl aqueous solution at 25 degrees C. To clarify the component of polysaccharide and protein in each fraction, the sample GM3 was treated with 0.2 M NaOH aqueous solution to degrade polysaccharide and trypsin to hydrolyze protein. The obtained products were analyzed by SEC combined with detectors such as UV, differential refractive index (DRI) and LLS. The results indicated that both the fractions I and II were protein-bound polysaccharide, but had different protein content and degree of branching, resulting in the difference of the chain conformation.     

Effect of molecular mass on antitumor activity of heteropolysaccharide from Poria cocos

A water-soluble heteropolysaccharide ac-PCM0 from Poria cocos was successfully fractionated using a preparative size exclusion chromatography (SEC) column, and their weight-average molecular mass (M(w)) was characterized by analytical SEC combined with laser light scattering (SEC-LLS). The results indicate that the fractions having relatively high M(w) exhibited higher inhibition ratio in vivo antitumor activity than those having M(w) below 3.29 x 10(4). However, the relatively low molecular mass was beneficial to the in vitro antitumor activity. Moreover, ac-PCM0 has a significantly higher enhancement ratio of the body weight than 5-fluorouracil, and its 50% lethal dose is above 1250 mg/kg, indicating a nontoxic nature.