Depolymerization Of Polysaccharides Research Articles

Closely related fungi employ diverse enzymatic strategies to degrade plant biomass

BackgroundPlant biomass is the major substrate for the production of biofuels and biochemicals, as well as food, textiles and other products. It is also the major carbon source for many fungi and enzymes of these fungi are essential for the depolymerization of plant polysaccharides in industrial processes. This is a highly complex process that involves a large number of extracellular enzymes as well as non-hydrolytic proteins, whose production in fungi is controlled by a set of transcriptional regulators. Aspergillus species form one of the best studied fungal genera in this field, and several species are used for the production of commercial enzyme cocktails.ResultsIt is often assumed that related fungi use similar enzymatic approaches to degrade plant polysaccharides. In this study we have compared the genomic content and the enzymes produced by eight Aspergilli for the degradation of plant biomass. All tested Aspergilli have a similar genomic potential to degrade plant biomass, with the exception of A. clavatus that has a strongly reduced pectinolytic ability. Despite this similar genomic potential their approaches to degrade plant biomass differ markedly in the overall activities as well as the specific enzymes they employ. While many of the genes have orthologs in (nearly) all tested species, only very few of the corresponding enzymes are produced by all species during growth on wheat bran or sugar beet pulp. In addition, significant differences were observed between the enzyme sets produced on these feedstocks, largely correlating with their polysaccharide composition.ConclusionsThese data demonstrate that Aspergillus species and possibly also other related fungi employ significantly different approaches to degrade plant biomass. This makes sense from an ecological perspective where mixed populations of fungi together degrade plant biomass. The results of this study indicate that combining the approaches from different species could result in improved enzyme mixtures for industrial applications, in particular saccharification of plant biomass for biofuel production. Such an approach may result in a much better improvement of saccharification efficiency than adding specific enzymes to the mixture of a single fungus, which is currently the most common approach used in biotechnology.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0285-0) contains supplementary material, which is available to authorized users.

Depolymerization of polysaccharides from Opuntia ficus indica: Antioxidant and antiglycated activities

The extraction, purification and degradation of polysaccharides from Opuntia ficus indica cladodes, as well as the evaluation of their antioxidant and antiglycated activities in vitro were investigated. The optimization of the extraction showed that extraction by ultrasound at 40°C presented the best carbohydrates yield. The degradation of the extracted polysaccharides was achieved by free radical depolymerization with H2O2 in the presence of copper(II) acetate for various reaction times. Sugar contents were determined by colorimetric assays. The macromolecular characteristics of the different isolated and degraded carbohydrates were carried by size exclusion chromatography (SEC/MALS/VD/DRI). These experiments showed that all samples are polysaccharides, which are probably pectins and that molecular weight (Mw) has decreased from 6,800,000 to 14,000g/mol after 3h of depolymerization without changing the structure. Preliminary antioxidant and antiglycated tests indicated that degraded polysaccharides for 2 and 3h showed even better antioxidant and antiglycated activities.

An alternative method for the determination of polysaccharide cleavage enzymes activities

Most of usual methods used to detect and quantify polysaccharide cleavage enzymes (PCE) are not adapted to high throughput. In this work, enzymatic activities of three enzymes, namely an endocellulase (EC 3.2.1.4), an exocellulase (EC 3.2.1.91) and an alginate lyase (EC 4.2.2.3) were characterized by classical Michaelian model (Km, Vmax). Enzymatic activities were then determined by the use of the Enzyme Ring Test® (ERT®) method which allowed following the depolymerization of polysaccharides by PCE. Kinetic parameters were determined and compared to the ones obtained by classical methods. Besides, the ERT® technology clearly showed the possibility to discriminate endolytic and exolytic enzymatic mechanisms.

Cellulose degradation by polysaccharide monooxygenases.

Polysaccharide monooxygenases (PMOs), also known as lytic PMOs (LPMOs), enhance the depolymerization of recalcitrant polysaccharides by hydrolytic enzymes and are found in the majority of cellulolytic fungi and actinomycete bacteria. For more than a decade, PMOs were incorrectly annotated as family 61 glycoside hydrolases (GH61s) or family 33 carbohydrate-binding modules (CBM33s). PMOs have an unusual surface-exposed active site with a tightly bound Cu(II) ion that catalyzes the regioselective hydroxylation of crystalline cellulose, leading to glycosidic bond cleavage. The genomes of some cellulolytic fungi contain more than 20 genes encoding cellulose-active PMOs, suggesting a diversity of biological activities. PMOs show great promise in reducing the cost of conversion of lignocellulosic biomass to fermentable sugars; however, many questions remain about their reaction mechanism and biological function. This review addresses, in depth, the structural and mechanistic aspects of oxidative depolymerization of cellulose by PMOs and considers their biological function and phylogenetic diversity.

A Direct Sulfation Process of a Marine Polysaccharide in Ionic Liquid.

GY785 is an exopolysaccharide produced by a mesophilic bacterial strain Alteromonas infernus discovered in the deep-sea hydrothermal vents. GY785 highly sulfated derivative (GY785 DRS) was previously demonstrated to be a promising molecule driving the efficient mesenchymal stem cell chondrogenesis for cartilage repair. This glycosaminoglycan- (GAG-) like compound was modified in a classical solvent (N,N′-dimethylformamide). However, the use of classical solvents limits the polysaccharide solubility and causes the backbone degradation. In the present study, a one-step efficient sulfation process devoid of side effects (e.g., polysaccharide depolymerization and/or degradation) was developed to produce GAG-like derivatives. The sulfation of GY785 derivative (GY785 DR) was carried out using ionic liquid as a reaction medium. The successful sulfation of this anionic and highly branched heteropolysaccharide performed in ionic liquid would facilitate the production of new molecules of high specificity for biological targets such as tissue engineering or regenerative medicine.

Plant-polysaccharide-degrading enzymes from Basidiomycetes.

Basidiomycete fungi subsist on various types of plant material in diverse environments, from living and dead trees and forest litter to crops and grasses and to decaying plant matter in soils. Due to the variation in their natural carbon sources, basidiomycetes have highly varied plant-polysaccharide-degrading capabilities. This topic is not as well studied for basidiomycetes as for ascomycete fungi, which are the main sources of knowledge on fungal plant polysaccharide degradation. Research on plant-biomass-decaying fungi has focused on isolating enzymes for current and future applications, such as for the production of fuels, the food industry, and waste treatment. More recently, genomic studies of basidiomycete fungi have provided a profound view of the plant-biomass-degrading potential of wood-rotting, litter-decomposing, plant-pathogenic, and ectomycorrhizal (ECM) basidiomycetes. This review summarizes the current knowledge on plant polysaccharide depolymerization by basidiomycete species from diverse habitats. In addition, these data are compared to those for the most broadly studied ascomycete genus, Aspergillus, to provide insight into specific features of basidiomycetes with respect to plant polysaccharide degradation.

Oligosaccharide production by hydrolysis of polysaccharides: a review

SummaryOligosaccharides are carbohydrates with a low molecular weight, which, when nondigestible, can produce bifidogenic activity and several other effects to human health. Oligosaccharides can be found naturally in foods or are produced by the synthesis from disaccharide substrates or by the hydrolysis of polysaccharides. Although it has yet to be improved, the hydrolysis of polysaccharides is the best choice for oligosaccharide production on a large scale, due to its reproducibility and smaller cost. This review concisely presents the main processes for the production of oligosaccharides by depolymerisation of polysaccharides (enzymatic, acid, and physical hydrolysis), taking into account their advantages, disadvantages, and perspectives.

A rapid quantitative activity assay shows that the Vibrio cholerae colonization factor GbpA is an active lytic polysaccharide monooxygenase

The discovery of the copper-dependent lytic polysaccharide monooxygenases (LPMOs) has revealed new territory for chemical and biochemical analysis. These unique mononuclear copper enzymes are abundant, suggesting functional diversity beyond their established roles in the depolymerization of biomass polysaccharides. At the same time basic biochemical methods for characterizing LPMOs, such as activity assays are not well developed. Here we describe a method for quantification of C1-oxidized chitooligosaccharides (aldonic acids), and hence LPMO activity. The method was used to quantify the activity of a four-domain LPMO from Vibriocholerae, GbpA, which is a virulence factor with no obvious role in biomass processing.

Insight into the Role of Substrate-binding Residues in Conferring Substrate Specificity for the Multifunctional Polysaccharide Lyase Smlt1473

Anionic polysaccharides are of growing interest in the biotechnology industry due to their potential pharmaceutical applications in drug delivery and wound treatment. Chemical composition and polymer length strongly influence the physical and biological properties of the polysaccharide and thus its potential industrial and medical applications. One promising approach to determining monomer composition and controlling the degree of polymerization involves the use of polysaccharide lyases, which catalyze the depolymerization of anionic polysaccharides via a β-elimination mechanism. Utilization of these enzymes for the production of custom-made oligosaccharides requires a high degree of control over substrate specificity. Previously, we characterized a polysaccharide lyase (Smlt1473) from Stenotrophomonas maltophilia k279a, which exhibited significant activity against hyaluronan (HA), poly-β-d-glucuronic acid (poly-GlcUA), and poly-β-d-mannuronic acid (poly-ManA) in a pH-regulated manner. Here, we utilize a sequence structure guided approach based on a homology model of Smlt1473 to identify nine putative substrate-binding residues and examine their effect on substrate specificity via site-directed mutagenesis. Interestingly, single point mutations H221F and R312L resulted in increased activity and specificity toward poly-ManA and poly-GlcUA, respectively. Furthermore, a W171A mutant nearly eliminated HA activity, while increasing poly-ManA and poly-GlcUA activity by at least 35%. The effect of these mutations was analyzed by comparison with the high resolution structure of Sphingomonas sp. A1-III alginate lyase in complex with poly-ManA tetrasaccharide and by taking into account the structural differences between HA, poly-GlcUA, and poly-ManA. Overall, our results demonstrate that even minor changes in active site architecture have a significant effect on the substrate specificity of Smlt1473, whose structural plasticity could be applied to the design of highly active and specific polysaccharide lyases.

Clean Fractionation Pretreatment Reduces Enzyme Loadings for Biomass Saccharification and Reveals the Mechanism of Free and Cellulosomal Enzyme Synergy

Enzymatic depolymerization of polysaccharides is often a key step in the production of fuels and chemicals from lignocellulosic biomass. Historically, model cellulose substrates have been utilized to reveal insights into enzymatic saccharification mechanisms. However, translating findings from model substrates to realistic biomass substrates is critical for evaluating enzyme performance. Here, we employ a commercial fungal enzyme cocktail, purified cellulosomes, and combinations of these two enzymatic systems to investigate saccharification mechanisms on corn stover deconstructed either via clean fractionation (CF) or deacetylated dilute sulfuric acid pretreatments. CF is an organosolv pretreatment method utilizing water, MIBK, and either acetone or ethanol with catalytic amounts of sulfuric acid to fractionate biomass components. The insoluble cellulose-enriched fraction (CEF) from CF contains mainly cellulose, with minor amounts of residual hemicellulose and lignin. Enzymatic digestions at both low and ...

Structure of a PL17 Family Alginate Lyase Demonstrates Functional Similarities among Exotype Depolymerases

Brown macroalgae represent an ideal source for complex polysaccharides that can be utilized as precursors for cellulosic biofuels. The lack of recalcitrant lignin components in macroalgae polysaccharide reserves provides a facile route for depolymerization of constituent polysaccharides into simple monosaccharides. The most abundant sugars in macroalgae are alginate, mannitol, and glucan, and although several classes of enzymes that can catabolize the latter two have been characterized, studies of alginate-depolymerizing enzymes have lagged. Here, we present several crystal structures of Alg17c from marine bacterium Saccharophagus degradans along with structure-function characterization of active site residues that are suggested to be involved in the exolytic mechanism of alginate depolymerization. This represents the first structural and biochemical characterization of a family 17 polysaccharide lyase enzyme. Despite the lack of appreciable sequence conservation, the structure and β-elimination mechanism for glycolytic bond cleavage by Alg17c are similar to those observed for family 15 polysaccharide lyases and other lyases. This work illuminates the evolutionary relationships among enzymes within this unexplored class of polysaccharide lyases and reinforces the notion of a structure-based hierarchy in the classification of these enzymes.

Graded inclusions of sodium metabisulphite in sorghum-based diets: II. Modification of starch pasting properties in vitro and beneficial impacts on starch digestion dynamics in broiler chickens

Seven sorghum-based broiler diets containing the reducing agent, sodium metabisulphite (SMBS) at levels of 0, 1.50, 2.25, 3.00, 3.75, 4.50 and 5.25g/kg were prepared. The in vitro starch pasting properties of the sorghum-based diets and starch extracted from the diets were assessed by rapid viscosity analysis (RVA). The seven dietary treatments were offered to 294 male Ross 308 chicks from 10 to 24 days post-hatch to determine SMBS effects on starch and nitrogen digestion dynamics. Digesta samples were collected, freeze-dried and weighed for determination of digestibility coefficients and mean retention times in the proximal jejunum, distal jejunum, proximal ileum and distal ileum. The digestion kinetics of starch and nitrogen (N) were determined using an exponential mathematical model to relate digestibility coefficients to mean retention times in each small intestinal segment. SMBS inclusions linearly reduced apparent starch digestibility coefficients in the proximal jejunum (r=−0.444, P<0.01), distal jejunum (r=−0.371, P=0.010) and predicted glycaemic responses (r=−0.474; P<0.001). SMBS linearly decreased (r=−0.413; P<0.01) starch disappearance from the proximal jejunum (abruptly digestible starch) and increased (r=0.483; P<0.001) starch disappearance from the three caudal small intestinal segments (gradually digestible starch). SMBS significantly (P<0.001) reduced final starch viscosity in diets from 2567 to 1935 cp and final extracted starch viscosity from 1035 to 441 cp. The SMBS-induced reductions in final viscosity of extracted starch were correlated with apparent metabolisable energy (AME) (r=−0.347; P<0.05), N-corrected AME (AMEn) (r=−0.376; P<0.01) and feed conversion ratio (FCR) (r=0.337; P<0.05). Also, gradually digestible starch was correlated (r=−0.291, P<0.05) with FCR improvements and abruptly digestible starch was negatively correlated (r=−0.282; P<0.05) with AMEn. It appears that the previously reported positive responses in AME, AMEn and FCR generated by SMBS stem from reductions in final starch viscosity that are probably attributable to SMBS-induced oxidative–reductive depolymerisation of starch polysaccharides. The small intestinal sites of starch digestion (and glucose absorption) were altered with higher levels of gradually digestible starch disappearing from the distal jejunum, proximal and distal ileum. Consideration is given to the possible mechanisms whereby SMBS generated more gradually digestible starch and how gradually digestible starch enhanced energy utilisation and efficiency of feed conversion.

Graded inclusions of sodium metabisulphite in sorghum-based diets: I. Reduction of disulphide cross-linkages in vitro and enhancement of energy utilisation and feed conversion efficiency in broiler chickens

Seven sorghum-based diets containing 0, 1.50, 2.25, 3.00, 3.75, 4.50 and 5.25g/kg of the reducing agent sodium metabisulphite (SMBS) were prepared in which sodium levels were balanced with sodium bicarbonate. A red sorghum (766.4g/kg starch, 78.6g/kg protein) was hammer-milled through a 3.2mm screen prior to being mixed into complete broiler diets containing 195g/kg protein with an energy density12.97MJ/kg, which were steam-pelleted at an 84°C conditioning temperature. Each of the seven dietary treatments was offered to a total of 294 male Ross 308 chicks in seven replicate cages (6 birds per cage) from 10 to 24 days post-hatch to determine the effects of SMBS on growth performance, nutrient utilisation and nitrogen (N) digestibility coefficients in four small intestinal segments. SMBS did not linearly influence (P>0.50) weight gains and feed intakes. However, SMBS quadratically improved (r=0.416; P<0.05) feed conversion ratios and it may be deduced from the quadratic equation that 4.75g/kg SMBS improved FCR by 3.06% (1.424 versus 1.469). At all positive inclusions, SMBS significantly enhanced apparent metabolisable energy (AME) and N-corrected AME (AMEn); at the lowest inclusion, 1.50g/kg SMBS significantly improved AME by 0.36MJ (13.81 versus 13.45MJ/kg; P=0.005), AMEn by 0.45MJ (12.30 versus 11.85MJ/kg; P=0.001) and FCR by 2.47% (1.422 versus 1.458; P=0.025) on the basis of pair-wise comparisons. SMBS linearly decreased concentrations of disulphide bonds (r=−0.775; P<0.001) and linearly increased free sulphydryl groups (r=0.890; P<0.001) and protein solubility of the diets (r=0.943; P<0.001). However, SMBS had little influence on N digestibility coefficients as the sole significant response was a 6.41% increase (0.797 versus 0.749; P<0.01) in the distal ileum at 3.75g/kg and SMBS did not influence small intestinal sites of protein disappearance. Consideration is given to the possibility that the sulphite reducing agent, SMBS, caused oxidative-reductive depolymerisation of starch polysaccharides which may have been responsible for the improvements in energy utilisation and feed conversion efficiency.

A Polysaccharide Lyase from Stenotrophomonas maltophilia with a Unique, pH-regulated Substrate Specificity

Polysaccharide lyases (PLs) catalyze the depolymerization of anionic polysaccharides via a β-elimination mechanism. PLs also play important roles in microbial pathogenesis, participating in bacterial invasion and toxin spread into the host tissue via degradation of the host extracellular matrix, or in microbial biofilm formation often associated with enhanced drug resistance. Stenotrophomonas maltophilia is a Gram-negative bacterium that is among the emerging multidrug-resistant organisms associated with chronic lung infections as well as with cystic fibrosis patients. A putative alginate lyase (Smlt1473) from S. maltophilia was heterologously expressed in Escherichia coli, purified in a one-step fashion via affinity chromatography, and activity as well as specificity determined for a range of polysaccharides. Interestingly, Smlt1473 catalyzed the degradation of not only alginate, but poly-β-D-glucuronic acid and hyaluronic acid as well. Furthermore, the pH optimum for enzymatic activity is substrate-dependent, with optimal hyaluronic acid degradation at pH 5, poly-β-D-glucuronic acid degradation at pH 7, and alginate degradation at pH 9. Analysis of the degradation products revealed that each substrate was cleaved endolytically into oligomers comprised predominantly of even numbers of sugar groups, with lower accumulation of trimers and pentamers. Collectively, these results imply that Smlt1473 is a multifunctional PL that exhibits broad substrate specificity, but utilizes pH as a mechanism to achieve selectivity.

Depolymerization of sulfated polysaccharides under hydrothermal conditions

Fucoidan and chondroitin sulfate, which are well known sulfated polysaccharides, were depolymerized under hydrothermal conditions (120–180°C, 5–60min) as a method for the preparation of sulfated polysaccharides with controlled molecular weights. Fucoidan was easily depolymerized, and the change of the molecular weight values depended on the reaction temperature and time. The degree of sulfation and IR spectra of the depolymerized fucoidan did not change compared with those of untreated fucoidan at reaction temperatures below 140°C. However, fucoidan was partially degraded during depolymerization above 160°C. Nearly the same depolymerization was observed for chondroitin sulfate. These results indicate that hydrothermal treatment is applicable for the depolymerization of sulfated polysaccharides, and that low molecular weight products without desulfation and deformation of the initial glycan structures can be obtained under mild hydrothermal conditions.

Modification of pectin and hemicellulose polysaccharides in relation to aril breakdown of harvested longan fruit.

To investigate the modification of cell wall polysaccharides in relation to aril breakdown in harvested longan fruit, three pectin fractions (WSP, water soluble pectin; CSP, CDTA-soluble pectin; ASP, alkali soluble pectin) and one hemicellulose fraction (4 M KOH-SHC, 4 M KOH-soluble hemicellulose) were extracted, and their contents, monosaccharide compositions and molecular weights were evaluated. As aril breakdown intensified, CSP content increased while ASP and 4 M KOH-SHC contents decreased, suggesting the solubilization and conversion of cell wall components. Furthermore, the molar percentage of arabinose (Ara), as the main component of the side-chains, decreased largely in CSP and ASP while that of rhamnose (Rha), as branch point for the attachment of neutral sugar side chains, increased during aril breakdown. Analysis of (Ara + Gal)/Rha ratio showed that the depolymerization of CSP and ASP happened predominantly in side-chains formed of Ara residues. For 4 M KOH-SHC, more backbones were depolymerized during aril breakdown. Moreover, it was found that the molecular weights of CSP, ASP and 4 M KOH-SHC polysaccharides tended to decrease as aril breakdown intensified. These results suggest that both enhanced depolymerization and structural modifications of polysaccharides in the CSP, ASP and 4 M KOH-SHC fractions might be responsible for aril breakdown of harvested longan fruit.

Establishment of near Infrared Archaeometry

Introduction I t is of great importance to investigate the degradation mechanism of antique wooden materials used for historical temples, buildings and crafts from the viewpoint of wood science and archaeology. Near infrared (NIR) spectroscopy is a powerful tool for measuring the physical, chemical and mechanical properties of wood whilst retaining the anatomical structure of the archaeological samples. Cell walls of wood are composed of cellulose, hemicelluloses, lignin and extractives. The concentration of extractives is relatively low. The cellulose molecules form microfibrillar aggregates which are embedded in a matrix of hemicelluloses as well as lignin. As physical, chemical and mechanical properties of wood are governed by the complex arrangement of these polysaccharides, their variation with degradation should strongly affect the wooden materials. Knowledge on the aging of archaeological wood has mainly been obtained from those preserved in saturated soils or bogs in water-logged conditions. On the other hand, many important wooden materials have been preserved and repaired in non-closed environments in Japan, i.e. the Horyuji temple (constructed in the 7th century). We examined the temporal change in properties of archaeological wood and the possibility of non-destructively evaluating archaeological wood properties (i.e. age, strength, chemical components) by NIR spectroscopy with the aim of establishing NIR archaeometry. One reasonable approach for investigation of the degradation mechanism of wood is to employ thermally-degraded wood samples. As thermal treatment is one degradation process, this technique is sometimes applied to archaeology when artificially degraded wood is utilised for the repair of historical wooden artefacts. In this article, the changes in mechanical properties and crystalline structure with aging or thermal treatment, as investigated by NIR spectroscopy, are explained. Prediction of compressive Young’s modulus of hydrothermally treated wood by NIR spectroscopy The changes in compressive Young’s modulus of hydrothermally treated softwood and hardwood as a function of thermal treatment time were measured. Hinoki cypresses (Chamaecyparis obtusa) and beech (Fagus creneta) were hydrothermally treated at 140°C under steamed atmospheric conditions in order to artificially degrade wood to create an analogue of archaeological objects. NIR spectra and compressive Young’s modulus of artificially degraded wood were systematically measured. Figure 1 shows the change in compressive Young’s modulus of (a) hinoki, (b) beech and the relationship between measured compressive Young’s modulus and that predicted from NIR spectra of (c) hinoki and (d) beech by partial least squares (PLS) regression analysis. Compressive Young’s modulus of hinoki decreased as a function of treatment time. Conversely, that of beech rapidly increased with treatment time and subsequently gradually decreased. These calibrations demonstrated that NIR spectroscopy could predict the compressive Young’s modulus of hydrothermally-treated hinoki and beech wood samples with sufficient accuracy, although this parameter varies greatly within the same thermal treatment time. Figure 2 shows the plot of the first and second PLS loadings. The first loading plot, ranging from 10,000 cm to 8800 cm, where absorbance of NIR spectra increased with increments in treatment time, showed a negative value. On the other hand, the loading ranging from 8800 cm to 5000 cm due to polysaccharide absorption bands, where absorbance of NIR spectra decreased with increments in treatment time, showed a positive value. Therefore, the first loading might be regarded as a parameter reflecting the degree of hydrothermal degradation (i.e. depolymerisation of polysaccharide). On the other hand, the second loading has characteristic positive peaks observed around 7000 cm due to the amorphous region in cellulose and 5200 cm due doi: 10.1255/nirn.1394

Sugars and sugar derivatives in ionic liquid media obtained from lignocellulosic biomass: Comparison of capillary electrophoresis and chromatographic analysis

Processing of woody lignocellulosic biomass, under heating in combination with ionic liquids (ILs) was studied in order to obtain simple (fermentable) sugars. Due to the new environmental challenges, finding greener ways to produce platform chemicals and/or bio-fuels has become a popular research area. Various industrial, pilot or laboratory scale technologies for the depolymerization or fractionation of lignocellulosic polysaccharides to monomers are known. One of the new, interesting, methods is to utilize ILs in biomass pre-treatment procedures with an aim to bypass other pre-treatment methods. Furthermore, in order even to initiate studies whether ILs can contribute to catalytic depolymerization, there has to be a robust way to analyze the IL-treated lignocellulosics. This is a major issue since woody samples that contain any salts such as ILs can indeed be quite challenging from the analytic point of view. The applied capillary electrophoresis was found to be an excellent analytical method providing substantial improvements compared to the earlier used chromatographic methods.In this work it was demonstrated that application of ILs, at elevated temperatures, contributes to hydrolysis and depolymerization of lignocellulose. The effect is time and temperature dependent: in harsh conditions sugars degrade but too low processing temperatures or short treatment times result in no meaningful effect. The formation of the degradation products of the monosaccharides is a good indicator of the harshness of the applied chemical conditions. Evidently, furfural and 5-(hydroxymethyl)furfural formed in rather short treatment times.

Degradation and Synthesis of β-Glucans by a Magnaporthe oryzae Endotransglucosylase, a Member of the Glycoside Hydrolase 7 Family

Plant pathogens secrete enzymes that degrade plant cell walls to enhance infection and nutrient acquisition. A novel endotransglucosylase catalyzes cleavage and transfer of β-glucans and decreases the physical strength of plant cell walls. Endotransglucosylation causes depolymerization and polymerization of β-glucans, depending on substrate molecular size. Enzymatic degradation of plant cell walls is required for wall loosening, which enhances pathogen invasion. A Magnaporthe oryzae enzyme, which was encoded by the Mocel7B gene, was predicted to act on 1,3-1,4-β-glucan degradation and transglycosylation reaction of cellotriose after partial purification from a culture filtrate of M. oryzae cells, followed by liquid chromatography-tandem mass spectrometry. A recombinant MoCel7B prepared by overexpression in M. oryzae exhibited endo-typical depolymerization of polysaccharides containing β-1,4-linkages, in which 1,3-1,4-β-glucan was the best substrate. When cellooligosaccharides were used as the substrate, the recombinant enzyme generated reaction products with both shorter and longer chain lengths than the substrate. In addition, incorporation of glucose and various oligosaccharides including sulforhodamine-conjugated cellobiose, laminarioligosaccharides, gentiobiose, xylobiose, mannobiose, and xyloglucan nonasaccharide into β-1,4-linked glucans were observed after incubation with the enzyme. These results indicate that the recombinant enzyme acts as an endotransglucosylase (ETG) that cleaves the glycosidic bond of β-1,4-glucan as a donor substrate and transfers the cleaved glucan chain to another molecule functioning as an acceptor substrate. Furthermore, ETG treatment caused greater extension of heat-treated wheat coleoptiles. The result suggests that ETG functions to induce wall loosening by cleaving the 1,3-1,4-β-glucan tethers of plant cell walls. On the other hand, use of cellohexaose as a substrate for ETG resulted in the production of cellulose II with a maximum length (degree of polymerization) of 26 glucose units. Thus, ETG functions to depolymerize and polymerize β-glucans, depending on the size of the acceptor substrate.

β-Lactoglobulin–sodium alginate interaction as affected by polysaccharide depolymerization using high intensity ultrasound

The purpose of this study was to provide insights into the interaction between β-lactoglobulin (BLG) and sodium alginate (ALG, before and after sonication) using isothermal titration calorimetry (ITC), streaming current detector (SCD), turbidity measurement, dynamic light scattering and electrophoretic mobility methods. High intensity ultrasound could effectively decrease the intrinsic viscosity of the ALG solution. Time and amplitude of the sonication treatment had a direct effect on the viscosity depression, while the sonication temperature had an inverse effect. ITC measurements indicated that the sonication decreased the interaction strength between ALG and BLG. The isoenthalpic plateau was obtained at higher ALG (US)/BLG weight ratio compared to ALG (IN)/BLG weight ratio. The zeta-potential of the nanoparticles produced from ALG (US)–BLG attractive interaction was lower than of those produced from ALG (IN)–BLG interaction. These differences were attributed to the lower charge density of the ALG (US) as a result of sonochemical interactions. Particle size measurements showed that the effect of the sonication treatment was the homogenization of the nanoparticles in the mixed dispersion. The biopolymeric nanoparticles formed may therefore be useful as a delivery system for fortification purposes of transparent liquid food products.