Analysis of unsaturated alginate oligosaccharides using high-performance anion exchange chromatography coupled with mass spectrometry.

Alginate is a commercially important polysaccharide composed of mannuronic acid and its C5 differential isomer guluronic acid. Comprehensive research on alginate and alginate lyases requires efficient and precise analytical methods for alginate oligosaccharides. In this research, high-performance anion exchange chromatography (HPAEC) in parallel with pulsed amperometric detection (PAD) and mass spectrometry (MS) was applied to the analysis of oligosaccharides obtained by alginate lyase. By optimizing the chromatographic conditions including mobile phase concentration, flow rate, and elution gradient, the analysis of a single sample could be completed in 30 min. Seven unsaturated alginate oligosaccharides were separated and identified through their analysis time observed with PAD, including all structurally different unsaturated disaccharides and trisaccharides. The quantitative analysis of seven oligosaccharides was performed based on the quantitative capability of PAD. The method exhibited adequate linearity and precision parameters. All the calibration curves showed good linearity at least in the concentration range of 0.002 to 0.1 mg/mL. The HPAEC-PAD/MS method provides a general and efficient online method to analyze alginate oligosaccharides.

Complete composition analysis of polysaccharides based on HPAEC-PAD coupled with quantitative analysis of multi-components by single marker.

INTRODUCTION: Monosaccharide compositions analysis (MCA) is indispensable for structural characterisations and structure-activity relationships of plant polysaccharides. OBJECTIVES: To develop a concise and direct MCA method, we established a quantitative analysis of the multi-monosaccharaides by single marker (QAMS) by high-performance anion-exchange chromatography with pulsed-amperometric detection (HPAEC-PAD) method. METHODOLOGY: A stable and reproducible HPAEC-PAD method for simultaneous determination of aldoses, ketoses and uronic acids (i.e., l-arabinose, d-xylose, d-ribose, l-rhamnose, d-fucose, d-mannose, d-glucose, d-galactose, d-fructose, d-glucuronic acid and d-galacturonic acid) was established by systematic optimisation of stationary phases, column temperatures and elution programmes. On this basis, the QAMS method was proposed through comprehensive investigations of relative correction factor (RCF) variations under different influencing factors, for example, sample concentrations, flow rates, and column temperatures. RESULTS: Using rhamnose as an internal reference standard, the contents of the other monosaccharide components in polysaccharides from Panax quinquefolium L. and Achyranthes bidentata Bl. samples were simultaneously determined by QAMS, and there was no significant difference between the results from the QAMS and external standard method (t test, P > 0.520). In addition, a MCA fingerprinting of 30 batches of P. quinquefolium polysaccharide was established by HPAEC-PAD, and six common peaks were assigned and determined. CONCLUSIONS: The established HPAEC-PAD-QAMS method was successfully applied to the MCA of polysaccharides from P. quinquefolium and A. bidentata after optimisation of hydrolysis conditions. HPAEC-PAD-QAMS was proposed and established for MCA of plant polysaccharides for the first time.

Plasma measurements of the dual sugar test reveal carbohydrate immediately alleviates intestinal permeability caused by exertional heat stress.

The aim of this set of randomised cross-over studies was to determine the impact of progressive heat exposure and carbohydrate or protein feeding during exertional stress on small intestine permeability using a dual sugar test. In our previous work, and typically in the field, recovery of lactulose and l-rhamnose is measured cumulatively in urine. This follow-up study exploits our novel high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) protocol to accurately quantify the sugars in plasma. Endurance-trained participants completed experimental trial A (ET-A; n = 8), consisting of 2 h running at 60% V ̇ O 2 max ${\dot V_{{{\mathrm{O}}_{\mathrm{2}}}{\mathrm{max}}}}$ in temperate, warm and hot ambient conditions, and/or experimental trial B (ET-B; n = 9), consisting of 2 h running at 60% V ̇ O 2 max ${\dot V_{{{\mathrm{O}}_{\mathrm{2}}}{\mathrm{max}}}}$ in the heat while consuming water, carbohydrate or protein. Blood samples were collected and plasma lactulose (L) and l-rhamnose (R) appearance, after dual sugar solution ingestion at 90 min of exercise, was quantified by HPAEC-PAD to measure plasma L/R and reveal new information about intestinal permeability immediately post-exercise and during recovery. In ET-A, plasma L/R increased immediately post-exercise in hot compared with temperate and warm conditions, while, in ET-B, carbohydrate alleviated this, and this information was otherwise missed when measuring urine L/R. Consuming carbohydrate or protein before and during exercise attenuated small intestine permeability throughout recovery from exertional heat stress. We recommend using the dual sugar test with quantification of plasma sugars by HPAEC-PAD at intervals to maximise intestinal permeability data collection in exercise gastroenterology research, as this gives additional information compared to urinary measurements. KEY POINTS: Intestinal permeability is typically assessed using a dual sugar test, by administering a drink containing non-metabolisable sugars (e.g. lactulose (L) and l-rhamnose (R)) that can enter the circulation by paracellular translocation when the epithelium is compromised, and are subsequently measured in urine. We demonstrate that our recently developed ion chromatography protocol can be used to accurately quantify the L/R ratio in plasma, and that measuring L/R in plasma collected at intervals during the post-exercise recovery period reveals novel acute response information compared to measuring 5-h cumulative urine L/R. We confirm that exercising in hot ambient conditions increases intestinal epithelial permeability immediately after exercise, while consuming carbohydrate or protein immediately before and during exercise attenuates this. We recommend using our dual sugar absorption test protocol to maximise intestinal epithelial permeability data collection in exercise gastroenterology research and beyond.

Acid hydrolysis conditions for quantification of meningococcal X polysaccharide in a pentavalent vaccine using HPAEC-PAD/ESI-MS.

A selective and sensitive method was evaluated for quantitation of meningococcal X (Men X) polysaccharide in pentavalent meningococcal A, C, W, Y and X conjugate vaccine using different acid hydrolysis conditions like HCl, TFA, HF, HF-TFA, and HF-HCl. High-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using CarboPac PA10 column was used to identify the hydrolyzed products based on retention time and its comparison with monosaccharide standards. Complete release of glucosamine (GlcN) from Men X in monovalent bulk and pentavalent vaccine samples was achieved using HF hydrolysis at 80 °C for 2 h. The Men X HF-hydrolyzed polysaccharide to glucosamine along with the reference standard was identified using collision-induced dissociation (CID) electrospray mass spectroscopy and the MS/MS fragments of m/z 162, m/z 144 and m/z 84. Meningococcal polysaccharide concentration was determined with a correlation coefficient r2 >0.99 using polysaccharide reference standard. The serogroups A, W, and Y were converted to their monosaccharides units and quantified using this method however, milder acid hydrolysis 0.1 M HCl 80 °C 2 h for release of sialic acid for Men C polysaccharide was found to be more suitable. These methods will provide necessary tools and prove to be beneficial to laboratories developing new saccharide-based vaccine combinations.

Neutral and Pectic Heteropolysaccharides Isolated from Opuntia joconostle Mucilage: Composition, Molecular Dimensions and Prebiotic Potential.

Opuntia joconostle is a semi-wild cactus cultivated for its fruit. However, the cladodes are often discarded, wasting the potentially useful mucilage in them. The mucilage is composed primarily of heteropolysaccharides, characterized by their molar mass distribution, monosaccharide composition, structural features (by vibrational spectroscopy, FT IR, and atomic force microscopy, AFM), and fermentability by known saccharolytic commensal members of the gut microbiota. After fractionation with ion exchange chromatography, four polysaccharides were found: one neutral (composed mainly of galactose, arabinose, and xylose) and three acidic, with a galacturonic acid content from 10 to 35%mol. Their average molar masses ranged from 1.8 × 105 to 2.8 × 105 g·mol-1. Distinct structural features such as galactan, arabinan, xylan, and galacturonan motifs were present in the FT IR spectra. The intra- and intermolecular interactions of the polysaccharides, and their effect on the aggregation behavior, were shown by AFM. The composition and structural features of these polysaccharides were reflected in their prebiotic potential. Lactobacilli and Bifidobacteria were not able to utilize them, whereas members of Bacteroidetes showed utilization capacity. The obtained data suggest a high economic potential for this Opuntia species, with potential uses such as animal feed in arid areas, precise prebiotic, and symbiotic formulations, or as the carbon skeleton source in a green refinery. Our methodology can be used to evaluate the saccharides as the phenotype of interest, helping to guide the breeding strategy.

HPAEC-PAD Analytical Evaluation of Carbohydrates Pattern for the Study of Technological Parameters Effects in Low-FODMAP Food Production.

BACKGROUND: "FODMAPs" (fermentable-oligo-, di-, monosaccharides, and polyols) are a group of fermentable carbohydrates and polyols largely diffused in food products. Despite their beneficial effects as prebiotics, people affected by irritable bowel syndrome manifest symptoms when eating these carbohydrates. A low-FODMAP diet seems to be the only possible therapy proposed for symptom management. Bakery products are a common source of FODMAPs, whose pattern and total amount can be affected by their processing. This work aims at studying some of the technological parameters that can influence the FODMAPs pattern in bakery products during the production process. METHODS: high-performance anion exchange chromatography coupled to a pulsed amperometric detector (HPAEC-PAD) was used as a highly selective system for carbohydrates evaluation analyses on flours, doughs, and crackers. These analyses were performed using two different columns, the CarboPac PA200 and CarboPac PA1, which are selective for oligosaccharide and simple sugar separation, respectively. RESULTS: emmer and hemp flours were selected to prepare doughs as they contained low oligosaccharide content. Two different mixes of ferments were used at different times of fermentation to evaluate the best conditions to achieve low-FODMAP crackers. CONCLUSION: the proposed approach allows carbohydrate evaluation during crackers processing and permits the selection of opportune conditions to obtain low-FODMAP products.

Chitin-Active Lytic Polysaccharide Monooxygenases Are Rare in Cellulomonas Species.

Cellulomonas flavigena is a saprotrophic bacterium that encodes, within its genome, four predicted lytic polysaccharide monooxygenases (LPMOs) from Auxiliary Activity family 10 (AA10). We showed previously that three of these cleave the plant polysaccharide cellulose by oxidation at carbon-1 (J. Li, L. Solhi, E.D. Goddard-Borger, Y. Mattieu et al., Biotechnol Biofuels 14:29, 2021, https://doi.org/10.1186/s13068-020-01860-3). Here, we present the biochemical characterization of the fourth C. flavigena AA10 member (CflaLPMO10D) as a chitin-active LPMO. Both the full-length CflaLPMO10D-Carbohydrate-Binding Module family 2 (CBM2) and catalytic module-only proteins were produced in Escherichia coli using the native general secretory (Sec) signal peptide. To quantify chitinolytic activity, we developed a high-performance anion-exchange chromatography-pulsed amperometric detection (HPAEC-PAD) method as an alternative to the established hydrophilic interaction liquid ion chromatography coupled with UV detection (HILIC-UV) method for separation and detection of released oxidized chito-oligosaccharides. Using this method, we demonstrated that CflaLPMO10D is strictly active on the ß-allomorph of chitin, with optimal activity at pH 5 to 6 and a preference for ascorbic acid as the reducing agent. We also demonstrated the importance of the CBM2 member for both mediating enzyme localization to substrates and prolonging LPMO activity. Together with previous work, the present study defines the distinct substrate specificities of the suite of C. flavigena AA10 members. Notably, a cross-genome survey of AA10 members indicated that chitinolytic LPMOs are, in fact, rare among Cellulomonas bacteria. IMPORTANCE Species from the genus Cellulomonas have a long history of study due to their roles in biomass recycling in nature and corresponding potential as sources of enzymes for biotechnological applications. Although Cellulomonas species are more commonly associated with the cleavage and utilization of plant cell wall polysaccharides, here, we show that C. flavigena produces a unique lytic polysaccharide monooxygenase with activity on ß-chitin, which is found, for example, in arthropods. The limited distribution of orthologous chitinolytic LPMOs suggests adaptation of individual cellulomonads to specific nutrient niches present in soil ecosystems. This research provides new insight into the biochemical specificity of LPMOs in Cellulomonas species and related bacteria, and it raises new questions about the physiological function of these enzymes.

Mobility shift-based electrophoresis coupled with fluorescent detection enables real-time enzyme analysis of carbohydrate sulfatase activity.

Sulfated carbohydrate metabolism is a fundamental process, which occurs in all domains of life. Carbohydrate sulfatases are enzymes that remove sulfate groups from carbohydrates and are essential to the depolymerisation of complex polysaccharides. Despite their biological importance, carbohydrate sulfatases are poorly studied and challenges remain in accurately assessing the enzymatic activity, specificity and kinetic parameters. Most notably, the separation of desulfated products from sulfated substrates is currently a time-consuming process. In this paper, we describe the development of rapid capillary electrophoresis coupled to substrate fluorescence detection as a high-throughput and facile means of analysing carbohydrate sulfatase activity. The approach has utility for the determination of both kinetic and inhibition parameters and is based on existing microfluidic technology coupled to a new synthetic fluorescent 6S-GlcNAc carbohydrate substrate. Furthermore, we compare this technique, in terms of both time and resources, to high-performance anion exchange chromatography and NMR-based methods, which are the two current 'gold standards' for enzymatic carbohydrate sulfation analysis. Our study clearly demonstrates the advantages of mobility shift assays for the quantification of near real-time carbohydrate desulfation by purified sulfatases, and will support the search for small molecule inhibitors of these disease-associated enzymes.

Knockdown of CXCL3-inhibited apoptosis and inflammation in lipopolysaccharide-treated BEAS-2B and HPAEC through inactivating MAPKs pathway.

BACKGROUND: CXCL3 (C-X-C motif chemokine ligand 3) is a member of chemokines family, which binds to the receptor to recruit neutrophils to lungs, thus participating in the pathogenesis of asthmatic lung. The role of CXCL3 in sepsis-induced acute lung injury is investigated here. METHODS: Human lung epithelial cell line (BEAS-2B) and human pulmonary artery endothelial cell line (HPAEC) were treated with lipopolysaccharides (LPS). MTT and flow cytometry were performed to detect cell viability and apoptosis, respectively. Enzyme-linked immunosorbent assay (ELISA) and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to assess the levels of inflammatory factors. RESULTS: Treatment with LPS resulted in the decrease of cell viability in BEAS-2B and HPAEC. CXCL3 was particularly upregulated in LPS-treated BEAS-2B and HPAE cells. Knockdown of CXCL3 enhanced viability and suppressed apoptosis i006E LPS-treated BEAS-2B and HPAE cells. Knockdown of CXCL3 also upregulated TNF-α, IL-1ß, and IL-18 in LPS-treated BEAS-2B and HPAE cells. Moreover, knockdown of CXCL3 suppressed the activation of mitogen-activated protein kinases (MAPKs) signaling in LPS-treated BEAS-2B and HPAE cells through downregulation of p-ERK1/2, p-p38, and p-JNK. On the other hand, overexpression of CXCL3 caused completely opposite results in LPS-treated BEAS-2B and HPAE cells. CONCLUSION: Knockdown of CXCL3 exerted antiapoptotic and anti-inflammatory effects against LPS-treated BEAS-2B and HPAE cells, at least partially, through inactivation of MAPKs signaling, suggesting a potential strategy for the intervention of sepsis-induced acute lung injury.

Comparative Study on Volatile Compounds and Taste Components of Different Durian Cultivars Based on GC-MS, UHPLC, HPAEC-PAD, E-Tongue and E-Nose.

In order to comprehensively evaluate the aroma-active substances and taste components of durian, solid-phase microextraction combined with gas chromatography mass spectrometry (SPME/GC-MS), high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and ultra-high-performance liquid chromatography (UHPLC) were used to test the key components of three popular durian cultivars. A total of 27 volatile compounds, 5 sugars, 27 organic acids and 19 free amino acids were detected in Black Thorn (BT) durian. A total of 38 volatile compounds, 4 sugars, 27 organic acids and 19 free amino acids were detected in Monthong (MT) durian. A total of 36 volatile compounds, 4 sugars, 27 organic acids and 20 free amino acids were detected in Musang King (MK) durian. Finally, the flavor differences of the three durians were evaluated using electronic nose (e-nose) and electronic tongue (e-tongue), and different cultivars were classified through principal component analysis (PCA).

Nutraceutical Content and Genetic Diversity Share a Common Pattern in New Pomegranate Genotypes.

The nutraceutical value of pomegranate in the treatment of many diseases is well-documented and is linked to its richness in phenolic compounds. This study aims to evaluate the nutraceutical and genetic diversity of novel pomegranate genotypes (G1-G5) in comparison to leading commercial pomegranate varieties, i.e., 'Wonderful', 'Primosole', 'Dente di Cavallo' and 'Valenciana'. Morphometric measurements were carried out on fruits, accompanied by chemical characterization (total phenolic content, antioxidant activity, carbohydrates and minerals) and the development of four new polymorphic SSR markers involved in the flavonoid pathway. The cultivars displayed a marked variability in the weight and shape of the fruits, as well as in the weight of the arils and juice yield. The highest level of total phenolic content and antioxidant activity was found in 'Wonderful' and G4, while the lowest was in 'Dente di Cavallo'. Furthermore, the results showed that pomegranate juice is an excellent source of minerals, especially potassium, which plays a key role in organ functioning. The new flavonoid-related markers effectively differentiated the cultivars with the same diversity pattern as morpho-chemical characterization, so the SSRs developed in the present study can be used as a rapid tool for the identification of pomegranate cultivars with relevant nutraceutical traits, such as the new genotypes investigated.

Characterization of glycosylation in monoclonal antibodies and its importance in therapeutic antibody development.

Glycosylation is one of the structurally diverse and complex forms of post translational modifications observed in proteins which influence the effector functions of IgG-Fc. Although the glycosylation constitutes 2-3% of the total mass of the IgG antibody, a thorough assessment of glycoform distribution present on the antibody is a critical quality attribute (cQA) for the majority of novel and biosimilar monoclonal antibody (mAb) development. This review paper will highlight the impact of different glycoforms such as galactose, fucose, high mannose, NANA (N-acetylneuraminic acid), and NGNA (N-glycoylneuraminic acid) on the safety/immunogeneicity, efficacy/biological activity and clearance (pharmacodynamics/pharmacokinetic property (PD/PK)) of biological molecules. In addition, this paper will summarize routinely employed reliable analytical techniques such as hydrophilic interaction chromatography (HILIC), high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and mass spectrometry (MS) for characterizing and monitoring glycosylation in monoclonal antibodies (mAbs). The advantages and disadvantages of each of the methods are addressed. The scope of this review paper is limited to only N-linked and O-linked glycosylation.

Comparison and Optimization of Quantification Methods for Shigella flexneri Serotype 6 O-antigen Containing Galacturonic Acid and Methyl-Pentose.

Shigella is a leading diarrheal cause of morbidity and mortality worldwide, especially in low- and middle-income countries and in children under five years of age. Increasing levels of antimicrobial resistance make vaccine development an even higher global health priority. S. flexneri serotype 6 is one of the targets of many multicomponent vaccines in development to ensure broad protection against Shigella. The O-antigen (OAg) is a key active ingredient and its content is a critical quality attribute for vaccine release in order to monitor their stability and to ensure appropriate immune response. Here, the optimization of two methods to quantify S. flexneri 6 OAg is reported together with the characterization of their performances. The optimized Dische colorimetric method allows a tenfold increment of the sensitivity with respect to the original method and is useful for fast analysis detecting selectively methyl-pentoses, as rhamnose in S. flexneri 6 OAg. Also, a more specific HPAEC-PAD method was developed, detecting the dimer galacturonic acid-galactosamine (GalA-GalN) coming from S. flexneri 6 OAg acid hydrolysis. These methods will facilitate characterization of S. flexneri 6 OAg based vaccines. The colorimetric method can be used for quantification of other polysaccharide containing methyl-pentoses, and the HPAEC-PAD could be extended to other polysaccharides containing uronic acids.

Uncover aldehydes in biomass hydrolyzates: disproportionation of aldehydes in alkaline solution and subsequent measurement using an automated HPAEC-PAD method.

High-performance anion exchange chromatography coupled to pulsed amperometric detection (HPAEC-PAD) was used for developing a method for identifying and quantifying aldehydes in biomass hydrolyzates. This method was optimized to the requirements of HPAEC-PAD in order to allow for a simultaneous determination of aldehydes by respective Cannizzaro alcohols. To this end, sodium hydroxide concentration (0.1 to 5.0 mol/L), temperature (30 to 40 °C), and reaction time (0 to 24 h) were investigated for sufficient and reproducible disproportionation of the biomass-derived aldehydes. The optimized method for aldehyde disproportionation and subsequent measurement are 1 mol/L sodium hydroxide, 40 °C, and 1 h reaction time. The detection limits resulting from this method are lower than 68.55 mg/L and the sensitivity above 0.024 (nC min)/(mg/L) for 3,4-dimethoxybenzaldehyde. Linearity for aldehyde calibration always exceeded 0.98. Thus, HPAEC-PAD analysis allows for the quantification of biomass-derived compounds from all natural polymers and, therefore, it has exemplarily been used to quantify aldehyde concentration of beech wood, orange peel, and algae biomass hydrolyzates. Graphical abstract.

Carbohydrate determination in honey samples by ion chromatography-mass spectrometry (HPAEC-MS).

Honey is a complex mixture of carbohydrates, in which the monosaccharides glucose and fructose are the most abundant compounds. Currently, more than 20 oligosaccharides have been identified in different varieties of honey normally at quite low concentration. A method was developed and validated using high-performance anion-exchange chromatography coupled to a mass spectrometry detector to investigate the composition of carbohydrates in honey samples. The method was tested for linearity range, trueness, instrumental and method detection and quantification limits, repeatability, and reproducibility. It was applied to determine seven monosaccharides, eight disaccharides, four trisaccharides, and one tetrasaccharide in various honey samples. The present work describes the composition of sugars in unifloral, multifloral, and some honeydew honey, which were produced and collected by beekeepers in the Trentino Alto-Adige region. Statistical techniques have been used to establish a relationship based on levels of carbohydrates among different Italian honey. The results emphasize that mono- and oligosaccharide profiles can be useful to discriminate different honeys according to their floral characteristics and inter-annual variability.

Molecular Analysis of Fetal and Adult Primary Human Liver Sinusoidal Endothelial Cells: A Comparison to Other Endothelial Cells.

In humans, Factor VIII (F8) deficiency leads to hemophilia A and F8 is largely synthesized and secreted by the liver sinusoidal endothelial cells (LSECs). However, the specificity and characteristics of these cells in comparison to other endothelial cells is not well known. In this study, we performed genome wide expression and CpG methylation profiling of fetal and adult human primary LSECs together with other fetal primary endothelial cells from lung (micro-vascular and arterial), and heart (micro-vascular). Our results reveal expression and methylation markers distinguishing LSECs at both fetal and adult stages. Differential gene expression of fetal LSECs in comparison to other fetal endothelial cells pointed to several differentially regulated pathways and biofunctions in fetal LSECs. We used targeted bisulfite resequencing to confirm selected top differentially methylated regions. We further designed an assay where we used the selected methylation markers to test the degree of similarity of in-house iPS generated vascular endothelial cells to primary LSECs; a higher similarity was found to fetal than to adult LSECs. In this study, we provide a detailed molecular profile of LSECs and a guide to testing the effectiveness of production of in vitro differentiated LSECs.

Fast automated online xylanase activity assay using HPAEC-PAD.

In contrast to biochemical reactions, which are often carried out under automatic control and maintained overnight, the automation of chemical analysis is usually neglected. Samples are either analyzed in a rudimentary fashion using in situ techniques, or aliquots are withdrawn and stored to facilitate more precise offline measurements, which can result in sampling and storage errors. Therefore, in this study, we implemented automated reaction control, sampling, and analysis. As an example, the activities of xylanases on xylotetraose and soluble xylan were examined using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The reaction was performed in HPLC vials inside a temperature-controlled Dionex™ AS-AP autosampler. It was started automatically when the autosampler pipetted substrate and enzyme solution into the reaction vial. Afterwards, samples from the reaction vial were injected repeatedly for 60 min onto a CarboPac™ PA100 column for analysis. Due to the rapidity of the reaction, the analytical method and the gradient elution of 200 mM sodium hydroxide solution and 100 mM sodium hydroxide with 500 mM sodium acetate were adapted to allow for an overall separation time of 13 min and a detection limit of 0.35-1.83 mg/L (depending on the xylooligomer). This analytical method was applied to measure the soluble short-chain products (xylose, xylobiose, xylotriose, xylotetraose, xylopentaose, and longer xylooligomers) that arise during enzymatic hydrolysis. Based on that, the activities of three endoxylanases (EX) were determined as 294 U/mg for EX from Aspergillus niger, 1.69 U/mg for EX from Bacillus stearothermophilus, and 0.36 U/mg for EX from Bacillus subtilis. Graphical abstract Xylanase activity assay automation.

Analysis of the substrate specificity of α-L-arabinofuranosidases by DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis.

Carbohydrate-active enzyme discovery is often not accompanied by experimental validation, demonstrating the need for techniques to analyze substrate specificities of carbohydrate-active enzymes in an efficient manner. DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) is utmost appropriate for the analysis of glycoside hydrolases that have complex substrate specificities. DSA-FACE is demonstrated here to be a highly convenient method for the precise identification of the specificity of different α-L-arabinofuranosidases for (arabino)xylo-oligosaccharides ((A)XOS). The method was validated with two α-L-arabinofuranosidases (EC 3.2.1.55) with well-known specificity, specifically a GH62 α-L-arabinofuranosidase from Aspergillus nidulans (AnAbf62A-m2,3) and a GH43 α-L-arabinofuranosidase from Bifidobacterium adolescentis (BaAXH-d3). Subsequently, application of DSA-FACE revealed the AXOS specificity of two α-L-arabinofuranosidases with previously unknown AXOS specificities. PaAbf62A, a GH62 α-L-arabinofuranosidase from Podospora anserina strain S mat+, was shown to target the O-2 and the O-3 arabinofuranosyl monomers as side chain from mono-substituted ß-D-xylosyl residues, whereas a GH43 α-L-arabinofuranosidase from a metagenomic sample (AGphAbf43) only removes an arabinofuranosyl monomer from the smallest AXOS tested. DSA-FACE excels ionic chromatography in terms of detection limit for (A)XOS (picomolar sensitivity), hands-on and analysis time, and the analysis of the degree of polymerization and binding site of the arabinofuranosyl substituent.

HPAEC-PAD for oligosaccharide analysis-novel insights into analyte sensitivity and response stability.

The rising importance of accurately detecting oligosaccharides in biomass hydrolyzates or as ingredients in food, such as in beverages and infant milk products, demands for the availability of tools to sensitively analyze the broad range of available oligosaccharides. Over the last decades, HPAEC-PAD has been developed into one of the major technologies for this task and represents a popular alternative to state-of-the-art LC-MS oligosaccharide analysis. This work presents the first comprehensive study which gives an overview of the separation of 38 analytes as well as enzymatic hydrolyzates of six different polysaccharides focusing on oligosaccharides. The high sensitivity of the PAD comes at cost of its stability due to recession of the gold electrode. By an in-depth analysis of the sensitivity drop over time for 35 analytes, including xylo- (XOS), arabinoxylo- (AXOS), laminari- (LOS), manno- (MOS), glucomanno- (GMOS), and cellooligosaccharides (COS), we developed an analyte-specific one-phase decay model for this effect over time. Using this model resulted in significantly improved data normalization when using an internal standard. Our results thereby allow a quantification approach which takes the inevitable and analyte-specific PAD response drop into account. Graphical abstract HPAEC-PAD analysis of oligosaccharides and determination of PAD response drop leading to an improved data normalization.

Automated chromatographic laccase-mediator-system activity assay.

To study the interaction of laccases, mediators, and substrates in laccase-mediator systems (LMS), an on-line measurement was developed using high performance anion exchange chromatography equipped with a CarboPac™ PA 100 column coupled to pulsed amperometric detection (HPAEC-PAD). The developed method was optimized for overall chromatographic run time (45 to 120 min) and automated sample drawing. As an example, the Trametes versicolor laccase induced oxidation of 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-1,3-dihydroxypropane (adlerol) using 1-hydroxybenzotriazole (HBT) as mediator was measured and analyzed on-line. Since the Au electrode of the PAD detects only hydroxyl group containing substances with a limit of detection being in the milligram/liter range, not all products are measureable. Therefore, this method was applied for the quantification of adlerol, and-based on adlerol conversion-for the quantification of the LMS activity at a specific T. versicolor laccase/HBT ratio. The automated chromatographic activity assay allowed for a defined reaction start of all laccase-mediator-system reactions mixtures, and the LMS reaction progress was automatically monitored for 48 h. The automatization enabled an integrated monitoring overnight and over-weekend and minimized all manual errors such as pipetting of solutions accordingly. The activity of the LMS based on adlerol consumption was determined to 0.47 U/mg protein for a laccase/mediator ratio of 1.75 U laccase/g HBT. In the future, the automated method will allow for a fast screening of combinations of laccases, mediators, and substrates which are efficient for lignin modification. In particular, it allows for a fast and easy quantification of the oxidizing activity of an LMS on a lignin-related substrate which is not covered by typical colorimetric laccase assays. ᅟ.