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|Substrate For (Enzyme):||endo-1,4-β-Xylanase|
High purity Xylotetraose for use in research, biochemical enzyme assays and in vitro diagnostic analysis.
McCleary, B. V. & McGeough, P. (2015). Appl. Biochem. Biotechnol., 177(5), 1152-1163.
The most commonly used method for the measurement of the level of endo-xylanase in commercial enzyme preparations is the 3,5-dinitrosalicylic acid (DNS) reducing sugar method with birchwood xylan as substrate. It is well known that with the DNS method, much higher enzyme activity values are obtained than with the Nelson-Somogyi (NS) reducing sugar method. In this paper, we have compared the DNS and NS reducing sugar assays using a range of xylan-type substrates and accurately compared the molar response factors for xylose and a range of xylo-oligosaccharides. Purified beechwood xylan or wheat arabinoxylan is shown to be a suitable replacement for birchwood xylan which is no longer commercially available, and it is clearly demonstrated that the DNS method grossly overestimates endo-xylanase activity. Unlike the DNS assay, the NS assay gave the equivalent colour response with equimolar amounts of xylose, xylobiose, xylotriose and xylotetraose demonstrating that it accurately measures the quantity of glycosidic bonds cleaved by the endo-xylanase. The authors strongly recommend cessation of the use of the DNS assay for measurement of endo-xylanase due to the fact that the values obtained are grossly overestimated due to secondary reactions in colour development.Hide Abstract
Versatile high resolution oligosaccharide microarrays for plant glycobiology and cell wall research.
Pedersen, H. L., Fangel, J. U., McCleary, B., Ruzanski, C., Rydahl, M. G., Ralet, M. C., Farkas, V., Von Schantz, L., Marcus, S. E., Andersen, M.C. F., Field, R., Ohlin, M., Knox, J. P., Clausen, M. H. & Willats, W. G. T. (2012). Journal of Biological Chemistry, 287(47), 39429-39438.
Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.Hide Abstract
In Vitro Assessment of the Prebiotic Potential of Xylooligosaccharides from Barley Straw.
Álvarez, C., González, A., Ballesteros, I., Gullón, B. & Negro, M. J. (2023). Foods, 12(1), 83.
Barley straw was subjected to hydrothermal pretreatment (steam explosion) processing to evaluate its potential as a raw material to produce xylooligosaccharides (XOS) suitable for use as a prebiotic. The steam explosion pretreatment generated a liquid fraction containing solubilised hemicellulose. This fraction was purified using gel permeation chromatography to obtain a fraction rich in XOS DP2-DP6. The sample was characterised through analytical techniques such as HPAEC-PAD, FTIR and MALDI-TOF-MS. The prebiotic activity was evaluated using in vitro fermentation in human faecal cultures through the quantification of short-chain fatty acid (SCFA) and lactate production, the evolution of the pH and the consumption of carbon sources. The total SCFA production at the end of fermentation (30 h) was 90.1 mM. Positive significant differences between the amount of XOS fromHide Abstract
Selfish uptake versus extracellular arabinoxylan degradation in the primary degrader Ruminiclostridium cellulolyticum, a new string to its bow.
Liu, N., Gagnot, S., Denis, Y., Byrne, D., Faulds, C., Fierobe, H. P. & Perret, S. (2022). Biotechnology for Biofuels and Bioproducts, 15(1), 1-16.
Background: Primary degraders of polysaccharides play a key role in anaerobic biotopes, where plant cell wall accumulates, providing extracellular enzymes to release fermentable carbohydrates to fuel themselves and other non-degrader species. Ruminiclostridium cellulolyticum is a model primary degrader growing amongst others on arabinoxylan. It produces large multi-enzymatic complexes called cellulosomes, which efficiently deconstruct arabinoxylan into fermentable monosaccharides. Results: Complete extracellular arabinoxylan degradation was long thought to be required to fuel the bacterium during this plant cell wall deconstruction stage. We discovered and characterized a second system of “arabinoxylan” degradation in R. cellulolyticum, which challenged this paradigm. This “selfish” system is composed of an ABC transporter dedicated to the import of large and possibly acetylated arabinoxylodextrins, and a set of four glycoside hydrolases and two esterases. These enzymes show complementary action modes on arabinoxylo-dextrins. Two α-L-arabinofuranosidases target the diverse arabinosyl side chains, and two exo-xylanases target the xylo-oligosaccharides backbone either at the reducing or the non-reducing end. Together, with the help of two different esterases removing acetyl decorations, they achieve the depolymerization of arabinoxylo-dextrins in arabinose, xylose and xylobiose. The in vivo study showed that this new system is strongly beneficial for the fitness of the bacterium when grown on arabinoxylan, leading to the conclusion that a part of arabinoxylan degradation is achieved in the cytosol, even if monosaccharides are efficiently provided by the cellulosomes in the extracellular space. These results shed new light on the strategies used by anaerobic primary degrader bacteria to metabolize highly decorated arabinoxylan in competitive environments. Conculsion: The primary degrader model Ruminiclostridium cellulolyticum has developed a “selfish” strategy consisting of importing into the bacterium, large arabinoxylan-dextrin fractions released from a partial extracellular deconstruction of arabinoxylan, thus complementing its efficient extracellular arabinoxylan degradation system. Genetic studies suggest that this system is important to support fitness and survival in a competitive biotope. These results provide a better understanding of arabinoxylan catabolism in the primary degrader, with biotechnological application for synthetic microbial community engineering for the production of commodity chemicals from lignocellulosic biomass.Hide Abstract
Characterization of a GH5 endoxylanase from Penicillium funiculosum and its synergism with GH16 endo-1, 3 (4)-glucanase in saccharification of sugarcane bagasse.
Ogunyewo, O. A., Okereke, O. E., Kumar, S. & Yazdani, S. S. (2022). Scientific Reports, 12(1), 1-17.
The production of second-generation fuels from lignocellulosic residues such as sugarcane bagasse (SCB) requires the synergistic interaction of key cellulose-degrading enzymes and accessory proteins for their complete deconstruction to useful monomeric sugars. Here, we recombinantly expressed and characterized unknown GH5 xylanase from P. funiculosum (PfXyn5) in Pichia pastoris, which was earlier found in our study to be highly implicated in SCB saccharification. The PfXyn5 has a molecular mass of ~ 55 kDa and showed broad activity against a range of substrates like xylan, xyloglucan, laminarin and p-nitrophenyl-β-D-xylopyranoside, with the highest specific activity of 0.7 U/mg against xylan at pH 4.5 and 50°C. Analysis of the degradation products of xylan and SCB by PfXyn5 showed significant production of xylooligosaccharides (XOS) with a degree of polymerization (DP) ranging from two (DP2) to six (DP6), thus, suggesting that the PfXyn5 is an endo-acting enzyme. The enzyme synergistically improved the saccharification of SCB when combined with the crude cellulase cocktail of P. funiculosum with a degree of synergism up to 1.32. The PfXyn5 was further expressed individually and simultaneously with a notable GH16 endoglucanase (PfEgl16) in a catabolite-derepressed strain of P. funiculosum, PfMig188, and the saccharification efficiency of the secretomes from the resulting transformants were investigated on SCB. The secretome of PfMig188 overexpressing Xyn5 or Egl16 increased the saccharification of SCB by 9% or 7%, respectively, over the secretome of PfMig188, while the secretome of dual transformant increased SCB saccharification by ~ 15% at the same minimal protein concentration.Hide Abstract
Role of Bifidobacterium pseudocatenulatum in Degradation and Consumption of Xylan-Derived Carbohydrates.
Drey, E., Kok, C. R. & Hutkins, R. (2022). Applied and Environmental Microbiology, 88(20), e01299-22.
Xylans, a family of xylose-based polysaccharides, are dietary fibers resistant to digestion. They therefore reach the large intestine intact; there, they are utilized by members of the gut microbiota. They are initially broken down by primary degraders that utilize extracellular xylanases to cleave xylan into smaller oligomers. The resulting xylooligosaccharides (XOS) can either be further metabolized directly by primary degraders or cross-feed secondary consumers, including Bifidobacterium. While several Bifidobacterium species have metabolic systems for XOS, most grow poorly on longer-chain XOS and xylan substrates. In this study, we isolated strains of Bifidobacterium pseudocatenulatum and observed that some, including B. pseudocatenulatum ED02, displayed growth on XOS with a high degree of polymerization (DP) and straight-chain xylan, suggesting a primary degrader phenotype that is rare in Bifidobacterium. In silico analyses revealed that only the genomes of these xylan-fermenting (xylan+) strains contained an extracellular GH10 endo-β-1.4 xylanase, a key enzyme for primary degradation of xylan. The presence of an extracellular xylanase was confirmed by the appearance of xylan hydrolysis products in cell-free supernatants. Extracellular xylanolytic activity was only detected in xylan+ strains, as indicated by the production of XOS fragments with a DP of 2 to 6, identified by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Additionally, in vitro fecal fermentations revealed that strains with a xylan+ phenotype can persist with xylan supplementation. These results indicate that xylan+ B. pseudocatenulatum strains may have a competitive advantage in the complex environment of the gastrointestinal tract, due to their ability to act as primary degraders of xylan through extracellular enzymatic degradation.Hide Abstract
Production of Xylooligosaccharide from Cassava Pulp’s Waste by Endo-β-1, 4-D-Xylanase and Characterization of Its Prebiotic Effect by Fermentation of Lactobacillus acidophilus.
Ratnadewi, A. A. I., Rahma, M. T., Nurhayati, N., Santoso, A. B., Senjarini, K., Labes, A. & Reza, M. (2022). Fermentation, 8(10), 488.
This study explores the production of prebiotic xylooligosaccharide (XOS) from cassava pulp waste and its effectiveness for the growth of Lactobacillus acidophilus (L. acidophilus). We successfully produced and characterized XOS from cassava pulp xylan using a Bacillus sp. endo-β-1,4-D-xylanase. The XOS was added to modify the MRS medium (MRSm) in various concentrations (0, 1, 3 and 5%) in which the L. acidophilus was inoculated. The growth of L. acidophilus was observed every 12 h for 2 days, and the fermentation products were analyzed for pH, sugar content, and short-chain fatty acids (SCFA) in terms of types and amount. The study showed that L. acidophilus grew well in MRSm. The optimum XOS concentration in MRSm was 5%, indicated by the highest growth of L. acidophilus (8.61 log CFU mL−1). The profile of SCFA products is 14.42 mM acetic acid, 0.25 mM propionic acid, 0.13 mM isobutyric acid, 0.41 mM n-butyric acid, 0.02 mM n-valeric acid, 0.25 mM isovaleric acid, and 25.08 mM lactic acid.Hide Abstract
Enzymatic bioconversion of beechwood xylan into the antioxidant 2′-O-α-(4-O-methyl-D-glucuronosyl)-xylobiose.
Miguez, N., Fernandez-Polo, D., Santos-Moriano, P., Rodríguez-Colinas, B., Poveda, A., Jimenez-Barbero, J., Ballesteros, A. O. & Plou, F. J. (2022). Biomass Conversion and Biorefinery, 1-12.
Acidic xylooligosaccharides (XOS), also called aldouronics, are hetero-oligomers of xylose randomly branched with 4-O-methyl-D-glucuronic acid residues linked by α(1 → 2) bonds, which display bioactive properties. We have developed a simple and integrated method for the production of acidic XOS by enzymatic hydrolysis of a glucurono-xylan from beechwood. Among the enzymes screened, Depol 670L (a cellulolytic preparation from Trichoderma reesei) displayed the highest activity (70.3 U/mL, expressed in reducing xylose equivalents). High-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) analysis revealed the formation of a neutral fraction (corresponding to linear XOS, mainly xylose and xylobiose) and a group of more retained products (acidic XOS), which were separated using strong anion-exchange cartridges. The acidic fraction contained a major product, characterized by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and mono- and two-dimensional nuclear magnetic resonance spectroscopy (NMR) as 2′-O-α-(4-O-methyl-α-D-glucuronosyl)-xylobiose (X2_MeGlcA). Starting from 2 g of beechwood xylan, 1.5 g of total XOS were obtained, from which 225 mg (11% yield) corresponded to the aldouronic X2_MeGlcA. The acidic XOS exhibited higher antioxidant activity (measured by the ABTS·+ discoloration assay) than xylan, whilst neutral XOS displayed no antioxidant activity. This work demonstrates that it is possible to obtain a safe and natural antioxidant by enzymatic biotransformation of hardwood hemicellulose.Hide Abstract
Debranching enzymes decomposed corn arabinoxylan into xylooligosaccharides and achieved a prebiotic regulation of gut microbiota in broilers.
Wu, W., Zhou, H., Chen, Y., Guo, Y., & Yuan, J. (2022), In Press.
BACKGROUND: Corn arabinoxylan (AX) is a complicated and multi-branched antinutritional factor, thereby proving the use of endo-xylanase (EX) to be marginally valid. This study focused on specific types of AX-degrading enzymes (ADE) to exert the synergy of debranching enzymes and track prebiotic potential of enzymatic hydrolysates. It aimed to investigate the effects of ADE on growth performance, intestinal histomorphology, absorption functions, changes of polysaccharide components, fermentation, and gut microbiota of broilers. To this purpose, 576 five-day-old Arbor Acres male broilers were randomly allocated into eight treatments with six replicates each. Corn basal diets supplemented with or without enzymes were fed for a 21-day period, specifically including EX, its compatible use with arabinofuranosidase (EXA) or ferulic acid esterase (EXF), and compound groups with the above three enzymes (XAF). RESULTS: Specific ADE stimulated the jejunal villus height and goblet cell number, and evidently decreased the crypt depth (P < 0.05), while the ratio of ileal villus height to crypt depth was significantly increased in EXF (P < 0.05). Maltase activities of ileal mucosa in XAF groups were extremely enhanced (P < 0.01), and EX boosted the activity of Na+ -K+ ATPase in the small intestine (P < 0.01). The insoluble AX concentrations comparatively lessened, thereby notably raising the sundry xylooligosaccharide (XOS) yield in the ileal chyme (P < 0.05), which was dominant in xylobiose and xylotriose. Improvements in the abundance and diversity of ileal microbial communities within EXA, EXF, XAF treatments were observed (P < 0.05). Positive correlations between microbiota and XOS were revealed, with xylobiose and xylotriose being critical for ten beneficial bacteria (P < 0.05). EXF escalated the BWG and FCR of broilers in this phase (P < 0.05), attributing to the thriving networks modified by Lactobacillus. The intracecal contents of acetic acid, butyric acid, and propionic acid were greatly enhanced in most ADE groups such as EXF (P < 0.05). CONCLUSIONS: Debranching enzymes appreciably targeted corn AX to release prebiotic XOS in the posterior ileum and facilitated intracaecal fermentation. It was beneficial for improving gut development, digestion and absorption, and modulating the microflora to promote early performance of broilers.Hide Abstract
Manufacturing of hemicellulosic oligosaccharides from fast-growing Paulownia wood via autohydrolysis: Microwave versus conventional heating.
Pablo, G., Pérez-Pérez, A., Garrote, G. & Gullón, B. (2022). Industrial Crops and Products, 187, 115313.
Microwave hydrothermal treatment (MHT) is considered a sustainable technology for the valorization of lignocellulosic materials, enabling the solubilization of hemicellulosic-derived compounds, especially in the form of oligosaccharides that may present potential in the chemical, pharmaceutical or nutraceutical industries. Hence, MHT at 200 and 230°C, at severity (S0) among 2.92-4.66 were performed. S0 = 3.98 permitted the recovery of about 80% of the initial xylan as xylooligosaccharides. In order to compare the effectiveness of MHT, conventional hydrothermal treatment (CHT) was performed at conditions leading to the maximum recovery of oligosaccharides (S0 =3.98, non-isothermal regime at 203°C). Despite the structural features of oligomers in the three liquors were very similar, the spent solids presented different enzymatic digestibility, which implied a different effect of the treatments, reaching up to 80% of glucan to glucose conversion for the solid after MHT at 230°C for 0.5 min. Additionally, CHT consumed 2.1-2.8-fold greater energy than MHT, reflecting that microwave-assisted autohydrolysis is a sustainable and efficient technology to process PW.Hide Abstract
Characterization of a novel GH10 alkali-thermostable xylanase from a termite microbiome.
Mon, M. L., Marrero Díaz de Villegas, R., Campos, E., Soria, M. A. & Talia, P. M. (2022). Bioresources and Bioprocessing, 9(1), 1-15.
The aim of the present study was to assess the biochemical and molecular structural characteristics of a novel alkali-thermostable GH10 xylanase (Xyl10B) identified in a termite gut microbiome by a shotgun metagenomic approach. This endoxylanase candidate was amplified, cloned, heterologously expressed in Escherichia coli and purified. The recombinant enzyme was active at a broad range of temperatures (37-60°C) and pH values (4-10), with optimal activity at 50°C and pH 9. Moreover, its activity remained at more than 80% of its maximum at 50°C for 8 h. In addition, Xyl10B was found to be stable in the presence of salt and several ions and chemical reagents frequently used in the industry. These characteristics make this enzyme an interesting candidate for pulp and paper bleaching industries, since this process requires enzymes without cellulase activity and resistant to high temperatures and alkaline pH (thermo-alkaliphilic enzymes). The products of xylan hydrolysis by Xyl10B (short xylooligosaccharides, xylose and xylobiose) could be suitable for application as prebiotics and in the production of bioethanol.Hide Abstract
Technical pipeline for screening microbial communities as a function of substrate specificity through fluorescent labelling.
Leivers, S., Lagos, L., Garbers, P., La Rosa, S. L. & Westereng, B. (2022). Communications biology, 5(1), 1-12.
The study of specific glycan uptake and metabolism is an effective tool in aiding with the continued unravelling of the complexities in the human gut microbiome. To this aim fluorescent labelling of glycans may provide a powerful route towards this target. Here, we successfully used the fluorescent label 2-aminobenzamide (2-AB) to monitor and study microbial degradation of labelled glycans. Both single strain and co-cultured fermentations of microbes from the common human-gut derived Bacteroides genus, are able to grow when supplemented with 2-AB labelled glycans of different monosaccharide composition, degrees of acetylation and polymerization. Utilizing a multifaceted approach that combines chromatography, mass spectrometry, microscopy and flow cytometry techniques, it is possible to better understand the metabolism of labelled glycans in both supernatants and at a single cell level. We envisage this combination of complementary techniques will help further the understanding of substrate specificity and the role it plays within microbial communities.Hide Abstract
Enterococcus faecium s6 enabled efficient homofermentative lactic acid production from xylan-derived sugars.
Abdel-Rahman, M. A. (2022). Fermentation, 8(3), 134.
A thermotolerant Enterococcus faecium s6 strain exhibited homoferementative lactic acid (LA) production at high xylose concentration (≥50 g/L). In batch fermentation at 45°C and controlled pH of 6.5, strain E. faecium s6 produced up to 72.9 g/L of LA with a yield of 0.99 g/g-consumed xylose and productivity of 1.74 g/L.h from 75 g/L xylose. An increased LA concentration and productivities with high yields were obtained in repeated batch fermentation that was conducted for 24 runs. In this mode, the strain could produce LA up 81.1 g/L within 5 h fermentation at a high productivity of 13.5 g/L.h and a yield of 1.02 g/g-consumed xylose. The strain was unable to consume birchwood xylan as sole carbon source. However, it could efficiently utilize xylooligosaccharides of xylobiose, xylotriose, xylotetraose, xylopentaose, and xylohexaose. The intracellular xylosidase activity was induced by xylose. Using xylooligosaccharide (50 g/L)/xylose (5 g/L) mixtures, the strain was able to produce maximum LA at 48.2 g/L within 120 h at a yield of 1.0 g/g-consumed sugar and productivity of 0.331 g/L.h. These results indicate that E. faecium s6 is capable of directly utilizing xylan-hydrolyzate and will enable the development of a feasible and economical approach to the production of LA from hemicellulosic hydrolysate.Hide Abstract
Green production of low-molecular-weight xylooligosaccharides from oil palm empty fruit bunch via integrated enzymatic polymerization and membrane separation for purification.
Kim, D., Yu, J. H., Hong, K. S., Jung, C. D., Kim, H., Kim, J. & Myung, S. (2022). Separation and Purification Technology, 293, 121084.
For xylooligosaccharides (XOS) production from agricultural residues, highly concentrated xylan liquor (116 g/L) was produced by crushing an oil palm empty fruit bunch (EFB) via continuous hydrothermal treatment and subsequent membrane-based concentration on a pilot scale. The yield of xylan extracted from the raw EFB was 53.7%. To produce XOS with a low degree of polymerization, xylanase-catalyzed hydrolysis was performed and the sum of xylobiose and xylotriose to extracted xylan was 81.6% under lab-scale optimal conditions and 69.3% under pilot-scale conditions. In addition, the crude XOS could be purified through a novel approach involving enzyme-mediated radical polymerization and membrane-based separation, while minimizing XOS loss and waste generation. As a result, 50.2% of the total phenolic compounds in the crude XOS material could be polymerized and precipitated through peroxidase-catalyzed reactions, and an additional 22.6% were removed using the membrane. Furthermore, the amount of activated carbon consumed and the XOS recovery rate after phenolic compound removal via the adsorption method using activated carbon were evaluated and compared with the integrated enzyme-membrane purification results. Consequently, this study provides an ecofriendly biomass refinery process for low-molecular-weight XOS production and purification that produces few toxic substances and little waste.Hide Abstract
Partial acid-hydrolysis of TEMPO-oxidized arabinoxylans generates arabinoxylan-structure resembling oligosaccharides.
Pandeirada, C. O., Speranza, S., Bakx, E., Westphal, Y., Janssen, H. G. & Schols, H. A. (2021). Carbohydrate Polymers, 275, 118795.
Arabinoxylans (AXs) display biological activities that depend on their chemical structures. To structurally characterize and distinguish AXs using a non-enzymatic approach, various TEMPO-oxidized AXs were partially acid-hydrolysed to obtain diagnostic oligosaccharides (OS). Arabinurono-xylo-oligomer alditols (AUXOS-A) with degree of polymerization 2-5, comprising one and two arabinuronic acid (AraA) substituents were identified in the UHPLC-PGC-MS profiles of three TEMPO-oxidized AXs, namely wheat (ox-WAX), partially-debranched WAX (ox-pD-WAX), and rye (ox-RAX). Characterization of these AUXOS-A highlighted that single-substitution of the Xyl unit preferably occurs at position O-3 for these samples, and that ox-WAX has both more single substituted and more double-substituted xylose residues in its backbone than the other AXs. Characteristic UHPLC-PGC-MS OS profiles, differing in OS abundance and composition, were obtained for each AX. Thus, partial acid-hydrolysis of TEMPO-oxidized AXs with analysis of the released OS by UHPLC-PGC-MS is a promising novel non-enzymatic approach to distinguish AXs and obtain insights into their structures.Hide Abstract
Lignocellulose degradation for the bioeconomy: the potential of enzyme synergies between xylanases, ferulic acid esterase and laccase for the production of arabinoxylo-oligosaccharides.
Schmitz, E., Leontakianakou, S., Norlander, S., Karlsson, E. N. & Adlercreutz, P. (2021). Bioresource Technology, 343, 126114.
The success of establishing bioeconomies replacing current economies based on fossil resources largely depends on our ability to degrade recalcitrant lignocellulosic biomass. This study explores the potential of employing various enzymes acting synergistically on previously pretreated agricultural side streams (corn bran, oat hull, soluble and insoluble oat bran). Degrees of synergy (oligosaccharide yield obtained with the enzyme combination divided by the sum of yields obtained with individual enzymes) of up to 88 were obtained. Combinations of a ferulic acid esterase and xylanases resulted in synergy on all substrates, while a laccase and xylanases only acted synergistically on the more recalcitrant substrates. Synergy between different xylanases (glycoside hydrolase (GH) families 5 and 11) was observed particularly on oat hulls, producing a yield of 57%. The synergistic ability of the enzymes was found to be partly due to the increased enzyme stability when in combination with the substrates.Hide Abstract
Production of thermostable xylanase using Streptomyces thermocarboxydus ME742 and application in enzymatic conversion of xylan from oil palm empty fruit bunch to xylooligosaccharides.
Khangkhachit, W., Suyotha, W., Leamdum, C., & Prasertsan, P. (2021). Biocatalysis and Agricultural Biotechnology, 102180.
Empty fruit bunch (EFB), the largest amount of biomass from a palm oil mill, is a promising renewable source for many value-added products. This study aimed to utilize EFB for production of xylanase by Streptomyces thermocarboxydus ME742 and applied the enzyme to the production of xylooligosaccharides (XOS) from EFB derived xylan (EFB-xylan). The strain ME742 produced the maximum xylanase (473.21 ± 6.55 U/mL) in a medium containing 50 g/L EFB supplemented with 3.0 g/L peptone at 40°C for 4 days. Xylanase ME742 was purified 23.68 fold with specificity of 1229 U/mg by acetone precipitation and DEAE-Sepharose chromatography. It was stable in a wide range of pH (4.0-9.5) and in elevated temperatures up to 55°C. Xylanase ME742 effectively hydrolyzed EFB-xylan. HPLC analysis revealed that the hydrolysate was composed of xylose and XOS with degree of polymerization between 2 and 5 and xylobiose was the predominant product. The XOS yield ranging in 95.01-151.19 mg/g EFB-xylan was generated from enzymatic processing for 12-72 h. Interestingly, arabinosyl-xylooligosaccharides were also predicted to be produced from EFB-xylan. XOS mixture exhibited high antioxidant activity (71.36 ± 0.57%) against DPPH at 100 μg/mL concentration and moderate activity (45.18 ± 1.56%) against ABTS radical. These findings reveal the potential use of Streptomyces thermocarboxydus ME742 and xylanase produced in a sustainable bioprocess to add value of EFB waste.Hide Abstract
Rapid profiling strategy for oligosaccharides and polysaccharides by MALDI TOF mass spectrometry.
Wang, J., Zhao, J., Nie, S., Xie, M. & Li, S. (2021). Food Hydrocolloids, 124, 107237.
The application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) in glycan was limited due to their poor ionization efficiency, compared with biomolecules such as proteins and peptides. Aiming to improve the ionization efficiency and simplify preparation procedure simultaneously during MALDI MS analysis, an on-target derivatization method using 3-aminoquinoline (3-AQ)/α-cyano-4-hydroxycinnamic acid (CHCA) as matrix was employed and it was conducted both in the positive and negative ion MALDI TOF MS. Results indicated that after on-target derivatization, the ions generated had substantially improved S/N ratios and sensitivity in the tandem mass spectra. The B/Y- type ions of 3-AQ-labeled glycans could be easily recognized, and cross-ring A- type ions provided additional information to reveal the linkage patterns. Specifically, positive ion mass spectra with protonated adduct as precursor ion produced a simple fragmentation pattern benefited for sequencing and observation of branches. Furthermore, this method was successfully applied in polysaccharides analysis, including arabinoxylan, xylan, arabinogalactan and dextran after enzymatic or acid degradation. This study demonstrated that it was feasible to analyze higher molecular weight polysaccharides by MALDI TOF MS using 3-AQ/CHCA matrix through appropriate hydrolysis, and it allowed much efficient structural interpretation with increased sensitivity and characteristic fragment ions.Hide Abstract
Ozone assisted autohydrolysis of wheat bran enhances xylooligosaccharide production with low generation of inhibitor compounds: A comparative study.
Sonkar, R. M., Gade, P. S., Bokade, V., Mudliar, S. N. & Bhatt, P. (2021). Bioresource Technology, 338, 125559.
In the present study, ozone assisted autohydrolysis (OAAH) was evaluated for enhanced generation of xylooligosaccharide (XOS) from wheat bran. The total XOS yield with optimum ozone dose of 3% (OAAH-3) was found to be 8.9% (w/w biomass) at 110°C in comparison to 7.96% at 170°C by autohydrolysis (AH) alone. Although, there was no significant difference in oligomeric composition (DP 2-6), significant decrease in degradation products namely furfural (2.78-fold), HMF (3.15-fold), acrylamide (nil) and acetic acid (1.06-fold), was observed with OAAH-3 as a pretreatment option. There was 1-fold higher xylan to XOS conversion and OAAH-hydrolysate had higher DPPH radical scavenging activity than AH. PCA plots indicated clear enhancement in XOS production and lower generation of inhibitors with decrease in treatment temperature. Results of the study therefore suggest OAAH can be an effective pretreatment option that can further be integrated with downstream processing for concentration and purification of XOS.Hide Abstract
Controlling autohydrolysis conditions to produce xylan-derived fibers that modulate gut microbiota responses and metabolic outputs.
Zhao, S., Dien, B. S., Lindemann, S. R. & Chen, M. H. (2021). Carbohydrate Polymers, 271, 118418.
Autohydrolysis is used for producing xylan-derived oligosaccharides from lignocellulosic biomass. Although numerous studies report optimized autohydrolysis conditions for various plants, few of these studies correlate process parameters with the resulting structural properties to their impact on intestinal bacterial communities. Thus, to further clarify these relationships, beechwood xylan (BWX)-derived substrates, processed under five conditions, were fermented in vitro by human gut microbiota. Autohydrolysis reduced the mean molecular size and substitutions of BWX. Distinct fermentation kinetics were observed with differing processing of BWX substrates, which correlated with impacts on community species evenness. The relative abundances of Bacteroides, Fusicatenibacter, Bifidobacterium, and Megasphaera within the fermentations varied with processing conditions. While the total short-chain fatty acid concentrations were the same among the treatments, processing conditions varied the extent of propionate and butyrate generation. Autolysis parameters may be an important tool for optimizing beneficial effects of xylan-derived fibers on human gut microbiota structure and function.Hide Abstract