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|Stability:||> 10 years under recommended storage conditions|
|Monosaccharides (%):||Xylose: Glucose: Galactose: Arabinose: Other sugars = 34: 45: 17: 2: 2|
|Main Chain Glycosidic Linkage:||β-1,4, α-1,6 and β-1,6|
|Substrate For (Enzyme):||endo-Cellulase, Xyloglucanase|
High purity Xyloglucan (Tamarind) for use in research, biochemical enzyme assays and in vitro diagnostic analysis.
Mapping molecular recognition of β1, 3-1, 4-glucans by a surface glycan-binding protein from the human gut symbiont Bacteroides ovatus.
Correia, V. G., Trovão, F., Pinheiro, B. A., Brás, J. L., Silva, L. M., Nunes, C., Cimbra, M. A., Liu, Y., Feizi, T., Fontes, C. M. G. A., Mulloy, B., Chai, W., Carvalho, A. L. & Palma, A. S. (2021). Microbiology Spectrum, 9(3), e01826-21.
A multigene polysaccharide utilization locus (PUL) encoding enzymes and surface carbohydrate (glycan)-binding proteins (SGBPs) was recently identified in prominent members of Bacteroidetes in the human gut and characterized in Bacteroides ovatus. This PUL-encoded system specifically targets mixed-linkage β1,3-1,4-glucans, a group of diet-derived carbohydrates that promote a healthy microbiota and have potential as prebiotics. The BoSGBPMLG-A protein encoded by the BACOVA_2743 gene is a SusD-like protein that plays a key role in the PUL's specificity and functionality. Here, we perform a detailed analysis of the molecular determinants underlying carbohydrate binding by BoSGBPMLG-A, combining carbohydrate microarray technology with quantitative affinity studies and a high-resolution X-ray crystallography structure of the complex of BoSGBPMLG-A with a β1,3-1,4-nonasaccharide. We demonstrate its unique binding specificity toward β1,3-1,4-gluco-oligosaccharides, with increasing binding affinities up to the octasaccharide and dependency on the number and position of β1,3 linkages. The interaction is defined by a 41-Å-long extended binding site that accommodates the oligosaccharide in a mode distinct from that of previously described bacterial β1,3-1,4-glucan-binding proteins. In addition to the shape complementarity mediated by CH-π interactions, a complex hydrogen bonding network complemented by a high number of key ordered water molecules establishes additional specific interactions with the oligosaccharide. These support the twisted conformation of the β-glucan backbone imposed by the β1,3 linkages and explain the dependency on the oligosaccharide chain length. We propose that the specificity of the PUL conferred by BoSGBPMLG-A to import long β1,3-1,4-glucan oligosaccharides to the bacterial periplasm allows Bacteroidetes to outcompete bacteria that lack this PUL for utilization of β1,3-1,4-glucans.Hide Abstract
Characterisation of the enzyme transport path between shipworms and their bacterial symbionts.
Pesante, G., Sabbadin, F., Elias, L., Steele-King, C., Shipway, J. R., Dowle, A. A., Li, Y., Busse-Wicher, M., Dupree, P, Besser, K., Cragg, S. M., Bruce, N. C. & McQueen-Mason, S. J. (2021). BMC Biology, 19(1), 1-18.
Background: Shipworms are marine xylophagus bivalve molluscs, which can live on a diet solely of wood due to their ability to produce plant cell wall-degrading enzymes. Bacterial carbohydrate-active enzymes (CAZymes), synthesised by endosymbionts living in specialised shipworm cells called bacteriocytes and located in the animal’s gills, play an important role in wood digestion in shipworms. However, the main site of lignocellulose digestion within these wood-boring molluscs, which contains both endogenous lignocellulolytic enzymes and prokaryotic enzymes, is the caecum, and the mechanism by which bacterial enzymes reach the distant caecum lumen has remained so far mysterious. Here, we provide a characterisation of the path through which bacterial CAZymes produced in the gills of the shipworm Lyrodus pedicellatus reach the distant caecum to contribute to the digestion of wood. Results: Through a combination of transcriptomics, proteomics, X-ray microtomography, electron microscopy studies and in vitro biochemical characterisation, we show that wood-digesting enzymes produced by symbiotic bacteria are localised not only in the gills, but also in the lumen of the food groove, a stream of mucus secreted by gill cells that carries food particles trapped by filter feeding to the mouth. Bacterial CAZymes are also present in the crystalline style and in the caecum of their shipworm host, suggesting a unique pathway by which enzymes involved in a symbiotic interaction are transported to their site of action. Finally, we characterise in vitro four new bacterial glycosyl hydrolases and a lytic polysaccharide monooxygenase identified in our transcriptomic and proteomic analyses as some of the major bacterial enzymes involved in this unusual biological system. Conclusions: Based on our data, we propose that bacteria and their enzymes are transported from the gills along the food groove to the shipworm’s mouth and digestive tract, where they aid in wood digestion.Hide Abstract
In vitro fermentation of onion cell walls and model polysaccharides using human faecal inoculum: effects of molecular interactions and cell wall architecture.
Lu, S., Flanagan, B. M., Mikkelsen, D., Williams, B. A., & Gidley, M. J. (2021). Food Hydrocolloids, 124, 107257.
Plant primary cell walls provide a primary dietary source of fermentable carbohydrates. They are typically based on a cellulose network cross-linked by xyloglucan, with pectin incorporated, but the relative contributions of components and their architectural arrangement to gut fermentation performance is incompletely understood. Onion cell walls (OCW) were isolated and used as a primary cell wall model. OCW, models for their main constituents (xyloglucan, pectin, cellulose) and the physical mixture of these model polysaccharides (Mix) were fermented in vitro up to 48h, using a human faecal inoculum. Each constituent in Mix was fermented to a similar extent as single-component substrates, with comparable gas and short chain fatty acid (SCFAs) production. The microbiota responded differently and specifically to each polysaccharide, with the microbial community for Mix reflecting both pectin and xyloglucan. OCW was degraded more slowly at the early stage of fermentation, with less SCFAs produced compared with Mix, though a more extensive fermentation occurred by the end of fermentation, due to the slow but essentially complete fermentation of cellulose in OCW but not Mix. Microbiota shifts for OCW were different to those for Mix. The architecture of OCW as well as polysaccharide composition determined both fermentation outcomes and microbial community shifts.Hide Abstract
High level expression of a xyloglucanase from Rhizomucor miehei in Pichia pastoris for production of xyloglucan oligosaccharides and its application in yoghurt.
Wang, N. N., Li, Y. X., Miao, M., Zhu, C. H., Yan, Q. J. & Jiang, Z. Q. (2021). International Journal of Biological Macromolecules, 190, 845-852.
The xyloglucanase gene (RmXEG12A) from Rhizomucor miehei CAU432 was successfully expressed in Pichia pastoris. The highest xyloglucanase activity of 25,700 U mL−1 was secreted using high cell density fermentation. RmXEG12A was optimally active at pH 7.0 and 65°C, respectively. The xyloglucanase exhibited the highest specific activity towards xyloglucan (7915.5 U mg−1). RmXEG12A was subjected to hydrolyze tamarind powder to produce xyloglucan oligosaccharides with the degree of polymerization (DP) 7-9. The hydrolysis ratio of xyloglucan in tamarind powder was 89.8%. Moreover, xyloglucan oligosaccharides (2.0%, w/w) improved the water holding capacity (WHC) of yoghurt by 1.1-fold and promoted the growth of Lactobacillus bulgaricus and Streptococcus thermophiles by 2.3 and 1.6-fold, respectively. Therefore, a suitable xyloglucanase for tamarind powder hydrolysis was expressed in P. pastoris at high level and xyloglucan oligosaccharides improved the quality of yoghurt.Hide Abstract
Influences of polysaccharides in wood cell walls on lignification in vitro.
Lyu, Y., Matsumoto, T., Taira, S., Ijiri, K., Yoshinaga, A., Shigetomi, K. & Uraki, Y. (2021). Cellulose, 28(15), 9907-9917.
To elucidate the effects of polysaccharides, cellulose, water-soluble xylan (WXY), galactoglucomannan (GGM) and xyloglucan (XG) on lignification in vitro, artificial polysaccharide matrices were prepared from a combination of cellulose and hemicelluloses, and dehydrogenation polymer (DHP) was synthesized from coniferyl alcohol in the presence of the matrices by using horseradish peroxidase (HRP). Prior to DHP formation, interactions between cellulose and hemicelluloses were investigated with equilibrium adsorptions of the hemicelluloses on bacterial cellulose (BC) films and with quartz crystal microbalance with dissipation technique (QCM-D) to determine their adsorption on cellulose nanofibers (CNFs). Both analyses showed that the order of adsorption amounts was XG > GGM > WXY. The QCM-D experiments also suggested that HRP strongly interacted with cellulose rather than hemicelluloses. The amount of DHP generated in the XG-BC matrix was the largest among the prepared matrices, and XG facilitated the formation of 5–5′ interunitary linkages. Thus, XG must be involved in the lignification in primary wood cell wall. On the other hand, the amount of DHP in the GGM-BC matrix was the smallest, indicating that GGM hampered lignification.Hide Abstract
Structural and functional analysis of a multimodular hyperthermostable xylanase-glucuronoyl esterase from Caldicellulosiruptor kristjansonii.
Krska, D., Mazurkewich, S., Brown, H. A., Theibich, Y., Poulsen, J. C. N., Morris, A. L., Koropatkin, N. M., Leggio, L. L. & Larsbrink, J. (2021). Biochemistry, 60(27), 2206-2220.
The hyperthermophilic bacterium Caldicellulosiruptor kristjansonii encodes an unusual enzyme, CkXyn10C-GE15A, which incorporates two catalytic domains, a xylanase and a glucuronoyl esterase, and five carbohydrate-binding modules (CBMs) from families 9 and 22. The xylanase and glucuronoyl esterase catalytic domains were recently biochemically characterized, as was the ability of the individual CBMs to bind insoluble polysaccharides. Here, we further probed the abilities of the different CBMs from CkXyn10C-GE15A to bind to soluble poly- and oligosaccharides using affinity gel electrophoresis, isothermal titration calorimetry, and differential scanning fluorimetry. The results revealed additional binding properties of the proteins compared to the former studies on insoluble polysaccharides. Collectively, the results show that all five CBMs have their own distinct binding preferences and appear to complement each other and the catalytic domains in targeting complex cell wall polysaccharides. Additionally, through renewed efforts, we have achieved partial structural characterization of this complex multidomain protein. We have determined the structures of the third CBM9 domain (CBM9.3) and the glucuronoyl esterase (GE15A) by X-ray crystallography. CBM9.3 is the second CBM9 structure determined to date and was shown to bind oligosaccharide ligands at the same site but in a different binding mode compared to that of the previously determined CBM9 structure from Thermotoga maritima. GE15A represents a unique intermediate between reported fungal and bacterial glucuronoyl esterase structures as it lacks two inserted loop regions typical of bacterial enzymes and a third loop has an atypical structure. We also report small-angle X-ray scattering measurements of the N-terminal CBM22.1–CBM22.2–Xyn10C construct, indicating a compact arrangement at room temperature.Hide Abstract
Poaceae-specific cell wall-derived oligosaccharides activate plant immunity via OsCERK1 during Magnaporthe oryzae infection in rice.
Yang, C., Liu, R., Pang, J., Ren, B., Zhou, H., Wang, G., wang, E. & Liu, J. (2021). Nature Communications, 12(1), 1-13.
Many phytopathogens secrete cell wall degradation enzymes (CWDEs) to damage host cells and facilitate colonization. As the major components of the plant cell wall, cellulose and hemicellulose are the targets of CWDEs. Damaged plant cells often release damage-associated molecular patterns (DAMPs) to trigger plant immune responses. Here, we establish that the fungal pathogen Magnaporthe oryzae secretes the endoglucanases MoCel12A and MoCel12B during infection of rice (Oryza sativa). These endoglucanases target hemicellulose of the rice cell wall and release two specific oligosaccharides, namely the trisaccharide 31-β-D-Cellobiosyl-glucose and the tetrasaccharide 31-β-D-Cellotriosyl-glucose. 31-β-D-Cellobiosyl-glucose and 31-β-D-Cellotriosyl-glucose bind the immune receptor OsCERK1 but not the chitin binding protein OsCEBiP. However, they induce the dimerization of OsCERK1 and OsCEBiP. In addition, these Poaceae cell wall-specific oligosaccharides trigger a burst of reactive oxygen species (ROS) that is largely compromised in oscerk1 and oscebip mutants. We conclude that 31-β-D-Cellobiosyl-glucose and 31-β-D-Cellotriosyl-glucose are specific DAMPs released from the hemicellulose of rice cell wall, which are perceived by an OsCERK1 and OsCEBiP immune complex during M. oryzae infection in rice.Hide Abstract
Prospection of Fungal Lignocellulolytic Enzymes Produced from Jatoba (Hymenaea courbaril) and Tamarind (Tamarindus indica) Seeds: Scaling for Bioreactor and Saccharification Profile of Sugarcane Bagasse.
Contato, A. G., de Oliveira, T. B., Aranha, G. M., de Freitas, E. N., Vici, A. C., Nogueira, K. M. V., de Lucas, R. C., de Almeida Scarcella, A. S., Buckeridge, M. S., Silva, R. N. & Polizeli, M. D. L. T. D. M. (2021). Microorganisms, 9(3), 533.
The lignocellulosic biomass comprises three main components: cellulose, hemicellulose, and lignin. Degradation and conversion of these three components are attractive to biotechnology. This study aimed to prospect fungal lignocellulolytic enzymes with potential industrial applications, produced through a temporal analysis using Hymenaea courbaril and Tamarindus indica seeds as carbon sources. α-L-arabinofuranosidase, acetyl xylan esterase, endo-1,5-α-L-arabinanase, β-D-galactosidase, β-D-glucosidase, β-glucanase, β-D-xylosidase, cellobiohydrolase, endoglucanase, lichenase, mannanase, polygalacturonase, endo-1,4-β-xylanase, and xyloglucanase activities were determined. The enzymes were produced for eight filamentous fungi: Aspergillus fumigatus, Trametes hirsuta, Lasiodiplodia sp., two strains of Trichoderma longibrachiatum, Neocosmospora perseae, Fusarium sp. and Thermothelomyces thermophilus. The best producers concerning enzymatic activity were T. thermophilus and T. longibrachiatum. The optimal conditions for enzyme production were the media supplemented with tamarind seeds, under agitation, for 72 h. This analysis was essential to demonstrate that cultivation conditions, static and under agitation, exert strong influences on the production of several enzymes produced by different fungi. The kind of sugarcane, pretreatment used, microorganisms, and carbon sources proved limiting sugar profile factors.Hide Abstract
Interaction of cellulose and xyloglucan influences in vitro fermentation outcomes.
Lu, S., Mikkelsen, D., Flanagan, B. M., Williams, B. A. & Gidley, M. J. (2021). Carbohydrate Polymers, 258, 117698.
To investigate the effects of interactions between cellulose and xyloglucan (XG) on in vitro fermentation, a composite of bacterial cellulose (BC) incorporating XG during pellicle formation (BCXG), was fermented using a human faecal inoculum, and compared with BC, XG and a mixture (BC&XG) physically blended to have the same BC to XG ratio of BCXG. Compared to individual polysaccharides, the fermentation extent of BC and fermentation rate of XG were promoted in BC&XG. XG embedded in the BCXG composite was degraded less than in BC&XG, while more cellulose in BCXG was fermented than in BC&XG. This combination explains the similar amount of short chain fatty acid production noted throughout the fermentation process for BCXG and BC&XG. Microbial community dynamics for each substrate were consistent with the corresponding polysaccharide degradation. Thus, interactions between cellulose and XG are shown to influence their fermentability in multiple ways.Hide Abstract
Four cellulose-active lytic polysaccharide monooxygenases from Cellulomonas species.
Li, J., Solhi, L., Goddard-Borger, E. D., Mathieu, Y., Wakarchuk, W. W., Withers, S. G. & Brumer, H. (2021). Biotechnology for Biofuels, 14(1), 1-19.
Background: The discovery of lytic polysaccharide monooxygenases (LPMOs) has fundamentally changed our understanding of microbial lignocellulose degradation. Cellulomonas bacteria have a rich history of study due to their ability to degrade recalcitrant cellulose, yet little is known about the predicted LPMOs that they encode from Auxiliary Activity Family 10 (AA10). Results: Here, we present the comprehensive biochemical characterization of three AA10 LPMOs from Cellulomonas flavigena (CflaLPMO10A, CflaLPMO10B, and CflaLPMO10C) and one LPMO from Cellulomonas fimi (CfiLPMO10). We demonstrate that these four enzymes oxidize insoluble cellulose with C1 regioselectivity and show a preference for substrates with high surface area. In addition, CflaLPMO10B, CflaLPMO10C, and CfiLPMO10 exhibit limited capacity to perform mixed C1/C4 regioselective oxidative cleavage. Thermostability analysis indicates that these LPMOs can refold spontaneously following denaturation dependent on the presence of copper coordination. Scanning and transmission electron microscopy revealed substrate-specific surface and structural morphological changes following LPMO action on Avicel and phosphoric acid-swollen cellulose (PASC). Further, we demonstrate that the LPMOs encoded by Cellulomonas flavigena exhibit synergy in cellulose degradation, which is due in part to decreased autoinactivation. Conclusions: Together, these results advance understanding of the cellulose utilization machinery of historically important Cellulomonas species beyond hydrolytic enzymes to include lytic cleavage. This work also contributes to the broader mapping of enzyme activity in Auxiliary Activity Family 10 and provides new biocatalysts for potential applications in biomass modification.Hide Abstract
Formation of Cellulose-Based Composites with Hemicelluloses and Pectins Using Komagataeibacter Fermentation.
Mikkelsen, D., Lopez-Sanchez, P., Wang, D. & Gidley, M. J. (2020). The Plant Cell Wall, 73-87.
Komagataeibacter xylinussynthesizes cellulose in an analogous fashion to plants. Through fermentation of K. xylinus in media containing cell wall polysaccharides from the hemicellulose and/or pectin families, composites with cellulose can be produced. These serve as general models for the assembly, structure, and properties of plant cell walls. By studying structure/property relationships of cellulose composites, the effects of defined hemicellulose and/or pectin polysaccharide structures can be investigated. The macroscopic nature of the composites also allows composite mechanical properties to be characterized. The method for producing cellulose-based composites involves reviving and then culturing K. xylinu in the presence of desired hemicelluloses and/or pectins. Different conditions are required for construction of hemicellulose- and pectin-containing composites. Fermentation results in a floating mat or pellicle of cellulose-based composite that can be recovered, washed, and then studied under hydrated conditions without any need for intermediate drying.Hide Abstract
Configuration of active site segments in lytic polysaccharide monooxygenases steers oxidative xyloglucan degradation.
Sun, P., Laurent, C. V., Scheiblbrandner, S., Frommhagen, M., Kouzounis, D., Sanders, M. G., van Berkel, W. J. H., Ludwig, R. & Kabel, M. A. (2020). Biotechnology for Biofuels, 13, 1-19.
This study investigated pilot-scale production of xylo-oligosaccharides (XOS) and fermentable sugars from Miscanthus using steam explosion (SE) pretreatment. SE conditions (200°C; 15 bar; 10 min) led to XOS yields up to 52 % (w/w of initial xylan) in the hydrolysate. Liquid chromatography-mass spectrometry demonstrated that the solubilised XOS contained bound acetyl- and hydroxycinnamate residues, physicochemical properties known for high prebiotic effects and anti-oxidant activity in nutraceutical foods. Enzymatic hydrolysis of XOS-rich hydrolysate with commercial endo-xylanases resulted in xylobiose yields of 380 to 500 g/kg of initial xylan in the biomass after only 4 h, equivalent to ~74 to 90 % conversion of XOS into xylobiose. Fermentable glucose yields from enzymatic hydrolysis of solid residues were 8 to 9-fold higher than for untreated material. In view of an integrated biorefinery, we demonstrate the potential for efficient utilisation of Miscanthus for the production of renewable sources, including biochemicals and biofuels.Hide Abstract
In vitro gastrointestinal digestion of crisphead lettuce: Changes in bioactive compounds and antioxidant potential.
Ketnawa, S., Suwannachot, J. & Ogawa, Y. (2020). Food Chemistry, 311, 125885.
In this study, the potential health benefits of crisphead lettuce (Lactuca sativa L.) before and after digestion were represented by the recovery, bioaccessibility, and change of bioactive compounds including total phenolic (TPC) and total flavonoids content (TFC), and bioactivities [in vitro antioxidant activities including 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2, 2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activities, ferric reducing antioxidant power (FRAP) and metal ion chelating activity (MIC)]. The release of bioactive compounds as well as bioactivities increased during gastric and intestinal digestion for 1 h and subsequently decreased when digestion was completed. The bioaccessibility of TPC and TFC at after digestion was 56–73 and 75–79%, respectively. Among all bioactivities, crisphead lettuce showed a residual activity of ABTS (61–95%) followed by FRAP (70–86%), DPPH (24–52%) and MIC (32–73%) during the digestion. Our study suggested that crisphead lettuce maintains stability in both bioactive compounds and bioactivities during the digestion.Hide Abstract
High-level production and characterization of a novel β-1, 3-1, 4-glucanase from Aspergillus awamori and its potential application in the brewing industry.
Liu, X., Jiang, Z., Ma, S., Yan, Q., Chen, Z. & Liu, H. (2020). Process Biochemistry, 92, 252-260.
A novel β-1,3-1,4-glucanase gene (AaBglu12A) from Aspergillus awamori was extracellularly expressed in Pichia pastoris. AaBglu12A showed amino acid identity of 96 % with a glycoside hydrolase family 12 cellulase from A. kawachii and 48 % with a β-1,3-1,4-glucanase from Magnaporthe oryzae. The highest β-1,3-1,4-glucanase activity of 159,500 ± 500 U/mL with protein concentration of 31.7 ± 0.3 g/L was achieved in a 5-L fermentor. AaBglu12A was purified until homogeneous with recovery yield of 92 %. Its maximal activity was found at 55°C and pH 5.0. The enzyme was stable up to 60°C and within the pH range of 2.0-9.0. It also demonstrated strict substrate specificity towards oat- and barley-glucans as well as lichenan. The Km values for oat-, barley-glucans, and lichenan were 2.82, 3.51, and 2.53 mg/mL, respectively. The Vmax values for oat-, barley-glucans, and lichenan were 12,068, 10,790, and 7236 μmol/min·mg, respectively. AaBglu12A hydrolyzed oat- and barley-β-glucans to produce tetra- and tri-saccharides. However, lichenan was hydrolyzed to yield trisaccharides as the main end product. The addition of AaBglu12A to the mashing process substantially decreased filtration time by 34.5 % and viscosity by 9.6 %. Therefore, the high-level production of AaBglu12A might be a promising strategy for the brewing industry owing to its favorable properties.Hide Abstract
Identification and characterization of α-xylosidase involved in xyloglucan degradation in Aspergillus oryzae.
Matsuzawa, T., Kameyama, A. & Yaoi, K. (2020). Applied Microbiology and Biotechnology, 104(1), 201-210.
Aspergillus oryzae produces hydrolases involved in xyloglucan degradation and induces the expression of genes encoding xyloglucan oligosaccharide hydrolases in the presence of xyloglucan oligosaccharides. A gene encoding α-xylosidase (termed AxyA), which is induced in the presence of xyloglucan oligosaccharides, is identified and expressed in Pichia pastoris. AxyA is a member of the glycoside hydrolase family 31 (GH31). AxyA hydrolyzes isoprimeverose (α-D-xylopyranosyl-(1→6)-D-glucopyranose) into D-xylose and D-glucose and shows hydrolytic activity with other xyloglucan oligosaccharides such as XXXG (heptasaccharide, Glc4Xyl3) and XLLG (nonasaccharide, Glc4Xyl3Gal2). Isoprimeverose is a preferred AxyA substrate over other xyloglucan oligosaccharides. In the hydrolysis of XXXG, AxyA releases one molecule of D-xylose from one molecule of XXXG to yield GXXG (hexasaccharide, Glc4Xyl2). AxyA does not contain a signal peptide for secretion and remains within the cell. The intracellular localization of AxyA may help determine the order of hydrolases acting on xyloglucan oligosaccharides.Hide Abstract
Characterization of an alkali-stable xyloglucanase/mixed-linkage β-glucanase Pgl5A from Paenibacillus sp. S09.
Cheng, R., Cheng, L., Wang, L., Fu, R., Sun, X., Li, J., Wang, S. & Zhang, J. (2019). International Journal of Biological Macromolecules, 140, 1158-1166.
Xyloglucans and mixed-linkage β-glucans are the major components of hemicelluloses in lignocellulosic biomass. In this study, a novel β-1,4-glucanase Pgl5A belonging to the glycoside hydrolase family 5 subfamily 4 (GH5_4), was identified from Paenibacillus sp. S09. Pgl5A is a 70.9-kDa protein containing an N-terminal GH5_4 module, a carbohydrate-binding module (CBM)_X2 and a CBM3. Full-length Pgl5A and its CBM deletion mutants Pgl5A∆C and Pgl5A-CD were expressed in E. coli. All three enzymes showed maximal activity at 55 °C and pH 4.5–5.0, and possessed similar activity toward xyloglucan, barley β-glucan, and lichenan. Deletion of the CBM modules can improve thermostability and acid-tolerant properties of Pgl5A. Circular dichroism (CD) and intrinsic fluorescence spectroscopy analysis verified that C-terminus truncation improves the enzyme acid-tolerant properties. Homology modeling and CD spectra indicated that Pgl5A has an architectural (β/α)8 fold of GH5_4 enzymes. The catalytic efficiency (kcat/Km) of Pgl5A toward xyloglucan, but not mixed-linkage β-glucan, was reduced due to C-terminus truncation. TLC and LC-MS analysis showed that Pgl5A cleaves xyloglucan and mixed-linkage β-glucan into a series of xyloglucan oligosaccharides and gluco-oligosaccharides, respectively. The favorable enzymatic characteristics and high catalytic activities toward both xyloglucan and mixed-linkage β-glucan make Pgl5A a promising candidate for biotechnological industrial applications.Hide Abstract
Novel endo-(1, 4)-β-glucanase Bgh12A and xyloglucanase Xgh12B from Aspergillus cervinus belong to GH12 subgroup I and II, respectively.
Rykov, S. V., Kornberger, P., Herlet, J., Tsurin, N. V., Zorov, I. N., Zverlov, V. V., Liebl, W., Schwarz, W. H., Yarotsky, S. V. & Berezina, O. V. (2019). Applied Microbiology and Biotechnology, 103(18), 7553-7566.
In spite of intensive exploitation of aspergilli for the industrial production of carbohydrases, little is known about hydrolytic enzymes of fungi from the section Cervini. Novel glycoside hydrolases Bgh12A and Xgh12B from Aspergillus cervinus represent examples of divergent activities within one enzyme family and belong to the GH12 phylogenetic subgroup I (endo-(1,4)-β-glucanases) and II (endo-xyloglucanases), respectively. The bgh12A and xgh12B genes were identified in the unsequenced genome of A. cervinus using primers designed for conservative regions of the corresponding subgroups and a genome walking approach. The recombinant enzymes were heterologously produced in Pichia pastoris, purified, and characterized. Bgh12A was an endo-(1,4)-β-glucanase (EC 184.108.40.206) hydrolyzing the unbranched soluble β-(1,4)-glucans and mixed linkage β-(1,3;1,4)-D-glucans. Bgh12A exhibited maximum activity on barley β-glucan (BBG), which amounted to 614 ± 30 U/mg of protein. The final products of BBG and lichenan hydrolysis were glucose, cellobiose, cellotriose, 4-O-β-laminaribiosyl-glucose, and a range of higher mixed-linkage gluco-oligosaccharides. In contrast, the activity of endo-xyloglucanase Xgh12B (EC 220.127.116.11) was restricted to xyloglucan, with 542 ± 39 U/mg protein. The enzyme cleaved the (1,4)-β-glycosidic bonds of the xyloglucan backbone at the unsubstituted glucose residues finally generating cellotetraose-based hepta-, octa, and nona-oligosaccharides. Bgh12A and Xgh12B had maximal activity at 55°C, pH 5.0. At these conditions, the half-time of Xgh12B inactivation was 158 min, whereas the half-life of Bgh12A was 5 min. Recombinant P. pastoris strains produced up to 106 U/L of the target enzymes with at least 75% of recombinant protein in the total extracellular proteins. The Bgh12A and Xgh12B sequences show 43% identity. Strict differences in substrate specificity of Bgh12A and Xgh12B were in congruence with the presence of subgroup-specific structural loops and substrate-binding aromatic residues in the catalytic cleft of the enzymes. Individual composition of aromatic residues in the catalytic cleft defined variability in substrate selectivity within GH12 subgroups I and II.Hide Abstract
Influence of solubility on the adsorption of different xyloglucan fractions at cellulose-water interfaces.
Kishani, S., Vilaplana, F., Ruda, M., Hansson, P. & Wågberg, L. (2019). Biomacromolecules, 21(2), 772-782.
Xylogucan (XG) fractions with different molar masses were prepared while preserving the natural structure of the XG. The solubility of the fractions was investigated using light scattering, chromatography, and microscopy techniques. The conformational changes of the XG molecules and their association and phase separation were investigated together with concentration and molar mass changes. The knowledge gained was then applied to investigate the interaction of different XG fractions at cellulose model surfaces using a quartz crystal microbalance with dissipation. The results indicate that there is a cluster formation and phase separation of the XG molecules at the cellulose/water interface induced by the increase in XG concentration close to the surface. Concomitantly, the adsorption regimes are altered for the XG fractions depending on the solubility properties, indicating that the insolubility, association, and phase separation of XGs in aqueous media affect their interaction with cellulose. The study is of vital importance for improving the functionality of sustainable materials made from xyloglucan/cellulose natural composites.Hide Abstract
Development of an extensive linkage library for characterization of carbohydrates.
Galermo, A. G., Nandita, E., Castillo, J. J., Amicucci, M. J. & Lebrilla, C. B. (2019). Analytical Chemistry, 91(20), 13022-13031.
The extensive characterization of glycosidic linkages in carbohydrates remains a challenge because of the lack of known standards and limitations in current analytical techniques. This study encompasses the construction of an extensive glycosidic linkage library built from synthesized standards. It includes an improved liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitation of glycosidic linkages derived from disaccharides, oligosaccharides, and polysaccharides present in complicated matrices. We present a method capable of the simultaneous identification of over 90 unique glycosidic linkages using ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC/QqQ MS) operated in dynamic multiple reaction monitoring (dMRM) mode. To build the library, known monosaccharides commonly found in plants were subjected to partial methylation to yield partially derivatized species representing trisecting, bisecting, linear, and terminal structures. The library includes glycosidic linkage information for three hexoses (glucose, galactose, and mannose), three pentoses (xylose, arabinose, and ribose), two deoxyhexoses (fucose and rhamnose), and two hexuronic acids (glucuronic acid and galacturonic acid). The resulting partially methylated monosaccharides were then labeled with 1-phenyl-3-methyl-5-pyrazolone (PMP) followed by separation and analysis by UHPLC/dMRM MS. Validation of the synthesized standards was performed using disaccharide, oligosaccharide, and polysaccharide standards. Accuracy, reproducibility, and robustness of the method was demonstrated by analysis of xyloglucan (tamarind) and whole carrot root. The synthesized standards represent the most comprehensive group of carbohydrate linkages to date.Hide Abstract
High molecular weight mixed-linkage glucan as a mechanical and hydration modulator of bacterial cellulose: Characterization by advanced NMR spectroscopy.
Muñoz-García, J. C., Corbin, K. R., Hussain, H., Gabrielli, V., Koev, T., Iuga, D., Round, A. N., Mikkelsen, D., Gunning, P., Warren, F. J. & Khimyak, Y. Z. (2019). Biomacromolecules, 20(11), 4180-4190.
Bacterial cellulose (BC) consists of a complex three-dimensional organization of ultrafine fibers which provide unique material properties such as softness, biocompatibility, and water-retention ability, of key importance for biomedical applications. However, there is a poor understanding of the molecular features modulating the macroscopic properties of BC gels. We have examined chemically pure BC hydrogels and composites with arabinoxylan (BC–AX), xyloglucan (BC–XG), and high molecular weight mixed-linkage glucan (BC–MLG). Atomic force microscopy showed that MLG greatly reduced the mechanical stiffness of BC gels, while XG and AX did not exert a significant effect. A combination of advanced solid-state NMR methods allowed us to characterize the structure of BC ribbons at ultra-high resolution and to monitor local mobility and water interactions. This has enabled us to unravel the effect of AX, XG, and MLG on the short-range order, mobility, and hydration of BC fibers. Results show that BC–XG hydrogels present BC fibrils of increased surface area, which allows BC–XG gels to hold higher amounts of bound water. We report for the first time that the presence of high molecular weight MLG reduces the density of clusters of BC fibrils and dramatically increases water interactions with BC. Our data supports two key molecular features determining the reduced stiffness of BC–MLG hydrogels, that is, (i) the adsorption of MLG on the surface of BC fibrils precluding the formation of a dense network and (ii) the preorganization of bound water by MLG. Hence, we have produced and fully characterized BC–MLG hydrogels with novel properties which could be potentially employed as renewable materials for applications requiring high water retention capacity (e.g. personal hygiene products).Hide Abstract