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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.
(Aspergillus niger) E-CELBA - Cellulase (endo-1,4-β-D-glucanase)
(Bacillus amyloliquefaciens) E-CELTE - Cellulase (endo-1,4-β-D-glucanase)
(Talaromyces emersonii) E-CELTH - Cellulase (endo-1,4-β-D-glucanase)
(Thermobifida halotolerans) E-CELTR - Cellulase (endo-1,4-β-D-glucanase)
(Trichoderma longibrachiatum) E-CELTM - Cellulase (endo-1,4-β-D-glucanase)
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 220.127.116.11) 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 18.104.22.168) 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
A processive endoglucanase with multi-substrate specificity is characterized from porcine gut microbiota.
Wang, W., Archbold, T., Lam, J. S., Kimber, M. S. & Fan, M. Z. (2019). Scientific Reports, 9(1), 1-13.
Cellulases play important roles in the dietary fibre digestion in pigs, and have multiple industrial applications. The porcine intestinal microbiota display a unique feature in rapid cellulose digestion. Herein, we have expressed a cellulase gene, p4818Cel5_2A, which singly encoded a catalytic domain belonging to glycoside hydrolase family 5 subfamily 2, and was previously identified from a metagenomic expression library constructed from porcine gut microbiome after feeding grower pigs with a cellulose-supplemented diet. The activity of purified p4818Cel5_2A was maximal at pH 6.0 and 50°C and displayed resistance to trypsin digestion. This enzyme exhibited activities towards a wide variety of plant polysaccharides, including cellulosic substrates of avicel and solka-Floc®, and the hemicelluloses of β-(1 → 4)/(1 → 3)-glucans, xyloglucan, glucomannan and galactomannan. Viscosity, reducing sugar distribution and hydrolysis product analyses further revealed that this enzyme was a processive endo-β-(1 → 4)-glucanase capable of hydrolyzing cellulose into cellobiose and cellotriose as the primary end products. These catalytic features of p4818Cel5_2A were further explored in the context of a three-dimensional homology model. Altogether, results of this study report a microbial processive endoglucanase identified from the porcine gut microbiome, and it may be tailored as an efficient biocatalyst candidate for potential industrial applications.
Adaptation of syntenic xyloglucan utilization loci of human gut Bacteroidetes to polysaccharide side chain diversity.
Déjean, G., Tauzin, A. S., Bennett, S. W., Creagh, A. L. & Brumer, H. (2019). Applied and Environmental Microbiology, 85(20), e01491-19.
Genome sequencing has revealed substantial variation in the predicted abilities of individual species within animal gut microbiota to metabolize the complex carbohydrates comprising dietary fiber. At the same time, a currently limited body of functional studies precludes a richer understanding of how dietary glycan structures affect the gut microbiota composition and community dynamics. Here, using biochemical and biophysical techniques, we identified and characterized differences among recombinant proteins from syntenic xyloglucan utilization loci (XyGUL) of three Bacteroides and one Dysgonomona species from the human gut, which drive substrate specificity and access to distinct polysaccharide side chains. Enzymology of four syntenic glycoside hydrolase family 5 subfamily 4 (GH5_4) endo-xyloglucanases revealed surprising differences in xyloglucan (XyG) backbone cleavage specificity, including the ability of some homologs to hydrolyze congested branched positions. Further, differences in the complement of GH43 alpha-L-arabinofuranosidases and GH95 alpha-L-fucosidases among syntenic XyGUL confer distinct abilities to fully saccharify plant species-specific arabinogalactoxyloglucan and/or fucogalactoxyloglucan. Finally, characterization of highly sequence-divergent cell surface glycan-binding proteins (SGBPs) across syntenic XyGUL revealed a novel group of XyG oligosaccharide-specific SGBPs encoded within select Bacteroides.Hide Abstract
Characterization of acidic endoglucanase Cel12A from Gloeophyllum trabeum and its synergistic effects on hydrogen peroxide–acetic acid (HPAC)-pretreated lignocellulose.
Oh, C. H., Park, C. S., Lee, Y. G., Song, Y. & Bae, H. J. (2019). Journal of Wood Science, 65(1), 1-10.
Gloeophyllum trabeum is a potent filamentous fungus that rapidly decomposes lignocellulose. In the present study, we cloned the G. trabeum cel12a gene and expressed it in Pichia pastoris strain GS115. The purified recombinant GtCel12A exhibited high pH stability and very high specific enzymic activity against β-glucan (6546 U mg−1) and carboxymethyl cellulose (1129 U mg−1) compared to GtCel5B, endoglucanases from Trichoderma reesei, and other glycoside hydrolase family 12 (GH12) enzymes. GtCel12A exhibited high enzymic activity with regard to hydrogen peroxide–acetic acid (HPAC)-pretreated lignocellulose biomass, and produced cellobiose as a major product with a small quantity of glucose. In combination with commercial cellulase, this enzyme also showed synergistic effects of 14.5, 16.1, 29.0, and 13.4% on filter paper, HPAC-pretreated pine, corn stover, and rice straw, respectively. The acidic endoglucanase GtCel12A from G. trabeum is a promising tool that can be used in combination with cellulase against HPAC-pretreated lignocellulose.Hide Abstract
A family AA5_2 carbohydrate oxidase from Penicillium rubens displays functional overlap across the AA5 family.
Mollerup, F., Aumala, V., Parikka, K., Mathieu, Y., Brumer, H., Tenkanen, M. & Master, E. (2019). PloS One, 14(5), e0216546.
Copper radical alcohol oxidases belonging to auxiliary activity family 5, subfamily 2 (AA5_2) catalyze the oxidation of galactose and galactosides, as well as aliphatic alcohols. Despite their broad applied potential, so far very few AA5_2 members have been biochemically characterized. We report the recombinant production and biochemical characterization of an AA5_2 oxidase from Penicillium rubens Wisconsin 54-1255 (PruAA5_2A), which groups within an unmapped clade phylogenetically distant from those comprising AA5_2 members characterized to date. PruAA5_2 preferentially oxidized raffinose over galactose; however, its catalytic efficiency was 6.5 times higher on glycolaldehyde dimer compared to raffinose. Deep sequence analysis of characterized AA5_2 members highlighted amino acid pairs correlated to substrate range and conserved within the family. Moreover, PruAA5_2 activity spans substrate preferences previously reported for AA5 subfamily 1 and 2 members, identifying possible functional overlap across the AA5 family.Hide Abstract
Molecular insights of a xyloglucan endo-transglycosylase/hydrolase of radiata pine (PrXTH1) expressed in response to inclination: Kinetics and computational study.
Morales-Quintana, L., Carrasco-Orellana, C., Beltrán, D., Moya-León, M. A. & Herrera, R. (2019). Plant Physiology and Biochemistry, 136, 155-161.
Xyloglucan endotransglycosylase/hydrolases (XTH) may have endotransglycosylase (XET) and/or hydrolase (XEH) activities. Previous studies confirmed XET activity for PrXTH1 protein from radiata pine. XTHs could interact with many hemicellulose substrates, but the favorite substrate of PrXTH1 is still unknown. The prediction of union type and energy stability of the complexes formed between PrXTH1 and different substrates (XXXGXXXG, XXFGXXFG, XLFGXLFG and cellulose) were determined using bioinformatics tools. Molecular Docking, Molecular Dynamics, MM-GBSA and Electrostatic Potential Calculations were employed to predict the binding modes, free energies of interaction and the distribution of electrostatic charge. The results suggest that the enzyme formed more stable complexes with hemicellulose substrates than cellulose, and the best ligand was the xyloglucan XLFGXLFG (free energy of −58.83 ± 0.8 kcal mol−1). During molecular dynamics trajectories, hemicellulose fibers showed greater stability than cellulose. Aditionally, the kinetic properties of PrXTH1 enzyme were determined. The recombinant protein was active and showed an optimal pH 5.0 and optimal temperature of 37°C. A Km value of 20.9 mM was determined for xyloglucan oligomer. PrXTH1 is able to interact with different xyloglycans structures but no activity was observed for cellulose as substrate, remodeling cell wall structure in response to inclination.Hide Abstract
A rapid-throughput adaptable method for determining the monosaccharide composition of polysaccharides.
Amicucci, M. J., Galermo, A. G., Nandita, E., Vo, T. T. T., Liu, Y., Lee, M., Xu, G. & Lebrilla, C. B. & Lebrilla, C. B. (2019). International Journal of Mass Spectrometry, 438, 22-28.
Polysaccharides make up the largest non-water component of plant-based foods. Their ability to manipulate the gut microbiome and modulate the immune system has increased interest in the rapid elucidation of their structures. A necessary component for the structural characterization of polysaccharides is the determination of their monosaccharide composition. Current methods of monosaccharide analysis are not suitable for analyzing large sample-sets and are limited by their inability to analyze polysaccharides. We have developed a 96-well plate hydrolysis and derivatization procedure followed by a rapid and sensitive 10-min ultra-high performance liquid chromatography triple quadrupole mass spectrometry analysis capable of the absolute quantitation of 14 plant monosaccharides. Four polysaccharide standards, inulin, xyloglucan, arabinogalactan, and rhamnogalacturonan-I, which are commonly found in plants, were used to optimize and validate the method. The optimized conditions were applied to eight foods to show the method’s reproducibility and ability to analyze complicated and insoluble polysaccharide mixtures. This approach will allow researchers to obtain accurate and absolute quantitation of monosaccharides in the large sample-sets that are required for agricultural, food, clinical, and nutrition-based studies.Hide Abstract
Arabidopsis thaliana α1, 2‐L‐fucosyltransferase catalyzes the transfer of L‐galactose to xyloglucan oligosaccharides.
Ohashi, H., Ohashi, T., Misaki, R. & Fujiyama, K. (2019). FEBS Letters, 593(2), 187-194.
l‐Galactose (L‐Gal) is one of the components of plant cell wall polysaccharides. In the GDP‐L‐fucose‐deficient Arabidopsis thaliana mutant mur1, L‐fucose (l‐Fuc) residues in xyloglucan are substituted by L‐Gal residues. L‐Gal only differs from L‐Fuc by the presence of an oxygen at C‐6. Thus, we hypothesized that the A. thaliana xyloglucan α1,2‐L‐fucosyltransferase (AtFUT1) is also responsible for the L‐galactosyl transfer to D‐galactose residues in xyloglucan. In this study, we heterologously produced AtFUT1 in fission yeast and carried out an in vitro assay for the activities of AtFUT1 on GDP‐l‐Gal and xyloglucan oligosaccharide. We show that the recombinant AtFUT1 catalyzes L‐Gal transfer to xyloglucan oligosaccharides although the initial velocity of L‐Gal transfer is 3.1 times lower than that of L‐Fuc transfer.Hide Abstract