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This product has been discontinued
|Stability:||> 10 years under recommended storage conditions|
|Viscosity:||~ 0.1 cSt|
|Monosaccharides (%):||Galactose: Arabinose: Other sugars = 81: 14: 5|
|Main Chain Glycosidic Linkage:||β-1,3|
|Substrate For (Enzyme):||endo-1,3-β-Galactanase|
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
Glycoside hydrolase family 2 exo-β-1, 6-galactosidase LpGal2 from Lactobacillus plantarum: Cloning, expression, and enzymatic characterization.
Zhang, X., Yu, G., Leng, J., Zhang, H., Zhou, Y., Yuan, Y. & Gao, J. (2021). Process Biochemistry, 102, 269-274.
Lactobacillus plantarum is a useful microorganism that metabolizes galactose-containing polysaccharides. Genome analysis has shown that L. plantarum contains four β-galactosidase-related genes. Here, we cloned the β-galactosidase gene that encodes the glycoside hydrolase family 2 (GH2) enzyme LpGal2. Recombinant LpGal2 (rLpGal2, 72 kDa) is a homodimer with maximal enzymatic activity at pH 7.0 and 50°C. Under these conditions, rLpGal2 hydrolyzes p-nitrophenyl-β-D-galactopyranoside (pNPβGal) with a specific activity of 2.16 × 10−3 U/mg and substrate specificity for β-1,6-galactobiose to produce D-Galactose. In addition, rLpGal2 can also hydrolyze β-1,6-galactan to D-Galactose, whereas other galactose-containing oligosaccharides and polysaccharides tested could not be hydrolyzed. This finding demonstrates that LpGal2 functions as an exo-β-1,6-galactosidase with narrow substrate specificity. To our knowledge, this is the first report of a β-galactosidase derived from L. plantarum with exo-β-1,6-galactosidase activity that has potential application for structure analysis of polysaccharides.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
A novel thermostable GH10 xylanase with activities on a wide variety of cellulosic substrates from a xylanolytic Bacillus strain exhibiting significant synergy with commercial Celluclast 1.5 L in pretreated corn stover hydrolysis.
Wang, K., Cao, R., Wang, M., Lin, Q., Zhan, R., Xu, H. & Wang, S. (2019). Biotechnology for Biofuels, 12(1), 48.
Background: Cellulose and hemicellulose are the two largest components in lignocellulosic biomass. Enzymes with activities towards cellulose and xylan have attracted great interest in the bioconversion of lignocellulosic biomass, since they have potential in improving the hydrolytic performance and reducing the enzyme costs. Exploring glycoside hydrolases (GHs) with good thermostability and activities on xylan and cellulose would be beneficial to the industrial production of biofuels and bio-based chemicals. Results: A novel GH10 enzyme (XynA) identified from a xylanolytic strain Bacillus sp. KW1 was cloned and expressed. Its optimal pH and temperature were determined to be pH 6.0 and 65°C. Stability analyses revealed that XynA was stable over a broad pH range (pH 6.0-11.0) after being incubated at 25°C for 24 h. Moreover, XynA retained over 95% activity after heat treatment at 60°C for 60 h, and its half-lives at 65°C and 70°C were about 12 h and 1.5 h, respectively. More importantly, in terms of substrate specificity, XynA exhibits hydrolytic activities towards xylans, microcrystalline cellulose (filter paper and Avicel), carboxymethyl cellulose (CMC), cellobiose, p-nitrophenyl-β-D-cellobioside (pNPC), and p-nitrophenyl-β-D-glucopyranoside (pNPG). Furthermore, the addition of XynA into commercial cellulase in the hydrolysis of pretreated corn stover resulted in remarkable increases (the relative increases may up to 90%) in the release of reducing sugars. Finally, it is worth mentioning that XynA only shows high amino acid sequence identity (88%) with rXynAHJ14, a GH10 xylanase with no activity on CMC. The similarities with other characterized GH10 enzymes, including xylanases and bifunctional xylanase/cellulase enzymes, are no more than 30%. Conclusions: XynA is a novel thermostable GH10 xylanase with a wide substrate spectrum. It displays good stability in a broad range of pH and high temperatures, and exhibits activities towards xylans and a wide variety of cellulosic substrates, which are not found in other GH10 enzymes. The enzyme also has high capacity in saccharification of pretreated corn stover. These characteristics make XynA a good candidate not only for assisting cellulase in lignocellulosic biomass hydrolysis, but also for the research on structure-function relationship of bifunctional xylanase/cellulase.Hide Abstract
Metabolism of polysaccharides in dynamic middle lamellae during cotton fibre development.
Guo, X., Runavot, J. L., Bourot, S., Meulewaeter, F., Hernandez-Gomez, M., Holland, C., Harholt, J., Willats, W. G. T., Mravec, J., Knox, P. & Ulvskov, P. (2019). Planta, 249(5), 1565-1581.
Evidence is presented that cotton fibre adhesion and middle lamella formation are preceded by cutin dilution and accompanied by rhamnogalacturonan-I metabolism. Cotton fibres are single cell structures that early in development adhere to one another via the cotton fibre middle lamella (CFML) to form a tissue-like structure. The CFML is disassembled around the time of initial secondary wall deposition, leading to fibre detachment. Observations of CFML in the light microscope have suggested that the development of the middle lamella is accompanied by substantial cell-wall metabolism, but it has remained an open question as to which processes mediate adherence and which lead to detachment. The mechanism of adherence and detachment were investigated here using glyco-microarrays probed with monoclonal antibodies, transcript profiling, and observations of fibre auto-digestion. The results suggest that adherence is brought about by cutin dilution, while the presence of relevant enzyme activities and the dynamics of rhamnogalacturonan-I side-chain accumulation and disappearance suggest that both attachment and detachment are accompanied by rhamnogalacturonan-I metabolism.Hide Abstract
Structural and functional characterization of a family GH53 β-1,4-galactanase from Bacteroides thetaiotaomicron that facilitates degradation of prebiotic galactooligosaccharides.
Böger, M., Hekelaar, J., van Leeuwen, S. S., Dijkhuizen, L. & van Bueren, A. L. (2019). Journal of Structural Biology, 205(1), 1-10.
Galactooligosaccharides (GOS) are prebiotic compounds synthesized from lactose using bacterial enzymes and are known to stimulate growth of beneficial bifidobacteria in the human colon. Bacteroides thetaiotaomicron is a prominent human colon commensal bacterial species that hydrolyzes GOS using an extracellular Glycosyl Hydrolase (GH) family GH53 endo-galactanase enzyme (BTGH53), releasing galactose-based products for growth. Here we dissect the molecular basis for GOS activity of this B. thetaiotaomicron GH53 endo-galactanase. Elucidation of its X-ray crystal structure revealed that BTGH53 has a relatively open active site cleft which was not observed with the bacterial enzyme from Bacillus licheniformis (BLGAL). BTGH53 acted on GOS with degree of polymerization ≤3 and therefore more closely resembles activity of fungal GH53 enzymes (e.g. Aspergillus aculeatus AAGAL and Meripileus giganteus MGGAL). Probiotic lactobacilli that lack galactan utilization systems constitute a group of bacteria with relevance for a healthy (infant) gut. The strains tested were unable to use GOS ≥ DP3. However, they completely consumed GOS in the presence of BTGH53, resulting in clear stimulation of their extent of growth. The extracellular BTGH53 enzyme thus may play an important role in carbohydrate metabolism in complex microbial environments such as the human colon. It also may find application for the development of synergistic synbiotics.Hide Abstract
Larsbrink, J., Tuveng, T. R., Pope, P. B., Bulone, V., Eijsink, V. G., Brumer, H. & McKee, L. S. (2017). Journal of Proteomics, 156, 63-74.
Together with fungi, saprophytic bacteria are central to the decomposition and recycling of biomass in forest environments. The Bacteroidetes phylum is abundant in diverse habitats, and several species have been shown to be able to deconstruct a wide variety of complex carbohydrates. The genus Chitinophaga is often enriched in hotspots of plant and microbial biomass degradation. We present a proteomic assessment of the ability of Chitinophaga pinensis to grow on and degrade mannan polysaccharides, using an agarose plate-based method of protein collection to minimise contamination with exopolysaccharides and proteins from lysed cells, and to reflect the realistic setting of growth on a solid surface. We show that select Polysaccharide Utilisation Loci (PULs) are expressed in different growth conditions, and identify enzymes that may be involved in mannan degradation. By comparing proteomic and enzymatic profiles, we show evidence for the induced expression of enzymes and PULs in cells grown on mannan polysaccharides compared with cells grown on glucose. In addition, we show that the secretion of putative biomass-degrading enzymes during growth on glucose comprises a system for nutrient scavenging, which employs constitutively produced enzymes. Significance of this study: Chitinophaga pinensis belongs to a bacterial genus which is prominent in microbial communities in agricultural and forest environments, where plant and fungal biomass is intensively degraded. Such degradation is hugely significant in the recycling of carbon in the natural environment, and the enzymes responsible are of biotechnological relevance in emerging technologies involving the deconstruction of plant cell wall material. The bacterium has a comparatively large genome, which includes many uncharacterised carbohydrate-active enzymes. We present the first proteomic assessment of the biomass-degrading machinery of this species, focusing on mannan, an abundant plant cell wall hemicellulose. Our findings include the identification of several novel enzymes, which are promising targets for future biochemical characterisation. In addition, the data indicate the expression of specific Polysaccharide Utilisation Loci, induced in the presence of different growth substrates. We also highlight how a constitutive secretion of enzymes which deconstruct microbial biomass likely forms part of a nutrient scavenging process.Hide Abstract
Thieme, N., Wu, V. W., Dietschmann, A., Salamov, A. A., Wang, M., Johnson, J., Singan, V. R., Grigoriev, I. V., Glass, N. L., Somerville, C. R., & Benz, J. P. (2017). Biotechnology for Biofuels, 10(1), 149.
Background: Pectin is an abundant component in many fruit and vegetable wastes and could therefore be an excellent resource for biorefinery, but is currently underutilized. Fungal pectinases already play a crucial role for industrial purposes, such as for foodstuff processing. However, the regulation of pectinase gene expression is still poorly understood. For an optimal utilization of plant biomass for biorefinery and biofuel production, a detailed analysis of the underlying regulatory mechanisms is warranted. In this study, we applied the genetic resources of the filamentous ascomycete species Neurospora crassa to screen for transcription factors that play a major role in pectinase induction. Results: The pectin degradation regulator-1 (PDR-1) was identified through a transcription factor mutant screen in N. crassa. The Δpdr-1 mutant exhibited a severe growth defect on pectin and all tested pectin-related poly- and monosaccharides. Biochemical as well as transcriptional analyses of WT and the Δpdr-1 mutant revealed that while PDR-1-mediated gene induction was dependent on the presence of L-rhamnose, it also strongly affected the degradation of the homogalacturonan backbone. The expression of the endo-polygalacturonase gh28-1 was greatly reduced in the Δpdr-1 mutant, while the expression levels of all pectate lyase genes increased. Moreover, a pdr-1 overexpression strain displayed substantially increased pectinase production. Promoter analysis of the PDR-1 regulon allowed refinement of the putative PDR-1 DNA-binding motif. Conclusions: PDR-1 is highly conserved in filamentous ascomycete fungi and is present in many pathogenic and industrially important fungi. Our data demonstrate that the function of PDR-1 in N. crassa combines features of two recently described transcription factors in Aspergillus niger (RhaR) and Botrytis cinerea (GaaR). The results presented in this study contribute to a broader understanding of how pectin degradation is orchestrated in filamentous fungi and how it could be manipulated for optimized pectinase production.Hide Abstract
An evolutionarily distinct family of polysaccharide lyases removes rhamnose capping of complex arabinogalactan proteins.
Munoz-Munoz, J., Cartmell, A., Terrapon, N., Baslé, A., Henrissat, B. & Gilbert, H. J. (2017). Journal of Biological Chemistry, jbc-M117.
The human gut microbiota utilizes complex carbohydrates as major nutrients. The requirement for efficient glycan degrading systems exerts a major selective selection pressure on this microbial community. Thus, we propose that this microbial ecosystem represents a substantial resource for discovering novel carbohydrate active enzymes. To test this hypothesis we screened the potential enzymatic functions of hypothetical proteins encoded by genes of Bacteroides thetaiotaomicron that were upregulated by arabinogalactan arabinogalactan proteins or AGPs. Although AGPs are ubiquitous in plants, there is a paucity of information on their detailed structure, the function of these glycans in planta and the mechanisms by which they are depolymerized in microbial ecosystems. Here we have discovered a new polysaccharide lyase family that is specific for the L-rhamnose-alpha1,4-D-glucuronic acid linkage that caps the side chains of complex AGPs. The reaction product generated by the lyase, delta4,5-unsaturated uronic acid, is removed from AGP by a glycoside hydrolase located in family GH105, producing the final product 4-deoxy-β-L-threo-hex-4-enepyranosyl-uronic acid. The crystal structure of a member of the novel lyase family revealed a catalytic domain that displays an (alpha/alpha6)6 barrel fold. In the centre of the barrel is a deep pocket, which, based on mutagenesis data and amino acid conservation, comprises the active site of the lyase. A tyrosine is the proposed catalytic base in the beta-elimination reaction. This study illustrates how highly complex glycans can be used as a scaffold to discover new enzyme families within microbial ecosystems where carbohydrate metabolism is a major evolutionary driver.Hide Abstract
Lukova, P. K., Karcheva-Bahchevanska, D. P.,Nikolova, M. M.,Iliev , I. N. & Mladenov, R. D. (2017). Bulgarian Chemical Communications, 49, 282-288.
In the current study for the first time were investigated the chemical composition and antioxidant activity of polysaccharides isolated from three indigenous for Bulgaria species of Plantago genus - Plantago major L., Plantago lanceolata L. and Plantago media L. Crude polysaccharides were extracted from fresh leaves with water and dilute acid and their yield was between 0.64% and 2.79%. The chemical composition of water-extractable polysaccharides (WEPs) and total acid-extractable polysaccharides (TAEPs) of Plantago leaves was evaluated by HPLC analysis. The phytochemical data revealed the presence of branched heteropolysaccharides with different neutral/acidic monosaccharide ratio. The predominant monosaccharide unit of WEPs was galacturonic acid (62.64% - 70.58%). Additionally, there were registered small amounts of arabinose and rhamnose. In TAEPs among with galacturonic acid (36.93% - 41.46%), significant amounts of neutral monosaccharides as galactose (22.80% - 46.11%) and rhamnose (16.96% - 35.74%) were determined. Two types of analyses were used to evaluate the antioxidant activity of Plantago isolated polysaccharides: DPPH and FRAP assay. Based on DPPH method, WEPs exhibited stronger radical scavenging ability (29.39% - 40.08%) compared to TAEPs (19.44% - 24.15%). In parallel, WEPs showed greater rate of ferric reducing power (103.71 - 137.83 µM TE/5 mg Ps) compared to TAEPs (34.63 - 117.66 µM TE/5 mg Ps). Although lower than synthetic BHT, Plantago polysaccharides revealed antioxidant potential and could be further explored as promising natural antioxidants for the nutraceutical and pharmaceutical industries.Hide Abstract
Ramasamy, U. R., Lips, S., Bakker, R., Gruppen, H. & Kabel, M. A. (2014). Carbohydrate Polymers, 113, 256-263.
During the industrial extraction of starch from potatoes (Seresta), some starch remains within undisrupted potato cells in the fibrous side-stream. The aim of this study was to investigate if enzymatic degradation of cell wall polysaccharides (CWPs) can enhance starch recovery and lower the water holding capacity (WHC) of the “fibre” fraction. The use of a pectinase-rich preparation recovered 58% of the starch present in the “fibre” fraction. Also, the “fibre” fraction retained only 40% of the water present in the non-enzyme treated “fibre”. This was caused by the degradation of pectins, in particular arabinogalactan side chains calculated as the sum of galactosyl and arabinosyl residues.Hide Abstract
Wegmann, U., Louis, P., Goesmann, A., Henrissat, B., Duncan, S. H. & Flint, H. J. (2014). Environmental Microbiology, 16(9), 2879–2890.
The recently isolated bacterial strain 80/3 represents one of the most abundant 16S rRNA phylotypes detected in the healthy human large intestine and belongs to the Ruminococcaceae family of Firmicutes. The completed genome sequence reported here is the first for a member of this important family of bacteria from the human colon. The genome comprises two large chromosomes of 2.24 and 0.73 Mbp, leading us to propose the name Ruminococcus bicirculans for this new species. Analysis of the carbohydrate active enzyme complement suggests an ability to utilize certain hemicelluloses, especially β-glucans and xyloglucan, for growth that was confirmed experimentally. The enzymatic machinery enabling the degradation of cellulose and xylan by related cellulolytic ruminococci is however lacking in this species. While the genome indicated the capacity to synthesize purines, pyrimidines and all 20 amino acids, only genes for the synthesis of nicotinate, NAD+, NADP+ and coenzyme A were detected among the essential vitamins and co-factors, resulting in multiple growth requirements. In vivo, these growth factors must be supplied from the diet, host or other gut microorganisms. Other features of ecological interest include two type IV pilins, multiple extracytoplasmic function-sigma factors, a urease and a bile salt hydrolase.Hide Abstract