Versatile high resolution oligosaccharide microarrays for plant glycobiology and cell wall research.
Pedersen, H. L., Fangel, J. U., McCleary, B., Ruzanski, C., Rydahl, M. G., Ralet, M. C., Farkas, V., Von Schantz, L., Marcus, S. E., Andersen, M.C. F., Field, R., Ohlin, M., Knox, J. P., Clausen, M. H. & Willats, W. G. T. (2012). Journal of Biological Chemistry, 287(47), 39429-39438.
Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.
A thermostable Gloeophyllum trabeum xylanase with potential for the brewing industry.
Wang, X., Luo, H., Yu, W., Ma, R., You, S., Liu, W., Hou, L., Zheng, F., Xie, X. & Yao, B. (2016). Food Chemistry, 199, 516-523.
A xylanase gene of glycoside hydrolase family 10, GtXyn10, was cloned from Gloeophyllum trabeum CBS 900.73 and expressed in Pichia pastoris GS115. Purified recombinant GtXyn10 exhibited significant activities to xylan (100.0%), lichenan (11.2%), glucan (15.2%) and p-nitrophenol-β-cellobiose (18.6%), demonstrated the maximum xylanase and glucanase activities at pH 4.5–5.0 and 75°C, retained stability over the pH range of 2.0–7.5 and at 70°C, and was resistant to pepsin and trypsin, most metal ions and SDS. Multiple sequence alignment and modeled-structure analysis identified a unique Gly48 in GtXyn10, and site-directed mutagenesis of Gly48 to Lys improved the temperature optimum up to 80°C. Under simulated mashing conditions, GtXyn10 (80 U) reduced the mash viscosity by 12.8% and improved the filtration rate by 31.3%. All these properties above make GtXyn10 attractive for potential applications in the feed and brewing industries.
Modulation of cellulosome composition in Clostridium cellulolyticum: Adaptation to the polysaccharide environment revealed by proteomic and carbohydrate‐active enzyme analyses.
Blouzard, J. C., Coutinho, P. M., Fierobe, H. P., Henrissat, B., Lignon, S., Tardif, C., Pages, S. & de Philip, P. (2010). Proteomics, 10(3), 541-554.
Clostridium cellulolyticum is a model mesophilic anaerobic bacterium that efficiently degrades plant cell walls. The recent genome release offers the opportunity to analyse its complete degradation system. A total of 148 putative carbohydrate-active enzymes were identified, and their modular structures and activities were predicted. Among them, 62 dockerin-containing proteins bear catalytic modules from numerous carbohydrate-active enzymes' families and whose diversity reflects the chemical and structural complexity of the plant carbohydrate. The composition of the cellulosomes produced by C. cellulolyticum upon growth on different substrates (cellulose, xylan, and wheat straw) was investigated by LC MS/MS. The majority of the proteins encoded by the cip-cel operon, essential for cellulose degradation, were detected in all cellulosome preparations. In the presence of wheat straw, the natural and most complex of the substrates studied, additional proteins predicted to be involved in hemicellulose degradation were produced. A 32-kb gene cluster encodes the majority of these proteins, all harbouring carbohydrate-binding module 6 or carbohydrate-binding module 22 xylan-binding modules along dockerins. This newly identified xyl-doc gene cluster, specialised in hemicellulose degradation, comes in addition of the cip-cel operon for plant cell wall degradation. Hydrolysis efficiencies determined on the different substrates corroborates the finding that cellulosome composition is adapted to the growth substrate.
Thermostable carbohydrate‐binding modules in affinity chromatography.
Johansson, R., Gunnarsson, L. C., Ohlin, M. & Ohlson, S. (2006). Journal of Molecular Recognition, 19(4), 275-281.
Affinity chromatography is routinely used mostly on a preparative scale to isolate different biomolecules such as proteins and carbohydrates. To this end a variety of proteins is in common use as ligands. To extend the arsenal of binders intended for separation of carbohydrates, we have explored the use of carbohydrate-binding modules (CBM) in affinity chromatography. The thermostable protein CBM4-2 and two variants (X-6 and A-6) thereof, selected from a newly constructed combinatorial library, were chosen for this study. The CBM4-2 predominantly binds to xylans but also crossreacts with glucose-based oligomers. The two CBM-variants X-6 and A-6 had been selected for binding to xylan and Avicel® (a mixture of amorphous and microcrystalline cellulose), respectively. To assess the ability of these proteins to separate carbohydrates, they were immobilized to macroporous microparticulate silica and analyses were conducted at temperatures ranging from 25 to 65°C. With the given set of CBM-variants, we were able to separate cello- and xylo-oligomers under isocratic conditions. The affinities of the CBMs for their targets were weak (in the mM–µM range) and by adjusting the column temperature we could optimize peak resolution and chromatographic retention times. The access to thermostable CBM-variants with diverse affinities and selectivities holds promise to be an efficient tool in the field of affinity chromatography for the separation of carbohydrates.
Functional characterization and target discovery of glycoside hydrolases from the digestome of the lower termite Coptotermes gestroi.
Cairo, J. P. L. F., Leonardo, F. C., Alvarez, T. M., Ribeiro, D. A., Büchli, F., Costa-Leonardo, A. M., Carazzolle, M. F., Costa, F. F., Paes Leme, A. F., Pereira, G. A. G. & Squina, F. M. (2011). Biotechnology for Biofuels, 4(1), 50.
Background: Lignocellulosic materials have been moved towards the forefront of the biofuel industry as a sustainable resource. However, saccharification and the production of bioproducts derived from plant cell wall biomass are complex and lengthy processes. The understanding of termite gut biology and feeding strategies may improve the current state of biomass conversion technology and bioproduct production. Results: The study herein shows comprehensive functional characterization of crude body extracts from Coptotermes gestroi along with global proteomic analysis of the termite’s digestome, targeting the identification of glycoside hydrolases and accessory proteins responsible for plant biomass conversion. The crude protein extract from C. gestroi was enzymatically efficient over a broad pH range on a series of natural polysaccharides, formed by glucose-, xylose-, mannan- and/or arabinose-containing polymers, linked by various types of glycosidic bonds, as well as ramification types. Our proteomic approach successfully identified a large number of relevant polypeptides in the C. gestroi digestome. A total of 55 different proteins were identified and classified into 29 CAZy families. Based on the total number of peptides identified, the majority of components found in the C. gestroi digestome were cellulose-degrading enzymes. Xylanolytic enzymes, mannan- hydrolytic enzymes, pectinases and starch degrading and debranching enzymes were also identified. Our strategy enabled validation of liquid chromatography with tandem mass spectrometry recognized proteins, by enzymatic functional assays and by following the degradation products of specific 8-amino-1,3,6-pyrenetrisulfonic acid labeled oligosaccharides through capillary zone electrophoresis. Conclusions: Here we describe the first global study on the enzymatic repertoire involved in plant polysaccharide degradation by the lower termite C. gestroi. The biochemical characterization of whole body termite extracts evidenced their ability to cleave all types of glycosidic bonds present in plant polysaccharides. The comprehensive proteomic analysis, revealed a complete collection of hydrolytic enzymes including cellulases (GH1, GH3, GH5, GH7, GH9 and CBM 6), hemicellulases (GH2, GH10, GH11, GH16, GH43 and CBM 27) and pectinases (GH28 and GH29).
Isolation and identification of phenolic glucosides from thermally treated olive oil byproducts.
Rubio-Senent, F., Lama-Muñoz, A., Rodríguez-Gutiérrez, G. & Fernández-Bolaños, J. (2013). Journal of Agricultural and Food Chemistry, 61(6), 1235-1248.
A liquid phase rich in bioactive compounds, such as phenols and sugars, is obtained from olive oil waste by novel thermal treatment. Two groups of fractions with common characteristics were obtained and studied after thermal treatment, acid hydrolysis, and separation by ultrafiltration, chromatography, and finally Superdex Peptide HR. In the first group, which eluted at the same time as oligosaccharides with a low DP (4–2), an oleosidic secoiridoid structure conjugated to a phenolic compound (hydroxytyrosol) was identified as oleuropeinic acid, and three possible structures were detected. In the second group, glucosyl structures formed by hydroxytyrosol and one, two, or three units of glucose or by tyrosol and glucose have been proposed. Verbascoside, a heterosidic ester of caffeic acid, in which hydroxytyrosol is linked to rhamnose–glucose or one of its isomers was also identified. Neutral oligosaccharides bound to a phenol-containing compound could be antioxidant-soluble fibers with bioactive properties.
Kinetics of the enzymatic cellulose hydrolysis by the endoglucanase from the extremophile S. solfataricus.
Bonhage, B., Seiferheld, B. & Spiess, A. C. (2013). In Kraslawski A, Turunen I (eds.). Proceedings of the 23rd European Symposium on Computer Aided Process Engineering, 23, 85-90.
The hydrolysis of cellulose is a necessary step to provide sugars from biomass, e.g. for fermentation. A promising approach is to hydrolyse cellulose enzymatically. Naturally, cellulolytic enzymes appear in mixtures of at least four different enzyme activities. Until now, research has focused on these enzyme mixtures. But, to accurately describe cellulose hydrolysis, it is essential to identify the individual kinetic parameters of the employed cellulases. Therefore, we investigated the behaviour of the extremophile endoglucanase (EG) SSO1354 from S. solfataricus on cello-oligomers (COs) to determine its kinetic performance. The properties of interest were the binding affinity as function of the chain length of the cellulose as well as inhibitory and activating effects of short COs. We monitored the evolution of the chain length distribution over the reaction time using thin layer chromatography and the formation of reducing sugars with a colorimetric assay. According to the measurements, the cellulase requires a chain length of four or more glucose units to be catalytically active and the enzyme gets more active with increasing chain length. Also, cellotriose (C3) is an inhibitor for the used EG, and cellobiose (C2) seems to be an enzyme activator, in contrast to literature. With the obtained results it should be possible to mechanistically describe the hydrolysis of cellulose.
Mutational and Structural Analyses of Caldanaerobius polysaccharolyticus Man5B Reveal Novel Active Site Residues for Family 5 Glycoside Hydrolases.
Oyama, T., Schmitz, G. E., Dodd, D., Han, Y., Burnett, A., Nagasawa, N., Mackie, R. I., Nakamura, H., Morikawa, K. & Cann, I. (2013). PloS one, 8(11), e80448.
CpMan5B is a glycoside hydrolase (GH) family 5 enzyme exhibiting both β-1,4-mannosidic and β-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196) in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity.
Mutational insights into the roles of amino acid residues in ligand binding for two closely related family 16 carbohydrate binding modules.
Su, X., Agarwal, V., Dodd, D., Bae, B., Mackie, R. I., Nair, S. K. & Cann, I. K. (2010). Journal of Biological Chemistry, 285(45), 34665-34676.
Carbohydrate binding modules (CBMs) are specialized proteins that bind to polysaccharides and oligosaccharides. Caldanaerobius polysaccharolyticus Man5ACBM16-1/CBM16-2 bind to glucose-, mannose-, and glucose/mannose-configured substrates. The crystal structures of the two proteins represent the only examples in CBM family 16, and studies that evaluate the roles of amino acid residues in ligand binding in this family are lacking. In this study, we probed the roles of amino acids (selected based on CBM16-1/ligand co-crystal structures) on substrate binding. Two tryptophan (Trp-20 and Trp-125) and two glutamine (Gln-81 and Gln-93) residues are shown to be critical in ligand binding. Additionally, several polar residues that flank the critical residues also contribute to ligand binding. The CBM16-1 Q121E mutation increased affinity for all substrates tested, whereas the Q21G and N97R mutants exhibited decreased substrate affinity. We solved CBM/substrate co-crystal structures to elucidate the molecular basis of the increased substrate binding by CBM16-1 Q121E. The Gln-121, Gln-21, and Asn-97 residues can be manipulated to fine-tune ligand binding by the Man5A CBMs. Surprisingly, none of the eight residues investigated was absolutely conserved in CBM family 16. Thus, the critical residues in the Man5A CBMs are either not essential for substrate binding in the other members of this family or the two CBMs are evolutionarily distinct from the members available in the current protein database. Man5A is dependent on its CBMs for robust activity, and insights from this study should serve to enhance our understanding of the interdependence of its catalytic and substrate binding modules.
Revisiting the Brønsted acid catalyzed hydrolysis kinetics of polymeric carbohydrates in ionic liquids by in situ ATR-FTIR spectroscopy.
Kunov-Kruse, A. J., Riisager, A., Saravanamurugan, S., Berg, R. W., Kristensen, S. B. & Fehrmann, R. (2013). Green Chemistry, 15(10), 2843-2848.
A new versatile method to measure rates and determine activation energies for the Brønsted acid catalysed hydrolysis of cellulose and cellobiose (and other polymeric carbohydrates) in ionic liquids is demonstrated by following the C–O stretching band of the glycoside bond with in situ ATR-FTIR. An activation energy in excellent agreement with the literature was determined for cellulose hydrolysis, whereas a distinctly lower activation energy was determined for cellobiose hydrolysis. The methodology also allowed to independently determine activation energies for the formation of 5-hydroxymethylfurfural in the systems.
Biochemical and mutational analyses of a multidomain cellulase/mannanase from Caldicellulosiruptor bescii.
Su, X., Mackie, R. I. & Cann, I. K. O. (2012). Applied and Environmental Microbiology, 78(7), 2230-2240.
Thermophilic cellulases and hemicellulases are of significant interest to the biofuel industry due to their perceived advantages over their mesophilic counterparts. We describe here biochemical and mutational analyses of Caldicellulosiruptor bescii Cel9B/Man5A (CbCel9B/Man5A), a highly thermophilic enzyme. As one of the highly secreted proteins of C. bescii, the enzyme is likely to be critical to nutrient acquisition by the bacterium. CbCel9B/Man5A is a modular protein composed of three carbohydrate-binding modules flanked at the N terminus and the C terminus by a glycoside hydrolase family 9 (GH9) module and a GH5 module, respectively. Based on truncational analysis of the polypeptide, the cellulase and mannanase activities within CbCel9B/Man5A were assigned to the N- and C-terminal modules, respectively. CbCel9B/Man5A and its truncational mutants, in general, exhibited a pH optimum of ∼5.5 and a temperature optimum of 85°C. However, at this temperature, thermostability was very low. After 24 h of incubation at 75°C, the wild-type protein maintained 43% activity, whereas a truncated mutant, TM1, maintained 75% activity. The catalytic efficiency with phosphoric acid swollen cellulose as a substrate for the wild-type protein was 7.2 s-1 ml/mg, and deleting the GH5 module led to a mutant (TM1) with a 2-fold increase in this kinetic parameter. Deletion of the GH9 module also increased the apparent Kcat of the truncated mutant TM5 on several mannan-based substrates; however, a concomitant increase in the Km led to a decrease in the catalytic efficiencies on all substrates. These observations lead us to postulate that the two catalytic activities are coupled in the polypeptide.
RP-UHPLC-UV-ESI-MS/MS analysis of LPMO generated C4-oxidized gluco-oligosaccharides after non-reductive labeling with 2-aminobenzamide.
Frommhagen, M., van Erven, G., Sanders, M., van Berkel, W. J., Kabel, M. A. & Gruppen, H. (2017). Carbohydrate Research, In Press.
Lytic polysaccharide monooxygenases (LPMOs) are able to cleave recalcitrant polysaccharides, such as cellulose, by oxidizing the C1 and/or C4 atoms. The analysis of the resulting products requires a variety of analytical techniques. Up to now, these techniques mainly focused on the identification of non-oxidized and C1-oxidized oligosaccharides. The analysis of C4-oxidized gluco-oligosaccharides is mostly performed by using high pressure anion exchange chromatography (HPAEC). However, the alkaline conditions used during HPAEC analysis lead to tautomerization of C4-oxidized gluco-oligosaccharides, which limits the use of this technique. Here, we describe the use of reverse phase-ultra high performance liquid chromatography (RP-UHPLC) in combination with non-reductive 2-aminobenzamide (2-AB) labeling. Non-reductive 2-AB labeling enabled separation of C4-oxidized gluco-oligosaccharides from their non-oxidized counterparts. Moreover, RP-UHPLC does not require buffered mobile phases, which reduce mass spectrometry (MS) sensitivity. The latter is seen as an advantage over other techniques such as hydrophilic interaction liquid chromatography and porous graphitized carbon coupled to MS. RP-UHPLC coupled to UV detection and mass spectrometry allowed the identification of both labeled non-oxidized and C4-oxidized oligosaccharides. Non-reductive labeling kept the ketone at the C4-position of LPMO oxidized oligosaccharides intact, while selective reducing agents such as sodium triacetoxyborohydride (STAB) reduced this ketone group. Our results show that RP-UHPLC-UV-ESI-MS in combination with non-reductively 2-AB labeling is a suitable technique for the separation and identification of LPMO-generated C4-oxidized gluco-oligosaccharides.
Characterization of a novel theme C glycoside hydrolase family 9 cellulase and its CBM-chimeric enzymes.
Duan, C. J., Huang, M. Y., Pang, H., Zhao, J., Wu, C. X. & Feng, J. X. (2017). Applied Microbiology and Biotechnology, 1-15.
In bacterial cellulase systems, glycoside hydrolase family 9 (GH9) cellulases are generally regarded as the major cellulose-degrading factors besides GH48 exoglucanase. In this study, umcel9A, which was cloned from uncultured microorganisms from compost, with the encoded protein being theme C GH9 cellulase, was heterologously expressed in Escherichia coli, and the biochemical properties of the purified enzyme were characterized. Hydrolysis of carboxylmethylcellulose (CMC) by Umcel9A led to the decreased viscosity of CMC solution and production of reducing sugars. Interestingly, cellobiose was the major product when cellulosic materials were hydrolyzed by Umcel9A. Six representative carbohydrate-binding modules (CBMs) from different CBM families (CBM1, CBM2, CBM3, CBM4, CBM10, and CBM72) were fused with Umcel9A at the natural terminal position, resulting in significant enhancement of the binding capacity of the chimeric enzymes toward four different insoluble celluloses as compared with that of Umcel9A. Catalytic activity of the chimeric enzymes against insoluble celluloses, including phosphoric acid-swollen cellulose (PASC), alkali-pretreated sugarcane bagasse (ASB), filter paper powder (FPP), and Avicel, was higher than that of Umcel9A, except for Umcel9A-CBM3. In these chimeric enzymes, CBM4-Umcel9A exhibited the highest activity toward the four tested insoluble celluloses and displayed 4.2-, 3.0-, 2.4-, and 6.6-fold enhanced activity toward PASC, ASB, FPP, and Avicel, respectively, when compared with that of Umcel9A. CBM4-Umcel9A also showed highest Vmax and catalytic efficiency (kcat/KM) against PASC. Construction of chimeric enzymes may have potential applications in biocatalytic processes and provides insight into the evolution of the molecular architecture of catalytic module and CBM in GH9 cellulases.