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.
Xylo-oligosaccharides are competitive inhibitors of cellobiohydrolase I from Thermoascus aurantiacus.
Zhang, J. & Viikari, L. (2012). Bioresource Technology, 117, 286-291.
The effects of xylo-oligosaccharides (XOS) and xylose on the hydrolytic activities of cellulases, endoglucanase II (EGII, originating from Thermoascus aurantiacus), cellobiohydrolase I (CBHI, from T. aurantiacus), and cellobiohydrolase II (CBHII, from Trichoderma reesei) on Avicel and nanocellulose were investigated. After the addition of XOS, the amounts of cellobiose, the main product released from Avicel and nanocellulose by CBHI, decreased from 0.78 and 1.37 mg/ml to 0.59 and 1.23 mg/ml, respectively. During hydrolysis by CBHII, the amounts of cellobiose released from the substrates were almost cut in half after the addition of XOS. Kinetic experiments showed that xylobiose and xylotriose were competitive inhibitors of CBHI. The results revealed that the strong inhibition of cellulase by XOS can be attributed to the inhibitory effect of XOS especially on cellobiohydrolase I. The results indicate the necessity to totally hydrolyze xylo-oligosaccharides into the less inhibitory product, xylose, to increasing hydrolytic efficiency.
Altered substrate specificity of the gluco‐oligosaccharide oxidase from Acremonium strictum.
Foumani, M., Vuong, T. V. & Master, E. R. (2011). Biotechnology and Bioengineering, 108(10), 2261-2269.
A gluco-oligosaccharide oxidase (GOOX) from Acremonium strictum type strain CBS 346.70 was cloned and expressed in Pichia pastoris. The recombinant protein, GOOX-VN, contained fifteen amino acid substitutions compared with the previously reported A. strictum GOOX. These two enzymes share 97% sequence identity; however, only GOOX-VN oxidized xylose, galactose, and N-acetylglucosamine. Besides monosaccharides, GOOX-VN oxidized xylo-oligosaccharides, including xylobiose and xylotriose with similar catalytic efficiency as for cello-oligosaccharides. Of three mutant enzymes that were created in GOOX-VN to improve substrate specificity, Y300A and Y300N doubled kCat values for monosaccharide and oligosaccharide substrates. With this novel substrate specificity, GOOX-VN and its variants are particularly valuable for oxidative modification of cello- and xylo-oligosaccharides. Biotechnol. Bioeng. 2011;108: 2261–2269.
Processivity, synergism, and substrate specificity of Thermobifida fusca Cel6B.
Vuong, T. V. & Wilson, D. B. (2009). Applied and Environmental Microbiology, 75(21), 6655-6661.
A relationship between processivity and synergism has not been reported for cellulases, although both characteristics are very important for hydrolysis of insoluble substrates. Mutation of two residues located in the active site tunnel of Thermobifida fusca exocellulase Cel6B increased processivity on filter paper. Surprisingly, mixtures of the Cel6B mutant enzymes and T. fusca endocellulase Cel5A did not show increased synergism or processivity, and the mutant enzyme which had the highest processivity gave the poorest synergism. This study suggests that improving exocellulase processivity might be not an effective strategy for producing improved cellulase mixtures for biomass conversion. The inverse relationship between the activities of many of the mutant enzymes with bacterial microcrystalline cellulose and their activities with carboxymethyl cellulose indicated that there are differences in the mechanisms of hydrolysis for these substrates, supporting the possibility of engineering Cel6B to target selected substrates.
Clostridium thermocellum cellulase CelT, a family 9 endoglucanase without an Ig-like domain or family 3c carbohydrate-binding module.
Kurokawa, J., Hemjinda, E., Arai, T., Kimura, T., Sakka, K. & Ohmiya, K. (2002). Applied Microbiology and Biotechnology, 59(4), 455-461.
The celT gene of Clostridium thermocellum strain F1 was found downstream of the mannanase gene man26B [Kurokawa J et al. (2001) Biosci Biotechnol Biochem 65:548–554] in pKS305. The open reading frame of celT consists of 1,833 nucleotides encoding a protein of 611 amino acids with a predicted molecular weight of 68,510. The mature form of CelT consists of a family 9 cellulase domain and a dockerin domain responsible for cellulosome assembly, but lacks a family 3c carbohydrate-binding module (CBM) and an immunoglobulin (Ig)-like domain, which are often found with family 9 catalytic domains. CelT devoid of the dockerin domain (CelTΔdoc) was constructed and purified from a recombinant Escherichia coli, and its enzyme properties were examined. CelTΔdoc showed strong activity toward carboxymethylcellulose (CMC) and barley β-glucan, and low activity toward xylan. The Vmax and Km values were 137 µmol min-1 mg-1 and 16.7 mg/ml, respectively, for CMC. Immunological analysis indicated that CelT is a catalytic component of the C. thermocellum F1 cellulosome. This is the first report describing the characterization of a family 9 cellulase without an Ig-like domain or family 3c CBM.
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.
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.
Sitosterol-β-glucoside as primer for cellulose synthesis in plants.
Peng, L., Kawagoe, Y., Hogan, P. & Delmer, D. (2002). Science, 295(5552), 147-150.
Cellulose synthesis in plants requires β-1,4-glucan chain initiation, elongation, and termination. The process of chain elongation is likely to be distinct from the process of chain initiation. We demonstrate that a CesA glucosyltransferase initiates glucan polymerization by using sitosterol-β-glucoside (SG) as primer. Cotton fiber membranes synthesize sitosterol-cellodextrins (SCDs) from SG and uridine 5′-diphosphate–glucose (UDP-Glc) under conditions that also favor cellulose synthesis. The cellulase encoded by the Korrigan (Kor) gene, required for cellulose synthesis in plants, may function to cleave SG from the growing polymer chain.
Two-stage statistical medium optimization for augmented cellulase production via solid-state fermentation by newly isolated Aspergillus niger HN-1 and application of crude cellulase consortium in hydrolysis of rice straw.
Sandhu, S. K., Oberoi, H. S., Babbar, N., Miglani, K., Chadha, B. & Nanda, D. (2013). Journal of Agricultural and Food Chemistry, 61(51), 12653–12661.
Cellulolytic enzyme production by newly isolated Aspergillus niger HN-1 was statistically optimized using Plackett–Burman and central composite design (CCD). Optimum concentrations of 2, 0.40, 0.01, and 0.60 g L-1 for KH2PO4, urea, trace elements solution, and CaCl2•2H2O, respectively, were suggested by Design-Expert software. The two-stage optimization process led to a 3- and 2-fold increases in the filter paper cellulase (FP) and β-glucosidase activities, respectively. FP, β-glucosidase, endoglucanase, exopolygalaturonase, cellobiohydrolase, xylanase, α-L-arabinofuranosidase, β-xylosidase, and xylan esterase activities of 36.7 ± 1.54 FPU gds-1, 252.3 ± 7.4 IU gds-1, 416.3 ± 22.8 IU gds-1, 111.2 ± 5.4 IU gds-1, 8.9 ± 0.50 IU gds-1, 2593.5 ± 78.9 IU gds-1, 79.4 ± 4.3 IU gds-1, 180.8 ± 9.3 IU gds-1, and 288.7 ± 11.8 IU gds-1, respectively, were obtained through solid-state fermentation during the validation studies. Hydrolysis of alkali-treated rice straw with crude cellulases resulted in about 84% glucan to glucose, 89% xylan to xylose, and 91% arabinan to arabinose conversions, indicating potential for biomass hydrolysis by the crude cellulase consortium obtained in this study.
A novel exo-cellulase from white spotted longhorn beetle (Anoplophora malasiaca).
Chang, C. J., Wu, C. P., Lu, S. C., Chao, A. L., Ho, T. H. D., Yu, S. M. & Chao, Y. C. (2012). Insect Biochemistry and Molecular Biology, 42(9), 629-636.
Wood feeding insects depends heavily on the secretion of a combination of cellulases, mainly endoglucanases and other glucanases such as exoglucanases and xylanases, for efficient digestion of the cellulosic materials. To date, although a high number of endoglucanases have been found in xytophagous insects, little is known about exoglucanases encoded in the genome of these insects. Here we report the identification and isolation of an exoglucanase, designated as AmCel-5B, from the white spotted longhorn beetle, Anoplophora malasiaca. The optimal condition of enzymatic activity was found to be 50°C and pH 4.0. Interestingly, this enzyme is not only exhibited exo-β-glucanase activity, but also with obvious endo-β-glucanase activity. Furthermore, this enzyme is unique in that, although it recognizes Avicel, evidenced as an exo-β-glucanase, it cannot recognize oligosaccharides smaller than cellohexaose. This may explain why longhorn beetle can well digest hard “living” wood, which contains primarily rigid long fibers. Although it is known that metal ions can enhance the activity of some cellulases, we further demonstrated that reducing agent could work synergistically with metal ions for significant activity enhancement of AmCel-5B. The discovery and investigation of an insect exoglucanase should lead to a greater understanding of the mechanism for efficient digestion of cellulosic materials by wood feeding insects, as well as facilitate their potential applications in the production of bioenergy and biomaterials from lignocellulosic biomass in the future.
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.
Unraveling the secretome of Termitomyces clypeatus grown on agroresidues as a potential source for bioethanol production.
Mukherjee, S. & Khowala, S. (2016). Process Biochemistry, 51(11), 1793-1807.
Termitomyces clypeatus MTCC 5091 is an edible mushroom and is prized for its nutritional value as well as for harboring plethora of enzymes essential for carbohydrate degradation. T. clypeatus when grown on agricultural based carbon sources efficiently induced high quantities of lignocellulolytic enzymes in the secretome. Optimization in tamarind kernal powder (TKP) media through response surface methodology enhanced the enzyme yields by several folds. Correlation between extracellular protein productions of the fungus with respect to its specific growth rate established that secreted proteins were produced most efficiently at low specific growth rates. Proteins released in the T. clypeatus secretome were quantified and identified using SDS-PAGE, 2D gel electrophoreses, zymography and matrix-assisted laser desorption mass spectrometry. 36 proteins identified from the protein spots belonged majority to glucosyl hydrolase family, transporters, uncharacterized and hypothetical proteins. The potential synergistic interactions between the cellulases and xylanases in enzyme preparations of T. clypeatus during hydrolysis of steam pretreated bagasse (SPB) showed improved hydrolysis efficiency and enhanced rate of hydrolysis as observed in high performance liquid chromatograpy. The changes in the ultra-structure of SPB after 12 h enzymatic hydrolysis were observed by scanning electron microscopy. The hydrolysates obtained produced ~7.2 g/L ethanol after 6 h fermentation determined by gas chromatography.
Heterologous expression of a GH3 β-glucosidase from Neurospora crassa in Pichia pastoris with high purity and its application in the hydrolysis of soybean isoflavone glycosides.
Pei, X., Zhao, J., Cai, P., Sun, W., Ren, J., Wu, Q., Zhang, S. & Tian, C. (2016). Protein expression and Purification, 119, 75-84.
Previous studies have shown isoflavone aglycones to have more biological effects than their counterparts, isoflavone glycones. Some β-glucosidases can hydrolyze isoflavone glucosides to release aglycones, and discovery of these has attracted great interest. A glycoside hydrolase (GH) family 3 beta;-glucosidase (bgl2) gene from Neurospora crassa was heterologously expressed in Pichia pastoris with high purity. The recombinant BGL2 enzyme displayed its highest activity at pH 5.0 and 60°C, and had its maximum activity against p-nitrophenyl-β-D-glucopyranoside (pNPG) (143.27 ± 4.79 U/mg), followed by cellobiose (74.99 ± 0.78 U/mg), gentiobiose (47.55 ± 0.15 U/mg), p-nitrophenyl-β-D-cellobioside (p NPC) (40.07 ± 0.87 U/mg), cellotriose (12.31 ± 0.36 U/mg) and cellotetraose (9.04 ± 0.14 U/mg). The kinetic parameters of Km and Vmax were 0.21 ± 0.01 mM and 147.93 ± 2.77 µM/mg/min for pNPG. The purified enzyme showed a heightened ability to convert the major soybean isoflavone glycosides (daidzin, genistin and glycitin) into their corresponding aglycone forms (daidzien, genistein and glycitein). With this activity against soybean isoflavone glycosides, BGL2 shows great potential for applications in the food, animal feed, and pharmaceutical industries.
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.
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.