Identification of features associated with plant cell wall recalcitrance to pretreatment by alkaline hydrogen peroxide in diverse bioenergy feedstocks using glycome profiling.
Li, M., Pattathil, S., Hahn, M. G. & Hodge, D. B. (2014). RSC Advances, 4(33), 17282-17292.
A woody dicot (hybrid poplar), an herbaceous dicot (goldenrod), and a graminaceous monocot (corn stover) were subjected to alkaline hydrogen peroxide (AHP) pretreatment and subsequent enzymatic hydrolysis in order to assess how taxonomically and structurally diverse biomass feedstocks respond to a mild alkaline oxidative pretreatment and how differing features of the cell wall matrix contribute to its recalcitrance. Using glycome profiling, we determined changes in the extractability of non-cellulosic glucans following pretreatment by screening extracts of the pretreated walls with a panel of 155 cell wall glycan-directed monoclonal antibodies to determine differences in the abundance and distribution of non-cellulosic glycan epitopes in these extracts and assess pretreatment-induced changes in the structural integrity of the cell wall. Two taxonomically-dependent outcomes of pretreatment were identified that both improved the subsequent enzymatic hydrolysis yields but differed in their impacts on cell wall structural integrity. Specifically, it was revealed that goldenrod walls exhibited decreases in all classes of alkali-extractable glycans indicating their solubilization during pretreatment, which was accompanied by an improvement in the subsequent extractability of the remaining cell wall glycans. The corn stover walls did not show the same decreases in glycan abundance in extracts following pretreatment, but rather mild increases in all classes of cell wall glycans, indicating overall weaker associations between cell wall polymers and improved extractability. The hybrid poplar walls were relatively unaffected by pretreatment in terms of composition, enzymatic hydrolysis, and the extractability of cell wall glycans due presumably to their higher lignin content and denser vascular structure.
Correlating lignin structural features to phase partitioning behavior in a novel aqueous fractionation of softwood Kraft black liquor.
Stoklosa, R. J., Velez, J., Kelkar, S., Saffron, C. M., Thies, M. C. & Hodge, D. B. (2013). Green Chemistry, 15(10), 2904-2912.
In this work, a set of softwood lignins were recovered from a Kraft black liquor using a novel pH-based fractionation process involving sequential CO2 acidification and separation of the solvated aqueous lignin fraction. These recovered lignins fractions were characterized with respect to properties that may be responsible for their phase partitioning behavior as well as properties that may render the lignins more suitable for materials applications. Lignin fractions were recovered between a pH range of 12.8 and 9.5 with the bulk of the lignin (90%) recovered between a pH of 11.1 and 10.0. While all the fractions were found to consist primarily of lignin as validated by sample methoxyl content, the first fractions to phase separated were found to be especially enriched in aliphatic extractives and polysaccharides. From the bulk of the lignin that was recovered between a pH of 11.1 and 10.0 a number of noteworthy trends were discernible from the data. Specifically, the phenolic hydroxyl content was found to exhibit a strong negative correlation to the fractionation pH and exhibited a nearly 50% increase with recovery at decreasing pH, while the GPC-estimated molecular weights and 13C NMR-estimated β-O-4 content showed strong positive correlations to the pH at recovery. The aliphatic hydroxyl content exhibited minimal differences between recovery conditions. Overall, these results suggest that this fractionation approach can generate lignin fractions enriched in select physical or structural properties that may be important for their application as feedstocks for renewable chemicals or materials.
Dietary Supplementation with Soluble Plantain Non-Starch Polysaccharides Inhibits Intestinal Invasion of Salmonella Typhimurium in the Chicken.
Parsons, B. N., Wigley, P., Simpson, H. L., Williams, J. M., Humphrey, S., Salisbury, A. M., Watson, A. J., Fry, S. C., O'Brien, D., Roberts, C. L., O'Kennedy, N., Keita, Å. V., Söderholm, J. D., Rhodes, J. M. & Campbell, B. J. (2014). PloS One, 9(2), e87658.
Soluble fibres (non-starch polysaccharides, NSP) from edible plants but particularly plantain banana (Musa spp.), have been shown in vitro and ex vivo to prevent various enteric pathogens from adhering to, or translocating across, the human intestinal epithelium, a property that we have termed contrabiotic. Here we report that dietary plantain fibre prevents invasion of the chicken intestinal mucosa by Salmonella. In vivo experiments were performed with chicks fed from hatch on a pellet diet containing soluble plantain NSP (0 to 200 mg/d) and orally infected with S.Typhimurium 4/74 at 8 d of age. Birds were sacrificed 3, 6 and 10 d post-infection. Bacteria were enumerated from liver, spleen and caecal contents. In vitro studies were performed using chicken caecal crypts and porcine intestinal epithelial cells infected with Salmonella enterica serovars following pre-treatment separately with soluble plantain NSP and acidic or neutral polysaccharide fractions of plantain NSP, each compared with saline vehicle. Bacterial adherence and invasion were assessed by gentamicin protection assay. In vivo dietary supplementation with plantain NSP 50 mg/d reduced invasion by S.Typhimurium, as reflected by viable bacterial counts from splenic tissue, by 98.9% (95% CI, 98.1–99.7; P <0.0001). In vitro studies confirmed that plantain NSP (5–10 mg/ml) inhibited adhesion of S.Typhimurium 4/74 to a porcine epithelial cell-line (73% mean inhibition (95% CI, 64–81); P <0.001) and to primary chick caecal crypts (82% mean inhibition (95% CI, 75–90); P <0.001). Adherence inhibition was shown to be mediated via an effect on the epithelial cells and Ussing chamber experiments with ex-vivo human ileal mucosa showed that this effect was associated with increased short circuit current but no change in electrical resistance. The inhibitory activity of plantain NSP lay mainly within the acidic/pectic (homogalacturonan-rich) component. Supplementation of chick feed with plantain NSP was well tolerated and shows promise as a simple approach for reducing invasive salmonellosis.
Optimization of a formic/acetic acid delignification treatment on beech wood and its influence on the structural characteristics of the extracted lignins.
Simon, M., Brostaux, Y., Vanderghem, C., Jourez, B., Paquot, M. & Richel, A. (2014). Journal of Chemical Technology and Biotechnology, 89(1), 128-136.
Background: In order to replace petrochemicals by bio-based lignin products in high value-added applications, a formic/acetic acid treatment was adapted to beech wood (Fagus sylvatica L.) for lignin extraction. Results: Beech wood particles were delignified at atmospheric pressure by a formic acid/acetic acid/water mixture. Cooking time and temperature were optimized for delignification, pulp yield and 2-furfural concentration. Response surface design analysis revealed that delignification yield increased with cooking time and temperature. Conclusion: The multi-criteria optimization of delignification was used to find the ideal cooking conditions (5 h 07 min, 104.2°C) to maximize delignification (70.5%) and pulp yield (58.7%) and, to a lesser extent, minimize 2-furfural production. Treatment conditions were found to influence the chemical structure of extracted lignins. Cooking time and temperature inversely influenced lignin molecular weights.
Fractionation and improved enzymatic deconstruction of hardwoods with alkaline delignification.
Stoklosa, R. J. & Hodge, D. B. (2015). BioEnergy Research, 8(3), 1224-1234.
In this work, an alkaline delignification was investigated for several industrially relevant hardwoods to understand the kinetics of xylan solubilization and degradation and the role of residual lignin content in setting cell wall recalcitrance to enzymatic hydrolysis. Between 34 and 50 % of the xylan was solubilized during the heat-up stage of the pretreatment and undergoes degradation, depolymerization, as well as substantial disappearance of the glucuronic acid substitutions on the xylan during the bulk delignification phase. An important finding is that substantial xylan is still present in the liquor without degradation. Cellulose hydrolysis yields in the range of 80 to 90 % were achievable within 24–48 h for the diverse hardwoods subjected to delignification by alkali at modest enzyme loadings. It was found that substantial delignification was not necessary to achieve these high hydrolysis yields and that hybrid poplar subjected to pretreatment removing only 46 % of the lignin was capable of reaching yields comparable to hybrid poplar pretreated to 67 or 86 % lignin removal. Decreasing the lignin content was found to increase the initial rate of cellulose hydrolysis to glucose while lignin contents under approximately 70 mg/g original biomass were found to slightly decrease the maximum extent of hydrolysis, presumably due to drying-induced cellulose aggregation and pore collapse. Pretreatments were performed on woodchips, which necessitated a “disintegration” step following pretreatment. This allowed the effect of comminution method to be investigated for the three hardwoods subjected to the highest level of delignification. It was found that additional knife-milling following distintegration did not impact either the rate or extent of glucan and xylan hydrolysis.
Kinetic properties of Rhizopus oryzae RPG1 endo-polygalacturonase hydrolyzing galacturonic acid oligomers.
Mertens, J. A. & Bowman, M. J. (2016). Biocatalysis and Agricultural Biotechnology, 5, 11-16.
The kinetic characteristics of Rhizopus oryzae endo-polygalacturonase, RPG1, hydrolyzing galacturonic acid oligomers (GalpA)n were determined. RPG1 generates (GalpA)3 as a dominant product of polygalacturonic acid and (GalpA)4-6 hydrolysis. The enzyme can hydrolyze (GalpA)3, but hydrolysis occurs at a significantly lower rate relative to oligomers with a higher degree of polymerization. Hydrolysis of the α-1,4 glycosidic bond by RPG1 is an endothermic process with a δHapp, of 1.03±0.04 kcal/mol. Determination of kinetic constants by isothermal titration calorimetry showed that for oligomers (GalpA)3-6, the Km decreased and the Kcat increased as the length of the (GalpA) oligomer increased. Fixed time point assays followed by chromatographic analysis provided apparent Kcat values similar to those found using isothermal titration calorimetry. Assays to determine to what extent the enzyme is subject to product inhibition demonstrated that the enzyme is competitively inhibited by (GalpA)2 when using (GalpA)4 as substrate. The apparent Ki of 767 µM is significantly higher than the Km values obtained for the series of galacturonic acid oligomers.
Characterisation of three fungal glucuronoyl esterases on glucuronic acid ester model compounds.
Hüttner, S., Klaubauf, S., de Vries, R. P. & Olsson, L. (2017). Applied Microbiology and Biotechnology, 1-11.
The glucuronoyl esterases (GEs) that have been identified so far belong to family 15 of the carbohydrate esterases in the CAZy classification system and are presumed to target ester bonds between lignin alcohols and (4-O-methyl-)D-glucuronic acid residues of xylan. Few GEs have been cloned, expressed and characterised to date. Characterisation has been done on a variety of synthetic substrates; however, the number of commercially available substrates is very limited. We identified novel putative GEs from a wide taxonomic range of fungi and expressed the enzymes originating from Acremonium alcalophilum and Wolfiporia cocos as well as the previously described PcGE1 from Phanerochaete chrysosporium. All three fungal GEs were active on the commercially available compounds benzyl glucuronic acid (BnGlcA), allyl glucuronic acid (allylGlcA) and to a lower degree on methyl glucuronic acid (MeGlcA). The enzymes showed pH stability over a wide pH range and tolerated 6-h incubations of up to 50°C. Kinetic parameters were determined for BnGlcA. This study shows the suitability of the commercially available model compounds BnGlcA, MeGlcA and allylGlcA in GE activity screening and characterisation experiments. We enriched the spectrum of characterised GEs with two new members of a relatively young enzyme family. Due to its biotechnological significance, this family deserves to be more extensively studied. The presented enzymes are promising candidates as auxiliary enzymes to improve saccharification of plant biomass.
Action of a GH115 α-glucuronidase from Amphibacillus xylanus at alkaline condition promotes release of 4-O -methylglucopyranosyluronic acid from glucuronoxylan and arabinoglucuronoxylan.
Yan, R., Vuong, T. V., Wang, W. & Master, E. R. (2017). Enzyme and Microbial Technology, 104, 22-28.
Glucuronic acid and/or 4-O-methyl-glucuronic acid (GlcA/MeGlcA) are substituents of the main xylans present in hardwoods, conifers, and many cereal grains. α-Glucuronidases from glycoside hydrolase family GH115 can target GlcA/MeGlcA from both internally and terminally substituted regions of xylans. The current study describes the first GH115 α-glucuronidase, AxyAgu115A, from the alkaliphilic organism Amphilbacillus xylanus. AxyAgu115A was active in a wide pH range, and demonstrated better performance in alkaline condition compared to other characterized GH115 α-glucuronidases, which generally show optimal activity in acidic conditions. Specifically, its relative activity between pH 5.0 and pH 8.5 was above 80%, and was 35% of maximum at pH 10.5; although the enzyme lost 30% and 80% relative residual activity after 24-h pre-incubation at pH 9 and pH 10, respectively. AxyAgu115A was also similarly active towards glucuronoxylan as well as comparatively complex xylans such as spruce arabinoglucurunoxylan. Accommodation of complex xylans was supported by docking analyses that predicted accessibility of AxyAgu115A to branched xylo-oligosaccharides. MeGlcA release by AxyAgu115A from each xylan sample was increased by up to 30% by performing the reaction at pH 11.0 rather than pH 4.0, revealing applied benefits of AxyAgu115A for xylan recovery and processing.
Fungal glucuronoyl esterases: genome mining based enzyme discovery and biochemical characterization.
Dilokpimol, A., Mäkelä, M. R., Cerullo, G., Zhou, M., Varriale, S., Gidijala, L., Brás, J. L. A.,Jütten, P., Piechot, A., Verhaert, R., Faraco, V., Hilden, K. S. & de Vries, R. P. (2017). New Biotechnology, In Press.
4-O-Methyl-D-glucuronic acid (MeGlcA) is a side-residue of glucuronoarabinoxylan and can form ester linkages to lignin, contributing significantly to the strength and rigidity of the plant cell wall. Glucuronoyl esterases (4-O-methyl-glucuronoyl methylesterases, GEs) can cleave this ester bond, and therefore may play a significant role as auxiliary enzymes in biomass saccharification for the production of biofuels and biochemicals. GEs belong to a relatively new family of carbohydrate esterases (CE15) in the CAZy database (www.cazy.org), and so far around ten fungal GEs have been characterized. To explore additional GE enzymes, we used a genome mining strategy. BLAST analysis with characterized GEs against approximately 250 publicly accessible fungal genomes identified more than 150 putative fungal GEs, which were classified into eight phylogenetic sub-groups. To validate the genome mining strategy, 21 selected GEs from both ascomycete and basidiomycete fungi were heterologously produced in Pichia pastoris. Of these enzymes, 18 were active against benzyl D-glucuronate demonstrating the suitability of our genome mining strategy for enzyme discovery.