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The natural catalytic function of CuGE glucuronoyl esterase in hydrolysis of genuine lignin–carbohydrate complexes from birch
© The Author(s) 2018
Received: 4 January 2018
Accepted: 9 March 2018
Published: 19 March 2018
Glucuronoyl esterases belong to carbohydrate esterase family 15 and catalyze de-esterification. Their natural function is presumed to be cleavage of ester linkages in lignin–carbohydrate complexes particularly those linking lignin and glucuronoyl residues in xylans in hardwood.
Here, we show for the first time a detailed product profile of aldouronic acids released from birchwood lignin by a glucuronoyl esterase from the white-rot fungus Cerrena unicolor (CuGE). CuGE releases substrate for GH10 endo-xylanase which results in significantly increased product release compared to the action of endo-xylanase alone. CuGE also releases neutral xylo-oligosaccharides that can be ascribed to the enzymes feruloyl esterase side activity as demonstrated by release of ferulic acid from insoluble wheat arabinoxylan.
The data verify the enzyme’s unique ability to catalyze removal of all glucuronoxylan associated with lignin and we propose that this is a direct result of enzymatic cleavage of the ester bonds connecting glucuronoxylan to lignin via 4-O-methyl glucuronoyl-ester linkages. This function appears important for the fungal organism’s ability to effectively utilize all available carbohydrates in lignocellulosic substrates. In bioprocess perspectives, this enzyme is a clear candidate for polishing lignin for residual carbohydrates to achieve pure, native lignin fractions after minimal pretreatment.
Efficient and complete enzymatic degradation is an essential prerequisite in the utilization of lignocellulosic material for production of energy and value-added biorefinery products. However, cross links in plant cell walls, such as lignin–carbohydrate complexes (LCCs) are obstacles for selective separation and isolation of lignin in enzymatic conversion of plant biomass .
Glucuronoxylan present in the secondary cell walls of hardwoods primarily consists of xylan (β-1 → 4-linked d-xylosyl) substituted with α-1 → 2 -linked 4-O-methyl-d-glucuronosyl residues [2, 3]. Lignin–carbohydrate ester linkages are formed between aliphatic alcohols in lignin and 4-O-methyl-d-glucuronic acid residues of glucuronoxylan [4, 5]. In addition, the bifunctional nature of ferulic acid  is assumed to contribute to linkages between the lignin moiety (via ether bonds) and arabinoxylan via esterifications to arabinosyls, hence representing another “LCC” component [7, 8]. It is hypothesized that the 4-O-methyl-d-glucuronoyl comprising ester linkages can be enzymatically hydrolyzed in nature by glucuronoyl esterases (GE), a relatively new family of esterases assigned to the CAZymes family 15 under carbohydrate esterase (CE15) (http://www.cazy.org/) . The first CE15 was discovered in the cellulolytic system of the basidiomycete fungus Schizophyllum commune in 2006 . At present, the CE15 family contains eight characterized proteins. Of these, the crystal structures of two have been determined [11, 12]. Until now, all glucuronoyl esterases have been characterized using a limited number of synthetic model substrates and very little is known about the biological function of these esterases. Studies on model substrates have revealed a significant importance of the 4-O-methyl group on the glucuronoyl residue for catalytic activity [13, 14]. Glucuronoyl esterase from Schizophyllum commune has been found to exclusively attack esters of 4-O-methyl-glucuronoyls and exhibits no acetyl xylan esterase or feruloyl esterase activity on model substrates . Similar catalytic activity on low and high molecular mass polymeric methyl esters of glucuronoxylan indicates the potential ability of glucuronoyl esterases to act on large substrates, potentially releasing high molecular weight products. This is supported by the active site being exposed on the surface of the protein . However, studies on the biological function of glucuronoyl esterases and any possible correlations between proposed groupings based on bioinformatics and functional differences have been hindered by the lack of natural substrates amendable to hydrolysis by this type of enzyme .
High heterogeneity and recalcitrance of lignocellulosic biomass together with low concentration of LCCs have made it difficult to evaluate the effect of glucuronoyl esterases on genuine biomass [1, 14]. The previous two attempts to show GE activity on natural substrates have not demonstrated direct product release. d’Errico et al. treated heat pre-treated corn fiber with complex commercial enzyme preparations Ultraflo® (from Humicola insolens) and Cellic® CTec (from Trichoderma reesei) supplemented with recombinantly produced glucuronoyl esterases derived from either Cerrena unicolor or T. reesei and observed a minor increase in the yield of monomeric sugars and glucuronic acid . GE activity has also been detected in several of the commercial complex glucanase preparations from lignocellulose degrading fungi  making it complicated to conclude a boosting effect of extra GE supplement. Bååth et al. used two substrates originating from spruce and birch for testing glucuronoyl esterase activity and interpreted a decrease in substrate size by SEC and an increase in carboxylic acid concentration by NMR as evidence for GE activity .
We hypothesized that the concentration of LCCs could be increased via biomass fractionation after pretreatment. Enrichment of LCCs in the substrate would make it possible to demonstrate that the glucuronoyl esterase catalyzes the release of products directly from a genuine lignin-rich fraction prepared from birchwood and verify the putative natural action of the enzyme.
The gene encoding the glucuronoyl esterase from the white-rot fungus Cerrena unicolor (CuGE) was successfully expressed in Pichia pastoris and purified (see Additional files 1, 2). Glucuronoyl esterase activity was confirmed by monitoring the degradation of benzyl d-glucuronate by LC–MS (see Additional file 3). Furthermore, no endo-xylanase or acetyl xylan esterase activity was observed (see Additional files 4, 5).
Comparison of relative composition of raw birchwood and lignin-rich precipitate (LRP) after pretreatment
4.49 ± 1.12
449.03 ± 26.33
297.08 ± 20.85
198.36 ± 1.97
2.75 ± 0.05
0.08 ± 0.02
2.39 ± 0.30
0.51 ± 0.07
11.43 ± 2.3
902.05 ± 27.91
0.81 ± 0.17
As expected, the GH10 endo-xylanase generated neutral xylo-oligosaccharides (Fig. 2) but it was less expected to observe neutral products released by CuGE and they appear to not originate from any background signal of free xylo-oligosaccharides (Fig. 2, no enzyme). One possible explanation could be that CuGE catalyzes release of neutral arabinoxylo-oligosaccharides after hydrolysis of feruloyl esters in arabinoxylan-like regions of the hemicellulose. According to the chemical composition of LRP, such regions are likely to be present in minor amounts (Table 1) since the compositional analysis of LRP showed minor amounts of arabinose supporting the presence of feruloyl substitutions (see Additional file 7). Previous literature suggests that feruloyl moieties may be incorporated into lignin by ether linkages  and if this is the case in the LRP, free feruloyl substituted xylo-oligosaccharides would not be products of GH10 treatment. Assessing the product release catalyzed by CuGE action on wheat arabinoxylan confirmed that the glucuronoyl esterase was capable of releasing ferulic acid in comparable amounts to a genuine CE1 feruloyl esterase (see Additional file 13). However, the CuGE had no activity on methyl ferulate which is in accordance with previous studies on glucuronoyl esterase activity . Because of the surprising feruloyl esterase side activity, it seems plausible that the neutral products released by CuGE could originate from the hydrolysis of feruloyl esters.
In this study, we have positively verified product release catalyzed by glucuronoyl esterase CE15 from Cerrena unicolor most likely as a result of cleavage of ester-linked LCCs in hardwood. The extraction method generated a substrate with relatively low background carbohydrate signal by the removal of all cellulose and most of the hemicellulose. Hence, the common recalcitrance parameters in lignocellulose were removed, enabling the detection of enzyme reaction products generated directly by glucuronoyl esterase activity.
The fact that CuGE can release aldouronic acids from the substrate strongly indicates the presence of ester-linked LCCs in the substrate, and is also supported by release of aldouronic acids after direct saponification. GH10 endo-xylanase alone can release aldouronic acids from LRP; hence, not all glucuronoyls are associated with lignin. The glucuronoxylan in LRP appears with a relatively high degree of polymerization as evaluated by HPAEC-PAD (Fig. 2) indicating that the substrate after ethanol extraction is a better representation of the original biomass compared to the synthetic substrates currently used for assessing GE activity. The glucuronoxylan is also highly acetylated as would be expected in the native glucuronoxylan. The degree of acetylation does not seem to affect the activity of CuGE as numerous sizes of aldouronic acids are released (Fig. 2) and various acetylated products and isomers are detected by LC–MS (Fig. 1). The influence of the lignin moiety of the ester linkage on CuGE’s activity is not evaluated here but the lignin structure in LRP is expected to be optimal for CuGE activity because the substrate closely resembles the naturally occurring structure. In previous studies, the ester linkages have been synthesized by esterification to simpler alcohols  with little resemblance to lignin. We observe a synergistic activity between CuGE and GH10 endo-xylanase because CuGE releases oligomeric products that serve as new substrate for GH10 endo-xylanase. The synergistic effect is directly observed in Fig. 3a for charged aldouronic acid products where the sum of released MeGlcA equivalents is significantly higher than an additive effect of the GE and GH10 individually. The fact that no apparent synergistic effect on release of neutral oligosaccharides is observed indicates that each xylo-oligomeric chain is linked by several closely positioned glucuronoyl ester linkages creating a heavily glucuronidated substrate for the endo-xylanase. According to the increase in short aldouronic acids, the glucuronoyl substitutions are likely to occur for every 2nd–4th xylosyl residue in certain block regions of the substrate. No oligomeric products carrying two MeGlcA substitutions were detected in the samples incubated with either CuGE alone or GH10 and CuGE together; such specimens might occur in CuGE-hydrolyzed samples but would have too high DP to be directly detected in this setup (Fig. 1). In samples with co-incubation of CuGE and GH10, the products would predominantly be mono-glucuronidated as a result of the endo-xylanase activity. If the distance between glucuronoyl esters in LRP was similar to the average glucuronoyl substitution on glucuronoxylan (1 out of 10 xylosyl residues), the synergistic effect would also be observed directly in the release of neutral xylo-oligosaccharides. Our results prove that the combined action of CuGE and a GH10 endo-xylanase is sufficient to obtain complete release of ester-linked glucuronoxylan from lignin.
It appears that the activity of CuGE is not restricted by steric obstacles such as acetylations compared to GH10 endo-xylanase [21, 22] and this could be related to a surface exposed active site  and might also explain why CuGE has feruloyl esterase activity on polymeric substrate. In relation to the natural function of the enzyme, it seems highly relevant that the enzyme possesses side activity towards other ester-linked LCCs in minor amounts that link carbohydrates to lignin  (i.e., feruloyl esters) instead of evolving a highly specific esterase for this purpose only. To the best of our knowledge, it has not been investigated if any of the known feruloyl esterases could have activity towards glucuronoyl esters. Altogether our findings contribute to the explanation of the biological function of glucuronoyl esterases as enzymes evolved to enable full utilization of the last remaining residues of carbohydrates in lignocellulosic biomass after removal of the main part of polysaccharides. The biological function of GEs corroborates the presence of several putative GE sequences identified by recent bioinformatics studies in genomes of saprophytic fungi that are highly specialized at growing on the most recalcitrant lignin-containing types of biomass [23, 16].
In an application aspect, glucuronoyl esterases can serve as a tool for enzymatic polishing of lignin in biomass residues where pretreatment has not destroyed ester-linked LCCs. Many pretreatment strategies are focusing on disrupting cellulose crystallinity by employing exposure to high temperatures and alkaline or acidic conditions. Such strategies will cause hydrolysis of most ester-linked LCCs and thereby eliminate the need for glucuronoyl esterases . However, such approaches also cause lignin to condense and thereby decrease the final value and applicability of the lignin product and inhibits the cellulolytic conversion . A pretreatment strategy that is based on the initial selective separation of a relatively pure, native lignin fraction from the major polysaccharides could potentially increase both the value and the applications of lignin and lower the costs of converting cellulose and hemicellulose [26, 27]. Glucuronoyl esterases are highly important enzymes for industrial applications that aim for selective lignin recovery in order to obtain a final high-quality lignin product from hardwood.
Glucuronoyl esterase CuGE from Cerrena unicolor releases aldouronic acids from a lignin-enriched fraction from birchwood most likely as a result of hydrolysis of ester linkages between lignin and glucuronoxylan. The enzyme also releases minor amounts of neutral xylo-oligosaccharides from hydrolysis of feruloyl ester linkages to arabinoxylan-like regions in the substrate. CuGE boosts the activity of GH10 endo-xylanase synergistically by releasing glucuronidated xylo-oligosaccharides that act as new substrate for the GH10 endo-xylanase. In this way, the two enzymes are capable of releasing all neutral and charged xylo-oligosaccharides associated with the lignin-rich birchwood fraction. Further insight into the kinetics and cooperative mechanism of the two enzymes will elucidate the exact biocatalytic degradation and should be pursued.
The action of CuGE demonstrates a biological function of the enzyme as part of the fungal enzyme battery necessary to retrieve all available carbohydrates from the most recalcitrant parts of plant cell walls. The fact that the enzyme exhibits feruloyl esterase side activity signifies that even linkages present in negligible amounts are of importance to the survival of the host organism. Glucuronoyl esterases are clear candidates for polishing of lignin from hardwood and we suggest an approach in pretreatment of hardwood where lignin is extracted prior to hydrothermal pretreatment. In this way, the purity and hence the value and applicability of lignin will increase.
Preparation of LRP
Lignin-Rich Precipitate (LRP) from Norwegian birchwood was prepared as previously reported . 15 g raw birchwood was mixed with 135 mL 50 v/v-% ethanol in a batch reactor (300 mL HC EZE-Seal, Parker Autoclave Engineers, Pennsylvania USA). The reactor was heated to 180 °C and kept at 180 °C for 1 h with stirring at 600 rpm. After extraction, the reactor was cooled and the material retrieved. The pre-treated liquor was separated from the solid biomass by filtration using Ashless 40 filter paper, 8 µm (Whatman). The solids (named Cellulose-Rich Precipitate) were washed with 50 v/v-% EtOH and freeze dried. The pre-treated liquor (280 mL in total including washings) was diluted with three volumes of water (840 mL) resulting in the precipitation of a Lignin-Rich Precipitate (LRP). pH in the pre-treated liquor was measured to 3.8. The suspension of lignin-rich precipitate and the pre-treated liquor was separated by centrifugation. The separated liquor phase consisted of a hemicellulose-rich liquid (HRL). The lignin-rich precipitate was washed in water and freeze dried prior to use for hydrolysis experiments. A schematic overview of the extraction process can be found in Additional file 6. All fractions were subjected to acid hydrolysis for determination of monomeric components (see Additional file 2). A small portion of the LRP was subjected to NaOH treatment in order to release all esterified carbohydrates from the lignin matrix. 0.5 M NaOH was added to approx. 11.25 mg of LRP in suspension in water to reach pH > 11 and kept at room temperature overnight. Hereafter, absolute ethanol was added to a final concentration of 90 v/v-% and kept overnight at 4 °C. The precipitate was retrieved by centrifugation, re-dissolved in water, and analyzed by HPAEC-PAD as LRP alkali and represented the majority of the carbohydrate moiety of the lignin-rich fraction. It is expected that the precipitation of short, linear, neutral xylo-oligosaccharides under these conditions is incomplete. The resulting concentration in the LRP alkali fraction analyzed by HPAEC-PAD originated from 37.5 mg/mL. As described below, the enzyme hydrolysis samples were performed with 5 mg/mL substrate and hence the final concentration of analytes in LRP alkali was approx. 7.5 times higher than in the enzyme hydrolysis samples.
Lignin-rich precipitate was suspended in 25 mM sodium acetate buffer pH 6 to a concentration of 5 mg/ml. CuGE (for expression and purification protocol see Additional file 2) and GH10 endo-β1,4-xylanase Shearzyme® 500 L (batch CDN00486) (donated by Novozymes A/S) were added either individually or together to the reactions to a final concentration of 30 mg enzyme protein/g dry matter and 10 mg enzyme protein/g dry matter, respectively, and incubated for 24 h at 50 °C. After reaction, the solid substrate was removed by centrifugation and the supernatant used for analysis.
Enzyme reaction products and substrate (LRP alkali) were analyzed by HPAEC-PAD using a CarboPac PA100 (4.6 × 250 mm) and guard (4.6 × 50 mm) on a Dionex ICS3000 system (Thermo Fischer Scientific, Sunnyvale, CA, USA). The column was operated at 1 mL/min with eluent A (water), eluent B (500 mM NaOH), and eluent C (500 mM sodium acetate) according to the following gradient: 0–2 min isocratic 40% B and 1% C, 2–35 min linear gradient to 40% B and 45% C, hereafter immediately shifted to 5% B and 90% C and these conditions were kept for 4 min and ended by shifting back to starting conditions and reconditioning for 6 min.
Identification and quantification of enzyme reaction products were performed by LC–MS analysis on a UHPLC Dionex UltiMate 3000 system (Thermo Fischer Scientific, Sunnyvale CA, USA) connected to an ESI-iontrap (model AmaZon SL from Bruker Daltonics, Bremen, Germany) operated in MRM mode (multiple reaction monitoring) or fullscan mode. The UHPLC was equipped with a porous graphitized carbon column (Hypercarb PGC, 150 × 2.1 mm; 3 µm, Thermo Fischer Scientific, Waltham, MA, USA) including a guard column of same brand (10 × 2.1 mm). The column was operated at 0.4 mL/min at 70 °C with eluent A (0.1% formic acid) and B (acetonitrile) according to the following gradient: 0–1 min 0% B, from 1 to 15 min linear gradient to 50% B, from 15 to 20 min linear gradient to 80% B, 20–30 isocratic 80% B, hereafter immediately back to starting conditions followed by reconditioning for 10 min.
The ESI was operated in positive mode with spray nebulizer at 3 bar nitrogen, a dry gas flow, and temperature of 12 L/min and 280 °C, respectively. The capillary cap voltage was set to 4.5 kV and end-plate offset of 0.5 kV. Target mass was set to 500 and for MRM conditions, a pre-determined list of ions representing the sodium adducts of exact masses of neutral and charged species was applied: Xyl2 (m/z 305), Xyl2Ac (m/z 347), Xyl2Ac2 (m/z 389), Xyl3 (m/z 437), Xyl3Ac (m/z 479), Xyl3Ac2 (m/z 521), Xyl3Ac3 (m/z 563), XylMeGlcA (m/z 231), Xyl2MeGlcA (m/z 495), Xyl2MeGlcAAc (m/z 537), Xyl3MeGlcA (m/z 627), Xyl3MeGlcAAc (m/z 669), Xyl3MeGlcAAc2 (m/z 711), Xyl4MeGlcA (m/z 759), Xyl4MeGlcAAc (m/z 801), and Xyl4MeGlcAAc2 (m/z 843). CID fragmentation was performed using SmartFrag enhanced amplitude ramping of 100% and helium as the colliding gas. Data analysis and quantification were performed by Compass DataAnalysis 4.2 and Compass QuantAnalysis 2.2 from Bruker Daltonics.
Quantification of enzyme reaction products was done against external calibration curves of authenticated standards of xylobiose, xylotriose, xylotetraose, and reduced aldotetrauronic acid all of which were purchased from Megazyme, Ireland. Samples for quantification purposes were treated with NaOH prior to analysis (and after enzyme reaction) in order to raise pH > 11 and hereby remove all acetylations. This procedure resulted in a much simplified mixture of reaction products mainly consisting of xylose, xylobiose, xylotriose, xylotetraose, aldotriuronic acid, aldotetrauronic acid, and aldopentauronic acid (see Additional file 14). The aldouronic acids were quantified relative to the signal response for reduced aldotetrauronic acid. Elaborate description of the quantification procedure is provided in the Additional files 2, 15.
Author CM and JWA conceived the conceptual ideas, conducted the experiments, preformed the analyses, interpreted the data, and prepared the manuscript. JH and AM participated in the conceptual development, the data interpretation, and in the preparation of the manuscript. All authors read and approved the final manuscript.
We thank Dr. Bjørge Westereng, Faculty of Chemistry, Biotechnology and Food Science at the Norwegian University of Life Sciences for providing the raw birchwood substrate.
The authors declare that they have no competing interests.
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This work was financed by Grant NNF15OC0015222 funded by the Novo Nordisk Foundation and also supported by the Bio-Value Strategic Platform for Innovation and Research, co-funded by The Danish Council for Strategic Research and The Danish Council for Technology and Innovation, Case No: 0603-00522B.
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