Jane Wittrup Agger

Discovery of LPMO activity on hemicelluloses shows the importance of oxidative processes in plant cell wall degradation

Significance Plant cell walls are recalcitrant copolymeric structures mainly comprising polysaccharides and lignin. Enzymatic degradation of the polysaccharides is a crucial step in biorefining of biomass. Recently, it was discovered that nature employs copper-dependent redox enzymes called lytic polysaccharide monoxoygenases (LPMOs) to promote degradation of the most recalcitrant and crystalline of these polysaccharides, cellulose. By carrying out oxidative cleavage of otherwise inaccessible cellulose chains, LPMOs create access points for classical hydrolytic enzymes such as cellulases. Intriguingly, the genomes of biomass degrading microorganisms encode a plethora of LPMOs (up to over 40). To our knowledge, we demonstrate for the first time that LPMOs act on hemicelluloses. This finding dramatically widens the scope of LPMOs and oxidative processes in plant cell wall degradation and biorefining. The recently discovered lytic polysaccharide monooxygenases (LPMOs) are known to carry out oxidative cleavage of glycoside bonds in chitin and cellulose, thus boosting the activity of well-known hydrolytic depolymerizing enzymes. Because biomass-degrading microorganisms tend to produce a plethora of LPMOs, and considering the complexity and copolymeric nature of the plant cell wall, it has been speculated that some LPMOs may act on other substrates, in particular the hemicelluloses that tether to cellulose microfibrils. We demonstrate that an LPMO from Neurospora crassa, NcLPMO9C, indeed degrades various hemicelluloses, in particular xyloglucan. This activity was discovered using a glycan microarray-based screening method for detection of substrate specificities of carbohydrate-active enzymes, and further explored using defined oligomeric hemicelluloses, isolated polymeric hemicelluloses and cell walls. Products generated by NcLPMO9C were analyzed using high performance anion exchange chromatography and multidimensional mass spectrometry. We show that NcLPMO9C generates oxidized products from a variety of substrates and that its product profile differs from those of hydrolytic enzymes acting on the same substrates. The enzyme particularly acts on the glucose backbone of xyloglucan, accepting various substitutions (xylose, galactose) in almost all positions. Because the attachment of xyloglucan to cellulose hampers depolymerization of the latter, it is possible that the beneficial effect of the LPMOs that are present in current commercial cellulase mixtures in part is due to hitherto undetected LPMO activities on recalcitrant hemicellulose structures.

A C4-oxidizing lytic polysaccharide monooxygenase cleaving both cellulose and cello-oligosaccharides

Background: Lytic polysaccharide monooxygenases (LPMOs) are recently discovered enzymes that cleave polysaccharides. Results: We describe a novel LPMO and use a range of analytical methods to characterize its activity. Conclusion: Cellulose and cello-oligosaccharides are cleaved by oxidizing the sugar at the nonreducing end in the C4 position. Significance: This study provides unequivocal evidence for C4 oxidation of the nonreducing end sugar and demonstrates a novel LPMO substrate specificity. Lignocellulosic biomass is a renewable resource that significantly can substitute fossil resources for the production of fuels, chemicals, and materials. Efficient saccharification of this biomass to fermentable sugars will be a key technology in future biorefineries. Traditionally, saccharification was thought to be accomplished by mixtures of hydrolytic enzymes. However, recently it has been shown that lytic polysaccharide monooxygenases (LPMOs) contribute to this process by catalyzing oxidative cleavage of insoluble polysaccharides utilizing a mechanism involving molecular oxygen and an electron donor. These enzymes thus represent novel tools for the saccharification of plant biomass. Most characterized LPMOs, including all reported bacterial LPMOs, form aldonic acids, i.e., products oxidized in the C1 position of the terminal sugar. Oxidation at other positions has been observed, and there has been some debate concerning the nature of this position (C4 or C6). In this study, we have characterized an LPMO from Neurospora crassa (NcLPMO9C; also known as NCU02916 and NcGH61–3). Remarkably, and in contrast to all previously characterized LPMOs, which are active only on polysaccharides, NcLPMO9C is able to cleave soluble cello-oligosaccharides as short as a tetramer, a property that allowed detailed product analysis. Using mass spectrometry and NMR, we show that the cello-oligosaccharide products released by this enzyme contain a C4 gemdiol/keto group at the nonreducing end.

Efficient separation of oxidized cello-oligosaccharides generated by cellulose degrading lytic polysaccharide monooxygenases

We present an evaluation of HPLC-based analytical tools for the simultaneous analysis of native and oxidized cello-oligosaccharides, which are products of enzymatic cellulose degradation. Whereas cello-oligosaccharides arise from cellulose depolymerization by glycoside hydrolases, oxidized cello-oligosaccharides are produced by cellobiose dehydrogenase and the recently identified copper dependent lytic polysaccharide monooxygenases (LPMOs) currently classified as CBM33 and GH61. The latter enzymes are wide-spread and expected to play crucial roles in further development of efficient enzyme technology for biomass conversion. Three HPLC approaches with well documented performance in the field of oligosaccharide analysis have been investigated: high-performance anion-exchange chromatography (HPAEC), hydrophilic interaction chromatography (HILIC) and porous graphitized carbon liquid chromatography (PGC-LC). HPAEC with pulsed amperometric detection (PAD) was superior for analysis of oxidized oligosaccharides, combining the best separation with superior sensitivity for oligosaccharide species with a degree of polymerization (DP) ranging from 1 to 10. Furthermore, the HPAEC method can be optimized for operation in a high-throughput manner (run time 10 min). Both PGC-LC and HILIC allow reasonable run times (41 and 25 min, respectively), with acceptable separation, but suffer from poor sensitivity compared to HPAEC-PAD. On the other hand, PGC-LC and HILIC benefit from being fully compatible with online mass spectrometry. Using an LC-MS setup, these methods will deliver much better sensitivity than what can be obtained with conventional detectors such as ultraviolet-, charged aerosol-, or evaporative light scattering and may reach sensitivities similar to or even better than what is obtained in HPAEC-PAD. Pure oxidized cello-oligosaccharide standards, ranging from DP2 to DP5, were obtained by semi-preparative PGC and characterized by MS and NMR analysis.

Enzymatic cellulose oxidation is linked to lignin by long-range electron transfer

Enzymatic oxidation of cell wall polysaccharides by lytic polysaccharide monooxygenases (LPMOs) plays a pivotal role in the degradation of plant biomass. While experiments have shown that LPMOs are copper dependent enzymes requiring an electron donor, the mechanism and origin of the electron supply in biological systems are only partly understood. We show here that insoluble high molecular weight lignin functions as a reservoir of electrons facilitating LPMO activity. The electrons are donated to the enzyme by long-range electron transfer involving soluble low molecular weight lignins present in plant cell walls. Electron transfer was confirmed by electron paramagnetic resonance spectroscopy showing that LPMO activity on cellulose changes the level of unpaired electrons in the lignin. The discovery of a long-range electron transfer mechanism links the biodegradation of cellulose and lignin and sheds new light on how oxidative enzymes present in plant degraders may act in concert.

Enzymatic Xylose Release from Pretreated Corn Bran Arabinoxylan: Differential Effects of Deacetylation and Deferuloylation on Insoluble and Soluble Substrate Fractions

In the present work enzymatic hydrolysis of arabinoxylan from pretreated corn bran (190 degrees C, 10 min) was evaluated by measuring the release of xylose and arabinose after treatment with a designed minimal mixture of monocomponent enzymes consisting of alpha-L-arabinofuranosidases, an endoxylanase, and a beta-xylosidase. The pretreatment divided the corn bran material approximately 50:50 into soluble and insoluble fractions having A:X ratios of 0.66 and 0.40, respectively. Addition of acetyl xylan esterase to the monocomponent enzyme mixture almost doubled the xylose release from the insoluble substrate fraction and gave release of 1 mol of xylose/mol of acetic acid released, whereas addition of feruloyl esterase promoted release of only approximately 0.4 mol of xylose/mol of ferulic acid released. For the soluble substrate fraction up to 36% of the xylose could be released by the enzymatic treatment. Acetyl xylan esterase addition on top of the minimal monocomponent enzyme mixture resulted in liberation of up to 0.5 mol of xylose/mol of acetic acid released, whereas feruloyl esterase addition released 1 mol of xylose/mol of ferulic acid released from the soluble substrate. The results imply that on the insoluble material the acetyl xylan esterase was more important for the enzymatic degradation than feruloyl esterase, whereas on soluble arabinoxylan the feruloyl esterase seemed to be more important for the release of xylose.

Simultaneous analysis of C1 and C4 oxidized oligosaccharides, the products of lytic polysaccharide monooxygenases acting on cellulose.

Lytic polysaccharide monooxygenases play a pivotal role in enzymatic deconstruction of plant cell wall material due to their ability to catalyze oxidative cleavage of glycosidic bonds. LPMOs may release different products, often in small amounts, with various oxidation patterns (C1 or C4) and with varying stabilities, making accurate analysis of product profiles a major challenge. So far, HPAEC has been the method of choice but it has limitations with respect to analysis of C4-oxidized products. Here, we compare various HPLC methods and present procedures that allow efficient separation of intact C1- and C4-oxidized products. We demonstrate that both PGC and HILIC (in WAX-mode) can separate C1- and C4-oxidized products and that PGC gives superior chromatographic performance. In contrast to HPAEC, these methods are directly compatible with mass spectroscopy and charged aerosol detection (CAD), which enables online peak validation and quantification with LOD levels in the low ng range. While the novel methods show lower resolution than HPAEC, this is compensated by easy peak identification, allowing, for example, discrimination between chromatographically highly similar native and C4-oxidized cello-oligomers. HPAEC-MS studies revealed chemical oxidation of C4-geminal diol products, which implies that peaks commonly believed to be C4-oxidized cello-oligomers, in fact are on-column generated derivatives. Non-destructive separation of C4-oxidized cello-oligosaccharides on the PGC column allowed us, for the first time, to isolate C4-oxidized standards. HPAEC fractionation of a purified C4-oxidized tetramer revealed that on-column decomposition leads to formation of the native trimer, which may explain why product mixtures generated by C4-oxidizing LPMOs seem to be rich in native oligosaccharides when analyzed by HPAEC. The findings and methods described here will aid in future studies in the emerging LPMO field.

Steam explosion pretreatment for enhancing biogas production of late harvested hay

Grasslands are often abandoned due to lack of profitability. Extensively cultivating grassland for utilization in a biogas-based biorefinery concept could mend this problem. Efficient bioconversion of this lignocellulosic biomass requires a pretreatment step. In this study the effect of different steam explosion conditions on hay digestibility have been investigated. Increasing severity in the pretreatment induced degradation of the hemicellulose, which at the same time led to the production of inhibitors and formation of pseudo-lignin. Enzymatic hydrolysis showed that the maximum glucose yields were obtained under pretreatment at 220 °C for 15 min, while higher xylose yields were obtained at 175 °C for 10 min. Pretreatment of hay by steam explosion enhanced 15.9% the methane yield in comparison to the untreated hay. Results indicate that hay can be effectively converted to methane after steam explosion pretreatment.

Mode of action of acetylxylan esterases on acetyl glucuronoxylan and acetylated oligosaccharides generated by a GH10 endoxylanase

BACKGROUND Substitutions on the xylan main chain are widely accepted to limit plant cell wall degradability and acetylations are considered as one of the most important obstacles. Hence, understanding the modes of action of a range of acetylxylan esterases (AcXEs) is of ample importance not only to increase the understanding of the enzymology of plant decay/bioremediation but also to enable efficient bioconversion of plant biomass. METHODS In this study, the modes of action of acetylxylan esterases (AcXEs) belonging to carbohydrate esterase (CE) families 1, 4, 5 and 6 on xylooligosaccharides generated from hardwood acetyl glucuronoxylan were compared using MALDI ToF MS. Supporting data were obtained by following enzymatic deacetylation by (1)H NMR spectroscopy. CONCLUSIONS None of the used enzymes were capable of complete deacetylation, except from linear xylooligosaccharides which were completely deacetylated by some of the esterases in the presence of endoxylanase. A clear difference was observed between the performance of the serine-type esterases of CE families 1, 5 and 6, and the aspartate-metalloesterases of family CE4. The difference is mainly due to the inability of CE4 AcXEs to catalyze deacetylation of 2,3-di-O-acetylated xylopyranosyl residues. Complete deacetylation of a hardwood acetyl glucuronoxylan requires additional deacetylating enzyme(s). GENERAL SIGNIFICANCE The results contribute to the understanding of microbial degradation of plant biomass and outline the way to achieve complete saccharification of plant hemicelluloses which did not undergo alkaline pretreatment.

Trichoderma reesei CE16 acetyl esterase and its role in enzymatic degradation of acetylated hemicellulose.

BACKGROUND Trichoderma reesei CE16 acetyl esterase (AcE) is a component of the plant cell wall degrading system of the fungus. The enzyme behaves as an exo-acting deacetylase removing acetyl groups from non-reducing end sugar residues. METHODS In this work we demonstrate this exo-deacetylating activity on natural acetylated xylooligosaccharides using MALDI ToF MS. RESULTS The combined action of GH10 xylanase and acetylxylan esterases (AcXEs) leads to formation of neutral and acidic xylooligosaccharides with a few resistant acetyl groups mainly at their non-reducing ends. We show here that these acetyl groups serve as targets for TrCE16 AcE. The most prominent target is the 3-O-acetyl group at the non-reducing terminal Xylp residues of linear neutral xylooligosaccharides or on aldouronic acids carrying MeGlcA at the non-reducing terminus. Deacetylation of the non-reducing end sugar may involve migration of acetyl groups to position 4, which also serves as substrate of the TrCE16 esterase. CONCLUSION Concerted action of CtGH10 xylanase, an AcXE and TrCE16 AcE resulted in close to complete deacetylation of neutral xylooligosaccharides, whereas substitution with MeGlcA prevents removal of acetyl groups from only a small fraction of the aldouronic acids. Experiments with diacetyl derivatives of methyl β-d-xylopyranoside confirmed that the best substrate of TrCE16 AcE is 3-O-acetylated Xylp residue followed by 4-O-acetylated Xylp residue with a free vicinal hydroxyl group. GENERAL SIGNIFICANCE This study shows that CE16 acetyl esterases are crucial enzymes to achieve complete deacetylation and, consequently, complete the saccharification of acetylated xylans by xylanases, which is an important task of current biotechnology.

A polysaccharide utilization locus from an uncultured bacteroidetes phylotype suggests ecological adaptation and substrate versatility.

ABSTRACT Recent metagenomic analyses have identified uncultured bacteria that are abundant in the rumen of herbivores and that possess putative biomass-converting enzyme systems. Here we investigate the saccharolytic capabilities of a polysaccharide utilization locus (PUL) that has been reconstructed from an uncultured Bacteroidetes phylotype (SRM-1) that dominates the rumen microbiome of Arctic reindeer. Characterization of the three PUL-encoded outer membrane glycoside hydrolases was performed using chromogenic substrates for initial screening, followed by detailed analyses of products generated from selected substrates, using high-pressure anion-exchange chromatography with electrochemical detection. Two glycoside hydrolase family 5 (GH5) endoglucanases (GH5_g and GH5_h) demonstrated activity against β-glucans, xylans, and xyloglucan, whereas GH5_h and the third enzyme, GH26_i, were active on several mannan substrates. Synergy experiments examining different combinations of the three enzymes demonstrated limited activity enhancement on individual substrates. Binding analysis of a SusE-positioned lipoprotein revealed an affinity toward β-glucans and, to a lesser extent, mannan, but unlike the two SusD-like lipoproteins previously characterized from the same PUL, binding to cellulose was not observed. Overall, these activities and binding specificities correlated well with the glycan content of the reindeer rumen, which was determined using comprehensive microarray polymer profiling and showed an abundance of various hemicellulose glycans. The substrate versatility of this single PUL putatively expands our perceptions regarding PUL machineries, which so far have demonstrated gene organization that suggests one cognate PUL for each substrate type. The presence of a PUL that possesses saccharolytic activity against a mixture of abundantly available polysaccharides supports the dominance of SRM-1 in the Svalbard reindeer rumen microbiome.