Modification of a Marine Pine Kraft Lignin Sample by Enzymatic Treatment with a Pycnoporus cinnabarinus Laccase.

Here, we report work on developing an enzymatic process to improve the functionalities of industrial lignin. A kraft lignin sample prepared from marine pine was treated with the high-redox-potential laccase from the basidiomycete fungus Pycnoporus cinnabarinus at three different concentrations and pH conditions, and with and without the chemical mediator 1-hydroxybenzotriazole (HBT). Laccase activity was tested in the presence and absence of kraft lignin. The optimum pH of PciLac was initially 4.0 in the presence and absence of lignin, but at incubation times over 6 h, higher activities were found at pH 4.5 in the presence of lignin. Structural changes in lignin were investigated by Fourier-transform infrared spectroscopy (FTIR) with differential scanning calorimetry (DSC), and solvent-extractable fractions were analyzed using high-performance size-exclusion chromatography (HPSEC) and gas chromatography-mass spectrometry (GC-MS). The FTIR spectral data were analyzed with two successive multivariate series using principal component analysis (PCA) and ANOVA statistical analysis to identify the best conditions for the largest range of chemical modifications. DSC combined with modulated DSC (MDSC) revealed that the greatest effect on glass transition temperature (Tg) was obtained at 130 U g cm-1 and pH 4.5, with the laccase alone or combined with HBT. HPSEC data suggested that the laccase treatments led to concomitant phenomena of oligomerization and depolymerization, and GC-MS revealed that the reactivity of the extractable phenolic monomers depended on the conditions tested. This study demonstrates that P. cinnabarinus laccase can be used to modify marine pine kraft lignin, and that the set of analytical methods implemented here provides a valuable tool for screening enzymatic treatment conditions.

Another building block in the plant cell wall: Barley xyloglucan xyloglucosyl transferases link covalently xyloglucan and anionic oligosaccharides derived from pectin.

We report on the homo- and hetero-transglycosylation activities of the HvXET3 and HvXET4 xyloglucan xyloglucosyl transferases (XET; EC 2.4.1.207) from barley (Hordeum vulgare L.), and the visualisation of these activities in young barley roots using Alexa Fluor 488-labelled oligosaccharides. We discover that these isozymes catalyse the transglycosylation reactions with the chemically defined donor and acceptor substrates, specifically with the xyloglucan donor and the penta-galacturonide [α(1-4)GalAp]5 acceptor - the homogalacturonan (pectin) fragment. This activity is supported by 3D molecular models of HvXET3 and HvXET4 with the docked XXXG donor and [α(1-4)GalAp]5 acceptor substrates at the -4 to +5 subsites in the active sites. Comparative sequence analyses of barley isoforms and seed-localised TmXET6.3 from nasturtium (Tropaeolum majus L.) permitted the engineering of mutants of TmXET6.3 that could catalyse the hetero-transglycosylation reaction with the xyloglucan/[α(1-4)GalAp]5 substrate pair, while wild-type TmXET6.3 lacked this activity. Expression data obtained by real-time quantitative polymerase chain reaction of HvXET transcripts and a clustered heatmap of expression profiles of the gene family revealed that HvXET3 and HvXET6 co-expressed but did not share the monophyletic origin. Conversely, HvXET3 and HvXET4 shared this relationship, when we examined the evolutionary history of 419 glycoside hydrolase 16 family members, spanning monocots, eudicots and a basal Angiosperm. The discovered hetero-transglycosylation activity in HvXET3 and HvXET4 with the xyloglucan/[α(1-4)GalAp]5 substrate pair is discussed against the background of roles of xyloglucan-pectin heteropolymers and how they may participate in spatial patterns of cell wall formation and re-modelling, and affect the structural features of walls.

Engineering the acceptor substrate specificity in the xyloglucan endotransglycosylase TmXET6.3 from nasturtium seeds (Tropaeolum majus L.).

KEY MESSAGE: The knowledge of substrate specificity of XET enzymes is important for the general understanding of metabolic pathways to challenge the established notion that these enzymes operate uniquely on cellulose-xyloglucan networks. Xyloglucan xyloglucosyl transferases (XETs) (EC 2.4.1.207) play a central role in loosening and re-arranging the cellulose-xyloglucan network, which is assumed to be the primary load-bearing structural component of plant cell walls. The sequence of mature TmXET6.3 from Tropaeolum majus (280 residues) was deduced by the nucleotide sequence analysis of complete cDNA by Rapid Amplification of cDNA Ends, based on tryptic and chymotryptic peptide sequences. Partly purified TmXET6.3, expressed in Pichia occurred in N-glycosylated and unglycosylated forms. The quantification of hetero-transglycosylation activities of TmXET6.3 revealed that (1,3;1,4)-, (1,6)- and (1,4)-ß-D-glucooligosaccharides were the preferred acceptor substrates, while (1,4)-ß-D-xylooligosaccharides, and arabinoxylo- and glucomanno-oligosaccharides were less preferred. The 3D model of TmXET6.3, and bioinformatics analyses of identified and putative plant xyloglucan endotransglycosylases (XETs)/hydrolases (XEHs) of the GH16 family revealed that H94, A104, Q108, K234 and K237 were the key residues that underpinned the acceptor substrate specificity of TmXET6.3. Compared to the wild-type enzyme, the single Q108R and K237T, and double-K234T/K237T and triple-H94Q/A104D/Q108R variants exhibited enhanced hetero-transglycosylation activities with xyloglucan and (1,4)-ß-D-glucooligosaccharides, while those with (1,3;1,4)- and (1,6)-ß-D-glucooligosaccharides were suppressed; the incorporation of xyloglucan to (1,4)-ß-D-glucooligosaccharides by the H94Q variant was influenced most extensively. Structural and biochemical data of non-specific TmXET6.3 presented here extend the classic XET reaction mechanism by which these enzymes operate in plant cell walls. The evaluations of TmXET6.3 transglycosylation activities and the incidence of investigated residues in other members of the GH16 family suggest that a broad acceptor substrate specificity in plant XET enzymes could be more widespread than previously anticipated.

Characterization of a long-chain α-galactosidase from Papiliotrema flavescens.

α-Galactosidases are assigned to the class of hydrolases and the subclass of glycoside hydrolases (GHs). They belong to six GH families and include the only characterized α-galactosidases from yeasts (GH 27, Saccharomyces cerevisiae). The present study focuses on an investigation of the lactose-inducible α-galactosidase produced by Papiliotrema flavescens. The enzyme was present on the surface of cells and in the cytosol. Its temperature optimum was about 60 °C and the pH optimum was 4.8; the pH stability ranged from 3.2 to 6.6. This α-galactosidase also exhibited transglycosylation activity. The cytosol α-galactosidase with a molecular weight about 110 kDa, was purified using a combination of liquid chromatography techniques. Three intramolecular peptides were determined by the partial structural analysis of the sequences of the protein isolated, using MALDI-TOF/TOF mass spectrometry. The data obtained recognized the first yeast α-galactosidase, which belongs to the GH 36 family. The bioinformatics analysis and homology modeling of a 210 amino acids long C-terminal sequence (derived from cDNA) confirmed the correctness of these findings. The study was also supplemented by the screening of capsular cryptococcal yeasts, which produce the surface lactose-inducible α- and ß-galactosidases. The production of the lactose-inducible α-galactosidases was not found to be a general feature within the yeast strains examined and, therefore, the existing hypothesis on the general function of this enzyme in cryptococcal capsule rearrangement cannot be confirmed.

Inactivation of Cellobiose Dehydrogenases Modifies the Cellulose Degradation Mechanism of Podospora anserina.

Conversion of biomass into high-value products, including biofuels, is of great interest to developing sustainable biorefineries. Fungi are an inexhaustible source of enzymes to degrade plant biomass. Cellobiose dehydrogenases (CDHs) play an important role in the breakdown through synergistic action with fungal lytic polysaccharide monooxygenases (LPMOs). The three CDH genes of the model fungus Podospora anserina were inactivated, resulting in single and multiple CDH mutants. We detected almost no difference in growth and fertility of the mutants on various lignocellulose sources, except on crystalline cellulose, on which a 2-fold decrease in fertility of the mutants lacking P. anserina CDH1 (PaCDH1) and PaCDH2 was observed. A striking difference between wild-type and mutant secretomes was observed. The secretome of the mutant lacking all CDHs contained five beta-glucosidases, whereas the wild type had only one. P. anserina seems to compensate for the lack of CDH with secretion of beta-glucosidases. The addition of P. anserina LPMO to either the wild-type or mutant secretome resulted in improvement of cellulose degradation in both cases, suggesting that other redox partners present in the mutant secretome provided electrons to LPMOs. Overall, the data showed that oxidative degradation of cellulosic biomass relies on different types of mechanisms in fungi. IMPORTANCE: Plant biomass degradation by fungi is a complex process involving dozens of enzymes. The roles of each enzyme or enzyme class are not fully understood, and utilization of a model amenable to genetic analysis should increase the comprehension of how fungi cope with highly recalcitrant biomass. Here, we report that the cellobiose dehydrogenases of the model fungus Podospora anserina enable it to consume crystalline cellulose yet seem to play a minor role on actual substrates, such as wood shavings or miscanthus. Analysis of secreted proteins suggests that Podospora anserina compensates for the lack of cellobiose dehydrogenase by increasing beta-glucosidase expression and using an alternate electron donor for LPMO.

Cell-free protein synthesis of membrane (1,3)-ß-d-glucan (curdlan) synthase: co-translational insertion in liposomes and reconstitution in nanodiscs.

A membrane-embedded curdlan synthase (CrdS) from Agrobacterium is believed to catalyse a repetitive addition of glucosyl residues from UDP-glucose to produce the (1,3)-ß-d-glucan (curdlan) polymer. We report wheat germ cell-free protein synthesis (WG-CFPS) of full-length CrdS containing a 6xHis affinity tag and either Factor Xa or Tobacco Etch Virus proteolytic sites, using a variety of hydrophobic membrane-mimicking environments. Full-length CrdS was synthesised with no variations in primary structure, following analysis of tryptic fragments by MALDI-TOF/TOF Mass Spectrometry. Preparative scale WG-CFPS in dialysis mode with Brij-58 yielded CrdS in mg/ml quantities. Analysis of structural and functional properties of CrdS during protein synthesis showed that CrdS was co-translationally inserted in DMPC liposomes during WG-CFPS, and these liposomes could be purified in a single step by density gradient floatation. Incorporated CrdS exhibited a random orientation topology. Following affinity purification of CrdS, the protein was reconstituted in nanodiscs with Escherichia coli lipids or POPC and a membrane scaffold protein MSP1E3D1. CrdS nanodiscs were characterised by small-angle X-ray scattering using synchrotron radiation and the data obtained were consistent with insertion of CrdS into bilayers. We found CrdS synthesised in the presence of the Ac-AAAAAAD surfactant peptide or co-translationally inserted in liposomes made from E. coli lipids to be catalytically competent. Conversely, CrdS synthesised with only Brij-58 was inactive. Our findings pave the way for future structural studies of this industrially important catalytic membrane protein.

A Putative Lignin Copper Oxidase from Trichoderma reesei.

The ability of Trichoderma reesei, a fungus widely used for the commercial production of hemicellulases and cellulases, to grow and modify technical soda lignin was investigated. By quantifying fungal genomic DNA, T. reesei showed growth and sporulation in solid and liquid cultures containing lignin alone. The analysis of released soluble lignin and residual insoluble lignin was indicative of enzymatic oxidative conversion of phenolic lignin side chains and the modification of lignin structure by cleaving the ß-O-4 linkages. The results also showed that polymerization reactions were taking place. A proteomic analysis conducted to investigate secreted proteins at days 3, 7, and 14 of growth revealed the presence of five auxiliary activity (AA) enzymes in the secretome: AA6, AA9, two AA3 enzymes), and the only copper radical oxidase encoded in the genome of T. reesei. This enzyme was heterologously produced and characterized, and its activity on lignin-derived molecules was investigated. Phylogenetic characterization demonstrated that this enzyme belonged to the AA5_1 family, which includes characterized glyoxal oxidases. However, the enzyme displayed overlapping physicochemical and catalytic properties across the AA5 family. The enzyme was remarkably stable at high pH and oxidized both, alcohols and aldehydes with preference to the alcohol group. It was also active on lignin-derived phenolic molecules as well as simple carbohydrates. HPSEC and LC-MS analyses on the reactions of the produced protein on lignin dimers (SS ßß, SS ßO4 and GG ß5) uncovered the polymerizing activity of this enzyme, which was accordingly named lignin copper oxidase (TrLOx). Polymers of up 10 units were formed by hydroxy group oxidation and radical formation. The activations of lignin molecules by TrLOx along with the co-secretion of this enzyme with reductases and FAD flavoproteins oxidoreductases during growth on lignin suggest a synergistic mechanism for lignin breakdown.

Screening New Xylanase Biocatalysts from the Mangrove Soil Diversity.

Mangrove sediments from New Caledonia were screened for xylanase sequences. One enzyme was selected and characterized both biochemically and for its industrial potential. Using a specific cDNA amplification method coupled with a MiSeq sequencing approach, the diversity of expressed genes encoding GH11 xylanases was investigated beneath Avicenia marina and Rhizophora stylosa trees during the wet and dry seasons and at two different sediment depths. GH11 xylanase diversity varied more according to tree species and season, than with respect to depth. One complete cDNA was selected (OFU29) and expressed in Pichia pastoris. The corresponding enzyme (called Xyn11-29) was biochemically characterized, revealing an optimal activity at 40-50 °C and at a pH of 5.5. Xyn11-29 was stable for 48 h at 35 °C, with a half-life of 1 h at 40 °C and in the pH range of 5.5-6. Xyn11-29 exhibited a high hydrolysis capacity on destarched wheat bran, with 40% and 16% of xylose and arabinose released after 24 h hydrolysis. Its activity on wheat straw was lower, with a release of 2.8% and 6.9% of xylose and arabinose, respectively. As the protein was isolated from mangrove sediments, the effect of sea salt on its activity was studied and discussed.

Polysaccharide microarrays for high-throughput screening of transglycosylase activities in plant extracts.

Polysaccharide transglycosylases catalyze disproportionation of polysaccharide molecules by cleaving glycosidic linkages in polysaccharide chains and transferring their cleaved portions to hydroxyl groups at the non-reducing ends of other polysaccharide or oligosaccharide molecules. In plant cell walls, transglycosylases have a potential to catalyze both cross-linking of polysaccharide molecules and grafting of newly arriving polysaccharide molecules into the cell wall structure during cell growth. Here we describe a polysaccharide microarray in form of a glycochip permitting simultaneous high-throughput monitoring of multiple transglycosylase activities in plant extracts. The glycochip, containing donor polysaccharides printed onto nitrocellulose-coated glass slides, was incubated with crude plant extracts, along with a series of fluorophore-labelled acceptor oligosaccharides. After removing unused labelled oligosaccharides by washing, fluorescence retained on the glycochip as a result of transglycosylase reaction was detected with a standard microarray scanner. The glycochip assay was used to detect transglycosylase activities in crude extracts from nasturtium (Tropaeolum majus) and mouse-ear cress (Arabidopsis thaliana). A number of previously unknown saccharide donor-acceptor pairs active in transglycosylation reactions that lead to the formation of homo- and hetero-glycosidic conjugates, were detected. Our data provide experimental support for the existence of diverse transglycosylase activities in crude plant extracts.

Structural characterization of the Pet c 1.0201 PR-10 protein isolated from roots of Petroselinum crispum (Mill.) Fuss.

The native dimeric Petroselinum crispum (Mill.) Fuss protein Pet c 1.0201 and a monomeric xyloglucan endotransglycosylase enzyme (Garajova et al., 2008) isolated from the root cells co-purify and share similar molecular masses and acidic isoelectric points. In this work, we determined the complete primary structure of the parsley Pet c 1.0201 protein, based on tryptic and chymotryptic peptides followed by the manual micro-gradient chromatographic separation coupled with offline MALDI-TOF/TOF mass spectrometry. The bioinformatics approach enabled us to include the parsley protein into the PR-10 family, as it exhibited the highest protein sequence identity with the Apium graveolens Api g 1.0201 allergen and the major Daucus carota allergen Dau c 1.0201. Hence, we designated the Petroselinum crispum protein as Pet c 1.0201 and deposited it in the UniProt Knowledgebase under the accession C0HKF5. 3D protein homology modelling and molecular dynamics simulations of the Pet c 1.0201 dimer confirmed the typical structure of the Bet v 1 family allergens, and the potential of the Pet c 1.0201 protein to dimerize in water. However, the behavioural properties of Pet c 1.0201 and the celery allergen Api g 1.0101 differed in the presence of salts due to transiently and stably formed dimeric forms of Pet c 1.0201 and Api g 1.0101, respectively.

The Podospora anserina lytic polysaccharide monooxygenase PaLPMO9H catalyzes oxidative cleavage of diverse plant cell wall matrix glycans.

BACKGROUND: The enzymatic conversion of plant biomass has been recently revolutionized by the discovery of lytic polysaccharide monooxygenases (LPMO) that catalyze oxidative cleavage of polysaccharides. These powerful enzymes are secreted by a large number of fungal saprotrophs and are important components of commercial enzyme cocktails used for industrial biomass conversion. Among the 33 AA9 LPMOs encoded by the genome of Podospora anserina, the PaLPMO9H enzyme catalyzes mixed C1/C4 oxidative cleavage of cellulose and cello-oligosaccharides. Activity of PaLPMO9H on several hemicelluloses has been suggested, but the regioselectivity of the cleavage remained to be determined. RESULTS: In this study, we investigated the activity of PaLPMO9H on mixed-linkage glucans, xyloglucan and glucomannan using tandem mass spectrometry and ion mobility-mass spectrometry. Structural analysis of the released products revealed that PaLPMO9H catalyzes C4 oxidative cleavage of mixed-linkage glucans and mixed C1/C4 oxidative cleavage of glucomannan and xyloglucan. Gem-diols and ketones were produced at the non-reducing end, while aldonic acids were produced at the reducing extremity of the products. CONCLUSION: The ability of PaLPMO9H to target polysaccharides, differing from cellulose by their linkages, glycosidic composition and/or presence of sidechains, could be advantageous for this coprophilous fungus when catabolizing highly variable polysaccharides and for the development of optimized enzyme cocktails in biorefineries.

Lytic polysaccharide monooxygenases disrupt the cellulose fibers structure.

Lytic polysaccharide monooxygenases (LPMOs) are a class of powerful oxidative enzymes that breakdown recalcitrant polysaccharides such as cellulose. Here we investigate the action of LPMOs on cellulose fibers. After enzymatic treatment and dispersion, LPMO-treated fibers show intense fibrillation. Cellulose structure modifications visualized at different scales indicate that LPMO creates nicking points that trigger the disintegration of the cellulose fibrillar structure with rupture of chains and release of elementary nanofibrils. Investigation of LPMO action using solid-state NMR provides direct evidence of modification of accessible and inaccessible surfaces surrounding the crystalline core of the fibrils. The chains breakage likely induces modifications of the cellulose network and weakens fibers cohesion promoting their disruption. Besides the formation of new initiation sites for conventional cellulases, this work provides the first evidence of the direct oxidative action of LPMOs with the mechanical weakening of the cellulose ultrastructure. LPMOs can be viewed as promising biocatalysts for enzymatic modification or degradation of cellulose fibers.

Action of lytic polysaccharide monooxygenase on plant tissue is governed by cellular type.

Lignocellulosic biomass bioconversion is hampered by the structural and chemical complexity of the network created by cellulose, hemicellulose and lignin. Biological conversion of lignocellulose involves synergistic action of a large array of enzymes including the recently discovered lytic polysaccharide monooxygenases (LPMOs) that perform oxidative cleavage of cellulose. Using in situ imaging by synchrotron UV fluorescence, we have shown that the addition of AA9 LPMO (from Podospora anserina) to cellulases cocktail improves the progression of enzymes in delignified Miscanthus x giganteus as observed at tissular levels. In situ chemical monitoring of cell wall modifications performed by synchrotron infrared spectroscopy during enzymatic hydrolysis demonstrated that the boosting effect of the AA9 LPMO was dependent on the cellular type indicating contrasted recalcitrance levels in plant tissues. Our study provides a useful strategy for investigating enzyme dynamics and activity in plant cell wall to improve enzymatic cocktails aimed at expanding lignocelluloses biorefinery.

Salt-responsive lytic polysaccharide monooxygenases from the mangrove fungus Pestalotiopsis sp. NCi6.

BACKGROUND: Lytic polysaccharide monooxygenases (LPMOs) belong to the "auxiliary activities (AA)" enzyme class of the CAZy database. They are known to strongly improve the saccharification process and boost soluble sugar yields from lignocellulosic biomass, which is a key step in the efficient production of sustainable economic biofuels. To date, most LPMOs have been characterized from terrestrial fungi, but novel fungal LPMOs isolated from more extreme environments such as an estuary mangrove ecosystem could offer enzymes with unique properties in terms of salt tolerance and higher stability under harsh condition. RESULTS: Two LPMOs secreted by the mangrove-associated fungus Pestalotiopsis sp. NCi6 (PsLPMOA and PsLPMOB) were expressed in the yeast Pichia pastoris and produced in a bioreactor with >85 mg L(-1) for PsLPMOA and >260 mg L(-1) for PsLPMOB. Structure-guided homology modeling of the PsLPMOs showed a high abundance of negative surface charges, enabling enhanced protein stability and activity in the presence of sea salt. Both PsLPMOs were activated by a cellobiose dehydrogenase (CDH) from Neurospora crassa, with an apparent optimum of interaction at pH 5.5. Investigation into their regioselective mode of action revealed that PsLPMOA released C1- and C4-oxidized cello-oligosaccharide products, while PsLPMOB released only C4-oxidized products. PsLPMOA was found to cleave polymeric cellulose in the presence of up to 6 % sea salt, which emphasizes the use of sea water in the industrial saccharification process with improved ecological footprints. CONCLUSIONS: Two new LPMOs from the mangrove fungus Pestalotiopsis sp. NCi6 were found to be fully reactive against cellulose. The combined hydrolytic activities of these salt-responsive LPMOs could therefore facilitate the saccharification process using sea water as a reaction medium for large-scale biorefineries.

Single-domain flavoenzymes trigger lytic polysaccharide monooxygenases for oxidative degradation of cellulose.

The enzymatic conversion of plant biomass has been recently revolutionized by the discovery of lytic polysaccharide monooxygenases (LPMOs) that carry out oxidative cleavage of polysaccharides. These very powerful enzymes are abundant in fungal saprotrophs. LPMOs require activation by electrons that can be provided by cellobiose dehydrogenases (CDHs), but as some fungi lack CDH-encoding genes, other recycling enzymes must exist. We investigated the ability of AA3_2 flavoenzymes secreted under lignocellulolytic conditions to trigger oxidative cellulose degradation by AA9 LPMOs. Among the flavoenzymes tested, we show that glucose dehydrogenase and aryl-alcohol quinone oxidoreductases are catalytically efficient electron donors for LPMOs. These single-domain flavoenzymes display redox potentials compatible with electron transfer between partners. Our findings extend the array of enzymes which regulate the oxidative degradation of cellulose by lignocellulolytic fungi.

Substrate specificity and regioselectivity of fungal AA9 lytic polysaccharide monooxygenases secreted by Podospora anserina.

BACKGROUND: The understanding of enzymatic polysaccharide degradation has progressed intensely in the past few years with the identification of a new class of fungal-secreted enzymes, the lytic polysaccharide monooxygenases (LPMOs) that enhance cellulose conversion. In the fungal kingdom, saprotrophic fungi display a high number of genes encoding LPMOs from family AA9 but the functional relevance of this redundancy is not fully understood. RESULTS: In this study, we investigated a set of AA9 LPMOs identified in the secretomes of the coprophilous ascomycete Podospora anserina, a biomass degrader of recalcitrant substrates. Their activity was assayed on cellulose in synergy with the cellobiose dehydrogenase from the same organism. We showed that the total release of oxidized oligosaccharides from cellulose was higher for PaLPMO9A, PaLPMO9E, and PaLPMO9H that harbored a carbohydrate-binding module from the family CBM1. Investigation of their regioselective mode of action revealed that PaLPMO9A and PaLPMO9H oxidatively cleaved at both C1 and C4 positions while PaLPMO9E released only C1-oxidized products. Rapid cleavage of cellulose was observed using PaLPMO9H that was the most versatile in terms of substrate specificity as it also displayed activity on cello-oligosaccharides and ß-(1,4)-linked hemicellulose polysaccharides (e.g., xyloglucan, glucomannan). CONCLUSIONS: This study provides insights into the mode of cleavage and substrate specificities of fungal AA9 LPMOs that will facilitate their application for the development of future biorefineries.

Effect of the label of oligosaccharide acceptors on the kinetic parameters of nasturtium seed xyloglucan endotransglycosylase (XET).

Fluorescently labeled derivatives of a xyloglucan (XG) nonasaccharide Glc(4)Xyl(3)Gal(2) (XLLG) were used as glycosyl acceptors in assays of xyloglucan endotransglycosylase (XET) from germinated nasturtium (Tropaeolum majus) seeds. We have investigated how the type of the oligosaccharide label influences the kinetic parameters of the reaction. The fluorescent probes used to label XLLG were anthranilic acid (AA), 8-aminonaphtalene-1,3,6-trisulfonic acid (ANTS), fluorescein isothiocyanate (FITC), and sulforhodamine (SR), respectively. The obtained data were compared with those of the reactions where aldose and/or alditol forms of tritium-labeled xyloglucan-derived nonasaccharide served as the respective acceptors. Modification at C-1 of the reducing-end glucose in XLLG by substitution with the fluorophore markedly affected the kinetic parameters of the reaction. The Michaelis constants K(m) for individual acceptors increased in the order [1-(3)H]XLLGXLLG-SR>XLLG-ANTS>[1-(3)H]XLLGol>[1-(3)H]XLLG>XLLG-AA. Catalytic efficiency (expressed as k(cat)/K(m)) with XLLG labeled with SR or FITC was 15 and 28 times, respectively, higher than with the tritium-labeled natural substrate [1-(3)H]XLLG. Comparison of the kinetic parameters found with acceptors labeled with different types of labels enables to select the most effective substrates for the high-throughput assays of XET.

Xyloglucan endotransglycosylases (XETs) from germinating nasturtium (Tropaeolum majus) seeds: isolation and characterization of the major form.

Five forms of xyloglucan endotransglycosylase/hydrolase (XTH) differing in their isoelectric points (pI) were detected in crude extracts from germinating nasturtium seeds. Without further fractionation, all five forms behaved as typical endotransglycosylases since they exhibited only transglycosylating (XET) activity and no xyloglucan-hydrolysing (XEH) activity. They all were glycoproteins with identical molecular mass, and deglycosylation led to a decrease in molecular mass from approximately 29 to 26.5 kDa. The major enzyme form having pI 6.3, temporarily designated as TmXET(6.3), was isolated and characterized. Molecular and biochemical properties of TmXET(6.3) confirmed its distinction from the XTHs described previously from nasturtium. The enzyme exhibited broad substrate specificity by transferring xyloglucan or hydroxyethylcellulose fragments not only to oligoxyloglucosides and cello-oligosaccharides but also to oligosaccharides derived from beta-(1,4)-d-glucuronoxylan, beta-(1,6)-d-glucan, mixed-linkage beta-(1,3; 1,4)-d-glucan and at a relatively low rate also to beta-(1,3)-gluco-oligosaccharides. The transglycosylating activity with xyloglucan as donor and cello-oligosaccharides as acceptors represented 4.6%, with laminarioligosaccharides 0.23%, with mixed-linkage beta-(1,3; 1,4)-d-gluco-oligosaccharides 2.06%, with beta-(1,4)-d-glucuronoxylo-oligosaccharides 0.31% and with beta-(1,6)-d-gluco-oligosaccharides 0.69% of that determined with xyloglucan oligosaccharides as acceptors. Based on the sequence homology of tryptic fragments with the sequences of known XTHs, the TmXET(6.3) was classified into group II of the XTH phylogeny of glycoside hydrolase family GH16.