Jacqueline Vigouroux

Isolation and partial characterization of feruloylated oligosaccharides from maize bran.

Maize bran contains phenolic acids [approximately 4% dry matter; mainly ferulic acid (Fe) and also diferulic acid], heteroxylans (approximately 50%), and cellulose (approximately 20%), but is devoid of lignin. Treatment of maize pericarp with 0.05 M trifluoroacetic acid at 100 degrees C for 2 h released approximately 90% of the heteroxylans and approximately 90% of the ferulic acid and its esters. After fractionation of the products with Amberlite XAD-2 and Sephadex LH-20 three main feruloylated oligosaccharides (F3-F7) were isolated. They represented approximately 30% of the ferulic acid, and approximately 2% of the neutral sugars contained in the hydrolysis supernatant. The compositions of F7, F6, and F3 were Fe-Ara (1:1), Fe-Ara-Xyl (1:1:1), and Fe-Ara-Xyl-Gal (1:1:1:1), respectively. The structures of the three oligomers were determined using chemical methods (methylation, acetalation, reduction) and 13C NMR spectroscopy: F7 was 5-O-(trans-feruloyl)-L-Araf;F6 was O-beta-D-Xyl p-(1-->2)-[5-O-(trans-feruloyl)-L-Araf]; and F3 was O-L-Gal p-(1-->4)-O-D-Xyl p-(1-->2)-[5-O-(trans-feruloyl)-L- Araf]. F7 has been previously isolated from other monocots especially from wheat bran and soluble arabinoxylans from wheat flour; this is the first report of feruloylated oligosaccharides F6 and F3. Our results suggest that these oligomers are side-chain constituents of heteroxylans in maize bran. Ferulic acid is probably partly responsible for the insolubility of heteroxylans by coupling polysaccharide chains through ferulic acid dimers.

Endo-β-1,4-d-galactanase from Aspergillus niger var. aculeatus: Purification and some properties

Abstract An endo-1,4-β- d -galactanase from the commercial preparation SP 249 (Novo Industri) originating from Aspergillus niger var. aculeatus was purified using affinity chromatography on laboratory cross-linked Sepharose 6B with divinyl sulfone, gel filtration on Sephadex G 75, and ion-exchange chromatography on DEAE-Sephadex A 50. Galactanase-apparent specific activity was increased 114-fold after purification and the enzyme is devoid of endopolygalacturonase, pectin-, pectate-lyase, arabinanase, β- d -galactosidase, and α- l -arabinofuranosidase activities. It migrates as a single band in sodium dodecyl sulfate (SDS) polyacrylamide-gel electrophoresis (PAGE) and has a molecular weight of 38 000. It is an acidic enzyme of pHi 2·8 containing a low amount of a putative isoenzyme of pHi 2·7 which also migrates as a minor band on PAGE without SDS. Enzymatic activity is optimum at pH 3·5–4·0 and at 50–55°C and the enzyme is most stable between ∼ pH 4·0–7·0 and below 30°C. It does not attack other galactans such as agarose, kappa- and iota-carrageenan nor type II arabinogalactans. Activity is inhibited by excess substrate and dissociation constants K D (0·6 mg ml −1 ) and K 1 D (10·7 mg ml −1 ), and maximal velocity V (8500 nkat) were obtained from Line-weaver-Burk and Khun plots. Endo-galactanase specific activity is 17·0 mkat mg −1 . Galactose and galactobiose are the end products of galactan hydrolysis.

Liquefaction of dulse (Palmaria palmata (L.) Kuntze) by a commercial enzyme preparation and a purified endo ,β-1,4-D-xylanase

Liquefaction of dry and freshPalmaria palmata by food grade enzyme preparations and a purified endo-β-1,4-D-xylanase was studied.The endo-β-1,4-D-xylanase (EC 3.2.1.8) was purified to homogeneity from a commercial food grade enzyme prepared fromAspergillus niger. It has a molecular weight of 22 500, a pI of 3.5, is inactive toward corn arabinoxylan,p-nitrophenyl-β-D-xylose, carboxymethyl cellulose but shows a weak activity toward microcrystalline cellulose. It hydrolyzes oat and dulse xylan equally well in seawater and deionized water essentially into xylose and xylobiose. It is stable between pH 5.5 to 9.0 and 0 to 30 °C and its activity is optimal at pH 4.5–5.5 and 40–60 °C. It has a Km of 2.2 and 2.8 mg ml-1 and Vmax of 3600 and 3900 nkat mg-1 of protein on oat and dulse xylan, respectively.Acetate buffer, deionized water and seawater alone extracted 62.6 to 64.5 % of the dry weight of dry dulse, but the use of commercial food grade enzyme preparations or the purified xylanase improved liquefaction to 81.2–87.1 %. Xylose and galactose were the only sugars present in the soluble extracts. Deionized and seawater extracted 58.8–52.7 and 39.1–42.2% of the dry weight of the fresh algae collected in fall and summer, respectively. Only galactose was found in the seawater extract, while some xylose with galactose were measured in the deionized water extract of the fresh autumn algal sample. Purified and crude xylanase improved liquefaction of fresh algae to 79.8–81.4 and 71.9–77.9% of the fresh dry weight (fall and summer, respectively) in deionized and seawater, respectively, and increased the xylose content of the soluble fractions. Polysaccharides in the soluble residues were composed of 1,3/1,4-linked xylose, 1-linked galactose (floridoside) and 1,4-linked glucose (cellulose) and contained essentially 1,4-linked xylose and 1,4-linked glucose in insoluble fractions obtained after enzymatic treatment.The use of xylanase-containing food grade enzyme preparations improves liquefaction ofPalmaria palmata, particularly from fresh alga. This study indicates that processing such as drying may modify markedly the solubility ofP. palmata cell wall polysaccharides, which would imply the existence of some organization and/or other components in the fresh cell wall that lower xylan solubility in seawater.

Preliminary characterization of a new exo-β-(1,4)-galactanase with transferase activity

As a prerequisite to the study of the fine chemical structure of the branched region of pectin, an exo-beta-(1,4)-galactanase was purified from a commercial preparation (Pectinex AR). Purification was carried out by precipitation with 70% saturated ammonium sulfate, preparative electrofocusing, anion-exchange chromatography and affinity chromatography on cross-linked alginate. Exogalactanase specific activity was 992 nkat mg-1 and the enzyme was devoid of beta-(1,3)- or beta-(1,6)-galactanase, arabinanase, beta-D-galactosidase and alpha-L-arabinofuranosidade activities. Residual exopolygalacturonase activity represented 2.9% of the galactanase activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing showed two close bands with molecular weights of 120,000 and 90,000 and pHi of 3.8 and 4.1, respectively. The enzyme acted in an exo manner and its activity was optimum at pH 3.5 and 60 degrees C. When incubated with galacto-oligosaccharides, new oligosaccharides with a higher degree of polymerization appeared, indicating the ability of the enzyme to transfer galactose residues.

Xylans Provide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat

Arabidopsis seed mucilage contains a population of RG-I with xylan side branches that mediate the adsorption of mucilage to cellulose microfibrils in vitro. Arabidopsis (Arabidopsis thaliana) seed coat epidermal cells produce large amounts of mucilage that is released upon imbibition. This mucilage is structured into two domains: an outer diffuse layer that can be easily removed by agitation and an inner layer that remains attached to the outer seed coat. Both layers are composed primarily of pectic rhamnogalacturonan I (RG-I), the inner layer also containing rays of cellulose that extend from the top of each columella. Perturbation in cellulosic ray formation has systematically been associated with a redistribution of pectic mucilage from the inner to the outer layer, in agreement with cellulose-pectin interactions, the nature of which remained unknown. Here, by analyzing the outer layer composition of a series of mutant alleles, a tight proportionality of xylose, galacturonic acid, and rhamnose was evidenced, except for mucilage modified5-1 (mum5-1; a mutant showing a redistribution of mucilage pectin from the inner adherent layer to the outer soluble one), for which the rhamnose-xylose ratio was increased drastically. Biochemical and in vitro binding assay data demonstrated that xylan chains are attached to RG-I chains and mediate the adsorption of mucilage to cellulose microfibrils. mum5-1 mucilage exhibited very weak adsorption to cellulose. MUM5 was identified as a putative xylosyl transferase recently characterized as MUCI21. Together, these findings suggest that the binding affinity of xylose ramifications on RG-I to a cellulose scaffold is one of the factors involved in the formation of the adherent mucilage layer.

Fractionation and structural characterization of LiCl-DMSO soluble hemicelluloses from tomato.

To prepare and explore the structure of native hemicellulose from tomato, extraction of the natively acetylated polysaccharides was achieved from partially depectinated cell walls by DMSO doped with LiCl. DEAE anion exchange chromatography of the LiCl-DMSO extract allowed the removal of residual acidic pectin and the isolation of acetylated glucuronoxylan. The hemicellulose neutral fraction from the anion exchanger was fractionated by size exclusion chromatography into xyloglucan (XyG) and galactoglucomannan (GgM) either as single major constituents or as mixtures of both. Residual hemicellulose in the cell wall was extracted by 4.0 M and not 1.0 M KOH. The fine structure of all LiCl-DMSO fractions and alkali extracts was assessed by coupling β-glucanase, β-mannanase and β-xylanase enzymatic degradations to the analysis of the resulting fragments by HPAEC and MALDI-TOF mass spectrometry. This approach revealed substitutions in part of the GgM fractions by pentose residues, presumably arabinose and/or xylose occurring in highly substituted block domains. It also demonstrated a different glucanase hydrolysis profile from 4.0 M KOH compared to LiCl-DMSO soluble fractions. The present extraction and purification scheme allow the recovery of several populations of acetylated hemicellulose families which emphasize the structural diversity and complexity of these polysaccharides.

Novel and diverse fine structures in LiCl-DMSO extracted apple hemicelluloses.

Hemicelluloses are key polysaccharides in the regulation of the mechanical properties of plant cell walls during organ development and in fruit texture. Their diverse compositions and structures are partially known, in particular with regard to their function in cell walls. To that end, apple hemicelluloses were sequentially extracted by DMSO doped by LiCl followed by potassium hydroxide. The weakly bounded hemicelluloses in the LiCl-DMSO soluble extract were fractionated by ion exchange (AEC) and size exclusion (SEC) chromatographies. The structure of all the extracts and fractions was established by enzymatic fingerprinting using β-glucanase, β-mannanase and β-xylanase. Molecular weight of the fraction was established by HPSEC. MS as well as HPAEC analyses of the enzyme digests revealed the remarkable diversity of apple hemicelluloses. Different xyloglucan (XyG), galactoglucomannan (GgM) and glucuronoarabinoxylan were isolated along the extraction and fractionation process. All LiCl-DMSO soluble fractions were acetyl-esterified. Besides, the LiCl-DMSO soluble XyG differed from the 4M KOH extracted one essentially on the basis of its molecular weight. At least two populations differing in their content and distribution of glucose and mannose composed GgM. Moreover, galactose ramifications occurred on mannose blocks in the glucose rich fraction. These results open the way for future studies on the complex structure-function relationship of hemicelluloses in plant cell walls.

Kinetic parameters of hydrolysis and transglycosylation catalyzed by an exo-β-(1,4)-galactanase

Abstract The action pattern and reaction mechanism of the exo-β-(1,4)-galactanase from Aspergillus niger var. aculeatus on galactooligosaccharides of degree of polymerization (dp) from 2–6 were investigated. The reaction was assayed at the optimum pH of both hydrolysis and transglycosylation, and HPLC analysis of the reaction mixtures was used to obtain kinetic parameters. Hydrolysis of oligosaccharides to galactose followed a Michaelis-Menten behavior whatever the dp. Catalytic activity was always higher for hydrolysis than for transglycosylation. At the optimum pH, the affinity of the enzyme increased for hydrolysis and decreased for transglycosylation with increasing dp. Transglycosylation was explained as a dependent binding of two similar molecules of substrates. The dependence of the kinetic parameters on the degree of polymerization of substrates was used to evaluate the subsite affinities. The active site of exogalactanase was concluded to possess three subsites with the catalytic site being located between subsites 1 and 2.

Induced mutations in tomato SlExp1 alter cell wall metabolism and delay fruit softening

Fruit ripening and softening are key traits for many fleshy fruit. Since cell walls play a key role in the softening process, expansins have been investigated to control fruit over ripening and deterioration. In tomato, expression of Expansin 1 gene, SlExp1, during fruit ripening was associated with fruit softening. To engineer tomato plants with long shelf life, we screened for mutant plants impaired in SlExp1 function. Characterization of two induced mutations, Slexp1-6_W211S, and Slexp1-7_Q213Stop, showed that SlExp1 loss of function leads to enhanced fruit firmness and delayed fruit ripening. Analysis of cell wall polysaccharide composition of Slexp1-7_Q213Stop mutant pointed out significant differences for uronic acid, neutral sugar and total sugar contents. Hemicelluloses chemistry analysis by endo-β-1,4-d-glucanase hydrolysis and MALDI-TOF spectrometry revealed that xyloglucan structures were affected in the fruit pericarp of Slexp1-7_Q213Stop mutant. Altogether, these results demonstrated that SlExp1 loss of function mutants yield firmer and late ripening fruits through modification of hemicellulose structure. These SlExp1 mutants represent good tools for breeding long shelf life tomato lines with contrasted fruit texture as well as for the understanding of the cell wall polysaccharide assembly dynamics in fleshy fruits.

Negative electrospray ionization mass spectrometry: a method for sequencing and determining linkage position in oligosaccharides from branched hemicelluloses

Xyloglucans of apple, tomato, bilberry and tamarind were hydrolyzed by commercial endo β-1-4-D-endoglucanase. The xylo-gluco-oligosaccharides (XylGos) released were separated on CarboPac PA 200 column in less than 15 min, and, after purification, they were structurally characterized by negative electrospray ionization mass spectrometry using a quadrupole time-of-flight (ESI-Q-TOF), a hybrid linear ion trap (LTQ)/Orbitrap and a hybrid quadrupole Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. In order to corroborate the fragmentation routes observed on XylGos, some commercial galacto-manno-oligosaccharides (GalMOs) and glucurono-xylo-oligosaccharides were also studied. The fragmentation pathways of the ionized GalMos were similar to those of XylGos ones. The product ion spectra were mainly characterized by prominent double cleavage (D) ions corresponding to the entire inner side chains. The directed fragmentation from the reducing end to the other end was observed for the main glycosylated backbone but also for the side-chains, allowing their complete sequencing. Relevant cross-ring cleavage ions from (0,2)X(j)-type revealed to be diagnostic of the 1-2-linked- glycosyl units from XylGos together with the 1-2-linked glucuronic acid unit from glucuronoxylans. Resonant activation in the LTQ Orbitrap allowed not only determining the type of all linkages but also the O-acetyl group location on fucosylated side-chains. Moreover, the fragmentation of the different side chains using the MS(n) capabilities of the LTQ/Orbitrap analyzer also allowed differentiating terminal arabinosyl and xylosyl substituents inside S and U side-chains of XylGos, respectively. The CID spectra obtained were very informative for distinction of isomeric structures differing only in their substitution pattern. These features together makes the fragmentation in negative ionization mode a relevant and powerful technique useful to highlight the subtle structural changes generally observed during the development of plant organs such as during fruit ripening and for the screening of cell wall mutants with altered hemicellulose structure.