Esterification and glycosydation of oligogalacturonides: examination of the reaction products using MALDI-TOF MS and HPAEC

Alebeek, G.J.W.M. van; Zabotina, Olga A.; Beldman, G.; Schols, Henk A.; Voragen, A.G.J.

doi:10.1016/s0144-8617(99)00207-6

Cited by 35 publications

(31 citation statements)

References 12 publications

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“…1, A and B, respectively). To determine the specificity of pectin lyase in more detail oligoGalpA were purified and derivatized as described before (11,12) (Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

“…⌬4,5 Unsaturated oligoGalpA were purified from a saponified pectin lyase digest of a 1% (w/v) 70% DM pectin solution. The oligoGalpA were separated on a source Q column and isolated as described before (12,15). The purity of the oligoGalpA (DP2-10) was determined using HPAEC, pH 12, equipped with PAD detection (12) and found to be in the range of 90 -99% based on area percentages.…”

Section: Methodsmentioning

confidence: 99%

“…Esterification of the (un)saturated oligoGalpA (DP3-10) was performed with acidic methanol (12). Partially methyl-esterified oligoGalpA were prepared by carefully controlled de-esterification through stepwise addition of 0.1 N sodium hydroxide at 4°C.…”

Section: Methodsmentioning

confidence: 99%

“…1B) or ethyl-esterified (Fig. 1C) (12). Subsequently, partially methyl-esterified oligoGalpA were prepared using limited alkaline de-esterification.…”

mentioning

confidence: 99%

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Mode of Action of Pectin Lyase A of Aspergillus nigeron Differently C6-substituted Oligogalacturonides

Alebeek¹,

Christensen²,

Schols³

et al. 2002

Journal of Biological Chemistry

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A thorough investigation of the mode of action of Aspergillus niger (4M-147) pectin lyase A (PLA) on differently C 6 -substituted oligogalacturonides is described. PLA appeared to be very specific for fully methyl-esterified oligogalacturonides: removal of the methyl-ester or changing the type of ester (ethyl esterification) or transamidation resulted in (almost) complete loss of conversion. The PLA activity increased with increasing length of the substrate up to a degree of polymerization (DP) of 8 indicating the presence of at least eight subsites on the enzyme. Product analysis demonstrated the formation of several ⌬4,5 unsaturated products and their saturated counterparts. The ⌬4,5 unsaturated trimer was the main product up to DP 8. For DP 9 and 10 ⌬4,5 unsaturated tetramer was the major product. Based upon the bond cleavage frequencies, a provisional subsite map was calculated, which supports the presence of eight subsites. By limited alkaline de-esterification of fully methyl-esterified pentamer and hexamer two sets of partially methyl-esterified pentamers (x and y methyl groups) and hexamers (a and b methyl groups) were prepared. Matrix-assisted laser desorption/ionization time of flight mass spectroscopy (MALDI-TOF MS) analysis demonstrated that the methyl-ester distribution was fully random. Using these partially methyl-esterified oligogalacturonides as substrates for PLA a 10-fold decrease in reaction rate was recorded compared with the fully methyl-esterified counterparts. Analysis of the methyl-ester distribution of the products showed that PLA tolerates carboxyl groups in the substrate binding cleft. At either subsite ؉2, ؉4, or ؊1 to ؊4 a free carboxyl group could be tolerated, whereas methyl-esters were obligatory at subsite ؉1 and ؉3. So PLA is capable to cleave the bond between a methyl-esterified and a nonesterified galacturonic acid residue, where the newly formed ⌬4,5 unsaturated non-reducing end residue always contains a methyl-ester.Pectin is found in the middle lamella of the primary cell wall of practically all higher plant tissues (1, 2). It is composed of a homogalacturonan backbone (smooth region) interrupted by heavily branched regions of rhamnogalacturonan (hairy region). Homogalacturonan consists of ␣-134-linked D-galacturonic acid (GalpA) 1 residues, which can be methyl-esterified (1-4).Both pectin lyase (EC 4.2.2.10) and pectate lyase (EC 4.2.2.2) are able to depolymerize the smooth region of pectin by ␤-elimination resulting in the formation of a ⌬4,5 unsaturated bond at the newly formed non-reducing end. Although pectate lyase cleaves between two ␣-1,4-D-GalpA residues and pectin lyase cleaves between two methyl-esterified ␣-1,4-D-GalpA residues, this distinction on substrate specificity is not strict. Pectate lyase is generally active on pectin with a moderate degree of methyl esterification (DM) (0 -50%), whereas pectin lyase activity often increases with increasing DM. The only discriminating property is that pectate lyases characterized to date need Ca 2ϩ ions for their acti...

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“…1, A and B, respectively). To determine the specificity of pectin lyase in more detail oligoGalpA were purified and derivatized as described before (11,12) (Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…1B) or ethyl-esterified (Fig. 1C) (12). Subsequently, partially methyl-esterified oligoGalpA were prepared using limited alkaline de-esterification.…”

mentioning

confidence: 99%

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Mode of Action of Pectin Lyase A of Aspergillus nigeron Differently C6-substituted Oligogalacturonides

Alebeek¹,

Christensen²,

Schols³

et al. 2002

Journal of Biological Chemistry

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“…HM lime pectin was prepared in the laboratory from lime pectin with the methanol/H 2 SO 4 method (Van Alebeek et al 2000).…”

Section: Microorganism Media Inocula and Culture Conditionsmentioning

confidence: 99%

Role of PPase-SE in Geotrichum klebahnii, a yeast-like fungus able to solubilize pectin

Rojas¹,

Cavalitto²,

Mignone³

et al. 2008

Electron. J. Biotechnol.

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Structural analysis of (methyl-esterified) oligogalacturonides using post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

et al. 2000

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The use of post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the structural analysis of ((partly) methyl-esterified) oligogalacturonides (oligoGalA) is described. The fragmentation behavior of purified (un)saturated oligoGalA (degree of polymerization 3-6), methyl-esterified and methyl-glycosydated oligoGalA was studied. General fragmentation patterns are described and used for the elucidation of the positions of methyl esters on partly methyl-esterified oligoGalA. This technique now permits the determination of the position of methyl esters or other substituents on pectic fragments, helping in understanding the mode of action of pectinolytic enzymes.

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Esterification and glycosydation of oligogalacturonides: examination of the reaction products using MALDI-TOF MS and HPAEC

Cited by 35 publications

References 12 publications

Mode of Action of Pectin Lyase A of Aspergillus nigeron Differently C6-substituted Oligogalacturonides

Mode of Action of Pectin Lyase A of Aspergillus nigeron Differently C6-substituted Oligogalacturonides

Role of PPase-SE in Geotrichum klebahnii, a yeast-like fungus able to solubilize pectin

Structural analysis of (methyl-esterified) oligogalacturonides using post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

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