Amidation of methyl-esterified oligogalacturonides: examination of the reaction products using MALDI-TOF MS

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

doi:10.1016/s0144-8617(00)00330-1

Cited by 15 publications

(12 citation statements)

References 14 publications

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“…Amidated saturated oligoGalpA were prepared from methyl-esterified oligoGalpA using either ammonia or methylamine as described before (13). A degree of methyl amidation of 95 was reached using methylamine, whereas saturated oligoGalpA could be amidated up to a degree of 60 using ammonia.…”

Section: Methodsmentioning

confidence: 99%

“…Whenever appropriate, an extra 0.2 l of 10 mM NaCl was added to the sample to improve ionization in the positive mode. The samples were analyzed with the Voyager DE TM -RP MALDI-TOF MS (Perseptive Biosystems Inc., Framingham, MA) as described before (13).…”

Section: Methodsmentioning

confidence: 99%

“…Partly methyl-esterified (GalpA) 5 and (GalpA) 6 were prepared by controlled alkaline de-esterification of the fully methyl-esterified equivalent. The DM was determined using MALDI-TOF MS as described before (13). The DM was lowered to 80 and 74 for the pentamer and to 86 and 79 for the hexamer, respectively (Table IV), which corresponds to 1-1.3 free carboxyl groups per molecule on average.…”

Section: Pla Mode Of Action On Partly Methyl-esterifiedmentioning

confidence: 99%

“…1D) or methylamine (Fig. 1E) were made (13). Here we report the activity of PLA on the various substrates and the mode of action on those substrates where significant activity was observed carried out by newly developed techniques like high performance anion-exchange chromatography (HPAEC) and mass spectrometry (14,15).…”

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confidence: 93%

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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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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Pla Mode Of Action On Partly Methyl-esterifiedmentioning

confidence: 99%

mentioning

confidence: 93%

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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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“…Using ammonia up to 70% of the methyl-esters could be arnidated, whereas amidation using methylamine resulted in conversions of 95-97% [19]. The amidated oligoGalpA, arnide-GalpA and 6-N-methyl GalpA, resemble GalpA and 6-0-methyl-GalpA, respectively, and may be able to rnimic these compounds in their digestibility.…”

Section: Preparation Of Model Substrates Of Defined Dpmentioning

confidence: 99%

Mode of Action Analysis and Structure-Function Relationships of Aspergillus Niger Pectinolytic Enzymes

Benen¹,

Alebeek²,

Voragen³

et al. 2003

Advances in Pectin and Pectinase Research

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As a result of the cloning and overexpression of individual Aspergillus niger pectinase genes the corresponding enzymes are now readily available for detailed characterisation using defmed substrates, including derivatised oligogalacturonides. In this chapter we give an overview of the results of these studies for a pectate and pectine lyase and a polygalacturonase. Detailed kinetic studies on pectate lyase have revealed that the substrate binds to the enzyme as a Ca 2 +-substrate complex, thus explaining the absolute requirement of pectate lyases for Ca 2 +-ions. Using derivatised oligogalacturonides it was shown that pectin lyase A cannot cleave substrates that are: 1) fully de-esterified, 2) fully ethyl-esterified, 3) fully amidated, and 4) fully methylamidated. Furthermore, partial de-esterification of fully methyl esterified oligogalacturonides, one methyl group on average, resulted in a strong reduction of the catalytic efficiency and a change in the preferred binding mode. From these studies it can be concluded that pectin lyase A is highly specific for pectin with a high degree of methylesterification. Endopolygalacturonases (endoPGs) were thouroughly characterised kinetically which allowed to ca1culate subsite affinities. Site directed mutagenesis of endoPGII in combination with the 3D-structure allowed to pin-point amino acids involved in catalysis as well as amino acids that are important for binding of the substrate. At subsite -5 the presence of an arginine appeared critical for displaying multiple attack attack on a single chain (processive) behavior.

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Pectin: A new perspective from the biorefinery standpoint

Ciriminna

Hernández

et al. 2015

Biofuels Bioprod Bioref

178

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Perspective besides having a number of fundamental biological functions (signaling, cell proliferation, diff erentiation, and cell adhesion). 1 Th e rhamnose-rich regions of pectin chains enhance molecular interactions between cells and the polysaccharide, while the branched galactose-rich hairy regions promote the formation of entangled structures. 3 From a functional viewpoint, pectin is a hydrocolloid, namely a substance capable of trapping water and forming gels at low concentration. Being soluble in water and thanks to its excellent health and safety profi le, pectin is widely used for adding a desirable texture to food and beverages. In 2010, the European Food Safety Authority recognized the scientifi c validity of nutrition and health claims regarding pectin as a nutritional supplement in the reduction of post-prandial glycemic responses and the maintenance of normal blood cholesterol concentrations. It also increases satiety leading to a reduction in energy intake. 4Abstract: Pectin is a natural product of central importance in the emerging biorefi nery that uses fruit waste as a raw material. Generally obtained from lemon peel or from apple pomace, pectin is mainly used as a thickener and a stabilizer in the food industry. Due to its unique chemical properties and biochemical activity, however, its use is growing in many other sectors. Providing a unifi ed view of the main research and utilization trends, this study identifi es open opportunities toward larger scale production of a valued biopolymer that, we argue, will shortly emerge as a central product of the biorefi nery, and of the citrus-based biorefi nery in particular.

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Amidation of methyl-esterified oligogalacturonides: examination of the reaction products using MALDI-TOF MS

Cited by 15 publications

References 14 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

Mode of Action Analysis and Structure-Function Relationships of Aspergillus Niger Pectinolytic Enzymes

Pectin: A new perspective from the biorefinery standpoint

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