Characterisation of cell-wall polysaccharides from mandarin segment membranes
Coll-Almela, L. ; Saura-Lopez, D. ; Laencina-Sanchez, J. ; Schols, H.A. ; Voragen, A.G.J. ; Ros-García, J.M. - \ 2015
Food Chemistry 175 (2015). - ISSN 0308-8146 - p. 36 - 42.
hairy ramified regions - cross-flow filtration - pectolytic enzyme - citrus-fruit - pectins - degradation - extraction - skin - rhamnogalacturonase - populations
In an attempt to develop a process of enzymatic peeling of mandarin segments suitable for use on an industrial scale, the cell wall fraction of the segment membrane of Satsuma mandarin fruits was extracted to obtain a chelating agent-soluble pectin fraction (ChSS), a dilute sodium hydroxide-soluble pectin fraction (DASS), a 1 M sodium hydroxide-soluble hemicellulose fraction (1MASS), a 4 M sodium hydroxide-soluble hemicellulose fraction (4MASS) and a cellulose-rich residue (3.1, 0.9, 0.4, 0.7 and 1.6% w/w of fresh membrane, respectively). The ChSS pectin consisted mainly of galacturonic acid followed by arabinose and galactose. The DASS fraction contained less galacturonic acid and more neutral sugars than ChSS. Eighty-nine percent of the galacturonic acid present in the segment membranes was recovered in the above two pectin fractions. The two hemicellulosic fractions consisted of two different molecular weight populations, which also differed in their sugar composition. Arabinose, xylose, mannose, galactose and glucose were the main sugar constituents of these hemicellulose fractions. In addition to an (arabino)xylan and a xyloglucan, the presence of an arabinogalactan is suggested by the sugar composition of both hemicelluloses. The pectin fractions were also characterised by their degradability by the pectic enzymes polygalacturonase, pectinmethylesterase and rhamnogalacturonan hydrolase. However the degree of degradation of the pectin fractions by enzymes differed, and the amount of the polymeric materials resistant to further degradation and the oligomeric products also differed. Using pectic enzymes it is possible to obtain peeled mandarin segments ready to eat or for canning.
A Bacillus licheniformis pectin acetylesterase is specific for homogalacturonans acetylated at O-3
Remoroza, C.A. ; Wagenknecht, M. ; Buchholt, H.C. ; Moerschbacher, B.M. ; Schols, H.A. ; Gruppen, H. - \ 2014
Carbohydrate Polymers 107 (2014). - ISSN 0144-8617 - p. 85 - 93.
hairy ramified regions - sugar-beet pectin - erwinia-chrysanthemi 3937 - rhamnogalacturonan acetylesterase - esterification - identification - deacetylation - aspergillus - extraction - oligomers
A recombinant acetylesterase from Bacillus licheniformis DSM13, belonging to carbohydrate esterase family 12, was purified and biochemically characterized. The purified enzyme, termed BliPAE, was capable of deacetylating acetylated pectins, e.g. sugar beet pectin (SBP). Contrary to its provisional annotation as rhamnogalacturonan acetylesterase, the enzyme specifically removed acetyl groups from the homogalacturonan region classifying it as a PAE. The recombinant enzyme has a molecular mass of 26.7 kDa and shows optimal activity at pH 8.0 and 50 °C. It is stable in the range pH 5.0–7.0 and below 50 °C. Methylesterification of the galacturonic acid (GalA) moieties reduces the deacetylation efficacy of BliPAE. The enzyme efficiently removes acetyl groups from SBPs with low degree of methylesterification (DM) 9-30, releasing about 75% of the acetyl groups present in the homogalacturonan. Furthermore, 1H NMR of polymer and LC-HILIC-MSn after endo-PGII and PL degradation were used to structurally characterize the BliPAE-modified pectins. The results show that BliPAE removes acetyl groups specifically when substituted at the O-3 position of GalA moieties.
Affecting osteoblastic responses with in vivo engineered potato pectin fragments
Kokkonen, H. ; Verhoef, R.P. ; Kauppinen, K. ; Muhonen, V. ; Jorgensen, B. ; Damager, I. ; Schols, H.A. ; Morra, M. ; Ulvskov, P. ; Tuukkanen, J. - \ 2012
Journal of Biomedical Materials Research Part A 100A (2012)1. - ISSN 1549-3296 - p. 111 - 119.
enzymatically-tailored pectins - hairy ramified regions - plant-cell wall - rhamnogalacturonan-i - citrus pectin - differentiation - vitro - mineralization - proliferation - architecture
Pectins, complex plant-derived polysaccharides, are novel candidates for biomaterial nanocoatings. Pectic rhamnogalacturonan-I regions (RG-I) can be enzymatically treated to so-called modified hairy regions (MHR). We surveyed the growth and differentiation of murine preosteoblastic MC3T3-E1 cells on Petri dishes coated with RG-Is from native or genetically engineered potato tubers. Uncoated tissue culture polystyrene (TCPS) and aminated (AMI) dishes served as controls. MHRPTR_GAL sample was depleted of galactose (9 mol % galactose; 23 mol % arabinose) and MHRPTR_ARA of arabinose (61 mol % galactose; 6 mol % arabinose). Wild-type (modified hairy region from potato pectin (MHRP)_WT) fragment contained default amounts (58 mol % galactose; 13 mol % arabinose) of both sugars. Focal adhesions (FAs) indicating cellular attachment were quantified. Reverse transcriptase polymerase chain reaction (RT-PCR) of alkaline phosphatase and osteocalcin genes indicating osteoblastic differentiation was performed along with staining the produced calcium with tetracycline as an indicator of osteoblastic differentiation. Osteoblasts proliferated on all the samples to some extent. The control surfaces performed better than any of the pectin samples, of which the MHRP_WT seemed to function best. FA length was greater on MHRPTR_GAL than on other pectin samples, otherwise the mutants did not significantly deviate. RT-PCR results indicate that differences between the samples at the gene expression level might be even subtler. However, tetracycline-stained calcium-containing mineral was detected merely only on uncoated TCPS. These results indicate the possibility to affect bone cell growth with in vivo-modified pectin fragments, consecutively providing information on the significance of certain monosaccharides on the biocompatibility of these polysaccharides.
Introducing porous graphitized carbon liquid chromatography with evaporative light scattering and mass spectrometry detection into cell wall oligosaccharide analysis
Westphal, Y. ; Schols, H.A. ; Voragen, A.G.J. ; Gruppen, H. - \ 2010
Journal of Chromatography. A, Including electrophoresis and other separation methods 1217 (2010)5. - ISSN 0021-9673 - p. 689 - 695.
treated eucalyptus wood - hairy ramified regions - capillary-electrophoresis - black-currants - polysaccharides - pectin - xylogalacturonan - quantification - nomenclature - ionization
Separation and characterization of complex mixtures of oligosaccharides is quite difficult and, depending on elution conditions, structural information is often lost. Therefore, the use of a porous-graphitized-carbon (PGC)-HPLC-ELSD-MSn-method as analytical tool for the analysis of oligosaccharides derived from plant cell wall polysaccharides has been investigated. It is demonstrated that PGC-HPLC can be widely used for neutral and acidic oligosaccharides derived from cell wall polysaccharides. Furthermore, it is a non-modifying technique that enables the characterization of cell wall oligosaccharides carrying, e.g. acetyl groups and methylesters. Neutral oligosaccharides are separated based on their size as well as on their type of linkage and resulting 3D-structure. Series of the planar ß-(1,4)-xylo- and ß-(1,4)-gluco-oligosaccharides are retained much more by the PGC material than the series of ß-(1,4)-galacto-, ß-(1,4)-manno- and a-(1,4)-gluco-oligosaccharides. Charged oligomers such as a-(1,4)-galacturonic acid oligosaccharides are strongly retained and are eluted only after addition of trifluoroacetic acid depending on their net charge. Online-MS-coupling using a 1:1 splitter enables quantitative detection of ELSD as well as simple identification of many oligosaccharides, even when separation of oligosaccharides within a complex mixture is not complete. Consequently, PGC-HPLC-separation in combination with MS-detection gives a powerful tool to identify a wide range of neutral and acidic oligosaccharides derived from various cell wall polysaccharides.
Inhibition of LPS-induced proinflammatory responses of J774.2 macrophages by immobilized enzymatically tailored pectins
Gallet, M. ; Vayssade, M. ; Morra, M. ; Verhoef, R.P. ; Perrone, S. ; Cascardo, G. ; Vigneron, P. ; Schols, H.A. ; Nagel, M.D. - \ 2009
Acta Biomaterialia 5 (2009). - ISSN 1742-7061 - p. 2618 - 2622.
hairy ramified regions - glinus-oppositifolius - rhamnogalacturonan-i - cell - polysaccharides - adhesion - activation - polymer - plant - vitro
The surface of an implant device can be modified by immobilizing biological molecules on it to improve its integration into the host tissue. We have previously demonstrated that enzymatically tailored plant pectins are promising nanocoatings for biomaterials. This study investigates whether a coating of modified hairy region (rhamnogalacturonan-I) from apple pectin (MHR-a) which has anti-adhesive properties can inhibit the generation of inflammatory mediators by lipopolysaccharide (LPS)-activated macrophages. For that purpose, J774.2 murine macrophages were cultured for 24 h on MHR-a-coated Petri dishes and tissue culture polystyrene controls, with and without LPS. Cell morphology, cell growth, nitrite and TNF-a secretion were studied. The results indicate that MHR-a coating inhibits the LPS-induced activation of macrophages.
Okra pectin contains an unusual substitution of its rhamnosyl residues with acetyl and alpha-linked galactosyl groups
Sengkhamparn, N. ; Bakx, E.J. ; Verhoef, R.P. ; Schols, H.A. ; Sajjaanantakul, T. ; Voragen, A.G.J. - \ 2009
Carbohydrate Research : an international journal 344 (2009)14. - ISSN 0008-6215 - p. 1842 - 1851.
hairy ramified regions - fat ingredient substitute - rhamnogalacturonan oligomers - structural-characterization - galacturonic acid - oligosaccharides - polysaccharides - degradation - substances - products
The okra plant, Abelmoschus esculentus (L.) Moench, a native plant from Africa, is now cultivated in many other areas such as Asia, Africa, Middle East, and the southern states of the USA. Okra pods are used as vegetables and as traditional medicines. Sequential extraction showed that the Hot Buffer Soluble Solids (HBSS) extract of okra consists of highly branched rhamnogalacturonan (RG) I containing high levels of acetyl groups and short galactose side chains. In contrast, the CHelating agent Soluble Solids (CHSS) extract contained pectin with less RG I regions and slightly longer galactose side chains. Both pectic populations were incubated with homogeneous and well characterized rhamnogalacturonan hydrolase (RGH), endo-polygalacturonase (PG), and endo-galactanase (endo-Gal), monitoring both high and low molecular weight fragments. RGH is able to degrade saponified HBSS and, to some extent, also non-saponified HBSS, while PG and endo-Gal are hardly able to degrade either HBSS or saponified HBSS. In contrast, PG is successful in degrading CHSS, while RGH and endo-Gal are hardly able to degrade the CHSS structure. These results point to a much higher homogalacturonan (HG) ratio for CHSS when compared to HBSS. In addition, the CHSS contained slightly longer galactan side chains within its RG I region than HBSS. Matrix-assisted laser desorption ionization-time of flight mass spectrometry indicated the presence of acetylated RG oligomers in the HBSS and CHSS enzyme digests and electron spray ionization-ion trap-mass spectrum showed that not only galacturonosyl residues but also rhamnosyl residues in RG I oligomers were O-acetylated. NMR spectroscopy showed that all rhamnose residues in a 20 kDa HBSS population were O-acetylated at position O-3. Surprisingly, the NMR data also showed that terminal a-linked galactosyl groups were present as neutral side chain substituents. Taken together, these results demonstrate that okra contained RG I structures which have not been reported before for pectic RG I.
Characterisation of cell wall polysaccharides from okra (Abelmoschus esculentus (L.) Moench)
Sengkhamparn, N. ; Verhoef, R.P. ; Schols, H.A. ; Sajjaanantakul, T. ; Voragen, A.G.J. - \ 2009
Carbohydrate Research : an international journal 344 (2009)14. - ISSN 0008-6215 - p. 1824 - 1832.
fat ingredient substitute - hairy ramified regions - europaea cv koroneiki - sugar-beet pectins - structural-characterization - oligosaccharides - acid - rhamnogalacturonan - xyloglucan - chromatography
Okra pods are commonly used in Asia as a vegetable, food ingredient, as well as a traditional medicine for many different purposes; for example, as diuretic agent, for treatment of dental diseases and to reduce/prevent gastric irritations. The healthy properties are suggested to originate from the high polysaccharide content of okra pods, resulting in a highly viscous solution with a slimy appearance when okra is extracted with water. In this study, we present a structural characterisation of all major cell wall polysaccharides originating from okra pods. The sequential extraction of okra cell wall material yielded fractions of soluble solids extractable using hot buffer (HBSS), chelating agent (CHSS), dilute alkaline (DASS) and concentrated alkaline (CASS). The HBSS fraction was shown to be rich in galactose, rhamnose and galacturonic acid in the ratio 1.3:1:1.3. The degree of acetylation is relatively high (DA = 58) while the degree of methyl esterification is relatively low (DM = 24). The CHSS fraction contained much higher levels of methyl esterified galacturonic acid residues (63% galacturonic acid; DM = 48) in addition to minor amounts of rhamnose and galactose. The ratio of galactose to rhamnose to galacturonic acid was 1.3:1.0:1.3 and 4.5:1.0:1.2 for HBSS and CHSS, respectively. These results indicated that the HBSS and CHSS fractions contain rhamnogalacturonan type I next to homogalacturonan, while the latter is more prevailing in CHSS. Also the DASS fraction is characterised by high amounts of rhamnose, galactose, galacturonic acid and some arabinose, indicating that rhamnogalacturonan I elements with longer arabinose- and galactose-rich side chains were part of this fraction. Partial digestion of HBSS and CHSS by pectin methyl esterase and polygalacturonase resulted in a fraction with a lower Mw and lower viscosity in solution. These samples were subjected to NMR analysis, which indicated that, in contrast to known RG I structure, the acetyl groups in HBSS are not located on the galacturonic acid residues, while for CHSS only part of the acetyl groups are located on the RG I galacturonic acid residues. The CASS fraction consisted of XXXG-type xyloglucan and 4-methylglucuronoxylan as shown by their sugar (linkage) composition and enzymatic digestion.
Fingerprinting complex pectins by chromatographic separation combined with ELISA detection
Verhoef, R.P. ; Lu, Y. ; Knox, J.P. ; Voragen, A.G.J. ; Schols, H.A. - \ 2009
Carbohydrate Research : an international journal 344 (2009)14. - ISSN 0008-6215 - p. 1808 - 1817.
polysaccharide rhamnogalacturonan-ii - enzymatically-tailored pectins - hairy ramified regions - cell-walls - plant-cell - arabinogalactan-proteins - extracellular polysaccharides - monoclonal-antibodies - structural features - galacturonic acid
Enzyme-resistant pectin or modified hairy regions were subjected to size exclusion (HPSEC) and weak anion exchange (WAX) chromatography. Fractions collected after separation were tested for the presence of different pectic epitopes using the monoclonal antibodies LM2, LM5, LM6, and JIM7. Separation by HPSEC showed that based on molecular weight the different epitopes were restricted to distinct molecular weight populations. WAX chromatography resulted in an even better separation of the different pectic epitopes present. A clear separation between arabino galactan type II epitopes and the RG I side chains, (1,5)-a-l-arabinan and (1,4)-ß-d-galactan, could be established. Arabinogalactan type II was found in the first populations eluting off the WAX column. The observations made within the ELISA assays of the collected fractions could be confirmed by determination of the sugar composition of the individual populations obtained. The sugar composition of the AGII positive populations eluting off the WAX column shows the presence of significant amounts of rhamnose and galacturonic acid. Together with the delay on an anion exchanger, this observation indicates a possible linkage between RGI and AGII. The volume of the individual fractions collected provides enough material for a maximum of 20 different antibodies to be tested from one analytical separation.
Pectin, a versatile polysaccharide present in plant cell walls
Voragen, A.G.J. ; Coenen, G.J. ; Verhoef, R.P. ; Schols, H.A. - \ 2009
Structural Chemistry 20 (2009)2. - ISSN 1040-0400 - p. 263 - 275.
hairy ramified regions - sugar-beet pectins - anion-exchange chromatography - flight mass-spectrometry - galacturonic acid oligomers - host-pathogen interactions - cultured sycamore cells - rhamnogalacturonan-i - side-chains - citrus pectin
Pectin or pectic substances are collective names for a group of closely associated polysaccharides present in plant cell walls where they contribute to complex physiological processes like cell growth and cell differentiation and so determine the integrity and rigidity of plant tissue. They also play an important role in the defence mechanisms against plant pathogens and wounding. As constituents of plant cell walls and due to their anionic nature, pectic polysaccharides are considered to be involved in the regulation of ion transport, the porosity of the walls and in this way in the control of the permeability of the walls for enzymes. They also determine the water holding capacity. The amount and composition of pectic molecules in fruits and vegetables and other plant produce strongly determine quality parameters of fresh and processed food products. Pectin is also extracted from suitable agro-by-products like citrus peel and apple pomace and used in the food industry as natural ingredients for their gelling, thickening, and stabilizing properties. Some pectins gain more and more interest for their health modulating activities. Endogenous as well as exogenous enzymes play an important role in determining the pectic structures present in plant tissue, food products, or ingredients at a given time. In this paper functional and structural characteristics of pectin are described with special emphasis on the structural elements making up the pectin molecule, their interconnections and present models which envisage the accommodation of all structural elements in a macromolecule. Attention is also given to analytical methods to study the pectin structure including the use of enzymes as analytical tools.
Modulating in vitro bone cell and macrophage behavior by immobilized enzymatically tailored pectins
Bussy, C. ; Verhoef, R.P. ; Haeger, A. ; Morra, M. ; Duval, J.L. ; Vigneron, P. ; Bensoussan, A. ; Velzenberger, E. ; Cascardo, G. ; Cassinelli, C. ; Schols, H.A. ; Knox, J.P. ; Nagel, M.D. - \ 2008
Journal of Biomedical Materials Research Part A 86a (2008)3. - ISSN 1549-3296 - p. 597 - 606.
hairy ramified regions - osteoblast adhesion - extracellular-matrix - surface modification - synthetic peptides - rhamnogalacturonan - growth - polysaccharides - biomaterials - activation
Previous work has reported the results of a multidisciplinary effort producing a proof-of-concept on the use of pectic polysaccharides in the surface modification of medical devices. This study was designed to learn more about the capability of engineered rhamnogalacturonan-I (RG-I) fractions of apple pectin to control bone cell and macrophage behavior. Thermanox® or polystyrene Petri dishes were surface modified with two different modified hairy regions (MHRs) obtained by different enzymatic liquefaction processes of apples differing in relative amounts and lengths of their neutral side chains: (long-haired) MHR- and (short-haired) MHR-B. Bone explants from 14-day-old chick embryos were cultured for 14 days on both pectic substrata. MHR-B promoted cell migration and differentiation, MHR- did not. On MHR-, J774.2 macrophages grew well, their percentage in G1 phase was decreased and in S phase increased, and they did not secrete either proinflammatory-cytokines or nitrites. Contrasting results were gained from macrophages on MHR-B, except for nitrite secretion. Thus, we conclude that coatings from tailored pectins show different biological activities in vitro and are potential innovative candidates for improving the biocompatibility of medical devices in various applications. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res, 2008
Enzymatically-tailored pectins differentially influence the morphology, adhesion, cell cycle progression and survival of fibroblasts
Nagel, M.D. ; Verhoef, R.P. ; Schols, H.A. ; Morra, M. ; Knox, J.P. ; Ceccone, G. ; Volpe, C.D. ; Vigneron, P. ; Bussy, C. ; Gallet, M. ; Velzenberger, E. ; Vayssade, M. ; Cascardo, G. ; Cassinelli, C. ; Haeger, A. ; Gilliland, D. ; Liakos, I. ; Rodrigues-Valverde, M. ; Siboni, S. - \ 2008
Biochimica et Biophysica Acta. General subjects 1780 (2008)7-8. - ISSN 0304-4165 - p. 995 - 1003.
hairy ramified regions - growth-factor receptors - surface-chemistry - fibronectin conformation - arabinogalactan-proteins - monoclonal-antibodies - human keratinocytes - integrin binding - adsorbed fibronectin - rhamnogalacturonan-i
Improved biocompatibility and performance of biomedical devices can be achieved through the incorporation of bioactive molecules on device surfaces. Five structurally distinct pectic polysaccharides (modified hairy regions (MHRs)) were obtained by enzymatic liquefaction of apple (MHR-B, MHR-A and MHR-), carrot (MHR-C) and potato (MHR-P) cells. Polystyrene (PS) Petri dishes, aminated by a plasma deposition process, were surface modified by the covalent linking of the MHRs. Results clearly demonstrate that MHR-B induces cell adhesion, proliferation and survival, in contrast to the other MHRs. Moreover, MHR- causes cells to aggregate, decrease proliferation and enter into apoptosis. Cells cultured in standard conditions with 1% soluble MHR-B or MHR- show the opposite behaviour to the one observed on MHR-B and --grafted PS. Fibronectin was similarly adsorbed onto MHR-B and tissue culture polystyrene (TCPS) control, but poorly on MHR-. The Fn cell binding site (RGD sequence) was more accessible on MHR-B than on TCPS control, but poorly on MHR-. The disintegrin echistatin inhibited fibroblast adhesion and spreading on MHR-B-grafted PS, which suggests that MHRs control fibroblast behaviour via serum-adhesive proteins. This study provides a basis for the design of intelligently-tailored biomaterial coatings able to induce specific cell functions.
CE-MSn of complex pectin-derived oligomers
Coenen, G.J. ; Kabel, M.A. ; Schols, H.A. ; Voragen, A.G.J. - \ 2008
Electrophoresis 29 (2008)10. - ISSN 0173-0835 - p. 2101 - 2111.
anion-exchange chromatography - capillary zone electrophoresis - trap mass-spectrometry - hairy ramified regions - maldi-tof ms - rhamnogalacturonan-i - 8-aminonaphthalene-1,3,6-trisulfonic acid - galacturonic acid - apple pectin - oligosaccharides
As pectin molecules are too large and heterogeneous to analyze as a whole, the polymer is usually degraded to smaller oligomers, which are often analyzed by high-performance anion exchange chromatography (HPAEC). However, the high salt concentration necessary to elute pectin oligomers by HPAEC is incompatible with online mass detection. To overcome such a disadvantage, a CE-IT-MS system was set up to further elucidate the fine structure of charged oligosaccharides. An effective separation of differently substituted galacturonic acid containing oligomers was obtained by low-pH CE-LIF analysis. By adapting the buffer and capillary online MS detection was enabled. Moreover, with MS/MS it was possible to localize sugar residues' substitutions. With this combined CE-MS approach LIF electropherograms of xylogalacturonan and rhamnogalacturonan I digests could be annotated. The method was further exemplified by a complex oligomer mixture of acid hydrolyzed apple pectin, which was separated and characterized by CE-MSn. Oligomers present in low amounts could be localized by their corresponding m/z, as was demonstrated by selected mass range representation.
Identification of the connecting linkage between homo- or xylogalacturonan and rhamnogalacturonan type I
Coenen, G.J. ; Bakx, E.J. ; Verhoef, R.P. ; Schols, H.A. ; Voragen, A.G.J. - \ 2007
Carbohydrate Polymers 70 (2007)2. - ISSN 0144-8617 - p. 224 - 235.
hairy ramified regions - cell-wall polysaccharides - flight mass-spectrometry - pectic polysaccharide - structural features - rhamnose residues - black-currants - cross-linking - apple pectin - side-chain
Pectin is of interest both as cell wall component and as food additive. The precise chemical structure of pectin remains under debate, although the structural elements of pectin are rather well described. In order to get more insight in the inter linkage between the various structural elements, apple pectin modified hairy regions were degraded by controlled acid hydrolysis. From the degradation products oligomeric fragments were selected which could represent interconnection points, and these oligomers were characterized using LC¿MS and NMR approaches. It was shown that the oligomers GalA3Rha1, GalA4Rha2, and GalA5Rha3 consisted out of a homogalacturonan and a rhamnogalacturonan type I segment connected via a GalAp¿-(1 ¿ 2) Rhap linkage. In addition, a GalA6Rha3Xyl1 oligomer was identified, which consisted out of a xylogalacturonan and a rhamnogalacturonan type I segment. These oligomers indicated that in apple pectin both homogalacturonan and xylogalacturonan were covalently linked to rhamnogalacturonan type I. With these new insights, currently used pectin models were refined. Keywords: Homogalacturonan; Xylogalacturonan; Rhamnogalacturonan I; Pectin model; Covalent linkage; Pectin structure
Cell wall polysaccharides in black currants and bilberries-characterisation in berries, juice, and press cake
Hilz, H. ; Bakx, E.J. ; Schols, H.A. ; Voragen, A.G.J. - \ 2005
Carbohydrate Polymers 59 (2005)4. - ISSN 0144-8617 - p. 477 - 488.
hairy ramified regions - rhamnogalacturonan-ii - pectic polysaccharides - dietary fiber - chromatography - substances - fractions - cellulose - fruit - ca2+
Cell wall polysaccharides from black currants and bilberries were characterised in three approaches. First, compositions of skin, pulp, and seeds show the distribution of polysaccharides over these tissues. A sequential extraction of cell wall material with different aqueous extractants informs about the extractability of the different polysaccharides, viz. pectins, hemicellulose, and cellulose. Finally, by isolation of cell wall polysaccharides from juice and press cakes obtained by the conventional juice manufacturing. The polysaccharide distribution was followed during juice processing. The main difference between bilberries and black currants is the dominant sugar residue in seeds: mannose for black currants and xylose for bilberries. Most of the hemicellulolytic sugars and cellulose can be found back in the press cake. The sugar composition of the press cake is similar to the composition of the residue after sequential extraction. Black currants contain more pectic sugars than bilberries. Consequently, a commercial enzyme used during processing releases more pectic material into the juice
Degradation of differently substituted xylogalacturonans by endoxylogalacturonan hydrolase and endopolygalacturonases.
Beldman, G. ; Vincken, J.P. ; Schols, H.A. ; Meeuwsen, P.J.A. ; Herweijer, M.A. ; Voragen, A.G.J. - \ 2003
Biocatalysis and Biotransformation 21 (2003)4/5. - ISSN 1024-2422 - p. 189 - 198.
hairy ramified regions - aspergillus-niger - pectins - enzyme - rhamnogalacturonase - polysaccharides - chromatography - oligomers - sugars
A method was developed to make xylogalacturonans (XGAs) with different degrees of xylosylation from gum tragacanth (XGA-25, XGA-29, XGA-35 and XGA-47), using alkali treatment at 4degreesC and acid treatment at 100degreesC. Ester linkages as well as fucose and arabinose substituents could selectively be removed by this procedure. Galactosyl- and xylosyl-linkages appeared to be more stable, while some backbone degradation of the galacturonan took place upon prolonged acid treatment. Using XGA-35, endoxylogalacturonan hydrolase (XGH) from Aspergillus tubingensis , expressed in Kluyveromyces lactis , was characterised with respect to kinetic parameters, temperature and pH effects. XGA-25 and XGA-47 were degraded with endopolygalacturonases (PGs) from Aspergillus niger (PG1, PG2), from A. tubingensis (PG-arf), from Kluyveromyces fragilis (PG-kluyv) and XGH from A. tubingensis . The activity of the different PGs decreased with increasing degrees of xylosylation. However, for each PG a different tolerance for the presence of side chains was observed. PG-arf and PG1 were hindered most by xylosyl branching, whereas XGH appeared to have a requirement for xylosylation and was almost not active towards polygalacturonic acid. The degradability of xylogalacturonans by XGH increased with higher degrees of xylosylation. Typically, a highly substituted xylogalacturonan from pea was almost resistant to XGH treatment. XGH produces a distinctive set of oligosaccharides from XGA, which is different from the hydrolysis products of PG action. Saponified modified hairy regions from apple (MHR-S), containing xylogalacturonan, were partially degraded by XGH. A combination of XGH and rhamnogalacturonan hydrolase was able to fully degrade the high molecular weight fraction of MHR-S. The two enzymes acted additively, no synergy being observed.