Characterisation of Pectic Substances from Hemicellulose Fractions of Apples

154 Pages·2008·5.78 MB·English

ETH Library
Characterisation of pectic
substances from hemicellulose
fractions of apples
Doctoral Thesis
Author(s):
Lutz, Marc Valentin
Publication date:
2003
Permanent link:
https://doi.org/10.3929/ethz-a-004555078
Rights / license:
In Copyright - Non-Commercial Use Permitted
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Diss. ETH No. 15069
Characterisation of Pectic Substances from
Hemicellulose Fractions of Apples
A dissertation submitted to the
SWISS FEDERAL INSTITUTE OF TECHNOLOGY
ZURICH
forthe degree of
Doctor of Natural Sciences
presented by
Marc Valentin Lutz
Dipl. Chem. UniversityofZurich
born January 6,1973
citizen of Basel (BS) and Thal (SG), Switzerland
accepted on the recommendation of
Prof. Dr. R. Amadö, examiner
Prof. Dr. A. G. J. Voragen, co-examiner
Zurich 2003

Dank
Herrn Prof. Dr. Renato Amadö danke ich dafür, dass ich in seiner Gruppe eine Doktor¬
arbeit machen durfte. Seine vorbehaltlose Unterstützung und die grosse Selbständig¬
keit, die er mir gewährte, waren für das Gelingen meinerArbeit sehr wertvoll.
Ein besonderer Dank geht an Prof. Dr. A. G. J. Voragen von der Universität Wageningen
(NL) für das dieser Arbeit entgegengebrachte Interesse und für die Übernahme des
Korreferates.
Ferner bedanke ich mich bei der Firma Novozymes A/S, insbesondere Dr. Lene V. Kofod
für die grosszügige Bereitstellung einiger Enzyme.
Ein grosser Dank geht an die gesamte Arbeitsgruppe für das angenehme Arbeitsklima
und die vielen heiteren Stunden. Insbesondere danke ich Rahel Oechslin für die Zusam¬
menarbeit im Zellwandprojekt und vor allem für die gute Zeit, die wir zusammen im La¬
bor D24 verbracht haben. Ein grosser Dank gebührt auch Berit Abt für die englischen
Korrekturen.
Weiter gilt ein Dank auch den Studenten Thomas M. Amrein, Barbara Berufter, Caspar
Buri, Marco Fallico, Jürg Gujan, Urs Scherrer, Monika Koch, Alexandra Kocher, Fabian
Rohner, Helen Schwarze und Claudia Weiss, die im Rahmen von Diplom- und Semester¬
arbeiten zum Gelingen meiner Arbeit beigetragen haben.
Stefan Wolf von sofie's Kommunikationsdesign, Zürich danke ich ganz herzlich für die
Gestaltung des Titelbildes.
Meiner Freundin Annekäthi Bühler danke ich von Herzen für die Unterstützung und die
Geduld während der Zeit meiner Dissertation. Ein herzlicher Dank gilt auch meinen
Eltern und meiner Schwester dafür, dass sie immerfür mich da sind.

Table of contents
Table of contents
List of abbreviations
Summary
Zusammenfassung
Introduction
i
2 Literature review
2.1 Structure ofthe cell wall
2.2 Components ofthe cell wall
2.2.1 Cellulose
2.2.2 Hemicelluloses
2.2.3 Pectic polymers
2.2.4 Proteins
2.2.5 Phenolic compounds
2.3 Evidence for a cross-link between xyloglucan and pectin
2.4 Plant growth and ripening
2.4.1 Plant growth
2.4.2 Changes ofthe cell wall polysaccharides during
2.4.3 Enzymes associated with cell wall degradation
2.4.3.1 Polygalacturonases
2.4.3.2 Pectin methyl esterase
2.4.3.3 ß-Galactosidase
2.4.3.4 Xyloglucan endotransglycosylase
2.4.3.5 £ndo-i,4-ß-glucanases
Material and methods
3
3.1 Material
3.2 Extraction ofthe hemicellulose fractions
3.3 Enzymatic degradation ofthe hemicellulose fractions
3.3.1 Degradation with endo-i,4-ß-D-glucanase
Table ofcontents
3.3.2 Degradation with a mixture ofendo-i,5-a-i_-arabinanase and
a-L-arabinofuranosidase 19
3.3.3 Degradation with enc/o-i,4-ß-D-galactanase 19
3.3.4 Degradation with a mixture ofendo-i,4-ß-D-galactanase,
endo-i,5-a-i_-arabinanase and a-L-arabinofuranosidase 20
3.3.5 Degradation with a rhamnogalacturonase A 20
3.4 Separation methods 20
3.4.1 Anion exchange chromatography 20
3.4.2 Dialysis 21
3.4.3 Ultrafiltration in a stirred cell 21
3.4.4 Ultrafiltration by centrifugal separation 21
3.5 Analytical methods 22
3.5.1 Photometric determination oftotal uronic acid content 22
3.5.2 Determination oftotal uronic acid and total neutral sugar contents
using an automated segmented flow analyser 22
3.5.3 Determination ofthe neutral sugar content as alditol acetates 22
3.5.4 Determination ofthe neutral sugar and uronic acid content by
methanolysis 23
3.5.5 Determination ofthe substitution pattern of neutral sugar and
uronic acid residues 26
3.5.5.1 CMC-activated reduction of uronic acids 26
3.5.5.2 Methylation analysis 26
3.5.6 Molecular weight determination with high performance size-
exclusion chromatography 27
3.5.7 Determination ofthe starch content 28
3.5.8 Determination ofenzyme activities 29
3.6 Identification ofthe fractions 30
Results and discussion
4 31
4.1 Characterisation ofthe pectic polymers present in the hemicellulose
fractions 32
4.1.1 Extraction ofthe depectinated residue 32
4.1.2 Characterisation ofthe 4M hemicellulosefraction 35
4.1.3 Anion exchange chromatography ofthe 4M hemicellulosefraction 41
4.1.4 Enzymatic degradation ofthe 4M hemicellulose fraction 45
4.1.5 Characterisation ofthe ultrafiltration-retentate XG3k after
xyloglucan degradation 49
4.1.6 Characterisation ofthe ultrafiltration-retentate XG5ok after
xyloglucan degradation 52
4.1.7 Degradation with specific enzymes acting on pectic polymers 56
Table ofcontents
4.1.7.1 Degradation with a mixture ofendo-i,5-a-i_-arabinanase
and a-L-arabinofuranosidase 58
4.1.7.2 Degradation with an enc/o-i,4-ß-D-galactanase 64
4.1.7.3 Degradation with a mixture ofendo-i,4-ß-D-galactanase,
endo-i,5-a-L-arabinanase and a-L-arabinofuranosidase 70
4.1.7.4 Degradation with a rhamnogalacturonase A 75
4.1.8 Structural model for the pectic substances present in the 4M
hemicellulosefraction 79
4.2 Ripening related changes in the 4M hemicellulose fractions 84
4.2.1 Extraction ofthe depectinated residue 85
4.2.2 Characterisation ofthe 4M hemicellulosefractions 86
4.2.3 Anion exchange chromatography ofthe 4M hemicellulose
fractions 89
4.2.4 Characterisation ofthe ultrafiltration-retentate XG5ok after
xyloglucan degradation 92
4.2.5 Degradation with specific enzymes acting on pectin 95
4.2.5.1 Degradation with a mixture ofendo-i,5-a-L-arabinanase
and a-L-arabinofuranosidase 96
4.2.5.2 Degradation with an enc/o-i,4-ß-D-galactanase 96
4.2.5.3 Degradation with a mixture ofendo-i,4-ß-D-galactanase,
endo-a-i,5-D-arabinanase and a-L-arabinofuranosidase 97
4.2.5.4 Degradation with a rhamnogalacturonase A 97
4.2.6 Ripening related changes in the structure ofthe pectic substances
present in the 4M hemicellulose fraction 99
4.3 Variety related differences in the hemicellulose fractions 101
4.3.1 Extraction ofthe depectinated residues 101
4.3.2 Characterisation ofthe 4M hemicellulosefractions 102
4.3.3 Anion exchange chromatography ofthe 4M hemicellulose
fractions 104
4.3.4 Characterisation ofthe ultrafiltration-retentate XG5ok after
xyloglucan degradation 105
4.3.5 Degradation with specific enzymes acting on pectin 108
4.3.6 Variety related differences in the structure ofthe pectic
substances present in the 4M hemicellulose fraction 109
Conclusions and Outlook
5 in
6 References
117
Appendix
IV
List of abbreviations
AFM Atomic force microscopy
AIR Alcohol-insoluble residue
Ara Arabinose
CDTA 7rans-i,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid
CMC N-Cyclohexyl-N'-(2-morpholinoethyl)carbodiimide methyl-p-toluene-
sulfonate
CR Cellulosic residue
DE Degree ofesterification
dm Dry matter
dp Degree of polymerisation
DMSO Dimethyl sulfoxide
ECR Effective carbon response
EG £ndo-i,4-ß-glucanase
El Electron ionisation
/ Furanose
Fuc Fucose
Gal Galactose
GalA Galacturonic acid
GLC Gas-liquid chromatography
Glc Glucose
HG Homogalacturonan
Hyp Hydroxyproline
HPSEC High performance size exclusion chromatography
kDa Kilodalton
LALS Low angle laser light-scattering detector
Lys Lysine
MALDI-TOFMS Matrix-assisted laser desorption/ionisation-time of flight mass spec¬
trometry
Man Mannose