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Remarks on multidetector HPSEC characterization of native and enzymatically modified cereal arabinoxylans
Leena Pitkänen, Liisa Virkki, Maija Tenkanen, Päivi Tuomainen
COST Action FP0901 ”Analytical tools for biorefinery”
Workshop: Current needs in biorefinery analytics
February 4, Vienna
Faculty of Agriculture and Forestry
04.02.2010Leena Pitkänen
Hemicelluloses
Large group of non-crystalline heteropolysaccharides found in the primary and the secondary cell walls of plants
The second most abundant polysaccharide after cellulose produced by plants
Arabinoxylans are the main hemicellulose component in cereals
Due to the abundant nature of arabinoxylans they could be
utilized industrially as polymers for biodegradable materials
04.02.2010Leena Pitkänen
Structure of arabinoxylans (AX)
Arabinoxylans consist of a linear (1→4) -linked β-D-xylopyranose backbone with α-L-arabinofuranosidase substituent at the positions of O-2, O-3 or both
Esterified acetyl, feruloyl and p-cumaroyl groups also exist
Ebringerova and Hromadkova 1997 Ebringerova and Heinze 2000
α-L-Araf(1→3)mono
α-L-Araf(1→2) and (1→3)di
β-D-Xylp(1→2)-α-L-Araf(1→3)
04.02.2010Leena Pitkänen
Arabinoxylan content in cereals (%)
Cereal husks and straw are rich in arabinoxylans barley husks may contain from one third to 46% of
arabinoxylan
15 - 25Bran
10 - 25Straw
15 - 40Husks (spelts, hulls)
5 - 10Whole grain
1 - 5Flour
Arabinoxylan content (%)
Cereal material
Henry 1987, Puls 1992
Composition of arabinoxylans varies in different cereals and their parts
0.48
0.11
0.36
0.55
HusksAra : Xyl
0.77Oats
0.59Rye
0.67Barley
0.56Wheat
EndospermAra : Xyl
Cereal
Henry, J. Cereal Sci. 1987
04.02.2010Leena Pitkänen
Cereal arabinoxylans in our studies
AX isolated from barley husks (BH) and barley fibre (BF)laboratory scale alkali-extraction
BH and BF side-stream fractions from industrial ethanol
process (Koskenkorva, Altia)
Commercially available arabinoxylans from wheat (WAX) and rye (RAX) endosperm
good polysaccharides for structure-function studies due to
the high level of purity
used for enzymatic modications
04.02.2010Leena Pitkänen
Why to use enzymes for modification of biopolymers?
Production of molecules with varying chemical structureConvenient way to produce chemically different molecules
(vs. extraction from plants)
Controlled modification with spesific enzymes
04.02.2010Leena Pitkänen
─X─X─X─X─X─X─X─X─X─X─X─X─\l
A A A AA
─X─X─X─X─X─X─X─X─X─X─X─X─\
A A AA A
a)
b)
\l \
\l \\ \A
A\
04.02.2010Leena Pitkänen
Action of enzymes (AXH-m and AXH-d3) used for enzymatic modification and for structure elucidation
Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl
AraAra Ara Ara AraAra
Xyl
Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl
Ara Ara AraAra
Xyl
AXH-m
Xyl
Ara
Xyl
Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl
AraAra Ara Ara AraAra
Xyl
Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl
Ara Ara AraAra
Xyl
AXH-m
Xyl
Ara
Xyl
Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl
AraAra Ara Ara AraAra
Xyl
Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl
Ara Ara
XylAra Ara
AXH-d3
Xyl
Ara
Xyl
Ara
Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl
AraAra Ara Ara AraAra
Xyl
Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl
Ara Ara
XylAra Ara
AXH-d3
Xyl
Ara
Xyl
AraVan Laere et al., (1999) AMB
Structural differences between wheat and rye arabinoxylans
Relative intensities (%) of α-anomeric protons of α-L-Arafunits
04.02.2010Leena Pitkänen
Sugar residue WAX-HV(%)
WAX-MV(%)
WAX-LV(%)
RAX-HV (%)
α-L-Araf(1→3) mono 34.2 34.8 32.8 66.1
α-L-Araf(1→3) diα-L-Araf(1→2) mono
33.3 32.1 40.2 17.3
α-L-Araf(1→2) di 32.5 33.1 27.0 16.6
WAX 1/3 monosubst., 2/3 disubst.RAX 2/3 monosubst., 1/3 disubst.
HV = high viscosity, MV = medium viscosity, LV = low viscosity
Structures of wheat and rye arabinoxylans
04.02.2010Leena Pitkänen
─X─X─X─X─X─X─X─X─X─X─X─X─\l
A A A AA
─X─X─X─X─X─X─X─X─X─X─X─X─\
A A AA A
a)
b)
\l \
\l \\ \A
A\
a) Wheat arabinoxylanb) Rye arabinoxylan
The effect of modification to the structure of arabinoxylans (1H NMR)
04.02.2010Leena Pitkänen
AXH-d3
a) b)
a) Wheat AXb) Rye AX
1 1 1
22
2
3
3 3
1) α-L-Araf(1→3)mono
2) α-L-Araf(1→2)mono and α-L-Araf(1→3)di
3) α-L-Araf(1→2)di
HPSEC analyses of native and enzymatically modified arabinoxylans
Many polysaccharides are known to form molecular assemblies in aqueous environment
Arabinoxylans with low ara:xyl ratio tend to precipitate
Aqueous solution are generally used in HPSEC analyses of arabinoxylans
Behaviour in aqueous solution often interesting due to
interfaces of arabinoxylans with various biological systems
04.02.2010Leena Pitkänen
We use both aqueous and DMSO-based solvents for analysis of arabinoxylans
HPSEC with multiple detection
Molar mass distribution with LS/VISC method
Simultaneous determination of intrinsic viscosityValuable information on the molecular density and possible
association of molecules
UV useful for detection of lignin/protein impurities
04.02.2010Leena Pitkänen
UV LS + VISC RI
HPSEC of barley arabinoxylans
UV detection reveals the presence of protein/lignin impurities
04.02.2010Leena Pitkänen
Retention Volume (mL)0.00 3.00 6.00 9.00 12.00 15.00 18.00 21.00 24.00 27.00 30.00
Retention Volume (mL)0.00 3.00 6.00 9.00 12.00 15.00 18.00 21.00 24.00 27.00 30.00
RI
VISC
RALS
UV
BHAX
BFAX
Pitkänen et al. 2008
Comparison of water and DMSO as solvents for arabinoxylan with low degree of substitution
Wheat arabinoxylan sample (WAX-LV, Megazyme)Ara:xyl ratio 0.32
04.02.2010Leena Pitkänen
23.38
25.70
28.02
30.34
32.65
Rig
ht A
ngle
Lig
ht S
catte
ring
(mV
)
Retention Volume (mL)0.00 5.00 10.00 15.00 20.00 25.00 30.00
64.49
65.24
66.00
66.75
67.51
Rig
ht A
ngle
Lig
ht S
catte
ring
(mV
)
Retention Volume (mL)0.00 5.00 10.00 15.00 20.00 25.00 30.00
water + 0.1 M NaNO3 DMSO + 0.01 M LiBr
Mw 8.3 · 104 g/mol Mw 4.9 · 104 g/mol
Pitkänen et al., 2009
Molar mass-intrinsic viscosity relationship of wheat and rye arabinoxylans in two solvents
04.02.2010Leena Pitkänen
-1.00
-0.50
0.00
0.50
1.00
1.50
Log
Intri
nsic
Vis
cosi
ty
Log Molecular Weight4.00 4.50 5.00 5.50 6.00 6.50 7.00
-1.00
-0.50
0.00
0.50
1.00
1.50
Log
Intri
nsic
Vis
cosi
ty
Log Molecular Weight4.00 4.50 5.00 5.50 6.00 6.50
WAX-LV
WAX-MV
WAX-HV
RAX-HV
DMSO + 0.01 M LiBr
0.1 M NaNO3 (aq.)
Determination of persistence lenght (Lp) using molar mass and intrinsic viscosity data
Bohdanecký modelModel dependence → useful for comparison of samples
04.02.2010Leena Pitkänen
Lp
WAX-MV = 1.8 nmRAX-HV = 2.3 nm
Enzymatic modification and HPSEC analysis of wheat and rye arabinoxylans
04.02.2010Leena Pitkänen
AX solution (buffer)
AXH treated AX solution
measurement of released arabinose
HPSEC in aqueous solution
determination of ara:xyl
ratiodialysed sample
dialysis
AXH treatment
freeze dried sample
freeze drying
HPSEC in DMSO solution
Samples were filtered with 0.45 µm filters before HPSEC analyses!
The effect of enzymatic treatments to molar mass distribution of wheat arabinoxylan sample with initial ara:xyl ratio of 0.51
Molar mass distributions in DMSO + 0.01 M LiBr
04.02.2010Leena Pitkänen
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00W
F / d
Log
MW
Log Molecular Weight3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50
WAX-HV00.51
WAX-HVd30.40
WAX-HVm0.34
Recoveries from HPSEC: WAX-HV0 = 85%WAX-HVd3 = 80%WAX-HVm = 47%
Mw values:
WAX-HV0 = 2.86 ·105 g/mol
WAX-HVd3 = 2.62 ·105 g/mol
WAX-HVm = 0.80 ·105 g/mol
Pitkänen et al., unpublished
Theoretical reduction of mass caused by enzymes:AXH-d3 25 000 g/molAXH-m 30 000 g/mol
Comparison of water and DMSO as solvents for AXH-m treated rye arabinoxylan (RAX-HV)
47% of AXH-m treated sample dissolved in DMSO after modification, freeze drying and redissolution
04.02.2010Leena Pitkänen
No peaks!
water + 0.1 M NaNO3 DMSO + 0.01 M LiBr
0.00
0.28
0.57
0.85
1.13
1.42
1.70
WF
/ dLo
g M
W
Log Molecular Weight4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00
Mw = 0.72 ·105 g/mol
longer arabinoxylan chains could not be dissolved after AXH-m treatment
Ara:xyl 0.21
Conclusions
UV and viscometric detection coupled to HPSEC very useful in characterization of arabinoxylans extracted from cereal sources (in addition to MALS and RI)Analysis of arabinoxylans with both aqueous and DMSO solutions reveals facts about association of xylose chainsArabinoxylan assemblies occur in aqueous solution when the amount of arabinose side groups is lowIn addition to low ara:xyl ratio, chain length seems to have an effect on the precipitation of arabinoxylan moleculesAXH-m enzyme caused partial precipitation of xylan molecules
Occurrence of longer regions of unsubstituted xylose
residues 04.02.2010Leena Pitkänen
Acknowledgements
Funding University of Helsinki Research Funds
Glycoscience Graduate School
Finnish Cultural Foundation 04.02.2010Leena Pitkänen