Remarks on multidetector HPSEC characterization of native...

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


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)


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


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


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


─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\


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


Ara Ara AraAra

Xyl

AXH-m

Xyl

Ara

Xyl



Xyl


Ara Ara AraAra

Xyl

AXH-m

Xyl

Ara

Xyl



Xyl


Ara Ara

XylAra Ara

AXH-d3

Xyl

Ara

Xyl

Ara



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


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


─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)


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


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


UV LS + VISC RI

HPSEC of barley arabinoxylans

UV detection reveals the presence of protein/lignin impurities


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


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


-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


Lp

WAX-MV = 1.8 nmRAX-HV = 2.3 nm

Enzymatic modification and HPSEC analysis of wheat and rye arabinoxylans


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


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


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

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