Substrate specificity of the α-L-arabinofuranosidase from Rhizomucor pusillus HHT-1

Article abstract of DOI:10.1016/S0008-6215(03)00203-9

The α-L-arabinofuranosidase (AF) from the fungus Rhizomucor pusillus HHT-1 released arabinose at appreciable rates from (1→5)-α-L-arabinofuranooligosaccharides, sugar beet arabinan and debranched arabinan. This enzyme preferentially hydrolyzed the terminal arabinofuranosyl residue [α-(1→5)-linked] of the arabinan backbone rather than the arabinosyl side chain [α-(1→3)-linked residues]. The enzyme-hydrolyzed arabinan reacted at and debranched the arabinan almost at the same rate, and the degree of conversion for both cases was 65%. Methylation analysis of arabinan showed that the arabinosyl-linkage proportions were 2:2:2:1, respectively, for (1→5)-Araf, T-Araf, (1→3, 5)-Araf and (1→3)-Araf, while the ratios for the AF-digested arabinan shifted to 3:1:2:1. Enzyme digestion resulted in an increase in the proportion of (1→5)-linked arabinose and a decrease in the proportion of terminal arabinose indicated this AF cleaved the terminal arabinosyl residue of the arabinan back bone [α-(1→5)-linked residues]. Peak assignments in the ¹³C NMR spectra also confirmed this linkage composition of four kinds of arabinose residues. Both ¹H and ¹³C NMR spectra are dominated by signals of the α-anomeric configuration of the arabinofuranosyl moieties. No signals were recorded for arabinopyranosyl moieties in the NMR spectra. Methylation and NMR analysis of native and AF-digested arabinan revealed that this α-L-arabinofuranosidase can only hydrolyse α-L-arabinofuranosyl residues of arabinan.

Full text of DOI:10.1016/S0008-6215(03)00203-9

Carbohydrate Research 338 (2003) 1469Á1476
/
www.elsevier.com/locate/carres
Substrate specificity of the a-
L
-arabinofuranosidase from Rhizomucor
pusillus HHT-1
A.K.M. Shofiqur Rahman,a Koji Kato,b Shingo Kawai,c Kazuhiro Takamizawaa,*
a Department of Bioprocessing, Faculty of Agriculture, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan
b Department of Food Science, Faculty of Agriculture, Gifu University, Gifu, Japan
c Department of Applied Bioorganic Chemistry, Faculty of Agriculture, Gifu University, Gifu, Japan
Received 1 November 2002; accepted 18 April 2003
Abstract
The a-
5)-a- -arabinofuranooligosaccharides, sugar beet arabinan and debranched arabinan. This enzyme preferentially hydrolyzed the
terminal arabinofuranosyl residue [a-(10 3)-linked
L
-arabinofuranosidase (AF) from the fungus Rhizomucor pusillus HHT-1 released arabinose at appreciable rates from (10
/
L
/
5)-linked] of the arabinan backbone rather than the arabinosyl side chain [a-(10
/
residues]. The enzyme-hydrolyzed arabinan reacted at and debranched the arabinan almost at the same rate, and the degree of
conversion for both cases was 65%. Methylation analysis of arabinan showed that the arabinosyl-linkage proportions were 2:2:2:1,
respectively, for (10
/
5)-Araf, T-Araf, (10
/
3, 5)-Araf and (10
/
3)-Araf, while the ratios for the AF-digested arabinan shifted to
5)-linked arabinose and a decrease in the proportion of
5)-linked residues].
3:1:2:1. Enzyme digestion resulted in an increase in the proportion of (10
/
terminal arabinose indicated this AF cleaved the terminal arabinosyl residue of the arabinan back bone [a-(10
/
Peak assignments in the 13C NMR spectra also confirmed this linkage composition of four kinds of arabinose residues. Both 1H and
13C NMR spectra are dominated by signals of the a-anomeric configuration of the arabinofuranosyl moieties. No signals were
recorded for arabinopyranosyl moieties in the NMR spectra. Methylation and NMR analysis of native and AF-digested arabinan
revealed that this a-
L
-arabinofuranosidase can only hydrolyse a-
L-arabinofuranosyl residues of arabinan.
# 2003 Elsevier Science Ltd. All rights reserved.
Keywords: a-L-Arabinofuranosidase; Rhizomucor pusillus; Substrate specificity; Arabinan; Methylation analysis; NMR spectroscopy
1. Introduction
and b-(10/4)-linked xylopyranose moieties, respectively.
Both polysaccharides are decorated at C-2 and/or C-3
with arabinofuranose molecules as side chains.2,3 These
side chains may restrict the enzymic hydrolysis of
Lignocellulosic biomass is a constant source of sustain-
able and renewable energy in Nature. It is comprised of
about 23% lignin, 40% cellulose, and 33% hemicellu-
lose.1 The monomers of various lignocellulosic agricul-
tural and industrial wastes are useful in the production
of different antibiotics, alcohols, animal feed, chemicals
respected polysaccharides. The a-
(exo-(105)-a- -arabinofuranosidase, AF, EC.3.2.1.55)
cleaves the arabinose side chains, allowing endo-arabi-
L-arabinofuranosidase
/
L
nase
(endo-(10
/
5)-a-
L
-arabinofuranosidase,
EC
3.2.1.99) to attack the arabinan backbone.4,5 These
enzymes act synergistically in degrading branched
and fuels. L-Arabinosyl residues are one of the mono-
mers and common components of some lignocellulosic
plant polysaccharides. Arabinose is present at high
concentrations in arabinans, arabinoxylans and arabi-
nogalactan. The arabinan and arabinoxylan backbones
arabinan to generate
a- -Arabinofuranosidase, purified for the first time
L
-arabinose.6
L
by Kaji and coworkers,7 has proven to be an essential
enzyme for the production of arabinose. Since then,
there have been many studies on AF. However, the
substrate specificity of the enzyme has not been studied
well in comparison with other glycosidases. Recently,
are comprised of a-(10/5)-linked arabinofuranose units
* Corresponding author. Tel./fax: ꢀ
/
81-58-2932906.
there is interest growing for L-arabinose as a possible
E-mail address: tak2003@cc.gifu-u.ac.jp (K. Takamizawa).
0008-6215/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0008-6215(03)00203-9

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A.K.M. Shofiqur Rahman et al. / Carbohydrate Research 338 (2003) 1469Á1476
/
food additive, due to its low uptake by the body and its
sweet taste. It has been proved that -arabinose
selectively inhibits intestinal sucrase in a competitive
Sugar beet arabinan, debranched arabinan and (10
a- -arabinofuranooligosaccharides were obtained from
Megazyme pty., Ltd. (North Rocks, Australia).
/
5)-
L
L
manner and reduces the glycemic response after sucrose
ingestion in animals.8 Based on these findings,
arabinose can be used as a physiologically functional
sugar that inhibits sucrose digestion. It has also been
L-
2.2. Gas
/
liquid chromatography (GLC)
Á
GLC was carried out on a Shimadzu GC-18A apparatus
equipped with a flame-ionization detector. A capillary
column of CBP-1-M25-025 (0.25 mmꢁ25 m) was used
and operated at 220 8C with gas flow rate of 60 mL/min
of nitrogen. Peak areas were measured with a Shimadzu
Chromatocorder-21.
reported that in mice,
suppressed the increase of blood glucose after the
ingestion of sucrose. Furthermore, -arabinose delays
L-arabinose dose-dependently
/
L
and reduces the digestion, absorption, and net energy
derived from sucrose when both are ingested simulta-
neously.9 In this way,
L
-arabinose is useful in preventing
postprandial hyperglycemia in diabetic patients when
foods containing sucrose are ingested. Effective
L
-
2.3. Infrared (IR) spectroscopy
arabinose production is therefore important in the
food industry. To achieve this goal, it is necessary to
IR spectra were recorded with a PerkinÁ
FTIR spectrometer system.
/
Elmer 2000
characterize a-
L-arabinofuranosidase by using defined
oligosaccharides, which is a powerful method for under-
standing the substrate specificity. In fact, AF in
combination with xylanases is being used for the
structural elucidation and bioconversion of hemicellu-
loses to biologically useful pentose sugars. These types
of synergism studies were also reported in our previous
works.10Á13
2.4. Gas chromatography
/
mass spectrometry (GC
/
MS)
Á
Á
GCÁ
/
MS was conducted with a Shimadzu GCÁ
/MS QP-
5000 apparatus with a class 5000 mass data system. A
methyl silicon DB-1 capillary column (30 mꢁ0.25
mmꢁ1 mm, J&W Scientific) was used. The column
/
/
The novel a-L-arabinofuranosidase (AF) from Rhizo-
temperature was programmed first at 150 8C for 2 min,
and then raised to 250 8C at 5 8C/min. The spectra were
recorded at an ionizing potential of 70 eV.
mucor pusillus HHT-1 was purified and characterized in
our previous work,14 but the specificity of the enzyme
towards arabinofuranose-containing oligosaccharides
was not studied. Therefore, we investigated the substrate
specificity of AF produced by R. pusillus HHT-1, to
2.5. High-performance liquid chromatography (HPLC)
develop an effective process of L-arabinose production
from some plant polysaccharides.
HPLC was performed using a JASCO apparatus (880-
PU pump, 860-CO column oven, and 830-RI detector).
A Wakobeads T-130-E (7.8ꢁ300 mm) column was used
/
2. Experimental
2.1. Enzyme and substrates
and operated at 60 8C with a flow rate of Milli Q water
at 0.7 mL/min. The peak areas were estimated with an
SIC Chromatocorder 11.
Extracellular a-L-arabinofuranosidase from R. pusillus
HHT-1 was purified as described in our previous
2.6. Effect of a-
L-arabinofuranosidase (AF) on hydrolysis
article14 and used throughout this study. The a-
arabinofuranosidase activity was determined by the
following method: the reaction mixture, consisting of
L
-
of (105)-a- -arabinofuranooligosaccharides
/
L
The reaction mixture contained 0.5 mL of AF solution
with different concentrations (1 U, 3 U, and 5 U/mL),
0.4 mL of 20 mM sodium phosphate buffer (pH 4.0) and
180 mL of 2.5 mM p-nitrophenyl a-L-arabinofuranoside
(dissolved in 50 mM citrate buffer, pH 5.0) and 20 mL of
enzyme solution, was incubated at 50 8C for 20 min,
followed by the addition of 100 mL of 1 M Na2CO3.
Absorbances were read at 414 nm with a microtitre plate
reader (Immuno Mini NJ-2300, System Instrument Co.,
Tokyo, Japan). One unit of enzyme activity was defined
as the amount of enzyme required to liberate 1.0 mmol of
p-nitrophenol from the substrate per minute under the
assay conditions and expressed as units per milliliter. p-
0.1 mL of 10% (10
/
5)-a-
L
-arabinofuranobiose, (10
/5)-
a- -arabinofuranotriose, and (10
L
/
5)-a- -arabinofura-
L
notetraose, respectively. After 40 min incubation at
50 8C, the reaction mixture was heated in boiling water
for 10 min to stop the reaction. The reaction mixture
was then centrifuged and filtered using the 0.2-mm pore
size syringe-driven filter unit (Millipore Corporation,
Bedford, USA). Ten micro-liter of filtered reaction
mixture was analyzed by HPLC for the characterization
of hydrolysis products.
Nitrophenyl a-
L-arabinofuranoside was purchased from
SigmaÁAldrich Chemical Co. (St. Louis, MO, USA).
/

A.K.M. Shofiqur Rahman et al. / Carbohydrate Research 338 (2003) 1469Á
/1476
1471
2.7. Time course of hydrolysis of (10
/
5)-a-
L
-
product was confirmed to show no absorption for the
hydroxyl group in its IR spectrum. It was then hydro-
lyzed with 90% formic acid, and then with 0.25 M
sulphuric acid. The partially methylated sugars thus
obtained were converted to their alditol acetates for
arabinofuranotetraose by AF
The reaction mixture contained 0.5 mL of AF solution
(1 U/mL), 0.4 mL of 20 mM sodium phosphate buffer
(pH 4.0) and 0.1 mL of 10% (10
/
5)-a-
L
-arabinofurano-
GLC and GCÁMS analyses. The glycosyl-linkage com-
/
tetraose. After 0, 10, 20, 30, 40, 50, and 60 min
incubation at 50 8C, the reaction mixtures were heated
in boiling water for 10 min to stop the reaction. Ten
microliters of filtered reaction mixture were analyzed by
HPLC for the characterization of the hydrolysis pro-
ducts.
positions were then analyzed by GCÁMS of the result-
/
ing partially methylated alditol acetates.16
2.11. NMR spectroscopy
1H and 13C NMR spectra were recorded at 399.956 (1H)
and 100.578 MHz (13C) with a Varian Unity Inova 400
FT instrument. The spectra were recorded at room
temperature in D2O. Sodium 4,4-dimethyl-4-silapent-
anoate-2,2,3,3-d4 (DSS) (adjusted to 0 ppm), was used as
the internal standard. Chemical shifts are given in d
values. The spectra of native and AF-digested arabinan
hydrolysates were interpreted on the basis of methyla-
tion analysis and literature data.
2.8. Time course of hydrolysis of arabinan and
debranched arabinan
For the time course of hydrolysis of sugar beet arabinan
(88:3:2:7
nic acid) and debranched arabinan (88:4:2:6
binoseÁgalactoseÁrhamnoseÁgalacturonic acid), the
L
-arabinoseÁ
/
galactoseÁ
/
rhamnoseÁ
/galacturo-
L-ara-
/
/
/
reaction mixture contained 0.5 mL of AF solution (5
U/mL), 0.4 mL of 20 mM sodium phosphate buffer (pH
4.0) and 0.1 mL of 10% arabinan or debranched
arabinan. After 6-, 12-, 24-, 48-, 72- and 96-h incuba-
tions at 50 8C, each of the reaction mixture was heated
in boiling water bath for 10 min to stop the reaction.
Ten microliters of the mixture were used for HPLC for
the characterization of hydrolysis products.
3. Results and discussion
3.1. Effect of a-
L-arabinofuranosidase (AF) on hydrolysis
of (105)-a- -arabinofuranooligosaccharides
/
L
The hydrolysis of (10
charides by the AF was carried out (data not shown).
AF from R. pusillus HHT-1 hydrolyzed the (105)-a-
arabinofuranooligosaccharides completely, indicating
its capability of hydrolysing the (105)-a- -linked
arabinosyl terminal residues.
A time course of hydrolysis of (10
/
5)-a-
L-arabinofuranooligosac-
2.9. Effect of AF concentration on hydrolysis of arabinan
and debranched arabinan
/
L-
/
L
The components of reaction mixture were same as above
except for the concentrations of AF used. The AF
concentrations were 0.2, 0.5, 1, 2, 3, 4 and 5 U/mL. After
24-h incubations at 50 8C, each of the reaction mixture
was heated in boiling water bath for 10 min to stop the
reaction. Ten microliters of the mixture were used for
HPLC for the characterization of hydrolysis products.
/
5)-a-
L-arabinofur-
anotetraose by AF was performed (Fig. 1). The AF
released 100% arabinose from the oligosaccharides at 40
min.
3.2. Time course of hydrolysis of arabinan and
debranched arabinan
2.10. Glycosyl-linkage composition of AF-digested
arabinan
The hydrolysis of sugar beet arabinan and debranched
arabinan by AF is presented in Fig. 2. Finally, only
arabinose was detected in the reaction product.
Although the initial rates of arabinose release from the
two substrates were almost the same, the rates of
degradation after 24 h differed significantly from each
other. This AF preferentially hydrolyzes terminal arabi-
nofuranosyl residue of the arabinan backbone rather
than the arabinosyl side chain. The arabinans that have
In the glycosyl-linkage analysis of arabinan hydrolysates
digested by AF, the reaction mixture contained 0.5 mL
of AF solution (1 U/mL), 0.4 mL of 20 mM sodium
phosphate buffer (pH 4.0), and 0.1 mL of 10% arabinan.
After 5-h incubation at 50 8C, the mixture was heated in
boiling water bath for 10 min to stop the reaction.
Thereafter, the reaction mixture was dialyzed by the
dialysis membrane (Wako Chemicals, USA, Inc.)
against the tap water overnight to remove arabinose.
Arabinan hydrolysates, after removal of arabinose, were
lyophilized. Glycosyl-linkage compositions of arabinan
hydrolysates were determined by methylation analysis
using a modification15 of the Hakomori procedure. The
been analyzed are highly branched with (10
/5) links
between the main chain residues, which are substituted
at O-3 with single or multiple-unit side chains.2 From
the results shown in Fig. 2, it is suggested that the AF
from R. pusillus HHT-1 has hydrolytic activity for both
a-(10
/
3)- and a-(10
/
5)-linked, nonreducing, terminal L-

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A.K.M. Shofiqur Rahman et al. / Carbohydrate Research 338 (2003) 1469Á1476
/
Effect of AF concentration on hydrolysis of arabinan
and debranched arabinan was studied (data not shown).
The optimum concentration of AF was 1 U/mL for
effective arabinose production after 24-h incubations for
both cases. There were no remarkable changes observed
even after prolonged incubation for 96 h and presence of
the excess amount of AF in the reaction mixture. The
limit of hydrolysis of arabinan and debranched arabinan
by the AF from R. pusillus HHT-1 was almost same
(65%). The range of limit of hydrolysis of arabinan by
the AF reported until now is 15Á
100%.17,22
/
3.3. Glycosyl-linkage composition of AF-digested
arabinan
GLC analysis gave several peaks (chromatograms not
shown), and six of them were identified as terminal
arabinofuranosyl (T-Araf), terminal arabinopyranosyl
Fig. 1. Time course of hydrolysis of 1,5,- -L-arabinofuranote-
traose by AF. A1-arabinose, A2-arabinobiose, A3-arabino-
triose, and A4-arabinotetraose.
(T-Arap), 5-linked arabinofuranosyl (10
5)-linked arabinofuranosyl (103,5-Araf), (20
arabinofuranosyl (102,5-Araf) and 3-linked arabino-
furanosyl (103-Araf) residues by their retention time
/
5-Araf), (30
/
/
/
5)-linked
arabinofuranose residues. This type of enzymatic de-
arabinosylation are comparable to those AFs from
Aspergillus niger,17,18 Aureobasidium pullulans,19 Strep-
tomyces sp. strain 17-1,20 Streptomyces diastaticus,21
Bacillus subtilis 3-6.22 This is in contrast to AF from A.
niger23 and S. purpurascens IFO 3389,24 which hydro-
/
/
and mass spectra. The relative proportions of glycosyl
linkage composition are summarized in Table 1. Methy-
lation analysis of arabinan showed that the arabinosyl-
linkage proportions were 2:2:2:1, respectively, for (10
5)-Araf, T-Araf, (103,5)-Araf and (103)-Araf, while
the rations for the AF-digested arabinan shifted to
/
lyzed either (10
/
5)- or (10/3)-arabinosyl linkages of
/
/
arabinan.
Fig. 2. (A) Primary structure of Arabinan and Debranched arabinan. (B) Time course of hydorlysis of Arabinan and Debranched
arabinan by Á -AF. (AF-1U/ml at 508 C) Arabinan, Debranched arabinan.
/
L

A.K.M. Shofiqur Rahman et al. / Carbohydrate Research 338 (2003) 1469Á
/1476
1473
Table 1
Glycosyl-linkage composition of arabinan hydrolysates by a-
L-arabinofuranosidase from R. pusillus HHT-1
Peak
Glycosyl linkage
Arabinan a (native)
Arabinan treated with a-
L
-AFase a
A
B
C
D
E
F
b T-Araf
b T-Arap
24.5
2.2
13.2
2.1
0
/
5)-Araf
30.0
26.1
6.6
42.9
24.2
6.1
0
/
3,5)-Araf
2,5)-Araf
3)-Araf
0
/
0
/
10.6
11.5
a
Values expressed as mol%.
b
Tꢂnonreducing terminal.
/
3:1:2:1. Enzyme digestion resulted in an increase in the
relative proportion of (105)-linked arabinose and a
decrease in the proportion of terminal arabinose. There-
fore, this AF is highly specific for a- -arabinofurano-
3.4. NMR spectroscopy
/
1H and 13C NMR analyses of native and AF-digested
arabinan hydrolysates were carried out. In the 1H NMR
spectra of native and AF-digested arabinans (data not
shown), signals at 5.16 and 5.10, were assigned to
L
sides and cleaves the terminal arabinosyl residue of the
arabinan backbone. The presence of 2,3,4-tri-O-methyl-
-arabinose in the methylation analysis is indicative of
anomeric protons of terminal-a-
L-arabinofuranosyl
L-arabinofuranosyl residues, respec-
L
and 5-linked a-
the occurrence of a few residues in the pyranose form.
Arabinopyranose residues have been reported in many
arabinans. In all instances, the proportion of the
arabinopyranose residues was only a few percent of
the arabinofuranose form and these residues appeared
only in terminal, nonreducing positions (T-Arap).17,18,25
T-Arap residues obtained in this analysis also accounted
for less than 2% of total residues. It is therefore
questionable as to whether the pyranose residues are
really part to the polysaccharides or if they are artifacts
arising during the methylation analysis. It has been
tively.38 In the 13C NMR spectra of native and AF-
digested arabinans (Fig. 3), signals at 110.3, 109.9 and
110.3, 109.7 were assigned to anomeric carbons of a-
L-
5)-
arabinofuranosyl residues38Á40, respectively. a-(10
/
Linked arabinofuranosyl units preponderate highly in
native arabinan as shown by methylation analysis, thus
an intense signals are expected for C-1 linked to C-5 of
this residue. Examination of the 13C NMR spectra of
arabinan confirmed this feature. The spectrum of AF-
digested arabinan showed five strong signals at d 110.3
(C-1), 85.1 (C-4), 83.7 (C-2), 79.6 (C-3) and 69.7 (C-5-
linked). These five signals correspond to the carbon
demonstrated26 that some methyl a-
side is formed on treatment of methyl a-
L
-arabinopyrano-
L
-arabinofur-
atoms of the a-
L
-(10/5)-linked arabinofuranosyl resi-
anoside with alkali. In an arabinan, the terminal
arabinofuranosyl residues may be regarded as simple
glycofuranosides, and consequently if any tautomeric
rearrangements were to take place, it would be on the
terminal residues, and not on the internal residues where
dues which, according to the methylation analysis, are
expected to be the most abundant in the arabinans.
Moreover, Fig. 3 shows the 13C NMR spectrum of a
mixture of the four kinds of arabinose e.g., T-a-
L
-Araf,
L-
5) linkages.27
(105)-a- -Araf, (103,5)-a- -Araf and (10
/
L
/
L
/
3)-a-
the furanose ring-form is fixed by the (10
The enzyme released arabinose by step-wise hydrolyzing
the (105) linkage of the arabinosyl residues in the main
/
Araf. Based on the literature data published for spectra
of arabinofuranosides,41 arabinan,42Á45 arabinogalac-
tan,40,46 it was possible to identify those four residues.
Peak assignments of 13C NMR of these four kinds of
arabinose residues are shown in Table 2. The little
discrepancy of chemical shifts observed as compared to
the results reported earlier,39,40,44Á46 can be due to
different NMR instrument and reference systems used
for recording the spectra. It may be mentioned here that
number of peaks for these four kinds of residues were
qualitatively similar but different in order of magnitude
in respect to the proportion of arabinosyl residues of
arabinan. It has been shown earlier43,47 that the
integrated intensities of signals in the 13C NMR
spectrum of polysaccharides permit conclusions on the
relative proportion of constituent sugars in the polymer
/
chain starting from the nonreducing terminal end of
arabinan. Similar specificity was also observed for the
AFI from Aspergillus awamori IFO 403328 hydrolyzed
the (10/5) linkage of the arabinosyl residues of arabino-
oligosaccharides. Whereas, the AFs from Streptomyces
diastatochromogenes 065,29 B. subtilis 3-6,22 A. awamori
IFO 4033 AFII,28 Trichoderma reesei,25 A. niger 5-16,17
A. niger (Megazyme),18 Streptomyces chartreusis
GS90130 preferentially hydrolyzed the (10
/3) linkage
of the arabinosyl side chain of arabinan. This AF also
released arabinose exowisely. On the other hand,
enzymes that release arabinose in endo-fashion (endo-
arabinase) also have been described for species of
Bacillus,31Á35 Aspergillus6,36 and Pseudomonas.37

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A.K.M. Shofiqur Rahman et al. / Carbohydrate Research 338 (2003) 1469Á1476
/
Fig. 3. 13C NMR spectrum of Native and AF-digested arabinan. The numbers above each peak refer to the carbon atoms
positioned across the Ara residues listed in the top left.
investigated. Thus, for
L
-arabinan (native and AF-
It is to be noted here that 13C NMR spectroscopy is
particularly suitable for the structural investigation of
arabinans, which exhibit very characteristic spectra,
digested arabinan), integration of the spectrum gave
the tentative values showed in both spectrums for above
four sugar residues, which were in good agreement with
the results of methylation analysis. However, the spectra
because of their a-L-furanoid constituents. As methyla-
tion analysis and NMRs depicted similar information
on the structure of sugar beet arabinan, the approach
presented here appears to be very effective for elucida-
tion of the structure of any type of polysaccharides, if
suitable model compounds or literature data are avail-
able.
of both cases are dominated by signals of a-
L-arabino-
furanosyl moieties. As it was stated earlier, whether the
pyranose residues are really part to the polysaccharides
or if they are artifacts arising during the methylation
analysis. This can be circumvented by the use of NMR
spectroscopy. So, no signals were recorded for a-
arabinopyranosyl (Arap) residue in this NMR. The C-1
signal for the a- -arabinopyranosyl residue resonated at
102Á
105 ppm.41,42,45 Evidences for the absence of a-
arabinopyranosyl residue in the tested arabinan are
from the 13C NMR spectrum, which did not show any
signal around at 102 and 105 ppm where C-1 resonance
L-
L
4. Conclusions
/
L-
AFs warrant substantial research efforts because they
represent potential rate limiting enzymes in xylan
degradation, particularly those substrates from agricul-
tural residues such as corn fiber, corn stover and rice
of an a-
L-arabinopyranosyl residue would be expected.

A.K.M. Shofiqur Rahman et al. / Carbohydrate Research 338 (2003) 1469Á
/1476
1475
Table 2
Peak assignments of 13C NMR of arabinan and AF-digested arabinan
Residues
Chemical shift in d (ppm)
C-1 C-2
C-3
C-4
C-5
Native arabinan
c
(10
T-a-A-Araf
(103,5)-a-
(105)-a- -Araf
/
3)-a-
L-Araf
110.3(110.25 )
109.9(110.9 a)
109.9(108.3 b)
110.3(110.25 c)
81.9(84.2 c)
83.7(83.0 a)
79.4(80.0 b)
83.7(84.2 c)
84.5(84.8 c)
79.4(78.2 a)
79.4(80.0 b)
79.4(79.4 c)
83.7(84.2 c)
85.1(85.5 a)
83.7(82.5 b)
85.1(84.2 c)
63.9(64.1 c)
63.9(62.9 a)
69.3(67.7 b)
69.3(72.3 c)
/
L-Araf
/
L
AF-digested arabinan
(103)-a- -Araf
T-a-A-Araf
(103,5)-a-
(105)-a- -Araf
/
L
110.3(110.25 c)
109.7(110.9 a)
109.7(108.3 b)
110.3(110.25 c)
79.6(84.2 c)
83.7(83.0 a)
79.6(80.0 b)
83.6(84.2 c)
84.2(84.8 c)
79.4(78.2 a)
79.6(80.0 b)
79.6(79.4 c)
83.6(84.2 c)
85.1(85.5 a)
83.6(82.5 b)
85.1(84.2 c)
63.9(64.1 c)
63.9(62.9 a)
69.7(67.7 b)
69.7(72.3 c)
/
L-Araf
/
L
a
b
c
Ref. 40.
Ref. 45.
Ref. 46.
straw.48,49 Thus, the high activity of the AF from R.
pusillus HHT-1 on both arabinan and debranched
6. Voragen, A. G. J.; Rombouts, F. M.; Searle-van Leeuwen,
M. F.; Schols, H. A.; Pilnik, W. Food Hydrocolloids 1987,
1, 423Á
7. Kaji, A.; Tagawa, K.; Matsubara, K. Agric. Biol. Chem.
1967, 31, 1023Á1028.
8. Seri, K.; Sanai, K.; Matsuo, N.; Kawakubo, K.; Xue, C.;
Inoue, S. Metabolism 1996, 45, 1368Á1374.
9. Sanai, K.; Seri, K.; Inoue, S. Nihon Eiyou Syokuryougaku
Kaishi 1997, 50, 133Á137 (in Japanese).
10. Rahman A. K. M. S.; Sugitani N.; Hatsu M.; Takamizawa
K. Can. J. Microbiol. 2003, 49, 58Á64.
/
437.
arabinan, its ability to release L-arabinose from arabi-
noxylan,14 thermotolerancy and vital role for structural
elucidation of polysaccharides make the enzyme a
promising candidate in food and chemical industries
for use in the production of fermentable sugars from
lignocellulosic biomass such as agricultural wastes, oat-
spelt xylan and to improve animal feed digestibility by
hydrolyzing arabinoxylans, a major component of
animal feed.50
/
/
/
/
11. Cho C. -H.; Hatsu M.; Takamizawa K. Proceedings of
Fifth International Symposium on Waste Management
Problems in Agro-Industries, 2001, Shiga-Japan, 16Á
November, pp. 235Á242.
12. Cho, C.-H.; Hatsu, M.; Takamizawa, K. The Japan
Society of Waste Management Experts 2001, 12, 266Á
/
18
/
Acknowledgements
/
274 (in Japanese).
13. Kubata, K. B.; Horitsu, H.; Takamizawa, K.; Suzuki, T.;
Kawai, K. The Japan Society of Waste Management
The authors are grateful to Professor Makoto Shimoya-
mada for kindly providing the NMR facilities. The
assistances of Dr Benedict C. Okeke and Md. Ali Asgar
for critical reading and discussion are gratefully ac-
knowledged.
Experts 1994, 5, 29Á
14. Rahman, A. K. M. S.; Kawamura, S.; Hatsu, M.; Hoq, M.
M.; Takamizawa, K. Can. J. Microbiol. 2001, 47, 767Á
/35.
/
772.
15. Harris, P. J.; Henry, R. J.; Blakeney, A. B.; Stone, B. A.
Carbohydr. Res. 1984, 127, 59Á
16. York, W. S.; Darvill, A. G.; McNeil, M.; Albersheim, P.
Methods Enzymol. 1985, 118, 3Á40.
17. Kaneko, S.; Shimasaki, T.; Kusakabe, I. Biosci. Biotech-
nol. Biochem. 1993, 57, 1161Á1165.
18. Kaneko, S.; Ishii, T.; Kobayashi, H.; Kusakabe, I. Biosci.
Biotechnol. Biochem. 1998, 62, 695Á699.
19. Saha, B. C.; Bothast, R. J. Appl. Environ. Microbiol. 1998,
64, 216Á220.
20. Kaji, A.; Sato, M.; Tsutsui, Y. Agri. Biol. Chem. 1981, 45,
925Á931.
21. Tajana, E.; Fiechter, A.; Zimmermann, W. Appl. Environ.
Microbiol. 1992, 58, 1447Á1450.
/
73.
References
/
1. Coughlan, M. P.; Hazlewood, G. P. Biotechnol. Appl.
Biochem. 1993, 17, 259Á289.
2. Bacic, A.; Harris, P. J.; Stone, B. A. In The Biochemistry of
Plants; Preiss, J., Ed.; Vol. 14; Academic press: San Diego,
/
/
/
1988; pp 297Á
3. Manin, C. F.; Shareek, R. M.; Kluepfel, D. Biochem. J.
1994, 302, 443Á449.
4. Dekker, R. F. H.; Richards, G. N. Adv. Carbohyd. Chem.
Biochem. 1976, 32, 277Á352.
5. Kaji, A. Adv. Carbohydr. Chem. Biochem. 1984, 42, 383Á
397.
/
371.
/
/
/
/
/
/

1476
A.K.M. Shofiqur Rahman et al. / Carbohydrate Research 338 (2003) 1469Á1476
/
22. Kaneko, S.; Sano, M.; Kusakabe, I. Appl. Environ.
Microbiol. 1994, 60, 3425Á3428.
23. Kaji, A.; Tagawa, K. Biochim. Biophys. Acta 1970, 207,
456Á464.
24. Komae, K.; Kaji, K.; Sato, M. Agri. Biol. Chem. 1982, 46,
1899Á1905.
36. Ramon, D.; van der Veen, P.; Visser, J. FEMS Microbiol.
Lett. 1993, 113, 15Á22.
37. McKie, V. A.; Black, G. W.; Millward-Sadler, S. J.;
/
/
Hazlewood, G. P.; Laurie, J. I.; Gilbert, H. J. Biochem.
/
J. 1997, 323, 547Á
38. Qin, X.; Yamauchi, R.; Aizawa, K.; Inakuma, T.; Kato,
K. Carbohydr. Res. 2001, 333, 79Á85.
39. Saulnier, L.; Brillouet, J. M.; Moutounet, M. Carbohydr.
Res. 1992, 224, 219Á235.
40. Gane, A. M.; Craik, D.; Munro, S. L. A.; Howlett, G. J.;
Clarke, A. E.; Bacic, A. Carbohydr. Res. 1995, 277, 67Á
/555.
/
/
25. Kaneko, S.; Kuno, A.; Matsuo, N.; Ishii, T.; Kobayashi,
H.; Hayashi, K.; Kusakabe, I. Biosci. Biotechnol. Biochem.
/
1998, 62, 2205Á
26. Janson, J.; Lindberg, B. Acta Chem. Scand. Ser. B 1982,
36, 277Á279.
27. Joseleau, J.-P.; Chambat, G.; Lanvers, M. Carbohydr.
Res. 1983, 122, 107Á113.
/2210.
/
/
85.
41. Akiyama, Y.; Mori, M.; Kato, K. Agric. Biol. Chem. 1980,
/
44, 2487Á
42. Joseleau, J.-P.; Chambat, G.; Vignon, M.; Barnoud, F.
Carbohydr. Res. 1977, 58, 165Á175.
43. Capek, P.; Toman, R.; Kardosova, A.; Rosik, J. Carbo-
hydr. Res. 1983, 117, 133Á140.
44. Swamy, N. R.; Salimath, P. V. Phytochemistry 1991, 30,
263Á265.
45. Cardoso, S. M.; Silva, A. M. S.; Coimbra, M. A.
Carbohydr. Res. 2002, 337, 917Á924.
46. Qin, X.; Yamauchi, R.; Aizawa, K.; Inakuma, T.; Kato,
K. J. Appl. Glycosci. 2000, 47, 155Á161.
47. Shaskov, A. S.; Chizhov, O. S. Bioorg. Khim. 1976, 437Á
496.
48. Saha, B. C. Biotechnol. Adv. 2000, 18, 403Á
49. Saha, B. C.; Bothast, R. J. Appl. Biochem. Biotechnol.
1999, 76, 65Á77.
50. Campbell, G. L.; Bedford, M. R. Can. J. Anim. Sci. 1992,
72, 449Á466.
/2489.
28. Kaneko, S.; Arimoto, M.; Ohba, M.; Kobayashi, H.; Ishii,
T.; Kusakabe, I. Appl. Environ. Microbiol. 1998, 64,
/
4021Á
29. Kaneko, S.; Higashi, K.; Yasui, T.; Kusakabe, I. J.
Ferment. Bioeng. 1998, 5, 518Á520.
30. Matsuo, N.; Kaneko, S.; Kuno, A.; Kobayashi, H.;
Kusakabe, I. Biochem. J. 2000, 345, 9Á15.
31. Takao, M.; Akiyama, K.; Sakai, T. Appl. Environ.
Microbiol. 2002, 68, 1639Á1646.
32. Kaji, A.; Saheki, T. Biochem. Biophys. Acta 1975, 410,
354Á360.
33. Sakai, T.; Sakamoto, T. Agric. Biol. Chem. 1990, 54, 879Á
/4027.
/
/
/
/
/
/
/
/
/
/
889.
34. Weinstein, L.; Albersheim, P. Plant Physiol. 1979, 63,
/423.
425Á
35. Yoshihara, O.; Kaji, A. Agric. Biol. Chem. 1983, 47,
1935Á1940.
/432.
/
/
/