Previous Article | Next Article 
Applied and Environmental Microbiology, October 1998, p. 4021-4027, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Purification and Substrate Specificities of Two
-L-Arabinofuranosidases from Aspergillus
awamori IFO 4033
Satoshi
Kaneko,1,
Mitsue
Arimoto,1
Misako
Ohba,1
Hideyuki
Kobayashi,2
Tadashi
Ishii,3 and
Isao
Kusakabe1,*
Institute of Applied Biochemistry, University
of Tsukuba, 1-1-1 Tennoodai, Tsukuba,1
National Food Research Institute, Ministry of Agriculture,
Forestry, and Fisheries,2 and
Forestry
and Forest Products Research Institute, Tsukuba Norin Kenkyu
Danchinai,3 Ibaraki 305, Japan
Received 30 January 1998/Accepted 13 July 1998
 |
ABSTRACT |
-L-Arabinofuranosidases I and II were purified from
the culture filtrate of Aspergillus awamori IFO 4033 and
had molecular weights of 81,000 and 62,000 and pIs of 3.3 and 3.6, respectively. Both enzymes had an optimum pH of 4.0 and an optimum
temperature of 60°C and exhibited stability at pH values from 3 to 7 and at temperatures up to 60°C. The enzymes released arabinose
from p-nitrophenyl--L-arabinofuranoside, O--L-arabinofuranosyl-(1
3)-O-
-D-xylopyranosyl-(14)-D-xylopyranose, and arabinose-containing polysaccharides but not from
O--D-xylopyranosyl-(12)-O--L-arabinofuranosyl-(13)-O--D-xylopyranosyl-(14)-O--D-xylopyranosyl-(14)-D-xylopyranose. -L-Arabinofuranosidase I also released arabinose from
O--D-xylopy-ranosyl-(14)-[O--L-arabinofuranosyl-(13)]-O--D-xylopyranosyl-(14)-D-xylopyranose. However, -L-arabinofuranosidase
II did not readily catalyze this hydrolysis reaction.
-L-Arabinofuranosidase I hydrolyzed all linkages that can occur between two -L-arabinofuranosyl residues in
the following order: (15) linkage > (13) linkage > (12) linkage. -L-Arabinofuranosidase II hydrolyzed
the linkages in the following order: (15) linkage > (12)
linkage > (13) linkage. -L-Arabinofuranosidase
I preferentially hydrolyzed the (15) linkage of branched
arabinotrisaccharide. On the other hand,
-L-arabinofuranosidase II preferentially hydrolyzed the
(13) linkage in the same substrate.
-L-Arabinofuranosidase I released arabinose from the
nonreducing terminus of arabinan, whereas
-L-arabinofuranosidase II preferentially hydrolyzed the arabinosyl side chain linkage of arabinan.
| |
INTRODUCTION |
Recently, it has been proven that
L-arabinose selectively inhibits intestinal sucrase in a
noncompetitive manner and reduces the glycemic response after sucrose
ingestion in animals (33). Based on this observation,
L-arabinose can be used as a physiologically functional
sugar that inhibits sucrose digestion. Effective
L-arabinose production is therefore important in the food
industry. L-Arabinosyl residues are widely distributed in
hemicelluloses, such as arabinan, arabinoxylan, gum arabic, and
arabinogalactan, and the -L-arabinofuranosidases (-L-AFases) (EC 3.2.1.55) have proven to be essential
tools for enzymatic degradation of hemicelluloses and structural
studies of these compounds.
-L-AFases have been classified into two families of
glycanases (families 51 and 54) on the basis of amino acid sequence
similarities (11). The two families of
-L-AFases also differ in substrate specificity for
arabinose-containing polysaccharides. Beldman et al. summarized the
-L-AFase classification based on substrate specificities
(3). One group contains the Arafur A (family 51) enzymes,
which exhibit very little or no activity with
arabinose-containing polysaccharides. The other group contains
the Arafur B (family 54) enzymes, which cleave arabinosyl side chains
from polymers. However, this classification is too broad to define the
substrate specificities of -L-AFases. There have
been many studies of the -L-AFases (3,
12), especially the -L-AFases of
Aspergillus species (2-8, 12-15, 17, 22, 23, 28-32,
36-39, 41-43, 46). However, there have been only a few
studies of the precise specificities of these
-L-AFases. In previous work, we elucidated the substrate specificities of -L-AFases from Aspergillus
niger 5-16 (17) and Bacillus subtilis 3-6 (16, 18), which should be classified in the Arafur A group
and exhibit activity with arabinoxylooligosaccharides, synthetic methyl 2-O-, 3-O-, and
5-O-arabinofuranosyl--L-arabinofuranosides (arabinofuranobiosides) (20), and methyl
3,5-di-O--L-arabinofuranosyl--L-arabinofuranoside (arabinofuranotrioside) (19).
In the present work, we purified two -L-AFases from a
culture filtrate of Aspergillus awamori IFO 4033 and
determined the substrate specificities of these
-L-AFases by using arabinose-containing polysaccharides
and the core oligosaccharides of arabinoxylan and arabinan.
| |
MATERIALS AND METHODS |
Substrates.
p-Nitrophenyl--L-arabinopyranoside,
p-nitrophenyl--D-galactopyranoside,
p-nitrophenyl--D-xylopyranoside, and larch
wood arabinogalactan were purchased from Sigma Chemical Company (St. Louis, Mo.). Gum arabic was obtained from Wako Pure Chemical Industries (Osaka, Japan). Nihon Syokuhin Kakoh Co., Ltd. (Fuji, Japan), supplied
corn hull arabinoxylan, and debranched arabinan (linear 15-linked
arabinan) was obtained from Megazyme Pty., Ltd. (North Rocks,
Australia). Sugar beet arabinan and arabinoxylooligosaccharides were
prepared by methods described previously (24, 25, 49). p-Nitrophenyl--L-arabinofuranoside
(PNP--L-Araf) was synthesized by the method
of Kelly et al. (21). Methyl 2-O-, methyl
3-O-, and methyl
5-O--L-arabinofuranosyl--L-arabinofuranosides
(arabinofuranobiosides) and methyl
3,5-di-O--L-arabinofuranosyl--L-arabinofuranoside (arabinofuranotrioside) were synthesized by methods described previously (19, 20). Figure 1
shows the structures of arabinoxylooligosaccharides and
arabinooligosaccharides.
-L-AFase assay and measurement of protein.
Each assay mixture contained 0.5 ml of a 2 mM
PNP--L-Araf solution, 0.4 ml of McIlvaine
buffer (0.2 M Na2HPO4, 0.1 M citric acid) (pH
4.0), and 0.1 ml of enzyme solution. The reaction was carried out at
50°C for 10 min and was stopped by adding 1.0 ml of a 0.2 M
Na2CO3 solution, and the amount of
p-nitrophenol (PNP) released was then determined at 408 nm
(standard method). One unit of enzyme activity was defined as the
amount of enzyme which released 1 µmol of PNP from
PNP--L-Araf per min under these conditions.
The -L-AFase activities with PNP glycosides were
determined like the activity with PNP--L-Araf
was determined except that various PNP glycosides were used.
The protein concentration during purification of the enzyme was
measured by determining the absorbance at 280 nm and assuming
that an
absorbance at 280 nm of 1.0 was equal to a concentration
of 1 mg per
ml. The protein concentration was also determined
by the method
described by Smith et al. (
34) by using a BCA
protein assay
kit (Pierce, Rockford, Ill.) with bovine serum albumin
as the
standard.
Preparation of the crude enzyme.
A. awamori IFO 4033, which was purchased from the Institute of Fermentation, Osaka (Osaka,
Japan), was cultured in five 500-ml shaking flasks containing 100 ml of
medium consisting of 2.0% arabinoxylan, 0.6% peptone, 0.3% yeast
extract, 1.0% KH2PO4, and 0.05%
MgSO4 · 7H2O. Preparations were
cultivated on a reciprocal shaker (125 oscillations per min) at 35°C
for 90 h. The culture broth was then filtered through filter paper
(type 2; Toyo Roshi Co. Ltd., Tokyo, Japan), and the culture filtrate
was used as the crude enzyme preparation.
Purification of -L-AFases.
All purification
procedures were performed at 6°C.
(i) Step 1 (both fractions).
The culture filtrate obtained
as described above was dialyzed against deionized water, and the
dialyzed enzyme was applied to a column (30 by 200 mm) containing
ECTEOLA-Cellulose (Wako) equilibrated with 50 mM phosphate buffer (pH
4.5). The column was then washed with the same buffer, and the enzyme
was eluted from the column with a linear 0 to 0.5 M NaCl gradient
(total volume, 2,000 ml) at a flow rate of 100 ml per h. The eluate was fractionated into 20-ml portions. The -L-AFase was
separated into two fractions, fraction I
(-L-AFase I) (fraction tubes 41 through 49) and
fraction II (-L-AFase II) (fraction tubes 56 through
75). Fraction I was dialyzed against deionized water, while fraction II
was concentrated by ultrafiltration (type YM 10 membrane filter; Amicon
Inc., Beverly, Mass.).
(ii) Step 2 (fraction I).
The dialyzed fraction I
(-L-AFase I) was applied to an SP-Sephadex C-50 column
(37 by 750 mm) equilibrated with McIlvaine buffer (pH 2.5). After the
column was washed with the same buffer, the enzyme was eluted from the
column with a pH 2.5 to 5.0 gradient (total volume, 1,000 ml) at a flow
rate of 50 ml per h. The eluate was collected in 10-ml portions. The
active fractions, fractions 40 to 44, were combined and dialyzed
against deionized water.
(iii) Step 3 (fraction I).
The dialyzed enzyme
(-L-AFase I) was applied to a Mono S HR 5/5 column (5 by
50 mm) which had been equilibrated with a 1/5 dilution of McIlvaine
buffer (pH 2.5). After the column was washed with the same buffer, the
enzyme was eluted from the column with a pH 2.5 to 4.25 gradient (total
volume, 60 ml) at a flow rate of 1 ml per min. The eluate was
fractionated into 1-ml portions. The active fractions, fractions 47 to
50, were combined and dialyzed against deionized water, and the
final preparation was used as purified -L-AFase I.
(iv) Step 2 (fraction II).
Concentrated fraction II
(-L-AFase II) was applied to an Ultrogel AcA 44 column (40 by 700 mm) equilibrated with 0.2 M NaCl in 50 mM phosphate
buffer (pH 6.5). An elution flow rate of 15 ml per h was used, and the
eluate was fractionated into 10-ml portions. The fractions with
-L-AFase activity (fraction tubes 47 through 53) were
combined and dialyzed against deionized water. The final preparation
obtained was used as purified -L-AFase II.
PAGE.
Sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis (PAGE) was carried out in a 12% polyacrylamide gel by
the method of Laemmli (27). The protein was stained with
Coomassie brilliant blue R-250 and then destained with 10% acetic acid
in 30% methanol. The molecular weight of the enzyme was determined by
SDS-PAGE by using molecular weight markers (SDS-PAGE Standard Low;
Bio-Rad). The N-terminal amino acid sequences of
-L-AFases I and II were determined with a model HP
G1005A protein sequence system.
The method described previously (
48) was used to determine
the isoelectric points of
-
L-AFases I and II. The
protein was
stained with Coomassie brilliant blue R-250, and the
isoelectric
focusing calibration kit (pH 2.5 to 6.5; Pharmacia,
Uppsala, Sweden)
was used as the standard proteins for the pI
measurements.
Enzymatic properties.
The effects of pH on the activity and
stability of -L-AFases were determined in a series of
McIlvaine buffers with pH values from 2 to 8. The activity of
-L-AFase was assayed by the standard method. To measure
the stability of -L-AFases, preparations were preincubated in the absence of substrate for 120 min at 30°C. The
residual activity was then assayed by the standard method.
The effects of temperature on the activity and stability of
-
L-AFases were determined with a series of water baths
at temperatures
ranging from 30 to 80°C. The activity of
-
L-AFase was assayed
by the standard method. To measure
the stability of the
-
L-AFases,
preparations were
preincubated at pH 4.0 for 120 min, and the
residual activity was then
assayed.
-L-AFase activity with arabinose-containing poly-
or oligosaccharides.
Each reaction mixture contained 0.5 ml of an
-L-AFase solution (0.3 U), 0.4 ml of McIlvaine buffer
(pH 4.0), and 0.1 ml of 1% substrate (arabinoxylan, arabinogalactan,
gum arabic, arabinan, or debranched arabinan). After 24 h of
incubation at 30°C, the reducing power formed from each substrate was
measured by the Somogyi-Nelson method (35).
The
-
L-AFase activity experiments with
arabinoxylooligosaccharides were identical to the activity experiments
with polysaccharides
except that the concentration of each
arabinoxylooligosaccharide
used was 10%. After 0, 1, 3, 6, 12, and
24 h of incubation at
30°C, the reaction mixtures were heated in
a boiling water bath
for 10 min to stop the reaction. One microliter of
each mixture
was used for thin-layer chromatography to characterize the
hydrolysis
products. Chromatography was performed by using the
ascending
method with HPTLC Alufolien Cellulose (Merck, Darmstadt,
Germany)
and a 1-butanol-pyridine-water (6:4:3, vol/vol/vol) solvent
system.
The sugars on each plate were detected by heating the plate at
140°C for about 5 min after it was sprayed with a 1% methanol
solution of
p-anisidine hydrochloride.
Reaction mixtures that contained 0.1-ml portions of 10%
arabinofuranobioside or arabinofuranotrioside solutions instead of
polysaccharides were used to determine the activity with
arabinooligosaccharides.
After 0, 10, 20, and 30 min of incubation at
50°C, the reducing
power formed from methyl
-
L-arabinofuranobioside was determined
by the
Somogyi-Nelson method (
35). Also, after 3 h of
incubation
at 30°C, reaction mixtures containing methyl
arabinofuranotrioside
were applied to a C
18 column
[LiChrospher 100 RP-18 (5 µm); Cica-Merck,
Darmstadt, Germany] that
had been preequilibrated with 1.7% CH
3CN
at a flow rate of
0.5 ml per min. The eluted sugars were detected
with a reflective index
detector.
Glycosyl linkage composition of -L-AFase-digested
arabinans.
The reaction mixture described above containing 1%
arabinan was incubated for 3 h at 30°C. Then 100 µl of the
mixture was applied to a Superdex peptide HR 10/30 (Pharmacia) column
which had been equilibrated with 200 mM HCOONH4 buffer (pH
7.0) to remove the arabinose. The arabinan hydrolysate was pooled and
lyophilized. The glycosyl linkage composition of the arabinan
hydrolysate was determined by using a modification of the Hakomori
procedure (9). The polysaccharide was
per-O-methylated with methylsulfinyl methyl potassium and
iodomethane, and the resulting products were isolated by using Sep-Pak
C18 cartridges (44). The glycosyl linkage
composition was then determined by gas chromatography-mass spectrometry
of the resulting partially methylated, partially acetylated alditol acetate derivatives (47).
| |
RESULTS |
Purification and enzymatic properties of the
-L-AFases.
A. awamori IFO 4033 produced
two extracellular -L-AFases. Purification of
-L-AFases I and II from A. awamori IFO 4033 is summarized in Table 1. These two
enzymes were completely separated by ECTEOLA-Cellulose column
chromatography (Fig. 2). Each of
the purified enzymes was resolved as a single band by SDS-PAGE
when the bands were visualized by Coomassie brilliant blue R-250
staining (Fig. 3). The
molecular weights of -L-AFases I and II
were estimated to be 81,000 and 62,000, respectively, by SDS-PAGE. The
-L-AFase I pI was 3.3, and the
-L-AFase II pI was 3.6 (Fig. 3). Figure 4 shows the amino acid sequences of
the amino termini of -L-AFases from various sources. The
amino acid sequences of -L-AFases I and II were quite
different but exhibited high levels of homology to the amino acid
sequences of A. niger -L-AFases A and B,
respectively. Both -L-AFase I and
-L-AFase II exhibited maximum activity at pH 4.0. Both
enzymes were also slowly inactivated at pHs above 7.0 and below
3.0. The maximum activity for each enzyme occurred at 60°C; however,
the enzymes were inactivated at temperatures above 60°C (data not
shown).
View larger version (22K):
[in this window]
[in a new window]
|
FIG. 2.
ECTEOLA-Cellulose column chromatography. The
experimental conditions used are described in Materials and Methods.
 , -L-AFase activity;  , absorbance at 280 nm; ---,
NaCl gradient. The bar indicates fractions that were pooled.
|
|
View larger version (53K):
[in this window]
[in a new window]
|
FIG. 3.
SDS-PAGE and isoelectric focusing of
-L-AFases from A. awamori IFO 4033. (A)
SDS-PAGE of -L-AFases I and II. Lanes 1 and 4, standard
proteins (2 µg each), including phosphorylase b (molecular
weight, 97,000), bovine serum albumin (66,200), ovalbumin (45,000),
carbonic anhydrase (31,000), soybean trypsin inhibitor (21,500), and
lysozyme (14,400); lane 2, purified -L-AFase I (2 µg);
lane 3, purified -L-AFase II (2 µg). (B) Isoelectric
focusing of -L-AFases I and II. Lanes 1 and 4, standard
proteins (1 µg each), including pepsinogen (pI 2.80),
amyloglucosidase (pI 3.5), glucose oxidase (pI 4.15), and soybean
trypsin inhibitor (pI 4.55); lane 2, purified -L-AFase I
(1 µg); lane 3, purified -L-AFase II (1 µg).
|
|
View larger version (26K):
[in this window]
[in a new window]
|
FIG. 4.
N-terminal amino acid sequences of
-L-AFases from A. awamori IFO 4033. The
search for amino acid sequences homologous to the amino acid sequences
of -L-AFases was performed with MPsrch
(mpsearch{at}dna.affrc.go.jp). The amino acid residues
that are identical in three sequences are shaded. The amino acid
residues that are completely conserved are indicated by white letters
on a black background. -L-AFase A from A. niger (accession no. A27979) belongs to glycosyl hydrolase family
51; -L-AFase B from A. niger (A27977),
-L-AFase from A. niger (U39942),
-L-AFase from Trichoderma reesei (Z69252),
and -L-AFase from Trichoderma koningii
(U38661) belong to family 54.
|
|
Substrate specificities.
Both enzymes from A. awamori IFO 4033 hydrolyzed PNP--L-Araf
but did not hydrolyze
p-nitrophenyl--L-arabinopyranoside,
p-nitrophenyl--D-xylopyranoside, and
p-nitrophenyl--D-galactopyranoside (data not
shown).
Table
2 shows the levels of
hydrolysis of arabinan, debranched arabinan, arabinoxylan,
arabinogalactan, and gum arabic by
the
-
L-AFases
from
A. awamori IFO 4033. Differences in the amounts
of
arabinose produced from these substrates were observed for
the two
enzymes.
Figure
5A shows the course of hydrolysis
of
O-
-
L-arabino-furanosyl-(1
3)-
O-
-
D-xylopyranosyl-(1
4)-
D-xylopyranose (A
1X
2),
O-
-
D-xylopyranosyl-(1
4)-[
O-
-
L-arabinofuranosyl-(1
3)]-
O-
-
D-xylopyranosyl-(1
4)-
D-xylopyranose (A
1X
3),
and
O-
-
D-xylopyranosyl-(1
2)-
O-
-
L-arabinofuranosyl-(1
3)-
O-
-
D-xylopyranosyl-(1
4)-
O-
-
D-xylopyranosyl-(1
4)-
D-xylopyranose
(A
1X
4) by
-
L-AFase I from
A. awamori
IFO 4033.
-
L-AFase I released
arabinose from
A
1X
2 and A
1X
3 (Fig. 5Aa
and Ab) but not from A
1X
4 (Fig. 5Ac). Figure
5B
shows the course of hydrolysis of A
1X
2,
A
1X
3, and A
1X
4 by
-
L-AFase II from
A. awamori IFO 4033.
-
L-AFase
II completely hydrolyzed
A
1X
2 and only slightly hydrolyzed
A
1X
3 (Fig. 5Ba and Bb), but arabinose was
not liberated from A
1X
4 (Fig.
5Bc).
View larger version (102K):
[in this window]
[in a new window]
|
FIG. 5.
Activities of -L-AFases from A. awamori IFO 4033 with arabinoxylooligosaccharides. (A)
-L-AFase I. (B) -L-AFase II. (a)
A1X2. (b) A1X3. (c)
A1X4. Lanes A1, authentic xylose to xylohexaose
from top to bottom; lanes A2, authentic arabinose.
Authentic xylose to xylohexaose was prepared by the method described
previously (26). The experimental conditions used are
described in Materials and Methods. After 0, 1, 3, 6, 12, and 24 h
of incubation at 30°C, 1 µl of each reaction mixture was subjected
to thin-layer chromatography.
|
|
Figure
6 shows the time course of
hydrolysis of arabinofuranobiosides by
A. awamori IFO 4033
-
L-AFases. Arabinofuranobiosides
were
hydrolyzed to arabinose and methyl
-
L-arabinofuranoside
by
-
L-AFases I and II. The order of
-
L-AFase I hydrolysis of
the substrate linkages was
(1
5) linkage > (1
3) linkage > (1
2)
linkage, and
the rates of hydrolysis for 30-min reactions were
8.7, 5.9, and 5.3%,
respectively (Fig.
6A). The order of
-
L-AFase
II
hydrolysis of the substrate linkages was (1
5) linkage > (1
2)
linkage > (1
3) linkage, and the rates of
hydrolysis for 30-min
reactions were 7.0, 5.6, and 5.0%, respectively
(Fig.
6B).
View larger version (18K):
[in this window]
[in a new window]
|
FIG. 6.
Hydrolysis of arabinofuranobiosides. (A)
-L-AFase I. (B) -L-AFase II.  , methyl
2-O--L-arabinofuranosyl--L-arabinofuranoside;
, methyl
3-O--L-arabinofuranosyl--L-arabinofuranoside;
 , methyl
5-O--L-arabinofuranosyl--L-arabinofuranoside.
The experimental conditions used are described in Materials and
Methods. After 0, 10, 20, and 30 min of incubation at 50°C, the
reducing powers formed from arabinose disaccharides were
measured by the Somogyi-Nelson method (35).
|
|
Figure
7 shows the activities of
-
L-AFases from
A. awamori IFO 4033 with
arabinofuranotrioside.
-
L-AFase I showed greater
hydrolysis of the (1
5) linkage than of the (1
3) linkage
(Fig.
7A), whereas
-
L-AFase II exhibited a higher
hydrolysis rate for
the (1
3) linkage than for the (1
5) linkage
(Fig.
7B). A total
of 92.4% of the trisaccharide was hydrolyzed
by
-
L-AFase I and
44.2% was hydrolyzed by
-
L-AFase II during 3-h incubations.
View larger version (9K):
[in this window]
[in a new window]
|
FIG. 7.
Activities of -L-AFases from A. awamori IFO 4033 with arabinofuranotrioside.
(A) -L-AFase I. (B) -L-AFase II. Peak 1, methyl
5-O--L-arabinofuranosyl--L-arabinofuranoside;
peak 2, methyl
3-O--L-arabinofuranosyl--L-arabinofuranoside;
peak 3, methyl
3,5-di-O--L-arabinofuranosyl--L-arabinofuranoside.
The experimental conditions used are described in Materials and
Methods. After 3 h of incubation at 30°C, the reaction mixtures
containing methyl arabinofuranotrioside were applied to a
C18 column.
|
|
Table
3 shows the glycosyl linkage
compositions of arabinan and
-
L-AFase-digested
arabinan.
-
L-AFase I hydrolyzed arabinan
from the
nonreducing terminus, and
-
L-AFase II
preferentially
hydrolyzed the arabinosyl side chain of arabinan.
| |
DISCUSSION |
Rombouts et al. (31) reported that A. niger
produced two extracellular -L-AFases,
-L-AFase A and -L-AFase B. The substrate specificities of these enzymes have been elucidated for
arabinofuranose-containing polysaccharides, 15-linked
arabinofuranooligosaccharides (31), and various kinds
of arabinoxylooligosaccharides (22). Flipphi et
al. have cloned the genes of these -L-AFases
(6-8). These -L-AFases were classified into
glycosyl hydrolase families 51 (-L-AFase A) and 54 (-L-AFase B) on the basis of amino acid similarities. It
is known that the substrate specificity of -L-AFase A is
quite different from that of -L-AFase B (31).
Based on the differences in the substrate specificities of these
enzymes, Beldman et al. classified them as Arafur A
(-L-AFase A) and Arafur B (-L-AFase B)
(3). Arafur A is defined as the enzyme which does not
exhibit activity with arabinose-containing polysaccharides. In
contrast, Arafur B exhibits activity with the polymers.
-L-AFase A released arabinose from
A1X2 and A1X3
(22), as did -L-AFase I from A. awamori IFO 4033 (Fig. 5A). The N-terminal amino acid sequences of
these enzymes were also very similar (Fig. 4). These results show that
-L-AFase I and -L-AFase A are similar.
-L-AFase B from A. niger preferentially
hydrolyzed arabinofuranosyl side chains from arabinan
(31) and released arabinose from
A1X2 but not from A1X3
(22). -L-AFase II from A. awamori IFO 4033 also hydrolyzed arabinosyl side chains in
preference to the 15-arabinofuranosyl backbone (Table 3). In
addition, -L-AFase II released arabinose
from A1X2 and only small amounts of
arabinose from A1X3 (Fig. 5B). It
is clear that these results are very similar to the results obtained
for -L-AFase B. These types of enzymes are
produced by several other organisms, including A. niger K1 (14), A. niger (Megazyme)
(29), radishes (10), and B. subtilis
(45). The optimum pH of -L-AFase II from
A. awamori was 4.0, and the molecular weight was 62,000 (Fig. 3); these data are almost identical to the data for
-L-AFase B from A. niger (31). The
isoelectric point of -L-AFase II, pI 3.6, was quite
different from the pI obtained for -L-AFase B (pI 4.5 to
5.5) (31). The amino-terminal amino acid sequence of
-L-AFase II (Fig. 4) was, however, very similar to the
amino-terminal amino acid sequence of -L-AFase B
(5).
The arabinans analyzed so far are highly branched, with (15) links
between the main-chain residues, many of which have substitutions at
the O-2 and/or O-3 position of single- or
multiple-unit side chains (1). Methyl 2-O-,
3-O-, and
5-O-arabinofuranosyl--L-arabinofuranosides and methyl
3,5-di-O--L-arabinofuranosyl--L-arabinofuranoside are therefore good substrates for elucidation of the substrate specificity of -L-AFases.
-L-AFase I acted on methyl
arabinofuranobiosides in the following order: (15)
linkage > (13) linkage > (12) linkage (Fig. 6A). The rates of hydrolysis of the arabinofuranobiosides
by -L-AFase II occurred in the following order: (15)
linkage > (12) linkage > (13) linkage (Fig. 6B). The
Km values for branched arabinan and debranched
arabinan for -L-AFase B from A. niger were
3.7 × 10
3 and 2.9 × 103
mol/liter, respectively (31). These results indicate that
-L-AFase B has a slightly higher affinity for the
-(15) linkages than has been observed in
-L-AFase II from A. awamori. The linkage preferences in arabinofuranobiosides of
-L-AFases I and II from A. awamori were quite
different from those of the -L-AFases from B. subtilis 3-6 [(12) linkage > (13) linkage > (15) linkage] (16) and from Scopolia japonica
[(13) linkage > (15) linkage] (40).
By using a branched arabinose trisaccharide, we found that
-L-AFase I hydrolyzed the (15) linkage faster than it
hydrolyzed the (13) linkage (Fig. 7A) and that
-L-AFase II hydrolyzed the (13) linkage faster than
it hydrolyzed the (13) linkage (Fig. 7B). Our results are consistent
with the enzymatic mode of action with arabinan (Table 3), as follows.
-L-AFase I hydrolyzed arabinan gradually from the
nonreducing terminus; in contrast, -L-AFase II
preferentially hydrolyzed the monosaccharide arabinosyl
side chains of arabinan. Based on these results, it is apparent that the linkages cleaved preferentially in the branched
arabinose trisaccharide by -L-AFase II are
not necessarily related to the linkages cleaved in the various
arabinose disaccharides.
-L-AFases are potentially important for
arabinose production from hemicelluloses, and we
demonstrated in this study that -L-AFases from A. awamori IFO 4033 may play a role in this process. We investigated
the substrate specificities of -L-AFases with a limited
number of substrates, and it appears that a more effective approach for
production of arabinose will be necessary for elucidation of the detailed structure of arabinan and the -L-AFases.
| |
ACKNOWLEDGMENTS |
We are grateful to Derek Watt and Pauric J. McGinty, an STA
fellow of the National Food Research Institute, for critically reading
the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Applied Biochemistry, University of Tsukuba, 1-1-1 Tennoodai, Tsukuba, Ibaraki 305, Japan. Phone: 81-298-53-6623. Fax: 81-298-53-4605.
Present address: National Food Research Institute, Ministry of
Agriculture, Forestry, and Fisheries, Ibaraki 305, Japan.
| |
REFERENCES |
| 1.
|
Bacic, A.,
P. J. Harris, and B. A. Stone.
1988.
Structure and function of plant cell walls, p. 297-371.
In
J. Preiss (ed.), The biochemistry of plants, vol. 14. Academic Press, San Diego, Calif.
|
| 2.
|
Beldman, G.,
M. J. F. Searl-van Leeuwen,
G. A. De Ruiter,
H. A. Siliha, and A. G. J. Voragen.
1993.
Degradation of arabinans by arabinanases from Aspergillus aculeatus and Aspergillus niger.
Carbohydr. Polym.
20:159-168.
|
| 3.
|
Beldman, G.,
H. A. Schols,
S. M. Piston,
M. J. F. Searl-van Leeuwen, and A. G. J. Voragen.
1997.
Arabinans and arabinan degrading enzymes.
Adv. Macromol. Carbohydr. Res.
1:1-64.
|
| 4.
|
Crous, J. M.,
I. S. Pretorius, and W. H. van Zyl.
1996.
Cloning and expression of the alpha-L-arabinofuranosidase gene (ABF2) of Aspergillus niger in Saccharomyces cerevisiae.
Appl. Microbiol. Biotechnol.
46:256-260[Medline].
|
| 5.
|
Fernández-Espinar, M. T.,
J. L. Peña,
F. Piñaga, and S. Vallés.
1994.
-L-Arabinofuranosidase production by Aspergillus nidulans.
FEMS Microbiol. Lett.
115:107-112[Medline].
|
| 6.
|
Flipphi, M. J. A.,
M. van Heuvel,
P. van der Veen,
J. Visser, and L. H. de Graaff.
1993.
Cloning and characterization of the abfB gene coding for the major -L-arabinofuranosidase (ABF B) of Aspergillus niger.
Curr. Genet.
24:525-532[Medline].
|
| 7.
|
Flipphi, M. J. A.,
J. Visser,
P. van der Veen, and L. H. de Graaff.
1993.
Cloning of the Aspergillus niger gene encoding -L-arabinofuranosidase A.
Appl. Microbiol. Biotechnol.
39:335-340[Medline].
|
| 8.
|
Flipphi, M. J. A.,
J. Visser,
P. van der Veen, and L. H. de Graaff.
1994.
Arabinase gene expression in Aspergillus niger: indications for coordinated regulation.
Microbiology
140:2673-2682[Abstract/Free Full Text].
|
| 9.
|
Hakomori, S.
1964.
A rapid permethylation of glycolipid and polysaccharide catalyzed by methylsulfinyl carbanion in dimethyl sulfoxide.
J. Biochem.
55:205-208[Free Full Text].
|
| 10.
|
Hata, K.,
M. Tanaka,
Y. Tsumuraya, and Y. Hashimoto.
1992.
-L-Arabinofuranosidase from radish (Raphanus sativus L.) seeds.
Plant Physiol.
100:388-396[Abstract/Free Full Text].
|
| 11.
|
Henrissat, B., and A. Bairoch.
1996.
Updating the sequence-based classification of glycosyl hydrolases.
Biochem. J.
316:695-696.
|
| 12.
|
Kaji, A.
1984.
L-Arabinosidases.
Adv. Carbohydr. Chem. Biochem.
42:383-394.
|
| 13.
|
Kaji, A., and K. Tagawa.
1970.
Purification, crystallization and amino acid composition of -L-arabinofuranosidase from Aspergillus niger.
Biochim. Biophys. Acta
207:456-464[Medline].
|
| 14.
|
Kaji, A.,
K. Tagawa, and T. Ichimi.
1969.
Properties of purified -L-arabinofuranosidase from Aspergillus niger.
Biochim. Biophys. Acta
171:186-188[Medline].
|
| 15.
|
Kaji, A.,
K. Tagawa, and K. Matsubara.
1967.
Studies on the enzymes acting on araban. VIII. Purification and properties of arabanase produced by Aspergillus niger.
Agric. Biol. Chem.
31:1023-1028.
|
| 16.
|
Kaneko, S., and I. Kusakabe.
1995.
Substrate specificity of -L-arabinofuranosidase from Bacillus subtilis 3-6 toward arabinofuranooligosaccharides.
Biosci. Biotechnol. Biochem.
59:2132-2133[Medline].
|
| 17.
|
Kaneko, S.,
T. Shimasaki, and I. Kusakabe.
1993.
Purification and some properties of intracellular -L-arabinofuranosidase from Aspergillus niger 5-16.
Biosci. Biotechnol. Biochem.
57:1161-1165[Medline].
|
| 18.
|
Kaneko, S.,
M. Sano, and I. Kusakabe.
1994.
Purification and some properties of -L-arabinofuranosidase from Bacillus subtilis 3-6.
Appl. Environ. Microbiol.
60:3425-3428[Abstract/Free Full Text].
|
| 19.
|
Kaneko, S.,
Y. Kawabata,
T. Ishii,
Y. Gama, and I. Kusakabe.
1995.
The core trisaccharide of -L-arabinofuranan: synthesis of methyl 3,5-di-O--L-arabinofuranosyl--L-arabinofuranoside.
Carbohydr. Res.
268:307-311[Medline].
|
| 20.
|
Kawabata, Y.,
S. Kaneko,
I. Kusakabe, and Y. Gama.
1995.
Synthesis of regioisomeric methyl -L-arabinofuranobiosides.
Carbohydr. Res.
267:39-47[Medline].
|
| 21.
|
Kelly, M. A.,
M. L. Sinnott, and D. Widdows.
1988.
Preparation of some aryl -L-arabinofuranosides as substrates for arabinofuranosidase.
Carbohydr. Res.
181:262-266.
|
| 22.
|
Kormelink, F. J. M.,
H. Gruppen, and A. G. J. Voragen.
1993.
Mode of action of (14)--D-arabinoxylan arabinofuranohydrolase (AXH) and -L-arabinofuranosidases on alkali-extractable wheat-flour arabinoxylan.
Carbohydr. Res.
249:345-353[Medline].
|
| 23.
|
Kroon, P. A., and G. Williamson.
1996.
Release of feruric acid from sugar-beet pulp by using arabinanase, arabinofuranosidase and an esterase from Aspergillus niger.
Biotechnol. Appl. Biochem.
23:263-267.
|
| 24.
|
Kusakabe, I.,
S. Ohgushi,
T. Yasui, and T. Kobayashi.
1983.
Structures of the arabinoxylo-oligosaccharides from the hydrolytic products of corn cob arabinoxylan by a xylanase from Streptomyces.
Agric. Biol. Chem.
47:2713-2723.
|
| 25.
|
Kusakabe, I.,
T. Yasui, and T. Kobayashi.
1975.
Some properties of araban degrading enzymes produced by microorganisms and enzymatic preparation of arabinose from sugar beet pulp.
Nippon Nogeikagaku Kaishi
49:295-305.
|
| 26.
|
Kusakabe, I.,
T. Yasui, and T. Kobayashi.
1975.
Preparation of crystalline xylooligosaccharides from xylan.
Nippon Nogeikagaku Kaishi
49:383-385.
|
| 27.
|
Laemmli, U. K.
1970.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature (London)
227:680-685[Medline].
|
| 28.
|
Matthew, J. A.,
G. M. Wyatt,
D. B. Archer, and M. R. A. Morgan.
1991.
Purification of a sugar beet pectin modifying enzyme system from Aspergillus niger by antibody affinity column chromatography.
Carbohydr. Polym.
16:381-390.
|
| 29.
|
McCleary, B. V.
1991.
Comparison of endolytic hydrolases that depolymerize 1,4--D-mannan, 1,5--L-arabinan, and 1,4--D-galactan, p. 437-449.
In
G. F. Leatham, and M. E. Himmel (ed.), Enzymes in biomass conversion. American Chemical Society, Washington, D.C.
|
| 30.
|
Ramón, D.,
P. van der Veen, and J. Visser.
1993.
Arabinan degrading enzymes from Aspergillus nidulans: induction and purification.
FEMS Microbiol. Lett.
113:15-22[Medline].
|
| 31.
|
Rombouts, F. M.,
A. G. Voragen,
M. F. Searl-van Leeuwen,
C. C. J. M. Geraeds,
H. A. Schols, and W. Plinik.
1988.
The arabinanases of Aspergillus niger purification and characterisation of two -L-arabinofuranosidases and an endo-1,5--L-arabinanase.
Carbohydr. Polym.
9:25-47.
|
| 32.
|
Sanchez-Torres, P.,
L. Gonzalez-Candelas, and D. Ramon.
1996.
Expression in a wine yeast strain of the Aspergillus niger abfB gene.
FEMS Microbiol. Lett.
145:189-194[Medline].
|
| 33.
|
Seri, K.,
K. Sanai,
N. Matsuo,
K. Kawakubo,
C.-Y. Xue, and S. Inoue.
1996.
L-Arabinose selectively inhibits intestinal sucrase in an uncompetitive manner and reduces glycaemic response after sucrose ingestion in animals.
Metabolism
45:1368-1374[Medline].
|
| 34.
|
Smith, P. K.,
R. I. Krohn,
G. T. Hermanson,
A. K. Mallia,
F. H. Gartner,
M. D. Provenzano,
E. K. Fujimoto,
N. M. Goeke,
B. J. Olson, and D. C. Klenk.
1985.
Measurement of protein using bicinchoninic acid.
Anal. Biochem.
150:76-85[Medline].
|
| 35.
|
Somogyi, M.
1952.
Notes on sugar determination.
J. Biol. Chem.
195:19-23[Free Full Text].
|
| 36.
|
Tagawa, K.
1970.
Kinetic studies of -L-arabinofuranosidase from Aspergillus niger. I. General kinetic properties of the enzyme.
J. Ferment. Technol.
48:730-739.
|
| 37.
|
Tagawa, K.
1970.
Kinetic studies of -L-arabinofuranosidase from Aspergillus niger. II. Stability of the enzyme.
J. Ferment. Technol.
48:740-746.
|
| 38.
|
Tagawa, K., and A. Kaji.
1969.
Preparation of L-arabinose-containing polysaccharides and the action of an -L-arabinofuranosidase on these polysaccharides.
Carbohydr. Res.
11:293-301.
|
| 39.
|
Tagawa, K., and G. Terui.
1968.
Studies on the enzymes acting on araban. IX. Culture conditions and process of arabanase production by Aspergillus niger.
J. Ferment. Technol.
46:693-700.
|
| 40.
|
Tanaka, M.,
A. Abe, and T. Uchida.
1981.
Substrate specificity of -L-arabinofuranosidase from plant Scopolia japonica calluses and a suggestion with reference to the structure of beet arabinan.
Biochim. Biophys. Acta
658:377-386[Medline].
|
| 41.
|
van der Veen, P.,
H. N. Arst, Jr.,
M. J. A. Flipphi, and J. Visser.
1994.
Extracellular arabinases in Aspergillus nidulans: the effect of different cre mutations on enzyme levels.
Arch. Microbiol.
162:433-440[Medline].
|
| 42.
|
van der Veen, P.,
M. J. A. Flipphi,
A. G. J. Voragen, and J. Visser.
1991.
Induction, purification and characterization of arabinanases produced by Aspergillus niger.
Arch. Microbiol.
157:23-28[Medline].
|
| 43.
|
van der Veen, P.,
M. J. A. Flipphi,
A. G. J. Voragen, and J. Visser.
1993.
Induction of extracellular arabinases on monomeric substrates in Aspergillus niger.
Arch. Microbiol.
159:66-71[Medline].
|
| 44.
|
Waeghe, T. J.,
A. G. Darvill,
M. McNeil, and P. Albersheim.
1983.
Determination, by methylation analysis, of the glycosyl-linkage compositions of microgram quantities of complex carbohydrates.
Carbohydr. Res.
123:281-304.
|
| 45.
|
Weinstein, L., and P. Albersheim.
1979.
Structure of plant cell walls. IX. Purification and partial characterization of a wall degrading endoarabanase and arabinosidase from Bacillus subtilis.
Plant Physiol.
63:425-432[Abstract/Free Full Text].
|
| 46.
|
Wood, T. M., and S. I. McCrae.
1996.
Arabinoxylan-degrading enzyme system of the fungus Aspergillus awamori: purification and properties of an alpha-L-arabinofuranosidase.
Appl. Microbiol. Biotechnol.
45:538-545[Medline].
|
| 47.
|
York, W. S.,
A. G. Darvill,
M. McNeil, and P. Albersheim.
1985.
Isolation and characterization of plant cell walls and cell wall components.
Methods Enzymol.
118:3-40.
|
| 48.
|
Yoshida, S.,
Y. Kawabata,
S. Kaneko,
Y. Nagamoto, and I. Kusakabe.
1994.
Detection of -L-arabinofuranosidase activity in isoelectric focused gels using 6-bromo-2-naphthyl--L-arabinofuranoside.
Biosci. Biotechnol. Biochem.
58:580-581.
|
| 49.
|
Yoshida, S.,
I. Kusakabe,
N. Matsuo,
K. Shimizu,
T. Yasui, and K. Murakami.
1990.
Structure of rice-straw arabinoglucuronoxylan and specificity of Streptomyces xylanase toward the xylan.
Agric. Biol. Chem.
54:449-457[Medline].
|
Applied and Environmental Microbiology, October 1998, p. 4021-4027, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Miyanaga, A., Koseki, T., Matsuzawa, H., Wakagi, T., Shoun, H., Fushinobu, S.
(2004). Crystal Structure of a Family 54 {alpha}-L-Arabinofuranosidase Reveals a Novel Carbohydrate-binding Module That Can Bind Arabinose. J. Biol. Chem.
279: 44907-44914
[Abstract]
[Full Text]
-
Shallom, D., Belakhov, V., Solomon, D., Shoham, G., Baasov, T., Shoham, Y.
(2002). Detailed Kinetic Analysis and Identification of the Nucleophile in alpha -L-Arabinofuranosidase from Geobacillus stearothermophilus T-6, a Family 51 Glycoside Hydrolase. J. Biol. Chem.
277: 43667-43673
[Abstract]
[Full Text]
-
Debeche, T., Cummings, N., Connerton, I., Debeire, P., O'Donohue, M. J.
(2000). Genetic and Biochemical Characterization of a Highly Thermostable alpha -L-Arabinofuranosidase from Thermobacillus xylanilyticus. Appl. Environ. Microbiol.
66: 1734-1736
[Abstract]
[Full Text]
Top
Abstract
Introduction
