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Applied and Environmental Microbiology, September 1999, p. 3964-3968, Vol. 65, No. 9
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
An 
-L-Arabinofuranosidase from
Trichoderma reesei Containing a Noncatalytic
Xylan-Binding Domain
Masahiro
Nogawa,
Kenji
Yatsui,
Akiko
Tomioka,
Hirofumi
Okada, and
Yasushi
Morikawa*
Department of Bioengineering, Nagaoka
University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata
940-21, Japan
Received 11 March 1999/Accepted 1 July 1999
 |
ABSTRACT |
L-Sorbose, an excellent cellulase and xylanase inducer
from Trichoderma reesei PC-3-7, also induced
-L-arabinofuranosidase (-AF) activity. An -AF
induced by L-sorbose was purified to homogeneity, and its
molecular mass was revealed to be 35 kDa (AF35), which was not
consistent with that of the previously reported -AF. Another
species, with a molecular mass of 53 kDa (AF53), which is identical to
that of the reported -AF, was obtained by a different purification
procedure. Acid treatment of the ammonium sulfate-precipitated fraction
at pH 3.0 in the purification steps or pepsin treatment of the purified
AF53 reduced the molecular mass to 35 kDa. Both purified enzymes have
the same enzymological properties, such as pH and temperature effects
on activity and kinetic parameters for
p-nitrophenyl--L-arabinofuranoside (pNPA). Moreover, the N-terminal amino acid sequences of these enzymes were
identical with that of the reported -AF. Therefore, it is obvious
that AF35 results from the proteolytic cleavage of the C-terminal
region of AF53. Although AF35 and AF53 showed the same catalytic
constant with pNPA, the former showed drastically reduced specific
activity against oat spelt xylan compared to the latter. Furthermore,
AF53 was bound to xylan rather than to crystalline cellulose (Avicel),
but AF35 could not be bound to any of the glycans. These results
suggest that AF53 is a modular glycanase, which consists of an
N-terminal catalytic domain and a C-terminal noncatalytic xylan-binding domain.
| |
INTRODUCTION |
Xylan is the most abundant renewable
biomass polymer next to cellulose and one of the major components of
plant cell walls. Xylan refers to any of a class of heterogeneous plant
polysaccharides whose compositions differ depending on their origins.
It consists of a backbone of 
-1,4-linked xylopyranose and side
chains of -L-arabinofuranoside, an acetyl group, and/or
4-o-methylglucuronic acid at the C-2 and C-3 positions of
the xylose units. Xylan-degrading enzyme systems of microorganisms
include endo--1,4-xylanase (EC 3.2.1.8),
-D-xylosidase (EC 3.2.1.37), and the side
chain-debranching enzymes -L-arabinofuranosidase (EC
3.2.1.55) (-AF), -glucuronidase (EC 3.2.1.139), and acetylxylan
esterase (EC 3.1.1.72) (20). In addition to xylanase, these
side chain-cleaving activities were required for effective degradation
of this polysaccharide because the side chains hinder xylanase from
accessing the xylan backbone (20).
Plant cell wall hydrolases often contain both catalytic and
noncatalytic domains linked by a hinge region. Most cellulose-binding domains (CBDs) are quite common not only in cellulases but also in
hemicellulases (24). The role of the CBDs located in
hemicellulases is unclear, although it is thought that they bring the
hemicellulase into contact with the plant cell wall. Recently,
xylan-binding domains (XBDs) were reported to be present in enzymes
isolated from Cellulomonas fimi, Thermomonospora
fusca, and Streptomyces lividans (2, 7, 8,
23). The amino acid sequences of these XBDs show some
similarities to those of bacterial CBDs.
An -AF can release terminal -L-1,2- and
-L-1,3-arabinofuranosyl residues from arabinoxylan.
These enzymes have been found in many microorganisms, and reports of
their purification and gene cloning have been published
(20). The filamentous fungus Trichoderma reesei
is one of the most potent cellulolytic organisms and also produces a
xylanolytic system, which contains multiple enzyme activities required
for the complete degradation of xylan (1, 10).
An -AF from T. reesei has been purified and partially
characterized, and its cDNA has been cloned (14, 18). In
this fungus, L-arabinose was reported to induce the enzyme
(11, 12). In our preliminary results, we observed that
-AF activity was also induced by L-sorbose, which is an
excellent inducer for the production of cellulase and xylanase by
T. reesei PC-3-7, a cellulase-hyperproducing mutant (9,
25). Comparison of the purified -AF induced by L-sorbose with that induced by arabinose proved that the
enzymes differed in their molecular masses. In the present paper, it is reported that the enzyme with the lower molecular mass is the truncated
form of the enzyme with the higher molecular mass. It is shown that the
latter possesses a noncatalytic XBD.
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MATERIALS AND METHODS |
Fungal strains and culture conditions.
The strain used in
this study was T. reesei PC-3-7, a cellulase-hyperproducing
mutant with an enhanced response to L-sorbose as an inducer
(9); it was obtained from Kyowa Hakko Kogyo Co. Ltd. (Tokyo,
Japan). This strain was maintained on a potato dextrose agar (PDA;
Difco) slant, and the conidia were obtained from a PDA plate culture.
Induction experiments were performed in a resting cell system
(16). To obtain mycelium, 106 conidia of
T. reesei PC-3-7 were inoculated into 50 ml of basal medium
with 0.3% (wt/vol) glucose as a carbon source (9) and were
incubated for 48 h at 28°C with vigorous shaking (220 rpm). For
induction, the mycelium was washed twice with 0.9% (wt/vol) NaCl and
the dry weight of the mycelium was determined. The washed mycelium was
transferred into an induction medium containing L-sorbose (500 µg/ml) or -sophorose (250 µg/ml) as previously described by
Kawamori et al. (9), giving a final dry weight of about 2.0 mg/ml for the mycelium. The induction was carried out for 24 h at
28°C on a reciprocal shaker at 120 strokes per min. For purification
of -AF, the mycelium obtained was transferred into 800 ml of the
induction solution containing L-sorbose in a 2-liter Erlenmeyer flask and was incubated for 60 h at 28°C with shaking (220 rpm).
Enzyme assay.
-AF and -D-xylosidase
activities were measured by monitoring the release of
p-nitrophenol at 420 nm from
p-nitrophenyl--L-arabinofuranoside (pNPA;
Sigma) and p-nitrophenyl--D-xylopyranoside
(pNPX; Sigma), respectively (4). The -AF activity that
liberated -arabinofuranose from xylan was measured as the amount of
reducing sugar liberated during the hydrolysis of oat spelt xylan
(Sigma) by the Somogyi-Nelson method (19).
-D-Glucosidase, -L-arabinopyranosidase,
-D-galactosidase, -D-mannosidase, and
-D-galactosidase activities were determined by
liberation of p-nitrophenol or o-nitrophenol from
p-nitrophenyl--D-glucopyranoside, p-nitrophenyl--L-arabinopyranoside,
p-nitrophenyl--D-galactopyranoside, p-nitrophenyl--D-mannopyranoside, and
o-nitrophenyl--D-galactopyranoside (all from
Sigma), respectively (4, 13, 17). Endoglucanase, xylanase,
mannanase, laminarinase, and amylase activities were measured by
evaluating the amounts of reducing sugar released from carboxymethyl
cellulose (Wako, Tokyo, Japan), oat spelt xylan, locust bean gum
(Fluka), laminarin (Sigma), and soluble starch (Wako) by the
Somogyi-Nelson method (5, 6, 9, 19, 25). One unit of enzyme
activity was defined as the amount of enzyme that liberated 1 µmol of
product from the substrate.
The Km and Vmax values
for -AF were determined from Lineweaver-Burk plots using pNPA as a
substrate at concentrations ranging from 0.2 to 5.0 mM.
Enzyme purification.
All operations were carried out at
4°C. The crude enzyme was precipitated from 800 ml of the filtrate
obtained from the induction medium with ammonium sulfate (40 to 60%
saturation) and was dissolved in 20 ml of 50 mM sodium acetate buffer
(pH 4.0) containing 30% saturated ammonium sulfate. The sample was
then applied onto a column (1.5 by 2.8 cm) of phenyl Sepharose HP
(Pharmacia) which had been equilibrated with the same buffer. The
column was washed with the buffer, and the enzyme was eluted with a
linear gradient of 30 to 0% saturated ammonium sulfate in the buffer
at a flow rate of 20 ml/h. The -AF fractions eluted with 17%
saturated ammonium sulfate were pooled and concentrated by
ultrafiltration (PM-10; Amicon). The concentrate was applied onto a
column (1 by 96 cm) of Sephacryl S-100 HR (Pharmacia) previously
equilibrated with 50 mM sodium acetate buffer (pH 4.0) containing 300 mM NaCl and was eluted with the same buffer at a flow rate of 14 ml/h. Fractions exhibiting -AF activity were pooled, desalted, and concentrated, and the buffer was changed to 10 mM citrate-phosphate (pH
6.0), by ultrafiltration (PM-10). The sample was subjected to
ion-exchange chromatography on a column of SP-Sepharose FF (Pharmacia)
(0.8 by 2 cm) previously equilibrated with the same buffer. The column
was washed with the buffer and eluted with a linear gradient of 0 to
0.2 M NaCl in the same buffer at a flow rate of 4 ml/h. The fractions
with -AF activity were pooled and then desalted and concentrated by
ultrafiltration (PM-10). The sample was applied onto a Q-Sepharose FF
column (0.8 by 2 cm) previously equilibrated with the same buffer (pH
6.0). The column was washed with the same buffer at a flow rate of 4 ml/h. The resulting active fraction without adsorption was concentrated and used as the purified enzyme preparation throughout this study.
The
-AF with a molecular mass of 35 kDa (AF35) was preliminarily
purified by SP-Sepharose FF column chromatography (pH 4.0)
as the first
step after precipitation with ammonium sulfate. As
this enzyme was
suggested to be an acid protease cleavage product
of AF53, the ammonium
sulfate precipitate (40 to 60% saturation)
was dissolved in a 50 mM
sodium acetate buffer (pH 3.0) and then
incubated at 28°C for 24 h for digestion. The enzyme was thereafter
purified as described above
for
AF53.
Endoglucanase I (EGI) used in this experiment was purified from a crude
preparation of
T. reesei cellulase (Kyowa Hakko) as
previously described by Morikawa et al. (
15).
pH and temperature optima and stability.
The optimum
temperature for the activity against pNPA was determined by performing
the assays (see above) at various temperatures between 30 and 70°C in
50 mM sodium acetate buffer, pH 4.0. For determination of temperature
stability, the purified enzyme preparation in 10 mM citric
acid-Na2HPO4 buffer (30 µg of AF53/ml; 50 µg of AF35/ml), pH 6.0, was incubated at different temperatures (0 to 60°C) for 1 h, and aliquots of the samples were thereafter
assayed for activity. The optimum pH value was determined by monitoring the activity at different pH values (3.0 to 11.0) at 50°C. The following buffers were used: 0.2 M citric
acid-Na2HPO4 (pH 3.0 to 8.0) and 0.2 M
H3BO4-Na2CO3 (pH 7.0 to 11.0). For pH stability, the preparation in 0.2 M citric
acid-Na2HPO4 buffers (pH 3.0 to 8.0) or in 0.2 M H3BO4-Na2CO3 (pH 7.0 to 11.0) (3.0 µl of AF53/ml; 5.0 µg of AF35/ml) was incubated at
25°C for 1 h. Aliquots of the samples were assayed immediately
for activity against pNPA at pH 4.0.
Acid and pepsin treatments (pH 3.0).
The ammonium
sulfate-precipitated sample was dissolved in 50 mM sodium acetate
buffer (pH 4.0), and the buffer was changed to 50 mM sodium acetate
buffer (pH 3.0) by ultrafiltration. After incubation at 28°C for
24 h, the samples were analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Purified AF53 (12.6 µg) was incubated with pepsin (1.4 µg) in 100 µl of 10 mM citric acid-Na
2HPO
4 buffer (pH
3.0) at 37°C
for 0.5, 2, 4, and 12 h. The reaction was stopped
by the addition
of 2 µl of 50 µM pepstatin A in 1 M citric
acid-NaOH buffer (pH
7.0). The digestion products were analyzed by
SDS-PAGE, and their
activities against pNPA and oat spelt xylan were
measured.
Substrate-binding assay.
Oat spelt xylan and crystalline
cellulose (Avicel) used for the substrate-binding assay were prepared
as follows: 0.75 g was washed three times with 50 mM sodium
acetate buffer (pH 4.0), and the resulting pellet was suspended in the
same buffer to give suspensions of 22.2 mg/ml (xylan) or 29.9 mg/ml
(Avicel). For the substrate-binding assay, 50 µl of AF53, AF35, or
EGI at various concentrations (0 to 12, 0 to 6, and 0 to 20 µg/ml,
respectively) was mixed with 450 µl of the substrate suspensions and
held at 4°C for 10 min. This mixture contained bovine serum albumin
(BSA) at a final concentration of 1 mg/ml to prevent inactivation
caused by dilution. The supernatant was then separated from the
insoluble substrate by centrifugation at 18,000 × g
for 2 min. Immediately after centrifugation, an aliquot of the
supernatant was taken, and the activity was assayed.
For analysis of proteins adsorbed to xylan, BSA was omitted from the
sample mixture. The pellet after incubation for binding
was washed
three times with 50 mM sodium acetate buffer (pH 4.0)
at 4°C, and the
resulting pellet was suspended in 50 µl of the
SDS-PAGE sample
buffer, immersed in a boiling water bath for 2
min, and then analyzed
by SDS-PAGE (10%
polyacrylamide).
Other analytical methods.
Protein concentration was
determined by the method of Bradford (3) with BSA as a
standard. SDS-PAGE was carried out by using a MINI-PROTEAN II
Electrophoresis Cell (Bio-Rad) according to the manufacturer's manual.
The proteins in the gel were stained with Coomassie brilliant blue
R-250. The N-terminal amino acid sequences of both of the purified
enzymes were identified by automated Edman degradation using a Shimadzu
PPSQ-21 protein sequencer.
| |
RESULTS |
Induction of -AF activity by L-sorbose and its
purification.
As shown in Table 1,
L-sorbose induced -AF activity, while -sophorose did
not. In order to compare this L-sorbose-induced -AF with
the previously reported enzyme, we purified it, using SP-Sepharose FF
chromatography as the first step. The molecular mass of the purified
enzyme was 35 kDa when it was analyzed by SDS-PAGE (data not shown),
which is in contrast to the previously reported 53 kDa (18).
We speculated that proteolysis of the enzyme by an acid protease may
have occurred during the SP-Sepharose chromatography step, because some
of the SDS-PAGE bands of L-sorbose-induced proteins
disappeared when the proteins were incubated under acidic conditions
(pH 3.0) (Fig. 1, lanes 3 and 4).
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FIG. 1.
SDS-PAGE of the purified -AFs and the acid-treated
sample (pH 3.0) of the crude enzyme induced by L-sorbose.
Lane 1, marker proteins (Pharmacia) containing soybean trypsin
inhibitor (20 kDa), carbonic anhydrase (30 kDa), ovalbumin (43 kDa),
BSA (67 kDa), and phosphorylase (94 kDa); lane 2, purified AF35 (2.0 µg of protein); lane 3, crude enzyme after an
(NH4)2SO3 precipitation step (12 µg of protein); lane 4, acid-treated crude enzyme (11 µg of
protein); lane 5, purified AF53 (2.1 µg of protein).
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|
To avoid proteolysis of the enzyme, we changed the purification step as
described in Materials and Methods. The
-AF was purified
32-fold to
a specific activity of 120 U/mg of protein with pNPA
as a substrate.
This enzyme preparation yielded a single band
in SDS-PAGE, from which
its molecular mass was estimated to be
53 kDa (Fig.
1, lane 5). We
named this enzyme AF53. We also purified
the enzyme after proteolysis
by incubation at pH 3.0. The purification
is summarized in Table
2. The enzyme incubated at pH 3.0 was
purified 65-fold to a specific activity of 196 U/mg of protein.
The
molecular mass of this enzyme was determined by SDS-PAGE as
35 kDa
(Fig.
1, lane 2), which is in a good agreement with that
of the
preliminarily purified enzyme described above. We designated
this
enzyme AF35. The N-terminal amino acid sequences of both
enzymes were
identical, G-P-X-D-I-Y-S-A-G-G (where "X" stands
for an
undetermined amino acid), and were also identical to that
of the
-AF
earlier purified from this fungus (
14). Therefore,
these
results show that AF35 is most probably a C-terminally truncated
form
of AF53.
Time course of digestion of AF53 by pepsin.
In vitro
proteolysis of purified AF53 with pepsin was performed to verify the
conversion of AF53 to AF35. As shown in Fig. 2A, a decrease in the 53-kDa band was
accompanied by an increase in the 35-kDa protein band on SDS-PAGE.
After an incubation of 6 h, the 53-kDa protein band was completely
converted to the 35-kDa band, and for a further 12 h no
proteolysis of the 35-kDa band was observed. The enzyme activity
against pNPA did not change during proteolysis, whereas that against
xylan strongly decreased (Fig. 2B). The time course of the activity
with xylan coincided with that of the conversion of the 53-kDa band to
a 35-kDa band on SDS-PAGE. Therefore, the conversion of the 53-kDa
protein to a 35-kDa protein by pepsin is likely responsible for the
decrease in the activity against xylan.
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FIG. 2.
(A) In vitro proteolysis of AF53 by pepsin. The course
of the remaining protein after the pepsin treatment was followed by
SDS-PAGE. Lane M, marker proteins. Numbers above the lanes are
incubation times (in hours). (B) Time course of the -AF activities
through in vitro proteolysis of AF53 by pepsin. The hydrolysis
activities for pNPA ( ) and oat spelt xylan ( ) were plotted.
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|
Enzymological analysis of -AFs.
The enzymological
properties of both -AFs are summarized in Table
3. The influences of pH and temperature
on enzyme activity and stability were largely similar for the two
enzymes. The Km and catalytic constant
(kcat) values of AF53 and AF35 were 0.9 and 1.1 mM and 269 and 259 s
1, respectively, although the
Vmax values of the two enzymes were different
from each other. However, the most striking difference between the two
enzymes was that the specific activity of AF35 against oat spelt xylan
was significantly lower (0.061 U/mg of protein) than that of AF53 (2.6 U/mg of protein) (Table 4). AF35 also
exhibited high activities against pNPA and pNPX, as did the 53 kDa
enzyme (196 and 1.8 U/mg of protein and 120 and 1.3 U/mg of protein,
respectively).
Xylan-binding assay.
To investigate the difference in action
on xylan between the two enzymes, their adsorption on xylan and Avicel
was tested. T. reesei EGI containing a CBD could bind to
Avicel (70% of the added enzyme was bound) but bound to xylan with
less avidity (less than 25% was bound) under the conditions used (Fig.
3). The activity of AF53 in the
supernatant of the xylan solution decreased appreciably, to 32% of the
initially added activity, whereas that in the supernatant of the Avicel
solution was reduced only to 74% (Fig. 3). On the other hand, almost
all the activity of AF35 remained in both supernatants. Furthermore,
the AF53 adsorbed on the xylan was eluted with SDS and detected by
SDS-PAGE (Fig. 4).
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FIG. 3.
Adsorption of the purified enzymes on insoluble glycans.
The remaining activities in the supernatant were determined by the
standard assay. The concentrations of oat spelt xylan and Avicel in the
binding mixture were 20 and 27 mg/ml, respectively, and various
concentrations of the AF53, AF35, and EGI in the mixture (0 to 1.2, 0 to 0.6, and 0 to 2.0 µg/ml, respectively) were used. Symbols
represent activity after incubation with oat spelt xylan ( ), with
Avicel ( ), and without substrate ( ).
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FIG. 4.
SDS-PAGE analysis of the xylan-bound enzymes. The
xylan-bound protein was detected by SDS-PAGE. Lane 1, purified AF53;
lane 2, xylan-bound fraction of AF53; lane 3, purified AF35; lane 4, xylan-bound fraction of AF35.
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| |
DISCUSSION |
We have previously reported that L-sorbose is an
excellent inducer of cellulase and xylanase formation of T. reesei PC-3-7 (9, 25), but we have been unaware whether
the compound also triggers the production of other glycanases.
Therefore, we compared the glycanase activities induced by
L-sorbose and by -sophorose, the most potent cellulase
inducer, in T. reesei PC-3-7. The results showed that
L-sorbose induced higher hemicellulase activities than
-sophorose did, particularly in the case of -AF, which was formed
only by L-sorbose. Recent studies showed that
L-arabinose and arabitol induced -AF expression in
T. reesei (11, 12). We show that
L-sorbose is also an inducer of -AF. The
L-sorbose-inducible -AF was therefore purified to
ascertain that it was the same enzyme as that induced by
L-arabinose. In the purification step, two enzymes, AF35
and AF53, were obtained with and without the acid treatment,
respectively. These enzymes share some enzymological properties, such
as effects of pH and temperature on enzyme activity and stability, and
kinetic properties (Km and
kcat) for pNPA. Both enzymes purified in this
study also possessed a slight -D-xylosidase activity,
which could be measured by using pNPX as a substrate (Table 4). The
-D-xylosidase activities of AF53 and AF35 were about 1%
of the -AF activities for pNPA, which agrees well with the findings
for the T. reesei -AF previously reported
(18). Moreover, the N-terminal amino acid sequences of the
two purified enzymes were identical to that of the deduced amino acid
from the cloned -AF cDNA from T. reesei RUT C-30
(14). These results clearly indicated that the enzymes were
identical to that previously reported for this fungus (14,
18).
Proteolysis of AF53 by the acid proteases led to a decrease in the
molecular mass to 35 kDa and to reduction of the activity against
xylan, whereas the activities against pNPA and pNPX were not altered
(Tables 3 and 4). The possibility that the AF53 activity on xylan is
due, at least in part, to a very slight amount of contaminating
xylanase activity cannot be ruled out. For further characterization of
the activities of AF53 and AF35 on xylan, it is preferable to use
recombinant enzymes produced by a non-xylanase-producing host. A
reduction of activity for insoluble substrates as a result of
proteolysis has been shown in some polysaccharide-degrading enzymes
with modular structures (24). These modular glycanases usually possess a catalytic domain responsible for the hydrolysis reaction itself and a substrate-binding domain promoting adsorption of
the enzyme onto insoluble substrates. The two domains of the glycanases
are joined by linker peptides (24). In cellobiohydrolase I
of T. reesei, limited proteolysis by papain separated the
CBD from the catalytic domain, which is fully active on small soluble substrates but has strongly reduced activity against insoluble celluloses (Avicel and phosphoric-acid-swollen cellulose)
(22). In our study, AF35, the truncated form in which the
C-terminal half of AF53 was removed, lost the binding activity for
xylan. Therefore, we suppose that the -AF from T. reesei
is a modular glycanase containing a catalytic domain and a
substrate-binding domain. This noncatalytic domain of the enzyme has a
higher affinity for xylan than for crystalline cellulose (Avicel).
Therefore, it is regarded as an XBD rather than a CBD. Most of the
substrate-binding domains of hemicellulases are commonly CBDs. On the
other hand, two bacterial xylanases, TfxA from T. fusca and
XYLD from C. fimi, and an -AF (AbfB) from S. lividans were thought to possess XBDs. The XBDs of bacterial
xylanases have some homology to CBDs of the same origin (2, 7, 8,
23). Unfortunately, the AF53 XBD was not isolated when AF53 was
treated with pepsin because no distinct band appeared except for the
AF35 band. Furthermore, no region homologous to the CBD and/or XBD and
linkers was found in the deduced amino acid sequence of the -AF
C-terminal half from T. reesei (14). In
preliminary experiments performed with various ionic strengths, the
amounts of AF53 that bound to xylan varied with the ionic strength
(28% of the activity initially added was bound to xylan in the buffer
with a concentration of 200 mM), as reported by Tenkanen et al.
(21). It is postulated, therefore, that electrostatic
interactions may have a significant role in binding to oat spelt xylan.
However, AF35 cannot be bound to xylan at all under the same
conditions, suggesting that the AF53 C-terminal half without catalytic
function can be bound to xylan. This indicates that the type of the XBD
would be different from that of CBDs reported so far. In any case, AF53
is, to our knowledge, the first eukaryotic hemicellulase containing an XBD.
We have cloned the -AF gene, using the PCR technique, from the
chromosomal DNA of T. reesei PC-3-7. Now we are trying to express this gene in the yeast Schizosaccharomyces pombe and
to use this expression system to determine the precise region of the
gene corresponding to the XBD in the C-terminal half of AF53.
| |
ACKNOWLEDGMENTS |
Special thanks go to H. Watanabe for critical reading of the manuscript.
This work has been supported by a Grant-in-Aid for Scientific Research
on Priority Areas (A), no. 11132218, from the Ministry of Education,
Science, Sports and Culture of Japan.
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FOOTNOTES |
*
Corresponding author. Mailing address: Nagaoka
University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-21, Japan. Phone: 81-258-479407. Fax: 81-258-479407. E-mail:
yasushi{at}nagaokaut.ac.jp.
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Applied and Environmental Microbiology, September 1999, p. 3964-3968, Vol. 65, No. 9
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
