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Appl Environ Microbiol, February 1998, p. 789-792, Vol. 64, No. 2
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Purification and Characterization of an Acetyl
Xylan Esterase from Bacillus pumilus
Giuliano
Degrassi,1
Benedict C.
Okeke,1
Carlo V.
Bruschi,2 and
Vittorio
Venturi1,*
Bacteriology Group1
and
Microbiology Group,2 International
Centre for Genetic Engineering and Biotechnology, I-34012 Trieste,
Italy
Received 11 August 1997/Accepted 10 November 1997
 |
ABSTRACT |
Bacillus pumilus PS213 was found to be able to release
acetate from acetylated xylan. The enzyme catalyzing this reaction has
been purified to homogeneity and characterized. The enzyme was
secreted, and its production was induced by corncob powder and xylan.
Its molecular mass, as determined by gel filtration, is 190 kDa, while
sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a
single band of 40 kDa. The isoelectric point was found to be 4.8, and
the enzyme activity was optimal at 55°C and pH 8.0. The activity was
inhibited by most of the metal ions, while no enhancement was observed.
The Michaelis constant (Km) and
Vmax for 
-naphthyl acetate were 1.54 mM
and 360 µmol min
1 mg of protein1,
respectively.
| |
TEXT |
Xylan is an important constituent of
hemicelluloses, and next to cellulose it is the most abundant renewable
polysaccharide in nature. It is a 
-1,4-linked D-xylose
polymer with arabinofuranose, glucuronic acid, methylglucuronic acid,
and acetyl side groups (22). Efficient and complete
degradation of xylan requires the cooperation of xylanases and
-xylosidases with the following accessory enzymes:
-arabinofuranosidase, -methylglucuronidase, acetyl xylan esterase
(AXE) (1), and ferulic acid esterase (12). The
AXE which liberates acetyl groups from the backbone of xylan has
recently been studied in several fungi, including Aspergillus
niger (11), Schizophyllum commune
(9), Trichoderma reesei (13) and
Penicillium purpurogenum (7), and also in bacteria such as Fibrobacter succinogenes (14),
Pseudomonas fluorescens (8), Streptomyces
lividans (6), and a Thermoanaerobacterium sp. (21).
Bacillus pumilus was reported to degrade xylan via xylanase
and -xylosidase enzymes (17, 18). Both genes coding for
these proteins have been isolated (16); however, no
accessory enzymes have yet been reported. In this report we describe
the purification and biochemical characterization of the AXE from
B. pumilus PS213, a strain which also has efficient xylanase
activity.
Enzyme production.
For monitoring the dynamics of xylanase and
AXE production, B. pumilus was grown in M9CA medium plus
0.5% corncob powder and in M9CA medium plus 0.5% oat spelt xylan.
Samples of the culture supernatant taken every 8 h were tested for
both acetylesterase activity and xylanase activity. B. pumilus PS213 produced AXE and xylanase simultaneously when grown
in the presence of corncob powder (Fig.
1). This enzyme released acetyl groups
from acetylated xylan, as demonstrated by treating the substrate with
the purified enzyme. The highest level of activity was found during the
stationary phase, between 40 and 72 h of growth in the medium
supplemented with corncob powder. In fungi maximal production of these
enzymes can take up to several days (4, 9). A lower level of
acetylesterase activity was detected in the medium containing 0.5%
xylan, and a much lower level was detected in the same medium devoid of
corncob powder. The majority (>60%) of the esterase activity was
found in the culture supernatant, while the remaining activity was cell associated and was detected in the crude extract prepared as described previously (5). Xylanase activity is also induced by corncob powder and to a lesser extent by xylan. Both xylanase and AXE activities were induced by xylan which is not acetylated, indicating that the two enzymes may be coregulated. The production patterns of
both enzymes are strongly related, suggesting cooperativity (Fig. 1).
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FIG. 1.
Induction of acetylesterase (A) and xylanase (B) from
B. pumilus PS213 grown on M9CA medium either alone ( ) or
with corncob powder ( ) or xylan ( ).
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Enzyme purification.
B. pumilus PS213 was grown in
800 ml of M9CA medium (20) supplemented with 0.5% (wt/vol)
corncob powder. Purification was performed at room temperature with a
low-pressure liquid chromatography system (GradiFrac; Pharmacia
Biotech, Uppsala, Sweden). The culture supernatant was subjected to
(NH4)2SO4 fractionation (30 and
70% saturation). The 70% pellet was resuspended in 100 mM sodium
phosphate (pH 7)-1.7 M (NH4)2SO4,
filtered through 0.45-µm-pore-size membrane, and fractionated by
hydrophobic interaction chromatography (phenyl Sepharose HP 16/10;
Pharmacia Biotech), as described previously (5). Active
fractions were pooled and dialyzed against 20 mM bis-Tris buffer (pH
7), concentrated by ultrafiltration with a YM30 membrane (Amicon Inc.,
Beverly, Mass.), applied to a Q Sepharose fast-flow column, and
fractionated (5). Active fractions were pooled, concentrated
to 1 ml, and loaded onto a gel filtration column (Sephacryl HR200,
column XK16; Pharmacia Biotech), previously equilibrated with 50 mM
sodium phosphate-150 mM NaCl (pH 7). Proteins were eluted at a flow
rate of 0.5 ml/min, and fractions of 2.5 ml were collected. The column
was calibrated with an MW-GF-200 kit (Sigma Chemical Co., St. Louis,
Mo.) for molecular weight estimation.
Enzyme purification is summarized in Table
1. After gel filtration, the enzyme was
purified to electrophoretic homogeneity
(Fig.
2). Only one band was obtained when the
purified protein
was loaded onto a sodium dodecyl sulfate (SDS)-12%
polyacrylamide
gel. Purification steps indicated the presence of a
single AXE
in the supernatant of the
B. pumilus culture;
multiple AXEs have
been found in the culture filtrate of some fungi
such as
T. reesei (
3) and
P. purpurogenum (
7). Also, in a
Thermoanaerobacterium sp., two distinct AXEs have been
purified and characterized (
21),
but they were cell
associated, with less than 25% in the culture
supernatant, whereas for
B. pumilus over 60% of the AXE is secreted.
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FIG. 2.
SDS-polyacrylamide gel electrophoresis (A) and
analytical isoelectric focusing (B) of the purified acetylesterase.
Lanes: 1, molecular mass standard; 2, 5 µg of acetylesterase; 3, pI
markers, consisting of trypsinogen (9.30), lentil lectin (8.65, 8.45, and 8.15), myoglobin (7.35 and 6.85), human carbonic anhydrase B
(6.55), bovine carbonic anhydrase (5.85), -lactoglobulin A (5.2),
soybean trypsin inhibitor (4.55), and amyloglucosidase (3.5); 4, 2.5 µg of acetylesterase.
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|
Enzyme assays.
Acetylesterase activity was measured by using 2 mM -naphthyl acetate as the substrate (19). AXE activity
was measured with acetylated oat spelt xylan, prepared in accordance
with the method of Johnson et al. (10). The reaction mixture
comprised 500 µl of acetylated xylan (5% suspension in 50 mM sodium
phosphate buffer; pH 7.0), 450 µl of 50 mM sodium phosphate
buffer (pH 7.0), and 50 µl of purified enzyme (0.79 µg of protein).
The incubation was at 37°C with orbital shaking (150 rpm) for 1 h. The deacetylation of xylose tetra-acetate and glucose penta-acetate
was determined as described for acetylated oat spelt xylan except that
500 µl of each substrate (1.25 mM; in 50 mM sodium phosphate buffer
[pH 7.0]) was used for the reaction. The activity with
p-nitrophenyl acetate and 4-methylumbelliferyl acetate was
determined by monitoring photometrically the release of
p-nitrophenol at a wavelength of 410 nm (
410)
(2) and of 4-methylumbelliferone at 354
(21). Xylanase activity was tested by measuring the release
of reducing sugar from oat spelt xylan by the dinitrosalicilic acid
method (15). The liberated acetic acid from acetylated
substrates was quantified with an enzymatic analysis kit from
Boehringer Mannheim (catalog no. 148261) according to the
manufacturer's instructions. One unit of enzyme released 1 µmol
of product min1 under the assay conditions. The protein
concentration was estimated with Bio-Rad protein assay kit I, with
bovine serum albumin as the reference.
The enzyme demonstrated a broad spectrum of activity on a variety of
substrates, including both aryl and carbohydrate acetyl
esters. The
activities against acetylated xylan and sugars are
presented in Table
2. The enzyme showed a high level of
specific
activity on acetyl xylan, 40 U/mg, which is comparable to the
value of 23 U/mg reported for
Schizophyllum commune
(
9). On
the other hand, lower levels of specific activity
were reported
for the AXEs of
Thermomonospora fusca,
F. succinogenes, and
Thermoanaerobacterium spp.
(esterases I and II): 0.6, 8.63, and 5.2 and 12.4 U/mg, respectively.
The purified enzyme hydrolyzed
p-nitrophenyl acetate and
4-methylumbelliferyl
acetate, releasing
p-nitrophenol and
4-methylumbelliferone, respectively,
with acetic acid.
Enzyme characterization.
SDS-polyacrylamide gel
electrophoresis (5% stacking gel, 12% resolving gel) was performed by
the method of Sambrook et al. (20). Protein bands were
stained with Coomassie blue R-250 after electrophoresis. The molecular
mass of the purified enzyme was estimated to be 190 kDa by Sephacryl
HR200 gel filtration with gel filtration molecular weight markers
MW-GF-200 (Sigma). The enzyme consisted of one type of subunit with a
molecular mass of 40 kDa on SDS-polyacrylamide gel (Fig. 2A). These
data suggested that AXE could be a homotetramer or a homopentamer.
The pI of the AXE was determined by using an Ampholine PAGplate precast
polyacrylamide gel (Pharmacia Biotech), with pH values
ranging from 3 to 10, and by using the broad-pI calibration kit
(Pharmacia Biotech) as
the pI marker, in accordance with the instructions
of the supplier. The
pI value was estimated to be 4.8 (Fig.
2B)
from a plot of migration
distance versus the pI values of the
standards, with the help of an
UltroScan XL laser densitometer
(Pharmacia Biotech).
The purified protein was subjected to N-terminal amino acid sequence
determination by automated Edman degradation on a pulsed
liquid-phase
protein sequencer (model 470A; Applied Biosystems,
Foster City, Calif.)
equipped with an on-line phenylthiohydantoin
amino acid analyzer (model
120A; Applied Biosystems). The amino
acid sequence of an internal
fragment of the purified protein
was also determined after trypsin
digestion. The band of 40 kDa
which belongs to the purified AXE
revealed the following N-terminal
amino acid sequence: MQLFD
LFLEE LG. The internal amino acid sequence
determined is the following:
ALEVI QSFPE VDEHR. The N-terminal
sequence, when subjected to
a FastA homology search, showed 70%
identity with those of two
xylanase precursors from
Clostridium thermocellum (XynX) and
Thermoanaerobacterium saccharolyticum (XynA) (data not
shown), while the internal amino acid sequence
is 80% identical to
that of a cephalosporin-C deacetylase from
Bacillus subtilis
(data not shown). At this stage we do not know
the significance of this
homology.
The optimal pH and temperature were determined in the range from pH 3 to 9.5 (50 mM sodium acetate, pH 3 to 5.5; sodium phosphate,
pH 6.0 to
7.0; Tris-HCl, pH 7.5 to 9.5) and 4 to 80°C, respectively.
For the pH
stability determination, samples were incubated in
buffers from pH 3.0 to 9.0 and at 37°C for 60 min. For the determination
of thermal
stability, temperatures of 37, 50, 60, 65, and 70°C
were used at pH
7.0 for 135 min, with measurement of residual
activity at 15-min
intervals. The remaining activity was assayed
under standard conditions
as described above. The optimal temperature
and pH were about 55°C
and 8.0, respectively. The optimal pH of
8.0 is similar to the pH
values of 7.7 reported for
Schizophyllum commune
(
9) and 7.5 for
Streptomyces lividans
(
6). The optimal
temperature and pH were about 55°C and
8.0, respectively. The
optimal pH of 8.0 is similar to the pH values of
7.7 reported
for
Schizophyllum commune (
9) and
7.5 for
Streptomyces lividans (
6). The optimal
temperature of 55°C is, however, lower than
those reported for other
microorganisms such as
Streptomyces lividans and a
Thermoanaerobacterium sp. (70°C and 80°C, respectively).
The AXE was stable at 50°C and was rapidly inactivated at
temperatures
higher than 60°C, with a half-life of about 1 h at
this temperature.
The pH stability results showed that AXE was stable
in the alkaline
pH range, exhibiting almost 100% of its total activity
between
pH 8.0 and 9.5 (data not shown).
The initial velocity of AXE was determined in 20 mM sodium phosphate
buffer (pH 7.0) at 37°C over the substrate concentration
range of
0.08 to 4 mM
-naphthyl acetate. A Lineweaver-Burk plot
showed a
linear response over this concentration range. The Michaelis
constant
(
Km) was 1.54 mM
-naphthyl acetate, and the
maximal
velocity (
Vmax) was 360 µmol
min
1 mg
1. The
Km
value of 1.54 mM determined for the purified esterase
is lower than the
value of 2.7 reported for
F. succinogenes (
14)
and higher than the
Kms of 0.45 and 0.52 of the
two esterases
from a
Thermoanaerobacterium sp.
(
21), which were determined
by using 4-methylumbelliferyl
acetate as the substrate. It is
much lower than the
Km of 23 reported for the acetylesterase from
A. niger with
p-nitrophenyl acetate as the
substrate (
11).
Little information has been produced to date on the effect of cations
on the AXEs. We found that the activity was not significantly
affected
by any of the cations tested at a 2 mM concentration,
while at 10 mM
many of the chemicals affected enzyme activity.
B. pumilus
AXE is markedly inhibited by Fe
3+, Cu
2+,
Zn
2+, Mn
2+, Co
2+, Ca
2+,
and Ag
+ at a concentration of 10 mM, which is in accordance
with the
behavior of AXE from
Schizophyllum commune
(
9). The greatest
inhibitory effect was recorded with
Fe
3+; Cu
2+ and Zn
2+ were the
divalent cations with the greatest inhibitory effect.
No stimulatory
effect was observed.
In conclusion, our results show that
B. pumilus PS213 has
potent AXE and xylanase activities. Moreover, the AXE purified and
characterized here has properties potentially useful for pulp
biobleaching by xylanases.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: International
Centre for Genetic Engineering and Biotechnology, Area Science Park, Padriciano 99, I-34012, Trieste, Italy. Phone: 39-40-3757317. Fax:
39-40-226555. E-mail: venturi{at}icgeb.trieste.it.
| |
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Appl Environ Microbiol, February 1998, p. 789-792, Vol. 64, No. 2
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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