Microbiol. Immunol. Vol. 36 (6), 643-647, 1992
Characterization
of an Exo-13-D-Fructosidase
from Streptococcus mutans Ingbritt
Takeshi
IGARASHI,* Ayako
YAMAMOTO, and
Nobuichi
GOTO
Departmentof Oral Microbiology,Showa UniversitySchoolof Dentistry, Shinagawa-ku,
Tokyo 142, Japan
(Accepted for publication, March 12, 1992)
Abstract
An
supernatant weight
of
the
trophoresis. linked
The by
enzyme
levan,
Fe3+,
chromatographic
9
inulin
enzyme
was also
5%
of
and
Zn2+ analysis
able
sucrose
optimally
as
to
were
not
revealed
for
by
by
55
either
C
that
anionic the
sucrose
enzyme
and
levan. or
6.0 The
non-ionic attacked
gel
raffinose pH
5.0,
enzyme
by
at
the
optima
respectively. was
detergents. levan
elec-
of ƒÀ-(2,6)-
The and
culture
molecular
consists
respectively. 5.5,
for
the
The
mainly
inulin, levan,
approximately at
from
SDS-polyacrylamide
which
hydrolyze
purified
characterized.
levan
hydrolyzing
reactive and
and
determined
specific
that
was
Ingbritt
127,000
was
and and
was Hg2+
was
and 13,
mutans
enzyme
D-fructose of
enzyme ƒÀ-D-fructosidase
Streptococcus
The
activities for
extracellular of
inhibited Paper
an
exo-type
mechanism.
Sucrose metabolism by oral streptococci is closely associated with the development of dental plaque and caries (9), and several streptococcal enzymes involved in the metabolism have been studied as virulence factors (5, 9, 16). Whereas the mechanism of the water-insoluble glucan synthesis from sucrose by glucosyltransferase (GTF) has been studied extensively by purification of the enzyme and cloning of the gene (1, 10, 11, 17), the roles of other enzymes, e.g., fructosyltransferase (FTF), dextranase, and fructanase, have not been clearly described. It has been reported that several oral bacteria produce fructans (levan and inulin) from dietary sucrose (3, 9, 16), and the fructans are regarded as one of the plaque matrix components from their physical properties such as size, shape, and viscosity (6). It has been suggested that fructans could also serve as storage polysaccharides in the plaque matrix (12, 18) and reported that a number of bacteria in human dental plaque including Streptococcusmutans produce fructanases which can degrade the fructans to metabolizable saccharides (7, 14, 16). Those studies, however, have been carried out only with crude enzymes, and hence little is known about the enzymes themselves (14, 16, 20). In the present study, we purified and characterized an extracellular fructanase of S. mutansIngbritt. S. mutans Ingbritt was grown in brain heart infusion (Difco Laboratories, Detroit, Mich., U.S.A.) with 1% yeast extract (Difco) and fructanase was purified from the culture supernatant. An amount of 48 liters of the culture supernatant 643
644
T. IGARASHI Table
was
precipitated
buffer
(PB,
facturing The
with pH
Co., bound
the
7.6) Tokyo)
with
mond,
Calif., were
dialysate
was
x 90
the
same
and
applied
cm)
then
fractions
was
The
was
1.
subjected
to
127,000
enzyme
was was levan
fructan),
sucrose,
respectively. that
glucans
dependent
S. and GTF
type
the
blue
of
raffinose mutan
mutans
produces
GTF,
S.
to
the
and GTF
of
band
This
however,
has
The column
was eluted fractions
(Bio-Rad)
with were column
carried
out
using
concentrated,
described
above.
All
phenylmethylsulfonyl gel
electrophoresis
Laemmli
the
and (9).
with
2%
the
enzyme, an
(15).
relative
Proteins
The
enzyme
efficiently
which has
activity melezitose
been in
the
that
purified
preferentially
able
13,
9, at
to
convert that
absence
of our
hy-
(ƒÀ-2,1-linked
100,
reported
whereas
weight
the
hydrolyzed
are
enzyme
molecular
inulin of
not
summarized
purified
apparent
activities
FTF,
as
the
indicated
were
It
recovery
also
less
melezitose
invertase hydrolyzes
active
7.0).
pooled,
of
of
with
result
and
respectively mutans
The
Tokyo)
was
as
method
107-fold
a single
with
m
were
column
and
staining.
weight
1).
1.5
Elution
presence
in eas
Rich-
(pH
Co.,
sulfate-polyacrylamide
fructan),
Dextran,
of
m the
homogeneous.
(ƒÀ-2,6-linked
fructans,
in
7.6).
substrat
6.0). PB
protein The active
A
6.0).
0.5
C
purified
(Fig.
Bio-Gel
(pH
A 4
molecular and
observed
a
the
Manu(pH
gradient
as
(pH
4 mm
and 7.0).
PB
concentrated,
Kogyo,
7.0), (pH
mm
Laboratories,
PB
fructanase
according
SDS-PAGE,
and
to
dodecyl
finally
assess
mm
against
phosphate Soda
levan
(Bio-Rad
20
mm
linear
pooled,
m
in
(pH PB
PB
at
electrophoretically
drolyzed
known
was
To
PB mm
Bio-Gel
Coomassie
using
(Seikagaku
mm
out
performed
with
fructanase
Table
a
carried Sodium
visualized
dialyzed
containing
with were
(SDS-PAGE)
cm)
and
0.5
10
M NaC1
were
A
x 90
applied 20
0-0.5
fractions
4 mm 4-200
with
The
(PMSF).
were
That
and
a
10 (Toyo
with
assayed
Bio-Gel
(2.5
with of
of
was
a hydroxyapatite
equilibrated
procedures
fluoride
to
on
buffer.
a
a 650
equilibrated
liters
enzyme
over
rechromatographed
these
of
and
cm) 2
concentrated
concentrated,
(2.5
enzyme
against
a DEAE-Toyopearl
20
The
column
pooled,
x with
purification
dialyzed
on
eluted
filtration
X 19 cm) equilibrated ml of a linear gradient
pooled,
in
(3.5
U.S.A.)
fractions
(1.7 700
column
(13),
gel
of enzyme
(NH4)2SO4, loaded
buffer. reported
separated
Summary
was
were
equilibration
previously
70%
and
proteins
1.
ET AL
the
primer enzyme
and all.
5%, It
sucrose primer(8, did
11). not.
is
NOTES
Fig.
1.
SDS-PAGE
pattern
of
the
purified
phoresed on a 10% SDS-polyacrylamide with Coomassie blue.
These
results
suggest
primer-dependent
that GTF.
It
substituted ƒÀ-fructofranoside, was fi-fructofranosidc,
the
was
not.
These
enzyme.
gel with
invertase
was
6 45
activity also
digested results
of
revealed by
The
the size
purified markers.
our that
the
indicate
enzyme The
enzyme
was
whereas
enzyme that
enzyme
electro-
was stained
not
due
raffinose,
, melezitose, the
was
protein
to an
a is
the un-
substituted a ƒÀ-D-fruc-
tosidase which cleaves 243-linkage of poly- and oligosaccharides of D-fructose, and that the enzyme preparation has activities of neither GTF nor FTF . To determine the mode of action of the enzyme against levan , the reaction end products of levan were analyzed by paper chromatography in 2-propanol-pyridine-acetic acid-water (8: 8: 4: 1). Sugars were detected by staining with AgNO 3-saturated acetone according to the method of Bailey and Bourne (2), and only fructose was detected zs the reaction products. The liberation of fructose as a sole end product indicated that the enzyme hydrolyzed levan with an exo-type mechanism. The substrate specificity and the mode of action of the enzyme indicate that the purified enzyme is an exo43-D-fructosidase (exo-fi-D-fructan fructohydrolase, EC 3.2.1.80). The maximum activities of the enzyme to levan, inulin, and sucrose were observed at approximately pH 5.5, 6.0, and 5.0, respectively. Levan-hydrolyzing activity was almost none at pH 4.0. The pH profiles for levan and inulin were almost same, but that for sucrose was broader than those for levan and inulin . In addition, a significant level (60%) of the sucrose-hydrolyzing activity remained even at pH 4.0. The activity of the enzyme for levan remarkably increased over 40 C, and reached to the maximum at 55 C, and 61°4, of the activity still remained even at 60 C. The effects of metal ions and chemicals on the enzyme activity were determined at a final concentration of 1 mm. Whereas Hg24, Fe3+ and Zn2+ inhibited the enzyme activity by approximately 100, 90, and 70%, respectively, the other materials (Ba2+, Ca2+, Co2+, Mn2- , Mg2+, NaF, EDTA, TLCK, PMSF, 2ME, and DTT) had no effect at the concentrations tested. The effect of the detergents to the enzyme was also examined at final concentrations of 0.01 to 0.05%. Neither the anionic
646
T.
detergents
(SDS
Triton
and
X-100,
far,
from
purified S.
When
to
5.5,
those are
5.5,
and
effect
of
effect
of
the
detergents the
(61
%
was
(Lubrol
enzyme
of
mutans
the
different
that et
Ingbritt
it
paper,
that
PX,
activity
S.
the
at
mutans
any
their
an
optimum
detergents
to
the
60
S.
the
C)
maxoptima
far
as
than
tested,
Hg2
more
was
temperature-
Takahashi's
one,
exo-ƒÀ-D-fructosidase
salivarius
in
KTA-19
whether
a
single
Therefore,
exact
of and
a
little
of
fructanase
of
inulin,
S.
levan,
were
revealed
mutans (20).
to As
and
levan-
purify
the
described
Ingbritt,
and
in
showed
raffinose-hydrolyzing
addition,
optimum
of
for
S.
both
necessary
mutans
effects
of
has
enzyme.
sucrose,
for
activity
is
the
In
temperature
fluid purification
enzyme
it
properties
levan,
culture
without
exo-ƒÀ-D-fructosidase.
enzyme
the
enzyme
not.
the
had
of
of
of
As
was
the
fructanase
study
extracellular
enzyme mode
substrates,
of
or
clear an
at
that
40%
different:
enzyme
Takaoptimal
temperature
characters.
al
characterand
85,000;
none, for
conclusion,
the
activities
a
a
from
presence
in
purified
in
As
distinct
and
et
KTA-19.
Burne's
significantly
remained
C.
several
4.0:
those
not
Burne
GS-5.
the
make
pH
by
salivarius
enzyme,
enzyme
Our
activity
S.
140,000,
test
enzymes.
60
has
at
was
characterized
to
purified
not
only
from
our
Burne's
did
different
unclear
order we
activities
enzymes
at
inulin-hydrolyzing
this
they
the three
reported
is
the
since
(19)
enzyme
activity
compare
all
partially
Apparently,
in
127,000,
much
of
al
and
enzyme
weight:
maximum
is
from Walker
among
inactive
Ingbritt
present
reported
al
the
to
to
entirely
et
differences
not
ions
been
Takahashi
major
detergents
inhibitor
resistant
three
non-ionic
influenced
have
by
levan-hydrolyzing
could
metal
common
and
the
80)
enzymes, the
7.0;
We
the
which
nor
Tween
Molecular
none.
and
and
and
respectively.
imum,
S.
GS-5,
Following
hashi's,
a
and
exo-fl-D-fructosidases
mutans
compared
istics.
pH:
acid)
P-40,
ET AL
tested.
So (4)
deoxycholic
Nonidet
concentration
IGARASHI
metal
the
pHs
ions,
first
time
for
chemicals, in
this
study.
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NOTES
8)
9) 10)
11) 12) 13)
14)
15) 16) 17)
18) 19) 20)
647
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