Microbiol. Immunol. Vol. 36 (9), 969-976, 1992
Characterization of the Dextranase Purified from Streptococcus mutans Ingbritt Takeshi
IGARASHI,* Ayako
YAMAMOTO, and Nobuichi
GOTO
Department of Oral Microbiology,Showa UniversitySchoolof Dentistry, Shinagawa-ku,
Tokyo 142, Japan
(Accepted for publication, June 22, 1992)
Abstract We purified dextranase from the culture supernatant of Streptococcus mutans Ingbritt by procedures including ammonium sulfate precipitation, ionexchange chromatography, and gel filtration. The molecular weight of the enzyme was estimated as 78 kDa by SDS-PAGE. The enzyme degraded dextran at the optimum pH of 5.5, but not other glucans and fructans at all. Paper chromatographic analysis revealed that the enzyme cleaved dextran by an endo-type mechanism. The enzyme was inhibited by Hg2+, Fe3+, Zn2+, and anionic detergents SDS and deoxycholic acid, but not inhibited by non-ionic detergents Triton X-100, Lubrol PX, Nonidet P-40, and Tween 80. SDS-blue dextran-PAGE analysis of the culture supernatant revealed that the enzyme activity detected in the 96 kDa band shifted gradually to the 78 kDa band during handling the supernatant. This shift was inhibited by phenylmethylsulfonyl fluoride, suggesting that the shift of the molecular size is due to proteolytic degradation of the enzyme by serine protease.
The by
water-insoluble
the
mutans
formation
of
dental
purification
be
involved
Meanwhile, believed more
to et
al
dextranase interactions
Murchison modify
with
displace
the
tranase
may
glucans (4, the mutans understand
17, 20). streptococci
dental
So
properties S.
mutans
al
far, due
there to the of
serotype
and
the
of
Walker
some for
that other
the
dextranase, c
produced important caries
glucans
the
in (7).
have
mutans
of
the
et
been
the The
studied
the
plaque
which
we has
plaque.
96•@ 9
been
(7,
(26)
suggested probably
of
the
purified most
that by
about (1,
6,
and frequently
also
Larrithat
the
through
dextranase
proposed bacteria
is 21).
forms role
authors
streptococci
glucans
al
have been few reports difficulty of purification
the
be
dental
water-insoluble
proposed
while sources
of
most
into al
of
processing
(16),
et
(25),
to
(15).
by
glucans
carbon
synthesis
and/or
Tanzer flora
provide
of
et the
GTF.
resident
the
dextranase human
(14),
the
forming
and ƒ¿-1,3-linkages
considered
pathogenesis
cloning
produced in
are
the
gene
dextranase
may
in
catalyzing and
of ƒ¿-1,6-
sucrose
and
(GTF) by
glucans
from
plaque
glucosyltransferases extensively
adherent
streptococci
is to the
degrading
dexthe
the dextranase 18). In order characterized isolated
of to the from
97 0
T.
IGARASHI
MATERIALS
AND
ET AL
METHODS
Bacterial strains and culture conditions. S. mutans Ingbritt (12) was grown in brain heart infusion (Difco Laboratories, Detroit, Mich., U.S .A.) supplemented with 1% yeast extract (Difco) and 2% glucose. Enzyme substrates. Mutan (water-insoluble glucan) was prepared by using cell-free GTF of Streptococcussobrinus OMZ 176 by the method of Ebisu et al (5) . Levan was prepared from the culture supernatant of Streptococcussalivarius KT-19 as has been described elsewhere (8). Other substrates were purchased from the following sources : dextran T2000 from Pharmacia Fine Chemical Co ., Uppsala, Sweden; nigeran from Sigma Chemical Co ., St. Louis, Mo., U.S.A. ; cellulose from Seikagaku Kogyo Chemicals Co., Tokyo . Enzyme assay. The dextranase activity was measured by the procedure described previously (9). Briefly, enzyme preparations were added to the substrates and incubated for 60 min at 37 C and the reducing sugar released was measured by the method as reported previously (9). One unit of the enzyme was defined as that releasing 1 pmol of reducing sugar per min . Unless otherwise noted, all assays were carried out in 20 mm sodium phosphate buffer (PB , pH 6.0). Purificationof the dextranase. The dextranase was purified from 37 liters of the culture supernatant of S. mutans Ingbritt as follows . The precipitate with 70%saturated ammonium sulfate was dissolved in a minimum volume of 10 mm PB (pH 7.6), and dialyzed against the same buffer. The dialysate, designated as crude enzyme, was applied to a DEAE-Toyopearl 650 (Toyo Soda Manufacturing Co., Tokyo) column (3.5 x 20 cm) which had been previously equilibrated with 10 mm PB (pH 7.6), and the proteins were eluted with a total volume of 2 ,000 ml of linear 0-0.5 M NaC1 gradient in the equilibration buffer . The fractions containing dextranase, which were eluted at around 0.21 M NaC1 , were pooled and concentrated. The concentrate was then applied to a Toyopearl HW60 (Toyo Soda) column (2.5 X 90 cm) equilibrated with 20 mm PB (pH 6.0) and eluted with the same buffer. The fractions containing dextranase were pooled and dialyzed against 4 mm PB (pH 7.0). The sample was then applied to a hydroxyapatite (Seikagaku Kogyo) column (1.7 x 18 cm) equilibrated with 4 mm PB (pH 7.0) and eluted with 500 ml of linear gradient of 4-300 mm PB (pH 7.0). The enzyme fractions, which were eluted at around 65 mm of PB (pH 7.0), were pooled, concentrated and applied to a Bio-Gel A 0.5 m (Bio-Rad Laboratories , Richmond, Calif., U.S.A.) column (2.5 x 90 cm) equilibrated with 20 mm PB (pH 6 .0). The enzyme fractions were eluted with the same buffer, and the active fractions were pooled, concentrated and then rechromatographed by a Bio-Gel A 0.5 m column as described above. All procedures were carried out at 4 C. Gel electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out using Laemmli's system (13). Protein bands were visualized by staining with Coomassie brilliant blue, with protein molecular weight standards from Bio-Rad. To monitor the dextranase activity, the samples were electrophoresed on a gel containing 0.5% blue dextran (supplier) and the enzyme
DEXTRANASE
OF
S. MUTANS
INGBRITT
97 1
in the gel was renatured by removing SDS by Triton X-100 and Lubrol PX as has been described previously (9). The dextranase activity was visible as a clear zone on blue background. Paper chromatography. Reaction end products of dextran were identified with paper chromatography in pyridine-2-propanol-water-acetic acid (8: 8: 4: 1). Sugars were detected by staining with AgNO3-saturated acetone as reported previously (10) . RESULTS
Purificationof the Enzyme The dextranase was purified 508-fold with 8% recovery from the culture supernatant, as summarized in Table 1. The purified dextranase appeared as a single band by SDS-PAGE, and the molecular weight was estimated as 78 kDa (Fig. 1A). In addition, this protein showed dextranase activity when it was analyzed by SDS-blue dextran-PAGE (Fig. 1B). These results indicate that the purified enzyme is electrophoretically homogeneous. To determine whether the purified dextranase of 78 kDa is the only extracellular dextranase product by S. mutans Ingbritt, the dextranase activity in the crude enzyme preparation was examined by SDS-blue dextran-PAGE. Surprisingly, the crude enzyme preparation contained only one enzyme of 96 kDa (Fig. 1C). When the crude enzyme was incubated at 37 C for 24 hr, however, the intensity of the 96 kDa band decreased and a new band of 78 kDa appeared (Fig. 1D). Only these two bands were observed even when the incubation was prolonged for over 24 hr. The shift of the molecular sizes was inhibited by a serine protease inhibitor, phenylmethylsulfonyl fluoride (PMSF) (Fig. 1E). Another serine protease inhibitor, tosyl-L-lysine-chloromethyl ketone (TLCK), however, did not inhibit the shift at all (Fig. 1F). These results suggest that PMSF-sensitive serine protease existing in the crude enzyme preparation cleaved the 96 kDa dextranase to produce the 78 kDa enzyme. SubstrateSpecificityand Mode of Action of the Enzyme The substrate specificity of the enzyme was examined using various poly- and oligosaccharides as substrates. This enzyme cleaved dextran (a-1,6-glucan), but Table
1.
Summary
of dextranase
purification
97 2
T.
IGARASHI
ET AL
Fig. I. SDS-PAGE patterns of S . mutans Ingbritt )reparations. The enzyme preparations were electrophoresed on a lfr• „ SDS-polyacrylamide gel and sunned with Coomassie brilliant blue (A), or a 10°,.;) SDS-polyacrylamide-blue dextral) gel and renatured (B (;). Lanes: A and B, purified dextranase (0 .6 pg); C, crude enzyme (not incubated) ; I) , crude enzyme (incubated); 1', crude enzyme -1 PMSF; crude enzyme C . crude enzyme PMSF-: TLCK. 256 pg of crude enzyme were used in C I) () were incu bated at 37 C for 24 hr before electrophoresis .
Fig. 2. Paper chromatogram of reaction end products from dextran hydrolysi s by purified enzyme. Reducing sugars were stained with silver nitrate reagent as described in "MATE RIALS AND METHODS." Lane A, reaction end products; lane 11, standard sample of glucose (20 pg).
DEXTRANASE
none
of
mutan
glucan),
( ƒ¿-1,3-glucan)
inulin
OF
,
of
that
the
n-glucose,
enzyme
and
that
the
as
products
of
were
dextran
hydrolyzed
the
imately pH
of
pH pH
5.2
or
The final
above effects
of
(Fig.
materials showed
activity (Ba2+, essentially
paper
endo-type
the
enzyme
S.
carry
mutans
(Fig.
of
the
(3,
11,
against
2).
of
enzymes
GTF, which
15). dextran,
the
and It
results
a-1,6-linkage
activities
-hydrolyzing
enzyme
(D-glucoThese
consists
chromatography,
products
an
3).
pH
The
6.4.
of
reaction
oligosaccharides
indicated
that
the
enzyme
as
approx-
mechanism.
of by
ions 1
mm.
no
Co2+,
activity
was
100,
96, Mn2+,
the
Fe3+, 96,
less
detected
to
Hg2+,
was to
chemicals
While
Mg2+,
dextran
decreased
no and
approximately
Ca2+,
against
activity
Almost
metal
concentration
enzyme
of the
not
inulin)
all.
4-
Enzyme
optimum 5.5
did
of by
end
by
the
action
analyzed
reaction
dextran
Properties The
the
of
at
which
(levan,
cellulose •@(ƒÀ-1,
Sucrose
dextran
enzymes
mode
were
as
for
fructan
extracellular the
detected
Other
the
determine
,
hydrolyzed
preparation
and
97 3
levan (ƒÀ-2,6-fructan). neither
specific
enzyme
(FTF),
known To
was
INGBRITT
(ƒ¿-1,3; ƒ¿-1,4-glucan)
and was
fructosyltransferase were
nigeran
0-2,1-fructan),
pyranosy1-(-D-fructofuranoside) indicated
S. MUTANS
and
at
enzyme Zn2+,
EDTA,
estimated than
97%, NaF,
50% pH were
and
SDS
either 4.0
and
pH
8.0.
examined
at
inhibited
respectively, PMSF,
below
the and
a the
other TLCK)
effect.
Fig. 3. Optimum pH of the dextranase. The activity is shown as percentage of the maximum activity. The purified enzyme (80 mU) was utilized in the experiment. Acetate and phosphate buffer (each, 20 mm) were used at pH 4.0 to 5.0 and pH 5.5 to 8.0, respectively.
T. IGARASHI
97 4
Fig.
4. in
Effects
of detergents
"MATERIALS
AND
experiment.
The
deoxycholic
acid; •œ
activity
on
the
dextranase.
METHODS." is
, Triton
shown X-100;
The The
as
ET AL
reactions
purified
percentage
of
A,
PX.
Lubrol
were
enzyme the
maximum
performed (70
mU)
as was
activity. •›,
described
used
in
the
SDS; •¢,
The dose effects of detergents to the enzyme activity are shown in Fig. 4. The enzyme was inhibited by the anionic detergents SDS and deoxycholic acid. In particular, SDS completely inhibited the enzyme at a concentration as low as 0.01 %. Deoxycholic acid finally inhibited the enzyme by 85% although a transient enhancement was observed at a concentration of 0.01 %. On the other hand, the non-ionic detergents Triton X-100 and Lubrol PX increased the enzyme activity up to 120% at any concentration tested. Other non-ionic detergents Tween 80 and Nonidet P-40 also showed a similar enhancing effect as Triton X-100 and Lubrol PX (data not shown). DISCUSSION
From the culture supernatant of S. mutans Ingbritt, we detected two forms of extracellular dextranases with sizes of 96 and 78 kDa, and we purified and characterized the 78 kDa enzyme. The 78 kDa enzyme was stable at 4 C throughout the enzyme purification without protease inhibitor although the 96 kDa enzyme was easily degraded to the 78 kDa enzyme within a few days. Barrett et al (1) purified an extracellular dextranase of S. sobrinusand reported that it had a size of 175 kDa, which was almost twice as large as our 96 kDa enzyme. The optimum pH (5.5) of our dextranase was close to that (5.4) of the S. sobrinus enzyme.
DEXTRANASE It
is known
enzymes sidase
gene
carried
of
out
the
those dextran of
dextranase
and
most
of
our
that
the
with
and
utilized
due
a
of (1),
we showed
lyzed
by
SDS-blue
have
be
due
to
induced of
of
oralis
6,
we
as
9,
18,
smaller
pH
(6.0)
several
the
groups implying
present
study
hydrolyzed by
across
of
optimum
22-24),
dextran
Russell
extracellular
to
was
The
in
suggested
transported
intra-
isomaltosac-
by
showed
by
in al
Ig4a
et
al
(20),
dextranase,
the glucose
the
several
and
cell
membrane
which
is
able
to
of
S.
sobrinus
protease
is
remains
has
and
in
the
of
serine
extracellular
common
(the
to
of
be
S.
S.
of
protease
whcih of
mutans.
The
mutans
anadata).
of are
S.
all
sensitive
multiple
S.
streptowere
enzymes These is
in report).
enzymes
mutans.
formation
The
only
(unpublished
forms
of
forms
kDa.
present
other
those paper
multiple
175
reported
Ingbritt
this
multiple
of
been
when
(3)
activity
far,
dextranase
shown
the
dextranase
conversion
on
that
mutans
extracellular
reports
the
largest
S.
and ƒÀ-fructosidase
that
Y.
in
as
proteolytic
Miss
the
and
supernatant
suggested
so
weight
other 19),
culture
They of
(9),
that
the
(1).
dextranase,
molecular
(15,
serine
thank
et
forms
phenomenon
We
ours.
specifically
that,
dextranases
Barrett
therefore, by
this
of
been
present
optimum
reported (2,
glucosidase
dextran-PAGE
the
evident,
Since Ingbritt
degradation
the
FTF
pH.
dextran
observed
been
(15),
been
mutans
is cleaves
(5.5)
glucohave
glucosidase
that
5.0-6.0
likely
enzyme
The
isomaltosaccharides
of
by
multiple
also
is
is
The
dextran
product
source.
Bacteroides
addition,
It
S.
it
proteolytic
cocci
to
of
are
forms
the
than
the
gene
dextran
have
optimum
to
the
between
dextran-hydrolyzing of
the
enzymes.
bacteria
of
preferentially
of
higher
are
intracellular
reported to
sobrinus
detected
oral pHs
normal
nutrient
occurrence
GTF
other
mechanism,
multiple
were
our
slightly
hydrolyzed
by
The been
of
classes
(20).
and
kDa)
isomaltosaccharides
as
have
cells,
al
97 5
Cloning of
et
(62
dextranase
first
cleaved
There
kDa)
optimum
endo-type is
Russell
Ingbritt
was
has
resulting
In
78
two
(26).
characterization
and
size
of
the
enzyme
an
(2)
mutans
extracellular
dextran
then
al
glucosidase
pHs
that
et
INGBRITT
have
glucosidase and
molecular
(96
streptococci
dextran Ingbritt
S.
The
than
mutans
mutans
Burne
in
charides.
the
the and
S.
by
cellularly
the
that
: dextranase
OF S. MUTANS
to
sizes
PMSF.
of
physiological
mutans: believed
enzymes
significance
unclear.
Yokoyama
for
her
technical
assistance.
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