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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Characterization of the dextranase purified from Streptococcus mutans Ingbritt.

We purified dextranase from the culture supernatant of Streptococcus mutans Ingbritt by procedures including ammonium sulfate precipitation, ion-excha...
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