Extracellular Dextran Hydrolase from Streptococcus mutans Strain 6715 DAVm W. ELLIS and CHRIS H. MILLER

Laboratory of Oral Microbiology, Indiana University School of Dentistry, Indianapolis, Indiana 46202, USA Streptococcus mutans strain 6715 was shown to produce an extracellular dextranase with endohydrolytic a-(1 6)-glucan-6-glucanohydrolase activity. The enzyme degraded soluble polymers produced by some oral streptococci but did not disperse the streptococcal plaques tested. Enzyme levels in the culture supernatant were elevated in sucrose and sucrose plus glucose cultures but remained at basal levels in glucose and dextran plus glucose cultures. The possible biological significance of plaque dextranase activity in the oral cavity is of particular importance in the ecology of plaque. Survival of plaque bacteria as well as a continuation of plaque acidogenesis could be enhanced by plaque dextranase activity during periods of low dietary carbohydrate intake. Whether plaque dextrans could be hydrolyzed to directly fermentable products would depend on specific mechanisms of enzymatic degradation of the dextrans. Walkerl suggested that plaque dextranase type of enzymes may also play a role in the regulation of glucan synthesis by oral streptococci. A recent study has indeed shown that commercial dextranases and those from Actinomyces israelii and Bacteroides ochraceus inhibit insoluble glucan synthesis and adherence to smooth surfaces by Streptococcus mutans.'

Several investigations have provided evidence of the presence of dextran hydrolase activity in dental plaque. Common plaque bacteria thus far shown to possess this activity are S mutans,47 Fusobacterium fusiforme,s A israelii, and B ochraceus.2'3 The investigations This investigation was based on a thesis required for partial fulfillment of the MS degree in microbiology by D. W. Ellis. Received for publication November 7, 1975. Accepted for publication April 20, 1976. * Obtained from Dr. J. Carlsson, University of

Umea, Swed. t Baltimore Biological Laboratories, Cockeysville, Md. Difco Laboratories, Detroit, Mi. § Pharmacia Fine Chemicals, Uppsala, Swed.

t

with S mutans have indicated that strains of this species predominantly produce either an endohydrolytic type of enzyme with oligosaccharides as the major end products or an exohydrolytic type of enzyme with glucose as the product. In this communication, we report on the characterization of an extracellular endohydrolytic dextranase from S mutans strain 6715. This strain is used in many caries-related investigations, but little is known about its dextranase activity.

Materials and Methods CULTURAL CONDITIONS-.S mutans 6715 and NCTC 10449, Streptococcus strains MPK 1, KPK 2, and KPK 3,* and S sanguis JC 804* and ATCC 10556 were stored at 4 C and transferred at monthly intervals in Trypticase-soy brotht containing an excess of calcium carbonate. The presence of microbial contaminants was checked regularly by microscopic examination. All bacteria were cultured in a complex medium of Trypticase,t yeast extract,$ and salts9 supplemented with various sugars. For routine preparation of extracellular dextran hydrolase, S mutans 6715 was cultured for 24 hours in 1-liter portions of the complex medium supplemented with 0.058 M sucrose. In the experiments designed to detect the effects of various carbon sources on enzyme production, the complex medium was supplemented with 0.03 M glucose, 0.03 M sucrose, 0.03 M glucose plus 0.03 M sucrose, 0.03 M glucose plus 0.5% dextran T-500,§ or 0.5% dextran. For enzyme preparation, cells were removed from the culture by centrifugation at 4 C at 15,000 X g for 20 minutes. The pro-tein was precipitated from the supematant fluid by the addition of ammonium sulfate tog 90% saturation, and the precipitate was harvested by centrifugation for 20 minutes at 15,000 X g. The precipitated protein was dis-solved in 15 ml of distilled water and the solution dialyzed for 48 hours at 4 C against 4 57-

Downloaded from jdr.sagepub.com at Bobst Library, New York University on June 4, 2015 For personal use only. No other uses without permission.

58

ELLIS AND MILLER

liters of 0.05 M sodium acetate buffer, pH 5.5; the buffer was changed daily. The protein content of all enzyme preparations was determined by the Lowry assay method.10

ENZYME ASSAY. The activity of dextran hydrolase was measured in standardized reaction mixtures (1 ml of 20% dextran T-500, 0.5 ml of 0.05 M sodium acetate buffer [pH, 5.5], and 1 ml of enzyme preparation). The reaction mixture was incubated 1 hour at 37 C. Enzyme activity was determined by measuring the production of reducing sugars with the Somogyi test.1' Presence of free glucose was detected by use of glucose oxidase.# Incubated control tubes included dextran in buffer and enzyme in buffer. The dextran controls consistently gave a reducing sugar background of 0.33 ,umole/ml reaction mixture; this value was always subtracted from the enzyme assays. Enzyme controls did not yield reducing sugar background. One unit of enzyme was defined as that amount of enzyme that produced 1.0 ,urmole of reducing sugar per minute under the standard assay conditions. A variety of polysaccharides produced by oral streptococci was substituted for dextran T-500 in the standard assay. S mutans 6715, Streptococcus MPK 1, KPK 2, and KPK 3, S mutans NCTC 10449, and S sanguis ATCC 10556 and JC 804 were cultured separately in 1 liter of the complex medium supplemented with 2 % sucrose at 37 C for 24 hours. Cells were removed by centrifugation at 4 C for 20 minutes at 15,000 X g. Two volumes of 95% ethanol were added to the supernatant fluid and the mixture was stored at 4 C for 48 hours. The precipitate was collected by centrifugation at 15,000 X g for 20 minutes at 4 C and washed twice with ethanol. Thirty milliliters of distilled water were added to the precipitate and the mixture was stored at 4 C for 48 hours. Insoluble material was removed by cenrtifugation for five minutes at 15,000 X g at 4 C, and the water-soluble material was reprecipitated by the addition of 2 vol of ethanol. After storage at 4 C for 24 hours, the precipitate was collected by centrifugation, dissolved in 10 ml of distilled water, and dialyzed against four liters of distilled water for 48 hours, with the water being changed once daily. Total hexose concentration of each polysaccharide prep# Glucostat Special, Worthington Biochemicals Corporation, Freehold, NJ. / 13179, Eastman Kodak, Rochester, NY. ¶ Fracto-mette, Buchler Instruments Inc., Fort Lee, NJ.

J Dent

Res

January 1977

aration was measured by the phenol-sulfuric acid method.'2 These preparations were used as potential substrates in the dextran hydrolase assay.

THIN-LAYER CHROMATOGRAPHY.-Reactants and products of the degradation of dextran by S mutans 6715 extracellular enzyme preparations were separated by thin-layer chromatography. Fifty microliters of enzyme reaction mixtures and equivalent substrate and enzyme controls as well as carbohydrate standards were spotted on 20X 20-cm silica gel thinlayer chromatogram sheets/ 2 cm from the bottom and at 2-cm intervals. The sheets were developed for five hours at room temperature in a chloroform-acetic acid-water (3.0:3.5:0.5 volume ratio) solvent system.'3 Sheets were removed from the glass chromatocab, air dried, and sprayed with a mixture of diphenylamine, aniline, phosphoric acid, and acetone.'3 Carbohydrate-positive spots were made visible by heating the sheets in an oven for 15 minutes at 100 C. Rf values of each spot were calculated as the ratio of the distance between the origin and the center of the spot to the distance between the origin and the solvent front.

SEPHADEX GEL FILTRATION. -Sephadex G-75, G-150, and G-200 gel filtration media were also used to separate reactants and products in dextran hydrolase reaction mixtures. Sephadex resins were swelled for the proper times in 0.05 M sodium acetate buffer (pH, 5.5), with 0.02% sodium azide and poured into 2 X 40-cm glass columns. The columns contained a scintered glass disk at the bottom which was covered with a small amount of glass wool to prevent clogging of the disk. The beads were allowed to settle to a height of 31 cm; each column was washed with 1.5 liters of the acetate buffer. Two milliliters of reaction mixtures, controls, or standards were uniformly applied to the surface of the resin and eluted with the acetate buffer. The height of the buffer reservoir was adjusted so that the distances from the constant fluid level in the reservoir to the bottom of the columns were 75 cm for the G-75, 36 cm for the G-150, and 16 cm for the G-200 Sephadex gels. This resulted in constant flow rates of 1.0, 0.7, and 0.4 ml/minute, respectively. Column eluates were collected in 5-ml amounts with the aid of an automatic fraction collector.! Fractions were analyzed for total hexose by the phenolsulfuric acid method.12

Downloaded from jdr.sagepub.com at Bobst Library, New York University on June 4, 2015 For personal use only. No other uses without permission.

Vol. 56 No. I

DEXTRAN HYDROLASE FROM S MUTANS

EFFECT OF DEXTRAN HYDROLASE ON IN PLAQUE.-Bacteria were allowed to accumulate on the surfaces of glass slides by a modification of the procedure described by McCabe, Keyes, and Howell.14 Glass microscope slides// (25 X 75 cm) were cut in half lengthwise and placed in separate 2OX 150 mm test tubes containing 10 ml of the complex medium supplemented with 2% sucrose and the system was sterilized. Inoculum cultures of S mutans 6715 and 10449, Streptococcus MPK 1, KPK 2, KPK 3, S sanguis 10556 and JC 804 were grown in the complex medium supplemented with 0.016 M glucose. Five tenths milliliter of each 24-hour-old inoculum culture was placed into duplicate glass slide tubes. At the end of 24 hours of incubation at 37 C, slides were aseptically removed and transferred to fresh sucrose-supplemented medium inoculated with the respective 24-hourold inoculum culture. This transfer of slides to fresh inoculated medium was repeated daily for three days. At the end of this fiveday plaque growth period, the slides were removed and carefully washed with sterile distilled water so as not to disturb the plaque. One of the duplicate slides for each of the seven test bacteria was placed in 10 ml of 0.05 M sodium acetate buffer (pH, 5.5). The other was placed in a 10-ml buffer solution containing a standard extracellular dextran hydrolase preparation from a S mutans 6715 culture at a final concentration of 300 ,tg protein/ml. Reaction mixtures were allowed to incubate for four hours at 37 C. Samples of reaction fluid were then assayed for the presence of reducing sugar by the Somogyi method.'1 Visible observations of gross plaque degradation were also made.

59

VITRO

Results ANALYSIS OF DEXTRAN HYDROLASE REACTION PRODUCTS.-Experiments were designed to characterize the dextran hydrolase from S mutans. The effect of varying the enzyme concentration between 0 and 320 jug protein per milliliter of reaction mixture was examined. All other reactants were at the standard concentrations and the incubation time was one hour. The velocity of the reaction was shown to be proportional to the amount of protein added up to at least 320 ,ug/ml reaction mixture (Fig 1). The specific activity of the enzyme preparation was 0.088. The effect of varying the incubation time was examined un// Becton, Dickinson and Co., Pansippany, NJ.

1.5

1.0 u

0)

0.

5

40

120 160 200 240 280 320

80

jig PROTEIN/ml

FIG 1.-Effect of protein concentration on velocity of dextran degradation. Standard reaction mixtures with varying amounts of protein were incubated for 60 minutes at 37 C and assayed for presence of reducing sugars.

der standard assay conditions with the exception that 0.02% sodium azide was added to prevent growth of microbial contaminants. Micromoles of reducing sugar per milliliter increased proportionally with time throughout the 24 hours of incubation (Fig 2). Sodium azide did not affect enzyme activity. At 0, 2, 6, and 24 hours, the reactions were stopped by placing tubes in a boiling water bath and each reaction mixture was tested for the presence of reducing sugars and free glucose (Table 1). Since dextran demonstrated a slight reducing sugar background, this value was subtracted from all assay tubes. Reducing sugar production in complete reaction mixtures increased with time yielding 37.1 ,umoles/ml of reaction mixture after 24 hours. Only 0.127 ,rmole/ml of free glucose (0.34% of the reducing sugar) was detected after 24TABLE 1 ANALYSIS

OF

REACTION PRODUCTS

Reducing Sugar*

Incubation Time (h)

(gumole/rnl)

0 2 6 24

0 2.67 8.33 37.22

Free

Glucoset

% Glucose of Total

(,pmole/ml) Reducing Sugar-

0 0.006 0.028 0.128

0.21 0.33 0.34

Somogyi method."

t Glucostat special reagent.

Downloaded from jdr.sagepub.com at Bobst Library, New York University on June 4, 2015 For personal use only. No other uses without permission.

60

ELLIS AND MILLER

J

Dent Res

20

24

January 1977

15 S I"

0

Ca

0 U) 10 0 0 A

5 Cn

A

04

5

10

15

TIME (HOURS) FIG 2.---- -Effect of incubation time on velocity of dextran degradation. Standard reaction mnixtures corntaining 240 gg protein/ml were incubated at 37 C for various timiie periods and assayed for presence of reducing sugars.

lours of inicubationi. In inicubated control tubes, conitaining glucose, enzymle, and buffer, complete recovery of the added glucose was obtained after incubation for 24 hours. Aliquots of 50 I from each of these reaction miixtures and control were spotted on thini-laycr chromatogram sheets and analyzed

(Fig 3 Clarbohydrate-positive material representative of dextran remained at the origin of all spots except that of the glucose control. No other spots were observed from the zerotime reactioin imiixture or the dextran control tubes. The glucose control had a single spot witli an R, value of 0.41. The inicubated re-

FIG 3. Thin-layer chromnatography of dextran hydrolase reaction mixtures incubated for various times. Silica gel thin-layer chromatogram sheet was

developed in chloroform-acetic acid-water solvent system and sprayed with diphenylainine-aniline-acetone-phosphoric acid mixture to visualize carbohydrate spots. Samples are, from left to right, 0, 2, 6, and duplicate 24-hour reaction miixtures, dextran standard, and glucose standard. Reducing sugar xalues of reaction miixtures are given in Table 1. Downloaded from jdr.sagepub.com at Bobst Library, New York University on June 4, 2015 For personal use only. No other uses without permission.

Vol. 56 No. 1

DEXTRAN HYDROLASE FROM S MUTANS

action mixtures demonstrated at least four between that of free glucose and the undegraded dextran at the origin. The intensity of these spots increased as incubation time increased. Only a slight amount of carbohydratepositive material with an Rf value similar to that of free glucose was observed in the 24hour reaction mixtures. This corresponded to the relatively low amount of free glucose detected by the Glucostat analysis (Table 1). In order to further describe the nature of the high molecular weight products suggested to be present by the thin-layer chromatography, reaction mixtures were analyzed by gel filtration through Sephadex columns. An elution standard containing 80 mg of dextran per milliliter plus 0.2% glucose was made in the sodium acetate buffer. Two milliliters of this mixture was passed through the Sephadex columns to establish the elution limits of the highest and lowest possible molecular size products in the reaction mixtures. Fractions were analyzed by the phenol-sulfuric acid assay.12 Two milliliters of a standard enzyme reaction mixture incubated for 24 hours was passed through the same washed and equilibrated columns in an attempt to separate reaction products. Although this reaction mixture contained 37.2 .moles of reducing sugar per milliliter, there was no apparent separation of products on analysis of the Sephadex G-75 or G-150 fractions. Passage of the same reaction mixtures through Sephadex G-200 did provide some separation of the products (Fig 4). The dextran control eluted from the column immediately after the void column of 30 ml and reached its peak concentration after 50 ml of effluent had passed through the column. Glucose eluted between 100 and 130 ml of effluent, with its highest concentration in the 110-ml fraction. The carbohydrate-positive material in the enzyme reaction mixture was present at varying concentrations between 40and 110-ml fractions, which again indicated that the molecular size of most of the reaction products was significantly greater than that of the free glucose but less than that of the dextran substrate.

spots

EFFECT OF pH ON ENZYMATIC ACTIVITY.The effect of pH on enzyme activity was determined in sodium acetate, potassium phosphate, and Tris-HCl buffers. Each reaction mixture contained 80 mg of dextran per milliliter enzyme (90% saturated ammonium sulfate fraction) at 240 tg of protein per milli-

30

61

A -

20

10

-

x : uz

30

B

0

20

10

I

. . . . . . . . 1= T 20 40

1

VI

60 ml

80

100

I

120

140

EFFLUENT

FIG 4. Separation of dextran hydrolase reacproducts by gel-filtration through Sephadex G-200 column. Column of resin 2 X 31 -= cm was washed, equilibrated, and eluted with 0.05M tion

sodium acetate, buffer (pH, 5.5), containing 0.02% sodium azide. It had a flow-rate of 0.4 ml/min. Column fractions were collected in 5-ml amounts and analyzed for hexose by phenolsulfuric acid method.12 A, elution pattern of standard mixture of dextran (at concentration equal to that in standard enzyme reaction mixture) and glucose. B, elution pattern of standard enzyme reaction mixture incubated at 37 C for 24 hours.

liter reaction mixture, and 0.05 M buffer. The reaction mixtures were incubated for one hour at 37 C. The effect of varying the pH was examined at every 0.5 pH unit between 4.5 and 7.0 in the potassium phosphate buffer and over a range from 6.5 to 9.0 in the Tris-HCI buffer. Enzyme activity was measured in micromoles of reducing sugar liberated per milliliter of reaction mixture (Fig 5A). The dextran hydrolase showed a pH optimum of 5.5 both in the sodium acetate and phosphate buffers. An increase in pH beyond 6.0 showed a sharp decrease in activity. The specific activity at optimum pH in sodium acetate was 0.104 ,Mmole/ min/mg protein compared with that in phosphate buffer of 0.093 j mole/min/mg. There was little activity detected in the Tris-HCI buffer.

Downloaded from jdr.sagepub.com at Bobst Library, New York University on June 4, 2015 For personal use only. No other uses without permission.

62

I Dent Res January 1977

ELLIS AND MILLER A

E

*

B

B

X~I w w

A

-r

x

x

U

B

1.5

0

-4

Zu

C~

w 0.5

I_

I

I

I

I

I

I

I

I

I

1

4.0

5.0

6.0

7.0

8.0

9.0

10

20

30

40

50

pH

TEMPERATURE (C)

FIG 5. Effect of pH and temperature on velocity of dextran degradation. A, standard reaction mixtures containing 80 mg of dextran and 240 ,g protein/ml were incubated for 60 minutes at 37 C in presence of 0.05 M sodium acetate buffer (solid circles joined by line), 0.05 M potassium phosphate buffer (solid squares joined by line), and 0.05 M Tris-HCIl buffer (solid triangles joined by line) at various pH values. B, standard reaction mixtures containing 80 mg dextran and 240 j,g protein were incubated in 0.05 M sodium acetate buffer (pH, 5.5), for 60 minutes at various temperatures. Reducing sugars were detected by Somogyi method "1.

EFFECT OF TEMPERATURE ON ENZYME ACTIVITY.-The effect of temperature on dextran hydrolase activity was measured in standard reaction mixtures. The Somogyill test for reducing sugar was used to determine activity. Reaction mixtures were analyzed after being

incubated at 5, 25, 37, and 55 C for one hour (Fig 5B). Optimum activity was seen at 37 C with a specific activity of 0.09 Jtmole/min/mg protein. EFFECT OF SUBSTRATE CONCENTRATION ON ENZYMATIC REACTIONS.-The effect of varying

1.0

0.8 U)

0.6

C~

0.4 0

0.2

20

40

60

80

100

DEXTRAN (mg/ml)

FIG 6. Effect of substrate concentration on velocity of dextran degradation. Standard reaction mixtures containing 240 ,ug protein/ml and various amounts of dextran were incubated at 37 C for 60 minutes and assayed for presence of reducing sugars. V, ttmoles reducing sugar per minute per milliliter. S, dextran concentration in milligrams per milliliter. Downloaded from jdr.sagepub.com at Bobst Library, New York University on June 4, 2015 For personal use only. No other uses without permission.

DEXTRAN HYDROLASE FROM S MUTANS

Vol. 56 No. I

dextran concentration on the reaction velocity was measured under standard assay conditions. The substrate was varied between 0.4 and 9.6 mg/ml reaction mixture. The reaction velocity increased at a constant rate with increasing concentrations of dextran through 1.6 mg/ml (Fig 6). The velocity then increased disproportionately with concentrations greater than 1.6 mg. Maximum velocity was achieved at 32 mg/ml and was calculated as being 0.015 Mumole/min/ml reaction mixture. The Lineweaver-Burk-double-reciprocal plot was used to determine the Michaelis-Menten constant or Km which was calculated as 8 mg dextran/ml of reaction mixture (Fig 6). Since the dextran used had an average molecular weight of 5X105, the calculated Km of 8 mg/ml would be equivalent to 1.6X 105 M. PRODUCTION OF ENZYME DURING GROWTH. -Twelve hundred milliliters of the complex medium supplemented with 0.05 M sucrose was inoculated (1% v/v) with a 24-hour culture of S mutans 6715 grown in the complex medium with 0.05 M sucrose. Because of cell aggregation in the presence of sucrose, estimation of the growth cycle was determined by measuring culture pH rather than optical density. Aliquots of 200 ml were removed at 4, 8, 12, 16, 20, and 24 hours, and the cellfree supematant was saturated to 90% with amI

I

4

0.03

I

0

-

I

9

46--4

0

0.02

0D

Ev Z

0.01 _

Po==--IF I 4

8

12

16 20

24 30

TIME (HOURS)

FIG 7. Specific activity of dextran hydrolase during growth of S mutans. Specific activity (solid circles joined by line) of extracellular enzyme was measured at various times during growth of S mutans in complex medium supplemented with 0.05 M sucrose. Estimation of growth was determined by measuring culture pH (solid squares joined by line). One unit of enzyme produces one jtmole of reducing sugar from dextran per minute under standard assay conditions.

63

monium sulfate. Protein precipitates were collected, dissolved in 5.0 ml of distilled water, and dialyzed. Each enzyme sample was assayed under standard conditions to determine specific activities. Figure 7 shows specific activity of the hydrolase with growth as a function of the pH. After four hours of growth, the specific activity reached only ane third of its maximum value. Maximum specific activity of this extracellular enzyme was seen after eight hours of growth and remained constant throughout the stationary phase of growth at 24 hours. EFFECT OF CARBON SOURCE ON SPECIFIC ACTIVITY.-Five hundred-milliliter portions of the complex medium supplemented with 0.03 M glucose, 0.03 M sucrose, 0.03 M glucose plus 0.03 M sucrose, 0.03 M glucose plus 0.5% dextran, and 0.5% dextran were inoculated (1%) with a culture of S mutans 6715 grown for 24 hours in each of the respective media. The initial pH of all cultures was 7.0. The cultures were incubated at 37 C and the pH values of the media were monitored every four hours. There was no change in the pH of the 0.5% dextran culture, but all other cultures showed a pH of 5.0 after 20 hours incubation. At 20 hours, cells were removed from all cultures and the supernatant fluids were saturated with ammonium sulfate to 90%. The protein precipitates were dissolved in distilled water, dialyzed for 48 hours against standard buffer, and the final protein concentrations measured. Standard reaction mixtures were prepared containing enzyme, 80 mg dextran/ ml, and buffer, and were allowed to incubate at 37 C for one hour. Reducing sugar formation was measured by the Somogyi assay1' (Fig 8). The highest specific activity was obtained in the preparation from the 0.03 M sucrose culture. Addition of 0.03 M glucose to the sucrose did not significantly affect the hydrolase specific activity. The specific activities in 0.03 M glucose alone and the glucose plus dextran culture were equivalent but were one-half the value of those from sucrose cultures. In the media supplemented with 0.5% dextran T-500 alone, only very slight growth occurred, as expected, and specific enzyme activity could not be measured. SUBSTRATE SPECIFICITY.-The activity of dextran hydrolase on the water-soluble polysaccharides produced in sucrose cultures of S mutans 6715 and NTCC 10449, Streptococcus MPK1, KPK2, and KPK3, and S sanguis

Downloaded from jdr.sagepub.com at Bobst Library, New York University on June 4, 2015 For personal use only. No other uses without permission.

64

Z

J

ELLIS AND MILLER

H z

0.03

0.02

u0.01

2

1

3

January 1977

standard conditions for one hour at 37 C, and hydrolysis was determined by detecting an increase in reducing sugars. Controls consisted of substrate alone and enzyme alone incubated for one hour at 37 C. Table 2 gives a comparison of the specific activity of the enzyme preparation with the various polysaccharides and with equivalent concentrations of dextran T500. No reducing sugars were released from the S mutans 6715 or Streptococcus MPK1, KPK2, and KPK3 strains under the assay conditions used. However, reducing sugar release was detected when polysaccharides from S mutans 10449 and both strains of S sanguis were added. Specific activities with polysaccharides from S mutans 10449 and S sanguis 10556 and JC 804 were 10.0, 17.1, and 43.2%, respectively, of those exhibited with equivalent concentrations of dextran T-500.

0 be

Dent Res

4

MEDIUM SUPPLEMENT

FIG 8.-Specific activity of dextran hydrolase from late-log cultures of S mutans grown in medium supplemented with 1, 0.03 M glucose; 2, 0.03 M sucrose; 3, 0.03 M glucose plus 0.03 M sucrose; 4, 0.03 M glucose plus 0.5% dextran. One unit of enzyme produces one umole of reducing sugar from dextran per minute under standard assay conditions.

IN VITRO PLAQUE DEGRADATION BY DEXTRAN HYDROLASE.-Since dextrans and other glucans have been shown to participate in aggregation and adhesion of certain oral streptococci, a dextran hydrolase preparation was tested for its ability to degrade various streptococcal plaques. Plaques from S mutans 6715 and 10449, Streptococcus MPK1, KPK2, and KPK3, and S sanguis 10556 and JC 804 were prepared and analyzed for degradation by dextran hydrolase. Although the enzyme preparation had a specific activity of 0.092 with dextran T-500, there was no visible degradation of any of the plaque samples tested after four hours of incubation. An assay for reducing sugar in these reaction mixtures showed no increase over the control tubes.

ATCC 10556 and JC 804 was measured. Because of the varying amounts of polysaccharides produced by each of these strains and the desire to use relatively high substrate concentrations, it was decided not to adjust each polysaccharide substrate to an equal concentration. Thus, total hexose12 of each substrate preparation was measured and 1 ml was added to a standard amount of enzyme. For comparison, reaction mixtures containing dextran T500 at concentrations equivalent to each streptococcal polysaccharide were used. All reaction mixtures were allowed to incubate under

BLE 2 ENZYME SPECIFIC ACTIVITY WITH STREPTOCOCCAL POLYSACCHARIDES

Polysaccharides Source

S mutans 6715 10449 Streptococcus MPK1

KPK2 KPK3 S san guis 10556 JC 804 *

Substrate Concentration

Specific Activity*

(mg/ml)

Streptococcal Polysaccharides

Dextran T-SOO5

3.2 12.0

0 0.0046

0.0173 0.0458

0

0

0.0387 0.0083 0.0424

0.0038 0.0042

0.0222 0.0097

9.0 1.3 10.2 4.4 1.5

0

Mvicrom3les reducing sugar per minute per milligram of protein. At same concentration as respective streptococcal polysaccharide.

Downloaded from jdr.sagepub.com at Bobst Library, New York University on June 4, 2015 For personal use only. No other uses without permission.

Vol. 56 No. I

DEXTRAN HYDROLASE FROM S MUTANS

Discussion Several investigations have reported that many strains of S mutans possess a cell-associated or extracellular dextranase activity.4-7 However, no information is available conceming dextranase activity of strain 6715. Since this strain is used in many important cariesrelated investigations,15-39 it is important to characterize its properties potentially related to caries etiology. The present study indicates that dextran with an average molecular weight of 5 X 105 daltons was readily degraded by an extracellular enzyme preparation from S mutans strain 6715. Analysis of the reaction products by ascending thin-layer chromatography and Sephadex gel filtration demonstrated that the dextran was enzymatically reduced to oligosaccharides and trace quantities of glucose. The amount of glucose produced ranged from only 0.21 to 0.34% of the total reducing sugars produced. The oligosaccharides and the dextran substrate dropped immediately through Sephadex G-75 and G-150 columns, but the oligosaccharides were retained on G-200 which suggests their molecular weight was less than 2 X 105. Since only slightly detectable amounts of glucose were present, these data would suggest an endohydrolytic activity. The S mutans 6715 enzyme preparation also degraded soluble polymers produced in the presence of sucrose by some other oral streptococci. However, degradation of several streptococcal plaques formed in vitro could not be demonstrated with the S mutans 6715 enzyme preparation. Thus, activity of S mutans 6715 dextranase in the oral cavity would seem to be directed toward the more soluble glucans, presumably those with a higher percentage of a-( 1-G6) linkages, and it would not seem to be an important plaque-dispersing agent. The S mutans 6715 enzyme seems to be similar if not identical in mechanism of action to the purified endo a- (1-* 6) -glucan-6-glucanohydrolase from S mutans strain OMZ 176.6 The extracellular dextran hydrolase from S mutans strain 6715 seems to be well suited for the environmental conditions present in the oral cavity. The pH of the oral cavity and the plaque-tooth interface will vary with the amount of fermentable carbohydrates but is generally between 4.5 and 6.0. The optimum pH for dextran hydrolase activity was found to be 5.5, although activity ranged from 5.0 to 6.0. The temperature optimum for the dextranase was 37 C. These data are in close

65

agreement with those of the purified dextran hydrolase from S mutans OMZ 176.6 To examine dextran hydrolase production, S mutans 6715 was grown in the complex medium supplemented with different carbohydrates. During a 24-hour incubation with sucrose, a maximum extracellular specific activity was reached just as the bacteria entered the logarithmic phase of growth and this activity continued through the stationary phase. Cellfree supernatants from glucose cultures showed a basal level of enzyme production. Supematants from sucrose or sucrose plus glucose cultures showed a 100% increase in enzyme specific activity over the basal levels. Since sucrose is required for the synthesis of dextran, it might be suggested that the presence of sucrose induced the production of dextranase through dextran synthesis. However, specific activity from cultures supplemented with glucose plus dextran T-500 remained at the basal level. One possible explanation for these findings would be that a dextran synthesized from sucrose and of a different molecular weight or structure from T-500 is required for the apparent enzyme induction. Dextran T-500 has few branches and a fairly uniform molecular weight and may not be able to function in this capacity, although it can serve as a substrate for the enzyme. The importance of dextranase activity of plaque microorganisms seems to be at least threefold: (1) degradation of dextran-producing plaques, (2) formation of readily fermentable hexose units within dextran-producing plaques, and (3) regulation of streptococcal glucan synthesis. Since Fitzgerald, Spinell, and Stoudt40 reported dextranase disruption of in vitro streptococcal plaques, several studies have shown varying degrees of success in the removal of dextran-producing plaques in vitro from the dentition of experimental animals and man.15'41-51 This variation seems to be the result of the differences in the type of dextran or glucan present in the plaques tested. The water-insoluble type of glucans synthesized from sucrose by S mutans seems to be responsible for the ability of this bacterium to form plaques, 6,16,52,53 and investiagtions suggest that an increased ratio of a-(1 >3) to a(l16) glucopyranosyl linkages decreases the water solubility of streptococcal glucans.5456 Thus, dextran with exohydrolytic a- ( 16) specificity such as that from Penicillium funiculosum have varying degrees of activity on glucans depending on the number of a-(tl->6)

Downloaded from jdr.sagepub.com at Bobst Library, New York University on June 4, 2015 For personal use only. No other uses without permission.

66

ELLIS AND MILLER

linkages present.4057'58 Other glucanases such as mutanase have a- ( 1- 3) specificity and have been shown to degrade some insoluble streptococcal glucans.49,59 The endohydrolytic dextranase from S mutans 6715 would not seem to be an adequate plaque dispersal agent since the enzyme preparation did not degrade in vitro plaques formed by several streptococci. The formation of fermentable hexose from plaque glucans by oral dextranases does seem plausible. The extracellular dextranases from S mutans strain OMZ 1766 and strain B 25 hydrolyze dextran to apparently nonfermentable oligosaccharides. However, crude cell extracts of S mutans strains NSW 1, JC 2, and IB 16 were shown to hydrolyze dextran to oligosaccharides and to free glucose.7 The free glucose produced was equivalent to 43.7 for NSW 1, 34.3 for JC 2, and 12.7% for IB 16 of the total reducing sugars produced in the enzyme reaction mixtures. These three strains also hydrolyzed isomaltose to free glucose. Thus, it seems that some strains of S mutans are capable of fermenting dextrans and may possess both endohydrolytic and exohydrolytic dextranases. The endodextranase of strain 6715 was unable to degrade dextran to a carbon source fermentable by the organism. However, in the mixed microbial population present in plaque, other oral bacteria that possess exohydrolytic extranases 1,2,3,7 may be capable of fermenting the oligosaccharide products of the endodextranase reaction. Several investigations have shown that the presence of dextranases during the synthesis of streptococcal glucans from sucrose affects the amount and type of glucans synthesized. Walker' has shown that in the presence of endodextranase, production of polysaccharides by glucosyltransferases produced by S mutans and S sanguis was suppressed. It was also demonstrated that an exohydrolytic dextranase produced by S mitis 439 was able to hydrolyze oligosaccharides that were possible intermediates in streptococcal dextran synthesis.' Schachtele, Staat, and Harlander3 have also shown that the endohydrolytic dextranase from A israelii and the combined exohydrolytic and endohydrolytic dextranases from B ochraceus caused a 58.1 and 60.4%, respectively, inhibition of water-insoluble glucan production by S mutans 6715 dextransucrase. Sucrosedependent adherence of S mutans 6715 was also shown to be inhibited by 80% in the presence of these dextranases. Since these investigators demonstrated that the dextranases de-

I Dent Res January 1977 graded only 15 to 16% of the preformed waterinsoluble glucans from S mutans 6715, it seems that the observed effect of the dextranases was mainly related to the synthesis of water-insoluble glucans rather than the hydrolysis of the completely formed water-insoluble molecules. A subsequent report has suggested that the A israelii endohydrolytic dextranase may have its effect on the synthesis of glucans by altering their structure since the rate of total polysaccharide synthesis by S mutans 6715 was not reduced in the presence of the dextranase.60 Thus, the total complement of streptococcal glucans synthesized in the presence of an endohydrolytic dextranase may contain a new structural type of glucan or a different proportion of the same structural types of molecules and this change results in a reduction in the adhesive nature of the organisms. Changes in the streptococcal glucans may involve insertion of the endodextranase oligosaccharide products into a growing branched type dextran molecule as a side chain. Such a mechanism of oligosaccharide incorporation by glucosyltransferases has been suggested by Staat and Schachtele.5 As pointed out by these investigators, studies with dextransucrases produced by Leuconostoc mesenteroides have indicated that low molecular weight dextrans could be inserted into a-( l-*6) chains as c-( 1-*3) branches.61 Water-insoluble glucan synthesized by S mutans in the presence of dextranase has been shown to contain a much higher percentage of a-( 1->3) linkages and to be much less adhesive than the glucan synthesized in the absence of dextranase.62 The increase in synthesis of the endodextranase produced by S mutans 6715 in the presence of sucrose but not in the presence of dextran T-500, which did serve as a substrate for the enzyme, may be related to the possible role of the dextranase in the sucrose-dependent synthesis of glucans. If, indeed, endodextranases are involved with the regulation of the types or amounts of glucans synthesized, then a system such as enzyme induction mediated by molecules synthesized from sucrose which would control the production of the endodextranase would seem to be an efficient mechanism for such regulation. Conclusions Culture filtrates of S mutans 6715 contain an enzyme that degrades dextran primarily to oligosaccharides with a trace amount of free glucose. The enzyme seems to be an a-(16)

Downloaded from jdr.sagepub.com at Bobst Library, New York University on June 4, 2015 For personal use only. No other uses without permission.

Vol. 56 No. I

DEXTRAN HYDROLASE FROM S MUTANS

glucan-6-glucanohydrolase with properties similar to the purified extracellular dextranase from strain OMZ 176. The S mutans 6715 enzyme was active toward some soluble sucrosedependent streptococcal polymers but did not degrade insoluble polymers associated with streptococcal plaques formed in vitro. S mutans strain 6715 did not grow or produce acids in media containing the dextran substrate as the sole source of carbohydrate. Glucose cultures yielded excellent growth and a basal level of dextranase specific activity. The specific activity in media with glucose and sucrose alone was twice that of the basal level. However, the amount of enzyme produced in the presence of glucose plus dextran remained at the basal level. Thus, the apparent induction of the dextranase by sucrose but not by high molecular weight dextran (T-500) which serves as the enzyme substrate might suggest that the apparent induction mechanism is more closely related to the possible function of dextranase in the sucrose-dependent synthesis of glucans than to the mere hydrolysis of large extracellular dextrans. Many extracellular hydrolases are inducible for the purpose of degrading the substrate to smaller units that can subsequently be used for the growth of the organism. However, induction of the extracellular dextranase for this sole purpose would not seem to be very efficient since the dextran was not able to serve as a fermentable carbohydrate for S mutans 6715.

References 1. WALKER, G.J.: Some Properties of a Dextranglucosidase Isolated from Oral Streptococci and Its Use in Studies on Dextran Synthesis, J Dent Res 51:409-414, 1972. 2. STAAT, R.H.; GAWRONSKI, T.H.; and SCHACHTELE, C.F.: Detection and Preliminary Studies on Dextranase-Producing Microorganisms from Human Dental Plaque, Infec Immun 8:1009-1016, 1973. 3. SCHACHTELE, C.F.; STAAT, R.H.; and HARLANDER, S.K.: Dextranases from Oral Bacteria: Inhibition of Water-Insoluble Glucan Production and Adherence to Smooth Surfaces by Streptococcus mutans, Infect Immun 12:309-317, 1975. 4. MAKINEN, K.K., and PAUNIO, I.K.: Exploitation of Blue Dextran as a Dextranase Substrate, Anal Biochem 39: 202-207, 197 1. 5. STAAT, R.H., and SCHACHTELE, C.F.: Evaluation of Dextranase Production by the Cariogenic Bacterium Streptococcus mutans, Infect Immun 9:467-469, 1974.

67

6. GUGGENHEIM, B., and BURKHARDT, J.J.: Isolation and Properties of a Dextranase from Streptococcus mutans OMZ 176, Helv Odontol Acta 18: 101-113, 1974. 7. DEWAR, M.D., and WALKER, G.J.: Metabolism of the Polysaccharides of Human Dental Plaque: I. Dextranase Activity of Streptococci, and the Extracellular Polysaccharides Synthesized from Sucrose, Caries Res 9:2135, 1975. 8. DA COSTA, T.; BIER, L.C.; and GAIDA, F.: Dextran Hydrolysis by a Fusobacterium Strain Isolated from Human Dental Plaque, Arch Oral Biol 19:341-342, 1974. 9. JORDAN, H.V.; FITZGERALD, R.J.; and BOWLER, A.E.: Inhibition of Experimental Caries by Sodium Metabisulfite and Its Effect on the Growth and Metabolism of Selected Bacteria, J Dent Res 39:116-123, 1960. 10. LOWRY, O.H.; ROSENBROUGH, N.J.; FARR, A.L.; and RANDALL, R.J.: Protein Measurement with the Folin Phenol Reagent, J Biol Chem 193:265-275, 1951. 11. SOMOGYI, M.: Determination of Blood Sugar, J Biol Chem 160:69-73, 1945. 12. DUBOIS, M.; GILES, K.A.; HAMILTON, J.K.; REBERS, P.A.; and SMITH, F.: Colorimetric Method for Determination of Sugars and Related Substances, Anal Chem 28:350-354, 1956. 13. DESTEFANIS, V.A., and PONTE, J.G., JR.: Separation of Sugars by Thin-Layer Chromatography, J Chromatogr 34:116-120, 1968. 14. MCCABE, R.M.; KEYES, P.H.; and HOWELL, A., JR.: An In Vitro Method for Assessing the Plaque Forming Ability of Oral Bacteria, Arch Oral Biol 12:1653-1656, 1967. 15. FITZGERALD, R.J.; KEYES, P.H.; STOUDT, T.H.; and SPINELL, D.M.: The Effects of Dextranase Preparation on Plaque and Caries in Hamsters: A Preliminary Report, JADA 76:301-304. 16. GIBBONS, R.J., and FITZGERALD, R.J.: Dextran-Induced Agglutination of Streptococcus mutans, and Its Potential Role in the Formation of Microbial Dental Plaques, J Bacteriol 98:341-346, 1969. 17. GIBBONS, R.J., and NYGAARD, M.: Interbacterial Aggregation of Plaque Bacteria, Arch Oral Biol 15:1397-1400, 1970. 18. GREER, S.B.; HSIANG, W.; MUSIL, G.; and ZINNER, D.D.: Viruses of Cariogenic Streptococci, I Dent Res 50:1594-1604, 1971. 19. VAN HOUTE, J.; GIBBONS, R.J.; and PULKKINEN, A.J.: Adherence as an Ecological Determinant for Streptococci in the Human Mouth, Arch Oral Biol 16:1131-1141, 1971. 20. GIBBONS, R.J.: Presence of an InvertaseLike Enzyme and a Sucrose Permeation System in Strains of Streptococcus mutans, Caries Res 6:122-131, 1972.

Downloaded from jdr.sagepub.com at Bobst Library, New York University on June 4, 2015 For personal use only. No other uses without permission.

68

ELLIS AND MILLER

21. SCHAGHTELE, C.F.; LOKEN, A.E.; and KNUDSON, D.J.: Preferential Utilization of the Glucose Moiety of Sucrose by a Cariogenic Strain of Streptococcus mutans, Infect Immun 5:531-536, 1972. 22. EVANS, R.T., and GENCO, R.J.: Inhibition of Glucosyltransferase Activity by Antisera to Known Serotypes of Streptococcus mutans, Infect Immun 7:237-241, 1973. 23. PITTS, G., and KEELE, B.B., JR.: Bacterial Degradation of Streptococcal Glucans, J Dent Res 52:1303-1309, 1973. 24. TANZER, J.M.; BROWN, A.T.; and MCINERNEY, M.F.: Identification, Preliminary Characterization, and Evidence for Regulation of Invertase in Streptococcus mutans, J Bacteriol 1 16: 192-202, 1973. 25. MILLER, C.H., and KLEINMAN, J.L.: Effect of Microbial Interactions on In Vitro Plaque Formation by Streptococcus mutans, J. Dent Res 53:427-434, 1974. 26. GERMAINE, G.R.; SCHACHTELE, C.F.; and CHLUDZINSKI, A.M.: Rapid Filter Paper Assay for the Dextransucrose Activity from Streptococcus mutans, J Dent Res 53:13551360, 1974. 27. CHLUDZINSKI, A.M.; GERMAINE, G.R.; and SCHACHTELE, C.F.: Purification and Properties of Dextransucrose from Streptococcus mutans, I Bacteriol 118:1-7, 1974. 28. FREEDMAN, M.L.; and TANZER, J.L.: Dissociation of Plaque Formation from GlucanInduced Agglutination in Mutants of Streptococcus mutans, Infect Immun 10:189-196, 1974. 29. TANZER, J.L.; FREEDMAN, M.L.; FITZGERALD, R.J.; and LARSON, R.H.: Diminished Virulence of Glucan Synthesis-Defective Mutants of Streptococcus mutans, Infect Immun 10: 197-203, 1974. 30. LINZER, R., and SLADE, H.D.: Purification and Characterization of Streptococcus mutans Group d Cell Wall Polysaccharide Antigen, Infect Immun 10: 361-368, 1974. 31. GERMAINE, G.R.; CHLUDZINSKI, A.M.; and SCHAGHTELE, C.F.: Streptococcus mutans Dextransucrase: Requirement for Primer Dextran, Infect Immun 120:287-294, 1974. 32. STRECKFUSS, J.L.; SMITH, W.N.; BROWN, L.R.; and CAMPBELL, M.M.: Calcification of Selected Strains of Streptococcus mutans and Streptococcus sanguis, J Bacteriol 120: 502-506, 1974. 33. NALBANDIAN, J.; FREEDMAN, M.L.; TANZER, J.M.; and LOVELACE, S.M.: Ultrastructure of Mutants of Streptococcus mutans with Reference to Agglutination, Adhesion, and Extracellular Polysaccharides, Infect Immun 10:1170- 1179, 1974. 34. SPINELL, D.M., and GIBBONS, R.J.: Influence of Culture Medium on the Glucosyl Transferase- and Dextran-Binding Capacity

I Dent Res January 1977 of Streptococcus mutans 6715 Cells, Infect Immun 10: 1448-1451, 1974. 35. SCHACHTELE, C.F.: Glucose Transport in Streptococcus mutans: Preparation of Cytoplasmic Membranes and Characteristics of Phosphotransferase Activity, J Dent Res 54: 330-338, 1975. 36. IACONO, V.J.; TAUBMAN, M.A.; SMITH, D.J.; and LEVINE, M.J.: Isolation and Immunochemical Characterization of the GroupSpecific Antigen of Streptococcus mutans 6715, Infect Immun 11: 117-128, 1975. 37. TERLECKYJ, B.; W1LLETT, N.P.; and SHOCKMAN, G.D.: Growth of Several Strains of Oral Streptococci in a Chemically Defined Medium, Infect Immun 11:649-655, 1975. 38. TERLECKYJ, B., and SHOCKMAN, G.D.: Amino Acid Requirements of Streptococcus mutans and Other Oral Streptococci, Infect Immun 11:656-664, 1975. 39. MICHALEK, S.M.; MCGHEE, J.R.; and NAVIA, J.M.: Virulence of Streptococcus mutans: A Sensitive Method for Evaluating Cariogenicity in Young Gnotobiotic Rats, InfectIImmun 12:69-75, 1975. 40. FITZGERALD, R.J.; SPINELL, D.M.; and STOUDT, T.H.: Enzymatic Removal of Artificial Plaques, Arch Oral Biol 13:125-128, 1968. 41. K6NIG, K.G., and GUGGENHEIM, B.: In Vivo Effects of Dextranase on Plaque and Caries, Helv Odontol Acta 12:48-55, 1968. 42. GUGGENHEIM, B.; K6NIG, K.G.; MUHLEMANN, H.R.; and REGOLATi, B.: Effects of Dextranase on Caries in Rats Harbouring an Indigenous Cariogenic Bacterial Flora, Arch Oral Biol 14:555-558, 1969. 43. BLOCK, P.L.; DOOLEY, C.L.; and HOWE,

E.E.: The Retardation of Spontaneous Periodontal Disease and the Prevention of Caries in Hamsters with Dextranase, J Periodontol 40:41-46, 1969. 44. BOWEN, W.H.: The Effect of Dextranase on Caries Activity in Monkeys (Macaca irus), Br Dent J 131:445-449, 197 1. 45. LOBENE, R.R.: A Clinical Study of the Effect of Dextranase on Human Dental Plaque, JADA 82:132-135, 1971. 46. CALDWELL, R.C.; SANDHAM, H.J.; MANN, W.V., JR.; FINN, S.B.; and FORMICOLA, A.J.: The Effect of Dextranase on Human Dental Plaque, JADA 82:124-131, 1971. 47. KEYES, P.H.; HIcKS, M.A.; GOLDMAN, B.M.; MCCABE, R.M.; and FITZGERALD, R.J.: Dispersion of Dextranous Bacterial Plaques on Human Teeth with Dextranase, JADA 82:136-141, 1971. 48. NYMAN, S.; LINDHE, J.; and JANSON, J.: The Effect of a Bacterial Dextranase on Human Dental Plaque Formation and Gingivitis Development, Odontol Revy 23:243252, 1972.

Downloaded from jdr.sagepub.com at Bobst Library, New York University on June 4, 2015 For personal use only. No other uses without permission.

Vol. 56 No. I

DEXTRAN HYDROLASE FROM S MUTANS

49. GUGGENHEIM, B.; REGOLATI, B.; and MtUHLEMANN, H.R.: Caries and Plaque Inhibition by Mutanase in Rats, Caries Res 62:289-297, 1972. 50. MINAH, G.E.; LOESCHE, W.J.; and DZIEWIATKOWSKI, D.D.: The In Vitro Effect of Fungal Dextranase on Human Dental Plaque, Arch Oral Biol 17:35-42, 1972. 51. MURAYAMR, Y.; WADA, H.; HAYASHI, H.; UCHIDA, T.; YOKOMIZO, I.; and HAMADA, S.: Effects of Dextranase from Spicaria violaceae (IFO 6120) on the Polysaccharides Produced by Oral Streptococci and on Human Dental Plaque, J Dent Res 52:658-667, 1973. 52. GIBBONS, R.J., and BANGHART, S.B.: Synthesis of Extracellular Dextran by Cariogenic Bacteria and Its Presence in Human Dental Plaque, Arch Oral Biol 12:11-24, 1967. 53. GIBBONS, R.J., and NYGAARD, M.: Synthesis of Insoluble Dextran and Its Significance in the Formation of Gelatinous Deposits by Plaque-Forming Streptococci, Arch Oral Biol 13:1249-1262, 1968. 54. GUGGENHEIM, B.: Enzymatic Hydrolysis and Structure of Water-Insoluble Glucan Produced by Glucosyltransferases from a Strain of Streptococcus mutans, Helv Odontol Acta 5:89-108, 1970. 55. HOLTZ, P.; GUGGENHEIM, B.; and SCHMID, R.: Carbohydrates in Pooled Dental Plaque, Caries Res 6:103-121, 1972.

69

56. EBISU, S.; MISAKI, A.; KATO, K.; and KOTANI, S.: The Structure of Water-Insoluble Glucans of Cariogenic Streptococcus mutans, Formed in the Absence and Presence of Dextranase, Carbohydr Res 38:374-381, 1974. 57. TSUCHIYA, H.M.; JEANNES, A.; BRICKER, H.M.; and WILHAM, C.A.: Dextran-Degrading Enzymes from Molds, I Bacteriol 64: 513-519, 1952. 58. CHAIET, L.; KEMPF, A.S.; HARMAN, R.; EACZKA, E.; WESTON, R.; NALLSTADT, K.; and WOLF, F.: Isolation of A Pure Dextranase from Penicillium funiculosum, Appl Microbiol 20:421-426, 1970. 59. GUGGENHEIM, B., and ROSEMARIE, H.: Purification and Properties of Alpha (1, 3) Glucanohydrolase from Trichoderma harzianum, J Dent Res 51:394-402, 1972. 60. STAAT, R.H., and SCHACHTELE, C.F.: Characterization of a Dextranase Produced by an Oral Strain of Actinomyces israelii, Infect Immun 12:556-563, 1975. 61. EBERT, K.H., and BROSCHE, M.: Origin of Branches in Native Dextrans, Biopolymers 5:123-130, 1967. 62. EBISU, S.; MISAKI, A.; and KOTANI, S.: The Structure of Water-Insoluble Glucans of Cariogenic Streptococcus mutans, Formed in the Absence and Presence of Dextranase, Carbohydr Res 38:374-381, 1974.

Downloaded from jdr.sagepub.com at Bobst Library, New York University on June 4, 2015 For personal use only. No other uses without permission.

Extracellular dextran hydrolase from Streptococcus mutans strain 6715.

Extracellular Dextran Hydrolase from Streptococcus mutans Strain 6715 DAVm W. ELLIS and CHRIS H. MILLER Laboratory of Oral Microbiology, Indiana Univ...
1MB Sizes 0 Downloads 0 Views