Canadian Journal of Biochemistry

Published by THENATIONAL RESEARCH OF CANADA

COUNCIL

VOLUME 48 MARCH, 1970 NUMBER 1

A rapid method for the assay of dextranase

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T. Y . KOHAND B. T. KHOW
Rwearch and Deueloprnent Laboratories, Canada Backers Limited, Toronto 167, Qnfario
Received August 18, 1969

KOH,T. Y., AND KHOUW,B. T. A rapid method for the assay of dextranase. Can. J. Biochem. 48,225-227
(1970).
A method, based on the liberation of a dye compIex from Blue Dextran 2000, is described for the assay
of dextranase. The reaction was conducted at 40 "C for 5 min at pH 4.8 in a mixture containing 0.5 "/,
Blue Dextran 2006 and the enzyme. The K, for the hydrolysis s f Blue Dextran 2000 was estimated at
2.5 x M.

Intraductf on Toronto. Fungal eellulase (type I), and a-amylase (type

II) were purchased from Sigma Chemical Company.
Dextranases (EC 3.2.1.1 1) are enzymes capable Blue Dextran 2000 was a product of Pharmacia. All other
For personal use only.

of hydrolyzing the a-l,6-glycosidic linkages of chemicals used were of reagent grade.

the bacterial polysaccharide dextran. Much
interest has been focused recently on the enzyme
because of its possible application as a caries-
preventing agent (1-3). A convenient and rapid
assay procedure is, however, lacking, although
methods have been described which are based
on the determination of the liberated reducing
sugars by iodometric titration (4) or by the 3,<
dinitrosalicylic acid reagent (5). Alternately,
dextranase can be assayed by measuring the
reduction of viscosity of dextran-dextranase
solutions on incubation (6). While these methods
are highly sensitive, they are both tedious and
time-consuming.
Blue Dextran 2000 is a dye-conjugated dextran
normally used in the calibration of gel filtration
columns. In the course of our purification work
on dextranase, we noticed that a dye complex
was released from Blue Dextran 2800 when the
latter was disgested with dextranase. This prog-
erty was subsequently used as the basis of a
method for the assay of dextranase, the results of
which are reported in this paper.
Materials and Methods
Enzymes
Partially purified fungal dextranase was obtained from
the Fine Chemicals Division, Canada Packers Limited,
Enzyme Assay
The rate of release of the dye complex from Blue Dex-
tran 200Q was used as a measure of enzyme activity.
Determinations of enzyme activity were made in 25-ml
stoppered conical flasks incubated in a Bubnoff metabolic
incubator maintained at 40 "C and shaken at 90 cycles/
min. The reaction mixture consisted of the enzyme in 1.0
ml 0.1 A4 sodium acetate buffer, pH 4.0, and 1.8 mB
freshly prepared 1 % Blue Dextran 2000 in the same
buffer. After temperature equilibration, the reaction was
started by the addition of the substrate and was termi-
nated after 5 min of incubation by the addition of 4.0 mB
95 "/, ethanol. The flasks were then immersed in an ice
bath for exactly 15 min, after which the contents were
centrifuged at 25808 x g for 5 min at 6°C. The absor-
bance of the supernatant was then read at 650 mp in a
Zeiss PMQ I1 spectrophotometer against a blank from
which the enzyme was omitted. The amount of the dye
complex released was calculated by using the relation:
El ,,I mglml = 0.855. One dextranase unit is defined as
that amount of enzyme which releases 1 rng dye complex
in 5 min under the specified conditions. The specific
activity is defined as the number of units per milligram
protein. Protein was determined by the phenol method
as described by Lowry ef a/. (71, using bovine serum
albumin as standard.
For the determination of the absorption spectrum of
the dye complex, the clarified hydrolysate was scanned
on a Beckman DK-2 spectrophotometer. For the estima-
tion of the extinction coefficient of the released dye
complex, the clarified reaction mixture was evaporated
to dryness; the residue, taken up in water, was filtered
through a short column of Sephadex G-25 in water. The
 226 CANADIAN JOURNAL OF BIOCHEMISTRY. VOL. 4$, 1970
eluate was lyophilized to recover the dye complex. The
dye complex was dissolved in 2 voIumes of 0.1 M sodium
acetate buffer, pH 4.0, and 4 volunles of 95 "/, ethanol,
and the absorbance values at several dilutions were read
in a Zeiss PMQ 11 spectrophotometer at 650 my.
Reslmlts and Discussion
Absorption Spectra
The absorption spectra of both the hydrolyzed
and the urmhydrolyzed Blue Dextran 2Q00showed
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maxima at 258 and 650 mp. The extinction
eseficient (El ,,I "g/ml at 650 rnp) of the released
dye complex at pH 4.0 was computed to be 0.855.
A strict proportionality between the absorbance
readings and the concentratisns of the dye corn-
plex was observed in the r a n g of 0.2-1.0 rngjrnl
(Fig. 1)
For personal use only.
FIG. 1. Absorbance at 650 mp of increasing concen-
trations of dye complex.
Validity of the Assay
The hydrolysis of the Blue Dextran 2Q00 by
dextranase yields a dye complex which was
extractable with 46 % aqueous ethanol. The rate
of release of the dye complex into the aqueous
ethanol phase was linear with incubation time
for the first 5 min, when less than 8.Qpg protein
of a partially purified dextranase preparation
was incubated with the Blue Dextran 2000 (Fig.
2). After 5 min, the reaction rate gradually de-
creased and linearity was no longer evident. At
16B geg protein, the reaction rate varied dispro-
portionately with the incubation time.
The effects of pH on dextranase activity are
shown in Fig. 3. The optimum activity for dex-
tranase was found at about pH 4.8, This is in
5
a
g 3
Z
2I
5
I):
2
e
1
TIME IN MINUTES
FIG.2. The of Blue Dextran 2M0 by
different levels of dextranase (micrograms dextranase
protein per 2 rnl reaction mixfur@-
FIG. 3. The eRect of hydrogen-ion concentrations
(0.1 A4 acetate buffer) on dextranase activity.
accord with the observations of Tsuchiya et al.
(4) who found the pH optima for mold dextra-
nases in the range of 4.0-5.5,
The effects of incubation. temperature on
dextranase activity are shown in Table I. The
dextranase activity increased with increasing
temperature of incubation. From the Arrhenius
equation, the activation energy was calculated at
13.4 kcaljmole. The effects of incubation tern-
perature above 45 OC were not investigated.
Figure 4 shows the effects of substrate concen-
tration on dextranase activity, as shown in the
kineweaver-Burk plot. The dextranase activity
increased with increasing substrate concentra-
 K8W AND KHBUW: DEXTRANASB ASSAY 227
TABLE I
The effect of incubation
temperature on dextranase
activity

Temperature, Dextranase
"C units
30 6.961
35 1.385
40 1.950
45 2.275
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NOTE: Each 2.0 ml reaction mixture

contained 8 pg dcxtranase protein.

PIG. 5. The relationskip be- dextranase activity

and dextranase concentration. The reaction mixture
contained 1.0 ml of 1.0% Blue Dextran 2000, and 1.0 ml
partial purified dextranase in 0.1 A4 acetate buffer, pH
4.0. Incubation was at 40 'C for 5 min with constant
shaking.

The hydrolysis s f Blue Dextran 2000 appears

to be specific for dextranase. Enzymes such as
cellulase and a-amylase, which might be expected
For personal use only.

to hydrolyze Blue Dextran 2000, had no effect

at equivalent or at 10 times higher protein con-
centration.
FIG. 4. The Lineweaver-BuPk plot of the effect of The method described above appears, to date,
substrate concentrations on dextranase activity at 40 "C
and pH 4.0 (0.1 M acetate buffer). Values for I/S are to be the most convenient and rabid procedure
based on fim1 substrate csncentratiorls expressed in for measuring dextranase activity. We have also
grams per liter. found that chis method is suitable for purity
tion, reaching a peak at about 2.5 g/l, beyond measurements during isolation work.
which it began to decline. The inhibitory effects
of Blue Dextran 2000 at high concentrations on Acknowledgment
dextranase activity could be due to substrate We thank Mr. Eugene Ziolkowski for his
inhibition but this has not been definitely estab- excellent technical assistance.
lished. From the Lineweaver-Burk plot, the K,
was estimated at 5 g/l, corresponding to 2.5 x 1. FITZGERALD, R. J., SPINELL,D. M., and Smma,T. H. :
M (kr. of Blue Dextran 2000 is 2 x 106, Arch. Oral Bisl. 13, 125 (1968).
2. FITZGERALD, R. J., KEYES, P. H., STOUDT, T. W.,and
Pharmacia Technical Bulletin). SPINELL, D. M. : 5. Am. Dent. Assoc. 78, 301 (1968).
When the assay was conducted at 40 "C with 3. K ~ N I GK., G., and GUGGENHBM, B.: Welv. Bdonto1.
continuous shaking at a final substrate concen- 4. TSUCHIYA,Acta, 12, 48 (1968).
H. M., JEANBS, A., BRICKER, H. M.,and
tration of 0.5 0/,, it was found that the dextranase WILHAM, C. A.: 9. Bacteriol. 64, 513 (1952).
activity, assayed between 2 and 10 pg protein, 5. JANSON, J. C., and BORATH, J.: In Methods in enzy-
mology. Voi. 9. Edited by Colsw~ck, S. P., and
was directly proportional to the enzyme concen- Kaplan, N. 8.Academic Press Inc,, New York. 1966.
tration (Fig. 5). It appears that the shaking was p. 615.
important; the reaction rate was reduced by as 6. BAILEY, R. W., and CLARKE, R. T. J.: Biochem. J. 72,
49 (1959).
much as 25 0/, if the incubation was carried out 7. LOWRY, 0.W.,ROSEBROUGK, N.J., FARR,A. L.,and
without shaking. RANDALL, R.J.: J. Biol. Ghem. 693,265 (1951).