Effect of Dilution on
Ca rboxymethylcelIuIase
and Xylanase Assays
Nicole Cauchon and Anh LeDuy*
Department of Chemical Engineering, Lava1 University,
Sainte-Foy, Quebec, Canada GIK 7P4
Accepted for Publication December 29, 1983
It has been demonstrated that the dilution of samples
prior to the carboxymethylcellulase and xylanase assays
causes serious discrepancies in the numerical values
obtained for the enzyme activities. Even when the sample
is assayed with the identical procedure, one could obtain
different numerical values of the enzyme activity U de-
pending on how much this sample has been diluted be-
fore the enzyme assay. Two crude commercial cellulase
samples of Aspergillus niger and Trichoderma viride as
well as the culture filtrate of our newly isolated acido-
philic fungus have been used for the demonstration. An
empirical method for reporting the cellulolytic activity by
taking into account this dilution effect is proposed.
INTRODUCTION
Assay procedures for activities such as the carboxy-
methylcellulase (CMCase) and xylanase activities of an
unknown sample usually include the following steps: 1)
addition of a known quantity of the sample (containing en-
zyme E) to a buffered solution containing an appropriate
substrate S (carboxymethylcellulose or xylan) at nonlimit-
ing concentration; 2) incubation of the mixture at the op-
timum temperature for an appropriate period of time; 3)
measurement of the concentration of the product P re-
leased, and 4) calculation of the enzymatic activity, U , of
the unknown sample using an appropriate formula.
An examination of procedures for the above assays
reported in recent literature studies reveals that these are
not standardized. Indeed, in assaying the same enzyme,
different laboratories report the use of substrate S of dif-
ferent quality, different assay conditions, and different
formulae for the calculation of the unit of enzyme activity
U . The nonexhaustive sample shown in Table I illustrates
the diversity of the CMCase assay procedures used by sev-
eral As a consequence, it is impossible to
compare the results reported from one laboratory with
those obtained elsewhere. The effects of the quality of sub-
strate and the assay conditions on the numerical values of
the enzyme activity U have been investigated for different
*To whom all correspondence should be addressed.
Biotechnology and Bioengineering, Vol. XXVI, Pp. 988-991 (1984)
0 1984 John Wiley &Sons, Inc.
crude and purified cellulolytic enzyme^.^.^ These studies
lead to the conclusion that the numerical value of the en-
zyme activity U is dependent on the nature of the substrate
S and on the assay conditions in an extremely sensitive
manner. This is due to the complex nature of both the en-
zyme (usually a complex of several enzymes) and the sub-
strate used for the assay.
An unknown sample for assay of cellulolytic or xylano-
lytic activity could be a cell-free fermented broth or a solu-
tion of either crude or purified enzyme. Such samples often
contain either too much or too little enzyme and should be
either diluted with the buffer solution or concentrated by
ultrafiltration prior to step 1 of the assay procedure. This
paper examines the effect of dilution of sample prior to
assay, on the numerical values of the enzyme activity U ob-
tained with otherwise identical procedures. The results ob-
tained for the CMCase and xylanase activities of two crude
commercial cellulase samples of Aspergillus niger and Tri-
choderma viride, as well as the culture filtrate of a newly
isolated acidophilic fungus, are used to illustrate the effects
of dilution of the enzymes.
MATERIALS AND METHODS
The commercially available crude cellulases of A. niger
and T. viride used in this study were received in powder
form as a gift from Dr. Stanley Parker of Biocon Inc. The
stock enzyme solutions were prepared as follows: 0.1 g en-
zyme powder was dissolved in 100 mL sodium-potassium
phosphate buffer solution at pH 5.0. The same buffer was
used to prepare various diluted samples for the study.
The acidophilic fungus was a newly isolated cellulolytic
fungus which could grow in a very acidic m e d i ~ m .The
~.~
strain NC-11 from our culture collection (Acidophilic
Fungus ATCC-20677) was used for this work. The cells
were grown in Erlenmeyer flasks containing 200 mL cul-
ture medium. The composition of the medium was [in 9’0
(w/v)] xylan (Sigma, from larchwood), 1%; (NH4),S04,
0.1%; and MgS04 . 7 H 2 0 , 0.016%. The initial pH was
adjusted to 2.0 with H2S04. Ten-day-old inoculum previ-
CCC 0006-35921841080988-04$04.00
ously grown in the same medium was used to seed the I A NIGER T VlRlDE AClDOPHlLlC FUNGU6
Erlenmeyer flasks at 2% (v/v). The cultures were incu-
bated on a rotary shaker at 150 rpm and at room tempera- 0.4 t /"
ture (28-30°C) for 14 days. The culture filtrate was used as
the stock enzyme solution for this study. The fresh culture

F:PI:
medium without substrate at pH 2.0 was used as buffer
solution for preparing subsequent samples from the stock
solution before the enzyme assays.
Cellulolytic and xylanolytic activities were measured 10 15
15
L
515
with Na-CMC (Sigma, low viscosity) or xylan (Sigma, from
r
larchwood) as substrate, as described by Khan and co- W
0 41

w o r k e r ~ .The
~ only modification was that 0.4M acetate ::
W
buffer was used instead of 0.2M as suggested by these au-
thors. In both cases, one unit of activitywas taken to be 1pg u)

glucose equivalents released/mL/min. W

9
U
U

RESULTS AND DISCUSSIONS I ir

The nonlinear effects of dilution are clearly observed in
the progress curves (Fig. 1)and the enzyme dilution curves
(Fig. 2) for both CMCase and xylanase activities of the
A . niger, T . viride, and acidophilic fungus enzyme solu-
tions. This phenomenon has recently been reported by
Lindner and co-workersI0 for a purified endoceilulase of
Sclerotium rolfsii. These authors suggested that a cryptic
substrate limitation might be the primary cause of non-
linearity in the CMCase progress curve with product inhi-
bition playing a secondary role. Galas et al." also reported
the nonlinear effects of sample dilution on the C1, C,,
avicelase, and xylanase activities of culture filtrates of
Aspergillus, Trichoderma, and Chaetomium while the
discrepancy in assay results due to the dilution effect has
been reported by Canevascini and GattlenI2 for the filter
paper activity (FPase) of crude enzyme preparations from
different cellulolytic fungi, namely T. viride, Trichoderma
2o A NIGER T ViRiDE/'
35, e
5
0
0 2 4 6 8 1 0 v 0 2 4 6 8 1 0 1 0 2 4 6 8 1 0
EN; LYME SOLUTION ( M L I
Figure 2. Enzyme dilution curves calculated from the data of Figure 1.
The same legends as in Figure 1.
koningii, Fusarium solani, Sporotrichum pulverulentum,
and Sporotrichum thermophile.
The most serious consequence of these nonlinear effects
of dilution of the samples is that the numerical value of the
enzyme activity U is nonlinearly dependent on the dilution
factor chosen prior to the assay. This implies that even
when the same sample is assayed with the identical pro-
cedure, different numerical values of the enzyme activity
U may be obtained depending on how much the sample is
diluted before the assay. The differences are enormous as
shown in Figure 3. For instance, the CMCase activity of
the stock A . niger solution is 77U if assayed without dilu-
tion, 480U if assayed with the 10-fold diluted sample, and
1400U if assayed with the 100-fold diluted sample. The
ACIDOPHILIC FUNGUS question raised is which one of these values ought to be re-
ported as the CMCase activity of the sample. This question
becomes more difficult to answer when the original sample

loo - --
-I-
A NIGER \ T VlRlOE AClDOPHlLlC FUNGUS

U
5

10
DILUTION ,
$08 \
a
" I
I
04 50 5
10
2
\\L
4
100 20

O 0 I0 20 30 6 I0 20
TIME I (MINI
30 - 0 10 20 30

0 20 40 60
Figure 1. Progress curves at different dilutions. Assay conditions in-
clude CMC = 2.5 g/L and xylan = 2.5 g/L. For the dilution, D = 1
means without dilution, D = x means the sample is diluted up to x Figure 3. Dilution-activity dependence. The same legends apply as in
times its initial volume prior to assay. Figure 1.

CAUCHON AND LEDUY: DILUTION EFFECTS ON ASSAY PROCEDURES 989

for assay, such as the cell-free fermented broth or the straight line expressed by eq. (l),and seems to depend on
eluant from purification devices, contains either too much the enzyme sources; and D is dilution. When D = 1, there
or too little enzyme and has to be diluted or concentrated is no dilution; when D = x, the stock sample is diluted up
before the assay procedure. t o x times its initial volume.
It should be noted that the incubation time t for step 1of This empirical equation, although it has no value in the
all current assay procedures as shown in Table I exceed the elucidation of the theory or the reaction mechanisms,
initial velocity phase of the enzymatic reaction as shown in would be very useful for expressing the enzymatic activities
Figure 1. In order to overcome the nonlinear effect of sam-
ple dilution, Lindner and co-workers1° suggested the use
of very diluted enzyme solution (sample) and a very short
incubation time (to measure the true initial velocity). This
method does not always work with crude culture filtrates
due to interference by the impurities present, especially
when the crude culture filtrate samples contain glucose or
other reducing sugars. For monitoring culture filtrates,
instrumental methods for automated enzyme assays are
attractive because of the high speed and the high precision
and accuracy of the apparatus. However, despite the suc-
cess reported in some case^,'^.'^ automated methods for
cellulolytic enzyme assays have not found wide application
because of the expense of the instruments and also because
assay procedures have not been standardized.
Thus, there is a real and urgent need for the develop-
ment of a standard method, either a manual or inexpensive
automated procedure, for cellulolytic and xylanolytic en-
zyme assays. In addition to standardization of the sub-
strates, assay conditions, and analytical procedures, a
uniform procedure is required to allow for the effects of
dilution of the enzyme sample.
In the meantime, it is recommended that all enzyme
assays be conducted and the results reported for several
dilutions over at least one order of magnitude as shown in
Figure 3. Within the linear or reasonably linearized re-
FERMENTATION TIME f DAY 1
gions of the curves shown in Figure 3, the cellulolytic ac-
tivity U can then be expressed by the empirical equation Figure 4. Extracellular xylanase production from batch fermentation
of an acidophilic fungus in CMC-xylan medium and at pH 2.0. The
UD=Uo-a log D (1) legends refer to: ( 0 )undiluted samples, ( A , A ) samples diluted 20 times
prior to assay, and (H. [I) samples diluted 30 times prior to assay; the (A)
where UDis the cellulolytic activity of the sample at dilu- curve was obtained by using linear or conventional correction for the
tion D; U , is the cellulolytic activity of the undiluted or the dilution and the (B) curve was obtained by using the nonlinear correction
stock sample; a is a coefficient, which is the slope of the for the dilution [eq. (1)).

Table I. Diversity of carboxymethylcellulase assay procedures.

Reference

Step 1 2 3 4 5 6

1) Enzyme, E 1-mL sample 0.5-mL sample 1-mL sample 0.5-mL sample 5-mL sample 0.1 -mL sample
Substrate, S 10 mg Na-CMC 30 mg Na-CMC 10 mg CMC (7 LT, 5 mg Na-CMC I 0 0 mg CMC 7.5 mg CMC
(Sigma) in 1 mL (Sigma) in 0.5 mL Hercules) in 1 mL (high viscosity in (Wako Pure Phar- (7 HF, Hercules)
citrate-phosphate sodium acetate sodium acetate 0.5 mL phos- maceutical Co.) in in 0.9 mL citrate
buffer 0.2M at buffer 0.01M at buffer 0.2M at phate buffer 5 mL acetate buf- phosphate buf-
pH 5.0 pH 4.8 pH 5.0 0.02M at pH 7.0 fer 0.1M at pH 5.5 fer at pH 4.5
2) Temperature, T 50°C 70°C 30°C 40°C 50°C 70°C
Time, t 30 min l h 10 min 30 min 1 day 30 min
3 ) Product, P dinitrosalicylic acid Somogyi-Nelson dinitrosalicylic acid Somogyi-Nelson phenol-sulfuric acid Somogyi-Nelson
4) Enzyme activity, 1 pg of P/min 1 mg of P/mL 1 pmol of P/min 1 pmol of P/mL h 1 mg of P/min increment of 0.10
U in absorbance at
560 nm

990 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 26, AUGUST 1984

by taking into account the effects of sample dilution. An
example in Figure 4 illustrates the usefulness of eq. (1) for
correcting the dilution effect. In this experiment, the batch
fermentation kinetics of the acidophilic fungus in a mix-
ture of Na-CMC and xylan was followed by measuring the
xylanolytic activity of the culture filtrate. After the seventh
day, because of the very high concentration of xylanase,
the samples had to be diluted 20 times (at the eighth and
ninth days) or 30 times (at the 10th day) prior to the assay
procedure. It is very clear that if the correction for the dilu-
tion of sample is made by the conventional or linear way,
e.g., by multiplying the data by 20 for samples previously
diluted 20 times and 30 for samples previously diluted 30
times, a false and over-estimated result is obtained (curve
A). The true result (curve B) was obtained by using eq. (1)
to correct the dilution effect, with an empirical coefficient
a = -31.546 (derived from Fig. 3) for the xylanolytic ac-
tivities of the acidophilic fungus enzymes under defined
assay conditions and procedure, as mentioned above.
CONCLUSIONS
The carboxymethylcellulase and xylanase assays re-
ported in the literature are not standardized, and it is im-
possible to compare the results obtained from one labora-
tory with those reported by others. There is a real and
urgent need for establishing one standard method for cel-
lulolytic and xylanolytic enzyme assays.
With current assay procedures, dilution of samples
prior to the carboxymethylcellulase and xylanase assays
causes serious discrepancies in the numerical values ob-
CAUCHON AND LEDUY: DILUTION EFFECTS ON ASSAY PROCEDURES
tained for the enzyme activities. These dilution effects
should be taken into account in the calculation and inter-
pretation of the results.
Financial support for this work W.IS received from Imperial Oil
Limited in the form of a Univers ty Research Grant, and from the
QuCbec Ministry of Energy and Resources.
References
I . J. N. Saddler and A. W. Khan, Can. J. Microbiol., 27, 288 (1982).
2. S . Sen, T. K. Abraham, and S. L. Chakrabarty, Can. J. Microbiol.,
28,271 (1982).
3. M. Desrochers, L. Jurasek, and M. G. Paicc, Dev. hid. Microbiol..
22,675 (1981).
4. B. H. Kim and J. W. T. Wimpenny, Can. J. Microbiol., 27, 1260
(1981).
5. S. Hayashida and H. Yoshioka, Agric. Bid. Chem., 44, 1721
(1980).
6. C. C. Tong, A. L. Cole, and M. G. Shepherd, Biochem. J . . 191,83
(1980).
7. J. Boa and A. LeDuy, Can. J. Chem. Eng., 60,532 (1982).
8. N. Cauchon and A. LeDuy, “Activitts Cellulolytiques d’un Fungus
Acidiphile Nouvellement 1~016,” Proceedings of the Stminaire sur
I’Hydrolyse des Mattriaux Ligno-cellulosiques, QuCbec Ministry of
Energy and Resources, Quebec, Canada, 1982, pp. 193-206.
9. A. W. Khan, D. Wall, and L. Van den Berg, Appl. Environ. Mi-
crobiol., 41, 1214 (1981).
10. W. A. Lindner, C. Dennison, and G. V. Quicke, Biotechnol. Bio-
eng., 25,377 (1983).
11. E. Galas, R. Pyc, K. Pyc, and K. Siwinska, Eur. J. Appl. Microbiol.
Biotechnol., 11, 229 (1981).
12. G. Canevascini and C. Gattlen, Biotechnol. Bioeng., 23, 1573
(1981).
13. K. A. Holm, Anal, Biochem., 84,522 (1978).
14. M. Leisola and J. Virkkunen, Enzyme Microbiol. Technol., 1, 117
( 1979).
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