Suggested improvements to the standard filter
paper assay used to measured
cellulase activity
Guillermo Coward-Kelly, Cateryna Aiello-Mazzari, Sehoon Kim,
Cesar Granda, and Mark Holtzapple*
Department of Chemical Engineering
Texas A&M University
College Station, TX 77843-3122
(979) 845-9708 (phone)
(979) 845-6446 (fax)
m-holtzapple@tamu.edu
*Corresponding author
Abstract
In 1976, Mandels et al. developed the filter paper assay to measure the hydrolytic activity of
cellulases. Although this assay is widely used, it has a reputation for not being reproducible. In
the literature, two suggestions have been made to improve the assay: add supplemental cellobiase
and increase the boiling time for color development.
This paper provides data that strongly supports adding supplemental cellobiase. Because
supplemental cellobiase converts cellobiose to glucose, there is no need for additional boiling
time – 5 min is sufficient. For maximum reproducibility, it is essential that the water bath
vigorously boil so that temperature excursions are minimized. Also, it is necessary to rigorously
follow the recommended procedure by adding citrate to the calibration curve and using 1 cm × 6
cm strips.
Key Words: DNS, filter paper assay, cellulase activity, supplemental cellobiase.
Introduction
Cellulose is a polymer of -1,4 linked glucose with amorphous and crystalline regions. It is
hydrolyzed by cellulase, a complex mixture of enzymes containing endo- and exo-glucanases
plus cellobiase. The complete hydrolysis of insoluble cellulose requires the synergistic action of
these components. Different cellulase preparations vary widely in the proportions of these
components, depending on the source, growing conditions of the organism, and harvesting and
handling procedures [Mandels et al., 1976].
Exo-glucanase hydrolyzes cellobiose, but also glucose, from the nonreducing end of the
polyglucose chain. Endo-glucanase acts randomly within the cellulose chain breaking glycosidic
bonds and generating free ends on which exo-glucanases can act. Cellobiase hydrolyzes highly
inhibitory cellobiose to less inhibitory glucose.
In 1976, Mandels et al. developed the filter paper assay to measure the hydrolysis activity of
cellulases. The substrate is filter paper, which is a readily available and reproducible substrate. In
the assay, a 50-mg filter paper strip (1 cm × 6 cm, Whatman No. 1), 0.5 mL of diluted enzyme,
and 1-h incubation time are used to produce 2.0 mg of glucose equivalents, as measured by the
dinitrosalicylic acid (DNS) reducing sugars assay.
The DNS reagent contains sodium potassium tartrate (Rochelle salt), which reduces the tendency
to dissolve oxygen by increasing the ion concentration in the solution. Phenol increases the
amount of color produced during the color-developing reaction. Sodium bisulphite stabilizes the
color obtained and reacts with any oxygen present in the medium. Finally the alkaline medium
(NaOH) is required for the red-ox reaction between DNS and glucose, or other reducing sugars.
In most cases, some of the reducing sugars degrade while the analysis is performed [Miller,
1959].
Although the filter paper assay is widely used, it has a reputation for not being reproducible. In
the literature, two suggestions have been made to improve the assay: add supplemental cellobiase
[Ghose et al, 1987] and increase the boiling time for color development [Miller, 1959].
Adding Supplemental Cellobiase The composition of sugars (oligosaccharides) produced during
the standard assay greatly influences the filter paper activity (FPA) obtained from the enzyme.
The reaction products are reported as glucose equivalents, even though the absorption
coefficients for glucose, cellobiose, and higher dextrins are different. Hydrolyzates containing a
significant amount of cellobiose or higher dextrins result in lower filter paper activities [Shwald
et al., 1988]. Low cellobiase activity in an enzyme preparation affects the FPA results;
unfortunately, the cellulase complex of most cellulolytic fungi tends to be deficient in cellobiase
[Breuil et al., 1985]. In 1987, the Commission of Biotechnology [Ghose et al, 1987]
recommended adding excess cellobiase to the filter paper assay so that all the cellobiose
produced by the enzyme is converted to glucose. Although they provided no quantitative data,
they reasoned that adding cellobiase would eliminate cellobiose inhibition and reduce the error
when measuring the total reducing sugars on the DNS assay.
Increasing Boiling Time In order to complete the color development reaction, Miller [1959]
recommends increasing the boiling time from 5 min to 15 min.
The purpose of this paper is to provide data that allows us to investigate the above issues in more
detail. Also, we will explore other features (e.g., paper strip size, tube material and size, etc.) of
the filter paper assay to determine if they affect the final results. Ultimately, our objective is to
make recommendations that will reduce the variability of the filter paper assay, as currently
practiced.
Materials and Methods
Chemicals and enzyme preparations
Cellulase (Genecor, Spezyme CP, lot # 301-00348-257) and cellobiase (Novozym 188, 250
CBU/g, lot # DCN00029) were a gift from the National Renewable Energy Laboratory (NREL).
When supplementing the cellulase with cellobiase, a volume ratio 1:1 was used to have excess
cellobiase in the mixture.
Assay methods
The enzyme (cellulase) activity was determined by the method described in Mandels et al.
(1976). The procedure uses 1 mL of citrate buffer (0.05 M, pH 4.8), 0.5 mL of each enzyme
dilution, and a 50-mg filter paper strip. This mixture is then incubated for 1 h at 50 oC. The
hydrolysis reaction is stopped by adding DNS reagent and the reducing sugars are measured as
glucose equivalents by DNS.
Sugar analysis was performed using an HPLC with refractive index detector (Perkin Elmer series
200), equipped with a Biorad Aminex HPX-87P column using reverse osmosis deionized water.
Peak areas as compared to external standards were used to quantify the sugars present.
Results and Discussion
This study was performed in two phases.
First, the effects of various parameters were
investigated on the color development by the DNS reagent.
Then, the effects of various
parameters were investigated on the filter paper assay itself.
DNS Color Development
Temperature effect During the DNS assay, color development occurs only under alkaline
conditions, but reducing sugars decompose under these conditions as well; therefore, there is a
competition between the red-ox reaction with DNS and the sugar decomposition [Miller, 1959].
Figure 1 shows the effect of two temperatures (60oC and 80oC) on color development. In each
case, glucose generates more color than cellobiose. As expected, the reaction occurs more
rapidly at higher temperatures, but, at lower temperatures, more color develops once the reaction
is complete.
Boiling effect The boiling rate of the water bath used to develop the color affects the results. In
this study, we used both “vigorously” and “gently” boiling water baths. In both baths, the
temperature was 100oC prior to adding the tubes containing the sugar/DNS mixture. In the
vigorously boiling bath, the bubble volume was about 20% and in the gently boiling bath, the
bubble volume was about 1%. Figure 2 shows glucose calibration curves for both vigorously
and gently boiling baths – the gentle bath develops more color. This unexpected result can be
explained as follows: When the tubes containing the sugar/DNS mixture are placed in the bath,
the boiling stops temporarily and the bath temperature reduces. As shown in Figure 1, a lower
temperature allows more color to develop.
In the gently boiling bath, the temperature is
depressed for a longer time than in the vigorously boiling bath, so more color develops. In the
vigorously boiling bath, the temperature quickly returns to 100oC so less color develops when
the reaction is complete.
Citrate effect Table 1 shows how absorbance is affected by replacing the recommended citrate
buffer with distilled water. The DNS is highly alkaline. In the distilled-water case, there is no
buffer to moderate the pH, so the pH increases substantially when DNS is added to the sugar
solution. In contrast, in the citrate-buffer case, the pH is moderated so it does not increase as
much. High pH favors more color development; hence, more color was developed in the
distilled-water case. To regulate pH, citrate buffer is essential for the enzyme preparations. To
ensure that the sugars in the calibration and enzyme preparations respond to DNS in the same
manner, it is important that the citrate concentration be identical in both the calibration and the
enzyme preparations.
Sugar effect Figure 3 shows calibration curves for both glucose and cellobiose. In both cases, the
reaction temperature was 100oC, the water bath was vigorously boiling, and citrate was added at
the recommended concentration. Glucose develops substantially more color than cellobiose.
Table 2 shows the absorbance for glucose and cellobiose at 5 min and 15 min. The cellobiose
reaction is not complete after 5 min in a vigorously boiling bath.
Filter Paper Assay
Cellobiase addition If an enzyme complex has low cellobiase activity, a high concentration of
cellobiose is produced; which lowers the absorbance measured in the DNS assay (see Figure 3).
This produces “false low values” for the filter paper activity when compared to a cellulase
complex with higher cellobiase activity.
Shwald et al. [1988] showed that, when evaluating sugars released during the filter paper assay
of an unsupplemented cellulase enzyme, the total glucose equivalents estimated by DNS are
lower than those measured by HPLC. This discrepancy can be explained by the fact that the DNS
assay is less sensitive to cellobiose, so the true glucose equivalents are underestimated by the
DNS assay.
The Commission of Biotechnology [Ghose et al, 1987] has suggested adding cellobiase when
measuring cellulase activity using the filter paper assay. Table 3 presents the results for the
absorbances of several enzyme systems. It shows that the cellobiase enzyme has no measurable
cellulolytic activity. It also shows higher absorbances for cellulase + cellobiase (Cases 9 and 10)
than for cellulase-only (Cases 6 and 7). This results from two effects: (1) the elimination of
cellobiose inhibition and (2) the conversion of cellobiose to glucose, which has higher
absorbance.
A filter paper assay run with a 1:160 cellulase dilution factor, gave the following results: For
cellulase-only (Case 6), the glucose equivalent measured by DNS was 4.5 mg/mL whereas that
measured by HPLC was 5.2 mg/mL (3.1 mg/mL cellobiose and 1.9 mg/mL glucose), an
underestimate of the total sugar content by the DNS method. For the cellulase + cellobiase (Case
9), the glucose equivalent measured was 5.6 mg/mL (DNS) and 5.75 mg/mL (HPLC) with no
cellobiose detected. There was better agreement between the two values and a greater amount of
glucose was produced.
In their studies of the filter paper assay, Sengupta et al. (2000) found that more color develops
with longer DNS boiling times; they used a 10-min boiling period. Table 3 shows the effect of
increasing DNS boiling times from 5 to 15 min (Case 6 vs 7 and Case 9 vs 10). In both these
comparisons, more color develops when the DNS boiling time increases, which is consistent
with Sengupta et al. In Cases 6 and 7, which lack supplemental cellobiase, the increased color
can be explained by the longer reaction time needed for cellobiose to fully develop color (see
Table 2). In Cases 9 and 10, which have supplemental cellobiase, we also see that longer boiling
times increase color development. There are three possible sources of extra reducing species
released during the longer boiling times:
1. Cellobiose As shown by the HPLC analysis described previously, the supplemental
cellobiase converts all the cellobiose to glucose; no cellobioase was detected.
So,
cellobiose cannot be the source of the extra color at longer DNS boiling times.
2. Cellulose The DNS directly contacts the cellulose strip; perhaps reducing species are
released from its degradation under the alkaline conditions. Cases 2 and 3 show that
color develops from filter paper alone, and that more color develops at longer times;
however, the amount of color released from the longer reaction times is not sufficient to
account for the differences between Cases 9 and 10.
3. Higher Dextrins As cellulose enzymatically hydrolyzes, higher dextrins are released.
Under the alkaline conditions from the DNS reagent, these will degrade to reducing
species that produce color. Logically, by the process of elimination, higher dextrins
must be responsible for the extra color development.
Because color development from cellulose and higher dextrins is a complicating factor, we do
not recommend extending the boiling time beyond 5 min. As clearly shown in Table 2,
provided the sugars are in the form of glucose, 5 min is sufficient to fully develop the color.
Figure 4 shows the effect of adding supplemental cellobiase to the filter paper assay. At the
same enzyme dilution, supplemental cellobiase significantly increases production of glucose
equivalents. Because cellobiose inhibition is reduced, and because DNS is more sensitive to
glucose than cellobiose, the equivalent glucose concentration is much higher when cellobiase is
added. When adding supplemental cellobiase to Spezyme CP, the measured filter paper activity
increased by a factor of 1.56 (Table 4). Adding supplemental cellobiase better represents the
true cellulolytic activity of the enzyme, and eliminates errors from comparing cellulase enzyme
preparations with different levels of cellobiase activity.
Figure 5 and Table 5 show the effect of the boiling condition on the filter paper activity
measured. Because the “vigorously” boiling condition is easier to replicate, the results obtained
with the vigorously boiling bath are more reproducible.
Filter paper shape Filter paper was cut into two shapes: 1 cm × 6 cm strips and 0.5 cm × 12 cm
strips. Although the masses were identical, the results differed, so it is important to cut the strips
in the recommended shape (1 cm × 6 cm).
Other factors The following factors had no significant effect on the filter paper assay: Pyrex
culture tubes 20 mL (16 mm × 150 mm) vs 25 mL (20 mm × 125 mm), glass tubes vs plastic
(polypropylene) tubes, DNS batches prepared by different researchers, and cooling method
(water-ice bath vs running water). The number of tubes placed in the water bath can significantly
affect the boiling condition and impact the final result. We recommend that the water bath be
large relative to the number of tubes.
Conclusion
Based on this study, we strongly recommend that supplemental cellobiase be added, which
supports the recommendation of the Commission of Biotechnology [Ghose et al, 1987]. Because
supplemental cellobiase converts cellobiose to glucose, there is no need for the additional boiling
recommended by Miller [1959] – 5 min is sufficient.
For maximum reproducibility, it is
essential that the water bath vigorously boil so that temperature excursions are minimized.
Also, it is necessary to rigorously follow the recommended procedure by adding citrate to the
calibration preparation and using 1 cm × 6 cm strips.
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