An economical and high throughput alternative for endoglucanase
activity determination
Gastón E.Ortiz1, Martín Blasco2, Edgardo Albertó1*
1Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico Chascomús (IIB-INTECH, UNSAM-
CONICET), Universidad Nacional de San Martín, San Martín, Buenos Aires, (ARGENTINA)
2Centro de Investigación y Desarrollo en Biotecnología Industrial, Instituto Nacional de Tecnología Industrial,
Av. General Paz 5445, Ediûcio 51, San Martín, Buenos Aires, (ARGENTINA)
E-mail: eoalberto@gmail.com
FULL PAPER
ABSTRACT
The search for new strains with good productivity of cellulolytic enzymes
is always necessary to improve the processes are usually employed.
Screening protocols used for these purposes require a low cost and high
throughput assay for determination of endoglucanase activity. However,
the test proposed by the IUPAC and other microadaptations not meet this
requirement. The aim of our work is to obtain an economical and high-
performance proposed by IUPAC for determination of endoglucanase
activity assay adaptation. For this we reduced the assay volume and
investigate the effect of this reduction on endoglucanase activity
determination. As a result a miniaturized assay statistically correlated with
the assay proposed by IUPAC reduced 10-fold lower scale was obtained.
As a result a new economic and high performance adaptation for
endoglucanase activity assay based on the IUPAC protocol was obtained.
 2016 Trade Science Inc. - INDIA
KEYWORDS
Miniaturized CMCase assay;
Cellulase;
Endoglucanase;
Carboxymethylcellulases.
INTRODUCTION
The development of efficient enzyme mixtures
for the treatment of lignocellulosic material is one
important goal of modern industrial biotechnology.
The screening of different enzyme activities is of
fundamental importance to pursue a rational design
for specific applications. Three major groups of en-
zyme activities have been described in Trichoderma
spp. cellulose degrading machinery: endo-â-
glucanase (EC 3.2.1.4; endoglucanase; EG), exo-â-
glucanase (EC 3.2.1.91; exoglucanases; CBH) and
â-glucosidase (EC 3.2.1.21)[1]. The filter paper assay
(FPA), generally applied for cellulose activity assess-
ment, accounts for the depolymerization of cellulose
considering the three activities as a whole[1,2]. Other
methods allow evaluating particular enzyme activities
within complex enzyme mixtures broadening the infor-
mation of the enzymatic profile within the mixture. In
particular, endoglucanase activity is often represented
by carboxymethylcellulase activity (CMCase) due to
the inability of cellobiohydrolases to attack substituted
cellulose substrates[3,4]. This activity is relevant in appli-
cations pursuing viscosity reduction of cellulosic sub-
BTAIJ, 12(2), 2016 [070-074]
BioTechnology
An Indian Journal
Volume 12 Issue 2ISSN : 0974 - 7435
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Edgardo Albertó et al. 71
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strates[5].
The screening for particular enzyme profiles in-
volves hundreds of strains and several rounds of se-
lection, and then efficient assay methods are re-
quired[1]. Dealing with large amounts of samples re-
quires methodological procedures able to produce
good quality results with minimal turnaround time,
cost and waste.
The miniaturization of already established tech-
niques is a viable approach to develop adequate as-
say procedures. The scaling down of good perfor-
mance known methods to around hundred microliter
volume and adaptation to a microplate format would
result in numerous advantages: reduced sample vol-
ume, less reagent use, reduction of reagent waste.
Furthermore, the laboratory space involved in the
assay procedure would be reduced, time for pipetting
shortened and then cost per assay[6,9,10]. In this con-
text, Xiao and co-workers[9] establish a microassay
protocol for endoglucanase activity determination
based on 96-well plate format, but this microassay
requires the use of atermocycler equipment per plate
thereby limiting the processing of multiple plates in
parallel.
The aim of this work is to find a new microsasay
protocol for endoglucanase activity determination
that allows multiple plates in parallel and studying
the correlation to the IUPAC assay widely used.
MATERIALS AND METHODS
Culture conditions and enzyme: The enzyme
preparations were obtained from culture superna-
tants from the hypercellulolyticTrichoderma
reeseimutant RUT C30. Cultures were performed in
a 5l lab scale bioreactor using modified Mandel’s
media with microcrystalline cellulose as carbon
source[2]. The reactor was inoculated with
1x106espores/mL obtained from 14 days old potato
dextrose agar (PDA) cultures. Fermentation was
performed at pH 4.8, 28 °C and 500 rpm agitation
for 7 days. The supernatant was recovered by
vacuum assisted filtration using WhatmanNº 1
filterpaper, then concentrated by tangential flow fil-
tration (10 KDa nominal cutoffpolyethersulphone
hollow fiber cartridge), and it was finally formulated in
0.1 % potassium sorbate; 10 % sodium chloride and
10% glycerol.
IUPAC CMCase assay
CMCase measurements were performed follow-
ing the IUPAC method compiled by Ghose[4]. Briefly,
0.5 mL of the enzyme dilution was mixed with 0.5
mL 2% carboxymethylcellulose in 0.05 M citrate
buffer, pH 4.8 and kept at 50°C for 30 min. Then, 1
mL of the mixture was assayed for glucose equiva-
lent of reducing sugar by DNS method[10].
Unit calculation
All determinations were carried out by tripli-
cate and all activities were calculated according to
IUPAC criteria using the formulas proposed in
Ghose[4]. Briefly, for enzyme formulations releasing
0.5 mg of glucose equivalents (concentrated enzyme
formulations), the CMC units were calculated using
the formula: CMC (units/mL) = 0.185 (units/mL)/
enzyme concentration to release 0.5 mg glucose,
where concentration = (vol. enzyme in dilution)/(to-
tal volume of dilution). For preparations displaying
glucose release lower than 0.5 mg (diluted enzyme
formulations), units were calculated as CMC (units/
mL) = mg glucose released x 0.37 (units/mL).
Miniaturized CMCase assay
CMCase measurements were performed with a
reduced volume version of the IUPAC protocol.
Briefly, 50 µL of the enzyme dilution and 50 µL 2 %
carboxymethylcellulose both prepared in 0.05 M
citrate buffer pH 4.8 was transferred to 96 wells
plated in separated wells and preheated for five min-
utes; then the well containing the enzyme dilution
was mixed with the substrate and mixed by pipetting
up and down. The enzymatic reaction was carried
out according IUPAC protocol at 50°C for 30 min.
Colorimetric assay in microplate
The colorimetric assay was performed accord-
ing to Miller´s protocol for IUPAC CMCase assay
and the reduced version of this protocol was as-
sayed in deep well plates. In the reduced protocol,
300 µL of DNS was added to each sample (mixed
by pipetting up and down) and then the plate was
held for 5 min at 100°C in water bath, finally 200 µL of

72 An economical and high throughput alternative for endoglucanase activity determination
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BTAIJ, 12(2) 2016
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each sample were diluted with 1 mL of deionized water
and absorbance measured at 540 nm.
Experimental design and statistical treatments
Twenty enzymatic dilutions were tested for glucose
equivalent release (n=3) by using the miniaturized
CMCase assay and the IUPAC assay. The results from
the two methods were compared by linear regression
and Pearson’s correlation test. The enzymatic activity
of samples was measure by using both methods in par-
allel. Four samples of concentrated and diluted enzymes
formulations were assayed. The results of all the deter-
minations were analyzed in the first place by a Fisher
test to evaluate homoscedasticity, and then the signifi-
cant difference between the two methods was analyzed
by Student’s test using the Statgraphics Software
(Statpoint Technologies, Inc.).
RESULTS
Evaluation of miniaturized CMCase assay
The assessment of the scaling down of the IUPAC
test consisted in a stepwise validation of the assay
procedures. Firstly, we studied the glucose equiva-
lents released in the enzymatic reactions performed by
using low and high enzyme concentrations in order to
obtain glucose equivalents values around the 0.5 mg
recommended by the IUPAC methodology[4] (Figure
1). Twenty enzymatic dilutions were tested for glucose
equivalent release (n=3) by using the miniaturized
CMCase assay and the IUPAC assay. The descriptive
statistical analysis accomplished to compare two sets
of data showed a normal distribution of data (results
not shown). When results from the two methods were
compared by linear regression and Pearson’s correla-
tion test, we founda R2= 99.06 %, a coefficient of cor-
relation 0.995 and a standard error of estimation of
0.027, all values were estimated with a p value < 0.05.
These results confirmed that there is a strong linear re-
lationship between the two methods along the data set
tested, with a confidence level greater than 95%.
Activity determination in both methods
To evaluate the performance of the miniaturized
CMCaseassay, the enzymatic activity of samples was
measuredby using both methods in parallel.
Samplesof concentrated and diluted enzymes formula-
tions were assayed (Figure 2). The p value obtained in
Student’s testwas higher than 0.05 (p> 0.05) confirm-
Figure 1 : Correlation model between glucose equivalents (mg) released by IUPAC assay (x-axis) and miniaturized
CMCase assay (y-axis). Each point represents the mean of three independent replicates of glucose equivalents
released in the enzymatic reactions performed using the miniaturized CMCase assay and the IUPAC assay respec-
tively. The straight line represents the linear regression showing the correlation between the two methods

Edgardo Albertó et al. 73
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ing that there were not significant differencesin the en-
zymatic determinationsbetween the methods(TABLE
1).
DISCUSSION
The IUPAC technique presented by Ghose[4] has
been used for decades in the determination of
endoglucanase activity. The precise assessment of
the CMCaseactivity within a sample by using this
method required several dilutions of the sample to
achieve a glucose equivalent release around 0.5 mg
Figure 2 : Comparison of activity determined by both methods. A) Concentrated enzyme formulation. B) Diluted
enzyme formulation
Concentrated sample

Miniaturized
CMCase assay (n=3) IUPAC Assay (n=3) Statistics
Sample Mean in CMC [units /mL]
Mean in CMC
[units /mL]
Fisher test
(p value)
Student test
(p value)
1 122 ± 17 118 ± 18 0,95 0,75
2 131 ± 9 116 ± 14 0,37 0,49
3 34 ± 3 31 ± 5 0,58 0,22
4 35 ± 2 33 ± 1 0,79 0,14
Dilutedsimple

Miniaturized
CMCaseassay (n=3) IUPAC Assay (n=3) Statistics
Sample Mean in CMC [units /mL]
Mean in CMC
[units /mL]
Fisher test
(p value)
Student test
(p value)
1 0,11 ± 0,01 0,11 ± 0,01 1,00 0,52
2 0,13 ± 0,01 0,14 ± 0,01 0,40 0,42
3 0,14 ± 0,01 0,14 ± 0,01 1,00 0,23
4 0,08 ± 0,01 0,08 ± 0,01 1,00 1,00
TABLE 1: Statistical summary of activity determinations for both methods, The p value higher than 0.05 (p> 0.05)
indicates that the homoscedasticity criteria is met (Fisher’s test) and there are not significant differences between
the methods (Student’s test)
of glucose[1,4].
The method presented here poses an order of
magnitude reduction in the reagent volume, space
usage, pipetting effort and the use of water bath
equipment allows multiples plates processing in
parallel (TABLE 2). Thus, allowing high throughput
determination of this activity more feasible and eco-
nomically. On the other hand during the method op-
timization it was observed that the geometric pa-
rameters of the reaction container greatly affected
the activity result, in particular when using PCR multiwell
plates (results not shown).

74 An economical and high throughput alternative for endoglucanase activity determination
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It is noteworthy that the correlation between the min-
iaturized method and the IUPAC method was observed
in the whole dynamic range of the standard assay;asa
consequence not only CMCunits but also international
units (IU) would be determined when the glucose re-
lease is lower than the critical value (0.5 mg of glucose
equivalents) as stated by Ghose[4].
CONCLUSION
The results obtained in this paper indicated that
a 10 fold scale down version of IUPAC CMCase
assay linearly correlateswith the classical assay
even at low concentrations of enzymes. The equip-
ment used and assay reduction in the present
work,diminishthe amount of DNS used (contaminant
reagent), increases the number of samples that can
be processed in parallel per assay in the same work
area. In conclusion, the assay reduction result in a
lower cost per test and then poses it as the preferred
option to carry out high-throughput screening.
ACKNOWLEDGEMENTS
This work was supported by a grant from the
National Agency for Science and Technology Pro-
motion from the National Ministry of Science and
Technology of the Argentina. We thankDr. Antoine
Margeot(IFP Energies nouvelles) who kindly pro-
vided theTrichoderma reesei RUT C30strain.
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TABLE 2 : Procedure comparison between standard IUPAC CMCase activity assay and miniaturized assay
Comparison of methods
Assay IUPAC assay Miniaturized CMCase assay
2% CMC in Buffer 0.5mL 50 µL
Buffer volume in blank and standard
curve
0.5 mL 50 µL
Enzyme Dilution or glucose standard
volume 0.5 mL 50 µL
Enzyme reaction at 50°C for 30 min Test tubes in water bath 96 deep-well microplate in water bath
Volume of DNS 3.0 mL 300 µL.
Color development 100°C for 5 min 100°C for 5 min
Amount of H2O added prior to
measurement 20 mL
1 mL of water to 200 µL of the reaction/DNS
mixture
Read at 454 nm 1 mL in spectrophotometer
cuvette 200 µL in standard optical 96 well plate