Q 2011 by The International Union of Biochemistry and Molecular Biology BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION 281

Vol. 39, No. 4, pp. 280–290, 2011

detergents. Thiol proteases (e.g. papain) would be oxidized albumin degradation capacity (BDC)1 revealed by sodium
by the bleaching agents, and metalloproteases (e.g. ther- dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
Laboratory Exercise molysin) would lose their metal cofactors due to its binding PAGE) and Coomassie Blue protein staining.
with the water softening agents or hydroxyl ions [6]. We aim for students to become aware, both theoretically
The important role of enzymes in several common prod- and practically, of a major application of enzymes in indus-
Characterization of the Protease Activity of Detergents ucts has usually only been described to students from a the- try and how various parameters affect the action of the
oretical point of view. To provide a practical approach to this final industrial product. Therefore, students learn that in
LABORATORY PRACTICALS FOR STUDYING THE PROTEASE PROFILE AND ACTIVITY OF VARIOUS
issue, we have introduced a series of easy laboratory exer- addition to temperature and pH there are other factors,
COMMERCIAL DETERGENTS cises that reinforce knowledge of the utility and the impact of such as ionic strength, surfactants, and oxidizing agents,
the biotechnological application of enzymes in our normal that can affect the enzymatic reaction, and how each of
Received for publication, June 29, 2010, and in revised form, October 21, 2010
life. Additionally, these exercises could also be used to revise these physical and chemical parameters must be consid-
several elementary enzymology concepts such as, for exam- ered and optimized in order for an enzymatic reaction to
Cristina Valls, Gerard Pujadas, Santi Garcia-Vallve, and Miquel Mulero* ple, the influence of temperature on protease activity (in this be accurate and reproducible. Furthermore, students learn
From the Nutrigenomics Group, Department of Biochemistry and Biotechnology, article) and the influence of pH and effect of surfactants and how to apply and synthesize biotechnological concepts
Rovira i Virgili University, Tarragona 43007, Spain oxidizers (proposed). The laboratory exercises presented and biochemical principles and to communicate ideas and
here are designed to get students involved in nearly every information about what they have learned in the course.
Detergent enzymes account for about 30% of the total worldwide production of enzymes and are one of aspect of the teaching process. Students, then, are asked to Discussions led by the course teacher and instructors,
the largest and most successful applications of modern industrial biotechnology. Proteases can improve prepare the project by reading the primary literature on plus supplementary lectures and bench experiments, help
the wash performance of household, industrial, and institutional laundry detergents used to remove enzyme detergents and their industrial applications [7–9]. to give the problem a context and conceptual framework.
protein-based stains such as blood, grass, body fluids, and food soils. This article describes two easy The first lab period is also used to discuss detergent-specific
EXPERIMENTAL PROCEDURES
and cheap laboratory exercises to study the presence, profile, and basic enzymology of detergent formulations, the influence of several parameters (substrate,
pH, and temperature) on detergent enzymatic activities, and Equipment
proteases. These laboratory practicals are based on the determination of the detergent protease
activity of various commercial detergents using the N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine quantitative and qualitative methods for evaluating enzy- The following equipment is necessary to carry out the
p-nitroanilide method and the bovine serum albumin degradation capacity. Students are also required to matic activity [10]. The next four lab periods are used to eval- practical exercises: table spectrophotometer, vertical mini-
elucidate the enzymatic subtype of detergent proteases by studying the inhibitory potential of several uate two commercial household laundry detergents [a stand- gel electrophoresis unit, electrophoresis power supply, ther-
types of protease inhibitors revealed by the same experimental methodology. Additionally, the results of ard detergent that contains proteases (S) and a detergent for mostatic water bath, light transilluminator, image acquisition
the exercises can be used to provide additional insights on elementary enzymology by studying the influ- delicate clothes that contains no enzymes (D)] by measuring, system or digital camera, micropipettes, and consumables,
ence of several important parameters on protease activity such as temperature (in this article) and the on the one hand, the influence of temperature on detergent such as cuvettes, tips, and tubes.
influence of pH and effects of surfactants and oxidizers (proposed). Students also develop laboratory protease activity and, on the other hand, by developing an
skills, problem-solving capacities, and the ability to write a laboratory report. The exercises are mainly approximate determination of the protease detergent profile
designed for an advanced undergraduate project in the biochemistry and biotechnology sciences. using several protease inhibitors. These evaluations are Materials and Solutions
Globally, these laboratory practicals show students the biotechnological applications of proteases in the done by quantitative and qualitative methods that are Reagents were purchased from Sigma-Aldrich (St. Louis,
detergent industry and also reinforce important enzymology concepts. performed quickly and easily. To this end, we quantitatively MO), if not otherwise specified, and were of analytical
Keywords: Detergents, proteases, bovine serum albumin degradation capacity, teaching protein chemistry and determined enzymatic activity by using standard measure- grade. The SDS-PAGE molecular weight standard used
industrial biochemistry, industrial enzymology. ments for the detergent protease enzyme [N-succinyl-L-ala- was Prestained SDS-PAGE Standard Low Range, catalog
nyl-L-alanyl-L-prolyl-L-phenylalanine p-nitroanilide (Suc-AAPF- 161-0305 from Biorad. In the one-dimensional SDS-PAGE,
pNA) method] [11] that overcome possible interferences in the stacking gel contained 4% acrylamide–bisacrylamide in
Enzymes have been used to improve the cleaning effi- depends on the application for which they are designed so enzymatic activity and stability by the complex detergent 0.5 M Tris hydrochloride buffer (pH 6.8) and 0.4% SDS. The
ciency of detergents for more than 50 years and are now their enzymatic content may differ. Nowadays, the most matrix. Therefore, by using chemically well-defined sub- resolving gel contained 12% of acrylamide–bisacrylamide
well-accepted ingredients in powder and liquid household common enzymes found in detergents are proteases that strates and an objective and reproducible measuring tech- in 0.5 M Tris hydrochloride buffer (pH 8.8) and 0.4% SDS.
detergents, stain removers and laundry prespotters, auto- assist in the removal of protein-based stains, amylases that nique, it is possible to measure the functional activity of The gels were stained in solution containing 0.15% Coo-
matic dishwashing detergents, and industrial and institu- decompose starch to small sugars, lipases that facilitate proteolytic enzymes using simple determinations. Chro- massie Brilliant Blue R-250, 9% acetic acid, and 45% meth-
tional cleaning products. The first soap that included the removal of fat and oil-based stains, and cellulases that mogenic peptide substrates have been designed to mimic anol solution. The nonstaining solution contained 7.5% ace-
enzymes was made in 1913 by Otto Röhm [1], who remove microfibrils formed during the washing and wearing the natural protein substrate. The choice of amino acids tic acid and 25% methanol. The running buffer was pre-
observed that a pancreas extract was able to degrade pro- of cotton and cotton blends and brighten and soften colors determines the affinity (Km) to the active-site region of the pared with 0.5 M Tris hydrochloride buffer (pH 8.8), 1.92 M
teins and could be used to optimize the process of washing [2]. However, there is a continuous need to formulate deter- enzyme as well as the reactivity (kcat). To make the assay glycine, and 1% SDS. The sample buffer contained 0.188
clothes. However, it was not until 1960 that enzymes effec- gent compositions, which provide a higher cleaning per- simple and practical, a chromogenic group is attached to M Tris hydrochloride, pH 6.8, 30% glycerol, 6.9% SDS, 2%
tively became available and could be routinely incorporated formance, and several other enzymes, such as peroxidases the peptide via an amide bond. When para-nitroaniline of mercaptoethanol, and bromophenol blue. A solution of
into detergents. Enzymes are now an essential component hemicellulases or mannanases, can be found in new deter- (pNA) is released from the peptide catalyzed by the Suc-AAPF-pNA (Sigma, S7388) was prepared at a concen-
of detergents, and their use provides consumers with well- gent compositions [3]. Proteases are the most common de- proteolytic enzyme, the wavelength is shifted and it turns tration of 1.25% w/v in DMSO and stored at 220 8C until
proven benefits, both in the washing process itself and in tergent enzymes and are found in all major brands of Euro- yellow. At 405 nm, which is the wavelength most used as substrate for protease activity assay.
terms of the wider environment. These benefits include the pean, American, and Japanese detergents, despite the frequently used with pNA substrates, the absorbance of
possibility of working at low temperatures, saving water considerable differences in cleaning regimens in these Hazards
the substrate is less than 1% of that of an equimolar
and energy, and removing the need for harsher chemicals countries [4]. Proteases convert their substrates into small, Acrylamide and bisacrylamide are potent neurotoxins
amount of pNA. The hydrolysis of the chromogenic
that have been proven to have a negative impact on the readily soluble fragments, which can be easily removed and are absorbed through the skin. Gloves and a lab coat
peptide substrate by the proteolytic enzyme follows a Line-
environment. In addition, enzymes are biodegradable and from fabrics. All the proteolytic enzymes found in deter- should be worn when handling the solution, and it must not
weaver–Burk relationship. This means that, if the substrate
leave no harmful residues. The composition of detergents gents are nonspecific serine endoproteases (e.g. subtilisin) be pipetted by mouth.
is present at a sufficiently high concentration or if a compa-
[5] with a preferred cleavage on the carboxyl side of hydro-
ratively small fraction of the substrate is hydrolyzed, the rate 1
* To whom correspondence should be addressed. E-mail: phobic amino acid residues, but capable of hydrolyzing The abbreviations used are: BDC, bovine serum albumin
of product (color) formation is practically constant and line- degradation capacity; S, standard detergent that contains pro-
miquel.mulero@urv.cat. most peptide links. Other proteases cannot be used in
arly proportional to the amount of enzyme [12]. We also teases; D, detergent for delicate clothes that does not contain
DOI 10.1002/bmb.20488 280 This paper is available on line at http://www.bambed.org developed a qualitative assay based on the bovine serum enzymes.
282
FIG. 1. Work distribution during the 5-day laboratory practicals.
Samples
Samples consist of two tubes with 10 mL of two kinds
of household liquid detergents: S and D. These deter-
gents can be brought by the students if desired. It must
be noted that specific protease activity can vary between
different detergent brands or uses. This means that
appropriate dilution factors for each detergent must be
assayed before the enzymatic activity assays. Different
detergents can also show a different optimal temperature
for the enzyme activity; therefore, a preliminary tempera-
ture test must be developed to determine the tempe-
rature range that should be tested in further analyses.
LABORATORY SESSIONS
As depicted in Fig. 1, the laboratory practicals took
place in five sessions of
3 hours each. The first was a
theoretical and experimental session. The four additional
sessions were used to carry out the laboratory practicals.
Laboratory Session 1
We used the first half of the first session in the labora-
tory (1.5 hours) for the theoretical discussion of several
aspects related to the use of enzymes in industry and
more specifically of the role of proteases. These aspects
include, for example, the following topics: detergent-spe-
cific formulations, influence of various parameters (sub-
strate, pH, and temperature) on detergent enzymatic
activities, and technical methods for evaluating enzy-
matic activity (quantitative and qualitative). After this dis-
cussion, the students were asked to do the following:
1. Calculate the protease activity of the two deter-
gents in the temperature test exercises. Make a
graphic representation.
2. Describe the results obtained in the BDC assay for
the two types of detergents in the temperature test
exercises.
BAMBED, Vol. 39, No. 4, pp. 280–290, 2011
3. Explain why only one detergent has protease in its
composition.
4. Calculate the protease activity of each of the prote-
ase inhibitor(s) and concentration(s) at times 0 and
15 minutes. Make a graphic representation of the
results of these protease inhibitor test exercises.
5. Determine whether all the protease inhibitors have
the same efficiency.
6. Determine the best temperature for the proteases
among the tested conditions (4, 40, and 60 8C). Is
it the same as the optimal washing temperature
advertised for the detergent?
7. Decide what kind of proteases the S detergent
contains.
8. Describe the results obtained in the BDC assay
concerning the protease inhibitors test exercises.
9. Determine whether the quantitative and qualitative
evaluations of the enzymatic activity correlate with
each other and justify the answer.
Students have to do these exercises after the end of
the fifth laboratory session and hand in their answers to
teachers 1 week later as final laboratory reports.
The second half of the session was used to prepare the
Coomassie Blue Solution and the polyacrylamide gels.
Once prepared, the gels were plastic sealed in a
humidity-rich environment (wet Whatman paper was
placed in the plastic bags to prevent the gel from drying
out) and stored at 4 8C until needed in laboratory
session 3.
Laboratory Session 2. Quantitative Measure of the
Effect of Temperature on the Protease Activity
(Temperature–pNA Assay)
The second laboratory session was used to carry out
the measurement of the protease activity concerning the
temperature test exercises. For this purpose, we specifi-
cally used the Suc-AAPF-pNA. This chromogenic peptide
substrate has been designed to mimic the natural protein
FIG. 2. Suc-AAPF-pNA method used for the measurement of protease activity. (a) pNA synthetic substrate contains specific
protease cleavage site. (b) Generation of para-nitroaniline (pNA) by proteolytic hydrolysis.
substrate for detergent proteases (Fig. 2a) and allows a
precise measurement because of its high sensitivity that it
is enough to ‘‘dilute’’ out virtually all product matrix effects.
The method (see Fig. 2b) is based on the specific generation
of a yellow chromophore, the pNA, when proteases cleave
the chromophore precursor Suc-AAPF-pNA.
Tubes containing 500 lL of a standard (S) and delicate
(D) detergent, properly diluted with distilled water, plus 50
lL of 1/10 diluted protease substrate solution (Suc-AAPF-
pNA, 0.02 M final concentration), were used as samples (5
mL, final volume). A blank reference was also used (500 lL
of distilled water). Triplicates were done for each condition.
Samples and blanks were incubated at several tempera-
tures (4, 40, and 60 8C) for 6 minutes. After rapid ice-cold
incubation (1 minute), 50 lL of 0.1 M phenylmethylsulfonyl
fluoride (PMSF) was added to each tube to stop the reac-
tion. The absorbance was recorded at 405 nm at room tem-
perature. The pNA concentration was calculated using the
Lambert–Beer law (e ¼ 8480 L mol21 cm21). For each tem-
perature and detergent, the protease activity was calcu-
lated. A unit of enzyme activity (U) was defined as the
amount of enzyme that catalyzes the transformation of 1
lmol of substrate per minute.
Laboratory Session 3. Qualitative Measure of the
Effect of Temperature on Protease Activity
(Temperature–BDC Assay)
The BDC assay concerning the temperature test exer-
cises was carried out in the third laboratory session. Using
this method, we studied the BSA degradation capacity for
each temperature and detergent from a qualitative point
of view. The proteases were shown to cleave the BSA
protein and lower 66-kDa molecular weight degradation
bands subsequently appeared in a SDS-PAGE electro-
phoresis after Coomassie Blue staining. For this test, we
prepared three different solutions in Eppendorf tubes:
• A no-detergent condition consisting of 40 lL BSA (2
mg/mL) and 10 lL distilled water. This no-detergent
control was used to show that in the absence of
detergent, the BSA was not degraded because of
the hypothetical presence of other proteases in the
solvent and that a single band of 66 kDa appeared.
• A detergent condition containing 40 lL BSA (2 mg/mL),
5 lL ethanol, and 5 lL detergent (S or D). This condi-
tion was the assay of the protease activity of the deter-
gent. The ethanol was added to demonstrate that the
283
inhibitory effect in the PMSF condition was not due to
the presence of alcohol.
• A PMSF condition containing 40 lL BSA (2 mg/mL), 5
lL PMSF (0.1 M), and 5 lL detergent (S or D). PMSF is
an inhibitor of the protease activity. With this assay,
the conclusion that proteases from detergents were
responsible for the BSA degradation was reinforced.
The tubes were incubated for 30 minutes in a water
bath at the corresponding temperature (4, 40, or 60 8C).
After incubation, the sample buffer was added, and the
tubes were placed in a boiling water bath for 5 minutes.
The Eppendorfs were ice cooled and a centrifuge spin
was carried out. The molecular marker was loaded into
the first well. The following wells were loaded with each
solution of 4, 40, and 60 8C treatments, respectively.
Each student group carried out the corresponding
SDS-PAGE for each detergent (S or D). Proteins were
electrophoresed through the stacking gel at 70 V; once
samples reached the resolving gel the voltage was
increased until 120 V.
The gels were stained in Coomassie brilliant blue solu-
tion for 30 minutes and initially destained with destaining
solution for 10 minutes followed by final destaining by an
overnight incubation in destaining solution with agitation
at room temperature.
The image was captured the following day using an
Alpha Innotech Fluochem FC2 image acquisition system.
Alternatively, a simple digital camera could be used.
During this lab session, students also prepared the poly-
acrylamide gels needed for the following laboratory ses-
sion.
Laboratory Session 4. Qualitative Inhibition Profile Test
(Inhibitors–BDC Assay)
During the fourth laboratory session, we carried out
the qualitative inhibition profile test using the BDC assay
described above. By this method, we studied the BSA
degradation capacity for each inhibitor (PMSF, aprotinin,
pepstatin, leupeptin, EDTA, and EGTA) using the S deter-
gent at 40 8C (the best tested temperature for the
enzymatic activity as determined in the two previous
experiments) and analyzed the specific protease
profile from a qualitative point of view. For this test,
we set 12 conditions: six protease inhibitors and two
concentrations, low (L) or high (H), respectively. We also
carried out a positive control without an inhibitor. A total
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FIG. 3. Temperature–BDC assay reveals the best tested temperature and the protease presence in S detergent. (a) S and
D detergent BSA degradation capacity under several temperatures (4, 40, and 60 8C) obtained by teachers during protocol setup.
(b) One representative result obtained by students.
of 1.6 lg/lL of BSA and 5 lL of detergent S was added
to the Eppendorf tubes for a 50-lL final volume; the
following concentrations of inhibitors were also added to
the corresponding Eppendorf tube: 2 lM (L) and 10 lM
(H) for PMSF; 0.05 g/mL (L) and 0.25 mg/mL (H) for apro-
tinin; 2 lM (L) and 10 lM (H) for pepstatin; 25 lM (L) and
100 lM (H) for leupeptin; 2.5lM (L) and 5 lM (H) for
EDTA; and 2.5 lM (L) and 5 lM (H) for EGTA. The inhibi-
tor concentrations used were the lower and upper ones
and were chosen using the recommended range of
useful concentrations provided by the manufacturer.
The tubes were incubated for 30 minutes in a water bath
at the corresponding temperature. After incubation, the
sample buffer was added and the tubes were placed in a
boiling water bath for 5 minutes. The Eppendorfs were ice
cooled and a centrifuge spin was carried out. The molecu-
lar marker was loaded into the first well. The following
wells were loaded with the corresponding type(s)/concen-
tration(s) of inhibitor(s), and two SDS-PAGEs were carried
out for detergent S. Proteins were electrophoresed
through the stacking gel at 70 V; once samples reached
the resolving gel the voltage was increased until 120 V.
The gels were stained in Coomassie brilliant blue solu-
tion, and the image was captured the following day.
Laboratory Session 5. Quantitative Inhibition Profile
Test (Inhibitors–pNA Assay)
The protease inhibitor test exercises for measuring the
protease activity were done during the fifth laboratory ses-
sion. For this purpose, we used the Suc-AAPF-pNA method
BAMBED, Vol. 39, No. 4, pp. 280–290, 2011
as previously described but in the following experimental
conditions: incubation at 40 8C for detergent S and addition
of the corresponding type(s)/concentration(s) of inhibitor(s).
We tested the aforementioned with six different kinds of
inhibitor proteases and studied the protease inhibition
capacity of these compounds to assess the protease pro-
file of the detergent tested. Samples and blanks were incu-
bated at 40 8C for 6 minutes. After rapid ice-cold incuba-
tion (1 minute), two concentrations of inhibitors [high (H) or
low (L)] were added to each tube to stop the reaction. The
absorbance was recorded at 405 nm at room temperature
at 0 and 15 minutes. The pNA concentration was calcu-
lated using the Lambert–Beer law. For each inhibitor and
concentration, the enzymatic activity was calculated. The
results were expressed as lmol/min. To do so, the follow-
ing inhibitors and concentrations were prepared in the cor-
responding assay tubes (5 mL, final volume): 2 lM (L) and
10 lM (H) for PMSF; 0.05 g/mL (L) and 0.25 mg/mL (H) for
aprotinin; 2 lM (L) and 10 lM (H) for pepstatin; 25 lM (L)
and 100 lM (H) for leupeptin; 2.5 lM (L) and 5 lM (H) for
EDTA; and 2.5 lM (L) and 5 lM (H) for EGTA. A positive
control protease tube without an inhibitor was also added.
Finally, the inhibition capacity for each inhibitor
and concentration was calculated, assuming that no
difference in the absorbance between 0 and 15 minutes
indicated 100% inhibition capacity.
RESULTS AND LABORATORY REPORTS
The results of the temperature–BDC assay obtained
by the teachers are shown in Fig. 3a. As can be seen,
FIG. 4. Temperature–pNA assay quantitatively confirmed 40 8C as the best tested temperature for S detergent and the
absence of protease activity in D detergent. (a) Units of enzyme activity (lmol/min) of S and D detergent under several tempera-
tures (4, 40, and 60 8C) obtained by the teachers during protocol setup. (b) One representative result obtained by students.
we found clear protease activity in S detergent but not
in D detergent. This evidence was found by using a
qualitative methodology, where the protease effect was
revealed as a cleavage of the BSA protein and the
subsequent appearance of lower 66-kDa molecular
weight degradation bands. This method also showed
that temperature had an important effect on the washing
efficiency concerning protease action: for detergents
containing these enzymes the highest protease activity
was at 40 8C.
As can be observed in Fig. 3b, the students also
carried out the same experimental protocol, and their
results were comparable to those obtained by teachers.
In the students’ image, we observed an experimental
mistake in the lane corresponding to detergent S,
temperature 40 8C, PMSF condition. This error is related
to one detected pitfall namely the task distribution, which
requires that a large number of different conditions be
tested by each student group. In the future, then, the
methodology could be improved by reducing the number
of experimental conditions to be tested by each group or
by distributing the work better by splitting the whole
experimental protocol into several experimental exercises
among several student groups. In this way, students
would be able to focus on only a few experimental
parameters and carry out the lab routine more efficiently
and in less time. Once they have finished, students
would be able to share their experimental results and
discuss them as a whole.
Figure 4 shows the results of the temperature–pNA
assay obtained by teachers (a) and students (b). This
confirms the previous results of the BDC assay, that is,
protease enzymes are absent in D detergents,
and the best tested condition for protease activity for
285
detergent S is 40 8C. When we compared the overall level
of enzymatic activity found by teachers and students, we
realized that, in general, it was lower for the students.
Again, this may be due to less effective experimental
work and a heavy work schedule, which could be
improved in the future by relaxing the experimental work-
ing assignment(s). Nevertheless, it could be seen that the
assay is robust and tolerates this obvious limitation in
student groups. Although the level of students’ total
enzymatic activity is significantly lower, the methodology
allows the observation of important differences. This
confirms that the protocol is suitable for studying this
industrial biotechnological enzyme application.
Overall, the temperature test exercises are a practical
way of teaching the different enzyme composition of two
detergents, and they make students aware that protease
activity is not always desirable: for example, in deter-
gents designed to wash delicate clothes the presence of
this enzyme means that proteases may not act only on
stains and that they may degrade textile fibers, thus
weakening or damaging the clothes.
Another important conclusion reached by students is
that some biotechnological industrial applications may
influence such routine aspects of our normal life as effi-
cient washing or the life span of clothes and that these
influences are sometimes related to classical enzymatic
parameters (e.g. temperature), which can affect catalytic
activity. Students, then, became aware that these para-
meters do not only affect biological concerns such
as metabolism and cell signaling; they may also be
important from an industrial point of view because
they affect both trivial and nontrivial aspects like output
efficiency, process performance, scale production, and
cost effectiveness.
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FIG. 5. Inhibitors–BDC assay results suggest the presence of subtilisin protease in S detergent. (a) S detergent BSA degra-
dation capacity at 40 8C in the presence of two concentrations (L and H) of PMSF, aprotinin, pepstatin, leupeptin, EDTA, and EGTA
obtained by teachers during protocol setup. (b) One representative result obtained by students.
Figure 5a shows the results of the inhibitors–BDC
assay obtained by teachers. These results enable the
protease profile of a standard detergent to be eluci-
dated. From a qualitative point of view, the BDC assay
shows that the protease inhibitors that had the maxi-
mal inhibition capacity were PMSF and aprotinin (two
serine protease inhibitors), although the lower concen-
tration of aprotinin was less efficient than the lower
concentration of PMSF. Additionally, both concentra-
tions of the metal chelating agents, EDTA and EGTA,
were less effective at abrogating the proteolytic degra-
dation of high concentrations of BSA, which suggests
that proteases from detergents are not metallopro-
teases. Neither pepstatin (an aspartic protease inhibitor)
nor leupeptin (which inhibits serine and cysteine pro-
teases with trypsin-like specificity) had an inhibitory
effect on protease action. This inhibition profile sug-
gests that the proteases in the detergent were not as-
partic proteases, metalloproteases, or cysteine pro-
teases, and that the proteases were closely related to
serine proteases, but not related to trypsin. These
results are compatible with subtilisin proteases, the
common detergent proteases used for the last 20
years. Subtilisins are serine proteases but are structur-
ally unrelated to the chymotrypsin type of serine
BAMBED, Vol. 39, No. 4, pp. 280–290, 2011
proteases, although both groups have the same cata-
lytic mechanisms involving a catalytic triad from a ser-
ine, a histidine, and an aspartic acid. Chymotrysin-like
proteases and subtilisins are, therefore, a classic exam-
ple of convergent evolution.
As can be observed in Fig. 5b, the students obtained
comparable results to the ones obtained by teachers.
We expressly show one students’ result image were we
can observe a representative mistake concerning the
aforementioned limitation. In this case, the mistake is
trivial and related to the alteration of the protocol gel
loading order.
In all cases, the mistakes detected were minor and not
‘‘result compromising’’ but they can suggest possible
improvements in the experimental protocol. This reinfor-
ces the suggestion that the experimental condition(s)
could be distributed differently among the groups. In this
case, for example, it could be possible to distribute the
different inhibitors or concentrations to be tested
between two groups; in this way, each student group
would only be responsible for one gel loading/staining.
Again, once they have finished, students would share
their experimental results.
The inhibitors–pNA assay also quantitatively confirmed
the protease profile obtained by the BDC assay.
287
FIG. 6. The protease activity inhibition percentages of several antiproteases on S detergent (inhibitors–pNA assay) rein-
force the possible subtilisin protease profile. (a) Units of enzyme activity (lmol/min) obtained by teachers during protocol setup
of S detergent at 40 8C in the presence of two concentrations (L and H) of PMSF, aprotinin, pepstatin, leupeptin, EDTA, and EGTA.
(b) One representative result obtained by students.
As shown in Fig. 6, the inhibition capacity was calcu- Finally, each group of students has to produce a labora-
lated using the protease activity data for each inhibitor/ tory report of at least 10 pages, which must include a brief
concentration. As expected, the inhibition capacity was introduction to the industrial application of detergents, all
strongest for PMSF and aprotinin (nearly 100%), and it the experimental results obtained, and a critical discussion
was also evident that the low concentration of aprotinin of the results. In these laboratory reports, they also had to
was less effective than the low concentration of PMSF. answer the questions presented in laboratory session 1.
The other inhibitors had an inhibition capacity between Students were also asked not to restrict the discussion
75 and 50%. The remaining protease activity was to their own results but include the results obtained by
enough to produce a significant decrease in the intensity the other groups, discuss all the results as a whole, and,
of the 66-kDa BSA band observed in BDC assay. It if possible, provide a descriptive statistic. This would
should be pointed out that in the case of pepstatin the improve their final laboratory report.
difference between the effect of high and low concentra- The students were also encouraged to discuss the role
that genetic engineering could play in the development
tions on the inhibition capacity was also evident in BDC
assay; on the other hand, the slight difference in band of new detergent proteases with interesting properties for
intensity between leupeptin, metalloprotease inhibitors final commercial application.
(EGTA and EDTA), and leupeptin was also reflected in
the pNA assay. DISCUSSION AND CONCLUSIONS
In this regard, another minor pitfall detected in the pro- This article is our attempt to develop a reliable, eco-
tocol is that the comparison between the BDC assay nomic, and easy method to train students in industrial
(qualitative) and the pNA assay (quantitative) was some- biochemistry/biotechnology applications. As can be seen
times not sufficiently clear to students. Therefore, we in Table I, proteases have several important applications
propose using the free software image J to perform band in industry. The detergents we have selected are a repre-
densitometry of the BSA band of the BDC assay. This sentative sample of, sometimes unknown, protease
will transform the data from qualitative to quantitative, industrial biotechnology applications. Additionally, the
and it will also give us the opportunity to train students fact that the product is in powder or liquid form makes it
in image processing and analysis. an ideal and relatively economic source of enzymes for
TABLE I
Application of proteases in industry
Industry Protease Application
Baking Neutral protease Dough conditioner
Beverage Papain Chill proofing, removal of haze in beverages
Dairy Fungal proteases, chymosin, Replacement of calf rennet, whey protein processing, production
other proteases of enzyme-modified cheese (EMC)
Detergent Alkaline proteases, subtilisin Laundry detergents for protein stain removal
Food processing Several proteases Modification of protein-rich material, that is, soy protein
or wheat gluten
Leather Trypsin, other proteases Bating of leather, dehairing of skins
Meat and fish Papain, other proteases Meat tenderization, recovery of protein from bones and fish waste
Medicine Trypsin Dead tissue removal, blood clot dissolution
Photography Several proteases Recovery of silver from used X-ray and photographic films
Sweetener Thermolysin Reverse hydrolysis in aspartame synthesis
Adapted from ref. 8.
288
the teaching process in comparison with other appli-
cations in which the protease is involved exclusively in
the industrial process (leather, meat, and fish) or in which
the recovery of the final industrial product for teaching
purposes is more complex.
As mentioned previously, proteases are one of the
standard ingredients of all kinds of detergents ranging
from those used for household laundering to reagents
used for cleaning contact lenses or dentures. The use of
proteases in laundry detergents accounts for
30% of the
total worldwide sales of enzymes. These enzymes, also
known as proteinases, proteolytic enzymes, or pepti-
dases, are enzymes that hydrolyze the peptide bond of
proteins; hence, they are all hydrolases. In general,
proteases accomplish two major functions: (i) a regulatory
function, which involves activation or inactivation of
specific proteins by selective proteolysis, and (ii) a general
proteolytic function, which is a less specific process,
resulting in the bulk breakdown of cellular proteins. These
degradative mechanisms remove denatured proteins as
well as facilitate adaptive responses by destroying native
proteins no longer needed by the cell. Both types of
proteolysis are highly regulated and usually occur in
response to specific extracellular signals, such as game-
togenesis, differentiation, and tissue development [13].
Proteases can be divided depending on the type of reac-
tion catalyzed into: (a) exopeptidases or peptidases, which
are proteases catalyzing the splitting of peptide bonds at ei-
ther the N- or C-terminus of the substrate and (b) endopep-
tidases or proteinases, which are proteases splitting the
peptide bonds within the protein substrate. Endopeptidases
act preferentially in the inner regions of the peptide chain
and are further classified according to their active site into:
serine, cysteine, aspartic, and metalloproteases. Serine
proteases are the most studied class of enzymes, which is
characterized by a serine residue at its active site. There are
two different families: the mammalian serine proteases and
the bacterial serine proteases. Subtilisin is the most repre-
sentative example from the latter family. On the other hand,
representative enzymes belonging to the mammalian family
are trypsin, chymotrypsin, elastase, and kallikrein. It is
believed that these enzymes evolved from a common
ancestor. They are assumed to have acquired different spe-
cificities by mutation of the genes descended from an evo-
lutionary precursor.
It is interesting to comment that the chymotrypsin family is
among the most extensively investigated and thoroughly
characterized model systems for the detailed study of
enzyme mechanism and structure/function relationships,
and the ‘‘artificial" substrate p-nitrophenyl acetate was used
extensively in elucidating the mechanism of catalysis of chy-
motrypsin [14]. p-Nitrophenol, the product of the proteolytic
cleavage of p-nitrophenyl acetate, is colorless. However, at
alkaline pH, it exists as an anion, p-nitrophenate, which is
yellow. Hence, the progress of hydrolysis is easy to follow by
determining the absorbance at 405 nm [15].
All detergent proteases currently used in the market
are serine proteases produced by Bacillus strains. The
ideal detergent protease should possess broad
substrate specificity to facilitate the removal of a large
BAMBED, Vol. 39, No. 4, pp. 280–290, 2011
variety of stains due to food, blood, and other body
secretions. Activity and stability at high pH and tempera-
ture and compatibility with other chelating and oxidizing
agents added to the detergents are among the major
prerequisites for the use of proteases in detergents [16].
Teaching students how biotechnological products such
as purified enzymes are applied in industry can sometimes
be difficult from a practical point of view because of the
lack of bench standard protocols and the fact that com-
mercial products suitable for practical teaching are difficult
to find. Although a vast array of methods has been
described that can be used as general detection of prote-
ase activity with industrial purposes [17], there are no clear
teaching proposals to study the enzymology of industrial
enzymes. Related to this, the proteolytic degradation of
proteins like hemoglobin and casein is the traditional way
of measuring proteolytic activity. The activity in this case is
determined by the amount of peptides that become solu-
ble in trichloroacetic acid, either by absorption at 280 nm
in alkaline solution or by a protein determination method.
All these methods are of medium sensitivity. Other meth-
ods with higher sensitivity are as follows: (i) dimethyl
casein: the activity in detergents is classically determined
by using derivatized proteins such as dimethylated casein
with subsequent determination of free amino groups gen-
erated by protease action. These methods became quite
popular within the detergent industry because of their
increased sensitivity and their potential for automatization.
(ii) Chromogenic substrates: versatile substrates that can
be used in single determinations for protease determina-
tions as well in high-throughput approaches in microliter
plates or flow injector analysis (i.e. Suc-AAPF-pNA). The
method can also be used for quantitative determination of
proteases, when the type of protease used is known and
has become a standard because of its versatility and
flexibility. (iii) Zymography is known as an electrophoretic
technique, commonly based on SDS-PAGE, which con-
tains a substrate copolymerized within the polyacrylamide
gel matrix, for the detection of an enzymatic activity.
Samples are normally prepared by the standard SDS-
PAGE treatment buffer under nonreducing conditions.
After the electrophoretic run, the SDS is soaked out from
the gel (zymogram) by incubation in a nonbuffered Triton
X-100 (or similar detergent), followed by incubation in an
appropriate activation buffer, for an optimized length of
time and temperature, depending on the type of enzyme
being assayed and the type of substrate being degraded.
The zymogram is subsequently stained, and areas of
digestion are distinguished.
Typical substrates for zymography are gelatin, casein,
and fibrin. The MW of the proteolytic bands can be deter-
mined by using appropriate MW standards. The type of
protease can be established by comparison with recombi-
nant proteins and the use of specific protease inhibitors
[18]. For our teaching purposes, the BDC assay presented
in this article is a methodology that could be considered
similar to zymography but presents several advantages.
For example, in the BDC assay, we do not need to treat
the gel with activation buffer, and we obtain specific band
disappearance instead of areas of digestion.
The major problem for detergent proteases is the fact
that detergent ingredients can disturb the assay. This is
the reason why fluorogenic substrates such as BODIPY-
FL-Casein substrates, although seen as an increasing
valuable tool of enzyme preparation, are not recom-
mended for the analysis of detergents because of its
negative interaction with a high number of potential
detergent ingredients [19]. Furthermore, incorporating
enzymes into detergent formulations poses numerous
practical problems. Proteases are susceptible to auto-
lytic degradation, oxidation, and denaturation processes
that are often enhanced by the surfactants, bleaches,
and water-softening builders that are included in any
laundry detergent product. Additionally, proteases
catalyze the lytic degradation of other detergent
enzymes that may also be present in the formulation. In
powdered formulations, many of these issues can
be overcome by physically isolating the enzymes in
separate particles.
In liquid formulations, however, physical isolation of
enzymes is more difficult, and the presence of solvent (i.e.
water) amplifies the detrimental effect of surfactants and
enhances the rate of undesirable reactions [20]. Neverthe-
less, considerable effort has been devoted to overcome
the challenge of formulating enzymes into liquid laundry
detergent products. One avenue of research has pursued
the development of various enzyme stabilization strat-
egies based on chemical additives. For example, a com-
bination of carboxylic acid salts and calcium chloride can
protect against protease degradation [21]. Boron com-
pounds (boric acid and borate salts), especially in con-
junction with polyols (propylene glycol and glycerol), also
have been shown to stabilize enzymes in liquid detergent
formulations. In this regard, it is also possible to adapt
these laboratory practicals for the study of the effect of
surfactants and oxidizing agents to test, for example, the
effect of 1.0% final concentration of different surfactants
and oxidizers (SDS in w/v, Tween-80, Triton-X-100 in v/v,
and H2O2 in v/v) on the proteolytic activity of the standard
detergent protease by preincubating it for 1 hour in the
above surfactant solutions at 40 8C before testing for pro-
tease activity under standard pNA assay conditions.
Interestingly, although because of time limitations we
were only able to test the effect of temperature on enzy-
matic activity, this protocol is also suitable for studying
the influence of other important enzymatic conditions/pa-
rameters. For example, it allows the study of the effect of
pH on the protease activity and stability. Therefore, the
optimum pH of the proteolytic activity could be studied
over a pH range of 7.0–13.0. For the measurement of pH
stability, the following buffer systems could be used as
detergent solvents: 100 mM acetate buffer, pH 4.0–6.0;
100 mM Tris-HCl buffer, pH 7.0–8.0; 100 mM glycine-
NaOH buffer, pH 9.0–10.0; 100 mM Na2HPO4-NaOH
buffer, pH 11.0; and 100 mM KCl-NaOH, pH 12–13. The
residual proteolytic activity could be determined under
standard pNA assay conditions.
It should also be pointed out that the same conceptual
framework and initial substrate (detergents S and D) could
also be used to study, by equivalently rapid and inexpen-
sive methods, the presence/absence and the dependence
289
on conditions of other very interesting enzymatic activities
in detergents such as cellulases or amylases.
Finally, with the experimental methodology presented
in this article, we intend not only to train students from a
theoretical and practical point of view in enzyme biotech-
nology but also to reinforce several important concepts
such as the influence of temperature and other parame-
ters on enzymatic activity and the impact that this can
have on industrial/home processes. It also raises aware-
ness of the specificity and action mechanism of several
protease inhibitors and how this can be used to elucidate
a protease enzyme profile from a complex biological
matrix/product. And, finally, we also aim to carry out com-
plementary but very different experimental approaches for
the study of a biotechnological application.
Students are made aware of the important role that
proteases play in the detergent industry, and why the
choice of bacterial enzymes to add is not trivial, some-
times because they facilitate downstream processing
and the preparation of a microbe-free enzyme or
because they have new interesting properties (e.g. low-
temperature performance).
It is also important to emphasize that students were
surprised at the presence of this enzymatic activity in the
standard commercial product (S). And, more important,
they discovered that it was absent in the more specific
detergent (D) because of its specific purpose. Students,
then, realized that industry is continuously evolving in its
attempts to find better products, and that biotechnology
has a strong impact on the development of this kind
of optimized products to present better industrial final
commercial outputs.
Acknowledgments— We thank Jonathan Levin from Labora-
toire de Différenciation Cellulaire et Croissance of Montpellier
and John Bates from Rovira i Virgili University for help in editing
this manuscript.
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