ISSN 0104-6632
Printed in Brazil
www.abeq.org.br/bjche
Vol. 28, No. 03, pp. 373 - 380, July - September, 2011
*To whom correspondence should be addressed
Brazilian Journal
of Chemical
Engineering
HIDE UNHAIRING AND CHARACTERIZATION
OF COMMERCIAL ENZYMES USED
IN LEATHER MANUFACTURE
A. Dettmer1*, M. A. Z. Ayub2 and M. Gutterres1*
1Chemical Engineering Department, Laboratory for Leather and Environmental Studies (LACOURO),
Phone: + (55) (51) 3308 3954, Fax: + (55) (51) 3308 3277, Federal University of Rio Grande do Sul,
Luiz Englert str., s/n°, 90.040-040, Porto Alegre - RS, Brazil.
E-mail: alinedet@enq.ufrgs.br, mariliz@enq.ufrgs.br
2Food Science and Technology Institute, BiotecLab, Federal University of Rio Grande do Sul,
Av. Bento Gonçalves, 9500, P.O. Box 15090, 91570-901, Porto Alegre - RS, Brazil.
(Submitted: September 6, 2010 ; Revised: March 25, 2011 ; Accepted: April 2, 2011)
Abstract - The enzymatic treatment of hides in tannery processes is a promising technology. However, the
reaction kinetics of commercial enzymes available to the leather industry are not fully understood and their
activities have been mainly determined with model proteins such as casein as substrate, which are not of
direct relevance for cattle hides. Therefore, it is important to determine their activities on collagen and keratin,
the main proteins of skin, in order to use these enzymes in leather processing. This work describes the study
of five proteases, used commercially in tanneries, to assess their ability to act upon collagen and keratin and to
determine their unhairing. Results showed that all commercial enzymes tested had more activity on collagen
than on keratin. Unhairing was also tested and four out of the five enzymes tested showed some unhairing
activity. Optima of the temperature and pH of the enzymes were very similar for all five enzymes, with
maximal activities around 55°C and pH 9 to 12, respectively.
Keywords: Unhairing; Commercial enzymes; Enzyme characterization; Leather tanning.
INTRODUCTION
The leather industry converts hide (putrescible)
into commercial leather (non-putrescible) using large
amounts of chemicals, generating an environmental
impact. Leather processing involves a series of unit
operations, of which the process of unhairing is the
first major step in leather making. The pelt has to be
freed from the epidermis and hair, including the hair
roots, and the keratinous material filling the hair
follicles, before proceeding to the next step, the
tanning (Sivasubramanian et al. 2008). During the
unhairing process, large quantities of water and toxic
chemicals such as sulfide are employed, generating
huge amounts of effluent that must be treated, as well
as solid wastes, that could be reused or better treated
to avoid soil and water contaminations (Zhi-Hua et
al., 2009; Priya et al. 2008; Galarza et al., 2009).
Despite its high environmental impact, the leather
industry is economically important in regions where
tanneries are prevalent and, in 2008, 343 million
pieces of leather were produced worldwide. In this
context, Brazil is one of the world’s largest leather
producers, with a production of 44 million pieces in
2009, representing 13% of the total global
production (Brazilian Guide of Leather, 2010).
The application of biotechnology in leather
manufacturing, with the replacement of chemicals by
enzymes, is an alternative for the reduction of the
environmental impact. Enzymes can be applied in
different steps of the process, such as in soaking,
liming, unhairing, bating, dyeing, and degreasing, as
374 A. Dettmer, M. A. Z. Ayub and M. Gutterres
Brazilian Journal of Chemical Engineering
well as for effluent treatment, both solid and liquid
(Kanth et al., 2009; Kanth et al., 2008; Macedo et al.,
2005; Dayanandan et al., 2003; Lutckmeier et al.,
2008; Kumar et al., 2008). Despite being consolidated
in other industrial applications, the use of enzymes in
the leather industry needs more research to be
effectively used in large scale operations and at
competitive costs. Enzymes have been used in tanning
industries for several years, but the majority of
enzymatic preparations do not present sufficient
specificity. Some of the expected advantages of using
enzymes in leather processing are a shorter wetting
time, better fiber opening, and solubilization and
removal of proteins, fat and carbohydrates
(Thanikaivelan et al., 2004). Enzymes could also be
used for the unhairing process, epidermis and hair
removal, removal of residual components,
removal/dispersion of adipose components, and
reduction of effluent load (Wang et al., 2009).
The currently marketed enzymes lack sufficient
specificity and their characteristics are not well
established in detail. Usually, their activities are
determined using casein as substrate (BASF, 1995),
while cattle hides do not have this protein in their
composition. Therefore, it is important to establish
their activities on collagen, the main component of
animal skin, and keratin in order to allow their use in
leather processing.
In this work, commercial enzymes available for
the soaking, liming, and bating processes were
characterized for their optimal pH, temperature,
thermal stability, and the influence of inhibitors.
Furthermore, due to the possibility of eliminating the
use of lime and sulfide, these commercial enzymes
were also tested in the hide unhairing process.
MATERIALS AND METHODS
Chemicals
Five commercial protease preparations were used
in this research and, for simplicity, they were identified
as “A” to “E”. Enzymes A (Buzyme 7703, Buckman
Laboratories), B, and C (Tanzyme RD 04 and CD 05,
respectively, both provided by Tanquímica) are
unspecified preparations of microbial proteolytic
enzymes, recommended for soaking and liming by
their producers. Enzyme D (Tanzyme P 10, from
Tanquímica), is a trypsin, while enzyme E (Buzyme
7706, from Buckman Laboratories) is a preparation of
proteolytic enzymes. They are both applied during the
bating process. The substrates for the enzymes used in
his work were azocasein, keratin azure, and azocoll,
bought from Sigma-Aldrich. All other reagents were
of analytical grade.
Proteolytic Assay
Proteolysis using azocasein as substrate was based
on Giongo et al. (2007). Enzymatic solutions were
prepared immediately before their use, with
concentrations of 5 mg of commercial enzyme/ml of
distilled water. The reaction mixture contained 100 µl
of substrate (azocasein 10 mg/ml), 100 µl of buffer
(0.1M sodium bicarbonate or sodium phosphate,
according to the pH value), and 100 µl of enzyme
solution (5 mg of enzyme/ml of distilled water).
Samples were incubated at 37°C for 30 min, and the
reaction was stopped using 500 µl of 10%
trichloroacetic acid (TCA). After centrifugation at
10,000 g for 5 min, 800 µl of the supernatant were
added to 200 µl of 1.8N NaOH and the absorbance
was determined at 420 nm. One unit of enzyme
activity was defined as the amount of enzyme causing
a change of absorbance of 0.01 at 420 nm in 30 min at
37°C. Reaction controls were prepared by adding the
enzyme solution, buffer and substrate solution to TCA.
Keratinolytic and Collagenolytic Assay
Enzymatic activities on keratin and collagen were
determined using keratin azure and azocoll as
substrates, respectively. The methodology was adapted
from Adigüzel et al. (2009) and Ionata et al. (2008).
Keratinolytic activity was determined by
incubating 500 µl of enzyme solution with 4 mg of
keratin azure and 500 µl of buffer (0.1 M sodium
bicarbonate or sodium phosphate, according to the pH
value). The reaction mixture was incubated at 55°C for
30 min under agitation. After centrifugation at 10,000 g
for 5 min, the absorbance was determined at 595 nm.
One unit of enzyme activity was defined as the amount
of enzyme causing a change of absorbance of 0.01 at
595 nm in 30 min at 55°C. The control was prepared
by adding enzymatic solution and buffer, without
substrate. The same procedure was used to evaluate
enzymatic activity for azocoll.
Effects of pH and Temperature on Enzymatic
Activities
The enzymes were characterized for their
optimum pH, temperature, and thermal stability,
using azocasein as substrate. The activities were
evaluated in 0.1M sodium phosphate (pH 6 – 8) or in
0.1M sodium bicarbonate (pH 9 – 13) buffers at
37°C. The effect of temperature on enzymatic
activities was tested between 28 and 75°C, with the
pH fixed at its best value, previously determined.
The thermal stability of the enzymes was determined
at their optimal pH, with enzymatic solutions being
exposed to temperatures of 37, 45, and 55°C for 15,
Hide Unhairing and Characterization of Commercial Enzymes Used in Leather Manufacture 375
Brazilian Journal of Chemical Engineering Vol. 28, No. 03, pp. 373 - 380, July - September, 2011
30, 60, and 120 min. The residual enzymatic
activities were determined as described above at
37°C and using azocasein as substrate.
Effect of Inhibitors and Some Chemical Products
on the Enzymatic Activity
The effects of some chemicals on the activities of
enzymes were tested. The chemicals were: 5 mM
EDTA; 0.1% (w/v) surfactant (Eusapon, Basf);
0.1% (v/v) fatty alcohols (Busperse 7769, Buckman
Laboratories); salts (0.5 and 1% w/v calcium
carbonate; 0.3% w/v sodium carbonate); and
1% (w/v)sulfides; which are normally used during
the leather production process. Although CaCO3 is
insoluble at these concentrations, it were used to
follow the practice by the leather industry, since the
excess of this salt provides for skin saturation.
Enzymatic solutions (50 ml of 5 mg enzyme/ml of
distilled water) were preincubated for 15 min at
room temperature with the tested chemicals at room
temperature. The residual enzymatic activities were
determined as described above at 37°C using
azocasein as substrate.
Unhairing Activity
Bovine skin pieces weighing approximately 40 g
were immersed in the enzyme solution (5 mg of
commercial enzyme/ml of distilled water, pH 8) and
incubated for 18 h at room temperature. About 2 ml
of enzyme solution/g of hide were used for these
experiments. The tests were conducted in a
laboratory cylindrical drum rotating reactor (which is
used for hide and leather processing) at 24 rpm. The
next day, the pieces were analyzed for the presence
or absence of depilated areas and change of color
after incubation with the enzymatic solution.
RESULTS AND DISCUSSION
Effects of pH and Temperature on the Proteolytic
Activities
The effects of pH on the enzymatic activities of the
five commercial enzymes are shown in Figure 1 (a).
As can be seen, proteolytic activities varied
markedly from enzyme to enzyme but, in general,
they followed the expected profile suggested by their
producers. Enzymes A, B, and C are recommended to
be used in the soaking and liming processes, where
the usual pH ranges from 7.0 to 13.0. While Enzymes
D and E are recommended for the bating process, in
which the pH varies between 7.5 and 10. Almost all
the tested enzymes showed a remarkable loss of
activity at pH lower than 7 and higher than 13,
showing a plateau of high activity for pH varying
from 7 to 12. These plateaus suggest the presence of
two or more isoforms of the enzymes with the same
specific substrate activities, but dependent on pH
variations. Mixtures of enzyme isoforms are common
in commercial enzymatic preparations for technical
applications such as in the leather industry. These
preparations can be used over a large range of pH,
which is interesting from the industrial point of view.
The profile of the influence of temperature on
proteolytic activities can be observed in Figure 1(b),
with maximal activities around 55°C for enzymes A,
B, and C, while enzymes D and E showed their best
activity at 37°C. Enzymes used for the bating
process (enzyme D) are from the pancreatic family,
such as trypsin, explaining their typically lower
activities at higher temperatures. Above 60°C, all
enzymes presented decreased activities. These
temperature profiles are similar to those found by
other authors. The enzyme described by Ionata et al.
(2008) had optimal activity at 55°C, while Farag and
Hassan (2004) also reported the highest activities for
a keratinase from Aspergillus oryzae at 55°C.
(a)
(b)
Figure 1: Effect of (a) pH and (b) temperature on the
proteolytic activity, using azocasein as substrate.
Enzyme (A) ( ), enzyme (B) ( ), enzyme
(C) ( ), enzyme (D) ( ), enzyme (E) ( ).
376 A. Dettmer, M. A. Z. Ayub and M. Gutterres
Brazilian Journal of Chemical Engineering
Thermal Stability of Enzymes
The thermal stability of enzymes is very important
for their industrial utilization. In Figure 2, the thermal
stability profiles of all enzymes tested are presented.
Enzyme A was the most stable, maintaining its activity
for 120 minutes at 37 and 45°C, although even short
times of incubation at 55°C were sufficient for its
inactivation. The remaining enzymes were less stable
at any tested temperature. Tatieni et al. (2008) reported
that the activity of an enzyme from Streptomyces sp.
was 80% stable at 50°C and completely inactivated at
70°C and higher temperatures. Ogino et al. (2008)
evaluated the thermal stability of a Bacillus sp.
enzyme. The authors found that the enzyme was stable
from 30 to 40°C and about 60% of the activity
remained at 50°C, losing completely its activity above
60°C for 10 min.
(a) (b)
(c) (d)
(e)
Figure 2: Thermal stability of protease preparations Enzyme A (a), Enzyme B (b), Enzyme C (c), Enzyme D (d)
and Enzyme E (e). 37°C ( ), 45°C ( ), 55°C ( ).
Hide Unhairing and Characterization of Commercial Enzymes Used in Leather Manufacture 377
Brazilian Journal of Chemical Engineering Vol. 28, No. 03, pp. 373 - 380, July - September, 2011
Enzymatic Activities with Different Proteic
Substrates: Azocoll, Keratin Azure, and Azocasein
Hides are mainly constituted of three layers:
epidermis, which is basically keratin, dermis that is
composed of collagen fibers, and hypodermis,
mainly fat, which is completely removed during the
fleshing process. Therefore, we carried out
experiments by testing the activities of the 5
enzymes with different types of substrates,
simulating different proteic structures. These were
azocoll, keratin azure, and azocasein and the results
are summarized in Table 1. Ratios between the
activities for the different substrates are presented in
Table 2.
Enzymes A, B, C and E exhibited the highest
activities for azocasein, but were also active with the
other substrates, keratin azure and azocoll. Keratin
azure, which is an insoluble substrate, was less
susceptible to enzymatic attack. According to Ionata
et al. (2008), data from soluble proteins cannot be
compared with those for insoluble proteins since the
mechanism of action of enzymes on this substrate
will be a function of the surface area. Enzyme A
showed the highest activities among all enzymes
tested, justifying its use in hide unhairing. However,
its high activity with collagen (azocoll) might cause
loss of skin mechanical strength and, therefore, its
use must be carefully controlled. Keratinolytic
proteases with mild collagenolytic and elastolytic
activities might be particularly suitable for enhancing
the dehairing process without harming the tensile
strength of leather (Gupta and Ramnani, 2006).
Results for collagenolytic activity suggest that
enzymes A and E should be applied carefully,
observing the time and amounts indicated in order to
avoid loss of mechanical strength of the leather.
According to the ratios between keratin
azure/azocasein and azocoll/azocasein, all 5 commercial
enzymes can be classified as collagenolytic enzymes.
All tested enzymes presented higher activities with
collagen than with keratin, as can be seen in the
results presented in Table 2. These results contrast
with other reports, which presented higher ratios
between keratin azure and azocasein than those for
the commercial enzymes described in this work, as
well as lower azocoll activities.
Inhibitors of Enzymes Activities
The salts and other chemicals present in industrial
solutions may interfere with enzyme activity and
therefore must be tested in order to prevent process
losses. Table 3 presents the results of the effects of
inhibitors and other chemical products on the
enzymatic activities of the tested enzymes.
EDTA caused reductions of the activities of
enzymes B and C, with little or no effect on enzymes A,
D, and E. This might indicate the presence of metallic
cations at the active centres of enzymes B and C,
susceptible to EDTA sequestration (Kumar and Takagi,
1999). According to the manufactureres, enzymes B and
C are non-specific preparations of microbial proteolytic
enzymes, probably a mix of metalloproteases (inhibited
by EDTA) and other proteases. These explain the
residual activity of enzymes A, D and E after contact
with EDTA, because these enzymes may not be
metalloproteases or may contain a minor fraction of
metalloproteases. Tatineni et al. (2008) reported that a
keratinase from Streptomyces sp. retains only 27% of its
original activity after incubation with EDTA.
Fatty alcohol, which is an organic solvent,
sodium carbonate, and the surfactant caused no
significant variations in enzymes activities, except
for enzyme D, which was more active in the
presence of the surfactant, possible because this
agent might have facilitated its action. Riffel et al.
(2003) determined the residual activities of bacterial
enzymes after incubation with organic solvents, the
remaining activities being between 56 and 70%.
Enzyme D was completely inhibited when incubated
with calcium carbonate. Enzymes B, C and D were
inhibited by sodium sulfide, while enzyme A was
activated by this chemical. Syed et al. (2009)
reported that a keratinase from S. gulbargensis was
totally inhibited by calcium compounds and its
activity increased in the presence of sodium sulfite.
Riffel et al. (2007) reported that a protease from
Chryseobacterium sp. showed a 3.5 fold increase in
its activity in the presence of calcium ions.
Table 1: Proteolytic activity with different substrates
Enzymatic activity (U/ml)
Enzyme
Substrate
A B C D E
Keratin azure 25±1.98 11±3.00 12±0.2 5±0.3 13±1.76
Azocoll 110±0.85 32±1.70 25±5.2 8±1.3 76±0.6
Azocasein 166±3.96 153±1.27 134±2.1 8±0.57 145±1.91
Values are means ± standard deviation for three samples.
378 A. Dettmer, M. A. Z. Ayub and M. Gutterres
Brazilian Journal of Chemical Engineering
Table 2: Ratios between proteolytic activities for different substrates
Ratios Between Proteolytic activities
Commercial Enzymes Other Works
Substrate
A B C D E Farag et. al.
(2004)
Pillai and
Archana (2008)
Macedo et al.
(2008)
Keratin azure/ Azocasein Ratio 0.15 0.07 0.09 0.63 0.09 0.92 0.94 0.012
Azocoll/ Azocasein Ratio 0.66 0.21 0.19 1.00 0.52 0.83 0.125 0
Table 3: Effect of some inhibitors and chemical products on the enzymatic activity
Residual Caseinolytic Activity (%)
Enzyme
Chemical Concentration
A B C D E
EDTA 5 mM 87±3.97 33±1.70 69±0.70 87±0.70 104±2.58
Fatty alcohol (Busperse 7769) 0.1% (v/v) 111±1.50 96±1.60 100±3.45 85±0.76 96±2.75
Sodium carbonate 0.3% (w/v) 103±0.20 97±1.37 97±3.97 103±0.21 98±2.90
Calcium carbonate 0.5% (w/v) 85±3.30 107±4.33 77±0.78 0 83±0.68
1% (w/v) 76±6.92 113±13.71 79±1.27 0 85±8.76
Surfactant (Eusapon) 0.1% (w/v) 87±7.20 92±1.77 87±1.31 161±1.60 82±4.34
Sodium sulfide 1% (w/v) 138±2.57 54±1.30 57±3.97 44±1.40 75±0.75
Values are means ± standard deviation for three samples.
(a) (b) (c)
(d) (e) (f)
Figure 3: Hide unhairing tests. Enzyme A (a); Enzyme B (b); Enzyme C (c); Enzyme D (d); Enzyme E (e);
Control, without enzyme (f). The unhaired box in the control was mechanically produced with a razor to
serve as an area of reference.
Enzyme Application for Leather Unhairing
After these activity characterizations, the
commercial enzymes were applied in the leather
unhairing process to see whether their applications
were promising. Figure 3 shows the results.
The enzymatic treatment of the hides produced
significant differences compared to the control.
Hides treated with enzymes A, B, C, and E presented
large depilated areas. These hides presented a dark
coloration, caused by the incomplete removal of
epidermis and pigments. According to Priya et al.
(2008), enzymatic processes are associated with
problems such as high cost, potential hide damage,
processing time, retention of ‘fine’ or ‘short’ hairs
after depilation, improper removal of epidermis and
Hide Unhairing and Characterization of Commercial Enzymes Used in Leather Manufacture 379
Brazilian Journal of Chemical Engineering Vol. 28, No. 03, pp. 373 - 380, July - September, 2011
pigments, and the inability to control the action of
the enzyme on the desired reticular structure of the
dermis. Enzyme D showed a weak activity on
leather, probably due to its low proteolytic,
colagenolytic, and keratynolitic activities. Galarza et
al. (2009) reported that the use of proteolytic
enzymes in the unhairing process causes the
digestion of the cells in the Malpighi’s layer, of the
basal cells of the hair bulb, degrading the medulla,
but not the cortex. The keratin in the cortex is called
hard keratin, as opposed to the soft keratin that is
found in the hair medulla. During enzymatic
unhairing (or hair loosening) there is a partial or total
destruction of the tissues sustaining the hairs, the
components of the epidermis surrounding the follicle
and the root sheats. These tissues contain few
disulfide bonds compared with hard keratins. It can,
therefore, be suggested that the commercial enzymes
tested in this work presented some capability to
digest the soft α-keratin. Pillai and Archana (2008)
reported that an enzymatic preparation from Bacillus
subtilis was ineffective in the hydrolysis of native α-
keratin, while under similar conditions, the feather β-
keratins were nearly completely degraded.
CONCLUSIONS
The results of this work may help to elucidate
some of the properties of enzymes used or
recommended for leather production, including the
determination of the ideal pH and temperature at
which the enzymes show the best stabilities and
activities. Activities on different substrates were
tested, showing that enzyme A could be efficiently
used for leather unhairing due to its good activity on
keratin azure and azocoll, with enzymes B, C, and E
showing a somewhat smaller unhairing activity. The
chemicals normally used during leather production
did not inhibit most of the enzymes tested.
ACKNOWLEDGEMENTS
The authors wish to thank the Brazilian Agencies
CNPq (CTAgro 40/2008) and CAPES for their
financial support of this study and for scholarships
for the first author.
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