Probing the Crucial Role of Leu31 and Thr33 of the Bacillus pumilus CBS Alkaline Protease in Substrate Recognition and Enzymatic Depilation of Animal Hide
et al. (2014) Probing the Crucial Role of Leu31 and Thr33 of the Bacillus pumilus CBS
Alkaline Protease in Substrate Recognition and Enzymatic Depilation of Animal Hide. PLoS ONE 9(9): e108367. doi:10.1371/journal.pone.0108367
Probing the Crucial Role of Leu31 and Thr33 of the Bacillus pumilus CBS Alkaline Protease in Substrate Recognition and Enzymatic Depilation of Animal Hide
Samir Bejar 0
Nadia Zara Jaouadi 0
Bassem Jaouadi 0
Hajer Ben Hlima 0
Hatem Rekik 0
Mouna Belhoul 0
Maher Hmidi 0
Houda Slimene Ben Aicha 0
Chiraz Gorgi Hila 0
Abdessatar Toumi 0
Nushin Aghajari 0
Pratul K. Agarwal, Oak Ridge National Laboratory, United States of America
0 1 Laboratory of Microorganisms and Biomolecules, Centre of Biotechnology of Sfax (CBS), University of Sfax, Sfax, Tunisia, 2 National Leather and Shoe Center (CNCC) , Me grine, Ben Arous , Tunisia , 3 Laboratory for Biocrystallography and Structural Biology of Therapeutic Targets, Molecular and Structural Bases of Infectious Systems, UMR 5086-CNRS-University of Lyon 1, Institute for the Biology and Chemistry of Proteins (IBCP) , FR3302, Lyon , France
The sapB gene, encoding Bacillus pumilus CBS protease, and seven mutated genes (sapB-L31I, sapB-T33S, sapB-N99Y, sapBL31I/T33S, sapB-L31I/N99Y, sapB-T33S/N99Y, and sapB-L31I/T33S/N99Y) were overexpressed in protease-deficient Bacillus subtilis DB430 and purified to homogeneity. SAPB-N99Y and rSAPB displayed the highest levels of keratinolytic activity, hydrolysis efficiency, and enzymatic depilation. Interestingly, and at the semi-industrial scale, rSAPB efficiently removed the hair of goat hides within a short time interval of 8 h, thus offering a promising opportunity for the attainment of a lime and sulphide-free depilation process. The efficacy of the process was supported by submitting depilated pelts and dyed crusts to scanning electron microscopic analysis, and the results showed well opened fibre bundles and no apparent damage to the collagen layer. The findings also revealed better physico-chemical properties and less effluent loads, which further confirmed the potential candidacy of the rSAPB enzyme for application in the leather industry to attain an ecofriendly process of animal hide depilation. More interestingly, the findings on the substrate specificity and kinetic properties of the enzyme using the synthetic peptide para-nitroanilide revealed strong preferences for an aliphatic amino-acid (valine) at position P1 for keratinases and an aromatic amino-acid (phenylalanine) at positions P1/P4 for subtilisins. Molecular modeling suggested the potential involvement of a Leu31 residue in a network of hydrophobic interactions, which could have shaped the S4 substrate binding site. The latter could be enlarged by mutating L31I, fitting more easily in position P4 than a phenylalanine residue. The molecular modeling of SAPB-T33S showed a potential S2 subside widening by a T33S mutation, thus suggesting its importance in substrate specificity.
Funding: This work was funded by the Tunisian Ministry of Higher Education, Scientific Research and Information and Communication Technologies Higher
Education and Scientific Research sector under the Contract Program LMB-CBS, grant no. LR10CBS04 20102013 and the Tunisian Ministry of Industry, Energy
and Mines under the National Programme for the Promotion of Technological Innovation, grant no. PNRI-ENZYMES 20122015. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Leather processing is one of the oldest industries known to
mankind. Despite its significant contributions to the
socioeconomic development of several countries around the world,
this industry has been the target of mounting criticism owing to the
pollution it causes to the environment. It has, therefore, been
under pressure to comply with increasingly stringent global
environmental regulations. In fact, the conventional leather
making process includes a complex set of operations, such as
pre-tanning, tanning, post tanning, and finishing, which involve
the application of various hazardous chemicals, notably lime and
sodium sulphide, that generate several environmental and waste
disposal problems. The application of those chemicals can also
lead to the destruction of the hair, thus causing high biological
oxygen demand (BOD), chemical oxygen demand (COD), and
total suspended solid (TSS) loads in the effluents. The search for
cleaner technologies that can help overcome the serious problems
associated with the conventional depilating methods has,
therefore, become a necessity in the leather industry .
In order to overcome the hazards caused by these effluents,
enzymes have often been proposed as viable alternatives. In fact,
enzymes have long been used as alternatives to chemicals to
improve the efficiency, safety, and cost-effectiveness of a wide
range of industrial systems and processes . Currently, the most
commonly used biotechnological applications cover all the stages
of leather making and waste treatment processes. An emerging
technology is the use of proteolytic enzymes in the depilation
process, which minimizes/replaces the use the major pollutant in
the tannery process, namely sulphide.
Microbial alkaline proteases are among the most important
hydrolytic enzymes. They play important roles in both cellular
metabolic processes and industrial sectors, accounting for more
than 65% of the global industrial enzyme sales . Alkaline
proteases produced from Bacillus can withstand high temperature,
pH, chemical denaturing agents, and non-aqueous environments.
They have attracted considerable attention particularly due to
their promising potential for application in a wide range of
industries, including the detergent, leather, and pharmaceutical
industries. The keratinolytic activity of these enzymes has also
been of interest in several biotechnological processes, such as the
management of waste from various food-processing industries, the
recovery of silver from used X-ray and photographic films, and
production of proteinaceous fodder from waste feathers or
keratincontaining materials .
Due to their efficiency, low cost, and eco-friendliness, microbial
alkaline proteases have attracted increasing attention for
application in enzymatic depilation as substitutes to chemical agents, such
as sodium sulphide, lime, and chromate, which have long been
used in conventional industrial depilation processes. Several
researchers have, therefore, focused on the isolation and
characterization of novel microbial strains with efficient depilation
activity. Alkaline proteases/keratinases selectively cleave the hair
on the skin without causing damage to the skin, thus leaving the
quality of the leather unaffected [5,6]. The adequacy of
keratinolytic proteases with mild elastinolytic but no collagenolytic
activities for the depilation process has also been highlighted in the
literature. Keratinases may participate in the selective breakdown
of the keratin tissues in hair follicles, thus pulling out intact hairs
without affecting the ductile strength of the leather .
Keratinases are robust enzymes that are active over a wide
range of temperature and pH. The sequence analysis of
keratinases indicates their close relatedness to subtilisin and serine
proteases, which are generally from bacterial origins. They are
specific to aliphatic or hydrophobic amino-acid residues, most
active at around pH 10, and with a molecular weight ranging from
10 to 35 kDa. This class of proteases also has a tight relationship
with subtilisins isolated from Bacillus strains, exhibiting similar
protein sequences and biochemical characteristics. Various
keratinases have been produced from Bacillus species, including B.
licheniformis, B. pumilus, B. subtilis, B. circulans, and
Brevibacillus brevis [4,7,8].
Although several subtilisins, including keratinases, have been
described in the literature, little data is currently available on the
distinction between the two types of hydrolytic enzymes. In fact,
while several subtilisins display efficient keratinase activity, some
others either have a very low or no keratinase activity.
Nevertheless, little work has so far been performed on the modes
or mechanisms of actions and structural differences between those
two types of proteases. Moreover, so far and to the authors
knowledge, no previous work has been performed to investigate
the conversion of a subtilisin into a keratinase or vice versa. In this
context, the authors have previously reported the purification and
characterization of a serine protease, called SAPB, from B.
pumilus CBS. The pure enzyme showed optimal activity at
pH 10.6 and 65uC. The sapB gene was cloned and expressed in
Escherichia coli . The laundry detergent stability and dehairing
ability of SAPB were also investigated . Furthermore, the
effects of five essential amino-acid residues on the biochemical
properties of the enzyme were also explored, allowing the
generation of seven efficient and thermostable mutant enzymes,
particularly SAPB-N99Y, SAPB-L31I/N99Y, SAPB-T33S/
N99Y, and SAPB-L31I/T33S/N99Y. Among those mutants,
and compared to the wild-type enzyme, the triple mutant was
noted to display the highest levels of specific activity, catalytic
efficiency and stability at elevated pH and temperature values with
casein as a substrate . The authors have also reported on the
overexpression of sapB and sapB-L31I/T33S/N99Y genes in B.
subtilis DB430 . The present study aimed to investigate the
overexpression of all SAPB mutant enzymes in B. subtilis DB430.
It also explored their substrate specificities and potential keratin
biodegradation and depilation activities. The effects of L31I and
T33S mutations on their animal hide depilation ability and the
relationship between keratin/casein activity ratios were also
Materials and Methods
2.1. Substrates and Chemicals
Unless otherwise specified, all substrates, chemicals and reagents
were of analytical grade or highest available purity, and purchased
from Sigma Chemical Co. (St. Louis, MO, USA).
2.2. Bacterial Strains, Plasmids, Media, and Cultivation Conditions
The two protease-deficient host strains used were the B. subtilis
DB430 strain (trpC npr apr epr bpf ispl), a generous gift from Dr.
Philippe Glaser and Dr. Marie Francoise Hullo (LGMP, Institut
Pasteur, Paris), and the E. coli DH5a strain (Invitrogen, Carlsbad,
CA, USA). The B. pumilus CBS strain  was used as a donor
for the SAPB enzyme and sapB wild-type gene. The plasmid pBJ4,
containing the sapB wild-type gene , and plasmids pBJ22,
pBJ25, pBJ28, pBJ31, pBJ34, pBJ37 and pBJ40, containing
sapBL31I, sapB-T33S, sapB-N99Y, sapB-L31I/T33S, sapB-L31I/
N99Y, sapB-T33S/N99Y, and sapB-L31I/T33S/N99Y,
respectively, were used as sources for mutated sapB genes . The E.
coli-Bacillus shuttle vector pBSMuL2, kindly provided by Dr.
Thorsten Eggert (Institut fur Molekulare Enzymtechnologie,
Heinrich-Heine Universitat Dusseldorf, Julich, Germany), was
used to construct the expression plasmid in E. coli DH5a. The
pNZ1 and pNZ2 plasmids, used for the production of rSAPB and
SAPB-L31I/T33S/N99Y proteins, respectively, were previously
described elsewhere . The different strains harboring the wild
and mutant type genes were routinely cultured in LB media
consisting of (g.l21): peptone, 10; yeast extract, 5; and NaCl, 5 at
pH 7.4. Skimmed milk media were used for the screening of
protease-producing recombinant strains. The production medium
consisted of a 2 6 LB and 10 g.l21 glucose at pH 7.4 . The
media were autoclaved for 20 min at 120uC and supplemented,
when required, with antibiotics at the following concentrations:
Ampicillin (100 mg.ml21) for E. coli; and Kanamycin (5 mg.ml21)
for B. subtilis.
2.3. DNA Manipulation and Sequencing
Plasmid DNA was isolated from E. coli and B. subtilis using
general molecular biology techniques as described by Sambrook
et al. . PCRs were performed using an Applied Biosystems
2720 thermal cycler. The amplification reaction mixtures (50 ml)
contained 20 pg of each primer, 200 ng of DNA template,
amplification buffer, and 2 U of Pyrococcus furiosus DNA
polymerase (Biotools, Madrid, Spain). The cycling parameters
were as follows: 94uC for 3 min, followed by 35 cycles of 94uC for
30 s denaturation, 54uC for 60 s primer annealing, and 72uC for
120 s extension). The PCR products were then purified using an
agarose gel extraction kit (Jena Bioscience, GmbH, Germany).
DNA sequencing was carried out by an automated DNA
sequencer ABI Prism 3100-Avant Genetic Analyser (Applied
Biosystems, Foster City, CA, USA) with the Big-Dye terminator
cycle sequencing kit recommended by the manufacturer
(Amersham Pharmacia Biotech, Buckinghamshire, UK).
2.4. Construction of Protease Overexpression Plasmids and Bacillus Transformation
To overproduce SAPB mutated proteases in B. subtilis DB430,
a 1260 bp EcoRI/HindIII DNA fragment from pBJ22, pBJ25,
pBJ28, pBJ31, pBJ34, and pBJ37 plasmids carrying the whole
sapB-L31I, sapB-T33S, sapB-N99Y, sapB-L31I/T33S,
sapBL31I/N99Y, and sapB-T33S/N99Y genes, respectively, were
subcloned in the pBSMuL2 (7559 bp) shuttle vector linearized by
EcoRI/HindIII to produce pNZ7 (8819 bp), pNZ8 (8819 bp),
pNZ9 (8819 bp), pNZ10 (8819 bp), pNZ11 (8819 bp), and pNZ12
(8819 bp) plasmids, respectively.
B. subtilis DB430 was transformed as previously described by
Zara Jaouadi et al. . After the suitable dilution of competent
cells, the pNZ7, pNZ8, pNZ9, pNZ10, pNZ11, and pNZ12
plasmids or pBSMuL2 plasmid DNA multimers were added, and
the samples were incubated at 37uC for 20 min. Transformation
mixtures were then spread on LB-Kanamycin agar. Kanamycin
resistant colonies the B. subtilis DB430 transformants were
screened for protease activity on LB-Kanamycin-skimmed milk
plates, based on the detection of cleared zone formations around
B. subtilis DB430 transformant colonies. The latter were further
screened by PCR to confirm the introduction of the sapB genes.
2.5. Enzyme Activity Assays
The keratinolytic activity of the recombinant SAPB enzymes
was determined under the optimal pH and temperature values of
the respective enzymes using keratin azure as a substrate . One
keratin unit was defined as the amount of enzyme causing an
increase of 0.1 in absorbance at 440 nm in one min. The same
protocol was used to determine enzyme activity on azo-casein.
Caseinolytic activity was measured using the Folin-Ciocalteu
method , with Hammersten casein (Merck, Darmstadt,
Germany) as a substrate. One casein unit was defined as the
amount of enzyme that produced 1 mg of amino-acid equivalent to
tyrosine per min. The same protocol was used to determine
enzyme activity on natural proteins, namely keratin, elastin-orcein,
2.6. Enzyme Purification Procedure
The SAPB enzymes were purified following the procedures
previously described by Zara Jaouadi et al. . In brief, five
hundred ml of a 72-h-old culture of B. subtilis DB430 harboring
the pNZ1, pNZ2, pNZ7, pNZ8, pNZ9, pNZ10, pNZ11, or
pNZ12 plasmids were harvested by centrifugation at 10,0006g for
35 min to remove microbial cells. For each culture, the cell-free
supernatant containing extracellular protease was used as the
crude enzyme preparation and submitted to the following
purification steps. Each supernatant was heat-treated at 65uC for
15 min, and insoluble material was removed by centrifugation at
10,0006g for 30 min. The clear supernatant was precipitated
between 40% and 60% ammonium sulphate saturation. The
precipitate was then recovered by centrifugation at 14,0006g for
20 min, resuspended in a minimal volume of 50 mM
2-(Nmorpholino) ethanesulfonic acid (MES) buffer containing 2 mM
CaCl2 at pH 5.5 (Buffer A), and dialyzed overnight against
repeated changes of buffer A. Insoluble material was removed by
centrifugation at 14,0006g for 20 min. The supernatant was
loaded on a fast performance liquid chromatography (FPLC)
system using an UNO S-1 column (Bio-Rad Laboratories, Inc.,
Hercules, CA, USA) equilibrated in buffer A. The column
(7 mm635 mm) was rinsed with 500 ml of the same buffer. After
being washed with the same buffer A, the unadsorbed protein
fractions were eluted. Adsorbed material was eluted with a linear
NaCl gradient (from 0 to 0.5 M in buffer A) at a rate of 60 ml.h21.
Fractions with protease activity were pooled and applied to high
performance liquid chromatography (HPLC) system using a
BioSil SEC 1255 column (7.8 mm6300 mm), pre-equilibrated with
25 mM HEPES buffer and supplemented with 2 mM CaCl2 and
150 mM NaCl at pH 7.5 (Buffer B). Proteins were separated by
isocratic elution at a flow rate of 30 ml.h21 with buffer B and
detected using a UV-VIS detector (Knauer, Berlin, Germany) at
280 nm. The pooled fractions (eluted at a void volume of 1.8),
exhibiting protease activity and a retention time of 20 min, were
concentrated in centrifugal micro-concentrators (Amicon Inc.,
Beverly, MA, USA) with 10 kDa cut-off membrane and stored at
20uC for further analysis.
2.7. Protein Measurement and Analytical Methods
Protein concentration was determined by the method of
Bradford , using a Dc protein assay kit purchased from
BioRad Laboratories (Inc., Hercules, CA, USA) with bovine serum
albumin as a reference. Analytical soduim dodecyl sulphate
polyacrylamide gel electrophoresis (SDS-PAGE) was performed
following the method of Laemmli . Protein bands were
visualized with Coomassie Brilliant Blue R-250 staining.
Caseinzymography analysis was performed as previously described
elsewhere . Low molecular weight markers from Amersham
Biosciences were used as protein marker standards. The molecular
masses of the selected purified SAPB-L31I, SAPB-T33S, and
SAPB-N99Y enzymes were analyzed in the linear mode by
MALDI-TOF/MS using a Voyager DE-RP instrument (Applied
Biosystems/PerSeptive Biosystems, Inc., Framingham, MA, USA).
Bands of purified enzymes (SAPB-L31I, SAPB-T33S, and
SAPBN99Y) were separated on SDS gels and transferred to a ProBlott
membrane (Applied Biosystems, Foster City, CA, USA).
Nterminal sequence analyses were performed by automated
Edmans degradation using an Applied Biosystem Model 473A
2.8. Substrate Specificities and Kinetic Studies
The substrate specificity of rSAPB (pH 10.6, 65uC), SAPB-L31I
(pH 11.5, 65uC), SAPB-T33S (pH 12, 65uC), SAPB-N99Y
(pH 11, 75uC), SAPB-L31I/T33S (pH 12, 65uC), SAPB-L31I/
N99Y (pH 11.5, 70uC), SAPB-T33S/N99Y (pH 12, 70uC), and
SAPB-L31I/T33S/N99Y (pH 12, 70uC) were determined using
natural (keratin, casein, elastin-orcein, and albumin) and modified
(keratin azure, azo-casein, and collagen types I and II FITC
conjugate) proteins as well as synthetic para-nitroanilide (pNA)
linked peptide chromogenic substrates. The synthetic substrates
were Succinyl-XXX or XXXX-pNA with XXX or XXXX
representing YLV, AAV, AAI, AAL, AAA, APA, and AAF or
FAAF, AAPF, LLVY, AAPM, AAPL, and AAVA. Peptides were
dissolved in 10% (v/v) dimethylformamide (DMF) before being
diluted in buffer, and prepared just before the experiment since
nitroanilide substrates show varying degrees of autolysis during
Kinetic parameters were calculated from the initial rate
activities of the eight purified SAPB enzymes using YLY and
FAAF as synthetic peptide substrates at different concentrations,
ranging from 0.2 mM to 50 mM. MichaelisMenten constant
(Km) and maximal reaction velocity (Vmax) values were calculated
by Lineweaver-Burk plots using Hyper32 software.
The pH and temperature values used in this study were adjusted
to the optimum conditions for each SAPB enzyme as reported
elsewhere . Enzymatic activities were determined on each
substrate according to standard conditions. For the natural
substrate, the complete hydrolysis of a protein unit was defined
as the amount of enzyme that produced 1 mg of amino-acid
equivalent to tyrosine per min, in absorbance at 660 nm, under
the experimental conditions described. For the modified substrate,
one protein azure unit was defined as the amount of enzyme
causing an increase of 0.1 in absorbance at 440 nm in one min
under the assay conditions described. Collagenolytic activities
were determined by measuring absorbance at 440 nm as
illustrated in the protocol of Sigma-Aldrich Co. LLC. Activity
with pNA substrates was tested at suitable pH and temperature for
10 min in 100 mM buffer containing 2 mM Ca2+. For the
synthetic peptide substrate, the amount of released pNA was
recorded at 405 nm. The initial rates measured as A405/min were
converted into velocity (mM21.min21) for each substrate
concentration using the molar absorbance coefficient for pNA
(9800 M21.cm21 at 405 nm). One unit of enzymatic activity
was defined as the amount of enzyme releasing 1 mmole of pNA
under standard assay conditions.
2.9. Zymorgam Gel Analysis
To confirm the substrate specificity profile of SAPB enzymes,
discontinuous substrate native-PAGE (Zymogram analysis) was
performed with a 4% stacking gel, except that 1 mg/ml keratin or
casein, as a substrate, were incorporated into the 10% separation
gel. Electrophoresis was performed at a constant current of 25 mA
under non-reducing conditions. The gels were then gently washed
and incubated at 50uC for 2 h in 100 mM Glycine-NaOH buffer
at pH 10 supplemented with 2 mM CaCl2, which produced a
keratin or casein cleared zone at the location of the proteolytic
band of each SAPB enzyme. A clear zone was visualized by fixing
the gel with ice-cold trichloroacetic acid 20% (w/v) for 1 h,
staining it with 0.1% Coomassie Brilliant Blue G-250 (Bio-Rad
Laboratories, Inc., Hercules, CA, USA) in water/methanol/acetic
acid 60:30:10, and distaining it in the same solution without dye.
2.10. Determination of Hydrolysis Degree
Keratin hydrolysis was carried out according to Zara Jaouadi
et al.  at 65uC and pH 10.6 (for rSAPB); 65uC and pH 11.5 (for
SAPB-L31I); 65uC and pH 12 (for SAPB-T33S), 75uC and pH 11
(for SAPB-N99Y), 65uC and pH 12 (for SAPB-L31I/T33S), 70uC
and pH 11.5 (for SAPB-L31I/N99Y), 70uC and pH 12 (for
SAPBT33S/N99Y), and 70uC and pH 12 (for SAPB-L31I/T33S/
N99Y). An amount of 4 g of keratin azure was dissolved in 100 ml
of assay buffer and then treated with 2,000 U of the purified target
2.11. Enzyme Preparation for Leather Processing
The cell-free supernatant of a 72-h-old culture of B. subtilis
DB430 harboring the pNZ1 plasmid was brought to 80% (w/v)
saturation with ammonium sulphate. The precipitated proteins
were centrifuged at 14,000 6 g for 20 min at 4uC. The resultant
pellet was dissolved in buffer B and dialyzed against the same
buffer. The dialyzed semi-purified enzyme was used for depilation
in leather processing.
2.12. Hide-Depilation Ability of SAPB Enzymes
Small pieces (about 6 cm 66 cm) of haired goat, rabbit, bovine,
and sheep skins, which were freshly obtained from a local
municipal slaughterhouse (Sfax municipal slaughterhouse,
permission was obtained from this slaughterhouse to use these animal
parts), and rinsed to remove excess blood, were placed into 20 ml
of buffer B containing a purified rSAPB, SAPB-L31I, SAPB-T33S,
SAPB-N99Y, SAPB-L31I/T33S, SAPB-L31I/N99Y,
SAPBT33S/N99Y, or SAPB-L31I/T33S/N99Y enzymes having
2,000 U of keratinase activity. After 8 h of incubation at 37uC,
the skins were taken out and the hair was gently hand-pulled to
test whether it had parted from the skin. Since, to the authors
knowledge, no quantitative method is currently available for the
determination of depilation effects, this ability was defined
qualitatively as no, yes or easily. Depilation efficiency was
assessed according to the depilated area of the skin at the end of
the process, and the quality of the depilated skin was estimated
according to naked-eye observations and microscopic
examinations made after treatments.
The handling of the skin from rabbit, goat, bovine and sheep
animals were carried out in strict accordance with the
recommendations in the Guide for the Care and Use of Laboratory
Animals issued by the University of Sfax, Tunisia. The protocol
was approved by the Tunisian Committee on the Ethics of Animal
2.13. Semi-Industrial Scale Test for Goat Skin Depilation
and Scanning Electron Microscopy Analysis
The depilation activity of rSAPB enzyme was tested in the SO.
SA. CUIR leather tannery (MSaken, Sousse, Tunisia) using a
100 kg fresh goat hides obtained from a local slaughterhouse. Wet
salted goat hides were used for depilating experiments. The
experiments were performed in a mini drum (cylindrical rotating
reactor, used for hide and leather processing) with shaking at
15 rpm. The skins were cut into two halves along the backbone.
The right half was used for enzymatic depilation; the left half was
used for conventional lime and sulphide-based depilation and
served as a control. Prior to depilation, the skins were washed and
soaked in water (200% v/wt) for 5 h with intermittent changing to
remove salt, dirt, and blood. The soaked weight of all the left and
right halves were recorded, and the percentages of chemicals and
enzyme used in the experiment was based on this soaked weight.
For the conventional process, the paste method was used wherein
10% lime and 2% sodium sulphide were mixed with 10% water,
and the thus prepared paste was applied on the flesh side
(Sivasubramanian et al., 2008a; Sivasubramanian et al., 2008c). It
was left overnight at room temperature and then depilated using
the conventional method. For the enzymatic group, the optimized
dip method was adopted wherein the soaked skins were dipped in
water float (100% v/wt) containing 2,000 U enzyme. The float
was left for 6 h and then depilated by conventional methods. After
depilation, both the chemical and depilated enzyme-treated pelts
were processed and finished as dyed crusts as per conventional
procedures. The depilated skins were visually assessed, and the
quality of the removed hair was studied by a light microscope.
Samples were cut from depilated pelts, and then washed and
fixed in formal saline. They were then dehydrated using a graded
ethanol series and freeze-dried. The dried samples from goat
leather treated with rSAPB were cut into approximately 5 mm
thick slices and fixed onto metallic sample holders with conducting
silver glue and then sputtered with a layer of gold (Edwards
E306). The micrographs were then observed using a scanning
electron microscope (SEM) (XL 30 ESEM with an integrated
EDAX system, Philips, Netherlands) operating at an accelerating
voltage of 12 kV. The dyed crust samples were cut and directly
coated with gold for SEM analysis.
2.14. Assessment of the Physical and Chemical Properties
of Dyed Crust
The dyed crusts from both the rSAPB-treated and
chemicallytreated groups were visually assessed for quality and tested for
strength characteristics as per standard procedures. After
conditioning at room temperature and relative humidity of 65% for
48 h, the properties of the crust leather, including tensile strength,
elongation at break, and tear strength were measured using
standard methods . The samples were also assessed for general
appearance and dyeing characteristics. The Cr2O3 content was
determined as per the standard procedure . The crusts were
also analyzed for their hide substance and grease content [18,19].
2.15. Measurements of Pollution Load
To assess the effect of enzymatic depilation on pollution load,
effluents were collected at the end of the depilation of the
rSAPBtreated and control groups and analyzed in terms of
wellestablished pollution parameters, viz. BOD, COD, TSS, sulphide,
calcium, conductivity, and pH following standard analytical
2.16. Bioinformatics and Homology Modeling
The automated comparative protein structure homology
modeling server, SWISS-MODEL (http://www.expasy.org/
swissmod/), was used to generate the three-dimensional model
of SAPB enzymes based on the crystal structure of subtilisin E
(PDB-code 1SCJ). The Deep View Swiss-PDB Viewer software
from the EXPASY server (http://www.expasy.org/spdbv) and
PyMOL v0.99 (http://www.pymol.org) were used to visualize and
analyse the three-dimensional model.
2.17. Statistical Analysis
All data were analyzed using Microsoft Excel. Values are
expressed as means 6 standard deviation of results from three
independent experiments. Data were considered as statistically
significant for P values of less than or equal to 0.05.
Results and Discussion
3.1. Overexpression of sapB Mutated Genes in ProteaseDeficient Bacillus Subtilis DB430
The plasmid constructs pNZ7, pNZ8, pNZ9, pNZ10, pNZ11,
and pNZ12 in which the sapB-L31I, sapB-T33S, sapB-N99Y,
sapB-L31I/T33S, sapB-L31I/N99Y, and sapB-T33S/N99Y
genes were inserted between the restriction sites for EcoRI/
HindIII at the multiple cloning site of pBSMuL2 were prepared in
E. coli, respectively. To guarantee the efficient DNA uptake for
the naturally competent B. subtilis DB430 cells, multimeric
plasmid DNA forms of generated plasmids and pBSMuL2 were
constructed by the in vitro ligation of the linearized plasmids and
used for transformation. Unlike the Kanamycin resistant colonies
of B. subtilis transformed with pBSMuL2, those transformed with
plasmid constructs showed clear zones of casein hydrolysis. This
was correlated with the PCR detection of the sapB genes being
introduced. The recombinant plasmids were identified through
restriction enzyme analysis and DNA sequencing .
3.2. Production of Recombinant and Mutated SAPB Enzymes
The production of SAPB enzymes was noted to start after a 6-h
lag phase and then to increase exponentially and concomitantly
with the cellular growth increases by B. subtilis DB430/pNZ1,
pNZ2, pNZ7, pNZ8, pNZ9, pNZ10, pNZ11, and pNZ12. The
SAPB-N99Y enzyme from B. subtilis DB430/pNZ9 was noted to
exhibit the highest keratinolytic activity of 18,000 U.ml21, with
4,500 U.ml21 of caseinolytic activity, followed by rSAPB
(13,750 U.ml21 of keratinolytic activity and 5,500 U.ml21 of
caseinolytic activity). Interestingly, the levels of protease
production that were obtained by the recombinant strains of B. subtilis
DB430/pNZ9 and B. subtilis DB430/pNZ1 were 21 and 37-fold
higher than those obtained by strains of E. coli DH5a/pBJ28
(210 U.ml21) and E. coli DH5a/pBJ4 (150 U.ml21), respectively,
using casein as a substrate . Unlike the case of the wild-type
strain , this production proved to be highly reproducible, and its
efficiency could presumably be attributed to the strong P59
constitutive promoter carried by the pBSMuL2 shuttle vector.
3.3. Purification and Identification of SAPB Enzymes
The results of the specific activities of the purified recombinant
and mutated SAPB enzymes at the last purification step are
summarized in Table 1. The findings revealed that the rSAPB and
SAPB mutated proteases overexpressed in B. subtilis exhibited
specific activities ranging from 24,950 U.mg21 (for rSAPB) to
45,500 U.mg21 (for SAPB-L31I/T33S/N99Y) when casein was
used as a substrate. In fact, these results are similar to those
reported for the rSAPB and SAPB mutated enzymes expressed in
E. coli whose specific activities were 25,500 U.mg21 and
46,310 U.mg21, respectively . Moreover, the SAPB mutated
proteases overexpressed in B. subtilis were noted to exhibit specific
activities ranging from 15,469 U.mg21 (for SAPB-L31I) to
139,012 U.mg21 (for SAPB-N99Y) when keratin was used as a
The SDS-PAGE analysis of the pooled fractions of each purified
SAPB enzyme overexpressed in the B. subtilis system showed a
single band corresponding to an apparent molecular mass of about
34 kDa . The exact molecular masses of the SAPB-L31I,
SAPB-T33S, and SAPB-N99Y mutant enzymes from the B.
subtilis DB430/pNZ7, B. subtilis DB430/pNZ8, and B. subtilis
DB430/pNZ9 strains were confirmed by MALDI-TOF mass
spectrometry as being 34579.10 Da, 34585.54 Da, and
34601.41 Da, respectively, which are similar to those previously
reported for the same proteases expressed in the E. coli system
. Zymographic analysis also revealed one zone of caseinolytic
activity for the purified sample co-migrating with proteins whose
molecular masses were of approximately 34 kDa . Taken
together, these findings indicate that the heterologous SAPB
enzymes overexpressed in B. subtilis DB430 are monomeric
proteins comparable to those previously reported for SAPB
proteases expressed in E. coli  and for SAPB produced by
B. pumilus CBS .
The N-terminal sequencing of the blotted purified SAPB-L31I,
SAPB-T33S, and SAPB-N99Y enzymes from the B. subtilis
DB430/pNZ7, B. subtilis DB430/pNZ8, and B. subtilis DB430/
pNZ9 strains allowed the identification of the first 21, 23, and 25
amino-acid residues, namely AQTVPYGIPQIKAPAVHAQGY,
AQTVPYGIPQIKAPAVHAQGYKGAN, respectively. These sequences showed
uniformity, indicating that they were isolated in a pure form. The
findings also revealed that the N-terminal amino-acid sequences of
SAPB-L31I, SAPB-T33S, and SAPB-N99Y from the B. subtilis
system were completely identical to those of the same SAPB
enzymes expressed in the E. coli system .
3.4. Substrate Specificity Profiles of SAPB Enzymes
The substrate specificity of proteases is often attributed to the
amino-acid residues preceding the peptide bond they hydrolyze.
The relative hydrolysis rates of various protein substrates were
Table 1. Specific activities and keratin/casein ratios of the purified wild-type and mutant SAPB enzymes using keratin and casein
SAPB Bacillus subtilis Specific activity with
enzyme DB430 keratin (U.mg21)a, b
Relative specific activity to
wild-type with keratinc
Specific activity with
casein (U.mg21)a, b
Relative specific activity to
wild-type with caseinc
aSpecific activity is defined as units (U) of activity per amount (mg) of protein. 1 U of protease activity was defined as the amount of enzyme that liberated 1 mg tyrosine
per min under the optimal temperature and pH values of the respective recombinant enzymes using keratin or casein as a substrate. Proteins were estimated by the
Bradford method using the Dc protein assay kit obtained from Bio-Rad Laboratories (Inc., Hercules, CA, USA).
bThe experiments were conducted three times and 6 standard errors are reported.
cThe relative activity is calculated by taking the specific activity of the wild-type as 1.00.
investigated to elucidate the amino-acid preference/substrate
specificity of SAPB enzymes (Table 2). Among the proteinaceous
substrates tested, rSAPB and SAPB-N99Y were noted to show
highest activity with keratin and keratin azure, followed by elastin.
However, a poor to moderate hydrolysis of casein and albumin
was observed. For the other recombinant SAPB enzymes, on the
other hand, the highest activities were observed with casein and
azo-casein. While a relatively high rate of hydrolysis was observed
with keratin and keratin azure, no collagenase activities were
detected on collagen types I and II, which provided further
support for the relevance of SAPB enzymes for hair removal in the
leather industry. In fact, the lack of collagenase activity is highly
valued in the leather industry because this advantage would reduce
the potential degradation of collagen, the major leather-forming
protein. This criterion was particularly satisfied by rSAPB and
SAPB-N99Y, which highlights their suitability for animal hide
The preferences of the SAPB enzymes for synthetic substrates
incorporating N-terminal residues to the cleavage site (P1, P2, etc.)
have also been elucidated. The Suc-P4-P3-P2-P1-pNA substrate
where, according to the nomenclature of Schechter and Berger
, Suc is a succinyl group and Pn represents the individual
amino-acids, was abbreviated to the 4 core amino-acid residues.
The amino-acids at position P1 exerted strong effects on the
catalytic action of the SAPB enzymes, see Table 2. The order of
the substrate specificity values of rSAPB and SAPB-N99Y
enzymes was almost the same i.e., YLV < AAV < AAI <
AAPF and FAAF. Hence, the rSAPB and SAPB-N99Y enzymes
showed preference for hydrophobic aliphatic amino-acids (valine,
isoleucine, and leucine) at position P1 and exhibited the highest
activity for YLV, AAV, AAI, and AAL. By analogy, most of the
amino-acids at position P1 in the native keratin could be assumed
to be aliphatic residues in nature. This preference for aliphatic
amino-acids could presumably be due to the active site cleft lined
up with aliphatic amino-acid residues.
Furthermore, low or very low hydrolysis was detected when
Met, Ala or Tyr where present at the P1 position. A decrease in
the hydrolysis rate was, however, observed for FAAF, and AAPF,
indicating that catalytic activity was also affected by the
aminoacid in position P2. When Suc-(Ala)n-pNA was used, a minimum
length of two residues was required for hydrolysis. Enzymatic
activity was largely dependent on secondary enzyme substrate
contacts with amino-acid residues (P2, P3, etc.) more distant from
the scissile bond, as illustrated by the differences observed between
the activity of YLV and AAVA, a quality that was previously
demonstrated for microbial keratinases KERUS , KERAB
, and SAPDZ . The SAPB-L31I, SAPB-T33S,
SAPBL31I/T33S, and SAPB-L31I/T33S/N99Y enzymes, on the other
hand, showed preference for aromatic amino-acid residues, such
as Phe and Tyr, and the carboxyl side of the splitting point in the
P1 and P4 positions of pNA substrates. They were, therefore,
active against phenylalanine (FAAF and AAPF) and tyrosine
(LLVY) peptide bonds. Their substrate specificity profile suggested
that they largely preferred hydrophobic substrates, especially those
with aromatic residues occupying the P1 and P4 positions, which
were almost the same i.e., FAAF < AAPF < LLVY.AAPM.
YLV. The nature of the amino-acid in position P2 also influenced
the specificity in position P1. This could be noted from the
differences in the activity of the FAAF and APA substrates; a
decrease in activity was observed when proline was substituted by
alanine, thus confirming the effect of the amino-acid present at
position P2. These characteristics, also reported for other
subtilisins from Bacillus origins [23,24], indicated that the
SAPB-L31I, SAPB-T33S, SAPB-L31I/T33S, and SAPB-L31I/
T33S/N99Y proteases were closely similar to subtilisins not only
in terms of specificity for position P1 but also with regard to the
effects of amino-acids residues neighboring the cleavage site.
Nevertheless, some differences were observed in side chain
specificity at P2, which could presumably indicate the presence
of an extended active site. Proline was also noted to promote
hydrolysis at the P2 position in these SAPB proteases, a feature
that was not observed for subtilisin E .
3.5. Kinetic Parameters
A kinetic study was performed using YLV and FAAF to
investigate the effects of amino-acid residues adjacent to valine and
phenylalanine in peptide substrates. Those pNA peptides were
cleaved at valines and phenylalanines, see Table 3. With pNA,
3 5 5 5
I/T .20 .12 .14 6.2 .23 6.2 .00 .00 .10 .13 .12 .12 .11 .11 .12 6.2 .25 .24 .21 .15 .14
1 6 6 6 0 6 0 6 6 6 6 6 6 6 0 6 6 6 6 6
3 5 0 0 0 1 0 6 6 7 1 8 1 6 4 9 0 6 3 2 8 0
L 7 4 5 1 9 1 0 0 1 4 3 3 2 2 2 1 9 9 8 5 5
1 0 6 6 6 0 6 0 0 6 6 0 6 6 6 6 6 6 6 6 6 6
3 0 8 2 5 0 3 6 6 2 0 0 0 0 7 5 7 9 1 7 5 8
L 1 4 5 8 1 7 0 0 7 8 1 9 7 5 5 7 4 3 1 2 3
.lSuea2Tb ttrsaeSub littrreauaopnN irtaeKn ili-rtscaEenon ilnubAm isaenC iiiftreeddoopnM irrtzaaeeKnu i-szcaeonA llItyaeenogpC llIItyaeenogpC iitttcyeeSdhnpp LYV VAA IAA LAA AAA PAA FAA FFAA FPAA LLYV PAAM LPAA VAAA alsaeuVbeTunh .:i011od
99 00 60 68 69 600 65 60 60 00 67 65 60 61 64 60 60 61 62 60 63 62 in
N 1 6 3 1 1 1 0 0 1 9 9 9 8 6 6 1 1 1 2 3 4
A a 6 6 2 2 2 2 1 1 0 0 0 1 1 1 . t
S le T 0 6 6 6 0 6 0 0 0 6 6 6 6 6 6 6 6 6 6 6 6 d a
t R W 01 25 03 54 01 73 60 60 01 89 29 68 77 06 15 11 21 51 42 53 44 rte ce
kinetic data also indicated that aliphatic and aromatic amino-acids
were by far the preferred residues at positions P1 and P1/P4 for
keratinases and subtilisins, respectively. All the purified SAPB
enzymes exhibited the classical Michaelis-Menten kinetics for the
pNA substrates used. Table 3 summarizes the kcat/Km value of
each enzyme. The findings revealed that rSAPB and SAPB-N99Y
exhibited the highest kcat/Km values of 399 min21.mM21 and
932.50 min21.mM21 when YLV was used as a specific tripeptide
substrate for keratinases. YLV was also the preferred substrate for
SAPB-N99Y, with kcat/Km that were at least 100-fold higher than
those observed for SAPB-L31I/T33S, SAPB-T33S, and
SAPBL31I. The differences between those enzymes were largely due to
the closely similar Km and kcat values (Table 3), indicating that
substrate binding had the greatest effect. This further confirmed
the promising candidacy of SAPB-N99Y for future industrial
application. Likewise, when FAAF was used as a specific
tetrapeptide substrate for subtilisins, SAPB-T33S, SAPB-L31I,
SAPB-L31I/T33S, and SAPB-L31I/T33S/N99Y exhibited kcat/
Km values that were 29, 38, 40, and 70 fold higher than those
recorded for SAPB, respectively.
3.6. Zymogram Analysis
In order to confirm the substrate specificity profile for SAPB
enzymes, the gel-based protease activity assays (zymograms),
which represent a sensitive and rapid assay method for the analysis
of protease activity, were performed using gel-incorporated keratin
or casein as a substrate as described in Section 2. The zymograms
revealed one clear zone against the blue background for the
purified samples, containing varied bands of intensity and with a
molecular weight of about 34 kDa (Fig. 1). When keratin was used
as substrate, the data confirmed that SAPB-N99Y exhibited the
highest activity, followed by the wild-type enzyme, the double
SAPB mutant L31I, and T33S. Poor to moderate keratin activities
were, however, observed with SAPB-L31I, SAPB-L31I/T33S,
Assays were performed using the purified proteases in 100 mM buffer containing 2 mM Ca2+, and 0.2 mM to 50 mM synthetic peptide substrates (YLV and FAAF) at
suitable pH. The samples were incubated for 10 min at suitable temperature. Results are mean values from triplicate experiments. 1 U of protease activity was defined
as the amount of enzyme that catalyses the transformation of 1 mM pNA per minute under standard assay conditions.
SAPB-L31I/T33S/N99Y, and SAPB-T33S (Fig. 1A). Hence, the
wild-type and SAPB-N99Y mutant enzyme displayed the profile of
true keratinases. Similarly, clearer keratinolytic activities were
observed in zymographic assays with the microbial keratinases
KERUS , KERAB , and SAPDZ . Moreover, when
casein was used as a substrate, the data confirmed that
SAPBL31I/T33S/N99Y exhibited the highest activity, followed by
SAPB-L31I/T33S, SAPB-L31I, and SAPB-T33S. Poor to
moderate casein activities were, however, observed with wild-type and
SAPB-N99Y enzymes (Fig. 1B). Accordingly, SAPB-L31I/T33S/
N99Y, SAPB-L31I/T33S, SAPB-L31I, and SAPB-T33S showed
the profile of true subtilisins. Likewise, clearer caseinolytic
activities were visualized on zymograms with other subtilisins
from Bacillus origins [23,25].
Overall, the data correlated well the results obtained by the
kinetics studies. The relative hydrolysis rates observed when
keratin or casein were used as protein substrates and high increase
of protease activity in zymograph assays elucidated the substrate
specificity of the SAPB enzymes.
3.7. Determination of Hydrolysis Degree
The hydrolysis curves of keratin azure after 3 h of incubation
are shown in Fig. 2. The purified enzymes were used at the same
levels of activity (2,000 U) for the production of protein
hydrolysates from keratin azure and for subsequent comparisons
of hydrolytic efficiencies. High hydrolysis rates were attained with
keratin azure (0.4 g.l21) during the first hour of incubation. The
enzymatic reaction rates were noted to decrease thereafter,
reaching a subsequent steady-state phase where no apparent
hydrolysis took place. As shown in Fig. 2, the purified
SAPBN99Y displayed the most efficient keratinase activity (33%),
followed by rSAPB (25%), with SAPB-L31I being the least efficient
(6%). These findings provided further support for the usefulness of
Relative catalytic efficiency to WT
Figure 1. Gel-based protease activity assays (zymograms) demonstrating substrate specificity of SAPB enzymes correlated with
their relative-activity. Zymogram gels were carried out under non-reducing conditions using keratin (left panel) and casein (right panel) as protein
substrates and 50 mg of each purified SAPB enzyme.
SAPB-N99Y and rSAPB in future industrial applications,
particularly for upgrading the nutritional value of keratin.
3.8. Enzymatic Depilation of Animal Hide With SAPB Enzymes
Enzymatic depilation involves the use of keratinolytic enzymes
to cleave the substances that hold the hair to the skin without
causing damage to the hide. Several efforts have been directed
towards developing novel proteases for animal hide depilation.
Most of the proteases so far identified have a collagen degrading
activity that destroys the collagen structure of the hide and are,
therefore, not suitable for depilation. Accordingly, it is important
to identify novel proteases that have no collagenolytic activity and,
hence, efficient depilating activity . In this respect, the
incubation of SAPB enzymes with goat, rabbit, bovine, and sheep
skin samples showed that the skins treated with rSAPB and
SAPBN99Y had their hairs removed very easily and with no visible
damage on the collagen after 8 h of incubation at 37uC as
compared to their control counterparts (Fig. 3). These findings
provided evidence that rSAPB and SAPB-N99Y, alone, could
accomplish the whole depilation process, and that SAPB-L31I/
N99Y and SAPB-T33S/N99Y had moderate depilatory effects.
The comparison with the specific activities illustrated in Table 1
strongly suggests a relationship between the depilation capacity
and the keratin/casein ratio of each SAPB enzyme. In fact the
highest ratio recorded was 4.5 for SAPB-N99Y followed by 2.5 for
rSAPB, the only two enzymes showing full depilation ability. All
the other mutant enzymes had moderate to low keratin/casein
ratios ranging from 1.40 for SAPB-L31I/N99Y to 0.55 for
SAPBL31I. The SAPB-L31I, SAPB-T33S, SAPB-L31I/T33S/N99Y,
and SAPB-L31I/T33S enzymes showed, however, keratin/casein
ratios of 0.55, 0.62, 0.63, 0.70, respectively, and, hence, lacked
In leather processing, depilation is generally carried out at pH
values between 8 and 12 . The fact that SAPB-N99Y meets
this operational criterion provides further support for the strong
candidacy of this enzyme for future application as a hair removal
agent in the leather industry. Comparatively, alkaline proteases
from B. pumilus were reported to have high keratinolytic activity
and to accomplish the depilation process on their own for bovine
hair , cow hides , and goat skins . Accordingly, further
studies, some of which are currently underway in our laboratories,
are needed to test the hide and skin depilation potential of
SAPBN99Y at an industrial scale.
Figure 2. Hydrolysis curves of keratin treated with purified SAPB enzymes. The purified proteases used were: rSAPB, SAPB-L31I, SAPB-T33S,
SAPB-N99Y, SAPB-L31I/T33S, SAPB-L31I/N99Y, SAPB-T33S/N99Y, and SAPB-L31I/T33S/N99Y. Each point represents the mean (n = 3) 6 standard
3.9. Depilating Studies at Semi-Industrial Level With rSAPB
Preliminary experiments were set up to standardize the optimal
conditions for the enzymatic depilation of goat hides by the rSAPB
protease preparation using the dip method. The optimal
conditions obtained were 2,000 U enzyme in water float (100%
w/v) for 8 h. The process time obtained in this study was shorter
when compared to previous results in the literature where the
enzymatic dehairing process was fulfilled in time intervals ranging
from 18 to 21 h for different animal skins [29,30,31,32].
Experiments were then set up to investigate enzymatic depilation
at a semi-industrial scale.
3.10. Visual Assessment
Visual observations revealed that the enzymatically depilated
pelts from goat hides showed white surfaces having hair pores with
no fine hair and that the hair was completely removed along with
the epidermis. The chemically depilated pelts, on the other hand,
were black and showed visible residual hairs in the hair pores. The
black color of the chemically treated skins was due to the use of
sulphide. The hair recovered from enzymatic depilation was intact
in both skins and hides, which was attributed to the absence of the
hair destructing sulphide in the depilating bath. The microscopic
analysis of the hairs obtained from the enzymatic depilation of
goat hides confirmed the presence of hair roots. Contrarily, the
hairs from chemical depilation were pulped with damaged ends,
and the hair roots were absent (data not shown). The intact hair of
good quality can be a value added saleable by-product useful in
organic fertilizers, poultry feed stuff, and felt manufacturing .
Furthermore, the chemically treated pelts were heavier and
more swollen than the enzymatically treated skins, which could be
ascribed to osmotic swelling brought about by the presence of
lime. In the conventional process, osmotic swelling caused by
exposure to high concentrations of lime leads to water absorption
by pelts and, hence, the increase in weight. The hydrostatic
pressure developed in the pelts by water absorption also enhances
the splitting of fibre bundles. The fact that the enzyme-treated
pelts were lighter can be attributed to the higher rates of
interfibriller protein removal from the collagen matrix by the
enzyme. Due to the elimination of lime from the process, the
osmotic swelling of the enzyme-treated skins was not adequate and
was, however, adjusted in subsequent steps of leather processing.
3.11. SEM Analysis
The enzyme-treated samples from the SO. SA. CUIR leather
tannery (MSaken, Sousse, Tunisia) were subjected to
morphological studies using SEM; the photomicrographs are shown in
Figs. 2EG. Compared to the conventional systems, the complete
absence of the above structural features was observed in the
depilated pelts of goat hides obtained after enzymatic dehairing.
While the sections from the chemically depilated pelt showed
remnants of hair root in the hair follicle, those from the
enzymatically treated ones showed that the hair was completely
removed from the root. The collagen structure in the
enzymetreated pelts was intact with no apparent damage to the collagen
fibres. The inactivity of protease on skin collagen is one of the
prerequisite for its application in depilation. Some of the proteases
previously reported for depilation need controlled application for
they were shown to bring relative degrees of damage to the
collagen at the grain layer, which can impart unfavorable
properties to finished leather [34,35].
Information on grain surface of depilated pelt and dyed crust
was obtained by SEM analysis (Figs. 2EG). The findings revealed
that the grain structure of the enzymatically depilated group was
cleaner and that their surfaces were smoother, with opened
collagen fibre bundles, than the chemically dehaired group.
Moreover, the hair pores on the enzyme-treated pelts did not show
residual hair, indicating hair removal from the root. The opening
of the collagen fibre structure was complete and regular in the
enzymatically dehaired pelts, with the degradation of the
interfibrillar substances. The extent of interfibrillar substance
removal is directly proportional to the degree of collagen fibre
bundle opening . This could further accelerate the penetration
of protease through the collagen matrix to act upon anchoring
proteins around the hair follicle and eventually facilitate hair
removal. The enzyme-treated samples also showed a clear surface
with no deposition of foreign particles or grain damage due to
Tensile strength (N.mm22)
Elongation at break (%)
Tear strength (N.mm21)
Stitch tear resistance (N.mm21)
Values represent means of three samples from three sets of experiments, and 6 standard errors are reported.
degradation of the adhering non-structural protein materials
surrounding the hair roots, thus resulting in complete hair
3.12. Physical and Chemical Properties of Dyed Crust
The physical testing of the dyed crust from the rSAPB-treated
goat hides showed that enzymatic treatment did not affect the
strength properties of the leather adversely. The physical
characteristics of the enzyme-treated dyed crusts viz. tear strength,
elongation percentage, tensile strength, and stitch tear resistance
were in good agreement with those of the conventional process
(Table 4). This was due to higher fibre opening in the enzyme
treated pelts, which resulted in higher degrees of softness for the
enzyme-treated leather. The softness of leathers is related to the
opening up of fibre bundles. The crusts of well-opened fibre
structure show more degrees of softness than the moderately or
incompletely opened up ones .
The analysis of the chemical properties of the dyed crusts
obtained from the enzymatic and chemical treatments showed
similar patterns (Table 4). The amount of hide substance recorded
in the enzyme-treated dyed crust from goat hides was 13.6%,
which was approximately equal to the amount registered for the
chemically treated group (20.1%), indicating that the collagen
content was not affected by enzyme treatment. The chrome
content in enzyme-treated goat leather (5.32% for buffalo and goat
skins, respectively) was higher than that of the chemical control
(7.15%), which indicated good fibre opening. The finished leathers
were also assessed for dyeing properties, softness and general
appearance. The results revealed that the enzyme-treated leather
was comparable to the chemically treated one. The oil and fat
content of the enzyme-treated leather was also comparable to that
of the chemically treated leather. In fact, grease gives suppleness
and handle to leather. While too little grease results in hard leather
that will tend to crack, too much grease not only makes the leather
feel greasy but creates subsequent problems during the
3.13. Analysis of Environmental Parameters
Pretanning processes generally account for 7080% of the total
pollution load from the whole leather making process , and the
reduction of pollution at this stage can be considered critical for
making the whole process ecofriendly. Accordingly, the enzymatic
depilation process of skins and hides was assessed in terms of
pollution control parameters, including BOD, COD, TSS,
sulphide, and calcium (Table 5). The effluent collected after the
depilation step from the enzyme-treated group contained
dislodged intact hair and epidermal substances whereas the
effluent from chemical depilation consisted of lime, sulphide,
epidermal substances, and degraded pulped hair. When compared
to chemically treated controls, the total Kjeldahl nitrogen (TKN)
Washing bath after depilation
Washing bath after depilation
Results were average of three different sets of experiments.
and TSS in effluents from the enzymatic depilation of goat hides
were reduced by 5977%. This can be attributed to the
elimination of lime from the process which resulted into sludge
formation. The BOD5 and COD of effluents from the enzymatic
depilation of goat hides were reduced by approximately 38%. This
significant reduction in BOD and COD was due to the removal of
lime and hair degrading sulphide from the process. Degraded
pulped hair, rich in nitrogen, also contributes to high BOD5 and
COD in the effluent from conventional processes. The elimination
of sulphide in enzymatic depilation facilitated the recovery of
intact hair, thus leading to low COD values in the effluent. In
similar studies, 25% reduction in BOD and COD was reported by
Sundarajan et al.  and approximately 50% reduction by
Dayanandan et al. . Moreover, as lime was eliminated from
the enzymatic process, it did not generate an alkaline effluent, and
the pH of the effluent was almost neutral, indicating its lower levels
3.14. Structural Interpretation
In the absence of an experimental three-dimensional structure,
the generation of a model structure through use of known
homologous enzyme structures is helpful for understanding the
roles of various mutations in improving the characteristics of
enzymes. Though no crystal structure is currently available for
SAPB, the 3D structure of this enzyme is likely to be similar to that
of the bacilli subtilisins E, BPN, and Carlsberg, which was
evidenced by their significant sequence identities of 69%, 66%,
and 65%, respectively. The 3D structure of the complex subtilisin
E-pro-peptide  was used as a template to build a model
structure for the SAPB-WT  and SAPB mutant enzymes
(Fig. 4). The possible effects of the three single mutations at
positions 31, 33, and 99 in the SAPB enzyme were studied, using
the homology-model structure along with the known structure of
subtilisin E (PDB-code 1SCJ) from B. subtilis . In fact, the
importance of residues Leu31 and Thr33 with regard to specific
activity and enzymes pH and temperature profile has already
been reported in the literature [11,24]. However, and to the
authors knowledge, no previous work has reported on the
potential roles of those two residues bordering the catalytic residue
Asp32 in substrate recognition and animal hide enzymatic
The generated SAPB models showed that Leu31 was located at
the C-terminal end of the b1 sheet, which was close to the catalytic
cavity (Figs. 3ABC). According to the model, Leu31 was not
directly involved in substrate binding but involved in a network of
hydrophobic interactions that could have shaped the active site of
the enzyme, especially with Ile107 (Figs. 3AD). This latter residue
was directly implicated in the binding cleft subsite S4. According
to the model of SAPB-L31I, an isoleucine in position 31 would not
have established this hydrophobic contact. As a result, the catalytic
cavity might have been enlarged (Fig. 4D) fitting more easily a
bulky residue, such as phenylalanine, which is present in most
subtilases . The kinetic parameters of this mutant
corroborated this hypothesis since the mutant enzyme became more specific
for a substrate that displayed a phenylalanine (FAAF) in position
Concerning the T33S mutation, Thr33 was directly involved in
the substrate binding site being located at the surface of subsite S2
(Figs. 3AE). In the SAPB model, residue P2 would fit exactly the
cavity site and establish a hydrogen bond with the catalytic residue
Asp32. The mutant enzyme model shown in Figs. 4AF, suggests
that subsite S2 was also enlarged and became more selective.
Though the S2 binding site would be expected to be better
adapted for large side chains, the opposite effect was observed. In
fact, substrate docking simulations with the best substrate of the
SAPB-T33S model (FAAF) revealed that it bound in a way that
was similar to that of the YLV tripeptide synthetic substrate in the
SAPB model (Fig. 4B), with the backbone trajectories of the two
peptides being almost identical. More interestingly, the S2 binding
pocket was noted to remain largely unoccupied by the methyl side
chain of the P2 alanine residue, and, consequently, no hydrogen
bonds would be established between the catalytic Asp32 and the
residue in P2. In fact, most of studies so far performed on the role
of catalytic subsites (S1, S2, S3, S4) concluded that the S2 site may
not be vital for defining substrate specificity as compared to the S1
and S4 sites . The findings of the present study, however,
clearly demonstrated the important role of subsite S2 in substrate
specificity, which was further consolidated by the fact that the
catalytic residues His64 and Asp32 were both involved in
maintaining the residue in position P2. Additionally, since Leu31
and Thr33 were close to the catalytic Asp32, this change could be
attributed to the enhanced keratin hydrolysis, which in turn could
explain the unchanged kinetic parameters of the SAPB-L31I and
SAPB-T33S mutants when compared to the wild-type enzyme.
The preference for longer substrates at both sides of the scissile
peptide bond suggests the suitability of keratinases for the
conversion of native and complex substrates. In fact, the cleavage
of peptide bonds in the compact keratin molecules is difficult due
to the restricted enzyme-substrate. Therefore, the hydrolysing
ability of keratinolytic proteases may be due to their ability and
specificity to bind to compact substrates, and more open active
Concerning Asn99, which was located in a turn preceding
bstrand 4 in SAPB (Fig. 4GH), the replacement Asn/Tyr in
SAPBN99Y influenced the shape and flexibility of the gate wall at the
substrate binding cleft, which resulted in high substrate selectivity
[11,24]. Thus, the polarity of the amino-acid at position 99 was an
important factor for substrate affinity due to its localization next to
the substrate binding cleft along with its ability to perform p-p
interactions with the nitroanilide parts of the synthetic pNA
In this study, extracellular alkaline rSAPB protease from B.
subtilis DB430/pNZ1 was purified and characterized, and an
ecofriendly enzymatic depilation process was developed for goat
hides, completely eliminating the use of lime and sulphide. The
process resulted in the complete removal of hair with well opened
collagen fibre bundles, which was further confirmed by SEM
analysis. The dyed crusts produced by enzymatic depilation
displayed better physico-chemical properties when compared to
the chemical group. Moreover the process resulted in a significant
reduction of pollution parameters, making it ecofriendly. The
results from the skin application trials at industrial level supported
the promising biotechnological potential of this enzyme for the
ecofriendly depilation of animal skins in the leather industry
without affecting the quality of the leathers produced. This study
also demonstrated that the Leu31 and Thr33 residues play
important roles in substrate recognition and hydrolysis. The
findings from molecular modeling analyses indicated that L31I
and T33S mutations could directly impinge on the dimensions of
S2 and S4 substrate binding sites affecting enzyme specificity.
They clearly demonstrated that the engineering of kinetic
performances of enzymes of interest is not restricted to the
amino-acids of the catalytic cluster but relates to the hydrophobic
environments near the active site. This study is the first to
demonstrate the promising keratinase and depilation activities of
SAPB and the transformation of subtilisin into keratinase.
The authors wish to express their gratitude to Dr. I. Hssairi, Mr. A. Zitoun,
Mrs I. Rekik, Mrs N. Ayadi, and Mr. F. Ben Kahlaoui (UVRR-CBS) for
their technical assistance and valuable help during the preparation of this
work. We extend our thanks to Pr. H. Belghith and Pr. A. Gargouri
(LVBPPE-CBS) for their constructive discussions and suggestions. Many
thanks are also owed to Pr. Z. Fakhfakh, Pr. A. Kallel, and Mrs S. Damek
(USCR-MEB/03-FSS) for their help with the scanning electron
microscopy. We acknowledge our industrial partner Mr. M. E. Moussa and Mr.
M. S. Bchir from SO. SA. CUIR leather tannery (MSaken, Sousse,
Tunisia) for granting us access to their technical facility to perform our
semi-industrial scale tests on animal hides using the SAPB enzymes. Special
thanks are also due to Pr. A. Smaoui and Mrs H. Ben Salem from the
English department at the Sfax Faculty of Science (Sfax, Tunisia) for their
constructive proofreading and language polishing services.
Conceived and designed the experiments: BJ SB. Performed the
experiments: NZJ HBH HR MB MH. Analyzed the data: NZJ HR.
Contributed reagents/materials/analysis tools: NA HSBA CGH AT.
Contributed to the writing of the manuscript: NZJ BJ SB.
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