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Journal of Cleaner Production xxx (2014) 1e9

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Journal of Cleaner Production


journal homepage: www.elsevier.com/locate/jclepro

Approach to ecofriendly leather: characterization and application of


an alkaline protease for chemical free dehairing of skins and hides at
pilot scale
Nancy George a, Prakram Singh Chauhan a, Vivek Kumar a, Neena Puri b, Naveen Gupta a, *
a
Department of Microbiology, BMS Block, Panjab University, Chandigarh, India
b
Department of Industrial Microbiology, Guru Nanak Khalsa College, Yamunanagar, Haryana, India

a r t i c l e i n f o a b s t r a c t

Article history: The objective of this work was to purify and characterize alkaline protease from Vibrio metschnikovii NG
Received 23 December 2013 155 and to study its applicability as a dehairing agent at pilot scale in leather industry. Relative molecular
Received in revised form weight of the purified protease was found to be 45 kDa with optimum temperature and pH of 65  C and
25 April 2014
10 respectively. Stability of enzyme in wide range of pH (7e11) and temperature (10e50  C) makes it
Accepted 13 May 2014
Available online xxx
suitable for industrial applications. The protease efficiently removed hair from goat skins and buffalo
hides, completely eliminating the use of lime and sulphide within short process duration of 8 h. Efficacy
of the developed process is supported by histology, scanning electron microscopic analysis and quan-
Keywords:
Vibrio metschnikovii
tification of different skin components. In addition, better physicochemical properties of dyed crusts and
Purification lesser pollution load in dehairing process, further affirms the potential of this enzyme for ecofriendly
Dehairing dehairing of animal skins in leather industry.
Leather industry © 2014 Elsevier Ltd. All rights reserved.

1. Introduction serious health hazards to the workers. Hence, need of the time is to
revamp the conventional dehairing process that could lead to a
Leather processing is one of the earliest industrial activities shift from chemical to enzymatic leather processing.
taken up by mankind. Despite of making significant contributions Enzyme based dehairing processes using proteases which lead
to economic development, leather industry is globally facing to substantial reduction of effluent load and toxicity in addition to
challenges owing to the pollution it causes to the environment. The improvement in leather quality is a viable alternative to the con-
close monitoring from the pollution control authorities and ventional chemical based process (Kamini et al., 1999). Although
increased awareness in the society is increasing pressure on the studies on the use of protease for dehairing (either free of lime or
industry to adopt cleaner processing methods. The commercial sulphide or both) are numerous, its application at industrial level is
leather-making process involves sequential set of unit operations not much prevalent. Tanners hesitate to use enzymes because of
which includes pretanning, tanning, post tanning and finishing. At certain limitations like inability to process different types of skins
each stage, variety of chemicals are used and expelled out. Among and removal of fine hair, instability in wide range of pH and
the pretanning operations, dehairing is an important step in which temperature, long duration of process, high production cost etc.
hair along with epidermis, noncollagenous proteins and other (Crispim and Moti, 2003). Apart from this, although many pro-
cementing substances are removed from the skin (Ramasami et al., teases can remove hair but they usually cause degradation of
1999). The conventional dehairing process, where saturated lime collagen at the same time (Najafi et al., 2005). Damage to collagen
and sodium sulphide are employed in high concentrations, layer by the protease can be either due to non specific reaction of
contribute to 50e60% of the total dissolved solids and chemical and enzyme on collagen or presence of collagen degrading enzyme in
biochemical oxygen demand in the effluent (Thanikaivelan et al., the protease preparation (Edmonds, 2008). Collagen fibres are
2004). Moreover, the extensive use of toxic sulphide imparts proteins that form the basic skin structure and are permanently
fixed against any degradation during the tanning process. Enzy-
matic treatment leading to the damage of collagen to any extent
would give unacceptable physical properties to the finished
* Corresponding author. Tel.: þ 91 9872692680; fax: þ91 172 2541770.
E-mail address: gupta27_naveen@yahoo.com (N. Gupta). leather, thus reducing the leather quality (Feigel, 1998). In addition,

http://dx.doi.org/10.1016/j.jclepro.2014.05.046
0959-6526/© 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: George, N., et al., Approach to ecofriendly leather: characterization and application of an alkaline protease for
chemical free dehairing of skins and hides at pilot scale, Journal of Cleaner Production (2014), http://dx.doi.org/10.1016/j.jclepro.2014.05.046
2 N. George et al. / Journal of Cleaner Production xxx (2014) 1e9

enzymatic treatment should also result in removal of interfibriller 2.4. Protein purification and molecular weight determination
cementing substances like sulphated glucoseamine glycans, uronic
acid, hexoseamines and other glycoproteins for opening up of The crude alkaline protease from V. metschnikovii NG 155 was
collagen fibre bundles leading to production of better quality purified to homogeneity by using combination of acetone precipi-
leather (Sivasubramanian et al., 2008a). Removal of these interfi- tation, gel filtration (Sephadex G-150) and ion exchange chroma-
briller components has not been studied for most of the proteases tography (DEAE-Cellulose). All the purification steps were
having dehairing ability. performed at 4  C. Chilled acetone (70%) was added to the cell-free
Some of the enzymatic dehairing processes use silicates culture supernatant. The enzyme precipitate obtained was har-
(Saravanabhavan et al., 2005) or inert material like kaolin in sig- vested by centrifugation at 7826 g for 10 min at 4  C, and the
nificant quantities (Dayanandan et al., 2003) which may contribute pellet was suspended in carbonate bicarbonate buffer (50 mM, pH
to rise in total dissolved solids (TDS), total suspended solids (TSS) 10) and dialysed against the same buffer. Further purification was
and chemical oxygen demand (COD) of effluent. done by gel filtration chromatography. Dialysed sample was loaded
Therefore, till now most of the reported proteases could not be on a sephadex G-150 column (30  1.5 cm2) equilibrated with
exploited for dehairing at industrial level. Consequently, the search 50 mM carbonate bicarbonate buffer (pH 10) and the protein was
is on to isolate/develop novel enzymes for dehairing, having eluted at a flow rate of 1.0 ml min1. The active fractions were
properties suitable for industrial applications. pooled and concentrated by polyethylene glycol (PEG) 6000 grade
Earlier an extracellular alkaline protease producing microor- at 4  C and then applied to a DEAE-cellulose column (12  1.2 cm2).
ganism Vibrio metschnikovii NG 155 was isolated from soil samples The bound enzyme was eluted with 0e1.0 M NaCl gradient at a flow
of leather industry (George et al., 2013). In preliminary experiment rate of 0.8 ml min1. The active fractions were pooled, concentrated
the crude enzyme could efficiently remove hair from goat skin, by PEG and dialyzed. The purified enzyme was analysed by sodium
without damaging the collagen. Present work describes hair saving dodecyl sulphateepolyacrylamide gel electrophoresis (SDSePAGE)
enzymatic dehairing of goat skins and buffalo hides using alkaline using 12.5% (w/v) gel, by the method of Laemmeli (1970). Gel was
protease from V. metschnikovii NG155 at pilot scale in leather in- stained with coomassie brilliant blue R-250 and destained with
dustry and detailed comparison of the physicochemical properties methanol: acetic acid: water (4:1:5). The molecular weight of the
of dyed crusts and pollution load of the enzymatic process with the purified protease was determined using medium range commercial
conventional chemical based process. protein markers (Bangalore Genie Pvt. Ltd.).
Zymography was performed in conjunction with SDS-PAGE
2. Materials and methods (Jellouli et al., 2009). After electrophoresis, the gel was sub-
merged in 50 mM carbonate bicarbonate buffer (pH 10) containing
2.1. Bacterial strain 2% Triton X-100 for 20 min at 4  C, with constant agitation to
remove SDS. Triton X-100 was then removed by washing the gel
Alkaline protease producing bacterium V. metschnikovii NG 155, two times with buffer. The gel was then incubated with 2% (w/v)
(MTCC No. 11401, GenBank Accession No. JN837684) isolated from casein in 50 mM carbonate bicarbonate buffer (pH 10) for 20 min at
soil samples of leather industry was used in this study. The culture 50  C. Then the gel was stained with coomassie brilliant blue R-250.
was grown and maintained in Horikoshi medium (Horikoshi and The development of clear zones on the blue background of the gel
Akiba, 1982) agar slants (pH 10) at 4  C and sub cultured at regu- indicated the presence of protease activity.
lar intervals.
2.5. Effect of temperature on activity and stability of protease

2.2. Enzyme production The optimal temperature for protease activity was determined
by performing enzyme assays in the temperature ranges of
Production of enzyme was done in optimized media (pH 10) 30e70  C (pH 10).
containing Starch (1%), Soybean meal (1%), Wheat gluten meal (1%), Thermostability of the protease was analyzed by incubating the
Cotton seed flour (1%) and KH2PO4 (0.1%) (George et al., 2014). enzyme at temperatures between 10 C and 65  C up to 4 h. Samples
Medium was inoculated with 1% inoculum of 18 h seed culture and were withdrawn sequentially at different time intervals and re-
fermentation was carried out at 25  C at shaking (150 rpm). Cell free sidual activity was measured under standard assay conditions.
supernatant was recovered after 24 h incubation by centrifugation
(7826 g) for 10 min at 4  C and used as source of enzyme. 2.6. Effect of pH on the activity and stability of protease

2.3. Protease activity and protein estimation The optimal pH of protease was determined by preparing the
substrate in the buffers of different pH values i.e. pH 7 (50 mM,
Protease activity was determined by casienolytic method (Atalo phosphate buffer), pH 8 (50 mM, triseHCl buffer), pH 9 (50 mM,
and Gashe, 1993) in which 100 ml of protease was mixed with 400 ml triseHCl buffer), pH 10 (50 mM, carbonate bicarbonate buffer), pH
of casein (6 mg ml1 in carbonate bicarbonate buffer, pH10). After 11 (50 mM, glycine-NaOH buffer) and pH 12 (50 mM, bicarbonate-
5 min of incubation at 65  C, reaction was stopped with 5% TCA. NaOH buffer) and performing the assay at 65  C.
Control was run with same procedure as test except that 5% TCA The pH stability of purified protease was determined by
was added before the addition of enzyme. Then the mixture was measuring the residual enzyme activity at 65  C under the standard
centrifuged and the supernatant was used to measure the tyrosine conditions after pre incubating the enzyme in various buffers
released by Lowry method (Lowry et al., 1951). One unit of protease (50 mM each) of pH 7 to 12 at room temperature up to 4 h without
activity was defined as the amount of enzyme that releases 1 mmol the substrate.
tyrosine per minute under the assay conditions.
Protein concentration was determined by Lowry method using 2.7. Enzyme preparation for leather processing
bovine serum albumin as standard. The proteins eluted after col-
umn chromatography was estimated by taking absorbance at The cell-free supernatant was concentrated by 80% (w/v) satu-
280 nm. ration with ammonium sulphate. The precipitated proteins were

Please cite this article in press as: George, N., et al., Approach to ecofriendly leather: characterization and application of an alkaline protease for
chemical free dehairing of skins and hides at pilot scale, Journal of Cleaner Production (2014), http://dx.doi.org/10.1016/j.jclepro.2014.05.046
N. George et al. / Journal of Cleaner Production xxx (2014) 1e9 3

centrifuged at 7826 g for 20 min at 4  C. The resultant pellet was glucoseamine glycans, uronic acid and hexosamines from skin
dissolved in carbonate bicarbonate buffer (pH 10.0) and dialyzed samples was carried out as per the methods given by Barbosa et al.
against the same buffer for removal of residual salt. This concentrated (2003), Bitter and Muir (1962) and Elson and Morgan (1933)
crude enzyme was used for dehairing in leather processing. respectively. Extraction and estimation of collagen were carried
out using dry fat free samples, according to the methods given by
2.8. Dehairing of goat skins and buffalo hides Neuman and Logan (1950a, 1950b). For all the estimations soaked
skin was taken as control.
Wet salted goat skins and buffalo hides were used for dehairing
experiments. A mini drum (cylindrical rotating reactor, used for hide
2.12. Assessment of physical and chemical properties of dyed crust
and leather processing) fitted at shaking (15 rpm) was used for the
experiment. The skins and hides were cut into two halves along the
The dyed crusts from both the enzyme treated and chemical
backbone. The left half was used for conventional dehairing using lime
treated groups were visually assessed for quality and tested for
and sulphide representing as control in the study and the right half
strength characteristics as per standard procedures. After condi-
was used for enzymatic dehairing. Prior to dehairing, the skins and
tioning the crust leather at room temperature at 65% relative hu-
hides were washed and soaked in water (300% v/wt) for 4e6 h with
midity for 48 h, the properties such as tensile strength, elongation
intermittent changing for removing salt, dirt and blood. The soaked
at break and tear strength and compression index were measured
weight of all the left and right halves were noted and the percentage of
using standard methods (IUP 6, 2000). The dyed crusts were also
chemicals and enzyme used in the experiment was based on this
assessed for general appearance and dyeing characteristics. The
soaked weight. For the conventional process paste method was used
chromium oxide (Cr2O3) content was determined as per the stan-
in which 10% lime and 2% sodium sulphide were mixed with 10%
dard procedure (IUP 8, 2002). Samples were also analyzed for their
water and the paste thus prepared was applied on the flesh side
hide substance and grease (oil and fat) content (IUC 10, 1984; IUC 4,
(Sivasubramanian et al., 2008b). It was left overnight at room tem-
2008).
perature and then dehaired by rubbing. For enzymatic group, opti-
mized dip method was adopted, wherein the soaked skins were
dipped in water float (pH 8 ± 0.5, 200% v/wt) containing 100 U ml1 2.13. Measurements of pollution load
enzyme. The float was left for 8 h at 25 ± 5  C and then dehaired by
rubbing. Depilated skins/hides were visually assessed and quality of To assess the effect of enzymatic dehairing on pollution load, the
hair removed was studied by light microscope. After dehairing, con- effluents were quantitatively collected at the end of the dehairing
ventional industrial procedures were followed to convert the chemi- process of control and enzyme treated group and analyzed for
cal and enzyme treated dehaired pelts into dyed crusts. pollution parameters viz. chemical oxygen demand (COD), biolog-
ical oxygen demand (BOD), total dissolved solids (TDS), total sus-
2.9. Histological studies pended solid (TSS), calcium, sulphide and pH by following the
standard analytical procedure (APHA, 1995).
Samples of 2 cm2 were cut from identical portions of dehaired
pelts of skins and hides, washed and fixed in formal saline (0.85%
2.14. Statistical analysis
sodium chloride in 10% formaldehyde solution). Samples were then
dehydrated with ethanol series and embedded in paraffin block to
All the experiments were carried out in triplicates and the
cut sections of 6 mm using microtome. Sections were then stained
mean ± standard deviation has been plotted. Data was analyzed
using hematoxylin and eosin (H&E) to analyse the histological
using analysis of variance (ANOVA) by Sigma Stat version 2.03 and
features (Bancroft and Gamble, 2004).
values which were statistically significant (p value  0.05) were
taken.
2.10. Scanning electron microscopy

Samples were cut from dehaired pelts, washed and fixed in 3. Result and discussion
formal saline. Then the samples were dehydrated using a graded
ethanol series and were finally freeze-dried. The dried samples V. metschnikovii NG 155 isolated earlier in our laboratory pro-
were cut into approximately 4 mm thickness, coated with 20 nm of duced extracellular alkaline protease, capable of dehairing the an-
gold and examined in a Scanning electron microscopy (SEM) unit imal skins efficiently with no damage to skin collagen. The non
(S-3400 N, Hitachi) operated at an accelerating voltage of 10 kV. collagenolytic nature of the crude enzyme was confirmed by
The dyed crust samples were cut and directly coated with gold for treating it with pure collagen, which did not show any degradation
SEM analysis. (George et al., 2013). The production of protease by this strain has
been optimised to 200 U ml1 using statistical methods (George
2.11. Quantitative analysis of skin components et al., 2014). In this study the enzyme was purified, characterized
and applied for dehairing at pilot scale in leather industry. Results
Dehaired skin/hide samples were cut into pieces, washed and were compared to the chemical based process in terms of quality of
dehydrated using ethanol series. Estimation of sulphated leather produced and pollution load.

Table 1
Summary of purification of alkaline protease from Vibrio metschnikovii NG155.

Purification step Total activity (U) Total protein (mg) Specific activity (Um g1) Purification fold Recovery (%)

Crude 40,000 644 62.10 1 100


Acetone precipitation 34,000 212 160.37 2.58 85
Sephadex G-150 Gel Filteration 22,400 28 800 12.88 56
DEAE-cellulose ion exchange chromatography 7680 8 960 15.45 19

Please cite this article in press as: George, N., et al., Approach to ecofriendly leather: characterization and application of an alkaline protease for
chemical free dehairing of skins and hides at pilot scale, Journal of Cleaner Production (2014), http://dx.doi.org/10.1016/j.jclepro.2014.05.046
4 N. George et al. / Journal of Cleaner Production xxx (2014) 1e9

The optimum pH for activity of alkaline protease from


V. metschnikovii NG 155 was found to be 10 and more than 40% of
the maximum activity was detected for the protease at pH 11.0 and
12.0 (Fig. 3A). The enzyme was highly stable between pH 7e11 as it
retained more than 70% of the activity up to 3 h (Fig. 3B). As leather
manufacturing is a water intensive process and pH and tempera-
ture of water varies depending on source, region and season
(Thanikaivelan et al., 2005), activity and stability of protease from
V. metschnikovii NG155 in wide range of pH and temperature makes
it suitable for its application at industrial level.

3.3. Dehairing studies at pilot scale in leather industry

Before going for pilot scale studies, experiments were set up to


standardize the optimal conditions for enzymatic dehairing of goat
skins and buffalo hides using the protease preparation by dip
method. The dehairing process was optimized in terms of pH,
Fig. 1. SDS-PAGE analysis of alkaline protease from V. metschnikovii NG155 at different temperature ( C), enzyme dose (U ml1) and duration (h) of
purification steps; Lane 1: molecular weight markers, Lane 2: crude enzyme, Lane 3: treatment. The optimized enzyme dose and time of the treatment
gel filtration fractions, Lane 4: DEAE-cellulose fractions, Lane 5: zymograph of purified
was 100 U ml1 and 8 h. The process time obtained in this study
protease.
was lesser when compared with other literature reports on enzy-
matic dehairing, ranging from 18 h to 21 h for different animal skins
3.1. Protein purification and molecular weight determination (Dayanandan et al., 2003; Nilegaonkar et al., 2007; Kumar et al.,
2011). Enzyme could remove hair with equal efficiency in wide
The V. metschnikovii NG155 alkaline protease was purified to range of pH and temperature. It could bring complete dehairing
homogeneity using sephadex G-150, gel filtration chromatography when diluted in buffers of pH 8, 9, 10 and even in tap water
followed by DEAE-cellulose anion-exchange chromatography. The (pH8 ± 0.5). Similarly complete dehairing was observed at tem-
results of the purification procedures are summarized in Table 1. peratures above 25  C by 8 h, at low temperatures (10  C, 15  C and
The enzyme was purified to 15.45 fold with a recovery of 19%. SDS- 20  C) dehairing could be achieved with little longer treatment.
PAGE analysis of purified enzyme showed a major band of 45 kDa Experiments were then set for enzymatic depilation at pilot scale in
(Fig. 1) which also coincided with active band on zymograph. leather industry with optimal dose of the enzyme and time in tap
Generally the molecular masses of alkaline proteases from micro- water (municipal water supply, pH8 ±0.5) at 25 ± 5  C as these are
organisms range from 15 to 30 kDa with few exceptions eg. alkaline the conditions generally prevalent in industry.
protease reported from V. metschnikovii ATCC700040, which had
molecular weight of 50 kDa (Park et al., 2012). 3.3.1. Visual assessment
After dehairing, visual observations of enzymatically dehaired
pelts from goat skins and buffalo hides revealed that there was
3.2. Effect of pH and temperature on the activity and stability of complete and uniform removal of hair from all over the area,
protease showing white surface having clean hair pores with complete
absence of fine hair. Contrarily, chemically dehaired pelt was black
The purified protease showed maximum activity at 65  C and and residual hair was visible in the hair pores. The black colour of
more than 40% of activity could be detected at temperatures be- the chemical treated skins could be due to the use of sulphide
tween 30  C and 70  C (Fig. 2A). Thermostability profile of the (Dayanandan et al., 2003). The hair recovered from enzymatic
enzyme showed that it could retain up to 70% of activity for 3 h at dehairing was intact in both skins and hides owing to the absence
10e50  C (Fig. 2B). The half-life of protease at 65  C was found to be of hair destructing sulphide in the dehairing bath. The microscopic
1 h. The enzyme was slightly more thermostable than protease analysis of the hair obtained from enzymatic dehairing skins as well
reported from some other V. metschnikovii strains which have as hides confirmed the presence of hair root. Contrarily hair
temperature optima at 60  C and half life of 30 min at 60  C (Jellouli recovered from the chemical dehairing were pulped with damaged
et al., 2009; Mei and Jhang, 2005). ends and hair root were also absent (Supplementary Fig. 1). The

Fig. 2. Determination of optimum temperature (A) and temperature stability (B) of protease from V. metschnikovii NG155.

Please cite this article in press as: George, N., et al., Approach to ecofriendly leather: characterization and application of an alkaline protease for
chemical free dehairing of skins and hides at pilot scale, Journal of Cleaner Production (2014), http://dx.doi.org/10.1016/j.jclepro.2014.05.046
N. George et al. / Journal of Cleaner Production xxx (2014) 1e9 5

Fig. 3. Determination of optimum pH (A) and pH stability (B) of purified protease from V. metschnikovii NG155.

intact hair of good quality can be a value added saleable by product Complete absence of the above structural features was observed in
for use in organic fertilizers, manufacture of felt, and poultry feed dehaired pelts obtained after enzymatic dehairing whereas,
stuff (Sivasubramanian et al., 2008b). The chemical treated pelts removal of these components was incomplete in chemically
looked heavier and swollen than the enzyme treated skins which dehaired pelts. The collagen structure in enzyme treated pelts was
can be due to osmotic swelling brought about by lime (Kamini et al., intact with no apparent damage to the collagen fibres. The inac-
1999). In the conventional process osmotic swelling brought about tivity of protease on skin collagen is one of the prerequisite for its
by high concentration of lime leads to absorption of water by the application in dehairing. Some of the reported proteases for
pelts and increase in weight. Due to the elimination of lime from dehairing need controlled application as they damaged the
the process, the osmotic swelling of the enzyme treated skins was collagen of grain layer to a certain extent, due to the presence of
not adequate, which was however, adjusted in subsequent steps of collagen degrading protease in the enzyme preparations (Najafi
leather processing. et al., 2005; Haddar et al., 2011). This can impart unfavourable
properties to finished leather.
3.3.2. Histology and scanning electron microscopy Further information on effect of enzymatic dehairing was ob-
The histological micrographs of pelts conformed the visual tained by scanning electron microscopic (SEM) analysis of dehaired
assessment. Hematoxylin and eosin (H&E) stained sections were pelts and dyed crusts (Figs. 5 and 6) which showed that the grain
analysed for the histological features like extent of removal of structure of enzymatically dehaired group was cleaner and surface
epidermis, glandular structures, hair shaft and hair root (Fig. 4). appeared to be smoother than chemically dehaired group.

Fig. 4. Hematoxylin and Eosin stained sections of pelts, from goat skin (A) chemically dehaired skin (B) enzymatically dehaired skin and buffalo hides (C) chemically dehaired hide
(D) enzymatic dehaired hide (100X).

Please cite this article in press as: George, N., et al., Approach to ecofriendly leather: characterization and application of an alkaline protease for
chemical free dehairing of skins and hides at pilot scale, Journal of Cleaner Production (2014), http://dx.doi.org/10.1016/j.jclepro.2014.05.046
6 N. George et al. / Journal of Cleaner Production xxx (2014) 1e9

Fig. 5. Scanning Electron Micrograph showing grain surface of dehaired pelts from goat skin (A) chemically dehaired skin (B) enzymatically dehaired skin and buffalo hides (C)
chemically dehaired hide (D) enzymatic dehaired hide (55 X).

Moreover, hair pores on enzyme treated pelts did not show residual the enzyme. The removal of interfibriiler proteins and inactivity of
hair, stipulating removal of hair from root. Opening of collagen fibre enzyme on collagen were further confirmed by histological analysis
structure was complete and regular in enzymatically dehaired using specific stains for different skin components, which showed
pelts. Well opened fibre bundles could further accelerate the significant removal of these proteins from the skin with no damage
penetration of protease through collagen matrix to act upon to the collagen layer (data not shown).
anchoring protein around the hair follicle eventually facilitating the
removal of hair (Thanikaivelan et al., 2002).
3.3.4. Physical and chemical properties of dyed crust
Physical testing of the dyed crust made from the enzyme treated
3.3.3. Quantitative analysis of skin components skin/hide showed that the enzymatic treatment did not affect the
To analyse the effect of enzymatic dehairing on different skin strength properties of the leather. Physical characteristics of the
components their quantitative estimation was done. Reduction in enzyme treated dyed crusts viz. tear strength, percent elongation
the levels of interfibriller constituents such as sulphated glucose- and tensile strength was in good agreement with the conventional
amine glycan (GAG), uronic acid and hexosamines was observed in process (Table 3). The compression index value of the enzyme
the dehaired pelts of both chemical as well as enzymatic processes treated dyed crusts were higher than the chemical controls. This
(Table 2). However, the reduction was more significant in the may be due to higher fibre opening of enzyme treated pelts which
enzymatically dehaired pelts. 60e70% of these constituents were resulted in higher softness of enzyme treated leather. Softness of
removed from skin/hide in the enzymatic process whereas it was leathers is related to opening up of fibre bundles, crusts of well-
approximately 40e60% for chemical process. Extent of removal of opened fibre structure had more softness than the ones, which
interfibriller substances is directly proportional to the degree of were moderately or incompletely opened up (Sivasubramanian
opening up of collagen fibre bundles. The interfibriller cementing et al., 2008b).
substances makes the collagen fibres compact and inhibits the Analysis of chemical properties of dyed crusts obtained from
diffusion of various agents, which need to penetrate during the enzymatic treatment and chemical treatment showed similar
making of leather (Cantera et al., 1996). Hence, elimination of these pattern (Table 4). Amount of hide substance in the enzyme treated
noncollagenous components leading to opening up of collagen fi- dyed crust for goat skins and buffalo hides were 62% and 65%
bres is necessary for production of better quality leather respectively, which was approximately equal to the chemical
(Sivasubramanian et al., 2008a). An important observation was that treated group (63% and 64% respectively) which indicated that the
the collagen content remained unaffected in enzymatically collagen content was not affected by enzyme treatment. The
dehaired pelts, which confirmed the non collagenolytic nature of chrome content in enzyme treated leathers (5%, 5.1% for buffalo and

Please cite this article in press as: George, N., et al., Approach to ecofriendly leather: characterization and application of an alkaline protease for
chemical free dehairing of skins and hides at pilot scale, Journal of Cleaner Production (2014), http://dx.doi.org/10.1016/j.jclepro.2014.05.046
N. George et al. / Journal of Cleaner Production xxx (2014) 1e9 7

Fig. 6. Scanning Electron Micrograph of dyed crusts from goat skin (A) chemically dehaired skin (B) enzymatically dehaired skin and buffalo hides (C) chemically dehaired hide (D)
enzymatic dehaired hide (370 X).

goat skins respectively) was higher than the chemical controls 3.3.5. Analysis of environmental parameters
(4.23%, 4.50% respectively). Enzyme treated leather was found Pretanning processes generally accounts for 70e80% of the total
comparable to chemical treated leather in terms of dyeing prop- pollution load from whole of the leather making processes
erties, general appearance, oil and fat content. Grease gives sup- (Thanikaivelan et al., 2004) and reduction of pollution at this stage
pleness and handle to leather. While too little grease results in hard can be decisive to make the whole process ecofriendly. Hence
leather that will tend to crack, too much not only makes the leather enzymatic dehairing process of skins and hides was assessed in
feel greasy but creates problems later, during the manufacturing terms of pollution control parameters such as BOD, COD, TDS, TSS,
process. calcium and sulphide (Table 5). The effluent collected after the

Table 2
Quantitative analysis of components of dehaired skins and hide.

Skin components Goat skin Buffalo hide

Control Chemical dehairing Enzymatic dehairing Control Chemical dehairing Enzymatic dehairing

Collagen (mg g1 of dry fat free skin) 330 ± 10 324 ± 12 323 ± 10 370 ± 15 365 ± 10 363 ± 12
Uronic acid (mg g1 of skin) 2580 ± 135 1565 ± 34 980 ± 13 2800 ± 25 1620 ± 56 1150 ± 25
Hexosamines (mg g1 of skin) 3500 ± 141 1640 ± 15 1100 ± 22 3660 ± 88 1750 ± 56 1280 ± 110
Sulphated GAGs (mg g1 of skin) 1650 ± 38 785 ± 49 550 ± 45 1720 ± 55 850 ± 14 680 ± 65

Values were mean ± standard deviation for three samples from three sets of experiments.

Table 3
Physical characteristics of dyed crusts produced from goat skins and buffalo hides.

Sample/method Tensile strength (N mm2) Elongation at break (%) Tear strength (N mm1) Compression index

Parallel Perpendicular Parallel Perpendicular Parallel Perpendicular

Goat/conventional 24.60 ± 0.82 17.20 ± 0.35 46.34 ± 1.46 68.08 ± 3.40 42.06 ± 2.61 37.82 ± 2.72 5.32 ± 0.4
Goat/enzymatic 25.78 ± 1.02 18.01 ± 0.40 49.01 ± 2.61 74.18 ± 2.84 43.06 ± 1.90 37.71 ± 1.44 5.78 ± 0.2
Buffallo/conventional 28.01 ± 1.21 24.01 ± 1.18 49.18 ± 2.87 58.98 ± 0.89 45.12 ± 0.76 37.88 ± 0.65 5.10 ± 0.3
Buffalo/enzymatic 28.33 ± 0.89 24.34 ± 2.54 49.11 ± 1.21 60.01 ± 2.10 45.77 ± 1.33 38.65 ± 0.26 6.01 ± 0.5

Values were mean ± standard deviation for three samples from three sets of experiments.

Please cite this article in press as: George, N., et al., Approach to ecofriendly leather: characterization and application of an alkaline protease for
chemical free dehairing of skins and hides at pilot scale, Journal of Cleaner Production (2014), http://dx.doi.org/10.1016/j.jclepro.2014.05.046
8 N. George et al. / Journal of Cleaner Production xxx (2014) 1e9

Table 4 Acknowledgment
Chemical Properties of dyed crust produced from goat skins and buffalo hides.

Properties Goat skin Buffalo hide Authors acknowledge University Grant Commission, Govern-
Conventional Enzymatic Conventional Enzymatic
ment of India, New Delhi for the financial assistance and J D Tan-
neries Pvt. Ltd. Jalandhar, India for supporting the research work.
% Hide substance 62.81 ± 2.10 63.50 ± 1.70 65.23 ± 1.02 64.86 ± 1.20
% Oils and fats 13.14 ± 0.21 14.31 ± 0.82 13.44 ± 0.86 13.02 ± 1.20
% Cr2O3 4.23 ± 1.01 5.01 ± 0.21 4.50 ± 0.10 5.10 ± 0.13 Appendix A. Supplementary data
Values were mean ± standard deviation for three samples from three sets of
experiments. Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.jclepro.2014.05.046.
Table 5
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Please cite this article in press as: George, N., et al., Approach to ecofriendly leather: characterization and application of an alkaline protease for
chemical free dehairing of skins and hides at pilot scale, Journal of Cleaner Production (2014), http://dx.doi.org/10.1016/j.jclepro.2014.05.046