Indian Journal of Chemical Technology

Vol. 11, September 2004, pp. 659-671

Enzyme technology applications in leather processing

R B Choudhary, A K Jana & M K Jha*
Department of Chemical and Bioengineering, National Institute of Technology, Jalandhar 144 011, India
Received 19 August 2003; revised received 30 April 2004; accepted 28 May 2004
The emphasis on the use of enzymes has come about because of the unique properties of the enzymes. The most
important properties are the catalysis of chemical reactions at high rate under mild environmental conditions of pH,
temperature and pressure, specificity of reactions, minimal side reactions, simple operations, non toxic nature and non
polluting effluent generations. The leather industry world over is coming under pressure from environmental regulations to
comply with the pollution and discharge legislation. The current activity in the area of leather processing is shifting towards
the design and utilization of cleaner and softer technology like enzymatically enhanced processes. The enzymes are
successfully employed for the better quality leather production with less pollution impact and also for the treatment of waste
discharged from the industry. The leather processing from the raw skins to the finished products required the various steps
like curing, soaking, liming, dehairing, bating, pickling, degreasing and tanning. The various processing principles have
been discussed in brief along with application of suitable enzymes, their properties and sources. It showed that leather
industries have enormous potential for the wide range of applications of several industrial enzymes.
IPC Code: C14 B 1/00
Keywords: Leather processing, enzyme technology

Interest in enzyme system is a result of a desire to

utilize their vast catalytic potential, high specificity
and high catalytic activity under mild environmental
conditions of pH, temperature and pressure. The use
of enzymes, although only recently understood, has
been going on for centuries. Since micro-organisms
are responsible for the fermentation of beer, wine,
bread, cheese and various vegetables, all these
processes are examples of cell-mediated conversions
or application of enzymes. Current technology makes
it possible to isolate, purify, even to immobilize (bind
to fixed support) the specific enzymes needed for a
desired function. Enzymes claim potential
applications in agriculture, leather, food, textile and in
pharmaceutical industries1-6. Enzymes play significant
role in industrial effluent treatment, water treatment,
petroleum sludge degradation, crude oil spill
treatment, fly ash dump reclamation, eco-restoration
of mine dumps and degraded eco-system7. Enzymes
are also used for many more typical applications
like fuel oil additive to improve dispersion and
flame temperature, mould release agent in
building and construction industry, removal of
dead tissue and dissolution of blood clots, reverse
__________
*For correspondence (E-mail: [email protected];
Fax: 0181 690320-690932)

hydrolysis in the aspartame synthesis, surfactant for

bitumen in the surfaced roads, modification of protein
rich materials, and removal of turbidity in the
beverage.
Animal skin goes through a series of operations
prior to the making of various leather goods. The
enzymatic action in leather production was reported to
have started at research level in the early of the 20th
century and first patent was taken by Rohm8 in 1910
for the use of enzyme in bating. It took more than
seventy years to apply them on industrial scale for
dehairing process. Later on enzymes were
successfully employed for the better quality leather
production and also for the waste treatment in the
leather industry. The prime stages in leather
processing are curing, soaking, liming, dehairing,
bating, pickling, degreasing and tanning. The
discharges and refuges disposed from all these
processing stages in the leather production, causes
severe health hazards and environmental problems to
the entire eco-system. The huge amount of industrial
effluents contain relatively higher amount of sulphide
and chromium for improving the quality of tanning in
the leather production. The leather industry world
over is therefore, coming under high pressure from
environmental regulation to comply with the pollution
and discharge legislation. Because of the restriction of

INDIAN J. CHEM. TECHNOL., SEPTEMBER 2004

660

the environmental protection agency (EPA), dealing

with dissolved solid levels in plant effluents,
processing of brine cured hide is sometimes
uneconomical. This is the reason, US exports a large
volume of the hides to other countries, where
environmental restrictions are less stringent, and it
buys finished leather from abroad, at a value added
price. As a result, the leather industry is looking for
cleaner option for the dehairing process. The current
activity in this area indicates that the trend is shifting
towards design and utilization of cleaner and safer
technology
like
enzymatically
enhanced
processes6,910. A number of different enzymes
(proteases, lipases, amylases) have been used in
leather processing in these directions.
Traditionally, enzymes found in dog were used to
treat leather to make it pliable by removing some
protein components. The reason behind the use of
protease lies in the fact that the protein is the major
constituent of hair and skins. Hair is composed of keratin fibres, insoluble protein molecules containing
a large fraction of cysteine residues and having an helix conformation. The -keratin is arranged in piles
of fibrils. Different skin layers are composed of
collagens, -keratin, and some elastin. Collagen
contains a large fraction of glycine, alanine, proline,
and hydroxy proline. These are arranged in a triple
helix conformation11. The use of enzymes with
different specific constituents in leather makes it
possible, selective hydrolysis of the noncollagenous
constituents of the skin.
Dehairing is the single largest process in leather
production, which requires huge amount of industrial
enzymes like proteases, amylases and lipases. Works
on enzymatic option for the dehairing process, were
carried out by Raju et al.12 using a strain belonging to
Bacillus isolated and evaluated for its efficacy. The
activity and the performance of the enzymes satisfied
the condition necessary for its application in

dehairing. Enzymes have good potential to be

exploited as an environmentally friendly option in
dehairing as the trend is shifting towards cleaner
technology. Therefore, the potential for the industrial
use of enzymes in leather processing is very high
because of their marked properties as highly efficient
and selective catalysis. The resulting saving in
process time increases efficiency and allows increased
leather output as well.
Enzyme applications
Bull hides, buffalo hides, steer hides, heifer hides,
calf hides, bovine skins, goat skins, sheep skins and
many more precious hides and skins have been the
concern of high interest for leather professionals all
around the world. Table 1 shows some typical events
in relation to the chronological development of the
leather technology and the enzymatic involvement.
From the data of chronological development, it is
observed that leather technology in the early of the
twenty-century rarely used any enzymatic treatment,
although the demand for quality leather has been the
matter of concern throughout the ages.
The various important processing methods involved
in the leather manufacture are curing, soaking, liming,
dehairing, bating, pickling, degreasing and tanning.
All these successive steps in the leather production
involve enzymatic action directly or indirectly for
facilitating the procedures and enhancing the leather
output of desired quality. Table 2 showed the extent
to which the enzymatic action have been involved at
different stages of leather processing. Enzymes are
mainly used in soaking, unhairing, bating, degreasing
and waste processing of leather industries.
Curing

Curing is the process of preserving the hides from

getting spoiled well before the exercise of various

Table1Major land marks in the leather technology development in 20th century

Year

Researchers

Some typical events in leather technology

1913
1914
1922
1931
1958
1960
1970
1972
1985

Rohm & Haas

Loveland F A
Mc Laughlin
Theis E R
Strandine & Connick
Mycek & Clarke
Folk, Cole & Chung
Chung
Blair & Sirolime

Attempts for enzymatic dehairing process were made

Concept of white weight and comparative were studied
Brine-cured hides were found much better white weight
Effect of salt in lime liquor with Na2Sn were studied
Effect of storage on brine-cured hides were studied
Research report on tranglutaminse were made public
Basic research on mechanism of enzyme action started
Isolation of transglutaminase from hair follicle done
Blair - Sirolime methods of dehairing process developed

CHOUDHARY et al.: ENZYME APPLICATIONS IN LEATHER PROCESSING

661

Table 2Enzymatic function and its involvement at different leather processing stages
Stages

Enzymes involved

Function of enzymes

Curing
Soaking
Dehairing
Degreasing
Bating
Tanning
Waste processing

Enzyme are directly not involved

Alkaline & pancreatic proteases
Alkaline & neutral proteases
Lipases & proteases
Trypsin & alkaline proteases
Enzyme are directly not involved
Trypsin & proteolytic enzymes

To preserve hides and skins

To remove non fibrillar proteins
To improve the waste water quality
To remove fats
To make soft, supple and pliable
To influence the quality of tanning
Chrome- tanned-waste processing

processing and to use them purposefully later on. If

the hides and skins are not cured just after flaying,
they get putrefied within two-three days. Therefore,
hides are required to immediate thorough curing to
stop them from deterioration. Hides are steeped in a
brine bath and dried in the sun and salt is added to the
flesh side. For curing, dry, airy and clean places are
preferred. Curing is done at controlled temperature,
pH, moisture and by using toxic materials. In curing,
use of biocides has been preferred although these are
inimical to the environment. Radiation curing is one
among several curing methods that is theoretically an
attractive and alternate method but practically this is
not feasible. Therefore, salt curing is still prevalent in
many countries. Some times the presence of
organisms on cured hides reduces the value of the
hides as a raw material for leather manufacture. The
quality of leather manufactured from the brine cured
cattle hides are known to deteriorate on prolonged
storage, particularly at elevated temperatures. The
deterioration is probably due to the presence of
proteolytic enzymes produced by micro-organisms
growing on the hide. Curing the hide with the salt to
overcome the degenerative process has been used for
long. If the hide is properly salt cured, the activity of
the organism is readily controlled13.
Soaking

Soaking is the first tanning operation for treatment

of hides and skins with water. Hides are first soaked
for rehydration before further processing. The better
the rehydration, superior the leather. In this stage
hides and skins are washed and soaked in surfactants
and anti-microbial compounds. This process is
preferred to facilitate the further processing of leather.
Green hides and skins are soft enough and therefore
do not require any soaking14. A salt-cured hide
requires a soaking process that raises the moisture
content from 45 percent to greater than 52 percent.
This is achieved through a carefully designed and
monitored soaking process. The longer the soak, the

more significant the bacterial threat is to the crude

material. The method of soaking for a pack of hides
and skins depends mainly upon its conditions and to a
less extent upon the type of leather which is going to
be produced. In developed countries like Europe and
America, proteolytic and amylolytic enzymes are
widely used for soaking. Use of enzymes in soaking
have been tried successfully since 1966 because
surfactants in excess amounts cause pollution15.
Several works on swelling for brine-cured hides with
enzyme soak has also been reported providing
important information about swelling versus
immature collagen16,17. In a study of various types of
soaks on the rehydration of brine cured hides,
Tancous et al.18 exercised six different soaking
methods (three enzymatic methods and three
conventional methods) and compared for their
effectiveness in rehydrating the center portion. The
ultimate goal of the study was to reduce the incidence
of hard spots. A 45 percent reduction in the soaking
time over conventional methods and a 40 percent
reduction in the amount of sulphide used for dehairing
was found. The enzymatic method, which employed
the use of, protease + surfactant, was the best of the
six methods attempted. The type of soaks used and
the soaking times are listed below:
Protease + surfactants 5 hours
Lipases + surfactants 5 hours
Proteases + lipase 5 hours
Soda ash + surfactants 9 hours
Regular process (sodium tetrasulphide +
surfactant) 9 hours
Modified process (sodium tetrasulphide +
surfactant) 9 hours
For the two set of experimental values determined,
the typical values for sulphide (mg/L) used in the
regular processing were 2490 and 2340 and in the
protease + surfactants were 1330 and 1490, a 41
percent reduction. Values for the protease + lipase

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INDIAN J. CHEM. TECHNOL., SEPTEMBER 2004

process were 1450 and 1340, a 42 percent reduction

over the regular process. Values for soda ash +
surfactant; regular process modified, and lipase +
surfactant were 2090, 2180; 2332, 2530; and 2410
and 2360. With regard to the effect of soak types, the
centre layers of the soaked hides varied considerably
depending upon the type of soak used. The protease +
surfactant appeared as the best rehydration of the
center layers at 59.9 percent, the other process were
less effective. The other soak type showed lower
values at 55.3, 56.6, 56.2 and 55.6 percent for the
protease + lipase, soda ash, regular processing and
regular processing modified. Zung's19 work has
recently shown that protease has positive impact in
the soaking of salt-cured hides. By using a special
historical techniques, Zung found that enzymes
definitely penetrated both sides of the hide to the
depth of one-twentieth to one-tenth of the thickness
and that some enzymes penetrated into the centre of
hide by means of the blood vessels. When leather was
produced, only a short, four-hour soaking time was
cleaner than that of the control leather. Protein
between the fibres of hides are removed by
proteinases. The removal of the proteins disturbs the
molecular structure and enables faster rehydration or
soaking.
Liming

Hides and skins are taken for the liming operation

after soaking. It is learned that most of the skins are
not sufficiently swollen and they need a liming
treatment for desired swelling. In this process, the
soaked hides and skins are treated with milk of lime.
It gives the desired swelling of the collagen structure
which helps to open up the fibre bundles. The
objective of this process is to remove the hairs, nails,
hooves and other keratinous matters and also to
remove the interfibrillary soluble proteins like
mucins. The quality of the finished leather is largely
controlled in the liming process. High abrasive
resistance of sole leather, high tensile strength of
picking band or belting leathers are largely dependent
on the process of liming. The liming method used on
sheep skins is the painting of the flesh side with a
lime paint, a mixture of slaked lime and sodium
sulphide solution or slaked lime and red arsenic.
Dehairing

Dehairing is the process of removing the hairs and

furs from the hides without any damage to them. The
process of dehairing largely depends upon the

phenomenon of hair loosening. Loosening of the hair

is due to the chemical reaction of lime liquor on the
hair root or base of the hair shaft. This weakening of
the hair is dependent on the breakdown of the
disulphide link of the amino acid, cystine, which is
characteristic of the keratin class of proteins e.g. wool
and hair. The process of dehairing achieved using a
variety of techniques was reviewed by Germann20.
The most commonly employed methods for dehairing
rely upon the use of sulphide during liming to destroy
keratin, the principal components of hair. This
produces an effluent with a chemical oxygen demand
of about 60,000 mg/L and constitutes the polluting
aspect of leather manufacture. The use of sulphide in
dehairing can be circumvented by the use of
proteolytic enzymes21,22. These are used as
supplement in the chemical dehairing processes. The
enzymes remove the hair by disturbing the
proteinaceous matter present at the base of the hair.
Proteolytic enzymes can also attack the dermal
collagen, damaging fine fibres in the grain
enamel2324. Therefore, there is a definite need to
identify specific proteases that can remove hair
without damaging the fibrous collagen. A number of
enzymes have been proposed and include proteases
from bacterial, fungal and vegetable origin23-25.
Dehairing is the process where enzymatic
involvement is the most important factor to expedite
the process. Enzymatic dehairing, either in the
alkaline range or in the acid range, has been widely
exercised26. The primary studies on dehairing by Raju
et al.12 with the extracellular protease secreted by the
Bacillus isolate, showed that it has a dual pH
maxima at pH 7.5 and 9.0 and the temperature
maxima at 37C. It requires the presence of complex
protein substrate in the medium for optimal enzymatic
action. In enzymatic dehairing, apart from pH,
temperatures also play significant role. At
temperatures ranging from 32-37C unhairing could
be accomplished between 18-24 h. At temperature
below 32oC, the duration of enzyme application needs
to be increased for complete dehairing. Further, below
25oC no appreciable enzymatic dehairing within a
reasonable period is envisaged. Studies conducted on
the temperature stability of the enzyme indicate that
the enzyme is stable between temperature ranging
from 20 to 50oC. Their studies also showed that
although even 2% (w/w) of crude enzyme was
sufficient for dehairing, 3% (w/w) of the enzyme was
preferred because at this concentration even the tough

CHOUDHARY et al.: ENZYME APPLICATIONS IN LEATHER PROCESSING

hair at the neck region was removed completely. Pal

et al.27 have experimentally carried out the
investigation for dehairing goat and ship skin using
the enzyme secreted by Rhizopus oryzae. They
studied the effect of units of activity of enzymes,
effect of pH, and effect of hydration on dehairing
process. Subsequently, they evaluated the physical
properties of wet blue leather for both goat skin and
sheep skin in the prevailing condition of limesulphide dehairing and enzymatic dehairing. An
enzyme powder was obtained after the drying of
enzyme extract and this was used to prepare a paste
that was painted on the flesh side of both the goat and
sheep skins. The skins were piled flesh to flesh and
left at room temperatures (33-35oC) overnight. The
hair was removed using a blunt knife. After dehairing,
the skins were further processed to the wet blue stage.
Their physical evaluation also included the general
appearance, feel, fullness, gloss and grain
smoothness. They finally concluded that an economic
viable enzymatic dehairing process can be favourably
compete with the chemical dehairing process. They
also observed that enzymatic dehairing could be
accomplished within 11-12 h provided the
temperature was maintained in the range of 30-37oC.
The enzyme used by them was stable from pH 3-11
and temperature up to 80oC. They optimized the
prevailing condition for dehairing hide and skin with
46% hydration, 83% humidity, pH 8.0 and 2 h of
incubation period.
Thangam et al.28 made an investigation of alkaline
protease isolated from Alcaligenes faecalis for
enzymatic dehairing in tanneries. The enzyme used by
them was relatively stable in the pH range of 8-11 and
at temperature up to 30oC for 24 h. Their results
indicated that the protease produced by Alcaligenes
faecalis was best suitable for dehairing and could be
exploited as an eco-friendly dehairing agent in leather
processing. Their results envisaged to reduce the
pollution load to the environment and help in bating
operations for improved yield and soft leather. In their
work goat-skins were washed and cut into two halves.
The paint method of the dehairing was followed in all
the studies. The enzyme powder obtained after
lyophilization was used to prepare a paste that was
painted on the flesh side of the goat skin. The
dehairing trials were conducted by following sulphide
free and less sulphide methods using various
concentrations of enzymes and sulphide. The right
halves of the skin were applied with the enzyme

663

preparations and treated as experimental skins. The

left halves were the control and dehaired by following
a conventional lime-sulphide method of using 2.5%
sodium sulphide and 10% lime. The efficacy of the
dehairing process in relation to different temperature,
pH, enzyme concentration and less sulphide methods
were measured by applying a scale such as difficult
dehairing, slightly difficult dehairing and moderate
dehairing. The complete removal of hair was achieved
even with 0.5% enzyme concentration. At 0.25%
enzymes concentration, only moderate dehairing was
noticed after 18 h of applications. Their findings
indicated that the use of 0.5% of crude powder was
sufficient to reduce sulphide from 2.5 to 1%.
A recent report communicated by Paul et al.29
involved the proteolytic enzymes for providing a
suitable alternative to destructive sulphide dehairing
which included the use of a diverse array of enzymes
many of which were rather non-specific. The enzymes
caused loosening of the hair, without damaging the
fibrous collagen of dermis. The enzymes included
were proteases from bacterial, fungal and vegetable
origin. Another enzyme for use in depilation was
dispase, a neutral proteinase of bacterial origin. The
enzymes have been used successfully for a number of
years in biomedical field to achieve separation of
epidermal and epithelial cells from the underlying
matrix through the destruction of the basement
membrane. In their study, incubation of bovine skin
with dispase caused detachment of the epidermis of
the level of the epidermal/dermal junction leaving a
clean grain surface. Sloughing of the epidermis was
observed initially at the surface but with prolonged
treatment extended into the hair follicles to bring
about in hair loosening. This is likely to be a result of
enzymatic cleavage of the collagenous components of
the basement membrane upon which these structures
reside. However, the microscopic examination did not
show any evidence of grain damage, indicating that
the fibrous collagen of the dermis has not been
degraded by the enzyme.
Ever since the onset of the industrial enzymatic
dehairing process in 1990, considerable amount of
work has been carried out and some researchers
presented the review report elaborately on the
phenomenon of enzymatic dehairing30. But most of
the enzymatic process development is propriety in
nature. Many people rationalize enzymatic dehairing
as a sound alternative to the lime-sulphide process
owing to the severe problem created by sulphide and

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INDIAN J. CHEM. TECHNOL., SEPTEMBER 2004

chromium in tanneries. The reason being, these two

components are the major pollutants of the leather
processing9. The advantage of enzymatic dehairing is
the reduction of sulphide content in the effluent, and
elimination of the bate in the deliming, reduction in
the COD and BOD of the effluent of toxic chemicals
and easy handling15,31. Therefore, economically viable
enzymatic dehairing process can compete well with
the chemical dehairing process. Disadvantage of
enzymatic dehairing are that enzymes are more
expensive than the conventional process chemicals,
require careful control, and because of the effect on
the structure of the hides and skin, may require
subsequent process changes32. Ultrafiltration of the
enzymatic dehairing using proteolytic enzyme was
carried out by Cassano et al.33 during the unhairing
operation thus creating an enzymatic reactor for the
production of dehaired skins. Ultrafiltration permits to
control the enzymatic action on skin because the
enzyme was rejected by the membrane and is
accumulated in the feed tank, while the sulphide
component (used in low concentration) is rejected
with the permeate. The advantages of the coupled
enzymatic/UF system were: control of enzyme action;
reduction of sulphide requirement; shortening of
unhairing-liming time; then possibility to recover hair
and to reduce pollution of waste water and cost of
cleaning up processes. Disadvantage for adopting
enzymatic dehairing is sometimes ruled out when a
cost effective enzyme is available.
Bating

Bating is the process of beating the leather cruelly

with heavy and sudden stroke using metal rods or
wooden logs in prevailing condition. The purpose of
bating is loosening and peptization of the noncollagenous skin structure through the removal of the
residues of the interfibrillary proteins, epidermis and
scuds. This makes them soft and supple and to
prepare them for tanning. Strong bating is required to
achieve a soft and pliable leather such as purses and
gloves, whereas slight bating is required for the soles
of shoes. Bating is in fact an offensive stage in the
preparation of quality leather. Bating brings about the
following effects in the pelts: removal of lime,
produce silky grain, remove swelling and plumbing,
increase the degree of stretch possessed by the
finished leather. Failure to remove the noncollagenous proteins causes a cementing together of
the fibres when the leather is dried and results in
firmness and lack of flexibility. It is well known that

the classical bating process in the alkaline condition

makes use of proteolytic enzymes, which are of
pancreatic or bacterial origin, and the efficiency of the
process depends on the enzyme concentration as well
as temperature, pH and time during the bating
process. The bating in alkaline conditions is today
universally recognized by the entire leather industry,
but to be effective it should be conducted at 95-100oF
and at pH 7.5-8.5, otherwise the enzyme efficiency
drops drastically34. The effect of bating enzymes
occur through the diffusion of the enzymes into the
hide but the greatest concentration is found on the
outer layers. On the unsplit pelts, penetration of the
bating enzymes to the inside of the hide are
insufficient to digest unwanted proteins, this is
particularly true in the neck and butt area. It is worth
to note that whether the enzymes are free or bound to
the particles. If the enzymes are free then the
particulate can be removed before adding the
substrate. If the enzymes are particle bound,
clarification may decrease the activity. In fact,
particulate interferes with the exposure of the
substrate, which is insoluble to the enzymes.
Degreasing

Hides and skins, specially domestic sheep skins,

contain large amount of natural grease which is
generally removed in the tannery by liming operation.
But sometimes it so happens that the hides and skins
contain appreciable amount of grease even after
liming. This residual grease is responsible for fatty
acid spues, uneven dyeing and finishing, waxy
patches in alum tanned leathers and pink stains in
chrome blues, etc. Tanners were therefore trying for a
long time to find a suitable means to get rid of this
residual grease in hides and skins. Treatment of
tanned leathers with a suitable solvent of fat makes
the former hard and horny and therefore, for
pliability, extra fat liquoring was necessary. On the
other hand, it was practically impossible to remove
grease from pelts by similar treatment due to the
presence of large percentage of water in the pelts.
Moreover, in hides and skins, grease always remains
inside fat cells made up of reticulin or other type of
tissues. Unless these cells are ruptured, grease cannot
be removed by any degreasing method. Sun drying,
ruptures the fat cells almost completely, and therefore
it is easy to remove grease from flints by any suitable
degreasing methods. But wet salted and fresh hides
and skins require special treatment, before degreasing,
to rupture the fat cells. With one percent sulphuric

CHOUDHARY et al.: ENZYME APPLICATIONS IN LEATHER PROCESSING

acid , ten percent common salt and storage for few

weeks after pickling rupture almost all the fat cells
and therefore, degreasing is done mostly after
pickling. Three methods used for the removal of the
grease are aqueous emulsification, solvent extraction
and pressure degreasing.
During the last five years enormous progress has
been made in the field of degreasing process in an
aqueous medium, both as regards to the use of
contaminating chemicals and in the application of
technology of the products. The best time for carrying
out the aqueous degreasing process is on pickled skin,
as the deposits are more accessible to the surfactants.
The application of the enzymes mainly of the lipase
type, in different stages of the leather process has
been studied35-38. When enzymes are applied in
aqueous medium (pickling phase), there was
combined action involving rupture of the membranes
surrounding the fat cells and triglyceride splitting, all
of which helps to improve the degreasing process.
Palop et al.39 studied the effectiveness of the
degreasing with lipase enzyme. They started with five
English domestic lamb skins which underwent a
conventional beam house operation (soaking,
unhairing, deliming and bating). Then these were
treated with a reference pickle, standard degreasing
with 8% ethoxylated fatty alcohol (EPA) and enzyme
treatments as follows:
Skin 1: Reference picklestandard degreasing
Skin 2: Reference pickle + 1.5% acid protease
standard degreasing
Skin 3: Reference pickle + 1.5% acid lipase
standard degreasing
Skin 4: Reference pickle + acid protease + acid
lipasestandard degreasing
Skin 5: Reference pickle + acid protease + acid
lipasestandard degreasing + 1.5% neutral lipase
Samples were taken from both pickled and
degreased skins for initial and residual fat
determination. Percent degreasing effectiveness was
expressed as [(% initial fat - % residual fat)/% initial
fat]100. It was observed that the reference
degreasing process (skin no.1) had an effectiveness of
58%. The addition of an acid protease (skin no.2) to
reference pickle has no influence on the degreasing
effectiveness, since it remains at 58%. Treatment with
acid lipase increased the degreasing effectiveness to
78% (skin no.3). The combine effect of protease and
lipase gave effectiveness 78.5% and further increase

665

in effectiveness up to 88% was produced by adding

lipase to the degreasing process in a neutral medium
(skin no. 5). Waters and Price40 observed that
treatment with acid lipase did not rupture the fat cells,
but it broke down the triglycerides of the natural fat
released from the cells due to reaction of the acid of
the pickle process. The natural fat was composed of
fatty acids 10, triglycerides 56, waxes 23,
phospholipids 6 and cholesterol, 5%. So with the
breakdown of the triglyceride with lipase the fatty
acid composition increased from 10 to 40% which
became of enormous assistance to emulsify the
diglycerides and monoglycerides, fatty acid and
glycerol which were formed in the rupture of the
triglycerides39.
Mitchell and Ouellette41 evaluated the combination
of lipase and protease to clear the surface of the
chrome tanned stock of grease, dirt, scud and other
stains for the purpose of making more uniformly
coloured leather. Lipase and protease were tried since
the undesired material on the surface of chrome
tanned blue stock are proteins, fats and oils. A
significant reduction in grease stain, neck wrinkle
discolouration was observed. Also improvement was
observed in brightness and uniformity in dyeing. This
was accomplished using extremely low amount of a
combination of two enzymes selected to remain
particularly active in the condition of retanning with
respect to pH, temperatures, running times and
presence of other chemicals. The low pH conditions
found in the retannage of blue stock required to select
enzymes active in low pH range. 0.015% acid lipase
and 0.3% acid protease was best combination.
Tanning

Tanning is the last stage in leather manufacturing.

It is the process of converting unstable raw hides into
leather, with adequate strength properties and
resistance to biological and physical attacking agents.
In fact, it is the process of introducing a tanning agent
into the hides. This is accompanied by the
introduction of additional cross links into collagen,
which bind the active group of the tanning agents to
the functional group of the protein. Tanning makes
collagen more resistant to the hydrolysis by acids and
enzymes, but not by alkalis. Thus, the tanned collagen
as a rule binds less water than the native one. Tanning
with chrome compounds or with tannins does not
prevent alkaline collagen hydrolysis14. Chrome
tanning is the most important tanning method to
obtain light, inexpensive leather of high thermal and

666

INDIAN J. CHEM. TECHNOL., SEPTEMBER 2004

bacterial resistance. It improves the colour,

appearance and look of the finished leather as well.
Almost all skins and hides are coloured, using chrome
tanning after these are treated with acid solution for
deliming purposes. Although, enzymes are not
directly involved at this stage, however, the enzymatic
treatment in the previous stage significantly
influences the quality of tanning.
Physical evaluation

The details which correlate the enzymatic

processing with the physical properties of the leather
is scanty in literature. The evaluation of physical
properties of the leather includes the measurements of
tensile strength, tearing strength, stitch tear strength,
and percent elongation. Raju et al.12 reported the
determination of elongation of break tongue tear
strength, brushing strength and tensile strength of the
dyed crust leather samples, cut from the identical
portion in the butt area. They conducted the
experiments of dehairing by the enzyme extra-cellular
protease secreted by the Bacillus spp. The enzyme
concentrations applied were 1 to 3 percent in the pH
optimum 7.5 to 9.0 and temperature optimum 32 to
37oC. Their specimens compared favorably after
enzymatic dehairing process with regard to tensile
strength, elongation of break and tongue tear
resistance. Pal et al.27 also proceeded for the physical
evaluation of the leather which included the
measurements of tensile strength, tearing strength,
stitch tear strength and percent elongation with the
application of enzymes as depilant. Their
measurement data are shown in Table 3. However,
they did not make any conclusion based upon their
enzymatic study. In the study conducted by Thangam
et al.28, they also measured the strength properties of
the leathers treated by the enzyme secreted from
Alcaligenes faecalis at pH optimum 9.0 and
temperature optimum 55oC. The properties of the
enzyme treated leathers were comparable to those of

control leather. The tear strengths of the experimental

leathers were considerably better when compared to
the corresponding control leather. These indicated that
the enzymes treatment did not have an adverse effect
on the strength of the leather. However, there is not
any scientific details in the literature which can
correlate the physical properties of leather and the
enzymatic action. This invites the attention of
researchers to make a dent in this direction for
improving the leather quality and facilitating the
process in leather production.
Waste processing

Effluent discharges from leather processing

industries create health hazards and environmental
problems unless these wastes are properly treated.
Fleshings, the major solid waste generated at the
pretanning operations of leather processing, were
hydrolyzed using pancreatic enzymes with a view to
evolve a simple method for solid waste management
by Kumarguru et al.42. The proteolytic activity of the
pancreatic homogenate with casein was found to be
80 units/mL. Fleshings treated with pancreatic
enzyme preparation showed a six fold increase in
proteolysis against the control at the end of 7 days.
The protein content, collagen and the free fatty acids
in the hydrolysate supernatant were 80.0, 10.64 and
72.86 mg/mL respectively. The optimum pH for the
enzyme preparation was 8.5. The hydrolysis was
observed by almost total liquefaction of the fleshing.
Bajza and Marcovic43 studied the effect of alkaline
protease on untanned leather (hide) waste. Trimmings
obtained after liming had the alkalinity that
corresponds to pH 10. The enzyme which was active
in this pH was used. The process was conducted at
constant temperature 55C favourable for the enzyme.
The enzyme was a commercial preparation of alkaline
protease Protoderm 100T, produced from submerged
cultivation of Bacillus genus. It was observed that
leather solubility increased by increasing the enzyme

Table 3Physical evaluation of wet blue leather

Experimental details

Tensile strength (Kg/cm2)

Elongation (percent)

Tearing strength
(Kg/cm)

Stitch tear strength

(Kg/cm)

Goat skins
I. Lime-sulphide dehairing
II. Enzymatic dehairing

1st
120
131

2nd
212
269

1st
90
60

2nd
60
60

34
56

38
89

Sheep skin
I. Lime-sulphide dehairing
II. Enzymatic dehairing

140
175

176
200

80
66

66
70

48
48

55
71

CHOUDHARY et al.: ENZYME APPLICATIONS IN LEATHER PROCESSING

concentration from 500 to 15,000 units per gram of

leather. Leather waste breakdown yielded a water
soluble hydrolysate, which could be concentrated on a
vacuum evaporator and then dried to fine flour for
various purposes.
The major pollutants in leather industry are
sulphide and chromium. Chromium in the effluent is a
serious threat to the water resources. In many
countries it is not allowed to exceed 5 ppm. But the
chrome letout in the effluent is about 40 percent of
that input offered. Therefore, reduction have been
made to the offered chrome from 2.5 percent Cr2O3 to
2 percent Cr2O3. But mere reduction does not do well.
Nowadays conversion of tannery by-products into
industrially useful products has been given prime
attention as an alternate solution. Enzymes have been
applied by Cabeza et al.44 for gelatine isolation from
chrome shavings. Chrome shavings were pretreated
for 6 to 24 h with enzyme solutions at the optimum
pH for the enzyme. Then gelatin was extracted at
70C, pH 8.0. To evaluate the effectiveness of the
process, the protein yield i.e. the amount of initial
protein recovered as gelatin was determined. Pepsin
rose to a maximum of 6.10% using 0.01% of enzyme,
higher than the control (without enzyme) value
(4.33%). Trypsin had a maximum of 14.70 % using
0.25% of enzyme in the extraction. This value was
much higher than the control and the values obtained
with pepsin. Kolomaznik et al.45 has reported on
enzymatic dechromation of chrome-tanned wastes by
using proteolytic enzyme (ALCALASE of Novo
Nordisk, Denmark). The mechanism of enzymatic
reaction was as follows.
Enzymatic hydrolysis

Chrome-tanned-wastes Gelatin/Protein hydrolyzate + Chrome sludge

They sought for the potential application of the
reaction products in different industrial sectors. The
chrome sludges were found suited for pigment in
glassmaking, heat-resistant bricks and alkaline
chromate. The use of hydrolysates was best suited for
concrete admixture, grinding of cement hydration of
lime, protective coating and plaster binder. The
chrome sludges were further processed and recycled
tanned salt were produced. The typical composition of
chrome cake thus produced was Na2O-0.42%, MgO6.40%, Al2O3-0.40%, SiO2-0.89%, P2O5-0.12%, SO31.50%, CaO-2.50%, Cl-0.33%, Cr2O3-15.90%, MnO0.13%, Fe2O3-2% and the Ignition loss - 68.81%.
Cabeza et al.46 studied the pilot plant trials of a

667

process to treat chrome shavings to isolate protein

products and purified chromium. The process used
two enzymes, pepsin and alkaline protease, in two
consecutive extractions with isolation of high quality
gelatin and a hydrolysate. Chrome shavings were
pretreated with 0.1% pepsin at pH 3 -3.5 and at room
temperature for 8 h and gelatin is then extracted at pH
8 and 70C. Gelatin removed by using filter press and
hydrolysed protein was then isolated by second
extraction from sludge using 0.005% alkaline protease
at pH 8 and 70C for 3 h. The remaining solid after
filter press, called chrome cake, was chemically
treated to prepare it for recycling in the tannery
industry.
Industrial enzymes

The commercial exploitation of enzymes is not

new. The use of yeast as bio-catalyst dates back to
about 6000 B C. However, the production of enzymes
at large scale cannot boast of so ancient a history. The
first large scale production of enzymes came about
only in 1874, with the first industrial batch of
chymosin. From 1913 onwards the detergent industry
became a prime concern of enzymes. The leather
industry in 1917 and the starch industry in 1950 set a
similar trend. The last three decades have been
marked by an explosive advancement in the field of
commercial production of enzymes. The increasing
demand of quality leathers in the country as well as in
the world is increasing fast. This invites new processefficient and clean technology in the leather
production. Industrial enzymes have appeared to
develop the so called clean technology and economic
process in this direction. Industrial enzymes related to
leather processing are usually mixture of different
enzymes and these are standardized with diluents.
Based upon the type of bio-chemical reaction,
enzymes are assigned to large groups.
Many industrial enzymes are being manufactured
from animal and plant system. However, major share
is obtained by cultivation of microbes. About twelve
categories of enzymes are used for industrial
purposes. Table 4 showed the global production of
industrial enzymes. Most of these are hydrolytic
enzymes used for the depolymerisation of natural
substrates to low molecular mass. The largest group
being proteolytic enzymes from bacteria (59%)
followed by carbohydrases (20%) in terms of the
relative sales value1. It is evident that the global
production of industrial enzymes is increasing fast

INDIAN J. CHEM. TECHNOL., SEPTEMBER 2004

668

Table 6Properties of proteases 1

Table 4Global production of industrial enzymes

Enzymes

Amount (Ton )

Bacillus protease
Aspergillus amyloglucosidase
Bacillus amylase
Glucose isomerase
Microbial rennet
Fungal amylase
Pectinase
Fungal protease

550
350
350
60
25
20
20
15

Table 5Country wise production of commercial protease

enzymes
Country
Denmark
Netherlands
USA
Japan
Germany
France
UK
Switzerland
Others

Amount (Ton)
249
100
64
42
32
16
11
11
5

owing to the multiplying demand of typical enzymes

in different industrial sector. Among the industrial
enzymes producing countries, Denmark top in
protease production with its annual production of 249
tones, which is approximately 47 percent of the global
production (Table 5). The worldwide market of
industrial bulk protease (trypsin) of the animal
pancreas origin are approximately 107 $ for their
comprehensive application in the leather production.
However, there is much more likelihood of increasing
its sales market in near future.
Proteases used by the detergent industry are also
suitable for soaking purpose since they are relatively
resistant to the increased pH of about 10 used in the
soaking bath. Among different industrial enzymes
pepsin, trypsin, rennin and transglutaminase are the
most frequently used enzymes in leather
manufacturing process. These are the typical enzymes
with their specific utility in soaking, bating and
dehairing process. The details for scores of industrial
enzymes and their applications are widely available
throughout the literature. Properties of some of the
important industrial enzymes are given in Tables 6-8.
Trypsin is formed in the intestine from trypsinogen
which in turn is formed in the pancrease. Trypsinogen
becomes trypsin when a splitting of Val-(ASP)4-Lys

Organisms

Aspergillus saitoi
Aspergillus oryzae
Panceilomyces varioti
Mucor pusillus

pH Range for
haemoglobin

pH stability

3.5 - 4.5
3.0 - 4.0
3.5 - 5.5
3.5 - 4.5

2.0 - 5.0
5.0
3.0 - 5.0
3.0 - 6.0

Table 7Properties of lipases1

Source
Porcine pancrease
Rhizopus species
Mucor javanicas
Candida cylindracea

Optimum pH

Temperature
stability oC

6.5 - 9.5
6.0 - 7.5
5.5 - 8.0
5.0 - 7.5

40 -45
35 - 40
40 - 45
40 - 45

Table 8Properties of amylases1

Enzymes
Bacterial -amylase
Fungal -amylase
Malt -amylase
Pancreatic amylase

Optimum pH

Optimum
temp oC

6.5 - 7.5
5.0 - 6.0
4.5 - 7.0
6.0 - 7.0

95.0
80.0
85.0
75.0

hexapeptide occurs. Many trypsin like enzymes are

also known. Their way of action and composition are
close to trypsin. They attack the denatured proteins.
According to the research report communicated by
Cabeza et al.44 and Taylor et al.47 trypsin proved to be
an effective enzyme for the isolation of protein
product during the treatment of chrome shavings.
They developed the commercial trypsin preparation
that proved to be not only efficient in solubilizing the
shavings but also cost effective.
Pepsin, has been reported being applied during
pickling and on chrome tanned hides and skins. This
acid enzyme has been extensively used on pigs and
sheep skins, which by nature are very greasy. The
proteolytic action of this acid enzyme hydrolyzes the
cell membranes and a good part of grease is
eliminated. Hence this enzyme also acts as a
degreasing agent. Pepsin is an enzyme characteristic
of the mammalian stomach structure, with molecular
weight of 35, 000 and a large amount of dicarboxylic,
aliphatic and aromatic amino acids. This enzyme has
an optimum activity at pH around 2.0, but is still
active up to pH 6.0, and becomes active in the
presence of HCl. The enzyme product has the

CHOUDHARY et al.: ENZYME APPLICATIONS IN LEATHER PROCESSING

advantage of a lower cost for the leather industry. The

hydrolyzed protein components of this enzyme
product assure a better stabilization of the pepsin
activity and do not require the addition of other
agents. According to a report communicated by
Deselnicu et al.34 pepsin enzymes work more gently
than the alkaline proteolytic enzyme and can be used
at lower temperature such as 70F. The diffusion and
the penetration of this acid pepsin enzyme through the
inner layer is much faster and more effective than
during the alkaline bating. This acid enzymatic
process also has tremendous advantages for those
tanneries which process wet blue hides from different
sources. This new acid pepsin enzymatic process is
covered by a US patent application48.
Another class of important industrial enzymes is
transglutaminases. Transglutaminase is an enzyme
capable of forming inter or intra-molecular crosslinks in many proteins and has the utility in the
modification of gelatin byproducts from the leather
industry. The enzymes catalyze an acyl transfer
reaction between the -carboxamide group of peptide
bound glutamine residues as acyl donors and primary
amines as acceptors. When the -amino group of
peptide bound lysine acts as acyl acceptor, an -(glutamyl) lysine cross-link is formed;
P-CH2-CH2-CO-NH2 + H2N-(CH2)4-P P-CH2-CH2CO-NH-(CH2)4-P + NH 3
This enzyme produced by fermentation is
commercially available, relatively inexpensive and
environmentally safe. In the absence of amine
substrate, transglutaminase can catalyze the
deamidation of glutamine residue during which water
molecules are the acyl acceptors.
P-CH2-CH2-CO-NH2+H2O P-CH2 -CH2 -CO-OH+
NH3
Transglutaminase can also catalyze an acyl transfer
reaction between the -carboxamide group of peptide
bound glutamine residue and various primary amines.
P-CH2-CH2CONH2+H2N-R P-CH2-CH2-CONHR+NH3
The earliest report on transglutaminase appeared in
1959-1960 when Mycek et al.49,50 and Clarke et al. 51
described their experiments in the incorporation of
primary amines into protein and in deamidations of

669

protein using this enzyme. During 1960 and 1970,

Folk et al.52-56 exercised basic research that resulted in
understanding the structural requirements of specific
substrate for this enzyme as well as the mechanism of
enzyme action. In 1980s Motoki and Nio57, Motoki et
al.58, Nio et al.59,60 and Ikura et al.61-64 published
several research papers describing practical
application of transglutaminase isolated from guinea
pig liver particularly with food proteins. The
description included cross-linking between different
proteins, studies on the mechanism of gelation of
protein solutions as effected by transglutaminase, and
incorporation of amino acids. Because of the isolation
problem, it was not commercially feasible to use this
enzyme preparation. In 1989, Ando et al.65 isolated
the enzymes from a Streptoverticillium species and,
based on this discovery two companies were jointly
granted a patent48 and shortly after this began
producing the enzyme mainly for modification of
food products. They demonstrated the feasibility of
using microbial transglutaminase to alter the physical
properties of gelatin by-products from the leather
industry and thus increasing the potential markets for
new value-added products. The characteristics of
microbial transglutaminase isozyme, as reported by
Taylor et al.9 that it is calcium independent and has
broad specificity. The enzyme is a single polypeptide
chain with a molecular weight of about 38000 and this
is about one half the molecular weight of the
transglutaminase derived from pig liver. The -SH
group of single cysteine is involved in the catalytic
reaction.
The most important feature of these industrial
enzymes is their activity. For all sorts of industrial
and research purposes, enzymatic activity is one of
the major determinants in their application. Various
complexing agents may decrease the activity of metal
dependent enzymes. However, in a report
communicated by Didato and Bryant66, they did not
observe any inhibition of enzymatic activity in
tannery soaking procedures when normal dosages of
microbicides up to 600 ppm Busan 1630 (2 bromo- 4'
- hydroxy acetophenone) were applied. Microbicides
are also essential along with the enzymes in the
processing of leather. Six commercial proteases were
assayed in the presence of four selected industrial
microbicides. None exhibited any significant
inhibition of the protease studied. The comparison of
enzymes based on specific activity must be
conducted. Except for proteases, -amylases and

670

INDIAN J. CHEM. TECHNOL., SEPTEMBER 2004

lipases, many more enzymes are still being developed.

However, their commercialization subjects to both the
technical feasibility and the economics of utilization.
Discussion
The emphasis on the use of enzymes in the industry
has come about because enzymes have unique
properties such as catalysis of chemical reactions at
high rate, operations at ambient conditions, selectivity
of substrate, minimal side reactions, simple
operations, availability in large amount from
microbial sources, reusability if immobilized,
nontoxic nature and non polluting effluent
generations. During the early days of enzyme
technology, development was slow. This was due to
impediments in the fields of enzyme stabilization,
production on large scale, cofactor regeneration and
lack
of
enzyme
immobilization
facilities.
Advancements in the various areas of biotechnology
have overcome most of the hurdles and currently, the
pace of development of enzyme technology is quite
rapid. The advancements in the techniques of genetic
engineering which permit the manipulation of cellular
DNA, have led to the opening up of a new field called
protein engineering. The structure of a protein can
now be altered by offering specific and precise
changes in the DNA molecule, which ultimately will
be reflected in the protein formed. The structurally
altered enzyme thus obtained has different physico
chemical properties which distinguishes it from its
normal cellular component. The physico chemical
differences engineered into the enzyme would, of
course, depend on the requirements of the relevant
industry. It has also been possible to increase
manifold production of microbial enzyme by inserting
extra copies of the gene responsible for producing the
enzymes. Capability has now been developed to make
use of microbes to express important enzymes of
animal and plant origin. Recently, many novel
industrial enzymes that prove to be efficient in the
process of dehairing, soaking, bating and solubilizing
the chrome shaving have been developed.
Leather industries have enormous potential for the
wide range of applications of several industrial
enzymes such as proteases (alkaline, neutral and
acidic), lipases, amylases, pepsin, trypsin, renin and
around glutaminase etc.67-69. Dehairing and dewooling
are the prominent stages where enzymatic application
can effectively govern the leather processing with
least environmental pollution. Enzymes also have
vital application in the effluent treatment from the

tanneries. The parameters involved in the enzyme

applications are the parameters like enzyme and
substrate concentration, pH and temperature. The cost
factor in the enzyme operation which are required to
be solved are enzyme production costs (or purchase
price), enzyme life, turn over number, equilibrium
constant for reaction, conversion efficiency,
temperature stability, operation concentration of
substrate,
and
microbial
contamination
or
maintenance of low bacterial counts. The future is still
waiting for the involvement of enzymes for their
successful utilization in the quality leather production.
Acknowledgement
The authors acknowledge the financial support
received from the Ministry of Human Resources and
Development, Government of India under research
program on Production, Recovery and Application of
Enzymes.
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