Review

13 l Advanced Biotech l October 2009

Cleaner Leather Processing By Using Enzymes: A Review

Abstract

Leather industry is facing tremendous pressure from the various pollution control bodies because of the huge amount of pollution associated with processing. Advancements in processing techniques and adoption of cleaner technologies have enabled the tanners to get rid off pollution from the leather processing. Though there are various cleaner technologies based on chemical methods are available but cleaner technologies based on enzymatic methods are viable, eco-friendly and form alternative to the existing technologies. Enzymes in leather industry became a part and parcel of the system to mitigate pollution problem in the leather processing operation. The enzymes find application in soaking, unhairing, degreasing and bating of leather processing operations for obtaining better leather qualities. Applications of enzymes in various stages of leather processing are discussed in this paper.

Key words: Enzymes in leather processing, soaking, unhairing, bating, degreasing and post tanning.

(Kanagaraj and Chandra Babu,2002). There is an enormous pressure from the various pollution control bodies to regulate and minimize the amount of pollution generated from the leather processing. The need for use of alternative to chemical methods to combat pollution problem have become necessary to protect the industry and to comply with the environmental norms (Taeger, 1985). The technologies based on enzyme offer excellent scope for the reduction of pollution in leather processing (Ludvik, 1996).

Enzymes in leather processing

Soaking

In the soaking stage, the enzymes make it possible to shorten production times considerably. The proteolytic and partially lipolytic enzyme treatments, thus facilitating a structural opening-up necessary for the chemical products to penetrate more rapidly, particularly those used in the unhairing-liming stage.

Enzymes during soaking accelerate the process of removing hyaluronic acid and improve quality through, more effective rehydration of the skin, fat degradation and dispersion, better removal of carbohydrates or protein components. Both proteases and lipases aid soaking processes. They are particularly helpful when processing fatty raw materials, very dry raw materials, fresh hides without salt, where the removal of non structural proteins and carbohydrates is very difficult (Ramasami, 1999c).

Removal of dung in the skin/hide is also a problem and this problem may be easily solved in soaking process by using Laccase. Laccase used in soaking evaluates the use of appropriate, targeting enzymes in soaking, to effect dung removal from hide. The major components of the composition of dung are lignocellulosic derivatives, cellulose, hemi cellulose and lignin. The effects of individual enzymes and enzyme mixtures on dung removal were investigated. The Laccase is more effective than individual enzyme treatments. The proposed mechanism for fast dung degradation is based on opening up the structure with lignin and hemicellulose degrading enzymes and breaking down the fibrous structure of cellulose. The studies have demonstrated that enzymatic dung removal is based on solublization of the dung as a whole and not of one or more of the lignocellulosic components (Tozan and Covington, 2002). NaCl, up to 3 molar concentrations, enhanced enzyme activity by 20% for cellulose and 100% for Xylanase (Auer et al.,1999). The role of NaCl is to dissociate interactions between protein-protein or generally to avoid any ion exchange effect thereby, solublising and increasing the enzyme activity.

Liming

The main objective of liming is to remove hair by using Lime and Sodium Sulphide in the conventional process. The mechanism is different if we use enzyme in liming process for unhairing purpose.

J.Kanagaraj

Introduction

Leather processing operation is categorized into three main parts i.e., a) Pretanning b) Tanning and C) Post tanning. Prior to pretanning, the raw animal skin and hides are preserved by application of salt that restrains microbial attack. This process of preserving skins is called as 'Curing' (Kanagaraj et al., 1988). Soaking, liming, deliming, bating, degreasing and pickling are the pre-tanning processes. The main objective of soaking is to rehydrate the skin and also to open up the contracted fibre structure of the skin. Similarly the main objective of liming is to remove hair, deliming is to reomve lime, bating is to split the fibre into fibril, degreasing is to remove the fat and pickling is to reduce the pH of the skin from 8 to 2.8-5.0. Tanning renders permanent stability to the skin/hide (Ramasami et al.,1999). The post-tanning operations include retanning, dyeing and fat liquoring.

During the conversion of skin/hide into leather, the skin/hide undergoes various treatments. From salt used for preservation of skin into top coats of finishing, the conversion part of leather involves variety of chemicals (Ludvik, J, 1997), (Ramasami, 2001). The chemicals used in tanning industry, are mainly, Lime, Sodium Sulphide, Ammonium salts, Sulphuric acid, Chromium salts and Vegetable tanning materials. These chemicals generate considerable amount of pollution in leather processing. A considerable part of this is discharged into the effluent (Mariappan, 1997).

The level of utilization of various chemicals used in leather processing is given in Table 1. Nearly 70% of the emission loads of BOD, COD, and Total Dissolve Solids (TDS) are generated from soaking, liming, degreasing, pickling and tanning process (Ramasami et al.,1999b),

achieved more quickly and completely as a result of the joint action of the chemicals, converting hair into hair pulp in conventional dehairing process (Ramasami, 1996), (Marshal, 2002). The Fig.1 shows the histology of raw skin/hide and the various composition of skin/hide.

The cementing substances, mostly represented by proteoglycans, participate in the formation and stability of the fibrous tissues by interacting with collagen molecule at the fibril level; the elimination of such substance is closely related to the opening up of the collagenous fibrous structure. The epidermis, dermis- transition area, the basement membrane is composed of an arrangement of complex organic macromolecules, which is vulnerable to enzymatic and chemical treatments; thus leads in the various unhairing process by the elimination of basement membrane, along with the epidermis, thus freeing the grain surface formed by densely packed collagen fibrils (Cantera, 2001a), (Cantera, 2001b).

Many researchers have carried out enzymatic dehairing with variety of proteolytic enzymes. The experiment carried out with Alcalase (homogenous mixture), bacterial proteinase by Novo industry, Denmark, showed that enzyme with proteolytic activity of broad specificity, is necessary to bring about depilation (Yates, 1972).

Enzymatic dehairing with alkali pretreatment is effective in depilation of skin. This includes protease with a narrower range of specificities may be sufficient to induce depilation. Cleavage of Proteoglycans and protein denaturation in strongly alkaline conditions would result in the exposure of more peptide bonds, facilitating proteases with narrow ranges of specificities to disrupt the integrity of proteins (Cantera, 2001c).

Enzymatic dehairing is also studied as a function of enzyme penetration when applied to the hair and flesh sides. There were differences in the results when the enzyme was applied to the flesh side and this probably reflects the effect of skin thickness. It is confirmed

that the 0.1% of enzyme (620mg/ml) applied on the flesh side of steer hide did not unhair the skin but on the other hand with application of same concentration of enzyme on the grain side is able to depilate the skin/hide very satisfactorily (Cordon and Windus Clarke, 1959).

In the other experiment of modeling for the proteolytic depilation put forward by Brady et al (1990). A synergistic depilatory combination of commercial proteases are used together in enzymatic dehairing. The dehairing mechanism is based on the disruption of basement membrane at the dermal-epidermal junction through the degradation of its proteinaceous compounds or the combination of multiple proteases with complementary specificities. The mechanism of enzymatic dehairing of skin by Keratinase was studied. The bacterial isolates were identified as Bacillus licheniformis for producing Keratinase activity. It has been found that the enzyme concentration (crude concentration) of 6%

o(380ug/ml) at 35 C for 48 h and at 100 rev/ m is optimum for enzymatic dehairing. The Scanning Electron Microscopic studies of leather showed that there is a possibility of reduction of chemical compounds mainly sulfide, to assure good skin quality (Cantera, 2001d), (Cantera, 2001e).

To determine the behavior of the enzymes, assessment of protein content, keratin bases, activity upon proteoglycans, inhibitions and activation, isoelectric focusing were carried out on diverse protein skin substrates. The results showed that total protein content of the enzyme was 47.5 mg/g of protein in enzyme preparation (the protein content is directly proportional to the enzyme activity) and at optimum pH 8.5-9.0

oat 362 C were ideal for the enzyme behavior that has to be used for unhairing (Cantera, et al.,2003), (Cantera, et al.,2004).

Depilation

Depilation stage is where the root of the hair is to be attacked by a selective proteolytic action. The mechanism of enzymatic dehairing is based on the capacity to break the number of peptide bonds that surrounds the basement membrane of the hair bulb that constitutes mainly proteoglycans such as Glycosaminoglycans and Dermatan sulphate, thereby, removing hair very safely. Degradation of hair is

Table 1 - Level of various chemicals used in Leather Processing

Process

Soaking Liming /unhairingWashing

Deliming

Degreasingpickling

Chrome tanning

Post tanning

Chemicals Offer g/kg of hide

Exhaustion of chemicals (%)

Unspent chemicals (g/kg of leather)

Wetting agentSod.SulphideLimeLimeSod.SulphideAmm.Chloride/ Amm.SulphateReaction products(CaCl/caSO.2 4NaCl/NaSO)2 4Solvent SaltSulfuric acidBasic Chromium SulphateSod.Formate/Sod.BicarbonateReaction saltsSuch as Sod. SulphateMineral Synatn

Sod.Formate/Sod.BicarbonateOrganic TanninsFatliquorsDyes Acids

0-230-40180-200-

-

13-26

-

3.9-6.591-10413-2091-104

13-20

-

52-63 80 10.4-12.621-32

105-15084-10521-4221-42

0510-

-

95

-

9002070

0

-

80

0

7580800

0-228-38160-1805-101-1.50.65-1.3

20-40

0.4-0.6591-10410.4-165.5-6.3

13-20

22-27

10.4-12.6

21-32

26-3817-214.2-8.421-42

Fig 1: Histlogy of raw skin/ hide. During enzymatic unhairing, basement membrane of hair bulb portion is degraded by using enzyme and hair is released very safely in the unhairing process. Source: Physical chemistry of leather making by Bienkiwickz.

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15 l Advanced Biotech l 2009October

Hair burning is conducted in the presence of Proteolytic enzyme (NUE-Novo Nordisk; 2g in 100ml water). It has been confirmed that the central portion of the hair, the medulla, is dissolved by causing the collapse of hair. Detaching and collapsing of the shaft allows the residual hair to be completely removed from the follicle by mechanical action (Elbaba, et al.,2005).

A Bacillus species has been isolated and evaluated for its dehairing efficacy. The activity of the enzyme (450ug/ml) was found to be high at

opH 9 and at the temperature of 37 C. The enzyme was tested in unhairing process at the various offer levels (1-3% based on fresh skin weight) with Kaolin 10% to fresh skin and the results showed that the enzyme level at 3% (w/w) is preferred because at this concentration, even the tough hair at the neck region is removed completely. The laboratory scale experiments showed that complete dehairing can be achieved without alkaline treatment. The physical strength of the leather was comparable to that of Sulphide treated leather (Annapurna Raju, et al.,1996).

Sirolime process is a multi-stage process involving impregnation, hair penetration, and hair loosening /hair removal stages. These three stages allow the soaked stock for the removal of hair within 3-4 h and subsequent operations in a lime comparable with that of the conventional lime-sulfide system. This typically uses 1% Sodium hydrosulfide (70% purity) based on soaked or green hide weight in 30% float, and once penetration is over, the liquor is drained and can be recovered and recycled if required. Hair protection stage is to remove the free Sodium hydrosulfide associated with the hair and hide in the drum, leaving sufficient concentration within the hair follicles so that hair loosening is achieved but ensuring relatively little attack on the hair fibres. Hair loosening stage is where hair is loosened by activating the Hydrosulfide within the follicles by addition of lime to the above float. Typically, this stage uses 1.0% lime and hair loosening begins within 15-20 min, for salted hides and completed within 1.0-1.25 h. The process is particularly suitable where the loosened hair can be removed continuously from the processing vessel during the hair removal stage. It can be easily used in regular production and reduction of 70-80% of BOD, COD, Suspended solids, total solids could be easily achieved. The recovered hair can be used for protease production and improved leather quality with cleaner grain and less 'growth marks' (Crankston et al.,1986).

Treating the hides with a Protease enzyme to loosen most of the hair present on the hides includes removal of loosened hair from the hides without destroying the hair and rinsing the hides of enzyme solution for complete removal of hair and softening the hides. Further the hides are treated in a Sulphide liquor of relatively low strength (2.0% by weight of the hides) and therefore adding Hydrogen peroxide to the liquor (0.25 to 1.0% by weight of the hides) is sufficient to oxidize the Sulphide ion present in the liquor (Fekete. et al.,1982).

Various experimental procedures for the extraction of protease from Jawasee powder were adopted to optimize conditions for leaching. Water

oat pH 7.0 and 37 C was found to extract maximum enzyme in 24 h. The Jawasee protease is optimally active at pH 6.0 with 2.5% egg albumin as substrate. Sodium chloride at higher concentration inhibits the enzyme considerably. Strong inhibition was observed Para Chloro Meta Cresol, Penta Chloro Phenate, Phenyl Mercuric Nitrate and Sodium tri Chloro Phenate.

Jawasee protease has been used for unhairing sheep skins. Soaked skins were immersed in a tub containing 15% Jawasee powder and 200% water, pH of the skin was adjusted to 6.0 with a little alkali. After 24 h, the skins were taken out and unhaired and relimed in a separate bath containing 400% water, 5% lime, 0.25% Sodium Hydroxide and 0.2% wetting agent for 2 days. The pelts were further processed into leather

and studied. The leathers gave comparable characteristics with very minor differences. The advantages of using enzyme for unhairing process are, reduction of pollution problem and for the recovery of good quality hair/wool (Yeshodha et al.,1976).

Extra cellular protease from Aspergillus flavus for depilation of skin has been investigated. The protease concentrate contained (300 units/ml) in relation to wet weight of the skin was 1% (v/w) was applied as a paste on the flesh side of the skin and dehairing efficacy was observed. The enzyme is able to dehair the skins at the pH 9.0-9.5 in 16 h. The leathers produced after enzymatic unhairing was comparable with lime-sulfide systems. Physical and chemical analyses have showed no major differences but there was a considerable reduction of BOD and sulfides upto 50% in the effluent (Puvanakrishnan and Dhar, 1998). The pollution load generated in conventional and hair saving process of enzymatic method is given in Table 2. It is seen that there was 50% reduction of BOD and 40% reduction of COD in the experimental process.

By using protease, some or all of decorin was removed in limimg process. Alkaline protease treated hides are, however, substantially softer and more flexible than the controls. The second protease

preparation was one that made from a halophile because it did not produce collagenase. This protease was active in 4M NaCl at mild alkaline pH. Bating with this protease resulted leather with satisfactorily physical characteristics (Mozersky et al.,2002).

Alkaline protease isolated from a new strain Alcaligenes faecalis was evaluated. The enzyme is relatively stable in the pH range of 8-11 and at

o o30 C for 24 h. The enzyme can be stored at 0-4 C up to 6 months without much loss of activity. Unhairing action was found to be best at pH 9 and

oat 30-37 C with 0.5% w/w enzyme concentration. Microscopic studies of the unhaired skins and the physical properties of the enzyme-treated finished leathers were well comparable with leather processed by conventional methods. These results indicate that the protease produced by Alcaligenes faecalis is suitable for unhairing (Berla Thangam et al.,2001).

The neural protease enzyme, disperse; which possesses such substrate specificity was used for hair loosening. Enzyme concentration (U/ml) of

o1.2 at 30 C showed better unhairing with gentle scudding action. Epidermis was also removed after the incubation period for 16h (Paul et al.,2001).

Proteases and amylases enzymes from various sources have been applied individually or in combinations to produce effective unhairing of hides and skins (Bienkiewicz, 1983). However Protease enzymes are seen to be more efficient and find wider application in enzymatic unhairing than amylase enzymes. Hair gets loosened by the action of autolytic or lysosomal enzymes present in the hides at pH 7.0-8.5 after giving acetic acid treatment or by the autolytic action of protease in the skin or hide.

Table 2- Pollution load generated in liming process (conventional technology and hair saving process)

Pollution Load(kg/t raw hide)

Technology Decrease (%)Conventional Hair saving (Conventional)

Bio-Chemical Oxygen Demand (BOD)

Chemical Oxygen Demand (COD)

SulphidesSuspended Solids

30-40

79-122

3.9-8.753-97

12-20

46-77

1.9-4.314-26

50

50

40

73

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16 l Advanced Biotech l 2009October

conventional dehairing process. The H O in the alkaline medium was able to remove hair better than in acid medium. The results showed that destruction of hair was led by cooperative effect of H O and NaOH/ 2 2Ca(OH) at pH 13 or higher (Marsal et al.,2003). The leather produced by 2these methods showed better tensile strength than the conventional method. This may be due to the cross-linking reaction of collagen induced by H O that increases carboxyl group derived from oxidation of 2 2amino acid residues containing hydroxyl group, of serine and threonine residue (Shi et al.,2003).

Calcium Peroxide is used to rapidly unhair cattle hides with efficiency similar to that of Sodium Sulphide. Under alkaline conditions (pH 13) 5-15% (w/w) Calcium Peroxide mixture applied in two back-to-back applications, resulted complete unhairing within 10 min of total reaction

otime at a reaction temperature of 45 C (Gehring et al.,2003).

Three different dehairing products using alkaline Sodium Perborate and alkaline H O amended with either Potassium Cyanate or Urea was 2 2effective in removing hair, but it also led to extensive damage to the hide. It has been concluded that leather produced from the chemicals showed similar physical properties as that of lime and sulphide one (Marmer and Dudley, 2003).

Bating

Efficient bating relies on the use of enzyme like proteases, amylases and lipases to clean the hide or skin of degraded hair or epidermis.

Pepsin, extracted from pig stomach mucosa is active in acid condition. It was applied during pickling and on chrome tanned hides and skins. It can

oalso be conducted at lower temperature, such as 21-29 C, while in the classical alkaline bating, the activity of enzyme drops sharply at

otemperature under 32 C. Pepsin is an enzyme characteristic of the mammalian stomach structure, with molecular weight of 35kDa and a large amount of dicarboxylic, aliphatic and aromatic acids. Also the enzyme is an active in the pH range of 2.0-6.0 and becomes active in the presence of HCl. If it is applied on chrome tanned hides, after the splitting and shaving operations, the result is higher surface yield, softer leather and more uniform quality of leather (Deselnicu and Bratulesco, 1994).

The Proteolytic activity of pancreatic bate was determined in media of low ionic strength and in the presence of NaCl or NH SO at an ionic 4 4strength of 4. A peptide yielding of P-Nitroaniline as the hydrolytic product was used as the substrate. It has been confirmed that bate concentration of 0.11-3.35 (g/l) of NaCl, NH SO at high concentration 4 4(eg. Ionic strength 4) subsequently stimulates (60-75%) the protease activity of pancreatic bates (Mozersky et al.,2005).

Degreasing

The degreasing is mainly to remove fat and hence it is imperative to know the fat composition of hide in ovine and bovine animal (Table 3). The degreasing process, takes place in three successive stages: break-down of the proteic membrane of the fat containing sac, removal of the fat and emulsification of the removed fat in water or solubilization in solvent. If one of these is carried out incorrectly or inadequately then the whole degreasing operation will prove to be insufficient.

Triglycerol lipase under alkaline condition is able to penetrate fat cell to effect hydrolysis. Result suggests that the lipase can penetrate the adipocyte plasma membrane to effect interfacial catalysis under alkaline condition. Hydrolysis of the intracellular lipid droplet generates fatty acids and intermediate acylglycerols. In the presence of divalent calcium ions, this lipid transported away from the active site favoring the hydrolysis reaction. The depletion of the lipid droplet through the

2 2The addition of salts into the system of Sodium Sulphide plus Sodium Hydroxide improves the grain quality in comparison to dehairing without added salts. Also it has been found that the addition of salts into the system increases the removal of dermatan sulphate and this means that using added salts is desirable when dehairing by a lime free method (Valeika et al.,2000).

Ultra filtration of the enzymatic dehairing bath was carried out during the dehairing operation thus creating an enzymatic membrane reactor for the production of dehaired skins. The advantages of using ultra filtration system coupled with enzymatic dehairing process were control of enzyme action; reduction of sulphide requirement; easy recovery of hair with consequent reduction of polluting load and of cleaning up costs. It has also been found that by carrying out ultrafiltration process, there was increased opening up of fibre structure and production of softer leather in the leather processing (Cassano et al.,2000).

A mixture of proteolytic enzymes derived from Streptomyces griseus, was used to unhair bovine hide. The enzymatic dehairing was assisted by a 30 min pretreatment of the hide with Carbonate buffer with amphoteric surfactant (N,N-Dimethyl 1-1-Dodecanamine oxide) in the enzyme formulation. The unhairing reaction consisted of drumming pretreated hide pieces with 0.5 mg/ml of the bacterial protease (in 1% N,N-

odimethyl 1-1-dodecananine oxide) for 4 h at 37 C in 200% float. The leather produced by this method showed comparable shrinkage temperature, physical properties, dye levelness and grain appearance to the conventionally unhaired hide. Another experiment carried out with Pronase, a proteinase of a very broad specificity which breaks large number of peptide bonds produced by Streptomyces griseus and the limited dehairing activity of Trypsin, which is known to have a narrower specificity, breaking only the bonds on the carboxyl side of Arginine and Lysine (Gehring et al.,2002). It has been concluded that the skin hydrolyzed by facultative anaerobic microbes (Bacillus and Pseudomonas species) are capable of dehairing the skin and hydrolysis of skin collagen was found less when the organisms were grown anaerobically (Sambasiva Rao et al.,1976).

Various authors have emphasized the importance of either prior or subsequent alkali treatment of skin in enzyme unhairing. Shaving the hair from cattle hides before unhairing with enzyme also helps for dissolution of central portion of hair, the medulla, causing collapse of the hair (Yeshodha et al.,1978).

Using protease for dehairing skin, 50% reduction of Sulphide was observed in the waste water, as well as 40% reduction in the suspended solids level. The reduction in Sulphide helps for reduction of odors in the final Sulphate waste water content (Nashy et al.,2005), (Crispim and Mota, 2003).

It has been performed successful unhairing of sheep skins and cattle hides with a neutral amylase containing metallic proteinase derived out of Streptomyces hygroscopicus to reduce the Sulphide loads in the waste waters of conventional unhairing process (Zhang and Jiandong, 1990), (Wolf, 1991). It has been also reported that the alkaline protease obtained from Aspergillus tamarii had shown reduction of BOD to the level of 40% and COD to the reduction of 50% by using enzyme as the alternate method for conventional dehairing process (Dayanandan et al.,2003).

The enzyme isolated from Rhizopus oryzae and Aspergillus flavus was used for dehairing of hides and the final quality of leather was good with conventional liming methods (Banerjee and Bhattacharya, 1996), (Malathi and Dhar, 1987). .

The other unhairing systems

Oxidizing unhairing process based on H O alone and in combination 2 2with Ca(OH) or NaOH has been studied as an alternative to the 2

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17 l Advanced Biotech l 2009October

Review

accomplished using extremely low amounts of a combination of two enzymes selected to be particularly active in the condition of retanning with respect to pH, temperature, running times and presence of other chemicals. It has also been found that acid lipase at 0.3% and acid protease at 0.015% in retanning process resulted in more uniform grain and flesh appearance for full grain leathers. Stains from fats and oils were reduced and colors appeared cleaner and brighter due to reduced undertone (Mitchell and Ouellette, 1998).

Conclusion

Enzyme based technologies for leather industry has potential advantages for the reduction of pollution problem. Enzymes used in soaking process, provides better rehydration of the skin and better opening up of the fiber structure. Similarly in unhairing process, specific bond sites are attacked by using enzyme that resulted in removal of hair or wool, the epidermis, residual and non-structural components. Enzymatic unhairing resulted in reduction in effluent load especially BOD, COD to the level of 50% and 40% over conventional processes. Enzymatic process also helps to achieve better leather properties such as smoother grain surface and increased area in unhairing. Removal of keratin degradation products in bating and removal of fatty components in degreasing with the help of enzyme have paved the way for eco-friendly leather processing.

References1. Addy, V. L., A.D.Covington, D.A.Langridge and A.Watts, 2001.

Microscopy methods to study Lipase degreasing. Part2: A study of the interaction of ovine cutaneous adipocyes with Lipase enzymes using microscopy. J. Soc. Leather Technol. Chem ., 85: 52-65.

2. Annapurna Raju, A., N.K.Chandrababu, N.Samivelu, C.Rose and N.Muralidhara Rao, 1996. Eco-friendly enzymatic dehairing using extra-cellular proteases from a Bacillus species isolate. J. Amer. Leather Chem. Ass., , 91: 115-118.

3. Auer, N., A.D.Covington, Evans, M. Natt and Tozan M, 1999. Enzymatic removal of dung from hides. J. Soc. Leather Technol. Chem ., 83: 215-219.

4. Banerjee, P.S. and Bhattacharya BC, 1996. Application of a Proteolytic enzyme in tanneries as a depilating agent. J. Amer. Leather Chem. Ass., 91: 59-63.

5. Berla Thangam, E., T.Nagarajam, G.Suseela Rajakumar, 2001. Appliacation of alkaline protease isolated from Alcaligenes Faecalis for enzymatic unhairing in tanneries. J. Amer. Leather Chem. Ass., 96: 127-147.

6. Bienkiewicz,K, 1983. Physical chemistry of leather making. Robert E. Krieger Publishing Company, Malabar, Florida, Original English Edition. 1:55.

7. Brady, D., J.R. Duncan and A.E.Russel, 1990. A model for proteolytic depilation of skins. J. Amer. Leather Chem. Ass., 85: 334-343.

8. Cantera, C, 2001. Hair saving unhairing process. Part 4. Remarks on the evolution of the investigations on enzyme unhairing. J. Soc. Leather Technol. Chem ., 85: 125-132.

9. Cantera, C,2001. Hair saving unhairing process. Part 4. Remarks on the evolution of the investigate.ons on enzyme unhairing. J. Soc. Leather Technol. Chem ., 85: 125-132.

10. Cantera, C.S, 2001. Hair saving unhairing process. Part 1: epidermis and the characteristics of bovine hair. J. Soc. Leather Technol. Chem ., 85: 1-5 .

11. Cantera, C.S, 2001. Hair saving unhairing process. Part 2: Immunization Phenomenon. J. Soc. Leather Technol. Chem ., 85: 47-51.

12. Cantera, C.S, L.Goya , B.Galarza, M.Garro and M/L.Lopez, 2003. Hair saving unhairing process. Part5: Characterization of enzymatic preparations applied in soaking and unhairing process. J. Soc. Leather Technol. Chem., 87: 69-77.

13. Cantera, C.S, M.L.Garro, L.Goya, C.Babeito and B.Galarza, 2004. Hair saving unhairing process. Part 6. Stratum corneum as a diffusion barrier:

continuous hydrolysis may be responsible for causing a change in the intracellular pressure. This may cause the membrane to collapse spontaneously into the locules encompassed within the loss of supporting plasma memebrane, will also subside and form process around the collapsed plasma membrane. These results show why lipase-mediated degreasing is inconsistent within the leather making process (Addy et al.,2001).

Degreasing using an unhydroxylated fatty alcohol has been studied on pickled sheep skin. It has been found that degreasing effectiveness increases when the pH was increased. Effectiveness increases when the surfactant concentration increases up to an offer of 6% active matter. Also it has been found that degreasing effectiveness increases with time at 6-8 h (Palop et al.,2000).

The use of lipases to degrease hides and pelts have been discussed. A highly synergistic effect in degreasing is achieved when special proteases and emulsifying systems are used at the same time. Protease break down the cell membranes of fat cells in hide and the new lipase reduces the amount of emulsifier needed. In addition to improved degreasing a better soaking and liming effect is obtained (Christner, 1992), (Marsal et al.,1998). The overall reduction of pollution by using enzyme from soaking to bating is given in Table 4. It was observed that there was a pollution reduction from 40 to 90% in different process (Post,

1997).

Enzyme in post tanning process

Evaluation and cleaning of chrome tanned stock for grease, dirt, scud and other skins, for the purpose of making more uniformly colored leather has been treated with a lipase and a mild protease. A significant reduction was found in grease stains, neck wrinkle discoloration and other stains. Also, an improvement was observed in the brightness and uniformity of dyeing-backbone to belly and side to side within a mill. This was

Table 3 -Fat composition of hide as a relative % of total fat

Different kinds of fat (%)

53 (6% in Epidermis and 47% in Corium)

11214101

100

Ovine (%)

Triglycerides

Wax estersPhospholipidsCholesterolFree fatty acidsHydro carbons Total fat

Bovine (%)

56 (12% in Epidermis and 44% in Corium)

23655-

100

Table 4-Minimum Pollution Reduction by using Enzymes in various stages of leather processing

Enzymes in various Process BOD

Overall pollution reduction by using enzymeCOD

Removal of Dung in Soaking process

Unhairing in Liming process (Hair saving method)

Degreasing

Bating

Solid Wastes

95%

50%

60%

75%

90%

90%

40%

40%

60%

80%

18 l Advanced Biotech l 2009October

37. Mozersky, S..M., Wildermuth and W.N.Marmer, 2005. The relative proteoltic activity of pancreatic bate in media of low and high salt content. J. Amer. Leather Chem. Ass., 100: 396-400.

38. Mozersky, S.M., O.D.Allen and W.Marmer, 2002. Vigorous Proteolysis:Reliming in the presence of an alkaline protease and bating with an extremophile protease. J. Amer. Leather Chem. Ass., 97: 150-155.

39. Nashy, E. H..A., S.A.Ismail, A.M.Ahmady A M, 2005. Enzymatic bacterial dehairing of bovine hide by a locally isolated strain of bacillus licheniformis. J. Soc. Leather Technol. Chem ., 89: 242-249.

40. Palop, R., A.Marsal and J.Cot J, 2000. Optimisation of the aqueous degreasing process with enzymes and its influence on reducing the contaminant load. J. Soc. Leather Technol. Chem ., 84: 170-176.

41. Paul, R. G., I.Mohamed, D.Davighi and A.D.Covington, 2001. The use of neutral protease in enzymatic unhairing. J. Amer. Leather Chem. Ass., 96: 180-185.

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Review

Leather Processing Division Central Leather Research Institute,Adyar, Chennai 600 020.

For Correspondence: E-Mail:jkraj68@yahoo.co.uk