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Enzymatic Removal of Melanin in Enzyme Based Dehairing and Fibre Opening

Article in Journal of the American Leather Chemists Association · July 2008

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 Enzymatic Removal of Melanin in Enzyme Based Dehairing and Fibre Opening

V. Punitha, P. Kannan, S. Saravanabhavan, P. Thanikaivelan#, J. Raghava Rao,

Balachandran Unni Nair and T. Ramasami

Chemical Laboratory,
#
Centre for Leather Apparel & Accessories Development,

Central Leather Research Institute, Adyar, Chennai 600 020, India.

Tel: 91-44-24411630; Fax: 91-44-24911589; Email: [email protected]

Abstract

Melanin is a natural pigment of skin and hair, which provides a protective function against

sunlight. The non-removal of pigments from the skin gives the finished leather a patchy

appearance. As leather industry is undergoing a paradigm shift towards bioprocessing,

enzyme based dehairing and fibre opening are becoming ecologically important. However,

the enzymatic dehairing and fiber opening of buff calfskins from certain origin results in non-

removal of pigments. In this study, an attempt has been made to remove the pigments from

buff calfskins using enzymes. The presence of melanin in buff calfskins was identified

through UV-visible spectral analysis. Preliminary trials have been carried out with various

concentrations of xylanase in enzymatic dehairing and fibre opening. Addition of xylanase

provides complete removal of melanin during enzymatic dehairing as well as fiber opening.

Semi-technical trials have been performed by employing xylanase during enzymatic

dehairing and fiber opening individually. The removal of melanin was found to be 100%.

The performance characteristics of the resulted leathers have been analyzed and found

satisfactory.

1
Introduction

The do-undo methods adopted in conventional leather processing generates huge amount of

pollutants, in view of the fact that they subject the skin/hide to wide variations in pH.

Pretanning and tanning processes alone contribute to more than 90% of the total pollution

generated in a tannery. Leather industry is undergoing a paradigm shift towards

bioprocessing. Present day innovations in biotechnology have proved that enzyme assisted

dehairing using proteolytic enzymes with low amounts of lime and sodium sulfide is

commercially feasible. Recently, a lime free enzymatic dehairing process along with reduced

amount of sodium sulfide has been standardized for cowhides, which ensures complete

dehairing within 18 hrs.1 An enzyme only dehairing method for goatskins without the use of

lime and sodium sulfide has also been established.2 The interfibrillary proteins, which are

mostly mucoids that contain carbohydrate as prosthetic groups, are removed during fibre

opening. These non-collagenous proteins are known as proteoglycans. Hence, in principle, it

should be possible to produce pelt by removing the protein-carbohydrate conjugates through

the action of substrate specific enzymes. It has been shown that α-amylase has specific

activity on carbohydrate-containing proteins such as proteoglycans. Thanikailvelan et al have

developed the enzyme-based fibre opening for cowhides using α-amylase without using lime

at pH 8.0.3 Aravindhan et al have developed enzyme-based fiber opening process for

sheepskins.4 Usually, after the enzymatic dehairing and fibre opening the grain of the skin

appears clean and white. However, in our early trials, similar enzymatic treatment on buff

calfskins resulted in removal of hair with incomplete removal of epidermal layer leading to

black or brown grain surface. This leads to patches in the surface of the final leather. This is

primarily due to the presence of excess pigmentation as melanin in the buff calfskins. It is

2
reported that the solubilization of melanin occurs at a pH greater than 10.5 The conventional

liming method results in clean and white pelt due to the operational pH being greater than 10

during lime based processing. However, in the lime free enzyme-only methods, the removal

of melanin is difficult during enzymatic dehairing and fibre opening, especially in buff

calfskins. This is mainly due to presence of higher pigmentation in buff calfskins, which is

difficult to be removed by solubilization of melanin at the operational pH of 8 during

enzymatic dehairing and fibre opening process.

Melanin is a specific class of polycyclic biopolymer related to the humic acids and found

throughout nature, importantly in humans and animals. Melanin is produced by the

melanocyte due to tyrosinase action on amino acids. Melanin is a primary colour agent in

hair, skin and eyes and it is believed to be a protective agent against the damaging effects of

UV radiation.6 There are three main types of melanins: 1) Eumelanins: brown black pigments

derived from tyrosine following its conversion to dopa (dihydroxyphenylalanine); 2)

Phaomelanins: reddish-brown pigments which are cystine derivatives of eumelanin and 3)

Allomelanin: black pigments (similar to eumelanins) formed from catechols via

polyhydroxynapthalene. Eumelanins involve the formation of indole 5, 6-quinone by several

steps. Melanin is then formed by polymerization. Melanins have the ability to readily

undergo reduction and oxidation reactions due to its ability to accept or donate an electron

very easily over a large pH range. Melanin is a negatively charged polymer mainly due to its

numerous -COO− groups. Therefore, it is likely that it can interact with positive charges on

proteins. Thus, charge interaction between proteins and melanin polymer may be one mode

of melanin–protein binding. This was tested using the zeta potential meter. 7,8

3
Xylanase is an enzyme widely used for the bleaching of pulp and wood.9 It hydrolyzes the

xylan present in the material. Xylan is the most abundant non-cellulosic polysaccharide

present in both hard wood and animal plants. Bio-bleaching of wood and pulps are well

practiced in the industries employing xylanase.9 Hence, the xylanse has been chosen to

bleach the pigments present in the calfskins. It is classified under the category of hydrolases

and it has acid catalytic behavior.

In the present study, the black material present in the buff calfskins were extracted and

identified using UV-visible spectroscopy. Xylanase has been used during dehairing and fibre

opening for the removal of pigment. The efficiency of removal of melanin by xylanase has

been studied. The final quality of the leathers has been assessed through organoleptic and

physical properties. Scanning electron microscopic analysis of leathers has been carried out

and the results will be presented later.

4
Experimental Methods

Raw Materials

Wet salted buff calfskins from Indian origin (average weight of 3 kg per skin) were chosen as

the raw material. Biodart (dehairing enzyme), α-amylase (fibre opening enzyme) and

xylanase (melanin removal) were procured from Southern Petrochemical Industries

Corporation (SPIC) Limited, India. All chemicals used for leather processing were of

commercial grade. The chemicals used for analytical techniques were of laboratory grade.

Analysis of Melanin

The black material from the skin is removed by gentle scrapping after standardized

enzymatic dehairing, which is described below. The black material was hydrolysed by using

1M NaOH in the presence of 3% H2O2 in a boiling water bath for 30 min.10 After cooling, the

absorption spectra of hydrolysed matter were recorded in a Perkin Elmer Lambda 35 UV-

visible spectrophotometer at room temperature in the wavelength range of 200-800 nm.

Standardization of Dehairing Process

In order to standardize the application of dehairing method for buff calfskins, three methods

of applications were chosen namely, dip and pile method, grain side application and drum

method. Two soaked buff calfskins were used for each trail. The sodium sulfide and the

dehairing enzyme concentration were fixed as 0.5 and 1% respectively. The depilatory

composition for different application is given in Table 1. In dip and pile method, a solution of

corresponding composition was made and the pieces were mixed with the solution for 10

min. Then the pieces were piled and left overnight. In grain side application, a thick paste

5
was prepared as per the composition and then applied on grain side, piled and left overnight.

In the case of drum dehairing, the depilatory composition was added with soaked calfskins.

The duration of treatment was 6 hours with 15 min running per hour. Subsequently the skins

were left in the bath overnight. Next day, the calfskins were dehaired and rated on the basis

of the average area without hair out of the total area.

Preliminary Trials on Pigment Removal

Effect of Pigment Removal During Enzymatic Dehairing

Ten buff calfskins were soaked conventionally. Soaked weight of the each skins was noted.

Two skins were used for each trail. The extent of removal of melanin during enzymatic

dehairing by using xylanase was studied on soaked buff calfskins. The process for the

xylanase treatment is given below.

Process Chemicals % offer Remarks

Enzymatic dehairing Water 15 The duration of treatment was 6

(drum method) Biodart (SPIC) 1 hours with 15 min running per hour
Sodium sulfide 0.5 and left overnight in the bath.
Xylanase X Next day, the extent of removal of
pigment was assessed visually
during manual dehairing.
X was varied as 0.1, 0.2, 0.3, 0.4 and 0.5%

Percentages based on soaked weight of calfskins.

Effect of Pigment Removal During Enzymatic Fibre Opening

6
Ten buff calfskins were soaked conventionally. Soaked calfskins were dehaired using

standardized dehairing process. Two dehaired calfskins were used for each trail. The extent

of removal of melanin during enzymatic fibre opening3 by using xylanase was studied on

dehaired buff calfskins. The process for the xylanase treatment is given below.

Process Chemicals % offer Remarks

Enzymatic fibre Water 100

opening α-amylase 1

Xylanase X Run for 3 hours.

Extent of pigment removal was

assessed visually.

X was varied as 0.1, 0.2, 0.25 and 0.3%

Percentages based on dehaired weight of calfskins.

Optimized Process for Pigment Removal

Two optimized trials were selected based on the preliminary xylanse treatment trials for the

removal of pigment during enzymatic dehairing and enzymatic fibre opening. Ten soaked

buff calfskins were used for each optimized trial. The offer of xylanase was 0.5 and 0.3% for

application during enzymatic dehairing and enzymatic fiber opening, respectively. In the case

of xylanase application during enzymatic dehairing, the fibre opening was carried out using

α-amylase without employing xylanase.3 A control trial was performed without the use of

xylanase on ten soaked buff calfskins. The efficiency of removal of xylanase was assessed

visually. Then the calfskins were chrome tanned using a conventional post-fibre opening

process without deliming. The chrome tanned leathers were converted into upper leathers

7
using commercial post tanning process with the offer of 14% syntan, 10% fatliquor and 2%

dye.

Physical Testing and Hand Evaluation of Leathers

Samples for various physical tests from experimental and control crust leathers were obtained

as per IUP method.11 Specimens were conditioned at 26.6±2.2oC and 65±2% relative

humidity over a period of 48 h. Physical properties such as tensile strength, % elongation at

break and tear strength were examined as per the standard procedures.12,13 Experimental and

control crust leathers were assessed for softness, fullness, grain tightness, grain smoothness

and general appearance by hand and visual examination. Two experienced tanners performed

the assessment.

Results and Discussion

The selection of raw materials has been based on the presence of higher amount of melanin

in epidermal layer. It is known that buff calfskins contain higher amount of melanin as

compared to other raw materials. Especially, buff calfskins from Northern part of India have

the problems of non-removal of pigments during enzymatic dehairing and fiber opening. This

study aims at complete removal of melanin during enzymatic dehairing and fiber opening.

Standardization of Enzymtaic Dehairing for Buff Calfskins

Trials have been performed in order to find the optimal application method for buff calfskins.

The efficiency of dehairing for the various methods of application is shown in Figure 1. It is

seen that the dip and pile as well as grain side application methods do not result in 100%

8
removal of hair. But, the drum method results in 100% removal of hair. This method of

application is different from those observed for cowhides during sodium sulfide assisted

enzymatic dehairing, where painting on grain side application1 provided complete dehairing

while dip and pile method14 in the case of sodium metasilicate assisted enzymatic dehairing.

This could be due to the structural and keratin-grain layer compaction difference between

cow and buff calf species. Further studies were carried out based on drum dehairing method.

Identification of Melanin

It is paramount important to ascertain that the non-removed material on the epidermis of buff

calfskins after the enzymatic dehairing and fibre opening process is melanin. Hence, the non-

removed material from the grain surface of enzymatically dehaired buff calfskins was

extracted using standard procedure. The UV-Visible absorption spectrum for the extracted

solution is presented in Figure 2. It is seen that the absorption maxima for the solution

extracted from the grain surface of buff calfskins is 300 nm. It has been reported that the

eumelanin shows absorption maxima at 300 nm.10 Hence, it is evident that the absorption

maxima at 300 nm for the extracted solution from buff calfskin is primarily due to the

presence of eumelanin. Therefore, it can be concluded that the material present after the

enzymatic dehairing and fibre opening is melanin.

Effect of Xylanase Treatment During Enzymatic Dehairing

The extent of removal of melanin by xylanase treatment during enzymatic dehairing is shown

in Figure 3. It is seen that the extent of removal of melanin increases with the increase in

concentration of xylanase. The complete removal of melanin during enzymatic dehairing is

9
achieved at a xylanase concentration of 0.5%. The removal of melanin may be due to the

action of xylanase on the adhering matter present between epidermal and melanin layer. The

adhering matter may be such as proteoglycans or globular proteins. It has been postulated

that the loosening of adhering substance containing glycans by xylanase results in the

removal of melanin. Hence, the optimized concentration of xylanase for the removal of

melanin is 0.5%.

Effect of Xylanase Treatment During Enzymatic Fibre Opening

The offer of xylanase during enzymatic fibre opening was varied from 0.1 to 0.3%. The

percentage removal of pigment by xylanase during enzymatic fibre opening is shown in

Figure 4. It is seen that the increase in the concentration of xylanase results in significant

increase in the percentage removal of melanin. At 0.3% offer of xylanase, melanin was

completely removed. It is interesting to note that the 100% removal of melanin is achieved

using low offer of xylanase in enzymatic fibre opening in comparison to enzymatic

dehairing. This is due to the fact that substrate specificity for α-amylase as well as xylanase

is almost similar. Both the enzymes act on glycans, although their mechanism of action is

different. Hence, the presence of α-amylase assists xylanase in complete removal of melanin

in spite of its low offer in comparison to during enzymatic dehairing.

Semi-Technical Trials for Melanin Removal Using Optimized Processes

The use of xylanase during enzymatic dehairing and enzymatic fibre opening resulted in

100% melanin removal at an offer of 0.5 and 0.3%, respectively. These optimized processes

were carried out at semi-technical level trials along with a control trial with ten buff calfskins

10
for each trial. The pelts from these processes were processed using conventional post-fibre

opening process. The crust leathers were evaluated for strength and physical properties.

Strength Characteristics

The resultant crust leathers from xylanase treatment during enzymatic dehairing and fibre

opening were subjected to physical testing using standard procedures. The physical testing

data is presented in Table 2. It is seen that all the strength properties of experimental leathers

are comparable to the control leather values. Importantly, tensile strength of the experimental

leathers is slightly better than the control leathers. All the strength values from control and

experimental are meeting the standard norms.15

Organoleptic Properties

The crust leathers derived from xylanase treatment during enzymatic dehairing and fibre

opening were assessed for softness, fullness, uniformity in color, grain smoothness and

general appearance. The data is presented in Figure 5. It is seen from that the leathers made

from xylanase treatment have better rating in uniformity in color indicating that they are

cleaner than control leathers. The grain smoothness is comparatively better than control

leathers. Softness and fullness of xylanase treated leathers are comparable or even better than

that of control leathers. The general appearance of the experimental leathers is much better

than the control leathers.

Mechanistic Insight in the Removal of Melanin Through Xylanase

Melanin is covalently linked to the skin protein. Degradation of melanin can be achieved by

using oxidizing or reducing agents. On the other hand, breaking of bond between melanin

11
and the skin protein by employing suitable enzymes may induce melanin degradation.16 In

this study, it has been hypothesized that the removal of melanin may be due to the action of

xylanase on the adhering matter present between the epidermal and the melanin layer.

Loosening of adhering substance containing glycans by xylanase resulted in the removal of

melanin. The catalytic activity of xylanase is based on a double-displacement

mechanism.16,17 It has been shown that the xylanases hydrolyze the β-(1,4) linked xylose

backbone of xylans.17 It is interesting to note that α-amylase assists xylanase during its

application in enzymatic fibre opening for the removal of melanin, which has almost similar

mechanism. Two different mechanisms are suggested for the action of α-amylase such as

breaking the O-linkage between the protein and carbohydrate moiety or catalyzing the

hydrolysis of the α-(1,4) glycosidic linkages in glycans.18,19

Conclusion

The non-removal of pigments from buff calfskins during enzymatic dehairing and fibre

opening resulted leathers with unacceptable surface. In this study, this non-removed material

was extracted from buff calfskin and identified as melanin using UV-Visible

spectrophotometry. Optimization trials have been carried to find the optimal concentration of

xylanase for its application during enzymatic dehairing and fibre opening. The complete

removal of melanin has been achieved using xylanase during enzymatic dehairing and

enzymatic fibre opening processes at 0.5 and 0.3%, respectively. Semi-technical level trials

revel that the crust leathers have similar or even better strength and bulk properties than the

control leathers.

12
Acknowledgment

The authors thank Mr. P. Saravanan, Tannery Division, Central Leather Research Institute

for his helpful suggestions in the development of this process.

References

1. Thanikaivelan P., Rao J.R. and Nair B.U. (2000) ‘Development of a leather

processing method in narrow pH profile. Part 1. Standardization of unhairing

process’, J. Soc. Leather Technol. Chem., Vol. 84, pp. 276–284.

2. Saravanabhavan S., Aravindhan R., Thanikaivelan P., Rao J.R., Chandrasekaran B.

and Nair B.U. (2003) ’An integrated eco-friendly tanning method for the manufacture

of upper leathers from goatskins’, J. Soc. Leather Technol. Chem., Vol. 87, pp. 149–

157.

3. Thanikaivelan P., Rao J.R., Nair B.U. and Ramasami T. (2002) ‘Zero discharge

tanning: A shift from chemical to biocatalytic leather processing’, Environ. Sci.

Technol., Vol. 36, pp. 4187–4194.

4. Aravindhan R., Saravanabhavan S., Thanikaivelan P., Rao J.R., Chandrasekaran B.

and Nair B.U. (2004) ‘A bio driven lime and pickle free tanning paves way for

greener garment leather production’, J. Amer. Leather Chem. Ass., Vol. 99, pp.53–

66.

5. Ito S and Wakamatsu K. (2003) ‘Quantitative analysis of eumelanin and pheomelanin

in humans, mice and other animals: A comparative review’, Pigment Cell Res., Vol.

16, pp. 523–531.

13
6. Wakamatsu K. and Ito S. (2002) ‘Advanced chemical methods in melanin

determination’, Pigment Cell Res., Vol. 15, pp. 174–183.

7. Mani I., Sharma V., Tamboli I. and Raman G. (2001) ‘Interaction of melanin with

proteins – The importance of an acidic intra melanosomal pH’, Pigment Cell Res.,

Vol. 14, pp. 170–179.

8. Liu Y. and Simon J.D. (2003) ‘Isolation and biophysical studies of natural

eumelanins: Applications of imaging technologies and ultrafast spectroscopy’,

Pigment Cell Res., Vol. 16, pp. 606–618.

9. Kertesz D. (1957) ‘Skin lighteners and bleach creams’ Cosmetics Sci. Technol., Vol.

3, pp 225.

10. Ito S., Wakamatsu K., Ozeki H. (1993) ‘Spectrophotometric assay of eumelanin in

tissue samples’, Anal Biochem., Vol. 215, pp. 273–277.

11. IUP 2 (2000) ‘Sampling’, J. Soc. Leather Tech. Chem., Vol. 84, pp. 303–309.

12. IUP 6 (2000) ‘Measurement of tensile strength and percentage elongation’, J. Soc.

Leather Tech. Chem., Vol. 84, pp. 317–321.

13. IUP 8 (2000) ‘Measurement of tear load – Double edge tear’, J. Soc. Leather Tech.

Chem., Vol. 84, pp. 327–329.

14. Saravanabhavan S., Thanikaivelan P., Rao J.R. and Nair B.U. (2005) ‘Silicate

enhanced enzymatic dehairing: A new lime-sulfide-free process for cowhides,

Environ. Sci. Technol., Vol. 39, pp. 3776–3783.

15. Specification for upper leather, ‘Acceptable quality standards in leather and footwear

industry ‘,United Nations Industrial Development Organization (UNIDO), Vienna,

Austria, 1994.

14
16. Bray M.R. and Clarke A.J. (1994) ’Identification of glutamate residue at the active

site of xylanase A from Schizophyllum Commune’, Eur. J. Biochem., Vol. 219, pp.

821-827.

17. Canals A., Vega M.C., Gomis-Ruth F.X., Diaz M., Santamaria R.I. and Coll M.

(2003) ’Structure of xylanase Xys1delta from Streptomyces halstedii’, Acta

Crystallogr. D Biol. Crystallogr., Vol. 59, pp. 1447 – 1453.

18. Butler W.T. and Cunningham L.W. (1966) ‘Evidence for the linkage of a

disaccharide to hydroxylysine in tropocollagen’, J. Biol. Chem., Vol. 241, pp. 3882-

3888.

19. Gilles C.B., Rousseau P., Rouge P. and Payan F. (1996) ‘Substrate mimicry in the

active center of a mammalian alpha-amylase: structural analysis of an enzyme-

inhibitor complex’, Structure, Vol. 4, pp. 1441-1452.

15
 Table 1. Composition of depilatory mixture

Application Water (%) Na2S (%) Enzyme (%)

Dip and pile 15 0.5 1

Drum 15 0.5 1

Grain side 7 0.5 1

Percentages were based on soaked weight of buff calfskins.

Table 2. Physical testing data of the crust leathers

Sample Tear strength Tensile strength % Elongation at

(Kg/cm) (Kg/cm2) break
UNIDO norms15 30 200 60-70
Control without 31±2 246±12 67±5
xylanase
0.3% xylanase during 32±3 252±18 73±4
enzymatic dehairing
0.5% xylanase during 35±4 263±14 69±7
enzymatic fibre
opening
Average of mean of along and across backbone values for five leathers

16
Figure Captions

Figure 1. Dehairing efficiency by various methods of application for buff calfskins

Figure 2. UV-Visible absorption spectrum of the extracted solution from the grain

surface of dehaired buff calfskins using enzymes

Figure 3. Removal of melanin using xylanase during enzymatic dehairing

Figure 4. Removal of melanin using xylanase during enzymatic fibre opening

Figure 5. Organoleptic properties of crust leathers

Figure 1.

100

90

Rating 80
(% hair
removal) 70

60

50
Dip and pile Grain side Drum
pasting

1% Enzyme and 0.5% sodium sulfide (based on soaked

weight) was used in all the methods of application
15% Water was used for dip and pile method
15% Water was used for drum method
7% Water was used for paste method

17
 Figure 2.

Figure 3.

100
% Melanin removal

80

60

40

20

0
0.1 0.2 0.3 0.4 0.5
% Xylanase

18
 Figure 4.

100
% Melanin removal

80

60

40

20

0
0.1 0.2 0.25 0.3
% Xylanase

Figure 5.

10
8
6
Rating
4
2
0
Softness Fullness Uniform in Grain General
color smoothness appearance

Control without xylanase

0.5% Xylanase during enzymatic dehairing
0.3% Xylanase during enzymatic fiber opening

19

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