FIELD OF THE INVENTION:
The present invention relates to a novel composition and the process for dehairing of hides exclusively by proteases in a lime free, sulfur free and microbe free conditions.
PRIOR ART
Dehairing is one of the main operations in the process of dehairing. The most commonly practiced method of dehairing of hides and skins is the chemical process using lime and sodium sulphide. The use of high concentrations of lime and sodium sulphide creates an extremely alkaline environment resulting in the pulping of hair and its subsequent removal.
Conventional leather dehairing process involves the use of lime and sodium disufide. The liming process contributes to 50-70% of the total biological oxygen demand (BOD) and chemical oxygen demand (COD). The extensive use of sulfide bears unfavourable consequences on the environment flora and fauna.
Several lime and disulfide free methods have evolved in the past century.
The main disadvantages of the above process of dehairing are:
ii) This process contributes for 70-80% of the total COD of effluent from all leather making
processes.
(ii) Sulphide is highly toxic with obnoxious odor.
(iii) The severe alkaline condition is a health hazard for the workers.
The cleaner process for the dehairing is the use of enzymes. Enzymatic dehairing is suggested as an environmentally friendly alternative to the conventional chemical process. The enzyme digests the basal cells of the hair bulb and the cells of the malphigian layer.
This is followed by loosening of hair with an attack on the outermost sheath and subsequent swelling and breakdown of the inner root sheath and parts of the hair that are not keratinized.
The main advantages of enzymatic dehairing are:
(i) Significant reduction or even complete elimination of the use of sodium sulphide.
(ii) Recovery of hair of good quality and strength with a good saleable value.
(iii) Creation of an ecologically conducive atmosphere for the workers.
(iv) Enzymatically dehaired leathers have shown better strength properties and greater
surface area.
(v) Simplification of pre-tanning processes by cutting down one step, viz. bating.
Microbial proteases derived from a wide variety of yeasts, molds, and bacteria are used for dehairing, and later it was reported that simultaneous dehairing and bating is possible with some of the proteases.
Major chemical and microbial products used in dehairing along with their advantages and disadvantages are listed in table.
Though leather-makers have improved the process, 'dehairing' and 'fibre-opening' steps are still noxious. Enzymatic processing has a 45 percent lower COD than traditional lime and sulphide processing. It cuts the amount of solid sludge that dehairing and fibre opening generates about 20 percent of that from the entire treatment cycle, by an impressive 95 percent.
Enzymatic dehairing has lots of advantages over chemical dehairing still they increase the microbial load of the effluent released by tannery industry. As micro organisms break down the organic waste produced by stripping the raw hides, they consume oxygen. The more oxygen they use, the higher the COD and the greater the waste in the effluent.
The dehairing of hides or skins by means of enzymes was first described around mid- to late 1960's. A typical representative prior art method is described in US patent 3840333 which describes dehairing of skins and hides in a strongly alkaline environment by means of certain alkali resistant proteolytic enzymes.
A drawback in relation to method for dehairing of this kind resulted in the dehaired hide or skin with either incompletely dehaired (if the enzyme used is small) or be damaged in the grain(if the enzyme is high)
US Patent No. 5834299 discloses, dehairing of hides or skins using enzymes. However the process of the present invention also uses at least one protein disulfide redox agent.
EP patent No. 0660879 discloses a method for de-hairing of hides or skins by soaking and subsequent de-hairing by controlled addition of alkaline proteolytic enzymes and a small amount of NA2S.
In US patent no. 3840433, dehairing of skins and hides in an alkaline medium of pH of about 12.5 such as by lime treatment is accelerated by the inclusion of certain alkali resistant proteolytic enzymes
US patent No 3623950 discloses a process for dehairing skins and hides by treatment thereof at a pH less than 5, with a mold fungus protease with pepsin, with papain or with a combination of these enzymes
Bose and Dhar (Leather Science 1955, 1974) have reviewed the use of proteolytic, amylolytic enzymes from various sources namely bacterial, fungal, animal and plant for dehairing of hides and skins. However these methods involve the use of lime. Morera et. al. (1977) have reported the use of hydrogen peroxide , however, there has not been much reduction in COD. Sehagal et. al. (1996) have developed a non-enzymatic sulfide free dehairing process using 1% nickel carbonate, 1% sodium hydroxide, 5% lime and kaolin along with water by painting. However, the use of nickel leads to serious health
hazards. Saranabhavan et. al (2005) have completely eliminated the use of lime and sodium sulfide by using silicate salts. Macedo et. al. (2005) have also completely eliminated the use of sulfite leading to hair saving green dehairing technology. However in this report crude enzyme extract has been used where live cells might work as a reductant.
Thus, till today keratinolysis has always been an enzyme-assisted process, however no enzyme- mediated process is known till date where dehairing becomes purely enzymatic process.
SUMMARY AND OBJECT OF INVENTION
The main objective of the invention is therefore to develop purely enzymatic composition for dehairing of hides.
Another objective of the invention is to develop purely enzymatic method for dehairing of hides.
Another objective is to provide methods for modifying catalytic behaviour of proteases so that they become true keratinases having both sulfitolytic and proteolytic activity.
Still another object of the invention is to develop sulphide and lime free process for dehairing of hides hence providing green-technology for dehairing of hides for leather processing.
Another objective is to provide chemical free method and hence decreasing the COD level of the effluent of the leather industry.
Another objective is to exclude the role of microbe in the dehairing process to decrease the microbial load on the effluent from the leather industry.
Yet another object of invention is to develop dehairing at acidic to neutral pH.
Yet another objective is to use the mixture of proteases with different catalytic specificity for dehairing of hide.
Other objective of the invention would be to test these keratinases for their affectivity as permanent hair remover, wool cleaning, skin fairness creams and collar washing solutions.
To achieve the aforementioned object, the present invention, provides a process for modifying the catalytic behavior of proteases by biochemical interactions comprising the step of which cleaves at hydrophobic amino acids (subtilisin and chymotrypsin) so that the protease acquires additional catalytic potential for reducing sulphide bonds along with proteolytic action.
DEATILED DESCRIPTION OF INVENTION
A process was developed for modifying the catalytic behaviour of proteases by biochemical interactions.
The proteases used in the invention were from bacterial, fungal and animal sources. Peptide sources may be from various enzyme hydrolyzed plant or animal proteins, eg. corn steep liquor, tryptone, meat protein, soyflour, soybean meal, papain. These were prepared by enzymatic digest of chymotrypsin, trypsin and papain by individual and mixed digestions. The two were mixed in various ratios:
Enzyme-0.1-5g (1500U/g) Peptide mix- 5-1 g
The enzyme and peptide mix was mixed in the inverse ratio (low enzyme and high peptide mix and vice versa) when required.
The pH of the mixture was checked to be acidic to neutral. The enzyme thus prepared was characterized with reference to caseinolytic and keratinolytic activity on casein and feather keratin Enzyme mix thus prepared was tested for reductase activity using glutathione as the substrate (pH 7, 37°C). Glutathione reductase activity of different enzyme preparations was tested.
Keratinolytic assay
Keratinase activity was measured as described by Dozie et al. (1994) with some modifications. The assay mixture containing 1ml_ appropriately diluted enzyme, 4mL glycine/ NaOH buffer (0.05 M, pH 10) and 20 mg feather was incubated at 60°C for 60 min. The reaction was terminated by adding 4 mL of 5% TCA and the tubes were incubated for 60 min at room temperature (25°C). Feather and insoluble residue was removed by filtration through glass wool and the filtrate was centrifuged at 3000 g for 5 min. An enzyme control was prepared in a similar manner except that 1 mL of 5% TCA and 3 mL buffer were added instead of 4mL buffer in the test.
Proteolytic products in the supernatant were determined by absorbance at 280 nm against controls (enzyme and substrate controls). An increase in absorbance of 0.01 was considered as 1 unit enzyme activity per mL per hour at 60°C.
Caseinolytic assay
The proteolytic activity was assayed at 60°C in glycine/ NaOH buffer (0.05 M, pH 10) as described previously (Beg and Gupta 2003). One mL of appropriately diluted enzyme was incubated with 1 mL of casein solution (prepared in glycine/NaOH buffer pH 10) for 10 min at 60°C. The reaction was stopped by addition of 4 mL of 5% trichloroacetic acid. The contents were centrifuged after 1 h at 3000 g for 10 min. One mL of the supernatant was taken and the products were measured by adding 5 mL Folins Ciocalteau reagent. The optical density of the samples was taken at 660 nm against an appropriate substrate and enzyme blank.
One unit of protease was equivalent to the amount of enzyme required to release 1 |ug of tyrosine per mL per min under standard assay conditions.
Disulfide reductase activity
The disulfide reductase activity was measured as described by Brown et al. (1996) with some modification. Enzyme and peptide mix were dissolved in 10mL phosphate buffer. 1 mL of the enzyme mixture was incubated with 1 mL of 2mM glutathione (oxidized) solution (prepared in phosphate buffer pH 7) for 20 min at 37°C in the presence of 5mM PMSF. The contents were centrifuged after 1 h at 3000 g for 10 min and the production of sulfydryls from oxidized glutathione were detected by addition of 50µL of DTNB to 1.5µL of the supernatant. The absorbance of the solution was measured at 420 nm after 2 min of stable colour development. Enzyme control was prepared in a similar manner except that 1 mL of phosphate buffer (pH 7) was added instead of substrate. Also, a substrate control containing 1mL glutathione and 1mL phosphate buffer (pH 7) was prepared. One unit of sulfydryl reductase was equivalent to the amount of enzyme required to release 1 famole of sulfydyl per mL per min at 37°C. The efficacy of this mix was also tested on dehairing.
Experiment 1: A 4 by 4-square cm hide was incubated with enzymes and peptides in the ratio 0.1:5 and the pH was adjusted to acidic to neutral. The paste was applied to the skin side of hide and two pieces were piled on one above the other with skin-to-skin side together. Complete dehairing was observed at pH 7 within 14-16 hrs by altering the ratio of the enzyme and peptide mixes. In another experiment sodium azide was added to prevent the activity of live cells and to demonstrate that the process is exclusively enzymatic. Controls without enzyme were kept where no dehairing was observed.
Experiment 2: A 4 by 4-square cm hide was incubated with enzymes and peptides in the ratio 0.2-1 g and peptide mix concentration decreased from 5g-1g in different ratios. The pH was adjusted to acidic to neutral. The paste was applied to the skin side of hide and two pieces were piled on one above the other with skin-to-skin side together. Complete dehairing was observed at pH 7 within 12-14 hrs by altering the ratio of the enzyme and peptide mixes. In another experiment sodium azide was added to prevent the activity of
live cells and to demonstrate that the process is exclusively enzymatic. Controls without enzyme were kept where no dehairing was observed.
Experiment 3: Same as experiment 1 but the hide was kept in 50mL water and incubated at 30°C at 50 rpm. Complete dehairing was observed after within 10-12 hrs.
Experiment 4: Same as experiment 2 but the hide was kept in 50mL water and incubated at 30CC at 50 rpm. Complete dehairing was observed after within 8-10 hrs.
Experiment 5: A 4 by 4-square cm hide was incubated with mixture of two groups of enzymes. Group I (chymotrypsin, subtilin) and Group II (trypsin, papain, pepsin) in the ratio 1 (50U) of the Group I and 2-6 (100-600U) of Group II. The pH was adjusted to acidic to neutral. The paste was applied to the skin side of hide and two pieces were piled on one above the other with skin-to-skin side together. Complete dehairing was observed at pH 7 within 10-12 hrs by altering the ratio of the group II enzyme. In another experiment sodium azide was added to prevent the activity of live cells and to demonstrate that the process is exclusively enzymatic. Controls without enzyme were kept where no dehairing was observed.
Experiment 6: Same as experiment 5 but the hide was kept in 50mL water and incubated at 30°C at 50 rpm. Complete dehairing time was reduced to 8hrs. Even the enzyme concentration reduced to 10U of group I with the same ratio results in complete dehairing.
The present invention is not to be limited in scope by the specific embodiments and examples, which are intended as illustrations of a number of aspects of the invention and any embodiments which are functionally equivalent are within the scope of this invention. Those skilled in the art will know, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. These and all other equivalents are intended to be encompassed by the following claims.
Table:

(Table Removed)
References:
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We claim:
1. A composition for dehairing of hides comprising an enzyme and a peptide in the ratio of 0.1: 6
2. A composition as claimed in claim 1 wherein the ratio of enzyme to peptide is 0.2: 1.
3. An enzymatic composition comprising two groups of enzymes on the basis of cleavage specificity,

a) Group I, which attacks hydrophobic residues, and
b) Group II, which attacks lysine arginine basic residues in the ratio 1: 2-6 (100-600U).

4. An enzymatic composition for dehairing as claimed in claim 3 wherein Group I enzyme is selected from at least one of chymotrypsin and subtilin.
5. An enzymatic composition as claimed in claim 3 wherein the group II enzyme is selected from at least one of trypsin, papain and pepsin.
6. A process for the enzymatic dehairing of hides comprising the steps of:
incubating the said hides with enzymes and peptides in the ratio of
0.2: 1;
adjusting the pH to acidic to neutral.
Applying the enzymatic composition to the skin side of hide and
two pieces were piled on one above the other with skin to skin
side together and
if desired, adding sodium azide to prevent the activity of live cells.
7. A composition for dehairing of hides substantially as herein described with reference to the foregoing examples.
8. A process for the enzymatic dehairing of hides substantially as herein described with reference to the foregoing examples.