AU676600B2 - Enzymatically-aided liming process - Google Patents

Description

P/00/011 Regulation 3.2

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

TO BE COMPLETED BY APPLICANT Name of Applicant; ROHM GmbH Actual Inventor(s): CECIL, Alan; CHRISTNER, JOrgen; and WICK, Gertrud Address for Service: CALLINAN LAWRIE, 278 High Street, Kew, 3101, Victoria, Australia Invention Title: "ENZYMATICALLY-AIDED LIMING PROCESS" The following statement is a full description of this invention, including the best method of performing it known to me:- 1 9/26/94GS7912.F10,1

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la 61674.579 Enzyatically-Aided Liming Process The invention relates to an improved enzymatically liming process in which the liming time is considerably reduced.

Proteases are being increasingly used in various steps of the process of leather manufacture (see E. Pfleiderer and R. Reiner in Rehm G. Reed, Biotechnology, Vol. 6b, 729 743 VCH 1988). Recently, the use of proteases which are stable in the alkaline range and which are mostly mutants of Bacillus subtilis or Bacillus licheniformis has become more and more widespread for the depilation and opening up of the animal skin. These proteases are mostly used in conjunction with alkalis such as lime, sodium carbonate or sodium hydroxide solution, and with reducing agents such as sulphides or mercaptans, at pH values of between and 14. Depending on the nature of the final leather product, the treatment takes between 6 and 24 hours (see K. Alexander, J.A.L.C.A. Vol. 3B, 287 316 (1988)).

Further proposals with regard to hair-maintaining liming processes are described in WO 92/17 613, US-A 4 960 428 and DE-A 41 09 826.

Enzymatically-aided liming processes help to increase the surface yield and facilitate the cleaning and dyeing of the leather. At the same time, by using such processes it is possible to considerably reduce the required amount of reducing agent such as sulphide (e.g.

1 1.4 wt-% of sodium sulphide, Here, the enzyme dose was 0.4 0.8 wt-% of a product with 2000 L6hlein- Volhard units (LVU), whilst the high dose (0.8 was chosen in the case of a 6 hour lime.

2 [For determining the activity according to L6hlein- Volhard in the TEGEWA modification see Das Leder 22, 121 126 (1971); N.C.J. Lamb, Journal of Society of Leather Technologists/Chemists 66: 110 113 (1982)] Apart from the known advantages of the enzymaticallyaided lime, certain weaknesses of the process do exist.

These are, for example, a lower operational reliability with longer running times and high temperatures, which lead to disintegration of the skin surface (occurrence of a nubuk effect, a loose grain structure and an empty leather feel).

Efforts to overcome these disadvantages have so far been unsuccessful. If the amount of the enzymes or the running time or temperature are reduced, then it is possible to overcome these disadvantages, but, at the same time, the effect of the enzymes is considerably reduced. Attempts to reduce the enzyme activity and thus the destructive effect on the collagen have also been largely unsuccessful.

o Targeted experiments with various enzyme types, which differ in their activity with respect to collagen and elastin, have shown that the sensitivity of the collagen and the grain membrane to enzymes is generated during the alkaline opening-up stage effected during the liming procedure, i.e. the longer and the greater the effect of alkali on the skin, the more sensitive the skin collagen to the enzymes. According to present findings, this factor is seen to be of greater importance than the collagenolytic activity of the liming enzymes.

Any attempt to overcome the disadvantages outlined above had to be considered in such a way that any changes to the process took account of the approved process sequences (see Kirk-Othmer, Encyclopedia of Chemical -I cl-eY9 P I~~ 3 Technology, Vol. pg. 292 299, Interscience Publ.

1952).

Surprisingly, it has now iaen found that the above disadvantages can be largely avoided if the liming process is carried out with a concentration of enzymes of at least 2 to 3 times that conventionally used.

Thus, according to one aspect, the present invention provides an enzymatically-aided liming process using conventional, especially alkaline, proteases as liming enzymes in an aqueous-alkaline float in the pH range of from 9 14, the liming enzymes being used in a concentration of from 2nEc to 5nEc (wherein nEc is the conventional enzyme concentration used in the lime in the state of the art) and being allowed to act for a maximum of 3 to 6, preferably 3 to 4 hours (known as "enzyme douche"). The liming float is then conveniently run off. This is, advantageously, followed by a washing process, for example with water, while adding non-ionic or anionic wetting agents. The opening-up of the skin is then preferably carried out in a new float containing sodium hydroxide solution and optionally a smaller amount of lime than conventionally used and without sulphide addition, during 10 to 20 hours.

**e The liming process of the invention may be carried out as a hair-destroying or hair-maintaining process. In the hair-destroying process, the proteases are used at the beginning of the liming in conjunction with lime and reducing agents, more particularly inorganic sulphides and/or mercapto compounds. In general, the float has a pH value within the range of 11 14 and contains e.g.

0.8 of sodium sulphide-solution (60% Na 2

S).

The hair-maintaining process may be carried out in such a way that a smaller amount of lime and sulphide (about 2% based on the weight of the float) is used and -I I 4 the liming process is allowed to run for about 2 hours before the enzyme and optionally the remaining amount of sulphide are added.

Examples of mercapto compounds for use in the hairdestroying process include compounds of formula I: R. SH 0 (wherein R represents an optionally branched, cyclic or acyclic C2- 24 alkyl group, in particular a

C

2 18 alkyl group, especially a C2- 12 alkyl group, which may be hydroxy- or thiolsubstituted, or R, represents a group

-(CH

2 )p-NR 2 R3 wherein R 2 and R 3 independently represent hydrogen or a C1-i alkyl group optionally interrupted by a nitrogen, oxygen or sulphur atom to form a (preferably saturated) heterocyclic group, or

R

2 and R 3 independently represent a group

-R

4 -COORs, and p represents an integer of from 2 to 6 wherein R 4 represents an optionally branched

C

2 -6 alkylene group which may be substituted by a -COORs group, in which Rs represents hydrogen or a C 1 alkyl group wherein -SH may be bonded to a primary, secondary or tertiary carbon atom of the group R 1 The mercapto group may also be formamidine sulphinic acid (thioureadioxide).

Preferred compounds of formula I include the mercaptans, especially n-alkylmercaptans such as n-butylmercaptan, n-amylmercaptan, n-dodecylmercaptan, mercaptans of s u ~C L LOROL®-types, n-tetradecylmercaptan as well as hydroxysubstituted alkylmercaptans such as 2-mercaptoethanol, 3-mercapto-2,3-propanediol, also amine-substituted alkylmercaptans.such as 3-(di-namylamino)ethylmercaptan. These compounds are conveniently used in the form of their salts depending on the pH value of the aqueous float used.

Particularly preferred compounds of Formula I are the mercapto-mono- and -dicarboxylic acids or the salts thereof, such as mercaptoacetic acid, 2mercaptopropionic acid, 3-mercaptopropionic acid and mercaptosuccinic acid.

:In addition to the mercapto compounds, hydrotropics may also be added to the liming float in amounts of 0.2 to o" 2 Suitable hydrotropics are disclosed, for example, by H. Rath et al. in Melliands Textilbericht, 43 (7):718 (1962).

as Preferred hydrotropics are those of formula II e

H

2 N C X (II)

R

(wherein R represents hydrogen, -NH 2

-CH

3 or -NH-CN and X represents oxygen, sulphur or NH, or X, together with R, forms a heterocyclic system with the proviso that the heterocyclic system contains only nitrogen heteroatoms) and the acid addition salts thereof such as hydrochlorides, sulphates, phosphates, as well as rhodanide.

Particularly preferred hydrotropics include urea, thiourea, acetamide, formamide, guanidine, melamine and dicyandiamide. Additionally, amines, in particular ethanolamines, may be used. These include, for example, -6mono-, di-, triethanolamine and aminoethylethanolamine.

The above compounds (hydrotropics, amines) are capable of enhancing the effect of the liming enzymes. This is due to their hair-loosening effect and also to their swelling-retarding effect which facilitates the diffusion of the enzymes. These compounds are conveniently introduced together with the liming enzymes or 1 2 hours before the liming enzymes are added. Suitable lirr'-g enzymes for use in the process according to the invention are alkaline proteases known per se and conventionally used, i.e. proteases which exhibit sufficient proteolytic activity and satisfactory stability within the pH range of between 9 and 14, preferably 0 between 9 and 1 3. These include neutral (E.C.3.4.24) and, in particular, alkaline proteases (E.C.3.4.21) [see Kirk-Othmer, 3rd.Ed. pp. 199 202, J. Wiley 1990; Ullmann's Encyclopedia of Industrial Chemistry, Vol., A9, pp. 409 414, VCH S 1987, L. Keay in "Process Biochemistry 17 21 (1971)].

Preferred liming enzymes are: alkaline proteases which exhibit their optimum activity within the pH range of from 8 to 14, preferably between 8.5 to 14. These include alkaline bacterial proteases which mostly belong to the serine type, and alkaline fungus proteases. Mention must be made in particular of the proteases from the Bacillus strains such as B.subtilis, B.licheniformis, B.firmus, B.alcalophilus, B.polvmixa, B.mensentericus, also Streptomyces strains such as S.alcalophilus. The most favourable working temperature with alkaline bacteria proteases is generally 40 600C, with fungus proteases about 20 400C. Alkaline fungus proteases include those from the Aspergillus strains such as A.oryzae, from the Penicillin strains such as P.cvanofulvum or 27/91WGS7912.SPl,6

IIA

7 from Paecilomyces persicinus and the like. The activity of the alkaline fungus proteases is mainly within the pH range of from 8.0 11.0. The enzyme tivity is normally expected to be between 8,000 10,000 L6hlein-Volhard units (LVU) per gram of enzyme.

Neutral proteases exhibiting optimal activity within the pH range of from 6.0 9.0. These include, in particular, neutral bacterial proteases which normally belong to the metalloenzymes and fungus proteases, for example neutral Bacillus proteases such as B.subtilis, B.natto and B.Dolymixa, Pseudomonas-proteases, Streptomycesproteases, Aspergillus-proteases from A.orzae, A.parasiticus and Penicillium glaucum. Neutral bacterial proteases exhibit their optimal activity at working temperatures of 20 50CC, the most favourable working temperature for neutral fungus proteases being about 35 Particularly preferred proteases are those which exhibit a distinct activity within the pH range of from 10 to 14. The proteolytic activity of the enzymes is normally determined using the Anson-Haemoglobin-method [M.L.

Anson, J. Gen. Physiol, 22, 79 (1939)] or the L6hlein- Volhard method [modified according to TEGEWA in Leder 22, 121 126 (1971)]. Here, under test conditions (1 hour, 37 0 a L6hlein-Volhard unit (LVU) corresponds to an amount of enzymes which produces, in 20 ml of caseine filtrate, an increase in the hydrolysis product according to an equivalent of 5.75 x 10' 3 ml of 0.1 n NaOH.

In addition to the enzymes, conventional excipients such as stabilisers may also be used. Stabilisers for liquid alkaline proteases include, for example, multi-valent 8 alcohols such as diethyleneglycol, glycerine, propyleneglycol, sorbitol, ether alcohols, dialkylformamide or dioxane in normal amounts.

Proteases which have proved particularly suitable for use in the process according to the invention are those which are also capable of removing phospholipids, triglycerides and fatty acids (in the sebaceous gland fat or sebum) from the grain layer of the hides or skins. This removal must be seen as secondary to the proteolytic activity and does _,ot take place under the direct effect of phospholipases or lipases. In any case, after this more intense enzyme treatment, clearly more phospholipids and fat are found in the scud than when the conventional method is used.

It is not absolutely clear which protein structures of the skin have to be particularly broken down in this operation. However, without wishing to limit the scope of the present invention in any way, it seems that the release is most probably closely linked with a gradual disintegration of the elastin. This is supported by the fact that those proteases which also have elastase activity exhibit the best effect. The elastase activity can be determined according to DE-A 42 20 838. When this relatively hydrophobic phospholipid layer is removed, the penetration of the aqueous liming chemicals is considerably more uniform and the grain is therefore more even. This also explains the seasonally occurring problems with respect to drawn grain: animals which are pregnant or ready to be slaughtered produce more sebaceous gland fat (sebum).

As already mentioned, the characteristic feature of this invention is the use of a concentration of liming enzyme of at least 2 x nEc to 5 x nEc, preferably 2.5 x nEc to x nEc, in the liming float in question, nEc representing the normal enzyme 9 concentration of the prior art. In the lime, this normal enzyme concentration is about 10,000 to 20,000 LVU per kg of skin (see e.g. WO 92/17 613). Prior art methods usually use an amount of enzymes of about 0.1 0.2 based on the weight of the skins and hides. The proteolytic activity of such products which are alkaline-stable proteases and mainly derived from Bac.subtilis, licheniformis, alcalophilus strains is about 10,000 LVU per kg of skin.

The liming process according to the invention may, advantageously, be carried out according to prior art methods (see F. Stather, Gerbereichemie und Gerbereitechnologie, 4th ed., Akademie-Verlag, Berlin 1967). In general, animal skins and hides may be limed using the process of the invention. Firstly, the preserved skins and hides are, advantageously, thoroughly soaked. Dried skins and hides are generally soaked overnight, but salted material is advantageously soaked for 4 to 6 hours. After the soak, the float is generally run off. It hab been found that depilation is easier if the hides and skins are fleshed (by machine) subsequent to the soak. The liming process according to the invention is then carried out to advantage by means of a fresh float. Depending on the type of skin or hide the process is carried out using 50 to 300 of water based on the initial weight of the skins and hides.

The liming process may be carried out both in the vat, in the tanning machine, in the mixer or in the paddle vat. In the process according to the invention the depilation takes less time and the treatment now requires at most 3 to 4 hours. The temperature of the bath is advantageously between 25 and 27*C. The enzyme preparations are, for example, added to the float in the form of a powder.

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10 When the hair-destroying method is used, the enzyme is mostly introduced at the beginning together with lime and reducing agents. Suitable reducing agents include sodium sulphide, sodium sulphydrate as well as mercapto compounds, as shown in formula I. As a further option, the previously mentioned hydrotropics and amine compounds may also simultaneously be added at this stage. When the hair-maintaining method is used, the enzyme is added together with a reduced amount of lime and optionally with reducing agents as in formula hydrotropics and amines, and the depilation lasts 1 2 hours. The depilation is then completed by adding inorganic sulphides such as Na-sulphide With the hair-maintaining method, the hair is separated from the skins. Sheepskins may, for example, be dewoolled by hand or machine. The bristles of pigskins are generally removed by machine. The loose hair of calf hides and bovine hides may be removed by tumbling or machine depilation. A simultaneous de-fleshing and depilation is also possible (for example using the Stehling machine).

When the method according to the invention is used, the depilation generally produces completely dehaired and pigment-free skins and hides. After the liming float has been run off the skins and hides are preferably washed with water, for example with 150 of water.

The opening-up of the skin is conveniently carried out in a new float, generally 50 to 200 based on the weight of the skins and hides, at a pH value within the range of 12 to 14, normally over a period of from 10 to hours. The pH value is set by means of sodium hydroxide solution and an amount of lime which is less than that normally used. The use of sodium hydroxide solution with lime may serve as a guide. The skins and ona 11 hides are then further processed in accordance with conventional techniques Contrary to all expectations, the method according to the invention produces a leather with a greater smoothness and without a nubuk effect or loose grains.

Furthermore, as a result of the high concentration of enzymes on the grain surface, any blood stains which may have been preserved are completely dissolved. The depilation occurs quicker and the amount of sulphide can be further reduced. This means that the hair is better maintained and may easily be removed from the float during the short treatment or at the end.

*e The following non-limiting Examples serve to illustrate the invention. The composition of the excipient described below as "Product A" is as follows: of P-mercaptopropionic acid 17 of triethanolamine 8 of urea ad 100 of water.

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12

XAMPLES

Example 1 Hair-destroying depilation of bovine skins Material: Salted bovine skins Working vessel: Vat, data based on the salted weight Putrid soak: 150.0 of water 0.1 of preservative based on potassium dimethyldithiocarbamate (ARACIT® KF) 0.1 of tenside C 13 .fatty alcohol ethoxylate having 8 9 Mol of ethylene oxide BORRON® N) stirring for 60 minutes running float off.

Main soak: 150.0 of water, 28°C 0.2 of alkaline protease with 4000 LVU from Bac.licheniformis ERHAZYM® S) 0.2 of tenside 0.1 of preservative based on potassium dimethyldithiocarbamate (ARACIT® KF) of sodium hydroxide solution, 33% 1 3 stirring for 300 minutes pH 9.2 temperature 27 29°C running float off.

Depilation: 90.0 of water, 27°C of alkaline protease from Bac.subtilis with ~e I-- 13 2000 LVU enzyme activity ERHAVIT® MM) of product A 0.1 of tenside based on an n-alkanesulphonate-Na salt BORRON® A) of sodium sulphide, of hydrate of lime stirring for 60 minutes, resting for 30 minutes stirring for 60 minutes, resting for 30 minutes stirring for 60 minutes pH 12.2 12.5 temperature 26 27°C running float off.

Lime: 100.0 of water, 27°C of hydrate of lime of sodium hydroxide solution, 33% 1 3 0.1 of tenside based on an n-alkanesulphonate-Na Ssalt BORRON® A) 0.05% of lubricant based on a highly molecular acrylate polymer o40% of active substance) stirring for 30 minutes, then alternately stirring for 2 minutes, resting for 30 minutes total duration: 12 14 hours pH 12.3 12.5 temperature 26 27°C running float off continue normal operation without after-liming Example 2 Hair-maintaining depilation of bovine skins Material: Salted bovine skins Working vessel: ii I 14 Vat, data based on the salted weight Putrid soak: 150.0 of water 30 C 0.1 of preservative based on potassium dimethyldithiocarbamate ARACIT® KF) 0.1 of tenside based on a C 13 -fatty alcohol with 8 9 Mol of ethyleneoxide BORRON® N) stirring for 60 minutes running float off Main srak: 150.0 of water 28°C 0.2 of enzymatic soaking agent from Bac.licheniformis with 3000 LVU (e.g.

'ERHAZYM® C) 0.2 of tenside 0.1 of preservative based on potassium dimethyldithiocarbamate ARACIT® KF) 0.5 of sodium hydroxide solution, 33% 1 3 stirring for 300 minutes pH 9.2 temperature 27 29 C runnin -at off.

Depilation: 90.0 of water, 27°C of product A of alkaline protease froc. Bac.subtilis with 2000 LVU enzyme activity ERHAVIT® MM) 0.1 of tenside based on an n-alkanesulphonate-Na salt BORRON® A) of hydrate of lime, stirring for 60 120 minutes 1.2 of sodium sulphide, stirring for 60 minutes, resting for 30 minutes stirring for 60 minutes, resting for 30 minutes -u II 15 stirring for 60 minutes pH 12.2 12.5 temperature 26 27 C running float off Lime: 100.0 of water, 27°C of hydrate of lime 0.7 of sodium hydroxide solution, 33% 1 3 0.1 of tenside based on an n-alkanesulphonate-Na salt BORRON® A) 0.05% of lubricant based on a highly molecular acrylate polymer (40% of active substance) ROHAGIT® 3995) stirring for 39 minutes, then alternately stirring for 2 minutes, resting for 30 minutes total duration: 12 14 hours pH 12.3 12.5 temperature 26 27°C running float off operation continued as usual without after-liming Instead of the alkaline protase from B.subtilis an alkaline protease from B.alcalophilus may be used in Example 1. Metering is carried out to ensure the same proteolytic activity. The results are comparable to those obtained in Example 1. The finished leathers are also characterised by very good softness, smoothness and eveness of colour.

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Claims (6)

1. A process for the liming of skins and hides using proteases as liming enzymes, in which the liming enzymes are used in a concentration of at least 2 nEc to 5 nEc in an aqueous alkaline float having a pH in the range of from 9 14, and in which the liming enzymes are allowed to act on the skins and hides for a maximum of from 3 to 6 hours.

2. A process as claimed in claim 1, in which thie liming enzymes are used in a concentration of from 2.5 nEc to nEc.

3. A process as claimed in claim 1, in which the liming enzymes are used in a concentration of from more than 20,000 to 100,000 LVU per kg of skin or hide.

4. A process as claimed in any one of claims 1 to 3, in which the liming enzymes are allowed to act on the skins and hides for a maximum of from 3 to 4 hours. a A process as claimed in any one of claims 1 to 4, in which the liming enzymes comprise alkaline proteases having an optimal activity within the pH range of from 8 14.

6. A process as claimed in any one of claims 1 to 5, in which the liming enzymes comprise alkaline proteases having an optimum activity within the pH range of from

14. A process as claimed in any preceding claim, in which the liming enzymes have an elastolytic activity. 8. A process as claimed in any preceding claim, in which the liming enzymes together with lime and reducing 1r agents are added to the float at the beginning of the L 17 liming process. 9. A process as claimed in any one of claims 1 to 7, in which the liming enzymes are added to the float after the start of the liming process. A skin or hide when treated in accordance with a process as claimed in any preceding claim. 11. A process for the liming of skins and hides as claime in claim 1 substantially as herein describe.d with reference to the Examples. day of to 1997. Dated this S s ROHM GmbH By their Patent Attorneys: CALLINAN LAWRIE O. O 18 ABSTRACT ENZYMATICALLY-AIDED LIMING PROCESS The invention relates to a process for the liming of skins and hides using proteases as liming enzymes, in which the liming enzymes are used in a concentration of at least 2 nEc to 5 nEc in an aqueous alkaline float having a pH in the range of from 9 14. i oo ee o* i