Title of Invention	A METHOD FOR PRODUCING LEATHER BY PROCESSING HIDES OR SKINS
Abstract	(57) Abstract: The present invention relates to a method of producing leather by processing skim and hides by subjecting the same to an enzymatic treatment with microbial protease exhibiting trypsin like protease activity. This enzymatic treatment may be effected during one or more wet processing stages of conventional leather manufacturing processes.

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A METHOD FOR THE PROCESSING OF HIDES OR SKINS INTO LEATHER TECHNICAL FIELD The present invention relates to a method for the processing of hides and skins into leather. More specifically, the present invention relatqs to a method for the processing of hides or skins into leather, comprising enzymatic trealment of the hide or skin with a trypsin acting microbial protease. BACKGROUND ART The traditional use of enzymes in a process for manufacturing leather is in the steps of soaking, unhairing and bating. In these processes, proteases are used to achieve a partial degradation of the proteins, thus making the pe t soft, supple and ready for subsequent tannage. Proteases attack proteins and cut them into small soluble chains. When Hides and skins are treated with proteases, the interfibrillary proteins and the protaoglucans covering the collagen fibrils are removed. Different proteases have different actions and optimum conditions.' Traditionally, the digestive enzymes, in particular trypsin and chymotrypsin, 3xtrac1ed from the pancreatic glands of slaughtered animals, usually pigs, have sound use in the manufacture of leather. Trypsin is the ideal proteolytic enzyme for use in bating. It is a highly specific proteolytic enzyme, acting on the lysyl and arginyl bonds of peptide chains only. Trypsin is regarded the most safe enzyme for use in the manufacture cf leather. The fibrillar molecules that make out the leather are collagen of types I and 3. In the processing of hides and skins Into leather it is important not to damage this fibre structure excessively. Elastin is another fibrous type of pro-tein, it is especially abundant in the regions in and just below the grain (the up¬permost part). Its presence confers rigidity to the leather. For the production nf certain types of leather, for instance like upholstery and garment leather, the removal of elastin can be seen as beneficial as the leather becomes softer. But in other types of leather (e.g. shoe upper leather) a certain amount of rigidity must be retained.' Pure trypsin is known not to break down native collagen, neither does 11 hydrolyse elastin. Therefore, pancreatins -with a high trypsin content are often preferred in order to maintain rigidity and confer what is called "stand" to the shoe upper leather. Trypsin appears as a byproduct from the insulin extraction from por¬cine pancreatic glands, a process which would be economically prohibitive if not insulin was of primary interest. However, developments within genetical engine¬ering have now enabled the production of recombinant insulin. These develop¬ments have resulted in a scarcity of trypsin preparations and increasing prices. These developments have initiated investigations to replace trypsin preparaiions with microbial protease preparations in the enzymatic processing of hides and skins into leather. Proteolytic enzymes hitherto suggested for use in the manufacture of leatfier are proteases of bacterial origin derived from Bacillus, and proteases of fungal origin derived from Aspergillus, Streptomyces, Microsporum, and F en ici Ilium. However, none of these microbial proteases sufficiently mimic the action of trypsin, and no microbial protease has hitherto been able to fully replace trypsin in the pYocessing of hides and skins into leather. SUMMARY OF THE INVEMTION It is an object of the present invention to provide an enzymatic pro-:ess for the preparation of leather, which process comprises the action of a mi-:robial protease able to n .:> nic the action of trypsin. Accordingly, in its first aspect, the present invention provides a method for the processing of hides or skins into leather, comprising enzymatic treatmont of the hide or skin with a trypsin acting microbial protease. In another aspect, the invention provides a bate comprising a trypsin acting microbial protease and suitable excipients. DETAILED DISCLOSURE OF THE INVENTION The present invention provides a method for the processing of hides or skins into leather, comprising enzymatic treatment of the hide or skin with a trypsin scting microbial protease. The processing of hides or skins into leather The hides and skins are usually received in the tanneries 'n the form of saltG d or dried raw hides or skins. The processing of hides or skins into leather comorises several different process steps including the steps of soaking, un-hairing and bating. These steps constitute the wet processing and are performed in the beamhouse. Enzymatic treatment according to the present invention may take place any time during the manufacture of leather. However, proteases are usually employed during the wet processing, i.e. during soaking, unhairing and/or batirg. In one specific embodiment of the invention, the enzymatic treatment with a trypsin acting microbial protease takes place during the wet processing. The object of the soaking step is to restore lost moisture to the salted and dried skins. In a more specific embodiment of the invention, the enzymatic treatment with a trypsin acting microbial protease takes place during soaking. A soaking process of the present invention may be performed at conventional soaking conditions, i.e. a pH in the range pH 6-11, preferably the range pH 7-10, a temperature in the range 20-30°C, preferably the range 24-28°C, and a reaction time in the range 2-24 hours, preferably the range 4-16 hours, and together with known tensides and preservatives, if needed. The object of trie.bating step is to remove residual unhairing chemi¬cals and non-leather making substances. The first phase of the bating step is termed deliming, during which phase lime and alkaline chemicals are eliminated. The second phase of the bating step usually commences with the addition of the bate itself. In another specific embodiment, the enzymatic treatment takes place during bating. In a most preferred embodiment, the enzymatic treatment takes place during bating, after the deliming phase. A bating process of the present Invention may be performed at conventional conditions, i.e. a pH in the range pH 6-9, preferably the range pH 6.5-8.5, a temperature in the range 20-30°C, prefer¬ably the range 25-28°C, and a reaction time in the range 30-90 minutes, preferab¬ly the -ange 40-80 minutes. Processes for the manufacture of leather are well known to the per¬son skilled in the art and have been described in e.g. WO 9406942, WO 901?'18, US 3840433, EP-A1-505920, GB-A 2233665 and US 3986926. Trypsin acting microbial proteases The process of the present invention comprises enzymatic treatment of the hide or skin with a trypsin acting microbial protease. In the context of this invention, a trypsin acting microbial protease is a prot 3olytic enzyme able to mimic the action of trypsin, in particular when Subjected to hides or skin under the conditions of a leather manufacturing process. The trypsin acting microbial protease of the invention preferably is a lysyl and/or arginyl specific proteolytic enzyme (endopeptidase), i.e. an enzyme capable of cleaving peptide bonds at the C-terminal side of lysine and/or arginine. The trypsin-like protease activity may e.g. be determined in an assay based on cleavage of a trypsin substrate such as N-Benzoyl-L-arginine-p-nitro-anilide (L-BAPA or L-BAPNA). Trypsin acting microbial proteases of fungal origin are known from i.a. Achromobacter, Aschersonia, in particular Asc/7erson/a aleyrodis, Aspergillus, Beauvaria, in particular Beauvaria bassiana, Fusarium, in particular Fusarium oxy-sporuin Fusarium merismoides, Fusarium redolens, Fusarium sambucinum, Fusarium solani, and Fusarium verticilloides, Metarhizium, in particular Metarhiziurri anisopliae, Streptomyces, in particular Streptomyces griseus, Tricho-derma and Verticillium, in particular Verticillium lecanii. Thus EP 335,023 discloses trypsin acting fungal proteases obtained from Aschersonia, Beauvaria, Metarhizium and Verticillium. In a preferred embodiment of the invention, the trypsin acting rnicrot)ial protease is one derived from the strain Fusarium oxysporum DSM 2672. Trypsin acting proteases derived from Fusarium oxysporum DSM 267 has been descrioed in International Patent Application Nos. WO 89/06270 and WO 94/25583. Trypsin acting microbial proteases of bacterial origin are known from e.g. Lysobacter. in particular Lysobacter enzymogenes, and Achromobacter, in particular Achromobacter lyticus. Thus Jekel et al. [Jekel P A, Weijer W J and Beimema J J, Anal. Biochem". 1983 134 347-354] describe a lysine specific endopeotidase obtained from Lysobacter. The trypsin acting microbial protease may be of serine, thiol, metal or aspartate type. The trypsin acting microbial protease of the invention may be enpiied in concentrations conventionally employed in leather manufacturing procej;ses. It is at present contemplated that, in particular during soaking and bating respectively, the ti; psin acting microbial protease may be added in ai i amount corresponding to from 0.1 to 100 [jg active enzyme protein per g of hide, preferably from 1 to 25 ^g of active enzyme protein per g of hide. A Bate comprising a Trypsin Acting Microbial Protease A bate is an agent or an enzyme containing preparation comprising the chemically active ingrecfonts for use in beamhouse processes, in particular in the baling step of a process for the manufacture of leather. In another aspect, the invention provides a bate comprising a trypsin acting microbial protease and suitable excipients. In the context of'this invention, suitable excipients include the auxiliary chemicals known and used in the art, e.g. diluents, emulgators, delimers and carriers. The bate may be formulated as known and described in the art, e.g. as described in GB-A 225026;9, The bate of the invention may contain of from 0.00005 to 0.01 g of active enzyme protein per g of bate, preferably of from 0.0002 to 0.004 g of active Drotein per g of bate. In a preferred embodiment, the bate of the present invention comprises a trypsin acting microbial protease which is derived from a strain of ^usarium. In a more preferred embodiment, the bate of the present invention comprises a trypsin acting microbial protease which is derived from the strain Fusarium oxysporum DSM 2762, or a mutant thereof. Proteolytic Activity Proteolytic activity may be determined with denatured haemoglobin as substrate. In the Anson-Haemoglobin method for the determination of proteolytic activity, denatured haemoglobin is digested, and the undigested hae¬moglobin is precipitated with trichloroacetic acid (TCA). The amount of TCA sol¬uble product is determined with phenol reagent, which gives a blue colour with tyrosine and tryptophan. One Anson Unit (AU) is defined as the amount of enzyme which under standard conditions (i.e. 25°C, pH 7.5 and 10 min. reaction time) digests haemoglobin at an initial rate so that there is liberated per minute an amount of TCA soluble product which gives the same colour with phenol reagent as one milli-equivalent of tyrosine. Determination of Anson Units and the proteolytic activity expressed by the Anson Units are concepts known by the person skilled in the art. However, a folder, AF 4/5, describing the analytical method in more detail is available upon request to Novo Nordisk A/S, Denmark, which folder is hereby included by refer¬ence. Accordingly, the present invention provides a method for producing leather by processing hides or skins comprising the step of subjecting hides or skins to enzymatic treatment with a microbial protease which exhibits trypsin like protease activity in an amount corresponding to 0.1 to 100 µg active enzyme protein per gram of hide during one or more steps of conventional wet processing of skin and hides such as soaking, unhairing and/or bating in leather manufacturing. EXAMPLES The invention is further illustrated with reference to the following examples, which are not intended to be in any way limiting to the scope of the invention as claimed. In this example, the action of a trypsin acting microbial protease is evaluated at three different dosages of the enzyme. The trypsin acting microbial protease used in this example is a Fu-sarium oxysporum protease, obtained according to International Patent Applica¬tion No, PCT/DK94/00177. Three dry salted Danish dark/white Friesian bull hides of a total of 31 kg were treated according to the below recipo. All dosages are calculated as percentage on weight of the salted dry hides. The operations are carried out in hermostated pilot tanning drums. The pelts are taken out for fleshing and splitting. After splitting the pelts are parted along the backbone in two halves and weighed separately. From pelt 1 the first half is treated without enzyme and the second half is treated with 3.6 µg of active trypsin-like enzyme protein per g of pelt. From pelt 2 the first half is treated with 3.6 µg of active trypsin-like enzyme protein per g of pelt, while the second half is treated with 21 µg of active trypsin-l ke protein per g of pelt. From pelt 3 the first half is treated with 21 µg of active trypsin-like enzyme protein per g of pelt, whereas the second half is treated without enzyme. This system of treatments allows comparisons of dosages within the same pelt. The halves are treated two and two together according to the follow¬ing recive. After bating tne hides are collected in a drum with a pickle float, and after all the batings have been finished the pelts are treated all together according 15 :O tne following recipe. The resulting product, called "wet blue", is pressed to remove excess ater and afterw.i.'ds shaved to a thickness of 1.1 to 1.2 mm. The wet blue was orocessed further Into crust leather as follows. The leather was air dried. The visual inspection of the beige coloured crust leathers after drying ihovved that compared to the untreated sides, the trypsin-like enzyme treated sides had much cleaner and lighter coloured grain with less wrinkles. The enzyme treated sides were 6 to 10% longer than the non-treated sides due to the leather being more relaxed and less wrinkly. Also did the trypsin-like enzyme treatment increase the tear strength 20 to 25% compared to the non-treated, when analyzed according to DIN 53 329, Colour measurements using a Minolta™ Chroma Meter showed that comparing within hides, the trypsin-like enzyme treated sides were significantly fighter in colour (higher L-star value) than no-treated sides, which is a result of the enzymie treated sides being cleaner than the untreated. There were found no signifi¬cant ciferences between the two enzyme dosages. During the visual inspections and. 'sing microscopy, no damages to the grain were observed, whatsoever, not even at the high 21 µg treatments. Example 2 . In this example, the action of a trypsin-like protease obtained from Fusari J n is compared with the actions of a pure trypsin bate (PTN~ Crystalline, avail¬able from Novo Nordisk A/S, Denmark) and a pancreatin bate (PTN™ 3.0S, available from rJovo Nordisk A/S, Denmark), respectively, in a bating process. Tlie trypsin-like microbial protease used in this example is a Fusarium oxyspoiur^ protease, obtained according to International Patent Ap'"^lication No. PCT/DK94/00177. Two black white Friesian bull hides are used in this example which is un in tA'o series. The hides are processed together in a pilot drum according to Recipe 1 of Example 1. Just before bating, each hide is cut in two halves and treated according to Recipe 5, below. The halves are bated separately as follows (before enzyme treatment they have been delimed): Different treatment is then applied on the same pelt ensuring the pos¬sibility of comparing effects. All dosages are calculated as percentage on weight of fleshed and splitted pelt. The protease dosage on pelt is equal to S/jg of active enzyme protein per g of pelt, and the protease dosage on pelt 2 Is equal to The resulting wet blue is then pressed to-remove excess water and ,ftenvards shaved to a thickness of 1.1 to 1.2 mm. Thereafter the wet blue is processed into crust leather according to Recipe 4 of Example 1. Then the leather was air dried. Evaluations on bated pelt and wet blues did not disclose any significant itferences between treatments. Neither did we find any systematic differences veen proteases evaluating the crust leather visually. The sides were all much alike. Samples of leather were cut out and measured for tear strength (DIN 53329), tensile strength (DIN 53328), lastometer test (DIN 53325) and flexometer test (DIN 53351), respectively. The results appear from tables 2 and 3 below. The lastometer test, giving the grain crack and burst figures, is a test for the expandability of the grain. It is measured on an apparatus in which a ball- is forced against the centre of a diaphragm of the material until it cracks and tursts. The force and distension are measured. The tensile strength is measured as the strength and the extension at break and the tear strength is the force applied to tear the leather both perpen¬dicularly and parallelly to the fibre orientation in the leather. The flexometer tests how leather responds to bending forces (like the lastometer test this test is especially oriented towards the shoe upper leather testing;. The leather is folded in a defined way and exposed to repeated flexing to and f'C( in a Bally FlexomeieF; The flexometer test is not, however, included in the belov :ables, as no cracks were observed on any of the leathers after 50,000 dry flexes. In order to interpret the data of tables 2-3 it is essential not to look on each figure only, but to take an overall view of the data. First of all, there is a large hide to hide variation, but even though the leather samples have been taken from the same or parallel area on the leather sides, there is of course still some natural variation in the leather from side to side within the same hide. Taking this into consideration, PTNTM Crystalline seems to, have the highe 5t performance overall, whereas PTNTM 3.0S and the trypsin-like protease of the invention are close. This example demonstrates that the trypsin acting protease according 1:0 tne invention is an excellent microbial alternative to the highly purified pancreatin and trypsin bates. WE CLAIM; 1. A method for producing leather by processing hides or skins comprising the step of subjecting hides or skins to enzymatic treatment with a microbial protease which exhibits trypsin like protease activity in an amount corresponding to 0.1 to 100 pg active enzyme protein per gram of hide during one or more steps of conventional wet processing of skin and hides such as soaking, unhairing and/or bating in leather manufacturing. 2. The method as claimed in claim 1, wherein said trypsin acting microbial protease is of fungal origin. 3. The method as claimed in claim 2, wherein said trypsin acting fungal protease is obtained from a strain of Achromobacter, a strain of Aschersonia, a strain of Aspergillus, a strain of Beauvaria, a strain of Fusarium, a strain of Metarhizium, a strain of Streptomyces, a strain of Trichoderma, or a strain of Verticillium. 4. The method as claimed in claim 3, wherein said trypsin acting fungal protease is obtained from a strain of Aschersonia aleyrodis, a strain of Beauvaria bassiana, a strain of Fusarium oxysporum, Fusarium merismoides, Fusarium redolens, Fusarium sambucinum, Fusarium solani, and Fusarium verticilloides, a strain of Metarhizium anisopliae, a strain of Streptomyces griseus, or a strain of Verticillium lecanii. 5. The method as claimed in claim 4, wherein said trypsin acting fungal protease is obtained from the strain Fusarium oxysporum DSM 2672, or a mutant or a variant thereof. 6. The method as claimed in claim 1, wherein said trypsin aamg protease is of bacterial origin. 7. The method as claimed in claim 6, wherein said trypsin acting bacterial protease is obtained from a strain of Achromobacter, preferably Achromobacter lyticus, or a strain of Lysobacter, preferably Lysobacter enzymogenes. 8. The method as claimed in any of claims 1 to 7, wherein said trypsin acting microbial protease is a serine protease. 9. The method as claimed in any of claims 1 to 8, wherein said trypsin acting microbial protease is a Lys and/or Arg specific protease. 10. The method as claimed in claim 9, wherein the enzymatic treatment is carried out during the soaking step of conventional leather manufacturing process. 11. The method as claimed in claim 9, wherein the enzymatic treatment is carried out during the bating step of conventional leather manufacturing process. 12. A method for producing leather by processing hides or skins substantially as herein described and exemplified.

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