EP0464531B1 - Enzymatic bating preparations - Google Patents

Abstract

The invention concerns enzymatic bating preparations for carbonic acid deliming using proteases, which bating preparations contain A) alkaline bacterial protease having a pH optimum between 9 and 11 in proportions of 10 - 20% by weight, based on the totality of the proteases P, B) pancreas protease having a pH optimum between 7 and 9 in proportions of between 40 - 70% by weight, based on the totality of proteases P, and C) fungal or bacterial protease having a pH optimum within the pH range 5 to 7 in proportions of from 20 to 40% by weight, based on the totality of proteases P, with the proviso that the proteolytic activities of the A') alkaline bacterial proteases at pH 10, B') the pancreatic enzymes at pH 8 and C') the acidic fungal or bacterial protease at pH 6.5 have been adjusted to the same level.

Description

The invention relates to new enzymatic pickling preparations for CO₂ decalcification.

State of the art

Conventional leather technology consists of a sequence of individual steps, the first section of which represents the so-called water workshop, followed by the actual tanning and finishing of the leather (cf. F. Stather, tanning chemistry and tanning technology, Akademie-Verlag, Berlin 1967). As part of the water workshop, the hides and skins are usually subjected to a liming process, which is usually followed by so-called descaling and pickling. The liming is usually a treatment in the alkaline range (pH 12-14), carried out either as a purely alkaline "hydroxyl ash" using calcium hydroxide and sodium hydroxide or in the majority of cases as a reducing "sulphide ash". Sulfide ash usually contain inorganic sulfide in the form of S²⁻ or SH⁻ ion.

Before the actual tanning, the swelling of the skins, which occurs due to the alkali in the liming, should be reversed to ensure sufficient tanning. During the so-called descaling, the pH of the bare skin is reduced from approx. 13 to approx. 8. However, caution should be exercised when using strong acids, since even a temporary excess of acid can cause acid swelling in the outer layers of the skin, especially in the sensitive scar layer. (see HJ Rehm and G. Reed Ed., Biotechnology Vol, 6b, pp. 736 - 739, VCH 1988).
For this reason, the use of acidic salts (ie salts of weak bases with strong acids) such as the ammonium salts of sulfuric acid or hydrochloric acid or the use of organic acids has been recommended.
However, these processes have ecological concerns.

Task and solution

Instead of using ammonium salts, which in the wastewater treatment lead to a strong consumption of oxygen and therefore act as wastewater poisons, carbon dioxide dissolved in water shows (cf. F. Stather, Ger borders chemistry and tanning technology, p. 214, Akademie Verlag Berlin 1967; EE Ochs J. Am. Leather Assoc. 48, 105 (1953); US-A 2 147 542) has no environmentally harmful effect. Due to the high solubility of CO₂, a saturated CO₂ solution is reached at an early stage which is capable of neutralizing the alkaline present in the skin via carbonic acid formed as an intermediate.

• 8,5 besitzen. Bei pH-Werten um 6,5 liegen üblicherweise nur noch ca. 60
• 80 % der Aktivität vor, so daß die Beizaktivität nicht in befriedigendem Umfang ausgenutzt wird.

Um diesen Nachteil zu umgehen, wird in der Patentliteratur (PCT/SE88/00313) vorgeschlagen, die CO₂-Dosierung so vorzunehmen, daß der pH-Wert nicht unter das Optimum der Beizaktivität abfällt (pH = 7,5 ) bzw. das Beizmittel erst dann zuzusetzen, wenn die Entkälkung mit CO₂ abgeschlossen ist und der pH-Wert so weit angestiegen ist, daß dieser in das pH-Optimum des Beizmittels fällt. Bei dieser Verfahrensweise muß aber mit einer verringerten Druchkälkungskinetik gerechnet werden. Es bestand daher die Aufgabe, nach Lösungen zu suchen, die bei Anwendung der CO₂-Entkälkung eine bessere Ausnutzung der Beizaktivität in der Beize ermöglichen.It has now been shown in practice that the decalcification is fastest if the CO₂ metering is carried out continuously.
At the start of CO₂ decalcification, the pH is 11.5 - 12.5 and drops to 6.5 after just 15 - 25 minutes. The subsequent pickling is generally carried out using proteolytic enzymes, for example with proteases (cf. HJ Rehm and G. Reed, loc.cit., F. Stather loc.cit). It is known that pickling agents based on pancreatic proteases have an optimum activity at pH 7.5

• Own 8.5. At pH values around 6.5, there are usually only about 60
• 80% of the activity before, so that the pickling activity is not used to a satisfactory extent.

To avoid this disadvantage, it is proposed in the patent literature (PCT / SE88 / 00313) to make the CO₂ dosage so that the pH does not drop below the optimum of the pickling activity (pH = 7.5) or the pickling agent only then add when the descaling is completed with CO₂ and the pH has risen so much that it falls within the pH optimum of the pickling agent. With this procedure, however, reduced liming kinetics must be expected. It was therefore the task to look for solutions that enable better use of the pickling activity in the pickling when using CO₂ decalcification.

The present object can be achieved by using an enzymatic pickling preparation for carrying out the pickling which has a balanced activity spectrum in the entire pH range of the CO₂ decalcification with simultaneous optimum activity at pH 6-8, especially at pH 6.5-7. 5.

• A) alkalische Bakterienprotease mit einem pH-Optimum zwischen 9 und 11, speziell 9,5 und 10,5 in Anteilen von 10 - 20 Gew.-% bezogen auf die Gesamtheit der Proteasen P
• B) Pankreasprotease mit einem pH-Optimum zwischen pH 7 und 9 in Anteilen von 40 bis 70 Gew.-% bezogen auf die Gesamtheit der Poteasen P und
• C) Pilzprotease oder Bakterienprotease mit ausgeprägtem pH-Aktivitätsoptimum im pH-Bereich 5 bis 7 in Anteilen von 20 bis 40 Gew.-% bezogen auf die Gesamtheit der Proteasen P mit der Maßgabe, daß die
  • A') alkalischen Bakterienproteasen bei pH 10, der
  • B') pankreatischen Enzyme bei pH 8 und der
  • C') sauren Pilz- oder Bakterienprotease bei pH 6,5 bezüglich ihrer proteolytischen Aktivität gleich stark eingestellt sind,

und gegebenenfalls daneben an sich bekannte Stabilisatoren und Hilfsmittel enthält.
Die Summe der Proteasen A) + B) + C) sei stets zu 100 Gew.-% angenommen.The present invention thus relates to an enzymatic pickling preparation containing active proteases, the enzymatic pickling preparation being used as enzymatically active components

• A) alkaline bacterial protease with a pH optimum between 9 and 11, especially 9.5 and 10.5 in proportions of 10-20% by weight based on the totality of the proteases P.
• B) pancreatic protease with a pH optimum between pH 7 and 9 in proportions of 40 to 70% by weight, based on the totality of the potentiates P and
• C) Fungal protease or bacterial protease with a pronounced optimum pH activity in the pH range 5 to 7 in proportions of 20 to 40% by weight, based on the totality of the proteases P, with the proviso that the
  • A ') alkaline bacterial proteases at pH 10, the
  • B ') pancreatic enzymes at pH 8 and
  • C ') acidic fungal or bacterial protease at pH 6.5 are equally adjusted with regard to their proteolytic activity,

and optionally also contains known stabilizers and auxiliaries.
The sum of the proteases A) + B) + C) is always assumed to be 100% by weight.

The proteases P

The classification of proteolytically active enzymes (EC3.4) can take place from different points of view, for example according to the origin (plant / animal / microbiological), according to the point of attack (exo-versus-endo-enzymes), using the "Active Site "of the proteases (" serine proteases ") and according to the pH optimum of the proteolytic activity. (See Kirk-Othmer, 3rd Ed. Vol. 9, 199-202, J. Wiley 1980; K. Anstrup in B. Spencer, Industrial Aspects of Biochemistry, Vol. 30, pp. 23 - 46, North Holland 1974; HJ Rehm & G. Reed Ed.Biotechnology Vol. 6b, 729 - 743, VCH 1988; Ullmann's Encyclopaedia of Industrial Chemistry 5th Ed. Vol A9, pp 295 - 397 VCH 1987; Ullmann's Encyclopedia of Technical Chemistry. 4th Edition, Vol. 10, 475 -561, Verlag Chemie 1975).

To A)

The alkaline bacterial proteases to be used according to the invention [EC3.4.21.14; see. L. Keay in Process Biochemistry 17-21 (1971) also mostly belongs to the serine type.
They are preferably the so-called subtilisins, which are derived from various Bacillus sp. are produced, in particular by B.subtilis, B.licheniformis, B.pumilis, B.alkalophilus, B.firmus, B.megaterium, B.cereus, B.amylosacchariticus. The alkaline proteases, the structure of which has been partially analyzed, generally have molar masses in the range from 26,000 to 34,000. They are predominantly quite stable in the pH range from 5 to 10, as long as work is carried out in the temperature range below 35 ° C. At temperatures of around 65 degrees C and above, however, activity is quickly lost. The subtilisins are more active against casein than against hemoglobin or bovine serum albumin.
Technical preparations often show activities between 1 and 6 ANSON units per g (while the activity of crystalline subtilisin is between 25 and 30 ANSON units / g).

To B)

The pancreatic proteases, as they are used industrially, are mixtures of trypsin, chymotrypsin and various peptidases. They can contain amylase and lipase as accompanying enzymes. They are usually obtained by salt precipitation from fresh pancreatic press juice or from residues from insulin production.
Compared to the substrates casein, hemoglobin and gelatin, the optimal pH value of the effect is 9.0 (30 - 40 degrees C), whereby it should be noted that the proteases are inactivated in the optimal range with increasing temperature.

To C)

• strikte Einhaltung des tolerierten pH-Bereichs
• Anwesenheit von Metallionen (ca⁺² bzw. Zn⁺²)
• Unterdrückung der Aktivität evtl. vorhandener alkalischer Proteasen (wegen deren Tendenz zur gegenseitigen proteolytischen Spaltung).

The fungal proteinases or bacterial proteases with a pronounced optimum activity in the range between pH 5 and 7 preferably originate from Aspergillus sp., Streptomyces sp., Microsporum sp .. A.sojae, A.niger or oryzae, A.parasiticus, S. are particularly mentioned. griseus, S.naraensis, Piricularia oryzae and the like or from Bacillus species such as B. subtilis, B. thermoproteolyticus, B. cereus, B. megaterium, B. polymixa. The molecular weight of these proteases, which are generally not considered to be very stable, is usually in the range from 35,000 to 45,000.
According to the literature, the use of enzymes of this type is required

• strict adherence to the tolerated pH range
• Presence of metal ions (ca⁺² or Zn⁺²)
• Suppression of the activity of possibly existing alkaline proteases (because of their tendency towards mutual proteolytic cleavage).

The proteolytic activity of enzymes is customarily determined using the Anson-Hemoglobin method (ML Anson J. Gen. Physiol. 22, 79 (1939) or the Löhlein-Volhard method (Löhlein-Volhard's method for determining the proteolytic method Activity.
Area. Paperback, Dresden-Leipzig, 1955) determined as "LVE" (Löhlein-Volhard unit).
A Löhlein-Volhard unit is the amount of enzyme that digests 1.725 mg casein under the specific conditions of the method. Furthermore, units which are derived from the Anson method are used for the activity determination of the enzymes active in the acidic range. These are referred to as "proteinase units (hemoglobin)" U _Hb . A U _Hb corresponds to the amount of enzyme which catalyzes the release of trichloroacetic acid-soluble fragments from hemoglobin equivalent to 1 »mole tyrosine per minute at 37 degrees C (measured at 280 nm). (1 mU _Hb = 10⁻³ U _Hb ).

Implementation of the invention

• A) alkalische Bakterienproteasen mit einer Enzymaktivität von 2 000 - 50 000 U_HB vorzugsweise mit 10 000 - 30 000 U_HB gemessen bei pH 10
• B) Pankreasproteinasen mit einer Enzymaktivität von 2 000 - 60 000, vorzugseise mit 5 000 - 15 000 U_HB gemessen bei pH 8
• C) Pilzproease oder saure Bakterienproteasen mit einer Enzymaktivität von 5 000 - 100 000 U_HB, vorzugsweise mit 10 000 - 20 000 U_HB gemessen bei pH 6,5.

The application of the pickling preparations according to the invention expediently starts from the following design rules:
Total content of the protease preparations A) - C) 0.1 to 2% by weight based on the skin material.
The protease preparations are preferably composed as follows:

• A) alkaline bacterial proteases with an enzyme activity of 2,000-50,000 U _HB, preferably with 10,000-30,000 U _HB measured at pH 10
• B) Pancreatic proteinases with an enzyme activity of 2,000-60,000, preferably 5,000-15,000 U _HB measured at pH 8
• C) Fungal protease or acidic bacterial proteases with an enzyme activity of 5,000-100,000 U _HB , preferably with 10,000-20,000 U _HB measured at pH 6.5.

The enzymatic pickling preparations to be used according to the invention can be in the form of solid mixtures or preferably as preparations in a liquid medium (DE-A 39 04 465; unpublished German patent application P 39 27 286.9).
In addition to the proteases A) - C), the pickling preparations advantageously also contain known additives such as stabilizers, adjusting agents and the like. Calcium salts, for example, are suitable as stabilizers. Alkali chlorides, sulfates or bicarbonates are recommended as adjusting agents.
Ammonium salts such as ammonium sulfate or chloride can be used in small amounts (<0.5% by weight, based on the skin material) to accelerate the decalcification.
The proportion of salts in the pickling agents is generally <90% by weight, based on the agents as a whole.
Furthermore, the mordant, especially in liquid form, can also contain emulsifiers and solvents, for example of the type described in DE-A 33 12 840 or DE-A 33 22 840. In general, the proportion of emulsifiers is in the range from 0.01 up to 5% by weight, preferably 0.1 to 0.5% by weight, based on the weight of the skin.

The use of nonionic emulsifiers, if appropriate in combination with anionic emulsifiers, is particularly preferred. The ethoxylates of the C11-C13 fatty alcoholates with 6 to 25 moles of ethylene oxide, preferably with 6-9 moles of ethylene oxide, should be mentioned above all.
Suitable solvents, which are generally used in amounts of 0.1 to 5% by weight, based on the weight of the decalcifying liquor, are conventional, for example from the group of mineral oils or mineral oil fractions, the lower alcohols and glycols and their etherified derivatives in question. For example, mineral oils with between 10 and 60, in particular 15 and 30,% by weight of aromatics may be mentioned.

Implementation of the invention

The implementation of the invention can closely follow the known procedures for CO₂ decalcification.
It is preferable to work in the tanning drum or in the tanning machine, a recirculation or pumping device for the fleet being advantageous.
The liquors used are generally in the range up to 150% based on the weight of the skin. For descaling, CO₂ gas is fed directly into the pumping system.
The dosage is advantageously 7-11 l / min.
In general, the pickling agent, for example product A according to the exemplary embodiments, is metered in right at the start. As a rule, the pH of the liquor then drops from close to 12 to a value around 8 in the first period, after about 20 minutes, after one a further period of about 10 minutes to a value of approx. 6.5 - 7. In general, the pelts with a thickness of 1.8 - 2.5 mm are completely decalcified after a descaling period of approx. 120 minutes.

Beneficial effects

With regard to their basic purity, smoothness and skin disintegration, the skins are to be rated better than when using a purely pancreatic mordant with the same activity (with optimum activity at pH 8) if the procedure is otherwise identical. On the other hand, it is positive that the process according to the invention does not require a fundamental change in the CO₂ decalcification and pickling process.

The following examples serve to explain the invention.

EXAMPLES Product A Enzyme preparations consisting of:

10% by weight

an alkaline bacterial protease from Bacillus licheniformis with an activity of 250 U _HB at pH 10

50% by weight

a pancreatic enzyme with an activity of 250 U _HB at pH 8

40% by weight

An acid fungal protease from Aspergillus niger with an activity of 250 U _HB at pH 6.5, the salt of the individual enzyme preparations is sodium sulfate. The total protease activity of the mixture at pH 8 is 180 U _HB .

Product B Production of a stable liquid enzyme preparation:

• a) 10% by weight of a liquid aqueous alkaline bacterial protease formulation from Bacillus licheniformis with an activity of 250 U _HB at pH 10 (preparation 1 see below) and
• b) 50% by weight of a liquid, aqueous prancreas enzyme preparation with an activity of 250 U _HB at pH 8 (preparation 2) and
• c) 40% by weight of a liquid, aqueous fungal protease formulation from Aspergillus niger with an activity of 250 U _HB at pH 6.5 (preparation 3) are mixed homogeneously.
  The preparation has a total activity at pH 8 of 180 U _HB and is produced by simply mixing the individual preparations a) to c).

Preparation 1 Production of liquid single preparations

100 kg of an aqueous clear solution of an alkaline bacterial protease from Bacillus licheniformis with an activity of 10,000 U _HB at pH10 are mixed with 50 kg of ammonium sulfate at 16 ° C., the enzymes being precipitated. The enzyme suspension has an activity of 9,800 U _HB at pH 10 and a density of 1.22 to 1.23 g / cm³. This enzyme suspension is then diluted with as much thickened isotonic ammonium sulfate solution (480 mPa s) with stirring so that an activity of 250 U _HB at pH 10 is achieved.

Preparation 2

100 kg of squeezed juice obtained after grinding, activating and extracting pancreatic glands is mixed with 50 kg of ammonium sulfate and the pancreatic enzymes are precipitated.
The pancreatic enzyme suspension has an activity of 8 400 U _HB at pH 8 and a density of approx. 1.22 to 1.23 g / cm³. This suspension is then treated with as much thickened isotonic ammonium sulfate solution (480 mPa s) diluted to achieve an activity of 250 U _HB at pH 8.

Preparation 3

100 kg of an aqueous clear solution of a fungal protease from Aspergillus niger are mixed with 50 kg of ammonium sulfate at 16 ° C., the enzymes being precipitated. The enzyme suspension with an activity of 8,800 U _HB at pH 6.5 and a density of 1.22 to 1.23 g / cm³ is diluted with so much thickened isotonic ammonium sulfate solution that an activity of 250 U _HB at pH 6.5 is reached becomes.

Preparation of the thickened isotonic ammonium sulfate solution

60.7 kg of water are placed in a stirred kettle and 1 kg of a xanthan-based thickener (e.g. K1A96, Kelco) is incorporated using a toothed disk stirrer (circumferential speed 2-3 m / sec.). If the viscosity of the solution has risen to> 1,000 mPa s, 38.3 kg of ammonium sulfate are added. The mixture is stirred until the ammonium sulfate is dissolved. The viscosity drops to 480 mPa s.

Product C Preparation of a liquid, non-aqueous, stable enzyme preparation.

• a) 10 Gew.-% einer flüssigen nichtwäßrigen alkalischen Bakterienprotease aus Bacillus licheniformis mit einer Aktivität von 250 U_HB bei pH 10 und
• b) 50 Gew.-% einer flüssigen nichtwäßrigen Pankreasenzympräparation mit einer Aktivität von 250 U_HB bei pH 8 und
• c) 40 Gew.-% einer flüssigen, wäßrigen Pilzproteasepräparation aus Aspergillus niger mit einer Aktivität von 250 U_HB bei pH 6,5

werden homogen vermischt. Die Gesamtformulierung hat eine Aktivität von 180 U_HB bei pH 8.

• a) 10% by weight of a liquid non-aqueous alkaline bacterial protease from Bacillus licheniformis with an activity of 250 U _HB at pH 10 and
• b) 50% by weight of a liquid non-aqueous pancreatic enzyme preparation with an activity of 250 U _HB at pH 8 and
• c) 40% by weight of a liquid, aqueous fungal protease preparation from Aspergillus niger with an activity of 250 U _HB at pH 6.5

are mixed homogeneously. The overall formulation has an activity of 180 U _HB at pH 8.

Production of single enzyme preparations

• a) alkalischen Bakterienprotease aus Bacillus licheniformis mit der Aktivität von 20 000 U_HB bei pH 10 eine Endaktivität von 250 U_HB bei pH 10
• b) Pankreasprotease mit der Aktivität von 20 000 U_HB bei pH 8 eine Endaktivität von 250 U_HB bei pH 8
• c) Pilzprotease mit der Aktivität von 20 000 U_HB bei pH 6,5 eine Endaktivität von 250 U_HB bei pH 6,5.

500 g of C₁₃ fatty alcohol ethoxylate with 6-7 moles of ethylene oxide units, 150.5 g of petroleum (bp. 110 degrees C) and 307 g of mineral oil (bp.> 250 degrees C) are added after adding 30 g of an organically modified bentonite (e.g. Bentone 27 , From Kronos Titan, Leverkusen) dispersed for 35 minutes in an Ultra-Turrax mixer at a peripheral speed of 16 m / sec. The dispersion is left to stand for 24 hours, after which a viscosity of 350 mPa s is reached. Under the same dispersion conditions, 12.5 g of powdered protease enzyme are then added and dispersed for a further 5 minutes. The single preparation has a powdery form when used

• a) alkaline bacterial protease from Bacillus licheniformis with the activity of 20,000 U _HB at pH 10 and an end activity of 250 U _HB at pH 10
• b) pancreatic protease with the activity of 20,000 U _HB at pH 8 a final activity of 250 U _HB at pH 8
• c) fungal protease with the activity of 20,000 U _HB at pH 6.5 a final activity of 250 U _HB at pH 6.5.

example 1 Descaling and pickling of ashed large cattle hides (weight class 30 - 39 kg, gap thickness 2.2 mm).

1,000 kg of ashed beef skin with 800 kg of water are placed in a tanning drum with a recirculation or pumping unit for the fleet.
For descaling, CO₂ gas is fed directly into the pumping system. The dosage of CO₂ is 8 to 9 l / min. Right at the start, 0.5% (based on the pelt weight) of the mordant (product A) is added. The initial pH in the liquor drops from pH 11.8 to pH 8.2-8 during the first 10 minutes and to pH 6.5 -7 after a further 10 minutes.
After a total of 120 minutes of descaling, the bare cross-sections are completely descaled. The skins are to be rated better in terms of basic purity, smoothness and skin disruption than in identical applications of a pure pancreatic stain with the same activity and optimum activity at pH 8.

Example 2

Analogously to Example 1, Product B was used with the same dosage and application. Since product B is liquid, it can be added automatically via a dosing device. In this case an additional opening of the tanning drum is not necessary. This avoids exposure to toxic hydrogen sulfide, which is released to a greater extent during CO₂ decalcification.

Example 3

Analogously to example 2, product C is used in the same application and dosage. Product C contains emulsifiers which further improve the basic purity of the skins.

Claims (4)

1. Enzymatic bating preparations for carbonic acid deliming using proteases, characterised in that the bating preparations contain, as proteases P:

   A) alkaline bacterial protease with an optimum pH of between 9 and 11, in amounts of 10 - 20 wt.%, based on all the proteases P,

   B) pancreatic protease with an optimum pH of between 7 and 9, in amounts of 40 - 70 wt.%, based on all the proteases P, and

   C) fungal protease or bacterial protease with an optimum pH in the range from 5 to 7, in amounts of 20 to 40 wt.%, based on all the proteases P,

   with the proviso that the proteolytic activity of the

   A') alkaline bacterial proteases is set at pH 10, that of the

   B') pancreatic enzymes is set at pH 8 and

   C') that of the acidic fungal or bacterial protease is set at pH 6.5, so as to be of equal strength.
2. Enzymatic bating preparations according to claim 1, characterised in that, in addition to the proteases, they also contain adjuvants known per se, selected from the group of the enzyme stabilisers, adjusting agents.
3. Enzymatic bating preparations according to claims 1 and 2, characterised in that they contain solvents known per se.
4. Liquid or solid enzymatic bating preparations according to claims 1 to 3, characterised in that they are used right at the start of the CO₂ deliming process or during it.