DE4020110A1 - NEW ENZYMATIC SEEDING 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

Field of the Invention

The invention relates to new enzymatic pickling preparations for CO ₂ 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 S ^2- or SH⁻ ion.

Before the actual tanning, the swelling of the Skins that occur in the liming due to the action of alkali, can be undone to an adequate To ensure tanning. With the so-called descaling the pH of the nakedness will decrease from approx. 13 to approx. 8 reduced. When using strong acids however, caution is advised, as it is only temporary Acid excess causes an acid swelling in the outer Layers of skin, especially in the sensitive Can cause scar layer. (see H.J. Rehm and G. Reed Ed., Biotechnology Vol, 6b, pp. 736-739, VCH 1988). For this reason, the use of acidic salts (i.e. of salts of weak bases with strong acids) such as B. the ammonium salts of sulfuric acid or hydrochloric acid or the use of organic acids is recommended. However, these processes encounter 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 21 47 542) has no environmentally harmful effect. Due to the high solubility of CO₂, a saturated CO ₂ 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.

It has now been shown in practice that through-decalcification takes place fastest if the CO ₂ metering is carried out continuously.

At the beginning of CO ₂ decalcification, the pH is 11.5-12.5 and drops to 6.5 after 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 7.5-8.5.

At pH values around 6.5 there is usually only approx. 60-80% the activity before, so that the pickling activity is not is used to a satisfactory extent.

In order to avoid this disadvantage, it is proposed in the patent literature (PCT / SE88 / 00313) that the CO _{2 be} metered in such a way that the pH does not drop below the optimum of the pickling activity (pH = 7.5) or the pickling agent only add when the decalcification is complete with CO ₂ and the pH has risen so far that it falls within the pH optimum of the pickling agent. With this procedure, however, reduced liming kinetics must be expected. The task therefore was to look for solutions which, when using CO ₂ decalcification, enable better use of the pickling activity in the pickling line.

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 CO ₂ decalcification with a simultaneous optimum activity at pH 6-8, especially at pH 6.5-7 , 5th

The present invention thus relates to an enzymatic Pickling preparation containing active proteases, the enzymatic pickling preparation being enzymatic effective 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 total of Proteases P
• B) Pancreatic protease with a pH optimum between pH 7 and 9 in proportions of 40 to 70 wt .-% based on the The totality of poteases 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 the
  C ′) acidic fungal or bacterial proteases at pH 6.5 are equally adjusted with regard to their proteolytic activity,
  and optionally also contains known stabilizers and auxiliaries.

The sum of proteases A) + B) + C) is always increasing 100% by weight assumed.

The proteases P

The classification of proteolytically active enzymes (E.C.3.4) can be done from different points of view take place, e.g. B. by origin (vegetable / animal / microbiological), after the Attack site (exo-versus-endo-enzymes), based on the "Active  Site "of the proteases (" serine proteases ") and after Optimal pH 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; H.J. 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 Techn. Chemistry. 4th edition, vol. 10, 475-561, Verlag Chemie 1975).

To A)

The allcalic to be used according to the invention Bacterial proteases (E.C. 3.4.21.14; see also L. Keay in Process Biochemistry 17-21 (1971)) mostly belong to that Serine type.

It is preferably the so-called Subtilisins derived from various Bacillus sp. produced , especially from B.subtilis, B.licheniformis, B.pumilis, B.alkalophilus, B.firmus, B.megaterium, B.cereus, B.amylosacchariticus. The alkaline proteases, whose structure z. T. have been analyzed as a rule Molar masses in the range 26,000-34,000. They are predominant fairly stable in the pH range 5-10, as long as Temperature range below 35 degrees C. At Temperatures of approx. 65 degrees C and above are possible the activity 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 (during the activity of crystalline subtilisin at 25-30 ANSON units / g lies).  

To B)

The pancreatic proteases as they are used technically come, make mixtures of trypsin, chymotrypsin and different peptidases. They can be used as accompanying enzymes Contain amylase and lipase. They are won usually by salt precipitation from fresh pancreatic Squeezed juice or from residues of insulin production. Compared to the substrates casein, hemoglobin and gelatin the optimal pH of the effect is 9.0 (30-40 degrees C), whereby it should be noted that the proteases in optimal effective range with increasing temperature be deactivated.

To C)

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

• - Strict adherence to the tolerated pH range
• - presence of metal ions (ca ⁺² or Zn ⁺² )
• - suppression of the activity of any alkaline proteases (because of their tendency to mutual proteolytic cleavage).

The proteolytic effectiveness of enzymes is commonly used by the Anson hemoglobin method (M.L. Anson J. Gen. Physiol. 22, 79 (1939)) or after the Löhlein-Volhard method (the Löhlein-Volhard method to determine the proteolytic activity. Area. Paperback, Dresden-Leipzig, 1955) as "LVE" (Löhlein-Volhard unit) determined.

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 in the following 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 μmol tyrosine per minute at 37 degrees C (measured at 280 nm). (1 mU _Hb = 10 ^-3 U _Hb ).

Implementation of the invention

The pickling preparations according to the invention can be used expediently based on 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 preferably settle as follows together:

• A) alkaline bacterial proteases with an enzyme activity of 2000-50,000 U _HB, preferably with 10,000-30,000 U _HB measured at pH 10.
• B) Pancreatic proteinases with an enzyme activity of 2000-60,000, preferably with 5000-15,000 U _HB measured at pH 8
• C) Fungal protease or acidic bacterial proteases with an enzyme activity of 5000-100,000 U _HB , preferably with 10,000-20,000 U _HB measured at pH 6.5.

The enzymatic to be used according to the invention Pickling preparations 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) are available.

In addition to the proteases A) -C), the pickling preparations contain expediently known additives such as Stabilizers, actuators and. Ä. As stabilizers come z. B. Calcium salts in question. As a means of positioning alkali chlorides, sulfates or are recommended bicarbonates.

To accelerate decalcification can be done in small Quantities (<0.5% by weight based on the skin material) Ammonium salts such as ammonium sulfate or chloride used will.

The proportion of salts in the mordants is usually at <90 wt .-% based on the agent as a whole. Furthermore, the mordant, especially in liquid form still contain emulsifiers and solvents, for example that in DE-A 33 12 840 or DE-A 33 22 840 described type. In general, the proportion is Emulsifiers in the range from 0.01 to 5% by weight, preferably at 0.1 to 0.5 wt .-% based on the weight of the skin.  

The use of nonionic is particularly preferred Emulsifiers optionally in combination with anionic Emulsifiers. The ethoxylates of the C11-C13 fatty alcoholates with 6 to 25 moles of ethylene oxide, preferably with 6-9 moles of ethylene oxide.

As a solvent, generally in amounts of 0.1 up to 5% by weight based on the weight of the descaling Fleet are used, come usual, for example from the group of mineral oils or Mineral oil fractions, lower alcohols and glycols and their etherified derivatives in question. For example Mineral oils with between 10 and 60, especially 15 and 30 wt .-% aromatics called.

Implementation of the invention

The implementation of the invention can closely follow the known procedures for CO ₂ 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 nakedness. For descaling, CO ₂ 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 of 8 in the first period, approximately after approximately 20 minutes, and to a value of approximately 6.5-7 after a further period of approximately 10 minutes. 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 disruption, 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 CO ₂ decalcification and pickling process.

The following examples serve to explain the Invention.  

Examples Product A

Enzyme preparations consisting of:
10% by weight of an alkaline bacterial protease from Bacillus licheniformis with an activity of 250 U _HB at pH 10
50% by weight of a pancreatic enzyme with an activity of 250 U _HB at pH 8
40% by weight of an acidic fungal protease from Aspergillus niger with an activity of 250 U _HB at pH 6.5
The setting 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 pancreatic 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 an overall 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 a clear aqueous solution of an alkaline bacterial protease from Bacillus licheniformis with an activity of 10,000 U _HB at pH 10 are mixed with 50 kg of ammonium sulfate at 16 ° C., the enzymes being precipitated. The enzyme suspension has an activity of 9800 U _HB at pH 10 and a density of 1.22 to 1.23 g / cm ³ .

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 pressed juice, such as is 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 8400 U _HB at pH 8 and a density of approximately 1.22 to 1.23 g / cm ³ . This suspension is then diluted with as much thickened isotonic ammonium sulfate solution (480 mPa × s) so that an activity of 250 U _HB at pH 8 is achieved.

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 8800 U _HB at pH 6.5 and a density of 1.22 to 1.23 g / cm ³ is diluted with so much thickened isotonic ammonium sulfate solution that an activity of 250 U _HB at pH 6.5 is reached becomes.

Production of the thickened isotonic Ammonium sulfate solution

60.7 kg of water are placed in a stirred kettle and with a toothed disk stirrer (circumferential speed 2-3 m / sec.) 1 kg of a thickener Xanthan base incorporated (e.g. K1A96, Kelco). Is the viscosity of the solution has risen to <1000 mPa × s, 38.3 kg of ammonium sulfate are added. You stir like this long until the ammonium sulfate is dissolved. The viscosity drops to 480 mPa × s.

Product C Production of a liquid, non-aqueous settling stable Enzyme preparation.

• 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

500 g of C ₁₃ 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. B. Bentone 27, from Kronos Titan, Leverkusen) for 35 minutes in an Ultra-Turrax mixer with 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).

In a tanning drum with recirculation or pumping unit for the fleet, 1000 kg of cremated cowhide with 800 kg of water introduced.

For descaling, CO ₂ gas is fed directly into the pumping system. The dosage of CO ₂ is 8 to 9 l / min. Right at the start, 0.5% (based on pelt weight) pickling agent (product A) is added. The initial pH in the liquor drops from pH 11.8 to pH 8-8.2 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 with regard to basic purity, smoothness and skin disintegration than with 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 CO ₂ decalcification.

Example 3

Analogously to example 2, product C is used in the same way and Dosage used. Product C contains emulsifiers, which further improves the basic purity of the skins becomes.

Claims (4)

1. Enzymatic pickling preparations for carbonic acid decalcification using proteases, characterized in that the pickling preparations as proteases P

• A) alkaline bacterial protease with a pH optimum between 9 and 11 in proportions of 10-20% by weight based on the totality of the proteases P
• B) pancreatic protease with a pH optimum between 7 and 9, in proportions of 40-70% by weight, based on the totality of the proteases P and
• C) fungal protease or bacterial protease with pH optimum in the pH range 5 to 7 in proportions of 20 to 40% by weight, based on the totality of the proteases P,
  included, with the proviso that the proteolytic activity of
  A ′) alkaline bacterial proteases at pH 10, the
  B ′) pancreatic enzymes at pH 8 and
  C ') the acidic fungal or bacterial protease is adjusted to the same strength at pH 6.5.

2. Enzymatic pickling preparations according to claim 1, characterized characterized that they are still present in addition to the proteases well-known aids selected from the group of the enzyme stabilizers, adjusting agents.   3. Enzymatic pickling preparations according to claims 1 and 2, characterized in that it is known per se Contain solvents. 4. Liquid or solid enzymatic pickling preparations according to claims 1-3, characterized in that they are used at the beginning of the CO ₂ decalcification or during this.