NL9101869A - PROTEASE AS A TAMINE COMPLEX CONTAINING ENZYME PREPARATIONS, THE USES INCLUDING THIS AND LEATHER, PREPARED USING THESE PREPARATIONS. - Google Patents

Description

Protease as tame complex containing enzyme preparations. the use thereof as well as leather prepared with the aid of these preparations.

Field of the invention

The invention relates to protease as active enzymes containing surfactant-free solid enzyme preparations for soaking, pickling and depilating skins in the leather preparation, which are obtained by precipitation of tannin.

State of the art

The technique of tannin precipitation has long been an incidentally employed method variant in isolating enzymes from solutions, for example, from plant juices or aqueous culture media. There is a relatively wide prior art for this (compare for example GB-C-1.156.900, DE-A-1.642.619). The aim is usually the purification of enzymes. The addition of gelatin resp. working in an acidic pH range (pH 3 ~ 5) · However, since tannin interferes with further enzyme production - after all, it forms an insoluble complex with the enzymes, which can be precipitated with it - it must be removed again, which usually takes place by treating the precipitate with organic solvents, for example with acetone or ethanol or by adding a surfactant (tenside) or by increasing the pH in tannin precipitation: all measures generally only on a laboratory scale without significant complications can be achieved.

The technical application of the tannin complexes themselves has been described in very few cases. For example, a tannin complex together with skimmed milk will be dried to form a stable α-amylase preparation (CS 141.233). · For medicinal use, an insoluble pancreatic preparation, which was prepared by precipitation of pancreas using tannic acid, is provided. The preparation is insoluble in the acidic gastric juice, but it has its effect in the alkaline parts of the intestine (DE-C-128 419).

Furthermore, DE-A-2,143-9 ^ 5 describes "water-insoluble, skin tolerant dried enzyme adducts", inter alia, in the form of tannin complexes. For practical application, the aim is, for example, to directly process the adduct of a bacterial proteinase obtained in tannin precipitation, into detergent powder. Since the tannin complex must be fully active during washing, it must be "integrated" in the washing bath at the time of use.

Problem and solution

As shown in the foregoing representation of the prior art, although the method of tannin precipitation of enzymes from an aqueous medium represents a useful precipitation method, the relative stability of these precipitated enzyme-tannin complexes seemed to be an industrial use thereof, with the exceptions mentioned, to get in the way so far.

For the preparation of solid enzyme preparations in the manufacture of leather, in particular of the proteinase-containing preparations for use as pickling and softening preparations, hitherto mainly the precipitation using ammonium sulfate or sodium sulfate from pancreatic juice or bacterial culture juice has been used (compare Ullmans Encyclopedia of Technical Chemistry, 4th edition, vol. 10, pp. 475 to 561, pp. 495 et seq., Verlag Chemie, 1975. DE-A-2,234.4l2).

However, this mode of operation is by no means ideal, particularly from an ecological point of view, as it usually involves a very considerable waste water load. This is apparent from the usual rule of thumb, which says that 100 kg of enzyme-containing juice require 50 kg of ammonium sulfate for the precipitation. Such a salt load is completely contrary to the current trend in wastewater management to limit the sulphate load as much as possible. The aim would be to have an efficient method of precipitation of enzyme, in which the waste water load would be considerably less and finally also cheaper, especially in view of the leather industry operating under very special cost pressure. It has now been found that the EP proteinase preparations according to the invention come very close to the said prior art representations. The invention relates to a protease as active enzyme-containing (substantially) free from surfactants, solid, ie powdered or granulated enzyme preparation EP, which is obtained as a tannin complex by precipitation of the active enzymes occurring in an aqueous environment by adding tannin with it is understood that the enzyme preparation comprises at least 50% by weight, preferably more than 90% by weight and at most 99.9% by weight, of one or more salts customary as extenders.

"Tannins" within the meaning of the present invention are to be understood to mean the polyphenols summarized under this name, usually of a natural nature, in particular the tannic acids. More precise statements can be found in Ullmann, Encyclopadie der Techn. Chemistry, 3rd ed., Vol. 11, pp. 593 to 594, Kirk-Othmer, Encyclopedia of Chemical Technology, 2nd ed. Vol. 12, pp. 319 to 325, J-Wiley 1967 »Zeichmeister Hersg. Fortschritte der Chemie more organic Naturstoffe, volume 4l, pp. 1-46, Springer Verlag).

Under "proteases" the ones under E.C.3-4. summarized enzymes are understood.

(Compare Kirk-Othmer, Encyclopedia of Chemical Technology, 3rd ed., Vol. 9 pp. 173-223. J. Wiley 19δ0, E. Pfleiderer, R. Reiner in HJ Rehm & G. Reed, Biotechnology, vol. 6b, 729-742, VCH 1988, K. Aunstrup in B. Spencer Ed Industrial Aspects of Biochemistry, vol. 30 (I) pp. 23 to 46, North Holland, 1974).

Various differentiation criteria are applied, including the distinction according to origin: a) Animal origin, such as a) Rennine (E.C.3-4.23.4) (5) Pancreatic proteases

Pancreatin, in particular trypsin, chymotrypsin (pH action range approx. 7-10), pepsin (EC3-4.23.1) (pH action range approx. 1.5-4.0), kathepsin (EC3.4.23.5) range of action approx. 4.0-5.0) b) Vegetable origin a) Papain (EC3-4.22.1) pH range of action approx. 5.0-8.0 β) Ficin (EC3-4.22.3) pH operating range approx. 4.0-9.0.

γ) Bromelain (EC3-4.22.4) and 3-4.22.5) pH action range ca. 5.0-7.0 c) Microbial origin (according to L. Keay in "Process Biochemistry" 1971; pp. 17 to 21).

a) from Bacillus species such as B. subtilis, B. licheniformis, B. alkalophilus, B. cereus, B. natto, B. vulgatus, B. mycoides.

β) from streptococcus species γ) from streptomyces species such as streptomyces fradiae, S. griseus, S. rectus <5) from Aspergillus species such as Aspergillus flavus oryzae, A. niger, A. saitoi. A. usamii (e) from Mucor and Rhizopus species such as Mucor pusillus, M. miehei if) Endothia species such as Endothia paraditica η) from Trametes species such as Trametes sanguinea

In addition to the distinction according to origin, the distinction also takes place according to the site of action (exo versus endoenzyme) and on the basis of the "active site" of the proteases (serine proteases, which are inhibited by DFP, sulfhydryl enzymes).

Furthermore, the pH dependence of the enzyme activity is of considerable practical importance. It is therefore primarily distinguished from a practical point of view: i) alkaline proteases with an action optimum approximately in the pH range of 7 * 5 to 13, in particular alkaline bacterial proteases (EC3 ^ .21.) (Which usually belong to the serine type) and alkaline fungal proteases.

ii) Neutral proteases with an action optimum in the pH range of 6.0 to 9 * 0, in particular neutral bacterial proteases (EC3.4.24) (all belonging to the metallozymes) and fungal proteases, for example Bacillus proteases, Pseudomonas proteases, Streptomyces proteases, Aspergillus proteases.

iii) Acid proteases with an action optimum in the pH range of 2.0 to 5 * 0 (E.C.3.4.23), especially fungal proteases, e.g. from Rhizopus spp., Aspergillus spp., Penicillium spp., Mucor spp. and Impex lacteus and Endothitia parasitica.

Particularly mentioned as alkaline proteases are the subtilises, alkaline bacterial proteinases of the serine type, which are stable in the pH range 9-10 and are somewhat insensitive to perborate.

The application of proteolytic enzymes since the introduction of the enzymatic pickling (tryptic digestive enzymes of the pancreas) in the 0R0P0N® pickling by Dr. Otto Röhm (DE-C-2.005.19) about eighty years ago a permanent part in the preparation of the leather, especially in the wet house: in addition to the application in the stains (DE-C-927.464; DE-C-976.107; DE- C-941.811; DE-C-974.813; DE-C-975-095; DE-C-976.928; DE-C-1,120,066; DE-C-1,134,474; DE-C-1,219,620; DE- A-1,282,837; US-A-3,939-040; US-A-4,273,876) also find enzyme preparations useful in the soaking bath (DE-C-288,095; DE-C-976,662; DE-C-1,022,748; DE-Cl.O34.3i7, DE-C-1,282,828; DE-C-2,059,453; US-A-4,278,432; US-A-4,344,762) and in addition when loosening the hair and the digestion of the skin (US-A-4,294,087)

The proteolytic activity of enzymes is usually determined by the Anson hemoglobin method (ML Anson J. Gen. Physiol. 22, 79 (1939) or by the method according to Löhlein-Volhard (die Löhlein-Volhard'sche Method zur Bestimmung der proteolytischen Aktivitat, Gerbereichem, Taschenbuch, Dresden-Leipzig, 1955) as "LVE" (Löhlein-Volhard unit) A Löhlein-Volhard unit means that amount of enzyme which digests 1.725 mg casein under the specific conditions of the method. (Compare RJ Beynon, JS Bond, Proteolytic Enzymes IRL press).

Furthermore, in the following, units derived from the Anson method are used to determine the activity of the enzymes active in the acidic range. These are referred to as "Proteinase Units" "hemoglobin" UHb. A UHb corresponds to the amount of enzyme which catalyzes the release of trichloroacetic acid soluble fractions from hemoglobin equivalent to 1 pmol tyrosine per minute at 37 ° C (measured at 280 nm). (1 mUHb = 10'3 UHb).

As already mentioned, the preparation of the proteinase preparations EP according to the invention advantageously finds directly from aqueous culture media or. enzyme containing juices. The recovery of the preparations EP from pancreas is of particular importance in this respect.

A. Extraction from the complex of the pancreas

The measures according to the invention may partly correspond to the isolation process according to the state of the art (compare Ullman, 4th edition, volume 10, loc.cit. P. 536-537).

Isolation advantageously proceeds from pancreatic glands immediately after slaughter, mainly from pork or beef. For example, it is possible to process about 100 pancreatic glands in one go by immediately removing fat and connective tissue from the glands immediately after slaughtering and then homogenizing the glandular tissue, for example with the aid of a meat grinder. Immediately afterwards, 0.25 n sulfuric acid is extracted with approximately twice the volume (approx. 60 l) at 5 ° C for 18 to 24 hours. After adding filter flakes, it is advantageous to press off via a packing agent press. The press plates can be discarded. The highly fat-free pressed juice thus obtained is the starting material for the recovery of the proteinase preparations.

B. Collection from other proteinase containing aqueous starting extracts

Instead of the pancreatic juice, other proteinase-containing culture liquids, for example from fungal or bacterial cultures, can be used (compare the above statement on the occurrence of enzymes as well as Ullmann, 4th edition, vol. 10, pp. 518-522, HJ Rehm, G. Reed Ed. Biotechnology, Vol. 7a, pp. 156 to 168, VCH 1987).

The culture liquids contain - as a general guide - between 0.01-3% by weight of protein. The recovery of the proteases according to the invention as active enzymes containing surfactant-free solid enzyme products EP in powder or granulate form can take place as follows:

First, the correct dosage of the precipitant has proved important. If the tannin is used in an undersized way, an incomplete precipitation is obtained, if an excess is used, the tannin complex formed is difficult to digest. As an effective technical rule, it has been found to use just so much precipitant that 0.5 to 3% of enzyme reactivity is still found in the supernatant. For example, in the precipitation from pancreatic juice (containing about 2% by weight of active protein), 4% by weight of Mimosa tannin is advantageously used. In general, the protein content in the tannin complexes is from 10 to 80% by weight.

In particular, it is advantageous to proceed in such a way that a low pH value of the aqueous juices is set (whereby, of course, attention must also be paid to the stability of the proteases concerned). A pH value of approx. 3 ~ 6 is mentioned as a reference, culture liquids resp. pancreas usually have a pH of about 6. Precipitation above pH 7 is less effective. In any case, it is advantageous to use fairly low temperatures (<. = 20 ° C). Furthermore, the addition of water-insoluble surfactants with an HLB value <6 has proven beneficial. Such surfactants are selected, for example, from the class of the long chain esters such as, for example, the sorbitan fatty acid esters. For example, mention is made of the sorbitan monooctadecanate and the like. Surfactants of this type are commercially available, for example, compare products from Fa. ATLAS Chemie, Essen of the type SPAN®.

According to the definition, a salt content of the enzyme preparations of at least 50% by weight is provided by one or more salts commonly used as extenders. These salts are preferably ammonium sulfate or sodium sulfate.

Economical operations

Advantageous effects are already recorded from the viewpoint of preparation and handling, not least, however, when the enzyme preparations EP according to the invention are used.

The preparation of the tannin complexes involves a very good utilization of the starting products, since it is a very effective and at the same time selective precipitation method. To be emphasized is the significantly lower environmental impact from the "mother liquors", for example, compared to the predominantly performed salt precipitation. The enzyme preparations can also be considered "improved" in the field of occupational hygiene, since they do not dust and have hardly any allergic effect on the skin.

From an application point of view, it can be advantageous that the enzyme preparations according to the invention can be used within the usual technical manner of operation, i.e. no substantial change of the process sequence in the water workshop is required. The use of the enzyme preparations EP according to the invention has proved particularly suitable in connection with traditional enzymatically assisted soaking and pickling. The presence of a high application pH value is particularly favorable (pH> 8), since it promotes "unlocking" the tannin complexes.

Application: carry out the soaking

Soaking of skin material, negating the hardening of the skin during the preservation with salt, is usually carried out at a pH> 7.0 to 10.0. The simultaneous use of enzymes, in particular of proteolytic enzymes, accelerates the softening action by "digesting" the water-soluble and other protein bodies, which do not belong to the collagen fiber structure of the skin. In general, enzymes with an action range (or pH optimum of the proteolytic action) at pH 7.0-10.0 are used for soaking. With the removal of the non-collagen proteins, a faster and more intensive moistening of the skin is guaranteed. The soaking water is advantageously made alkaline (see above), but the pH value must always remain less than 12. In addition, plasticizers such as nonionic and anionic surfactants in combination with substituted phenols or di-thiocarbamates in the usual concentration ranges (0.1-10 g / l) are advantageous. Suitable enzymatic additives for the enzyme formulations according to the invention are, for example, the aforementioned proteases, in particular the proteases mentioned under c): especially microbial proteases in the action range 7-H.0, in particular Bacillus proteases, Streptomyces proteases, as well as fungal proteases, for example from Aspergillus species such as A. saitoi and A. usamii, further such from A. oryzae with a pH range from 7.0 to 9.5 further from A. niger and A. flavus with a pH range from 9.5 to 11.0. Generally, the concentration of the proteolytic activities of the proteinases used is in the range of Q] Q | -0.03 Anson units, respectively. 1000-3000 Löhlein-Volhard units per 1 week liquid. The amounts of enzyme preparation EP used will therefore correspond to these concentrations depending on their enzyme content. Finally, the soaking liquids can still contain amylases. For example, the amylases act as companion enzymes to fungal proteinases. They favor the splitting of glucosidic bonds in the proteoglucans and glucoproteins of the skin. Soaking usually involves passing through the wet house, followed by descaling, followed by descaling and, mainly enzymatic, pickling.

The enzyme preparations EP can also be used to advantage in these subsequent steps.

The application: pickling

Descaling traditionally serves to reduce the alkalinity of the blots from pH values from 13 to 14 to pH values in the range from 7 to 8. For descaling, it is preferable not to use highly dissociated, but weak organic acids, for example of the type of the dicarboxylic acids or weakly acidic salts. During pickling, epidermis, hair and pigment residues must be removed and, moreover, an opening of the skin must be effected. In addition, non-collagenous protein components are removed. (Compare Ullmann, 4th edition, vol. 16 loc.cit., Pp. 119-120). Pickling is conventionally carried out at one pH 7.5 to 8.5. DE-A ~ 3,108,428 discloses the use of cyclic carbonates in descaling.

At the same time, lipases, for example pancreatic lipases with an action range at pH 7.0 to 9.0, can also be used in the pickling. Also amylases according to B), for example pancreatic amylases with an activity range at a pH 5.5 to 8.5, which favor the splitting of glycosidic bonds during pickling, are especially beneficial as accompanying enzymes of trypsin and chymotrypsin). pickling.

The following examples serve to illustrate the invention. Example I

Preparation of a preparation from pancreas

To 100 kg of filtrate from defatted pancreatic glands containing 1.4% protein, a solution of 5 kg of mimosa tannin in 20 kg of water is added with stirring at room temperature. The directly occurring precipitate of the protein is stirred for an additional 2 hours at room temperature (20 ° C) and then filtered. The filter cake is pressed in a square baler and then made somewhat small. After this, it is dried under mild conditions at temperatures of 60 to 80 ° C and mixed in a ratio 1:10 with ammonium sulfate. The resulting mixture can be used directly for soaking or pickling leather, for hair removal and for loosening wet blues.

Example II

Preparation of a preparation from an A. oryzae fermentation

From a fungal culture, a proteinase concentrate containing about 2% protein is recovered by ultrafiltration and this concentrate is cooled to + 15 ° C. To improve the precipitability, 100 g of the concentrate are added with 100 g of SPAN®40 (product of Atlas Chemie, Essen) and then 4 kg of Mimosa tannin in 16 kg of water. The precipitation of the protein starts immediately. After an hour of rest, the mixture is filtered with the addition of 2 kg of perlite-based filter aid (DICALITE®44o8, Fa. Dicalite, Neuss), (60 to 80 l / h and m2 filter area). The pressed filter cake is carefully dried (60 to 80 ° C) and mixed in a 1:30 ratio with a mixture of sodium sulfate and ammonium sulfate.

The resulting mixture can be used directly for soaking or pickling leather, for hair removal and for loosening wet blues.

Claims (9)

Protease as an active enzyme, surfactant-free, powdered or granulated enzyme preparation, characterized. that in this enzyme preparation EP the protease is present as a tannin complex with the proviso that the enzyme preparation EP consists of at least 50 and at most 99 * 9 wt.% of one or more salts customary as extenders. Enzyme preparation EP according to claim 1, characterized in that the excipient consists of ammonium or sodium sulfate. Use of the enzyme preparations EP according to claim 1 in the enzymatic soaking bath. Use of the enzyme preparations EP according to claim 1 in the enzymatic pickling bath. The use of the enzyme preparations EP according to claim 3, characterized in that the initial pH value>. 7 to 11. Use of the enzyme preparations EP according to claim 5, characterized in that the initial pH value>. 8 to 11. Use of the enzyme preparations EP according to claim 4, characterized in that the initial pH value is at least> 5 to 11. The use of the enzyme preparations EP according to claim 7, characterized in that the initial pH value is> 6 to 9. Leather, prepared with the aid of the enzyme preparations according to claims 1 and 2, respectively. obtained using claims 3-8.