JPH04273000A - Powdery or granular enzyme preparation which contains protease as working enzyme and does not contain surface active agent, and enzymatic softening agent and bating agent which contain the preparation - Google Patents

Abstract

PURPOSE: To obtain the subject preparation used for the soaking, bating and dehairing of hides in leather manufacture by allowing protease to be present in an enzyme preparation EP present as an extender comprising a usual salt itself as a tannin composite.

CONSTITUTION: A surfactant-free powdered or granulated enzyme preparation EP contains protease as acting enzyme in the form of a tannin complex so that about 50-99.9 wt.% of one or more salts (e.g. ammonium sulfate, sodium sulfate) are present as an extender. This preparations is useful for soaking, bating and dehairing of hides in leather manufacture and can be obtained as the tannin composite by precipitating acting enzyme present in aq. environment by adding about 50-99.9 wt.% of usual salt as one or more extenders.

COPYRIGHT: (C)1992,JPO

Description

[Detailed description of the invention]

0001

FIELD OF INDUSTRIAL APPLICATION The present invention relates to a surfactant-free solid enzyme preparation containing a protease as an active enzyme for softening, fermentation and depilation of leather in leather production. can get.

0002

[Prior Art] The tannin precipitation method has been used for quite some time using solutions,
For example, this is a method often used to isolate enzymes from plant sap or aqueous media. There is a relatively wide range of known technology for this [e.g.
B-C) No. 1156900, West German Patent (D
E-A) No. 1642619]. The purpose is generally the purification of the enzyme. Guarantees for good precipitation include the addition of gelatin or the acidic pH range (p
Examples include operations in H3-5). However, the tannins are destroyed in the subsequent enzyme purification (eventually forming an insoluble complex with the enzymes with which they are precipitated) and have to be removed again, usually by removing the precipitate with an organic solvent, e.g. acetone. or by treatment with ethanol or by addition of surfactants or by increasing the pH value with tannin precipitation: these measures can generally be carried out without significant complications only on a laboratory scale. It's nothing more than that.

The industrial use of tannin complexes as such has been sparsely described in some cases. That is, by drying the tannin complex together with skim milk, it is processed into a stable α-amylase preparation (CS141233). For medical use, insoluble pancreatic preparations are prepared by precipitating the pancreas with tannic acid. This preparation is insoluble in the acidic gastric juices, but exerts its action in the alkaline stomach region (West German Patent No. 12
8419 specification).

[0004] In addition, West German Patent (DE-A) No. 2
No. 143,945 describes "water-insoluble, skin-compatible dry enzyme adducts", in particular in the form of tannin complexes. For practical use, for example, the bacteria-proteinase adduct obtained from the tannin precipitation method is incorporated directly into the detergent powder. The tannin complexes have a sufficient cleaning action so that they can be "disappeared" during use, ie in the washing bath.

[0005]

[Problems to be Solved by the Invention] From the above-mentioned known techniques, although the method of tannin precipitation of an enzyme from an aqueous environment is a usable precipitation method, it is difficult to use the precipitated enzyme-tannin complex industrially. - with the exceptions noted above - it appears to be hampered by relative stability under the expected conditions of use.

In order to produce solid enzyme preparations in leather production, in particular proteinase-containing preparations for use as fermentation and softening preparations, ammonium sulfate or sodium sulfate, mainly from pancreatic juice or bacterial cultures, have so far been used. The precipitate used was [Ullmanns E
cyclopadie der technis
Chen Chemie, 4th edition, Volume 10, 475
〜Page 561, from page 495, Verlag Chem
ie, 1975, West German Patent (DE-A) No. 223
4412].

However, this method is by no means ideal - especially from an ecological point of view - and generally entails a very significant load of waste water. This is clear from the usual rule of thumb, which means that 50 kg of ammonium sulphate are required for precipitation per 100 kg of enzyme-containing liquid. Such salt levels run counter to current trends in wastewater management that limit sulfate loads as much as possible. Especially bearing in mind that the leather industry operates under extremely severe cost constraints,
On the contrary, efforts are being made to develop efficient enzyme-precipitation processes which have a substantially lower wastewater load and are thus also cost-effective.

[0008]

It has now been found that the proteinase preparation EP according to the invention satisfies the above-mentioned requirements of the technology very well. The present invention relates to (mainly) surfactant-free solid, i.e. powdered or granular, enzyme preparations EP containing proteases as active enzymes, which preparations contain proteases present in an aqueous environment. 1 formulation
Obtained as a tannin complex by precipitation by addition of tannins in an amount consisting of at least 50% by weight, advantageously from 80% to 99.9% by weight of salts customary as fillers or fillers. It is something that can be done.

"Tannins" according to the invention are generally polyphenols of natural origin and summarized under this name, in particular tannic acid. For more information, please refer to “Ullmann's Enziklopedie der Technischen Chemie”
llmann encyclopedia de
r Technischen Chemie) 3rd
Edition, Volume 11, pp. 593-594; Kirk-Othmer (
Kirk-Othmer's Encyclopedia
of chemical technology (Encyclop)
aedia of Chemical Technology
2nd edition, Vol. 12, pp. 319-325, J.
Wiley, 1967, Zechmeister
Hersg. Fortschrit
te der Chemie organisc
her Naturstoffe, Volume 41, 4-4
6 (Springer Verlag).

"Protease" refers to E. C. 3.4.
These enzymes are summarized in

(Kirk-Othmer, Encycle
opaedie of Chemical Te
Chnology, 3rd edition, Volume 9, 173-223;
J. Wiley1980;E. Pfleiderer,
R. Reiner in H. J. Rehm &
G. Reed, Biotechnology, 6b
Volume, 729-742, VCH1988; K. Aunst
rup, B. Spencer Ed. Industry
ial Aspects of Bioch
emistry, Volume 30 (I), pp. 23-46,
(See North Holland, 1974).

There are various classification criteria, among them classification by origin: a) Animal origin, for example α) Rennin (E.C. 3.4.23.4) β)
Pancreatic - protease pancreatin, especially trypsin, chymotrypsin (p
H-Action range approx. 7-10), Pepsin (E.C. 3.4)
．． 23.1) (pH - range of action approximately 1.5 to 4.0), cathepsin (E.C. 3.4.23.5) (range of action approximately 4
．． 0-5.0) b) Plant origin α) Papain (EC3.4.22.1) pH-
Range of action approximately 5.0-8.0 β) Phytin (EC3.4.22.3) pH-
Range of action approximately 4.0-9.0 γ) Bromelain (E.C. 3.4.22.4 and 3.4.22.5) pH-range of action approximately 5.0-7.0c
) Microbial origin (L.Key “Process
Biochemistry”1971;17-2
1 year) α) Bacilli such as B. subtilis, B. B. licheniformis, B. licheniformis;
B. alkalophilus,
B. B. cereus Natto (B.
Natto), B. B. vulgatus
), B. mycoides β) Streptococcus γ) Streptomyces, such as Streptomyces fradiae
ptomyces fradiae), S. S. griseus, S. Recus (S. rac
tus) δ) Aspergillus species, such as Aspergillus flavus oryzae (As
pergillus flavus-oryzae)
, A. niger, A. s
aitoi), A. A. usamii ε) Mucor and spider molds such as Mucor pusil
lus), M. M. miehei ζ) Endothia such as Endothia parasitica
parasitica) η) Trametes such as Trametes sanguinia (Trametas sanguinia)
sanguinea) In addition to differences due to origin, there are also differences due to location of attack (exo-vs. endoenzyme) and differences due to “site of action” of proteases (D
Serum proteases inhibited by FP (sulfhydryl enzymes) are also used.

Furthermore, the pH dependence of enzyme activity is of great practical importance. Therefore, particularly from a practical point of view, we classify: i) Alkaline proteases, especially alkaline bacterial proteases (E.C. 3.4.21.), with optimal action in the pH range 7.5-13 (mostly belongs to the serine-type) and alkaline fungal protease ii)
Neutral proteases, especially neutral bacterial proteases (E.C.3.
4.24) (belonging to metalloenzymes) and fungal proteases, e.g.
-Proteasen), Pseudomonas-Protease (Pseudomonas-Proteasen),
Streptomyces protease
yces-Proteasen), Aspergillus-Protease
en).

iii) Acidic protease (E.C. 3.4
．． 23) Especially acidic fungal proteases, such as Rhizopus s.
pp. (Rhizopus spp.), Aspergillus spp. (Aspergillus spp.)
, Penicillium spp. (Penicillinm
spp. ), Mucor spp. (Mucor spp.
) and Impex lacteus (Impex I
acteus) and Endosythia parasitica (En
dothia parasitica).

[0015] As alkaline protease, p
Subtilisin is a serine-type alkaline bacterial proteinase that is stable in the H range of 9-10 and is fairly stable to perborate.

The use of proteolytic oxygen was introduced approximately 80 years ago by Dr. Otto Roehm in OROPON (trade name), an enzymatic lysate (pancreatic tryptic enzyme) (West German Patent No. 20051).
Since its introduction (DE 927 464; DE 976 107) it has been a definite component in leather production, especially in water treatment plants: use in fermentation agents (DE 927 464; DE 976 107
Specification; West German Patent No. 941811; West German Patent No. 974813; West German Patent No. 97
West German Patent No. 5095; West German Patent No. 976928; West German Patent No. 1120066; West German Patent No. 1134474; West German Patent No. 121
Besides DE 9620; DE 1282837; US Pat. No. 3,939,040; US Pat. Patent No. 976662; West German Patent No. 102
West German Patent No. 1,034,317; West German Patent No. 1,282,828; West German Patent No. 2,059,453; US Pat. No. 4,278
432; U.S. Patent No. 4,344,762)
, and also for hair loosening and skin swelling (US Pat. No. 4,294,087).

The proteolytic action of enzymes is usually carried out by the Anson-Haemoglobin method (M.L. Anson J. Gen. Physiol).
．． 22, p. 79, 1939) or the Loehlein-Volhard method (
die Loehlein-Volhard'sch
e Method zur Bestimmu
ng die Proteolytishen
Aktivitat. Gerbereichem. Ta
schenbuch Dresden-Leipzi
G., 1955) as “LVE” [Loehlein-Volhard units]. What is Lehline-Forhard unit?
The amount of enzyme is such that under the specific conditions of this method 1725 mg of casein is consumed (R.J. Beynon, J.
S. Bond, Proteolytic Enzyme
(see esIRL press).

[0018] Furthermore, in the following, enzyme units having an action in the acidic range derived from the Anson method are used for activity measurements. These are called "proteinase units" (hemoglobin). 1 unit contacts the release of the trichloroacetic acid-soluble part of hemoglobin corresponding to 1 μmol of trypsin per minute at 37°C (measured at 280 nm). ) corresponding to the amount of enzyme

[0019]

[Outside 1]

As already mentioned, the production of the proteinase preparation EP according to the invention is advantageously carried out directly starting from an aqueous medium or an enzyme-containing solution.

It is particularly advantageous here to produce the preparation EP from the pancreas.

A. Preparation from pancreatic complexes The method according to the invention relies in part on isolation methods known in the art (Ullma et al.
nn, 4th edition, Volume 10, 536-537 in the above cited text
(see page).

Isolation is advantageously carried out immediately after slaughter, primarily from the pancreas of pigs or cows.

For example, by removing as much fat and connective tissue as possible from the glands immediately after slaughter and then homogenizing the glandular tissue using, for example, a meat grinder, about 10 min.
Process 0 pancreases into one batch.

Immediately thereafter, advantageously about 3
18 at 5℃ with twice the volume (approximately 60L) of 0.25N sulfuric acid.
Extract for ~24 hours. After adding filter floc,
It is preferably compressed in a packaging press. The compression plate can be discarded.

The sufficiently fat-free pressed yeast juice obtained in this way is the starting material for the production of proteinase preparations.

B. Production from other proteinase-containing aqueous crude extracts Instead of pancreatic-pressed yeast juice, other proteinase-containing broths can be used, for example from fungal or bacterial cultures (see above for the origin of the enzymes and Ullm.
Ann, 4th edition, Vol. 10, pp. 518-522, H. J
．． Rehm, G. Reed Ed. Biotechn
ology Vol. 7a, pp. 156-168, VCH198
(see 7).

The culture medium contains - as a rough guide - 0.01-3% by weight of protein. The production of a surfactant-free powdered or granular solid enzyme preparation EP containing a proteinase as active enzyme according to the invention can be carried out as follows: first, the correct addition of the precipitant; was found to be important. If insufficient tannins are used, the precipitation is incomplete, and if excessively used, the tannin complexes produced are difficult to dissipate. It has proven to be an advantageous technical principle to use such an amount of precipitant that there is still 0.5-3% enzyme activity in the supernatant. Thus, for example, in the case of precipitation from pancreatic compress (which contains approximately 2% by weight of active protein), 4% by weight of mimosa tanning agent is preferably used. Generally, the protein content in the tannin complex is 10-80% by weight.

[0029] In particular, it is advantageous to prepare aqueous solutions with low pH values (taking into account, of course, the stability of the protease in question). A guideline is a pH value of about 3 to 6, with the culture fluid or pancreas usually having a pH value of about 6. Precipitation above pH=7 is less advantageous. The use of low temperatures (<20° C.) is advantageous in all cases. Furthermore, the addition of water-insoluble surfactants with an HLB value <6 has proven advantageous. This type of surfactant is
For example selected from the group of long chain esters, such as sorbitan fatty acid esters. For example, sorbitan monooctadecanate and the like can be mentioned. Surfactants of this type are commercially available, for example Atlas Chemie (Fa.AT)
LAS Chemie, Essen) Type S
Please refer to the product PAN (product name).

By definition, the salt content of the enzyme preparation consists of at least 50% by weight of one or more salts commonly used as fillers. These salts are preferably ammonium sulfate or sodium sulfate.

Advantageous Effects The advantageous effects apply not only to the use of the enzyme preparation EP according to the invention, but also to the production and handling aspects.

The production of tannin complexes is a very efficient and at the same time selective precipitation method and therefore makes very good use of resources. Compared to the commonly practiced salt precipitation methods, it is notable that the environmental pollution caused by the "mother liquor" is significantly lower. Enzyme preparations do not generate dust and do not have allergic effects on the skin, so they are evaluated as an "improvement" in terms of work hygiene.

In terms of use, the enzyme preparation according to the invention can be used within traditional industrial handling, ie:
Does not require fundamental changes to the process at the water treatment plant. The use of the enzyme preparation EP according to the invention has proven particularly effective in traditional enzyme-assisted softeners and fermenters. At that time, “dissipation (A
It is particularly advantageous for the pH used to be high (pH>8), as this promotes "nfgehen)".

Use: Implementation of Softeners Softening of the skin material to reverse the hardening of the skin that occurs during salting is generally carried out at a pH>7.0 to 10.0. By using together enzymes, especially proteolytic enzymes,
This softening effect is aided by the "digestion" of other water-soluble proteins that do not belong to the skin's collagen fiber tissue. Generally, enzymes are used that have an active range (or optimum pH value for proteolytic action) at pH 7.0 to 10.0 in softeners. With the removal of non-collagen proteins, rapid and intense moisturization of the skin is ensured. Advantageously, the softening water is prepared alkaline (see above), but the pH value is always <12
should be kept at Additionally, softening aids (e.g. nonionic and anionic surfactants) can be combined with substituted phenols or dithiocarbamates in a conventional concentration range (0.1-1
0 g/l). Enzyme additives in the enzyme preparations according to the invention include, for example, the proteases mentioned above, in particular the proteases mentioned under c): in particular microbial proteases with an action range of 7 to 11.0, in particular Bacillus protease, Streptomyces protease and Bacterium proteases. Proteases, such as Aspergillus,
For example, A. A. saitoi and A. saitoi. A. usamii, further e.g. pH working range 7
．． A. having a value of 0 to 9.5. A. oryzae
), and A. A. niger and A. niger. Flavus (A. fla)
Vus).

Generally, the proteolytic concentration of the proteinase used is 0.01 to 0.0 per liter of softening juice.
3 Anson units or 1000 to 3000 Lehlein-Forhard units. Therefore, the amount of enzyme preparation EP to be used will correspond to these concentrations depending on the extent of its enzyme content. Finally, softener juices can contain amylase. Amylase is, for example, a companion enzyme to fungal proteinases. Amylase assists in the breakdown of glycosidic bonds in skin proteoglycans and glycoproteins.

Following softening, water treatment plant processes generally include lime treatment followed by demineralization and - primarily enzymatic - fermentation.

[0037] Also in this step, the enzyme preparation EP can be advantageously used.

Use: Fermentation and demineralization traditionally reduce the alkalinity of rawhide to a pH of 13-14.
It helps to lower the pH value from 7 to 8. For deashing, preference is given to using weak organic acids or weakly acidic salts rather than strongly dissociated ones, for example of the dicarboxylic acid type. During fermentation, epidermal, hair and pigment residues should be removed and additional skin lysis should occur. Additionally, non-collagen protein components are removed (Ullmann, vol. 4).
Edition, Vol. 16, supra, pp. 119-120). During fermentation, the pH is generally 7.5 to 8.5. The use of cyclic carbonates in deashing is known from DE-A 31 08 428.

At the same time, during the fermentation it is possible to use lipases having an action range between pH 7.0 and 9.0, such as pancreatic lipase. B. has an active range between pH 5.5 and 8.5. Amylases such as pancreatic amylases, which assist in the breakdown of glycosidic bonds in fermentation, also have a beneficial influence on fermentation (in particular as companion enzymes for trypsin and chymotrypsin).

Next, the present invention will be explained in detail with reference to examples.

[0041]

EXAMPLES Example 1 Preparation of preparations from pancreas 100 kg of filtrate containing 0.4% protein from defatted pancreatic glands were mixed with 5 kg of tanning agent from Nastyrida in 20 kg of water under stirring at room temperature. Add solution. The immediately formed protein precipitate is further stirred for 2 hours at room temperature (20° C.) and then filtered. The filter cake is compressed in a Packpress and then pre-milled. Then carefully dry at a temperature of 60-80℃, 1:10
Mix with ammonium sulfate.

The resulting mixture is immediately added to leather softeners, leather decomposers for depilation and wet blue (Wet Blue).
tblues) can be used for relaxation.

Example 2 A. Preparation of preparations from Y. oryzae yeast A proteinase concentrate containing approximately 2% protein is obtained from the bacterial culture by ultrafiltration and cooled to +15°C. To improve sedimentation, the concentrate was coated with SPAN 40 (Atla
sChemie, trade name of Essen product) 100g
, followed by 4k of Nemurixa tanning agent in 16kg of water.
Add g. Protein precipitation occurs immediately. After standing for 1 hour, the batch was treated with a perlite-based filter aid (DIC).
ALITE 4408, Fa. Dicalite, N
euβ) (60-80 l/m2 of filter area for 1 hour). Carefully dry the compressed filter cake (60-80°C) and mix 1:3
Add a mixture consisting of sodium sulfate and ammonium sulfate in a ratio of 0.

The mixture obtained can be used immediately in leather softeners, leather decomposers, for depilation and for relaxing ewett blue.

Claims (8)

[Claims] 1. The protease is present as a tannin complex in the enzyme preparation EP, which enzyme preparation EP consists of at least 50% by weight and up to 99.9% by weight of one or more salts customary per se as fillers. A surfactant-free powder or granular enzyme preparation, characterized in that it contains protease as active enzyme, characterized in that it is present in such a degree. 2. Enzyme preparation EP according to claim 1, characterized in that the bulking agent consists of ammonium sulfate or sodium sulfate. 3. An enzymatic softener containing the enzyme preparation EP according to claim 1. 4. An enzymatic fermentation agent containing the enzyme preparation EP according to claim 1. 5. Enzymatic softener, characterized in that the enzyme preparation EP according to claim 3 is used at a starting pH value of >7-11. 6. Enzymatic softener, characterized in that the enzyme preparation EP according to claim 5 is used at a starting pH value of >8-11. 7. Enzymatic fermentation agent, characterized in that the enzyme preparation EP according to claim 4 is used at a starting pH value of >5 to 11. 8. An enzymatic fermentation agent, characterized in that the enzyme preparation EP according to claim 7 is used at a starting pH value of 6 to 9.