DE2033822A1 - Hydrolytic enzyme mixture and processes for its production - Google Patents

Description

Priority: July 9 19 69 due to the British patent application Jk 696/69

The present invention relates to new and valuable hydrolytic enzymes and a process for their production, isolation and purification. Specifically, the invention relates to proteolytic enzymes which can be obtained by culturing Cephalosporium sp. ATCC 1155 ° or mutations thereof. The microorganism is on the American Type Culture Collection, 2112 M Spreet NW, Washington 7, DC-. (USA) and is referred to below as Cs 32. In another aspect, the invention relates to the use of the proteolytic enzymes in various fields of technology. Examples of such areas of application are the use of the products according to the invention as a component in detergents, their use as agents for cell lysis, such as in bacteria, red and white blood cells and platelets, and the use of the products in the breakdown of biopolymers such as proteins, Xohlenhy- .

drates and lipids. 1Ö9816 / 181S

BATH ORIGINAL

The term "detergents" means a wide variety of detergents containing surface active ingredients, optical brighteners, perfumes and other ingredients known per se in the art. The enzyme preparation according to the present invention should not be used in combination with components in detergents which contain the see enzymatic activity inactivate · ^s omit are chlorine-containing petitioner * Gentien not intended for use in combination with the enzyme preparation of the present invention in general

suitable"

The enzymatic «product that is identified below rand referred to as the "crude product" is the product of this invention that is most suitable for use on an industrial scale Scale is suitable. Of course, the fractions of the crude product identified below, which with IP 4.1, IP 6.1 and IP 10.5 are also used technically for different purposes, and so is for example the preferred use is that as a component in detergents, but is general for reasons of nationality prefers the use of the raw product. The raw product can be used as an additive to detergents in amounts of about 0.01 to 5 wt. be used·

The areas of technology specifically mentioned above, in which the enzymes can be used in accordance with the relocating invention, serve only as illustrative examples.

109818 / 181S

BAD ORiGiNAL

2033R72

Licher areas of application, the enzymes with the dock have extremely valuable and surprising properties, which are described in detail below, also numerous other technological applications that all are in the spirit of the invention. Technical areas to which the enzyme preparations can be used are, for example, the food technologies such as the manufacture of bakery products and breweries, for example for the purpose of modifying gluten in Baok processes and clarification and prevention of sediments in soft drinks and fermented liquids »

When cultivating the microorganism Cs 32 in organic media it was found to be able to produce various hydrolytic enzymes such as proteases, amylases and lipases. Some of these enzymes can be very useful on different ones Areas of technology. For example, certain of these enzymes can be used as ingredients in detergents Certain enzymes can be used as a means of cell lysis certain others are used to solubilize biopolymers will. Of particular interest are some proteases which are produced by the organism in question and which are used in elevated temperatures up to about 60 C are stable and are therefore particularly suitable as ingredients in detergents. It it can be seen that such properties are particularly advantageous for the considered use of the proteases as additives to detergents. Another characteristic of the Proteases, which are particularly valuable for their envisaged Use as additives to detergents, their use is

10 9 8 1 6 / 1B 1 NC

BATH ORIGINAL

2033322

borrowed a high pH optimum, around pH 10-11,

In the case of a broad implementation of the method, which is more suitable Way can be used for the production of the enzymes according to the present invention, according to the organism in question under aerobic, submerged conditions in a suitable " clay grow medium. It is preferred that the nutrient medium an assimilable carbon source, nitrogen source and contains inorganic salts. Growth conditions, such as time, temperature and hydrogen ion concentration, can be varied widely without departing from the concept of the invention. After the growth period is over, the enzymes can or the material of interest from the mash by various methods, such as by centrifuging the mash, Dialysing the solution and spray drying the resulting solution can be obtained.

The following methods of isolating the enzyme preparation obtained from the cultivation can be used

1. Precipitation of the enzymes after filtration of the maize heels obtained from the breeding wagon. The precipitation can take place with the help of

a) tannin

b) lignin.

c) organ! see solvents such as acetone or isopropyl alcohol

BATH ORIGINAL

109816/1818

d) inorganic salts such as sodium sulfate or ammonium » sulfate,

2. Precipitation of the enzyme · after filtration of the corn cows and evaporation of the solution. The precipitation can occur with the following Funds are carried out *

a) tannin

b) lignin

o) organic solvents such as acetone or isopropyl

alkehel, d) inorganic salts, such as sodium sulfate or ammonium

sulfate.

3 · Spray drying of the · product dureh

a) Filtration of the mash, evaporation of the obtained Solution and spray drying of the resulting solution,

b) Filtration of the mash, evaporation of the mash obtained Solution, Dialysieron the solution thus obtained and Spray drying the resulting solution,

c) Filtration of the mash, evaporation therefrom Solution, precipitation of an ensymatisohen product, as described, for example, under 1 and 2, and Spray drying of the product thus obtained

The methods designated with Ia) ₉ 2a) and 3a) have been shown to be highly valued and are more sophisticated methods.

1 Ö Ö 8 1 6/1 8 1 Ö "BAD ORIGINAL

Its great variety of substances can be used as carbon sources

in 1 · η can be used in the high-temperature medium. Piping can make water Either soluble or insoluble, only mentioning that the compounds used can be readily assimilated by the organism. As examples are Pentoses, such as arabinose, ribose and xylose, the monosaccharides, like mannose, lftvulose and galactose, the disaccharides like trfthalese, maltose, laetose, gelloblose and sucrose, the polysaccharides like starch, the higher alcohols like glycerine, Mannitol, sorbitol and inositol, and various sources such as ethyl alcohol and calcium carbonate. Nitrogen in one assimilable fora can be derived from animal or vegetable proteins, soybean meal, casein, peptones, polypeptides Amino acids are added.

The adjustment of pH · β can be done with any suitable one Base, vie sodium hydroxide, phosph & tpmffex », sodium nitrate, Vatrluzmeetat can be made in any other suitable material fe that the pH-value in the chosen area

Bio temperature of the Nfthraediun "wlteremi <i" r Zfte ^ twig the fungus can advantageously swisehen about 2 # w & ü 35 ° CS g «You preferred temperature is at §6 =» 2® C

Time required for the production of a maximum esa Menu "proteeiytisoher Snzyme can largely -wariiert w®Tümn ₉ Depending on the Hatur specifically verv« a «l @ t" a Bestandteil® in dmm.

Nihrmedlum. However, Gewilmlieb «iae Seit w & m © twm, 3 ©

120 hours as appropriate

10 9 816/1818

BATH ORIGINAL

Antifoam agents can be added to the nutrient medium will. Examples of such anti-foaming agents are soybean oil, caetor oil, sulfonated oils, lard oil and brominated castor oil.

As stated above, the Stame Cs 32 can be grown in a culture medium without hydrolytic enzymes ku produ-

in grace. The culture medium can be any of a number of Technique of known media, since the organism in question is able to assimilate many sources of energy. However, in order to obtain economical production and maximum enzyme yield, certain culture media are preferred. For example, the currently preferred sources of carbohydrates in the culture medium are sucrose, starch, glucose, dextrin, and the like. The preferred nitrogen sources are soybean meal, brewer's yeast, cornflour & liquid, casein, fish meal, and the like. The organic nutrient salts _; which can be incorporated into the medium are, for example, the salts which are able to transfer ions such as ammonium, sodium, potassium, potassium, magnesium, phosphate, chloride, sulfate, nitrate and the like To yield ions. Essential trace elements can also be incorporated into the culture medium. However, such trace elements are usually contained as impurities in other components of the culture medium.

For maximum growth and production of Cs 32 should be the culture medium before inoculation to a pH value between about 4.5 and about 9.5 »preferably to pH 6-7

109816/1818

BATH ORIGINAL

can be set. The pH at the end of the growing season depends on the buffer substances present and the initial pH value

The rate of production of proteases in the culture medium can vary during the growth period of the organism can easily be followed by examining their caselnolytic activity in samples from the medium. The caseinase determinations are described in one part.

The crude product obtained after isolation of the enzyme preparation, which was obtained from the cultivation of Cs 32, is through the following properties are marked

a) It is a mixture of proteins

b) it is soluble in water, salt solutions and buffer solutions such as phosphate, borate and citrate buffers,

c) it is stable and retains its enzymatic activity at room temperature for more than a year in dry powder form,

d) its ultraviolet absorption spectrum is characteristic of proteins, the aromatic amino acids contain,

e) it is proteolytically active against casein, fibrin, hemoglobin, albumin, gelatine and elastin «,

f) it has a proteolytic optimum with Caeein as a substrate at a pH value higher than 10,

g) it is able to hydrolyze esters, such as Na-Tosyl-L-arginine ethyl ester (TAEE),

109B1 R / 1 818

BAD ORIQiNAL

N-dC-Benzoyl-L-arginine methyl ester (BAMB) and N- ^ C-Benzoyl-D-L-arglnin-y ^ -naphthylamide (BANA),

h) it contains esterases, lipases and amylases in addition to proteolytic enzymes,

l) the proteolytic activity can be precipitated with organic solvents such as alcohols, such as ethanol, acetone, tannins, lgnines or salts, such as ammonium sulfate or sodium sulfate,

j) it has a temperature optimum with casein as substrate at about 60 ° C at pH 7, k and at pH 9.0,

k) it contains a proteolytically active fraction an isoelectric point at pH 4.1,

l) it contains a proteolytically active fraction an isoelectric point at pH 6.1,

m) it contains a proteolytically active fraction an isoelectric point at pH 10.5 »

n) it has a pH optimum against casein, elastins, Albumin, BANA, TASE and BAMS each at pH 11 or higher, 10, 11.5 »9» 5 »10.5 or higher and 7»

o) it has no or only a very weak peptidase activity against the substrates listed in Table V,

p) the caselnelytisohe activity remains essentially

2+ 2+ unaffected by the presence of ven Mn, Co,

Ca ²⁺ , Mg ²⁺ and EDTA, q) the caseinolytic activity remains essentially

unaffected by the presence of Trasylel ** - · ' r) the easeinolytic activity becomes almost complete

inhibited by the presence of PMSF.

"1Ö9816 / 1818 BADORIGfMAL

Other characteristic features of the raw product and also characteristic features of the products XP 4.1 »IP 6.1 and IP 10.5, which are identified below, “ground” apparent from the following description · ■

The term "crude product", as it is used below, means the enzyme preparation which is produced in the cultivation of Cs 32

in holds, as described in examples h and 5

In order to identify the product according to the invention, that was Enzyme preparation of a treatment labeled "crude product" subjected to the following scheme «

Fractionation of proteolytic enzymes obtained by culturing Cs 32 with diethylaminoethyl cellulose (BEAEj ₁

5 g proteolytic enzyme crude product precipitated with tannin (15t3 CE./mg), suspended in 200 ml of water, pH to 7 * ° set .

Centrifuge Residue (discarded) solution Fraction 1, 1.8 g (6.0 CE / mg) 5 S diethyleneminoethyl -

cellulose

109816/1818 bad origswal

Centrifuge

solution

Auagefllt with 500 ■! acetone and iaoelectric separation

subjected (Flg. 2)

Fraction 2, 1.4 g (2O ₉ Z CE / mg)

Back and washed with 2 x 10 nl Vaaaer

Back stand

suspended in 100 ml of 1 M * ammonium acetate

Auatauacher

suspended in fjO ml 1 m Aasaonium Acetate

Solution

precipitated .It 300 «1 acetone and iaoelectriaeHer separation;

subjected (Fla; 3)

Fraction 3, 0.6 g (5.5 Ci, / i |) Centrifuge Rtiekatand

In the beliefU ^ th Drawings Figure 1 shows the in Heaaungen the optical density at 280 nm obtained results, the Carbobenxoxy-L-leuoin _t -. ^ - nitrophenyleeteraktlvitat, measured as optical density at 410 nm, and the pH of the in isoelektriecb .cn separation of the crude product fractions ^ Fig, 2 shows obtained in analeger Veiae the results which one _y for measurements of the optical density at 200 nm obtained "the Esteraaeaktivität that Carbobensoxy-L-leucine 4-nitrophenylesteraktivitat and the pH at ¥ ert Fractions obtained from isoelectric separation of fraction 2 according to the Torigen fractionation scheme. 3 shows in an analogous way the Xrgeniaae which are obtained by measurements of the optical density at 280 nm, the Esteraae -akti-rltKt, the carbobenzoxy-L-leucine-4-nitrophenyleeteraktivitÄt and the pH-value with fractions which one is at ieo ^ ektrlach ^, separation of the faction

BATH ORIGINAL

2033322

according to the fractionation scheme above.

An electrical bundling device LKB 8100 Ampholine ^ = * 'was used for the isoelectric separation of the material.

The activity of the enzyme preparations on * carbobenzoxy-L-leucine-pnitrophenyl ether was measured in the following way!

Reagent

ψ 10 mg of carbobenzoxy-L-leucine nitrophenyl ester in 10 ml of isopropanol were suspended in 200 ml of 0.03 M phosphate buffer, and the pH was adjusted to 7 # 8.

To 100 μl of enzyme solution diluted to 1/10 or more, 2.5 ml of substrate was added, the whole was mixed and kept at 37 ° C. for 10 minutes, followed by cooling to room temperature. Then 1 ml of ethanol was added, the resulting mixture blended, and the tubes were read immediately in a spectrophotometer at kiO nra against a blank.

Blind sample t Substrate solution at 37 C without enzyme.

The method was used for qualitative and comparative experiments.

The material from the tubes k0 - kk in Fig. 2 was pooled and deionized by dialysis against tap water or by passage through a Sephadex ^ - 'column. The ejitioni-

10 981-6 / 18-10

BATH ORIGINAL

Alerte material was then freeze-dried. This material had an activity of 25 C proteolytiache _a seineinheiten per mg at pH 7 »4 and an isoelectric point at pH 10.5. In the following and in the tables, this material is referred to as IP 10.5. In the same way, the material from the tubes 15-19 in FIG. 3 was combined, deionized, freeze-dried and examined with regard to the proteolytic activity and the isoelectric point. This material had a proteolytic activity of 1.0 Cae inclusions per mg at pH 7-4 and an isoelectric point at pH 4.1. This material is used below and in the tables with IP. 4.1. Similarly, the material of the tubes 24-27 in FIG. 3 was found to have a proteolytic activity of 0.24 casein contents per mg at pH 7.4 and a leoelectric point at pH 6.1.

The present invention is further explained with the aid of the tables below.

Table I shows the pH optimum of the protease and esterase activity of

a) the crude product that is obtained from the isolation of the enzyme preparation is referred to in the table as "raw",

b) the pro t "as efr alt tion with a leoelectric point at pH 4.1, in Table 1 P 4.1",

c) the protease fraction with an isoelectric point at pH 6.1, referred to in the table as "I P 6.1",

and d) the pro teas efraction with an isoelectrical point

109 816/1810

BATH ORIGINAL

2033B22

at pH 10.5, labeled ¹¹ IP 10.5 in the “a®r table”.

The activity against elastin, Casein® albumin, BAMB and BANA was measured. The activity smessiasigen was measured at 37 C using casein, elastin and albumin as substrate, at hO ° C using TAEE as substrate and at 23 ° C using carried out by BANA and BAMS as a Safestrat.

EAD ORiGiMAL

10 9 816/1818

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1 0 9 8 1 6/1 8 1 8

ORIGINAL INSPECTfD

2033322

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OBIGIMAL INSPECTED

10 9 8 16/1818

Table I (port network)

Percent of maximum activity against

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pH BAKE IP 10.5 raw ΒΑΝΑ raw TAEE IP 4.1 IP 4.1 raw 2 2.5 3 3.5 1 3 4th 2 ... 4th 1 K5 5 1 9 39 5 10 28 22nd 4th 20th 5.5 25th 53 49 30th 6th 90 82 74 44 55 9 6.5 95 92 100 57 77 7th 98 100 98 77 88 7.5 97 96 97 82 91 19th 8th 100 96 93 89 95 8.5 99 93 47

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109816/1818

The activity against TASE was only determined for the crude product. The products IP 6.1 and IP 10.5 were not active against BANA. The product IP 6.1 was not active against BAMD.

The activity of the preparations against albumin was measured in the following Veiae

Reagent

Albumin, bo ν. plasm, eryst »A quality O _t 55 & in buffer trichloroacetic acid (TCi), pa ninhydrin according to Moore-Stein

Puff

Teorell-Stenhagen BZ 299, page 417 (1939)

200 μl of enzyme solution were mixed with 200 μl of substrate of the desired pH value. The mixture was incubated at 37 ° C. for 15 minutes and then precipitated with 250 μl TGA and centrifuged.

Blind sample

Enzyme and substrate were kept separately at 37 ° C and after the addition of TCA mixed.

500 yul of the floating layer above were mixed with 1 ml of ninhydrin and kept at 100 ° C. for 20 minutes, after which the mixture was cooled and 5 ml of 50% n-propanol were added.

1098 1 f / mi 8

Each tube was shaken and then placed in a spectrophotometer measured at 575 nm against the blank sample.

The activity of the preparations against BAME was stimulated in the following way

The method uses the difference in UV absorption at nm between benzoylarginine and its methyl ester

Rea gentient N-ÖC-BensBoyl-L-arginine methyl ester + 2 HCL 32.9 ml in 10 ml

Buffer bufferι

Sodium phosphate + glycine 0.2 iii 1 + 1

Am part of BAME + J? Parts of the buffer were mixed and added to the set the desired pH. To 3 ml of substrate were 100 / ul enzyme solution added, the whole was mixed and the absorption measured directly in a spectrophotometer at 25Ί nm

After 15 minutes at room temperature (25 ° C) a new Reading made.

The difference in absorbance between these two determinations is a measure of the esterase activity of the enzyme.

The activity of the preparations against BANA was measured in the following way!

109816/1818 _{0R1QINÄL iN8PEC} T _S 0

Reagent N - $ {-Benzoyl-D, L-arginine-V-naphthylamide · HCl 8 mg in 10 ml

■ '.' ■■ buffer

Trichloroacetic acid (TCA), pa kO ^ I » Sodium Nitrite, Freshly Prepared $ 0.1 Ammonium sulfamate $ 0.5 N- (1-naphthyl) ethylenediamine 5 mg in 10 ml

Ethanol

Buffer:

Teorell-Stenhagen BZ 299t page k17 0939)

The substrate solution was adjusted to the desired pH with 2 η HCl adjusted and two samples of 2 x 200 / µl were prepared for each measurement, one for the blank.

200 µl enzyme solution was added to each sample to be tested and the rigid tubes were heated to 37 ° C for 30 minutes and then precipitated with 250 / µl TCA.

Blank sample t

The enzyme and substrate were kept separately at 37 ° C and mixed after TCA was added.

The tubes were centrifuged and 500 µl of the top layer were mixed with 500 / ul of sodium halide. After three minutes 500 / ul ammonium sulfamate were added, the whole was mixed carefully, and two minutes later the color was off developed by adding one ml of N- (1-naphthyl) -ethylenediamine. After 15 minutes the tubes were placed in a spectrometer

read the photometer at 575 * im against the blank.

The elastolytic activity was determined in the following catfish 5 »O ml Teorell-Stenhagen buffer of the desired pH value with a content of 1 $ elastin was homogenized. An appropriate amount of enzyme was dissolved in 1.2 ml of buffer and the mixture was shaken at 37 ° C. for various lengths of time. 3ml TCA was added to the withdrawn samples. After centrifugation, the optical density became <s determined at 280 mn. The activity was set as the difference in optical density per hour of incubation time to a concentration of the Enssympräparatee of 1 rag per ml, expressed.

From table I is egg © Batli @ £ i, da® the pH-Optiaram of the activity of the preparations against dia vesewemiteiaB. Substrates often with an "i" alieh h © Is. © n pH-Wea-t, which is advantageous for the use of the predmtete mash d®rv © ri®geiaden invention as additives to Betargemtiasü considered here. ± at ₀ So the pH optimum of the activity dos Ütoi & prodmkiQs against Oaooin is pH 11 or higher, against elastin at pH 19 _and against albumin at pH 11.5 »« «against BANA at pH 9t> 5 ^ md gassed TAEE b © i pH 1O ₉ 5, or higher. The pH optimum of the Reiaproeliaktea given BMME is pH 7.

Table II shows the ¥ 1σ · μθ stability the K © kpr @ dBktas ₀ of IP 4.1, IP 6.1 and IP 10 ₈ 5 at pH% _S 7 and pH 9 _s © o Di «samples were without substrate at the specified pH ¥ ®rt and the specified temperature 30 minutes incubiarto Bi © determination of the

10 9 816/181 «

proteolytic activity was thereafter, at a temperature of 37 ° C using casein as a substrate.

1098 16/1818

Table II

Thermal stability

temperature Percent of maximum activity IP 4.1 pH 7, ¹ IP 10.5 raw pH 9.0 IP 4, 1 IP 6.1 IP 10.5 raw ° C 100 IP 6.1 96 100 99 100 98 100 98 95 100 100 100 93 100 97 20th 81 100 94 89 51 36 76 63 37 12th 83 33 37 2 0 7th 5 50 1 10 2 3 1 4th 3 60 2 2 2 1 1 4th 1 70 2 1 2 6th 80 2 0 0 0 90 2 100

From Table II it can be seen that the preparations at pH 7.4 are still essentially stable at 50 ° C and that IP 10.5 and the crude product is remarkable even at 60 ° C at pH 7.4 Keep activity.

Table III shows the temperature optimum at pH 7t ^ and pH 9.0 for the crude product, for IP 4.1, IP 6.1 and IP 10.5. Casein was used as a substrate for the activity seeds at pH 7 »4 and pH 9.0 used.

ORIGINAL INSPECTED 1098 16/1818

■ 2033322

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! MSPECTED

109816/1818

From Table III it can be seen that the preparations investigated have a temperature optimum at about 60.degree.

Table IV shows the pH stability of the crude product, of IP 4.1, IP 6.1 and IP 10.5. The enzyme preparations were incubated for 30 minutes at 37 ° C. in Teorell-Stenhagen buffer which had been adjusted to the desired pH. At the end of the incubation, the samples were mixed with phosphate buffer of pH 7 _f k , and the remaining activity was determined by casein digestion.

Table IV

Stability at different pH values

pH IP 4.1 percent maximum activity IP 10.5 raw 2 15th 1 0 1 3 24 86 59 Λ 34 99 77 5 84 100 80 6th 95 93 73 7th 100 98 77 8th 98 94 77 9 too 100 95 10 97 YES 100 11 95 98 68 IP 6, 3 7th 15th 95 93 93 100 98 99 81

From Table IV it can be seen that the crude product and the Product IP 10.5 are more stable than IP 4.1 and IP 6.1 at pH 3.

109816/18 18 '■ - _Λ

JNSPECTED

It can also be seen that all products are essentially stable at pH 10.

The peptidase activity of the crude product was compared to the in Measured substrates listed in Table V «

Table V in O ₉ 2 m F h © @ 1 mg / ml N “J 0.1 H ti 0.3 η ti o, 3 η η o, 3 st S3 0.3 II ? 1 η K 3 «■ I « 3

Glycyl glycine Gly-gly-glycine Gly-L-leuoin L-leu-glycine Gly-L-proline L-pro-glyοin Z-gly-L-phenylalanine Z-L-hlu-L-zyro sin Z-gly-L-leu-amide

100 / UI enzyme in water and 100 λιΙ substrate were mixed mitein other and given in water at 37 ° C for 30 minutes.

Thereafter, 1 ml of ninhydrin in 2% solution was added ₉ and the tubes were kept at 100 ° C. for 20 minutes. Bad daraaoli cooled to room temperature.

Blank sample t

The enzyme and substrate were kept separately at 37 ° C. and mixed after the addition of ninhydrin.

5 ml of 50% n-propanol in water were. dajaa © li _$ added the resulting mixture was blended, wan the absorbance at 575 mn in a spectrophotometer against di © blind "sample measured. It was found that the @ crude product does not have any

109816/1818

ORIGINAL IMSPEGTED

or only a very weak peptidase activity against the in Substrates listed in Table V possessed.

The proteolytic activity against casein was determined as described mainly by Bergkvist in Acta Chem. Sοand 17 0963) »pages 1521-1540. 15 g of casein (Merck, Hammarsten-Casein) were suspended in 400 ml of water with vigorous stirring. Sodium hydroxide was added until dissolved. The pH was adjusted to the desired pH and the solution was diluted to 500 ml with water. 3 ml of casein solution were mixed in test tubes with 3 ml of Theorell-Stenhagen buffer (Biochemisches Taschenbuch, Rouen HM Springer Verlag (1956), page 651), which had the desired pH value.

In parallel, other test tubes were equilibrated to the appropriate temperature in water baths. An appropriate amount of the dissolved enzyme was added to the tea tubes. From one of the parallel experiments, 2 ml were pipetted directly into 3 ml of a 10-liter trichloroacetic acid. The other H _e ihe of Teetröhren was incubated for exactly 30 minutes, whereupon 2 ml withdrawn and was added to 3ml 1Obiger trichloroacetic acid. After standing for at least 30 minutes at room temperature, the tubes were centrifuged and filtered through small funnels plugged with a loose plug of cotton wool. The optical density at 280 m / u of the filtered centrifugate was measured.

The activity of the enzyme, expressed in Caein units

(C.E. per mg) is obtained in the following way

Optical density of the incubated sample minus optical density of the nloht-incubated sample, divided by the amount of enzyme in the tubes (expressed in mg).

When examining the stability of the enzyme, the Ensym dissolved in buffer and incubated without casein at a certain temperature for a certain time. determinations at the desired pH and temperature carried out.

In the determination of the proteolytisekoa activity with the application of Na-Toeyl ~ L-ar @ ± s & in ° - & tSi.yl © at @ 3 ^> (Τ & ΕΈ) as substrate - dia folae »d @ K CSaGEailsalisn iss, u'qt BsadlSstang uses «> TAEE la an O, © 5 is © lax ^ä © m Kemzemtratioia rad Q $ 9 $ is * NaCl. The added Meage the EassyalSsiamg feetsmg Q ₉ 2 ml, "the volume of ad ToetlSaunff was 10 e b1 _s © ie titration with ρ 0.05 M NaOH at ¥ erweadaag © imss · aiatesaatiseiaea Titrationeeinrichtungf a Radioraetertitrati @ 2is © ^ins> i © liL -feismg'Type TTA4 carried out.

The effect on the ease of use of the crude product © a by adding various metals and inhibitors © »was studied. The dissolved in 3 ml buffer enzyme preparation was mixed with 0 ₉ 1 ml of a 0.1 m solution of asu untersnichend @ H lldthode bsw _e Xthylendiamlntetraessigsäure (EDTA) in 0.1 m-Lii © iLmg O _{9 9} 5 ^ phenylmethylsulfonyl fluoride (PMSF) -L8sung and 0 ₉ 1 ml © iner Traeylol solution * ^ incubated. The incubation time was 10

10 9 8 16/1818

Minutes at 37 ° C. 3 ml of Caaein solution were then added and the proteolytic activity was determined. The results are summarized in Table YI. The caaeinolytic activity obtained was compared with the caseinolytic activity in control samples without added test material. AIa buffer was O ₁ 2 m tris (hydroxymethyl) aminomethane of pH 7 »^ and pH 10, O used *

Table VI

Effect of metals and inhibitors on the oaeeinolytiaohe activity of the crude product · »

Added material

0.03 m Mn 0.03 m Zn

2+

2+

0.03

Co

2+

2+

0.03 a Ca 0.03m Mg ²⁺ 0.01 ml traaylol * 0.03 m EDTA 0.1 al PMSF EDTA + PMSF

Change of activity

- 30

- 1 © + 10

0 0

- 10

- 95

- 95

From Table VI it can be seen that the addition of PMSF is a Almost complete inhibition of caaeinolytiachen activity results, but that the submerged metal ions as well as traaylol and EDTA result in practically no inhibition "

1 0 9 8 1 G / 1st fl 1 8

2+

Zn may have some inhibitory activity Ee is of particular value for the use in question here de * Product according to the invention as an additive to detergents, since calcium ions obviously reduce the caseinolytic activity of the Do not influence the enzyme preparation.

Table VII shows the results of the addition of .....,., ~~~ and soybean inhibitor to the crude product and to IP 4.1 and IP 10.5, whereby the activities are recorded with BAME

Table VII

raw 80 IP 4.1 10 IP 10.5 90

Effect on the eterolytic activity when using BAME through the addition of Trasylol ^ - * and soybean inhibitor

Test product percent activity ± m comparison with the control sample

(λ)
Trasylol ^ - 'soybeans

80 10 90

From Table VII it can be seen that the product IP k, 1 to 905t is inhibited by Trasylol ^v * - ^{> /} and soybean inhibitor, - »The inhibition obtained with the crude product can be compared to the inhibition - ° of the product IP 4.1, which in the Raw product is included, -j, to be added ·

- "From the above it is clear that the crude product of this

Invention contains various proteolytic enzymes · These

proteolytic enzymes can, for example, be separated from one another by ion exchange, as explained in the scheme given above Separation also DBAS, ion exchangers with acid groups, such as Carboxymethyl groups and sulfoethyl groups are used, and the separation can also be carried out by other methods such as by zone electrophoresis, gel filtration and fractional precipitation.

Compatibility of the enzyme preparation (raw product) with detergent __ tien

Three commercial detergent compositions were used; Sine contained perborates and about $ 50 (weight by weight) phosphate, one contained about $ 7 (weight by weight) phosphate and no perborate, and one was a soap and contained about $ 5 (weight each) Weight) phosphate. The pH optimum of the proteolytic activity, the pH stability of the proteolytic activity, the temperature optimum of the proteolytic activity and the thermal stability of the crude product of the enzyme preparation were investigated

A. pH optimum of the caseinolytic activity of the crude enzyme preparation in combination with detergents

The following solutions were prepared: Sine solution contained 5 g detergent in 100 ml water (detergent solution). One solution contained 5 »8 crude product enzyme preparation per ml of water (enzyme solution). One solution contained yf> casein.

109 8 16/181

The pH was adjusted using Teorell-Stenhagen buffer solution.

2 ml of buffer solution de · desired pH ¥ ®rte "and 1 ml cleaning * mlttell8sung were 3 ^m l casein mixed" The. resulting solution was heated to 37 ° C, after which 0.1 ml of Bnzymlöaung prepared and was measured eaeeinolytieehe activity "For comparison, a blind 18sung that no Reinigungsiaittel oxatfe ± elt ₈ .untersucht · The results are shown in Table VIII

Table VIII Caseinolytic activity of Combinations with pure mangamittol

pH Caseinolytic activity ;,> the maximum activity
the detergent composition with buckets of Phos
phat content of Ti 55t Blind Air Sunir 6.0 KO - 5OfI 7th 5 © 24 6.5 56 kl 7.0 55 55 55 kk 7.5 72 52 8.0 19th 6k 77 . 50 8.5 79 77 9.0 80 71 97 58 9.5 75 97 10.0 8th? 100 1Θ0 76 10.5 100 too 11.0 80 68 100

109816/1818 '

B. pH stability of the crude product enzyme preparations in combination with detergent compositions

Identical solutions were used as those according to test A. 2 ml of buffer solution and 1 ml of detergent solution were mixed. Thereafter O ₄ was 1 ml of the enzyme solution was added and the solution thus obtained was biert 30 Hinuten at 37 ° C incubators ·, after which the caseinolytic activity at pH measured 7.4 and 37, the 0th The results are shown in Table IX.

Table IX

Influence of pH on the stability of the crude product enzyme preparation in combination with detergent compositions

pH Caeeinolytic activity, percent of maximal activi
ity of the detergent composition with a Phos
phat content of 7 * 5 * Blind solution 2.0 ko - 505 & 2 2 1 3.0 k 0 59 k, 0 k 10 79 77 5.0 5 15th 80 6.0 18th 100 100 73 7.0 23 O ₃ 80 77 8.0 ko 99 81 77 9.0 95 95 82 95 to »ο 97 72 67 100 ι 1.0 100 50 38 68 71

1098 1 ß / 1 818

C. Temperature optimum of the caseinolytic activity of the crude product enzyme preparation in combination with detergent compositions

The detergent solution and casein solution used were identical to those described in Tinter A. above. * A crude product enzyme solution containing 3 mg of crude product enzyme preparation was used. Adjustment of the pH value β was used when using 0.2 M phosphate buffer to adjust to pH 7, k and Teorell-Stenhagen buffer to adjust to pH 9.0.

2 ml buffer solution and 1 ml detergent solution were with

Mix 3 ml casein solution and heat it in a water bath at the desired temperature for 3 minutes. After that, 0.1 ml Enzyme solution added and the caseinolytisohe activity determined. The results are shown in Table X below.

109816 / 1R18

Table X

Temperatoroptimam the caseinolytic activity of the Rotiproduct preparation »in combination ait detergent compositions

temperature
C. Casein © Iytisehe activity, percent of maximum activity
medium composition with a phosphate content of 505C 7 # pH 7.4 pH 9.0 5% pH 7.4 pH 9.0 the cleaning pH 9.0 40 - pH 9.0 12th 10 10 60 Blind solution 6th pH 7.4 7th 26th 26th 35 52 pH 7 * 4 30th 20th 4th 34 58 67 94 100 7th 67 37 23 85 100 100 100 86 25th 100 50 100 100 51 22nd 23 27 77 25th 60 95 30th 13th 9 15th 9 100 70 50 - ■ 15th 12th - 54 80 6th - 13th 16 - - 13th "MM" 90 . ■ -.- ■■. - - 100. -

D. Heat stable! did of the crude product ^ esizym preparation in.

tion with cleansing agentziasamensetiEiaiigea

Solutions were used which were identical to those of test C. 2 ml buffer solution and 1 ml detergent solution were mixed with 0.1 ml EnsyralSsiaiig vernrf.son.tg raad - the resulting solution was 30 minutes at the? The temperature was incubated, after which the caseinolytiaclie activity was determined at 37 ° C. The results s ± 2n <ä in Table 3El
posed.

10 9 816/1818

Table XI

CD

CO σ?

co CD

Heat stability de * crude product enzyme preparation in combination
set with detergent together pH 9.0 7 # pH 7.4 pH 9.0 5 * pH 7.4 pH 9.0 Blind charge pH 9.0 temperature Caseinolytic activity, percentage of maximum activity of cleaning with ~
telztieammensetzting with a phosphate content of 100 100 75 100 100 . ^T PH, 7.4: 100 20th 40 - 50 # 56 98 100 69 82 100 97 37 pH 7.4 1 52 4th 57 30th 100 63 50 100 - 7th 2 20th - 89 5 60 21st ■ - ■■ '"" 1 -. - 37 3 70 2 - ■ - - 3 1 80 - 1 -

VO I

CD GJ

OO NJ

- 4ο -

E. The relative caseinolytic activity of the crude enzyme preparation in combination with detergent compositions compared with the c as a »JSjti see activity that obtained without the addition of detergent compositions, was measured at pH 7.4 and 37.degree. The results are in Table XXI compiled.

Table XII

Relative caseinolytic activity of the raw product enzyme preparation

Detergent type Relative caseinolytic

activity

Phosphate content kO - 50 $ 126

Contains perborate

Phosphate content 7% 128

Phosphate content% ikh

(Soap)

no detergent additive 100

The test results listed in Tables VIII - XII show that the enzyme preparation crude product of the present invention is compatible with detergent compositions with high (kO - 50,, weight s- #) and low (5-10 weight- $) phosphate content and that the Detergent compositions can also contain perborates as optical brighteners * The results also show that the enzyme preparation is compatible with soaps. The pH optimum in soaps is given as unaffected in T _a ble VIII. The pH

109816/1818

Optimal in the compositions containing 4O - 50 $ phosphate resp. Containing 7 $ phosphate is lowered to about pH 10.

The maximum stability for the detergent composition is, as shown in TabellaiX, with a phosphate content of 40-50 $ at pH 10 and with a phosphate content of 7 $ and for the soap at pH 6. As Table X shows, this is

Temperature optimum for all compositions examined
θ

practically unaffected.

The stability to heat is, as Table XI shows, am most affected by detergents with a phosphate content of $ 40 to $ 50. Table XII shows that the total amount of caseinolytic activity in the enzyme preparation im Mixture with the investigated cleaning agent compositions did not decrease.

The following examples serve to further illustrate the invention.

example 1

In each of a series of 500 ml Erlenmeyer flasks were 100 ml of one of the following media is given.

I suorose 24 g

Pharmamedia 20 g

KH ₂ PO ₄ 19.5 C

Tap water to 1000 ml

109816/1818

II cornmeal Soybean meal

KH * 2 ^PO 4

tap water III sucrose So j bean flour tap water

IV Dried Beef Blood Sucrose

tap water

Corn soak faws Pharmamedia (J9

20th 5 G 20th 9 13, e on 1000 ml 20th 5 G 20th « 13, β on 1000 ml 30th 6th S. 40 G 13, β on 1000 ml 40 6th β 20th 8th 13, S. on 1000 ml

tap water

The flasks were placed in an autoclave at 120 ° C. for 20 minutes treated The pH was adjusted to about 6.8 set. After cooling, the flasks were vaccinated with an aqueous suspension of the organism for incubation was carried out at a temperature of 26-30 ° C, and it was stopped when that proteolytic activity a Maximuni achieved what it did in 96 hours. »The results are summarized in Table XIII.

Table XIII Organism activity in the medium as C base units per ml

I II III IV V

Os 32 32 1 * 5.9 46.0 21.4 34.5

109816/1818

Example 2

Soybean meal (100 mg). Sucrose (1? 5 g), KH ₂ ^PO 4 (67.5 g), anti-foaming emulsion RD (1 ml) in tap water (5 l) was adjusted to pH 6.9 with potassium hydroxide and 30 minutes in a 10 l fermentation vessel sterilized at 127 ° C.

The nutrient medium was cooled and added to 100 ml of a 24 hour culture inoculated by Ca 32, a mutation strain, and incubated at 28 ° C with agitation and aeration. After 72 hours if the proteolytic activity was estimated to be 65 CU / ml, was the nutrient medium cooled

Example 3

Soybean meal (100 g) _r sucrose (200 g), KH-POj, (67.5 g) tmd RD antifoam emulsion (i ml) in tap water (5 × ΐ) were adjusted to pH 6.9 with potassium hydroxide and 30 minutes at 127 ⁰ C sterilized in a 10-1 fermentation vessel. The nutrient medium was cooled and inoculated with 100 ml of a 24 hour culture of Cs 32 and incubated with agitation and aeration for about 36 hours at 28 ° C.

Soybean meal (3,000 g), sucrose (6,000 g), KHgPO ^ (2,025 g) and RD antifoam emulsion (10 ml) in tap water (150 l) were adjusted to pH 6.9 with potassium hydroxide and in a 300 liter fermentation vessel for 30 minutes at 127 ° C sterilized.

10 9 8 16/1818 ORIGINAL INSPECTED

2033322

The nutrient medium was cooled and with the above-mentioned inoculum inoculated and then incubated at 28 ° C with agitation and ventilation. After 72 hours, - when the proteolytic Activity estimated at UO CE / ml was fermentation broth chilled.

example k Obtaining an enzyme preparation by precipitation

For the isolation of the enzyme preparation from the fermentation broth of Example 3, this broth was in a Seitz filter press with arches 3/1250 and with 1 $ Dicalite as a filter aid filtered. The clear solution (15O 1, activity 38 C.E./ml) was adjusted to pH 5 with phosphoric acid, and the enzymes were treated with a solution of tannin (9OO g) in water (3 l) filled in · After stirring for one hour, the suspension was centrifuged in a centrifuge.

fc The moist tannin enzyme precipitate (4,600 g) was washed with cold Acetone (-10 C, 1,000 ml) treated slowly under Stirring was added. After the precipitate had partially dissolved, a further portion of acetone (20,000 ml) was added all at once and the mixture was filtered. The solid was washed with acetone and in air at room temperature dried overnight «

Yield 615 g with an activity of 7.8 C.U./mg.

ORiGSNAL INSPEGTiD 1 0 9 8 1 ß / 1 R 1 8

Example 5 Obtaining an enzyme preparation by spray drying

To isolate an enzyme preparation from a fermentation broth according to Example 3, the broth was filtered in a Seitz filter press with arches 3/1250 with 1 $ Dicalite as a filter aid. The clear solution (41.5 l, activity 33 CE./ml) was evaporated in a thin film evaporator at 15 C to 4.8 1 with an activity of 175 C.U./ml.

The solution was spray dried and yielded 970 g with a Activity of 0.95 CE./mg.

The proteolytic activity of the crude enzyme preparation of the The present invention was made with a proteolytic enzyme from Aspergillus oryzae, Protease I (R. Bergkvist, Acta Chem. Scand., 17 (1963), pp. 1521-1540). The examined The substrates were casein, gelatin, elastin and TAEE. A viscometric method was used for gelatin (S. Lindvall, 0. Magnusson and K. Orth, Acta Chem. Scand, 23, (1969), p. 2165). The results are compiled in Table XIV below.

ORIGINAL INSPECTED

109816/1818

" ^ke " 2033 * 322

Table XIV

Relationship between the proteolytic activity for a crude enzyme preparation obtained from Cs 32 according to the method described in Example h and the proteolytic activity of protease I (Bergkvisty _, loc. Cit

_,,. Proteolytic Activity of Crude Enzyme Preparation Proteolytic Activity of Protease I.

Casein 0.48

Gelatin O _t kk

Elastin k _t 0

TAEE 0.13

The activity of the crude enzyme preparation against elastin is higher than that of Protease I, while the activity of the crude enzyme preparation against gelatin, casein and TAEE is lower than that of protease I.

Example 6

Soybean meal (100 g), sucrose (200 g), KHgPO ^ (67.5 g), RD Soap Prevention Emulsion (1.00 ml) in tap water (5 L) was adjusted to pH 6.8-7.0 with potassium hydroxide and 30 minutes in a 10-1 fermentation vessel at 125 ° C sterilized.

The nutrient medium was cooled and inoculated with 100 ml of a 2 ^ hour culture of Ce 32 and incubated for about 30 hours at 28 ° C. with agitation and aeration.

ORIGINAL INSPECTlD

10 9 8 16/1818

- ^Λ7 ■ "■■ '2033322

Soybean flour (3,000 g), sucrose (6,000 g), KH "POj, (2,025 g) and RD antifoam emulsion (50 ml) in tap water (150 l) were adjusted to pH 6.8-7.0 with potassium hydroxide and at 125 ° C for 30 minutes in a 300 liter fermentation vessel sterilized. The nutrient medium was cooled and with the above mentioned inoculation culture and then incubated with agitation and ventilation at 28 ° C.

After 90 hours, when the proteolytic activity drops to 80 C.E./ ml was estimated, the fermentation broth was cooled.

Example 7 Obtaining an enzyme preparation by precipitation

To isolate the enzyme preparation from a fermentation broth according to Example 6, the broth was in a Seitz filter press filtered with sheets 3 / 125Ο without filter aid, and the pH value was adjusted to 5 »5 with phosphoric acid. the clear solution (i40 1, activity 79 CE./ml) was added to 10 bis Chilled to 15 ° C.

The enzymes were precipitated with a solution of tannin (900 g) in water (3 »0 l). After stirring for one hour, the suspension was centrifuged in a centrifuge. The moist tannin enzyme precipitate (1.83O g) was treated with cold acetone (-10 C _t 500 ml), which was slowly added while stirring »-" ^r ■ - "- ■ - :,». ■ - '^;

ORIGINAL! NSRECTED 1098 16/1818

- kB

After the precipitate had dissolved partly _s a further quantity of acetone (S ₀ OOO ml) was eye set at a time _c and the mixture was filtered after 60 minutes'

The solid was washed with acetone and in vacuo at Room temperature dried overnight

Yield: ^ 05 g with an activity of 18 C ₀ Ee

t example 8

Obtaining an enzyme preparation by precipitation

To isolate the enzyme preparation from a fermentation broth according to Example 6, the broth was filtered in a Seitz filter press with arches 3 / 125Ο without filter aid, and the pH was adjusted to 5-5 with phosphoric acid. The clear solution (i * _{10 I 1} activity 71, CE / ml) was cooled to 10 to 15 ° C. and then _{evaporated in a thin-film evaporator at 20 ° C. to 30 1 with an activity of 3OO CE 8} / mg.

The enzymes were precipitated with a solution of tannin (875 g) in water (2.9 l). After stirring for one hour it was the suspension centrifuged in a centrifuge The moist tannin enzyme precipitate (3,256 g) was washed with acetone (-10 ° C, 2,000 ml), which is slowly added while stirring became.

After the precipitate had partially dissolved ₈ a further amount of acetone (18®6 © Q ml) was added all at once ^ and

1 0 9 8 1 B / 1 8 1 8 _ncrrm

! MSPEGTEO

- k9 -

2033327 '.

the mixture was filtered after 60 minutes.

The solid was washed with acetone and dried in vacuo at room temperature overnight.

Yield: 612 g with an activity of 11.0 C.U./mg.

Example 9

Soybean meal (2.0 kg), sucrose (6.0 kg), KH ^ PO ^ (2.0 kg) and RD antifoam emulsion (0.5 L) in tap water (100 L) was adjusted to pH 6.9 with potassium hydroxide and at 125 ° C for 30 minutes in a 360-1 fermentation vessel sterilized.

The nutrient medium was cooled and inoculated with 400 ml of a 24-hour culture of Cs 32 and incubated for k8 hours at 28 ° C. with agitation and aeration. . . ':

Soybean meal (200 kg), sucrose (350 kg), KOH (32 kg), concentrated H-POr (57 kg) and RD anti-foaming emulsion (2.0 L) in tap water (8,000 L) were made with potassium hydroxide adjusted to pH 6.8-7.0 and 30 minutes at 125 ° C in sterilized in a 17 »000 l fermentation vessel.

The nutrient medium was cooled and with the above-mentioned inoculum inoculated and then incubated at 28 ° C with agitation and ventilation.

ORSGiWAL INSPECTED 10 9 8 16/1818

After 83 hours, when the proteolytic activity was estimated to be kO CE ₀ / ml, the fermentation broth was cooled.

Example 10 Obtaining an enzyme preparation durcji_Spj? ÜhJ <ro_ctoung;

To isolate an enzyme preparation from a fermentation broth according to Example 9, the broth was on a rotating Vacuum drum filter pre-coated with Dicalite (100 kg) filtered.

The clear solution (10,000 l; activity ^ O C.E./ml) was in a thin film evaporator at 25 C to 11,000 1 with a Activity of 3OO C.E./ml evaporated.

The solution was spray dried and yielded 318 ^k g with an activity of 1.0 CE / mg.

As an example of a detergent composition containing the crude enzyme preparation according to the present invention the following composition can be mentioned »

Example 11

Detergent composition with a content of the raw product enzyme preparation (Weight- ^ calculated on the final product).

ORIGINAL SMSPECTED 109816/1818

"* * Vi 2 O 3 - ^ S 2

Dodecylbenzene sulfonate 14

Sodium tripolyphosphate 7

Sodium silicate 6

Gluconate 5

Enzymatic product (crude product) 1

Carboxymethyl cellulose 1

Sodium sulfate

66 sodium carbonate

If desired, additives such as optical brighteners, Perfumes, dyes and the like can be added. Similar compositions that the enzyme preparation according to the present invention are:

a) Soap surfactant, about 20% by weight Perborate, calculated as NaBO "· ^ H" 0; and conventional components, such as optical brighteners, perfume, paint, sodium sulfate, sodium carbonate, sodium triphosphate and sodium silicate,

b) Dodecylbenzenesulfonate surfactant, about 33% by weight perborate calculated as NaBO · ^ H ₃ O, and conventional ingredients listed under a). These, too, are only examples of cleaning agent compositions according to the invention. A soap as mentioned under a) can contain about 5% by weight of sodium triphosphate. A composition like her

1098 16/1018

ORlGiNAL fMSPECTID

under b) mentioned above _p can contain up to kO - 50 weight sodium triphosphate »

ORIGINAL INSPECTED '

1 0 9 8 1 Π / 1 B 1 8

j «

Claims (1)

Claims b) it is soluble in water, salt solutions and buffer solutions such as phosphate, borate and citrate buffers, ^ it is stable in dry powder form at room temperature for more than 1 year and retains its enzymatic activity, d) its ultraviolet absorption spectrum is characteristic of proteins containing aromatic amino acids, e) it is proteolytically active against casein, fibrin ,. Is hemoglobin, albumin, gelatin and elastin, f) has a proteolytic optimum with casein as substrate at a pH higher than 10, g) it is able to hydrolyze esters such as Na-tosyl-L-arginine sat ^ l ester, NO ^ -Benzoyl-L-arginine methyl ester and N-Ot-benzoyl-DL-arginine-ß-naphthylamine, h) it contains esterases, lipases and amylasts in addition to probabilistic enzymes, i) the proteolytic activity with organic solvents such as alcohols such as ethanol, acetone, tannins, lignins or salts such as ammonium sulfate or sodium sulfate, noticeably iafc, j) there is a temperature optimum with casein as substrate ■ '1 09 8 16/1818 ORlQiNAL INSPECTID - 5h - 2 η 3 ' ^{} (} ) 2 about 60 ° C at pH 7 Λ and at pH 9.0, k) it contains a proteolytically active fraction with an isoelectric point at pH 4.1, l) it contains a proteolytically active fraction with an isoelectric point at pH 6.1, m) there is a proteolytically active fraction with an isoelectric fraction Contains point at pH 10.5, n) there is a pH optimum with casein, elastin, albumin, BANA, TAEE and BAME as substrate at pH 11 or higher, or. 10, or 11.5 »or where ₉ » 5 »or 10.5 or higher 9 or? owns, o) there is no or only a very weak paptidase activity against glycylglycine, Gly-Gly-glycine, Gly-L-leucine, L-Leu-glycine, Gly-L-proline, L-Pro-glycine, Z-Gly-L-phenylalanine , ZL-GIu-L-tyrosine and Z-Gly-L-leu-amide, caseinolytic activity due to the presence is essentially not influenced by Mn ²⁺ , Co ²⁺ , Ca ²⁺ , Mg ^{2+ and EDTA,} q) the caseinolytic activity remains practically unaffected by the presence of Trasylol ^ - ^ and r) the caseinolytic activity is almost completely inhibited by the presence of PNSF. 2.) Process for the preparation of a hydrolytic enzyme mixture according to claim I _1, characterized in that one 1 0 9 8 1 6/1818 ORiQiNAL IWSPEGTfD 2G3? the microorganism under aerobic, submerged conditions Cephalosporium ATGC 11 550 grows in a nutrient medium and the hydrolytic product produced from the • Culture wins. 3.) Method according to claim 2, characterized in that a nutrient medium is used which contains assimilable carbon and contains nitrogen sources and inorganic salts. k.) Process according to Claim 2 and 3, characterized in that the fermentation is started at a pH of 4.5 to 915. 5.) Process according to claim k, characterized in that the fermentation starts with a pH of 6 to 7. 6.) Method according to claim 2 to 5 »characterized in that that cultivation is carried out at a temperature of 20 to 35 ° C. 7.) The method according to claim 6, characterized in that the cultivation at a temperature of 26 to 28 C. carries out ο 8.) Method according to claim 2 to 7 ». characterized, that the mash obtained in the cultivation is filtered 1 0 9 8.1 B / 1 Π Τ 8 ORIGINAL INSPECTED ~ ⁵⁶ ~ 2 03 " and from the resulting solution the hydrolytic enzymes wins through precipitation " 9.) The method according to claim 8, characterized in that one precipitates with tannin. 10.) The method according to claim 8, characterized in that one with an organic solvent, with lignin or precipitates with an inorganic salt. 11.) Process according to claim 1O _9, characterized in that acetone or isopropyl alcohol is used as the organic solvent. 12.) Method according to claim 2 to 7 «characterized in that that the mash is filtered and the resulting solution is subjected to spray drying » ^ 13 ·) Method according to claim 12, characterized in that the solution is evaporated before spray drying » l4.) Use of a hydrolytic Ensymgamisches according to claim 1 as an additive to a cleaning agent! · 15) Use according to claim lh in a Zusatsnieng © of 0.01 to 5j preferably 0.1 to 2 wt _e -jt hydrolytic enzyme mixture · 10 9 B 1 Π / 1 Γ 1 8 ORIGINAL INSPECTED 16,) Use of substantially thermostable hydrelytic enzymes according to claim Ik and 15 ο 1098 16/1818 Blank page