DE2021909A1 - Proteolytic enzyme mixture production - Google Patents

Abstract

Heat stable hydrolytic (primary proteolytic) enzyme mixtures are made by culturing Streptomyces globisporus NRRL 3762 under submerged aerobic conditions at pH 6.0 - 7.0 (6.8) and 26-30 degrees C, heating the fermentation liquor for ca. 2 h. at 55-75 degrees C, and pptg. with org. liquid (acetone) or salting-out a thermostable proteolytic fraction, which is then purified by gel filtration and isoelectric separation. The product is used in detergents.

Description

Process for preparing a mixture of enzymes The present invention concerns new and valuable hydrolytic enzymes and processes for their production, Isolation and purification. In particular, the invention is concerned with proteolytic ones Enzymes produced by growing a newly discovered strain of Streptomyces globisporus are available, and with methods for obtaining the desired products by Breeding this organism. The microorganism is referred to here as Astrn-Stam 19.

The invention also relates to the use of the proteolytic enzymes in various fields of technology. Examples of such applications are Use of the products according to the invention as ingredients in detergents that use as Means for cell lysis and the use of the products for solubilizing biopolymers.

The special areas of technology mentioned above on which the enzymes May be useful in accordance with the present invention are illustrative only Examples of possible uses are given, and of course the very valuable enzymes with unexpected properties, which are detailed below are still to be described, are also used in other technological areas, all of which are covered by the invention.

That in the preparation of the enzymes according to the present invention The microorganism used was isolated from a soil sample collected in Södertälje was adopted in Sweden. The classification features of the organism were according to Bergey's Manual of Determinative Bacteriology, 7th Edition, Baltimore, Williams & Wilkins, 1957, and Selman A. Waksman's The Actinomycetes, Volume II, Baltimore, Williams & Wilkins, 1961, determined and considered characteristic of the genus Streptomyces globisporus found.

A study of the literature revealed that no strains of Streptomyces globisporus are known to be satisfactory with the ones listed below Properties coincide, and thus the organism is that of the present Invention is used to be viewed as a new strain.

The classification requirements for the microorganism were made according to those in Bergey's Manual of Determinative Baoteriology, 7th Edition, Baltimore, Williams & Wilkins, 1957, and Selman A, Waksman, rnhe Actinomycetes, Volume II, Paltimore, Wiliams & Wilkins, 1961, carried out the methods described. Received get the following results: Vegetative growth: flat, colorless colonies. It No pigment or a slightly yellow diffusible pigment is formed.

Well developed, airborne Mycell, especially on synthetic Agar, such as starch KNO3 agar. The airborne mycell is powdery, white, sometimes Slightly yellowish with a gray tinge when the spores are formed.

The spore bearers are just round broom-like.

The spores are spherical and have a diameter of - 1.0 micron, Milk is quickly peptonized and gives a slight a] -calic reaction.

gelatine is slowly liquefied.

Starch is hydrolyzed.

The growth is acceptable on cellulose.

Growth is good on cane sugar and on glucose.

Antagonistic properties were observed.

to the potatoes there is a trace of what is living in the air Iycell at 280, but at 220 there is copious sporulation. With all examined At high temperatures, the growth turns brown, but no soluble pigments were observed.

The microorganism Astra strain 19 was deposited with ARS Culture Collection Investigations Fermentation Laboratory, US Department of Agriculture, Northern Utilization Research and Development Division, 1815 North University Street, Peoria, Ill. 61604, USA, deposited under number NRRL 3762.

When the microorganism Astra strain 19 is grown in organic media was found that he is in the days of various hydrolytic enzymes, such as proteases, amylases and lipases. Some of these enzymes are very open useful in various areas of technology be. So can certain of the enzymes used as ingredients in detergents, certain Enzymes are used as a coat for cell lysis and certain enzymes for solubilization. from Bioyiolymer suitable. The enzyme preparation can also be used for food treatment can be used, for example to modify gluten in baking processes. Of particular interest are some proteases produced by the organism in question are produced and are stable at elevated temperatures and so especially as Components in detergents are suitable. The stability and activity of this Proteases seem independent of the concentration of calcium ions, Ca2 +, and others Ions in a solution containing the proteases, and it is clear that such Properties particularly advantageous for the stated use of the proteases as additives to detergents. Another property of proteases that particularly valuable for the use considered here as additives to detergents its pH optimum is quite high.

In a wide execution of the process that sur making the Enzymes according to the present invention can be used becomes the one in question Organism under aerobic, submerged conditions in a suitable Grown nutrient medium. It is preferred that the nutrient medium be an assimilable carbon source, Contains nitrogen source as well as inorganic salts. The growing conditions, such as Time, temperature and hydrogen ion concentration can vary widely without abandoning the inventive idea. At the end of the growing season know the enzymes or the material of interest from the mash one after the other obtained by methods such as centrifuging the mash, dialyzing the solution or spray drying the resulting solution.

A wide variety of substances can be used as a source of carbon in the Nutrient medium can be used. These substances can be either water-soluble or water-insoluble be, it is only desirable that the compounds used from the organism can be easily assimilated, as examples are the pentoses, such as arabinose, Ribose and xylose, the monosaccharides, like mannose, levulose and galactose, the Disaccharides, such as trehalose, maltose, lactose, cellobiose and sucrose, the polysaccharides, like starch, the higher alcohols like glycerin, mannitol, sorbitol and inositol, as well mixed sources called ethyl alcohol and calcium carbonate. nitrogen in an assimilable form can by animal or vegetable proteins, soybean meal, Casein, peptones, polypeptides or amino acids, are supplied to the classification the pH can be done with any suitable base, such as sodium hydroxide, Phosphate buffer, sodium citrate, sodium acetate or any other suitable Material that brings the pH into the desired range.

The temperature of the culture medium during the cultivation of the fungus can are expediently kept between about 26 and 300 ° C.

The enzymes that are necessary for the production of a maximum amount of proteolytic enzymes time required can be varied widely, depending on the nature of the particular one used Components in the nutrient medium. A time of about 70 to about 160 hours will be however, usually considered appropriate.

Antifoam agents can also be added to the culture medium. Examples are soybean oil, castor oil, sulfonated oils, lard oil and brominated Castordl.

As stated above, the new tribe of Streptomyces globisporus can be grown in a culture medium to be hydrolytic To produce enzymes.

The culture medium can be any of the number of known media, since the organism in question is able to assimilate many sources of energy. To a poisonous production and an economical and maximum yield a enzymes however, certain culture media are preferred. For example, are the currently preferred sources of carbohydrates in the culture medium sucrose, starch, Glucose, dextrin, molasses and the like. The preferred sources of nitrogen are soybean meal, Brewer's yeast, cereal soaking liquid, casein, fish meal, Scotaferm # from Distillers, Pharmamedia # et al. The organic nutrient salts incorporated into the medium are, for example, salts that -ammonium-, sodium-, potassium-, calcium-, Magnesium, phosphate, chloride, sulfate, nitrate and similar ions can result. Essential trace elements can also be added to the culture medium. Such However, trace elements are usually present as impurities in other components of the culture medium included.

For maximum growth and production of Astra strain 19 should be the culture medium before inoculation to a pH between about 6.0 and about 7.5, preferably to pH 6.8 pH at the end of the growing season depends on the buffer substances present and the initial pH.

Never speed production of proteases in the culture medium is easily followed by taking samples during the growth period of the organism of the culture medium for their caseinolytic activity. The caseinase regulations are described in detail below.

The crude product that is obtained after the isolation of the cultivation from Astra strain 19 received enzyme preparation. is characterized by that it is a) a mixture of proteins, b) in water, salt solutions and usual Buffer solutions is soluble, c) at room temperature for more than a year in a dry place Powder form is stable and retains its enzymatic activity; d) its ultramriolet absorption spectrum is characteristic of proteins containing aromatic amino acids, e) it is proteolytically active against casein, hemoglobin and gelatin, f) has a proteolytic optimum with casein as substrate at pH 9-10, g) in the Is able to hydrolyze N-x-tosyl-1-arginine ethyl ester (TAEE), h) by heating contains initiated proteolytic activity, i) in addition to proteolytic enzymes also contains esterases, lipases and amylases, j) the heat-resistant enzyme or the heat-resistant enzymes can be precipitated with organic solvents or ammonium sulfate are, k) its stability and activity essentially independent of the concentration which is calcium ions in a solution at an elevated temperature, 1) the mixture Temperature optimum with casein as substrate at around 600 C at pH 7.4 and a temperature optimum at about 50 ° a at pH 9.0 and m) the mixture has a temperature optimum with TAEE possesses as a substrate in the region of 50 ° C at pH 7.4.

The terms "crude product", "first acetone precipitate" and "second Acetone precipitate "that appear here in the description denote the various Products, dia one in the isolation and purification of the Cultivation of Astra strain 19 obtained enzyme preparations and are in the Way defined as they are described in Example 8 '.

The present invention is accomplished with the aid of the following tables Drawings further explained.

Table I shows the pH optimum of the protease activity a) in the crude product, obtained during the isolation of the enzyme preparation, b) in the first acetone precipitate and c) in the second acetone precipitate. The measurements of the proteolytic activity were done at 370 C using aein as a substrate and at 40 ° C using of N-α-tosyl-1-arginine ethyl ester (TAEE).

Table I pH Optimum Proteolytic Activity Percent of Maximum Proteolytic Activity Casein as a substrate TAEE as a substrate pH crude product First acetone- second acetone crude product precipitation precipitation 4.0 4 5.0 5 6.0 48 44 36 15 7.0 61 55 64 45 8.0 81 78 76 92 8.5 95 9.0 100 97 95 100 10.0 89 100 100 55 10.5 42 11.0 31 69 47 Table II shows the heat stability at pH 7.4 and pH 9.0 a) of the crude product obtained on isolation of the enzyme preparation, b) of the first acetone precipitate and c) of the second acetone precipitate.

The samples were given without substrate at the given pH and the stated pH Incubated temperature for 30 minutes.

The determination of the proteolytic activity was then carried out in a Temperature of 370 C using casein as substrate.

Table II Heat Stability Percentage of Maximum Activity Temperature pH 7.4 pH 9.0 ° C crude product first acetone- tube product ester acetone- second acetone- precipitation precipitation precipitation 20 100 100 100 98 100 37 94 99 73 100 76 50 87 95 13 87 52 60 20 82 10 66 50 70 15 78 6 57 45 80 13 77 2 3 31 90 2 2 5 100 3 0 2 Table III shows the temperature optimum at pH 7.4 pH 9.0 a! of the crude product obtained during the isolation of the enzyme preparation, b) the first acetone precipitate and c) the second acetone precipitate.

Casein was used as a substrate in the activity measurements at pl 7.4 and 9.0 used. The time for the activity determination was 15 minutes. TAEE was used as a substrate at pH 7.4.

Table III Optimum temperature for proteolytic activity Percentage of maximum activity Casein as substrate pH 7.4 pH 9.0 Tempera- crude product 1. Acton- 2. Acetone- crude product 1. Acetone- 2. Acetone- temperature ° C precipitation precipitation precipitation precipitation 20 29 14 6 35 8 10 37 28 15 45 46 25 50 40 50 76 51 100 100 66 100 60 100 77 96 70 76 52 70 80 93 70 77 87 57 80 84 100 72 79 100 55 90 30 53 56 27 35 15 100 10 14 14 7 9 0 Table III (continued) TAEE as a substrate pH 7.4 Temperature of the crude product First acetone- Second acetone ° C ice precipitation 20 39 53 48 37 40 49 69 77 50 100 92 - 100 - 60 39 100 58 - 70 O 29 0 80 - 90 100 Table IV shows the relationship between the proteolytic activity and the time and temperature for the incubation of crude product obtained in isolating the enzyme preparation. The proteolytic activity is measured as the light absorption at 280 nm, which is proportional to the proteolytic activity. The experiment was carried out by incubating the test sample (10 mg per ml) for the specified number of minutes, after which the proteolytic activity was measured on an equal proportion of the incubation mixture with casein as substrate at pH 7.4.

Table IV Relationship between heat stability, incubation time and temperature Optical density of material soluble in trichloroacetic acid Temperature (° C) 50 ° C 60 ° C 65 ° C 70 ° C 65 ° C incubation period (Minutes) 0 1.503 1.364 1.456 1.389 1.318 5 1.540 1.236 1.299 1.042 0.612 10 1.503 1.191 0.075 0.708 0.242 15 1.495 1.193 0.930 0.504 0.217 17 1.474 1.102 0.885 0.443 0.212 20 1.530 1.103 0.833 0.397 0.197 22 1.513 1.060 0.871 0.353 0.165 25 1.540 1.110 0.838 0.320 0.173 27 1.517 1.059 0.838 0.299 0.172 30 1.613 1.100 0.802 0.314 0.181 32 1.478 1.063 0.743 0.297 0.181 35 1.523 1.064 0.698 0.255 0.180 37 1.490 1.038 0.662 0.240 0.164 40 1.538 1.039 0.636 0.240 0.164 Table IV (continued) Temperature 50 ° C 60 ° C 65 ° C 70 ° C 75 ° C incubation period (Minutes) 42 - - 0.574 0.210 0.136 45 1.548 1.033 0.581 0.234 0.184 47 - - 0.529 0.218 0.133 50 1.520 0.996 0.539 0.196 0.151 52 - - 0.501 0.202 0.149 55 1.468 1.003 0.499 0.195 0.125 60 1.525 - - - - Table V shows the relationship between proteolytic activity, time and pH at 650 ° C. for the crude product. The tester used for the incubation was Teorell-Stenhagen universal buffer. The test was carried out with incubation of the test sample (10 mg / ml) for the specified number of minutes at the specified pH, whereupon the proteolytic activity at pH 7.4 was measured as light absorption at 280 nm for an equal proportion of the incubation mixture with casein as substrate which is proportional to the proteolytic activity.

Table V Relationship between proteolytic activity, incubation time and pH at 65 ° C. for the crude product optical density of material soluble in trichloroacetic acid pH pH 4 pH 5 pH 6 pH 7 pH 8 pH 9 pH 10 Incubation time (minutes) 0 0.471 0.501 0.526 0.540 0.525 0.201 0.180 5 0.127 0.546 0.462 0.450 0.136 0.094 0.069 10 0.078 0.405 0.418 0.338 0.112 0.097 0.072 15 0.071 --- 0.370 0.262 0.115 0.91 0.062 17 0.068 0.342 0.350 0.265 0.092 0.064 0.062 20 0.056 0.312 0.334 0.219 0.084 0.075 0.074 22 0.058 0.301 0.303 0.194 0.078 0.066 0.063 25 0.050 0.278 0.282 0.182 0.067 0.082 0.054 27 0.043 0.274 0.277 0.166 0.082 0.075 0.058 30 0.035 0.270 0.252 0.134 0.093 0.076 0.052 Table V (continued) pH pH 4 pH 5 pH 6 pH 7 pH 8 pH 9 pH 10 Incubation time (minutes) 32 0.051 0.257 0.241 0.144 0.115 0.078 0.044 35 0.025 0.242 0.231 0.158 0.105 0.083 0.026 37 0.022 0.230 0.218 0.117 0.105 0.078 0.034 40 0.039 0.219 0.215 0.105 0.101 0.086 0.043 42 0.048 0.224 0.210 0.113 0.092 0.077 0.032 45 0.034 0.209 0.188 0.116 0.091 0.084 0.043 47 0.034 0.200 0.180 0.106 0.086 0.072 0.037 50 0.027 0.212 0.171 0.104 0.089 0.065 0.040 52 0.030 0.209 0.142 0.089 0.084 0.075 0.031 55 0.033 0.216 0.142 0.081 0.070 0.054 0.040 Table VI shows the pH stability of the crude product and the first acetone precipitate, further purified by ion exchange chromatography on DEAE cellulose (see Table IX) using 0.01 M CH3COONH4, pH 7.2, as eluent. The enzyme preparations were 30 minutes incubated at 370 a in Teorell-Stenhagen buffer which had been adjusted to the desired pH. After incubation, the samples were mixed with phosphate buffer of pH 7.4 and the remaining activity was determined by casein digestion.

Table I 'pH stability of crude product and first exposure to acetone Percent of maximum activity pH crude product First acetone precipitate out DEAE cellulose eluted 2 4 99 3 4 97 4 37 90 5 70 92 6 97 93 7 100 100 8 93 96 9 90 95 10 50 95 11 14 86 Table VII shows the temperature optimum of the thermostable fractions 16, 18 and the non-thermostable fraction 31, which can be found in the iso-electric separation (see Sect.

Fig. 2 and 3) receives. It was used as a substrate for casein at pH 7.4 the activity regulations used.

Table VII Optimum temperature of fractions obtained by isoelectric separation Percent of maximum activity Temperature fraction 16 fraction 18 fraction 31 ° C in Fig. 3 in Fig. 3 in Fig. 2 37 6 5 5 50 12-13 13 60 47 37 44 70 70 70 100 80 100 100 3S 90 37 31 100 23 27 Table VIII shows the pH optimum of the thermostable fractions 16, 18 and the non-thermostable fraction 31, which were obtained in the isoelectric separation (see FIGS. And 3). Casein at pH was used as the substrate for the activity determinations.

Table aZIII pH optimum of the fractions obtained by isoelectric separation Percent of maximum activity PH parliamentary group 16 parliamentary group 18 parliamentary group 31 in Fig. 3 in Fig. 3 in Fig. 2 6 58 7 32 95 67 8 45 92 74 9 100 100 80 10 54 '83 - 86 -11 58 76 100 1a shows the results of a gel filtration of 300 mg of the product obtained during the isolation of the enzyme preparation using Sephadex # G-50, 2 × 112 cm. Elution was carried out with H2O at a rate of two fractions of 4.5 ml per hour.

Fig. 1b shows the results of a gel filtration of 500 mg of the first Aeetone precipitate from heat-treated raw product.

From FIGS. La and ib it can be seen that this is achieved by gel filtration material obtained from the first acetone precipitate contains proteolytic material, that is not contained in the raw material. This shows that the heating of the raw material apart from a denaturation of the proteolytic enzymes and other proteins, the formation initiates proteolytic activity with other properties. The results according to Tables IV and V show that the heating of the crude product to 65 to 70 0C at pE 7 - 8 its proteolytic activity during certain time intervals Slightly increased compared to a shorter period of heating the crude enzyme solution.

Purification of the heat-stable product through a column filled with 50 g DEAE cellulose, which is adjusted to pH 7.4 with 0.01 molar ammonium acetate was filled, 2.7 g of the first acetone precipitate were dissolved in 35 ml Buffer, filtered. Fractions of 10 ml were collected. If not a UV absorbing one Material and no more material with proteolytic activity was eluted the low ionic strength increased to 0.1 molar ammonium acetate, and then became some UV absorbing and proteolytic material still eluted. After that it was the pH value is reduced to pH 5.5 when only very little UV-absorbing d proteolytic material was eluted. The results are shown in Table IX.

Table IX Fraction weight 37 ° C, pH 7.4 after 30 minutes Casein heating to 70 ° C Units / mg remaining activity vity Source material, 1.Acetone precipitate 2700 mg 0.3 65% Eluted with 0.01 m ammonium acetate, pH 7.4, 1st part 430 mg 0.4 90% Eluted with 0.01 m ammonium acetate, pH 7.4, 2nd part 135 mg 0.6 85% Eluted with 0.1 M ammonium acetate, pH 7.4 320 mg 0.05 50% Eluted with 0.1 M ammonium acetate, pH 5.5 <20 mg 77% The crude product and the first part of the material, which had been eluted from DEAE-oilulose at pH 7.4 and with 0.01 molar ammonium acetate, was examined by isoelectric separations with a pH-ST gradient of 3-10.

The results are shown in Figs.

The crude product, Fig. 2, contains various proteolytically active Enzymes. You can see at least 7 enzymes that are active against casein at pH 7. Three of these are dominant. One has an isoelectric point at 5, -das others at 5.5 and the third at pH 10-11. That migrating to a pH between 4 and 6 Material contains thermostable enzymes.

The material is chromatographically purified by ion exchange by essentially two thermostable enzymes with isoelectric points of 5.2 and 5.7 (Fig. 3). The pE optimum and the temperature optimum of these two Enzyme preparation fractions 16 and 18 (Fig. 3) and the thermolabile fraction 31 (Fig. 2) were determined in Tables VII and 5TIII.

From Fig. 2 and 3 it can be seen that at 700 C there is practically no degradation of the thermally stable material takes place, but that the whole activity of the thermolabile enzyme disappears under the same conditions.

The influence of cobalt ions (Co2 +) on protecytic activity in different fractions, which are obtained from isoelectric treatment of the crude product obtained, and the enzymatic activity of such fractions on various synthetic Substances were studied.

The fractions from the isoelectric separations were kept overnight dialyzed. Appropriate narrow fractions of the dialyzed fractions were used to determine the Activity against benzoyl-d, l-arginine-naphthylamide (BATA), carbobenzoxy-glycine-l-leucylamide, Carbobenzoxy-l-glutamyl-ltyrosine, carbobenzoxy-l-leucyl-p-nitrophenyl ester, carbobenzoxy-glycine-l-phenylalanine, Determine l-leucine-glycine and casein at pH 7.4 and pH 10.0.

The measurements were essentially when EiNA was used as the substrate those which Reidel and Wünsch-Seit in Phys. Chem. 316, page 61 (1959) and by Blackwood and Mandl in Anal. Biochem. 2, page 370 (1961).

The measurements were made using l-leu-gly, Z-gly-l-phe, Z-glu-l-tyr, Z-gly-l-leu-amide as substrate with ninhydrin reaction of the incubates after 3 minutes Incubation at 370 C of the enzyme solution and the substrate in question are determined. as Blank was used enzyme plus ninhydrin solution plus substrate.

The measurements were made using Z-leu-pnitro-phenyl ester carried out so that the optical density at 405 nm of the incubate of the enzyme and dos esters was measured for 15 minutes. 2+ solutions were used as blanks used without enzyme. The concentration of Co was 0.1 molar; 0.1 ml of this solution was added to the enzyme solution. This mixture was allowed to stand for 10 minutes before the substrate was added. The results are shown in Table X below.

Table X Activity with and without Co2 + of fractions obtained by isoelectric separation of the crude product (FIG. 2) Casein fraction Casein Bz-dl-arg-Z-gly-1-leu-NH2 Z-1-glu-1-tyr-OH Tube No. pH 7.4 pH 10.0 naphth-NH2 + CO2 + + CO2 + 7 - 9 (I) 30 12 82 0 100 0 7 12-13 (II) 195 44 12 325 1560 80 183 15-16 (III) 308 275 6 465 2340 25 58 18-19 (IV) 393 245 4 530 1990 635 1090 24 - 26 (V) 52 29 6 0 225 17 2 28 - 29 (VI) 61 55 14 0 40 0 10 30 - 32 (VII) 154 122 0 0 57 0 12 Table X (continued) Fraction Zl-leu-nitro-phenol Z-gly-1-phe-OH 1-leu-gly-OH Tube No. + Co2 + + Co2 + + Co2 + 7-9 (I) 14 10 0 0 17 60 12-13 (II) 72 90 7 45 0 0 15-16 (III) 15 75 5 20 0 1350 18-19 (IV) 115 115 105 283 70 650 24 - 26 (V) 27 28 0 10 5 10 28-29 (VI) 120 135 0 0 0 80 30-32 (VII) 380 325 10 10 320 455 From Table X it can be seen that the activity against casein at pH 7.4 was the highest in fractions IV and III, but that the activity was present in all fractions. The activity against casein at pH 10.0 was the highest in fraction III and was also highest in fraction IV. The activity against benzoyl-dl-arginine-naphthylamide, which is only present in the crude product, was found in fraction I. The Activity towards Zl-leucyl-p-nitrophenyl ester, which is not influenced by CO2 + but is inhibited by FMSF (phenylmethylsulfonyl fluoride), is present in all fractions, but was highest in fraction VII. The activity against l-leucine-glycine, which strongly requires Co2 + or e (divalent metal ions) and is enhanced by Co2 +, is highest in fraction III. The activity in fraction VII is not so strongly influenced by Co2 +. The activity towards Z-gly-1-phenylalanine, which is strongly influenced by Co2 +, is highest in fraction IV. This activity is only present in the crude product. The activity towards Zl-glu-l-tyrosine, which Me2 + requires and is somewhat enhanced by Co 2+, is present in fraction IV. The neutral proteolytic activity, such as the activity against Z-gly-l-leu-NH2, which is very strongly enhanced by Co2 +, is about the same as in fractions II, III and IV, but highest in fraction II.

The different maxima that one can find in isoeic electrical separations mainly show the following activities: Fraction No. 7-9: Activity against benzoyl-d, larginine-naphthylamide fraction No. 12-13s activity against Z-gly-l-leu-NH2 fraction No. 15 - 16s activity against l-leu-gly and Z-gly-l-leu-NH2 Fraction No. 18 - 19s activity towards Z-gly-l-phenylalanine Fraction No. 30 - 32: Activity against l-leu-p-N02 The effect on caseinolytic activity by adding various metals and inhibitors, the crude product and studied the heat-stable enzyme preparations. There. Enzyme preparation, dissolved in 3 ml buffer, became with 0.1 ml of a 0.1 M solution of the metal to be examined incubated, especially with ethylenediaminetetraacetic acid (0.1 m), phenylmethylsulfonyl fluoride solution (0.5%) or 0.1 ml of a Trasylol C solution. The incubation time was 10 minutes at 37O C.

Then 3 ml of a casein solution were added, and the proteolytic Activity is determined. The results are shown in Table XI. the Activity was compared to activity in control samples with no added material. As a buffer, 0.2 M tris (hydroxymethyl) aminomethane with a pH of 7.4 and 10.0 used.

Table XI Effect of metals and inhibitors on caseinolytic activity Change in proteolytic activity (%) PH 7.4 buffer, pH 10.0 buffer Added material crude product first acetone crude product first acetone precipitation precipitation 0.03 m Mn2 + -30 -10 -80 -50 0.03 m Zn2 + -50 -20 -70 -20 0.03 m CO2 + -30 -10 -50 -50 0.03 m Ca2 + # 0 # 0 -10 -10 0.03 m Mg2 + # 0 # 0 # 0 +40 0.1 m Trasylol # -10 # 0 # 0 0.03 m EDTA -80 -20 -80 # 0 0.1 ml phenylmethyl sulphonyl fluoride (0.5%) -30 -70 -10 -10 EDTA + phenylmethyl- sulphonyl fluoride -90 -80 -80 -50 Casein proteolytic activity was determined essentially as described by Bergkvist in Acta Chem. Scand. 17 (1963), 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 value and the solution was diluted to 500 ml with water. 3 ml of casein solution were mixed in test tubes with 3 ml of Teorell-Stenhagen buffer of the desired pH (biochemical pocket book by Rouen H. x Springer Verlag (1956), page 651).

In parallel, test tubes were placed in water baths on the appropriate Temperature set. An appropriate amount of the dissolved enzyme was added to the test tubes added. From one of these test tubes, 2 ml were immediately transferred to 3 ml of a 10 show trichloroacetic acid pipetted. The other set of test tubes turned out exactly Incubated for 30 minutes, after which 2 ml were withdrawn and added to 3 ml of 10% trichloroacetic acid were added. After standing for at least 30 minutes at room temperature the test tubes were centrifuged and filtered through small funnels fitted with closed with a loose wad of cotton wool. The optical density at 280 mm of the filtered centrifugate was measured The activity of the enzyme expressed in casein units per mg, one obtains in the following way: Optical density of the incubated sample minus the optical density of the non-incubated sample by the amount of enzyme in the test tubes (expressed in mg).

When examining the stability of the enzyme, it was in buffer dissolved and without casein for a given time and at a certain time Incubated temperature. Thereafter, at a desired pH and a desired Temperature carried out the caseinase determinations.

When determining proteolytic activity using of Na-Tosyl-1-arginine ethyl ester (TAEE) als.Substrat were the following chemicals used in the final solution. TAEE in a 0.05 molar concentrate and in NaCl in a 0.9 show concentration. The amount of the enzyme solution added was 0.2 ml and the volume of the test solution 10 ml. The titration was 0.05 molar NaOH using automatic titration using a radiometer titration unit carried out by type TTA4.

Gel filtration was performed using Sephader # columns the conditions described above in connection with the figures are carried out.

The following examples serve to further illustrate the invention.

Example 1: Each of 7 rows of 500 ml Erlenmeyer flasks were made 100 ml of one of the following media was added: I sucrose 24 g Pharmamedia 20 g KH2PO4 13.5 g tap water 1000 ml II corn flour 20 g soybean flour 20 g KH2PO4 13.5 g Tap water 1000 ml III starch 30 g spray-dried bovine blood 15 g KH2P04 13.5 g tap water 1000 ml IV soybean meal 35 g starch 24 g tH2P04 13.6 g V Actiferment # 25 g sucrose 35 g KH2PO4 13.6 g VI soybean meal 24 g gelatin 10 g sucrose 20 g KH2PO4 13.6 g VII corn steep liquor 30 g aTIIT oatmeal 30 g KH2PO4 13.6 g IX soybean meal 15 g corn steep liquor 40 g KH2PO4 13.6 g x Scotaferm # 50 g molasses 40 g H2O to 1000 ml pH 5.4 before sterilization of the culture medium The flasks were treated in an autoclave at 1200 C for 20 minutes, after the pH was adjusted to 6.8. After cooling, the flasks became with an aqueous suspension of the specified in Table XII organism inoculated, which had been kept on conventional inclined ear agar plates. Incubation took place at a temperature of 26 - 300 C and was after 113 hours, or if the proteolytic activity went through a maximum, interrupted. The results are listed in Table XII.

Table XII organism activity in casein units each from one the medium of the following numbers obtained solution with a content of the thermostable Component I II III IV V VI VII VIII IX X Astra- 4.6 2.0 0.1 0.8 0.2 0.3 0.2 4.6 0.3 7.5 strain 19 Example 2: Pharmamedia # (100 g), sucrose (150 g), KH2PO4 (70 g), RD antifoam emulsion (1 ml), paraffin oil (2.6 ml) and cetanol (0.4 g) in tap water (5 1) were adjusted to pH 6.8 with sodium hydroxide (15 ml) and at 1240 during The culture medium was sterilized in a culture inoculation container for 30 minutes 100 ml of a 24 hour old o culture of Streptomyces globisporus inoculated Incubated at 28 C with aeration and stirred for 24 hours.

Pharmamedia # (1000 g), sucrose (1500 g), KH2PO4 (700 g), RD antifoam emulsion (5 ml), lard oil (40 ml), paraffin oil (6 ml) and cetanol (4 g) in tap water (50 1) were adjusted to pH 6.8 with 45% sodium hydroxide (150 ml), and the solution was sterilized in a fermentation vessel at 1240 ° C. for 30 minutes. The culture medium was cooled and then inoculated with the above-mentioned inoculum incubated at 286 ° C. with stirring and aeration. After 140 hours when the proteolytic Activity was estimated to be 6.1 casein units per ml, the culture broth was cooled.

Example 3: Pharmamedia # (100 g), sucrose (150 g), KH2PO4 (70 g), RD antifoam emulsion (1 ml), lard oil (17 ml), paraffin oil (2.6 ml) and cetanol (0.4 g) in tap water (5 1) were adjusted to pH 6.8 with sodium hydroxide (15 ml) and sterilized in an inoculation fermentation vessel at 1240 C for 30 minutes. The nutrient medium was mixed with 100 ml of a 24 hour old culture of Streptomyces globisporus inoculated and incubated at 280 C with aeration and stirring for 24 hours.

Pharmamedia # (3000 g), sucrose (4500 g), KH2PO4 (2100 g), RD antifoam emulsion (15 ml), lard oil (120 ml), paraffin oil (18 ml) and cetanol (12 g) in tap water (150 ml) were adjusted to pH 6.8 with 45% sodium hydroxide solution (about 450 ml) and Sterilized for 30 minutes at 124 ° a. The nutrient medium was cooled and with the above mentioned inoculation culture and then inoculated at 280 C with stirring and aeration incubated. After 136 hours when the proteolytic activity drops to 5.5 casein units per ml was estimated, the fermentation broth was cooled.

Example 4: The fermentation experiment was carried out according to Example 2B. After 140 hours the proteolytic activity was 5.2 casein units per ml, and the fermentation broth was cooled.

Example 5: Isolation of the Enzyme Preparation For the isolation of the Enzyme preparation from the fermentation broth that was obtained in Example 2, if the broth was separated in a centrifuge, after separating the solution was clarified in a Se.itz filter press with arches 3/1250. The clear one Solution (45 1, activity 6.0 casein units per ml) was in a thin film evaporator concentrated at 150 e to 8 l with an activity of 34 casein units per ml. the Solution was dialyzed against tap water for 24 hours and spray dried and yielded 210 g with an activity of 0.9 casein units per mg.

Example 6: For the isolation of the enzyme preparation from the fermentation broth obtained in Example 3, the broth was separated in a centrifuge, and after separation the solution was in a Seitz filter press with arches 3/1250 clarified. The clear solution (120 l), activity 4.8 casein units per ml) was in a thin film evaporator at 150 C to 17.5 1 with an activity of 38 casein units evaporated per ml. The solution was cooled, and acetone (13 L) with a temperature of -10 ° C was added with stirring. After stirring for 30 minutes, the precipitated one became Material removed by centrifugation in a Sharples centrifuge.

The clear solution was in a thin film evaporator at 150 ° C 11.8 1 with an activity of 60.5 casein units per ml evaporated.

The solution was spray dried to give 525 g of activity of 0.50 casein units per ml.

Example 7: To isolate the enzyme preparation from the fermentation broth, obtained in Example 4, the broth was separated in a centrifuge, and after the separation, the solution was gelatinized in a Seitz filter press with arches 3/1250. The clear solution (137 1, activity 5.2 casein units per ml) was in a thin film evaporator evaporated at 150 C to 16.0 1 with an activity of 4? s5 casein units per ml, The solution was gektühlt to about + 100 C, and acetone (8 1) with a temperature of -10 ° C was added with stirring. After stirring for 30 minutes, the precipitated one became Material removed by centrifugation in a Sharples centrifuge.

The clear solution was in a thin film evaporator at 10 to 150 C evaporated to 8.3 l with an activity of 81.5 casein units per ml.

After desalting on large-grain Sephadex # G 25 in a column K 100/100 (Pharmacia, Uppsala, Sweden, the volume was increased to 2.3 1 at +150 C in a thin film evaporator. The activity was 286 Caesin units each ml.

The solution was spray dried to give 289 g of activity of 1.9 casein units per mg.

Example 8: Production of the heat-stable component 25 g of a spray-dried Sample of a culture filtrate from Astra strain 19, in this description as a crude product designated, with a protease activity of 0.56 casein units per mg, were in 500 ml of water at 600 C and held at this temperature for 30 minutes. After the solution was centrifuged after cooling. The clear, top layer was mixed with 1000 ml of ice-cold acetone. The precipitation that is in the description referred to here as the first acetone precipitate, was removed by centrifugation. A further 750 ml of acetone was added to the clear upper solution, and the resulting mixture was centrifuged again. The so received Precipitation is referred to in this description as the second acetone precipitate. The precipitates were dissolved in water and freeze-dried. The ones when trying with the two acetone precipitates, a spray-dried sample of culture filtrate, referred to as Rohpro product, and incubated at 60 ° C for 30 minutes Results obtained from the crude product are shown in Table XIII.

3 Example 9: Production of the heat-resistant component The process according to Example 8, using 10 g of spray-dried sample of culture filtrate and repeated with incubation at 650 ci. The results are also in the table XIII listed.

Table XIII Substance Weight Proteolytic Total proteolytic Activity activity in Casein units Crude product example 8 25 g 0.56 CE / mg 14000 Crude product example 9 10 g 0.56 CE / mg 5600 Crude product, 30 minutes at 60 ° C in 25 g per cubed, example 8 500 ml 7.8 CE / ml 3900 Crude product, 30 minutes at 65 ° C in cubed, example 9 10 g per 1.6 CE / ml 800 First Acetone Deposits Example 8 5.6 g 0.39 CE / mg 2200 First Acetone Deposits Example 9 3.9 g 0.26 CE / mg 1000 Second Acetone Precipitation Example 8 3.2 g 0.57 CE / mg 1800 Second precipitate of acetone Example 9 1.0 g 0.15 CE / mg 150 From Table XIII it can be seen that heating the crude protease solution at 650 ° C. for 30 minutes causes a significant loss of total proteolytic activity. For the experiments reported in the table, the actual loss in activity was about 85%. The remaining activity can be precipitated by adding, for example, acetone.

The first acetone precipitate is associated with proteolytic activity a higher temperature optimum and, to a certain extent, better heat stability enriched as the second acetone precipitate and the crude protease mixture, like when looking at Tables II and III it can be seen in which the properties of the first acetone precipitate are clearly demonatrated.

From Table I it can be seen that the pE optimum when measured compared to Casein for the crude product, the first acetone precipitate and the second acetone precipitate is similar. The optimal values appear at pH 9-10.

In addition to acetone, other organic solvents such as ethanol, Propanol and other alcohols and the like, and salts such as ammonium sulfate and the like Precipitation of the heat-stable enzyme preparation can be used Heating period for the aqueous solution of the enzyme mixture can be up to about two hours at a temperature of about 55-750 C. Through this heating process a proteolytic activity is induced, which in the original enzyme preparation does not exist.

Compatibility of the enzyme preparation with detergents Three commercially available Detergent compositions were used. One contained perborates and about 40 bs.

50 4 (weight / weight phosphate, one contained about 7 t ((- weight / weight) Phosphate and no perborate, and one contained soap at about 5 levels % (W / w) phosphate. The pH optimum of the proteolytic activity that pH stability of the proteolytic activity, the temperature optimum of the proteolytic The activity and heat stability of the crude enzyme preparation product were determined.

A) pH optimum of the caseinolytic activity of the crude enzyme preparation product in combination with detergents The following solutions were made: One solution containing £ 5 detergent composition in 100 ml water (detergent solution). A solution with a content of 5 mg enzyme preparation raw product per ml of water (enzyme solution). A 3% casein solution.

The pH was determined using Teorell-Stenhagen buffer solution set.

Two ml of buffer solution of the desired pH value and 1 ml of detergent solution 3 ml of casein solution were mixed together. The resulting solution was increased to 7 ° (t adjusted, after which 0.1 ml of the enzyme solution was added and the caseinolytic activity was measured, For comparison, a blank solution was tested, which is no detergent contained. The results are shown in Table XIV.

Table XIV Caseinolytic activity of the crude enzyme preparation product in omhination with detergents pH Caseinolytic activity in% of maximum activity ity of the detergent composition with a Phosphate content of 40 - 50 4 7% 5% blank solution 6.0 70 58 55 38 6.5 82 60 7.0 82 74 91 61 7.5 88 90 8.0 92 90 95 82 8.5 100 93 9.0 92 100 99 100 9.5 92 100 10.0 80 45 85 89 10.5 45 55 11.0 9 31 B) pH stability of the enzyme preparation raw product in combination with detergents Identical solutions as in test A) were used.

and 2 ml of buffer solution and 1 ml of detergent solution were mixed with each other mixed. Thereafter, 0.1 ml of enzyme solution was added, and the solution thus obtained was incubated for 30 minutes at 370 C, after which the oaseinolytic activity at pH 7.4 and 370 C was measured. The results are shown in Table XV.

Table XV Influence of pH on the stability of the crude enzyme preparation product in combination with detergent compositions pH Caseinolytic activity in% of the maximum activity vity of the detergent composition with one Phosphate content of 40 - 50% 7% 5% blank solution 2.0 5 6 7 4 3.0 9 12 4 4.0 14 54 91 37 5.0 87 65 70 6.0 100 99 100 97 7.0 60 85 91 100 8.0 33 100 42 93 9.0 30 63 25 90 10.0 27 28 20 50 11.0 27 23 18 14 C) Temperature optimization of the caseinolytic activity of the enzyme preparation raw product in combination with detergents. The detergent solution and casein solution used were the same as those under A). A crude product enzyme solution containing 3 mg of crude enzyme prenar product was used. The pH was adjusted to 7.4 using 0.2 M phosphate buffer and to pI q; O using Teorell-Stenhagen buffer.

9 ml of buffer solution and 1 ml of dispersant solution were mixed with 3 ml of casein solution mixed and heated to the desired temperature in a water bath for three minutes. Then 0.1 ml of enzyme solution was added and the caseinolytic activity was determined. The results are shown in Table XVI.

Table XVI Temperature optimum of the caseinolytic activity of the crude product preparation in combination with detergents Temperature Caseinolytic activity in% of the maximum activity of the detergent ° C agent composition with a phosphate content of 40 - 50% 7% 5% blank solution pH 7.4 pH 9.0 pH 7.4 pH 9.0 pH 7.4 pH 9.0 pH 7.4 pH 9.0 20 18 28 20 54 36 30 29 35 37 39 58 47 86 45 76 28 45 50 70 96 71 95 52 50 77 100 60 100 87 100 100 87 82 100 70 70 87 100 71 93 100 100 80 77 80 82 85 78 65 99 85 78 D) Heat stability of the crude enzyme preparation product in combination with detergents The same solutions as in test C were used. Two ml of buffer solution and 1 ml of detergent solution are mixed with 0.1 ml of enzyme solution, and the resulting solution was incubated for 30 minutes at the desired temperature, after which the caseinolytic activity was determined at 37 ° C. The results are shown in Table XVII.

Table XVII Heat stability of the crude enzyme preparation in combination with detergent compositions Temperature Caseinolytic activity in% of the maximum activity of the ° C composition with a phosphate content of 40 - 50% 7% 5% blank solution pH 7.4 pH 9.0 pH 7.4 pH 9.0 pH 7.4 pH 9.0 pH 7.4 pH 9.0 20 100 100 100 100 100 100 100 100 37 36 71 99 71 84 55 95 74 50 25 34 70 35 47 54 87 13 60 9 2 31 21 15 22 20 10 70 3 2 15 10 4 7 15 7 80 2 0 4 0 2 0 13 2 E) The relative caseinolytic activity of the enzyme preparation was measured in combination with the detergent compositions in comparison with the caseinolytic activity obtained without the addition of detergent compositions. The results are shown in Table XVIII.

Table XVIII Relative caseinolytic activity of the crude enzyme preparation Detergent type Relative caseinolytic activity Phosphate content 40 - 50% 89 Perborate detergents Phosphate content 7% 107 no detergent 5 o "o (soap)" - 130 no detergent 100 The test results in Tables XIV-XVIIT show that the enzyme preparation according to the present invention is compatible with detergents with high (40-50 weight / weight) as well as with low (5-10% weight / weight) phosphate contents and that the detergents are also compatible Perborates may contain as optical brightening agents. The results also show that the enzyme preparation is compatible with soap detergents. The heat stability appears to be somewhat affected by a high phosphate or perberate content, but as can be seen from Table XVI, the temperature optimum is essentially unaffected by the presence of detergent compositions.

Claims (11)

Claims 1. Process for the production of a hydrolytic enzyme mixture, characterized in that the microorganism Streptomyces globisporus NRRL 3762 grows in a culture medium under submerged aerobic conditions. 2. The method according to claim 1, characterized in that one is a Uses nutrient medium that contains assimilable carbon sources, nitrogen sources and contains inorganic salts. 3. The method according to claim 1 and 2, characterized in that one culture begins at pH 6.0 - 7.0. 4. The method according to claim 3, characterized in that the Culture begins at pH 6.8. 5. The method according to claim 1-4, characterized in that the Culture at 26-30 ° C. 6. The method according to claim 1-5 for obtaining a proteolytic Enzyme fraction with high thermal stability, characterized in that one the resulting aqueous solution of the enzyme mixture up to about two hours to about 55 to tio C heated and the thermally stable proteolytic fractionating agent or one precipitates by adding an organic solvent or a salt. 7. The method according to claim 1-6 for obtaining a proteolytic Enzyme fraction with high thermal stability, characterized in that one the enzyme mixture obtained using a modified dextran with a Fractionation range for peptides and spherical proteins of molecular weights subjected to gel filtration in the range of 1500-30,000 and the modified Dextran eluted with water. 8. The method according to claim 6, characterized in that the The solution obtained of the enzyme fraction is treated with diethylaminoethyl cellulose and the diethylaminoethyl cellulose eluted with ammonium acetate at pH 7.4. 9. The method according to claim 1-5 for obtaining a proteolytic Enzyme fraction with high thermal stability, characterized in that one the enzyme mixture obtained is subjected to an isoelectric separation and dE at pH 4-6 eluted fraction wins. 10. The method according to claim 8, characterized in that the The enzyme mixture obtained is subjected to an isoelectric separation and the pH 5.2 and / or fraction eluted at pH 5.7 wins. 11. Use of a hydrolytic prepared according to claims 1-10 Enzyme mixture as an additive to detergents.