SE409725B - DETERGENT AND WASHING COMPOSITION CONTAINING PROTEOLYTIC ENZYMES WITH HIGH PROTEOLYTIC ACTIVITY IN THE HIGH PHO-WORLD WHICH NORMALLY OCCURS IN A WASHING SOLUTION - Google Patents

Description

7 2066 504: During their metabolism, a large number of hitherto unknown bacteria form proteolytic enzymes, which show optimal proteolytic activity against hemoglobin at high pH values up to 10-12, and which have other properties that make them extremely well suited as ingredients in detergent and detergent compositions.

From samples of soil, animal manure and a number of other sources in nature, the inventors have isolated about 100 strains of bacteria, carried out systematization studies and found that all the hitherto unknown bacteria belong to the genus Bacillus but that none of them belonged to any known to the inventors. species, and that as far as known by the inventors they do not belong to the same species. In addition, within the same species, in most cases there were different strains and several varieties.

In order to isolate the above-mentioned, hitherto unknown bacteria, a new technology has been used. Samples of soil, manure or other natural sources have been spread on nutrient media with a high pH value (9-1) and the bacteria that could grow under such alkaline conditions have then been isolated and subjected to further studies regarding species and enzyme formation.

In most cases, a number of different enrichment methods have also been used.

Enrichment methods are known in the art. refer to Hayaishi, Methods in Enzymology, Vol. l, l26-131. is to allow a sample from nature to grow in a substrate, which has a special and selected composition, which promotes the growth of a microorganism, which produces metabolic products which have sought Another principle is to store the sample from nature to- In this context principle properties. together with a compound, such as an inorganic salt, which promotes the development of the desired microorganism, cf. M.A. El-Nakeeb and H.A. Leche- valier, Appl. Microbiol., Vol. 11, 75 (1963), and then spreading the sample on a suitable substrate adjusted to a pH in the range 8-12.

Some of the hitherto unknown members of the genus Bacillus, which have been systematically isolated and tested for the production of proteolytic enzymes, are summarized in Table I below, in which the first column contains the inventors' reference number, the second column the number under which the bacterium was deposited. at The National Collection of Industrial Bacteria, Torry Research Station, Aberdeen, Scotland, the third column the source of isolation and the fourth column the enrichment procedure used. 10 15 20 25 30 35 40 kr! 7116685-4 Table_l Ref. No. Isolation no. NCIB source Enrichment procedure 0 300 10144 Soil from cemetery in Starch casein medium (pH Copenhagen increased gradually from 10-12) C 301 10145 Soil from cemetery i) Spread of soil sample Copenhagen g on agar with) sesquicarbonate ß - PH 9 6 -9 8 G 302 10146 Éårânšââ fi f1 ° dstrand 1 g Test of hydrolysis zones P on neutral agar with foam- C 303 10147 Pile of soil and leaves from) milk. cemetery in Copenhagen g, 0 304 10148 Wood sand from B1okhus, Jutland C 311 10281 Wooden soil from Ascheberg, Perboratagar Holstein C 323 10282 Field soil from the city of Denmark Perboratagar C 325 10284 Infection on plate with Alkaline skim milk gags with perborate holes Riverside soil from the Danish city Perboratagar 0 335 10287 Parkland from the Danish city Perboratagar G 336 10288 Horse and elephant manure Perboratagar C 339 10291 Riverside soil from the Danish city Perboratagar city C 341 10293 Field soil from the Danish city Perboratagar C 351 1030ik 5211 pH s, ß-9.7> C 552 10302 StPUtSgÖdSêl från ZOO Natïgiuypsgskvikarbgnat- C 554 10304 Kyoklinggårdjord Éäifšgïn å nitrate vid 40 CG 356 10306 Parkbark Anrikn. på baâis av glucos- nitrat på 50 CGn glujj 30. - nitrate at 50 G 0 358 10308 Chicken farm soil Enriched with glucose nitrate based on 50 O 0 360 10309 Park soil from the Danish city of Perb oratagar C 364 10310 Scraping from toilet- Thermofil sesquicarbonate- container rich. (50 ° C, pH 8.8-9.7) 0 365 10311 Liquid from tanning glue Enrich. with bran. soda C 366 10312 Baby excrement Starch enrichment. (pH ll) with inorganic nitrogen C 367 10313 Manure from elephant Thermophilic sesquicarbonate (5000) 8.8 "9.7) G 369 10314 Ostrich manure from Zoo Proteospeptone (shake bottles) PH 9: 7 C 370 10315 Scrap from containers for Alkaline mannito1-KNO, - tanning bath enrichment 10 15 20 25 30 40 1116635-4 74 cont. 0 371 110316 C 372 10317 0 573 10310 G 374 10319 0 375 10320 G 376 10321 0 377 10522 378 10325 0 410 10524 0 411 10325 0 412 10326 0 415 10327 7 Manure from elephant Clay from grass fi ít from gAscheberg, Holstein Park soil from Danish city Clay from grass field from Ascheberg, Holstein Ostrich manure from Zoo Manure from elephant Water from hippopotamus basin Scrape from container for tanning lodge Duckling Tig Ascheberg, Holstein Proteospeptone (shake flasks PH 9.7 - Starch casein detergent enriched.

Perboratagar Anrikn. m. the sodium salt of ethylenediaminetetraacetic acid starch casein Sodium sesquicarbonate. (pH 9.2-9.6) Sodium sesquicarbonate enriched. (pH 9.2-9.6) Thermophilic casein starch enriched. with NaOH Mannitol-KNO3-enriched.

Thermophilic locking carbon carbonate.

Storage in potato flour and sodium sesquicarbonate Starch enriched. (pH II) with inorganic nitrogen The systematization studies of all these members of the genus Bacillus have been carried out using the procedures described by Smith, Gordon & Clark in "Aerobic Spore-forming Bacteria", U.S. Pat. Department of Agr., Monograph No. 16 (1952). These procedures have hitherto been considered to be the most suitable, but it was necessary to modify them in view of the fact that all substrates must be adjusted to a much higher pH value than indicated by Smith, Gordon & Clark because all in Table I listed Bacillus species fillet grows at elevated pH values. '' The bacteria can be divided quite accurately into morphological groups.

These groups differ from each other to such an extent that they in fact represent different species. chemical reactions.

Within the morphological groups, variations are found in the bio- On the basis of these variations, the groups have been divided into varieties, which are represented by one or more strains.

] The systematization studies are described in detail in Patent No. 367,021. 'On the basis of the inventorisation studies of the inventors, the members of the genus Baeillus listed in Table I should be classified as shown in Table II below. g TABLE II 7116685-4 Species Varieties Strains 300, C 301, C 360, 572, C 574 302, C SBU I 525, C 559, C 552, 369 BOH, C 311, C 3364) II 535, C 5141 C 505 , C 55 ", C 557, C 566, C 557, C 571, C 575; C 578 ïV _ C 351, C 356, C 3614, C 575, C 577, C" ll c 358, c H10 VC 365, The proteolytic enzymes which form constituents of detergent and detergent compositions according to the invention may be prepared by aerobic cultivation of any of the compounds. species and strains listed in Tables I and II. The cultivation is carried out in accordance with principles known in the art, except that during cultivation the nutrient medium containing sources of assimilable carbon and nitrogen is kept at a higher pH value than hitherto, preferably in the range of 1.5. The yields obtained have been determined by Anson's well known method of hemoglobin, cf. Journal of General Physiology, 22, 79-89 (1959) .An Anson unit in the present preparation refers to the amount of prot. eolytic enzyme which consumes hemoglobin at a pH of 10.1 and a temperature of 25 ° C for a reaction time of 10 minutes. at such an initial rate that per minute such an amount of cleavage products is formed which cannot be precipitated with trichloroacetic acid so that these cleavage products give the same color with phenolic reagents as one milliequivalent of tyrosine.

The nutrient medium is formulated in accordance with the principles of the prior art. Suitable sources of assimilable carbon are carbohydrates, such as sucrose, glucose, starch, grain flour, malt, rice, sorghum, etc. The carbohydrate concentration can vary within fairly wide limits, eg up to 25% and down to 1-5%, but usually 8-10% is suitable, the percentage being calculated as dextrose. It has been found that the presence of carbohydrates in the substrate gives rise to the formation of acidic components, which leads to a reduction in the pH value during the cultivation. Since it is essential to maintain a pH of the substrate that is in the range of 7-12 during cultivation, measurements should be made so that the pH does not fall below 7 during any significant period of cultivation.

To keep the pH within the required range, a limited amount of carbohydrates can be used together with a buffer substance, which can maintain the required pH. It has been found that carbonates and especially sesquicarbonates used in a concentration of up to 0.2 M in the substrate are capable of creating a pH of about 10.5 and 10.5, respectively. 9.3. Other buffer systems, such as phosphate buffers, can also be used.

It is also possible to start the cultivation with a low carbohydrate content and add small amounts of carbohydrates successively during the cultivation.

A third possibility is to use automatic pH control by adding various alkaline reactants used in the present art.

The use of carbonates and sesquicarbonates as pH regulators is very suitable, and it is surprising that it is possible during cultivation to use these compounds in specified concentrations. BO v 71166854 »The nitrogen source in the substrate may be of inorganic and / or organic nature. Suitable inorganic nitrogen sources are in some cases the nitrate and ammonium salts, and among the organic nitrogen sources there are a large number known for use in fermentation processes and in cultivation. of bacteria. Illustrative examples are soy flour, cottonseed meal, peanut meal, casein, corn steep liquor, yeast extract, urea, albumin, etc.

In addition, the substrate should of course contain the usual trace elements.

The temperature at which the cultivation takes place is normally within the same range as in known cultivation of known species of the genus Bacillus.

Generally, a temperature of between 25 and 55 ° C is suitable. The temperature is preferably 30-40 ° C. a When the cultivation must be carried out under aerobic conditions, it is necessary to use artificial aeration when using fermentation tanks. procedures.

Maximum yields of the proteolytic enzymes are generally reached. The amount of air is the same as that used in known cultures - after a culture time of 1-5 days.

Illustrative examples of suitable culture media are: l) Medium BPFA with the following composition = Potato flour 50 g per liter of tap water Sucrose 50 g "" "Barley flour 50 g" "" Soy flour 20 g "" "Sodium caseinate 10 g" "" Na2HP04.l2H20 9 g "" "Pluronic 0, lg" "" 2) Medium BSX with the following composition: Barley flour 100 g per liter of tap water Soy flour 30 g "" "Pluronic 0, lg" "" Both of these media were adjusted to the desired pH by the addition of sesquicarbonate or soda under sterile conditions The starch contained in the above media was liquefied by alpha-amylase before sterilization.

The two culture media BPFA and BSX were then used for cultivation in 550 liter stainless steel tanks under submerged conditions and artificial aeration, obtaining the results summarized in Table III below, which also lists the strains used and the cultivation conditions. 10 15 20 25 '50 35 40 1116605-4 8 Table III Strain 0 335 0 559 0 351 Medium BPFA BPFA ESX pH before 0 ° C temp. i “o 34 34 3 Air, m5 / min. 0.25 0.25 0, 3 Cultivation time in hours. 84 97 83 Final pH 9.2 9.1 9.55 Final proteolytic activity expressed in Anson units / kg substrate 40 29 33 With strain G 503 in four runs and under different conditions, the results summarized in Table IV are obtained.

EàïflLllT Cultivation No. l 2 5 4 _ Barium flour, g / liter 100 100 150 200 soybean meal, g / liter 30 50 45 60 Pluronic, ml / liter 0.03 0.03 0.05 0.03 Na 003 (sterile additive fo e inoculation) ad 0.2 M 0.2 M 0.2 M 0.4 M pH before inoculation 10.0 10.0 10.35 10.1 Odlinastemp., ° 0 54 54 34 34 Air, m5 / m1n. 0.3 0, 3 0.3 0.5 Cultivation time in hours 125 104 ll? 126 PH at maximum 993 '9: 3 9: 6 9: 3 Maximum Anson unit. per kg I 67 80 77 66 The proteolytic enzymes can be recovered from the culture solution by subjecting it to centrifugation, precipitating the enzyme from the liquid thus obtained by adding Na 2 SO 4 or ethanol, separating the precipitate from the liquid by filtration with kieselguhr as filter additive and drying of the precipitate to form a powder containing the active proteolytic enzymes. More detailed information on the preparation and recovery of the proteolytic enzymes which form constituents of the detergent and detergent compositions of the invention is found in Patent No. 367,021.

Examination of the proteolytic enzymes produced by the strains listed in Table II has shown that there are significant differences between the enzymes. a number of their properties. With respect to proteolytic activity, it appears that the enzymes can be divided into two groups or types when the proteolytic activity is measured at pH 12 by the Anson method and expressed in% of maximum activity, thus Type 1: 100 to 80% Type 2: ao to 50%.

According to the invention, the type 1 proteolytic enzymes are preferred, but the type 2 enzymes are also useful.

It is known in the art that calcium ions stabilize the activity of most proteolytic enzymes. The new enzymes, which are formed by bacteria listed in Table I and divided into species and varieties in Table II, have been investigated for the stabilizing effect of calcium ions, at a concentration of 0.01 M at pH 10.5 or 11, and the stabilization has been stated as a percentage of residual activity after storage for 30 min. at 5000. The results of the enzyme type experiments and the calcium ion stabilization effect are summarized in Table V, where + means that the residual proteolytic activity in the absence of calcium ions is below 80% of the corresponding activity of the control sample in the presence of potassium ions, and - means that the residual proteolytic activity in the absence of calcium ions, more than 80% of the corresponding activity in the control sample is in the presence of calcium ions.

Table V Art Var. Strains Enzyme CA ++ - type stabilization a C 300, C 501, C 360, 0 372, G 574 1 ++++ I b C 302; C 334 l ++ c c 523: Û c 552: G 369 2 ++++ d C 304, C 311, C 336 1 +++ II C 335; C 341 2 ++ and C 305, C 354, C 357 C 365 »C 367; C 371 »C 375, C 378 1 ---- -? IV b G 551, C 356, C 364, G 576, C 377, G 411 1 - ++ --- + C c 358: C 410 l - + V C 565 »C 412 1 -? VI a G 573 1 - b C 325, G 415 1 ?? VII G 570 l - The proteolytic enzymes with optimal activity in the absence of calcium ions are preferred according to the invention. The proteolytic activity of enzymes prepared from strains listed in Table V has been tested not only at pH 12 but also at lower pH values to obtain a more detailed information. the proteolytic activity at different pH values and additional information regarding the activity of the three types of enzymes. For illustrative purposes, reference is made to the accompanying drawing, in which Fig. 1 shows the proteolytic activity of strain C 311, which belongs to type 1, Fig. 2 similarly shows the activity of strain C 335 formed of enzyme 2.

It should be noted that the purpose of the activity curves shown in the drawing is to illustrate in principle the difference in activity between the two types of proteolytic enzymes at varying pH values, and that the activity curve for each type may vary slightly without losing its characteristic appearance. The usability of the new enzymes in detergent and detergent compositions is shown by the following tests: a) Stability against tripolyphosphate §TPP) The stability of the enzymes in a solution containing tripolyphosphate in an amount of 0.2% was determined. Stability was expressed as a percentage of residual activity after 30 min. at 5000 and at pH 10. The enzyme concentration was 0.1 Anson unit per liter and the analysis method was the Anson method.

The results are summarized in Table VI below.

Corresponding solutions with 0.01 M 0aOl 2 showed in all cases a residual activity of 80-100%. b) Stability against perborate The activity of a solution containing the enzyme and sodium perborate in an amount of 0.1% was determined. Stability was expressed as a percentage of residual activity after 30 min. at 5000 and pH 10. The enzyme concentration and analysis method were as in sample a.

The results are summarized in the following Table VI. c) Stability against surfactants The stability of solutions containing the enzyme and various surfactants was determined using three typical surfactants in concentrations corresponding to those used in washing solutions: 1. mvåi 0.25 s / liter 2. rss - an aikyi- aryl sulfonate (50%) 3. IAS - tallow alcohol sulphate (25%) 5.0 g / liter The enzyme concentration was 0.1 Anson unit per liter. the conditions were 30 min. at 5000 at pH 10 and the analysis method was nitro- 2.5 g / liter The experimental casein method, cf. E.v. Pechmann, Biochemische Zeitschrift, Vol. 521, 248-260 (1950). 10 15 20 25 11 71166854 »The figures in Table VI are to be interpreted as follows: The figure before the slash shows the percentage of residual activity, when the analysis is carried out immediately after the addition of the surfactant, i.e. this figure gives a measure of the initial deactivation rate.

The number after the slash is the difference between this original residual activity and the percentage of residual activity after 30 min. d) Temperature optimum Table VI indicates the temperature in OC at which maximum activity is obtained at pH io; e) älaxaizr It has been found that all enzyme preparations are momentarily and completely inhibited by phenylmethylsulfonyl fluoride, which means that all the enzymes have serine in the active center. f) Rlï fi iaäililêi In connection with five enzyme preparations, the stability was determined at different pH values under the following conditions = The analysis method was the Anson method.

Storage: 24 hours at 2500 pH values = 5 - 7 - 8 - 10 - 12 Enzyme concentration: 0.2 Anson unit per liter.

Table Vï indicates the pH range within which a residual activity of 80-100% could be recorded.

Testing of the enzyme preparations produced by strain G 503 against sodium sulphite has shown that the enzyme is not sensitive to this reducing agent, which may indicate that S-S bridges are not essential for the tertiary structure of the enzyme. 12 Inn o 7116685-4 _ __ + om o. \ O mwm mNEN _ N Nm N S; m; N ... mmm o. _ M.oN | mm + _ mm Nm \ mm mm \ mm oH \ mm mm mm N Mm.mM _ oN mHN mmm o _ + _ mm Hm \ mm Nm \ oo, mm \ mm mN om N mm mo oom fi mmm o H _ wm mm \ M% mN \ mm wW \ ooN mm mm _N ñmnm mN mHN mmm on eoïš ._ om Mmmm mä .ämm æ Mm m ä .. K omv www. ._ + om mm \ mm ïåom omšm mm mm A _ mš otm om Hmm o. 1 _ H om om \ mm_ mm \ mm o \ mm mm H. mn ».f mno ooom omm oo Hm om om Nm \ H> om \ mm N \ mm oom om H mm _ NN oom mmm o + om Nm \ mm oN \ mm No \ Nm mm oom N _ mm m mo mN mmm o. _ + om mo \ Nm om \ No o \ ooH mm _om H _ m.m m o.o. oooN mmm o _ + om .m \ mH NH \> N Nm \ mm, om om H Mm.> _ mo mmm omm o N _ + om mm \ m> om \ m> om \ mm mm o_ _ H mm _ . m.o mmm omm n _ + om mm \ om mmäm. mmšm Nm ... m .A m; m.o No mmm o ____ _ + om mïmm omšm mmmm mm .mmm N m; må m Hmm o .N _ _ + _ o ... mïmm mNäm mmwoom mm mm N m; ma. _ oom fi .mmm _o _ _ m _ 1 _ w om. o \ m fl m \ om mm \ mm _ N_ H m.m m.o m.N oom o m oN_o m + om mH \ mN om \ oo om \ mm mm mm N m.m o.m oom mom o _ + om .im iom mm \ mm mo. N. H om m6 m _ .Nom o f. _ + om .N \ o Hm \ mm mo \ om _ o Hu Moa m.o N mom o N + _ __ om. N mb. mïmm áïmm N N BN m.o N oom o oo mom mmm Hm> o _ w m m. _. a subjects. m .när _ snowhøm ». : BB nosen> .. a | m. ._ 1 m. »Mšm fi mßmvm. : mama m. . . o. N mvwma. _ .Ho Hma 93. nco .... mmw.um fl ~ .cw fl omaow. . ... SQ _ “Gmhom: Zmâm fi. , “Fomf flflfifl mvw xšns fi m .æoæ fi fwæm fl á lih fl z fl Envw N _ _. . - ». »> M fi open _ - _ _. _ i - 1. IIIJ; 1. 4 ß. v. 1 _. 10 15 20 25 30 40 13 '71166354 Table VI shows that the number of enzyme preparations shows surprising stability properties.

In general, the enzyme preparations or compositions according to the invention consist of a solid or liquid mixture of the proteclytic enzymes prepared according to the invention and other constituents, the amount and composition of which depend on the purpose and technical or scientific field in which the enzyme compositions are to be prepared. used. When the enzyme preparations or compositions according to the invention are in solid form, they may consist of granules in which the enzymes are incorporated, for example together with other enzymes or substances which have properties other than enzymatic activity useful for the usefulness of the enzyme compositions. When the enzymes are not used in crystalline form, they may be accompanied by impurities of an organic nature, such as proteins and carbohydrates from the substrate.

The enzyme compositions in liquid form may consist of solutions or suspensions which, if necessary, may contain stabilizers.

The novel enzymes of the invention are usually used in small amounts. In view of this, the enzyme preparations or compositions for industrial use have an enzyme content not exceeding about 10% by weight.

The following washing experiments show the results that can be achieved by incorporating the new enzymes into detergent and washing compositions.

The washing tests were performed using EMEA test strips or patches 166 resp. 112. In other words, in the washing experiments, test strips contaminated with blood, milk and carbon black (heading 116) or with cocoa, milk and sugar (heading ll2) have been used. The experiments were performed with the two types of test strips separately and each experiment was repeated three times at 5000 and 6000. The enzyme concentration in the washing solution was 0.048 Anson units per liter.

The detergent which was to represent a detergent composition had the following composition.

Nansa S 40% sodium alkylaryl sulfonate, 60% sodium sulphate) 250 g Nonylphenyl, 10 E0 30 g Soap (80%) so g Sodium tripolyphosphate 300 g cmc (as) 16 g Sodium carbonate, anhydrous 80 g Sodium sulphate, anhydrous 74 g Sodium perborate (NaBO 3, 4H20) §gQ_g Total 1ooo g 10 15 20 25 50 40 7116585'4 14 Other conditions were as follows = Water hardness in German units l0 ° Relationship between fabric and water 1240 Experimental "50 min. PH value about 10 Detergent concentration 4.0 g / liters of washing solution The hydrogen procedure carried out at 50 DEG C. was as follows: Using a pipette, 20 ml of the enzyme solution with an activity of 0.288 Anson per liter was added to a 150 ml beaker, the pH being pre-set to 10.0 and the temperature of 2000. At time zero, 100 ml of detergent solution adjusted to pH 10.5 and 5600 were added.The detergent concentration was 4.8 g per liter.The beaker was immediately placed in a water thermostat at 5000 and 6 circular ElIPA sample patches were added to a total of 3.0 g. Stirring was performed with a gl asspatel for 10 sec., and the beaker stood in the thermostat for a total of 30 min., stirring. Then the washing solution was separated. The test pads were washed in a gutter for 10 sec. every 4 min. and the pH was measured after cooling. the tap water for 10 min. and then dried between two towels and ironed. Each sample patch was subjected to remission measurements in a Beckman spectrophotometer at 0.460 .mu.m.

In addition to these experiments, control experiments were performed in which the enzyme solution was replaced with water.

In the 600 experiments performed, the temperature of the detergent solution was set at 68 ° C.

The results of these experiments are summarized in the following Table VII. ggpell VII Enzyme ll6Remission from EMPA sample patch 112 from H. H _ after blotting after blotting strain Untreated and Rinsing Untreated and Rinsing 5000 6000 5000 60 ° C None ll 9 14 6 17 8 54 0 41 1 40 5 c 303 '3216 3514' 4115 4519 C 539 2710 5: 0 39 :; 59.7 C 551 50.0 27.7 40.5 40.2 0 567 54.0 5.0 59.7 41.5 C 577 54.0 55.2 41.7 45.8 0 554 55.5 51.0 41.9 45.5 C 572 58.1 29.7 45.6 42.5 C 576 52.7 52.7 42.4 45.2 C 556 55.8 55.5 - 41.2 41 .5 The remission values are averages of measurements of each of the six test pieces in each of the experiments. 10 15 20 25 50 55 40 15 7116685-4 Another series of washing experiments was performed with EMA test patches No. 116 which had been pre-treated with a solution containing an anionic surfactant and sodium perborate at a temperature of 4000 for 20 minutes.

After the treatment, the specimens were rinsed and dried. The treatment with perborate results in a strong fixation of the dirt, which would then be more difficult to remove.

The detergent had the following composition fl Nonylphenol, 10 EO 80 g Sodium tripolyphosphate 400 g GMO (45%) 20 g Sodium carbonate, anhydrous 150 g Sodium sulphate, anhydrous §âQ_g Total 1000 g Additional conditions: water 1o ° German hardness Relationship between fabric and water 1:40 Period 30 min.

Temperature 4500 pH about 10 Detergent concentration 4.0 g / liter washing solution The procedure was carried out as according to the above washing experiments except that the washing solution had a temperature of 5000 before it was mixed with the enzyme solution.

Three experiments were performed with each of the enzymes, and the results are shown in the following Table VIII.

Table V11; åïššm Remission from EMEA test patch 116 strain Untreated after soaking and rinsing None 16.4 18.7 C 505 b 56.8 0 559 52.5 C 351 31.0 C 567 54.5 0 577 57.6 G 564 54, 6 0 572 45.2 C 576 56.8 0 566 57.5 The remission values are averages of the measurements of each of the six specimens in each of the experiments, except that the values in connection with the experiments with enzymes from strain G 539 and C 351 are based on a measurement. Experiments have also been performed using different enzyme concentrations in the wash solution, thus in the range between 0.02 and 0.16 Anson units per liter of wash solution. In the experiments EMEA test patches No. 116 were used and the detergent was the same as that used in connection with the first washing experiments, thus Nansa (40% sodium alkylarylsulfonate, 60% sodium sulphate) 250 g Nonyifenpl, io no 30 g Soap (8072) 50 g Sodium tripolyphosphate 300 g GMC (60%) 16 g Sodium carbonate, anhydrous BO g Sodium sulphate, anhydrous 74 g Sodium perborate (NaBO 3. 4 H 2 O) _ggQ_g Total l000 g The experimental conditions were as follows: water 1o ° German hardness Relationship between fabric and water 1:40 Time period 50 my.

Temperature, 4500 and 6000 pH about 10 Detergent concentration 4.0 g / liter of washing solution The procedure is the same as stated in the first experiment except that the concentration of the enzyme solution was varied so that the activity in the washing solution obtains the following values: 0.02 - 0.04 - 0, 08 - 0.16 Anson unit per liter.

Before the detergent solution was mixed with the enzyme solution, it was adjusted to 50 resp. 6800, corresponding to a temperature of 45 resp. 6000 in the ready-to-use washing solution.

Each enzyme preparation was examined at both temperatures. The results from the remission measurements are summarized in the following Table IX. .7í1eess-4 A17 \ m.wn¶ o.mm o. ~ N o.m fi ow m.mm m.mm + .æw o.mH om HÅww. ïmw »ÄN 1 o-É .. om m.mn o.mn> .m ~ æ.o ~ om, + .en w.mn¶ ø.> N .v_> H om _.

H. «n w.Nn w.mN n.r fl øm L. ... så .Én 0: a .ïä \ 8 m.æw_ <.m ~ _ mo ~ m. @ Fi om fl. Wn H.en m.mN. m.mH. om üm ... ma »._ mon _» GH. 3 ma »2%. ... om a. .E 3 fi dv. .o fi e I Oån Nå fl. _. mw. ñâ »än Nå QS 3. wa fl w må Nåm må fl me n.wm> .Hn - w.m ~ ø. + H. m +; n.> n ~. + n o.w ~; m. ..> .m ~ m + o.mn m. ~ n w.m ~. Nowm fl. mw m.mn m.nn æ.m ~ o.mH mv .mon a mH.0 «æo.o« 0.0 wo.o. . o. ¶ 0 ¿Emvm: maa wc «= mmH # wm> v nøww fi Hmm vmmnolcommw H .. _ Høumnmm fi ma. Ehucm w fifi HNQQNHÃVHQIÅMEN cm fi wvco fi www fi w fi. . . _. n xH¶ñ fi @ pma. . _. . .. g Poon uuAuTv 10 15 20 7116685-4 18 The remission values are averages from six measurements.

Finally, the following has been performed. In storage stability test (service life) Each enzyme preparation was mixed in a Turbola mixer with the detergent mentioned on page 16. Corresponding experiments were performed with the same detergent except that the perborate was exchanged for anhydrous sodium sulfate.

The water content of the perborate-free detergent was 2.7%. The water content of the detergent-containing perborate was, of course, correspondingly higher due to the fact that the perborate contains about 47% of crystal water.

All mixtures were analyzed for proteolytic activity immediately after preparation and then placed in sealed glass containers at 40 ° C. The assay for proteolytic activity was repeated at certain intervals. The analyzes were performed as follows: of A sample weighing 12.5 g was taken from twelve different places in the glass containers, which was transferred to one liter of volumetric ash. With respect to the perborate-containing mixture, 4.4 g of sodium sulfite was added for the purpose of neutralizing the perborate. Deionized water was added up to the mark, and the solution was then kept for 30 minutes. at 2506 under mechanical agitation. The proteolytic activity was then determined in Anson units.

The mixtures examined and the residual proteolytic activity measured after different storage times are summarized in the following table X. 711668541 19 vøvH> HvMm xwwææ fi owvonm wwnmnmwHm> # H fi mßvnæoonm u &. Fi% <; Emaw Hmm Hwvmßmm | comQ <u M \ D <. \ _ R e: | I nu * I om mo.o mm Hm.o om o> .o om om.o .om om.o ooH om.o. _ + mm 36 mm 56 äH mm ... äH .m6 .__ mm moå ooH om.o 1 .mono Ho om.m Hm oo.m om o> .m om o> .m ._ mm omww ..6m . ~ o. +. _ Hm. oH.w mm .oo.o _ooH om.w ooH om.w .m om. o> .m ooH oo.æ. 1 omm o om moå mv oÉH E. oHå S. oH.m om mmfw. ooH .Hmmm + - ooH oo.m .ooH mm. ~ ooH.oo.m ooH oo.m _ ooH> m. ~ .ooH »mmm 1 mmm o Ho: m« .o om. m «.o .Hm mm.o moH Nmmo __, ooH o> .o _ooH omno +. . ooH om.o mm mo.o moH ~ m.o moH mm.o _ _ H ooH om.o .ooH ommm .r _ mmm o mm m «.H .mm> m.o .Ho _ ~ o.H» W _. _.ww om.m _ ooH.mo. ~ .ooH momw. _ + .m », _ M m fl m. om Hm. ~ Hm om. ~ .om o «. ~ Ä V; _ om mm. ~ _.mm _om, m. ooH mo. ~ fl v, _ _. >> m_o _ mm om.H me. oo.m. .mm _omJ ~ fm .H - om _omw «__ooH om.« ooH.ow. «_. . »» ~ Wf.h.

.H mm mm.m om ow.m. omr mmwm _ .M .om oo.m. mm: oo. «ooH omkm __... .mom o _ S mmå .om omå mo. omæ - ooHfomæ om. mïm .SH omä. + .. _ _. moH oo.m mm om.m _HoH mm. ~ moH oo.m -_ _ ooH mmmm HoH mm. ~ ooH om. ~ _ 1 mom o .m ... m .IW mm _ m. . THAN. ..m ___. f «_ \ o <» m <\ o <_ æm <\ ø <»HH \ o <» H <\ o <px <\ o «» H <\ = <ox <m \ = <_. = mHo om ammo Hm H .omm HH H ammo w H: mo m H .omm m H owmo H_H. H: Ho o H .xomn emmw om fi m o cv øH> mcwnwm fi Hmvwm vm # fi> H »Mw H fi wñvcmoonm soc Ehwcw Emnw som Hmvmncæ |: om§ <loßnwm Eæscw mø fi m o. -. _ .hmøowhmmmwn fi nwm fi .. _. _ _ _ vnowhmæo fl .. Ä, wwwwmmmw. . . The stability of some of the enzymes in the presence of perborate is considerable. The enzyme preparations intended for incorporation into detergent and detergent compositions are usually provided as a powder, of which the active enzyme or enzymes constitute a minor amount, the remainder of the powder consisting of inorganic salts such as sodium sulfate, calcium phosphate and sodium chloride, sometimes as well as other substances which form constituents of the final detergents.

As proteolytic enzymes have already found use as constituents in detergent compositions, all those skilled in the art know how to incorporate the enzyme formulations into the detergent and detergent compositions.

It is a significant advantage that many of the enzymes show optimal proteolytic activity at elevated pH values and improved stability in the presence of perborates.

The results of a comparison between the proteolytic activity of the known enzyme ALCALASE and the enzyme according to the present invention are presented in diagrammatic form. The diagrams refer to the use of two substrates, namely casein and hemoglobin, and show the proteolytic activity as a percentage as a function of pH. 21 7116685-4% activity I I 100 - '. _ '(/ ,, f ”\ - RI ~ / x 75 _ M! _ X ß fl w __ _ n \ l 25» - - ALCÅLÅSE ® J * Casein substrate v | 1 | 1 | PH! 7 8 9 10' H 12% activity UIII l I - xx _ ~ Åt tj x 75- - x 50- - 25- - ® ALCALASE Hemoglobin substrate 1 | 1 1 | | DH: 7 8 9 10 11 12 I 7116685-AL É 'L 22 ä activity l II 100 »K- x X 50-- fl -» - ¿- "“ ”'ø _ x 25" Enzyme according to the invention Casein substrate | I 1 1 1 1 pn; 7 s 9 mn 12% activity 50- Enzyme according to the invention Hemoglobin substrate lt FI 100 »x fi- unn-ui-o-Xnnm-øx-x“ ““ -e pH: 7 8 9 10 ll 12

Claims (6)

71166854 »23 PATENT REQUIREMENTS. 1. Detergent and detergent composition containing proteolytic enzymes having high proteolytic activity at the high pH values normally present in a washing solution, characterized in that one or more of the enzymes exhibits optimal activity against hemoglobin at a pH value exceeding 9. 2. A composition according to claim 1, characterized in that one or more of the enzymes exhibits a proteolytic activity which is 80-100% of the maximum activity measured by the Anson method at a pH of 12. 3. A composition according to claim 1, characterized in that one or more of the enzymes exhibits a proteolytic activity which is 50-80% of the maximum activity measured by the Anson method at a pH of 12. 4. A composition according to any one of claims 1-5, characterized in that one or more of the enzymes exhibits their optimum proteolytic activity in the absence of calcium ions. A composition according to any one of claims 11, characterized in that it contains one or more enzymes formed by culturing species of Bacillus capable of growing and yielding said enzymes at a pH in the nutrient medium within area 9-ll. Composition according to any one of claims 1 to 5, characterized in that it contains one or more enzymes formed by culturing one or more of the bacteria deposited with the National Collection of Industrial Bacteria, Torry Research Station, Aberdeen, Scotland, under the numbers 10lUH to l0lH8 and the numbers 10281, 10282, 102814, 10286 1111 10288, 10291, 10293, 10301, 10302, 10304 and 10306 to 10327, and mutants thereof. MENTIONED PUBLICATIONS: