NL194443C - Bacillus strain, basic protease produced by this Bacillus strain and detergent containing this protease. - Google Patents

Description

1 194443

Bacillus tam, basic protease produced by this Bacillus strain and detergent containing this protease

The invention relates to a Bacillus strain, a basic protease produced by this Bacillus strain and a detergent containing this protease.

Basic protease is widely used in, for example, the leather industry, food industry, textile industry and pharmaceutical industry because it would be more heat-resistant than neutral protease. It has also been used a lot in detergents recently.

The basic proteases used in detergent compositions are derived from bacteria belonging to the genus Bacillus.

In general, there is a need for enzymes for detergents that are active at relatively low temperatures.

For example, U.S. Pat. No. 3,723,250 describes basic proteases from various Bacillus strains, some of which have a pH optimum of 7.5 and 10 and a temperature optimum of 40, 45, 50 and 55 ° C (see Table IX).

The invention provides a new Bacillus strain, deposited and available from the Fermentation Research Institute (FRI) in Japan as Bacillus sp. FERM BP-93, which is capable of producing a protease under basic conditions.

The invention further relates to a basic protease designated by API-21, produced by the aforementioned new Bacillus strain, which protease has the following properties: 1. activity: hydrolyses casein under basic conditions, optimum activity at pH 10-11 and a temperature of 45-50 ° C, 2. stability: no reduction in enzymatic activity at 10 minutes heating to 40 ° C at a pH 10 in the absence of substrate, reduction in enzymatic activity of 90% or more at 10 minutes heating 25 at 50 ° C and a pH 10, starting deactivation at 10 min. heating at 50 ° C and a pH 8 and complete or almost complete deactivation at 10 min. heating at 60 ° C and a pH 8, stable under basic conditions, in in particular at a pH of 7-11.5, and resistant to freezing and freeze-drying, and 3. enzyme molecule: the protease is a protein with a molecular weight of approximately 22,000 as estimated by a gel filtration method, an isoelectric isch point of 7.4 as estimated by an electrofocusing method, an absorption peak in the UV spectrum at 275-282 nm, an IR absorption spectrum as shown in Figure 5, and a serine residue at the active site.

The invention further relates to an enzyme-containing detergent, which detergent comprises the basic protease API-21.

For an explanation of the properties of the enzyme according to the invention, reference is made to the figures.

In Figure 1 the relative activity is plotted against the pH. The graph shows that the optimum activity is at pH 1011.

Figure 2 relates to the resistance of the enzyme in the absence of substrate to different pHs.

40 In Figure 3, the relative activity is plotted against the temperature. The graph shows that the optimum activity is at a temperature of approximately 50 ° C.

Figures 4A and 4B relate to the resistance of the enzyme in the absence of substrate to different temperatures at a pH of resp. 10 and 8.

Figure 5 shows the Fourier Transform Infrared Spectrum of the enzyme.

45

The new microorganism from which the basic protease API-21 is extracted, namely Bacillus sp. Nov, NKS-21, hereinafter referred to as NKS-21, was isolated from soil material at Fuchu-shi,

Tokyo, Japan. It has been found that NKS-21 is a new, aerobic sporangium bacterium belonging to the genus Bacillus. The species NKS-21 was deposited on February 3, 1982 at the Fermentation Research Institute (FRI) in Japan as Bacillus sp. FERM BP-93. This collection is recognized by the Budapest Treaty. The microorganism is made available upon request to third parties to test the present invention.

The microbiological properties of NKS-21 are listed below. The reagents and isolation methods used are according to Bergey's Manual of Determinative Bacteriology, 8th edition, 1974. 55 The bacterium was isolated from soil material by adding a small amount thereof to a peptone medium and performing the cultivation on a shaking table at a temperature of 10 -40 ° C and a pH 7-10 for 40-150 hours. A portion of the culture product was spread on agar plates 194443 2, after which the strain was isolated in the usual manner.

Microbiological properties of NKS-21 a. Morphological characteristics 1. size of vegitative cells: 0. 6-0.8 µm x 2.5-3.5 µm 2. cell pleomorphism: cells sometimes expand to a size of 0, 6-1.0 µm x 4.5 µm.

3. Gram staining: 10 positive but quickly discolored.

4. motility: motile with peritrically flagellae.

5. spores: oval to elliptical, subterminal, 0.8 μ x 1.2 p.

15 6. sporangia: slightly swollen.

7. acid resistance: negative b. Cultivation characteristics in different media 20 1. Pepton broth:

a) pH 9.5: good growth at 20 ° C and 30 ° C

pH 7.2: poor growth at 20 ° C and good growth at 30 ° C

b) growth temperature at pH 9.5: growth between 14 and 41 ° C

No growth above 43 ° C or below 12 ° C

2. other media: oblique meat extract - pepton agar: good growth; meat extract-pepton broth with 7% NaCl: 30 good growth meat extract-pepton broth: good growth oblique casein-meat extract-pepton agar good growth 35 casein-meat extract- pepton broth: good growth starch-pepton broth: good growth glucose-pepton broth: 40 good growth tri pton yeast extract agar plate: good growth of starch-yeast extract broth: growth 45 meat extract gelatin plate: a) pH 10.0, 30 ° C: growth and liquefaction b) pH 10.0, 20 ° C: weak surface growth and poor liquefaction 50 c) pH 7 , 2.30 ° C: growth and flow d) pH 7.2, 20 ° C: weak surface growth and flow 3. Growth under anaerobic conditions: 55 no growth under anaerobic but growth under aerobic conditions.

c. Physiological characteristics 1. reduction of nitrate to nitrite: 3 194443 no reduction 2. anaerobic gas formation from nitrate: negative 3. methyl red (MR) test: 5 negative 4. Voges-Proskauer (VP) test: negative 5. indole formation: none 10 6. formation of H2S: none 7. starch hydrolysis: positive 8. utilization of citrate: positive 9. utilization of inorganic nitrogen source: utilization of nitrates and ammonium salts 10. pigmentation: none 11. urease test: negative 12. cytochrome oxidase test: positive 13. catalase test: 25 positive 14. OR test (Hugh and Leifson's medium): weak fermentation 15. acid formation and gas evolution from hydrocarbons: 30 acid formation gas development L-arabinose - - D-xylose - - D-giueose + D-mannose + - 35 D-fructose - - D-galactose + - maltose + - sucrose + - lactose - - 40 trehalose + - D-sorbitol + - D-mannitol + - inositol + glycerol + - 45 starch + ribose - - raffinose + - cellobiose + - 50 + ·. positive -: negative 16. Other characteristics: a) phenylalanine deamination: positive 55 b) casein hydrolysis: positive c) tyrosine decomposition 194443 4 positive

From the above it appears that the strain NKS-21 is an aerobic sporangium bacterium that is believed to belong to the genus Bacillus. The characteristics of this strain are that it grows in neutral to basic medium and that the pH is 8-10 for optimum growth. These microbiological properties are similar to those for the known Bacillus pasteurii and Bacillus alcaiophiius strains. In addition to similarities, there are also differences in microbiological properties. For example, the strains Bacillus pasteurii and Bacillus alcaiophiius both grow in basic environment but the latter does not grow at pH 7. In contrast, NKS-21 grows not only well in basic environment but also in neutral environment. In this respect, NKS-21 is therefore different from Bacillus alcaiophiius. Furthermore, Bacillus pasteurii does not hydrolyze starch, but NKS-21 does. In addition, there are the following differences between NKS-21 and Bacillus pasteurii: 1. Bacillus pasteurii does not reduce nitrates and NKS-21; 2. Bacillus pasteurii has spherical to almost oval endospores and NKS-21 oval to elliptical endospores; and 3. Bacillus pasteurii does not decompose urea and NKS-21.

This shows that the microbiological properties of NKS-21 differ greatly from those of Bacillus pasteurii.

The classification characteristics of Bacillus alcaiophiius are not described in detail in Bergey's Manual of Determinative Bacteriology, 8th edition 1974. Because the standard strain Bacillus alcaiophiius was not available, the microbiological properties of NKS-21 were experimentally compared to those of the original strains Bacillus alcaiophiius NCIB 10436 and NCIB 10438 cited in Bergey's Manual of Determinative Bacteriology, 8th edition 1974. NCIB 10436 and NCIB 10438 are both slightly positive in Gram staining and are rapidly decolorized, so just like NKS-21.

For the morphological characteristics of the vegetative cells, electron microscopy examination of NCIB 10436 shows short bars with a size of 0.7 x 2.0 µm and long bars with a size of 0.8 x 4.6 µm. NCIB 10438 has vegetative cells with a size of 0.7 x 1.5 µm under the electron microscope. In contrast, under the electron microscope NKS-21 has vegetative cells with a size of 0.6-0.8 x 2.5-3.5 µm and the endospores have an oval to elliptical shape with a size of 0.8 x 1.2 p.m. Motility for NCIB 10436 and NCIB 10438 was not observed but 30 for NKS-21. Furthermore, there are the following differences in physiological properties between NCIB 10436 and NCIB 10438 on the one hand and NKS-21 on the other: 1. Bacillus alcaiophiius NCIB 10436 and NCIB 10438 do not grow in a mannitol medium and NKS-21 does; and 2. NCIB 10436 does not grow in a meat extract-pepton broth with 7% NaCl and NCIB 10438 and NKS-21 35.

The above-mentioned differences show that there is a clear distinction between NKS-21 on the one hand and Bacillus alcaiophiius NCIB 10436 and NCIB 10438 on the other, although NKS-21 in some microbiological properties corresponds to Bacillus alcaiophiius.

Furthermore, NKS-21 corresponds to Bacillus subtilis in the size of the vegetative cells which for 40 Bacillus subtilis is 0.7-0.8 x 2-3 µm and in the starch hydrolysis. But NKS-21 distinguishes itself from Bacillus subtilis on the following points: 1. Bacillus subtilis grows well at a pH 5.58.5 but does not grow at a pH 9-10 such as NKS-21; and 2. The temperature for optimum growth of Bacillus subtilis is 45-55 ° C while NKS-21 does not grow above 43 ° C.

45 NKS-21 is thus clearly different from Bacillus pasteurii, Bacillus alcaiophiius and Bacillus subtilis, and any strain with the same microbiological properties as NKS-21 has not been described in Bergey's Manual of Determinative Bacteriology, 8® edition 1974. It can be concluded that NKS-21 is a new strain that belongs to the genus Bacillus.

The new basic protease API-21 with a high activity at relatively low temperature is obtained by cultivating the new species NKS-21 in a basic culture medium and recovering the protease formed from the culture liquid. In addition to NKS-21 itself, natural and artificial mutants thereof can also be used to form protease API-21. These mutants fall within the scope of the invention.

The cultivation of NKS-21 can be carried out aerobically in, for example, a peptone culture medium with a pH of 8-10 on a shaking table or aerobically with stirring of the culture after inoculation with NKS-21, at a temperature of 10-40 ° C for 40- 150 hours. The cells formed are easily separated and the protease API-21 can be precipitated easily from the clarified supernatant 194443 with an organic liquid such as ethanol. The precipitate is then separated by centrifugation and freeze-dried in vacuo.

The activity of the basic protease API-21 thus obtained can be determined as follows:

The clear culture fluid or enzyme solution is suitably diluted with 0.1 M sodium carbonate-0.1 M boric acid - potassium chloride buffer solution (pH10.0). 0.5 ml of 2% milk casein solution with a pH of 10.0 is added to 0.5 ml of the resulting solution, after which an enzymatic reaction is carried out at 30 ° C for 10 minutes. The reaction is stopped by adding 2 ml of 0 1 M trichloroacetic acid containing 0.2 M acetic acid and 0.2 M sodium acetate. After standing for 10 minutes or more at 30 ° C, the mixture is filtered on filter paper. To 1 ml of the filtrate, 5 ml of 0.4 M sodium carbonate and 1 ml of 5x diluted phenol reagent are added, after which the mixture is left at 30 ° C for 20 minutes with color development. The absorbance is measured at 660 nm on the colored mixture. The enzyme unit is determined according to the guidelines of the Commission on Biochemical Nomenclature (Comprehensive Biochemistry vol. 13, 1973, pages 26-27). According to these guidelines, the amount of basic protease that releases substances soluble in trichloroacetic acid, which substances develop the color at 660 nm, corresponds to 1 mole of tyrosine from 1% casein substrate having a pH of 10.0 at 30 ° C for 1 second. defined as 1 katal.

The physicochemical properties of the basic protease API-21 according to the invention are described in more detail below.

1. Activity API-21 hydrolyses proteins such as casein, hemoglobin, albumin, globulin, meat protein, fish protein and soybean protein.

2. Influence of the pH on the enzymatic activity. As appears from figure 1, the optimum relative activity is at a pH of about 10-11. The relative activity is determined as follows:

Each of the buffer solutions (0.5 ml) indicated below containing 2% milk casein is added to 50 μΙ of the enzyme solution.

PH buffer solution

6-9 0.1 M potassium phosphate - 0.05 M

sodium borate

9-11 0.1 M sodium carbonate - 0.1 M

boric acid potassium chloride 35 11-12 0.1 M disodium hydrogen phosphate sodium hydroxide

The enzymatic activity is determined on the test solution as described above. The relative activity (%) is calculated, the enzymatic activity at a pH of 10.0 being set at 100%.

3. Influence of the pH on the stability in the absence of substrate

As shown in Figure 2, the enzyme is stable at a pH of about 7-11.5. The stability of the enzyme is determined as follows: 45 Each of the buffer solutions (0.5 ml) indicated below is added to 50 μΙ of the dialyzed and suitably diluted enzyme solution, after which the mixture is incubated at 30 ° C for 10 minutes.

pH buffer solution

50 3-8 0.1 M citric acid - 0.2 M

disodium hydrogen phosphate

8-11 0.1 M sodium carbonate - 0.1 M

boric acid - potassium chloride 11-12 0.15 M disodium hydrogen phosphate 55 - sodium hydroxide 194443 6

At the end of the incubation, 9.5 ml of 0.1 M carbonate buffer solution containing 2% casein and having a pH of 10.0 is added. The enzymatic activity of the mixture is determined as described above. The relative activity (%) is calculated, the enzymatic activity at a pH of 10.0 being set at 100%.

It is apparent from Figure 2 that the enzyme is the most stable at a pH of about 10-11. At this pH, the original activity of the enzyme is fully preserved. Between pH 7 and 11.5, 80% or more of the original activity is retained.

4. Influence of the temperature on the enzymatic activity. As can be seen from figure 3, the enzyme hydrolyses casein at a temperature of 5-65 ° C and a pH 10.0. The optimum activity is at a temperature of 45-50 ° C.

5. Influence of the temperature on the stability in the absence of substrate

4A and 4B show that the activity of the enzyme is fully preserved by heating for 10 minutes to 40 ° C at a pH of 10.0, but most of the activity is lost at 50 ° C. Furthermore, the activity is maintained by heating for 10 minutes to 45 ° C at a pH of 8, but deactivation occurs at 50 ° C and the enzyme is completely or substantially completely deactivated at 60 ° C.

6. Storage stability The enzyme is resistant to freezing and freeze-drying. After storing a freeze-dried sample for 2 weeks at room temperature, no significant deterioration in activity is observed. When a freeze-dried sample was stored in an exsicator at room temperature, the activity was fully preserved.

25 7. Inhibition

The enzyme is strongly inhibited by an active serine residue inhibitor such as diisopropyl fluorophosphoric acid and phenyl methanesulfonyl fluoride. It is also inhibited by benzyloxyearbonylphenylalanine chloromethyl ketone. The enzyme, on the other hand, is not inhibited by tosyl-phenylalanine-chloromethyl ketone which is an inhibitor of an animal serine protease, chymotrypsin and tosyl-lysine-chloromethyl ketone which is a trypsin inhibitor.

Furthermore, the activity of the enzyme is not adversely affected by p-chloro-mercury (tl) benzoate and ethylene diamine tetraacetate. Pepstatin also has no adverse effect on activity. It follows from these inhibition data that the enzyme is a protease with a serine residue at the active site.

35 8. Enzymatic activity

For the determination method: see above.

9. Molecular weight

The molecular weight of the enzyme is approximately 22,000 as estimated by gel filtration through Sephadex G-75 40 at pH 10.

10. Isoelectric point

The isoelectric point of the enzyme is 7.4 as estimated by electrofocusing.

45 11. UV absorption

Absorption peak at 275-282 µm in the anorption spectrum.

12. IR absorption spectrum As shown in Figure 5.

50 13. Elemental analysis

Not determined because this is not well possible for substances with such a high molecular weight.

14. Amino acid composition 55 Preliminary calculation based on the results of acid hydrolysis of the enzyme except for cysteine / cystine and tryptophan, of decomposition with permic acid for cysteine / cystine, and of basic decomposition for tryptophan, according to the method described in Methods in Enzymology vol. XI, 1967, 7 194443

Academic Press, New York. The composition is given in Table A below together with that for other basic proteases for which the data are derived from the literature. It appears from Table A that there are large differences between API-21 and the other enzymes in amino acid composition with regard to, inter alia, serine, arginine, lysine, vlaine, alanine, tryptophan and praline.

5

TABLE A

Amino acid composition of API-21 and other basic proteases 10 Amino acid API-21 subtilisin subtilisin Carονοί) Carlsberg2) lysine 4 11 9 15 histidine 8 6 5 arginine 8 2 4 tryptophan 0 3 1 aspartic acid 11 9 / asparagine 27 17 19 20 threonine 10 13 19 serine 17 37 32 glutamic acid 4 5 / glutamine 14 11 praline 7 14 9 25 glycine 30 33 35 alanine 26 37 41 valine 20 30 21 methionine 4 5 5 isoleucine 10 13 10 30 leucine 14 15 16 tyrosine 12 10 13 phenylalanine 4 3 4 cysteine 0 0 / cystine 0 0 0 35 1) J. Biol. Chem., 243,5296 (1968) 2) J. Biol. Chem., 243.2134 (1968) 40 Subtilisin Novo in Table A above is from Bacillus subtilis, and subtilisin Carlsberg from Bacillus licheniformis. Both known basic proteases contain a serine residue at the active site and have a molecular weight of about 27,000-30,000 and an isoelectric point of 9-11. Most other known additional cellular basic proteases from bacteria of the genus Bacillus also have a molecular weight of 25,000 - 30,000 and an isoelectric point of 9-11. Most of them are not deactivated at 10 minutes heat-treated at 50 ° C and a pH 10 such as the enzyme according to the invention. Known lower molecular weight basic proteases from bacteria of the genus Bacillus are E-1 and E-2 from Bacillus D-6. Both enzymes have a molecular weight of 20,000 and when heated at 50 ° C and a pH 9.0 their activity does not decrease (see Japanese patent publication 56-4236). On the other hand, the aforementioned basic protease from Bacillus alcolophilus 50 NCIB 10436 and NCIB 10438 retain approximately 90% of their activity at 10 minutes heating at 50 ° C and a pH 10. And the aforementioned basic proteases subtilisin Carlsberg and subtilisin Novo 70-80% of their activity with heating at 50 ° C and a pH 10 for 10 minutes. The known basic proteases originating from bacteria containing the genus Bacillus such as Bacillus subtilis are thus relatively stable at high temperature in contrast to the enzyme according to the invention 55 that at 10 minutes heating at 50 ° C and a pH 10 loses most of its activity. API-21 is stable at 10 minutes heating to 40 ° C and a pH 10. At 10 minutes heating at 50 ° C and a pH 8 deactivation occurs and after 10 minutes heating at 60 ° C and a pH is 8 the activity for the most part 194443 8 disappeared. The above-mentioned differences between the known basic proteases and API-21 are summarized in Table B below.

TABLE B

5 - basic protease from molecular isoelectric maintenance of weight point activity (%) at 10 minutes of heating at a pH of 10

. 50 ° C 60 ° C

Bacillus licheniformis 27.2873) 9.43> 78 2

Bacillus subtilis 27.5374) 9.14) 70 1 15 Bacillus alcalophilus NCIB 10436 - 94 73

Bacillus alcalophilus NCIB 10438 25,000 - 85 63

Bacillus NKS-21 (API-21) 22,000 7.4 6 1 20 3) Fette Seiten Anstrichmittel. 75, p. 49 (1973) 4) The Enzymes vol III p. 564 (1971) API-21 can be used in detergents as a builder because it is stable at pH 7-11.5 and the highest enzymatic activity is achieved at a pH of 10-11. Even at a relatively low temperature of, for example, 20-30 ° C, the high activity is maintained in contrast to the known enzymes in detergent compositions. API-21 is therefore very suitable in washing agents for room temperature washing in countries such as Japan. Furthermore, the enzyme can be used to treat waste (household waste) and waste water to rapidly decompose the protein material contained therein. Another application is in the food industry because after use the enzyme can be rapidly deactivated by heating at a relatively low temperature of 50-60 ° C. This makes the enzyme suitable for quality control of the processed food.

When API-21 is included in detergents, it is usually in an amount of 5-500 nKatal (or 10'9 Katal), preferably 10-100 nKatal per gram of detergent, but these limits are not mandatory.

In addition to API-21 in the amount indicated above, the detergent compositions in which it is used also contain conventional ingredients. Examples thereof are 10-50% by weight of surfactant, up to 50% by weight of builder, 1-50% by weight of a basic agent or inorganic electrolyte, 0.1-5% by weight of an anti-redeposition agent, an enzyme , a bleaching agent, an optical or fluorescent dye, an anti-caking agent, and an antioxidant. To prevent spraying, it is recommended to include API-21 as a solution in the detergent.

EXAMPLE 1

The new strain NKS-21 was grown on an oblique culture medium. The culture product was suspended in sterilized water and 0.5 ml of the suspension was used to inoculate.

An enzyme-forming culture medium containing 1% by weight of casein, 1% by weight of meat extract, 1% by weight of polypeptone and 1% by weight of sodium hydrogen carbonate was adjusted to pH 9.5 or 7.2 by adding aqueous sodium hydroxide or hydrochloric acid.

A. 100 ml, 200 ml or 300 ml of the prepared media with a pH of 9.5 were placed in 1 I Erlenmeyer flasks. After sterilization, inoculation was carried out with 0.5 ml of the NKS-21 suspension prepared above. The cultivation was carried out 50 on a shaking table at 20 ° C and 200 rpm. for 41.65 or 89 hours.

B. 100 ml of the prepared medium with a pH 9.5 or 7.2 were placed in a 500 ml Sakaguchi flask. After sterilization, inoculation was carried out with 0.5 ml of the NKS-21 suspension prepared above. Culturing was performed on a shaking table at 30 ° C and 110 rpm. for 66 or 90 hours.

After the culture was completed, the culture fluid was centrifuged for 15 minutes at 28,000 x g. The supernatant liquid was separated and the enzymatic activity of the solution was determined as described above. The results are given in Tables C and D below.

9 194443

TABLE C

Results of A) 5 (20 ° C, starting medium pH 9.5) enzymatic activity (microcatal / liter) amount of medium (ml) 41 hours 65 hours 89 hours 100 1.3 2.6 2.7 10 200 2 0 4.3 4.0 300 2.5 5.6 5.4

TABLE D

^ results of B) (30 ° C) enzymatic activity (microcatal / liter) starting pH of the medium 66 hours 90 hours 20 9.6 4.5 3.0 7.2 4.9 3.4

When growing NKS-21 on a shaking table at 30 ° C, the growth was good not only at a starting pH of the medium of 9.5 but also at a starting pH of 7.2 while the rate at which API-21 was formed in both was high. And similar results as with cultures at 20 ° C and an initial pH of 9.5 were obtained with cultures at 20 ° C and an initial pH of 7.2.

7 l of the solution obtained after centrifugation of the culture liquid and separation were cooled to 3 ° C. Ethanol previously cooled to 3 ° C was added in 2 x the volume amount to form a precipitate. The precipitate was centrifuged in a cooled centrifuge at 11,000 x g and immediately freeze-dried in vacuo. Thus, 15.7 g (33% yield) of the enzyme was obtained as a powder. The specific activity was 0.71 pKatal / kg of protein (determination according to Lowry).

The freeze-dried enzyme was dissolved in 0.01 M sodium carbonate boric acid potassium chloride buffer (pH 10.0) containing 0.2 M sodium chloride. The enzyme solution was subjected to gel filtration over a column (diameter 2 cm, length 74 cm) packed with Sephadex G-75 previously equilibrated with the aforementioned buffer (from Pharmacia Fine Chemicals AB, Sweden). The eluate was collected in 3 ml fractions and the purified enzyme was obtained from the main fractions with the highest enzymatic activity. Molecular weight: approximately 22,000 as estimated by gel filtration on Sephadex G-75 at pH 1). Isoelectric point: 7.4 as estimated by an electrofocusing method.

The specific activity of the enzyme was increased by about 10 x due to gel filtration and was 7.0 pKatal / kg of protein. The yield was 70%.

EXAMPLE II

A culture medium containing 1% by weight of casein, 1% by weight of meat extract, 1% by weight of polypeptone and 1% by weight of sodium hydrogen carbonate was adjusted to pH 9.5 by adding aqueous sodium hydroxide. 300 ml of the medium was placed in a 1 liter Erlenmeyer flask. After sterilization, inoculation was carried out with 0.5 ml of an NKS-21 suspension as prepared in Example 1. The cultivation was carried out at 20 ° C for 3 days.

Afterwards, 300 ml of the culture product was placed in a sterilized 30 I fermenter containing 10 I of the culture medium prepared above with a pH of 9.5. The fermentation was carried out aerobically with stirring at 20 ° C for 3 days at a stirring speed of 100 rpm. and aeration rate of 12 l / min.

101 of the culture product was placed in a sterilized 500 liter fermenter containing 220 liters of the culture medium prepared above with a pH of 9.5. The fermentation was again aerobic and carried out with stirring at 20 ° C but for 4 days at a stirring speed of 400 rpm. and an aeration rate of 55 l / min.

After completion, the culture fluid was centrifuged at 11,000 x g in a cooled, continuous centrifuge.

The supernatant was separated in an amount of 200 l and cooled to 3 ° C. Ethanol cooled before 194443 to 3 ° C was added in 2 x volume by precipitation of API-21.

The enzyme was centrifuged off at approximately 11,000 x g in a cooled, continuous centrifuge and immediately freeze-dried in vacuo. Thus, 1230 g (70% yield) of API-21 was obtained with a specific activity of 0.52 pKatal / kg of protein.

The enzyme was purified in the same manner as in Example I. Molecular weight: approximately 22,000. Isoelectric point: at pH 7.4.

EXAMPLE III

API-21 and a commercially available basic protease were included in a detergent with which tissues with blood stains were washed.

A. Preparation of cotton fabrics stained with whole blood

Tissues turned into fresh total bovine blood under the conditions specified below, to which immediately after collection 1 part by volume of sodium citrate solution per 9 vol. parts of blood were added to prevent ecagulation, immersed, after which they were wrung with a mangle for 70% to obtain unevenly stained tissues. The fabrics were air dried with shielding from direct sunlight and then stored in the dark at 0-5 ° C. The fabrics were then cut into 10 x 5 pieces.

20 Tissues: 10 cm x 15 cm bovine blood: 100 ml per 10 tissues

Stain temperature: 10 ± 2 ° C

staining time: 5 minutes (the stirring direction was changed every 30 seconds) B. Washing procedure

The fabrics were washed under the conditions indicated below and rinsed (3x) in a Terg-O-Tometer.

30

Washing conditions stained fabrics: 5 cm x 10 cm detergent: 0.133% (0.7 nKat / ml enzyme bath ratio: 1:85 stirring: 105 rpm.

washing time: 10 min.

Rinse conditions stained fabrics: 5 cm x 10 cm bath ratio: 1:85 40 stirring: 105 rpm.

rinsing time: 3 min.

After rinsing, the fabrics were air dried while shielding against direct sunlight.

45 C. Used detergent and enzyme

The following six phosphate and non-phosphate detergents were used

Composition with phosphate without phosphate surfactant 15% 15% 50 sodium tripolyphosphate 17 zeolite - 17 sodium silicate 5 5 sodium carbonate 3 3 carboxymethyl cellulose 1 1 55 water 8 8 sodium sulfate remainder remainder 11 194443

The pH of each detergent at a concentration of 0.133% by weight and a temperature of 25 ° C had the following value:

Surfactant with phosphate without phosphate 5 NAS type1) 10.18 10.18 NDS type2) 9.76 9.86 AAS type3) 9.95 10.17 1) NAS: sodium linear alkyl benzene sulfonate 10 2) NDS: sodium dodecyl sulfate 3) AAS: alpha olefin sulfonate

API-21 and a commercially available basic protease were used as enzymes.

The cleaning effect was determined as follows:

The stained fabrics were extracted with 100 ml of a 0.1 N for 2 hours before and after washing

aqueous sodium hydroxide solution with a temperature of 90 ± 2 ° C. The extract was stained with a copper Folin reagent and the absorbance was measured at a wavelength of 750 nm. The cleaning effect (D) was calculated from the relation: OW-SE ^ 1'100

Da = absorption of stained tissue extract (1 g) washed without enzyme Db = absorption of stained tissue extract (1 g) before washing with enzyme Dc = absorption of stained tissue extract - (1 g) after washing with enzyme 25

TABLE E

surfactant phosphate wash-cleaning effect,% temperature

30 ° C

water without API-21 commercial enzyme protease 35 NAS type with 68.6 85.7 78.6 without 68.8 82.0 77.3 NDS type with 10eC 35.0 68.0 85.6 77.7 without 70.2 85.8 80.9 AAS type with 69.4 87.0 80.4 40 without 68.0 86.9 80.0 NAS type with 73.9 89.8 88.6 without 72.0 90.0 88, 5 AAS type with 75.9 91.6 91.6 without 74.5 90.0 91.6 45 NAS type with 73.8 91.7 90.0 without 74.8 94.3 92.1 AAS type with 80 3 92.6 90.5 without 79.0 91.2 91.0 50

EXAMPLE IV

The washing test in Example III was repeated with the non-phosphate API-21 containing detergent and based on the NAS type surfactant, and with different protease activities. The results are shown in Table F below.

194443 12

TABLE F

Cleaning effect,% protease activity wash temperature 5 in detergent -

(nKat / ml) 10 ° C 25 ° C 40 ° C

0 68.8 72.0 74.8 0.7 78.2 87.8 89.4 10 2.1 80.5 88.6 92.8 3.5 80.8 89.4 92.8 7 82, 0 89.8 94.3

Example V

Dirty neckbands were washed with various detergents containing API-21 or a commercially available basic protease.

A. Preparation of dirty neck collars.

200 dirty neck straps of 7 x 37 cm made from standard cotton fabric were cut into 1 x 5 cm pieces. The neckbands were soiled by wearing for 1 week according to the method described in Seni Seihin Shohi Kagaku 19 (1978), pages 106-115. Of the neckband pieces, 10 were sewn together to obtain a test fabric of 5 x 10 cm. The test tissues were stored in the dark at a temperature of 0-5 ° C.

B. Washing Procedure The fabrics were washed under the conditions indicated below and rinsed (3x) in a Terg-0-Tometer.

Washing conditions for dirty fabrics: 5 cm x 10 cm of detergent: 0.133% (0.7 nKat / ml enzyme bath ratio: 1:85 stirring: 105 rpm.

washing time: 10 min.

Rinse conditions 35 dirty fabrics: 5 cm x 10 cm bath ratio: 1:85 stirring: 105 rpm.

rinsing time: 3 min.

40

After rinsing, the fabrics were air dried while shielding against direct sunlight.

C. Detergent and enzyme used.

The following four phosphate and non-phosphate detergents were used: 45 Composition with phosphate without phosphate surfactant 15% 15% sodium tripolyphosphate 17 zeolite (4A) - 17 sodium silicate 5 5 50 sodium carbonate 3 3 carboxymethyl cellulose 1 1 water 8 8 sodium sulfate remainder remainder

The pH of each detergent at a concentration of 0.133% by weight and a temperature of 25 ° C had the following value: 55

Claims (3)

5 13 194443 surfactant with phosphate without phosphate NAS type ’> 10.18 10.18 AAS type1 2 9.95 10.17 1. NAS: sodium linear alkyl benzene sulfonate A basic protease API-21 produced by the Bacillus strain of claim 1, which protease has the following properties: 1. activity: hydrolyses casein under basic conditions, optimum activity at a pH 10-11 and a temperature of 45-50 ° C, 2. stability: no reduction in enzymatic activity at 10 min. Heating to 40 ° C at pH 10 45 in the absence of substrate, reduction in enzymatic activity of 90% or more at 10 min. 2 heating at 50 ° C and a pH 10, starting deactivation at 10 minutes of heating at 50 ° C and a pH 8 and complete or almost complete deactivation at 10 minutes of heating at 60 ° C and a pH 8, stable under basic conditions, in particular at a pH 7-11.5, and resistant to freezing and freeze-drying, and 194443 14 3. enzyme molecule: the protease is a protein with a molecular weight of approximately 22,000 as estimated by a gel filtration method, an isoelectric point of 7.4 as estimated by a e electrofocusing method, an absorption peak in the UV spectrum at 275-282 nm, an IR absorption spectrum as shown in Figure 5, and a serine residue at the active site. 2. AAS: alpha-olefin sulfonate API-21 and a commercially available basic protease were used as enzymes. The results are given in Table G below. TABLE G 15 cleaning effect,% wash - surfactant phosphate tempera water without API-21 trade, ° C enzyme protease 20 - NAS type with 70.1 82.0 77.9 without 66.7 82, 1 73.6 AAS type with 10 49.3 80.4 87.4 86.4 without 75.0 86.8 83.3 25 NAS type with 78.7 85.7 79.0 without 78.0 80.1 74.2 AAS type with 25 52.1 80.4 89.6 87.3 without 80.2 91.2 88.1 NAS type with 80.3 86.8 86.9 30 without 81.0 91.2 89.7 NAS type with 40 58.3 82.6 90.1 89.0 without 86.1 89.1 90.4 35 Bacillus strain, deposited and available from the Fermentation Research Institute (FRI) in Japan as Bacillus sp. FERM BP-93, which is capable of producing a protease under basic conditions. An enzyme-containing detergent composition, which detergent composition comprises the basic protease API-21 of claim 2. Hereby 4 sheets of drawing