DE3328619C2 - - Google Patents

Description

The invention relates to a novel alkaline protease of Designation API-21, a process for their preparation and their use as a constituent of a detergent (cf. the claims).

Alkaline protease is widely available at various levels Used in areas such as the leather industry, Food industry, fiber or textile industry or in medicine and the pharmaceutical industry. It is to assume that the reason for this is that alkaline Protease fairly stable compared to neutral protease against heat. In addition, lately the demand for enzymes as a detergent additive has risen.  

Enzymes that are used in practice as detergent additives are alkaline proteases that are different from Bacteria of the genus Bacillus derive an activity maximum at a pH of about 9 to about 10 demonstrate. Most of the enzymes mentioned above show maximum activity at a relatively high temperature, in particular at about 60 ° C, but are inactive or less active at relatively low temperatures, in particular in the range of room temperature. This means that in a country like Japan, where the laundry is in generally washed at room temperature, the properties not fully exploited by enzymes become. The development of enzymes that their enzymatic Maintain activity even at relatively low temperature, In addition, lately also because of increased use of synthetic fiber garments, have relatively low heat resistance, and because of the recent efforts to save Energy has become necessary.

The invention is therefore the object of the above explained disadvantages of the known alkaline Turn off protease and a novel alkaline To provide protease that is highly enzymatic Activity even at relatively low temperature has. The object of the invention is also to provide a method for Preparation of a new alkaline protease with high enzymatic activity from a new bacterium able to create this alkaline protease to create.

The invention relates to an alkaline protease the term API-21, the following physicochemical properties  having:

• (1) Activity: hydrolysis of casein under alkaline Conditions, at an optimum pH range of 10 to 11 and has an optimal temperature range for the activity from 45 ° C to 50 ° C;
• (2) Stability: No reduction in enzymatic activity is observed when heating without substrate for 10 minutes at a pH of 10 up to 40 ° C, but when heated at 50 ° C for 10 minutes at a pH of 10 observed a 90% or greater reduction in enzymatic activity. Deactivation occurs when heated to 50 ° C for 10 minutes at a pH of 8, and complete deactivation is essentially observed upon heating to 60 ° C for 10 minutes at pH 8.
  The protease is stable under alkaline conditions, especially in the pH range of 7 to 11.5, and is also stable against freezing and freeze-drying; and
• (3) Enzyme molecule: The protease is a protein having a molecular weight of about 22,000 as determined by gel filtration on Sephadex G-75 (trade mark) at a pH of 10, an isoelectric point of 7.4 as determined by aid of the electrofocusing method, an absorbance maximum of 275 to 282 nm in the UV absorption spectrum, IR absorption characteristics shown in Fig. 5, and an active site serine residue.

According to the invention, there is also provided a process for the preparation of the alkaline protease API-21 which comprises the following process steps:
Breeding Bacillus sp. FERM BP-93 or a mutant induced in a known manner which is capable of forming alkaline protease API-21 in the alkaline culture medium, and recovering the alkaline protease API-21 from the culture product.

The invention will be described below with reference to FIGS Drawings explained. Clarified in these drawings

Fig. 1 shows graphically the optimum pH range of the enzyme according to the invention;

Fig. 2 shows graphically the stability of the enzyme according to the invention without substrate as a function of the pH;

Fig. 3 graphically illustrates the temperature range for the activity and the optimum temperature of the enzyme of the invention;

Figures 4A and 4B graphically illustrate the stability of the enzyme of the present invention without substrate at pH's of 10 and 8, respectively, as a function of temperature, and

Fig. 5 shows the Fourier transformed infrared spectrum of the enzyme according to the invention.

The invention will be described below with reference to preferred embodiments described. The invention is investigations underlying bacteria, the alkaline protease produce with high activity at relatively low temperature can. As a result of classification and investigation numerous bacterial strains that are derived from nature, d. H. were isolated from the soil, became a new bacterium of the genus Bacillus sp. nov. NKS-21 (hereafter also known as NKS-21), which is capable of alkaline protease with high activity at relatively low levels To produce temperature. This strain was dating natural soil at Fuchu-shi, Tokyo, Japan, isolated. Based on the microbiological properties of this strain it was found that the strain NKS-21 according to the invention,  which is capable of the alkaline protease of the invention API-21, a new aerobic sporangium bacterium which belongs to the genus Bacillus.

This bacterium NKS-21 has been on 3 February 1982 at Fermentation Research Institute (FRI), Japan, as Bacillus sp. FERM BP-93 under the conditions of the Budapest Treaty on the International Recognition of Deposit of microorganisms for the purpose of Patent procedure deposited.

The microbiological properties of this bacterium are explained below. The reagents and isolation methods used to determine the microbiological properties of the bacterium NKS-21 are as described in Bergey's Manual of Determinative Bacteriology, 8th Edition (1974). The isolation of the bacterium from the soil was carried out in the following way:
A small amount of the soil was added to a peptone medium, and culturing was carried out in a shaking fermenter at a pH of 7 to 10 and a temperature of 10 to 40 ° C for 40 to 150 hours. A portion of the culture product was spread on agar plates and the strain was isolated in pure form by a conventional isolation method.

Microbiological properties of NKS-21

• (a) Morphological properties
  • (1) Size of vegetative cells:
    0.6-0.8 μm × 2.5-3.5 μm
  • (2) Pleomorphism of cells:
    The cells sometimes enlarge to a size of 0.6-1.0 μm × 4.5 μm.
  • (3) Gram stain:
    Positive, however, is easily discolored.
  • (4) agility:
    Agile with peritrichic flagella.
  • (5) Spores:
    Oval to elliptic, subterminal, 0.8 μm × 1.2 μm.
  • (6) Sporangia:
    Slightly thickened.
  • (7) Acid resistance:
    Negative.
• b) Cultural properties in different media
  • (1) Peptone broth:
    • (i) pH 9.5: Good growth both at 20 ° C and at 30 ° C;
      pH 7.2: sparse growth at 20 ° C and good growth at 30 ° C;
    • (ii) growth temperature at a pH of 9.5
      Growth between 14 ° C and 41 ° C
      No growth above 43 ° C or below 12 ° C.
  • (2) Other media:
    Meat extract-peptone agar slants:
    Good growth
    Meat extract peptone broth containing 7% sodium chloride:
    Good growth
    Meat extract-peptone broth:
    Good growth
    Casein meat extract-peptone slant:
    Good growth
    Casein meat extract-peptone broth:
    Good growth
    Strength peptone broth:
    Good growth
    Glucose-peptone broth:
    Good growth
    Tripton-yeast extract agar plate:
    Good growth
    Strength yeast extract broth:
    growth
    Meat extract gelatin stab culture:
    • (i) pH 10.0, 30 ° C:
      Growth and liquefaction
    • (ii) pH 10.0, 20 ° C:
      Weak surface growth and low liquefaction
    • (iii) pH 7.2, 30 ° C:
      Growth and liquefaction
    • (iv) pH 7.2, 20 ° C:
      Weak surface growth and low liquefaction.
  • (3) Growth under anaerobic conditions:
    No growth under anaerobic conditions, but growth under aerobic conditions.
• (c) Physiological properties
  • (1) Reduction of Nitrate to Nitrite:
    No reduction
  • (2) Anaerobic formation of gas from nitrate:
    negative
  • (3) Methyl red (MR) test:
    negative
  • (4) Voges-Proskauer (VP) test:
    negative
  • (5) Formation of indole:
    None
  • (6) Formation of H₂S:
    None
  • (7) Hydrolysis of starch:
    positive
  • (8) Utilization of citrate:
    positive
  • (9) Utilization of inorganic nitrogen sources:
    Uses nitrates and ammonium salts
  • (10) Formation of pigment:
    None
  • (11) urease test:
    negative
  • (12) cytochrome oxidase test:
    positive
  • (13) Catalase test:
    positive
  • (14) OF test (Hugh and Leifson medium):
    Weak fermentation
  • (15) Acid formation and gas production from carbohydrates:
  • (16) Other properties:
    • (i) Deamination of phenylalanine:
      positive
    • (ii) hydrolysis of casein:
      positive
    • (iii) Decomposition of Tyrosine:
      positive

As shown above, the inventively used Strain NKS-21 an anaerobic sporangium bacterium that is clearly attributable to the genus Bacillus. The characteristic Properties of this strain are that the stem grows in the neutral and alkaline medium and that the optimal pH range for growth is 8 to 10 is. These microbiological properties are similar those of the known strains Bacillus pasteurii and Bacillus alcalophilus. The strain NKS-21 differs However, from the above two Strains for some microbiological properties, although some of the microbiological properties of the strain NKS-21 the same as that of the The above-mentioned known strains are. To grow For example, both strains Bacillus pasteurii and Bacillus alcalophilus in the alkaline pH range, the however, the latter can not grow at a pH of 7. In contrast, NKS-21 grows well in the alkaline pH range, However, it still grows well with a neutral one pH. Thus, NKS-21 is different with respect to this point of Bacillus alcalophilus. In addition, it is Bacillus pasteurii not capable of hydrolysis of starch, however, NKS-21 hydrolyzes starch. also NKS-21 differs from Bacillus pasteurii in the following Properties:

• (1) Bacillus pasteurii reduces nitrates, while NKS-21 Nitrates not reduced;
• (2) Bacillus pasteurii has endospores in spherical or almost oval shape, while the endospores of NKS-21 are oval to elliptical, and
• (3) Bacillus pasteurii decomposes urea while NKS-21 Urea does not decompose.

With regard to the aforementioned different microbiological properties, NKS-21 is significantly different of Bacillus pasteurii.

The classification data of Bacillus alcalophilus are in the aforementioned "Bergey's Manual of Determinative Bacteriology "is not described in detail since a standard strain Bacillus alcalophilus was not available the microbiological properties of NKS-21 experimentally with those of the original strains Bacillus alcalophilus NCIB 10436 and NCIB 10438 were compared in "Bergey's Manual of Determinative Bacteriology. "Both Strains NCIB 10436 and NCIB 10438 showed a slightly positive Reaction at the Gram stain and became easy discolored. These properties are similar to those of NKS-21.

With regard to the morphological properties of the vegetative Cells were under an electron microscope for NCIB 10436 short rods of a size of 0.7 μm × 2.0 μm and long rods of size 0.8 μm x 4.6 μm observed in NCIB 10438 vegetative cells of a Size of 0.7 microns × 1.5 microns were observed.

In contrast, the observation under the electron microscope revealed at NKS-21 a size of the vegetative Cells of 0.6-0.8 μm × 2.5-3.5 μm and showed that the endospores oval to elliptical shape and one size of 0.8 μm × 1.2 μm. At NCIB 10436 and 10438 was  no mobility observed while NKS-21 is mobile is. In addition, the physiological differ Properties of NKS-21 from those of strains NCIB 10436 and NCIB 10438 clearly in the following points:

• (1) Bacillus alcalophilus NCIB 10436 and NCIB 10438 did not grow in a mannitol medium while NKS-21 grew in this medium; and
• (2) NCIB 10436 did not grow on meat extract peptone broth containing 7% sodium chloride while NCIB 10438 and NKS-21 in this medium grew.

Distinguished by the differences explained above NKS-21 from Bacillus alcalophilus NCIB 10436 and NCIB 10438, albeit with some microbiological properties of NKS-21 similar to those of Bacillus alcalophilus are.

In addition, NKS-21 resembles Bacillus subtilis in that the Size of vegetative cells of Bacillus subtilis 0.7 to 0.8 microns wide and 2 to 3 microns long and that Bacillus subtilis starch decomposes.

However, NKS-21 is different in terms of the following points of Bacillus subtilis:

• (1) Bacillus subtilis grows well at a pH from 5.5 to 8.5, but does not grow at one pH from 9 to 10, with the NKS-21 good Growth shows, and
• (2) the maximum growth temperature of Bacillus subtilis is 45 to 55 ° C, while NKS-21 at a temperature of more than 43 ° C not grows.

NKS-21 is therefore distinctly different from Bacillus subtilis.  

Thus, NKS-21 differs in terms of microbiological Properties clearly from the known strains Bacillus pasteurii, Bacillus alcalophilus and Bacillus subtilis and in "Bergey's Manual of Determinative Bacteriology "is not described a strain that the has the same microbiological properties as NKS-21. NKS-21 was therefore the new strain of the genus Bacillus identified.

According to the invention, a new alkaline protease API-21, the high activity at relatively low temperature Owned by the new strain NKS-21 in one alkaline culture medium is grown and the alkaline Protease is recovered from the culture product. The invention Process for the preparation of the alkaline protease is not limited to the breeding of NKS-21, but can also be done by natural ones or artificial mutants of NKS-21 are grown, capable of producing alkaline protease API-21 are.

The following is the new alkaline protease API-21, obtained by culturing NKS-21 according to the invention will be explained in detail.

NKS-21 can be bred aerobically, for example in one Peptone culture medium with a pH of 8 to 10 with the help of a shaking culture or an aerobic stirring culture at a temperature of 10 ° C to 40 ° C during 40 to 150 hours after NKS-21 in the culture medium was inoculated. After completion of the breeding can be the cultured microbial cells easily from the culture medium and it becomes a clear supernatant culture solution receive. The alkaline protease API-21 can be in easier  Way by adding an organic solvent, such as ethanol, to the supernatant solution, to be precipitated. The precipitated alkaline protease API-21 is subjected to centrifugal separation and in vacuo freeze-dried. In this way, the desired Enzyme, alkaline protease API-21.

The activity of the alkaline protease thus obtained API-21 is determined in the following way.

The clear culture solution or the enzyme solution is dissolved in suitably with 0.1 M sodium carbonate-0.1 M boric acid-potassium chloride buffer solution (pH 10.0). To 0.5 ml the resulting solution is 0.5 ml of a 2% milk casein solution added with a pH of 10.0 and the enzymatic reaction is then at 30 ° C for 10 minutes carried out. After completion of the reaction, 2 ml a 0.1 M trichloroacetic acid containing 0.2 M acetic acid and 0.2 M sodium acetate, added to the reaction mixture, to stop the reaction. The mixture becomes 10 Let stand for minutes or more at 30 ° C and then through Filtered filter paper. 5 ml of 0.4 M sodium carbonate and 1 ml to 5 times diluted "phenol reagent" to 1 ml of the filtrate obtained above and the mixture is allowed to stand at 30 ° C for 20 minutes, to develop the color. After that, the absorption becomes of the colored mixture measured at 660 nm. The unit of the enzyme is determined according to "Commission on Biochemical Nomenclature "(Comprehensive Biochemistry Vol. 13, p. 26/27, 1963), according to which the amount of alkaline protease which is soluble in trichloroacetic acid Substances at a pH of 10.0 at 30 ° C during one second from 1% casein substrate released at 660 nm to a color development corresponding to 1 mole Tyrosine leads, defined as "1 katal".

The physico-chemical properties of the alkaline Protease API-21 (hereinafter referred to as the enzyme of the present invention  will be described in more detail.

(1) activity

Hydrolyzes proteins such as casein, hemoglobin, albumin, Globulin, meat protein, fish protein and soybean protein.

(2) Influence of pH on enzymatic activity

As shown in Fig. 1, the optimum pH is about 10 to about 11. The relative activity was determined as follows.

0.5 ml of each of the buffer solutions listed below containing 2% milk casein were added to 50 μl of Enzyme solution added to a test solution manufacture.

pH buffer solution 6-9 0.1M potassium phosphate 0.05M sodium borate 9-11 0.1M sodium carbonate-0.1M boric acid potassium chloride 11-12 0.1 M disodium hydrogen phosphate sodium hydroxide

The enzymatic activity of the test solution was in the above determined manner. The relative activity (%) was calculated at which the enzymatic activity at a pH of 10.0 was defined as 100%.

(3) Influence of pH on stability without substrate

As shown in Figure 2, the enzyme of the present invention is stable in the pH range of about 7 to about 11.5.

The stability of the enzyme according to the invention was in the following Way determined.

0.5 ml of each of the buffer solutions listed below was added to 50 μl of the enzyme solution containing dialyzed and diluted appropriately, and that Mixture was incubated at 30 ° C for 10 minutes.

pH buffer solution 3-8 0.1 m citric acid 0.2 m disodium hydrogen phosphate 8-11 0.1M sodium carbonate-0.1M boric acid potassium chloride 11-12 0.15 M disodium hydrogen phosphate sodium hydroxide

After incubation, the mixture was 9.5 ml of a 0.1M carbonate buffer solution containing 2% casein and having a pH of 10.0. The enzymatic Activity of the mixture has been described in the above Way determined. The relative activity (%) was calculated at which the enzymatic activity at a pH of 10 was defined as 100%.

As shown in Fig. 2, the enzyme of the present invention was most stable at a pH of about 10 to about 11. In this pH range, the original activity of the enzyme of the present invention was completely retained. In addition, the enzyme showed 80% or more retained activity at a pH of 7 to 11.5.

(4) Influence of temperature on the enzymatic activity

As shown in Fig. 3, the enzyme of the present invention was active against casein at a temperature of 5 to 65 ° C and a pH of 10.0. The optimum temperature was 45 to 50 ° C.

(5) Influence of temperature on stability without substrate

As shown in Figs. 4A and 4B, the activity of the enzyme of the present invention is fully maintained up to 40 ° C when it is heated without a substrate at a pH of 10.0 for 10 minutes; however, most of the activity was lost when heated to 50 ° C. In addition, the activity of the enzyme of the present invention is maintained at a pH of 8 for 10 minutes up to a temperature of 45 ° C, but a deactivation occurred at 50 ° C and the enzyme was substantially deactivated at 60 ° C.

(6) Shelf life

The enzyme of the invention is resistant to Freezing and freeze-drying. No significant waste the activity of a freeze-dried sample observed when the sample is at room temperature 2 weeks was left. When the freeze-dried Sample was stored at room temperature in a desiccator, the activity of the enzyme according to the invention was completed maintained.

(7) Inhibition

The enzyme according to the invention was replaced by an inhibitor for the active serine residue, such as diisopropylfluorophosphoric acid (DFP) and phenylmethanesulfonyl fluoride (PMSF) significantly inhibited. It was also characterized by benzyloxycarbonylphenylalanine chloromethyl ketone (BPCK) inhibited. The However, the enzyme according to the invention was not replaced by tosylphenylalanine chloromethyl ketone (TPCK) inhibits one Inhibitor of an animal serine protease, chymotrypsin, and was not inhibited by tosyl lysine chloromethyl ketone (TLCK), a trypsin inhibitor.

In addition, the activity of the enzyme of the invention was by adding p-chloro-mercury benzoate (PCMB) and  Ethylenediaminetetraacetate (EDTA) is not affected. In addition, the activity of the enzyme of the invention was not affected by the addition of pepstatin. The aforementioned inhibition experiments clearly show the enzyme according to the invention is a protease which has a Serine residue in the active center.

(8) Method for determination of enzymatic activity

As explained above.

(9) Molecular weight

The molecular weight of the enzyme according to the invention is about 22,000 as determined by gel filtration on Sephadex (trademark) G-75 at a pH of 10.

(10) Isoelectric point

The isoelectric point of the invention Enzyme is 7.4, determined by the Elektrofocusiermethode.

(11) Ultraviolet (UV) absorption

The absorption maximum lies in the UV absorption spectrum at 275 to 282 μm.

(12) Infrared (IR) absorption spectrum

As shown in Fig. 5.

(13) Elemental analysis

Not performed because of characteristic differences of high molecular substances of this kind, such as  represents the enzyme of the invention, by comparison the contents of elements, such as carbon, nitrogen or Hydrogen, not expressed.

(14) Amino acid composition

The composition of API-21 from each amino acid residue was tentatively calculated based on the results of acid hydrolysis of the enzyme except for cysteine / cystine and tryptophan. The determination of cysteine / cystine was carried out by performic acid degradation and the determination of tryptophan by alkaline decomposition according to the method described in the literature (Methods in Enzymology, vol. XI, 1967, Academic Press, New York). The values obtained are shown in Table 1 together with the composition of other alkaline proteases whose data were taken from the literature ^{* 1, * 2)} .

As shown in Table 1, there are major differences between API-21 and the other specified enzymes in the Regard to the amino acid composition, such as serine, Arginine, lysine, valine, alanine, tryptophan, proline etc.  

Table 1

Comparison of amino acid composition between API-21 and other alkaline proteases

From the results shown above it is clear show that the enzyme of the invention, its optimal pH is 10 to 11, its activity at relative maintains low temperature, but at a relative high temperature (i.e., at 50 ° C or above) becomes.

The enzyme of the invention, this physico-chemical Features is below with compared to similar alkaline proteases.

Among extracellular alkaline proteases produced by Gram-positive bacteria are derived, for example the alkaline protease subtilisin Carlsberg, derived from Bacillus licheniformis, and the alkaline ones Protease Subtilisin Novo, derived from Bacillus subtilis well known.

These enzymes are proteases that have a serine residue on the having active center, a molecular weight of about 27,000 to 30,000 and an isoelectric point of 9 to 11 have. Most of the others described extracellular alkaline proteases, different from the genus Derive Bacillus have a molecular weight of 25,000 to 30,000 and an isoelectric point from 9 to 11. Most of the enzymes mentioned above will not when deactivated at a pH of 10 during Are heated to 50 ° C for 10 minutes. Well-known alkaline Lower molecular weight proteases released by the Genus Bacillus are derived, are the alkaline proteases E-1 and E-2 derived from Bacillus No. D-6. Both proteases have a molecular weight of 20,000 and its activity is when heated to 50 ° C at a pH value of 9.0 not diminished at all (JP-AS (Kokoku) 56-4 236). In addition, in this field are as  alkaline proteases derived from the genus Bacillus, one derived from Bacillus alcalophilus NCIB 10436 alkaline protease and one of Bacillus alcalophilus NCIB 10438 derived alkaline protease known. However, these enzymes retain about 90% of their Activity in when they are at a pH for 10 minutes from 10 to 50 ° C, as in Table 2 is shown. As also shown in Table 2 alkaline proteases derived from Bacillus licheniformis and Bacillus subtilis are derived, 70 to 80% of their Activity in when they are at a pH for 10 minutes be heated from 10 to 50 ° C. The known alkaline Proteases derived from bacteria of the genus Bacillus, such as Bacillus subtilis, are thus in one relatively high temperature range stable.

As mentioned above, in contrast, the enzyme according to the invention most of its activity, if it is 10 minutes at a pH of 10 to 50 ° C is heated while it is stable when it is 10 minutes long at a pH of 10 up to a temperature of 40 ° C is heated. In addition, deactivation occurs of the enzyme according to the invention when heated for 10 minutes at about 50 ° C at a pH of 8 and the largest Part of its activity goes below the same at 60 ° C Conditions lost. In terms of differences in the characteristic properties of temperature resistance, molecular weight and isoelectric Point is the enzyme of the invention significantly different from the known alkaline proteases, which derived from bacteria of the genus Bacillus, and this Enzyme has not been described in the literature yet. The enzyme according to the invention is therefore used as a new enzyme and referred to as "alkaline protease API-21".  

Table 2

The enzyme of the invention, API-21, can be used together with be applied to a detergent builder and can thus be used as a detergent additive, since it is stable at a pH of 7 to 11.5 and since, as explained above, the maximum enzymatic Activity is achieved at a pH of 10 to 11. In particular, the enzyme according to the invention retains its high activity even at relatively low temperature (For example, 20 to 30 ° C) compared with conventional Enzyme for detergent at. The enzyme of the invention As a result, it is a particularly desirable type of one alkaline protease, which is used as an additive for household detergents suitable in countries where the laundry is included Room temperature is washed, as in Japan. About that In addition, the enzyme according to the invention can also be more effective Waste treatment and wastewater treatment method for a rapid decomposition of proteins and reuse be used by nitrogen sources, since the protein content household and domestic waste water have risen in recent times. In addition, it is the use of the protease according to the invention in the Food industry, how to food processing advantageous because the enzyme after use by heating at a relatively low temperature, for example 50 to 60 ° C, easily deactivated. The use of the The enzyme according to the invention is therefore in view of Quality control of processed foods extremely effective.

When the enzyme API-21 of the present invention is added to a detergent, the enzyme API-21 will generally be in an amount of 5 to 500 n-catal (or 10 ^-9 catal), desirably 10 to 100 n-catal, based on 1 g of the detergent used, although the amount of the enzyme to be added is not particularly limited. In addition to the enzyme according to the invention API-21, the enzyme-containing detergent may contain all conventional detergent ingredients without their added amount changed over that in conventional detergents. Typical examples of such detergent ingredients include 10 to 50% by weight of a surfactant, 0 to 50% by weight of builder, 1 to 50% by weight of an alkaline agent or inorganic electrolyte, and 0.1 to 5% by weight % of a dirt repellent preventing agent, an enzyme, a bleaching agent, an optical brightener (fluorescent dye), a baking inhibitor and an antioxidant. The incorporation of the enzyme API-21 can be done in any conventional manner. However, it is desirable to add the enzyme in the form of a solution or in such a form to the detergent that dusting is avoided, which is more advantageous in terms of environmental protection.

The invention is illustrated below by the following examples explained. In these examples, all percentages are by weight on the weight, unless otherwise stated.

example 1

The new strain NKS-21 was in a Cultured slant culture medium. The resulting culture product was suspended in sterilized water and 0.5 ml This suspension was used as a seed.

A culture medium for enzyme formation, 1% casein, 1% meat extract, 1% polypeptone and 1% sodium bicarbonate  was added by the addition of aqueous sodium hydroxide or hydrochloric acid to a pH of 9.5 and 7.2, respectively.

In a shaking culture (A) in a rotary shaker were each 100 ml, 200 ml and 300 ml of the above-prepared medium having a pH of 9.5 in a 1 liter Erlenmeyer flask. To sterilization was 0.5 ml of the preparation prepared above NKS-21 suspension in the culture medium and inoculated the cultivation was at 20 ° C and with shaking at 200 rpm performed during 41, 65 and 89 hours.

On the other hand, a shake culture in a Geradschüttelvorrichtung (B), in which 100 ml of the above-mentioned culture medium having a pH of 9.5 or 7.2 in a 500 ml Sakaguchi flask were. After sterilization, 0.5 ml of the above inoculated NKS-21 suspension in the culture medium and the Geradschüttel cultivation was under Shake at 110 cpm (cycles per minute) at 30 ° C during 66 and 90 hours.

After culturing, the culture product became 15 minutes long centrifuged at 28,000 x g and the resulting supernatant Solution was won. The activity of alkaline protease in this solution has been described in the above Way determined.

The results obtained are shown in Tables 3 and 4 shown.  

Table 3

Results of Rotary Shaker Cultivation (A)

(20 ° C, initial pH of the medium: 9.5)

Table 4

Results of the Geradschüttel Culture (B)

(30 ° C)

When NKS-21 is in a straight-shake culture at 30 ° C good growth of NKS-21 was not only at an initial pH of the medium of 9.5, but also at an initial pH of the medium of 7.2 and the desired alkaline protease API-21 was in any case formed in high rate. As in the case of the starting medium with a pH of 9.5 were the same results achieved even if NKS-21 in a straight shaker at 20 ° C and at an initial pH  of the medium of 7.2 was bred.

7 L of after centrifugal separation of the NKS-21 culture product were obtained supernatant solution cooled to 3 ° C. Then it became 2 times the volume Ethyl alcohol previously cooled to 3 ° C, added to the cooled supernatant solution to one To form precipitation. The formed precipitate was centrifuged in a cooled centrifugal separator separated at 11,000 × g and then immediately Freeze-dried under vacuum. In this way were 15.7 g of the desired enzyme in powder form.

The specific activity of the thus obtained alkaline Protease, based on 1 kg of protein, was 0.71 microcatal (0.71 μ katal / kg protein), determined by the Lowry method, and the yield of enzyme was 33%.

The freeze-dried enzyme sample thus obtained was in a 0.01 M sodium carbonate-boric acid-potassium chloride buffer solution (pH 10.0) containing 0.2M sodium chloride, solved. The enzyme solution became gel filtration subjected to a column (2 cm diameter x 74 cm), those with Sephadex G-75 (trademark) was filled, that before equilibrated with the buffer solution indicated above had been. Fractions of 3 ml each were taken. The purified enzyme became major fractions, the high showed enzymatic activity. The thus obtained Enzyme had a molecular weight of about 22,000 by gel filtration on Sephadex G-75 (trademark) at a pH of 10, and an isoelectric point of 7.4, measured by the electrofocusing method. The specific activity of the enzyme thus obtained was in  Comparison with that of the crude enzyme without gel filtration on the Increased 10 times: it became a specific activity based on 1 kg of protein, obtained from 7.0 microcatal. The yield was 70%.

Example 2

The production of alkaline protease in a 500 L fermentation tank and the subsequent production of the Enzyme preparation was made using the alkaline Protease-producing strain NKS-21.

A culture medium for enzyme production, 1% casein, 1% meat extract, 1% polypeptone and 1% sodium bicarbonate was added by the addition of aqueous sodium hydroxide adjusted to a pH of 9.5. 300 ml of the thus prepared Medium were placed in a 1 liter Erlenmeyer flask given. After sterilization, 0.5 ml of the in the in Example 1 described NKS-21 suspension into the culture medium and the culture was carried out at 20 ° C for 3 days. 300 ml of the formed Culture product was placed in a sterilized 30 liter jug fermenter given, the 10 l of the above Culture medium, which had a pH of 9.5 contained. Then, an aerobic culture was added while stirring 3 days at 20 ° C, with a stirring speed of 400 rpm and aeration rate of 12 l / min were complied with.

After breeding in the 30 l jug fermenter for 3 days were 10 l of the resulting culture product in a sterilized 500 l fermentation tank transferred, the 220 l of the culture medium prepared above with a pH of 9.5. Then became an aerobic breeding while stirring for 4 days at 20 ° C at a stirring speed of 400 rpm and aeration rate of 200 l / min  carried out.

After culturing, the culture product was chilled continuous centrifugal separator at Centrifuged 11 000 × g, with 200 l of the supernatant Solution were obtained. This supernatant solution was cooled to 3 ° C. Then it became 2 times the volume Ethyl alcohol previously cooled to 3 ° C, added to the cooled supernatant solution to the precipitate desired alkaline protease. The precipitated alkaline protease API-21 was refrigerated continuous centrifugal separator at about 11 000 × g recovered by centrifugal separation and then immediately freeze-dried in vacuo.

In this way, a freeze-dried enzyme preparation was prepared alkaline protease API-21 with the following characteristics receive.

Weight of the recovered enzyme (g) 1230 specific activity (microcatal / kg protein) 0.52 Recovery yield (%) 70

The enzyme thus obtained was purified in the same manner as in Example 1, wherein alkaline protease with a molecular weight of about 22,000 and an isoelectric Point was obtained at a pH of 7.4.

Example 3

The alkaline protease API-21 obtained above and, as a comparative sample, a commercial alkaline Detergents for detergents were tested by different Detergent compositions were added. With the resulting detergents was then a washing test using blood-stained substances in the following  Manner performed:

A. Preparation of whole blood spotted cotton fabrics

Swatches were tested under the following conditions dipped in fresh bovine whole blood, immediately after this had been won. The blood was 1 volume of one Sodium citrate solution was added to 9 volumes of blood, to prevent clotting. The soiled ones Samples were squeezed to 70% using a defect, so that uniformly polluted substances are obtained were.

Thereafter, the swatches were placed in a room without any effect dried and dried in direct sunlight finally in a cold dark place at one Temperature kept 0 to 5 ° C. After the preparation The swatches were cut into pieces of size 10 cm × 5 cm cut.

Swatches: 10 cm × 15 cm polluting liquid: 100 ml per 10 fabric samples Temperature at pollution: 10 ± 2 ° C Duration of exposure to pollution: 5 minutes (the stirring direction was changed every 30 seconds)

B. washing process

The swatches were 3 times under the following conditions washed and rinsed in a Terg-O-Tometer.

washing conditions soiled pieces of fabric: 5 cm × 10 cm Laundry detergent: 0.133% (0.7 nkat / ml) enzyme Bath ratio: 1: 85 Stir: 105 rpm Washing time: 10 mins

flushing soiled pieces of fabric: 5 cm × 10 cm Bath ratio: 1: 85 Stir: 105 rpm Flushing time: 3 minutes

After rinsing, the swatches were in a room dried in the air without exposure to direct sunlight.

C. Detergent used and enzyme

The following six phosphate-containing and phosphate-free Detergents were used as detergents.

Composition of the detergent

The pH of each detergent, measured in one Concentration of 0.133% at 25 ° C, is in the following Table specified.

pH of the detergents

The enzyme used was a commercial alkaline Protease for detergent or the one prepared above alkaline protease API-21. The results obtained in the experiments Results are shown in Table 5.

The detergent efficiency for protein contamination was determined in the following way:

The polluted substances were before and after washing a heat extraction with 100 ml of a 0.1 N aqueous NaOH solution at a temperature of 90 ± 2 ° C during 120 Minutes, obtaining test fluid samples were. Each test fluid sample became a chemical Subjecting dyeing reaction with Copper Folin reagent and the absorption of the colored sample was at a Wavelength of 750 nm measured.  

The detergent efficiency (D) was as follows Equation calculated:

where Dc = absorbance of the extract from a standard cotton fabric (1 g)
Ds = absorption of the extract from the polluted substance (1 g) before washing
Dw = absorption of the extract from the polluted substance (1 g) after washing

Table 5

Example 4

The washing test procedure according to example 3 was repeated, with the modification that protease activity in the washing solution and the washing temperature in the case of Phosphate-free detergent of the LAS type changed which were the alkaline protease of the invention API-21 included.

The results are shown in Table 6.

Table 6

Example 5

Wash tests on soiled cotton collar or neck fabric samples were using different detergents carried out, which obtained the above alkaline protease API-21 contained and which one commercial alkaline protease for detergent contained. The tests were carried out in the following way.  

A. Preparation of soiled collar fabrics

200 pieces of soiled collar fabrics, made from standard cotton fabrics for detergent tests one Size of 7 cm × 37 cm were in small pieces one Size of 1 cm × 5 cm cut. The used Collar fabrics were worn for one week according to a Method by Motoi Minagawa and Ikuko Okamoto in "Seni Seihin Shohi Kagaku" 19, pp. 106 to 115 (1978) is described. Each 10 small pieces were under Formation of test substances of a size of 5 cm × 10 cm sewn together and at a temperature of 0 to 5 ° C kept in a cool dark place.

B. washing process

The test substances were under the following conditions washed in a Terg-O-Tometer and rinsed (3 times).

washing conditions polluted substances: 5 cm × 10 cm Laundry detergent: 0.133% (0.7 nkat / ml enzyme) Bath ratio: 1: 85 Stir: 105 rpm Washing time: 10 mins

flushing polluted substances: 5 cm × 10 cm Bath ratio: 1: 85 Stir: 105 rpm Flushing time: 3 minutes

After rinsing, the swatches were placed in a room at the  Air dried without being exposed to direct sunlight were suspended.

C. Detergent used and enzyme

The following four phosphate-containing and phosphate-free Detergents were used as detergents.

Composition of the detergent

The pH of each detergent measured in 0.133% Concentration at 25 ° C, had the following values:

pH of the detergent

The enzyme used was a commercial alkaline Protease for detergent or the one described above alkaline protease "API-21". The results achieved are shown in Table 7 below.  

Table 7

Claims (3)

1. Alkaline Protease API-21, characterized by the following properties:

• 1) Activity: Hydrolyzes casein under alkaline conditions, has an optimum pH range of 10 to 11, and has an optimum temperature range of activity of 45 to 50 ° C;
• 2) Stability: When heated without substrate for 10 minutes at a pH of 10 to 40 ° C no reduction in enzymatic activity, when heated for 10 minutes at a pH of 10 to 50 ° C, however, a 90% observed greater or lesser reduction of enzymatic activity; Deactivation when heated for 10 minutes at a pH of 8 to 50 ° C and complete deactivation when heated for 10 minutes at a pH of 8 to 60 ° C;
  the protease is stable under alkaline conditions, especially in the pH range of 7 to 11.5 and is stable to freezing and freeze-drying; and
• 3) Properties of the Enzyme Molecule: Protein of molecular weight about 22,000 as determined by gel filtration on Sephadex® G-75 at pH 10, isoelectric point of 7.4 as determined by the electrofocusing method; Absorption maximum at 275 to 282 nm in the UV absorption spectrum; IR absorption spectrum according to Fig. 5, which is part of this claim, contains a serine residue at the active site.

2. Process for the preparation of the alkaline protease API-21 according to claim 1, characterized in that Bacillus sp. FERM BP-93 or one in a known manner induced mutant responsible for the formation of alkaline API-21 is capable of being in the alkaline Culture medium breeds and the alkaline protease API-21 from the culture product wins. 3. Use of alkaline protease API-21 according to Claim 1 as part of a detergent.