JPS6119679B2 - - Google Patents

Description

[Detailed description of the invention]

TECHNICAL FIELD The present invention relates to enzyme-containing detergent compositions, and more particularly to detergent compositions containing the enzyme alkaline protease, which is capable of hydrolyzing proteins in the alkaline region. PRIOR ART It has been known for a long time to mix proteolytic enzymes into detergent compositions to decompose and remove proteins and other dirt adhering to objects to be washed. Proteolytic enzymes made from bacterial strains are commonly used. However, such alkaline proteases derived from Bacillus bacteria are generally highly active enzymes at high temperatures, and generally have an optimal operating condition around pH 9 to 10, and peak activity at high temperatures, especially around 60°. Many of them lose their enzymatic activity, or at least their activity decreases significantly, at low temperatures, particularly around room temperature. Therefore, in countries like ours where laundry is customary at room temperature, the above-mentioned properties of the enzyme are not fully exhibited. In addition, low-temperature washing is becoming popular not only in Japan but also in countries where washing at high temperatures is customary from the viewpoint of energy conservation, and the need for detergents suitable for low-temperature washing is increasing. However, even in the same country or region, people differ in whether they perform low-temperature washing or high-temperature washing, so it is better to use the same detergent composition than one suitable for low-temperature washing or one suitable for high-temperature washing. Detergents that are suitable for use in a wide range from low to high temperatures are preferred, and there is therefore a desire to develop detergents that are suitable for both low-temperature and high-temperature cleaning. Object and Structure of the Invention The object of the present invention is to provide a plurality of types of alkaline proteases that can clean objects with high cleaning efficiency in a wide temperature range from room temperature or lower to 60°C or higher. An object of the present invention is to provide a detergent composition containing the following. The alkaline protease-containing detergent composition according to the present invention hydrolyzes casein in an alkaline region, has an optimum pH of 10 to 11, an optimum action temperature of 45 to 50°C, a molecular weight of about 22,000 as determined by gel filtration, and a maximum absorption of 275 in the ultraviolet absorption spectrum. The alkaline protease API-21 has the characteristics of ~282 nm and the red line absorption spectrum shown in Figure 5, and the optimal action temperature is API-21.
It comprises 21 or more alkaline proteases. As for the latter alkaline protease,
Alcalase (registered trademark of Novo Industri A/S, Denmark), Esperase (registered trademark of Novo Industri, Denmark)
A/S Company registered trademark), Superase (USA Chas.
Pfizer & CO. (registered trademark), Maxatase (Netherlands)
Subtilisin Carlsberg, subtilisin BPN′, subtilisin contained in commercially available detergent enzyme products such as Gist Brocades NV (registered trademark), Bioplase (Nagase Seikagaku Co., Ltd. registered trademark)・
Novo (subtilisin Novo), subtilisin amylosaccharitakes (subtilisin
Amylosacchariticus), subtilisin A
(subtilisin-A), etc. Description of structure and effects of the invention Among the alkaline proteases blended into the detergent composition of the present invention, the optimum action temperature is API-
21 and more of the alkaline proteases are already well known (e.g. PDBoyer
Edited by The Enzymes vol P561-608 (1971)
(Academic Press)). Another type of alkaline protease API mixed in the present invention
21 is a new enzyme, and as a result of searching for microorganisms that are productive of API-21, such as alkaline protease-producing bacteria in various soils, a new bacterial species belonging to the genus Bacillus, genus Bacillus NKS-, was isolated from soil.
21 (Bacills sp.nov.NKS−21, hereinafter simply NKS−
21), and the applicant previously filed a patent application for this enzyme and its manufacturing method (Patent Application No. 1982-1980, filed on February 8, 1982).
(Refer to Special Publication No. 17596, Publication No. 60-55118). That is, as described in the application specification, the bacterial strain producing alkaline protease APL-21 in the present invention was confirmed to be an aerobic spore-bearing paulownia strain and a new bacterial species belonging to the genus Bacillus based on its mycological properties. did. Based on the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedures, this new strain of Bacillus NKS-21 was transferred to the Institute of Microbial Technology, Agency of Industrial Science and Technology on February 3, 1981. The deposit was made and the deposit number is ``Feikokenjo Deposit No. 93''. Note that the alkaline protease used in the present invention
The microorganism that produces API-21 is the genus Bacillus.
Not only NKS-21 but also natural or artificial mutant strains thereof are naturally included as long as they have productivity of alkaline protease API-21 having the characteristics described below. Next, the novel alkaline protease API-21 used in the present invention obtained by culturing the Bacillus genus NKS-21 will be explained. As the culture conditions, for example, a peptone medium adjusted to pH 8 to 10 is inoculated with the NKS-21 bacteria, and culture is carried out aerobically with shaking or aerated stirring. For example, culture with shaking at 15°C to 40°C for 40 to 150 hours. After the culture was completed, the bacterial cells were separated from the culture to obtain a clear culture supernatant. Alkaline protease API-21 was precipitated by adding an organic solvent such as ethanol to this supernatant.
The precipitated alkaline protease API-21 was centrifuged and vacuum lyophilized to obtain alkaline protease.
Obtain API-21 enzyme preparation. Alkaline protease obtained in this way
The activity of API-21 is measured as follows. The culture clear liquid was mixed with 0.1M sodium carbonate and 0.1M boric acid.
Add 0.5 ml of a 2% milk casein solution (PH 10.0) to 0.5 ml of a solution appropriately diluted with potassium chloride buffer (PH 10.0).
was added, and the enzymatic reaction was carried out at 30°C for 10 to 30 minutes. The enzymatic reaction was terminated by adding 2 ml of 0.1M trichloroacetic acid containing 0.2M acetic acid-0.2M sodium acetate buffer. After being left at 30°C for 10 minutes or more, it was filtered using filter paper. 5 ml of 0.4M soda carbonate to 1 ml of filtrate, 5
Add 1 ml of the diluted phenol reagent and store at 30℃ for 20 minutes.
After leaving it for a minute to develop color, measure the absorbance at 600 nm. The enzyme unit is a trichloroacetic acid-soluble substance that develops a color at 660 nm equivalent to 1 mole of tyrosine in 1 second using 1% casein with a pH of 10.0 as a substrate at 30°C, in accordance with the International Enzyme Committee's "Enzyme Nomenclature". The amount of alkaline protease released is 1 katal. Next, the physical and chemical properties of alkaline protease API-21 used in the present invention will be described. (1) Action and substrate specificity Decomposes proteins such as casein, hemoglobin, albumin, globulin, meat protein, fish protein, and soybean protein. (2) Optimal PH As is clear from Figure 1, the optimal PH is 10 to 11.
has. Note that the relative activity is determined as follows. 0.5ml of each buffer solution (PH6
-9 is 0.1M potassium phosphate - 0.05M sodium borate; PH9-11 is 0.1M sodium carbonate -
0.1M boric acid-potassium chloride; PH11-12 is
A test solution was prepared by adding 2% milk casein solution containing 0.1 M disodium phosphate (caustic soda), and the enzyme titer was measured by the method described above,
Relative activity is determined by setting the enzyme titer at PH10.0 as 100%. (3) Stable PH range As is clear from Figure 2, API-21 has a PH of 7~
It has a stable PH range of 11.5. This evaluation followed the following method. Various buffer solutions (PH3-8: 0.1M citric acid - 0.2M disodium phosphate; pH8-11.0: 0.1M sodium carbonate)
0.1M boric acid-potassium chloride; PH11.0-12.0 is
A test solution was prepared by adding 2% milk casein as a substrate to 0.15M disodium phosphate (caustic soda). Dialyzed with distilled water and appropriately diluted
0.5 ml of the various pH buffer solutions described above were added to 50 µ of the enzyme solution containing API-21, and the mixture was heated at 30°C for 10 minutes. After heating, add 9.5ml of 0.1M carbonate buffer with pH 10.0.
is added, the enzyme is treated with various PHs, the enzyme titer is measured by the method described above, and the relative activity is determined by setting the enzyme titer at PH 10.0 as 100%. As shown in Figure 2, API-21 was most stable at pH 10 to 11, and remained completely active in this pH range. Moreover, it shows residual activity of 80% or more at pH-7 to 11.5. (4) Range of operating temperature As shown in Figure 3, API-21 is 5 to 65℃.
It acts on casein within the range of . Optimum temperature is 45~
It is 50℃. The operating temperature range is PH as above.
Measurements were performed using 10.0. (5) Conditions for inactivation (temperature stability) As shown in Figure 4, API-21 has a pH of 10,
The activity is completely maintained up to 40°C under conditions of 10-minute heat treatment, but most of the activity is lost at 50°C. In addition, under the conditions of PH8 and 10 minutes of heat treatment,
It maintains its activity up to 45°C, but begins to lose its activity at 50°C, and is almost completely inactivated at 60°C. In either case, the samples were left for 10 minutes without adding any substrate. (6) Storage stability API-21 is stable when frozen or freeze-dried. There is almost no loss of activity even if the freeze-dried preparation is left at room temperature (21-22° C.) for two weeks, and if the preparation is stored at room temperature in a desiccator, almost no inactivation is observed. (7) Inhibition and activation API-21 is significantly inhibited by active serine residue inhibitors of proteases such as diisopropylfluorophosphate (DFP) and phenylmethanesulfonyl fluoride (PMSF). However, API-21 is an inhibitor of the animal serine protease chymotrypsin, tosyl-phenylalanine-chloromethyl ketone (TPCK).
Alternatively, it is not inhibited at all by tosyl-lysine-chloromethylketone (TLCK), an inhibitor of trypsin. However, APL-21 is benzyloxycarbonyl-phenylalanine-
Inhibited by chloromethyl ketone (ZPCK). Further, the activity of API-21 is not affected at all by the addition of parachloromercury benzoate (PCMB), and the activity is not affected at all by the addition of ethylenediaminetetraacetate (EDTA). Furthermore, addition of pepstatin to API-21 has no effect on activity. From the above experimental results of inhibitors, it is clear that API-21 is a serine protease with a serine residue in its active center. (8) Method for measuring titer As described above. (9) Molecular weight The molecular weight of API-21 determined by gel filtration method is approx.
22000 (PH using Sephadex G-75
(measured and calculated at 10). (10) Isoelectric point API-21 obtained from the active fraction with a molecular weight of approximately 22,000 by gel filtration method (Sephadex G-75, PH10)
The isoelectric point according to the electrofocusing method is
It is 7.4. (11) Ultraviolet absorption spectrum Maximum absorption exists between 275 and 282 nm. (12) Infrared absorption spectrum The Fourier transform infrared absorption spectrum of API-21 is shown in Figure 5. (13) Elemental analysis Even if you perform elemental analysis of a polymeric substance such as API-21 and calculate the ratio of carbon, hydrogen, nitrogen, and oxygen, it is impossible to show its characteristics.
Elemental analysis measurements were not performed. (14) Amino acid composition The amino acid composition of API-21, excluding cysteine/cystine and tryptophan, is as follows:
“Methods in Enzymology”VOI.XI, 1967
(Academic Press, New York) by enzymatic acid hydrolysis, cysteine/cystine by performic acid decomposition analysis, and tryptophan by alkaline decomposition analysis. It is shown in the table below. This table also lists the compositions of other alkaline proteases described in the literature. As is clear from these results, there are significant differences in amino acid composition between API-21 and other enzymes, such as serine, arginine, lysine, valine, alanine, tryptophan, and proline.

[Table] As is clear from the above results, API-21 retains its activity especially at low temperatures, and
It is relatively easily inactivated and has an optimum pH of 10-11. Novel enzyme API-21 used in the present invention
As mentioned above, it is stable at pH 9 to 10 and exhibits maximum activity at pH 10 to 11, so it can be used in combination with detergent builders. Therefore, it is effective as a detergent enzyme. Compared to conventionally known detergent enzymes, API-21 provides extremely superior cleaning effects when incorporated into detergents, especially at room temperature or lower temperatures. Therefore, in countries like ours where laundry is customary with room temperature water, API-21 is the most preferred type of alkaline protease for use as an auxiliary agent in general household detergents. However, since the optimal temperature for API-21's action is lower than that of other alkaline proteases known to date, at temperatures of 60°C or higher, the enzyme deactivates more quickly than other alkaline proteases. Therefore, even if high-temperature cleaning is performed using API-21, it is difficult to expect a better cleaning effect than when using other alkaline proteases. The inventor has determined that API-21 and the optimum working temperature are API-21.
The detergent composition containing the above-mentioned alkaline protease of -21 or higher cleans objects with high cleaning efficiency in a wide temperature range from room temperature or lower to 60°C or higher. They found that it is possible to do this and completed the present invention. Conventionally, when multiple enzymes coexist, especially proteases, they each exert a digestive action (hydrolytic action) using the other as a substrate, leading to the deactivation of both or one of the enzymes.
It is said that the action (performance) is inferior to when any one of these enzymes is used alone.
However, the present inventors have found that in a combination of API-21 and the other alkaline proteases mentioned above, for example, in an enzymatic reaction using casein as a substrate, the hydrolysis reaction proceeds without deactivating either or both of them. Furthermore, the inventor has proposed API-21
When a cleaning test was conducted using a detergent composition containing both API-21 and the other alkaline proteases mentioned above, the effect of improving cleaning efficiency due to the combination of enzymes was compared at the same temperature. It has been found that this is superior to the case where either one is blended alone, and that it has an excellent cleaning effect over a wide temperature range. This is thought to be due to the effect based on differences in basic enzymatic properties such as substrate specificity between the two types of alkaline proteases. There is no particular limitation on the amount of alkaline protease API-21 or the other alkaline proteases mentioned above that is blended into the alkaline protease-containing detergent composition of the present invention, but in general, the enzyme-containing detergent composition 1
5 to 500 nkatal per gram (1 nkatal = 1
×10 ^-9 katal), preferably 10 to 100 nkatal. If this amount is too small, a sufficient improvement in the cleaning effect cannot be obtained, and if it is too large, the improvement in the cleaning effect is not large compared to the amount of enzyme added, which is not preferred from an economic point of view. Furthermore, there is no particular limitation on the blending ratio of API-21 and the other alkaline proteases mentioned above, but the enzyme activity ratio is generally 0.05 to
20, preferably at a ratio of 0.2 to 5. According to the invention, said alkaline protease
API-21 and other alkaline proteases can be incorporated into any conventionally known detergent composition without changing the composition of the detergent composition; There are no limitations. Typical examples of such detergent compositions include, based on the dry weight of the detergent composition, 10 to 50% by weight of surfactant, 0 to 50% by weight of builder, 1 to 50% by weight of alkaline agent or inorganic electrolyte, and 0.1 to 50% by weight of alkaline agent or inorganic electrolyte. 5% by weight
Examples include detergent compositions comprising an anti-restaining agent, an enzyme, a whitening agent, a fluorescent paint, an anti-caking agent, and an antioxidant. Surfactants include soaps, such as linear or branched alkyl or alkenyl sulfates, amide sulfates, linear or branched alkyl or alkenyl groups, and ethylene oxide, propylene oxide and butylene oxide alone or Aliphatic sulfates such as alkyl or alkenyl ether sulfates with multiple components attached, alkyl sulfonates, amidosulfonates, dialkyl sulfosuccinates, α-olefins, vinylidene-type olefins, and internal olefin sulfonates. aliphatic sulfonates such as straight-chain or branched alkylbenzene sulfonates, aromatic sulfonates having straight-chain or branched alkyl or alkenyl groups, ethylene oxide, propylene oxide and butylene oxide. Alkyl or alkenyl ethers, carboxylic acid salts or amides, α-sulfo fatty acid salts or esters, amino acid type surfactants, alkyl or alkenyl acidic phosphates, alkyl or alkenyl phosphates, Alternatively, phosphate ester surfactants such as alkyl or alkenyl phosphate ester salts, sulfonic acid type amphoteric surfactants, betaine type amphoteric surfactants, linear or branched alkyl groups or alkenyl groups, and ethylene oxide , an alkyl or alkenyl ether or alcohol to which one or more components of propylene oxide and butylene oxide are added, a linear or branched alkyl group, and one or more components of ethylene oxide, propylene oxide and butylene oxide Detergent compositions such as polyoxyethylene alkyl phenyl ether, higher fatty acid alkanolamide or its alkylene oxide adduct, sucrose fatty acid ester, fatty acid glycerin monoester, alkyl or alkenyl amine oxide, tetraalkylammonium salt type cationic surfactant Any commonly used surfactant can be used, and in the case of an anionic surfactant, the counter ion is preferably a sodium ion or a potassium ion. These surfactants may be used alone or as a mixture of two or more. Builders and alkaline agents or inorganic electrolytes include phosphates such as orthophosphates, pyrophosphates, tripolyphosphates, metaphosphates, hexametaphosphates, phytates, and ethane-1.
1-diphosphonic acid, ethane-1.2-triphosphonic acid, ethane-1-hydroxy-1.1-diphosphonic acid and its derivatives, ethane-1.
Phosphonates such as 1,2-triphosphonic acid, ethane-1,2-dicarboxy-1,2-diphosphonic acid, methanehydroxyphosphonic acid, 2-phosphonobutane-1,2-dicarboxylic acid, 1-phosphonobutane-2, Phosphonocarboxylic acid salts such as 3,4-tricarboxylic acid and α-methylphosphonosuccinic acid, amino acid salts such as aspartic acid and glutamic acid, aminopolyacetic acid salts such as nitrilotriacetate, ethylenediaminetetraacetate, and diethylenetriaminepentaacetate. , polyacrylic acid, polyitaconic acid, polymaleic acid, maleic anhydride copolymer,
Polyelectrolytes such as carboxymethylcellulose salts, non-dissociated polymers such as polyethylene glycol and polyvinyl alcohol, carboxymethylated compounds such as diglycolic acid, oxydisuccinic acid, carboxymethyloxysuccinic acid, citric acid, lactic acid, tartaric acid, sucrose, and lactose. , carboxymethylated pentaerythritol, carboxymethylated gluconic acid, benzene polycarboxylic acid,
Organic salts such as oxalic acid, malic acid, oxydisuccinic acid, and gluconic acid, aluminosilicates such as zeolites, and inorganic salts such as carbonates, sesquicarbonates, sulfates, and metasilicates can be used as alkali metal salts. Also, organic substances such as starch and urea, and inorganic compounds such as sodium chloride and bentonite can be used. Furthermore, triethanolamine, diethanolamine, monoethanolamine, triisopropanolamine, etc. can be used as organic alkali agents. Other ingredients required include anti-recontamination agents such as polyethylene glycol, polyvinyl alcohol, polypynylpyrrolidone, and carboxymethyl cellulose, bleaching agents such as sodium percarbonate and sodium perborate, as well as anti-caking agents and antioxidants. It can be used depending on the situation. As mentioned above, the enzyme-containing detergent composition of the present invention essentially contains a surfactant, alkaline protease API-21, other alkaline proteases whose optimal action temperature is higher than that of API-21, and an alkaline agent or an inorganic electrolyte. Contains as a component, but may also contain amphoteric surfactants, whitening agents, pigments,
Builders, anti-recontamination agents, anti-caking agents, antioxidants, enzymes other than alkaline protease, etc. can be included. Any method may be used to incorporate the alkaline protease into the enzyme-containing detergent composition of the present invention, but incorporating it in fine powder form is
Dust generation during handling of detergents is not desirable in terms of health and safety for detergent users and workers in the detergent industry, so it is preferable to form them in a solution state or in a shape that suppresses dust generation in advance. This shaping may be carried out by any of the commonly used methods such as quince granulation, extrusion granulation, fluidized granulation, centrifugal fluidized granulation, and other methods. The shape is not particularly limited to these methods. Examples Examples of the present invention will be described below, but it goes without saying that the scope of the present invention is not limited to these examples. In the examples, "%" represents "% by weight" unless otherwise specified. Example 1 Alkaline protease API-21 and a commercially available alkaline protease for detergent (Subtilisin Carlsberg) were blended with various detergents and a cleaning test on cotton collar-stained cloth was conducted as follows. A. Preparation of collar-contaminated cloth (cotton) The collar-contaminated cloth was a 7cm × 7 cm cloth worn for one week according to the method described in Hajime Minagawa, Ikuko Okamoto; Orizu Shi, Vol. 19, pp. 106-115 (1978). Cut 200 pieces of 37cm collar cloth (standard cotton cloth for cleaning power test) into 1cm x 5cm pieces, and sew any 10 pieces together to make a 5cm x 10cm sample fabric.
Stored in a cool dark place at ℃. B. Cleaning method Using a tergotometer, cleaning and rinsing (3 times) were performed under the following conditions. <Washing conditions> Contaminated cloth: 5 cm x 10 cm Detergent: 0.133% (enzyme amount: 0.7 nkat/ml) Bath ratio: 1:85 Number of inversions: 105 rpm Washing time: 10 minutes <Rinse conditions> Contaminated cloth: 5 cm x 10 cm Bath Ratio: 1:85 Number of inversions: 105 rpm Rinse time: 3 minutes The rinsed cloth was air-dried indoors avoiding direct sunlight. C. Detergents and enzymes used Four types of phosphorus-containing and phosphorus-free detergents having the compositions shown below were used as detergents.

【table】

[Table] As the enzymes, API-21 and commercially available detergent alkaline protease (Subtilisin Carlsberg) were used as described above. The results obtained are shown in Table 1. The cleaning efficiency for protein stains was measured as follows. Dip the contaminated cloth before and after cleaning into 0.1N NaOH aqueous solution.
The sample solution obtained by thermal extraction (90±2℃, 120 minutes) with 100ml was colored with a copper-Folin reagent, and its absorbance was measured at a wavelength of 750nm, and the cleaning efficiency (D) for protein stains was calculated using the following formula. I asked for D (%) = {(Ds - Dw) / (Ds - Dc)} × 100 Dc: Absorbance of extract on standard cotton cloth (per 1 g) Ds: Absorbance of extract on contaminated cloth (per 1 g) before washing Dw: Absorbance of extract from contaminated cloth (per 1g) after washing

【table】

【table】 [Brief explanation of the drawing]

Figure 1 is a graph showing the optimum PH of the enzyme API-21 used in the present invention, Figure 2 is a graph showing the PH stability of the enzyme API-21 used in the present invention, and Figure 3 is a graph showing the PH stability of the enzyme API-21 used in the present invention. It is a graph showing the action temperature range and optimal temperature of the enzyme API-21 used in the present invention,
FIG. 4A and FIG. 4B are graphs showing the temperature stability of the enzyme API-21 used in the present invention at PH10 and PH8, respectively. FIG. 5 is a graph showing the Fourier transform infrared absorption spectrum of the enzyme API-21 used in the present invention.

Claims (1)

[Claims] 1 Hydrolyze casein in an alkaline region, with an optimum pH of 10 to 11, an optimum working temperature of 45 to 50°C, a molecular weight of about 22,000 by gel filtration, a maximum absorption of ultraviolet absorption of 275 to 282 nm, and an infrared absorption spectrum shown in Figure 5. 1. An enzyme-containing detergent composition comprising an alkaline protease API-21 having the characteristics of API-21 and another type of alkaline protease having an optimum action temperature higher than that of API-21.