JPH0255034B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a novel alkaline protease obtained by culturing a new strain of Bacillus. In particular, the present invention relates to a novel alkaline protease that has excellent stability when incorporated into general detergents and contributes to improved detergency. [Prior art and its problems] In recent years, in order to further improve the detergency of detergents, especially liquid detergents, the pH of the detergent stock solution has been made more alkaline, and various types of hydration have been added to protease, amylase, lipase, cellulase, etc. Attempts have been made to incorporate degrading enzymes. Among these, proteolytic enzymes, especially alkaline protease, decompose protein stains that are difficult to remove with detergents alone and contribute to improving detergency. Therefore, it is essential to add the enzyme to cleaning agents. Generally, Alcalase (manufactured by Novo, hereinafter referred to as enzyme A in the text) and Maxatase (manufactured by Gist) produced by Bacillus licheniformis are often used.
However, since these enzymes are immediately deactivated in high-PH detergent solutions, it is difficult to stably incorporate them into fluid detergents. Other species include Bacillus subtilis, Bacillus alcalophilus, Streptomyces, Aspergillus, Arthrobactor, and Fusarium. Alkaline proteases produced by microorganisms such as Alkaline Protease are also known, but as mentioned above, they cannot be used because of stability problems. [Means for Solving the Problems] An object of the present invention is to provide a novel alkaline protease that has excellent stability under highly alkaline conditions in the coexistence of detergent components and contributes to improved detergency. do. In order to obtain an alkaline protease that overcomes the above-mentioned problems, the present inventors conducted a wide search for alkaline protease-producing bacteria in nature, and as a result, one species belonging to the genus Bacillus was found to be superior to known alkaline proteases in the above-mentioned properties. It was discovered that alkaline protease was produced in the culture medium.
The strain producing this enzyme is disclosed in Japanese Patent Application No. 123022/1986.
It is the same strain as Bacillus sp. Y, No. 1029, Bacillus sp., which produces the Ya enzyme. The present inventors isolated and purified this enzyme by the method described below, and examined various properties while comparing it with Ya enzyme and known enzymes. The method for isolating and purifying the alkaline protease (hereinafter referred to as Yb enzyme) of the present invention is as shown in FIG. First, add the microbial culture solution at 10,000 rpm for 5 minutes.
Centrifugation was performed for a minute to obtain a supernatant. Then the supernatant was 70%
It was subjected to saturated ammonium sulfate salting out. Furthermore, the obtained precipitate
20mM Tris-HCl buffer (2mM Ca ion added,
pH7.2) and dialyzed against the same buffer.
Subsequently, the solution was subjected to anion exchange chromatography using diethylaminoethyl (DEAE)-53 cellulose, and the Ya enzyme was eluted as a non-adsorbed fraction with 20mM Tris-HCl buffer (2mM of Ca ion added, pH 7.2). Using the same buffer containing 0 to 0.5M sodium chloride, elution was performed with a linear concentration gradient to obtain a crude fraction of the Yb enzyme. The elution curve of the chromatography is shown in FIG. Furthermore, the corresponding Yb
The crude fraction was again subjected to ammonium sulfate salting out at 70% saturation. The obtained precipitate was dissolved in 50 mM phosphate buffer (PH7.0) and dialyzed against the same buffer. Furthermore, the solution was subjected to hemoglobin-agarose affinity column chromatography, eluted with 50 mM phosphate buffer (PH7.0), and active fractions were collected. Furthermore, the fraction was subjected to ammonium sulfate salting out at 70% saturation, and the resulting precipitate was dissolved in 20 mM Tris-HCl buffer (2 mM Ca ion added, pH 7.2) and dialyzed against the same buffer. Furthermore, the solution was subjected to gel filtration chromatography using Toyopearl HW-55 (trademark, manufactured by Toyo Soda Kogyo Co., Ltd.), and 20mM Tris-
It was eluted with a hydrochloric acid buffer (added 2mM of Ca ions, PH7.2), and active fractions were collected. Furthermore, the fraction
The precipitate obtained was subjected to ammonium sulfate salting out at 70% saturation.
20mM Tris-HCl buffer (2mM Ca ion added,
PH7.2) and dialyzed against the same buffer to obtain purified Yb enzyme. [Effect] Various properties of the Yb enzyme obtained according to the above method were investigated. The action of the Yb enzyme is protein hydrolysis.
The substrate specificity of the enzyme is shown in Table 1. In addition, the substrate specificity of the Ya enzyme produced simultaneously from Bacillus sp. Y and the mixture with the Ya enzyme was similarly evaluated and compared.

[Table] Relative decomposition rate when enzyme A activity is set as 100* Conditions: Temperature: 35°C PH10.5 (50mM borate buffer) Reaction time: 60 minutes, except for keratin, 30 minutes Substrate concentration: 1%, but for hemoglobin: 0.4
% Enzyme usage amount 100APU/ml However, egg white
500APU/ml *Measurement of proteolytic rate or activity followed a modified Anson-Hagiwara method. After the reaction, the absorbance of the filtered reaction solution was measured at 275 nm. The enzyme activity that releases 1 μg of tyrosine per minute was defined as 1 alkaline protease unit (APU). From this table, it can be seen that the present Yb enzyme has strong specificity for egg white, but weak for keratin, which is an insoluble protein. In addition, it has different characteristics from the Ya enzyme produced at the same time, complements it when used in combination with the Ya enzyme, and has the characteristic of acting more strongly on a wider range of substrates than the known A enzyme. Next, FIG. 3 shows a graph of the optimum PH and stable PH range of this Yb enzyme. The buffer solution used is as follows. PH range Buffer 3.5-5.5 Acetic acid 4.5-7.0 Citric acid 6.0-8.0 Phosphoric acid 7.5-9.0 Tris-HCl 8.0-9.0 Boric acid-HCl 9.0-10.5 Glycine-NaOH 9.5-11.0 Boric acid-NaOH 11.0-12.0 Phosphoric acid- NaOH 12.0−13.0 KCl−NaOH When investigating the optimal pH, use casein 0.6%
Approximately 400 APU/each enzyme was added to each 20mM buffer containing
ml and reacted at 35°C for 10 minutes to measure activity. The relative activity at each PH was determined when the activity at the optimal PH was set as 100. To investigate the stable PH range, add approximately each enzyme to 20mM of each buffer.
After adding 400 APU/ml and incubating at 25°C for 24 hours, the activity was measured. Relative activity was determined at each PH, setting the activity before incubation as 100. As can be seen from Figure 3, the optimal pH of this Yb enzyme is 9.0 to 10.0, and the stable pH range is 6.5.
or 12.0. Furthermore, the optimum temperature and heat resistance of this Yb enzyme are shown in FIG. To investigate the optimal temperature, each enzyme was added to a pH 10.5 buffer containing 0.6% casein as a substrate, and allowed to react at each temperature for 10 minutes. The relative activity at each temperature was determined with the activity at 35°C as 100.
Heat resistance was examined as follows. 50mM boric acid
Approximately 400 APU/ml in NaOH buffer (PH10.5 at 35℃)
of enzyme was added, heat treated at each temperature for 10 minutes, cooled on ice, and the activity was measured. As can be seen from Figure 4,
The optimum temperature for this Yb enzyme is in the range of 65 to 70°C, and 100% activity is maintained up to a temperature of 50°C. Next, FIG. 5 shows the ultraviolet absorption spectrum of this Yb enzyme. When the sample was dissolved in 50mM Tris-HCl buffer (PH8.0) and the ultraviolet absorption spectrum was measured, it showed maximum absorption at a wavelength of 278nm. The extinction coefficient E ¹ % _1cn at that wavelength was calculated to be 9.5. Next, we investigated the effect of metal ions on the activity of this Yb enzyme. The results are shown in Table 2.
This Yb was added to 20mM boric acid-NaOH buffer (PH10.5).
Add enzyme at approximately 400 APU/ml and also various metal salts.
It was added at a concentration of 1mM, and the residual activity after treatment was measured under each predetermined condition. The numerical value is expressed as a relative activity, with the activity at 0 minutes being 100.

[Table] This table shows that the activity of the Yb enzyme is inhibited by the addition of copper sulfate, silver nitrate, and mercuric chloride. Alkaline proteases produced by bacteria belonging to the genus Bacillus generally increase their thermostability with Ca ²⁺ , so to examine ^{the effect of Ca 2+ ,} a 50 mM boric acid-NaOH buffer (35 PH at °C
Add about 400 APU/ml of enzyme to 10.5) and at each temperature.
After heat treatment for 10 minutes and cooling on ice, activity was measured. For comparison, evaluation was also conducted under conditions in which Ca ²⁺ was not added.
The results are shown in Table 3. The numerical value indicates the activity of 0 minutes.
The relative activity is expressed as 100.

[Table] From this table, it can be seen that the addition of Ca ions improves the thermal stability by about 10°C. Next, we investigated the effects of various inhibitors on this Yb enzyme. The conditions and method are as follows. 50mM Tris-HCl buffer (PH7.2)
Yb enzyme was prepared at 800 APU/ml. After adding each inhibitor and incubating at 35°C for 30 minutes, residual activity was measured. The value was expressed as relative activity with the value without inhibitor added as 100. The results are shown in Table 4.

【table】

[Table] As can be seen from this table, when casein is used as a substrate, the activity of this Yb enzyme is not inhibited by EDTA, PCMB, antipain, and chymostatin, but the activity is inhibited by DFP and PMSF. It is a protease with a serine in the center. Furthermore, the stability of the present Yb enzyme in a liquid surfactant is shown in FIG. Glycine for each enzyme
After storing at 40°C in a liquid heavy detergent stock solution whose pH was adjusted to 11 with a NaOH buffer, the degrading activity of each enzyme against keratin was measured over time. As a result, as shown in Figure 6, enzyme A was
In contrast, this Yb enzyme loses 80% activity on the 3rd day and completely loses its activity on the 7th day.
% activity remains. In this way, this Yb enzyme is
Compared to known alkaline proteases, it is found to have superior stability in high PH liquid surfactants. Subsequently, the molecular weight of this Yb enzyme was investigated by gel filtration chromatography. Toyopearl HW-55 (trademark manufactured by Toyo Soda Kogyo Co., Ltd.) was used as a packing material, and 20mM Tris-HCl buffer (Ca ion 2mM
addition, pH 7.2) was used as the eluent. A calibration curve was created using the following proteins (molecular weights) as standard proteins. Ovalbumin (43000), thermolysin (37500), subtilisin (27300), chymotrypsinogen (25700), myoglobin (17200), and cytochrome C (11700) were used. With this method,
The molecular weight of this Yb enzyme was determined to be 40,000. Next, the isoelectric point of this Yb enzyme was investigated by isoelectric focusing. Pharmalite 3-10 (trademark, manufactured by Pharmacia Co., Ltd.) was used as the column carrier. By this method, the isoelectric point of this Yb enzyme was determined to be 5.1. Furthermore, the amino acid composition of this Yb enzyme was investigated using an amino acid analyzer JLC-200A (JEOL Ltd.). Incidentally, tryptophan was measured by the alkaline decomposition method, and cysteine was measured by the performic acid oxidation method. Its composition is shown in Table 5 in comparison with that of known proteases. As a result, the Yb enzyme of the present invention shows significant differences in amino acid composition, such as tryptophan, histidine, arginine, aspartic acid, glycine, and alanine, compared to other enzymes.

[Table] Next, Table 6 shows the elemental analysis values of this Yb enzyme.

[Table] Finally, as a summary, various properties of this Yb enzyme are shown for Ya enzyme, A enzyme, and A enzyme produced simultaneously from the same strain.
Table 7 shows a comparison with known alkaline proteases produced by alkalophilic bacteria of the genus Bacillus. Ya enzyme produced from the same strain, and other similar known alkaline proteases (E-
1, E-2, ADI-21, No. 221), the optimum pH of this Yb enzyme is 9 to 10, whereas that of A enzyme, E-1, E-2
and API−21 are high at 10 to 11, and No. 221 is high at 11 to 12.
Furthermore, the Ya enzyme has a high pH range of 10 to 12.5.
They differ in that they are wider on the sides. Next, the optimum temperature for this Yb enzyme and Ya enzyme is 70℃.
Enzyme A, No. 221, is at 60°C while it is nearby.
API-21 has a low temperature of 45 to 50°C, while E-1 and E-2 have a temperature of 75°C, which is higher than this enzyme, and they are different in this respect as well. In addition, this Yb enzyme was activated in the presence of 5mMCa ²⁺ ions.
Like the Ya enzyme, A enzyme, No. 221, and API-21 enzymes, the heat resistance improves by about 5 to 10 degrees Celsius, but Bacillus No.
E-1, E- produced by 6 strains (see notes to Table 7)
2 differs in that no increase in thermal stability due to Ca ²⁺ ions is observed. Furthermore, the molecular weight of this Yb enzyme is 40,000, which is larger than that of known alkaline proteases, and the isoelectric point is pI5.1.
It can be said that it is clearly a different species from the fact that it is low. From the above, this enzyme is different from any of the conventionally known alkaline proteases. Therefore, it is appropriate to conclude that this enzyme is a new enzyme, and we named it alkaline protease Yb.

[Table] [Example] Cultivation of bacterial strains Soluble starch 2%, magnesium sulfate 0.02%
and a liquid medium containing 1% dry yeast and 0.1% dipotassium hydrogen phosphate, respectively.
After sterilizing separately for 20 min at °C, each 20 ml was divided into 500 ml
Then, sterilized sodium carbonate was added to the flask to give a final concentration of 1% to prepare 50 ml of culture solution. Bacillus sp (Bacillus sp) Y strain is inoculated into the culture solution,
The culture solution was cultured at 30°C for 15 hours to prepare a seed culture solution. Add 100 ml of the seed culture solution to a fermentation tank containing 3.5 ml of medium of the same composition, and incubate the tank at 30°C every minute.
The culture was carried out with aeration for 70 hours while supplying 3.5 liters of air. The obtained culture solution 3.5 (2500 APU/ml) was sterilized by centrifugation to obtain a supernatant of about 3.0. Purification of Yb enzyme When 1250 g of ammonium sulfate was added to 2650 ml of the culture supernatant thus obtained while cooling and stirring, alkaline protease was precipitated. The precipitate was collected by centrifugation, dissolved in 500ml of 20mM Tris-HCl buffer (PH7.2, containing 2mM Ca ions), and the solution was placed in a dialysis membrane and incubated against the same buffer overnight. Dialyzed. Here, 890ml of crude enzyme solution (6600APU/
ml, specific activity 435 OAPU/mg protein). Due to the removal of the Ya enzyme produced at the same time,
20mM Tris-HCl buffer (2mM Ca ion added,
The solution was developed on a column packed with DEAE-53 (cellulose) equilibrated with PH7.2), and the same buffer solution was used to develop the solution.
Ya enzyme was eluted. Then, the Yb enzyme was eluted with the same buffer containing 0-0.5M sodium chloride.
When the active fractions were collected, the total volume was 350 ml, the activity was 4200 APU/ml, and the specific activity was 1350 APU/mg protein. Next, the crude Yb fraction was subjected to hemoglobin-agarose affinity column chromatography, eluted with 50mM phosphate buffer (PH7.0),
Active fractions were collected. The recovery rate was 15%, and the specific activity increased to 7400 APU/mg protein. Furthermore, the solution was subjected to gel filtration chromatography using Toyopearl HW-55 and developed with 20 mM Tris-HCl buffer (2 mM Ca ion added, pH 7.2). The obtained active fraction was subjected to ammonium sulfate salting out, and the precipitate was dissolved in 3 ml of the same buffer and dialyzed against the same buffer. After dialysis, activity 94000APU/ml, specific activity
7 ml of a solution of 8500 APU/mg protein was obtained. This purification process is summarized in Table 8.

[Table] Next, FIG. 7 shows the elution curve of gel filtration chromatography using the purified Yb enzyme as a sample. The Yb enzyme was completely purified by the above purification. Detergent power of this Yb enzyme The standard composition of the detergent is sodium alkyl polyethoxy sulfate 20, alcohol ethoxylate.
10, ethanol 6, sodium toluene sulfonate 6, alkali builder (glycine 6.6,
A formulation of NaOH3.3) and water (balance) was prepared. All numerical units are weight percent. A sample containing only the above composition was designated as Sample-1, and a sample in which 5000 APU/g of the present Yb enzyme was added to the above composition was designated as Sample-2. Sample 3 was prepared by adding the same amount of Ya enzyme produced from the same strain. Also, specific activity prepared according to the above purification method
Crude enzyme of 4350APU/mg protein (Ya/Yb=2/1
Sample 4 was prepared by adding the following ingredients: Furthermore, samples of commercially available liquid cleaning agents were sampled.
It was set to 0. The cleaning equipment is US-Testing's Terg-O.
- Using a Tometer, add 10 sheets of protein-containing wet soiled cloth, sebum cloth, and washed knitted cloth, adjust the bath ratio to 30 times, and heat at 25℃.
Washed at 120 rpm for 10 minutes. 900 ml of a cleaning solution with a detergent concentration of 0.1% was used, and rinsing was performed for 3 minutes with 900 ml of water. The water used was 3°DH. In addition, the detergency index is oil chemical, 30 , 432
(1981). The results are shown in Table 9.

[Table] From this result, the sample containing this Yb enzyme is
The cleaning action was clearly stronger than that of the sample that did not contain the present Yb enzyme, indicating that the present Yb enzyme contributed to improving the cleaning power of liquid heavy detergents. In addition, since the cleaning power of Sample 4 with crude enzyme added tended to be slightly stronger, this Yb
Although the enzyme can be used alone, it is desirable to use it in combination with the Ya enzyme, which has a different substrate specificity, from a performance and economic standpoint. Incidentally, it can be naturally predicted that the combined use of the present Yb and Ya enzymes with known enzymes is also effective in further improving the detergency, since the physical and chemical properties are different from those of the known enzymes.

[Brief explanation of drawings]

FIG. 1 is a flow sheet showing the purification steps of the present Yb enzyme. FIG. 2 is a graph showing the elution curve of the present Yb enzyme when it was subjected to anion exchange chromatography on DEAE-53 cellulose. FIG. 3 is a graph showing the optimum PH and stable PH range of the present Yb enzyme. FIG. 4 is a graph showing the optimum temperature and heat resistance of the present Yb enzyme. Figure 5 is a book
FIG. 2 is a graph showing an ultraviolet region absorption spectrum curve of Yb enzyme. FIG. 6 is a graph showing the stability of the present Yb enzyme in a surfactant. Figure 7 is a book
FIG. 2 is a graph showing a gel filtration chromatography elution curve of Yb enzyme.

Claims (1)

[Claims] An alkaline protease having the following physical and chemical properties. (a) Action: Decomposes various proteins under highly alkaline conditions. (b) Substrate specificity: Shows remarkable specificity for egg white. (c) Optimal pH: When reacting for 10 minutes at 35°C using casein as a substrate, the action is optimal at pH 9.0 to 10.0. (d) Stable PH range: 24 at 25℃ using casein as a substrate
When treated over time, the action is stable in the pH range of 6.5 to 12.0. (e) Optimal temperature: When reacting at pH 10.5 using casein as a substrate, the action is optimal at a temperature in the range of 65 to 70°C. (F) Heat resistance: 90% or more of the activity remains when heat treated at 60°C for 10 minutes at pH 10.5. (g) Absorption spectrum: Shows maximum absorption at 278 nm in the ultraviolet region in 50mM Tris-HCl buffer with pH 8.0. (H) Effect of metal ions: When casein is used as a substrate, Hg ions inhibit the activity, and Ca ions increase the thermal stability of the activity. (li) Effects of inhibitors: When casein is used as a substrate,
EDTA (ethylenediaminetetraacetic acid) and
The activity is not inhibited by PCMB (p-chloromercurybenzoic acid), but the activity is inhibited by DFP (diisopropylfluorophosphate) and PMSF (phenylmethanesulfonyl fluoride). (n) Effect of surfactant: When stored at 40°C for one week in a surfactant adjusted to pH 11, 50% activity remains. (le) Molecular weight: 40000 (gel filtration method). (w) Isoelectric point: 5.1 (isoelectric focusing method).