JPH03997B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention is obtained by culturing a new strain of Bacillus. Concerning a novel alkaline protease. 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. In general, alkaline proteases include Bacillus subtilis, Bacillus licheniforumis, Bacillus alcalophilus, other species of the genus Streptomyces, Aspergillus, and Aspergillus. Those produced by microorganisms such as Bacter genus (Arthrobacter) and Fusarium genus (Fusarium) are known.
As a specific example, alkaline protease isolated from Bacillus licheniformis [trade name Alcalase, Novo Corporation (hereinafter referred to as Enzyme A in the text)] is well known in the art. However, these enzymes are difficult to incorporate into liquid detergents due to their rapid deactivation in high PH and surfactant solutions. In the present invention, together with the above enzyme A, Bacillus alcalophilus No. 221 (ATCC
An alkaline protease isolated from 21522) [Horikoshi et al., RIKEN (hereinafter referred to as enzyme B in the text)] was used as a comparative example. [Means for Solving the Problems] An object of the present invention is to provide an alkaline protease that has excellent stability under highly alkaline conditions in the coexistence of detergent components and contributes to improved detergency. 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 found that alkaline protease was produced in the culture medium. The strain that produces the enzyme is Bacillus sp. The present inventors isolated and purified the enzyme by the method described below, and examined various properties while comparing it with conventional enzymes. The method for isolating and purifying the alkaline protease (hereinafter referred to as Ya enzyme) of the present invention is as shown in FIG. First, add the microbial culture solution at 10,000 rpm for 5 minutes.
The supernatant was obtained by centrifugation for a minute. Next, the supernatant was
% saturated ammonium sulfate salting out. Furthermore, the obtained precipitate was dissolved in 20mM Tris-HCl buffer (added with 2mM of Ca ions, PH7.2) and dialyzed against the same buffer. The solution was then subjected to anion exchange chromatography on diethylaminoethyl (DEAE)-53 cellulose in 10mM borate buffer (PH9.3).
The non-adsorbed fraction was obtained. Subsequently, the non-adsorbed fraction was again subjected to ammonium sulfate salting out at 70% saturation. The obtained precipitate was again dissolved in 20mM Tris-HCl buffer (added with 2mM of Ca ions, PH7.2) 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 eluted with 20mM Tris-HCl buffer (2mM of Ca ion added, pH7.2) to determine the activity. A certain fraction was collected. Furthermore, the fraction was subjected to ammonium sulfate salting out at 70% saturation, and the resulting precipitate was dialyzed against 20mM Tris-HCl buffer (2mM of Ca ions added, pH 7.2). Finally, to remove denatured proteins, the active fraction after dialysis was filtered using a Millipore filter to obtain purified Ya enzyme. [Effect] Various properties of the Ya enzyme obtained according to the above method were investigated. The action of the Ya enzyme is protein hydrolysis.
The substrate specificity of the enzyme is shown in Table 1.

[Table] Relative decomposition rate when the activity of enzyme A is set as 100* Conditions: Temperature: 35℃ PH 10.5 (50mM borate buffer) Reaction time: 60 minutes (30 minutes for keratin) Substrate concentration 1% (0.4 for hemoglobin)
% Enzyme usage amount 100APU/ml However, egg white
500 APU/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). This table shows that the present Ya enzyme has strong specificity for keratin. Next, FIG. 2 shows a graph of the optimum PH and stable PH range of this Ya 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 checking the optimal pH, 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 2, the optimal pH of the Ya enzyme is 10.0 to 12.5, and the stable pH range is 6.5 to 13.0. Furthermore, the optimum temperature and heat resistance of this Ya enzyme are shown in Figure 3. 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 3,
The optimum temperature of this Ya enzyme is 70°C, and its activity is maintained up to a temperature of 55°C. Next, FIG. 4 shows the ultraviolet absorption spectrum of the Ya 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 276nm. The extinction coefficient E1% 1 cm at that wavelength was calculated to be 7.4. Next, we investigated the effect of metal ions on the activity of the Ya enzyme. The results are shown in Table 2.
Add this Ya to 20mM boric acid-NaOH buffer (PH10.5).
Add enzyme at about 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 Ya enzyme is inhibited by the addition of copper sulfate, silver nitrate, mercury chloride, and cadmium chloride, but the stability of the activity against heat increases by the addition of calcium chloride. Next, we investigated the effects of various inhibitors on this Ya enzyme. The conditions and method are as follows. This Ya in 50mM Tris-HCl buffer (PH7.2)
The 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 3.

[Table] As can be seen from this table, the present Ya enzyme is a protease that has serine in its active center. Furthermore, the stability of the present Ya enzyme in liquid surfactant is shown in FIG. After each enzyme was stored in a liquid heavy detergent stock solution at 40°C for one month, the degrading activity of each enzyme against keratin was measured. The pH was changed with a glycine-NaOH buffer. In addition, each enzyme was stored in a liquid heavy detergent stock solution at varying temperatures for one month, and then the decomposition activity of each enzyme against keratin was measured. The pH was set at 11 with a glycine-NaOH buffer. As can be seen from Figure 5, the stability of this Ya enzyme in a liquid surfactant shows that when stored at 40°C for one month, the PH
At pH 9.0, 100% activity remains, and at pH 10.5, 50% activity remains. Regarding temperature dependence, when stored at PH11 for one month, 100% activity remains below 25°C, and 65% activity remains at 35°C. Thus, it can be seen that the present Ya enzyme has superior stability in liquid surfactants compared to known alkaline proteases. Subsequently, the molecular weight of the Ya enzyme was determined 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. The calibration curve is shown in FIG. By this method, the molecular weight of the Ya enzyme was
It was decided to be 21000. Next, the isoelectric point of the Ya enzyme was investigated by isoelectric focusing. Pharmalite 3-10 (trademark, manufactured by Pharmacia Co., Ltd.) was used as the column carrier. The isoelectric focusing pattern of this enzyme is shown in FIG. By this method, the isoelectric point of the Ya enzyme was determined to be 10.1. Furthermore, the amino acid composition of the Ya enzyme was investigated using an amino acid analyzer JLC-200A (JEOL Ltd.). Its composition is shown in Table 4 in comparison with that of other proteases.

【table】

[Table] Next, Table 5 shows the elemental analysis values of the present Ya enzyme.

[Table] Finally, as a summary, various properties of the present Ya enzyme are compared with those of the A enzyme and B enzyme and are shown in Table 6.

[Table] Quoted.
** Quoted from Biochemistry Data Book Volume 1.
[Example] Culture of bacterial strain 2% soluble starch, 0.02% magnesium sulfate
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 Y strain was inoculated into the culture solution, and the culture solution was cultured at 30° C. for 15 hours to prepare a seed culture solution. 100ml of the seed culture solution was added to media 3 and 5 with the same composition.
The mixture was added to a fermentation tank containing a 100% aqueous solution, and cultured with aeration for 70 hours at 30°C while supplying air at a rate of 3.5 per minute. The obtained culture solution 3.5 (2500 APU/ml) was sterilized by centrifugation to obtain a supernatant of about 3.0. Purification of Ya 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 500 ml 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, 890 ml of crude enzyme solution (6600 APU/ml, specific activity 4350 APU/mg protein) was obtained. 10mM for removal of contaminant proteins and decolorization
DEAE-53 equilibrated with borate buffer (PH9.3)
The solution was developed on a column filled with the same buffer and eluted with the same buffer. When the non-adsorbed active fractions were collected, the total volume was 420 ml, the activity was 11,300 APU/ml, and the specific activity was 11,800 APU/mg protein. The degree of purification so far was 6.2 times, and the recovery rate was 72%. Furthermore, 198 g of ammonium sulfate was added to the fraction to salt out the protein.
Subsequently, the precipitate was dissolved in 30 ml of 20 mM Tris-HCl buffer (PH7.2, containing 2 mM Ca ions),
The solution was subjected to gel filtration chromatography using Toyopearl HW-55 (trademark, manufactured by Toyo Soda Kogyo Co., Ltd.).
It was developed with the same buffer. The obtained active fraction was salted out with ammonium sulfate, and the precipitate was dissolved in 10 ml of the same buffer and dialyzed against the same buffer. After dialysis, insoluble denatured proteins are removed using a Millipore filter and activated
16.4 ml of a solution with a protein concentration of 210,000 APU/ml and a specific activity of 15,900 APU/mg was obtained. This purification process is summarized in Table 7.

[Table] Next, FIG. 8 shows the elution curve of gel filtration chromatography using the purified Ya enzyme as a sample. The resin used was Toyopearl HW-55, and the eluent was
20mM Tris-HCl buffer (PH7.2, Ca ion 2mM
), and the expansion was carried out by the ascending method.
Furthermore, the elution curve of high performance liquid chromatography using the purified Ya enzyme as a sample is shown in FIG. The model used is Waters WISP-710B,
Two −125 columns were connected in series. Elution was performed with 50mM phosphate buffer (PH7.0). As is clear from these two figures, the Ya enzyme was completely purified by the above purification. Detergent power of this Ya 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 having only the above composition was designated as Sample-1, and a sample in which 5000 APU/g of the present Ya enzyme was added to the above composition was designated as Sample-2. Further, a sample of a commercially available liquid cleaning agent was designated as Sample-0. The cleaning equipment includes
Using a US-Testing Terg-O-Tometer, add 10 protein-containing wet soiled cloths, sebum cloth, and washed knitted cloth.
Washing was performed at 25° C. and 120 rpm for 10 minutes at a bath ratio of 30 times. 900ml of cleaning solution with a cleaning agent concentration of 0.1%
Rinsing was performed with 900 ml of water for 3 minutes. The water used was 3°DH. The detergency index was calculated according to the formula shown in Yukagaku, 30 , 432 (1981). The results are shown in Table 8.

[Table] From this result, the sample containing this Ya enzyme is
The cleaning action was clearly stronger than that of the sample that did not contain the present Ya enzyme, indicating that the present Ya enzyme contributed to improving the cleaning power of liquid heavy detergents.

[Brief explanation of the drawing]

FIG. 1 is a flow sheet showing the purification steps of the present Ya enzyme. FIG. 2 is a graph showing the optimum PH and stable PH range of the present Ya enzyme. Figure 3 is a book
FIG. 2 is a graph showing the optimum temperature and heat resistance of Ya enzyme. FIG. 4 is a graph showing the absorption spectrum curve of the present Ya enzyme in the ultraviolet region. Figure 5 is a book
FIG. 2 is a graph showing the stability of Ya enzyme in a surfactant. FIG. 6 is a graph showing a calibration curve for determining the molecular weight of the present Ya enzyme. Figure 7 is book Ya
FIG. 2 is a graph diagram showing an isoelectric focusing pattern of an enzyme. FIG. 8 is a graph showing the gel filtration chromatography elution curve of the present Ya enzyme. FIG. 9 is a graph showing the high performance liquid chromatography elution curve of the present Ya enzyme.

Claims (1)

[Scope of Claims] An alkaline protease having the following physical properties. (a) Action: Decomposes various proteins under highly alkaline conditions. (b) Substrate specificity: Shows remarkable specificity for insoluble proteins, especially keratin. (c) Optimal pH: When reacting with casein as a substrate at 35°C for 10 minutes, the action is optimal at pH 10.0 to 12.5. (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 13.0. (e) Optimal temperature: When reacting with casein as a substrate at pH 10.5, the action is optimal at a temperature of 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: 50mM Tris at PH8.0
It exhibits maximum absorption in the ultraviolet region at 276 nm in hydrochloric acid buffer. (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). (v) Effect of surfactant: When stored in a surfactant at 40°C for one month, 100% activity remains at pH 9.0, and 50% activity remains at pH 10.5. (le) Molecular weight: 21000 (gel filtration method). (wo) Isoelectric point: 10.1 (isoelectric focusing method).