JPH0670765A - Protease, gene coding the same, method for producing the protease and its use - Google Patents

Abstract

PURPOSE:To provide a new alkali protease useful for cleanser compositions, etc. CONSTITUTION:(a) The optimal pH: >=11 (stable at a pH of 5-11.5), when reacted with a synthetic substrate: Suc-Ala-Ala-Pro-Phe-MCA as a substrate at 30 deg.C for 10 min, (b) the optimal temperature: approximately 50 deg.C (perfectly stable up to 55 deg.C and perfectly inactivated at 65 deg.C), when reacted in a 1.0% casein as a substrate at a pH of 10 for 10min, (c) substrate specificity: decomposes the above-mentioned synthetic substrate casein, hemoglobin, clupeine, salmine, and gelatin, (d) isoelectric point; <2.8, (e) mol.wt.: approximately 37000 (SOS- polyacrylamide gel electrophoresis method), (f) perfectly inhibited with phenylmethanesulfonyl fluoride, chymostatin, and antipain. The protease has e.g. the amino acid sequence of the formula. The protease is obtained from the culture solution of a microorganism produced by partially decomposing the genom DNA of the Bacillus NKS-21 strain (FERM P-93) and subsequently transforming with a DNA having a sequence of the formula by a shot gun method.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel alkaline protease, a gene encoding the same, and a method for producing the protease and its use. More specifically, a novel alkaline protease having excellent stability to a surfactant and heat, which is obtained by culturing a transformed microorganism into which an unexpressed portion of genomic DNA of Bacillus genus NKS-21 is introduced, and encodes it The present invention relates to a method for producing a gene and its protease, and uses.

[0002]

2. Description of the Related Art Alkaline protease, which is a typical extracellular protein produced by Bacillus bacteria, is widely used for leather processing, food industry, detergent addition, pharmaceutical use and the like. However, the wild-type alkaline protease derived from microorganisms obtained from the natural world is not sufficiently satisfactory in its characteristics, and for industrial use,
Temperature, pH, oxygen, solvent, pressure, oxidizer, surfactant,
It is necessary to improve the stability against other additives in the reaction solution.

[0003] In particular, it is desired that protease added as a cleaning auxiliary agent exhibits sufficient stability not only in powder type detergent but also in liquid type detergent and exhibits sufficient function. However, in liquid type detergents, the stability of protease is lower than in powder type detergents, and there are problems such as decomposition during storage as a product and deactivation during washing. It has not been raised.

Therefore, many techniques have been disclosed for improving the stability of enzymes in liquid detergents by developing new surfactants, adding stabilizers, encapsulating enzymes, etc. (Japanese Patent Laid-Open No. 41398/1990). U.S. Pat.No. 4287082
No. 2021142, JP-A-62-596, JP-A-63
-137996 etc.).

However, along with the technology for stabilizing the enzyme, it has been strongly demanded that the stability of the enzyme itself in a liquid detergent is superior to that of the conventional product, and a novel method for producing an alkaline protease having excellent stability is obtained. The search for various microorganisms is being conducted. Further, there is always a demand for proteases having higher stability and high application value, and so-called protein engineering has disclosed the improvement of the stability of proteases using site-specific modification of amino acid sequence (European Patent). No. 0130756, US Pat. No. 4760025).

Alkaline proteases for detergent addition are mostly active at relatively high temperatures.
Bacillus sp. NKS-21 strain (hereinafter referred to as Bacillus NKS-21) is an alkaline protease API-21 (hereinafter referred to as “Bacillus NKS-21”) having activity at a relatively low temperature.
It is called API-21. ) Is known to be produced outside the bacterial cell (Japanese Patent Publication No. 60-55118), and the SD-515 strain (Microtechnical Research Institute No. 9968), which is a mutant strain of this bacterial cell, is the alkaline protease SDP. -515 (hereinafter, SDP-51
5 ) Is produced (see Japanese Patent Laid-Open No. 1-281084). Furthermore, the applicant is
It has been proposed to produce a more stable protease by modifying the amino acid sequence of API-21 derived from the S-21 strain with a specific site using a gene modification technique (see Japanese Patent Application No. 3-280313).

[0007]

The object of the present invention is to provide an alkaline protease which is excellent in stability by expressing an unexpressed part in the genomic DNA of Bacillus NKS-21.
Another object of the present invention is to provide a gene encoding the protease, a method for producing the protease, and use of the protease, particularly for a detergent composition. In order to achieve the above-mentioned object, the present inventors have known Bacillus NKS-, which is known as a bacterium producing a cold alkaline protease.
A fragment obtained by partially digesting the genomic DNA of 21 bacteria with a restriction enzyme was introduced by the shotgun method, and the transformed microorganism was cultured and screened.

The Bacillus NKS-21 used in the present invention is
It is an aerobic spore bacterium belonging to the genus Bacillus and its detailed mycological characteristics are described in the literature (O. Tsuchida et al., Curr. M
icrobiol. 14, 7-12 (1986) and Japanese Patent Publication No. 60-55118). In the present invention, Bacillus NKS-
The genomic DNA obtained from No. 21 was partially digested with the restriction enzyme Sau3AI to obtain a fragment, which was then subjected to agarose gel electrophoresis to obtain GENECLEAN (Bio10).
1) was used to prepare only a 2 to 6 kb DNA fragment. Next, this was transformed into Escherichia coli HB101 strain by the shotgun method and cultured in LB-skim milk agar medium containing ampicillin, and ampicillin-resistant transformants were selected using the clear zone formation around the colony as an index.

This transformant was cultivated on a new identical plate to form a clear zone, and the clear zone forming portion on the medium was cut out, embedded in an agarose gel for electrophoresis and electrophoresed. After electrophoresis, the substrate Suc-
A plotting filter paper containing Atkins-Pantin buffer (pH 10.0) containing Ala-Ala-Pro-Phe-MCA (succinyl alanyl alanyl prolyl phenyl alanyl-4-methylcoumaryl-7-amide) was used. Place, 10-30 at room temperature
After reacting for a minute, known API-21 (isoelectric point 7.4) and S
A novel protease having an isoelectric point (less than 2.8) different from DP-515 (isoelectric point 2.8) was obtained, and it was confirmed that fluorescence was generated on the transilluminator.

The protease-producing strain thus screened is inoculated into a medium and cultivated, and the obtained culture solution is centrifuged to obtain a crude enzyme solution as a supernatant. The crude enzyme solution is purified by column chromatography. A purified protein-homogeneous enzyme was obtained. It was confirmed that this purified enzyme exhibits a substrate specificity similar to API-21.

Further, with respect to this substrate specificity, it is a major feature of the present protease that it decomposes arginine-rich basic proteins such as crupane and salmine very well, which means that the protease is very low. It is thought to be related to having an electric point. Therefore, due to the above properties, the protease of the present invention is Bacillus NKS-
API-21 and SDP- produced from the 21-derived strain
It was confirmed to be a novel protease different from 515.

When the structural analysis of the gene encoding this protease was carried out, as shown in SEQ ID NO: 1, TTG
We found a 0.97 kb open reading frame starting from. Furthermore, when the amino acid sequence deduced from this DNA sequence was examined, it was confirmed to be a serine protease in which serine exists in the active center.

When this was compared with the amino acid sequence of the subtilisin group, which is the best known extracellular serine protease of Bacillus bacterium, it usually shows about 80% homology between subtilisins. , AP
The homology including I-21 and SDP-515 was about 40%. In addition, a search for the most homologous serine proteases known so far revealed high homology with the intracellular serine protease (ISP-1) of Bacillus subtilis. It showed only about 50% homology.

No amino acid sequence was found at the N-terminal which seems to correspond to the signal sequence. Therefore, it was confirmed that the present protease is a novel protease which has never existed in terms of structure, and that it has higher stability against heat or a surfactant.

[0015]

Means for Solving the Problems The present invention comprises: 1) an alkaline protease having the following properties: (1) optimum pH: 10 at 30 ° C. using a synthetic substrate Suc-Ala-Ala-Pro-Phe-MCA as a substrate. Optimal action when reacting for minutes p
H is 11 or more; (2) pH stability: synthetic substrate Suc-Ala-Ala-Pro-Phe-MCA
PH of 5 when reacted at 30 ℃ for 10 minutes
It is stable in the range of ~ 11.5; (3) Optimum temperature: pH 10 with 1.0% casein as substrate
When the reaction is carried out for 10 minutes at 50 ° C, the optimum working temperature is around 50 ° C; (4) Thermal stability: 55 when treated at pH 10 for 10 minutes.
It is completely stable up to ℃ and completely inactivated at 65 ℃; (5) Substrate specificity: synthetic substrate Suc-Ala-Ala-Pro-Phe-MCA
Decomposes casein, hemoglobin, curpain, salmine and gelatin;

(6) Isoelectric point: Isoelectric point by agarose gel electrophoresis is less than 2.8; (7) Molecular weight: Molecular weight is about 37,000 when measured by SDS-polyacrylamide gel electrophoresis; (8) Effects on chemical substances: completely inhibited by phenylmethanesulfonyl fluoride, chymostatin and antipain, 2) the alkaline protease according to 1) having the amino acid sequence represented by SEQ ID NO: 1, 3) the 2 The gene represented by SEQ ID NO: 1 encoding the alkaline protease described in

4) Culturing a microorganism obtained by partially degrading the genomic DNA of Bacillus genus NKS-21 (Microtechnical Laboratory Article No. 93) and transforming the DNA having the sequence shown in SEQ ID NO: 1 by the shotgun method. Then, an alkaline protease is obtained from the culture solution, and the method for producing the alkaline protease according to 1) or 2) above, 5) The transformed microorganism into which the gene according to 3) above is introduced is cultured. And then obtaining the alkaline protease from the culture broth, and the method for producing the alkaline protease according to 1) or 2) above, and 6) containing the alkaline protease according to 1) or 2) above. The present invention provides a detergent composition characterized by the following.

The novel protease of the present invention can be obtained by introducing a target gene into a microorganism, culturing the microorganism in a medium to produce the protease, and recovering the protease from the culture solution. DN used at this time
As A, an unexpressed gene obtained from Bacillus NKS-21 is used, and in this DNA, a nucleotide sequence encoding at least the present protease gene represented by SEQ ID NO: 1 or the polypeptide represented by SEQ ID NO: 1 is used. The DNA of Bacillus N is necessary.
It is not necessary to include all unexpressed genes from KS-21. Further, a DNA fragment in which another promoter region, signal sequence, terminator region, etc. are bound to the protease structural gene portion can also be used. Any promoter region, signal sequence, terminator region, etc. may be used as long as they can express and produce alkaline protease in the host bacterium into which the gene is introduced.

As the host bacterium, any host bacterium can be used as long as it can introduce the target protease gene and can express and produce it. From the viewpoint of its growth rate and protease productivity, In industrial production, it is desirable to use Bacillus bacterium or Escherichia coli.
A method for transforming a host bacterium can be a commonly used method. For example, it can be carried out by a protoplast method, an electroporation method, a competent cell method, or the like. The introduced protease gene may be stably retained by a method of existing and replicating extrachromosomally using a plasmid or the like, or may be incorporated into the chromosome.
In the case of extrachromosomal presence or a method of replicating, a vector replicable in the host bacterium is used. For example, when Escherichia coli is used as a host bacterium, pBR322, pUC18, Col
E1 or the like can be used. On the other hand, in the method of integrating into the host chromosome of a bacterium of the genus Bacillus, it is desirable to use no vector or one that does not replicate in the host bacterium. For example, the above plasmid or pE194 can be used. Not limited.

The transformants can be selected by, but not necessarily limited to, clear zone formation on an agar medium containing skim milk or casein and expression of a gene marker linked to the transgene.
The medium for culturing the transformant obtained as described above is not particularly limited, and any medium can be used as long as the transformant can grow. Separation and purification of protease from the culture broth can be performed according to a general method for separating and purifying protein. For example, salting out, ion chromatography, hydrophobic chromatography and the like are possible, but not limited thereto.

[0021]

[Method of measuring enzyme activity] In the present invention, the activity of the enzyme was measured by the following method. 1.5 ml of 50 mM Atkins-Pantin buffer (pH 10.0) was mixed with 5 μl of the synthetic substrate Suc-Ala-Ala-Pro-Phe-MCA dissolved in dimethyl sulfoxide (DMSO) so that the concentration was 10 mM. Add 10 μl of enzyme solution to the mixture. Reaction temperature 30
7-amino-4 generated by excitation light at 345 nm
Fluorescence of methylcoumarin (AMC) is detected at 440 nm. The amount of enzyme that produces 1 mol of AMC per second is 1 ka
tal.

[0022]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited thereto. Example 1: Production of novel alkaline protease by Escherichia coli
Genomic D cloned from production Bacillus NKS-21
Transformation of NA into E. coli was performed using the shotgun method. That is, genomic DN of Bacillus NKS-21
A was partially digested with the restriction enzyme Sau3AI to obtain a fragment.
Next, this is subjected to agarose gel electrophoresis and 2 to 6 using GENECLEAN (manufactured by Bio101).
Only a kb DNA fragment was prepared.

The plasmid pUC119 was used as a vector and digested with the restriction enzyme HindIII. After this was treated with phenol and alkaline phosphatase, it was electrophoresed on agarose gel, and similarly treated with GENECLEAN.
A cleavage fragment was prepared using (manufactured by Bio101). Genomic DNA cloned from Bacillus NKS-21 containing a non-expressed gene by ligating a restriction enzyme digestion fragment of genomic DNA with a vector plasmid
A plasmid DNA incorporating the fragment of

Using this plasmid DNA, E. coli HB
101 strain was transformed and ampicillin (50 μl
/ Ml) on LB-skimmed milk agar
After culturing at 7 ° C. for 24 to 28 hours, ampicillin-resistant transformants were selected using the clear zone formation around the colony as an index. These transformants were cultured on a new identical plate to form a clear zone. Fragments were cut out from the clear zone forming portion on the medium with a punch, mounted on an agarose gel for electrophoresis, and directly subjected to agarose electrophoresis.

After completion of electrophoresis, 40 μM Su was used as a substrate.
0.1 M Atkins-Pan containing c-Ala-Ala-Pro-Phe-MCA
Placing a plotting filter paper containing tin buffer (pH 10.0) and reacting at room temperature for 10 to 30 minutes, known API-21 (isoelectric point 7.4) on the transilluminator.
And SDP-515 (isoelectric point 2.8) are different from the isoelectric point (2.
Suc-Ala-Al, a novel protease having
It was confirmed that fluorescence of AMC (7-amino-4methylcoumarin) generated by cleavage of a-Pro-Phe-MCA was generated.
This novel protease-producing strain was obtained at a ratio of about 1 in 10,000 strains.

100 ml of medium (pH 7.2) containing 1% yeast extract, 0.1% skim milk, and 0.5% NaCl
In a 500 ml Sakaguchi flask at 15 ° C for 15
Sterilize in an autoclave for minutes. Inoculating the new protease-producing strain obtained by screening into the medium,
The culture was performed at 26 ° C. for 48 to 72 hours with shaking. The obtained culture solution was centrifuged at 13,600 g for 10 minutes, and the supernatant liquid (90 ml)
When the enzyme activity of was measured, it was 2 nkatal / ml. The crude enzyme powder was obtained by freeze-drying this solution.

Example 2 Purification of Novel Protease All the following purification operations were carried out at 4 ° C. or lower. Example 1
1 g of the crude enzyme powder obtained in step 1 was added to a 10 mM Tris-HCl buffer solution (pH 7.
2) Dissolved in 50 ml (hereinafter referred to as buffer solution A). This enzyme solution was previously equilibrated with buffer solution A, DEAE.
-TOYOPEARL 650S column (3 x 8 cm)
And eluted with a linear gradient of 0-1.0 M NaCl. The active fraction was collected, precipitated by adding 30% saturated ammonium sulfate, and the supernatant was taken and equilibrated with Buffer A containing 30% saturated ammonium sulfate in advance.
ofine) (3.4 × 12 cm) and eluted with a linear concentration gradient of 30 to 0% ammonium sulfate. The active fraction is collected and 30%
After dialyzing against a buffer solution A containing saturated ammonium sulfate, the solution was applied to the same column again and eluted under the same conditions. The active fractions were combined and dialyzed against buffer A to remove ammonium sulfate. This was again applied to a DEAE-TOYOPEARL 650S column that had been equilibrated with buffer A in advance, and eluted with a linear concentration gradient of 0 to 1.0 M NaCl in the same manner to obtain a protein-uniform purified enzyme (activity yield 6%).

Example 3: Properties of purified enzyme (1) Measurement of pH stability and optimum pH The pH stability was measured by the following method. The alkaline protease obtained in Example 2 was adjusted to each pH.
Mix 1 M Britton-Robinson Wide Area Buffer in equal volume,
After incubating at 30 ℃ for 10 minutes, 0.1 M Atki
The activity was measured in ns-Pantin buffer using Suc-Ala-Ala-Pro-Phe-MCA as a substrate. The activity was determined by a colorimetric method after the enzymatic reaction was stopped and the color was developed with a phenol reagent or ninhydrin. The results are shown in FIG. 1, where the activity at pH 10.0 is expressed as 100. Also, the optimum p
H was determined by measuring the activity at 30 ° C. using the same buffer solution having the same pH as that used in the above measurement and also using Suc-Ala-Ala-Pro-Phe-MCA as a substrate. The results are shown in FIG. 2, in which the activity at pH 11.0, which showed the highest activity, was expressed as 100. From FIGS. 1 and 2, the alkaline protease is stable in the range of about pH 5 to 11.5, and the optimum p
It was found that H was 11 or more.

(2) Measurement of thermal stability and optimum temperature Thermal stability was measured by the following method. That is, the novel protease obtained in Example 2 was added to pH 10.0.
After incubating for 10 minutes at each temperature with Suc-Ala-
The activity was measured at 30 ° C. and pH 10.0 using Ala-Pro-Phe-MCA as a substrate. 100% activity when treated at 30 ℃
The result displayed as is shown in FIG. From Fig. 3, this alkaline protease is completely stable up to about 55 ° C,
It turns out that it will be completely deactivated. The optimum temperature is p
It was determined by measuring the activity at each temperature when reacting with H10.0 using 1.0% casein as a substrate for 10 minutes. The result of displaying the value at 30 ° C. as 100 is as shown in FIG. 4, and the optimum temperature is about 50 ° C.

(3) Measurement of Isoelectric Point The isoelectric point of the novel protease obtained in Example 2 was measured by agarose gel electrophoresis.
It was less than 2.8. (4) Measurement of molecular weight Similarly, the novel protease obtained in Example 2 was treated with SDS.
-The molecular weight measured by polyacrylamide gel electrophoresis was about 37,000.

(5) Activity measurement against various substrates Casein, ovalbumin, bovine serum albumin, hemoglobin, curpain, salmine, lysozyme, gelatin, and collagen were used as substrates and adjusted to 1% each, pH 10.0. The activity was measured at. After the reaction was stopped, the activity was measured by a method using a phenol reagent (method of Folin et al.) And a ninhydrin method. The result displayed by the relative activity which made the activity with respect to casein 100 is shown in FIG. From FIG. 5, the synthetic substrate Suc-
It can be seen that the activity against substrates such as Ala-Ala-Pro-Phe-MCA, hemoglobin, curpain, salmine, and gelatin is high. The present protease particularly acts effectively on basic proteins rich in basic amino acids such as arginine.

(6) Stability to Surfactants In the presence of 0.1% of each of the various surfactants listed in Table 1,
After incubating for 60 minutes at 30 ° C and pH 10.0,
The activity was measured using casein. Color development was performed using a phenol reagent. The results are shown in Table 1.

[0033]

[Table 1]

It can be seen from Table 1 that it is stable against surfactants, especially nonionic surfactants. (7) Inhibition of activity by chemical substances In the presence of various chemical substances at the concentrations shown in Table 2, 3
After incubating at 0 ℃ for 10 minutes, the synthetic substrate Suc-Al
The activity was measured by fluorescence spectroscopy using a-Ala-Pro-Phe-MCA. The results are shown in Table 2, where the activity when no chemical substance was added was set to 100.

[0035]

[Table 2]

From these results, it was found that the activity of this protease was completely inhibited by phenylmethanesulfonyl fluoride (PMSF), chymostatin and antipain.

Example 3: Wash containing the present protease
Agent composition The enzyme of the present invention was mixed with various detergents, and the washing test of blood-contaminated cloth was conducted as follows. A. Preparation of cotton cloth contaminated with whole blood Using fresh bovine whole blood (9 volumes of blood plus 1 volume of 3.8% sodium citrate solution to prevent coagulation) immediately after blood sampling, the test cloth was immersed and contaminated under the following conditions. 7 by mangle
After squeezing it to 0% to uniformly contaminate it, it was air-dried indoors while avoiding direct sunlight, and stored in a cool dark place of 0 to 5 ° C. After preparation,
It was cut into 10 cm × 5 cm and used for cleaning. <Conditions> Sample cloth: 10 cm × 15 cm, Contamination liquid: 100 ml per 10 sample cloths, Contamination temperature: 10 ± 2 ° C., Contamination time: 5 minutes (reversed every 30 seconds).

B. Washing Method Using a Terg-O-Tometer, washing and rinsing (three times) were performed under the following conditions. <Washing conditions> Contaminated cloth: 5 cm x 10 cm, Detergent: 0.133% (enzyme 5 nkatal / ml), Bath ratio: 1:85, Inversion number: 105 rpm, Washing time: 10 minutes, Washing temperature: 40 ° C. <Rinse conditions> Contaminated cloth: 5 cm × 10 cm, bath ratio: 1:85, inversion number: 105 rpm, rinse time: 3 minutes. The rinsed cloth was air-dried indoors, avoiding direct sunlight.

C. Detergents used and enzymes As the detergents, three types of phosphorus-free detergents having the compositions shown below were used. <Composition of detergent> Surfactant (LAS type, SDS type or AOS type) 15% Zeolite 17% Sodium silicate 5% Sodium carbonate 3% Carboxymethyl cellulose 1% Moisture 8% Sodium sulfate balance Washing results are shown in Table 3.

[0040]

[Table 3]

[0041]

INDUSTRIAL APPLICABILITY By the method of the present invention, a non-secreted alkaline protease which is not expressed under normal conditions can be produced. Moreover, this protease is a novel alkaline protease having a low isoelectric point and more excellent stability against heat and a surfactant. The protease of the present invention is useful as an industrial enzyme such as a cleaning aid.

[0042]

[Sequence list]

SEQ ID NO: 1 Sequence length: 972 Sequence type: Nucleic acid and amino acid Number of chains: Double strand Topology: Linear Sequence type: Genomic DNA sequence TTG AGT AAA GTG AGC CTG ATT CCA TTC AAA GTT GAG AAG GTT CTA AAT 48 Met Ser Lys Val Ser Leu Ile Pro Phe Lys Val Glu Lys Val Leu Asn 1 5 10 15 GAC ACA AAG GTT ATT CCG CCC GGT ATT GAA ATG ATT GAA GCA CCA GCC 96 Asp Thr Lys Val Ile Pro Pro Gly Ile Glu Met Ile Glu Ala Pro Ala 20 25 30 GTA TGG GAG GCT GGA TAT AAG GGT GGT AAT ACT GTT GTA GCT GTT CTA 144 Val Trp Glu Ala Gly Tyr Lys Gly Gly Asn Thr Val Val Ala Val Leu 35 40 45 GAT ACA GGG TGT GAA ACG ACC CAC ATC GAA TTT AAG GAT CAA ATT ATT 192 Asp Thr Gly Cys Glu Thr Thr His Ile Glu Phe Lys Asp Gln Ile Ile 50 55 60 GAC GGT CGT AAC TTT ACT ACA GAT GAT AAC AGC GAC CCT GAT AAT GTA 240 Asp Gly Arg Asn Phe Thr Thr Asp Asp Asn Ser Asp Pro Asp Asn Val 65 70 75 80 GAA GAT TCT AAC GGT CAT GGT ACT CAC GTA TGC GGA CCC GTT GCT GCC 288 Glu Asp Ser Asn Gly His Gly Thr His Val Cys Gly Pro Val Ala Ala 85 90 95 TGT GAG AAT GAC AAG GGC GTC ATT GGT ACC GCC CCA AAA GCG AAA CTG 336 Cys Glu Asn Asp Lys Gly Val Ile Gly Thr Ala Pro Lys Ala Lys Leu 100 105 110 CTC GTT GTA AAG GTG CTT AGC GGA CAA GGG TAC GGA GAT ACA AAA TGG 384 Leu Val Val Lys Val Leu Ser Gly Gln Gly Tyr Gly Asp Thr Lys Trp 115 120 125 GTC ATT GAA GGG GTT CGT TAT GCG ATA AAT TGG CGT GGA CCA AAC AAT 432 Val Ile Glu Gly Val Arg Tyr Ala Ile Asn Trp Arg Gly Pro Asn Asn 130 135 140 GAA CGA GTT CGT GTC ATT TCT ATG TCA CTC GGG GGA AGA ATT GAT ACT 480 Glu Arg Val Arg Val Ile Ser Met Ser Leu Gly Gly Arg Ile Asp Thr 145 150 155 160 CCT GAA CTT CAT CAA GCG ATA AAA CAT GCT GTA GCT GAG GAT ATT TTA 528 Pro Glu Leu His Gln Ala Ile Lys His Ala Val Ala Glu Asp Ile Leu 165 170 175 GTT GTA TGT GCA GCT GGA AAT GAA GGG GAT GGC AAT CAT GAC ACA GAT 576 Val Val Cys Ala Ala Gly Asn Glu Gly Asp Gly Asn His Asp Thr Asp 180 185 190 GAA TAT GCC TAC CCT GGA GCT TAT CCG GAA GTC GTT CAA GTA GGC TCT 624 Glu Tyr Ala Tyr Pro Gly Al a Tyr Pro Glu Val Val Gln Val Gly Ser 195 200 205 GTC AAT CTA GAA GGC GAG ATC TCT AGA TTC AGC AAT ACA AAT TGT GCG 672 Val Asn Leu Glu Gly Glu Ile Ser Arg Phe Ser Asn Thr Asn Cys Ala 210 215 220 ATT GAC CTT GTC GCA CCA GGC GAA GAA ATT ATT TCA ACT TAT CTT AAC 720 Ile Asp Leu Val Ala Pro Gly Glu Glu Ile Ile Ser Thr Tyr Leu Asn 225 230 235 240 AAC GGC TAC GCT GTC TTA TCC GGT ACT TCA ATG GCT ACA CCG CAT GTA 768 Asn Gly Tyr Ala Val Leu Ser Gly Thr Ser Met Ala Thr Pro His Val 245 250 255 TCC GGT GCG GCA GCC CTG TTA ATT GAA CAA GTA GAA AAA GAG TTT GAA 816 Ser Gly Ala Ala Ala Leu Leu Ile Glu Gln Val Glu Lys Glu Phe Glu 260 265 270 AGA AAG TTG ACG GAA CCA GAA ATT TTC GCA CAA CTG ATC AAA CAC ACC 864 Arg Lys Leu Thr Glu Pro Glu Ile Phe Ala Gln Leu Ile Lys His Thr 275 280 285 GTT TCT CTT AAC TTC AGC CGC CGC GCA CAA GGA AGC GGG CTG TTG AAA 912 Val Ser Leu Asn Phe Ser Arg Arg Ala Gln Gly Ser Gly Leu Leu Lys 290 295 300 TTA TCA TCA AGC GTT GTA TCA GTA GAG GAT GCC GAA TAT ACA ACT AGC 960 Leu Ser Ser Se r Val Val Ser Val Glu Asp Ala Glu Tyr Thr Thr Ser 305 310 315 320 TCT ATT AAA TAG 972 Ser Ile Lys *

[Brief description of drawings]

FIG. 1 is a graph showing the pH stability of the enzyme of the present invention.

FIG. 2 is a graph showing the optimum pH range of the enzyme of the present invention.

FIG. 3 is a graph showing the thermostability of the enzyme of the present invention.

FIG. 4 is a graph showing the optimum temperature range of the enzyme of the present invention.

FIG. 5 is a graph showing the relative activity of the enzyme of the present invention for each substrate.

Claims (6)

[Claims] 1. An alkaline protease having the following properties. (1) Optimum pH: optimum action p when the synthetic substrate Suc-Ala-Ala-Pro-Phe-MCA is used as a substrate and reacted at 30 ° C for 10 minutes
H is 11 or more. (2) pH stability: synthetic substrate Suc-Ala-Ala-Pro-Phe-MCA
PH of 5 when reacted at 30 ℃ for 10 minutes
Stable in the range of ~ 11.5. (3) Optimum temperature: pH 10 with 1.0% casein as a substrate
When the reaction is performed for 10 minutes, the optimum working temperature is around 50 ° C. (4) Thermal stability: 55 when treated at pH 10 for 10 minutes
It is quite stable up to ° C and completely deactivates at 65 ° C. (5) Substrate specificity: synthetic substrate Suc-Ala-Ala-Pro-Phe-MCA
Decomposes casein, hemoglobin, curpain, salmine and gelatin. (6) Isoelectric point: The isoelectric point by agarose gel electrophoresis is less than 2.8. (7) Molecular weight: The molecular weight measured by SDS-polyacrylamide gel electrophoresis is about 37,000. (8) Effects on chemical substances: Completely inhibited by phenylmethanesulfonyl fluoride, chymostatin and antipain. 2. The alkaline protease according to claim 1, which has the amino acid sequence represented by SEQ ID NO: 1. 3. The gene represented by SEQ ID NO: 1 which encodes the alkaline protease according to claim 2. 4. A microorganism obtained by partially degrading genomic DNA of Bacillus genus NKS-21 (Microtechnical Laboratory Article No. 93) and transforming the DNA having the sequence represented by SEQ ID NO: 1 by the shotgun method. Then, alkaline protease is obtained from the culture solution.
The method for producing an alkaline protease according to 1. 5. The method for producing an alkaline protease according to claim 1, wherein the transformed microorganism introduced with the gene according to claim 3 is cultured to obtain an alkaline protease from the culture solution. . 6. A detergent composition comprising the alkaline protease according to claim 1 or 2.