JPH0795882A - Alkaline protease and gene coding for the same - Google Patents

Abstract

PURPOSE:To obtain an alkaline protease having stability to alkali and high temperature and useful as an industrial enzyme such as a washing assistant. CONSTITUTION:An alkaline protease having the following properties is obtained by expressing a part of the genom DNA of a bacterial strain of Bacillus sp. THS-1001: (1) the optimum pH, 9.5-10.5; (2) a stable pH range, 3.5-12; (3) the optimum active temperature, about 50 deg.C; (4) stability at pH 10, entirely stable at 30 deg.C, comparatively stable up to 60 deg.C, completely inactive at 70 deg.C; (5) decomposes Sue-Leu-Leu-Val-Tyr-MCA; (6) isoelectric point (electrofocusing), >=11; (7) molecular weight (SDS-PAGE), about 29,000; (8) inhibited by chymostatin, antipain and phenylmethanesulfonyl fluoride(PMSF). The gene coding for the enzyme is also provided.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel alkaline protease and a gene encoding the same. More specifically, Bacillus sp. The present invention relates to a novel alkaline protease excellent in heat stability obtained by culturing THS-1001 strain and a gene encoding the same.

[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 (hereinafter referred to as Bacillus NKS-21) is an alkaline protease API-21 (hereinafter referred to as A) having an activity at a relatively low temperature.
It is called PI-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-515
Say. ) Is produced (see Japanese Patent Laid-Open No. 1-281084).

[0007] The applicant of the present invention has also found that Bacillus NKS-21
A more stable protease production has been proposed by modifying the amino acid sequence of the strain-derived API-21 with a specific site using a gene modification technique (see Japanese Patent Laid-Open No. 5-91876).

Furthermore, the present inventors have found that Bacillus NKS-2
To the stability obtained by expressing the unexpressed part of the genomic DNA of Bacillus NKS-21 by a screening method in which a fragment obtained by partially decomposing the genomic DNA of No. 1 by a restriction enzyme is introduced by the shotgun method and the transformed microorganism is cultured. An excellent alkaline protease, a gene encoding the protease, a method for producing the protease, and an application to the detergent composition have been previously proposed (Japanese Patent Application No. 4-296360).

[0009]

An object of the present invention is to provide a novel alkaline protease derived from Bacillus bacterium with excellent stability, and a gene encoding the protease.

The Bacillus sp. Used in the present invention. THS-1
The 001 strain was collected from soil in Tokyo, and p
Can grow in an alkaline region of H10 or higher, but has a neutral pH
It is an alkalophilic bacterium that cannot grow in.

This bacterium is a Gram-positive bacillus and forms spores, and thus it has been clarified to be an alkalophilic bacterium belonging to the genus Bacillus. The bacterium is Bacillus sp.
It is named THS-1001 strain and has been deposited as FERMP-13869 at the Institute of Biotechnology, Institute of Biotechnology, AIST.

[0012]

Means for Solving the Problems The present invention comprises: 1) an alkaline protease having substantially the amino acid sequence represented by SEQ ID NO: 2 and an isoelectric point of 11 or more; 2) the amino acid sequence represented by SEQ ID NO: 2. And 3) a gene represented by SEQ ID NO: 3 which encodes an alkaline protease having an amino acid sequence represented by SEQ ID NO: 2.

The novel alkaline protease according to the present invention has substantially the amino acid sequence shown in SEQ ID NO: 2. That is, it does not have to be the same as this,
Amino acid substitution, deletion, or insertion forms are also included as long as the basic characteristic that the isoelectric point is 11 or more does not change.

The novel alkaline protease according to the present invention is Bacillus sp. It can be obtained by culturing the THS-1001 strain, or by introducing the gene of the present invention into a microorganism and culturing the microorganism in a medium to produce it, and recovering the protease from the culture solution.
The DNA used at this time is Bacillus sp. TH
The gene obtained from the S-1001 strain is used, and this DNA contains at least the DNA of the nucleotide sequence encoding the present alkaline protease gene represented by SEQ ID NO: 3 or the amino acid sequence represented by SEQ ID NO: 2. Suffice it to say that Bacillus sp. It is not necessary to include all genes from the THS-1001 strain. Further, a DNA fragment in which another promoter region, a signal sequence, a terminator region or the like is bound to the present alkaline 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 may 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 not to use a vector or to use one that does not replicate in the host bacterium. For example, the above plasmid or pE194 can be used. Not limited.

Selection of transformants can be performed 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 transformant obtained as described above, or Bacillus sp. The medium for culturing the THS-1001 strain is not particularly limited, and any medium that can grow can be used. 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.

[0018]

[Method of measuring enzyme activity] In the present invention, the activity of the enzyme was measured by the following method. Synthetic substrate succinyl-leucyl-leucyl-valyl-tyrosyl-4-methylcoumaryl-7-amide (hereinafter referred to as Suc-Le) was added to 1.5 ml of 50 mM Atkins-Pantin buffer (pH 10.0) so that the concentration was 10 mM.
Abbreviated as u-Leu-Val-Tyr-MCA. ) Is dissolved in dimethylsulfoxide (DMSO) and mixed with 5 μl, and 10 μl of the enzyme solution is added to this mixed solution. Reaction temperature 30 ℃
The fluorescence of 7-amino-4-methylcoumarin (AMC) generated by the excitation light of 345 nm is detected at 440 nm. The amount of enzyme that produces 1 mol of AMC per second is 1 ka
tal.

[0019]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited thereto. Example 1: Purification of novel alkaline protease In a 500 ml Sakaguchi flask, 4 g of soybean powder and K ₂ HPO _{4 were} added.
Add 0.8 g, 0.08 g of MgSO ₄ and 90 ml of water and add 1
It was sterilized by autoclaving at 15 ° C for 15 minutes. Separately, 10 ml of 50% maltose solution and 1 ml of 10% Na ₂ CO ₃ solution sterilized under the same conditions were aseptically added to the Sakaguchi flask. Bacillus sp. The THS-1001 strain was inoculated into the above-mentioned medium and shake-cultured at 30 ° C. for 44 hours. The obtained culture broth was centrifuged at 9000 xg for 15 minutes, and the enzyme activity of the supernatant (92 ml) was measured.
It was 125 nkatal. The crude enzyme powder was obtained by freeze-drying this solution.

Hereinafter, all the purification operations were performed at 4 ° C. or lower. 1 g of the above crude enzyme powder was added to 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 A and DEAE-TOY
It was applied to an OPEARL 650S column (3 × 8 cm) and eluted with the above buffer solution A. The non-adsorbed active fraction is collected and 10 mM
After dialysis with Atkins-Pantin buffer (pH 10.0), equilibrated with CM-Sephadex C-50 column (2 x 2)
5 cm) and eluted with a linear gradient of 0 to 1.0 M NaCl. The active fraction was collected and 10 mM Atkins-Pan
HW-50F-gel filtration equilibrated with tin buffer (pH 10.0) was performed to obtain a purified protein-uniform enzyme (activity yield 10%).

Example 2: Properties of purified enzyme (1) Measurement of pH stability and optimum pH The pH stability was measured by the following method. The novel alkaline protease obtained in Example 1 was mixed with an equal amount of 0.1 M Britton-Robinson broad range buffer solution adjusted to each pH and incubated at 30 ° C. for 10 minutes, and then 0.1 M.
40 μM Suc-Leu with Atkins-Pantin buffer (pH 10.0)
The activity was measured using -Leu-Val-Tyr-MCA as a substrate.
Figure 1 shows the results when the activity at pH 10.0 is displayed as 100.
Shown in. In addition, the optimum pH is the same as that used in the above-mentioned measurement, using the same pH buffer solution at 30 ° C.
It was determined by measuring the activity using -Leu-Val-Tyr-MCA as a substrate. The results are shown in FIG. 2, in which the activity at pH 10.0, which shows the highest activity, is expressed as 100. 1 and 2
From this, it was found that the present alkaline protease is stable in the range of about pH 3.5 to 12, and the optimum pH is 9.5 to 10.5.

(2) Measurement of thermal stability and optimum temperature Thermal stability was measured by the following method. That is, the novel alkaline protease obtained in Example 1 was
After incubating at pH 10.0 for 10 minutes at each temperature,
Suc-Leu-Leu-Val-Tyr-MCA as substrate at 30 ℃, pH1
The activity was measured at 0.0. The results are shown in FIG. 3, in which the activity when treated at 30 ° C. is expressed as 100. From FIG. 3, it was found that this alkaline protease is quite stable up to about 30 ° C., is quite stable up to 60 ° C., but is completely inactivated at 70 ° C. The optimum temperature is pH 10.0.
40 μM Suc-Leu-Leu-Val-Tyr-MCA was used as a substrate for 10 minutes and the activity was measured at each temperature. 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 alkaline protease obtained in Example 1 was measured by electrofocusing (focusing electrophoresis method). First, perform agarose gel electrophoresis, and after completion, place a plotting filter paper containing Atkins-Pantin buffer (pH 10.0) containing the substrate Suc-Leu-Leu-Val-Tyr-MCA at room temperature. The reaction was performed for 10 to 30 minutes. As a result, YaB (Jou), which is an alkaline elastase showing the highest isoelectric point (10.6) among the conventionally known ones.
rnal of Bacteriology, 171, 5232-5236, 1989), showing a higher isoelectric point (11 or more). The isoelectric point of alkaline protease 221 having a value of about 70%, which has the highest correlation with the enzyme of the present invention and a primary structure, is pH 9.4 (Dainori Tsuru, Microorganisms, page 5,322 (1989)). The enzyme is obviously different. (4) Measurement of molecular weight The molecular weight of the novel alkaline protease obtained in Example 1 was measured by 15% SDS-polyacrylamide gel electrophoresis, and it was about 29,000.

(5) Comparative measurement of activity of various serine proteases against synthetic substrate Using the present invention and various known serine proteases, MCA substrates shown in Table 1 were used as substrates and prepared to be 1% each, The activity was measured at pH 10.0 (Table 1
Among them, the amino acids that make up the substrate are indicated by the one-letter abbreviation. Table 1 shows the results displayed by relative activity with the activity against Suc-Leu-Leu-Val-Tyr-MCA being 100. The activity of the novel alkaline protease on various synthetic substrates shows a unique specificity not found in known alkaline proteases.

[0025]

[Table 1]

In addition, the present invention and eight known serine proteases [enzyme 221 are also used with the formate-insulated insulin B chain having the amino acid sequence shown in SEQ ID NO: 4 as a substrate.
(H. Tamaki et al., Biosci. Biotech. Biochem., 56,333
-334 (1992)), Elastase YaB (same as above), AH101
(Heat-resistant alkaline protease of Bacillus sp. No. AH-101 (same as above)), BYA (Alkali-resistant protease produced by Bacillus sp. Y (H. Shimogaki et a
l., Agric. Biol. Chem., 55, 2251-2258 (1991))), ALPI
(Alkaline protease produced by Bacillus sp. NKS-21 (O. Tsuchida et al., Curr. Microbiol., 1
4, 7-12 (1986)), Subtilisin Carls-berg (Ta above)
maki et al.), Subtilisin BPN '(same as above), NA
T (serine protease produced by Bacillus natto of Bacillus natto (E. Ichishima et al., Biochim. Biophys. Acta,
869, 178-184 (1986))], and the peptides formed are analyzed to determine the action binding site. The results are shown in FIG. It is represented by a one-letter abbreviation consisting of, where C ^* represents cysteine).

From the results shown in Table 1 and FIG. 5, it can be seen that the alkaline protease of the present invention has a substrate specificity different from that of known serine proteases. (6) Inhibition of activity by chemical substances In the presence of various chemical substances at the concentrations shown in Table 2, 4
After incubating at ℃ for 20 hours, the synthetic substrate Suc-Leu-
The activity was measured by fluorescence spectroscopy using Leu-Val-Tyr-MCA (40 μM). Table 2 shows the results when the inhibition rate when no chemical substance was added (control group) was 0 (%).

[0028]

[Table 2]

From these results, this alkaline protease was almost completely inhibited by chymostatin, antipain and phenylmethanesulfonyl fluoride (PMSF), and tosylphenylalanyl chloromethyl ketone (T
PCK), which was found to be inhibited by elastatinal.

(7) Stability against surfactants To the enzyme solution according to the present invention, the surfactants (10% solution) shown in Table 3 were added so as to have respective set concentrations (0.05, 0.1, 1%). After incubation for 60 minutes at 30 ° C. and pH 10.0, the activity was measured using Suc-Leu-Leu-Val-Tyr-MCA. The results are shown in Table 3.

[0031]

[Table 3]

From Table 3, it can be seen that the present alkaline protease is stable to nonionic surfactants.

Example 3: A novel alkaline protease
Isolation of the DNA that carries Bacillus sp. Cloning from the THS-1001 strain was performed using the shotgun method. That is, Bacillus sp. The genomic DNA of the THS-1001 strain was partially digested with the restriction enzyme Sau3AI to obtain a fragment. Next, this is subjected to agarose gel electrophoresis, and Geneclean (GENECLE
AN) (manufactured by Bio101) using 2 to 6 kb of DNA
Only fragments were prepared.

The plasmid pUC119 was used as a vector and digested with the restriction enzyme BamHI. After this was treated with phenol and alkaline phosphatase, it was electrophoresed on an agarose gel and the same as GENECLEAN.
A cleavage fragment was prepared using (manufactured by Bio101). Ligation of a restriction enzyme digestion fragment of genomic DNA with a vector plasmid was performed, and Bacillus sp. THS-1
A plasmid DNA in which a genomic DNA fragment derived from strain 001 was incorporated was obtained.

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, the desired transformant was selected from the ampicillin-resistant transformants, 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, 0.1 M At containing 40 μM of the synthetic substrate Suc-Leu-Leu-Val-Tyr-MCA as a substrate.
Put a plotting filter paper containing kins-Pantin buffer (pH 10.0), and let it react at room temperature for 10 to 30 minutes.
A protease having an isoelectric point of 11 or more on the transilluminator equivalent to the novel alkaline protease obtained in Example 1 was obtained, and Tyr-M of Suc-Leu-Leu-Val-Tyr-MCA was obtained.
It was confirmed that the fluorescence of 7-amino-4-methylcoumarin (AMC) generated by the cleavage of the bond between CAs was generated. This protease producing strain was obtained at a ratio of about 15,000 strains.

Example 4: DNA of alkaline protease
Determination of Nucleotide Sequence and Amino Acid Sequence The nucleotide sequence of the gene encoding the alkaline protease obtained in Example 3 was determined by Sanger's method and analyzed. As shown in SEQ ID NO: 1, it begins with TTG.
We have found an open reading frame of 1.15 kb.
Further, 10 residues from the N-terminal of the novel alkaline protease obtained in Example 1 were determined by 473A Protein Sequencer manufactured by Applied Biosystems, and as a result, the first 1 of the amino acid sequence shown in SEQ ID NO: 1 was determined.
It was confirmed that the gene completely matched with 0 residue and encodes the novel alkaline protease obtained in Example 1.

When the homology with known proteases was examined, the alkalophilic Bacillus sp. 221 alkaline serine protease (isoelectric point: 9.4, Daisuke Tsuru, microorganism,
5,322 (1989)), the homology is about 70%, revealing that this enzyme is a novel enzyme.

This enzyme is a protein consisting of 269 amino acid residues and having a molecular weight of 27,232. Asparagine is present at the N-terminal of the protein, and it was revealed that it is a novel serine protease structurally unprecedented.

[0040]

INDUSTRIAL APPLICABILITY The alkaline protease of the present invention is a novel alkaline protease and is useful as an industrial enzyme such as a cleaning aid. In addition, the gene of the present invention is industrially useful because the alkaline protease of the present invention can be efficiently obtained by culturing a transformed microorganism into which the gene has been introduced, and can also be used as a sample for protein engineering. is there.

[0041]

[Sequence list]

SEQ ID NO: 1 Sequence length: 1152 Sequence type: Nucleic acid and amino acid Number of chains: Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Bacillus sp. THS-1001 (Bacillus s
p.THS-1001) SEQ TTGAAAAAGTTATTTACAAAGGTA 24 GTGGCAAGTGCCGCGTTATTATTGTCCATCTCTTTAACCGCAACATCCGTCTCAGCCGAGGAA 88 CAAAAGAAACAGTATTTAATTGGTTTTGAAAATCAACTACAAGTAACGGAATTTGTAGAAAGC 152 AGTGATAAAGGGCAAAGTGAAATGTCTCTTTTTGCTGAAGTGAATGATGAATCAATTGAAATG 216 GAACTTCTTTATGAATTCGAGGACATTCCAGTTGTATCAGTTGAATTGAGTCCAGAAGATGTA 280 AAGGACTTAGAAAAGGACCCTTCGATTACTTATATTGAGGAAGATATAGAAGTCACTATTACC 344 AAT CAA GTG ACT CCT TGG GGG ATT ACA CGT GTA CAA GCC CCT ACG GCA 392 Asn Gln Val Thr Pro Trp Gly Ile Thr Arg Val Gln Ala Pro Thr Ala 1 5 10 15 TGG ACT AGA GGA TAT ACT GGT ACG GGA GTT CGG GTA GCG GTA CTT GAT 440 Trp Thr Arg Gly Tyr Thr Gly Thr Gly Val Arg Val Ala Val Leu Asp 20 25 30 ACA GGG ATT TCT ACA CAT CCT GAT TTA AAT ATT CGA GGT GGA GTA AGT 488 Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Val Ser 35 40 45 TTT GTT CCT GGT GAA CCA TCC TAT CAA GAT GGA AAT GGT CAT GGA ACA 536 Phe Val Pro Gly Glu Pro Ser Tyr Gln Asp Gly Asn Gly His Gly Thr 50 55 60 CAT GTG GCA GGT ACA ATT GCT GCT TTA AAT AAT TCG ATT GGT GTT GTT 584 His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Val 70 70 75 80 GGT GTA GCT CCA AAT GCG GAG CTC TAT GCC GTG AAA GTA CTA GGG GCA 632 Gly Val Ala Pro Asn Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala 85 90 95 AAT GGT TCA GGC TCT GTT AGT TCA ATT GCT CAG GGA TTA CAA TGG ACA 680 Asn Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Gln Trp Thr 100 105 110 GCC CAA AAT AAT ATT CAT GTT GCT AAT TTA AGT TTA GGT AGT CCA GTA 728 Ala Gln Asn Asn Ile His Val Ala Asn Leu Ser Leu Gly Ser Pro Val 115 120 125 GGA AGT CAA ACA CTA GAG TTA GCT GTT AAT CAA GCT ACA AAT GCA GGT 776 Gly Ser Gln Thr Leu Glu Leu Ala Val Asn Gln Ala Thr Asn Ala Gly 130 135 140 GTT TTA GTG GTA GCA GCA ACT GGT AAT AAT GGT TCT GGT ACA GTT TCT 824 Val Leu Val Val Ala Ala Thr Gly Asn Asn Gly Ser Gly Thr Val Ser 145 150 155 160 TAT CCC GCT CGT TAT GCG AAT GCT TTA GCG GTA GGT GCA ACA GAT CAA 872 Tyr Pro Ala Arg Tyr Ala Asn Ala Leu Ala Val Gly Ala Thr Asp Gln 165 170 175 AAT AAT AAT CGT GCT AGC TTT TCA CAA TA T GGG ACA GGT TTA AAT ATC 920 Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Thr Gly Leu Asn Ile 180 185 190 GTA GCT CCA GGA GTC GGA ATT CAG AGT ACG TAT CCT GGT AAC AGA TAT 968 Val Ala Pro Gly Val Gly Ile Gln Ser Thr Tyr Pro Gly Asn Arg Tyr 195 200 205 GCA AGT TTA AGT GGA ACA TCA ATG GCT ACT CCT CAT GTT GCT GGG GTC 1016 Ala Ser Leu Ser Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Val 210 215 220 GCT GCA CTC GTT AAG CAG AAG AAC CCA AGC TGG TCT AAT ACA CAA ATT 1064 Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Thr Gln Ile 225 230 235 240 AGA CAA CAT CTT ACG TCT ACG GCC ACT TCA CTT GGG AAT TCA AAC CAG 1112 Arg Gln His Leu Thr Ser Thr Ala Thr Ser Leu Gly Asn Ser Asn Gln 245 250 255 TTT GGC AGT GGA CTT GTG AAT GCA GAA GCT GCA ACA AGG TAA 1152 Phe Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg * 260 265

SEQ ID NO: 2 Sequence length: 269 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide sequence Asn Gln Val Thr Pro Trp Gly Ile Thr Arg Val Gln Ala Pro Thr Ala 16 Trp Thr Arg Gly Tyr Thr Gly Thr Gly Val Arg Val Ala Val Leu Asp 32 Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Val Ser 48 Phe Val Pro Gly Glu Pro Ser Tyr Gln Asp Gly Asn Gly His Gly Thr 64 His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Val 80 Gly Val Ala Pro Asn Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala 96 Asn Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Gln Trp Thr 112 Ala Gln Asn Asn Ile His Val Ala Asn Leu Ser Leu Gly Ser Pro Val 128 Gly Ser Gln Thr Leu Glu Leu Ala Val Asn Gln Ala Thr Asn Ala Gly 144 Val Leu Val Val Ala Ala Thr Gly Asn Asn Gly Ser Gly Thr Val Ser 160 Tyr Pro Ala Arg Tyr Ala Asn Ala Leu Ala Val Gly Ala Thr Asp Gln 176 Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Thr Gly Leu Asn Ile 192 Val Ala P ro Gly Val Gly Ile Gln Ser Thr Tyr Pro Gly Asn Arg Tyr 208 Ala Ser Leu Ser Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Val 224 Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Thr Gln Ile 240 Arg Gln His Leu Thr Ser Thr Ala Thr Ser Leu Gly Asn Ser Asn Gln 256 Phe Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg * 269

SEQ ID NO: 3 Sequence length: 810 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Genomic DNA sequence AAT CAA GTG ACT CCT TGG GGG ATT ACA CGT GTA CAA GCC CCT ACG GCA 48 TGG ACT AGA GGA TAT ACT GGT ACG GGA GTT CGG GTA GCG GTA CTT GAT 96 ACA GGG ATT TCT ACA CAT CCT GAT TTA AAT ATT CGA GGT GGA GTA AGT 144 TTT GTT CCT GGT GAA CCA TCC TAT CAA GAT GGA AAT GGT CAT GGA ACA 192 CAT GTG GCA GGT ACA ATT GCT GCT TTA AAT AAT TCG ATT GGT GTT GTT 240 GGT GTA GCT CCA AAT GCG GAG CTC TAT GCC GTG AAA GTA CTA GGG GCA 288 AAT GGT TCA GGC TCT GTT AGT TCA ATT GCT GAT GGA TTA CAA TGG ACA 336 GCC CAA AAT AAT ATT CAT GTT GCT AAT TTA AGT TTA GGT AGT CCA GTA 384 GGA AGT CAA ACA CTA GAG TTA GCT GTT AAT CAA GCT ACA AAT GCA GGT 432 GTT TTA GTG GTA GCA GCA AACT GGT AAT GGT TCT GGT ACA GTT TCT 480 TAT CCC GCT CGT TAT GCG AAT GCT TTA GCG GTA GGT GCA ACA GAT CAA 528 AAT AAT AAT CGT GCT AGC TTT TCA CAA TAT GGG ACA GGT TTA AAT ATC 576 GTA GCT CCA GGA GTC GGA ATT CAG AGT ACG TAT CCT GGT AAC AGA TAT 624 GCA AGT TTA AGT GGA ACA TCA ATG GCT ACT CCT CAT GTT GCT GGG GTC 672 GCT GCA CTC GTT AAG CAG AAG AAC CCA AGC TGG TCT AAT ACA CAA CAATT 720 AGA CAT CTT ACG TCT ACG GCC ACT TCA CTT GGG AAT TCA AAC CAG 768 TTT GGC AGT GGA CTT GTG AAT GCA GAA GCT GCA ACA AGG TAA 810

SEQ ID NO: 4 Sequence length: 30 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Sequence Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 16 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Ala 30

[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 shows peptide binding sites that are hydrolyzed when the present invention and eight known serine proteases are used as a substrate with a formate-insulated insulin B chain as a substrate.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location C12R 1:07) (C12N 15/09 C12R 1:07) C12R 1:07)

Claims (3)

[Claims] 1. An alkaline protease having an amino acid sequence substantially represented by SEQ ID NO: 2 and an isoelectric point of 11 or more. 2. A gene encoding an alkaline protease having the amino acid sequence shown in SEQ ID NO: 2. 3. A gene represented by SEQ ID NO: 3 which encodes an alkaline protease having the amino acid sequence represented by SEQ ID NO: 2.