JP4212340B2 - Alkaline protease - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel alkaline protease useful as various industrial enzymes including detergents and a gene encoding the same.
[0002]
[Prior art and problems to be solved by the invention]
Alkaline proteases blended in various detergents including clothes detergents have played an important role as one component indispensable for improving detergency. Alkaline protease is the enzyme that is produced in large quantities among industrial enzymes because it is used in a mass consumer such as detergent.
[0003]
Currently used alkaline proteases for detergents include, for example, sabinase (registered trademark; manufactured by Novozymes), cannase (registered trademark; manufactured by Novozymes), durazyme (registered trademark; manufactured by Novozymes), maxacal (registered trademark; Genencor) ), Blap (registered trademark; manufactured by Henkel), KAP (manufactured by Kao Corporation), etc., all of which are derived from bacteria belonging to the genus Bacillus, belonging to the subtilisin family, and homologous to each other's amino acid sequences The property is very high (see Patent Document 1, Non-Patent Document 1, etc.).
[0004]
However, these alkaline proteases have advantages and disadvantages in alkali resistance, surfactant resistance, oxidant resistance, low-temperature activity, heat resistance, detergency, low substrate specificity, chelating agent resistance, metal ion resistance, etc. There is a need for a protease having superior properties as a cleaning enzyme.
[0005]
The present invention has a low amino acid sequence homology with the alkaline detergent for detergents as described above or conventional intrinsic subtilisin, and is not affected by calcium ions in stability to heat and pH, and various other industries including detergents. It is an object to provide an alkaline protease for use and a gene encoding the same.
[0006]
[Patent Document 1]
JP-A-6-292577 [Non-Patent Document 1]
Siezen, RJ & Leunissen JAM, Protein Science, 6 , 501-523 (1997)
[0007]
[Means for Solving the Problems]
The present inventors obtained a gene encoding an alkaline protease having a low homology in amino acid sequence with a known subtilisin from genomic DNA of an alkalophilic Bacillus bacterium, and conducted various studies. We succeeded in producing a novel alkaline protease that is not affected by.
[0008]
That is, the present invention provides the following protein (a) or (b):
(A) a protein comprising the amino acid sequence of SEQ ID NO: 1,
(B) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of (a) and having alkaline protease activity, a gene encoding the protein, and the gene And a transformant having the recombinant vector.
[0009]
DETAILED DESCRIPTION OF THE INVENTION
The protein of the present invention (hereinafter also referred to as “alkaline protease”) is (a) a protein consisting of the amino acid sequence of SEQ ID NO: 1, or (b) one or more amino acids deleted in the amino acid sequence of (a) , A protein consisting of a substituted or added amino acid sequence and having alkaline protease activity, and the gene of the present invention is DNA encoding the protein.
[0010]
Here, in the amino acid sequence of SEQ ID NO: 1, the amino acid sequence in which one or more amino acids are deleted, substituted or added means an amino acid sequence equivalent to the amino acid sequence of SEQ ID NO: 1, one or several , Preferably an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted or added, and still retain the alkaline protease activity. For addition, 1 to several amino acids at both ends are added. Includes appends.
[0011]
The base sequence encoding the equivalent amino acid sequence can be prepared using a known technique such as site-directed mutagenesis. For example, it can be prepared by introducing mutation using a mutagenesis kit [Mutan-super Express Km kit (Takara)] using site-directed mutagenesis.
[0012]
When compared to the alkaline protease consisting of the amino acid sequence of SEQ ID NO: 1 of the present invention and the amino acid sequences of other known alkaline proteases, subtilisin BPN 'is 67.4%, and subtilisin Carlsberg is 69.6% of the medium. It only shows homology. Furthermore, it shows only 58.8% homology with sabinase used as a detergent enzyme, 56.4% with esperase, and 58.0% with KAP (M-protease). Furthermore, the alkaline protease LD1 (GenBank Accession No AB085752) derived from the Bacillus sp. KSM-LD1 strain from which the gene of the present invention was obtained is 63.3%, and the homologue is 75.0% with the alkaline protease SA (GenBank Accession No AB090334). Showed sex. Moreover, it showed 77.9% homology with SprC derived from the alkalophilic Bacillus LG12 strain and 86.2% with SprD (Schmidt et al., Appl. Environ. Microbiol., 61, 4490-4493, 1995). , Was not an exact match. These results suggest that the alkaline protease of the present invention is a novel enzyme. For reference, the homology in the prepro sequence (for example, the amino acid sequence from the -101st position to the -1st position of SEQ ID NO: 2) is the same as that of the alkaline protease prepro sequence or other known alkaline protease prepro sequences. When compared, it shows only 15-51% homology, and no highly homologous sequences are found.
[0013]
That is, the alkaline protease of the present invention includes an alkaline protease having 90% or more homology when the corresponding sequence in the amino acid sequence of SEQ ID NO: 1 is appropriately aligned. Those having homology of preferably 90% or more, more preferably 95% or more are desirable.
The homology search described here can be calculated using the search homology program of GENETYX-WIN (software development) or the maximum matching method.
[0014]
As described above, the alkaline protease gene of the present invention encodes a protein consisting of the amino acid sequence of SEQ ID NO: 1 or a protein consisting of an amino acid sequence equivalent to the amino acid sequence. For example, from the 304th position of SEQ ID NO: 2. And DNAs represented by the nucleotide sequence up to the 1131st position, preferably (a) DNA represented by the nucleotide sequence of SEQ ID NO: 2 or (b) DNA comprising the nucleotide sequence of (a) under stringent conditions And a DNA encoding a protein having an alkaline protease activity.
[0015]
Here, the “stringent conditions” include, for example, conditions described in Molecular cloning-a Laboratory manual 2nd edition (Sambrook et al., 1989). For example, in a solution containing 6 × SSC (composition of 1 × SSC: 0.15M sodium chloride, 0.015M sodium citrate, pH 7.0), 0.5% SDS, 5 × Denhart and 100 mg / mL herring sperm DNA A condition for hybridization with a probe at 65 ° C. for 8 to 16 hours is mentioned.
[0016]
The alkaline protease of the present invention can be produced, for example, by cloning from the chromosomal DNA of Bacillus sp. KSM-LD1 strain and using a suitable vector and host fungus in large quantities. Here, the mycological properties of the KSM-LD1 strain are shown below.
[0017]
A. Morphological properties (a) Cell shape and size: Neisseria gonorrhoeae (0.7 × 2-3.0 μm)
(B) Cell polymorphism: None (c) Motility: None (d) Spore: Existence (0.6-0.7 × 1.0-1.2 μm)
(E) Gram staining: indefinite [0018]
B. Growth state on various media (a) Growth state on gravy agar medium: Forms round, low-convex, and smooth colonies. The surface of the colony is matte. Cream color (b) Meat broth liquid culture: Cloudy (c) Meat broth gelatin puncture culture: Good growth and liquefaction (d) Litmus milk: No growth (e) Egg yolk medium: No growth (f) Tyrosine agar medium: No growth 0019
C. Physiological properties (a) Reduction of nitrate: negative (b) MR test: negative (c) VP test: positive (d) production of hydrogen sulfide: negative (e) production of indole: negative (f) hydrolysis of starch: Positive (g) Use of citrate: Not used (h) Urease: Negative (i) Oxidase: Positive (j) β-galactosidase: Positive (k) Arginine hydrolase: Negative (l) Lysine decarboxylase: Negative (m) Ornithine decarboxylase: Negative (n) Tryptophan deaminase: Negative (o) Gelatinase: Positive (p) Catalase: Positive (q) Growth temperature range: 5 to 40 ° C. (r) Growth pH range: pH 7-10 (S) Anaerobic culture: Cannot grow under anaerobic conditions (t) Production of acid from saccharides (+; produced-; not produced)
Glycerol:-
Erythritol:-
D-arabinose:-
L-arabinose:-
Ribose:-
D-xylose:-
L-xylose:-
Adonitol:-
β-methyl-D-xylose:-
Galactose:-
Glucose: +
Fructose: +
Mannose: +
Sorbose:-
Rhamnose ： −
Zunositol:-
Inositol:-
Mannitol: +
Sorbitol:-
α-methyl-D-mannose:-
α-methyl-D-glucose:-
N-acetylglycosamine:-
Amygdalin:-
Arbutin:-
Esclin: +
Salicin: +
Cellobiose: +
Maltose: +
Lactose:-
Melibiose:-
White sugar: +
Trehalose: +
Inulin:-
Meletetose:-
Raffinose:-
Starch: +
Glycogen: +
Xylitol:-
Gentiobiose:-
D-Turanorose:-
D-lyxose:-
D-Tagatose:-
D-Fucose:-
L-Fucose:-
D-arabitol:-
L-arabitol:-
Gluconate:-
2-ketogluconic acid:-
5-ketogluconic acid: +
[0020]
The above-mentioned KSM-LD1 strain was examined according to the description of “Bergey's Manual of Systematic Bacteriology” (Williams & Wilkins, 1984) and found to belong to the genus Bacillus. However, since the species does not completely match the properties of the known species of the genus Bacillus, this strain is designated as a new microorganism by the National Institute of Advanced Industrial Science and Technology Patent Biology Center. Deposited as LD1 ( Bacillus sp. KSM-LD1; FERM P-18576).
[0021]
The gene of the present invention, the alkaline protease-producing bacteria described above, for example, microorganisms belonging to the genus Bacillus, preferably cloned from the use of Bacillus sp. KSM-LD1 strain (FERM P-18576) or the like, for example, shotgun method, the PCR method can do.
[0022]
In order to produce alkaline protease using the gene of the present invention, the alkaline protease gene (for example, DNA represented by the nucleotide sequence of SEQ ID NO: 2) is added to any vector suitable for expressing the gene in the target host. And transforming the host using the recombinant vector, culturing the obtained transformant, and collecting alkaline protease from the culture solution.
The culture may be carried out by inoculating a medium containing a carbon source, a nitrogen source and other essential nutrients that can assimilate microorganisms, and following a conventional method.
[0023]
Collection and purification of alkaline protease from the thus obtained culture can be performed according to a general method. That is, the cells can be removed from the culture by centrifugation or filtration, and the target enzyme can be concentrated from the obtained culture supernatant by conventional means. The enzyme solution or dry powder thus obtained can be used as it is, but can be further crystallized or granulated by a known method.
[0024]
As the characteristics of the alkaline protease of the present invention, the following properties are preferable. That is, it has a molecular weight of about 30,000 (SDS-polyacrylamide gel electrophoresis), an optimum reaction pH of around 10.5 (glycine / sodium hydroxide buffer), and an optimum temperature of around 60 ° C. It is desirable that calcium ions are not required for stabilization, and it is more preferable to have the properties shown in paragraphs [0030] to [0034].
[0025]
【Example】
Determining Bacillus subtilis cloning and nucleotide sequence of Example 1 gene is a region that is highly conserved in the serine protease family 1 produced 64 th His and around 221 amino acid sequence H near Ser GTHVAG and GT S MASPHVAG On the other hand, for the purpose of amplifying between these regions, primer 1 (SEQ ID NO: 5′-CAYGGNANCCAYGTNGCNGG-3 ′), primer 2 (SEQ ID NO: 5′-CCNGCNACRTGNGGNGNGGCCATNGANGTNCC-3 ′), primer 3 (SEQ ID NO: 5: 5′-CCNGCNACRTGNGNGNGNGCCATRCTNGTNCC-3 ′) was synthesized. The amino acid numbers are shown based on BPN ′. Genomic DNA was prepared from Bacillus sp. KSM-LD1 strain according to the method of Saito and Miura (Biochim. Biophys. Acta, 72, 619-629, 1963), and PCR was performed using primers 1 and 2 and primers 1 and 3. Was done. PCR conditions were 10 to 100 ng of genomic DNA, 20 nmol primer using 20 nmol as a template, Pyrobest (Takara) as a polymerase to make a 50 μL reaction system, and heat denaturation at 94 ° C. for 1 minute with a Biometra: T-Gradient Thermocycler (Whatman). Thereafter, 30 cycles were performed with 94 ° C. for 1 minute, 40 to 60 ° C. for 1 minute, and 72 ° C. for 3 minutes. The obtained PCR fragment was purified with a High Pure PCR Product Purification kit (Roche), ligated to pUC18 digested with Sma I, and introduced into Escherichia coli HB101. Plasmids were prepared using the High Pure Plasmid Isolation Kit (Roche) from the transformant, to confirm the presence or absence and magnitude of the Eco RI, insert the 1.2% agarose gel electrophoresis after digestion with Hin dIII. The base sequence was determined using a BigDye Terminator Cycle Sequencing kit (Perkin Elmer) and a DNA sequencer (type 377: Applied Biosystems) for inserts having a size of about 200 bp to 1500 bp. In addition, Bca BEST Sequencing Primer RV-M (Takara) or Bca BEST Sequencing Primer M13-47 (Takara) was used as a primer. By comparing base sequences and selecting different ones, primers suitable for base sequence determination in the upstream and downstream directions (same species, two types in the same direction: outside and inside) were synthesized. Bacillus sp. Genomic DNA of KSM-LD1 strain was digested with Eco RI or Hin dIII, respectively connecting the Eco RI or Hin dIII degradation fragments Eco RI or Hin dIII cassette in LA PCR in vitro cloning kit (Takara) PCR was performed with each outer primer and attached primer C1. PCR conditions were 0.1 to 1.0 ng of DNA fragments linked to the cassette, 10 to 20 pmol of primer as a template, heat denaturation at 94 ° C. for 1 minute in a 50 μL reaction system, then 94 ° C. for 1 minute, 55 ° C. For 1 minute and at 72 ° C. for 4 minutes for 30 cycles. Thereafter, the amplified DNA fragment was amplified by PCR with each inner primer and attached primer C2. The PCR conditions at this time were as follows: 1 μL of the amplified DNA fragment obtained previously was diluted 10,000 times with 10 to 20 pmol of the template as a template, 50 μL reaction system, heat denaturation at 94 ° C. for 1 minute, 1 at 94 ° C. 1 cycle at 55 ° C for 1 minute and 2 minutes at 72 ° C for 30 cycles. The base sequence of the amplified fragment was determined using the BigDye Terminator Cycle Sequencing kit (Perkin Elmer) and a DNA sequencer (type 377: Applied Biosystems) in the same manner as described above. Based on the newly obtained nucleotide sequence information, synthesize primers for determining the upstream and downstream nucleotide sequences, and use the same method to encode the protease and all bases in the upstream and downstream regions. The sequence was determined.
[0026]
Example 2 Production of Alkaline Protease by Bacillus subtilis Transformant Primer 4 (SEQ ID NO: 6: 5′-GTCACG GGATCCC TCTTCATTTTCCC-3, underlined portion was newly added as the gene encoding the alkaline protease determined in Example 1 Bam HI site), primer 5 (SEQ ID NO: 7: 5'- AACATTCTCATA TTGAATCATTCCCACC-3, underlined indicates newly added Xba I site) 20 pmol each, 0.1 to 1.0 ng of Bacillus PCR was performed using the genomic DNA of sp. KSM-LD1 strain as a template. PCR conditions were as follows: heat denaturation at 94 ° C. for 1 minute, 30 cycles of 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 3 minutes. The obtained PCR amplified fragment was purified with a High Pure PCR Product Purification kit (Roche), digested with Bam HI and Xba I, and then ligated to the similarly treated vector pHA64 to prepare a recombinant plasmid. Using this, the B. subtilis ISW1214 strain was transformed by the protoplast method. The resulting transformed strain was 3% (w / v) polypeptone S, 0.5% fish meat extract, 0.1% yeast extract, 0.1% monopotassium phosphate, 0.02% magnesium sulfate, 3% maltose. When aerobic shaking culture was performed at 30 ° C. for 72 hours in a medium consisting of (separately sterilized), production of protease could be confirmed in the culture supernatant. In addition, the production amount per culture solution was 25-30 U / mL.
[0027]
Example 3 Preparation Method of Protease 200 mL of the culture solution obtained in Example 2 was equilibrated with 50 mM Tris-HCl buffer (pH 8) containing 1 mM calcium chloride on a Super Q Toyopearl column (Tosoh: 2.5 × 8 cm). After adhering and washing with the same buffer, the adsorbed protease active fraction was eluted by a concentration gradient elution method using 0 to 0.5 M potassium chloride. This fraction was hydro-concentrated by ultrafiltration (Amicon: YM3 membrane) and DEAE-Toyopearl column (Tosoh: 1) previously equilibrated with 10 mM borate buffer (pH 9.6) containing 1 mM calcium chloride. Elution of the adsorbed protease activity fraction by concentration gradient method using 0 to 0.1M potassium chloride and further concentration gradient method using 0.1 to 0.25M potassium chloride did. The active fraction was hydroconcentrated by ultrafiltration (YM3 membrane) and then attached to a hydroxyapatite column (Biorad: 1.5 × 7 cm) equilibrated with 10 mM phosphate buffer (pH 7.0). did. Non-adsorbed protease activity fractions were collected and concentrated, and then subjected to SDS-polyacrylamide electrophoresis. As a result, it was confirmed that the protein was a uniform protein having a molecular weight of about 30,000. The specific activity of the purified enzyme was 121 U / mg. The amount of protein was measured using a DC protein assay kit (BioRad) with bovine serum albumin as a standard.
[0028]
EXAMPLE 4 activity assay of the purified alkaline protease obtained in KSM-LD1 strain derived alkaline protease enzymatic properties Example 3 N--succinyl-Ala-Ala Pro -Phe- p -nitroanilide ( abbreviated as AAPF) This was carried out by the synthetic substrate method using
[0029]
[Synthetic substrate method]
A reaction solution consisting of 0.25 mL of 0.1 M borate buffer (pH 10.5), 0.05 mL of 20 mM calcium chloride, 0.1 mL of deionized water and 0.025 mL of 50 mM AAPF was incubated at 30 ° C. for 3 minutes. Thereafter, 0.025 mL of enzyme solution was added to start the reaction. After incubating at 30 ° C. for 10 minutes, 2 mL of 5% (w / v) citric acid was added to stop the reaction. Absorbance at 420 nm was measured, and liberated p-nitroaniline was quantified. The enzyme 1 unit was an amount that liberates 1 μmol of p-nitroaniline per minute under the above reaction conditions.
[0030]
(1) Optimum reaction pH
The enzyme activity was measured by reacting in various buffers (pH 7 to 12) at 30 ° C. for 10 minutes. The protease of the present invention showed the highest activity in 50 mM glycine / sodium hydroxide buffer (pH 10.5).
[0031]
(2) Optimum temperature The activity was measured at 30 to 80 ° C. in a 50 mM borate buffer solution (pH 10.5). As a result, the optimum temperature of the protease of the present invention was 60 ° C.
[0032]
(3) An enzyme was added to a heat-resistant 50 mM borate buffer (pH 10.0), heat-treated at 30 to 70 ° C. for 15 minutes, and then rapidly cooled in ice water. As a result of measuring the residual activity, the protease of the present invention was stable up to 50 ° C. with or without 5 mM calcium chloride.
[0033]
(4) Substrate specificity Hemoglobin, keratin, and gelatin (0.25%) were dissolved / suspended in 50 mM borate buffer (pH 10.5), and an enzyme reaction was performed. When the degradation activity against casein was 100%, the relative activities were 36%, 5% and 460%, respectively.
[0034]
(5) The isoelectric point was determined from the mobility of the standard protein (Broad pI Calibration kit: Pharmacia) and the protease of the present invention by isoelectric focusing using a PAG plate (Multiphor II system: Pharmacia). However, it was around pH 4.5.
[0035]
【The invention's effect】
The alkaline protease of the present invention has low amino acid sequence homology with conventional subtilisins and is not affected by calcium ions in the stability to heat, so it is necessary to add calcium to the production, purification and granulation of the enzyme It can also be applied to systems that avoid the addition of metal salts to the reaction, and is useful as an enzyme for various industries including detergents for clothing.
[0036]
[Sequence Listing]

Claims (6)

The following protein (a) or (b):
(A) a protein comprising the amino acid sequence of SEQ ID NO: 1,
(B) A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of (a) and having alkaline protease activity.   A gene encoding the protein according to claim 1. Alkaline protease gene comprising the following DNA (a) or (b):
(A) DNA represented by the nucleotide sequence of SEQ ID NO: 2,
(B) DNA that hybridizes with DNA comprising the base sequence of (a) under stringent conditions and encodes a protein having alkaline protease activity.   A recombinant vector containing the gene according to claim 2 or 3.   A transformant comprising the recombinant vector according to claim 4.   The transformant according to claim 5, wherein the host is a microorganism.