JP2590462B2 - Method for producing thermostable protease - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a thermostable neutral protease gene obtained from Bacillus stearothermophilus, which is introduced into another species of Bacillus subtilis using genetic engineering techniques to perform transformation. The present invention relates to a production method suitable for mass production of a heat-resistant protease, in which a transformant is used, a culture solution is heated, and a protein derived from a host bacterium is heat-denatured and heat-selective protease is selectively collected.

[Prior Art] Proteases of Bacillus bacteria are widely used for industrial applications, and the development of useful proteases and protease production methods has industrial significance. The thermostable neutral protease produced by the thermophilic bacterium is stable not only against heat but also against various chemicals, and its activity does not relatively decrease even after long-term use. Therefore, it is not only used as a research reagent, but also as a detergent protease that can be placed in detergent to increase detergency.
It is used over a wide range of fields, such as for decomposing soy protein and for tanning leather.

[Problems to be Solved by the Invention] Conventionally, a thermostable protease is produced based on a bacterium producing the same (a structural gene (usually one gene) contained in chromosomal DNA of a thermophilic bacterium). Usually, productivity is low. Attempts have been made to obtain high-producing strains by mutagenesis in order to compensate for the drawbacks, but at most a 2- to 5-fold increase is the limit.

[Means and Actions for Solving the Problems] The present inventors have conducted studies to increase the method for producing a thermostable protease. As a result, when the Bacillus stearothermophilus-derived thermostable protease structural gene was cloned using Bacillus subtilis as a host, The gene is amplified and the productivity of the enzyme is improved, and the Bacillus subtilis-derived protein is heat-inactivated by producing a thermostable protease to produce Bacillus subtilis.
The present inventors have found that only a heat-resistant protease derived from llus stearothermophilus is selectively extracted and completed the present invention.

The present invention provides a heat-resistant protease structural gene derived from Bacillus stearothermophilus cloned using Bacillus subtilis as a host, improves the heat-resistant protease productivity by the gene amplification effect, and selectively heat-treats Bacillus stearothermopus.
A method for producing a heat-resistant protease, comprising recovering only a heat-resistant protease derived from hilus.

Bacteria belonging to the genus Bacillus used as the chromosome DNA donor used in the present invention include Bacillus stearothermophilus MK-232 (Bacillus stearothermophilus) and Bacillus stearothermophilus (Bacillus stearothermophilus).
hilus) NCA1503 (Microtechnical Laboratory No. 8978).

Bacillus stearothermophilus MK-232 has the following mycological properties.

Morphology Cell shape Haemophilus fungus Cell size 1.0 × 1.2μ × 3.0〜5.0μ Polymorphism No Mobility Yes Spores forming Spore shape Circular Spore size 0.8〜1.0μ × 1.2μ Antiacid None Gram staining + Growing condition Broth cultivation plate culture Good or bad growth Good colony shape Circular colony surface shape Smooth / ring ring Colony uplifting Flat colony rim Whole margin Colony content Uniform colony color Cream color Colony transparency Opacity Culture Good growth Good colony shape Filamentous colony surface shape Smooth colony transparency Opaque colony color Cream color juice liquid culture Surface growth None Turbidity Slightly turbid sedimentation Viscosity Gelatin puncture culture Gelatin liquefaction Yes, 50 ° C, 5 days Litmus milk No liquefaction coagulation Physiological properties Nitrate reduction Negative Denitrification negative MR test Positive VP test Negative Dole formation Negative hydrogen sulfide formation Positive starch hydrolysis Positive use of citric acid Negative Use of inorganic nitrogen source Pigment formation None Urease negative Negative oxidase Negative Catalase Negative Growth range Optimum around pH 7.0 50 ° C is optimal for oxygen Attitude Negative OF test Whether or not acid and gas are generated from saccharides. Acid is generated from arabinose, xylose, mannose, fructose, and galactose, but no gas is generated. Acid does not produce gas from maltose, sucrose, trehalose, sorbitol, mannitol, inosit, glycerin, or starch.

Growth on medium supplemented with 7% sodium chloride No growth.

Decomposition of phenylalanine deaminase tyrosine negative Decomposition of casein Positive Growth on medium supplemented with 0.02% sodium asiate Does not grow.

Bacillus stearothermophilus MK-232 produces a thermostable protease, the properties of which are as follows.

(1) Molecular weight 37,000 (due to gel filtration) (2) Action temperature According to the activity measurement method using casein as a substrate (the activity is indicated by the amount of tyrosine residue released after 10 minutes of reaction), the activity is in the range of 5 to 90 ° C. Have. The optimal temperature is 70-75 ° C.

(3) Working pH The preferred pH is 5 to 10, but it is a neutral protease having an optimum pH of pH 7 to 8.

(4) Thermal stability 90% or more of the activity remains at 75 ° C for 30 minutes (pH 7.5) (C
aCl ₂ 10mM present).

 No activity remains at 100 ° C for 30 minutes (pH 7.5).

(5) pH stability Under the condition of 25 ° C. for 24 hours, almost 100% activity remains in the pH range of 5 to 10.

(6) Inactivated by 5 mM EDTA as a blocking agent.

Bacillus stearothermophilus NCA1503 is a known strain, and its bacteriological properties are described in Journal of the Applied Bacteriology (J. Appl. Bact.) 38
Vol., Pp. 301-304 (1975).

Bacillus stearothermophilus NCA1503 is Showa 61
It was deposited with the Research Institute of Microbial Industry and Technology of the Ministry of International Trade and Industry on September 27, 2000, and has been given Microorganisms Bacteria No. 8978.

The enzyme produced by Bacillus stearothermophilus NCA1503 has the following properties.

(I) Molecular weight: 26,000 (SDS-PAGE) (ii) Thermal stability: treatment at 80 ° C. for 30 minutes shows 100% residual activity.

 Treatment at 90 ° C for 30 minutes shows 100% residual activity.

 A treatment at 100 ° C for 30 minutes shows about 30% residual activity.

(Iii) pH action: The preferred pH of the proteolytic activity using casein as a substrate is 6-7, optimally pH 7.0-7.5
Has a pH.

(Iv) pH stability: 7 hours at 37 ° C. for 30 minutes
Stable with 0% or more activity.

(V) Optimal temperature: The optimal temperature for proteolytic activity using casein as a substrate is 35 ° C to 80 ° C, and the optimal temperature is 35 to 80 ° C.
° C, and the optimal temperature is about 70 ° C.

(Iv) Inhibition: Inhibited by EDTA.

On the other hand, it is hardly inhibited by PMSF (pheny1-methy1-sulfony1-fluoride).

(Vii) Substrate specificity In addition, titer and substrate specificity were measured as follows.

Measurement method of titer 1 ml of 2% casein solution (50 mM Tris-1 mM CaCl ₂ )
1 ml of each sample, and after a certain period of time, stop reaction solution (0.33 M
Acetic acid, 0.22M sodium acetate, 0.1M trichloroacetic acid) 2ml
The mixture was left at room temperature for 30 minutes. I spent this on Whatman No. 1 paper.

Separately, the reaction was stopped in a casein solution, A _{275 nm} was measured based on the _excess of the sample, and a calibration curve of A ₂₇₅ was prepared based on the tyrosine solution to measure protease activity. One unit was the amount of the enzyme that released 1 μg of the tyrosine-equivalent substance to the trichloroacetic acid-soluble fraction at 37 ° C. for 1 minute.

Substrate reactivity The ability of each protease to degrade various proteins was shown as casein as 100%. 2% enzyme activity measurement
After a certain period of reaction using the substrate, the reaction was stopped with trichloroacetic acid, and the amount of free tyrosine was measured.

In addition, Bacillus subtilis (Bacillus subtilis) MT-2 and the like are examples of the host Bacillus subtilis.

As a method for cloning a thermostable protease structural gene, a shot gun method can be used, and transformation of Bacillus subtilis can be performed by a competent cell method.

According to the method of the present invention, for example, the heat-resistant protease structural gene of Bacillus stearothermo-philus was incorporated into the vector plasmid pTB53 (kanamycin resistance and tetracycline resistance) by the shotgun method. Thereafter, the resulting recombinant plasmid is used to transform Bacillus subtilis by a competent cell method, whereby a high heat-stable protease-producing strain can be obtained.

Preparation of chromosomal DNA from a thermostable neutral protease-producing bacterium was performed using the Saito-Miura method using phenol (Biochim. Biop.
tys.Acta. 72 619.'63) or Marmur method using a chloroform-isoamyl alcohol (J.Mo1.Biol. 3 20
8.'61) or Warrick method (Proc.Nat.Acad.Scl.USA 72 .6 separated by density inclination bye '75) can be carried out in a generally used method such as.

The prepared donor chromosomal DNA is then cut for ligation with the vector. Cleavage of the donor chromosomal DNA is usually performed by a method using a restriction endonuclease. In this case, any restriction endonuclease having no cleavage site in the thermostable protease gene can be used, and all kinds of restriction endonucleases can be used if reaction conditions that cause only partial cleavage are used. Can be used. As described above, various restriction endonucleases can be selected depending on the conditions to be used. However, it is preferable that the vector to be used has only one cleavage site from the viewpoint of easy connection with the vector. As a vector, any plasmid or phage can be used as long as it can replicate in Bacillus bacteria used as a host. In particular, those having a unique cleavage site by a specific restriction endonuclease and having a marker such as antibiotic resistance are preferable from the viewpoint of binding to a donor chromosomal DNA and ease of selection of a transformant. Examples of such plasmids include Bacillus stearothermophilus (Bacillus
pTB19 and pTB53 (J. Bacte) with kanamycin resistance and tetracycline resistance markers derived from sterothermophilus
riol. 146 1091 (1981)) Staphylococcus
pC194, pC221, pC232 (Proc. Nat1. Acad, Scl USA74) having a chloramphenicol resistance marker derived from P. lococcus)
1680 (1977)) pUB1 with a kanamycin resistance marker
10 (J. Bacteriol 134 318 (1978)) and the like. In particular, pTB1, a low-copy plasmid vector
9, pTB53 is stably maintained in Bacillus subtilis and can be a suitable vector.

When using a phage as a vector, the phage
Any DNA can be used as long as it can be replicated in Bacillus bacteria as a host. Examples of such phage are φ1,
φ3T, φ150, ρ11 and the like can be mentioned.

In order to insert and ligate the cut donor chromosomal DNA to the above-mentioned vector, it is common to cut the donor chromosome and the vector with the same restriction endonuclease, and then ligate them using DNA ligase.

The recombinant DNA molecule thus obtained (donor chromosome D
The conjugate of the NA cleavage and the vector) is then introduced into the host, Bacillus subtilis. In order to selectively accumulate a large amount of the desired heat-resistant protease in the culture solution, Bacillus subtilis having a low extracellular enzyme accumulation amount may be used as a host. In particular, a protease-deficient strain of a host bacterium is desirable because only a recombinant can be easily selected at the time of selecting a recombinant.

In order to perform the transformation by introducing the recombinant DNA molecule into the Bacillus subtilis which is the host cell selected in this way, a transformation method using competent cells (Mol. Gen. Genet. 16
7 251 (1979)) or transformation method using protoplasts (Mol.Gen.Genet. 168 111 (1979) ) can be carried out by using a. Donor chromosome DNA containing the desired thermostable neutral protease gene from the resulting transformant
No specific method is required for selective isolation of the microorganism into which the cleavage has been introduced, but temporary selection is carried out by expressing a marker such as antibiotic resistance possessed by the vector, and further, selection is made based on changes in the protease activity of the host. Easy to do.

No specific method is required for accumulating the desired thermostable neutral protease in the culture using the selected transformant.

That is, as the medium, a conventional one containing a carbon source, a nitrogen source, inorganic ions, and, if necessary, trace nutrients such as vitamins can be used. The cultivation is carried out under aerobic conditions while appropriately adjusting the pH and temperature of the medium until the thermostable protease accumulates.

Recovery of the heat-resistant neutral protease from the culture solution after the cultivation is performed at a temperature of 60 ° C. or higher, preferably at 65 ° C. or higher, by subjecting the target heat-resistant neutral protease to a heat treatment at a temperature at which deactivation does not occur for 10 to 20 minutes. It is possible to specifically recover only the thermostable neutral protease by inactivating the protein derived from Bacillus subtilis, which is the host, and removing the protein by centrifugation.

As described in detail above, in the present invention, any thermophilic bacterium chromosomal DNA donor is used, and Bacillus subtilis, a host bacterium arbitrarily selected from extracellular heat-stable neutral proteases produced by the donor, is used. This has the advantage that only a thermostable neutral protease can be produced. Therefore, in the present invention, the objective is to select a thermophilic bacterium having high heat-resistant neutral protease high productivity as a chromosome DNA donor, and a Bacillus bacterium which hardly accumulates extracellular enzymes as a host Bacillus subtilis. This makes it possible to produce a large amount of heat-resistant neutral protease with a low level of mixed enzyme.

As described above, the contribution to the production of the protease by improving the productivity of the thermostable neutral protease and simplifying the purification process is large.

EXAMPLES Hereinafter, the present invention will be further described with reference to Examples.

Example 1 (1) Preparation of chromosomal DNA of MK-232 strain Bacillus stearothermophilus (Bacillus stear)
Othermophilus strain MK-232 was cultured overnight at 55 ° C. in a 500 ml volumetric flask containing 100 ml medium. About 20 ml after centrifugation
Of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.5), and then 6 ml of 15% sucrose-50 mM Tris (pH 8.5) -50 mM ED.
The suspension was suspended in TA-1 mg / ml lysozyme and allowed to stand in water for 30 minutes. 6 ml of 2% sarkosyl-50 mM Tris (pH 8.5)
-50 mM EDTA was added and left at room temperature for about 15 minutes to completely lyse the cells. Next, 11.0 g of CsCl was placed in a 50 ml volumetric flask, and further 12 ml of the lysate was added. Thereafter, 0.6 ml of a 10 mg / ml ethidium bromide solution was added. This solution was dispensed into two centrifuge tubes, and separated in an ultracentrifuge using an RP65T rotor (manufactured by Hitachi, Ltd.). After the ultracentrifugation, the DNA section was extracted with a syringe needle, and extraction was repeated three times with n-butanol to remove ethidium bromide. This DNA sample was dialyzed against 10 mM Tris (pH 7.5) -0.1 mM EDTA and stored at 4 ° C.

(2) Restriction enzyme treatment / ligase treatment Using Bacillus subtilis as a host, a vector / plasmid pTB53 capable of maintaining replication (described above) was prepared by a conventional method. This pTB53
And separately prepared MK-232 strain chromosomal DNA with restriction enzyme Pst I
Was completely decomposed. The reaction conditions were as follows.

Pst I buffer (10 mM Tris (pH 7.4) -10 mM MgCl ₂ -5
Use a 10-fold concentrated solution of 0 mM NaCl-1 mM dithiothreitol (10 × PstI buffer) MK-232 DNA 30 μ 10 × Pst I buffer 10 μ bovine serum albumin (5 mg / ml) 2 μ distilled water 56 μ Pst I 2 μ pTB53 20 μ 10 × Pst I buffer 10 μ bovine serum albumin (5 mg / ml) 2 μ distilled water 66 μ Pst I 2 μ was reacted at 37 ° C. for 1-2 hours.

The two DNAs were mixed, 5M sodium acetate was added, and then 0.45 ml of ethanol was added. The mixture was left at -20 ° C for 10 to 15 minutes, centrifuged, and the supernatant was discarded. The precipitate was washed with ethanol, dehydrated and desalted, and dried in a desiccator. 10 x ligase for dry matter
Buffer (660 mM Tris (pH7.5) 66 mM MgCl) 5μ, 1
After adding and mixing 5 μm of 00 mM dithiothreitol, 5 μm of 10 mM ATP, and 34 μm of distilled water, 1 μm of DNA ligase was added and kept at 4 ° C. for 16 hours. This was used for the next transformation.

(3) Preparation of competent cells The culture solution cultured overnight at 37 ° C in an L culture medium was subjected to TFI (Spizige
n's Salt × 10 (NH ₄ ) ₂ SO ₄ 2%, K ₂ HPO ₄ 14%, KH ₂ PO ₄ 6
%, Na-citrate 1%) 2 ml, 2% casamino acid 0.2 ml, 1 mg / ml amino acid solution 1 ml, distilled water 14.8 ml, 5% glucose + 0.2% MgSO ₄
2 ml) and shake culture for 3 hours and 45 minutes. TF II (Spizige
n's Salt × 103.6ml, 2% casamino acid 0.18ml, 1mg / ml amino acid solution 0.18ml, distilled water 28.44ml, 5% glucose + 0.2% MgSO ₄
3.6 ml) and cultured with shaking for 1 hour and 30 minutes to obtain competent cells.

(4) Cloning of Heat-Stable Neutral Protease 40 μl of the DNA solution of the above (2) and 1 ml of competent cells were mixed and vigorously stirred at 37 ° C. for 30 minutes. After centrifugation, 3 ml of medium was added, and the mixture was gently stirred at 37 ° C. for 2 hours. 0.1 ml thereof is mixed with 1% casein in tetracycline (5 μg
/ ml) or kanamycin (5 μg / ml). The plate was kept at 37 ° C. for 16 hours, and colonies forming halos were obtained. The transformant thus obtained was named Bacillus subtilis MT-2 / PTZ-232.

Bacillus subtilis MT-2 / PTZ-232 was sold in September 1986
It was deposited with the Institute of Microbial Industry and Technology of the Ministry of International Trade and Industry on March 30 as Microorganism Research Bacteria No. 8982.

Bacillus subtilis MT-2 / PTZ-232 was cultured to obtain a plasmid DNA, PTZ-232, by a conventional method. FIG. 1 shows the thermostable protease structural gene derived from Bacillus stearothermophilus MK-232 of this plasmid and the restriction enzyme cleavage site in the vicinity thereof.

(5) Preparation of heat-resistant protease The colony obtained from the above (4) was inoculated into a 1 L medium, and cultured with shaking at 37 ° C for 16 hours. The culture was centrifuged, and the supernatant was salted out using 70% saturated ammonium sulfate. After centrifugation, the precipitate was added with 10 ml of 50 ml M Tris (pH 7.5) -1 mM CaCl ₂ and then heat-treated at 65 ° C. for 15 minutes. After centrifugation, remove the supernatant for 5 minutes.
Dialysis was performed against 0 mM Tris (pH 7.0) -1 mM CaCl ₂ .

The enzyme activity of the obtained enzyme solution was measured. For comparison, Bacillus stearothermophilus MK-232 strain and Bacillus subtilis MT-2 were similarly cultured, and the culture solution was treated to measure the enzyme activity. First result
It is shown in the table.

Example 2 Example 1 was repeated using Bacillus stearothermophilus NCA1503 as a chromosomal DNA donor in place of Bacillus stearothermophilus MK-232 to obtain the neutral protease NCA1503 efficiently. FIG. 2 shows the NCA1505 structural gene of the transformed plasmid and restriction enzyme cleavage sites in the vicinity thereof.

[Effect of the Invention] According to the method for producing a thermostable protease according to the present invention,
The production amount of the thermostable enzyme, which could only be raised at most about 2 to 5 times compared to the production in the chromosome DNA donor by the conventional method, can be increased to 10 times or more, and the purification thereof can be performed very efficiently. It can be carried out.

[Brief description of the drawings]

FIG. 1 and FIG. 2 are diagrams illustrating the heat-resistant protease structural gene of the plasmid of the transformant which can be used in the present invention.

────────────────────────────────────────────────── (5) Continuation of the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical indication location C12R 1:07) (56) References JP-A-63-502959 (JP, A) JP-A-58 -162291 (JP, A) JP-A-59-159778 (JP, A) Prcc. Natl. Acad. So i. VSA. , 83, (1986) p. 576- 580

Claims (1)

(57) [Claims] [Claim 1] A thermostable extracellular neutral protease structural gene derived from Bacillus stearothermophilus is cleaved, and the obtained DNA fragment is treated with a vector to obtain another Bacillus subtilis (37).
(Optimum growth temperature of 100 ° C.) as a host, selecting a strain into which the desired heat-resistant extracellular neutral protease structural gene fragment was introduced from the transformed strain, amplifying the gene, and amplifying the gene. Bacillus s is produced by releasing extraneous proteases, releasing them out of the host, and removing the Bacillus subtilis-derived proteins that are thermally denatured by heating the culture broth.
A method for producing a thermostable protease, comprising selectively collecting only a thermostable extracellular neutral protease derived from tearothermophilus.