JPH0767639A - New protease and its production - Google Patents

Abstract

PURPOSE:To obtain the subject enzyme having specified physical properties, exhibiting a decomposition activity against gelatin and azocol and capable of cutting insulin B chain at more numerous positions than other proteases by culturing a specified bacterium. CONSTITUTION:A protease-producing bacterium belonging to Bacillus, e.g. Bacillus sp.-WF-2 strain (FERM-P-13802) is cultured and a protease having following physical properties [action: having a remarkable activity against decomposition of gelation or azocol and an activity against casein; having no activity against undenaturated collagen but having an activity against a synthetic substrate (Cbz-Gly-Pro-Leu-Gly-Pro, Cbz-Gly-Gly-Pro-Gly-Pro-Ala, etc.) for collagenase; capable of cutting insulin B chain at many positions, e.g. Leu-Cys, Arg-Gly, etc.; optimum pH: 7 to 9; optimum temperature: 40 to 55 deg.C; molecular weight: about 30000] is collected from the cultured material, thus producing the objective enzyme.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel proteolytic enzyme and a method for producing the same. More specifically, it shows remarkable activity in degrading gelatin and azochol, acts on casein,
It does not act on native collagen but is a synthetic substrate for collagenase (Cbz-Gly-Pro-Leu-Gly-
Pro, Cbz-Gly-Pro-Gly-Gly-
Pro-Ala, Pz-Pro-Leu-Gly-P
ro-Arg), and the Leu- of insulin B chain
Cys, Arg-Gly, Thr-Pro, Pr
The present invention relates to a novel proteolytic enzyme that cleaves many sites such as o-Lys and a method for producing the proteolytic enzyme using the proteolytic enzyme-producing bacterium.

[0002]

2. Description of the Related Art For the degradation of cortical proteins, there are cases where a widely used proteolytic enzyme cannot obtain a satisfactory effect.

[0003]

[Problems to be Solved by the Invention] A proteolytic enzyme characterized by its action on cortical proteins is expected.
Conventional collagenase is one of them, and a proteolytic enzyme characterized by further specificity is expected.

Therefore, the present inventors isolated a large number of microorganisms in order to obtain a strain capable of producing a novel proteolytic enzyme by culturing under aerobic conditions, and the activity against cortical proteins produced by them was determined. As a result of the examination, it was confirmed that the strain belonging to the genus Bacillus isolated from the fish skin has a strong activity against the degradation of denatured collagen (gelatin) and azochol, and that it acts on a synthetic substrate of collagenase, and other proteolytic enzymes. It has been found that it cleaves many sites in the insulin B chain rather than in the insulin B chain, thus completing the present invention.

Accordingly, an object of the present invention is to provide this novel proteolytic enzyme and a method for producing the same.

[0006]

The present invention relates to a novel proteolytic enzyme and a method for producing the same. The proteolytic enzyme is obtained by culturing a production bacterium belonging to the genus Bacillus and has a strong activity on gelatin. , And is characterized by substrate specificity.

The microorganism used for the production of the proteolytic enzyme of the present invention is a proteolytic enzyme-producing bacterium belonging to the genus Bacillus, and a particularly effective strain is, for example, Bacillus sp. It is the WF-2 strain. This strain was transferred to the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry on August 17, 1993, under the contract number FERM P-13.
Deposited as No. 802.

This strain was isolated from fish skin, and its mycological characteristics are as follows. 1) Morphology It is a bacillus with a length of 1.6 μm and a width of 0.9 μm. It has periflagellates, and spores are elliptical and develop in the central part of the cell. 2) Growth condition Proliferate on ordinary agar medium, and colonies are grayish white and irregular. Aerobic, no anaerobic growth. Optimum growth temperature is 37 ℃ to 40 ℃, and growth temperature is 18 ℃ to 47 ℃.
Is. 3) Gram stainability: (-) 4) Physiological properties OF test: Oxidative degradation Acid production from sugar: Glucose, xylose, arabinose, mannose, maltose, lactose, melibiose, raffinose, mannitol, inositol produce acid. . No acid is produced from dulcitol.

Gas production test: (-) Forges-Proscavell test: (+) Catalase: (+) From the above properties, it was confirmed that this bacterium belongs to the genus Bacillus.

This novel proteolytic enzyme is produced by subjecting the above-mentioned production strain to an appropriate aerobic culture condition, for example, by shaking culture or aeration stirring culture.

The medium contains nitrogen and inorganic salts which can be taken up by the producing strain. Producing bacteria are usually 25 ℃ ~ 3
It is desirable to culture at 7 ° C. All nitrogen sources used for bacterial growth can be used to produce the enzymes of the invention. For example, peptone, meat extract, yeast, yeast extract, legume powder, corn steep liquor, rice bran,
Bread, gelatin, fish meal, meat meal, inorganic nitrogen and the like can be used. Especially, for the production of the enzyme of the present invention, a medium containing 0.1 to 0.4% gelatin is suitable. The culture is carried out by inoculating into an appropriate medium in which the production amount of the proteolytic enzyme is large, and the pH of the medium is preferably around neutral.

The proteolytic enzyme of the present invention can be purified and separated by appropriately combining the means generally used for the purification and separation of the product of the microorganism. For example, salting out with ammonium sulfate, acetone, methanol, precipitation with organic solvents such as isopropanol, adsorption and elution with various adsorbents, adsorption with ion exchangers, elution and chromatography, gel filtration with various carriers,
Various electrophoresis methods, ultrafiltration methods, freeze-drying methods, freezing methods,
The dialysis method, chromatofocusing, hydrophobic chromatography and the like. The proteolytic enzyme of the present invention can be isolated by combining and repeating these means.

More specifically, the culture supernatant is subjected to ultrafiltration, and ammonium sulfate (ammonium sulfate) is added to collect the fractions which precipitate at 30 to 70% saturation. This precipitate is dialyzed against a phosphate buffer solution and applied to a column of CM Sepharose FF for adsorption. This column was eluted with a NaCl gradient system consisting of the same phosphate buffer used in the above dialysis and 0.5M NaCl added to the same buffer to give 0.15M Na
The component eluted from around the Cl concentration is the enzyme of the present invention. Then, the obtained enzyme was added to Super Rose 6
Purify by gel filtration with HR column.

The physicochemical and enzymatic chemical properties of the novel proteolytic enzyme of the present invention are shown below. 1) Action: Remarkable degrading activity is shown with respect to denatured collagen such as gelatin and azochol (modified collagen standard, manufactured by Sigma).
It also shows high activity against casein. Collagenase synthetic substrate [Cbz-Gly-Pro-Leu-Gly
-Pro (sometimes abbreviated as Z-GPLGP), Cb
z-Gly-Pro-Gly-Gly-Pro-Ala
(Sometimes abbreviated as Z-GPGPGPA), Pz-Pr
o-Leu-Gly-Pro-Arg (PZ-PLGP
R may be abbreviated)]]. However, it does not act on native collagen. However, in the above formula, Cb
z represents a carboxybenzoyl group, and Pz means a phenylazobenzoxycarbonyl group.

The proteolytic enzyme of the present invention is insulin B
It cleaves many sites in the chain, but notably Leu-Cy
s, Arg-Gly, Thr-Pro, Pro-Lys
Disconnect between. 2) Optimum temperature: 40 ° C-55 ° C 3) Optimum pH: 7.0-9.0 4) Thermal stability: Stable below 40 ° C 5) pH stability: 6.0-9.0 6) Inhibitor: Not inhibited by EDTA but partially by p-APMSF (60% residual activity). Where p-
APMSF is para-aminophenylmethanesulfonyl fluoride. 7) Purification method: The proteolytic enzyme of the present invention can be purified to a uniform degree by polyacrylamide electrophoresis by ammonium sulfate fractionation of culture supernatant, CM sepharose column chromatography, and gel filtration method. 8) Molecular weight: 30,000 (SDS-polyacrylamide gel electrophoresis). 9) Titration method: 50 mM Tris / HCl buffer (pH
7.5 ml of 0.4 mM CaCl _{2 (} containing 4 mM CaCl ₂ ), the enzyme solution (0.05 ml) and Azocol (Sigma: 5 m)
After reacting with g) at 30 ° C. for 10 minutes, 0.5 ml of pure water is added and the mixture is centrifuged. The supernatant is taken and the absorbance is measured at 530 nm. The titer is 1 unit of the amount of enzyme that changes A ₅₃₀ by 1.00 in 1 minute. 10) Isoelectric point: pI 8.3 11) Ultraviolet absorption: Absorption maximum (λmax): 280 nm A ₂₈₀ / A ₂₆₀ : 1.5

[0016]

[Equation 1] 12) Amino acid composition (moles of residues): Aspartic acid 37 Methionine 2 Threonine 19 Isoleucine 13 Serine 39 Leucine 19 Glutamic acid 17 Tyrosine 10 Proline 14 Phenylalanine 8 Glycine 39 Lysine 14 Alanine 41 Histidine 10 Cystine N. D. Tryptophan 6 Valine 33 Arginine 4 Total 325 Note) D. Indicates that it is not detected.

[0017]

EXAMPLES Next, examples of the present invention will be described. Various means for collecting the novel proteolytic enzyme of the present invention from the culture solution based on the various properties disclosed in the present specification as described above. However, the present invention is not limited to this. (Example 1) In the medium shown in Table 1, a production strain belonging to the genus Bacillus, Bacillus sp. WF
-2 strain (FERM P-13802) was subjected to shaking culture (110 rpm) at 25 ° C. for 24 hours.

[0018]

[Table 1] After culturing, remove the cells and add ammonium sulfate to the filtrate to add 30-70%
Fractions that saturate and precipitate were collected. The obtained precipitate was dissolved, dialyzed against 10 mM phosphate buffer (pH 8.0), and applied to a CM Sepharose Fast Flow column for adsorption. Then, elution was carried out by a linear gradient system using the same buffer solution as described above and 0.5M NaCl added to the same buffer solution. 0.15M NaC
The active fraction eluted from around 1 (corresponding to the fraction of the proteolytic enzyme of the present invention) was collected. Furthermore, 0.1 M of this fraction was added to 10 mM phosphate buffer (pH 8.0).
Gel filtration chromatography was performed on a Superrose 6HR column in a system containing NaCl to obtain a purified fraction. This fraction gave a single band by polyacrylamide gel electrophoresis. The results are shown in the attached FIG. 1 by a photograph substituted for a drawing. The specific activity of the enzyme thus obtained by purification in the azochol was 8.03 units / m 2.
The specific activity of the enzyme in the filtrate after culturing was 0.16 unit / mg, and the specific activity was increased 50 times by purification.

Example 2 The proteolytic enzyme of the present invention obtained in Example 1, subtilisin BPN '(Sigma), subtilisin Carlsberg (Sigma) and subtilisin novo (Novo), and Various proteins for thermolysin (Sigma), trypsin (Sigma) and Clostridium collagenase (Sigma), namely gelatin (Nippi), Azocol (Sigma), casein (Merck), collagen (Sigma). , And a synthetic substrate for collagenase, Z-GPL
The effects on GP (Sigma), Z-GPPGPA (Sigma) and PZ-PLGPR (Sigma) were compared. For comparison, the reaction was performed under the optimal conditions for each enzyme, and the final concentration was 1
It was performed by quantifying the produced peptide soluble in 0% trichloroacetic acid (TCA) by the ninhydrin method. The results are shown in Table 2.

[0020]

[Table 2] As can be seen from the results in Table 2, the proteolytic enzyme of the present invention showed a remarkable decomposing activity for gelatin which is denatured collagen, as compared with other proteolytic enzymes. It also showed high activity against azochol (denatured collagen) and casein. However, it had no effect on native collagen. The proteolytic enzyme of the present invention also showed an action on the above-mentioned collagenase synthetic substrate, but the action of other proteolytic enzymes on them was weak or not observed.

(Example 3) The cleavage site in the insulin B chain of the proteolytic enzyme of the present invention obtained in Example 1 was changed to another proteolytic enzyme derived from a microorganism, that is, subtilisin BPN 'and subtilisin Carlsberg. And subtilisin novo and thermolysin. For comparison, each enzyme was reacted with insulin B chain, and the produced peptides were fractionated by high performance liquid chromatography, and their amino acid compositions were analyzed to determine the cleavage site (E. Ichishima et al., Agric. Bio).
l. Chem. 55 , pp. 2191-2193, 1991.
See the method described in the year). The results are shown in Table 3.

[0022]

[Table 3] As can be seen from the results in Table 3, the proteolytic enzyme of the present invention was found to cleave more sites than other proteolytic enzymes. In particular, Leu-Cys, Arg-
It showed a different behavior from other proteolytic enzymes derived from the genus Bacillus, such as cleaving the bonds of Gly, Thr-Pro and Pro-Lys. (Example 4) The amino acid compositions of the proteolytic enzyme of the present invention obtained in Example 1 and subtilisin BPN ', thermolysin and trypsin, which are other proteolytic enzymes derived from the genus Bacillus, were measured and compared. The amino acid composition of each enzyme is shown in Table 4.

[0023]

[Table 4] As can be seen from Table 4, the enzyme of the present invention showed a different amino acid composition from other enzymes derived from the genus Bacillus.

[Brief description of drawings]

FIG. 1 is a purification step of the proteolytic enzyme of the present invention (culture filtrate, ammonium sulfate precipitation, CM sepharose, gel filtration).
3 is an electrophoretic photograph of an SDS polyacrylamide gel in FIG.

Front page continuation (72) Inventor Koteru Suzuki 1-1, Senju Midoricho, Adachi-ku, Tokyo 1 Share company Nippi

Claims (2)

[Claims] 1. A proteolytic enzyme derived from a microorganism belonging to the genus Bacillus, which has the following physicochemical properties. (1) Action: It exerts remarkable activity on the decomposition of gelatin and azochol and acts on casein. It does not act on native collagen but is a synthetic substrate for collagenase (Cbz-Gl
y-Pro-Leu-Gly-Pro, Cbz-Gl
y-Pro-Gly-Gly-Pro-Ala, Pz
-Pro-Leu-Gly-Pro-Arg). Insulin B chain Leu-Cys, Arg-G
It cleaves many sites such as ly, Thr-Pro, Pro-Lys. (2) Optimum pH: 7-9 (3) Optimum temperature: 40 ° C-55 ° C (4) Molecular weight: about 30,000 2. A method for producing a proteolytic enzyme, which comprises culturing a proteolytic enzyme-producing bacterium belonging to the genus Bacillus and collecting a proteolytic enzyme having the following physicochemical properties from the culture. (1) Action: It exerts remarkable activity on the decomposition of gelatin and azochol and acts on casein. It does not act on native collagen but is a synthetic substrate for collagenase (Cbz-Gl
y-Pro-Leu-Gly-Pro, Cbz-Gl
y-Pro-Gly-Gly-Pro-Ala, Pz
-Pro-Leu-Gly-Pro-Arg). Insulin B chain Leu-Cys, Arg-G
It cleaves many sites such as ly, Thr-Pro, Pro-Lys. (2) Optimum pH: 7-9 (3) Optimum temperature: 40 ° C-55 ° C (4) Molecular weight: about 30,000