JPH0763369B2 - Novel metal protease and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel metalloprotease which specifically cleaves the N-side peptide bond of consecutive basic amino acid residues in a peptide chain by hydrolysis and its production. Regarding the method.

[Conventional technology]

The importance of limited protein degradation by proteases has been well studied and clarified in blood proteases such as blood coagulation, kinin system, and renin-angiodensin system. It has also become known that specific proteases act on the maturation and inactivation of neuropeptides and hormone peptides. Further, the specific signal peptide peptidase also acts when various proteins are secreted extracellularly or localized in a certain site in the cell. In order for the proteins that have undergone such biosynthesis (precursor proteins, propeptides) to express their unique functions, it is necessary to process precursor proteins or propeptides into mature proteins or peptides with specific proteases. Is said.

Recently, it has been known that two consecutive basic amino acids exist in prohormone, which is a precursor of many mammalian peptide hormones, and that site is the first processing site for producing active hormone. There are reports of possible cases (Docherty, k et al. Ann.Rev.P
hysiol.44,625-635 (1982), Mains et al. Trends Neur
osdi., 6,226-235 (1983), Kurjan.J et al. Cell 30,93
3-943 (1982)). It has also been suggested that an enzyme that cleaves the N-side, an enzyme that cleaves the C-side, and an enzyme that cleaves the middle of these continuous basic amino acids play an important function in the production of active hormone from prohormone.

There have been many research reports on the search for enzymes that prove this.

Several proteases that hydrolyze intermediate peptide bonds of consecutive basic amino acids in animals have already been found (Loh YP et al. Proc. Natn. Acad. Scl. USA7).
9,108-112 (1982), Mizuno et al. Biochem. Biophys. Re
s.Commun. 108, 1235-1242 (1982), Lindberg et al. Bio
chem.Biophys.Res.Commun.106,186-193 (1982), Evang
elists R et al. Biochem, Biophys, Res.Commun. 106,895
-902 (1982)), or the role of those enzymes in prohormones has also been reported. Since these enzymes are of animal origin, it is difficult to collect a large amount of these enzymes, and their practicality is poor.

On the other hand, research on yeast enzyme search has also been actively conducted, and a protease (Jullus, d et al. C) that cleaves the C-side peptide bond of consecutive basic amino acids from the yeast membrane fraction has been actively conducted.
ell 37,1075-1080 (1984), Achstetter et al The EM
BO Jourual 4,173-177 (1985)), a metalloprotease that processes a precursor protein from the mitochondrial matrix fraction has been reported. (McAda, PCet a
lJBiol.Chem.257, 3177-3182 (1983), Behni, PCet
al. J. Biol. Chem. 258, 4973-4943 (1983)) However, these enzymes are capable of cleaving the C-side peptide bond of a basic amino acid in the former and recognizing a certain amino acid sequence in the latter. Rather, it recognizes the three-dimensional structure of the substrate protein, and there is a clear difference in the molecular weight and the like from the protease of the present invention.

Mizuno et al. Have reported a serine protease that hydrolyzes peptide bonds between consecutive basic amino acids derived from the yeast soluble fraction (Mizuno et al. Nature 309,
558-560 (1984) differs from the protease of the present invention in its action, substrate specificity and enzymatic chemistry.

To date, no enzyme such as the present enzyme that cleaves the N-side peptide bond of consecutive basic amino acids has been reported.

[Problems to be solved by the invention]

The inventors of the present invention conducted comprehensive research to search for a novel protease that induces the produced protein or peptide into an intended active substance, and as a result, yeast belonging to the saccharomyces genus was found to have its intracellular mitochondria. It recognizes two basic amino acid sequences consecutive to
It was found that a novel metalloprotease that specifically hydrolyzes a side peptide bond is produced and accumulated, and the present invention has been completed.

[Means for solving problems]

(Enzymatic Chemical Properties) That is, the novel metalloprotease of the present invention has the following enzymatic chemical properties.

(1) Molecular weight 90,000-10,000 SDS polyacrylamide gel electrophoresis)
(See Fig. 1) 80,000 to 90,000 (gel filtration method by high performance liquid chromatography) (2) Isoelectric point 5 to 6 (3) Optimum pH and stable pH Optimum pH is 6.5 to 7.5 (see Fig. 2) .

Almost no deactivation after treatment at pH 5.5-8.5 for 15 minutes.

(4) Optimal temperature range and temperature stability It works in the range of 20 ℃ to 45 ℃, and the optimum temperature is about 37 ℃.

Almost inactivated by temperature treatment at 50 ° C for 30 minutes.

(5) Properties of substance The freeze-dried product is a white powder.

(6) Action and substrate specificity The N-side peptide bond of consecutive basic amino acid residues in the peptide chain is specifically hydrolyzed.

For example, the portion shown by the arrow in the synthetic peptide shown below is hydrolyzed.

↓ Boc-Gln-Arg-Arg-MCA (t-butoxycarbonyl-glycalyl-arginyl-
Arginyl-4-methylcoumaryl-7-amide) ↓ Cbz-Ala-Arg-Arg-MCA (carbobenzoxy-alanyl-arginyl-arginyl-4-methylcoumaryl-7-amide) ↓ Boc-Gln-Lys-Lys-MCA ( (t-Butoxycarbonyl-glitaryl-lysine-lysyl-4-methylcoumaryl-7-amide) (7) Reactivity to various inhibitors and metal ions Ethylenediaminetetraacetic acid (EDTA), ethyleneglycolbis (2-ethylether) tetraacetic acid ( EGTA), orthophenanthroline, parachloromercuribenzene sulfonic acid, zinc, copper.

It is not inhibited by the serine protease inhibitor phenylmethylsulfonyl fluoride.

Not inhibited by soybean trypsin inhibitor.

Ph is a microbial low-molecular-weight protease inhibitor
osphoramidon, Antipain, Leupeptin, Elastatin, E64, Peps
Not inhibited by tatin and Bestatin.

Activated with manganese, cobalt, calcium and magnesium.

The enzyme activity deactivated by the metal chelating agent is manganese,
Recovered by the addition of cobalt.

It has ionic strength dependence and its optimum ionic strength is 0.1 ~
It is 1.

(8) Amino acid composition (mol%) Table 1 shows the analysis results.

From the above results, this enzyme belongs to a metalloprotease, and it is a protease whose activity can be reversibly controlled by removal and addition of metal, and reversible by EDTA after activation by addition of metal such as manganese and cobalt. It is a protease that can be inactivated.

(Production Method) The yeast of the genus Saccharomyces used for production of the protease of the present invention may be any yeast that produces the protease of the present invention. For example, Saccharo
Examples include myces cerevisie. These strains can be obtained without any restrictions.

The medium used for producing the protease of the present invention may be an ordinary medium used for culturing yeast, and may be any medium as long as it produces the protease of the present invention. Glucose, dextran and the like can be used as the carbon source, and proteolytic substances such as peptone and animal extracts such as meat extract can be used as the nitrogen source.
Inorganic salts, vitamins and the like can be added if necessary.

Commercially available yeast such as live yeast (Saccharomyces cerevisie HANSEN; manufactured by Oriental Yeast Co., Ltd.) can also be used.

In culturing, a general yeast culturing method is applied. In order to obtain a large amount of cells, it is preferable to carry out aeration stirring culture or shaking culture using a liquid medium. When performing large-scale culture, first perform a small-scale pre-culture,
It is desirable to inoculate the obtained medium with the obtained bacterial cells. Other culturing conditions such as culturing temperature and period are appropriately selected and adjusted so that the production amount of the present enzyme is maximized, but usually under aerobic conditions at 25 to 30 ° C for 2 to 3 days. It is preferable to carry out the culture at about pH 5.

Since the enzyme of the present invention is extracted from the matrix fraction of mitochondria in yeast cells, in recovering and purifying the enzyme, the separated bacterial cells are first destroyed and the protease is eluted from the mitochondrial fraction. Collect. The separated bacterial cells are destroyed by an enzymatic treatment method or a physical method in an isotonic solution or the like, and a mitochondrial fraction is obtained by centrifugation. Then, the resulting mitochondrial fraction is eluted with a physical method to elute the protease, and purified by using various combinations of column chromatography.

The purification procedure is as follows. The protease-containing eluate from the mitochondrial fraction is centrifuged to remove insoluble matter. The obtained supernatant is treated with phenylmethanesulfonyl fluoride (PMSF) and then loaded on DEAE-cellulose to obtain an active fraction. Then, the active fraction is loaded on zinc chelate sepharose, and the resulting active fraction is dialyzed and then loaded on protamine sepharose for elution. The treatment with protamine sepharose is repeated twice. The active fraction thus obtained is loaded on hydroxyapatite to obtain an active fraction. This process is also repeated twice.
A single final purified sample can be obtained by concentrating the active fraction by ultrafiltration and finally performing gel filtration by high performance liquid chromatography. Purification can be carried out efficiently at a low temperature of 0 to 4 ° C. without losing enzyme activity, and can be efficiently carried out by using a buffer solution containing glycerin or the like effective as a stabilizer, a dialysate and a lysate.

(Activity measuring method) The protease activity of the present invention is determined by the synthetic substrate t-butoxycarbonyl-glycalyl-arginyl-arginyl-4-.
Methylcoumaryl-7-amide (Boc-Gln-Arg-Arg-MC
It is measured by the following two methods using A) as a substrate.

The procedure of activity measurement is as follows.

[Activity Assay Method I] 0.5 ml of 50 mM Tris-HCl buffer containing 10 nmol of substrate (p
H7.5) with 1.25 μmol MgCl ₂ , aminopeptidase M
(; EC3.4, 11.2, manufactured by sigma) 9.6 mU and a test enzyme solution are added and reacted at 30 ° C. for 1 hour.

Boc-Gln-Arg-Arg-MCA was the first enzyme in the enzyme to generate arginyl-arginyl-4-methylcoumaryl-7-amide (-Ar
g-Arg-MCA) and then sequentially hydrolyzed by aminopeptidase M from the N side of the peptide to release 7-amino-4-methylcoumarin (AMC). This A
The activity is measured by measuring the fluorescence of MC at an excitation wavelength of 380 nm and a fluorescence wavelength of 460 nm.

As for the enzyme activity unit (unit (U)), 1 unit (U) is the amount of enzyme that liberates 1 nmole of 7-amino-4-methylcoumarin per minute in the above reaction system.

[Activity measuring method II] 0.1M mops buffer, pH 7.0, 0.1 ml containing 10 nmol of substrate (same as the activity measuring method I) and purified enzyme solution is kept at 30 ° C to carry out the enzyme reaction. The reaction is stopped by adding 0.9 ml of 5 mM hydrochloric acid solution. Degradation product Arg-Arg-MC produced as a result of enzymatic reaction
A was loaded on high performance liquid chromatography using a cation exchanger, Monos HR5 / 5 (Pharmacia), and the excitation wavelength was 325n.
m, fluorescence wavelength 395 nm to identify and quantify.

The enzyme activity unit (unit (U)) is defined as 1 unit (U) of the amount of enzyme that liberates 1 nmole of Arg-Arg-MCA in 1 minute in the above reaction system.

(Use of Protease of the Present Invention) Since the protease of the present invention specifically hydrolyzes the N-side peptide bond of consecutive basic amino acid residues in the peptide chain, it specifically cleaves such a site in the peptide. Widely used when you need to.

Today, various useful proteins and peptides can be produced by genetic engineering and cell engineering techniques, but the produced protein has high specificity as a processing enzyme which is a physiologically active substance having a desired function. It is useful as a protease. It is also useful as a peptide chain-cleaving enzyme reagent when determining the amino acid sequence of a protein.

〔Example〕

 Next, examples of the present invention will be described.

Example 1 3 kg of live yeast (Saccharomyces cerevisiae HANSEN; manufactured by Oriental Yeast Co., Ltd.) was washed with 0.6 M sorbitol-10 mM Tris-HCl isotonic buffer, pH 7.5, and then suspended in 0.3% bovine serum albumin combined buffer 5. The lowered pH was adjusted to pH 7.5 with potassium hydroxide. Dyno-Mill KD
L) large-scale preparation method (Labbe et al. Anal. Biochem. 8
1,416-424 (1977)) to destroy yeast and release intracellular organelles, and the resulting disrupted solution is made up to 2 volumes with isotonic buffer and centrifuged (3000 rpm, 10 minutes) to remove mitochondria. Obtain the supernatant liquid containing it and further centrifuge (10000 rpm, 40 minutes)
Then, the mitochondria were precipitated. 2 this centrifugation
Repeatedly, the resulting mitochondrial precipitate was suspended in 10 mM Tris-HCl buffer pH 7.5 and sonicated (Branson
Mitochondria were disrupted with a sonifier, Max Power 15 sec. × 4), and centrifugation (14000 rpm, 40 minutes) followed by ultracentrifugation (40000 rpm, 40 minutes) was performed to obtain 1600 ml of mitochondria-soluble fraction. This fraction was adjusted to pH 7.5 and the final concentration was 1 mM.
PMSF was added so that

This fraction was loaded onto a DEAE cellulose column (φ6 × 19 cm) equilibrated with 10 mM Tris-HCl buffer pH 7.5, washed with a buffer containing 10% glycerin, and then subjected to a linear concentration gradient of 0 to 0.5 M sodium chloride. Elution was performed to obtain an enzyme active fraction.

To this fraction, add PMSF to a final concentration of 1 mM, add sodium chloride to a final concentration of 0.5 M, and add 0.5 M
Sodium chloride containing 10% glycerin 20 mM Tris-hydrochloric acid buffer solution Loaded onto a zinc chelate sepharose column (φ6 × 20 cm) equilibrated with pH 7.5 and washed with the same buffer, then containing 0.5 M sodium chloride-10% glycerin A linear concentration gradient to 10 mM sodium dihydrogen phosphate, pH 4.3 was applied, and elution was performed with the pH gradient to obtain an enzyme active fraction.

This active fraction was dialyzed (10% glycerin-containing 20 mM Tris-HCl buffer pH 7.5), and then loaded onto a protamine sepharose column (φ1.6 × 20 cm) equilibrated with the same buffer, and 4 M chloride was added from the same buffer. Sodium-10% glycerol containing 10 mM
Elution was performed with a linear concentration gradient to sodium dihydrogen phosphate, pH 4.3. After dialysis (10 mM glycerin-containing 20 mM Tris-HCl buffer pH 7.5), it was loaded again on the protamine sepharose column (φ1.6 × 20 cm) equilibrated with the same buffer, and 1 M
Elution was performed with a linear concentration gradient to 10 mM sodium dihydrogen phosphate containing 10% glycerin, pH 4.3 to obtain an enzyme active fraction. Marc was used to prepare protamine sepharose.
The method of H. et al. (Anal. Biochem. 60, 149-152 (1974)). The protamine sepharose chromatography was performed while cooling with ice.

Then, 20 mM Tris-HCl buffer pH 7.5 containing 10% glycerin
Hydroxyapatite column (φ1 × 25
cm) loaded with the enzyme active fraction, and eluted with a linear concentration gradient from 0.005% Pridi58 containing the same buffer to 0.005 Pridi58-10% glycerin containing 0.3M potassium phosphate buffer pH 7.5. Got After dialysis (0.005 prigi 58-10% glycerol containing 20 mM Tris-HCl buffer 7.5), equilibrated with the same buffer hydroxyapatite column (φ1 × 25
cm) and the enzyme was eluted with a linear concentration gradient using the same buffer containing 0.5 M sodium sulfite.

The enzyme-active fraction thus obtained was subjected to ultrafiltration.
Concentrate to 0.6 ml, 0.15 ml each Superose 12 column,
(Made by Pharmacia), 0.2M sodium chloride −0.0
05% Prizi 58-10mM Mops buffer pH 7.5
Gel filtration was performed by PLC. The enzyme active fractions were collected and concentrated to 1.5 ml by ultrafiltration to give a final purified sample.

As described above, a purified product of this enzyme can be obtained by 7-step column chromatography, and the degree of purification at each step is shown in Table 2. The total enzyme activity was measured by the above-mentioned activity measuring method (I), and the protein amount was measured by the method of Lowry et al. (J. Biol. Chem. 193, 265-27).
5 (1951)).

Example 2 Molecular Weight Measurement An example of molecular weight measurement by SDS polyacrylamide gel electrophoresis is shown.

The purified sample was prepared by the method of Laemmii et al. (Nature 227,680-685 (19
70)), and the molecular weight was measured by SDS gel electrophoresis under reducing conditions. Ovalbumin (MW 43000), bovine serum albumin (MW 68000) and phosphorylase B (MW 97400) were used as standard proteins. First result
Shown in the figure. The enzyme had a molecular weight of 9,000-100,000.

Example 3 Substrate Specificity Test A substrate specificity test was conducted using various synthetic substrates. The activity was measured by the above-mentioned activity measuring method (I). The results are shown in Table 3. The activity ratios of various substrates are shown when the activity against Boc-Gln-Arg-Arg-MCA is set to 100. As a result, almost no activity was shown for the substrate containing no continuous basic amino acid.

Example 4 Reaction Test with Various Inhibitors and Metal Ions The purified sample was treated with various inhibitors and metal ions to examine its activity. The activity was measured by the aforementioned activity identification method (II). The results are shown in Table 4. The activity when no inhibitor was added was defined as 100, and the activity after each inhibitor treatment was expressed as the ratio. As a result, this enzyme is a metal chelating agent EDTA, EG
It was shown to be a metalloprotease that is inhibited by TA and orthophenanthroline. In addition, phenylmethylsulfonyl fluoride (PMS which is a serine protease inhibitor)
It was not inhibited in F). It was also not inhibited by microbial small molecule protease inhibitors and soybean trypsin inhibitor, but it was activated by manganese, cobalt, calcium and magnesium. On the other hand, zinc and copper were deactivated.

Furthermore, arginine itself did not show any inhibitory effect,
Protamine sulfate and triarginine, which have a continuous sequence of arginine, showed inhibitory effects.

[Brief description of drawings]

FIG. 1 (a) is a photograph showing the results of subjecting the protease of the present invention to SDS polyacrylamide gel electrophoresis.
FIG. (B) is a graph showing the results of the photograph, and FIG. 2 is a graph showing the results of measuring the optimum pH of the protease of the present invention.

Claims (2)

[Claims] 1. A novel metalloprotease having the following enzymochemical properties: (a) molecular weight 90,000-100,000 (by SDC polyacrylamide gel electrophoresis) (b) action and substrate specificity in the peptide chain It specifically hydrolyzes the N-side peptide bond of a basic amino acid residue. (C) Optimum pH Optimum pH is 6.5 to 7.5 (d) Inhibition Ethylenediaminetetraacetic acid, ethylene glycol bis (2
-Ethyl ether) tetraacetic acid, orthophenanthroline,
Parachloromercurin Benzene Inhibited by sulfonic acid, zinc and copper. 2. A novel metalloprotease having the following enzymatic chemistry: (a) molecular weight 90,000-100,000 (by SDC polyacrylamide gel electrophoresis) (b) action and substrate specificity in the peptide chain It specifically hydrolyzes the N-side peptide bond of a basic amino acid residue. (C) Optimum pH Optimum pH is 6.5 to 7.5 (d) Inhibition Ethylenediaminetetraacetic acid, ethylene glycol bis (2
-Ethyl ether) tetraacetic acid, orthophenanthroline,
Parachloromercurin Benzene Inhibited by sulfonic acid, zinc and copper. In the method for producing a metalloprotease, the method for producing a metalloprotease described above comprises culturing a yeast of the genus Saccharo-myces having the ability to produce the above-mentioned metalloprotease, and collecting the yeast from the mitochondria of the cells. .