JPH01191683A - Novel protease - Google Patents

Abstract

NEW MATERIAL:The protease having the following physical and chemical properties. Action and substrate specificity, especially highly hydrolyzing a C-terminal peptide bond of Y of a compound of formula X-Y- (X is Arg, Lys or Pro which have or have not peptide bond at N-terminal; Y is Arg; - is peptide bond); optimum pH, about pH 7.0 with tris-HC1 buffer solution; stable pH, 6.0-7.0; optimum temperature, 40-47 deg.C (pH7.0); heat-stability, stable at <=38 deg.C (pH 7.0 for 10min). USE:Agent for cutting the C-terminal side of a continuing basic amino acid residue in the synthesis of protein. PREPARATION:The protease can be produced by culturing a yeast belonging to genus Sporobolomyces (preferably Sporobolomyces odrus IFO 1597).

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel protease, and more particularly to a basic amino acid residue-specific protease produced by yeast belonging to the genus Sporobolomyces.

[Conventional technology]

It has been suggested that basic amino acid residue-specific proteases are involved in the biosynthesis of hormones from prohormones, and we have clarified their enzymatic chemical properties and physiological functions, and are interested in the synthesis of proteins from fusion proteins. Attempts have been made to purify it from various sources in order to utilize it. The protease that has been completely purified and reported to date is porcine pituitary gland protease (IRCM-Serine Protease 1).
; J, Biol.

Chem, , 2f) J, 10850 (1986)
), protease from bovine pituitary gland (POMC-converting enzyme; J, Biol, CheIll.).

17194 (1985), J. Biol, Che.
m, 261 14392 (1986)), protease from yeast (Phoces in Y-1; N
ture 309, 558 (1984)), and in the classification of proteases, IRCM-3erine
Protease.

Phorcesin is a serine protease, POM
C-converting enzymes are classified as aspartic proteases. The cleavage site is IRCM-3erine Pr
oteasel, POMC-converting enzyme, converts the C-terminal side of consecutive basic amino acids into Phorcesin Y-1
specifically hydrolyzes between consecutive basic amino acid residues. In addition, although it is partially purified, Saccharomyces cerevisiae (Saccharomyces cerevisiae)
C of consecutive basic amino acid residue pairs from
Ca''-dependent Th1o that selectively hydrolyzes the terminal side
lProtease has also been reported (Biochem
, Biophys.

Res, Commun, 144.807 (1987
)).

[Problem to be solved by the invention]

When a protease is used to synthesize a protein from a fusion protein, it is desirable that the protease specifically cleaves the C-terminal side of consecutive basic amino acid residues, and the search for such a protease is expected. ing.

[Means to solve the problem]

In view of the above-mentioned current situation, the present inventor selected yeast as the origin because it can be prepared in large quantities, and searched for proteases from yeast that have the above-mentioned properties. They discovered that a protease with unique properties can be produced, purified this protease, clarified its physicochemical properties, and completed the present invention.

That is, the present invention provides a protease having the following physicochemical properties (1) to (5).

■ Action and substrate specificity One X-Arg-sequence (X is Ar) present in the peptide chain.
The peptide bond on the C-terminal side of Arg in Lys or Pro) is hydrolyzed.

↓ (Hydrolyze the arrow part of -X-Arg-)■ Optimum pH: )'J Susu-acid buffer pH around 7.0.

■ PN stability: Most stable at pH 6.0 to 8.0 (at 30℃1 at pH 6 to 8)
(Has more than 80% residual activity even after 30 minutes of treatment).

■ Optimum temperature: around 40-470°C (pH 7.0).

■ Thermal stability: Stable below 38°C (p)l 7.0. No decrease in activity after 10 minutes of heat treatment).

The physicochemical properties and enzymatic properties of the protease of the present invention other than those described above are as follows.

■ Activator: Activated by calcium chloride, cobalt chloride, manganese chloride, magnesium chloride, nickel chloride, etc. In particular, it is most activated by low concentrations of calcium chloride. It is also activated by surfactants such as Lubrol PX and Triton X-100.

■ Harmful agents: Metal chelators such as ethylenediaminetetraacetic acid (hereinafter abbreviated as EDTA), ethylene glycol bis(2-aminoethyl ether)tetraacetic acid (hereinafter abbreviated as Efl; TA), copper sulfate, zinc chloride, mercury chloride. It is inhibited by heavy metals such as. Also, parachloromercuric benzoic acid (hereinafter p-C)
It is also inhibited by paraamidinophenylmethanesulfonyl fluoride (hereinafter abbreviated as p-APMSF).

■ Molecular weight: Approximately 56,000 by gel filtration with TSK gel G30005WxL, and approximately 47,000 by 5O5-PAGE■ Isoelectric point: 4.5 by isoelectric focusing This was done by the method shown below.

Activity measurement method Nidris-hydrochloric acid buffer pH 7,050 μmol, Lubrol PX 10 mg, calcium chloride Q, 5 μmol
.

Boc-Gin-Arg-Arg-MCA (Boc is t-Butoxycarbonyl
bony l) group, MCA is 4-methylcoumarin-
7-amide (4-Methyl-coumarin-7
-an+1de)) 7- produced by reacting at 30°C in a reaction solution containing 0.1 μmol and enzyme.
Amino-4-methylcoumarin (7-Amino-4-m
ethylcoumarin; hereinafter abbreviated as AMC)
(excitation wavelength 380 nm, emission wavelength 460 nm)
nm) was measured over time. IU is 1 nmo per minute
The amount of enzyme was defined as the amount of enzyme that catalyzes the release of 1 of AMC. Hereinafter, these measurement conditions will be referred to as standard reaction conditions.

The microorganisms used in the present invention are all those belonging to the genus Sporobolomyces that can produce basic amino acid residue-specific proteases.

Including strains, mutants, and variants. Among them, the preferred strain is Sporobolomyces odorus (Sporobolomyces odorus).
olomyces odrus) IFO1597.

The basic amino acid residue-specific protease of the present invention can be obtained by culturing, for example, a strain having the ability to produce a basic amino acid residue-specific protease belonging to the genus Sporobolomyces in a conventional medium such as YM medium. It can be obtained from things. No particular inducer is required in the culture medium. Further, the culture temperature is preferably 25 to 30°C, and the culture time is preferably about 1 to 3 days.

The produced basic amino acid residue-specific protease is purified by a combination of conventional methods. For example, the culture is centrifuged to collect the bacterial cells, the bacterial cells are disrupted using guinomil, etc., the crushed liquid is centrifuged at low speed to separate the bacterial cell residue, and the resulting supernatant is ultracentrifuged. Membrane fractions are prepared by separation (eg 105.000 g, 60 minutes). The enzyme is solubilized from this membrane fraction with a surfactant, and the solubilized enzyme is purified by ammonium sulfate fractionation, heat treatment, ion exchange chromatography, affinity chromatography, gel filtration, and the like.

〔Example〕

Hereinafter, the present invention will be explained in detail with reference to Examples, but the present invention is not limited thereto.

Example 1 (Purification of basic amino acid residue-specific protease from Sporobolomyces odorus IPO1597) Sporobolomyces odorus IFO1597 was cultured in YM medium (glucose 10g1 Bacto peptone 5g1 yeast extract 3g, malt extract 3g/133p) Cultured for 2 days in
The culture solution was centrifuged to collect 265 g (wet weight) of bacterial cells. The bacterial cells were mixed with 300@7 buffer 1 (10mM
) Lis-hydrochloric acid pl+'7. o, o, 5mM Ca
Cells were suspended in C1z) and disrupted with guinomil, the crushed solution was centrifuged at 1,000g for 10 minutes to remove bacterial cell residues, and the resulting supernatant was ultracentrifuged at 105,000g for 60 minutes. A membrane fraction was prepared as a precipitate. This membrane fraction was added to 60mf of extraction buffer (10mM).
Hydrochloric acid pt+7.0.0.5mM CaC1z, 1%
Luprol px.

The enzyme was extracted by suspending it in 0.1M NaCl) and stirring overnight. Ultracentrifugation was performed under the above conditions to obtain a membrane extract as a supernatant.

Ammonium sulfate was added to this membrane extract until it reached 30% saturation.
．． A supernatant was obtained by centrifugation at 000g for 20 minutes, and ammonium sulfate was further added to this supernatant until it reached 70% saturation.6. O
QOg, the precipitate fraction was reduced to 30% by centrifugation for 20 minutes
It was recovered as a 70% ammonium sulfate fraction.

This precipitate was mixed with a small amount of buffer 2 (10mM) Lis-HCl + 0.5mM CaC1z + 0.2% Lubrol P.
X) After dissolving at pH 8.0 and dialyzing against the same buffer,
Heat treatment was performed for IO minutes at 0°C. The resulting precipitate was 39.0
The fraction was removed by centrifugation at 00g for 20 minutes to obtain a heat-treated fraction.

Both volumes were injected into DEAE-Toyopearl 650M (manufactured by Tosoh Corporation) (2.5 x 45 cm) equilibrated with buffer solution 2 (pH 8.0), thoroughly washed with the same buffer solution, and then injected into a gradient of IM NaC1 from 0. The enzyme was eluted using an elution method to obtain an active fraction. The elution pattern at this time is shown in FIG.

After concentrating the active fraction by ultrafiltration, buffer solution 2 (p
Arg dialyzed against l+7.0) and pre-equilibrated with the same buffer.
inine-Sepharose (manufactured by Pharmacia)
(2.5 x 10 cm). After thoroughly washing the column with the same buffer, elution was performed using a gradient elution method from 0 to 0.5M NaCl. This elution pattern is shown in FIG. The active fraction was concentrated and used as an Arg-Sepharose fraction.

Arg-Sepharose fraction was treated with 0.5M NaCl
Con equilibrated with buffer 2 (pH 7,0) containing
A-Sepharose (manufactured by Pharmacia) (1,6
x25cm). After washing thoroughly with the same buffer, 0.5M NaCl, 0.5M α-Methyl-D-mannoside (α-Methyl-D-mannos 1d
Elution was performed with buffer 2 (pH 7,0) containing e).

The active fraction was concentrated and used as a Con A-Sepharose fraction.

Con A-Sepharose fraction was added to buffer 1 (p
Mono dialyzed against H7,0) and equilibrated with the same buffer.
Inject into Q (manufactured by Pharmacia), 0 to Q, 6M
Elution was performed using a NaCl gradient elution method. Concentrate the active fraction and M
It was set as ono 0 fraction.

The Mono 0 fraction was gel-filtered with 5uperose 12 (manufactured by Pharmacia) equilibrated with buffer solution 1 (pH 7,0) to obtain an active fraction, which was concentrated and 5uperose
It was divided into 12 fractions. This purified enzyme was purified by gel filtration and
Both were uniform on OS-PAGE.

A summary of the purification is shown in Table 1.

Table 1 Sporobolomyces odorus IFO1597
Purification Example 2 of Basic Amino Acid Residue-Specific Protease (Substrate Specificity of Basic Amino Acid Residue-Specific Protease) The activity was measured by changing the fluorescent substrate under standard reaction conditions, and the activity against each substrate was measured. Boc-Gin-Ar
The activity was expressed as a relative activity with the activity against g-Arg-MCA set as 100, and is shown in Table 2.

Example 3 (Optimal pH of basic amino acid residue-specific protease) Activity was measured under standard reaction conditions by varying the type of buffer and pH. As a buffer solution, 50IIIM Tris-HCl buffer pH 6.0-9.0.14.3mM
Br1tton and Robinson buffer pH
4.5 to 10.0, and the activity under each condition was expressed as relative activity with the activity in Tris-HCl buffer pH 7.0 set as 100, and is shown in FIG.

Example 4 (pH stability of basic amino acid residue-specific protease) 11.9 mM Br1tton and Robin
son buffer pH 4-11, 0.2% Luprole PX,
After incubation of the enzyme in 0.5 mM CaC1z for 130 min at 30°C, residual activity was determined by adding an equivalent volume of 100 mM ) Lis-HCl (p)l 7.0 to return pi to 7. The residual activity after each treatment was expressed as relative activity, with the untreated activity set as 100, and is shown in FIG.

Example 5 (Optimal temperature of basic amino acid residue-specific protease) Activity was measured under standard reaction conditions by changing only the reaction temperature from 25 to 60°C. The activity at each temperature is 45
The relative activity is shown in FIG. 5, with the activity at 100° C. being taken as 100.

Example 6 (Thermostability of basic amino acid residue-specific protease) The enzyme was incubated at each temperature for 10 minutes, then rapidly cooled in ice water, and the residual activity was measured under standard reaction conditions. The residual activity at each temperature is expressed as a relative activity with the untreated activity as 100, and is shown in FIG.

Example 7 (Behavior of basic amino acid residue-specific protease toward various inhibitors) In the reaction condition solution containing various inhibitors in the absence of a substrate,
The enzyme was incubated at 25° C. for 130 minutes, and then the substrate was added and reacted. The residual activity was expressed as a relative activity based on the enzyme activity after the same treatment in the absence of an inhibitor as 100, and is shown in Table 3.

Example 8 (Influence of surfactant on the activity of basic amino acid residue-specific protease) A reaction was carried out by changing the type and concentration of surfactant in the standard reaction solution, and the activity in the presence of 3% Luprole PX was 100%. It is expressed in relative activity as shown in FIG.

Example 9 (Influence of CaC1z on the activity of basic amino acid residue-specific protease) The activity was measured in the presence of 0.1% EDTA by varying the concentration of CaC1g. The relative activity is shown in FIG. 8, with the activity in the presence of 0.5mM CaC1z set as 100. In addition, the concentration of free CaC1g is 1g of CaC in EDTA.
Log 1 to the apparent dissociation constant for K, = 7.3
It was calculated as follows.

Example 10 (Influence of various metal ions on recovery of basic amino acid residue-specific protease activity after EDTA treatment) Enzymes were treated for 930 minutes at 25°C in a standard reaction solution containing 1 mM EDTA in the absence of substrate. , various metal ions 1.
It was added to a concentration of 5 mM, incubated at 30°C for 5 minutes, and then the activity was measured. The activity under each condition was expressed as relative activity with the activity under no treatment as 100, and is shown in Table 4.

Table 4 Effect of various metal ions on the activity recovery of EDT^-treated basic amino acid residue-specific protease Example 11 (Molecular weight and isoelectric point of basic amino acid residue-specific protease) Using 7.5% gel The apparent molecular weight was determined to be approximately 47,000 by 5O5-PAGE (Fig. 9). Also, TSK
In gel filtration using gel G30005Wxt, it was about 56,000 (Figure 10).

The pl of this enzyme was found to be 4.5 by isoelectric focusing using IEFge139 (Figure 11).

[Brief explanation of the drawing]

Figure 1 shows Sporobolomyces odorus IFO1597.
It is an EDAE-Toyopearl 650M chromatogram during purification of derived basic amino acid residue-specific protease. Figure 2 shows Sporobolomyces odorus IF01597.
Basic amino acid residue-specific protease derived from Arg-
Sepharose chromatogram. FIG. 3 is a diagram showing the optimum pl+ of the basic amino acid residue-specific protease of the present invention. FIG. 4 is a diagram showing the pH stability of the basic amino acid residue-specific protease of the present invention. FIG. 5 is a diagram showing the optimum temperature of the basic amino acid residue-specific protease of the present invention. FIG. 6 is a diagram showing the temperature stability of the basic amino acid residue-specific protease of the present invention. FIG. 7 is a diagram showing the influence of surfactants on the activity of the basic amino acid residue-specific protease of the present invention. FIG. 8 is a diagram showing the influence of CaClg on the activity of the basic amino acid residue-specific protease of the present invention. FIG. 9 is a diagram showing the results of molecular weight measurement of the basic amino acid residue-specific protease of the present invention in electrophoresis using a 7.5% gel. FIG. 10 is a diagram showing the results of molecular weight measurement by gel filtration using TSK get G30005Wxt of the basic amino acid residue-specific protease of the present invention. FIG. 11 is a diagram showing the results of isoelectric focusing of the basic amino acid residue-specific protease of the present invention using IEF gel 3-9.

Claims (1)

[Scope of Claims] 1. A protease having the physical and chemical properties shown in the following (1) to (5). (1) Action and substrate specificity: -X-Arg- sequence (X is Ar
The peptide bond on the C-terminal side of Arg of G, Lys or Pro) is hydrolyzed. (2) Optimal pH: Tris-HCl buffer pH around 7.0. (3) pH stability: Most stable at pH 6.0 to 8.0. (4) Optimal temperature: around 40 to 470°C (pH 7.0). (5) Thermal stability: Stable below 38°C (pH 7.0, 10
minutes). 2. Sporobolomyces
s) A protease having the properties shown in (1) below, obtained from a culture of yeast belonging to the genus. (1) Action and substrate specificity: -X-Arg- sequence (X is Ar
The peptide bond on the C-terminal side of Arg of G, Lys or Pro) is hydrolyzed. 3. The yeast belonging to the genus Sporobolomyces is Sporobolomyces odorus.
3. The protease according to claim 2, which is IFO1597.