JPS5816683A - Protease and its preparation - Google Patents

Abstract

PURPOSE:To prepare a novel protease, protease MTE-56 having improved anti- inflammatory enzymatic activity, by cultivating a bacterium belonging to the genus Bacillus. CONSTITUTION:A bacterium such as Bacillus iwakuniensis MTE-56 (FERM-P 6034) belonging to the genus Bacillus, capable of producing protease MTE-56, is inoculated into a nutrient medium preferably containing 0.1-0.5wt% sodium chloride in the medium components, cultivated under aerobic conditions and protease MTE-56 is collected from the culture.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a protease and a method for producing the same. More specifically, the present invention relates to a protease with excellent anti-inflammatory effects and a method for producing this protease using microorganisms.

Conventionally, proteases produced by microorganisms are known to be produced by fungi, bacteria, and actinomycetes.

In addition, anti-inflammatory proteases include Dazen■ from Serratia n (Takeda Pharmaceutical KK), Enbinase■ from Streptomyces griseus (Kaken Kagaku KK), and protease 55 from Streptomyces subvarsogenes.
-11 A, Bacillus sphaericus, a novel protease, Bacillus pebutitase 0, etc. are known.

When using these proteases as anti-inflammatory agents,
Since these are proteins, there is a restriction that they cannot be administered in large quantities.
In other words, a protease with high specific activity has been sought.

In addition, the optimal pH of anti-inflammatory protease has been required to be close to neutral, just as it is in living organisms.

The present inventors paid attention to these points and investigated neutral proteases with high specific activity, and as a result, discovered a protease with excellent anti-inflammatory enzyme activity, leading to the present invention.

The present invention also relates to a suitable method for producing this protease, and relates to a method for producing a protease with excellent anti-inflammatory properties using a microorganism belonging to the genus Bacillus.

That is, the present invention provides protease M having the following characteristics.
It is TK-56.

(a) Molecular weight: 56.000 (gel p-filtration method) (
b) Ultraviolet absorption spectrum: maximum absorption at 275-280 nm and minimum absorption at 245-255 nm.

(C) Isoelectric point: pI=8.9t-0,1(d)
Solubility: Soluble in water, but insoluble in acetone, benzene to hexane, and chloroform.

(e) pH effect: The preferred Ill for proteolytic activity using casein as a substrate is 6 to 8;
・It has an optimum pH of 5 to 7.5.

(t) pH stability: 57° C., 311 minutes + 7) Stable, showing activity of 80% or more in the pH range of 6 to 10.

(g) Range of suitable temperature for action = The suitable temperature for proteolytic action using casein as a substrate is 55°C to 6o°C,
The optimum temperature is about 50°C.

(1,) Inhibition: Inhibited by ffDTA and dipyridyl, the degree of inhibition is EDTA>dipyridyl.

5 chloromercuric benzony), (POMB)
, diisopropyl fluorophosphate, and soybean-derived trypsin inhibitor are the protease MIC-56.
does not affect the activity of Also Hg′+, Fe family and O
It is also inhibited by a++, but the extent is Hg+F>
F e+F~a a” Theal.

(1) pH and temperature inactivation c7) conditions” 1)
More than 70% of H4.0% is deactivated at 57°C for 30 minutes. On the other hand, teno inactivation at pHjD, 37°C for 10 minutes was 2
Less than 0% teal. Sarani pH 7, 70°C, completely inactivated in 10 minutes.

The present invention also relates to a suitable method for producing protease, which belongs to the genus Bacillus and contains protease MTK-
The present invention relates to a method for producing protease MTE-56, which comprises culturing bacteria that produce MTE-56 in a medium under aerobic conditions, and collecting protease MTK-56 from the culture.

The protease MTE-56 producing microorganism used in the present invention includes all strains, mutants and variants belonging to the genus Bacillus that are capable of producing the protease MTRnee-56. Among these, a particularly preferred strain is strain MT2051, which was collected from soil by the present inventor and has extremely high anti-inflammatory enzyme activity.

The mycological properties of this strain are described below.

(a) Morphological properties Vegetative cells cultured on bouillon agar for 37°02 days
．． 7-1.2 x 5-10 micron rods.

One or two cells exist connected together. 30℃ 2 days~
Spore formation is observed after 6 days of culture.

Spores are formed mainly in the center of the sporangium, but also at the edges. The size is cylindrical with a diameter of 0.7 to 1.2 x 1.2 to 2.5. It is motile, Durham staining is negative, and it is acid-fast.

(b) Growth status in each culture medium (1) Broth agar plate and broth agar slant culture Colonies with smooth surfaces and gloss grow at 28°C. Colonies have golden edges and become 8-10 mm in diameter after two weeks of culture at 28°C. The color is dull yellow (buff)
), noticeably viscous.

(2) Sweetened agar slant culture Growth is better than on ordinary agar slopes, and the surface is also noticeably glossier.

(3) Salt-added meat juice culture Will not grow with addition of 5% salt.

(4) Meat juice becomes a film after 1 to 2 days of liquid culture, and eventually becomes a fairly thick, viscous, flat gel-like film. There is very little precipitation.

(5) Meat juice gelatin puncture culture It liquefies in one week at 22°C.

(6) Promptly decolorize lidmus milk. A film is formed and eventually turns into peptone. In addition, milk coagulation occurs within 2 to 3 days, producing white lumps.

(7) Growth on glucose and asparagine agar.

(2) MR test negative (3
) VP test Negative (4) Production of indole Negative (5) Production of hydrogen sulfide Negative (6) Hydrolysis of starch Positive (7) Utilization of citric acid (Kos
er's) Negative (8) Use of inorganic nitrogen sources Nitrate Do not use.

(Wickelham's synthetic medium on the ground) Use ammonium salt.

(Same as above) (9) Formation of pigment was not observed.

(10) Urease production positive (significant) (11
) Catalase positive (growth range of 1) Grows at pH 4, 5 to 8.5, optimally at pH 6 to 8. Optimum temperature is 5ON at 38°C, grows up to 45°C, but grows at 50°C If it exceeds it, it will not grow.

(13) # Attitude towards basics It is aerobic. However, it can also grow under anaerobic conditions.

(14) Sugar assimilation, acid production, and gas production The various properties of this strain were determined using Bergey's Manual Op Determinative Bacteriology (Bergey's M
annual of DeterminativeBaa
According to the 1975 edition,
It belongs to the genus Bacillus, but there is no known equivalent species.

Therefore, this strain was determined to be a new species of the genus Bacillus, and
Iwakuniensis novo subbases MT2031 (
Bacillus iwakuniensis-n,
Sp.

It was named MT2031).

To produce the protease MTE-56 of the present invention, for example, the above-mentioned new species of Bacillus, Bacillus iwakuniensis MT-2031, is inoculated into a medium containing a carbon source, a nitrogen source, salts and other substances, and an appropriate temperature, e.g. about 28
There is a method of producing the enzyme by culturing with shaking or aeration at ~35°C for an appropriate period of time until the enzyme titer reaches its maximum. In particular, the production amount of protease MTK-56 can be increased by using a medium containing yeast cells or a medium containing soybean meal.

The culture cultured as described above is clarified by centrifugation, filtration, or the like, and then, for example, ammonium sulfate is added to the clarified liquid to precipitate the enzyme of interest, and the enzyme can be collected. Further, the above precipitate was purified using an ion exchange resin and a gel filtration method, and a novel protease MT with extremely high activity was obtained.
The physical and chemical properties of E-56 are shown below.

(1) Preferred p for proteolytic action using casein as a substrate
As shown in FIG. 2, H is 6 to 8, and the optimum pH is 6,!
5-7.5 neutral protease.

(2) Substrate specificity Relative degradation rates for various proteins are shown in Table 2. In addition, the literature values of the commercially available enzyme Dazen ■ are also listed in Table 2. Relative Decomposition Rate" (3) Optimal pH Figure 2 shows the influence of pH on proteolysis using casein as a substrate. The optimal pH is 6.5 to 7.5.

(4) pH stability The residual activity after heating at 57°C for 30 minutes at various pH values was measured, and the results are shown in Figure 3. It showed residual activity of 90% or more.

(5) Measurement of titer The proteolytic activity was measured by partially modifying the Kunitz method (by changing the pH from 9.0 to 7.5).

That is, 2.0% Hammarsten casein solution 1-0
m/s (L2M) Lis-HCl buffer (pH 7,5)
A reaction solution consisting of 0-5 J and 0-5 ml of enzyme solution was used and incubated at 157°C for 10 minutes. 5 immediately after the reaction
% trichloroacetic acid (TOA) solution 3. Add OJ, stir to stop the reaction, leave for 50 minutes, and centrifuge (3
000rpm). Measure the OD275nm of the supernatant,
A TOA supernatant obtained by adding 0.5c++5 of water to the reaction solution instead of the enzyme solution was used as a control, and the difference in OD from the control was determined. 27575 nm of tyrosine The value of OD from the extinction coefficient at nm is 275 nm of tyrosine.
The activity was expressed in terms of n weight, and the amount of enzyme that produced 1 μg of tyrosine in the TCA supernatant per minute under the above conditions was defined as 1 unit. In addition, 1 μg/ml tyrosine solution (3
%TOA) is 0.0073575n
It was m.

(6) Optimal temperature and threat temperature range Figure 4 shows the temperature dependence of proteolytic action using casein as a substrate. The optimum temperature for the action of this enzyme is about 50°C.

Furthermore, temperature stability is shown in FIG. This enzyme is stable at temperatures below 40°C, and has approximately 90% residual activity at 50°C.

(Conditions for inactivation by pH and temperature as shown in Figure 3)
, rapidly deactivated below pH 4, pH 4.0, 37℃, 3
Approximately 80% of the activity was inactivated in 0 minutes.

On the other hand, it is relatively stable on the alkaline side, with pH 10 and 37.
Only about 10% is deactivated in 60 minutes at ℃.

Moreover, as shown in FIG. 5, it is completely inactivated at 70° C. for 10 minutes.

(8) Effect of ion concentration The effect of phosphate buffer concentration is shown in FIG.

(9) Effect of inhibitors Table 5 shows the effects of inhibitors. Furthermore, Table 4 shows the influence of metal salts.

Table 3 Table 4 (10) Protease purification method The crude enzyme sample obtained by the method shown in Example 1 can be separated and purified by the following method.

That is, 1 g of crude enzyme sample was dissolved in 10 m/ml of water,
30mM potassium phosphate buffer with pH 6,7 (hereinafter referred to as KP)
A sp-Sephadex column (abbreviated as B) equilibrated with an sp-Sephadex column (abbreviated as B)
2,5X 60α) and elute all active p7):3p with 30mM KPB (pH 6,7). The active fraction obtained here is Sephadex G-10
0 gel p application column (2,5×100 CIII) for further purification. The active fraction was extracted with ammonium sulfate salting out (
70% saturation), dialyzed, and lyophilized.

(11) Molecular weight The molecular weight measured by gel p-filtration method is approximately 56. ．． It is 000.

(12) Elemental analysis value 0 44.3% H6.1% N 15.5% 0 25.5% The elemental analysis value may vary somewhat depending on the purity of the enzyme.

(13) Ultraviolet absorption spectrum As shown in Figure 1, the absorption maximum is approximately 275 to 280 nm, and the absorption minimum is approximately 245 to 255 nm.

λ "278nm ax x', 4 (278nm) = 14.1 (14) Amino acid composition Table 5 shows the amino acid composition of this enzyme.

The amino acid composition ratio may vary somewhat depending on the purity of the enzyme and analytical accuracy.

Table 5 (15) Carrageenin edema inhibitory activity measurement method (a) Subcutaneous administration method Wistar male rats purchased from 7 associations were preliminarily bred for one week, with 7 rats per group and 10 rats per group in the control group), and the enzyme was administered. It was dissolved in physiological saline and administered subcutaneously at a rate of 5.0 ml/ktiC rat).

1 hour after enzyme solution administration, 1% carrageenan solution 0.05 m
/ was injected subcutaneously into the right hind leg of the rat, and 3 hours later,
The ankle joints of both hind legs were amputated, and the edema suppression rate of the enzyme was calculated using the weight difference between both ankles as the edema intensity.

(b) Duodenal administration method 7 Wi
In ster, 1% male rats, an approximately 1 incision was made along the abdominal midline under ether anesthesia, and the duodenal region was pulled out. The enzyme solution was administered into the duodenum by lightly pressing the pyloric region of the stomach with a finger to prevent the enzyme solution from flowing back into the stomach. After administration,
The incision site was sutured in two places and the anesthesia was removed.

1 hour after administering the enzyme solution, 1% carrageenin solution 0.1
ml was injected subcutaneously into the rat's right leg, and 5 hours later, the weight difference between the left and right ankles was determined. However, in the preliminary test, due to the effects of anesthesia and surgery, only about 60% of the edema was obtained compared to normal rats, so the amount of inflammatory agent was subcutaneously administered.From this, the edema suppression rate in the treated group compared to the control group was calculated. did. In addition, 10 animals were used in each group for the control group, and 7 animals were used in each group for the treatment group.
The average value was calculated.

(16) Carrageenin edema inhibitory activity (anti-inflammatory activity) (10)
Protease MTE-56 purified by the method of (1
5) The anti-inflammatory activity was measured by administering subcutaneously to rats by the method shown in (a). The results are shown in Table 6.

The enzyme of the present invention has an ED5o value of about 4 times or more as compared to commercially available Enbinase 0 and Dazen ■, and exhibits extremely excellent carrageenan edema suppressing activity.

Similarly, the purified protease of the present invention (15
) By the method shown in (b), the carrageenan was administered to the duodenum of rats, and the carrageenan edema-inhibiting activity by intestinal absorption was measured.

The results are shown in Table 7.

The enzyme of the present invention exhibits approximately 6.5 times more activity than the commercially available Empinase ■ to Dazen ■. protease MTE-5
As shown in Table 1, No. 6 has a significantly high specific activity as a protease. Furthermore, as shown in Tables 6 and 7, the anti-inflammatory activity is significantly higher than that of conventionally known enzymes.

Furthermore, the protease of the present invention has a high titer and can be used for purposes such as digestive agents, cosmetics, detergents, meat tenderizers, production of meat extracts, silk scouring, leather production, and plastin production.

Next, examples will be shown.

Example 1 400 ml of a medium having the composition shown in Table 9 is placed in a 24 volume Sakaro flask and sterilized at 120°C for 15 minutes. Add 50 ml of preculture solution of Bacillus iwakuniensis MT2031 cultured in a medium of the same composition, and
Cultured with shaking for hours. Protease in the culture solution (5)
Measured by the titer measurement method shown in 2 s 300un1t
It was s/m1. After culturing, add 30% ammonium sulfate.
To saturation (17.6% (-/v) to the culture solution)
) and stirred to precipitate impure proteins, which were removed by centrifugation (10,000 rpm, 10 minutes).

Ammonium sulfate was further added to the obtained supernatant to a saturation of 90% (44.9% relative to the supernatant) to precipitate the enzyme active fraction, followed by centrifugation (10+000 rpm).
0 minutes) and collected. Suspend the collected enzyme in as little water as possible, pack it in a cellophane tube for dialysis, tie tightly at both ends, and soak in cold water (15°C) for 24 to 48 hours.
(below). After dialysis, insoluble matter was removed by centrifugation and freeze-dried, and the resulting powder was used as a crude enzyme sample. This was purified according to the protease purification method shown in text (10), and 0.1 g of protease MTE-56 was obtained from the culture solution of 11 as shown in Table 9. Using a medium containing 1% yeast or 5% soybean meal as the main component, Bacillus iwakuniensis MT2031 was inoculated using a No. 21 Sakalo flask as in Example 1, and the pH was adjusted to pH 6.5 to 7 at 28°C.
．． 48 Table 10. while controlling to the range of 5. Effect of main components on protease production Example 3 Salt was added to the petroleum shown in Example 1, and the effect on protease production was investigated. Bacillus, Iwakuniensis MT
After inoculating 2051, culturing was carried out in the same manner as in Example 1. The results are shown in Table 11, with sodium chloride ranging from 0.1 to 0.
By adding 5%, especially 0.25%, a protease activity equal to or higher than that of dry yeast (Example 2) can be obtained even when using compressed bread yeast.Table 119 Effect of salt on protease production

[Brief explanation of drawings]

Figure 1 shows an aqueous solution of protease MTE-56 (MTE-56).
56325mg/ltn#distilled water). Figure 2 shows protease MTJ regarding caseinolytic activity.
The optimum pH of C-56 is shown. Figure 3 shows protease MTE with respect to caseinolytic activity.
-56 pH stability. Figure 4 shows protease MTE-56 at various temperatures.
The relative proteolytic activity of Figure 5 shows Protease MTI! It exhibits a temperature stability of 1-56 (treated for 10 minutes at each temperature). Figure 6 shows the effect of ion concentration (phosphate buffer) on the activity of one protease, MTK-56. Applicant Mitsui Petrochemical Industries Co., Ltd. Agent Kazu Yamaguchi Figure 2 Optimal IH = H Figure 4 Optimal humidity temperature ('C) Figure 3 MiH stability H Figure 5 Humidity stability humidity ('C) m6, ion Concentration L Tonic acid slowing/effect

Claims (4)

[Claims] (1) Protease MTK-56 (
IIL) Molecular weight: 56,000 (gel p-filtration method) (
b) Ultraviolet absorption spectrum: maximum absorption at 275-280 nm and minimum absorption at 245-255 nm. (,) Isoelectric point: pl28.9±0.1 (d) Solubility: Soluble in water, but insoluble in acetone, benzene, hexane, chloroform (1) (e) pH effect: Casein The preferred pH for proteolytic activity using as a substrate is 6 to 8;
It has an optimum pH of ~7.5. (f) I) H stability: After treatment at 37°C for 30 minutes, it exhibits 80% or more activity in the pH range of 6 to 10 and is stable. (C) Range of suitable temperature for action: The suitable temperature for proteolytic activity using casein as a substrate is 55°C to 60°C,
The optimum temperature is about 50°C. (h) Inhibition: Inhibited by EDTA and dipyridyl, the degree of inhibition is KDTA>dipyridyl. Parachloromercuric benzoate (POMB), diisopropyl fluorophosphate, and soybean-derived trypsin inhibitors do not affect the activity of the subject protease MTE-56. It is also inhibited by Hg, Fe, and Oa, but the degree of inhibition is Hg++>ye++
~Oa′+. (1) Conditions for inactivation due to pH and temperature? pH4.0
, 70% or more is inactivated at 37°C for 60 minutes. On the other hand, the deactivation at pH 10, 57°C for 10 minutes was 20%.
It is as follows. Furthermore, it is completely inactivated at pH 7 and 70°C for 10 minutes. (2) Belongs to the genus Bacillus and has protease MTffi-5
A method for producing protease MTK-56, which comprises culturing bacteria producing protease MTK-6 in a medium under aerobic conditions, and collecting protease MTF K-56 from the culture. (3) Bacillus iwakuniensis MT-2 belongs to the genus Bacillus and produces the protease MTB-56.
osi (Feikoken Bibori No. 6034, FIRM p-10
Claim No. 34)
The manufacturing method described in section. (4) The production method according to claim (2), wherein a medium containing 0.1 to 0.5% by weight of sodium chloride in the medium components is used as the medium.