JPH0331436B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a novel arginine deiminase, its production method, and its uses. [Prior art] Arginine deiminase (enzyme classification number
3.5.3.6) is an enzyme that catalyzes the enzymatic reaction that produces L-citrulline and ammonia from L-arginine and water, and its existence is known in animals, bacteria, and yeasts [Enzyme Handbook, p. 602, Asakura Shoten, 1984 edition, 1st edition, 3rd printing]. Among these enzymes, those that have been reported to have been purified include Pseudomonas putida and Mycoplasma hominis.
hominis) and Mycoplasma a arthritidis [Kakimoto, T. Kakimoto, T. etal FEBS Lett. VOL.19, P 166
-168, (1971); Schimke, R. Tee.
(Shimke, RT) Method of Enzymology (Meth.Enzymol.) VOL.17A, P 310−
313, (1970); Wijkman, J. L. and Falny, D. E. (Weickman,
J.L. & Fahrney, DE) Journal of Biological Chemistry (J.Biol.Chem.)
VOL.252, P2615-2620, (1977)]. In addition, L-arginine is an enzyme that decomposes L-arginine.
- Since arginase is known to have antitumor effects, there were expectations for a similar effect on arginine deiminase [JP-A-Sho
No. 48-58187]. [Problems to be Solved by the Invention] However, there are several problems in production using these conventional arginine deiminase producing bacteria. In other words, these arginine deiminase-producing bacteria have low arginine deiminase productivity; Mycoplasma hominis, which belongs to Mycoplasma;
Mycoplasma allusridis
When the enzyme is collected from A. arthritidis, culture management is complicated, and there are problems such as difficulty in using it as an enzyme source in mass production of the enzyme; and extremely difficult purification. Furthermore, there are no clear reports showing that mycoplasma-derived arginine deiminase inhibits tumor cell growth. [Means for Solving the Problems] The present inventors discovered a cell line among animal tumor cells whose culture medium exhibits a remarkable growth-inhibiting effect, and as a result of conducting various studies on that line, It was discovered that a protein produced by a parasitic strain belonging to the genus Mycoplasma (TGIF) inhibits tumor cell growth. The researchers also found that the protein was obtained homogeneously by culturing the aforementioned Mycoplasma strain (TGIF), and isolated and purified it, and discovered that this protein was a novel arginine deiminase, and completed the present invention. . That is, the present invention is an arginine deiminase having the following physical and chemical properties: (a) Action: catalyzes an enzymatic reaction that produces citrulline and ammonia from L-arginine and water; (b) Substrate specificity: L-arginine (c) Optimum pH; 7.0-7.5 (d) PH stability; 5.0 or more (e) Molecular weight; 47000±5000 (by SDS-PAGE method) (f) Isoelectric point; 7.4±0.5 Arginine belonging to Mycoplasma orale
A method for producing arginine deiminase, which is characterized by culturing deiminase-producing bacteria in a medium and collecting the arginine deiminase from the culture, and further comprising growing tumor cells having arginine deiminase activity derived from microorganisms belonging to the genus Mycoplasma. It provides an inhibitory active substance. The taxonomic properties of the novel arginine deiminase producing bacterium of the present invention are as follows. Growth status in medium Liquid medium of Mycoplasma orale (TGIF) [Medium composition; PPLO broth W/O in medium 1
(manufactured by DIFCO) 15g, L-arginine hydrochloride 5
200 ml of horse serum, 25 g of yeast extract, 0.25 g of thallium acetate, 100,000 units of potassium penicillin G, and ⁵ mg of phenol red]. [This is an abbreviation for Colony Foaming Unit] As a result of inoculation and static culture at 37℃, the number of bacteria enters the logarithmic growth phase following the lag phase, and the number of bacteria reaches its maximum 4 to 5 days after the start of culture. A stationary phase is reached. The period of the stationary phase is short and varies depending on the culture, and the growth state enters the meiotic phase in approximately a few hours. Physiological conditions PH for growth: 6-8 Optimal PH: 7.4-7.6 Temperature for growth: 30-37°C Optimal growth temperature: 37°C Morphological characteristics under the microscope Agar medium [Medium composition; medium 1 , Bactagar (manufactured by DIFCO) 10g, PPLO broth W/
O CV (manufactured by DIFCO) 14.5g, L-arginine hydrochloride 5g, L-glutamine 3g, DNA (crude)
20 mg, NAD 150 mg, glucose 10 g, Eagle vitamin solution (x100) 10 ml, horse serum 200 ml, yeast extract 25 g].
It is 10 to 500 μm, thicker in the center and thinner in the periphery.
It has a so-called fried egg shape. Staining is Dienes solution [methylene blue 2.5g; Azure
1.25 g; maltose 10.0 g; Na ₂ CO ₃ 0.25 g; distilled water 100 ml]. The staining solution was poured onto the agar surface, and after 1 to 2 minutes, the excess solution was discarded and microscopic observation was performed. The center of the village appears to be dyed dark blue and the surrounding area appears to be dyed pale blue. Under 100x microscope magnification,
The peripheral area appears granular. Growth inhibition by antibodies Using antiserum of each Mycoplasma species, we used the literature by Wallace A. Clyde, JR [J.Immunol. VOL.92,
P.958-965, (1964)] [at the Animal Experiment Facility attached to the Faculty of Medicine, University of Tokyo], it was identified as Mycoplasma orale. Glucose, arginine, urea decomposition test Test bacteria in heart infusion broth supplemented with 10% (V/V) horse serum Difco
1 ml of the culture was inoculated into a test medium and a control medium containing glucose, arginine, and urea, respectively. Seal the culture test tube tightly.
The cells were cultured at 37°C for one week. The control that did not contain the test substance and those that showed a decrease or increase in PH of 0.5 or more were judged as positive, and the results were as follows. Glucose degrading activity; Negative arginine degrading activity; Positive urea degrading activity; Negative or higher results indicate complete absence of cell wall, diameter 0.1~
It has an elementary body of 0.2 μm, and its colony form is characterized by embedding it in agar medium and multiplying, forming fried egg-shaped colonies, and because it requires serum for growth, it belongs to the genus Mycoplasma. Therefore, through a growth inhibition test using antibodies, this strain (TGIF) was confirmed to be Mycoplasma orale (TGIF).
orale (TGIF)]. This strain has been designated as Mycoplasma orale (TGIF) by the Institute of Microbial Technology, Agency of Industrial Science and Technology.
[Mycoplasma orale (TGIF)]
It has been deposited as No. 1970 (FERM BP-1970). In producing the arginine deiminase of the present invention, the arginine deiminase producing bacterium is cultured by a conventional method for producing enzymes and the like. The culture may be carried out by liquid culture or solid culture, but from an industrial perspective, it is advantageous to inoculate arginine deiminase-producing bacteria into a production medium and perform static culture. As the medium composition for culturing arginine deiminase, those commonly used for culturing microorganisms, particularly mycoplasma, are widely used. Any nitrogen compound that can be used as a nitrogen source may be used, such as yeast extract, lactalbumin hydrolyzate, urea, peptone, and meat extract. The carbon source may be any carbon compound that can be assimilated, such as glucose.
In addition, serum or sterols essential for culturing mycoplasma may be added as appropriate, and various inorganic salts may be used as necessary. The culture temperature can be changed as appropriate within the range that allows the bacteria to grow and produce arginine deiminase, but it is 30 to 37°C, preferably 35 to 37°C. The culture time varies depending on the conditions, but is usually 4 to 8 days, preferably 5 days.
~6. However, it is a matter of course that the culture should be terminated at an appropriate time by determining the time when the growth of this strain reaches its maximum titer. After the culture is completed, the present enzyme can be collected from the culture using a conventional enzyme collection method. Since this enzyme is an intracellular enzyme, it can be crudely purified using conventional means for collecting enzymes from within bacteria, such as ultrasonication, frozen cell impact disruption (low-temperature cell disruption), and methods using various drugs. Obtain the enzyme solution. The crude enzyme solution thus obtained further contains known proteins,
Purified arginine deiminase is obtained by purification using isolation and purification means such as enzymes. For example, a crude arginine deiminase-containing solution is buffered with a buffer such as Tris-HCl buffer,
This is purified by an appropriate combination of chromatography using an ion exchange resin such as carboxycellulose, carboxymethyl-dextran gel, sulfopropyl-dextran gel, or a gel filtration agent such as dextran gel or polyacrylamide, and then, if necessary, A purified arginine deiminase solution can be obtained by concentrating the arginine deiminase and dissolving it in a buffer solution. The physicochemical properties of the arginine deiminase thus obtained are as described below. (1) Arginine Deiminase Activity Assay Preparation of substrate solution: 200mM Tris-HCl buffer (PH7.2) 200μ
50mMDTT (deithiothreitol) 80μ,
Prepare the substrate solution by dissolving 40μ of 100mM arginine/hydrochloric acid adduct. Preparation of enzyme solution: The arginine deiminase solution of the present invention obtained in the example described below was diluted with 10 mM Tris-HCl buffer (PH7.0) to give an enzyme solution of OD ₂₈₀ =0.005. Activity measurement: Take 320μ of the above substrate solution into a small test tube,
After standing for 2 minutes in a water bath at 37℃, remove the enzyme solution.
Add 80μ and start the reaction. After reacting at 37 °C for exactly 30 min, stop the reaction by adding 100 µl of 5M perchloric acid. After that, a redox buffer [9.0g NH ₄ Fe(SO ₄ ) ₂ .
12H ₂ O, 11.0g (NH ₄ ) ₂ Fe (SO ₄ ) ₂・6H ₂ O
Solution dissolved in 1N sulfuric acid solution to 100ml〕125μ
and then add the acid mixture [200ml of distilled water,
H ₃ PO ₄ (d=1.74) 300ml, H ₂ PO ₄ (d=1.84)
Add 250μ of diacetylmonoxime solution [a solution of 0.75g of diacetylmonoxime dissolved in distilled water to make 100ml], heat in boiling water for 20 minutes in the dark, and then cool.
Measure the absorbance at 490 nm. The standard line for calculating the amount of citrulline produced is at various concentrations (0.01-0.05 μmole/ml) adjusted with 1M perchloric acid.
Using 500 μl of the L-citrulline solution, the following operations were performed after adding 125 μl of the redox buffer, and the relationship between absorbance at 490 nm and citrulline concentration was plotted, and the linear region was used as a standard calibration curve. (2) Substrate specificity: To examine the specificity of the reaction to free amino acids, an amino acid mixture (10mM Tris-HCl buffer PH7.0) containing 200mg of L-arginine, 200mg of L-arginine,
Asparagine/ _H2O 56.8mg, L-aspartic acid 20mg, L-cystine 50mg, L-glutamic acid 20mg, L-glutamine 300mg, glycine 10mg,
L-histidine 15mg, L-hydroxyproline
20mg, L-isoleucine 50mg, L-leucine 50
mg, L-lysine/HCl40mg, L-methionine 15
mg, L-phenylalanine 15 mg, L-proline
20mg, L-serine 30mg, L-threonine 20mg,
Contains 20mg L-tyrosine, 20mg L-valine, mg L-tryptophan) 800μ to 200μ
After adding the arginine deiminase solution of the present invention (OD _{280 o} m = 0.016) and incubating at 37°C for 18 hours, changes in amino acid concentration were analyzed using an automatic amino acid analyzer (Hitachi Model L-8500). As a control, the same operation was carried out by adding 10 mM Tris-HCl buffer instead of the arginine deiminase solution of the present invention. As a result, as shown in Table 1, the arginine deiminase of the present invention was found to be an enzyme that specifically acts on L-arginine to catalyze the reaction that produces citrulline.

【table】

[Table] (3) Enzyme action: Catalyzes the following reaction. L-Arginine + H ₂ O → Citrulline + NH ₃ (4) PH stability: 10μ of the present enzyme solution (OD _280o m = 0.8)
After mixing with 40μ of test PH buffer and incubating for 3 hours at 37°C, 950μ of PBS was added to dilute 20 times, and the remaining enzyme activity was measured according to the enzyme activity measurement method described above. As a result, PH
It was stable from 5.0 to 10.0. In addition, the test PH buffer solution is 0.1M from PH3.0 to 6.0.
Citric acid-sodium citrate buffer, PH7.0
0.1M Tris-HCl buffer until ~9.0, PH10.0
A 0.1M glycine-NaOH buffer was used. (5) Optimal PH: As a result of measurement according to the enzyme activity measurement method described above using buffer solutions with various PH values, it was found that the optimal PH was in the range of PH7.0 to 7.5. (6) Molecular weight: 47000±5000 (SDS-PAGE method) 55000±5000 (gel filtration method) (7) Isoelectric point Measured by isoelectric focusing using AMPHOLINE PAGPLATE (manufactured by LKB) As a result,
It has an isoelectric point at p17.4±0.5. (8) N-terminal amino acid sequence analysis Using the enzyme of the present invention (total OD _280o m = 0.01) obtained based on the Examples described below, a liquid phase protein sequencer (manufactured by Beckman:
As a result of analyzing the amino acid sequence from the N-terminal side using BECKMAN System 890ME), first,
The sequence up to the fifth position is revealed as shown below, and X-Ser-Val-Phe-Ser-Asp- (in the formula, X represents a _hydrogen atom or Met). As a result, the sequence up to number 30 was revealed as shown below. X-Ser-Val-Phe-Ser-Asp-Lys-Phe
−Asn−Gly−Ile−His−Val−Tyr−Ser−
Glu−Ile−Gly−Asp−Leu−Glu−Ser−Val−
Leu−Val−His−Glu−Pro−Gly−Lys−Glu
- (In the formula, X represents a hydrogen atom or Met) (9) Amino acid composition analysis Purified enzyme preparation of the present invention (OD _{280 o} m = 0.08)
Add an equal amount of 12N hydrochloric acid to 1 ml and hydrolyze at 105℃ for 24 hours.
-8500 model). The molecular weight of the enzyme of the present invention is approximately 47,000 according to SDS electrophoresis, and if the average molecular weight of amino acids is 110, the constituent amino acids are approximately 427 residues, so the analysis results are expressed as the number of amino acid residues per 427 residues. The results are shown in Table 2.

[Table] Comparing the above properties with known arginine deiminase, it can be seen that it is different from any other enzyme. Furthermore, since the enzyme of the present invention exhibits strong growth-inhibiting activity against tumor cells, it can be used as an anticancer agent. The physicochemical properties of the enzyme of the present invention against tumor cells are shown below. (a) Method for measuring cell proliferation inhibitory activity Cells used for measurement: CCRF-CEM cells (human lymphoid leukemia cells; purchased from Dainippon Pharmaceutical Co., Ltd.) were selected as cells with high inhibition sensitivity to the enzyme of the present invention. Used for testing. Culture medium: 10% fetal calf serum (FCS) added
RPMI-1640 medium (100 mg/penicillin G
and dihydrostreptomycin sulfate). Measurement method: CCRF-CEM cells in the logarithmic growth phase cultured in the above culture medium were collected, suspended in fresh culture medium, and the cell concentration was adjusted to 1.11×10 ⁴
Adjust to 2 pieces/ml. After that, the obtained cell-containing solution was poured into a 24-well multiplate (MULTIDISH;
A homogeneous cell suspension is seeded at 900 μ/well in a tube (manufactured by NUNC). Filter (MILLEX GV filter; manufactured by Millipore)
Add 100 μ/well of the test solution filter-sterilized using 100 μl/well and stir gently. as a control
PBS (phosphate buffered saline) 100μ/well
Added. Each of the 24-well multiplates seeded with cell suspensions was placed in 5% CO ₂ , 95% air, and 100%
After culturing in an incubator at 37°C and % humidity for 4 days, the number of cells in each well is counted using a Coulter counter (manufactured by Coulter, USA). Calculate the growth inhibition rate (%) according to the following formula. Growth inhibition rate (%) = 1-A-B/C-B x 100 A; Number of cells in test solution B; Number of cells seeded C; Number of cells in control solution In this system, the proliferation of CCRF-CEM cells was suppressed to 50%.
The inhibiting activity was defined as 1 unit (u). (b) PH stability for cell growth inhibition 10μ of the present enzyme solution (10000u/ml)
After mixing with 40μ of test PH buffer and incubating at 37°C for 3 hours, 950μ of PBS was added to dilute 20 times, and the remaining cytostatic activity was shown in the method for measuring cytostatic activity described above. Measurements were made according to the method. As a result, the pH was stable from 5.0 to 10.0. In addition, the test PH buffer solution is 0.1M from PH3.0 to 6.0.
Citric acid-sodium citrate buffer, PH7.0
0.1M Tris-HCl buffer until ~9.0, PH10.0
A 0.1M glycine-NaOH buffer was used. (c) Thermostability against cell growth inhibition 10μ of the present enzyme solution (10000u/ml)
In addition to 990μ of 0.05M Tris-HCl buffer (PH7.0), treat at 37℃ for 24 hours, 60℃ for 1 hour, and 100℃ for 5 minutes, cool on ice, and measure the remaining cell growth inhibitory activity. Evaluated by. The results are shown in Table 3 below.

[Table] (d) Stability of cell proliferation inhibitory effect against various enzymes.
After reacting at ℃ for 6 hours, the remaining cell proliferation inhibitory activity was evaluated. The results are shown in Table 4 below.

[Table] (e) Stability of cell proliferation inhibitory effect against various organic acids and organic solvents Various organic acids and organic solvents were added to the enzyme solution of the present invention (2000u/ml) at various concentrations (V/V%). The mixture was reacted at 25°C for 1 hour. After that, dilute the reaction solution with PBS to adjust the concentration of the enzyme of the present invention.
After adjusting the concentration to 100 u/ml, the remaining cell proliferation inhibitory activity was evaluated. The results are shown in Table 5 below.

[Table] (f) Growth inhibitory effect on various tumor cells The growth inhibitory effect of the enzyme of the present invention on tumor cells in various culture systems was investigated using human lymphoid leukemia-derived cell lines (CCRF-CEM, CCRF-HSB2,
CCRF-SB, RPMI-8226), human myeloid leukemia-derived cell line (K-562, HEL92・1・7),
Using a human kidney cancer-derived cell line (TRC-29R), a human lung cancer-derived cell line (TLC-7NC3), and a mouse breast cancer-derived cell line (C-1271), the effects on the proliferation of these cell lines were investigated. The culture medium used was E-MEM medium for C-1271 cell line.
% fetal bovine serum was used, and for other cell lines, a medium containing RPMI-1640 medium supplemented with 10% fetal bovine serum was used. Each cell line was cultured in each medium, and these tumor cell lines in the logarithmic growth phase were collected and suspended in fresh culture medium, and the cell concentration was adjusted to 1×10 ⁴ cells/ml. After seeding 1 ml/well of uniformly suspended cell suspension in a 24-well multi-plate, PBS
dilutions of the enzyme of the invention at various concentrations dissolved in
Added 100μ/well, 5% CO ₂ , 95% air,
The cells were cultured in an incubator at 100% humidity and 37°C for 4 days. As a control, culture was performed by adding PBS instead of the diluted solution of the enzyme of the present invention. To measure the number of cells after culturing, floating cells (leukemia-derived cells) are diluted 20 times with ISOTON (USA; Coulter), and adherent cells are diluted with 0.1% trypsin solution after removing the medium by suction. 1 ml,
Detach the cells from the culture substrate and incubate with ISOTON for 20 min.
A sample for measurement was diluted twice, and measured using a Coulter Counter (manufactured by Coulter Inc., USA). Furthermore, the growth inhibition rate (%) is calculated according to the following formula. Growth inhibition rate (%) = 1-A-B/C-B x 100 A; Number of cells in test solution B; Number of cells seeded C; Number of cells in control solution As a result, all tumor cell lines used in the test proliferated. As shown in Table 6, the concentration of the enzyme of the present invention at which an inhibitory effect was observed and 50% growth inhibition was observed, the OD value at 280 nm was in the range of 10 ^-5 to 10 ^-6 , indicating that it acts in extremely small amounts. It became clear.

[Table] (g) Effect of the enzyme of the present invention on general normal cells Although mouse 3T3 cells are capable of long-term division and proliferation, they do not have tumorigenic ability, so they are considered to be cells close to normal cells and are susceptible to cell mutations and cytotoxicity. It is widely used in research, and this time it was used as an indicator of the effect of the enzyme of the present invention on general normal cells. Eagle containing mouse 3T3 cells with 10% fetal bovine serum
The cells were suspended in MEM medium, adjusted to 5×10 ⁴ cells/ml, and seeded in 2 ml portions in φ35 mm tissue culture plastic dishes. Furthermore, the enzyme solution of the present invention having an OD _{280 o} m value of 4.7 x 10 ^-3 dissolved in PBS was added to a final concentration of OD _{280 o} m value of 2.35 x 10 ^-4 (10.2 u/ml).
5% CO ₂ , 95% air,
Culture was performed in an incubator at 100% humidity and 37°C. Instead of the enzyme solution of the present invention as a control
PBS was added and cultured. After culturing for 2 and 4 days, the cells were detached using a 0.1% trypsin solution and resuspended in a culture medium to obtain a single cell suspension. After adding 9 volumes of 0.1% trypan blue solution dissolved in PBS to 1 volume of the cell suspension, the number of cells was counted using a Neubauer hemocytometer. Cells that are stained blue by trypan blue are dead cells, and cells that are not stained are live cells.As a result of counting the number, as shown in Table 7, in the case of culture with the enzyme of the present invention and culture without additives, No significant difference in cell survival rate was observed, indicating that the enzyme of the present invention has no direct toxicity to normal cells.

〔Example〕

Next, examples of the present invention will be given, but the present invention is not limited thereto. Example 1 7 volumes of PPLO broth (manufactured by DIFCO), 2 volumes of horse serum, 1 volume of yeast extract, 1000 u/ml of penicillin G,
500ml of liquid medium containing 500μg/ml of thallium acetate
10 ml of Mycoplasma orale (TGIF) at 1×10 ⁶ CFU/ml was inoculated into the tube and cultured stationary at 37° C. for 5 days. Transfer the culture solution to a high-speed refrigerated centrifuge (Hitachi
SCR-20BA type), 4℃, 11000r.pm, 30
The cells were centrifuged for a minute to collect the bacteria into a pellet. After suspending 1 g (wet weight) of the obtained Mycoplasma orale TGIF cells in 30 ml of PBS,
The bacterial cells were disrupted by ultrasonication (120 seconds, 4 to 5 times) under ice cooling. The disrupted solution was centrifuged at 4° C. and 18,000 rpm for 40 minutes, and the supernatant was separated. The precipitate is again
After suspending in 30ml of PBS, perform the same operation as above to separate the supernatant, mix it with the supernatant obtained earlier, put it in a cellulose tube for dialysis (manufactured by VISKASE, USA), and add 10mM Tris-HCl buffer (PH7.0)
Transmission analysis at 4°C was repeated twice for 24 hours each. The obtained bacterial cell extract was adsorbed onto a DEAE-Sepharose column (φ16 x 200 mm) equilibrated in advance with the same buffer [10mM Tris-HCl buffer (PH7.0)], and the same buffer was added to 3 times the column volume. After washing with NaCl in the same buffer,
Elution was performed with a 200mM linear gradient. The eluate was collected into fractions of 6 ml each, and the cell proliferation inhibitory activity of each fraction was examined using the method described in the activity measurement method described above. As a result, activity was observed to elute in fractions with NaCl concentrations of 20 mM to 100 mM. Ta. The cell proliferation inhibitory activity fraction purified by DEAE-Sepharose ion exchange chromatography was collected and filtered using an ultrafiltration membrane (YM-10; manufactured by Amicon, USA).
After concentrating 9.5 ml using
It was charged to a column (φ26 x 930 mm) manufactured by LKB Co., Ltd., and eluted using the same buffer. Eluate is 5ml
The cell proliferation inhibitory activity of each fraction was examined using the method described in the activity measurement method described above, and as a result, cell proliferation inhibitory activity was observed in the eluate volume range of 240 ml to 310 ml. A 61 ml solution was obtained by collecting the cytostatic activity fractions purified by gel filtration.
The obtained solution was concentrated to 5 ml using an ultrafiltration membrane (YM-10; manufactured by Amicon, USA), and then diluted with 10mM Tris-HCl buffer (PH7) using a cellulose tube for dialysis (manufactured by VISKASE, USA). .0) 2 was subjected to dialysis twice (48 hours) at 4°C. A part of the solution after dialysis was preliminarily added to 10mM Tris-HCl buffer (PH7.0).
After adsorption onto an ion exchange chromatography column (MonoQ HR5/5 column; manufactured by Pharmacia, Sweden) equilibrated with , it was eluted with a linear concentration gradient from 0 to 200 mM using NaCl dissolved in the same buffer. 20 ml of solvent was used at a flow rate of 1 ml/min. While observing changes in the protein concentration of the eluate using ultraviolet absorption at 280 nm, each protein elution peak was fractionated, and the cell proliferation inhibitory activity of each fraction was examined using the method shown in the activity measurement method above. As a result, the NaCl concentration was determined. but
It was revealed that the protein peak eluted from 55mM to 65mM. In order to further purify the active protein peak fraction purified by the above ion exchange chromatography (MonoQ HR5/5 column; manufactured by Pharmacia, Sweden), the obtained active protein peak fractions were collected and The solution was filtered using a cellulose tube for dialysis (manufactured by VISKASE, USA).
10mM Tris-HCl buffer (PH7.0) 3
After diafiltration at 4°C for 24 hours, a portion of the diafiltration solution was subjected to ion exchange chromatography (MonoS) equilibrated in 10mM Tris-HCl buffer (PH7.0).
It was adsorbed onto an HR5/5 column (manufactured by Pharmacia, Sweden). Elution of adsorbed protein is
The concentration of NaCl was increased from 0 to 0 after 20 minutes at a flow rate of 1 ml/min.
Elution was performed with a linear concentration gradient up to 100mM. While observing changes in the protein concentration of the eluate using ultraviolet absorption at 280 nm, each protein elution peak was fractionated, and the cell proliferation inhibitory activity of each fraction was examined using the method shown in the activity measurement method above. As a result, the NaCl concentration was determined. was found to elute locally in a single peak around 60mM, and that fraction was collected. Ion exchange chromatography (MonoQ
As a result of repeated purification operations using HR5/5 column (manufactured by Pharmacia, Sweden) and ion exchange chromatography (MonoS HR5/5 column; manufactured by Pharmacia, Sweden), the enzyme preparation of the present invention was obtained. About 3 mg was obtained. Test example (purity test) (1) Verification by reversed phase high performance liquid chromatography A column for reversed phase chromatography (ODS) using 500μ of a 0.1% trifluoroacetic acid solution (100μg/ml) of the enzyme of the present invention as a carrier on silica gel. High performance liquid chromatography was performed over the course of the process.
Elution was carried out using 0.1% trifluoroacetic acid as a solvent at a flow rate of 1 ml/min, and a linear concentration gradient of acetonitrile ( _CH3CN ) from 0 to 60% at a rate of 1%/min. As a result, it was eluted as a single peak at an acetonitrile concentration of 43 to 45%.
It turned out to be a homogeneous substance. (2) Assay by polyacrylamide gel electrophoresis SDS electrophoresis of the enzyme of the present invention using a 12% polyacrylamide slab gel is performed using a conventional method [edited by the Electrophoresis Society, Electrophoresis Experimental Methods, p. 208, (1976) Bunkodo]
I followed. The enzyme preparation of the present invention subjected to electrophoresis was incubated in the presence of 1% SDS (sodium lauryl sulfate).
It was prepared by incubating and denaturing at 37°C for 20 hours with and without the addition of 0.1M 2-mercaptoethanol. After the electrophoresis is complete, use a silver staining kit manufactured by Nippon Bio-Rats Laboratories, Inc.
Electrophoretic protein staining was performed according to the instructions attached to this. As a result, the enzyme preparation of the present invention was found to be a homogeneous protein, as it formed a single band in the presence and absence of a reducing agent. Furthermore, the molecular weight of the enzyme of the present invention was found to be approximately 47,000 from the relative mobility with the molecular weight marker that was electrophoresed at the same time. [Effects of the Invention] The present invention provides a novel arginine deiminase, and this arginine deiminase exhibits a tumor cell growth inhibiting effect and is expected to be used as an antitumor agent.

Claims (1)

[Scope of Claims] 1. Arginine deiminase having at least the following physical and chemical properties. (a) Action: Catalyzes the enzymatic reaction that produces citrulline and ammonia from L-arginine and water (b) Substrate specificity: Has substrate specificity for L-arginine (c) Optimal pH: 7.0-7.5 (d ) PH stability: 5.0 or more (e) Molecular weight: 47000±5000 (according to SDS-PAGE method) (f) Isoelectric point: 7.4±0.5 2. Claim 1, which is an enzyme derived from a strain belonging to the genus Mycoplasma. Arginine deiminase as described in Section. 3. Claim 1 comprising a polypeptide having an amino acid sequence of at least X-Ser-Val-Phe-Ser-Asp- (wherein X represents a hydrogen atom or Met) from the N-terminal side. Arginine deiminase as described. 4. A method for producing arginine deiminase, which comprises culturing an arginine deiminase-producing bacterium belonging to Mycoplasma orale in a medium, and collecting the arginine deiminase from the culture. 5. The production method according to claim 4, wherein the arginine deiminase is the arginine deiminase according to claim 1. 6. The production method according to claim 4, wherein the arginine deiminase-producing bacterium belonging to Mycoplasma orale is Mycoplasma orale (TGIF) (FERM BP-1970). 7. A tumor cell growth inhibiting substance having arginine deiminase activity with at least the following physical and chemical properties. (a) Action: Catalyzes the enzymatic reaction that produces citrulline and ammonia from L-arginine and water (b) Substrate specificity: Has substrate specificity for L-arginine (c) Optimal pH: 7.0-7.5 (d ) PH stability: 5.0 or more (e) Molecular weight: 47000±5000 (by SDS-PAGE method) (f) Isoelectric point: 7.4±0.5