JPH0518556B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a novel aminoacylase that specifically decomposes specific aromatic acylamino acids. Various types of aminoacylases are known, but most of them show high decomposition activity for aliphatic acylamino acids, and some that decompose aromatic acylamino acids are of microbial origin. Only. The present inventors have developed a method for measuring the activity of angiotensin-converted oxygen (Japanese Patent Publication No. 58-23080, etc.), which is used for the diagnosis of sarcoidosis, or a method for measuring the activity of carboxypeptidase A (Japanese Patent Application Laid-Open No. 59-1989).
In order to utilize aminoacylases in the production of aminoacylases (e.g., 85299), we conducted a wide search for microorganisms in order to obtain aminoacylases with high activity for decomposing aromatic acylamino acids, and first investigated Corynebacterium equi H-7. It was discovered that the strain produced aminoacylase with high decomposition activity for N-benzoyl-L-alanine (Japanese Patent Application Laid-open No. 1983-1998).
−86292). Further research led to the discovery that Pseudomonas putida (Pseudomonas putida No. C692-3) produces a novel aminoacylase that has high degrading activity for N-benzoylglycine (Japanese Patent Application No. 120255/1982). (Problems to be solved by the invention) The aminoacylase produced by Corynebacterium equi has a relatively low activity compared to the aminoacylase produced by Pseudomonas putida.
On the other hand, aminoacylase produced by Pseudomonas putida is unstable at alkaline pH of 7.5 to 9.0, so the above-mentioned angiotensin-converting enzyme and carboxypeptidase A whose optimal pH is on the alkaline side
It is not suitable as a conjugated enzyme for measuring the activity of (Means for Solving the Problems) Therefore, the present inventors extensively searched the natural world in order to obtain an aminoacylase that has a high activity of decomposing aromatic acylamino acids and is stable in the alkaline pH range. It was discovered that a different microorganism from the above-mentioned species produces an enzyme suitable for this purpose, and based on this finding, the present invention was completed. The physicochemical properties of the purified enzyme preparation of aminoacylase of the present invention are shown below. (1) Action This enzyme hydrolyzes N-benzoylglycine to produce benzoic acid and glycine. (2) Substrate specificity Table 1 shows the results of enzyme activity measurements using N-acyl derivatives of various amino acids as substrates. The enzyme activities in the table are expressed as relative activities with the activity against N-benzoylglycine as 100.
As shown in this table, this enzyme decomposes N-benzoylglycine and N-benzoyl-L-alanine well, and has a particularly high activity of decomposing N-benzoylglycine, and a high activity of degrading N-acetylglycine. This is less than 1/100 of the activity that decomposes benzoylglycine.

[Table] (3) Optimal PH and stable PH range Using N-benzoylglycine as a substrate,
0.1M acetate buffer PH4.0-5.5 (triangle) 0.1M phosphate buffer PH6.0-7.5 (black circle), 0.2M Tris-HCl buffer 7.5-8.5 (white circle) and 0.1M carbonate buffer PH9.0
~10.0 (square) was used to react at 37°C for 10 minutes, and the optimum pH was measured. The results are shown in Figure 1. As is clear from the figure, the optimal pH of this enzyme is 8.0 to 8.5.
It is in. Next, add this enzyme to 0.1M acetate buffer with pH 4.0 to 5.0.
(triangle), 0.1M phosphate buffer PH5.5-8.5 (black circle),
0.2M HEPES buffer PH6.0~10.0 (white circle) and
Fig. 2 shows the results of heating at 37°C for 1 hour in 0.1M carbonate buffer pH 9.0 to 10.0 (squares) and measuring the remaining enzyme activity. This enzyme has a pH of 6.5~
It is most stable in the range of 8.5. On the other hand, FIG. 3 shows the results of similarly measuring the PH stability of aminoacylase produced by Pseudomonas putida. As shown in the figure, this enzyme is most stable near pH 7.0. (4) Measurement method for titer Add 0.45 ml of 100 mM sodium phosphate buffer (PH8.0) to 0.05 ml of enzyme solution, preheat at 37°C for 5 minutes, then add 1.0 ml of 30 mM N-benzoylglycine solution. Incubate at ℃ for 10 minutes. Add 1.0 ml of trichloroacetic acid to the reaction solution to stop the reaction, and quantify the produced glycine using the ninhydrin method. Quantification using the ninhydrin method was performed by Moore et al. (S.Moore and WHStein, J.Biol.Chem.,
vol.211, P907 (1954)) as follows. That is, the reaction solution is centrifuged, 0.2 ml of ninhydrin reagent is added to 1.0 ml of the supernatant, heated in a boiling water bath for 15 minutes, and 2.0 ml of 50% ethanol is immediately added. After cooling with water, compare the colors at 575nm,
The amount of glycine produced was quantified from the glycine standard curve obtained in the same manner. For enzyme activity, one unit was the amount of enzyme that produced 1 μmol of glycine per minute. (5) Conditions for inactivation due to pH, temperature, etc. As shown in Figure 2, this enzyme is almost completely inactivated by treatment at 37℃ for 1 hour at pH below 4 in acetate buffer and pH 10 or above in carbonate buffer. do. Furthermore, the activity is approximately halved at pH 5.5 in phosphate buffer and pH 9.0 in carbonate buffer. Next, this enzyme was heated in 0.1M sodium phosphate buffer pH7.0 at each temperature for 30 minutes, and the residual activity was measured. The results are shown in FIG. As shown in the figure, this enzyme is not inactivated by heating at 40°C for 30 minutes, but 80% is inactivated by heating at 50°C for 30 minutes.
It is almost completely inactivated after 30 minutes of treatment. This enzyme and the aminoacylase produced by Pseudomonas putida were each adjusted to 12U/ml.
HEPES with 10mM 4-aminoantipyrine
-(N-2-hydroxyethylpiperazine-N'-2
-ethanesulfonic acid) buffer (PH7.8). This was once lyophilized, immediately reconstituted with water and stored at 6°C, and changes in activity over time were measured.
The results obtained are shown in FIG. In the figure, the black circles represent the present enzyme, and the white circles represent the enzyme of Pseudomonas putida. (6) Inhibition, activation, and stabilization The effects on the activity of this enzyme when various metal ions, metal chelating agents, and SH inhibitors were added to the reaction solution at the final concentrations shown in Table 2 were evaluated. They are summarized in Table 2. The relative activity in this table is expressed with the activity in the absence of additives as 100.

[Table] (7) Purification method After culturing, the enzyme solution extracted from the bacterial cells was diluted with 0.01M
The enzyme is adsorbed on a diethylaminoethylcellulose column equilibrated with a sodium phosphate buffer, eluted with the same buffer containing 0.2M sodium chloride, and the active fraction is collected. Add ammonium sulfate to this active fraction to a saturation of 0.25 and remove the precipitate. Ammonium sulfate is further added to the supernatant to a saturation of 0.35, and the precipitate formed is collected and dialyzed against the same buffer overnight. This dialysate is passed through a hydroxyapatite column equilibrated with the same buffer to adsorb the enzyme, and eluted with 0.2M sodium phosphate buffer (PH7.0) to obtain a purified enzyme preparation. get. (8) Molecular weight The molecular weight determined by gel filtration method was 170,000. (9) Isoelectric point As a result of measurement using Ampholine (PH3-10) (manufactured by LKB), the isoelectric point was 4.4. (10) Electrophoresis Davis method (Ann.NMAcad.Sci.,
vol.21, P404.1964) using a standard acrylamide gel disc, 50mM Tris-glycine buffer (PH8.3), 3m per column.
Migration was performed at a constant current of A. As a result, the relative mobility of this enzyme to bromophenol blue was 0.65. Among the above-mentioned physicochemical properties, this enzyme differs from conventional aminoacylases in substrate specificity. That is, most of the conventional aminoacylases show high decomposition activity only for aliphatic acylamino acids, and only a few of microbial origin are known to decompose aromatic acylamino acids. And anything that decomposes this aromatic acylamino acid also decomposes acyl-D-amino acid. On the other hand, this enzyme exhibits high degrading activity for aromatic acylamino acids such as N-benzoylglycine, but it also exhibits high degrading activity for aromatic acylamino acids such as N-acetylglycine and acyl-D-amino acids such as N-benzoyl-D-alanine. Degrading activity for amino acids is low. Next, among the enzymes already applied by the present inventors, the enzyme produced by Corynebacterium equi has a high degrading activity against aromatic acylamino acids, and this enzyme is particularly effective against N-benzoyl-L-alanine. It has a high decomposition activity, and its activity against N-benzoylglycine is not very large. However,
This enzyme has particularly high degrading activity for N-benzoylglycine among aromatic acylamino acids. On the other hand, the enzyme produced by Pseudomonas putita has the above properties in common with this enzyme, but it has poor stability on the alkaline side of pH 7.5 to 9.0.
For example, if heated for 1 hour at pH 8.5 and 37°C, the residual activity will be less than 50. However, this enzyme is stable even in alkaline conditions, and is 50% stable under the above conditions.
The difference is that the above activities remain. Therefore, we concluded that this enzyme is novel. The enzyme of the present invention can be produced, for example, from the microorganism Pseudomonas sp. No. C684-2, which the present inventors isolated from a clay pot in Okinawa Prefecture. This Pseudomonas sp. No. C684-2 has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as FERM P-7716. The mycological properties of Pseudomonas sp. No. C684-2 are shown below. 1 Morphology (1) Cell shape and size The cells are rod-shaped (0.8-1.0 x 1.3-1.6 μm) and do not show pleomorphism. (2) Motile (3) Spores: Not formed (4) Gram staining: Negative 2 Growth status in various media (1) Broth agar plate culture Colonies are disc-shaped, with whole edges, translucent, and soft. , the surface is shiny. No soluble pigments are produced. Colony size is 1.2-1.5 in diameter
mm. (2) Juicy agar slant culture Grows well in a linear shape and is soft. The surface is shiny. It also grows well in condensed water. No soluble pigments are produced. (3) Meat juice liquid culture Grows well and becomes turbid. Does not produce sediment. No bacterial membrane is formed. (4) Meat juice gelatin puncture culture Grows well in the upper layer. Does not liquefy. Does not produce sediment. (5) Litmus milk: Does not coagulate or peptonize. PH is slightly alkaline. 3 Physiological properties (1) Nitrate reduction: Negative (2) MR test: Negative (3) VP test: Negative (4) Indole production: Negative (5) Hydrogen sulfide production: Negative (on SIM medium) (6 ) Starch hydrolysis: Negative (7) Utilization of citrate: Koser medium: Positive Christensen medium: Positive (8) Pigment production: Not produced in KingA medium. KingB medium produces a yellow fluorescent dye. (9) Oxidase: positive (10) Catalase: positive (11) Attitude towards oxygen: aerobic (12) OF test: oxidative (13) Production of acids and gases from sugars D-glucose, D-galactose, L-
Acid is produced from arabinose, D-xylose, D-fructose, D-mannose and glycerol. No acids are produced from maltose, trehalose, sucrose, inositol, D-mannitol, lactose, D-sorbitol and soluble starch. No gas formation is observed. The above properties are described in Bergey's Manual of Determinative Bacteriology.
Bacteriology) 8th edition, 1974 edition, and as a result, this bacterium was identified as a microorganism belonging to the genus Pseudomonas, as the properties of this bacterium closely matched those of microorganisms of the genus Pseudomonas. A method for culturing such microorganisms to produce the enzyme of the present invention can be carried out according to a general method for culturing microorganisms. That is, a culture medium containing a carbon source, a nitrogen source, inorganic salts, other nutritional substances, etc. is used. As a carbon source, sugars such as glucose and maltose, and alcohols such as glycerol can be used, but inducing substrates of this enzyme such as hippuric acid and N-benzoylalanine are particularly effective. The nitrogen source may be inorganic such as ammonium sulfate, ammonium chloride, or sodium nitrate, or organic such as yeast extract, meat extract, or peptone. In addition to sodium chloride, common inorganic salts such as monopotassium phosphate, disodium phosphate, and magnesium sulfate can be used as the inorganic salts. As a culture method, liquid culture is usually preferred, at 15 to 37°C, preferably 25 to 35°C, and pH 5 to 8.
Culture under aerobic conditions for 10-50 hours. Through this culture, most of the enzyme is accumulated within the bacterial cells. After completion of the culture, the culture may be used as it is as an enzyme source, but separation and purification are usually performed. As for the isolation method, the bacterial cells are first separated by a conventional method such as centrifugation, and the bacterial cells are destroyed by grinding, lysis with a lytic enzyme such as lysozyme, ultrasonication, pressure shock method, autolysis method, etc. Extract the enzyme. The destroyed bacterial cell residue is separated to obtain an enzyme extract, which is then subjected to salting-out method using ammonium sulfate, etc., solvent precipitation method using acetone, ethanol, etc., gel filtration method using Sephadex, Sepharose gel, etc., adsorption using ion exchange resin, etc. Purification is performed using an appropriate combination of methods to obtain an enzyme preparation of the desired purity. (Action and Effect of the Invention) The enzyme of the present invention contains N-benzoylglycine and N-benzoylglycine.
It is characterized by being able to degrade -benzoyl-L-alanine well and being stable even in alkaline conditions, and is useful in methods for measuring angiotensin-converting enzyme activity and carboxypeptidase A activity. (Example) 90 volumes of a medium 60 with a pH of 7.0 consisting of 1% hippuric acid, 0.3% yeast extract, 0.2% sodium nitrate, 0.1% dipotassium phosphate, 0.05% magnesium sulfate, and 0.05% sodium chloride. It was placed in a mentor and sterilized at 120°C for 20 minutes. After cooling, Pseudomonas sp. No. 684-2 (FERM P-
7716) and cultured with aeration at 28°C for 10 hours. The amount of ventilation during this time was 0.5vvm, and the rotation speed of the stirring blade was 200rpm. After the culture was completed, the culture solution was centrifuged to obtain about 1 kg of wet bacterial cells. This bacterial body
Suspend in 10mM sodium phosphate buffer (PH7.0) 3 and use Dyno Mill (KDL type).
Bacterial cells were disrupted at a flow rate of 3000 rpm and 30 ml/min. The bacterial cell residue was removed by centrifugation to obtain enzyme extract 3.5.
Diethylaminoethylcellulose 1 equilibrated with 10mM sodium phosphate buffer was added to this enzyme extract.
Kg was added and stirred at 6°C for 2 hours to adsorb the enzyme. This diethylaminoethyl cellulose was thoroughly washed with the same buffer, and the enzyme was eluted with the same buffer containing 0.2M sodium chloride. Ammonium sulfate was added to the eluate to give a saturation of 0.25, the resulting precipitate was removed, and ammonium sulfate was added to the supernatant to give a saturation of 0.35, and the resulting precipitate was collected. This precipitate was dialyzed against the same buffer overnight.
The dialysate was adsorbed onto a hydroxyapatite column equilibrated with the same buffer, and eluted with 0.2M sodium phosphate buffer (PH7.0). Ammonium sulfate was added to the eluted fraction to saturation of 0.35, and the resulting precipitate was collected and dialyzed against the same buffer overnight to obtain a purified sample with a specific activity of 150 U/mg in a yield of 27%.

[Brief explanation of the drawing]

FIG. 1 is a curve showing the optimum pH of the enzyme obtained in the Examples, and FIG. 2 is a curve showing the PH stability. FIG. 3 is a curve showing the PH stability of the enzyme of the previous application obtained by measurement under the same conditions. FIG. 4 is a curve showing the thermostability of the enzyme obtained in the example,
Figure 5 shows the pH of the enzyme obtained in the example and the enzyme of the earlier application.
7.8 shows the results of comparing storage stability.

Claims (1)

[Claims] 1. An aminoacylase having the following physical and chemical properties. Action: Hydrolyzes N-benzoylglycine to produce benzoic acid and glycine. Substrate specificity: specifically decomposes N-benzoylglycine and N-benzoyl-L-alanine, but
The activity against the former is higher than the activity against the latter, and the activity against N-acetylglycine and N-benzoyl-D-alanine is less than 1/100 of the activity against N-benzoylglycine. Optimum PH: PH8.0-8.5 Stable PH: PH6.5-8.5 Suitable temperature for action: Approximately 37°C Inactivation conditions: Almost completely inactivated at PH of less than 4 and PH of 10 or more. Inhibition, activation and stabilization: α,α′-dipyridyl 1
8-hydroxyquinoline, monoiodoacetic acid, p-chloromercuric benzoic acid,
1m each of Hg ⁺⁺ , Cu ⁺⁺ , Ni ⁺⁺ , Mn ⁺⁺ and Ba ⁺⁺
It is inhibited in M solution. Molecular weight: The molecular weight determined by gel filtration method is 170,000 Daltons. Isoelectric point: 4.4.