JP3471301B2 - Cyclohexanecarboxylic acid phenyl ester hydrolase and method for producing the same - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a cyclohexane catalyst.
Rubonic acid phenyl ester hydrolase and bacterial cells
And a method for producing the enzyme.

[0002]

2. Description of the Related Art The presence of aryl esterases is known as a phenolic ester hydrolase. For example, Clin. Chim. Acta, 7
Volume 560 (1962), human blood arylesterases and biokimica biophysical actors (Bioc
him. Biophys. Acta), 276, page 180 (1972), and the like are known.

[0003]

However, these aryl esterases are those purified from animal tissues, and the productivity of the enzyme is low, and it is difficult to industrially provide the enzyme.

Further, when these aryl esterases are used, cetraxate and liquid crystalline ester compounds which are important as pharmaceuticals cannot be produced.

[0005]

Means for Solving the Problems As a result of intensive studies to solve the above-mentioned conventional drawbacks, the present inventors have found that cyclohexanecarboxylic acid derived from a microorganism that strongly hydrolyzes a phenolic ester and has high enzyme productivity. A method for efficiently producing a phenyl ester hydrolase can be found, and the present invention has been completed.

That is, the present invention has the following physicochemical properties:
Hydrolysis of cyclohexanecarboxylic acid phenyl ester
It is an enzyme. [Physical and chemical properties] Action: In the presence of water, 1 mol of cetraxate
1 mol of tranexamic acid and 1 mol of hydroxypheny
Produces lupropionic acid. Substrate specificity: N-acetylcetraxate, tiger
Phenyl nexamic acid, cetraxate, 4- (2-cal
Boxyethyl) phenylcyclohexanecarboxylate
Acts on hexyl tranexamate,
For myristyl samnate and methyl tranexamate
I don't show you. Optimum pH: pH 8.0 to 9.0 pH stability: When stored at 4 ° C. for 24 hours,
80% or more residual activity at pH 6.0 to 11.0
Have. Optimum temperature: 0.1M phosphate buffer (pH
55-60 ° C in 7.5) Temperature stability: 0.
01M phosphate buffer (pH 7.5) at each temperature
90% or more residual activity at 50 ° C or below when held for 5 minutes
Have.

Another invention belongs to the genus Pseudomonas.
Cyclohexanecarboxylic acid phenyl ester water content
Is it a culture obtained by culturing cells having the ability to produce degrading enzymes?
It collects from the cyclone according to claim 1.
Production of xanthylcarboxylic acid phenyl ester hydrolase
Is the way.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, cyclohexanecarboxylic acid phenyl ester means cyclohexanecarboxylic acid phenyl ester and derivatives thereof, and specifically, for example, 4- (2-carboxyethyl) phenyl trans-4 -Aminomethylcyclohexanecarboxylate [commonly referred to as cetraxate], phenyl tranexamate, N-acetylcetraxate, 4- (2
-Carboxyethyl) phenylcyclohexanecarboxylate and the like.

In the present invention, the above cyclohexane calc
Manufacture an enzyme that hydrolyzes boric acid phenyl ester
However, at this time, as a microorganism, Pseudomonas pertussi
Nogena IFO 14163 (Pseudomonas pertucinogena),
Iudomonas aeruginosa IFO 3080 (Pseudomonas ae
ruginosa) and other Pseudomonas genera
Cyclohexanecarboxylic acid phenyl ester water content
A microorganism having a degrading enzyme producing ability is used.

In the present invention, these Pseudomonas
(Pseudomonas) belonging to the genus cyclohexanecarboxylic acid phenyl ester hydrolase culturing cells, isolate the cells from the culture, and extract cyclohexanecarboxylic acid phenyl ester hydrolase from the isolated cells To do.

The culture of these cells in the present invention can be carried out using a medium. The medium used for culturing the cells is not particularly limited. For example, carbon sources such as glucose, sugar-tight, soluble starch and the like, nitrogen sources such as meat extract, yeast extract, polypeptone and other nitrogen sources, inorganic salts such as potassium dibasic phosphate, dibasic potassium phosphate, sodium chloride, magnesium sulfate. Any combination of inorganic salts such as ammonium sulfate and the like may be used.

As for the culture conditions, the culture temperature is in the range of 15 to 40 ° C., preferably 25 to 40 ° C. under aeration and stirring conditions.
A method of culturing in the range of 35 ° C is suitable. P during culture
The H condition is in the range of 4.0 to 9.0, preferably pH.
The range of 5.0 to 8.0 is suitable. The culturing time is not particularly limited, but in view of economic efficiency such as enzyme productivity, it is appropriate that the culturing time is within a range of 10 hours from the time of reaching the latter stage of growth to the resting state. Cyclohexanecarboxylic acid phenyl ester hydrolase is accumulated both in the culture medium and on the culture solution side.

As a method for separating the culture solution and the bacterial cells from the culture obtained by culturing, conventionally used methods such as centrifugation and filtration can be used, but the method of centrifugation is preferred. is there.

In the present invention, the cyclohexanecarboxylic acid phenyl ester hydrolase accumulated in the separated bacterial cells is extracted from the bacterial cells.
Use conventional methods such as ultrasonic cell disruption, ultrasonic cell disruption using a Dynomill disruptor that rotates with glass beads, or disruption of cell membranes by enzymes such as lysozyme and organic solvents such as toluene. You can A solution (also referred to as a crude enzyme solution) containing the enzyme can be obtained by selecting an appropriate method from these and extracting the enzyme from the bacterial cell.

When it is necessary to further purify the cyclohexanecarboxylic acid phenyl ester hydrolase from the crude enzyme solution extracted by these methods, the ammonium sulfate precipitation method, which is a commonly practiced enzyme purification method, is used. , Ion exchange column chromatography method, gel filtration method, hydroxyapatite column chromatography method, affinity chromatography method, hydrophobic column chromatography method, preparative electrophoresis method, etc. are appropriately combined or repeated for purification. You can do it.

Depending on the type of cells used, cyclohexanecarboxylic acid phenyl ester hydrolase can be separated from components other than the cells of the culture. In this case, the culture solution is subjected to a conventional enzyme purification method. The method used for the above, for example, treatment such as salting out and dialysis may be performed.

The cyclohexanecarboxylic acid phenyl ester hydrolase thus obtained has the following characteristics:
It has specific physicochemical properties. The enzyme is, for example, an enzyme producing
Highly active Pseudomonas pertussinogena (IFO14163)
Produced by.

[ Physical and Chemical Properties ] In the presence of working water, 1 mol of tranexamic acid and 1 mol of hydroxyphenylpropionic acid are produced from 1 mol of cetraxate. Substrate Specificity Phenyl tranexamate, cetraxate, N-acetylcetraxate, 4- (2-carboxyethyl) phenylcyclohexanecarboxylate, acting on hexyl tranexamate, myristyl tranexamate,
It has no effect on methyl tranexamate. The strength of the action on these esters is shown in Table 1 as a relative activity value with the action on cetraxate being 100.

[0019]

[Table 1]

Optimum pH: pH 8.0 to 9.0 ( Fig. 1
PH stability: pH when stored at 4 ° C. for 24 hours
It has a residual activity of 80% or more at 6.0 to 11.0. ( Shown in FIG. 2 ) Optimum temperature: 55 ° C. to 60 ° C. in 0.1 M phosphate buffer (pH 7.5) ( shown in FIG. 3 ) Temperature stability: 0.01 M phosphate buffer (pH 7.
In 5), when it is kept at each temperature for 5 minutes, it has a residual activity of 90% or more at 50 ° C. or less. ( Shown in FIG. 4 ) Molecular weight: 220,000 ± 20,000 {(trade name: HPLC column TSK-gel G3000SWXL manufactured by Tosoh Corporation) by gel filtration method} ( shown in FIG. 5 ) Km value: 0.1 M Tris -Hydrochloric acid buffer (pH 8.
The Km value for cetraxate under condition 0) is 0.5M. Inhibitors Various reagents and metal ion concentration 1 mM or 0.1 m
The effect of M on the enzyme of the present invention is shown in Table 2 . It can be seen from Table 2 that the enzyme of the present invention is strongly inhibited by mercury ions.

[0021]

[Table 2]

[0022]

EXAMPLES The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples.

The method for measuring the activity of the cyclohexanecarboxylic acid phenyl ester hydrolase and the method for displaying the enzyme activity value in each example are as follows.

[Measurement of Hydrolytic Activity of Cyclohexanecarboxylic Acid Phenyl Ester] 2.0 mM cetraxate solution (in 0.1 M citrate buffer (pH 6.0) 0.5 ml, enzyme solution 0.1 ml, and distilled water 0.4 ml) After thoroughly stirring 1.0 ml, the mixture was incubated at 37 ° C. for 1 hour to prepare a reaction solution. Immediately add 50 to this reaction mixture.
% 0.1 ml of trichloroacetic acid was added, and the mixture was allowed to stand for 15 minutes under ice cooling and then centrifuged to collect 0.55 ml of the supernatant. To 0.55 ml of the deproteinized supernatant, 0.5 ml of 0.5 M sodium acetate and 0.45 ml of distilled water were added, and the mixture was stirred and used as a sample for HPLC analysis. As a blank, the enzyme solution was replaced with distilled water and the same operation was performed.
HPLC analysis column is Shim-pack CLC-ODS (6.0mmφ × 150m
m) (trade name: manufactured by Shimadzu Corporation) was used. Further, the detection of tranexamic acid was carried out by a fluorescence detection method. This HPLC
If the concentration of tranexamic acid in the enzyme solution system (E μmol / l) and the concentration of tranexamic acid in the blank system (B μmol / l) measured by the analysis are T μmol / l, the concentration of tranexamic acid produced by the enzymatic reaction is T μmol / l. = Eμmol / l
-B μmol / l. The enzyme activity value was calculated from the concentration of tranexamic acid by the following calculation formula (1).

Enzyme activity calculation formula: If the concentration of the produced tranexamic acid is T μmol / l, the enzyme activity value (u / ml) per 1.0 ml of the enzyme solution is: enzyme activity value = (3/1000) × (1/60 ) × (1 / 0.1) × T
(1) However, the enzyme activity unit u (unit) was defined as the amount of enzyme capable of producing 1 μmol of tranexamic acid per minute.

The analytical conditions of the HPLC for the determination of tranexamic acid were as follows.

[HPLC analysis conditions] 1. Equipment 1) Liquid chromatograph main unit: Waters 600E 2) Column constant temperature chamber (50 ° C): Shimadzu CTO-6A 3) Reaction liquid feed pump: Waters 510 4) Fluorescence detector: Shimadzu RF -535 5) Recorder and data processing ・ ・ ・ Shimadzu C-R4A 6) Autosampler ・ ・ ・ Waters 700 7) Reaction tank (50 ℃) ・ ・ ・ Shimadzu CRB-6A 8) UV detector ・ ・ ・ Waters 484 2 ． Column ・ ・ ・ Shimadzu Shim-pack CLC-ODS (6.0mm
φ × 150mm) 3. Mobile phase solution I 0.1 M sodium perchlorate 10 mM sodium hexanesulfonate (pH 4.0) solution II solution I 25% methanol 75% (pH 3.0) Concentration gradient elution method using solution I and solution II Reaction liquid Brij-35 ・ ・ ・ 0.4g Disodium carbonate ・ ・ ・ 40.0g Boric acid ・ ・ ・ 13.4g Potassium sulfate ・ ・ ・ 18.4g Orthophthalaldehyde / 12ml Ethanol ・ ・ ・ 0.80g β-Mercaptoethanol ・ ・ ・2.0 ml (pH 10.5) 5. Flow rate: Mobile phase: 1.1 ml / min Reaction solution: 0.4 ml
/ min.

Example 1 1.0% polypeptone, 0.5% yeast extract, 0.5% salt, 0.0
1,000 ml of medium (pH 7.2) consisting of 1% defoamer was placed in a 5 l Erlenmeyer flask and autoclaved at 120 ° C for 15 minutes, and then Pseudomonas pertucinogena (IFO14163) was inoculated into this medium at 30 ° C. To do. After shaking culture at 30 ° C for 24 hours, this culture solution was
170 liters of a medium having the same composition as above was charged in advance and inoculated into a steam-sterilized jar fermenter for main culture.

After culturing for 22 hours, the hydrolytic activity of cyclohexanecarboxylic acid phenyl ester in the culture was measured and found to be 0.03 unit / ml-culture.

The culture was centrifuged to collect bacterial cells. 100 g (wet cell weight) of the obtained bacterial cells was added to 1 L of 0.01M
The cells were suspended in a phosphate buffer (pH 7.5), and the suspension was continuously passed through a Dynomill cell disruptor to disrupt the cells. The disrupted liquid was centrifuged using a centrifuge to obtain a supernatant liquid. The total activity of cyclohexanecarboxylic acid phenyl ester hydrolase in this supernatant was 352 units, and the specific activity was 0.009 units / mg-protein. This supernatant was added to 0.01M phosphate buffer (pH 7.5) in advance.
The column (9.0 × 30 cm) packed with the DE52 ion exchange resin (trade name: manufactured by Whatman) equilibrated in 1. was loaded.
After thoroughly washing the column with 2 l of 0.01 M phosphate buffer (pH 7.5), the adsorbed protein was eluted with a linear concentration gradient (total volume: 16 l) with a salt concentration of 0.1 M to 0.5 M,
The cyclohexanecarboxylic acid phenyl ester hydrolysis active fraction was collected. The total activity of cyclohexanecarboxylic acid phenyl ester hydrolase in this active fraction was 230
The unit and specific activity were 0.04 unit / mg-protein.

The eluate was desalted and concentrated by ultrafiltration,
Slowly mix saturated ammonium sulfate solution under ice cooling
Add until 20% saturated ammonium sulphate solution. After standing for about 1 hour, centrifugation was performed. This supernatant was similarly used as a 50% saturated ammonium sulfate solution. After standing for about 1 hour, centrifugation was performed. This precipitate was ice-cooled 0.01M
100 ml of phosphate buffer (pH 7.5) was added and dissolved. This solution was desalted by ultrafiltration to obtain a cyclohexanecarboxylic acid phenyl ester hydrolyzing active fraction. The total activity of cyclohexanecarboxylic acid phenyl ester hydrolase in this active fraction was 210 units, and the specific activity was 0.18 unit / mg.
-It was protein.

Next, 0.01 M phosphate buffer (p
The sample was passed through a column (3.2 cm 28 cm) packed with DEAE-Toyopearl (trade name: manufactured by Tosoh Corporation) equilibrated with H7.5). 1
After washing the column with 1 L phosphate buffer (pH 7.5) containing 0.1 M sodium chloride, the adsorbed protein was eluted with a linear concentration gradient (total volume: 6 L) with a salt concentration of 0.1 M to 0.35 M. After that, a cyclohexanecarboxylic acid phenyl ester hydrolysis active fraction was collected. The total activity of cyclohexanecarboxylic acid phenyl ester hydrolase in this active fraction was 182 units, and the specific activity was 0.42 units / mg-protein.

This active fraction was desalted and concentrated by using an ultrafiltration device, and then 0.1M sodium chloride was added in advance.
Sephacryl S equilibrated with 01M phosphate buffer (pH 7.5)
A column (2.2 cm 120 cm) packed with -300 (trade name: Pharmacia) was loaded and gel filtration was performed. The cyclohexanecarboxylic acid phenyl ester hydrolysis active fraction was collected. The total activity of cyclohexanecarboxylic acid phenyl ester hydrolase in this active fraction was 142 units,
The specific activity was 4.73 units / mg-protein. This active fraction was collected, desalted with an ultrafiltration device, and then used as a 20% saturated ammonium sulfate solution. This was loaded onto a column (2.2 cm 20 cm) packed with phenyl sepharose CL-4B (trade name: Pharmacia) equilibrated with 0.01 M phosphate buffer (pH 7.5) containing 20% saturated ammonium sulfate in advance. After washing the column with 1 L of 0.01 M phosphate buffer (pH 7.5) containing 20% saturated ammonium sulfate, the ammonium sulfate concentration was 20%.
The adsorbed protein was eluted with a linear concentration gradient (total volume: 2 l) of 0 to 0%, and the cyclohexanecarboxylic acid phenyl ester hydrolyzing active fraction was collected. The total activity of cyclohexanecarboxylic acid phenyl ester hydrolase in this active fraction was 127 units, and the specific activity was 12.5.
Unit / mg-protein. Then, after desalting with an ultrafiltration device, it was passed through a column (2.0 x 20 cm) packed with DEAE-Toyopearl (trade name: manufactured by Tosoh Corporation) that had been equilibrated with 0.01 M phosphate buffer (pH 7.5) in advance. did. 300 ml
After washing the column with a phosphate buffer (pH 7.5) containing 0.15M sodium chloride, elute the adsorbed protein with a linear concentration gradient (total volume: 2 liters) with a salt concentration of 0.15M to 0.35M. The cyclohexanecarboxylic acid phenyl ester hydrolysis active fraction was collected to obtain a purified enzyme solution. The total activity of cyclohexanecarboxylic acid phenyl ester hydrolase in this purified enzyme solution was 102 units, and the specific activity was 18.3.
Unit / mg-protein.

As a result of examining the purity of the thus obtained enzyme by polyacrylamide gel electrophoresis, only one band was observed and it was confirmed that the enzyme was pure cyclohexanecarboxylic acid phenyl ester hydrolase. Using the obtained purified enzyme, the substrate specificity, optimum pH, pH stability, optimum temperature, temperature stability and molecular weight of this enzyme were investigated.

[Substrate Specificity] Various substrates shown in Table 1 were dissolved in 50 mM phosphate buffer to a concentration of 2 mM. 0.1 ml of the enzyme solution was added to 0.9 ml of this substrate solution, stirred and reacted at 37 ° C. for 5 minutes. The subsequent treatment was the same as the usual enzyme activity measuring method. However, for each reaction, the one prepared by adding distilled water instead of the enzyme solution was prepared and operated in the same manner. The analysis after the reaction was a usual HPLC analysis. After the analysis, the enzymatic activity was calculated by subtracting the produced amount of tranexamic acid or hydroxyphenylpropionic acid in the distilled water system from the produced amount of tranexamic acid or hydroxyphenylpropionic acid in the enzyme solution system. The results at that time show that the activity when using cetraxate as a substrate is 100
The relative activity is shown in Table 1 above .

[Optimal pH] The purified enzyme preparation was diluted with distilled water to about 0.1 u / ml to prepare a sample. The pH of the enzyme reaction was adjusted using the buffer solution shown below. The enzymatic reaction was performed by a usual method. However, the reaction time was 30 minutes, and for each reaction, one prepared by adding distilled water instead of the enzyme solution was prepared and operated in the same manner. The analysis after the reaction was a usual HPLC analysis. After analysis, the enzymatic activity was calculated by subtracting the amount of tranexamic acid produced in the distilled water system from the amount of tranexamic acid produced in the enzyme solution system.

Various buffers used in the reaction system (all buffers were 0.1 M) Citrate buffer: pH 2.5,3.0,3.5,4.0,4.5,5.0,5.5,6.0,
6.5 phosphate buffer: pH: 6.1,6.6,7.1,7.5,8.0 Tris buffer: 7.4,8.0,8.5,9.0 glycine buffer: 8.3,8.6,9.0,9.4,9.7,10.9,11.8 figure results of this Shown in 1 .

[PH stability] A fixed amount of the purified enzyme preparation was placed in each buffer and stored at 4 ° C for 24 hours. Thereafter, the residual activity of the enzyme was measured by a usual enzyme activity measuring method. However, the reaction time was 30 minutes.

Various buffers used for storage (all buffers are
It was set to 0.1M. ) Citrate buffer: pH2.5,3.0,3.5,4.0,4.5,5.0,5.5,6.0,
6.5 phosphate buffer: pH: 6.1,6.6,7.1,7.5,8.0 Tris buffer: 7.4,8.0,8.5,9.0 glycine buffer: 8.3,8.6,9.0,9.4,9.7,10.9,11.8 figure results of this 2 shows.

[Optimum temperature] The purified enzyme preparation was diluted with distilled water to about 0.1 u / ml to prepare a sample. The enzymatic reaction was performed by a usual method. However, the reaction temperatures were 20, 30, 40, 50, 55, 60, 70, 80 and ° C, respectively. The reaction time was 5 minutes. As a control for each reaction, distilled water was added instead of the enzyme solution, and the same operation was performed. The analysis after the reaction was a usual HPLC analysis. After analysis, the enzymatic activity was calculated by subtracting the amount of tranexamic acid produced in the distilled water system from the amount of tranexamic acid produced in the enzyme solution system. The result at this time is shown in FIG .

[Temperature Stability] The enzyme-purified preparation was subjected to 20, 30, 4 in 10 mM phosphate buffer (pH 7.5).
Hold at 0, 50, 55, 60, 80, ° C for 5 minutes. Immediately thereafter, it was cooled with ice. This sample is 10 mM phosphate buffer (pH 7.5)
It was diluted 50-fold with and used as a sample for measuring residual enzyme activity. The enzyme activity was measured by a usual method. However, the reaction time
30 minutes. The result at this time is shown in FIG . The molecular weight was measured by the molecular weight gel filtration method. The column used was an HPLC column TSK-gel G3000SWXL. As the mobile phase, 50 mM phosphate buffer (pH 7.1) and 0.2 M sodium sulfate were used.
The flow rate was 0.5 ml / min. The marker protein used was that shown in FIG . HPLC detection is performed at UV280 nm,
The elution position of the standard was also measured for its enzyme activity. The results are shown in Fig. 5 .

[0042]

EFFECTS OF THE INVENTION According to the production method of the present invention, a cyclohexanecarboxylic acid phenyl ester hydrolase which can be suitably used for hydrolysis of phenolic esters such as cetraxate or for synthesis (using an inverse enzyme reaction) is obtained. It becomes possible to industrially produce a solution containing the same.

[Brief description of drawings]

FIG. 1 is a view showing a pH activity curve of a cyclohexanecarboxylic acid phenyl ester hydrolase of Example 1.

FIG. 2 is a diagram showing pH stability of the enzyme.

FIG. 3 is a view showing a temperature activity curve of the same enzyme.

FIG. 4 is a view showing temperature stability of the enzyme.

FIG. 5 is a view showing a molecular weight of the enzyme. (●: enzyme of Example 1, ◯: marker protein)

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ identification code FI C12R 1:38) (58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 9/00-9/99 C12N 1 / 00-1/38 BIOSIS / MEDLINE / WPID S (STN)

Claims (2)

(57) [Claims] 1. Cyclohexa having the following physicochemical properties :
Carboxylic acid phenyl ester hydrolase . [Physical and chemical properties] Action: In the presence of water, 1 mol of cetraxate
1 mol of tranexamic acid and 1 mol of hydroxypheny
Produces lupropionic acid. Substrate specificity: N-acetylcetraxate, tiger
Phenyl nexamic acid, cetraxate, 4- (2-cal
Boxyethyl) phenylcyclohexanecarboxylate
Acts on hexyl tranexamate,
For myristyl samnate and methyl tranexamate
I don't show you. Optimum pH: pH 8.0 to 9.0 pH stability: When stored at 4 ° C. for 24 hours,
80% or more residual activity at pH 6.0 to 11.0
Have. Optimum temperature: 0.1M phosphate buffer (pH 7.5)
55-60 ° C temperature stability: 0.01M phosphate buffer (pH 7.
In 5), when kept at each temperature for 5 minutes, 50 ° C or less
Has a residual activity of 90% or more. 2. Cyclohexa belonging to the genus Pseudomonas
Carboxylic acid phenyl ester hydrolase production ability
To collect from the culture obtained by culturing the cells
The cyclohexanecarboxylic acid according to claim 1, characterized in that
Method for producing phenyl ester hydrolase .