JPH06153941A - New ester hydrolases i and ii and production thereof - Google Patents

Abstract

PURPOSE:To obtain a new ester hydrolases having high stereoselectivity. CONSTITUTION:A microorganism belonging to the genus Bacillus, especially Bacillus.licheniformis IFO12202 is cultured to produce new ester hydrolases I and II having high stereoselectivity. The enzymes can advantageously be used for resolving, e.g. a stereoisomeric mixture of esters.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel enzyme for selectively hydrolyzing an ester having a stereoisomer and a method for producing the same.

[0002]

It is widely known that stereoisomers exist in many biologically active compounds. The desired biological activity is usually predominantly in one stereoisomer, the other stereoisomer may not have the desired biological activity or may have undesired physiological effects, including toxicity. In the absence of suitable techniques for resolving stereoisomers of biologically active compounds with stereoisomers, mixtures of the stereoisomers are often used neat for agricultural and pharmaceutical applications. In this case, the biological activity of the mixture may be halved, or the mixture may have undesirable physiological effects including toxicity. In order to overcome this problem, various stereoisomer resolution methods have been investigated. As one of the methods, a method has been proposed in which various ester derivatives of stereoisomers to be resolved are prepared, an enzyme that stereoselectively hydrolyzes the ester is allowed to act, and separation is performed by the difference in the solubility of the reaction system in a solvent. . In order to realize this method, development of esterases having various stereoselectivities is desired.

[0003]

Therefore, an object of the present invention is to provide a novel esterase having high stereoselectivity and a method for producing the same.

[0004]

Means for Solving the Problems For the purpose of industrial production of ester-degrading enzyme having high stereoselectivity, the present inventors searched for a source of the enzyme and searched for a strain producing the enzyme.・ Likeniformis ( Bacillus
licheniformis ) IFO 12202 has the ability to produce esterases with high stereoselectivity,
The present invention has been completed.

That is, the novel esterases of the present invention (designated as esterases I and II) have the following physicochemical properties.

[A] Ester Degrading Enzyme I (1) Action It has the ability to hydrolyze an ester under weakly acidic-alkaline conditions (pH 5 to 11.5). (2) Substrate specificity It shows high substrate specificity for 2-arylpropionic acid esters, but does not act on p-nitrophenyl esters of linear aliphatic organic acids. (3) Optimum pH Using (R, S) -2- {4- [2- (3-methyl) thienyl] phenyl} methyl propionate as a substrate, 30
When the reaction is carried out at ℃, the optimum pH is 10.5-11. (4) Stable pH range The residual activity after treatment for 16 hours at 30 ° C at various pHs was measured using methyl (R, S) -2- {4- [2- (3-methyl) thienyl] phenyl} propionate as a substrate. When measured, it is stable in the pH range of 7-10.5,
At pH 5 or lower and pH 11.5 or higher, marked deactivation is observed. (5) Optimum temperature (R, S) -2- {4- [2- (3-methyl) thienyl] phenyl} methyl propionate as a substrate, pH
When the reaction is carried out at 8.0, the optimum temperature is 35 ° C. (6) Temperature stability The residual activity after heat treatment at pH 8.0 and 40 ° C. for 10 minutes was measured using methyl (R, S) -2- {4- [2- (3-methyl) thienyl] phenyl} propionate as a substrate. 100% activity remains when measured as. (7) Effect of metal ions Ni ²⁺ , Fe ³⁺ , Zn ²⁺ , Al ³⁺ , Pb ²⁺ remarkably inhibits the activity. (8) Effects of inhibitors and activators The activity is specifically inhibited by DFP and PMSF. (9) Molecular weight about 100,000 (by gel filtration method)

[B] Ester Degrading Enzyme II (1) Action It has the ability to hydrolyze an ester under weakly acidic-alkaline conditions (pH 5 to 11.5). (2) Substrate specificity It shows high substrate specificity for 2-arylpropionic acid esters, but does not act on p-nitrophenyl esters of linear aliphatic organic acids. (3) Optimum pH Using (R, S) -2- {4- [2- (3-methyl) thienyl] phenyl} methyl propionate as a substrate, 30
When the reaction is carried out at ℃, the optimum pH is 11 to 11.5. (4) Stable pH range The residual activity after treatment at 30 ° C. for 16 hours at various pHs was measured using methyl (R, S) -2- {4- [2- (3-methyl) thienyl] phenyl} propionate as a substrate. When measured, it is stable in the pH range of 7-10.5,
At pH 5 or lower and pH 12 or higher, marked deactivation is observed. (5) Optimum temperature (R, S) -2- {4- [2- (3-methyl) thienyl] phenyl} methyl propionate as a substrate, pH
When the reaction is carried out at 8.0, the optimum temperature is 40 ° C. (6) Temperature stability The residual activity after heat treatment at pH 8.0 and 40 ° C for 10 minutes was measured using methyl (R, S) -2- {4- [2- (3-methyl) thienyl] phenyl} propionate as a substrate. 100% activity remains when measured as. (7) Effect of metal ions Ni ²⁺ , Fe ³⁺ , Zn ²⁺ , Al ³⁺ , Pb ²⁺ remarkably inhibits the activity. (8) Effects of inhibitors and activators The activity is specifically inhibited by DFP and PMSF. (9) Molecular weight about 25,000 (by gel filtration method)

According to the present invention, the novel esterases I and II belong to the genus Bacillus and have the ability to produce esterases, in particular Bacillus licheniformis IFO 12202, which is cultivated from the culture. It can be produced by collecting the esterase.

Microorganisms that can be used to carry out the method of the present invention include Bacillus lichenif.
ormis ) Not limited to IFO 12202,
Belonging to the genus Bacillus and having the above-mentioned esterase I and
Any microorganism that can produce II may be used. Further, natural or artificial mutant strains of these organism species, or gene fragments necessary for expression of the esterase activity are artificially extracted, and other organism species incorporating the same can also be used in the present invention. You can

The above strain Bacillus licheniformis ( Bac
illus licheniformis ) IFO 12202, a method for producing ester-degrading enzymes I and II having high stereoselectivity will be described. Since the bacterium accumulates the enzyme in the cells by liquid culture in a nutrient medium, The enzyme powder can be obtained by purification and drying by the method. More specifically, the present bacterium is cultured in a suitable medium, for example, a medium containing natural fats and oils and various ester compounds in order to enhance enzyme production with appropriate sugars, nitrogen, inorganic salts and the like, and accumulates the enzyme. Excuse me. Here, monosaccharides and disaccharides such as glucose and sucrose, starch, and starch hydrolysates can be used as the sugar. As the nitrogen source, organic nitrogen sources such as yeast extract, peptone, meat extract, corn steep liquor, soybean powder and inorganic nitrogen sources such as ammonium sulfate and ammonium nitrate are effective.
As the inorganic salts, sodium chloride, potassium monophosphate, magnesium sulfate, manganese chloride, calcium chloride, ferrous sulfate and the like can be used. The pH of the medium is 5.5
9 to 20 to 55 for aerobic culture such as aeration and stirring culture.
Perform at 1 to 6 days at ℃.

The cells are collected from the culture solution containing the enzyme obtained by the above method by filtration or centrifugation, and the cell-free enzyme solution is obtained by a known method such as cell crushing, autolysis, lysis, or ultrasonic treatment. After that, insoluble components are removed by filtration or centrifugation, and the filtrate or supernatant is subjected to ammonium sulfate salting out, or solvent precipitation using acetone, alcohol or the like and ultrafiltration (for example, Diaflow membrane YC, manufactured by Amicon). A crude enzyme preparation is obtained by a known method such as concentration. From the crude enzyme preparation, ester-degrading enzymes I and II purified by adsorption-elution method applying ion exchange, hydrophobic chromatography and the like, gel filtration method and electrophoresis method are obtained. In order to obtain the enzyme powder, vacuum drying or vacuum freeze drying may be performed.

The enzymatic and physicochemical properties of the esterases I and II of the present invention will be described in detail below.

(Action) Ester degrading enzymes I and II have the ability to hydrolyze esters under mildly acidic-alkaline conditions (pH 5 to 11.5).

(Method for measuring titer) 0.5M Tris-
0.1 ml of the enzyme solution was added to 0.5 ml of HCl buffer solution (pH 8.0).
5 ml was added, methyl (R, S) -2- {4- [2- (3-methyl) thienyl] phenyl} propionate was added to a final concentration of 100 mM, and the mixture was shaken and stirred at 30 ° C. for 12 hours, React. After the reaction, acidify the reaction solution with hydrochloric acid (pH 2 to 5), add 2.5 ml of ethyl acetate,
The substrate and reaction product are extracted into the ethyl acetate layer. After extraction, the mixture was separated by centrifugation and the ethyl acetate layer was used.
The conversion rate and the optical purity of the produced acid are determined by PLC analysis. In the blank, a buffer solution (10 mM Tris-HCl buffer solution (pH 8.
0)) is used for the same measurement. One unit is (R, S) -2- {4- [2- (3-
It was defined as the amount of enzyme that converts methyl (thienyl) phenyl} methyl propionate to (R, S) -2- {4- [2- (3-methyl) thienyl] phenyl} propionic acid at a conversion rate of 1%. HPLC analysis conditions Column: CHIRALCEL OD (manufactured by Daicel Chemical Industries, Ltd.) Mobile phase: Hexane / isopropyl alcohol = 94/6 Flow rate: 1 ml / min Detection: UV, 272 nm

(Substrate Specificity) Table 1 shows the ester-decomposing action when 2-arylpropionic acid esters and p-nitrophenyl esters of linear aliphatic organic acids are used as substrates.

[0016]

[Table 1]

As is clear from this result, both enzymes were 2
-Higher substrate specificity for arylpropionic acid esters, but not for p-nitrophenyl esters of linear aliphatic organic acids.

(Optimal pH) Britton-Robinso of each pH
n Wide range buffer (1 / 25M (H ₃ PO ₄ , CH ₃ COO
_{H, H 3 BO 3) -N} / 5 NaOH) in 5 units / m
Esterases I and II were added to give 1 and the activity was measured. The relative activity at each pH when the activity at the optimum pH is 100% is shown in FIG. As is clear from this figure, the optimum pH of the esterase I of the present invention is 1
0.5-11, optimum pH of esterase II is 11-
11.5.

(Stable pH range) Britton-Robinson wide range buffer (1 / 25M (H ₃ PO ₄ , CH ₃ COOH,
H ₃ BO ₃ ) -N / 5 NaOH) was added with esterases I and II at 10 units / ml, and the temperature was 30 ° C.
And incubated for 16 hours. This treatment solution is 0.5M
After diluting 2-fold with Tris-HCl buffer (pH 8.0), the residual activity was measured. Enzyme activity before treatment is 100%
The residual activity of the sample treated in each pH range was relatively expressed. The result is shown in FIG. As is clear from this figure, the esterases I and II of the present invention are p
It is stable in the range of H7 to 10.5, and is markedly deactivated at pH 5 or lower and pH 12 or higher.

(Optimal temperature) The activity of the esterases I and II at each temperature was measured, and the activity at the optimal temperature was determined to be 10
The activity at each temperature was relatively expressed as 0%. The result is shown in FIG. As is clear from this figure, the optimum temperature of the esterase I of the present invention is 35 ° C, and the optimum temperature of the esterase II is 40 ° C.

(Temperature stability) 0.5M Tris-HC
1 buffer solution (pH 8.0) with esterases I and II
Was added at 5 units / ml, heat-treated at each temperature for 10 minutes and ice-cooled, and the residual activity was measured. The enzyme activity before treatment was defined as 100%, and the residual activity of the sample treated at each temperature was relatively expressed. The result is shown in FIG. As is clear from this figure, the activity of the esterases I and II of the present invention was maintained at 100% up to the temperature of 40 ° C.

(Effect of Metal Ions) Table 2 shows the results of investigations on the effects of metal ions on the enzyme activity.
Each metal salt was added so that the final concentration during the reaction was 2 mM, and the activity was measured. The effect of each metal ion was represented by the relative value when the activity was taken as 100%, using a system without addition of metal salt as a control. As a result, the esterases I and II of the present invention were found to contain Ni ²⁺ , Fe ³⁺ , Zn ²⁺ , Al ³⁺ ,
Approximately 70-80% activity was inhibited by Pb ²⁺ .

[0023]

(Effects of Inhibitors and Activators) Table 3 shows the results of investigations on the effects of inhibitors and activators on enzyme activity. An inhibitor and an activator were added so that the final concentration during the reaction was 2 mM, and the activity was measured. A system without addition of an inhibitor and an activator was used as a control, and the effect of the inhibitor and the activator was expressed as a relative value when the activity was 100%.

[0025] As a result, the esterases I and II of the present invention were DF
Since it is inhibited by P and PMSF, it is found that the enzyme has serine at the active center.

(Molecular weight) The molecular weight of the esterase A was measured by gel filtration chromatography using Superdex 200 (Pharmacia). In this gel filtration, the eluent contains 0.15M NaCl 2
0 mM Tris-HCl buffer (pH 8.0) was used, and ferritin (Mw: 44000) was used as a standard protein.
0), catalase (Mw: 232000), aldolase (Mw: 158000), bovine serum albumin (Mw:
67000) Ribonuclease A (Mw: 13700)
Was used. As a result, the molecular weight of the esterase I of the present invention was estimated to be about 100,000, and the molecular weight of the esterase II was estimated to be about 25,000.

[0027]

The present invention will be further described below with reference to examples, but these examples do not limit the scope of the present invention.

Example A bacterium fermented by the present invention, Bacillus licheniformis (IFO 12202), was inoculated into a 3 liter jar fermenter containing 2.5 liter of broth medium (pH 7.0), and aeration stirring was performed at 37 ° C. for 72 hours. (1vvm, 600r
(pm) After culturing, cells were collected from the culture solution by centrifugation. 27 g of the obtained microbial cell moist product 3.7 potassium monophosphate
g, EDTA.2Na 0.1 g, Triton X-
100 0.2 ml, lysozyme chloride 80 mg (titer)
Was dispersed in 270 ml of dissolved water and lysed by leaving it at 37 ° C. for 4 hours. The lysate was centrifuged to remove insoluble matter, 55 (w / v)% ammonium sulfate was added to the supernatant to dissolve it, and the mixture was left at 4 ° C. overnight, and then the salted-out product was collected by centrifugation. The obtained salted out product was treated with 20 mM Tris
-Dissolve in HCl buffer solution (pH 8.0) and dialyse overnight against the same buffer solution to desalt, 40 ml of crude enzyme solution
Got DEAE-Cellulofine in which the crude enzyme solution was equilibrated with 20 mM Tris-HCl buffer (pH 8.0)
A-500 (manufactured by Chisso Corporation) is subjected to anion exchange resin column chromatography to obtain 0-0.5M NaC.
Elution was performed with a linear gradient of l. The desired enzyme activity was eluted near 0.3 M NaCl. The active fractions thus eluted were collected and desalted by dialysis against 20 mM Tris-HCl buffer (pH 8.0),
As a result of vacuum freeze-drying, 300 mg of crudely purified enzyme powder was obtained. 150 mg of crude enzyme powder was added to 1.5 ml of 0.15
Super dissolved in 20 mM Tris-HCl buffer (pH 8.0) containing M NaCl and equilibrated with the same buffer
It was subjected to gel filtration with dex 200 (Pharmacia LKB). The elution volume of the desired enzyme activity is as follows: Column: 1.6c
Under conditions of mφ × 60 cm and a flow rate of 7.0 ml / hr, ester degrading enzyme I was about 70 ml and ester degrading enzyme II was about 92 ml. The eluted fractions of esterase I and esterase II were separately collected, and each was desalted by dialyzing it against 10 mM Tris-HCl buffer (pH 8.0), and then vacuum freeze-dried, 22 mg of purified esterolytic enzyme I and 35 mg of purified esterolytic enzyme II were obtained.

Reference Example 1 Production of (S) -2- {4- [2- (3-methyl) thienyl] phenyl} propionic acid using esterase I 0.5M Tris-HCl buffer solution (pH 8.0) ) 10
100 mg of the esterase I obtained in the example in 0 ml,
2.46 g (10 m) of methyl (R, S) -2- {4- [2- (3-methyl) thienyl] phenyl} propionate
mol) was added, and the mixture was shaken and stirred at 30 ° C. for 24 hours.
The reaction solution was centrifuged, and a mixture of the ester and the generated acid was collected from the reaction solution. To this, add 2 ml of toluene, 15
2 ml of (w / v)% sodium carbonate aqueous solution was added, and the mixture was stirred and separated. Acidify the aqueous layer with hydrochloric acid (pH 2-5),
The precipitated crystals were collected by filtration and dried to give an optically active (S) -2- {4
0.44 g of-[2- (3-methyl) thienyl] phenyl} propionic acid was obtained (yield 17.9%). The optical purity by HPLC was 99% or more. HPLC analysis conditions Column: CHIRALCEL OD (manufactured by Daicel Chemical Industries, Ltd.) Mobile phase: Hexane / isopropyl alcohol = 94/6 Flow rate: 1 ml / min Detection: UV, 272 nm

Reference Example 2 Production of (S) -2- {4- [2- (3-methyl) thienyl] phenyl} propionic acid using ester degrading enzyme II The ester degrading enzyme II obtained in Example was used. When (S) -2- {4- [2- (3-methyl) thienyl] phenyl} propionic acid was produced under the same conditions as in Reference Example 1, the optically active (S) -2- {4- [ 2- (3-methyl)
0.47 g of thienyl] phenyl} propionic acid was obtained (yield 19.1%). 99% optical purity by HPLC
That was all.

[0031]

INDUSTRIAL APPLICABILITY The novel esterase I of the present invention and
Since II is stable in a wide pH range and has high stereoselectivity, it can be used for resolution of a mixture of stereoisomers. Further, according to the present invention, such esterases I and II can be produced in large quantities by culturing a microorganism of the genus Bacillus.

[Brief description of drawings]

FIG. 1 shows the esterases I and II of the present invention at 30 ° C.
3 is a graph showing relative activity at each pH in FIG.

FIG. 2: 30 ° C. of esterases I and II of the present invention
The graph showing the stability at each pH in.

FIG. 3 pH of esterases I and II of the present invention
The graph showing the activity at each temperature at 8.0.

FIG. 4 pH of esterases I and II of the present invention
The graph which shows the activity after being kept at 8.0 at each temperature for 10 minutes.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Shunji Kamiyama 2-3 2-3 Muroya, Nishi-ku, Kobe-shi, Hyogo Nagase & Co., Ltd. (72) Chie Miura 2-3-2 Muroya, Nishi-ku, Kobe-shi, Hyogo Nagase Sangyo Co., Ltd.

Claims (4)

[Claims] 1. A novel esterase I (1) having the following physicochemical properties: It has the ability to hydrolyze an ester under weakly acidic-alkaline conditions (pH 5 to 11.5). (2) Substrate specificity It shows high substrate specificity for 2-arylpropionic acid esters, but does not act on p-nitrophenyl esters of linear aliphatic organic acids. (3) Optimum pH Using (R, S) -2- {4- [2- (3-methyl) thienyl] phenyl} methyl propionate as a substrate, 30
When the reaction is carried out at ℃, the optimum pH is 10.5-11. (4) Stable pH range The residual activity after treatment at 30 ° C. for 16 hours at various pHs was measured using methyl (R, S) -2- {4- [2- (3-methyl) thienyl] phenyl} propionate as a substrate. When measured, it is stable in the pH range of 7-10.5,
At pH 5 or lower and pH 11.5 or higher, marked deactivation is observed. (5) Optimum temperature (R, S) -2- {4- [2- (3-methyl) thienyl] phenyl} methyl propionate as a substrate, pH
When the reaction is carried out at 8.0, the optimum temperature is 35 ° C. (6) Temperature stability The residual activity after heat treatment at pH 8.0 and 40 ° C for 10 minutes was measured using methyl (R, S) -2- {4- [2- (3-methyl) thienyl] phenyl} propionate as a substrate. 100% activity remains when measured as. (7) Effect of metal ions Ni ²⁺ , Fe ³⁺ , Zn ²⁺ , Al ³⁺ , Pb ²⁺ remarkably inhibits the activity. (8) Effects of inhibitors and activators The activity is specifically inhibited by DFP and PMSF. (9) Molecular weight about 100,000 (by gel filtration method) 2. A novel ester-degrading enzyme II (1) having the following physicochemical properties: It has the ability to hydrolyze an ester under mildly acidic-alkaline conditions (pH 5 to 11.5). (2) Substrate specificity It shows high substrate specificity for 2-arylpropionic acid esters, but does not act on p-nitrophenyl esters of linear aliphatic organic acids. (3) Optimum pH Using (R, S) -2- {4- [2- (3-methyl) thienyl] phenyl} methyl propionate as a substrate, 30
When the reaction is carried out at ℃, the optimum pH is 11 to 11.5. (4) Stable pH range The residual activity after treatment at 30 ° C. for 16 hours at various pHs was measured using methyl (R, S) -2- {4- [2- (3-methyl) thienyl] phenyl} propionate as a substrate. When measured, it is stable in the pH range of 7-10.5,
At pH 5 or lower and pH 12 or higher, marked deactivation is observed. (5) Optimum temperature (R, S) -2- {4- [2- (3-methyl) thienyl] phenyl} methyl propionate as a substrate, pH
When the reaction is carried out at 8.0, the optimum temperature is 40 ° C. (6) Temperature stability The residual activity after heat treatment at pH 8.0 and 40 ° C for 10 minutes was measured using methyl (R, S) -2- {4- [2- (3-methyl) thienyl] phenyl} propionate as a substrate. 100% activity remains when measured as. (7) Effect of metal ions Ni ²⁺ , Fe ³⁺ , Zn ²⁺ , Al ³⁺ , Pb ²⁺ remarkably inhibits the activity. (8) Effects of inhibitors and activators The activity is specifically inhibited by DFP and PMSF. (9) Molecular weight about 25,000 (by gel filtration method) 3. A method for producing an ester degrading enzyme, which comprises culturing a microorganism belonging to the genus Bacillus and having the ability to produce ester degrading enzymes I and II, and collecting the ester degrading enzymes I and II from the culture. 4. The microorganism is Bacillus licheniformis ( B
The method according to claim 3, which is acillus licheniformis ) IFO 12202.