JPH0761263B2 - Novel esterase and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel esterase derived from a microorganism and a method for producing the same. More specifically, the present invention relates to Baci
The present invention relates to a novel esterase obtained by culturing a microorganism belonging to the genus llus) and a method for producing the same.

In general, esterase is a general term for enzymes that hydrolyze a substrate having an ester bond, and acts on a carboxylic acid ester, acts on a thiol ester, acts on a phosphoric acid ester, and acts on a sulfate ester depending on its substrate specificity. Are classified as those that act on. In a narrow sense, among those that act on carboxylic acid esters, those that hydrolyze lower fatty acid esters are called esterases, and those that hydrolyze glycerol esters are called lipases. The crude preparation of lipase has an esterase action in addition to the lipase action, and both actions are practically distinguished by heat resistance, trypsin and alkali sensitivity, and difference in chain length of fatty acid of substrate ester. .

However, it is said that the difference between the two actions is due to the difference in the physical existence state of the substrate. That is, in the esterase reaction, the enzyme acts on the substrate in the aqueous solution, but the lipase reaction is said to proceed at the interface between the water-insoluble substrate and water. (For example Sarda et al., Biochem. Bi
See ophys.Acta .30 513 (1958)). The esterase referred to in the present invention means the above-mentioned narrowly defined esterase.

In recent years, many attempts have been made to apply enzymes to organic synthesis, and various ester compounds are hydrolyzed using the enzyme esterase to obtain optically active compounds, or to create chiral compounds from prochiral compounds. Many attempts to do things have been reported. Many of these reports are based on pig liver esterase (Pig liver esterase or Porc).
ine liver esterase) (eg La
umen et al. Tetrahedron Lett. 26 407-410 (1985) and Wang
J. Am. Chem. Soc. 106 3695 (1984)) Pig liver esterase is expensive and has a limited quantitative supply, which is disadvantageous for industrial use. Therefore, instead of pig liver esterase, A method using an esterase of microbial origin has also been tried. In that case, it is general to use esterase-producing microbial cells, and there are few examples in which the enzyme esterase is purified to clarify its properties. (Eg Kota
ni et al., Agric. Biol . Chem .47 1363-1365 (1983); Fujimot.
O. et al., J. Chem. Soc. Chem. Commun. 1333-1334 (1985); and Brannon et al., J. Antibiot 29 121-124 (1976)).

On the other hand, Bacillus subtilis ATCC 6633 (Biochem.Biophys.Act
485 367-378 (1977)), Bacillus subtilis SR22
(Biochem. Biophys. Acta. 429 191-197 (1976)), B
acillus subtilis NRRL-B-558 (Appl. Microbiol. 30
413-419 (1975)), Bacillus stearothemophilus
(Archiv.Biochem.Biophys .160 504-513 (1974)) ,
Pseudomonas aeruginosa (J. Biochem .86 643-656 (1
979), Pseudomonas cepacia (J. Bacteriol. 118 880
-889 (1974), Pseudomonas fluorescens (J. Bioc
hem. 95 1047-1054 (1984) and JP-A-60-30681),
Escherichia coli K-12 (J. Bacteriol. 149 6-14 (198
2)), Aspergillus niger NRRL337 (Agric.Biol.Che
m. 47 1865-1868 (1983) and Mycobacterium smegmat
is ATCC14468 (J. Bacteriol. 155 1249-1259 (1983))
Esterases of origin have been reported, but B. subtilis
In all cases, no mention is made of the application to the organic synthesis reaction, except for the case where the esterase derived from NRRL-B-558 is applied to the synthesis of cephalosporin derivatives.

Under the circumstances, the present inventors have conducted extensive research to obtain an esterase of microbial origin, which has a wide range of applications to organic synthetic reactions like pig liver esterase. The more newly separated Baci
llus) a new microorganism belonging to the genus
(Bacillus sp. DC-1) (Ministry of Industrial Research, Microbiology Research Institute No. 8719;
61-92595) has been found to have such excellent properties, and the present invention has been completed.

That is, the present invention uses the esterase in the field of synthetic organic chemistry by supplying a novel esterase derived from a microorganism having such excellent properties, to produce an optically active compound and to produce a chiral compound from a prochiral compound. It can be carried out in a targeted manner.

The present invention firstly relates to a novel esterase having the following physicochemical properties.

1) Action: Hydrolyze the ester bond of the organic carboxylic acid ester.

2) Substrate specificity: It acts mainly on alcohol esters of organic carboxylic acids and has high activity on water-soluble short-chain fatty acid esters.
It works on short chain mono- and triacyl glycerides but not on long chain mono-, di- and triacyl glycerides. It acts on palmitoyl CoA but not acetyl CoA, choline esters, and cholesterol esters.

3) Optimum pH and pH stability: P- of dichlorovinyl chrysanthemic acid (hereinafter referred to as DCP-I).
The optimum pH for hydrolysis using nitrophenyl ester as a substrate is 8.5 to 9.0. 10 mM Tris-HCl buffer (pH 7.
0 to 9.0) and 10 mM phosphate buffer (pH 5.0 to 7.0), the residual activity after 4 hours at 4 ° C was about 10 at pH 7.0 to 9.0.
It is about 45% at 0%, pH 6.0 and about 20% at pH 5.0.

4) Optimum temperature and thermal stability: When using P-nitrophenyl ester of DCPI as a substrate, the optimum temperature for hydrolysis is 50 in 100 mM Tris-HCl buffer pH 9.0.
It is ~ 55 ° C.

Residual activity after treatment in the same buffer for 10 minutes at each temperature is 35 ° C.
100%, about 80% at 40 ° C, about 15% at 50 ° C.

5) Molecular weight: 54,000 ± 2,000 (by gel filtration and SDS-polyacrylamide gel electrophoresis) 6) Isoelectric point: 4.8 ± 0.1 7) Deactivation condition: a) 100 μM phenylmethylsulfonyl fluoride (PMSF) or 80 μM diisopropyl Fluorophosphoric acid (DI
FP) is completely inactivated when treated at 37 ° C for 10 minutes.

b) 100 mM Tris-HCl buffer (pH 9.0) containing 1 mM sodium deoxycholate and 5 mM sodium lauryl sulfate
Treatment at 37 ° C for 10 minutes decreases the activity by about 25% and about 70%, respectively.

The esterase of the present invention has no subunit structure 1
This chain peptide has a molecular weight of 54,000 ± 2,000, which means that Bacillus subtilis ATCC6633 (150,000), Bacillus
subtilis NRRL-B-558 (190,000), Bacillus subtil
is SR-22 (160,000), Bacillus stearothemophilus (4
2,000 ~ 47,000), Pseudomonas cepacia (34,500), Pseu
dmonas fluorescens (48,000), Aspergillus niger NRR
L337 (23,000, 27,800, 29,500, 127,000) and Mycobac
It is clearly different from the esterase derived from terium smegmatis ATCC 14468 (36,000-41,000). Also Pseudomo
The esterase of nas aeruginosa origin is not inactivated by diisopropyl fluorophosphate and sodium lauryl sulfate, whereas the esterase of the present invention is completely inactivated by about 70% at 5 mM sodium lauryl sulfate and 80 μM diisopropyl fluorophosphate. Different. Furthermore Escheric
The esterase derived from hia coli K-12 should be called a carboxylpeptidase existing in the membrane, and this is also clearly different from the esterase of the present invention in terms of intracellular localization. Therefore, the esterase of the present invention is a novel esterase that has hitherto been unknown.

Secondly, the present invention cultivates a microorganism which belongs to the genus Bacillus and has the ability to produce a novel esterase having the above-mentioned properties, accumulates the enzyme in the cultured cells, separates and collects it. The present invention relates to a method for producing esterase characterized by the above.

The novel esterase of the present invention is produced by using a microorganism, and as a producing bacterium thereof, a strain belonging to the genus Bacillus and having the ability to produce the enzyme having the above-mentioned properties may be used, for example, Bacillus sp. DC-1 ( Bacillus
sp.DC-1). This strain is Microtechnology Research Institute
It has been deposited as an issue and its mycological properties are as follows.

(A) Morphology 1) Cell morphology and size: Rod-shaped (0.5-0.6) μm × (1.2-1.7) μm. A single chain or 2-3 chains are formed.

2) Polymorphism: None 3) Motility: Yes Has periflagellates.

4) Spore formation: Yes Spherical or slightly oval, 0.4-0.6 μm in diameter. It is formed at the end of vegetative cells and has a bulge.

5) Gram stain: Negative 6) Anti-acidity: None (b) Growth condition in various media 1) Meat broth agar plate medium (35 ° C, 24 hours) Shape: Circular rim: None Ridge: Convex gloss: Yes Surface: Smooth color tone : Translucent and yellowish white 2) Broth agar slope culture (35 ° C, 24 hours) Growth rate: Normal Spread or juliform growth Surface: Smooth color tone: Translucent yellowish white gloss: Yes 3) Broth liquid culture (35 (° C, 24 hours) Growth rate: Normal Coloring / decolorization: None Surface growth: No bacterial ring is formed Sediment: Occurs 4) Meat juice gelatin stab culture (35 ° C, 14 days) Do not liquefy gelatin.

5) Litmus milk medium (35 ° C, 14 days) Slightly alkalinized, not coagulated or peptone.

(C) Physiological properties Culture at 35 ° C. for 1 to 5 days. Negative ones are observed for up to 14 days.

1) Reduction of nitrate: Positive nitric acid is reduced to produce nitrous acid.

2) Denitrification reaction: negative 3) MR test: negative 4) VP test: negative 5) Indole formation: negative 6) Hydrogen sulfide formation: negative 7) Starch hydrolysis: negative 8) Use of citric acid Koser's Medium: Negative Christensen's medium: Positive 9) Utilization of inorganic and nitrogen sources Modified method of Stanier's medium by Yamazato et al .: (Yamazato et al. J. Gen. Appl. Microbiol. (1982) 28: 1.
95-213) and sodium succinate was used as the carbon source.

Nitrate: Not used Ammonium salt: Used

10) Pigment formation: No formation 11) Urease Christensen's urea medium: positive 12) oxidase: positive 13) catalase: positive 14) Range of growth Growth temperature: 10 to 45 ° C (optimal 30 to 35 ° C) Growth pH: 6.0 ~ 9.5 (optimal 8.5-9.0) 15) Attitude toward oxygen: grows only aerobically 16) OF test: Negative 17) Production of acid / gas from sugars Acid gas L-arabinose-D-xylose-D -Glucose-D-mannose-D-fructose-D-galactose-maltose-sucrose-lactose-trehalose-D-sorbitol-D-mannitol-inosit-glycerin-starch − − For bacteria having the above-mentioned mycological properties, Barjay's
Manual of Determinative Bacterio (Bergey's Manual of Determinative Bacterio
logy) 8th edition (1974), and as a result, it was identified as a strain belonging to the genus Bacillus because it is a spore bacillus that grows under aerobic conditions. In addition, when this strain is compared with strains belonging to the same genus, it is similar to Bacillus sphaericus and Bacillus pasteurii, but in terms of Table 1, these strains are Is different.

From the above, this strain was identified as a new strain belonging to the genus Bacillus, and Bacillus sp. DC-1 (Bacillus sp.
us sp. DC-1).

The microorganism used in the present invention is not limited to the strain of the present invention and its variants and mutants, and any microorganism having the above-mentioned enzyme may be used.

The novel esterase-producing bacterium of the present invention can be cultured according to a conventional method known in the field of fermentation. As the medium to be used, any of synthetic medium and natural medium can be used as long as it contains a suitable amount of carbon source, nitrogen source, inorganic substance and other nutrients, and it can be cultured using liquid medium or solid medium. Specifically, as the carbon source, glucose, fructose, maltose, galactose, ribose, saccharose, starch, starch hydrolyzate, molasses, sugars such as molasses, wheat, natural carbohydrates such as rice, glycerol, mannitol, methanol, Alcohols such as ethanol, fatty acids such as gluconic acid, pyruvic acid, acetic acid and citric acid, hydrocarbons such as normal paraffin and kerosene, general carbon sources such as glycine, glutamic acid, amino acids such as glutamine, alanine and asparagine In consideration of the assimilation property of the microorganism to be used, one kind or two or more kinds may be appropriately selected and used. As the nitrogen source, meat extract, peptone, yeast extract, dry yeast, soybean hydrolyzate, soybean powder, milk casein, casamino acid, various amino acids, corn steep liquor, fish meal or its hydrolyzate, other animals, Use of organic nitrogen compounds such as hydrolysates of plants and microorganisms, ammonia, ammonium salts such as ammonium nitrate, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium acetate, nitrates such as sodium nitrate, inorganic nitrogen compounds such as urea Considering sex, one or more kinds are appropriately selected and used.

Furthermore, as inorganic salts, trace amounts of magnesium, manganese,
One or more of phosphates such as iron, zinc, copper, sodium, calcium and potassium, hydrochlorides, sulfates, carbonates, acetates and the like are appropriately added, and vegetable oils, surfactants and the like are added as necessary. An antifoaming agent may be added.

The culturing can be carried out in a liquid medium containing the above-mentioned medium components using a usual culturing method such as shaking culturing, aeration stirring culturing, stationary culturing, continuous culturing and the like.

The culture conditions may be appropriately selected depending on the type of medium and the culture method, and are not particularly limited as long as the strain can grow and produce esterase. Usually, the pH at the start of culture is 8 ~
It is preferable to adjust to 9 and culture under the temperature condition of 30 to 35 ° C. The appropriate number of days for culturing is usually 1 to 2 days.

The esterase produced and accumulated in the culture as described above can be collected and separated by the following method.

Since this esterase accumulates in the microbial cells, after culturing, the microbial cells are collected by a method such as filtration or centrifugation and washed with water or a buffer solution, for example, freeze-thaw treatment, ultrasonic treatment, pressure treatment. , Physical treatment such as osmotic pressure difference treatment, grinding treatment and acetone air-drying treatment, or biochemical treatment such as cell wall lysing enzyme treatment such as lysozyme and cellulase, or chemical treatment such as contact treatment with a surfactant. By applying these alone or in combination, the bacterial cells can be crushed and the esterase can be extracted.

An example is as follows.

That is, the cells collected by centrifugation were washed several times with 100 mM potassium phosphate buffer (pH 9.0), suspended in the same buffer, and the cells were pressure-crushed with a pressure-type crusher French press. To extract esterase. The crude esterase thus obtained from the bacterial cell extract is subjected to column chromatography such as salting out, fractional precipitation with an organic solvent, ion exchange chromatography, gel filtration, hydrophobic chromatography, hydrogen bond chromatography, affinity chromatography and high performance liquid chromatography. It is possible to purify using means known in the field of biochemistry such as chromatography and electrophoresis, alone or in combination.

An example is as follows.

That is, the treated bacterial cells crushed by the French press treatment were centrifuged at 10,000 g for 10 minutes, and subsequently the supernatant was treated with 100,000.
Use the supernatant obtained by ultracentrifugation for 60 minutes as a crude extract.

The extract was salted out with 40 to 70% ammonium sulfate to precipitate the esterase, the precipitate was dissolved in 20 mM Tris-HCl buffer (pH 7.0), and DEAE-Sepharose equilibrated with the same buffer.
Pass through CL-6B to adsorb esterase. After that, esterase is eluted by a 0 to 0.5 M Nacl linear gradient method. The fraction having an esterase activity eluted at around 0.25 M is concentrated and then dialyzed against 50 mM Tris-HCl buffer (pH 8.0). The concentrate was passed through Sephacryl S-200 Superfine equilibrated with the same buffer, the eluted esterase fraction was concentrated, and then passed through a Sephadex G-150 Superfine column equilibrated with the same buffer. Pour out and elute the esterase. The obtained esterase fraction was concentrated, dialyzed against 20 mM Tris-hydrochloric acid buffer (pH 8.0), passed through QAE-Sephadex equilibrated with the same buffer to adsorb esterase, and then 0- 0.5MNa
Elute the esterase with a linear gradient of cl.

When this eluate was concentrated and subjected to SDS-polyacrylamide gel electrophoresis, this esterase was electrophoretically purified into a single band with a molecular weight of 54,000 and a single-chain peptide with no subunit structure. I knew there was.

Table 2 shows an example of this purification process.

Next, the method for measuring the activity of esterase used in the present invention will be described.

Esterase solution is added to 100 mM Tris-HCl buffer of pH 9.0 and preincubated at 40 ° C. for 1 minute.
Then, the P-nitrophenyl ester of DCPI represented by the following general formula (I) dissolved in acetone was added to a final concentration of 1 mM.
After addition and stirring, the increase in absorbance at 405 nm accompanying the release of P-nitrophenol is measured by a spectrophotometer. The amount of enzyme that liberates 1 nmole of P-nitrophenol per minute is 1 unit.

Next, the present invention will be described with reference to Examples, but it goes without saying that the scope of the present invention is not limited by these.

Example 1 Bacillus sp. DC-1 (Bacillus sp. DC-1) (Microtechnology Research Institute No. 8719) was used as soluble starch 1%, polypeptone.
0.5%, 0.5% yeast _{extract, K 2 HPO 4 0.1%,} MgSO 4 · 7H 2 O
0.02%, and CuSO ₄ , 5H ₂ O 5mg / l, MnCl ₂ as trace metals
・ 4H ₂ O 5 mg / l, ZnSO ₄・ 7H ₂ O 1 mg / l and FeSO ₄・ 7H ₂ O 2 mg / l
One platinum loop was inoculated into 50 ml of a pH 9.0 medium containing 1 l and shake-cultured at 30 ° C. for 24 hours. The seed culture obtained in this pre-culture,
Furthermore, 5 liters of the above-mentioned composition was inoculated and cultured at 35 ° C. for 24 hours with an aeration rate of 5 l / min and a stirring speed of 600 rpm. After culturing,
The cells obtained by centrifugation at 9,000 g for 10 minutes were washed twice with 100 mM potassium phosphate buffer pH 9.0, and 100 ml of the culture solution was suspended in 4 ml of the same buffer solution.・ Cels Press (Aminco) at 4 ℃, 38.000
The cells were disrupted at a pressure of psi.

Centrifugation of the obtained treated cells (10,000g, 10 minutes)
And then the supernatant is ultracentrifuged (100,000g, 60 minutes).
Thus, a crude esterase extract was obtained. As a result, specific activity
6560 mg of 4.86 units / mg of crude enzyme was obtained.

Example 2 100m of crude esterase extract obtained according to Example 1
Protein concentration 10 to 20 mg / m in M potassium phosphate buffer (pH 9.0)
After adjusting to l, the ammonium sulfate was saturated to 40% and the formed precipitate was removed by centrifugation (10,000 xg for 10 minutes). Then, the supernatant was saturated to 70% ammonium sulfate and the precipitate was collected (10,000). × g1
(0 min), dissolved in 20 mM Tris-HCl buffer pH 7.0 40 ml,
It was dialyzed against the same buffer. As a result, total activity 17848u
A crude enzyme solution with nits and a specific activity of 6.80 units / mg was obtained.

Of this 43.5 ml of esterase solution, 10.2 ml (4199unit
s, 615mg prot.) equilibrated with the same buffer 14
After passing through 0 ml of DEAE-Sepharose CL-6B (2.6 x 26 cm) to adsorb the esterase to DEAE-Sepharose CL-6B, the column was washed with the same buffer and a linear concentration gradient of 0-0.5 M NaCl was applied. Esterase was eluted around 0.25M NaCl. As a result, 3734 units (89%) were recovered in the peak portion and 4115 units (98%) were recovered in the whole eluate, and the specific activity was increased 8.2 times.

After concentrating the esterase active fraction with an ultrafiltration membrane, the concentrate was dialyzed against 50 mM Tris-HCl buffer pH 8.0 to obtain Sephacryl S-200 Superfine (2.6 × 100 cm) equilibrated with the same buffer. The solution was passed through and gel filtration was performed. After concentration of the eluted esterase fraction, 50 mM Tris-HCl buffer pH8.0
The solution was passed through a Sephadex G-150 Superfine column (2.6 × 100 cm) equilibrated with, and the esterase was eluted with the same buffer solution. After concentrating the obtained esterase fraction,
Bed volume 70 ml equilibrated with mM Tris-HCl buffer pH 8.0
After passing through QAE-Sephadex (1.6 × 35 cm) to adsorb esterase, it was washed with the same buffer. Then 0
Elution of esterase by linear concentration gradient elution method of ~ 0.5M NaCl, 745 unit activity was recovered at NaCl 0.25 ~ 0.3M, specific activity was 413.8units / mg, or activity recovery from crude extract The rate was 9.9%.

After concentrating this esterase solution about 5 times, 10% SDS-
When subjected to polyacrylamide gel electrophoresis, esterase was recognized as a single band with a molecular weight of 54,000. This is in good agreement with the result of gel filtration with Sephacryl S-200 Superfine and Sephadex G-150 Superfine (52,000 to 58,000), suggesting that the esterase is a single-chain peptide having no subunit structure. Was done. However, phosphorylase B (molecular weight 92,500), bovine serum albumin (66,200), ovalbumin (45,000), carbonic anhydrase (31.000), soybean trypsin inhibitor (21,500) and lysozyme (14,400)
Was used as a standard substance for determination of esterase molecular weight by SDS-PAGE, and blue dextran (200,000), bovine serum albumin (66,200), ovalbumin (45,000), chymotrypsinogen A (25,000), ribonuclease A (13,7)
00) was used as a standard substance for the determination of esterase molecular weight by gel filtration.

Example 3 Purified esterase obtained according to Example 2 LK
B polyacrylamide gel (concentration 5%, crosslinking degree 3%,
Isoelectric focusing (constant power: 15 W) was performed in the range of pH 4.0 to 6.5 using amphiline concentration of 2.2%) for 90 minutes at 10 ° C. After the electrophoresis, staining with Coomassie Brilliant Blue R-250 was performed to detect the protein band. As a result, the isoelectric point of this esterase was 4.8 ± 0.1. In addition, as a standard substance for electrophoresis, PI marker (PI
4.65,5.10,6.00,6.50) and PI markers (PI4.10,4.90,6.40) from Oriental Yeast Co. were used.

Example 4 After the purified esterase obtained according to Example 2 was incubated with each of the following reagents at 37 ° C. for 10 minutes, the residual enzyme activity was measured according to the above-mentioned activity measuring method. Third
The results are shown in the table.

In Table 3, the residual activity was expressed as a relative value (%) when the untreated enzyme activity was 100. Further, in Table 3, 1) indicates phenylmethylsulfonyl fluoride, and 2) indicates diisopropylfluorophosphoric acid.

Example 5 The purified esterase obtained according to Example 2 was treated with 100 mM.
Each substrate was allowed to act on 2 to 20 mM of Tris-HCl buffer (pH 9.0), and its hydrolysis activity was measured by pH stat (Radiometer). Also, the hydrolytic activity of choline ester, cholesterol ester and thiol ester was measured.
The results are shown in Tables 4 and 5.

In Table 4, the hydrolytic activity was expressed as a relative activity (%) with the ethyl butyrate degrading activity of the present esterase being 100. still,
Each substrate was emulsified with 0.2% polyvinyl alcohol before use. In Table 5, choline ester and cholesterol ester hydrolyzing activities were measured using cholinesterase B-test (Wako) and free cholesterol B-test (Wako), respectively, and thiol ester degrading activity was measured using glutathione as a standard substance. , 5,5'-Dithiobis (2-nitrobenzoic acid) (DTNB) was used to measure the increase in absorbance at 412 nm with a spectrophotometer. The activity was expressed by the number of moles of choline, cholesterol and CoA released in 1 minute.

Example 6 Using the purified esterase obtained according to Example 2,
The optimum pH and pH stable range of the enzyme were measured when P-nitrophenyl ester of DCPI was used as a substrate. The result is first
Shown in Figures and 2. In Figure 1, activity measurement is 40 ℃
PH 5 to 7 with 100 mM potassium phosphate buffer,
100 mM Tris-HCl buffer for 7-9, 100 mM for pH 9-13
Dipotassium phosphate / potassium hydroxide buffer solution was used.
The activity was expressed as a relative activity (%) when the activity value at pH 9.0 was 100.

In FIG. 2, the enzyme solution was stored in 20 mM potassium phosphate buffer at pH 5 to 7 and 20 mM Tris-HCl buffer at pH 7 to 9 at 4 ° C. at each time. Residual activity was measured at 40 ° C. in 100 mM Tris-HCl pH 9.0. The activity was represented by the residual activity rate (%) when the activity value at 0 hours was 100.

Example 7 The optimum temperature for hydrolyzing p-nitrophenyl ester of DCPI and thermal stability of the purified esterase obtained according to Example 2 were measured. The results are shown in FIGS. 3 and 4. In FIG. 3, the horizontal axis represents the reciprocal of the absolute temperature, and the vertical axis represents the logarithm of the initial velocity. The activity was measured using 100 mM Tris-HCl buffer (pH 9.0).

In Fig. 4, the enzyme solution was pretreated for 15 minutes at each temperature shown on the horizontal axis, and the activity was measured in 100 mM Tris-HCl buffer pH 9.0 at 40 ° C. The activity was expressed as the residual activity rate (%) when the untreated esterase activity was 100.

Reference Example 1 Crude enzyme solution of esterase obtained according to the ammonium sulfate salting-out method described in Examples 1 and 2 (ammonium sulfate 40-70% fraction, specific activity 6.
80 units / mg) was used to carry out the hydrolysis reaction of ethyl dichlorovinyl chrysanthemate (hereinafter abbreviated as DCPE in the present specification).

That is, 1.0 ml of the crude enzyme solution (410.0 units, 60.3 mg) was added to 2%
Add (±) -cis, trans DCP to 8.0 ml of 100 mM Tris-HCl buffer of pH 9.0 containing 1.0 ml of polyvinyl alcohol.
E (cis / trans ratio = 45/55) was added so that the final concentration was 2 mM, and the mixture was reacted at 35 ° C. for 96 hours with stirring. After 96 hours, 0.1 ml of 35% HCl was added to this reaction solution to stop the reaction, and then DCPI formed with diethyl ether and unreacted DCPE were acidified and extracted.

Gas chromatography of the extract (column: 30% Thermon
3000, 1.1 m 140 ° C), and the yield (%) was calculated from the peak area ratio of DCPI and DCPE.

Next, 2.0 ml of IN NaOH was added to the above extract to extract only DCPI as a sodium salt into the aqueous layer, and the aqueous layer was extracted with 35% HCl.
The pH was adjusted to 2 or less with, the released DCPI was extracted with methyl isobutyl ketone, and then concentrated. Toluene was added to the obtained DCPI.
It was dissolved in 5 ml, and thionyl chloride, pyridine and 3,5-dichloroaniline in equimolar amounts to DCPI were added and reacted at 80 ° C to give an anilide, which was then subjected to high performance liquid chromatography (column: SUMIPAX OA-2100, mobile phase n- Isomer analysis was performed with hexane: dichloroethane (10: 3 V / V), flow rate 1.0 ml / min). The results are shown in Table 6.

1) Yield represents the molar yield of (+)-trans DCPI obtained relative to (+)-trans DCPE in the raw material.

[Brief description of drawings]

FIG. 1 shows the optimum pH of the esterase of the present invention. FIG. 2 shows the pH stability of the esterase of the present invention. FIG. 3 shows the optimum temperature of the esterase of the present invention. FIG. 4 shows the thermostability of the esterase of the present invention.

Claims (3)

[Claims] 1. A novel esterase having the following physicochemical properties 1) Action: Hydrolyze the ester bond of an organic carboxylic acid ester. 2) Substrate specificity: It acts mainly on alcohol esters of organic carboxylic acids and has high activity on water-soluble short-chain fatty acid esters.
It works on short chain mono- and triacyl glycerides but not on long chain mono-, di- and triacyl glycerides. It acts on palmitoyl CoA but not acetyl CoA, choline esters, and cholesterol esters. 3) Optimum pH and pH stability: When p-nitrophenyl ester of dichlorovinyl chrysanthemic acid (DCPI) is used as a substrate, the optimum pH for hydrolysis is 8.5-
It is 9.0. When using 10 mM Tris-HCl buffer (pH 7.0 to 9.0) and 10 mM phosphate buffer (pH 5.0 to 7.0), the residual activity after 48 hours at 4 ° C is about 100% at pH 7.0 to 9.0. , At pH 6.0 about 45%,
It is about 20% at pH 5.0. 4) Optimum temperature and thermal stability: The optimum temperature for hydrolysis when using p-nitrophenyl ester of dichlorovinyl chrysanthemic acid (DCPI) as a substrate is 50 to 55 ° C in 100 mM Tris-HCl buffer pH 9.0. Is. Residual activity after treatment in the same buffer for 10 minutes at each temperature is 35 ° C.
It is 100% at 40 ° C, about 80% at 40 ° C, and about 15% at 50 ° C. 5) Molecular weight: 54,000 ± 2,000 (by gel filtration and SDS-polyacrylamide gel electrophoresis) 6) Isoelectric point: 4.8 ± 0.1 7) Inactivation condition: a) 100 μM phenylmethylsulfonyl fluoride (PMSF) or 80 μM diisopropyl Fluorophosphoric acid (DI
When it is treated at 37 ℃ for 10 minutes in the presence of FP), it is completely inactivated. b) 100 mM Tris-HCl buffer (pH 9.0) containing 1 mM sodium deoxycholate and 5 mM sodium lauryl sulfate
Treatment at 37 ° C for 10 minutes reduces the activity by about 25% and about 70%, respectively. 2. A method for producing a novel esterase, which comprises culturing a novel esterase-producing bacterium belonging to the genus Bacillus and separating and recovering the novel esterase from the culture. 3. A Bacillus (Bacillus) is a new esterase producing bacteria belonging to the genus Bacillus sp DC-1 (Bacillus s
p.DC-1) (Ministry of Microbiological Research, No. 8719), The method according to claim 2.