# Patent

## Title
Esterase, its DNA, its overexpression and production of optically active aryl propionic acids using the same

## Abstract

The present invention relates to an esterase, its DNA, its overexpression and a method for preparing an optically active aryl propionic acid of formula (1) using the same in high yield,




wherein R1 represents an aryl group; and R2 represents a hydrogen atom.

## Background
This application is a continuation-in-part of application Ser. No. 10/118,047, currently now abandoned, as originally filed on Apr. 9, 2002, and incorporated herein by reference.


FIELD OF THE INVENTION
The present invention relates to an esterase, its DNA, its overexpression and a method for preparing optically active aryl propionic acids using the same in high yield. More particularly, the present invention relates to an esterase having a stereoselective hydrolyase activity, its manufacturing method for mass production by using recombinant E. coli expression system and a method for preparing optically active aryl propionic acids expressed by the following formula (1) using the same,




wherein R1 represents an aryl group; and R2 represents a hydrogen atom.

BACKGROUND OF THE INVENTION
Indeed, FDA (Food and Drug Administration)'s Policy Statement for Development of New Drugs recommends “that the pharmacokinetic profile of each isomer should be characterized in animals and later compared to the clinical pharmacokinetic profile obtained in Phase I” drug testing. Thus, the demand for racemic switch technologies to produce each pure isomer has been rapidly increased in recent years.
Aryl propionic acids are non-steroidal anti-inflammatory drugs and known as profen drugs such as ibuprofen, ketoprofen, naproxen, flurbiprofen, fenoprofen, suprofen and the like. It is generally believed that (S)-profens have higher pharmacological effects of the racemic mixture of profens bearing at least one benzene ring. A method for preparing optically pure (S)-profen drugs involves the conversion of a racemic mixture of profen ester to optically active profen carboxylic acid by reacting with a stereoselective chiral enzyme.
However, it has been recently reported that (R)-enantiomers of profens also exhibit therapeutic effects. Particularly, U.S. Pat. No. 6,255,347 discloses that (R)-enantiomer of ibuprofen may be used as a prophylactic and therapeutic agent in the treatment of diseases such as cancers, Alzheimer's and Alzheimer's-related diseases. In the method for preparing (R)-enantiomer of aryl propionic acid, a racemic mixture of aryl propionic acid is treated with an esterase to produce an ester of (S)-enantiomer of aryl propionic acid and un-reacted (R)-enantiomer of aryl propionic acid is recovered.
Further, inventors of the present invention have identified the presence of a stereoselective hydrolase activity in Pseudomonas sp. and its use in the preparation of (S)-profen (KR Patent Application No. 2000-02565). U.S. Pat. No. 6,201,151 discloses a process for preparing an optically active (S)-aryl propionic acid by hydrolyzing racemic thioester of aryl propionic acid in the presence of a (S)-stereoselective lipase. KR Patent Application No. 2001-0044879 discloses a process for preparing optically pure acetylmercaptoisobutylate using an esterase isolated from Pseudomonas aeruginosa. KR Patent Application No. 1996-14399 discloses a process for preparing optically pure aryl carboxylic acid stereoselectively from a racemic mixture of α-aryl carboxylic acid using S-(-)-α-ethyl benzylamine. KR Patent Application No. 1999-0042314 discloses a process for preparing optically active carboxylic acids and esters as drugs for the treatment of hypertension using a hydrolase isolated from Klebsiella pneumoniae. U.S. Pat. No. 5,516,690 discloses that (S)-ketoprofen can be produced with a purity greater than 95% using isolated Trichosporon laibacchii. 
However, the above-mentioned enzymes were not shown suitable for the selective production of optically pure (S)- or (R)-enantiomers of aryl propionic acid and there has been no report on mass production of those enzymes.
SUMMARY OF THE INVENTION
Accordingly, an object of the present invention is to provide an esterase having excellent stereoselectivity and its DNA sequence.
Another object of the present invention is to provide a method for producing the esterase in a mass production scale by overexpression of the esterase in recombinant E. coli. 
Further object of the present invention is to provide a process for preparing optically pure aryl propionic acid in high yield using the esterase,




wherein R1 represents an aryl group; and R2 represents a hydrogen atom.




BRIEF DESCRIPTION OF THE DRAWINGS
The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the accompanying drawings, in which:
FIG. 1 represents a manufacturing process of an esterase expression vector, pEESTa;
FIG. 2 represents a manufacturing process of an esterase expression vector, pEUbiESTa;
FIG. 3 represents a manufacturing process of an esterase expression vector, pErxESTa;
FIG. 4 represents an acryl amide gel electrophoresis of an esterase expression;
FIG. 5 represents an acryl amide gel electrophoresis of an esterase purified via anion exchange chromatography; and
FIG. 6 represents an acryl amide gel electrophoresis of an esterase purified via gel chromatography.



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention relates to an esterase having excellent stereoselectivity, its DNA and its mass production by transformation thereof. In accordance with one aspect of the invention, there is provided a method for preparing optically pure aryl propionic acid of formula (1) using the same esterase,




wherein R1 represents an aryl group; and R2 represents a hydrogen atom.

The present invention is described in detail as set forth hereunder. The esterase of the present invention is identified by the SEQ. ID. NO: 1 for its gene and the SEQ. ID. NO: 2 for its amino acid sequence and has a molecular weight of 41 kDa.
Further, the esterase derived from Pseudomonas sp. BHY-1 hydrolyzes racemic ester of a carboxylic acid unsymmetrically to produce the corresponding optically pure carboxylic acid. On the other hand, Pseudomonas sp. BHY-1 has a stereoselective hydrolase activity to convert racemic ester of aryl propionic acid to one-enantiomer aryl propionic acid. The racemic ester of aryl propionic acid used as a substrate may be prepared from racemic profen by a conventional method. Examples of the profen include ketoprofen, ibuprofen, naproxen, flurbiprofen, fenoprofen, suprofen and the like.
The inventors of the invention have selected Pseudomonas sp. BHY-1 exhibiting excellent stereoselectivity from soil and analyzed gene sequence of the esterase to obtain optically pure aryl propionic acid. Further, the present invention provides a construction of a recombinant E. coli expression vector to produce the esterase in an industrial scale. A novel esterase expression vector named as a pEESTa is constructed by introducing an NdeI restriction site to the N-terminal of the esterase and an XhoI restriction site to the C-terminal, performing PCR (Polymerase chain reaction) to amplify DNA fragments, and incorporating a T7 promoter and a T7 terminator. Other pEUbiESTa and pETrxESTa vectors are also constructed by introducing ubiquitin and thioredoxin to improve production efficiency. These vectors are also incorporated with T7 promoter and T7 terminator and produce an active esterase more effectively. Theses expression vectors are then transformed into E. coli to produce E. coli transformants BL21(DE3)/pEESTa, BL21/pEUbiESTa, and BL21/pETrxESTa.
Thus obtained E. coli transformants BL21(DE3)/pEESTta are cultured and the cultured E. coli is then recovered. Examples of the profens used as a substrate to identify an activity of the obtained recombinant esterase include ethyl esters of ibuprofen, ketoprofen, naproxen, and flurbiprofen. As a result, an enantiomeric excess (eep) of (S)-enantiomer profen produced by using the recombinant esterase of the present invention is not lower than 98%. It is preferable to maintain the pH in the range of from 6.0 to 12.0, more preferably from 8.0 to 10.0 and a temperature of from 15 to 80° C., more preferably 30 to 80° C. during resolution of aryl carboxylic acids. Thus obtained recombinant esterase may be purified by ion exchange chromatography, metal affinity chromatography or gel chromatography.
This invention is explained in greater detail based on the following Examples but they should not be construed as limiting the scope of this invention.
PREPARATION EXAMPLE
Preparation of Racemic Profen Ethyl Ester
Racemic profen (30 g) and ethanol (100 mL) were mixed and reacted in the presence of hydrosulfuric acid (2.5 mL) at 90° C. for 5 hours. The unreacted ethanol was removed by evaporation under the pressure. The reaction mixture was extracted with 1 M sodium bicarbonate solution three times to obtain racemic profen ethyl ester.




  
    
      
        
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Example 1
Sequence Analysis of a Novel Esterase Gene
Chromosomal DNA isolated from Pseudomonas sp. BHY-1 was partially digested with Sau3A, ligated with BamHI-digested pUC119 vector and then was transformed into E. coli DH5α. One of the clones, carrying a plasmid named as pT7HY (about 3 kb), exhibited enzymatic activity producing (S)-ketoprofen from (R, S)-ketoprofen ester and was chosen for further study. And also, the results showed that the novel esterase gene has a molecular weight of about 41 kDa. Transformants were selected based on a tributyrin hydrolysis as well as a stereoselectivity towards ketoprofen ester. The results showed that the novel esterase gene has a molecular weight of about 41 kDa and consists of 1,143 bp nucleotides (381 amino acids). The gene was registered in Genbank of NCBI and was assigned the Reg. No. AF380303 but has not been published yet. The novel esterase is identified by the SEQ. ID. NO: 1 for its gene and the SEQ. ID. NO: 2 for its amino acid sequence.
Example 2
Construction of an Expression Vector for a Novel Esterase Gene
Chromosomal DNA isolated from Pseudomonas sp. BHY-1 was partially digested with Sau3A, ligated with BamHI-digested pUC119 vector and then transformed into E. coli DH5α. One of the clones, carrying a plasmid named as pT7HY (about 3 kb), exhibited enzymatic activity producing (S)-ketoprofen from (R, S)-ketoprofen ester. The novel estererase cDNA coding sequence was amplified by PCR using pT7HY as a template. The primers used in the above PCR are as follows.










N-terminal primer
[SEQ. ID. NO: 3]









5′-GGG AAT TTC CAT ATG CAG ATT


CAG GGA CAT TAC GAG CTT CAA TTC-3′


 








C-terminal primer
[SEQ. ID. NO: 4]







5′-CCG CTC GAG TTA CAG ACA AGT GGC


TAG TAC CCG CGC CAG-3′






The N-terminal primer was introduced with an NdeI restriction site and also ATG was introduced as an initiation codon in place of GTG, whereas the C-terminal primer was introduced with an XhoI restriction site. The product with a size of about 1,100 bp obtained from the above PCR was double-digested with NdeI and XhoI and then separated on an agarose gel. The novel esterase gene fragment isolated from the above agarose gel was ligated into a 5,400 bp DNA fragment of pET22b (Novagen Co., Ltd., U.S.), an E. coli expression vector, double-digested with NdeI and XhoI by using a ligase. Then, an expression vector was constructed so that the novel esterase gene can be expressed, wherein its gene translation is carried out by T7 promoter and T7 terminator, and was named as pEESTa (FIG. 1). The vector pEESTa was then transformed into E. coli BL21(DE3) according to Simanis. Thus transformed E. coli BL21(DE3)/pEESTa was deposited to the Genebank of KRIBB on Nov. 20, 2001 and assigned the Accession No. KCTC 10122BP.
Example 3
Expression of a Novel Esterase Gene
The above E. coli transformant BL21(DE3)/pEESTa (KCTC 10122BP) was cultured in a solid LB medium (yeast extract 0.5%, tryptone 1%, and NaCl 1%). Thus cultured E. coli was inoculated into a liquid LB medium containing ampicillin (50 μg/mL), and then re-cultured at 37° C. until the OD600 reached 0.6. Then, the culture was added with isopropylthio-β-D-galactoside (IPTG) to the final concentration of 1 mM and cultured further for 4 hr for the expression of an esterase gene. Cold shock response was employed for the production of an active esterase because an esterase becomes in the form of an insoluble inclusion body, which has little enzyme activity, when E. coli transformant BL21(DE3)/pEESTa (KCTC 10122BP) is produced by culturing at 37° C. (FIG. 4). Cold shock response is a method to produce an active enzyme wherein a given culture is incubated at 37° C. until the expression is induced by IPTG followed by lowering the culturing temperature to 5–25° C. (Pamela G. Jones & Masayori Inouye, The cold-shock response, Mol. Microbiol., 11, 5, 1994).
Example 4
Construction of an Expression Vector for a Novel Ubiquitin-fused Esterase Gene
A 228 bp fragment encoding ubiquitin (76 amino acids) was amplified by PCR using Saccharomyces cerevisiae genomic DNA as a template. The primers used in the PCR are as follows.










N-terminal primer
[SEQ. ID. NO: 5]









5′-GGG AAT TTC CAT ATG CAC CAC CAC


CAC CAC CAC CAA ATT TTC GTC AAA ACT CTA ACA-3′


 








C-terminal primer
[SEQ. ID. NO: 6]


5′-ACC ACC CCT CAA CCT CAA GAC-3′






The N-terminal primer was introduced with an NdeI restriction site while the C-terminal primer, where a novel esterase is to be ligated, was treated to be blunt-ended. The product (fragment 1: 228 bp) obtained from the above PCR was digested with NdeI and then separated on an agarose gel.
The coding region of novel esterase was isolated by PCR. The primers used in the above PCR are as follows.










N-terminal primer
[SEQ. ID. NO: 7]









5′-CAG ATT CAG GGA CAT TAC GAG CTT CAA TTC-3′


 








C-terminal primer
[SEQ. ID. NO: 4]







5′-CCG CTC GAG TTA CAG ACA AGT GGC TAG TAC CCG-3′






The N-terminal primer was treated to be blunt-ended so that it can be ligated to ubiquitin sequence and then introduced with an XhoI restriction site. The product (fragment 2: 1,100 bp) obtained from the above PCR was digested with XhoI and then separated on an agarose gel.
The novel esterase gene fragment as well as the ubiquitin gene fragment (PCR-amplified product) isolated from the above agarose gels were ligated into a 5,400 bp DNA fragment of pET22b (Novagen Co., Ltd., U.S.), which was digested with NdeI and XhoI by using a ligase. Then, an expression vector was constructed so that an esterase can be expressed, wherein its gene translation is carried out by a T7 promoter and a T7 terminator, and was named as pEUbiESTa (FIG. 2).
Example 5
Construction of an Expression Vector for a Novel Thioredoxine-fused Esterase Gene
In order to increase the rate of production and expression of the novel esterase having an activity, an expression vector introduced with thioredoxine was constructed. The novel estererase cDNA coding sequence was amplified by PCR using pT7HY as a template. The primers used in the above PCR are as follows.









N-terminal primer
[SEQ. ID. NO: 8]








5′-CCG GAA TTC CAG ATT CAG GGA CAT


TAC GAG CTT CAA TTC-3′


 








C-terminal primer
[SEQ. ID. NO: 4]







5′-CCG CTC GAG TTA CAG ACA AGT GGC TAG TAC CCG-3′






N-terminal of primers were treated with EcoRI so that they can be ligated to thioredoxine sequences and then introduced with an XhoI restriction site. The PCR product (1,100 bp) was gel purified and digested with EcoRI and Xho.
The novel esterase gene fragment isolated from the above agarose gel was ligated into a 5,900 bp DNA fragment of pET32b (Novagen Co., Ltd., U.S.), an E. coil expression vector that contains thioredoxine which was double-digested with EcoRI and XhoI, by using a ligase. Then, an expression vector was constructed so that the esterase can be expressed, wherein its gene translation is carried out by a T7 promoter and a T7 terminator, and was named as pETrxESTa (FIG. 3). The two fusion partners have six histidine tags and are thus easily purified and are also characterized in that they have special cleavage sites for ubiquitin hydrolase and enterokinase (FIGS. 2 and 3).
Example 6
Expression of an Esterase
The above E. coli transformant BL21(DE3)/pEESTa (KCTC 10122BP) was inoculated into an LB medium and cultured at 37° C. until the OD600 reached 0.6. Then, the culture was added with IPTG to the final concentration of 1 mM and cultured further for 4 hr to induce the expression of the fused esterase gene. The expressed fused esterase was identified on an SDS-PAGE gel (12% acrylamide) (see Example 3) and compared with the esterase in the Example 3 (FIG. 4). Cold shock response was employed for the production of an active esterase because an esterase becomes in the form of an insoluble inclusion body, which has little enzymatic activity, when E. coli transformant BL21(DE3)/pEESTa (KCTC 10122BP) is produced by culturing at 37° C. (FIG. 4). It is noteworthy that the culture is incubated at 37° C. until the expression is induced by IPTG followed by lowering the culturing temperature to 20° C., whereby the esterase is produced in an active form. The result showed that the above two fused proteins of ubiquitin-esterase and thioredoxine-esterase, which were both produced by cold shock response, were shown to retain their optical selectivity and hydration capability.
Example 7
Identification of a Novel Esterase Expression
The culture was centrifuged for 20 min at 7,000 rpm and the cells were recovered. To study the expression level of the esterase that is expressed, the whole cells were divided into a soluble fraction and an insoluble fraction via sonication and its expression was examined. Three samples such as a whole fraction, a soluble fraction and an insoluble fraction, was dissolved in 100 μL of protein solubilizing buffer solution (12 mM Tris-HCl pH 6.8, 5% glycerol, 2.88 mM mercaptoethanol, 0.4% SDS, 0.02% bromophenol blue) and then heated for 5 min at 100° C. Ten μL each of thus formed solutions was loaded onto a polyacrylamide gel, wherein a 0.75 mm thick 12% gradient separating gel (pH 8.8, 20 cm(W)×10 cm(H)) was covered with a 5% stacking gel (pH 6.8, 10 cm(W)×12 cm(H)). Then, electrophoresis was performed for 80 min (120 V, 60 mA) and the gel was stained with Coomassie Blue. The gel scanning (BioRad, Imaging Densitometer GS-700, U.S.) result of the esterase revealed that the expression level after IPTG induction was 46.7%, and 94.2% of the total expression was present in the form of an insoluble inclusion body.
Example 8
Purification of a Novel Esterase Via Anion Exchange Chromatography
Ion exchange chromatography was performed to purify the novel esterase produced from the recombinant E. coli. The chromatography was performed by using Q-Sepharose (Pharmacia Co., Ltd., Sweden) as a resin at pH 8.5 at the rate of 4.0 mL/min. Samples were prepared by crushing cell walls of E. coli by using a sonicator followed by filtering thus obtaining a soluble fraction through micro filter (0.2 μm). Q-Sepharose was equilibrated with 50 mM Tris-HCl (pH 8.5) buffer solution. The esterase was fractioned by using NaCl linear gradient of an eluent buffer solution (1N NaCl/50 mM Tris-HCl, pH 8.5) wherein the sample was first put into the chromatography column followed by a thorough rinse with an equilibrium buffer solution. Thus purified esterase was identified on an SDS-PAGE gel electrophoresis as in the Example 6 (FIG. 5).
Example 9
Purification of a Novel Esterase Via Gel Chromatography
Gel chromatography was performed by using the fraction obtained from the above anion exchange chromatography. The chromatography was performed by using Sephacry S-200-HR (Pharmacia Co., Ltd., Sweden) as a resin at pH 8.5 at the rate of 0.3 mL/min. Samples were prepared by filtering the fraction obtained from the ion exchange chromatography through micro filter (0.21 μm). Sephacry S-200-HR was equilibrated with 50 mM Tris-Cl/10 mM NaCl buffer solution. The esterase was fractioned after putting the sample into the chromatography column and flowing it at the rate of 0.3 mL/min. Thus purified esterase was identified on an SDS-PAGE gel as in the Example 6(FIG. 6).
Example 10
Effect of Optical Resolution Conditions on Optical Resolution of a Novel Esterase
1. Effect of pH
The hydration by a novel esterase is mostly performed in a buffered solution and thus the structure of the enzyme can be influenced much by the pH and chemical properties of a buffer solution being used. When using Pseudomonas sp. BHY-1 as a whole cell enzyme, the optimal enzyme activity was observed at pH 8.5. In the case of the novel esterase, the enzyme activity was shown to have a relatively wide pH range of 7–11 and the optical selectivity was shown to be optimal at pH 10.0 as shown in the following Table 1.







TABLE 1







Optimal pH of Esterase









PH














7.0
8.0
8.5
9.0
10.0
11.0

















Conversion (%)
1.8
2.4
2.0
2.2
7.9
5.4


Enantiomeric excess
100
100
100
100
100
41


(%)









2. Effect of Temperature

Optimal temperature for optical resolution is affected by the fictive temperature, defined as racemic temperature, and the optical selectivity in response to a temperature increase tends to vary depending on the kind of an enzyme. The novel esterase of the present invention is shown to have an excellent optical selectivity and the following shows the reaction rate of the enzyme. The reaction rate was observed at 10° C.–90° C., a temperature range for culturing Pseudomonas sp. BHY-1, and the optimal reaction rate was observed at 60° C.







TABLE 2







Optimal temperature of Esterase









Temperature (° C.)















30
40
50
60
70
80
90


















Conversion (%)
7.9
8.9
11.5
13.1
12.8
10.9
9.9


Enantiomeric excess
100
100
100
100
100
41
41


(%)









3. Type of Reaction Substrates

Reaction substrates are in the form of ester and are mostly water insoluble. Therefore, it becomes necessary to mediate the reaction substrate to bind the enzyme for a desired enzyme reaction. In general, organic solvents such as dimethylsulfoxide, dimethylformamide, tetrahydrofuran, cyclohexane, benzene, etc., or a non-ionic surfactant are used to serve the above mediation purpose. It is important to determine an organic solvent or a surfactant suitable for a given substrate. In profen pharmaceuticals, for example, Triton X-100 and dimethylsulfoxide were shown most effective.
Example 11
Optical Resolution of Aryl Propionic Acid by Using a Novel Recombinant Esterase
Hydration was performed using 20 mM esters of ibuprofen, ketoprofen, and flurbiprofen to produce optically active ibuprofen, ketoprofen, and flurbiprofen. The reaction was performed at 37° C. (pH 8.5) with a reaction volume of 500 μL. Twenty four hours after the enzyme reaction, there was about 40% of conversion and enantiomeric excess (eep) was higher than 98.5% of optical selectivity as shown in the following Table 3.











TABLE 3







Substrate
Conversion (%)
Enantiomeric excess (%)




















Ibuprofen
40.9
>99



Ketoprofen
39.3
>99



Flurbiprofen
41.4
99










The novel esterase of the present invention derived from Pseudomonas sp. BHY-1 can be used in producing optically pure (S)- or (R)-type of aryl propionic acid having a pharmaceutical activity with high efficiency from racemic aryl propionic acid.

## Claims

1. An isolated esterase gene identified by SEQ. ID. NO:1.

2. An expression vector containing the esterase gene in claim 1.

3. The expression vector according to claim 2, wherein said expression vector is pEESTa, pEUbiESTa or pETrxESTa.

4. An isolated cell culture transformed by the expression vector in claim 2.

5. The cell culture according to claim 4, wherein said cell culture is E. coli BL21(DE3)/pEESTa, L21/pEUbiESTa, or BL21/pETrxESTa.

6. A method for preparing an esterase for mass production by isolation and purification of expressed esterase from the transformed cell culture in claim 4.