WO2001032847A9 - Novel esterases derived from pseudomonas aeruginosa, its gene and process for production of optically active carboxylic acids using them - Google Patents

Abstract

The present invention relates to a novel strain, Pseudomonas aeruginosa, the gene encoding esterase, and the method for the production of RAM from (R,S)-ester using this esterase. In detailly described, this invention discloses the novel esterase and the nucleotide sequence producing optical active RAM, the expression vector overproducing RAM from (R,S)-ester, transformant containing expression vector, and the method of enzyme solution for the production of RAM.

Description

NOVEL ESTERASES DERIVED FROM PSEUDOMONAS AERUGINOSA, ITS GENE AND PROCESS FOR PRODUCTION OF OPTICALLY ACTIVE CARBOXYLIC ACIDS USING THEM

TECHNICAL FIELD

The present invention relates to a novel strain, Pseudomonas aeruginosa, the esterases therefrom, and the method for the production of optically active carboxylic acids of following formulae I and esters of their enantiomers via stereospecific hydrolysis of racemic carboxylic esters. In detailed description, the present invention disclosed the novel esterase derived from Pseudomonas aeruginosa; the gene encoding the esterase; the recombinant expression vector containing its gene; the transformant containing the expressing vector; and the method of enzyme solution for production of optically active carboxylic acids by using the esterases from culture of the transformant.

FORMULAE I

RiCOS— (CH₂)n— C fH-COOH

wherein RI is alkyl, arylalkyl, or aryl, and R2 is alkyl, and n denotes 1 or 2. BACKGROUND ART

The optically active carboxylic acids are used as intermediates for production of various biologically active pharmaceuticals, especially captopril (Ondettii et al., Science 196, 441(1977)) or analapril (Japanese Patent publication

No. 63-18599) known as drugs for hypertension treatment. The Policy Statement or the Development of New Stereoisomeric Drug, announced by the FDA in 1992, recommends that novel chiral drugs should be stereochemically single enantiomers for the approval of FDA. In case of production of drugs as racemic form, the provision requires their inventor to demonstrate that novel drugs have no harm to animals and human when used in the form of each enantiomer as well as racemate. In other words, the production of drugs or their intermediates in the racemic forms is indirectly but ultimately restricted now. Main reason of these trends is that in most of chiral drugs, one enantiomer shows positive biological activity while the other show ill effects such as toxicity, or an adverse reaction.

Therefore, the research and studies for chiral compounds useful in production of chiral drugs become more and more necessary in the aspect of development of competitive power of the pharmaceutical industry and improvement of national health. Because Enalapril and Captopril, ACE-type inhibitors (angiotension- converting enzyme inhibitors), have important position to occupy about 30% of world markets of optically active pharmaceuticals, the development for biotechnological production of the inhibitor's intermediates such as (R)-2- hydroxy-4-phenyltanoate and (R)-β -acetylmercaptoisobutyrate is urgently required. Because the production costs of optically active drugs depends on the unit costs of their intermediates and properties of their intermediates, the import of the intermediates has the same effect, compared to the import of finished products. Due to high added-value of production technology for them, only the transfer of the technology for the intermediates is not avoided, but also the export of their intermediates themselves is occasionally difficult. Consequently, the introduction of the technology for production of the optically active drugs or their intermediates is almost impossible. Therefore, the development of production of the intermediates, if possible, closer to final products is the best solution, and development of the production system for commercially useful intermediate of optically active drugs, or (R)-β -acetylmercaptoisobutyrate, can provide very productive and significant results in this view, wherein (R)-β - acetylmercaptoisobutyrate is used as a precursor for production of several major chiral drugs such as captopril, tocopherol, rasalosid A, calcimycin, β -lactam antibiotics, and (R)- or (S)-muscone. The above-mentioned optically active carboxylic acids can be obtained by asymmetric hydrolysis from racemic carboxylic acid esters represented in formulae II.

FORMULAE II RjCOS— (CH₂)n— CH— COOR₃

, wherein RI, R2, and n are as defined in formulae I, and R3 is alkyl. In general, production methods of optically active carboxylic acids by chemical synthesis have encountered many difficulties, such as low selectivity, complicated chiral resolution, causing serious environmental pollution, or decrease of economical efficiency therefrom. For that reason, as alternatives for chemical synthesis methods, development of production system for chiral compounds using biocatalysts such as microorganisms or enzymes have been and is recently attempted by many world wide research groups. The strain, which has specific activity of selectively hydrolyzing only

(R)- or (S)-carboxylic acid esters, has been reported(Japanese Patent publication

No. 1-222798). But according to above patent, the esterases derived from the strain itself were of too small amount to be used for overproduction of optically active carboxylic acids. So our focus of research and development is adjusted on cloning of the gene encoding esterase and the overexpression in E. coli using recombinant DNA techniques. Japanese Patent publication No. 64-67190 discloses the gene encoding esterase producing optically active (R)-carboxylic acids from racemic carboxylic acid esters by specific activity on (R)-esters from Pseudomonas βuorescens

IFO3018, the nucleotides and amino acid sequences, and techniques producing recombinant esterase from E. coli. It is reported the cloning of gene encoding esterase from Pseudomonas putida FERM BP-3846, the nucleotides and amino acid sequences, and expression in recombinant E. coli, and physical properties of recombinant esterase (US Patent No. 5308765, US Patent No. 5482847, Ozaki et al., Biosci. Biotech. Biochem. 59, 1204(1992)). The esterases from above strains is different in nucleotide and amino acid sequences, and the esterase from P. fluorescens IFO3018 consists of 218 amino acids, while the esterase from P. putida FERM BP-3846 consists of 276 amino acids. In thermal stability of the above two esterases, the esterase from P. putida FERM BP-3846(70°C) is more stable than the esterase from P. fluorescens IFO3018(50°C). The esterase from P. putida FERM BP-3846 has the molecular weight of about 30,000 Da and the isoelectric point of pH 3.90 ± 0.1, and its optimum pH is 7.0 and this esterase is stable in pH 6.0~8.0. Present inventors tried to screen novel strain and novel esterase which can produce optically active carboxylic acids. As a result, novel esterase, which has stability at temperature range above 70 ^°C and about 35 kDa of molecular weight, was found from Pseudomonas aeruginosa isolated from soils, and its encoding gene was separated. The present invention has goals to serve novel thermostable esterase producing optically active carboxylic acids from carboxylic esters; and the gene encoding this esterase. Also this invention discloses novel E. coli strain producing the esterase; and the physicochemical properties of the esterase. The present invention includes the transformation of E. coli using recombinant expression DNA plasmid containing esterase-coding gene; and methods producing esterase from culture broth of recombinant E. coli strain. Finally the present invention provides the method of producing optically active carboxylic acid by the esterase.

DETAILED DESCRIPTION OF THE INVENTION

To accomplish above-mentioned purpose, this invention discloses novel esterase from Pseudomonas sp. strain. This esterase has the amno acid sequence described in SEQ ID No. 2, the molecular weight of about 35,000 Da, and the isoelectric point of pH 6.4. The esterase was isolated from Pseudomonas aeruginosa with the activity producing optically active (R)- or (S)-carboxylic acid ester specifically by the asymmetric hydrolysis of racemic carboxylic acid esters. The amino acid sequences of esterase in this invention were compared with those of triacylglycerol lipase from Moraxella sp. and Psychrobacter immobilis and carboxyl esterase from Acinetobacter calcoaceticus. As the result of comparing them, there is consensus sequences, G-X-S-X-G, containing 169-Ser in the amino acid of esterase. The serine residue is positioned at the active site of enzymes containing esterases and lipases, and also shows its typical feature that there is another concensus sequences, dipeptide containing His-Gly at 70 amino acids upstream from G-X-S-X-G. Further this invention includes the gene encoding novel esterase. The gene has the nucleotide sequences described in SEQ ID No. 1 and CDS(coding sequence) described in SEQ ID No. 3. The ratio of G+C in the entire nucleotide sequences is 67.41%. The inventors have submitted the nucleotide sequences of novel esterase to GenBank in accession No. AF170828. The present invention discloses Pseudomonas aeruginosa producing novel esterase. During culture in the first-selection medium including (R,S)- acetylmercaptoisobutyric acid methyl ester and an indicator, the strains active to carboxylic acids were screened from soil samples gathered in several sites of Korea and India, on criteria of the color change of the culture medium. From the first selected strains, the superior strain in the production of (R)-carboxylic acids was finally selected by quantitative analysis using GC(gas chromatography). The finally selected strain by the above described method has been identified. In the chemical taxonomic analysis of the cell wall(particularly quinone is an important component in chemotaxonomic analysis), the cell was observed to contain ubiquinone-9, and in the cellular fatty acid analysis, C18:l and C16:0 were found to be the major components. From the results, the strain of this invention was identified as a typical Pseudomonas sp. and in the analysis for the identification of species, 16S rRNA sequences of the selected strain were matched with those of Pseudomonas aeruginosa in 99.9% of homology. This strain, identified as Pseudomonas aeruginosa, was deposited in Korean Collection for Type.Cultures(KCTC) with the collection number KCTC8953P in August 4, 1999. For the overproduction of esterase, the inventors prepared recombinant esterase expression vector. First, for the amplification of the estA DNA fragment, PCR was done by using two designed primers described in SEQ ID No. 4 and No. 5. In N-terminal primer, Ncol site was coded, and His-tag residue (6 histidine) and BamΑl site were coded in C-terminal primer. After pT7ES vector was prepared by inserting the extracted DΝA fragments into the blunt cleavage site of the restriction enzyme EcoRI of pT7blue, the prepared pT7ΕS was partially digested with Ncol and combined with pET22b vector, resulting in recombinant esterase expression vector, pES22b (FIG. 1). The restriction site map of pES22b is described in FIG. 4. The pES22b was transferred to E. coli BL21 and the recombinant E. coli was named to E. coli BL21 PES, and the inventors deposited E. coli BL21 PES in KCTC with the collection number KCTC8952P in August 4, 1999. The present inventors obtained the cell from culture-broth of the recombinant E. coli BL21 PES and isolated recombinant esterase from cell- disrupted supernatant. The stereochemical activity of the recombinant esterase in selective hydrolysis of (R,S)-acetylmercaptoisobutyric aicd methyl ester was investigated on the basis of GC analysis of the chemically synthesized diastereomer. It was ascertained that the recombinant esterase catalyzed asymmetrical hydrolysis of the ester bond of (R)-form of racemic ester. In the chemical synthesis of (R)-acetylmercaptoisobutyric acid from (R,S)- acetylmercaptoisobutyric acid methyl ester, the product is generally a mixture(racemic form) with (S)-form, while, in the hydrolysis of the esteratse presented in this invention, we can obtain specifically (R)-form of the carboxylic acid, which can be used in the preparation of various kinds of pharmaceuticals. Thermal stability of the recombinant esterase produced by the recombinant E. coli BL21 PES has been investigated. The supernant containing the esterase, which was prepared by disrupting and centrifuging the cultured cell, was incubated at each temperature, and its activity test was performed at 30^°C . As a result, the esterase of this invention maintain its activity at 70 ^°C and from the fact, we can demonstrate that the esterase should be thermally stable above 70 ^°C

DESCRIPTION OF THE FIGURES

FIG.1 shows the cloning strategy of gene encoding esterase producing (R)-carboxylic acid. FIG.2 shows the restriction map and the localization of gene encoding esterase producing (R)-carboxylic acid. (The pTBL7, pTBL71, and pTBL72 are the insert ligated into pBluescript KSII+ plasmid vector. The other plasmids are the insert ligated into pUC119 plasmid vector. The symbol, '+' or '-', means ability of the production of RAM) FIG.3a and 3b show the comparison with the homology of amino acid sequence producing (R)-carboxylic acid to those of other esterase. 1 : estA, the gene encoding esterase in present invention 2: triacylglycerol lipase from Moraxella sp. 3, 4: triacylglycerol lipase from Psychrobacter immobilis 5, 6: carboxyl esterase from Acinetobacter calcoaceticus FIG.4 shows the construction of the expression vector, pES22b. FIG.5 shows SDS-PAGE showing proteins expressed by recombinant E. coli PES containing pES22b 1: 4 hr, 2: 2 hr, 3: 1 hr, 4: 0 hr, M: molecular marker FIG.6 shows GC chromatogram of the diastereomers of RAM producing recombinant E. coli in present invention and the authentic RAM and SAM. (a): the diastereomer of RAM producing recombinant E. coli in the present invention (b): the diastereomer of the authentic SAM(ii) and RAM(iii). In the Figures, the symbols are defined as followed: B = BamHl H = Nhel N = Ncol R = Nrul K = Kpnl Bl = BamΑllSaύik RAM = (R)-acetylmercaptoisobutyric acid SAM = (S)-acetylmercaptoisobutyric acid Ap = ampicillin resistant gene lac I = β -galactosidase gene EXAMPLES

The following examples are presented to illustrate this invention and are not to be considered as limiting the scope of the appended claims.

EXAMPLE 1

Screening of Strains Producing Esterase with Asymmetric Hydrolyzing Activity

Microorganisms were screened from soil samples collected near the oil industries of Korea and India. The soil samples (1.0 g) were suspended in distilled water (9.0 ml), and 200 1 of different diluted suspensions were poured on nutrient agar plates (Bacto beef extract 3g/l, Peptone 5g/l, Agar 15 g/1) and incubated at 30 ^°C for 24 hr. Single colonies grown on the plates were isolated and screened optical active producing strain using primary selection liquid medium. The composition of medium is 1% (v/v) of (R,S)- acetylmercaptoisobutyric acid methyl ester, 0.1% (w/v) of bromocresol purple as a pH indicator in 0.05M potassium phosphate buffer, pH 7.0. Isolated strains incubated in primary selection liquid medium at 30 ^°C for 4 hr, and were observed the change of color in the medium. Strains capable of producing the enzyme hydrolyzing (R,S)-ester produce the carboxylic acid, which changed the color of bromocresol purple from blue to yellow, due to decrease in pH. Many isolates could change the color of the reaction mixture and were selected as (R,S)-ester consumers (Akihiro et al. 1992). For the isolation of RAM-producing microorganisms, RAM-producing bacteria were selected among (R,S)-ester consumers from GC and HPLC analysis of RAM production from (R,S)-ester by the isolates. Twenty one strains changed the color of the reaction mixture from blue to yellow and were considered to be the positive strains capable of hydrolyzing both the ester-bond and the thioester-bond in (R,S)-ester. For the screening of best producing strain, 21strains were cultured in glucose medium (glucose 2.66%, ammonium sulfate 0.5%, yeast extract 0.066%, corn steep powder 2.0%, pH 6.8-

7.0) at 150rpm and 30 °C for 24 hr. Cells obtained by the centrifugation of 3 ,000 rpm for 30 min were used for the bioconversion. One strain, named as IS 1001, was selected as the best producer of RAM from (R,S)-ester from the GC and HPLC analysis of RAM-formation.

EXAMPLE 2

Identification of Screened Strain

IS 1001 strain, screened in Example 2, was identified by measuring the fatty acid and quinone compound composition, and comparing with database (Yoon et al., Int.J.Syst.Bacteriol., 47, 933, 1997). In the chemical taxonomic analysis of the cell wall (particularly quinone is an important component in chemotaxonomic analysis), the cell was observed to contain ubiquinone-9, and in the cellular fatty acid analysis, C18:l (oleic acid) and C16:0 (palmitic acid) were found to be the major components. In addition to this, the cellular fatty acids also contain 3-hydroxy C10:0 (capric acid) and C12:0 (lauric acid), specially. From the above mentioned results, IS 1001 strain was identified as a typical Pseudomonas sp. For molecular systematic analysis of IS 1001 strain, 16S rRNA analysis of IS 1001 strain was done. 16S rRNA sequence of the best isolated strain (IS 1001) was 1491-nt described in SEQ ID No. 6. Sequencing of 16S rRNA genes of IS 1001 revealed affiliation with Pseudomonas cluster with the y -subclass of the Proteobacteria (Table 1). TABLE 1

% similarity in 10 11 12 13 14 15 16 17 Strain IS 1001 Pseudomonas 99. aeruginosa 9 Pseudomonas 96. 96. mendocina 8 7 Pseudomonas 97. 97. 97. alcaligenes 7 7 2 Pseudomonas 97. 97. 97. 97. fragi 0 0 0 5 Pseudomonas 97. 96. 95. 96. 95. resinovorans 0 9 2 6 7 Pseudomonas 97. 97. 97. 97. 99. 95. stulzeri 3 2 4 7 7 9 Pseudomonas 95. 95. 95. 95. 97. 96. 97. balearica 9 9 4 7 3 2 6 Pseudomonas 96. 96. 95. 96. 96. 95. 96. 95. citronellolis 8 8 3 6 0 6 3 4 Pseudomonas 95 95 97 96 96 94 97 95 95 putida 3 2 6 5 7 1 0 2 0 Pseudomonas 94 94 96 95 95 94 95 95 94 97 chlororaphis 7 6 0 0 3 6 5 8 1 2 Pseudomonas 93 93 95 94 95 93 95 94 94 97 97 vmdiflava 9 8 5 4 0 3 2 7 2 0 4 Pseudomonas 94 94 95 94 94 93 94 94 93 96 97 97 fluorescens 3 2 4 8 8 6 9 6 7 7 4 5 Acmetobacter 87 87 88 87 87 86 88 87 87 88 88 87 88 calcoaceticus 9 9 3 7 8 9 0 5 0 0 6 9 4 Oceanospmllum 88 88 89 88 89 87 89 89 88 89 89 89 89 87 hnum 9 9 2 2 0 7 0 6 4 3 3 0 4 6 Escherwhiacoli 85 85 86 85 86 84 85 86 85 86 86 86 86 85 85 4 4 0 4 1 9 9 2 7 2 2 6 9 9 9 Chromatium 85 85 84 84 84 84 84 85 84 84 85 85 85 83 83 83 tepidum 0 0 4 0 7 1 5 5 6 4 0 1 0 5 6 7 Legionella 86 86 86 86 87 85 86 87 86 87 88 87 87 85 87 83 85 pneumophila 9 9 8 7 1 9 9 5 4 3 0 2 8 9 4 9 0 Comamonas 82 82 82 82 82 82 82 83 83 81 82 82 82 80 81 82 82 811 lerrigena 6 6 0 3 7 0 3 4 4 9 7 4 5 6 4 0 4

The 16S rRNA levels of similarity for all strains used in this analysis ranged from 81.1 to 99.9%, especially from 93.6 to 99.9% with the strains in Pseudomonas cluster. IS 1001 strain was related to Pseudomonas aeruginosa with a sequence similarity of 99.9%. So IS 1001 strain was identified as Pseudomonas aeruginosa 1001, and deposited in Korean Collection for Type Cultures (KCTC) with the collection number KCTC8953P to apply for a Korean Patent.

EXAMPLE 3

Isolation of Genomic DNA from Pseudomonas aerueinosa Chromosomal DNA was isolated from Pseudomonas aeruginosa identified in Example 2 using G-NOME isolation kit (Bio 101, La Jolla, Calif., USA). Pseudomonas aeruginosa was inoculated in 5ml of LB medium containing bacto trypton 10%, bacto yeast extract 5%, NaCl 10%, pH 7.0 and cultured at 30 °C for 24 hr. Cells were obtained by centrifugation for 5 min and suspended in lOmM Tris-Cl, pH 8.0, 0.1M EDTA, pH 8.0, and finally bring the cell to a final volume of 1.85ml in cell suspension solution. Then 50 fd of RNase and 100 fd of the cell lysis/denaturation solution, 0.5% SDS were added to the cell suspension solution, mixed well, following the incubation at 55 ^°C for 15 min. For the removal of protein in the solution, 25 fd of protease was added and incubated at 55 °C for 1 hr, and 500 fd of salt out mixture was added and mixed gently. Genomic DNA solution was divided into 1.5 ml tubes and refrigerated at 4^°C for 10 min. Pellets were obtained by the centrifugation for 10 min and the supernatant was transferred into 15 ml of tube. DNA precipitation was occurred by the addition of 2 ml of TE buffer, pH 7.5 and 8 ml of 100% ethanol. DNA was obtained by the centrifugation of 12,000 rpm for 15 min and dried in the air, and the dried DNA was dissolved in TE buffer. The DNA was partially digested with SaύiA and genome DNA library from Pseudomonas aeruginosa was obtained by the elution of DNA from gel slice having partially digested genomic DNA after electrophoresis using 10% agarose gel.

EXAMPLE 4 Preparation of Recombinant Plasmid

As decribed in Example 3, the genomic DNA was partially digested with

Sαw3Al and then ligated with BamR I-digested pBluescript^®IIKS+ vector (Stratagene Inc., USA), following to transfer to E. coli DH5α (Stratagene Inc.,

USA). For the screening of strains containing gene encoding esterase among the transformants, the following experiment was performed. Initial screening of clones were transferred onto tributyrin plate (lOg/1 of tributyrin, 15g/l of agar, lOg/1 of trypton, 5g/l of yeast extract, lOg/1 of NaCl) and incubated at 37 ^°C for 24 hours. The enzyme activity is indicated by clear zone around the colonies. The hollow zone may be due to any lipolytic enzyme. For secondary screening, the colonies with the hollow zone were transferred to an agar plate containing (R, S)-ester (1%, w/v) and bromocresol purple (0.1%, w/v) as a pH indicator. When (R, S)-ester is converted to RAM, the carboxyl acid produced resulting in pH decrease. Hence the color of bromocresol purple changes from purple (pH 6.2) to yellow (pH 4.5). Finally the enzyme activity and the RAM production were detected by GC and HPLC. RAM was analyzed by high-performance liquid chromatography (HPLC) using CHIREX(R) NGL & DNB column 250 x 4.6mm (Phenomenex Co., USA). The mobile phase containing 20 mM ammonium acetate in methanol at a flow rate of 1.0 ml/min was used. RAM and (R,S)-ester were detected by measuring UN absorbance at 254 nm. Products were identified by comparison of retention time with those of authentic standards. The hydrolyzed products, the carboxylic acid, and RAM production were separated and determined by gas chromatography (HEWLLET PACKARD 5890) using a SUPELCOWAX-10 capillary column, 30 m x 0.53 mm, 0.5 μm film (Supelco Co., USA), equipped with a flame ionization detector (FID). The column temperature was programmed from 100^°C to 220 ^°C in 20°C/min increasing speed after 1 min initially.

Injector and detector temperature was 170^°C and 180 ^°C, respectively. Nitrogen was used as carrier gas at flow rate of 20 m³/min and n-Butanol was used as an internal standard. One of the clones, carrying a plasmid termed pTBL7 (about 4.5-kb), exhibited enzyme activity producing RAM from (R, S)-ester and was chosen for the further study.

EXAMPLE 5

Screening and Analysis of Recombinant Plasmid and Subcloning

The strategy of cloning is described in FIG. 1. The preparation of plasmid DNA from transformed E. coli was carried out by the general boiling method (Sambrook et al, Molecular cloning, 1989) and Plasmid Midi Kit (Quiagen Inc. USA). The analysis was done with the electrophoresis using 0.8 -

1.0% gel after the digestion with the restriction emzymes (Kpnl, BamHl, Nhel, Nrul, Ncol, Seal et al.). The restriction map and the localization of gene encoding esterase were described in FIG. 2. In FIG. 2, the esterase gene was inserted into pBluescript^®IIKS+ vector (Stratagene Inc., USA) in pTBL7, pTBL71, and pTBL72, and in the other plasmids inserted into pUCl 19 (Stratagene Inc., USA). For the localization of estA gene on the 4.5-kb DNA fragment of pTBL7, the subcloning was done, yielding pTBL71, pTBL72, pTBL73, pTBL74, pTBL75, pTBL76, and pTBL77 (Fig.2). The RAM-producing activity of each plasmid was measured by the formation of RAM from (R,S)-ester using its lysate (Fig.2). As a result of subcloning, estA gene was located within Nhel-Nrul DNA fragment of pTBL76 (about 1.1 -kb).

EXAMPLE 6

Nucleotides and the Derived Amino Acids Sequences of Cloned Esterase

The DNA fragment (pTBL73) to be analyzed was transferred to pUCl 19 and the plasmid DNA was prepared. The plasmid DNA was digested with two enzymes, Xbal and Sphl, one leaving a 3 " single strand overhang and the other a blunt end or 5 ^*" overhang. The digested DNA was then incubated at 37^°C in a reaction mixture containing about 10 μg DNA, 100 μg of the exonuclease III buffer (2 x lOOmM Tris-HCl (pH 8.0), lOmM HgCl₂), and 1 μl (about 180 units) exonuclease III. Reaction mixture (100 μl) was transferred in 10 μl aliquots at one minute intervals to the 100 μl Mung Bean nuclease buffer (10 x 300mM NaOAc (pH 5.0), 500mM NaCl, lOmM ZnCl₂, 50% (v/v) glycerol) and then inactivated by incubation at 65 °C for 5 minutes. Mung bean nuclease (2 μl, 50 units) was added and incubated at 37°C for 1 hr. After DNA was extracted with phenol, it was dissolved in 50 μl Klenow buffer and incubated at 37C for 15 minutes with 1 1 (2 units) of Klenow fragment. Then the ligation step was carried out and transformed into E. coli. Plasmid DNA prepared from the transformants was digested with two restriction enzymes, EcoRI and Hindlϊl. The size of the deletion created was determined by agarose gel (0.8%) electrophoresis. Clones having different sizes of insert DNA with about 300-bp intervals were selected for nucleotide sequencing. And the sequencing was performed with

ABI PRISM 377 autosequencer (Perkin-Εlmer, USA). The length of pTBL73 was found to be 2455-bp by nucleotide sequencing. And open reading frame (ORF) search is performed using DNASIS program (MacDNASIS Pro V3.5, Hitachi Software Engineering Co., LTD.) and Editseq program in DNASTAR program package. Major ORF was found within about 2.4-kb BamHl/Kpnl pTBL73 DNA fragment (Fig. 3). And ORF2 has 315 amino acids (molecular weight = 34,836 Da). From the amino acid homology search in NCBI blast, 38% of homology to triacylglecerol lipase precursor from Psychrobacter immobilis, 29% of homology to dihydrolipoamide acetyltransferase from Pseudomonas putida, and 28% of homology to esterase2 from Acetobacter pasteurianus. This ORF was named to estA and deposited to GenBank in accession no. AF170828. Deletional variants and internal primers were used for determination of the nucleotide sequence of the 2.5-kb BamHl-Kpnl fragment from plasmid pTBL73. Primary sequence editing was performed with LAGERGENE software package (DNASTAR Inc., Madison, Wis.). The BLAST program (31) was used for similarity searches of the nonredundant NCBI sequence database (National Center for Biotechnology Information, National Institutes of Health, Bethesda, Md.). The multiple alignment was designed by using the MegAlign Program, version 4.00, in DNASTAR and GeneDoc Program, version 2.3.00. The estA exits within an ATG codon at nucleotide 1 and terminatory with a TGA codon at nucleotide 948 in the pTBL73. The nucleotide and deduced amino acid sequence of the estA gene, is shown in Fig. 4. The estA gene had 948-bp nucleotides and 316 amino acids derived by computer analysis using the DNASTAR program (Madison, Wis.). The estA gene had an A+T composition of 32.59% and C+G composition of 67.41% and derived isoelectric point and the molecular weight were pH 6.4 and

34,836 Daltons, respectively. The molecular weight calculated from the deduced amino acid sequence (316 amino acids) was about 35-KDa, which agreed well with the value obtained by SDS-PAGE (Fig. 7). Esterase described in present inventor has more large size than Pseudomonas flourescence and Pseudomonas putida, and proved to be novel esterase having the different coding sequences. The amino acid sequence of the estA gene was compared with those of triacylglycerol Upases in Moraxella sp. and Psychrobacter immobilis, carboxyl esterases from Acinetobacter calcoaceticus, as described in FIG. 3a and 3b. Several consensus sequences within the homology alignment results (black shadowed in Fig. 5) are present. Among these consensus sequences shown in

Fig. 5, the deduced amino acid sequence contained the Upases and esterases active-sites H-G and G-X-S-X-G, which is found in the majority of bacterial and eucaryotic Upases and esterases. There are only a few reports related to esterase in P. aeruginosa, in contrast with a lot of references about esterases in P. fluorescence and P. putida. The enzyme (the estA gene product) from P. aeruginosa 1001 in this work is clearly related to a group of enzyme having a serine active triad, which is identical in many esterases and Upases. Amino acid sequences of the estA gene product from P. aeruginosa 1001 has consensus sequences , Gly-Asn-Ser-Met-Gly (Fig. 5) containing active serine residue. This Gly-Xl-Ser-X2-Gly is the consensus sequence for the active site of esterases, Upases, and serine proteases (35). However, there was no overall homology between them. Their catalytic triads were believed to be composed of Ser, His and Asp/Glu by analogy with the serine hydrolase whose active site structure were well established (36, 37). As shown in Table 2, several esterases and Upases have consensus sequences, H-G and G-X1-S-X2-G, and the distances (about 65-70 amino acids) between above consensus sequences is the similar. X means any amino acid. This result is in good agreement with the case of the estA gene product from P. aeruginosa

1001. The human hormone-sensitive lipase (HSL) has the role in energy homeostasis by catalyzing the hydrolysis of triacylglycerol and cholesterol ester, and transport the fatty acid to tissues. Triacylglycerol lipase has the activity to be controlled by reversible phosphorylation. These HSLs and triacylglycerol Upases have the same pentapeptide consensus sequences, G-X1-S-X2-G, containing the catalytic serine. Also at the 70-100 amino acids upstream from the consensus sequences, another consensus sequences, H-G, dipeptides, exist and this motif does the role to support the pentapeptide consensus sequences, G-Xl-S- X2-G, as a hydrophobic wing. The enzymes having the catalytic serine residue were acetylcholine esterases, thioesterases, Upases, and serine protease (35) in microorganismsm, animals and human. On the basis of the conserved amino acid, Ser-137 and His-286 were tentatively assigned as the residues of catalytic triad of P. aeruginosa 1001 esterase. Another consensus sequence of esterase in Pseudomonas sp. had been reported by Shimada et al. (36). According to this report, histidine residue exits at the downstream of G-X1-S-X2-G This concensus sequence was found in 2- hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HPDA) hydrolase (38), tropinesterase, 2-hydroxymuconic semialdehyde (HMC) hydrolase (39) from P. putida and Pseudomonas CF600 (40), too. This fact suggests that the RAM-producing esterase is a serine esterase and the Ser 137 is the active serine. Therefore it seems likely that Ser 165 of E. coli esterase, which is located at the center of the G-X1-S-X2-G motif, is involved in the catalytic triad of the enzyme (41). In the aspect of primary structure of enzyme, the RAM-producing enzyme from P. aeruginosa 1001 is belonging to esterase having the active serine motif. Example 7

Development of Transformed E. coli Strain Overproducing Recombinant Esterase

For the papration of expression vector (pES22b) containing estA gene, the estA gene was inserted in Ncol and BamHI site of pET22b plasmid vector (Novagen Inc., USA). To express the estA gene in E. coli, the estA gene was amplified by PCR using pTBL74 plasmid as a template. The PCR products were analyzed by agarose gel electrophoresis and confirmed by DNA fragment size (about 1-kb).

N-terminal primer, Ncol site was coded, described in SEQ ID Νo.4 and C- terminal primer, His-tag residue (6histidine) and BamΑl site, described in SEQ ID No.5 were used. The DNA was denatured at 95 °C for 30 sec, primers were annealed at 52 °C for 1 min, and the products were extended at 72 °C for 1 min. Thirty cycles were performed for the amplification of DNA. After amplification of DNA insert, PCR product was purified with GelClean III kit. And the DNA fragment was ligated with Ncol and Rα HI-digested pET22b, as described in restriction map (FIG. 4). Expression vector constructed from pET22b vector is shown in Fig. 4. For recombinant E. coli strain producing esterase, the expression vector, pES22b was mixed to the competent cell of E. coli

BL21 (DE3) for protein expression and stored on the ice for 30 min, and then heated at 42 °C for 30 sec. The pES22b was transferred to E. coli BL21 and the recombinant E. coli was named to E. coli BL21 PES, and the inventors deposited E. coli BL21 PES in Korean Collection for Type Cultures (KCTC) with the collection number KCTC8952P.

Example 8

Expression of Esterase in Recombinant E. coli

Expression of estA gene in recombinant E. coli was determined by

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS- PAGE was performed on 10% polyacrylamide gel with a Tris-glycine buffer system. The gels were stained with Coomassie blue and the molecular weight of the enzyme was determined from the relative mobilities of standard protein. The high-range prestained standards were purchased from GIBCO BRL (LIFE

TECHNOLOGIES Inc., Gaithersburg, USA). E. coli BL21 PES was cultured to O.D. = 0.5 in LB medium at 37 ^°C, and ITPG was added. After 1, 2, and 4 hr of induction, the cell was harvested and resuspended in chilled sonication buffer. Cell disrupted solution by sonication was centrifuged, and 501 of the supernatant containing esterase and 501 of sample buffer were mixed and boiled for 5 min, and loaded in 10% polyacrylamide gel. After ITPG induction, the recombinant having expression plasmid, pES22B, showed higher RAM-producing enzyme activity than control. As shown in SDS-PAGE of FIG. 5, esterase produced from E. coli PES is about 3.5-kD, which is in agreement with molecular weight deduced from amino acid sequence.

Example 9

Activity of Recombinant Esterase

One ml of (R,S)-ester-hydrolyzed mixture of by the whole cells was evaporated to remove solvent. One ml of 10% thionyl chrolide solution in 25 ml of n- hexane containing 2.5 1 of dimethylforamide was added to a vial, mixed thoroughly on a vortex mixer and incubated at room temperature for at least 30 min. The reagent was subsequently evaporated under a gentle stream of nitrogen at 50 °C . 0.3 ml of </-2-octanol was added to the dried residue and mixed thoroughly on a vortex mixer. The mixture was incubated at 60 ^°C for 30 min in a heating block. The reactants were evaporated under a stream of nitrogen at 50°C and the residue was dissolved in 0.5 ml of methylene chloride (Patel et al. 1995). One microliter of residual solution was injected for the GC analysis of diasteromers. As shown in FIG. 6, the diastereomers of authentic RAM and SAM were detected at 28.1 min and 23.9 min of the retention time, respectively.

Diastereomer of acetylmercaptoisobutyric aicd hydrolyzed by the enzyme of the whole cells of Pseudomonas aeruginosa 1001, described in Example 7, was also detected at 28.0 min of the retention time in the same condition. The enzyme in the whole cells of Pseudomonas aeruginosa 1001 catalyzed asymmetric hydrolysis of the ester bond of (R)-form of (R,S)-ester and converted it into RAM.

Example 10

Thermostability of the Recombinant Esterase

The stability of esterase from Pseudomonas aeruginosa in recombinant E. coli BL21 PES, prepared in Example was investigated using (R,S)-ester as a substrate. E. coli containing pTBL72 harboring estA was cultured in LB medium at 37°C for 24 hr and the cells were harvested by the centrifugation. And the cells were resuspended in 0.2 volume of sonication buffer (50 mM potessium phosphate buffer, pH 7.8, 300mM NaCl, Quiagen Inc., USA), and disrupted by the sonicator. The test of thermostability was carried out using the supernatant after centrifugation. Thermal stability of the enzyme in the whole cells was determined by measuring the residual enzyme activity of cells after incubation for 1 hr at the desired temperature ranging from 30 ^°C to 80 ^°C . Enzyme activity in the lysate and pellet was determined as follows. Activity was determined by the amount of RAM produced in enzyme reaction mixture.

One unit was expressed as lmol RAM-formation per minute per ml of enzyme solution at 37^°C . The activity of esterase in the whole cell is units/mg dry cell weight (DCW) and described in Table 2. Temperature (^°C) Activity (U/mg DCW) 30 404.09 40 426.36 50 438.63 60 429.09 70 420.91 As shown in Table 2, the activity of esterase in present invention is not dependent on the changes of temperature, and is stable at 70 ^°C .

INDUSTRIAL APPLICABILITY Since novel esterase from Pseudomonas a in this invention has the excellent ability of production of optically active carboxylic acids from carboxylic acid esters and the thermal stability at high temperatrue above 70 ^°C , the esterase can be useful for production of various kinds of physiologically active pharmaceuticals, specially hypertension treatment drugs like analapril or captopril and the synthetic method of optically active carboxylic acids using the esterase of this invention is highly more selective than prior chemical methods, simple, and environmentally favorable as well.

Claims

30

CLAIMS 1. A esterase having the amino acid sequences described as SEQ ID No. 2, and producing optical active carboxylic acid and enantiomeric isomer by the assymmetric hydrolysis of the carboxylic ester racemates. 2. A esterase, in the claim 1, producing optical active carboxylic acid as described in formulae I. FORMULAE I

RiCOS— (CH₂)n— CH— COOH

(wherein RI is alkyl, arakyl, or aryl, R2 is alkyl, n is 1 or 2) 3. A esterase, in the claim 1, producing optical active (R)-carboxylic acid.

4. A DNA, according to the claim 1, consisting essentially of the nucleotide sequence as described in SEQ ID No. 1 and encoding esterase.

5. A DNA, according to the claim 1, consisting essentially of the nucleotide sequence as described in SEQ ID No. 3 and encoding esterase.

6. A expression vector harboring gene encoding esterase described in the claim 5.

7. A expression vector according to the claim 6, PES22b, with the restriction enzyme map described in FIG. 4.

8. A transformant containing the expression vector described in the claim 5. 31

9. A transformant E. coli BL21 PΕS(KCTC 8952P) described in the claim 8 10. A preparation method producing optical active carboxylic acid from carboxylic acid ester racemates by esterase described in the claim 1 11. A Pseudomonas aeruginosa(KCTC 8953P) producing esterase described in the claim 1