1Department of Molecular Science and Technology, College of Engineering, Ajou University, San 5, Woncheon-dong, Paldal-gu, Suwon 442-749 and 2Institute of Biotechnological Industry, College of Engineering, Inha University, 253 Yonghyun-dong, Nam-gu, Incheon 402-751, Korea
3 To whom correspondence should be addressed. e-mail: geunkim{at}inha.ac.kr
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Abstract |
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Keywords: bioinformatics/chiral resolution/esterase/gene mining/(S)-ketoprofen
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Introduction |
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Non-steroidal anti-inflammatory drugs (NSAIDs) have been widely used for alleviating pain and inflammation associated with tissue injury (Hayball, 1996). Among them, ketoprofen [(R,S)-2-(3-benzoylphenyl)propionic acid], as an in vitro inhibitor of prostaglandin synthesis, is one of the most prevalent anti-inflammatory agents belonging to the class of 2-arylpropionic acids (Mauleon et al., 1996
; Patal, 2000
). Since ketoprofen has been produced by chemical synthesis and thus sold as a mixture of stereoisomers, much effort is currently being devoted to enzymatic synthesis of optically pure ketoprofen for pharmacological use, because the activity is mainly exerted by the S-enantiomer (Caldwell et al., 1988
). In this context, with their abundance and great versatility in mediated reactions, esterases have recently been considered as a possible candidate for the chiral resolution of ketoprofen (Shen et al., 2001
; Bornscheuer, 2002
; Kim et al., 2002
).
We have recently screened various ecological niches and found a strain that expresses a novel esterase with relatively high activity and enantioselectivity (Kim et al., 2002). Although the results had partly seemed promising for practical applications, further attempts to obtain more potential enzymes failed, because the known information and also screening procedures were restricted to the whole cell level, mainly owing to the absence of established genetic data and sequence information. Therefore, we have designed a protocol to extend the scope for the screening of potential enzymes from natural sources.
In this paper, we present a systematic approach to a screening procedure for the isolation of potential enzymes from the natural pool of strains by combining pre-existing tools. The principle and practical application of this systematic approach are demonstrated by employing (R,S)-ketoprofen ethyl ester as a target compound for esterase-mediated chiral resolution.
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Materials and methods |
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Fast Blue RR, (R,S)-ketoprofen, -naphthyl acetate, tributyrin, Triton X-100 and ethoxyethanol were purchased from Sigma, acrylamide stock (30%) and protein assay solutions from Bio-Rad and agarose from Promega. Restriction enzymes, Taq DNA polymerase and T4 DNA ligase were obtained from KOSCO (Korea) and Vent DNA polymerase from New England Biolabs. (R,S)-Ketoprofen ethyl ester was prepared using a general procedure for esterification (Kim et al., 2000
).
Strain enrichment, selection and identification
Hundreds of samples were collected from various ecological niches, such as compost, forest, contaminated soils and sewage sludge, and suspended in a phosphate buffer (0.85% saline solution) or a previously formulated TS medium without tributyrin (Kim et al., 2002). Enrichment for the activity screening was also performed by using TS medium, as described previously (Kim et al., 2002
). The screened colonies were then inoculated into 50 ml of TS medium with or without 10 mM ketoprofen ethyl ester. After cultivation at 30°C for 24 h, the cells were harvested and then confirmed for activity to rac-ketoprofen ethyl ester. The strains showing a high ester-hydrolyzing activity were finally screened and systematically identified by general procedures from Bergeys Manual of Systematic Bacteriology and 16S rRNA sequencing, and further classified by morphological and genetic traits.
Cell growth and fractionation of crude extracts
After sorting the positive strains, the strain pool of Pseudomonas was cultivated at 30°C in 500 ml baffled flasks containing 100 ml of culture medium supplemented or not with ester derivatives as an inducer. Inoculating cells were prepared by pre-culture of a well-isolated colony in TS medium (10 ml) for 24 h. During the cultivation, an aliquot of culture broth was sampled and analyzed for cell growth and enzyme activity.
To obtain information at the protein level, crude extracts were fractionated to prepare the fraction with an activity to rac-ketoprofen ethyl ester. Pseudomonas cells from 0.5 l of culture broth were harvested and resuspended in a buffer A (20 mM TrisHCl, pH 8.0) containing protease inhibitor cocktail solution (Sigma) and disrupted using a sonicator. Cell debris was removed by centrifugation at 14 000 g for 30 min and the supernatant was treated with protamine sulfate (2%). After centrifugation, the supernatant was desalted with a prepacked column (Sephadex G-25) and then loaded on to an ion-exchange column (Resource Q, 6 ml) using an FPLC system (Pharmacia). The column was washed with 10 volumes of buffer A and eluted with a linear gradient of 00.5 M NaCl. The active fractions were pooled and then stored for further characterization.
Molecular mass, specific activity and enantioselectivity
The molecular masses and oligomeric structures of probable esterases in active fractions were determined on an FPLC system with a gel filtration column (Superdex-75). The flow rate of the mobile phase, containing 20 mM TrisHCl and 150 mM NaCl, was 0.5 ml/min. A molecular mass calibration curve was established by plotting the elution volumes of protein markers (Sigma) versus the known molecular masses on semi-logarithmic paper. Aliquots of eluted fractions were analyzed for the activity to rac-ketoprofen ethyl ester.
To determine the specific activity and enantioselectivity, the activity on the ketoprofen ethyl ester was determined at 30°C for 3060 min with the fractionated enzyme (3050 mg) in 3 ml of reaction mixture containing 50 mM TrisHCl (pH 8.5), 0.3% Triton X-100 and 5 mM substrate. The reaction was stopped by the addition of four volumes of ethanol (>99%) and then analyzed for the conversion yield and chiral selectivity.
Native gel electrophoresis and activity staining
For activity staining on native PAGE, protein samples were mixed with 0.2 vol. of a native sample buffer and resolved on a 12% gel under non-reducing conditions (Kim et al., 2002). After gel electrophoresis, the separating gel was washed twice with 20 ml of 50 mM TrisHCl buffer and then soaked in the same buffer (100 ml) containing 4 mg of
-naphthyl acetate dissolved in 0.5 ml of ethoxyethanol. The band corresponding to the active enzyme was visualized by the addition of Fast Blue RR (2 mg/ml) and then sliced. The activity was further confirmed with the sliced gel by using rac-ketoprofen ethyl ester as a substrate.
Protein database search and sequence alignment
With the information obtained from the fractionated crude extracts, enzymes and their sequences were searched for in databanks, including the complete genome sequence of Pseudomonas aeruginosa PA01 (Stover et al., 2000) and then analyzed further using the BLAST network service at the National Center for Biotechnological Information (NCBI). To access closely to the best candidate genes or proteins, valuable data, such as N-terminal amino acid sequence, ORF size, quaternary structure, apparent PI and substrate spectrum, were fully considered for gene mining. Primarily selected probable sequences were compared manually or automatically on annotated sequences of an esterase pool from various genomes and the protein data bank. The resulting sequences were aligned by hierarchical clustering of the individual sequences based on the pairwise similarity scores. The conserved patterns of amino acid sequences in related enzymes were analyzed with the Clustal W program (Thompson et al., 1994
) and then confirmed by visual inspection. The distribution of the secondary structural elements in these sequences was obtained by using a previous algorithm (Cuff and Barton, 2000
).
Probing the related genes by low-stringency PCR
The amplification of probable genes was performed by PCR with two wobble primers, N-terminal ESTF, 5'-A(G)TGCAG(C)ATTCA(G)G(A)GGT(C)CAT(C)TAC(T) GAA(G,C)-3', and C-terminal primer ESTR, 5'-TTACAG ACAA(C)C(G)C(T)G(C)G(C)CCA(G)A(G)TAT(C)(T,A)TCC(G)-3', which were designed based on terminal sequences of systematically mined esterase genes. Chromosomal DNA was isolated from each strain of Pseudomonas by using a kit (Promega). The partially digested or intact genomic DNA (2.520 ng) was used as the template for PCR (1 cycle, 94°C, 7 min; 40 cycles, 94°C, 1 min, 37.5 ± 0.4°C/cycle, 1 min, 72°C, 90 s; 1 cycle, 72°C, 7 min). To expand the amplifying sequence, the stringency at the annealing step was further modulated (Kim et al., 2001).
Cloning and selection of a potential esterase
The amplified DNA fragments under low-stringency conditions were reamplified by PCR with two primers, ESTF1 and ESTR1, which introduced the BamHI and PstI sites into the N- and C-terminal primer, respectively. For rapid purification and high-level expression, the reamplified genes were subcloned into the BamHI and PstI sites of a series vector pQE 30, 31 and 32 and thus expressed as poly-His-tag fusion proteins at their N-termini using a different reading frame, according to the general procedure of the manufacturer.
The positive clones were screened from transformants by activity staining using an overlaid soft agar (0.6%) containing Fast Blue RR (15 mg/ml) and -naphthyl acetate (45 mg/ml).
-Naphthyl acetate was first dissolved in ethoxyethanol and then added to soft agar solution. The positive clones rapidly (<5 min) developed a deep brown color around the colony, as reported previously (Kim et al., 2002
).
Enzyme purification and characterization
Escherichia coli cells expressing the fusion protein were grown in 200 ml of LB medium at 30°C to an OD600 nm of 0.40.5 and then induced with 0.2 mM IPTG for 2 h. After centrifugation at 10 000 g for 10 min, the cells were resuspended in 10 ml of phosphate buffer (50 mM, pH 8.0) containing 10 mM imidazole. The suspended cells were sonicated and then centrifuged at 18 000 g for 30 min. The resulting supernatant was loaded on to Ni-NTA resin (Qiagen) and the bound protein was eluted with a buffer containing 250 mM imidazole.
The enzyme properties were analyzed in terms of molecular mass, specific activity, substrate spectrum, enantioselectivity and conversion yield, by using the purified enzyme, according to the same procedures as above and from elsewhere (Fernandez et al., 2000; Kim et al., 2002
). All experiments were conducted in duplicate and mean values were calculated.
Analyses
The concentrations of (R,S)-ketoprofen ethyl ester and (R)- and (S)-ketoprofen were determined by HPLC (Waters). The column and mobile phase used were Chirex Phase 3005 (Phenomenex) and methanol containing 30 mM ammonium acetate, respectively. At a constant flow rate (0.8 ml/min), the eluate was monitored at 254 nm. One unit of esterase activity was defined as the amount of enzyme producing 1 mmol of ketoprofen from the corresponding ethyl ester per minute under the specified conditions. Protein concentration was measured by using a protein assay solution (Bio-Rad).
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Results and discussion |
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Step 1. Strain enrichment and activity screening.
By considering the previous results that established a screening procedure for the strains expressing an ester-hydrolyzing enzyme, the activity screening was repeated to broaden the strain pools (Kim et al., 2002). From various environmental samples, thousands of strains were first enriched and then analyzed for activity by using a TS agar plate or a selective plate overlaid with soft agar (0.6%) containing Fast Blue RR and
-naphthyl acetate as an activity indicator. Based on the developed clear zone around colonies,
150 strains were readily detected and further confirmed the activity to (R,S)-ketoprofen ethyl ester under favorable conditions for cell growth and enzyme assay. The strains showing a high growth rate and distinct clear zone were compared again for their potential for activity and enantioselectivity in a solid or solution culture and then finally the strains that had a strict selectivity to an S- or R-enantiomer form werescreened. As a result, 24 strains were chosen as possible candidates for further analyses.
Step 2. Strain identification and sorting. The next step was a further comparison with respect to the phenotypic and genotypic characters for strain classification. Among the screened isolates, the strain pool (nine out of 24 strains) was motile, rod-shaped, Gram-negative, no endospore-forming, pyruvate-utilizing and aerobic microorganisms. All strains showed both catalase- and oxidase-positive reactions and either fluorescence or not. They also utilized glucose, fructose, galactose and citrate as carbon sources. The optimal growth temperature was 2530°C and they could not grow at 40°C. The G + C content of the genomic DNA was in the range 5266%. The 16S rRNA sequence analyses revealed a fairly high homology (>96%) to the typical species of Pseudomonas, including P.fluorescens, putida and aeruginosa. The level of 16S rRNA identity, therefore, together with physiological and taxonomic properties, strongly suggested that the nine strains might be related to one of the typical strains of Pseudomonas. No distinct differences between the analyzed and documented characters of the genus were found as an ambiguous trait. Therefore, these strains were taxonomically identified as Pseudomonas strains according to Bergeys Manual of Systematic Bacteriology. The results were also confirmed by systematic approaches in the Korea Collection for Type Culture (KCTC). The remaining strains (15 out of 24) that had a relatively low activity were taxonomically identified as various strains including Bacillus, Mycobacterium, Streptococcus and Rhodococcus (data not shown).
Step 3. Data gathering from the strain pool of identified genus.
Although the activity from Pseudomonas on the rac-ketoprofen ethyl ester was not fully established to date, the screening results provided the possibility that most Pseudomonas strains could hydrolyze the ethyl ester in a stereospecific manner, which was also supported by our previous work (Kim et al., 2002). A further attempt to confirm the hydrolyzing activity on the rac-ketoprofen ethyl ester was carried out by employing various species of Pseudomonas. For this purpose, two fluorescens (KCTC1767 and 2344), two aeruginosa (KCTC1636 and 2450) and a putida (KCTC1642) were randomly chosen from the KCTC as representative strains. The two isolates screened, Pseudomonas sp. S34 (Kim et al., 2002
) and HS12 (Park and Kim, 2000
), were also employed as possible sources for the activity. Among these strains, only one case (S34) has been reported to possess an activity to rac-ketoprofen ethyl ester.
The seven strains were first analyzed as possible sources for potential esterases by using a selective TS plate. All strains tested were well grown and developed a clear zone distinctly, although the colony size and clear zone were somewhat different. Further experimental results that exhibited the rac-ketoprofen ethyl ester hydrolyzing activity to the R- or S-enantiomer or both, strongly suggested the presence of an esterase in these strains. As shown in Table I, when the cells were grown in TS and LB media, different enantioselectivity and conversion yield were observed, with a similar trend in the relative activity. A relatively high activity and enantioselectivity were found in the three strains S34, KCTC2450 and HS12. In contrast, two strains, KCTC1636 and 1767, revealed a minor activity to ketoprofen ethyl ester. Two strains, KCTC2450 and S34, fully retained the enantioselectivity to the S-enantiomer, whereas the enantioselectivity in other strains was partly affected or reversed by the culture conditions. Other factors, such as temperature, inducers, carbon and nitrogen sources, that probably affected the cell growth and enzyme production, did not improve the results significantly.
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Step 4. Bioinformatic analyses and gene(s) mining.
Gene mining using only preliminary biochemical and taxonomic data is partly troubleshooting. Fortunately, however, these data are valuable for a typical sequence that is predicted as an open reading frame from whole genome sequencing. In this context, data obtained from probable esterases could be sufficient to mine a sequence, at least to deduce a representative from the complete genome sequence of P.aeruginosa PA01 (Stover et al., 2000). In order to find candidate genes encoding an esterase, the whole genome sequence of PA01 was analyzed and then compared with data obtained above, yielding a sequence (NP_249738) as a possible one for the ketoprofen ethyl ester-hydrolyzing enzyme. In addition, the search results in protein data banks revealed a subfamily comprising a related set of esterases (AF228666, JC2091, A44832 and 2006221A), all of which also originated from the identical genus Pseudomonas. The first one is still uncharacterized to date and the last four enzymes have already been identified as a (carboxyl)esterase (McKay et al., 1992
; Kim et al., 1994
). However, not all cases have been reported on the activity to ketoprofen ethyl ester.
In accordance with the properties of crude enzymes, all mined enzymes were composed of a similar number of amino acid residues (377392) and oligomeric structure (monomer). From the reported cases (McKay et al., 1992; Kim et al., 1994
), the analyzed properties for optimal pH and substrate spectrum were also found to be similar to those of the crude extracts. The superimposable features in the nucleotide and amino acid sequences further supported the assumption. Consistent with these views, the N-terminal amino acid sequence (VQIQGHY) of the esterase from S34 also supported the contention that the mined enzymes might be able to hydrolyze rac-ketoprofen ethyl ester, because the sequence was also found in the mined enzymes.
Step 5. PCR amplification for functional expression.
From a further analysis of the mined sequences at the molecular level, the nucleotide and amino acid sequence identities at their N- and C-termini were also found to be rigidly conserved and thus sufficient to amplify the whole ORF from related chromosomes by using the degenerated primers (Figure 2). To verify and access the related genes, PCR amplifications were carried out using the chromosomal DNA of seven strains and a set of degenerated primers. Because the reaction yielded a smeared or no amplified DNA under normal conditions, low-stringent PCRs were selected to enhance the amplification of related genes (Kim et al., 2001; Yuen et al., 2001
). Under these relaxed conditions, the expected size of DNA fragments (
1.11.3 kb) were amplified by PCR from the chromosomal DNA of KCTC1642, 1767, 2344, 2450 and S34 (data not shown). When using two chromosomes (HS12 and KCTC1636) as templates, the reactions were inefficient and thus resulted in a minor or no band corresponding to the expected size.
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Characterization of a potential esterase
The crude extracts of E.coli cells harboring the gene from KCTC1767 showed a distinct protein band (>7% of total cell protein), corresponding to the expected size when induced with 0.25 mM IPTG for 2 h (Figure 3A). The enzyme was mainly expressed in the soluble fraction, while a minor portion was detected in the insoluble fraction (<5%). With the crude extracts of induced cells, the enzyme was easily purified to apparent homogeneity by using an affinity resin, Ni-NTA, in a single step (Figure 3A).
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To compare the primary structure with other mined sequences, the nucleotide sequence of the PCR-amplified gene from KCTC1767 was completely determined on both strands. Analysis of the DNA sequence showed a complete ORF (1146 bp) of 381 amino acid residues with a calculated molecular mass of 41 kDa (Figure 4). As expected, its amino acid sequence revealed a close identity to the mined sequences used here for primer design. As reported previously in the related genes, the ORF did not utilize ATG but GTG as the start codon (Gold et al., 1981; Kim et al., 1994
) and ended with TAA. The calculated molecular mass was in good agreement with those determined by SDSPAGE and gel filtration. A relatively high G + C content (65%) of the ORF also supported its origin being from a strain of Pseudomonas (Stover et al., 2000
). As expected, the sequence showed a distinct amino acid homology (8293%) and similar structural organization to the mined enzymes through the entire region, as revealed in the identical or counterpart enzymes. The consensus sequences conserved in the majority of esterases, HG and GXSXG, were also found at the identical positions. These results also strongly suggest that the systematically mined esterases might be able to hydrolyze rac-ketoprofen ethyl ester to (R)- or (S)-ketoprofen, although this remains to be proven. These results will assist us in exploiting the enzymes to obtain further information about their structure and function (Henke and Bornscheuer, 1999
).
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Given the need to hydrolyze stereospecifically an unnatural substrate, such as rac-ketoprofen ethyl ester, that could not induce activity and also could not be used easily as a indicative substrate, the principal question is what strategy is likely to be most effective. In instances in which the esterase-producing cell does not grow well in culture with such a substrate and does not correlate well between the potential and activity of an enzyme, alternative approaches must be used. The enzyme selected here may not be found if the screening is conducted by an activity-based approach, especially when using ketoprofen ethyl ester for the primary screening. As a promising result, we anticipate that it is indeed possible, by using the mined enzymes, to produce an economically valuable drug, (S)-ketoprofen, and also equally valuable products, ibuprofen and naproxen.
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Acknowledgements |
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Received July 4, 2002; revised January 20, 2003; accepted April 4, 2003.