Indian Patents. 268709:POLYPEPTIDE HAVING ESTERASE ACTIVITY AND RECOMBINANT ESTERASE AND USE THEREOF

Title of Invention	POLYPEPTIDE HAVING ESTERASE ACTIVITY AND RECOMBINANT ESTERASE AND USE THEREOF
Abstract	Polypeptide and recombinant protein having esterase activity which exhibit the amino acid sequence SEQ. ID. No. 1 and the use thereof.

Full Text	Novel polypeptide having esterase activity and recombinant esterase and use thereof The invention relates to a novel polypeptide having esterase activity, especially having 2-alkyl-5-halopent-4-enecarboxylesterase activity, and to an enzymatically active recombinant protein having esterase activity and to the use thereof for resolving racemates of 2-alkyl-5-halopent-4-enecarboxylic ester enantiomer mixtures. Enantiomerically enriched 2-alkyl-5-halopent-4-enecarboxylic acids and their esters are valuable intermediates for preparing pharmaceuticals, such as, for instance, for delta-amino-gamma-hydroxy-omega-arylalkanecarboxamides, which have renin-inhibiting properties and can be used as antihypertensive agents in pharmaceutical preparations. Esterases are generally employed in the resolution of racemates and asymmetrization. However, only very few esterases suitable for preparing chiral compounds are commercially available. The use of esterase extracts from pig liver is known on the preparative scale. Pig liver esterase (PLE) was isolated long ago from natural sources, and its activity has also been known for a long time (Simonds, J.P. (1919) Amer. J. Physiol. 48, 141; Bamann, E. et al. (1934) Hoppe-Seyler Z. 229, 15; Falconer J.S. and Taylor, D.B. (1946) Biochem. J. 40, 831-834) . Various studies have also already been carried out in order to characterize PLE (Heymann, E. and Junge, W. (1979) Eur. J. Biochem. 95, 509-518; Lehner, R. and Verger, T. (1997) Biochemistry 36, 1861-1868) . It has further been possible to show, for example in WO 01/09079, that esterase extracts from pig liver can selectively hydrolyze the (R) enantiomer of methyl 5-chloro-2-(1-methylethyl)-4-pentenoate. However, the use of such esterase extracts -from natural sources, such as pig liver, is associated with disadvantages. In the first place, the qualities of the different batches vary and thus make it difficult to optimize industrial processes. Secondly, the use of animal resources in the manufacture of pharmaceutical products is undesired because the presence of viruses and prions cannot always be precluded. For these reasons there is a need to produce recombinant pig liver esterases of standardized quality in microorganisms. The cloning of putative esterase genes is described for example in FEES Lett. (1991), 293, 37-41. The first functional expression of an active pig liver esterase enzyme was described for the first time in WO 02/48322. WO 2004/055177 describes the preparation of further recombinant esterases by site directed mutagenesis of the recombinant pig liver esterase of seq. ID No. 1 (rPLE) - from WO 02/48322. As is evident from the description of WO 2004/055177 and from the article authored by the same inventors in Protein Engineering, 16, 1139-1145, 2003, the modifications of the rPLE sequence from WO 02/48322 were chosen so that a recombinant intestinal pig esterase (PICE) disclosed in David et al. , (1998) Eur. J. Biochem. 257, 142-148, is obtained. The resolution of racemic 2-alkyl-5-halopent-4-enecarboxylic esters is not described in any of these articles. However, since the need for esterases which have the desired stereoselective activity for 2-alkyl-5-halopent-4-enecarboxylic esters and which can easily be prepared biotechnologically is not met, it was an object of the present invention to provide a corresponding novel recombinant esterase. In an attempt to isolate and to clone the gene described in FEBS Lett. (1991), 293, 37-41 and WO 02/48322 for known pig liver esterase (PLE) as cDNA starting from mRNA from pig liver, a second, novel esterase sequence was found in addition to the known PLE sequence. Following expression of the two sequences, in which the corresponding proteins or esterases, namely the known rPLE and a novel, recombinant "alternative" esterase (rAPLE), was prepared, it unexpectedly emerged that only the rAPLE is capable of selective resolution of racemic 2-alkyl-5-halopent-4-enecarboxylic esters. It was thus possible to achieve the object of the present invention by a novel polypeptide having esterase activity and a novel recombinant esterase (rAPLE) , whose amino acid sequence differs in 21 of a total of 548 amino acids from the known PLE sequence. The novel rAPLE differs in the amino acid sequence also from the known pig intestinal carboxylesterase (PICE) in 12 of a total of 548 amino acids. However, PICE is found in the pig intestinal tract, The present invention accordingly relates to a polypeptide having esterase activity, which comprises the amino acid sequence SEQ. ID. No. 1. The present invention further relates to a novel recombinant protein having esterase activity, which comprises the amino acid sequence SEQ. ID. No. 1. The polypeptide and the recombinant rAPLE of the invention have the ability to resolve stereoselectively racemic 2-alkyl-5-halopent-4-enecarboxylic esters of the formula (I) (Formula Removed) in which R is a C1-C6-alkyl radical, R2 is C1-C4-alkyl and X is chlorine, bromine or iodine. The polypeptide of the invention having esterase activity, and the novel recombinant esterase rAPLE differ, as stated above, in 21 of a total of 54 8 amino acids of the known sequence disclosed in FEBS Lett. (1991), 293, 37-41 and in 12 of a total of 548 amino acids from the known PICE protein disclosed in David et al., (1998) Eur. J. Biochem. 257, 142-148. The sequence of the protein of the novel rAPLE of the invention differs in the following amino acid positions from the known sequence of, the PLE protein: APLE Position PLE Glu 73 Asp lie 75 Val Gly 76 Val Gly 77 Glu Leu 80 Thr Arg 87 Gly lie 92 Thr Pro 93 Leu Val 129 Leu Ser 133 Pro Thr 134 Met Leu 138 Val Ala 139 Val Phe 234 Leu Ala 236 Val Gly 237 Ala Phe 286 Leu Ala 2 87 Thr Leu 290 Phe Pro 2 94 Gin Thr 302 Pro The protein of the invention and the novel recombinant rAPLE may moreover be in the form of a modified sequence as shown in SEQ ID No 1, which can be obtained for example by usual modifications such as, for instance, exchange, deletion or attachment of amino acid(s) in the sequence at the N or C terminus, such as, for instance, GluAlaGluAla from the a factor signal sequence, or by fusion to other proteins. The invention also further includes muteins having modifications within the protein sequence of the enzyme of the invention having the appropriate activity, in particular on 2-alkyl-5-halopent-4-enecarboxylic esters. Muteins can be obtained for example by modifications of the DNA which codes for the enzyme of the invention, by known mutagenesis techniques (random mutagenesis, site-directed mutagenesis, directed evolution, gene shuffling etc.) so that the DNA codes for an enzyme which differs at least by one amino acid from the enzyme of the invention, and subsequent expression of the modified DNA in a suitable host cell. The invention thus also includes modified DNA sequences as shown in SEQ ID. No 1, obtained by the mutations, deletions, extensions, fusions described above, and which code for enzymes having the desired esterase activity. Esterase activity, especially 2-alkyl-5-halopent-4-ene-carboxylesterase activity, is defined in this connection as the ability to resolve racemates of 2-alkyl-5-halopent-4-enecarboxylic esters of the formula (I). (Formula Removed) The polypeptide of the invention and the recombinant rAPLE can be prepared as described below: Firstly, mRNA is isolated from pig liver using a suitable kit, and then the cDNA is generated by reverse transcription based on the mRNA extract. Subsequently, specific PCR primers based on the sequence of the known pig liver esterase gene of GenBank accesssion No. X63323 (Matsushima et al. , 1991) is prepared, followed by amplification and cloning. These specific primers are: Primer 1: 5'-CAGAATTCATGGCTATCGGGCAGCCAGCCTCGC-3' Primer 2 : 5'-CCGGAATTCAGCCTCCCCTTCACAGCTCAG-3' This part of the primers which comprises the appropriate nucleotide sequences coding for the PLE protein and which is obligatorily present in the primers is in bold script. The other sequence part of the primers comprises for example information for cleavage sites for restriction endonucleases (in italics) or sequence elements which are important for expression. This part may vary in the preparation of the rAPLE of the invention. Amplification then takes place with primers 1 and 2 by prior art PCR methods. The PCR product is subsequently used to prepare by prior art methods expression constructs for heterologous expression of the encoded rAPLE protein in suitable host organisms. This preferably entails the PCR product being initially cloned into suitable plasmid vectors. The recombinant plasmids obtained in this way are then transformed into a suitable host, for example Escherichia coli. Inserts of several resulting clones are then sequenced. Unexpectedly, 2 groups of recombinant clones with different sequences were identified therein, one being 100% identical to the expected sequence for PLE according to Matsushima et al. , (1991) FEBS Lett. 293, 3 7-41, and a novel nucleotide sequence as shown in SEQ. ID. No. 2 (APLE sequence) which leads after expression to the amino acid sequence SEQ. ID. No. 1 of the invention. The present invention further relates to a nucleic acid or nucleotide sequence which codes for the polypeptide of the invention and the recombinant esterase rAPLE. For example, such a nucleic acid has the nucleotide sequence shown in SEQ. ID. No. 2. The invention also relates further to nucleotide sequences which include a nucleotide sequence which codes for the polypeptide of the invention and the recombinant esterase rAPLE, or comprises the nucleotide sequence shown in SEQ. ID. No. 2. A further possibility is to prepare appropriate oligonucleotides corresponding to nucleic acid sequences according to the present invention which code for the esterase of the invention by standardized synthetic techniques, for example with use of automated DNA synthesizers. The purely synthetic preparation of the nucleic acid sequences which code for the esterase of the invention is particularly advantageous for use in the production of pharmaceuticals or their intermediates, because enzymes are thus not obtained from animal sources. Expression of the two sequences found (PLE and APLE sequences) then takes place. The known pig liver esterase (PLE, Swiss-Prot ID Q2 9550) comprises an N-terminal signal sequence and a C-terminal ER retention signal, the last 4 amino acids HAEL. In order to express the known PLE and the novel APLE, vectors in which the sequences are introduced into suitable expression systems constructed. These expression constructs are then transformed into suitable host cells. Suitable host cells in this connection are for example microorganisms, animal cell lines and plants. Both prokaryotic and eukaryotic microorganisms can be employed. Preferred prokarytic hosts (bacteria) are Escherichia coli, and strains from the genera Bacillus (e.g. B.subtilis, B. licheniformis, B.amyloliquefaciens) , Pseudomonas (e.g. P.fluorescens, P.putida), or Streptomyces (e.g. S.lividana, S. tendae) Eukaryotic microorganisms are preferred, and fungi are particularly preferred. Examples thereof are Saccharomyces cerevisiae, Pichia pastorls, Kluyveromyces lactis or Aspergillus sp. . Expression may be secretory or intracellular and both inducible and constitutive. For bacterial expression a choice of species-specific signals can be obtained, a.o. ELS commercially available strains and vectors for protein expression (e.g provided by companies like Invitrogen, Novagen, New England Biolabs), that allow inducible or constitutive expression, intracellular and secretory localization of the target protein; in addition, technology to enable or promote the correct folding of proteins in order to result in soluble and active protein may be applied. The proteins are preferably expressed in a secretory manner, in which case vectors in which the sequences of PLE and APLE are linked N-terminally to the a factor signal sequence of S. cerevisiae are preferably constructed. It is further possible to prepare constructs in which the C-terminal tetrapeptide HAEL, which serves as ER retention signal as described for example in Hardwick et al., (1990) EMBO J. 9, 623-630, is additionally deleted. A further preferred expression is inducible expression of constructs with or without ER retention signal. Unexpectedly, constructs having the ER retention signal can also be expressed and lead to an rAPLE which is capable of selective resolution of racemic 2-alkyl-5-halopent-4-enecarboxylic esters. The amino acid sequence of the novel esterase rAPLE which is derived from the nucleotide sequence of the APLE gene is depicted in SEQ ID No. 1. It has unexpectedly been possible to find that the novel polypeptide or the rAPLE protein is able, in contrast to the known rPLE, in each case obtained by expression of the DNA segments coding for APLE and PLE, respectively, for example in P. pastoris cells, to resolve racemic 2-alkyl-5-halopent-4-enecarboxylic esters stereoselectively. The invention accordingly further relates to the use of the polypeptide having esterase activity and of the recombinant esterase (rAPLE) of the invention,' which have at least 8 0% identity to the sequence shown in SEQ ID No. 1, for resolving racemates of 2-alkyl-5-halopent-4-enecarboxylic esters of the formula (I) (Formula Removed) in which R is a C1-C6-alkyl radical, R1 is C1-C4-alkyl and X is chlorine, bromine or iodine. The polypeptide having esterase activity and ' the recombinant esterase (rAPLE) of the invention preferably have at least 90%, particularly preferably at least 98%, identity to the sequence of the protein shown in SEQ ID No. 1. It is also possible to employ polypeptide having an esterase activity or the recombinant esterase (rAPLE) of the invention with modified DNA sequences shown in SEQ ID. No. 1, obtained by usual modifications such as, for instance, mutations, deletions, extensions, fusions, which code for enzymes having the desired esterase activity. In this connection, enantiomerically enriched 2-alkyl-5-halopent-4-enecarboxylic acids or their esters of the formula (II) (Formula Removed) in which R is a C1-C6-alkyl radical, A is equal to H, R1, where R1 may be C1-C4-alkyl, or R2, where R2 is an alkyl group, but is not equal to Rl, and X is chlorine, bromine or iodine, are obtained by an enantiomeric mixture of a 2-alkyl-5-halopent-4-enecarboxylic ester of the formula (I) (Formula Removed) in which R, R1 and X are as defined above, being converted by means of the polypeptide of the invention or of the rAPLE of the invention in the presence of water or an alcohol of the formula R2OH, where R2 is an alkyl group which is not equal to RL, as nucleophile, and a) either the remaining enantiomerically enriched 2-alkyl-5-halopent-4-enecarboxylic ester of the formula (II) with A equal to R1 being isolated or b) if an alcohol is employed as nucleophile, the resulting enantiomerically enriched 2-alkyl-5- halopent-4-enecarboxylic ester of the formula (II) with A equal to R2 being isolated, or c) if water is employed as nucleophile, the resulting 2-alkyl-5-halopent-4-enecarboxylic acid of the formula (II) with A equal to H being isolated. R in the formula (II) is a C1-C6-alkyl radical such as, for instance, methyl, ethyl, n- and i-propyl, n-, i- and t-butyl, pentyl and hexyl. Ci-C4-Alkyl radicals are preferred, and the i-propyl radical is particularly preferred. A is H, R1, where R1 is a C1-C4-alkyl radical, preferably a C1-C2-alkyl radical and particularly preferably a methyl radical, or R2, where R2 is an alkyl radical which is not equal to R1, R2 is particularly preferably a C1-C6-alkyl radical. X is chlorine, bromine or iodine, preferably chlorine. Enantiomerically enriched compounds mean in this connection those which exhibit an enantiomeric excess (ee) of >80%, preferably of >90% and particularly preferably of >97%. The enzyme of the invention can moreover be used in any form. For example as dispersion, as solution, immobilized, as crude enzyme, as enzyme which has been obtained from its source by a combination of known purification methods, as whole cells (where appropriate immobilized and/or permeabilized) which have the required enzymatic activity (naturally or through genetic modifications) or in a lysate of such cells. The reaction temperature for the conversion of the invention is normally between 0 and 90°C, preferably between 10 and 60°C. The pH of the reaction solution is between 4 and 11, preferably between 6 and 9. The choice of the solvent depends on the nucleophile employed. If, for example, water is the nucleophile, solvents which can be employed are water, a mixture of water with a water-miscible solvent, for example with an alcohol such as, for instance, methanol, ethanol, isopropanol, t-butanol, etc., dioxane, tetrahydrofuran, acetone or dimethyl sulfoxide or a two-phase system of water and of a water-immiscible solvent, for example an aromatic compound such as, for instance, toluene, xylene, etc., an alkane such as, for instance, hexane, heptane, cyclohexane, etc., ether such as, for instance, diisopropyl ether, methyl t-butyl ether, etc. If the nucleophile is an alcohol, the solvent preferably employed is the alcohol R2OH where R2 is an alkyl group which is not, however, equal to R1. However, it is also possible to use mixtures of the alcohol with an organic solvent such as, for instance, tetrahydrofuran, heptane, toluene, hexane, CH3CN, methyl t-butyl ether etc. After the enzymatically catalyzed racemate resolution has taken place, the desired final product is isolated. This may be either the remaining enantiomerically enriched 2-alkyl-5-halopent-4-enecarboxylic ester of the formula (II) with A equal to R1, or if water is the nucleophile the enantiomerically enriched 2-alkyl-5-halopent-4-enecarboxylic acid of the formula (II) with A equal to H which has formed, or if the alcohol is the nucleophile the enantiomerically enriched 2-alkyl-5-halopent-4-enecarboxylic ester of the formula (II) with A equal to R2. The isolation can take place for example by conventional methods such as, for instance, extraction, crystallization, column chromatography, distillation, etc. The method of the invention results in the corresponding acids or esters of the formula (I) in theoretical yields of up to 98% yield and with an e.e. of up to >99%. Example 1: mRNA isolation and generation of cDNA 0.7 g of liver from a freshly slaughtered pig, obtained from a local abattoir, was frozen in liquid nitrogen and homogenized with a mortar, and the liberated mRNA was isolated or extracted using the Fast Track mRNA extraction kit 2.0 (Invitrogen, Carlsbad, Calif., USA) in accordance with the statements made by the manufacturer (Fast Track 2.0 kit manual; version J; 082301; 25-0099). The extraction afforded a total amount of 12.9 µg of mRNA. 0.26 µg of this mRNA was then used as template for generating cDNA using the Superscript III First-Strand synthesis system for RT-PCR according to the manufacturer's statements. Example 2: Amplification and cloning of cDNA fragments from pig liver Specific primers based on the sequence of the pig liver esterase gene of GenBank accession No. X63323 (Matsushima et al., 1991) were prepared: Primer 1: 5'-CAGAATTCATGGCTATCGGGCAGCCAGCCTCGC-3' (SEQ ID. No. 3) Primer 2: 5'-CCGGAATTCAGCCTCCCCUCACAGCTCAG -3' (SEQ ID No. 4) Bases homologous to the known PLE sequence are in bold script. Recognition sequences for restriction endonucleases are italicized for emphasis. The amplification took place in a 50 µl mixture with 1 U of Phusion DNA polymerase (Finnzymes, Espoo, Finland) , with 500 ng of cDNA as template, 20 µmol each of primer 1 and 2, 5 fil of a dNTP mix (2 mM each), all in lxPhusion HF buffer in accordance with 'Phusion High-Fidelity DNA Polymerase' manual (Finnzymes) , starting with a 3 0 second denaturation step at 98°C, followed by 30 cycles (10 sec 98°C, 20 sec 68°C, 1 rnin 72°C) for amplification and a final incubation at 70°C for 8 min to prepare complete products. This PCR resulted in a DNA fragment with a size of 1.8 kb (found by agarose gel electrophoresis), This PCR product was then purified using the Qiaguick kit (Qiagen, Hilden, Germany) in accordance with the manual included. About 0.1 µg of the purified PCR product was cut with the restriction endonuclease EcoRI and cloned into the plasmid vectors pHILZ and pHIL-D2 via the EcoRI cleavage sites. The vectors were then transformed into TOP10 electrocompetent cells prepared in accordance with 'Current Protocols in Molecular Biology'. Inserts of several resulting clones were sequenced using the 'Dye" Deoxy Terminator Cycle Sequencing' kit (Applied Biosystems Inc., Porster City, Calif., USA) . Two sequences were identified thereby, one corresponding 10 0% to the expected sequence published by Matsushima et al., (1991) FEES Lett. 293, 37-41, and the other sequence corresponding to SEQ ID No. 2. Example 3: Introduction of the a factor signal sequence and variations of the C-terminal end. In order to enable secretory expression of the known protein PLE and the protein rAPLE of the invention, vectors in which the sequence of PLE and APLE was connected N-terminally to the a factor start sequence of the cloning vector pPICZ a (Invitrogen) were constructed. " In addition, constructs in which the C-terminal tetrapeptide HAEL was deleted were prepared. PCR I: The EcoRIalphal/alphaPLE2 primer pair was used to amplify the a factor signal sequence of the cloning vector pPICZ a (Invitrogen) . The PCR was carried out in a 50 µl mixture (2 ng of template, 0.5 µM of each primer, 0.2 mM dNTPs, 1 U of the-Phusion DNA polymerase (Finnzymes) all in IxPhusion HF buffer in accordance with the 'Phusion High-Fidelity DNA Polymerase' manual (Finnzymes) ) . Denaturation at 95°C for 3 minutes was followed by amplification in 30 cycles (30 sec 95°C, 30 sec 57°C, 15 sec 72°C) and a final step at 72°C for 7 min. PCR II.- The PLE and APLE sequences were amplified from pHILZ plasmids using either the PLEalphal/EcoRIPLE+ER2 primer pair or the PLEalphal/EcoRIPLE2 (deletion of the C-terminal HAEL tetrapeptide) primer pair. These PCRs were again carried out in 50 til mixtures (2 ng of template, 0.5 µM of each primer, 0.2 mM dNTPs, 1 U of the Phusion DNA polymerase (Finnzymes) all in IxPhusion HF buffer in accordance with the 'Phusion High-Fidelity DNA Polymerase' Manual (Finnzymes)). A denaturation at 95°C for 3 minutes was followed by amplification in 30 cycles (30 sec 95°C, 30 sec 57°C, 15 sec 72°C) and a final step at 72°C for 7 min. PCR III: 3 µ1 of the products from PCR 1 and PCR II were used to combine these two products by primerless PCR. The extension was carried out in a 4 5 µl mixture with 0.2 mM dNTPs, 1 U of the Phusion DNA polymerase (Finnzymes) all in IxPhusion HF buffer. The reaction mixture was heated at 95°C for 3 minutes and then 10 cycles with 30 sec at 95°C and 45 sec at 72°C were carried out. To amplify these overlapping extension products, 5 µl of primer mix (3 µl of water, 1 µl of 5 µM EcoRIalphal primer and 1 µl of 5 µM EcoRIPLE+ER2 or EcoRIPLE2 primer) were added. The products were amplified with 20 PCR cycles (3 0 sec 95°C, 30 sec 57°C, 1 min 72°C) and a single temperature stop at 72°C for 7 min. Primer sequences: EcoRlalphal: 5'-TCTTCGAAGAA 7TCACGATGAGAT7TCCTTCAATTTTTACTGC-3' (SEQ ID No. 5) alphaPLE2:5'-GAGGCTGGCTGCCCAGCTTCAGCCTCTCTTTTCTCG-3' (SEQ ID No. 6) PLEalphal: 5'-AGAGAGGCTGAAGCTGGGCAGCCAGCCTCGCCG-3, (SEQ ID No. 7) EcoRIPLE+ER2:5,-ATGGTACCGAATTCTCACAGCTCAGCATGCTTTATCTTG-3• (SEQ ID No. 8) EcoRtPLE2:5'-ATGGrACCGAATTCTCACTTTATCTTGGGTGGCTTCTrTG-3' (SEQ ID No. 9) Regions with homology to the templates in bold, recognition sequences for restriction endonucleases in italics. Example 4: Construction of expression constructs for the heterologous expression of pig liver esterases in Pichia pastoris The overlapping extension PCR products from example 3 were purified using the Qiaquick kit (Qiagen, Hilden, Germany) in accordance with the manual included. About 0.1 µg of the purified PCR products was cut using the EcoRI restriction endonuclease and cloned via the EcoRI cleavage site into the plasmid vector pGAPZ A (Invitrogen). Correct orientation of the insert in relation to the promoters was checked with the aid of control cleavages, for example with Ncol. In each case, a clone with correctly oriented insert was selected, sequenced and preserved. The corresponding plasmids were named as follows: Plasmids which contained the known PLE sequence as disclosed in Matsushima et al. , (1991) FEBS Lett. 293, 37-41, were called pGAPZ A PLE-ER (the HAEL tetrapeptide was deleted) and pGAPZ A PLE+ER (HAEL tetrapeptide still present). Plasmids derived from the novel APLE sequence were called pGAPZ A APLE-ER (the HAEL tetrapeptide was deleted) and pGAPZ A APLE-ER (HAEL tetrapeptide still present). Example 5: Constitutive expression. of pig liver esterases in Plchia pastoris The plasmids pGAPZ A PLE-ER, pGAPZ A PLE+ER, pGAPZ A APLE-ER and pGAPZ A APLE+ER were transformed into P. pastoris X-33. The transformation took place in accordance with the instructions of the protocol for the Pichia Expressions kit from Invitrogen. The transformants were selected on YPD plates (1% yeast extract, 2% peptone, 2% D-glucose, 2% agar) which contained 100 mg/1 zeocin. 52 zeocin-resistant clones were streaked onto YPD plates with 100 mg/1 zeocin and preserved in 15% glycerol. Example 6: Qualitative analysis of the esterase activity P. pastoris transformants were cultured on YPD plates with 100 mg/1 zeocin at 30°C for 48 h. The cells were lifted onto Whatman 541 hardened ashless 70 mm0 filters and air-dried. The filters were incubated with a solution of 6 mg of a-naphthyl acetate (Sigma, dissolved in 500 µl of acetone), 2.5 mg of tetrazotized o-dianisidine (Past Blue Salt BN, Sigma, dissolved in 125 µl of water) and 5 ml of 0.1 M potassium phosphate buffer, pH 7, in order to visualize the esterase activity by a color reaction. Activities were detected in all transformants which had integrated one of the 4 plasmids pGAPZ A PLE-ER, pGAPZ A PLE+ER, pGAPZ A APLE-ER and pGAPZ A APLE+ER. This proves the expression of functional proteins having esterase activity. As a check, a clone with integrated empty vector was also tested in the same way. In this case, no significant esterase activity was visible in the comparable reaction period. Example 7: Stereoselective esterase activity in relation to methyl 5-chloro-2-(1-methylethyl)-4-pentenoate P. pastoris transformants as described in example 6 were cultured on YPD plates with 100 mg/1 zeocin at 30°C for 48 h. The cells were lifted onto Whatman 541 hardened ashless 70 mm0 filters and air-dried. The filters were incubated either with substrate solution A (100 µl of racemic methyl 5-chloro-2- (1-methylethyl) -4-pentenoate, 200 µl of 0.1 M potassium phosphate buffer, pH 8; 150 µl of 10 mg/ml phenol red; 450 µl of DMSO; 650 µl of H2O) or with substrate solution B (identical to solution A but employing methyl (2S,4E)-5-chloro-2-(1-methylethyl)-4-pentenoate instead of the racemate) . Owing to the specific esterase activity on the substrates, the liberation of acid on hydrolysis of the ester substrate results in a pH decrease which in turn leads to a change in the color of the phenol red indicator to yellow.' It emerged from this that trans formants containing the plasmid pGAPZ A APLE-ER gave signals (yellow coloration around the colony) after incubation for 3 to 4 hours if they were tested on substrate solution A, whereas no significant conversion could be found with substrate solution B (figure 1). Transformants obtained with the plasmids pGAPZ A PLE-ER or pGAPZ A PLE+ER showed no reaction with substrate solutions A or B under the same conditions. This showed that the recombinant rAPLE , has a substrate specificity different from recombinant rPLE and that hydrolysis of methyl 5-chloro-2-(1-methylethyl)-4-pentenoate using rAPLE takes place stereoselectively for the (R) enantiomer. Example 8: SDS polyacrylamide gel electrophoresis 10 µl of a 2x SDS sample buffer (125 mM Tris-HCl, pH 6.8; 4% SDS, 20% glycerol, 5% P-mercaptoethanol, 0.05% bromophenol blue) were added to 10 µl of commercially available pig liver esterase or 10 µl of the 60-fold concentrated (Centricon Ultrafiltrations-Spin Columns, from Sartorius) supernatants of the P. pastoris cultures (72 h at 100 rpm and 28°C in 250 ml of YPD medium in 2 1 Erlenmeyer flasks with baffles) which contained either the plasmid pGAPZ A APLE-ER or an empty pGAPZ A plasmid (control strain). After the samples had been heated at 95 °C for 5 minutes, the proteins were separated on a 12.5% polyacrylamide gel (4% stacking gel) and stained with Coomassie Brilliant Blue R250 for detection. The SDS-PAGE shows a protein band with the expected size for rAPLE (-60 kDa) in the yeast strain having the pGAPZ A APLE plasmid, but not in the control strain (figure 2) . The commercially available pig liver esterase which was also analyzed for comparison showed two protein bands in the same size range (arrow in figure 2). Example 9: Induced expression of pig liver esterases with the A0X1 promoter The plasmids pGAPZ A PLE-ER, pGAPZ A PLE+ER, pGAPZ A APLE-ER and pGAPZ A APLE+ER were cut with the restriction endonuclease Xhol, and the respective fragments coding for APLE and PLE proteins, with or without ER retention signal, were cloned via the Xhol cleavage site into the pPIC9 vector (Invitrogen). Correct orientation of the fragments in relation to the AOX1 promoter was checked by means of control cuts with the restriction endonuclease Ncol. The vectors having the AOX1 promoter were named, in analogy to the plasmids named in example 4, pPIC9 PLE-ER, pPIC9 PLE+ER, pPIC9 APLE-ER and pPIC9 APLE+ER, linearized with Sail and transformed into P. pastoris KM71. Transformation and selection for His prototrophy took place in accordance with the instructions of the Pichia expression kit from Invitrogen. Selected transformants and the KM71 strain were cultured on complete medium in accordance with the Pichia expression kit from Invitrogen overnight and induced with 1% methanol for 48 h. The resulting cultures were analyzed by means of the qualitative pH-shift assay described in example 7, testing 2 fil of the cultures in the mixtures in each case. Expression under the control of the inducible A0X1 promoter led to very much higher, by comparison with the situation described for constitutive expression in example 7, rAPLE enzymic activities in relation to racemic methyl 5-chloro-2-(1-methylethyl) -4-pentenoate. The phenyl red color change (red to yellow) was detectable after only a few minutes (figure 3). Unexpectedly, the rAPLE activity was independent of the presence of the ER retention signal HAEL at the C terminus, i.e. even cells which expressed rAPLE with ER retention signal showed activity. By contrast, yeast strains for producing rPLE had no activity in relation to racemic methyl 5-chloro-2-(1-methylethyl)-4-pentenoate. Claims A polypeptide or recombinant protein having esterase activity and exhibiting an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ. ID. No. 1. A polypeptide or recombinant protein which exhibits at least 98% identity to the amino acid sequence shown in SEQ ID No. 1 and has activity for racemate resolution of 2-alkyl-5-halopent-4-enecarboxylic esters of the formula (I) (Formula Removed) in which R is a C1-C6-alkyl radical, R1 is C1-C4-aikyl and X is chlorine, bromine or iodine. A polypeptide or recombinant protein having e-sterase activity according to claims 1-2, which exhibits an amino acid sequence modified by a modification selected from the group consisting of mutation, deletion, insertion, extension and fusion. A nucleotide sequence which codes for a polypeptide or recombinant protein according to claims 1 -3. A nucleotide sequence comprising the sequence shown in SEQ ID No. 2. A nucleotide sequence obtainable by isolating mRNA from an organism followed by amplification of a nucleic acid fragment using a first primer comprising the nucleotide sequence GGGCAGCCAGCCTCGC and a second primer comprising the nucleotide sequence GGGCAGCCAGCCTCGC. The use of a polypeptide or a recombinant protein according to claims 1-3 for racemic resolution of 2-alkyl-5-halopent-4-enecarboxylic esters of the formula (I) (Formula Removed) in which R is a C1-C6-alkyl radical, R1 is C1-C4-alkyl and X is chlorine, bromine or iodine. A method for preparing enantiomerically enriched 2-alkyl-5-halopent-4-enecarboxylic acids or esters thereof of the formula (II) (Formula Removed) in which R is a C1-C6-alkyl radical, A is equal to H, R1, where R1 may be C1-C4-alkyl, or R2, where R2 is an alkyl group, but is not equal to R1, and X is chlorine, bromine or iodine, which comprises a mixture of enantiomers of a 2-alkyl-5-halopent-4-enecarbcxylic ester of the formula (I) (Formula Removed) in which R, R and X are as defined above, being converted by means of a polypeptide or a recombinant esterase according to claims 1-3 in the presence of water or an alcohol of the formula R2OH, where R2 is an alkyl group which is not equal 10 Rl, as nucleophile, and b) a) either the remaining enantiomerically enriched 2-alkyl-5-halopent-4-enecarboxylic ester of the formula (II) with A equal to R1 being isolated or if an alcohol is employed as nucleophile, the resulting enantiomerically enriched 2-alkyl- halopent-4-enecarboxylic ester with A equal to R2 being isolated, or c) if water is employed as nucleophile, the resulting 2-alkyl-5-halopent-4-enecarboxylic acid of the formula (II) with A equal to H being isolated.

Full Text

Novel polypeptide having esterase activity and recombinant esterase and use thereof
The invention relates to a novel polypeptide having esterase activity, especially having 2-alkyl-5-halopent-4-enecarboxylesterase activity, and to an enzymatically active recombinant protein having esterase activity and to the use thereof for resolving racemates of 2-alkyl-5-halopent-4-enecarboxylic ester enantiomer mixtures.
Enantiomerically enriched 2-alkyl-5-halopent-4-enecarboxylic acids and their esters are valuable intermediates for preparing pharmaceuticals, such as, for instance, for delta-amino-gamma-hydroxy-omega-arylalkanecarboxamides, which have renin-inhibiting properties and can be used as antihypertensive agents in pharmaceutical preparations.
Esterases are generally employed in the resolution of
racemates and asymmetrization.
However, only very few esterases suitable for preparing
chiral compounds are commercially available.
The use of esterase extracts from pig liver is known on
the preparative scale. Pig liver esterase (PLE) was
isolated long ago from natural sources, and its
activity has also been known for a long time (Simonds,
J.P. (1919) Amer. J. Physiol. 48, 141; Bamann, E. et
al. (1934) Hoppe-Seyler Z. 229, 15; Falconer J.S. and
Taylor, D.B. (1946) Biochem. J. 40, 831-834) .
Various studies have also already been carried out in order to characterize PLE (Heymann, E. and Junge, W. (1979) Eur. J. Biochem. 95, 509-518; Lehner, R. and Verger, T. (1997) Biochemistry 36, 1861-1868) . It has further been possible to show, for example in WO 01/09079, that esterase extracts from pig liver can selectively hydrolyze the (R) enantiomer of methyl 5-chloro-2-(1-methylethyl)-4-pentenoate.
However, the use of such esterase extracts -from natural sources, such as pig liver, is associated with disadvantages.
In the first place, the qualities of the different batches vary and thus make it difficult to optimize industrial processes. Secondly, the use of animal resources in the manufacture of pharmaceutical products is undesired because the presence of viruses and prions cannot always be precluded.
For these reasons there is a need to produce recombinant pig liver esterases of standardized quality in microorganisms.
The cloning of putative esterase genes is described for example in FEES Lett. (1991), 293, 37-41. The first functional expression of an active pig liver esterase enzyme was described for the first time in WO 02/48322. WO 2004/055177 describes the preparation of further recombinant esterases by site directed mutagenesis of the recombinant pig liver esterase of seq. ID No. 1 (rPLE) - from WO 02/48322. As is evident from the description of WO 2004/055177 and from the article authored by the same inventors in Protein Engineering, 16, 1139-1145, 2003, the modifications of the rPLE sequence from WO 02/48322 were chosen so that a recombinant intestinal pig esterase (PICE) disclosed in David et al. , (1998) Eur. J. Biochem. 257, 142-148, is obtained.
The resolution of racemic 2-alkyl-5-halopent-4-enecarboxylic esters is not described in any of these articles.
However, since the need for esterases which have the desired stereoselective activity for 2-alkyl-5-halopent-4-enecarboxylic esters and which can easily be prepared biotechnologically is not met, it was an object of the present invention to provide a corresponding novel recombinant esterase.
In an attempt to isolate and to clone the gene described in FEBS Lett. (1991), 293, 37-41 and WO 02/48322 for known pig liver esterase (PLE) as cDNA starting from mRNA from pig liver, a second, novel esterase sequence was found in addition to the known PLE sequence. Following expression of the two sequences, in which the corresponding proteins or esterases, namely the known rPLE and a novel, recombinant "alternative" esterase (rAPLE), was prepared, it unexpectedly emerged that only the rAPLE is capable of selective resolution of racemic 2-alkyl-5-halopent-4-enecarboxylic esters.
It was thus possible to achieve the object of the present invention by a novel polypeptide having esterase activity and a novel recombinant esterase (rAPLE) , whose amino acid sequence differs in 21 of a total of 548 amino acids from the known PLE sequence. The novel rAPLE differs in the amino acid sequence also from the known pig intestinal carboxylesterase (PICE) in 12 of a total of 548 amino acids. However, PICE is found in the pig intestinal tract,
The present invention accordingly relates to a polypeptide having esterase activity, which comprises the amino acid sequence SEQ. ID. No. 1.
The present invention further relates to a novel recombinant protein having esterase activity, which comprises the amino acid sequence SEQ. ID. No. 1.
The polypeptide and the recombinant rAPLE of the invention have the ability to resolve stereoselectively racemic 2-alkyl-5-halopent-4-enecarboxylic esters of the formula (I)
(Formula Removed)
in which R is a C1-C6-alkyl radical, R2 is C1-C4-alkyl and X is chlorine, bromine or iodine.
The polypeptide of the invention having esterase activity, and the novel recombinant esterase rAPLE differ, as stated above, in 21 of a total of 54 8 amino acids of the known sequence disclosed in FEBS Lett. (1991), 293, 37-41 and in 12 of a total of 548 amino acids from the known PICE protein disclosed in David et al., (1998) Eur. J. Biochem. 257, 142-148.
The sequence of the protein of the novel rAPLE of the invention differs in the following amino acid positions from the known sequence of, the PLE protein:
APLE Position PLE

Glu 73 Asp
lie 75 Val
Gly 76 Val
Gly 77 Glu
Leu 80 Thr
Arg 87 Gly
lie 92 Thr
Pro 93 Leu
Val 129 Leu
Ser 133 Pro
Thr 134 Met
Leu 138 Val
Ala 139 Val
Phe 234 Leu
Ala 236 Val
Gly 237 Ala
Phe 286 Leu
Ala 2 87 Thr
Leu 290 Phe
Pro 2 94 Gin
Thr 302 Pro
The protein of the invention and the novel recombinant rAPLE may moreover be in the form of a modified sequence as shown in SEQ ID No 1, which can be obtained for example by usual modifications such as, for instance, exchange, deletion or attachment of amino acid(s) in the sequence at the N or C terminus, such as, for instance, GluAlaGluAla from the a factor signal sequence, or by fusion to other proteins. The invention also further includes muteins having modifications within the protein sequence of the enzyme of the invention having the appropriate activity, in particular on 2-alkyl-5-halopent-4-enecarboxylic esters. Muteins can be obtained for example by modifications of the DNA which codes for the enzyme of the invention, by known mutagenesis techniques (random mutagenesis, site-directed mutagenesis, directed evolution, gene shuffling etc.) so that the DNA codes for an enzyme which differs at least by one amino acid from the enzyme of the invention, and subsequent expression of the modified DNA in a suitable host cell. The invention thus also includes modified DNA sequences as shown in SEQ ID. No 1, obtained by the mutations, deletions, extensions, fusions described above, and which code for enzymes having the desired esterase activity.
Esterase activity, especially 2-alkyl-5-halopent-4-ene-carboxylesterase activity, is defined in this connection as the ability to resolve racemates of 2-alkyl-5-halopent-4-enecarboxylic esters of the formula (I).
(Formula Removed)
The polypeptide of the invention and the recombinant rAPLE can be prepared as described below:
Firstly, mRNA is isolated from pig liver using a
suitable kit, and then the cDNA is generated by reverse
transcription based on the mRNA extract.
Subsequently, specific PCR primers based on the
sequence of the known pig liver esterase gene of
GenBank accesssion No. X63323 (Matsushima et al. , 1991)
is prepared, followed by amplification and cloning.
These specific primers are:
Primer 1: 5'-CAGAATTCATGGCTATCGGGCAGCCAGCCTCGC-3'
Primer 2 : 5'-CCGGAATTCAGCCTCCCCTTCACAGCTCAG-3'
This part of the primers which comprises the appropriate nucleotide sequences coding for the PLE protein and which is obligatorily present in the primers is in bold script.
The other sequence part of the primers comprises for example information for cleavage sites for restriction endonucleases (in italics) or sequence elements which are important for expression. This part may vary in the preparation of the rAPLE of the invention.
Amplification then takes place with primers 1 and 2 by prior art PCR methods.
The PCR product is subsequently used to prepare by prior art methods expression constructs for heterologous expression of the encoded rAPLE protein in suitable host organisms. This preferably entails the PCR product being initially cloned into suitable plasmid vectors.
The recombinant plasmids obtained in this way are then transformed into a suitable host, for example Escherichia coli. Inserts of several resulting clones are then sequenced.
Unexpectedly, 2 groups of recombinant clones with different sequences were identified therein, one being
100% identical to the expected sequence for PLE according to Matsushima et al. , (1991) FEBS Lett. 293, 3 7-41, and a novel nucleotide sequence as shown in SEQ. ID. No. 2 (APLE sequence) which leads after expression to the amino acid sequence SEQ. ID. No. 1 of the invention.
The present invention further relates to a nucleic acid or nucleotide sequence which codes for the polypeptide of the invention and the recombinant esterase rAPLE. For example, such a nucleic acid has the nucleotide sequence shown in SEQ. ID. No. 2.
The invention also relates further to nucleotide sequences which include a nucleotide sequence which codes for the polypeptide of the invention and the recombinant esterase rAPLE, or comprises the nucleotide sequence shown in SEQ. ID. No. 2.
A further possibility is to prepare appropriate oligonucleotides corresponding to nucleic acid sequences according to the present invention which code for the esterase of the invention by standardized synthetic techniques, for example with use of automated DNA synthesizers.
The purely synthetic preparation of the nucleic acid sequences which code for the esterase of the invention is particularly advantageous for use in the production of pharmaceuticals or their intermediates, because enzymes are thus not obtained from animal sources.
Expression of the two sequences found (PLE and APLE
sequences) then takes place.
The known pig liver esterase (PLE, Swiss-Prot ID
Q2 9550) comprises an N-terminal signal sequence and a
C-terminal ER retention signal, the last 4 amino acids
HAEL.
In order to express the known PLE and the novel APLE,
vectors in which the sequences are introduced into
suitable expression systems constructed. These
expression constructs are then transformed into suitable host cells.
Suitable host cells in this connection are for example
microorganisms, animal cell lines and plants.
Both prokaryotic and eukaryotic microorganisms can be
employed. Preferred prokarytic hosts (bacteria) are
Escherichia coli, and strains from the genera Bacillus
(e.g. B.subtilis, B. licheniformis,
B.amyloliquefaciens) , Pseudomonas (e.g. P.fluorescens,
P.putida), or Streptomyces (e.g. S.lividana, S. tendae)
Eukaryotic microorganisms are preferred, and fungi are
particularly preferred. Examples thereof are
Saccharomyces cerevisiae, Pichia pastorls,
Kluyveromyces lactis or Aspergillus sp. .
Expression may be secretory or intracellular and both inducible and constitutive.
For bacterial expression a choice of species-specific signals can be obtained, a.o. ELS commercially available strains and vectors for protein expression (e.g provided by companies like Invitrogen, Novagen, New England Biolabs), that allow inducible or constitutive expression, intracellular and secretory localization of the target protein; in addition, technology to enable or promote the correct folding of proteins in order to result in soluble and active protein may be applied. The proteins are preferably expressed in a secretory manner, in which case vectors in which the sequences of PLE and APLE are linked N-terminally to the a factor signal sequence of S. cerevisiae are preferably constructed.
It is further possible to prepare constructs in which the C-terminal tetrapeptide HAEL, which serves as ER retention signal as described for example in Hardwick et al., (1990) EMBO J. 9, 623-630, is additionally deleted.
A further preferred expression is inducible expression of constructs with or without ER retention signal. Unexpectedly, constructs having the ER retention signal can also be expressed and lead to an rAPLE which is
capable of selective resolution of racemic 2-alkyl-5-halopent-4-enecarboxylic esters.
The amino acid sequence of the novel esterase rAPLE which is derived from the nucleotide sequence of the APLE gene is depicted in SEQ ID No. 1.
It has unexpectedly been possible to find that the novel polypeptide or the rAPLE protein is able, in contrast to the known rPLE, in each case obtained by expression of the DNA segments coding for APLE and PLE, respectively, for example in P. pastoris cells, to resolve racemic 2-alkyl-5-halopent-4-enecarboxylic esters stereoselectively.
The invention accordingly further relates to the use of the polypeptide having esterase activity and of the recombinant esterase (rAPLE) of the invention,' which have at least 8 0% identity to the sequence shown in SEQ ID No. 1, for resolving racemates of 2-alkyl-5-halopent-4-enecarboxylic esters of the formula (I)

(Formula Removed)
in which R is a C1-C6-alkyl radical, R1 is C1-C4-alkyl and X is chlorine, bromine or iodine.
The polypeptide having esterase activity and ' the recombinant esterase (rAPLE) of the invention preferably have at least 90%, particularly preferably at least 98%, identity to the sequence of the protein shown in SEQ ID No. 1. It is also possible to employ polypeptide having an esterase activity or the recombinant esterase (rAPLE) of the invention with modified DNA sequences shown in SEQ ID. No. 1, obtained
by usual modifications such as, for instance, mutations, deletions, extensions, fusions, which code for enzymes having the desired esterase activity.
In this connection, enantiomerically enriched 2-alkyl-5-halopent-4-enecarboxylic acids or their esters of the formula (II)
(Formula Removed)
in which R is a C1-C6-alkyl radical, A is equal to H, R1, where R1 may be C1-C4-alkyl, or R2, where R2 is an alkyl group, but is not equal to Rl, and X is chlorine, bromine or iodine, are obtained by an enantiomeric mixture of a 2-alkyl-5-halopent-4-enecarboxylic ester of the formula (I)
(Formula Removed)
in which R, R1 and X are as defined above, being converted by means of the polypeptide of the invention or of the rAPLE of the invention in the presence of water or an alcohol of the formula R2OH, where R2 is an alkyl group which is not equal to RL, as nucleophile, and
a) either the remaining enantiomerically enriched 2-alkyl-5-halopent-4-enecarboxylic ester of the formula (II) with A equal to R1 being isolated or
b) if an alcohol is employed as nucleophile, the resulting enantiomerically enriched 2-alkyl-5-
halopent-4-enecarboxylic ester of the formula (II) with A equal to R2 being isolated, or c) if water is employed as nucleophile, the resulting 2-alkyl-5-halopent-4-enecarboxylic acid of the formula (II) with A equal to H being isolated.
R in the formula (II) is a C1-C6-alkyl radical such as,
for instance, methyl, ethyl, n- and i-propyl, n-, i-
and t-butyl, pentyl and hexyl.
Ci-C4-Alkyl radicals are preferred, and the i-propyl
radical is particularly preferred.
A is H, R1, where R1 is a C1-C4-alkyl radical,
preferably a C1-C2-alkyl radical and particularly
preferably a methyl radical, or R2, where R2 is an alkyl
radical which is not equal to R1, R2 is particularly
preferably a C1-C6-alkyl radical.
X is chlorine, bromine or iodine, preferably chlorine.
Enantiomerically enriched compounds mean in this connection those which exhibit an enantiomeric excess (ee) of >80%, preferably of >90% and particularly preferably of >97%.
The enzyme of the invention can moreover be used in any form. For example as dispersion, as solution, immobilized, as crude enzyme, as enzyme which has been obtained from its source by a combination of known purification methods, as whole cells (where appropriate immobilized and/or permeabilized) which have the required enzymatic activity (naturally or through genetic modifications) or in a lysate of such cells.
The reaction temperature for the conversion of the invention is normally between 0 and 90°C, preferably between 10 and 60°C. The pH of the reaction solution is between 4 and 11, preferably between 6 and 9.
The choice of the solvent depends on the nucleophile employed.
If, for example, water is the nucleophile, solvents which can be employed are water, a mixture of water with a water-miscible solvent, for example with an alcohol such as, for instance, methanol, ethanol, isopropanol, t-butanol, etc., dioxane, tetrahydrofuran, acetone or dimethyl sulfoxide or a two-phase system of water and of a water-immiscible solvent, for example an aromatic compound such as, for instance, toluene, xylene, etc., an alkane such as, for instance, hexane, heptane, cyclohexane, etc., ether such as, for instance, diisopropyl ether, methyl t-butyl ether, etc. If the nucleophile is an alcohol, the solvent preferably employed is the alcohol R2OH where R2 is an alkyl group which is not, however, equal to R1. However, it is also possible to use mixtures of the alcohol with an organic solvent such as, for instance, tetrahydrofuran, heptane, toluene, hexane, CH3CN, methyl t-butyl ether etc.
After the enzymatically catalyzed racemate resolution has taken place, the desired final product is isolated. This may be either the remaining enantiomerically enriched 2-alkyl-5-halopent-4-enecarboxylic ester of the formula (II) with A equal to R1, or if water is the nucleophile the enantiomerically enriched 2-alkyl-5-halopent-4-enecarboxylic acid of the formula (II) with A equal to H which has formed, or if the alcohol is the nucleophile the enantiomerically enriched 2-alkyl-5-halopent-4-enecarboxylic ester of the formula (II) with A equal to R2.
The isolation can take place for example by conventional methods such as, for instance, extraction, crystallization, column chromatography, distillation, etc.
The method of the invention results in the corresponding acids or esters of the formula (I) in theoretical yields of up to 98% yield and with an e.e. of up to >99%.
Example 1: mRNA isolation and generation of cDNA
0.7 g of liver from a freshly slaughtered pig, obtained from a local abattoir, was frozen in liquid nitrogen and homogenized with a mortar, and the liberated mRNA was isolated or extracted using the Fast Track mRNA extraction kit 2.0 (Invitrogen, Carlsbad, Calif., USA) in accordance with the statements made by the manufacturer (Fast Track 2.0 kit manual; version J; 082301; 25-0099). The extraction afforded a total amount of 12.9 µg of mRNA.
0.26 µg of this mRNA was then used as template for generating cDNA using the Superscript III First-Strand synthesis system for RT-PCR according to the manufacturer's statements.
Example 2: Amplification and cloning of cDNA fragments from pig liver
Specific primers based on the sequence of the pig liver
esterase gene of GenBank accession No. X63323
(Matsushima et al., 1991) were prepared:
Primer 1: 5'-CAGAATTCATGGCTATCGGGCAGCCAGCCTCGC-3' (SEQ ID. No. 3)
Primer 2: 5'-CCGGAATTCAGCCTCCCCUCACAGCTCAG -3' (SEQ ID No. 4)
Bases homologous to the known PLE sequence are in bold script. Recognition sequences for restriction endonucleases are italicized for emphasis. The amplification took place in a 50 µl mixture with 1 U of Phusion DNA polymerase (Finnzymes, Espoo, Finland) , with 500 ng of cDNA as template, 20 µmol each of primer 1 and 2, 5 fil of a dNTP mix (2 mM each), all in lxPhusion HF buffer in accordance with 'Phusion High-Fidelity DNA Polymerase' manual (Finnzymes) , starting with a 3 0 second denaturation step at 98°C, followed by 30 cycles (10 sec 98°C, 20 sec 68°C, 1 rnin 72°C) for amplification and a final incubation at 70°C for 8 min to prepare complete products.
This PCR resulted in a DNA fragment with a size of 1.8 kb (found by agarose gel electrophoresis),
This PCR product was then purified using the Qiaguick kit (Qiagen, Hilden, Germany) in accordance with the manual included.
About 0.1 µg of the purified PCR product was cut with the restriction endonuclease EcoRI and cloned into the plasmid vectors pHILZ and pHIL-D2 via the EcoRI cleavage sites.
The vectors were then transformed into TOP10 electrocompetent cells prepared in accordance with 'Current Protocols in Molecular Biology'. Inserts of several resulting clones were sequenced using the 'Dye" Deoxy Terminator Cycle Sequencing' kit (Applied Biosystems Inc., Porster City, Calif., USA) . Two sequences were identified thereby, one corresponding 10 0% to the expected sequence published by Matsushima et al., (1991) FEES Lett. 293, 37-41, and the other sequence corresponding to SEQ ID No. 2.
Example 3: Introduction of the a factor signal sequence and variations of the C-terminal end.
In order to enable secretory expression of the known protein PLE and the protein rAPLE of the invention, vectors in which the sequence of PLE and APLE was connected N-terminally to the a factor start sequence of the cloning vector pPICZ a (Invitrogen) were constructed. " In addition, constructs in which the C-terminal tetrapeptide HAEL was deleted were prepared.
PCR I: The EcoRIalphal/alphaPLE2 primer pair was used to amplify the a factor signal sequence of the cloning vector pPICZ a (Invitrogen) . The PCR was carried out in a 50 µl mixture (2 ng of template, 0.5 µM of each primer, 0.2 mM dNTPs, 1 U of the-Phusion DNA polymerase (Finnzymes) all in IxPhusion HF buffer in accordance with the 'Phusion High-Fidelity DNA Polymerase' manual (Finnzymes) ) .
Denaturation at 95°C for 3 minutes was followed by amplification in 30 cycles (30 sec 95°C, 30 sec 57°C, 15 sec 72°C) and a final step at 72°C for 7 min.
PCR II.- The PLE and APLE sequences were amplified from pHILZ plasmids using either the PLEalphal/EcoRIPLE+ER2 primer pair or the PLEalphal/EcoRIPLE2 (deletion of the C-terminal HAEL tetrapeptide) primer pair. These PCRs were again carried out in 50 til mixtures (2 ng of template, 0.5 µM of each primer, 0.2 mM dNTPs, 1 U of the Phusion DNA polymerase (Finnzymes) all in IxPhusion HF buffer in accordance with the 'Phusion High-Fidelity DNA Polymerase' Manual (Finnzymes)). A denaturation at 95°C for 3 minutes was followed by amplification in 30 cycles (30 sec 95°C, 30 sec 57°C, 15 sec 72°C) and a final step at 72°C for 7 min.
PCR III: 3 µ1 of the products from PCR 1 and PCR II were used to combine these two products by primerless PCR.
The extension was carried out in a 4 5 µl mixture with 0.2 mM dNTPs, 1 U of the Phusion DNA polymerase (Finnzymes) all in IxPhusion HF buffer.
The reaction mixture was heated at 95°C for 3 minutes and then 10 cycles with 30 sec at 95°C and 45 sec at 72°C were carried out. To amplify these overlapping extension products, 5 µl of primer mix (3 µl of water, 1 µl of 5 µM EcoRIalphal primer and 1 µl of 5 µM EcoRIPLE+ER2 or EcoRIPLE2 primer) were added. The products were amplified with 20 PCR cycles (3 0 sec 95°C, 30 sec 57°C, 1 min 72°C) and a single temperature stop at 72°C for 7 min.
Primer sequences: EcoRlalphal: 5'-TCTTCGAAGAA 7TCACGATGAGAT7TCCTTCAATTTTTACTGC-3' (SEQ ID No. 5)
alphaPLE2:5'-GAGGCTGGCTGCCCAGCTTCAGCCTCTCTTTTCTCG-3' (SEQ ID No. 6)
PLEalphal: 5'-AGAGAGGCTGAAGCTGGGCAGCCAGCCTCGCCG-3, (SEQ ID No. 7)
EcoRIPLE+ER2:5,-ATGGTACCGAATTCTCACAGCTCAGCATGCTTTATCTTG-3• (SEQ ID No. 8)
EcoRtPLE2:5'-ATGGrACCGAATTCTCACTTTATCTTGGGTGGCTTCTrTG-3' (SEQ ID No. 9)
Regions with homology to the templates in bold, recognition sequences for restriction endonucleases in italics.
Example 4: Construction of expression constructs for the heterologous expression of pig liver esterases in Pichia pastoris
The overlapping extension PCR products from example 3
were purified using the Qiaquick kit (Qiagen, Hilden,
Germany) in accordance with the manual included. About
0.1 µg of the purified PCR products was cut using the
EcoRI restriction endonuclease and cloned via the EcoRI
cleavage site into the plasmid vector pGAPZ A
(Invitrogen).
Correct orientation of the insert in relation to the
promoters was checked with the aid of control
cleavages, for example with Ncol.
In each case, a clone with correctly oriented insert
was selected, sequenced and preserved.
The corresponding plasmids were named as follows:
Plasmids which contained the known PLE sequence as
disclosed in Matsushima et al. , (1991) FEBS Lett. 293,
37-41, were called pGAPZ A PLE-ER (the HAEL
tetrapeptide was deleted) and pGAPZ A PLE+ER (HAEL
tetrapeptide still present).
Plasmids derived from the novel APLE sequence were
called pGAPZ A APLE-ER (the HAEL tetrapeptide was
deleted) and pGAPZ A APLE-ER (HAEL tetrapeptide still present).
Example 5: Constitutive expression. of pig liver esterases in Plchia pastoris
The plasmids pGAPZ A PLE-ER, pGAPZ A PLE+ER, pGAPZ A APLE-ER and pGAPZ A APLE+ER were transformed into P. pastoris X-33. The transformation took place in accordance with the instructions of the protocol for the Pichia Expressions kit from Invitrogen. The transformants were selected on YPD plates (1% yeast extract, 2% peptone, 2% D-glucose, 2% agar) which contained 100 mg/1 zeocin. 52 zeocin-resistant clones were streaked onto YPD plates with 100 mg/1 zeocin and preserved in 15% glycerol.
Example 6: Qualitative analysis of the esterase activity
P. pastoris transformants were cultured on YPD plates with 100 mg/1 zeocin at 30°C for 48 h. The cells were lifted onto Whatman 541 hardened ashless 70 mm0 filters and air-dried. The filters were incubated with a solution of 6 mg of a-naphthyl acetate (Sigma, dissolved in 500 µl of acetone), 2.5 mg of tetrazotized o-dianisidine (Past Blue Salt BN, Sigma, dissolved in 125 µl of water) and 5 ml of 0.1 M potassium phosphate buffer, pH 7, in order to visualize the esterase activity by a color reaction.
Activities were detected in all transformants which had integrated one of the 4 plasmids pGAPZ A PLE-ER, pGAPZ A PLE+ER, pGAPZ A APLE-ER and pGAPZ A APLE+ER. This proves the expression of functional proteins having esterase activity. As a check, a clone with integrated empty vector was also tested in the same way. In this case, no significant esterase activity was visible in the comparable reaction period.
Example 7: Stereoselective esterase activity in relation to methyl 5-chloro-2-(1-methylethyl)-4-pentenoate
P. pastoris transformants as described in example 6 were cultured on YPD plates with 100 mg/1 zeocin at 30°C for 48 h. The cells were lifted onto Whatman 541 hardened ashless 70 mm0 filters and air-dried. The filters were incubated either with substrate solution A (100 µl of racemic methyl 5-chloro-2- (1-methylethyl) -4-pentenoate, 200 µl of 0.1 M potassium phosphate buffer, pH 8; 150 µl of 10 mg/ml phenol red; 450 µl of DMSO; 650 µl of H2O) or with substrate solution B (identical to solution A but employing methyl (2S,4E)-5-chloro-2-(1-methylethyl)-4-pentenoate instead of the racemate) . Owing to the specific esterase activity on the substrates, the liberation of acid on hydrolysis of the ester substrate results in a pH decrease which in turn leads to a change in the color of the phenol red indicator to yellow.'
It emerged from this that trans formants containing the plasmid pGAPZ A APLE-ER gave signals (yellow coloration around the colony) after incubation for 3 to 4 hours if they were tested on substrate solution A, whereas no significant conversion could be found with substrate solution B (figure 1).
Transformants obtained with the plasmids pGAPZ A PLE-ER or pGAPZ A PLE+ER showed no reaction with substrate solutions A or B under the same conditions. This showed that the recombinant rAPLE , has a substrate specificity different from recombinant rPLE and that hydrolysis of methyl 5-chloro-2-(1-methylethyl)-4-pentenoate using rAPLE takes place stereoselectively for the (R) enantiomer.
Example 8: SDS polyacrylamide gel electrophoresis
10 µl of a 2x SDS sample buffer (125 mM Tris-HCl, pH 6.8; 4% SDS, 20% glycerol, 5% P-mercaptoethanol, 0.05%
bromophenol blue) were added to 10 µl of commercially available pig liver esterase or 10 µl of the 60-fold concentrated (Centricon Ultrafiltrations-Spin Columns, from Sartorius) supernatants of the P. pastoris cultures (72 h at 100 rpm and 28°C in 250 ml of YPD medium in 2 1 Erlenmeyer flasks with baffles) which contained either the plasmid pGAPZ A APLE-ER or an empty pGAPZ A plasmid (control strain).
After the samples had been heated at 95 °C for 5 minutes, the proteins were separated on a 12.5% polyacrylamide gel (4% stacking gel) and stained with Coomassie Brilliant Blue R250 for detection. The SDS-PAGE shows a protein band with the expected size for rAPLE (-60 kDa) in the yeast strain having the pGAPZ A APLE plasmid, but not in the control strain (figure 2) . The commercially available pig liver esterase which was also analyzed for comparison showed two protein bands in the same size range (arrow in figure 2).
Example 9: Induced expression of pig liver esterases with the A0X1 promoter
The plasmids pGAPZ A PLE-ER, pGAPZ A PLE+ER, pGAPZ A APLE-ER and pGAPZ A APLE+ER were cut with the restriction endonuclease Xhol, and the respective fragments coding for APLE and PLE proteins, with or without ER retention signal, were cloned via the Xhol cleavage site into the pPIC9 vector (Invitrogen). Correct orientation of the fragments in relation to the AOX1 promoter was checked by means of control cuts with the restriction endonuclease Ncol. The vectors having the AOX1 promoter were named, in analogy to the plasmids named in example 4, pPIC9 PLE-ER, pPIC9 PLE+ER, pPIC9 APLE-ER and pPIC9 APLE+ER, linearized with Sail and transformed into P. pastoris KM71. Transformation and selection for His prototrophy took place in accordance with the instructions of the Pichia expression kit from Invitrogen. Selected transformants and the KM71 strain were cultured on complete medium in
accordance with the Pichia expression kit from Invitrogen overnight and induced with 1% methanol for 48 h. The resulting cultures were analyzed by means of the qualitative pH-shift assay described in example 7, testing 2 fil of the cultures in the mixtures in each case. Expression under the control of the inducible A0X1 promoter led to very much higher, by comparison with the situation described for constitutive expression in example 7, rAPLE enzymic activities in relation to racemic methyl 5-chloro-2-(1-methylethyl) -4-pentenoate. The phenyl red color change (red to yellow) was detectable after only a few minutes (figure 3). Unexpectedly, the rAPLE activity was independent of the presence of the ER retention signal HAEL at the C terminus, i.e. even cells which expressed rAPLE with ER retention signal showed activity. By contrast, yeast strains for producing rPLE had no activity in relation to racemic methyl 5-chloro-2-(1-methylethyl)-4-pentenoate.

Claims
A polypeptide or recombinant protein having esterase activity and exhibiting an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ. ID. No. 1.
A polypeptide or recombinant protein which exhibits at least 98% identity to the amino acid sequence shown in SEQ ID No. 1 and has activity for racemate resolution of 2-alkyl-5-halopent-4-enecarboxylic esters of the formula (I)
(Formula Removed)
in which R is a C1-C6-alkyl radical, R1 is C1-C4-aikyl and X is chlorine, bromine or iodine.
A polypeptide or recombinant protein having e-sterase activity according to claims 1-2, which exhibits an amino acid sequence modified by a modification selected from the group consisting of mutation, deletion, insertion, extension and fusion.
A nucleotide sequence which codes for a polypeptide or recombinant protein according to claims 1 -3.
A nucleotide sequence comprising the sequence shown in SEQ ID No. 2. A nucleotide sequence obtainable by isolating mRNA
from an organism followed by amplification of a
nucleic acid fragment using a first primer comprising
the nucleotide sequence GGGCAGCCAGCCTCGC and a second
primer comprising the nucleotide sequence
GGGCAGCCAGCCTCGC.
The use of a polypeptide or a recombinant protein according to claims 1-3 for racemic resolution of 2-alkyl-5-halopent-4-enecarboxylic esters of the formula (I)
(Formula Removed)
in which R is a C1-C6-alkyl radical, R1 is C1-C4-alkyl and X is chlorine, bromine or iodine.
A method for preparing enantiomerically enriched 2-alkyl-5-halopent-4-enecarboxylic acids or esters thereof of the formula (II)
(Formula Removed)
in which R is a C1-C6-alkyl radical, A is equal to H, R1, where R1 may be C1-C4-alkyl, or R2, where R2 is an alkyl group, but is not equal to R1, and X is chlorine, bromine or iodine, which comprises a mixture of
enantiomers of a 2-alkyl-5-halopent-4-enecarbcxylic ester of the formula (I)

(Formula Removed)

in which R, R and X are as defined above, being converted by means of a polypeptide or a recombinant esterase according to claims 1-3 in the presence of water or an alcohol of the formula R2OH, where R2 is an alkyl group which is not equal 10 Rl, as nucleophile, and
b)
a) either the remaining enantiomerically enriched 2-alkyl-5-halopent-4-enecarboxylic ester of the formula (II) with A equal to R1 being isolated or if an alcohol is employed as nucleophile, the resulting enantiomerically enriched 2-alkyl-
halopent-4-enecarboxylic ester
with A equal to R2 being isolated, or c) if water is employed as nucleophile, the resulting 2-alkyl-5-halopent-4-enecarboxylic acid of the formula (II) with A equal to H being isolated.

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Patent Number

268709

Indian Patent Application Number

5611/DELNP/2008

PG Journal Number

38/2015

Publication Date

18-Sep-2015

Grant Date

14-Sep-2015

Date of Filing

27-Jun-2008

Name of Patentee

DSM FINE CHEMICALS AUSTRIA NFG GMBH & CO KG

Applicant Address

C/O DSM INTELLECTUAL PROPERTY, P.O.BOX 9, 6160 MA, GELEEN, THE NETHERLANDS.

Inventors:

#	Inventor's Name	Inventor's Address
1	STEINBAUER, GERHARD	LORCH 21, A-4470 ENNS, AUSTRIA.
2	STANEK, MICHAEL	PILLWEINSTRASSE 43, A-4020 LINZ.AUSTRIA.
3	POJARLIEV, PETER	ROTENMüHLGASSE 44/23, A-1120 WIEN, AUSTRIA.
4	SKRANC, WOLFGANG CHEMIKER	OBERFELDSTRASSE 23, A-1220 WIEN, AUSTRIA.
5	SCHWAB, HELMUT	MARIAGRüNERSTRASSE 91, A-8043 GRAZ, AUSTRIA.
6	WUBBOLTS, MARCEL GERHARDUS	PREVOTLAAN 9, NL-6132 BM SITTARD, THE NETHERLANDS.
7	KIERKELS, JOANNES	MARGRIETLANN 17, NL-6133 BH SITTARD THE NETHERLANDS.
8	PICHLER, HARALD	HOLLENEGGERSTRASSE 42, A-8530 DEUTSCHLANDSBERG, AUSTRIA.
9	ZENZMAIER, CHRISTOPH	FREISINGSTRASSE 8, A-6020 INNSBRUCK, AUSTRIA.
10	HERMANN, MANUELA	KLEEGASSE 8, A-8042 WERNDORF, AUSTRIA.

PCT International Classification Number

C12N 9/18

PCT International Application Number

PCT/EP2006/011832

PCT International Filing date

2006-12-08

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	A 2081/2005	2005-12-27	Austria