MICROORGANISM AND ESTERASE OBTAINED THEREFROM

Abstract: A novel microorganism is characterised by the ability to produce (S)-N-benzoyl-2-azetidinecarboxylic acid, in at least 99 % enantiomeric excess, from racemic N-benzoyl-2-azetidinecarboxylic acid methyl ester. An enzyme has been obtained from the organism, and has the same or opposite enantioselectivity.

Description: MICROORGANISM AND ESTERASE OBTAINED THEREFROM Field of the Invention This invention relates to a microorganism and to a stereoselective esterase obtained therefrom. Background of the Invention Bioresolution is a procedure which is known to be of use generally in the production of enantiomerically-pure compounds. However, the potential utility of a specific biocatalyst in the resolution of a specific compound is almost impossible to predict. Azetidine-2-carboxylic acid and analogues are useful synthons in the production of certain therapeutic agents. If the synthon is chemically synthesised, it is racemic. By conventional synthesis, the ultimate drug will also be a mixture of enantiomers, which causes regulatory concerns if one of the enantiomers is not very active or causes unwanted side-effects. There is a need therefore to put a step into the synthesis where either of the two enantiomers of a racemic synthon can be isolated and the rest of the drug then built on it. An effective way of doing this is to use an enzyme to selectively hydrolyse one enantiomer of an ester of the acid to give the acid as one enantiomer, and leave the other enantiomer as the ester. The components of the mixture can then be readily separated. WO-A-9802568 discloses that enantiomerically-enriched azetidine-2-carboxylic acid may be obtained in enantiomerically-purified form and in high yield via a novel and efficient bioresolution process. Summary of the Invention According to the present invention, a novel organism produces an enzyme that is useful for biocatalysis, e.g. for the process described in WO-A-9802568. Surprisingly, it has been found that a strain of Aspergillus is of particular value. A novel organism identified as Aspergillus tamarii (CMC 3242), has been deposited at the International Mycological Institute, Egham, Surrey, UK, on 8th July 1997, under the terms of the Budapest Treaty. Its accession number is IMI 375930. The gene encoding the esterase has been isolated and sequenced (see SEQ ID NO: 1), and the enzyme's amino-acid sequence derived (see SEQ LD NO:2). This invention relates to compounds having this structure, and fragments thereof having the same activity, as will be readily evident to one of ordinary skill in the art. The novel wild-type enzyme is characterised by the ability to convert racemic N-benzoyl-2-azetidinecarboxylic acid and give (S)-2-azetidinecarboxylic acid in at least 99% ee; the novel recombinant enzyme gives the opposite enantiomer, also in at least 99% ee. These results may be achieved under the conditions given herein. Description of the Invention By way of illustration, the novel organism and isolated enzyme may be used in a process for obtaining an enantiomerically-enriched N-acylazetidine-2-carboxylic acid, by the biotransformation of a racemic N-acylazetidine-2-carboxylic acid ester. By "enantiomerically enriched" we mean any mixture of the enantiomers in which one is present in a greater proportion than the other, e.g. at least 50% ee, preferably at least 70% and more preferably at least 90%. If this is not reached by resolution, the ee (enantiomeric excess) may often be enhanced by crystallisation. The process according to the invention comprises the preferential hydrolysis of one acid ester enantiomer to the corresponding acid. The acid and residual ester may be readily separated. The remaining ester (if undesired) may be recovered, racemised (using for example a base, such as sodium methoxide), and reused in the resolution process. Suitable substrates for use in the invention are esters of chiral acids. Such acids include profens such as naproxen. Esters which may be mentioned include aryl (e.g. phenyl) or linear or cyclic alkyl (especially lower alkyl) esters. Particular esters which may be mentioned include propyl, ethyl and especially methyl esters. The new organism is characterised by its ability to produce (S)-N-benzoylazetidine- 2-carboxylic acid by bioresolution of racemic JV-benzoylazetidinecarboxylic acid ester. The enzyme activity may be isolated in conventional manner. Surprisingly, it has been found that a truncated enzyme has the opposite enantiospecificity, thereby allowing the production of either acid enantiomer in excess from racemic ester substrate. The enzyme may be used in whole cell or isolated form. It may be immobilised, if desired, by methods known to those of ordinary skill in the art. The enzyme may be produced from the deposited organism. Alternatively, it may be produced by recombinant techology. Using the DNA and amino-acid sequence provided herein, a person skilled in the art can readily construct fragments or mutations of the genes and enzymes disclosed herein. These fragments and mutations, which retain the activity of the exemplified enzyme, are within the scope of the present invention. Also, because of the redundancy of the genetic code, a variety of different DNA sequences can encode the amino-acid sequences disclosed herein. It is well within the skill of one of ordinary skill in the art to create these alternative DNA sequences encoding the same, or similar, enzymes. These DNA sequences are within the scope of the present invention. As used herein, reference to "essentially the same" sequence refers to sequences which have amino-acid substitutions, deletions, additions or insertions which do not materially affect activity. Fragments retaining activity are also included in this definition. The genes of this invention can be isolated by known procedures and can be introduced into a wide variety of microbial hosts. Expression of the gene results, directly or indirectly, in the intracellular production and maintenance of the enzyme. The gene may be introduced via a suitable vector into a microbial host. A wide variety of ways are available for introducing the gene into the microorganism host under conditions which allow for stable maintenance and expression of the gene. A DNA construct may include the transcriptional and translational regulatory signals for expression of the gene, the gene under their regulatory control and a DNA sequence homologous with a sequence in the host organism, whereby integration will occur, and/or a replication system which is functional in the host, whereby integration or stable maintenance will occur. In the direction of transcription, namely in the 5' to 3' direction of the coding or sense sequence, the construct can involve the transcriptional regulatory region, if any, and the promoter, where the regulatory region may be either 5' or 3' of the promoter, the ribosomal binding site, the initiation codon, the structural gene having an open reading frame in phase with the initiation codon, the stop codon(s), the polyadenylation signal sequence, if any, and the terminator region. This sequence as a double strand may be used by itself for transformation of a microorganism host, but will usually be included with a DNA sequence involving a marker. The gene can be introduced between the transcriptional translational initiation and termination regions, so as to be under the regulatory control of the initiation region. This construct can be included in a plasmid, which could include at least one replication system, but may include more than one, where one replication system is employed for cloning during the development of the plasmid and the second replication system is necessary for functioning in the ultimate host. In addition, one or more markers may be present, as described above. Where integration is desired, the plasmid will desirably include a sequence homologous with the host genome. The transformants can be isolated in accordance with conventional ways, usually employing a selection technique, which allows for selection of the desired organism as against unmodified organisms or transferring organisms, when present. The transformants then can be tested for activity. Suitable host cells include prokaryotes and eukarotes. An example is E. coli. The following Examples illustrate the invention. Identification of an Esterase-containing Strain Selected microbial strains from the Chirotech Technology strain collection with known esterase activity were grown in a medium consisting of an aqueous solution of KH 2 PO 4 (7 g/L), K 2 HPO 4 (2 g/L), (NH 4 ) 2 SO 4 (1 g/L), yeast extract (10 g/L), a trace elements solution (1 ml / L) and glucose (10 g/L). The medium was made up at 25mL per 250mL Erlenmeyer flask and was adjusted to pH 6.0 (for fungi and yeasts) and pH 7.0 (for bacteria) prior to sterilisation at 121°C for 20 minutes. The trace elements solution consisted of CaCl 2 .2H 2 O (3.6 g/L), CoCl 2 .6H 2 O (2.4 g/L), CuCl 2 .2H 2 O (0.85 g/L), FeCl 3 .6H 2 O (5.4 g/L), H 3 BO 4 (0.3 g/L), cone. HCl (333 ml L), MnCl 2 .4H 2 O (2.0 g/L), Na 2 MoO 4 .2H 2 O (4.8 g/L), and ZnO (2.0g/L). An amount of lOOμl of glycerol stock of each strain was inoculated into the flasks and grown at 25°C in a New Brunswick controlled environment incubator shaker (Model No. G-25) at 250 rpm for 24 to 72 hours. lOmL of each culture were then harvested by centrifugation and the pellets resuspended in 4 mL 50mM KH 2 PO 4 , pH 7.0. In a scintillation vial 860μL of 50mM KH 2 PO 4 , pH 7.0 was mixed with 40μL of 50% w/v N-benzoyl-2-azetidinecarboxylic acid methyl ester (2.0g N-benzoyl 2- azetidinecarboxylic acid methyl ester + 2 mL H 2 O + 0.02g Tween 80, sonicated at 15- 18μm at 4°C for 10 minutes at 10 seconds on and 3 seconds off) and lOOμL of resuspended culture, grown as described above. Reactions were carried out in a New Brunswick controlled environment incubator shaker (Model No. G-25) at 25°C, 250 rpm. Samples were taken over up to 7 days and assayed for conversion by HPLC. Thus, samples were diluted, as appropriate, and 20μl injected onto a 5 cm Hypersil BDS C18 column. The elution buffer was 50% v/v MeOH + lg/L H 3 PO 4 . Flow rate was 1.5 mL.min " 1 and detection was at 225nm, with a run time of 3 minutes. For those reactions showing significant hydrolysis, enantiomeric excesses (ee) of the ester and product were determined. The enantiomeric excess (ee) of the ester was determined by GC. The pH of the samples was adjusted to pH 9.5 with NaOH and extracted into ethyl acetate, dried with Mg 2 SO 4 and injected onto a 25m x 0.25mm Chirasil DEX CB column. The oven temperature was maintained at 125°C during the analysis. The enantiomeric excess (ee) of the product was determined by HPLC. The pH of the samples was adjusted to pH 9.5 with NaOH and extracted four times into ethyl acetate to remove the ester. The pH was then adjusted to 1.5 with H 3 PO 4 and the product extracted into ethyl acetate, dried with Mg 2 SO 4 , and 20μl injected onto a 25cm Chiralcel OD column. The elution buffer was 92:8:1 heptane: propan-2-ol: trifluoroacetic acid. The flow rate was 1.0 mL.min "1 and detection was at 254nm. One strain, Aspergillus tamarii (CMC 3242), in the initial screen achieved 30% conversion of the added substrate after 48 hours biotransformation. The residual ester was shown to be the (R)-enantiomer with a ee in excess of 99% and the product to be the (S)-enantiomer with an ee in excess of 74%. The strain has been deposited at the IMI, as described above. Fermentation of Aspergillus tamarii, CMC 3242 For preparation of spore suspension inoculum, a culture of Aspergillus tamarii was spread-plated onto a PDA plate ( 39g/L potato dextrose agar (Oxoid CM139) sterilised at 121°C for 20 minutes, cooled to 50°C and poured into 140mm petri dishes) and incubated at 25°C for 7 days. The spores of Aspergillus tamarii were then resuspended in sterile (sterilised at 121°C for 20 minutes) 10% w/v glycerol + 0.1% w/v Tween 80. 1 mL samples were aliquoted into 2 mL cryovials and stored at -80°C. The following medium was used in the fermenters: KH 2 PO 4 (7 g/L), K 2 HPO 4 (2 g/L), (NH 4 ) 2 SO 4 (1 g/L), MgSO 4 .7H 2 O (1 g/L), Trace elements solution (1 mL/L), polypropylene glycol (1 mL/L), yeast extract (20 g/L), and sucrose (20 g/L). The medium was made up to a final volume of 1.5 L per fermenter and the pH adjusted to 6.0 prior to sterilisation (60 minutes at 121°C). The sucrose was sterilised separately as a 50% w/v solution and added to the fermenter after cooling. The fermenter was inoculated with 1 mL of the spore suspension. The temperature was maintained at 25°C and pH controlled between 5.8 and 6.2. Agitation was 1000 rpm and air flow set at 1.0 LΛnin. A feed of 100 mL 34% w/v sucrose + 100 mL 34 % w/v yeast extract + 1.7g/L (NH 4 ) 2 SO 4 (sterilised separately at 121°C for 60 minutes) was added to the fermenters after 48 hours. The fermenters were harvested after 72 hours growth by filtration and stored as a cell paste at -20°C. A total wet bio ass of 250g was collected, with an activity of 41.7 U.g "1 of cells (1U = 1 mg product produced in 1 hour). Whole Cell Biotransformation Frozen cell paste (50 g) was thawed in 200 mL 0.1M Na 2 HPO 4 /NaH 2 PO 4 buffer, pH 6.4. The cells were disrupted using a mortar and pestle. lOOg of racemic N-benzoyl-2- azetidinecarboxylic acid methyl ester was added to the reaction and the volume made upto 1000 mL with 200 mL 0.1M Na 2 HPO 4 /NaH 2 PO 4 buffer, pH 6.4. The reaction was run at 25°C and pH controlled at 6.4. A further 50g cells was added after 4.5 hours. After 12 hours, the biotransformation broth was filtered through a Celite pad. The residues were washed with dichloromethane (500 mL), the biphasic filtrate partitioned and the aqueous solution extracted with further dichloromethane (4 x 750 mL). The combined dichloromethane solutions were washed with brine (200 mL), dried (MgSO 4 ), filtered and the solvent evaporated in vacuo to yield an orange oil (56.1 g, 56.1% recovery, 54 % ee). The original aqueous solution was acidified (pH2, c.HCl) and extracted again with dichloromethane (3 x 750 mL). The organic (lightly emulsified) solutions were separated each time and combined. These combined extracts separated out on standing (1 hour) and were put through a separating funnel. The organic layer was washed with brine (100 mL), dried (MgSO 4 ), filtered and the solvent evaporated in vacuo to yield a semi-crystalline solid (33.7 g, 36% yield, 94 % ee). The semi-crystalline acid (33 g) was stirred in ethyl acetate (75 mL) for a total of 20 minutes at room temperature and the white solid (18 g, 55 % yield, >99 % ee) collected by suction filtration. The filtrate was evaporated to dryness and a second crop of product isolated (2 g, 6 % yield, >99 % ee). *H nmr (CDC1 3 ) was consistent with the structure of the product. (S)-N-Benzoylazetidine-2-carboxylic acid (5.90 g, 28.78 mmol) was dissolved in a solution of sodium hydroxide (6.92 g, 0.173 mol) in water (92 mL), at room temperature with stirring. The reaction was heated to 75°C for 22 hours and then allowed to cool to room temperature. The reaction mixture was adjusted to pH 2 (c.HCl) and extracted with ethyl acetate (3 x 100 mL). The aqueous solution was then evaporated to dryness, yielding a white solid (13 g). This was slurried in absolute ethanol (150 mL) at 50°C for 1 hour and then allowed to cool to room temperature (~1 hour). The salt was removed by suction filtration and the ethanolic solution evaporated to dryness, yielding a white solid (1.85g, 47% yield). The isolate was re-dissolved in water (100 mL) and neutralised by stirring with Amberlite IRA-67 ion exchange resin (5 g) for 0 mins ( to pH 7.1). The resin was removed by suction filtration and the filtrate evaporated to dryness, yielding a slightly off-white solid (1.38 g, quantitative yield). The off-white solid was slurried with refluxing MeOH (10 mL) for 5 minutes, allowed to cool to room temperature and the purified product collected by suction filtration (905 mg, 31% yield, 98% ee). ! H nmr (D 2 O) was consistent with the structure of the product. Total RNA Preparation A 125ml culture of Aspergillus tamarii CMC 3242 was incubated at 30°C for 48 hr. Cells were harvested by filtration (Whatman 2.7μm GF/D glass microfibre). The recovered cells (12g wet weight) were placed in a RNase-free pestle and mortar with liquid N 2 and homogenised. The homogenisation was repeated 3 times incorporating 28 ml of Extraction buffer. The suspension was transferred to RNase-free centrifuge tubes and cell debris was pelletted by centrifugation (5000g, 30 min, 15°C). The supernatant was transferred to a clean, RNase-free tube, where the DNA was sheared by passing the supernatant through a 16 gauge syringe needle at least ten times. Aliquots (6.5ml) of the sheared solution where layered upon 6.0 ml of caesium trifluoroacetate in 13ml ultracentrufuge tubes. The tubes were then place in a 'swing-out' ultracentrifuge rotor, centrifugation (125000g, 15 hr, 20°C) formed a gradient to resolve the RNA. The gradient was aspirated down to the RNA pellet, which was then resuspended in lOOμl TE buffer. The RNA was then heated to 65°C for 10 min, clarified by centrifugation (16000g, 10 min, 20°C), the supernatant was mixed with lOOμl TE and heating and clarification repeated. The RNA concentration of the supernatant was determined with ethidium bromide by fluorescence against standards. The total RNA preparation was precipitated by the addition of 1/10 sample volume of 3M sodium acetate and 2.5 sample volumes of 100% (v/v) ethanol, and the precipitate was stored at -20°C until required. mRNA Purification The total RNA precipitate was recovered by centrifugation (21 OOOg, 30 min, 4°C), and the pellet was washed with RNase free 70% (v/v) ethanol (21 OOOg, 10 min, 4°C). The RNA pellet was dried and resuspended in 300μl TE buffer. From the resuspended total RNA preparation, mRNA was purified using the Pharmacia Biotech QuickPrep mRNA Purification Kit # 27-9254B. Two oligo (dT) cellulose columns were equilibrated and 150μl of the RNA preparation was mix with 3.85 ml of Elution buffer and applied to each column. The mRNA was allowed to bind by centrifugation (350g, 2 min, 20°C) and residual contaminants were removed by 3 x 3ml High Salt buffer washes (350g, 2 min, 20°C). Two subsequent washes were then carries out with 3ml of Low Salt buffer (350g, 2 min, 20°C). The bound mRNA was eluted by the addition of 2 x 250μl of Elution buffer (pre- warmed at 65°C) and centrifugation (350g, 2 min, 20°C). The eluted mRNA was then precipitated by the addition of lOμl glycogen, 1/10 ampoule volume potassium acetate solution and 1ml of 100% (v/v) ethanol, and the sample was placed at -20°C for 2 hr to complete the precipitation. The precipitate was recovered by centrifugation (21 OOOg, 30 min, 20°C). The mRNA pellet was washed with 70% (v/v) ethanol (21 OOOg, 2 min, 20°C) and dried. The mRNA pellet was then resuspended in 25 μl DEPC H 2 O, ready for subsequent cDNA synthesis. cDNA Synthesis The conversion of prepared mRNA to cDNA was carried out employing the Stratagene Zap-cDNA Synthesis kit #200400. First-strand synthesis was initialised by the binding of a poly d(T)-linker primer to the poly A tract of the mRNA and first-strand cDNA was reverse transcribed by MMLV-RT (reverse transcriptase). Incorporation of radio-labelled 35 P-dATP was used to monitor cDNA synthesis. At the completion of first- strand synthesis, the mRNA was nicked to form primers for second-strand synthesis, which was completed by DNA polymerase. An autoradiograph confirmed the production of sufficient quantities of double-stranded cDNA of sizes that flanked the 2.1 kb control. cDNA fragment preparation was completed by the "blunting", phosphorylation, ligation of an EcόRI linker and finally Xhol digestion, to form an asymmetric fragment which gives the correct orientation for expression. Size fractionation of cDNA Given that the desired cDNA fragment should encode a gene product (esterase) of around 83 kDa, cDNA fragments of 1.8-2.4 kb would be favourable for cloning and screening. Therefore, to produce separate size pools of cDNA's, the heterologous population was fractionated through a Sepharose C1-2B gel filtration column. The elution of cDNA and unincorporated 32 P-dATP was monitored by Geiger-counter. cDNA fragments were eluted largest to smallest as the matrix retained smaller fragments. Four eluted fragment pools were extracted with phenohchloroform (21000g, lOmin, 20°C) and precipitated with ethanol. The cDNA precipitated was recovered by centrifugation (21000g, 30min, 4°C), pellets were dried and resuspended in 2.5μl H 2 O. N-terminal A ino Acid Sequence-Derived Oligonucleotide Primers From the purification of the vA\d-type Aspergillus tamarii CMC 3242 esterase, one N-terminal amino acid sequence (SEQ LD NO.3) was determined to be KPPQTKK. From this sequence, 5 degenerate oligonucleotide primers (SEQ ID NOS:4-8) were synthesised. Another 3 degenerate oligonucleotide primers (SEQ LD Nos. 9-11) were synthesised based on another N-terminal sequence, i.e. DQAQWFK (SEQ ID NO: 12). Polymerase Chain Reaction (PCR) Using a synthesised poly 20) oligonucleotide primer and each of the primers above, PCR was carried out on cDNA prepared from Aspergillus tamarii CMC 3242. Employing variations on temperature and Mg 2+ concentrations, a range of PCR fragments was obtained. Fragments of between 1.5 and 2.5kb were isolated, purified and cloned into pCR2.1 (Invitrogen Ltd.) and pCR-script (Stratagene). In addition, PCR fragments were blunted and ligated into pUC19. Ligation reactions were transformed into E. coli strain INVα F', XLl-Blue MRF' and DH5α, respectively. The transformed cells were then plated onto Tryptone Soya Broth Agar (Oxoid Ltd. #CM129), with ampicillin (100 μg ml "1 ), lmM LPTG, and Xgal (50μg ml "1 ). Transformation experiments that were to be overlaid with N-benzyloxycarbonyl-2-azetidinecarboxylate were plated onto Tryptone Soya Broth Agar plus supplements in glass (borosilica) Petri dishes. Screening off. coli Esterase Expressing Transformants Transformed E. coli colonies were overlaid with methyl N-benzyloxycarbonyl- (S)-phenylalanine, methyl N-benzyloxycarbonyl-2-azetidinecarboxylate or methyl (S)-benzyloxycarbonyl-2-azetidinecarboxylate, by the following respective procedures. Oxoid Bactoagar was prepared 1% (w/v) in 200 mM KH 2 /K 2 HPO 4 pH 6.4, whilst molten (~60°C), Tween 80 (-0.5 mg.mT 1 ) and methyl N-benzyloxycarbonyl-(S)- phenylalanine (2mg.ml "1 ) were added. The emulsion was formed by vigorous shaking. Once cooled (48°C) the ester suspension was poured over the E. co/z ' -transformed colonies. Plates were left at room temperature for 24-48 hr, and observed for zones of clearance. This procedure was varied only to the extent of using 100 mM KFL/^HP t pH 6.4, with methyl N-benzyloxycarbonyl-2-azetidinecarboxylate (δmg.ml '1 ) and with methyl (S)-N-benzyloxycarbonyl-2-azetidinecarboxylate (δmg.ml "1 ). Isolation of Recombinant Esterase The overlay of PCR transformants identified 147 transformants which gave the desired clearance zones when overlaid with the racemic N-benzyloxycarbonyl-2- azetidinecarboxylate. Of these 147 isolates, 10 clones were able to produce significant clearing upon overlay with (S)-N-benzyloxycarbonyl-2-azetidinecarboxylate. Plasmid DNA was prepared from these 10 putative esterase clones. Agarose gel electrophoresis showed all 10 clones harbouring the same insert, with regards to size and restriction profile. A 3.0 kb band comprised the pCR-script cloning vector (2.9 kb) plus a 100 bp of the cloned PCR fragment; bands apparent at 900 bp and 2x 350 bp were Xhol fragments of the PCR insert. DNA Sequence Analysis of Putative Esterase DNA sequence analysis of the putative esterase clone showed there to be a 1.5kb insert (SEQ ID NO:l). The longest deduced open reading frame extends to 1283bp, translating a peptide of 427 amino acid residues (SEQ LD NO:2; approx. 43 kDa.). No sequence homologous to any of the permutations of N-terminal derived sequence was identified. Indeed, deletions within the multiple cloning site of the cloning vector had occurred, as well as rearrangement within the cloned fragment. In addition to the truncation, the putative recombinant esterase was shown to have the opposite selectivity from the wild-type Aspergillus esterase. A possible explanation of the deletion of a polylinker (-60 bp) and the absence of any N-terminal coding sequence could be recombination brought about by stem-loop formation caused by DNA homology around the point of insertion. TheE. coli host DNA mismatch repair mechanism may cleave the stem-loop in order that the plasmid can successfully replicate, thereby deleting the 5' region of the PCR insert and a portion of the pCR-script polylinker. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Chirotech Technology Limited (B) STREET: Cambridge Science Park, Milton Road (C) CITY: Cambridge (D) STATE: N/A (E) COUNTRY: United Kingdom (F) POSTAL CODE (ZIP) : CB4 4WE (ii) TITLE OF INVENTION: MICROORGANISM AND ESTERASE OBTAINED THEREFROM (iii) NUMBER OF SEQUENCES: 12 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO) (v) CURRENT APPLICATION DATA: APPLICATION NUMBER: WO Not yet known (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1532 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Aspergillus tamarii (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:!..1281 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: ATG ACC ATG ATT ACG CCA AGC TCA GCC ATG GGA ATG TCG TCT ACA TTT 48 Met Thr Met lie Thr Pro Ser Ser Ala Met Gly Met Ser Ser Thr Phe 1 5 10 15 GAA TTT GAG CGT GTC ATT ACC AAG GCG GTC GAG GAC CGC GTC ATT CCT 96 Glu Phe Glu Arg Val lie Thr Lys Ala Val Glu Asp Arg Val lie Pro 20 25 30 GGC GTC GTG CTG CTG GCC GAA AAC TCA TCG GGC TCG TAT CAC TAT GAA 144 Gly Val Val Leu Leu Ala Glu Asn Ser Ser Gly Ser Tyr His Tyr Glu 35 40 45 AAG GTA CTC GGC TAC AGC TCC ATC GAG GCG GGC AAC GAG AAA AAG CTC 192 Lys Val Leu Gly Tyr Ser Ser lie Glu Ala Gly Asn Glu Lys Lys Leu 50 55 60 GAG CGC GAC AGC GTG TTC ACG TTC ATG TCC ATG ACC AAG TTC ATA ACA 240 Glu Arg Asp Ser Val Phe Thr Phe Met Ser Met Thr Lys Phe lie Thr 65 70 75 80 GCC ATT GTG GCC ATG CAG GCC GTC GAG CGT GGT CTC TGG GAC CTC GAT 288 Ala lie Val Ala Met Gin Ala Val Glu Arg Gly Leu Trp Asp Leu Asp 85 90 95 GCC GAT GTC GCG CCT CTG CTG CCC GAA CTG GCG GCC CTG CCT GTC CTC 336 Ala Asp Val Ala Pro Leu Leu Pro Glu Leu Ala Ala Leu Pro Val Leu 100 105 110 AAG GGT TTC TCA GAC GAT GGC GTG CCC GAG CTG GTG CCG CGC GAG TCG 384 Lys Gly Phe Ser Asp Asp Gly Val Pro Glu Leu Val Pro Arg Glu Ser 115 120 125 GCC ATT ACG CTG CGC CAG CTG TTG TCG CAC ACG TCG GGC GCA GCC TAC 432 Ala lie Thr Leu Arg Gin Leu Leu Ser His Thr Ser Gly Ala Ala Tyr 130 135 140 GAC TTC TTG TCG CCC GAT CTG ATC AAC TAC CAC GCG TGG GTA CGC AAG 480 Asp Phe Leu Ser Pro Asp Leu lie Asn Tyr His Ala Trp Val Arg Lys 145 150 155 160 CAG CCT CCA TCG GCC GGT CTT GAG CAG CCA CCG GCA ATG ACT GTG GCA 528 Gin Pro Pro Ser Ala Gly Leu Glu Gin Pro Pro Ala Met Thr Val Ala 165 170 175 CCT CCG TCT GTT GAG GAG CGG TTC CGG TTT CCG CTC GTA TTC CAG CCT 576 Pro Pro Ser Val Glu Glu Arg Phe Arg Phe Pro Leu Val Phe Gin Pro 180 185 190 GGC CAA GGC TGG CAG TAC GGC TCG AGC CTG GAC TGG GTC GGC CGT CTT 624 Gly Gin Gly Trp Gin Tyr Gly Ser Ser Leu Asp Trp Val Gly Arg Leu 195 200 205 GTT GAA GCG CCT GGA CGC CAA GAC ACA GGG GCA AGA CTG AAG AAG GAA 672 Val Glu Ala Pro Gly Arg Gin Asp Thr Gly Ala Arg Leu Lys Lys Glu 210 215 220 GCA GGC ACC AAG CTG CCG TCG GTG CCT CTG GAA GAG ATT GTG ATC CGT 720 Ala Gly Thr Lys Leu Pro Ser Val Pro Leu Glu Glu lie Val lie Arg 225 230 235 240 GAT GTG CTC ACA CCC CTG GGG CTG CCT GCT GGC GCC TTG ACG TTC TCG 768 Asp Val Leu Thr Pro Leu Gly Leu Pro Ala Gly Ala Leu Thr Phe Ser 245 250 255 CCG GAG CGC TAT CCC GAC GTG TTC GCA CGA ATG TGG CCA TCG CTG CCC 816 Pro Glu Arg Tyr Pro Asp Val Phe Ala Arg Met Trp Pro Ser Leu Pro 260 265 270 GTG CGT GTC GGC AAC AAC GGT GCA CTG GAT GGC GGA CCA GTT GTG CAT 864 Val Arg Val Gly Asn Asn Gly Ala Leu Asp Gly Gly Pro Val Val His 275 280 285 GGT CCG TCC GTG TAC AAA AAG GCA CCT GCT GCT CTG GGC GGA CAG GGC 912 Gly Pro Ser Val Tyr Lys Lys Ala Pro Ala Ala Leu Gly Gly Gin Gly 290 295 300 ATG TAC GGC GAC ATG CCA TCG TTT TTC AAG GTG GCA CTC TCT ATT TTC 960 Met Tyr Gly Asp Met Pro Ser Phe Phe Lys Val Ala Leu Ser lie Phe 305 310 315 320 CGC GAC GAC GGC AAG CTG CTG AAG CCC GAG TCA ACG AAG CTA TTC TTT 1008 Arg Asp Asp Gly Lys Leu Leu Lys Pro Glu Ser Thr Lys Leu Phe Phe 325 330 335 GAG CCG CAG CTG GCG TCC AAG GCC GCA CAC GCG GGC ATT ATG CAT GGC 1056 Glu Pro Gin Leu Ala Ser Lys Ala Ala His Ala Gly lie Met His Gly 340 345 350 ACC GAG AAC TCT GGA TGG ATT ACT GGC GAT GTG CCC GAC ACC AAG GAG 1104 Thr Glu Asn Ser Gly Trp He Thr Gly Asp Val Pro Asp Thr Lys Glu 355 360 365 TAC TGC TGG AGT GTT GCT GGC CTT TTG GTG ACG GGC GAC TCG CAC CCA 1152 Tyr Cys Trp Ser Val Ala Gly Leu Leu Val Thr Gly Asp Ser His Pro 370 375 380 TTC CGG AAG AGG GGT GCT GTT CTG TGG GCC GGC GCG TTC AAC CTG ACA 1200 Phe Arg Lys Arg Gly Ala Val Leu Trp Ala Gly Ala Phe Asn Leu Thr 385 390 395 400 TGG ATC ATA GAC AAG GAG GCC GAT GTG TGC GCC GTG TTT GGA TCC AAC 1248 Trp He He Asp Lys Glu Ala Asp Val Cys Ala Val Phe Gly Ser Asn 405 410 415 TAC CAG CCG CCA GGC GAC CAG CAG GCA AGG CGC TGATGCGGCA GTGGGAGGAG 1301 Tyr Gin Pro Pro Gly Asp Gin Gin Ala Arg Arg 420 425 TTTGTCTACT CGCAAGCCAA GACGGCGAAG CTGTAGGCGG GAGGTATATG TCACAGTTTT 1361 GTCAAGTCTC AAGTTTGGAT AATATATTAA AAATGTAAAT TGCAGTAAAT TTCAAAAAAA 1421 AAAAAAAAAA ACTCGAGGGG GGGGGATCCG TCGGAAATAC AGGAACGCAC GCTGGATGGC 1481 CCTTCGCTGG GATGGTGAAA CCATGAAAAA TGGTAGCTTC AGTGGATTAA G 1532 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 427 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Thr Met He Thr Pro Ser Ser Ala Met Gly Met Ser Ser Thr Phe 1 5 10 15 Glu Phe Glu Arg Val He Thr Lys Ala Val Glu Asp Arg Val He Pro 20 25 30 Gly Val Val Leu Leu Ala Glu Asn Ser Ser Gly Ser Tyr His Tyr Glu 35 40 45 Lys Val Leu Gly Tyr Ser Ser He Glu Ala Gly Asn Glu Lys Lys Leu 50 55 60 Glu Arg Asp Ser Val Phe Thr Phe Met Ser Met Thr Lys Phe He Thr 65 70 75 80 Ala He Val Ala Met Gin Ala Val Glu Arg Gly Leu Trp Asp Leu Asp 85 90 95 Ala Asp Val Ala Pro Leu Leu Pro Glu Leu Ala Ala Leu Pro Val Leu 100 105 110 Lys Gly Phe Ser Asp Asp Gly Val Pro Glu Leu Val Pro Arg Glu Ser 115 120 125 Ala He Thr Leu Arg Gin Leu Leu Ser His Thr Ser Gly Ala Ala Tyr 130 135 140 Asp Phe Leu Ser Pro Asp Leu He Asn Tyr His Ala Trp Val Arg Lys 145 150 155 160 Gin Pro Pro Ser Ala Gly Leu Glu Gin Pro Pro Ala Met Thr Val Ala 165 170 175 Pro Pro Ser Val Glu Glu Arg Phe Arg Phe Pro Leu Val Phe Gin Pro 180 185 190 Gly Gin Gly Trp Gin Tyr Gly Ser Ser Leu Asp Trp Val Gly Arg Leu 195 200 205 Val Glu Ala Pro Gly Arg Gin Asp Thr Gly Ala Arg Leu Lys Lys Glu 210 215 220 Ala Gly Thr Lys Leu Pro Ser Val Pro Leu Glu Glu He Val He Arg 225 230 235 240 Asp Val Leu Thr Pro Leu Gly Leu Pro Ala Gly Ala Leu Thr Phe Ser 245 250 255 Pro Glu Arg Tyr Pro Asp Val Phe Ala Arg Met Trp Pro Ser Leu Pro 260 265 270 Val Arg Val Gly Asn Asn Gly Ala Leu Asp Gly Gly Pro Val Val His 275 280 285 Gly Pro Ser Val Tyr Lys Lys Ala Pro Ala Ala Leu Gly Gly Gin Gly 290 295 300 Met Tyr Gly Asp Met Pro Ser Phe Phe Lys Val Ala Leu Ser He Phe 305 310 315 320 Arg Asp Asp Gly Lys Leu Leu Lys Pro Glu Ser Thr Lys Leu Phe Phe 325 330 335 Glu Pro Gin Leu Ala Ser Lys Ala Ala His Ala Gly He Met His Gly 340 345 350 Thr Glu Asn Ser Gly Trp He Thr Gly Asp Val Pro Asp Thr Lys Glu 355 360 365 Tyr Cys Trp Ser Val Ala Gly Leu Leu Val Thr Gly Asp Ser His Pro 370 375 380 Phe Arg Lys Arg Gly Ala Val Leu Trp Ala Gly Ala Phe Asn Leu Thr 385 390 395 400 Trp He He Asp Lys Glu Ala Asp Val Cys Ala Val Phe Gly Ser Asn 405 410 415 Tyr Gin Pro Pro Gly Asp Gin Gin Ala Arg Arg 420 425 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Lys Pro Pro Gin Thr Lys Lys 1 5 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: AARCCNCCNC ARACNAARAA R 21 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: AAAARCCNCC NCARACNAAR AAR 23 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: AAAAARCCNC CNCARACNAA RAAR 24 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Oligonucleotide" (ix) FEATURE: (A) NAME/KEY: misc_difference (B) LOCATION: replaced..21, "n") (D) OTHER INFORMATION: /standard name= "Inositol" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: AANCCNCCNC ANACNAANAA N 21 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: ACCNCCNCAR ACNAARAAR 19 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: GAYCARGCNC ARTTYAARCC 20 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: AGAYCARGCN CARTTYAARC C 21 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: AAGAYCARGC NCARTTYAAR CC 22 (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: Asp Gin Ala Gin Trp Phe Lys 1 5

Claims: CLAIMS 1. A microorganism having the characteristics of that available as IMI 375930. 2. A microorganism characterised by the ability to produce (S)-N-benzoyl-2- azetidinecarboxylic acid, in at least 99% enantiomeric excess, from racemic N-benzoyl-2- azetidinecarboxylic acid methyl ester. 3. An enzyme having esterase activity, obtainable from a microorganism according to claim 1, or a fragment thereof having the same or opposite enantioselectivity. 4. An enzyme comprising the amino-acid sequence shown in SEQ ID NO:2, or a fragment thereof, capable of hydrolysing an enantiomer of N-benzoylazetidine-2- carboxylic acid methyl ester. 5. An enzyme according to claim 3 or claim 4, in immobilised form. 6. An isolated nucleotide encoding an enzyme according to claim 4. 7. A nucleotide according to claim 6, having all or part of the sequence shown in SEQ ID NO:l. 8. A microorganism transformed to express an enzyme according to claim 4. 9. A method for producing an enzyme according to claim 3, which comprises culturing a microorganism according to any of claims 1, 2 and 8.

Inventors: BROWN ROBERT CHRISTOPHER, KEENE PHILIP ALEXANDER, TAYLOR STEPHEN JOHN CLIFFORD, BROWN, ROBERT, CHRISTOPHER, KEENE, PHILIP, ALEXANDER, TAYLOR, STEPHEN, JOHN, CLIFFORD
Applicants: BROWN ROBERT CHRISTOPHER, CHIROTECH TECHNOLOGY LTD, KEENE PHILIP ALEXANDER, TAYLOR STEPHEN JOHN CLIFFORD, BROWN, ROBERT, CHRISTOPHER, CHIROTECH TECHNOLOGY LIMITED, KEENE, PHILIP, ALEXANDER, TAYLOR, STEPHEN, JOHN, CLIFFORD
Application year: 1998
Main classification: C12N15
IPC codes: C12N1, C12N5, C12N9, C12N15, C12P17, C12P41, C12R1