WO1999004015A1 - Micro-organisme et esterase obtenue a partir de ce micro-organisme - Google Patents

Micro-organisme et esterase obtenue a partir de ce micro-organisme Download PDF

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Publication number
WO1999004015A1
WO1999004015A1 PCT/GB1998/002089 GB9802089W WO9904015A1 WO 1999004015 A1 WO1999004015 A1 WO 1999004015A1 GB 9802089 W GB9802089 W GB 9802089W WO 9904015 A1 WO9904015 A1 WO 9904015A1
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gly
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PCT/GB1998/002089
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English (en)
Inventor
Philip Alexander Keene
Robert Christopher Brown
Stephen John Clifford Taylor
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Chirotech Technology Limited
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Publication date
Application filed by Chirotech Technology Limited filed Critical Chirotech Technology Limited
Priority to EP98933810A priority Critical patent/EP1003885A1/fr
Priority to JP2000503221A priority patent/JP2001510049A/ja
Priority to AU83503/98A priority patent/AU8350398A/en
Publication of WO1999004015A1 publication Critical patent/WO1999004015A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/003—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions
    • C12P41/005—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions by esterification of carboxylic acid groups in the enantiomers or the inverse reaction
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/66—Aspergillus

Definitions

  • This invention relates to a microorganism and to a stereoselective esterase obtained therefrom.
  • Bioresolution is a procedure which is known to be of use generally in the production of enantiomerically-pure compounds.
  • 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.
  • 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.
  • 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.
  • CMC 3242 A novel organism identified as Aspergillus tamarii (CMC 3242), has been deposited at the International Mycological Institute, Egham, Surrey, UK, on 8th July
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • one or more markers may be present, as described above.
  • 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.
  • 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
  • ZnO 2.0g/L
  • 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 20 ⁇ l 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.
  • 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 L ⁇ nin.
  • Whole Cell Biotransformation 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
  • 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.
  • a 125ml culture of Aspergillus tamarii CMC 3242 was incubated at 30°C for 48 hr. Cells were harvested by filtration (Whatman 2.7 ⁇ m 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.
  • 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.
  • 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 300 ⁇ l 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 150 ⁇ l 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 250 ⁇ l 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 lO ⁇ l 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 ⁇ l DEPC H 2 O, ready for subsequent cDNA synthesis.
  • cDNA 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.
  • 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 Ec ⁇ RI linker and finally Xhol digestion, to form an asymmetric fragment which gives the correct orientation for expression. Size fractionation of cDNA
  • 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.
  • phenohchloroform (21000g, lOmin, 20°C
  • 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)
  • 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 INV ⁇ F', XLl-Blue MRF' and DH5 ⁇ , respectively.
  • the transformed cells were then plated onto Tryptone Soya Broth Agar (Oxoid Ltd. #CM129), with ampicillin (100 ⁇ g ml "1 ), lmM LPTG, and Xgal (50 ⁇ g 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-
  • 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.
  • 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.
  • 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.
  • MOLECULE TYPE DNA (genomic)

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Abstract

Un nouveau micro-organisme est caractérisé par son aptitude à produire de l'acide (S)-N-benzoyl-2-azétidinecarboxylique, avec au moins 99 % d'énantiomères en excès, à partir d'un méthyl-ester d'acide N-benzoyl-2-azétidinecarboxylique racémique. Une enzyme présentant la même sélectivité énantiomère ou une sélectivité énantiomère opposée a été extraite de ce micro-organisme.
PCT/GB1998/002089 1997-07-15 1998-07-15 Micro-organisme et esterase obtenue a partir de ce micro-organisme WO1999004015A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP98933810A EP1003885A1 (fr) 1997-07-15 1998-07-15 Micro-organisme et esterase obtenue a partir de ce micro-organisme
JP2000503221A JP2001510049A (ja) 1997-07-15 1998-07-15 微生物および該微生物から得られるエステラーゼ
AU83503/98A AU8350398A (en) 1997-07-15 1998-07-15 Microorganism and esterase obtained therefrom

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GB9714877.9 1997-07-15
GBGB9714877.9A GB9714877D0 (en) 1997-07-15 1997-07-15 Microorganism and its use

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1057894A2 (fr) * 1999-06-04 2000-12-06 Sumitomo Chemical Company, Limited Gènes d'esterase et ses utilisations
US7759110B2 (en) 2004-08-11 2010-07-20 Taylor Ian N Process for the production of (S)-5-chloro-2-isopropylpent-4-enoic acid esters
WO2011149938A2 (fr) 2010-05-24 2011-12-01 Dr. Reddy's Laboratories Ltd. Préparation de (3as, 7ar)-hexahydroisobenzofuran-1- (3h) -one par résolution biologique catalysée du diméthylcyclohexane-1,2-dicarboxylate
DE112004000565B4 (de) * 2003-04-02 2014-03-27 Disco Corporation Schalten einer Fluidströmung durch Umleitung

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EP0552041A2 (fr) * 1992-01-15 1993-07-21 E.R. Squibb & Sons, Inc. Procédés enzymatiques pour la résolution d'un mélange d'énantiomères efficaces comme intermédiaires dans la préparation de taxanes
EP0634492A1 (fr) * 1993-07-14 1995-01-18 Bristol-Myers Squibb Company Procédés enzymatiques pour la résolution d'un mélange d'énantiomères efficaces comme intermédiaires dans la préparation de taxanes
WO1998002568A1 (fr) * 1996-07-15 1998-01-22 Astra Aktiebolag Bioresolution d'acides n-acylazetidine-2-carboxyliques

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DAVIES J S ET AL: "CONFORMATIONAL FEATURES OF BENZOYL N-ALKYLATED AMINO-ACIDS (N-ALKYLATED BENZAMIDO-ACIDS) DETERMINED BY NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY", JOURNAL OF THE CHEMICAL SOCIETY, PERKIN TRANSACTIONS 1, vol. 11, 1978, pages 1157 - 1163, XP002027667 *
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1057894A2 (fr) * 1999-06-04 2000-12-06 Sumitomo Chemical Company, Limited Gènes d'esterase et ses utilisations
EP1057894A3 (fr) * 1999-06-04 2002-01-02 Sumitomo Chemical Company, Limited Gènes d'esterase et ses utilisations
DE112004000565B4 (de) * 2003-04-02 2014-03-27 Disco Corporation Schalten einer Fluidströmung durch Umleitung
US7759110B2 (en) 2004-08-11 2010-07-20 Taylor Ian N Process for the production of (S)-5-chloro-2-isopropylpent-4-enoic acid esters
WO2011149938A2 (fr) 2010-05-24 2011-12-01 Dr. Reddy's Laboratories Ltd. Préparation de (3as, 7ar)-hexahydroisobenzofuran-1- (3h) -one par résolution biologique catalysée du diméthylcyclohexane-1,2-dicarboxylate
EP2576803A2 (fr) * 2010-05-24 2013-04-10 Abbott Products Operations AG Préparation de (3as, 7ar)-hexahydroisobenzofuran-1- (3h) -one par résolution biologique catalysée du diméthylcyclohexane-1,2-dicarboxylate
EP2576803A4 (fr) * 2010-05-24 2014-08-27 Abbott Products Operations Ag Préparation de (3as, 7ar)-hexahydroisobenzofuran-1- (3h) -one par résolution biologique catalysée du diméthylcyclohexane-1,2-dicarboxylate
US9080193B2 (en) * 2010-05-24 2015-07-14 Abbvie Inc. Preparation of (3aS,7aR)-hexahydroisobenzofuran-1(3H)-one by catalyzed biological resolution of dimethyl cyclohexane-1,2-dicarboxylate

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EP1003885A1 (fr) 2000-05-31

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