Classifications

• • 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
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
• • 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)
  • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
• • 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
  • C12P41/006—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C2601/00—Systems containing only non-condensed rings
  • C07C2601/06—Systems containing only non-condensed rings with a five-membered ring
  • C07C2601/10—Systems containing only non-condensed rings with a five-membered ring the ring being unsaturated

Definitions

• the present invention relates to a process for the preparation of 5-norbornene-2-carboxylic acid from 5-norbornene-2-endo-carbonotril and / or 5-norbornene-2-exo carbonitrile.
• the invention relates to a process in which 5-norbornene-2-carboxylic acid can be prepared at a high substrate concentration.
• the invention relates to a polypeptide suitable for the enzymatic conversion of 5-norbornene-2-carbonitrile to 5-norbornene-2-carboxylic acid, in particular also at a high substrate concentration and a nucleic acid encoding the polypeptide, a composition comprising 5-norbornene-2 carbonitrile to 5-norbornene-2-endo-carboxylic acid and 5-norbornene-2-exo-carboxylic acid, and the use of the polypeptide.
• 5-norbornene-2-carboxylic acid is used as a substrate for a variety of organic syntheses and is particularly suitable for the preparation of cyclic olefin copolymers (COC), pharmaceutical intermediates, pesticides or fragrances.
• COC cyclic olefin copolymers
• 5-norbornene-2-carboxylic acid can be produced substantially only chemically chemically.
• a particular disadvantage is that the known processes lead to mixtures of isomers from which the I-someren have to be isolated by complex purification processes.
• the invention therefore an object of the invention to provide a method to produce the fermentative economically 5-norbornene-2-carboxylic acid.
• the invention relates to a process for the enzymatic preparation of
• R1-R9 may each independently be: H. linear or branched alkyl of one to six carbon atoms, cycloalkyl of two to six carbon atoms, unsubstituted, amino, hydroxy or halo-substituted aryl of from 3 to 10 carbon atoms, and wherein
• R5 and R7 as well as R8 and R9 can also form a cycloalkyl having 3 to 6 carbon atoms, e.g. Cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl;
• R8 and R9 as well as R5 and R7 may also bear exocyclic double bonds with optional substituents;
• R3 and R4 may form a ring (4,5,6) or may be part of a fused aromatic;
• compound I in particular 5-norbornene-2-carbonitrile
• compound II in particular 5-norbornene-2-carboxylic acid
• arylacetonitrilases EC 3.5.5.5
• Nitrilases are enzymes which catalyze the hydrolysis of nitriles into the corresponding carboxylic acids and ammonium ions (Faber, Biotransformations in Organic Chemistry, Springer Verlag Berlin / Heidelberg, 1992). Nitrilases were first described in plants (Thimann and Mahadevan (1964) Arch Biochem Biophys 105: 133-141) and later found in many microorganisms as well.
• Nitrilases have different substrate specificities, but can roughly be classified into three groups: nitrilases specific for aliphatic nitriles, nitrilases, specific for aromatic nitriles and nitrilases specific for arylacetonitriles.
• nitrilases The enzymatic synthesis of chiral and achiral carboxylic acid and ⁇ -hydroxycarboxylic acids with nitrilases is described in the prior art. Most nitrilases are very substrate specific and can only react a few substrates; Thus, their application is limited to the implementation of only one or a few nitriles with economic efficiency. It is therefore advantageous to provide nitrilases which can react new compounds with high efficiency or favorable conditions.
• nitrilase as used herein includes all polypeptides that possess nitrilase activity.
• nitrilase activity means the ability to hydrolyze nitriles in their corresponding carboxylic acids and ammonium.
• nitrilase activity means the ability of an enzyme to catalyze the addition of two molar equivalents water to a nitrile group, so that the corresponding carboxylic acid is formed: R-CN + 2 H 2 O ⁇ R-COOH + NH 3.
• nitrilase preferably includes enzymes of the EC classes 3.5.5.1 (nitrilases), 3.5.5.2 (ricinone nitrilases), 3.5.5.4 (cyanoalanine nitriles), 3.5.5.5 (aryl acetonitrile), 3.5.5.6 (bromoxynil), and 3.5.5.7 (aliphatic nitrilases). Most preferred are arylacetonitrilases (EC 3.5.5.5).
• Arylacetonitrilases (EC 3.5.5.5) are usually mixed with aliphatics, e.g. Propionitrile or Korkklarenitril and benzonitriles hardly to not at all active. Therefore, it was surprising that an arylaceto-nitrilase was found, with which 5-norbornene-2-carbonotril can be converted with a high activity.
• each of R 1 -R 9 may be independently: H. linear or branched alkyl of one to six carbon atoms, cycloalkyl of two to six carbon atoms, unsubstituted, amino, hydroxy or halo-substituted aryl of from 3 to 10 carbon atoms, and in which
• R 5 and R 7 and R 8 and R 9 can also form cycloalkyl having 3 to 6 carbon atoms, for example cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl; R 8 and R 9 and R 5 and R 7 may also carry exocyclic double bonds with optional substituents, such as shown in compound IIb with R 5 , R 7 , R 10 , 11 each independently equal to H, alkyl or aryl having one to six carbon atoms; and
• R 3 and R 4 may form a ring ( 4, 5, 6) or may be part of a fused aromatic;
• the enzymes having activity according to the invention can be used to convert compound I to II in the process according to the invention as processed microorganisms or cells, eg as digests, free or immobilized enzymes, microorganisms or cells, or as partially or completely purified enzyme preparations, for example free or immobilized, used.
• growing cells can also be used for the method according to the invention which contain the nucleic acids, nucleic acid constructs or vectors according to the invention.
• dormant or open cells can be used.
• open cells are meant, for example, cells which have been rendered permeable by treatment with, for example, solvents, or cells which have been treated by enzyme treatment, e.g. lysed, disrupted by mechanical treatment (e.g., French Press or ultrasound) or otherwise.
• the crude extracts thus obtained are advantageously suitable for the process according to the invention.
• purified or purified enzymes can be used for the process.
• immobilized microorganisms or enzymes that can be used advantageously in the reaction.
• free organisms or enzymes are used for the process according to the invention, they are expediently removed before extraction, for example by filtration or centrifugation.
• a microorganism according to this invention can be cultured or propagated in a medium which allows the growth of this microorganism.
• the medium can be of synthetic or natural origin.
• Various media for microorganisms are known.
• the medium contains a carbon source, a nitrogen source, inorganic salts, and optionally, small amounts of vitamins and / or trace elements.
• Preferred carbon sources are e.g. Polyols, e.g. Glycerol, sugars such as e.g. Mono-, di- or polysaccharides (eg glucose, fructose, manose, xylolose, galactose, ribose, sorbose, ribulose, lactose, maltose, succose, rafinose, starch or cellulose) complex sugar sources (eg molasses), sugar phosphates (eg fructose-1 -ex- biphosphate), sugar alcohols (eg mannitol), alcohols (eg methanol or ethanol), carboxylic acids (eg soybean oil or linseed oil), amino acids or amino acid mixtures (eg casamino acids, Difco) or certain amino acids (eg glycine, asparagine) or amino sugars, where the latter can also serve as sources of nitrogen.
• sugars such as e.g. Mono-
• Preferred nitrogen sources are organic and inorganic nitrogen compounds or materials containing these compounds.
• ammonium salts eg NH 4 Cl or (NhU) 2 SO 4
• nitrates eg NH 4 Cl or (NhU) 2 SO 4
• urea e.g., urea
• complex nitrogen sources such as heelylsate, soybean meal, wheat gluten, yeast extract, pepdone, meat extract, casein hydrolysates, yeast or potato protein
• good nitrogen sources the latter also known as Serve carbon sources.
• inorganic salts include calcium, magnesium, sodium, cobalt, manganese, potassium, zinc, copper and iron salt.
• anions chloride, sulfate, sulfite and phosphate ions are particularly preferred.
• Important for good productivity is the control of the Fe2 + or Fe3 + ion concentration in the medium.
• the medium may additionally contain growth factors, e.g. Vitamins or growth enhancers such as biotin, 2-keto-1-gulonic acid, ascorbic acid, thiamine, folic acid, amino acids, carboxylic acids or substances such as e.g. DTT.
• growth factors e.g. Vitamins or growth enhancers such as biotin, 2-keto-1-gulonic acid, ascorbic acid, thiamine, folic acid, amino acids, carboxylic acids or substances such as e.g. DTT.
• the fermentation and growth conditions are selected so that a high yield of the desired product can be achieved (e.g., high nitrilase activity, especially high arylacetonitrilase activity).
• Preferred fermentation conditions are between 15 ° C and 40 ° C, preferably 25 ° C to 37 °.
• the pH is preferably regulated in the range of pH 3 to 9, more preferably between pH 5 and 8.
• the fermentation lasts between a few hours and a few days, preferably between 8 hours and 21 days, more preferably 4 hours and 14 days.
• the method according to the invention is carried out such that the enzymatic conversion of compound I to compound II is effected by incubation with a polypeptide or a medium containing a polypeptide and wherein the polypeptide is characterized in that the polypeptide is encoded by a nucleic acid molecule which comprises a nucleic acid molecule selected from the group consisting of:
• nucleic acid molecule whose sequence, due to the degeneracy of the genetic code, can be derived from a polypeptide sequence encoded by a nucleic acid molecule of (a) or (b); (d) a nucleic acid molecule encoding a polypeptide whose sequence has an identity of at least 60% to the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) or (b); (e) a nucleic acid molecule encoding a polypeptide derived from an aryl acetonitrile polypeptide wherein up to 25% of the amino acid residues are altered from SEQ ID NO: 2 by deletion, insertion, substitution or a combination thereof, and that still at least 30 has% of the enzymatic activity of SEQ ID NO: 2; and
• nucleic acid molecule encoding a fragment or epitope of an arylaceto-nitrilase encoded by any of the nucleic acid molecules of (a) to (c);
• Preferred enzymes having activity according to the invention comprise an amino acid sequence according to SEQ ID NO: 2 or 4.
• the nitrilase according to the invention saponifies very well phenylacetonitrile> phenylpropionitrile> mandelonitrile (moderate enantioselectivity) and is hardly or not at all active with aliphatics (for example propionitrile, subericinitrile) or benzonitriles. Therefore, in particular activity with norbornenitriles is surprising.
• the stability and productivity of the enzyme of the invention under reactor condition is enormous and the handling simple, as a wide temperature and pH range is available and the enzyme has a high nitrile tolerance, i. no nitrile dosage is necessary.
• “Functional equivalents” or analogues of the specifically disclosed enzymes in the context of the present invention are different polypeptides which furthermore have the desired biological activity, such as substrate specificity.
• “functional equivalents” are understood to mean enzymes derived from compound I to implement compound II and having at least 50%, preferably 60%, more preferably 75%, most preferably 90% or more of the activity of an enzyme having the amino acid sequence shown in SEQ ID NO: 2.
• Functional equivalents are also preferably stable at temperatures of 0 0 C to 70 0 C and advantageously have a pH optimum between pH 5 and 8 and a temperature optimum in the range of 10 0 C to 50 ° C.
• “functional equivalents” are in particular also understood as meaning mutants which are present in at least one sequence position of the abovementioned amino acids. Resequenzen have a different than the specifically mentioned amino acid but still possess one of the above biological activities. "Functional equivalents” thus include the mutants obtainable by one or more amino acid additions, substitutions, deletions, and / or inversions, which changes can occur in any sequence position as long as they result in a mutant having the property profile of the invention Equivalence is also given in particular when the reactivity patterns between mutant and unchanged polypeptide match qualitatively, ie, for example, identical substrates are reacted at different rates.
• “functional equivalents” are in particular also understood as meaning mutants which, in at least one sequence position of the abovementioned amino acid sequences, have a different amino acid than the one specifically mentioned but nevertheless possess one of the abovementioned biological activities.
• Lente encompass the mutants obtainable by one or more amino acid additions, substitutions, deletions and / or inversions, said changes occurring in any sequence position as long as they lead to a mutant having the property profile of the invention Functional Equivalence especially if the reactivity patterns between mutant and unchanged polypeptide match qualitatively, ie, for example, identical substrates are reacted at different rates, the rate being not less than 30% of that of the unchanged polypeptide, preferably more than 100%, in particular more than 150 %, more preferably a speed increased by a factor of 2, 5, or 10.
• Precursors are natural or synthetic precursors of the polypeptides with or without the desired biological activity.
• Salts are understood as meaning both salts of carboxyl groups and acid addition salts of amino groups of the protein molecules according to the invention.
• Sacks of carboxyl groups can be prepared in a manner known per se and include inorganic salts, such as, for example, sodium, calcium and ammonium salts. , Iron and zinc salts, and salts with organic bases, such as amines, such as triethanolamine, arginine, lysine, piperidine, etc.
• Acid addition salts such as salts with mineral acids, such as hydrochloric acid or sulfuric acid and salts with organic acids, such as acetic acid and Oxalic acid are also the subject of the invention.
• “Functional derivatives” of polypeptides of the invention may also be produced at functional amino acid side groups or at their N- or C-terminal end by known techniques
• Such derivatives include, for example, aliphatic esters of carboxylic acid groups, amides of carboxylic acid groups obtainable by reaction with ammonia or with a primary or secondary amine; N-acyl derivatives of free amino groups prepared by reaction with acyl groups; or O-acyl derivatives of free hydroxy groups prepared by reaction with acyl groups.
• “functional equivalents” also include polypeptides that are accessible from other organisms, as well as naturally occurring variants. For example, it is possible to determine regions of homologous sequence regions by sequence comparison and to determine equivalent enzymes on the basis of the specific requirements of the invention. "Functional equivalents” likewise include fragments, preferably individual domains or sequence motifs, of the polypeptides according to the invention which, for example, have the desired biological function.
• Fusion equivalents are also fusion proteins which have one of the above-mentioned polypeptide sequences or functional equivalents derived therefrom and at least one further functionally distinct heterologous sequence in functional N- or C-terminal linkage (ie without substantial substantial functional impairment of the fusion protein moieties)
• heterologous sequences are, for example, signal peptides or enzymes.
• homologs to the specifically disclosed proteins which have at least 60%, preferably at least 75%, in particular at least 85%, such as 90%, 95% or 99%, homology to one of the specifically disclosed amino acid sequences, comprising "functional equivalents" Calculated according to the algorithm of Pearson and Lipman, Proc Natl Acad, Sci. (USA) 85 (8), 1988, 2444-2448
• a percent homology of a homologous polypeptide of the invention means, in particular, percent identity of the amino acid residues relative to the total length of one of specifically described herein.
• “functional equivalents” include proteins of the type described above in deglycosylated or glycosylated form as well as modified forms obtainable by altering the glycosylation pattern.
• Homologs of the proteins or polypeptides of the invention can be generated by mutagenesis, e.g. by point mutation or shortening of the protein.
• Homologs of the proteins of the invention can be identified by screening combinatorial libraries of mutants such as truncation mutants.
• a variegated library of protein variants can be generated by combinatorial mutagenesis at the nucleic acid level, such as by enzymatic ligation of a mixture of synthetic oligonucleotides.
• methods that can be used to prepare libraries of potential homologs from a degenerate oligonucleotide sequence. The chemical synthesis of a degenerate gene sequence can be performed in a DNA synthesizer, and the synthetic gene can then be ligated into a suitable expression vector.
• degenerate gene set allows for the provision of all sequences in a mixture that encode the desired set of potential protein sequences.
• Methods for the synthesis of degenerate oligonucleotides are known to the person skilled in the art (eg Narang, SA (1983) Tetrahedron 39: 3; Itakura et al. (1984) Annu. Rev. Biochem. 53: 323; Itakura et al., (1984) Science 198: 1056; Ike et al. (1983) Nucleic Acids Res. 1 1: 477).
• REM Recursive ensemble mutagenesis
• the process according to the invention is carried out at a reaction temperature of 5 to 75.degree.
• the reaction temperature is room temperature or environmental dozenss- or more, for example 30 ° C or more but less than 70 0 C, preferably 60 0 C, 50 0 C or less.
• the reaction temperature is about 35 to 45 ° C, for example 40 ° C, for xNon production.
• the reaction temperature is between ambient and 50 ° C for the production of eNon.
• Compound I can be both an enantiomeric mixture, for example R 1 S or end / exo enantiomers, and also enantiomerically pure, ie contain predominantly one enantiomer.
• an enantiomerically pure substrate is reacted in the process according to the invention.
• enantiomers which show an enantiomeric enrichment.
• R-5-norbornene-2-endo-carbonitrile, S-5-norbornene-2-endo-carbonitrile, R-5-norbornene-2-exo-carbonitrile, and / or S-5 Norbornene-2-exo-carbonitrile to the corresponding S-5-norbornene-2-exo saponified carboxylic acid, S-5-norbornene-2-endo-carboxylic acid, R-5-norbornene-2-exo-carboxylic acid and R-5-norbornene-2-endo-carboxylic acid.
• compound I is R-5-norbornene-2-endocarbonitrile and S-5-norbornene-2-endo-carbonitrile or R-5-norbornene-2-exo-carbonitrile and S- ⁇ -norbornene exo-carbonitrile.
• compound I is R-5-norbornene-2-endocarbonitrile or S-5-norbornene-2-endocarbonitrile or R-5-norbornene-2-exo-carbonitrile or S-5-norbornene- 2-exo-carbonitrile.
• the invention also relates to a process in which an enantiomerically pure product is obtained.
• the invention relates to a process in which, at a substrate concentration, at least 20 ⁇ m, preferably 50 ⁇ m, 70 ⁇ m, 10 ⁇ m, 15 ⁇ m, 20 ⁇ m, 25 ⁇ m, 30 ⁇ m, 40 ⁇ m, 50 ⁇ m, 70 ⁇ m, 100 ⁇ m, 200 ⁇ m, or more, and wherein the substrate, ie compound I, in particular R-5-norbornene-2-endocarbonitrile, S-5-norbornene-2-endocarbonitrile, R-5-norbornene-2-exo-carbonitrile, and / or S-5-norbornene 2-exo-carbonitrile to at least 50%, preferably to 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more to compound II is reacted.
• the substrate ie compound I, in particular R-5-norbornene-2-endocarbonitrile, S-5-norbornene-2-endocarbonitrile, R
• the substrate used is an isomer mixture, in particular a mixture of enantiomers, of compound I as the substrate, and the product is enriched in an isomer, in particular an enantiomer of compound II.
• an endo- and exo- Enantiomer of the compound I is used and there is an accumulation of the endo or exo enantiomer of the compound II.
• Particularly preferred in the method according to the invention for enrichment is a mixture of R-5-norbornene-2-endo-carbonitrile and / or S- 5-norbornene-2-endo-carbonitrile and R-5-norbornene-2-exo-carbonitrile and / or S-5-norbornene-2-exo-carbonitrile to the corresponding S-5-norbornene-2-exo-carboxylic acid and / or R-5-norbornene-2-exo-carboxylic acid and R-5-norbornene-2-endo-carboxylic acid and / or S-5-norbornene-2-endo-carboxylic acid saponified, wherein it preferably to an enrichment of the endo-enantiomers norbornene acid comes.
• the pH in the process according to the invention is advantageously maintained between pH 6 and 10, preferably between pH 7 and 9, particularly preferably between pH 7.5 and 8.5.
• the product prepared in the process according to the invention for example R and / or S-5-norbornene-2-exo-carboxylic acid and / or R and / or S-5-norbornene-2 endo-carboxylic acid j .
• suitable extractants are solvents such as, but not limited to, toluene, methylene chloride, butyl acetate, diisopropyl ether, benzene, MTBE or ethyl acetate.
• the products can generally be obtained in good chemical purities, ie greater than 80%, preferably 85%, 90%, 95%, 98% or more, chemical purity.
• the organic phase with the product can only be partially concentrated and the product crystallized out.
• the solution is advantageously cooled to a temperature of 0 ° C to 10 ° C.
• the crystallization can also be carried out directly from the organic solution or from an aqueous solution.
• the crystallized product can be taken up again in the same or in another solvent for recrystallization and crystallized again.
• the enantiomeric purity of the product can be further increased if necessary.
• the product of the process according to the invention can be obtained in yields of from 60 to 100%, preferably from 80 to 100%, particularly preferably from 90 to 100%, based on the substrate used for the reaction, e.g. of R-5-norbornene-2-endo-carbonitrile, R-5-norbornene-2-exo-carbonitrile S-5-norbornene-2-endo-carbonitrile, and / or S-5-norbornene-2-exo-carbonitrile isolate.
• the isolated product is characterized by a high chemical purity of> 90%, preferably> 95%, particularly preferably> 98%.
• the products have a high enantiomeric purity, which can be advantageously further increased if necessary by the crystallization.
• the process according to the invention can be operated batchwise, semi-batchwise or continuously.
• the operation of the process may advantageously be carried out in bioreactors, e.g. in Biotechnology, Volume 3, 2nd Edition, Rehm et al. Ed., (1993), especially Chapter II.
• the invention also relates to a polypeptide which is suitable for the enzymatic saponification of the compound I to the compound II.
• the polypeptide encodes a nitrilase, especially an arylacetonitrilase.
• the polypeptide is characterized in that it is encoded by a nucleic acid molecule comprising a nucleic acid molecule selected from the group consisting of:
• nucleic acid molecule comprising at least the polynucleotide of the coding sequence of Seq. ID No .: 1 or 3;
• nucleic acid molecule whose sequence can be deduced from a polypeptide sequence encoded by a nucleic acid molecule according to (a) or (b) due to the degeneracy of the genetic code;
• nucleic acid molecule encoding a polypeptide whose sequence has an identity of at least 60% to the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) or (b); (e) a nucleic acid molecule encoding a polypeptide derived from an aryl acetonitrile polypeptide wherein up to 15% of the amino acid residues are altered from SEQ ID NO: 2 or 4 by deletion, insertion, substitution, or a combination thereof has at least 30% of the enzymatic activity of SEQ ID NO: 2 or 4; and (f) a nucleic acid molecule encoding a fragment or epitope of an arylaceto-nitrilase encoded by any of the nucleic acid molecules of (a) to (c);
• the polypeptide does not have the sequence according to Seq ID No .: 2 and / or 4. In one embodiment, the polypeptide also does not have the sequence described in Eur. J. Biochem. 182, 349-156, 1989 called nitrilase. In one embodiment, the polypeptide also does not have the sequence of database entry AY885240.
• the polypeptide according to the invention has the property of producing compound II, in particular norbornene acid, to a high percentage, even at a high substrate concentration, ie at a high concentration of compound I in the medium.
• the polypeptide can be the substrate is reacted to at least 50%, preferably to 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of compound II, wherein the substrate, ie compound I, in particular R-5 Norbornene-2-endo-carbonitrile, S-5-norbornene-2-endo-carbonitrile, R-5-norbornene-2-exo-carbonit
• the invention also relates to a nucleic acid molecule encoding the polypeptide of the invention.
• the present invention relates to a nucleic acid molecule comprising a polynucleotide encoding a polypeptide of the invention.
• the nucleic acid molecule does not have the sequence of Seq. ID No. 1.
• the nucleic acid molecule does not encode the nitrilase of Eur. J. Biochem. 182, 349-156, 1989.
• the nucleic acid molecule also does not have the sequence of database entry AY885240.
• the invention particularly relates to nucleic acid sequences (single and double-stranded DNA and RNA sequences, such as cDNA and mRNA) which code for an enzyme having activity according to the invention or can be used in the method according to the invention.
• nucleic acid sequences which are e.g. for amino acid sequences according to SEQ ID NO: 2 or 4 or characteristic partial sequences thereof, or nucleic acid sequences according to SEQ ID NO: 1 or 3 or characteristic partial sequences thereof.
• nucleic acid sequences mentioned herein can be prepared in a manner known per se by chemical synthesis from the nucleotide units, for example by fragment condensation of individual overlapping, complementary nucleic acid units of the double helix.
• the chemical synthesis of oligonucleotides can be carried out, for example, in a known manner by the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897).
• the attachment of synthetic oligonucleotides and filling of gaps with the aid of the Klenow fragment of the DNA polymerase and ligation reactions and general cloning methods are described in Sambrook et al. (1989), Molecular Cloning: A laboratory manual, CoId Spring Harbor Laboratory Press.
• the invention also relates to nucleic acid sequences (single and double stranded DNA and RNA sequences, such as cDNA and mRNA) encoding one of the above polypeptides and their functional equivalents, e.g. accessible using artificial nucleotide analogs.
• the nucleic acid sequence according to the invention differs in at least one base from that of Seq. ID No. 1 or 3.
• the nucleic acid molecule also does not have the sequence described in Eur. J. Biochem. 182, 349-156, 1989 called nitrilase.
• the nucleic acid molecule also does not have the sequence of database entry AY885240.
• the invention relates both to isolated nucleic acid molecules which code for polypeptides according to the invention or proteins or biologically active portions thereof, as well as nucleic acid fragments which are suitable, for example, for use as hybridization probes or Primers can be used for the identification or amplification of coding nucleic acids according to the invention.
• nucleic acid molecules according to the invention may additionally contain untranslated sequences from the 3 'and / or 5' end of the coding gene region
• the invention further comprises the nucleic acid molecules complementary to the specifically described nucleotide sequences or a portion thereof.
• the nucleotide sequences of the invention enable the generation of probes and primers useful for the identification and / or cloning of homologous sequences in other cell types and organisms.
• probes or primers usually comprise a nucleotide sequence region which is under "stringent" conditions (see below) at least about 12, preferably at least about 25, such as about 40, 50 or 75 consecutive nucleotides of a sense strand of a nucleic acid sequence of the invention or a corresponding nucleic acid sequence Antisense strands hybridizes.
• nucleic acid molecule is separated from other nucleic acid molecules present in the natural source of the nucleic acid and, moreover, may be substantially free of other cellular material or culture medium when produced by recombinant techniques, or free of chemical precursors or other chemicals when chemically synthesized.
• a nucleic acid molecule according to the invention can be isolated by means of standard molecular biological techniques and the sequence information provided according to the invention.
• cDNA can be isolated from a suitable cDNA library by using one of the specifically disclosed complete sequences or a portion thereof as a hybridization probe and standard hybridization techniques (such as described in Sambrook, J., Fritsch, EF and Maniatis, T. Molecular Cloning: A Laboratory
• nucleic acid molecule comprising one of the disclosed sequences or a portion thereof can be isolated by polymerase chain reaction, using the oligonucleotide primers prepared on the basis of this sequence.
• the thus amplified nucleic acid can be cloned into a suitable vector and characterized by DNA sequence analysis.
• the oligonucleotides of the invention may be further purified by standard synthetic methods, e.g. with an automatic DNA synthesizer.
• the nucleic acid sequences according to the invention can be identified and isolated in principle from all organisms.
• bacteria are called gram-negative and gram-positive bacteria.
• the nucleic acids according to the invention from gram-negative bacteria are preferably made from ⁇ -proteobacteria, ⁇ -proteobacteria or v-proteobacteria, more preferably from bacteria of the orders of Burkholde ⁇ ales, Hydrogenophilales, Methylophilales, Neisseriales, Nitrosomonadales, Procabacterial or Rhodocyclales. Most preferably from bacteria of the family Rhodocyclaceae.
• Nucleic acid sequences according to the invention can be prepared, for example, by conventional hybridization methods or the PCR technique from other organisms, e.g. isolate via genomic or cDNA libraries. These DNA sequences hybridize under standard conditions with the sequences according to the invention. For hybridization, it is advantageous to use short oligonucleotides of the conserved regions, for example from the active site, which can be determined by comparisons with a nitrilase according to the invention, in particular arylacetonitrilases, in a manner known to the person skilled in the art. However, it is also possible to use longer fragments of the nucleic acids according to the invention or the complete sequences for the hybridization.
• nucleic acid hybrids are about 10 ° C lower than those of DNA: RNA hybrids of the same length.
• the invention also relates to derivatives of the specifically disclosed or derivable nucleic acid sequences.
• nucleic acid sequences according to the invention can be derived from SEQ ID NO: 1 or 3 and differ therefrom by addition, substitution, insertion or deletion of individual or several nucleotides, but furthermore code for polypeptides having the desired property profile.
• nucleic acid sequences which comprise so-called silent mutations or are modified according to the codon usage of a specific source or host organism, in comparison to a specifically mentioned sequence, as well as naturally occurring variants, such as splice variants or allelic variants thereof.
• the subject is also afforded by conservative nucleotide substitutions (ie, the amino acid in question is replaced by an amino acid of like charge, size, polarity, and / or solubility).
• the invention also relates to the molecules derived by sequence polymorphisms from the specifically disclosed nucleic acids. These genetic polymorphisms may exist between individuals within a population due to natural variation. These natural variations usually cause a variance of 1 to 5% in the nucleotide sequence of a gene.
• Derivatives of a nucleic acid sequence according to the invention are, for example, allelic variants which have at least 50% homology at the derived amino acid level, preferably at least 75% homology, most preferably at least 80, 85, 90, 93, 95, 98 or 99% homology over the entire sequence - have area (with respect to homology at the amino acid level, see above comments on the polypeptides referred to). About partial regions of the sequences, the homologies may be advantageous higher.
• derivatives are also to be understood as meaning homologs of the nucleic acid sequences according to the invention, for example fungal or bacterial homologs, truncated sequences, single stranded DNA or RNA of the coding and noncoding DNA sequence.
• a homology of at least 50%, preferably of 75% or more, more preferably of 80%, most preferably of 90%, most preferably 95%, especially 98%, or more over the entire specified DNA range e.g.
• nucleic acid or amino acid sequence of a DNA molecule or a protein to at least 40%, preferably at least 50%, more preferably at least 60%, also preferably at least 70%, more preferably at least 90%, most preferably at least 95% and most preferably at least 98% to the nucleic acid or amino acid sequences of the arylacetonitrilases, in particular to SEQ ID No. 1, 2, 3 or 4 or its functionally equivalent parts, preferably the homology over the entire sequence length of the arylacetonitrilases, in particular to SEQ ID No. 1, 2, 3 or 4 is determined.
• identity between two proteins is meant the identity of the amino acids over a particular protein region, preferably the entire protein length, in particular, the identity obtained by comparison with the laser gene software of DNA Star Inc., Madison, Wisconsin (USA) using the CLUSTAL method (Higgins et al., 1989), Comput. Appl. Biosci., 5 (2), 151). Homologies can also be computed using the laser gene software of DNA Star Inc., Madison, Wisconsin (USA) using the CLUSTAL method (Higgins et al., 1989). Appl. Biosci., 5 (2), 151).
• the homology is thus preferably calculated over the entire amino acid or nucleic acid sequence range.
• other programs are available to the person skilled in the art for the comparison of different sequences, which are based on different algorithms.
• the algorithms of Meedleman and Wunsch or Smith and Waterman provide particularly reliable results.
• the program PiIe Aupa can also be used for the sequence comparisons (J.Mol.Evolution. (1987), 25, 351-360, Higgins et al., (1989) Cabgos, 5, 151-153) or the programs Gap and Best Fit (Needleman and Wunsch, (1970), J. Mol. Biol., 48, 443-453 and Smith and Waterman (1981), Adv., Appl.
• homology with the Gap program is determined via the full-length cDNA sequence.
• the homology with the program Gap is determined over the entire genomic sequence.
• homology with the program Gap is determined over the coding full-length sequence.
• the promoters upstream of the indicated nucleotide sequences may be altered by one or more nucleotide exchanges, insertions, inversions and / or deletions, but without impairing the functionality or efficacy of the promoters.
• the promoters can be increased in their activity by changing their sequence or can be completely replaced by more effective promoters of alien organisms.
• Derivatives are also to be understood as variants whose nucleotide sequence has been changed in the range from -1 to -1000 bases upstream of the start codon or 0 to 1000 bases downstream after the stop codon in such a way that gene expression and / or protein expression is changed, preferably increased.
• the invention also encompasses nucleic acid sequences which hybridize with the abovementioned coding sequences under “stringent conditions.”
• stringent conditions thus refers to conditions under which one nucleic acid sequence binds preferentially to a target sequence, but does not or at least substantially reduces it to others sequences.
• polynucleotides can be found in the screening of genomic or cDNA libraries and optionally multiply therefrom with suitable primers by means of PCR and then isolate, for example, with suitable probes.
• polynucleotides of the invention can also be chemically synthesized. This property is understood as the ability of a poly- or oligonucleotide to bind under stringent conditions to a nearly complementary sequence, while under these conditions nonspecific binding between non-complementary partners is avoided.
• the sequences should be 70-100%, preferably 90-100%, complementary.
• the property of complementary sequences to be able to specifically bind to one another for example, in the Northern or Southern Blot technique or in the primer binding in PCR or RT-PCR advantage.
• oligonucleotides are used from a length of 30 base pairs.
• the hybridization conditions are for DNA: DNA hybrids at 0.1 x SSC and temperatures between about 20 ° C to 45 ° C, preferably between about 30 ° C to 45 ° C.
• the hybridization conditions are advantageously 0.1 ⁇ SSC and temperatures between about 30 ° C. to 55 ° C., preferably between about 45 ° C.
• temperatures for the hybridization are exemplary calculated melting temperature values for a nucleic acid with a length of about 100 nucleotides and a G + C content of 50% in the absence of formamide.
• the experimental conditions for DNA hybridization are described in relevant genetics textbooks, such as Sambrook et al., "Molecular Cloning", CoId Spring Harbor Laboratory, 1989, and can be made dependent upon length, for example, by formulas known to those skilled in the art of the nucleic acids, type of hybrids or G + C content. Further information on hybridization can be found in the following textbooks by the person skilled in the art: Ausubel et al.
• Stringent conditions are understood, for example, in the Northern blot technique, the use of a 50 - 70 0 C, preferably 60 - 65 0 C warm wash, for example, 0.1x SSC buffer with 0.1% SDS (2Ox SSC: 3M NaCl , 0.3M Na citrate, pH 7.0) for the elution of nonspecifically hybridized cDNA probes or oligonucleotides.
• a 50 - 70 0 C preferably 60 - 65 0 C warm wash
• 0.1x SSC buffer with 0.1% SDS (2Ox SSC: 3M NaCl , 0.3M Na citrate, pH 7.0) for the elution of nonspecifically hybridized cDNA probes or oligonucleotides.
• SDS 2Ox SSC: 3M NaCl , 0.3M Na citrate, pH 7.0
• complementarity is meant the ability of one nucleic acid molecule to hybridize to another nucleic acid molecule due to hydrogen bonding between complementary bases.
• the person skilled in the art knows that two nucleic acid molecules do not have to have 100% complementarity in order to be able to hybridize with one another.
• Preference is given to a nucleic acid sequence which is intended to hybridize with another nucleic acid sequence to this at least 40%, at least 50%, at least 60%, preferably at least 70%, particularly preferably at least 80%, likewise particularly preferably at least 90%, more preferably at least 95%, and most preferably at least 98% or 100% complementary.
• homology, complementarity and identity levels are to be determined over the entire protein or nucleic acid length.
• Nucleic acid molecules are identical if they have identical nucleotides in the same 5'-3 'order.
• the invention also relates to a method for producing a vector or expression construct comprising the insertion of the nucleic acid molecule according to the invention into a vector or expression construct.
• the invention also relates to a nucleic acid construct or vector containing the nucleic acid molecule according to the invention or prepared in the process according to the invention or comprising a nucleic acid construct suitable for use in the process according to the invention.
• the invention therefore relates to expression constructs comprising, under the genetic control of regulatory nucleic acid sequences, a nucleic acid sequence coding for a polypeptide according to the invention; and vectors comprising at least one of these expression constructs.
• Such constructs according to the invention preferably comprise a promoter 5'-upstream of the respective coding sequence and a terminator sequence 3'-downstream and optionally further customary regulatory elements, in each case operatively linked to the coding sequence.
• “Operational linkage” is understood to mean the sequential arrangement of promoter, coding sequence, terminator and optionally further regulatory elements in such a way that each of the regulatory elements can fulfill its function in the expression of the coding sequence as intended.
• Other regulatory elements include selectable markers, amplification signals, origins of replication, etc. Suitable regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990) ).
• a nucleic acid construct according to the invention is in particular to be understood as meaning those in which the gene for a reaction according to the invention differs with one or more regulatory signals for the control, e.g. Increased, the gene expression was operatively or functionally linked.
• the natural regulation of these sequences may still be present before the actual structural genes and may have been genetically altered so that natural regulation is switched off and the expression of the genes was increased.
• the nucleic acid construct can also be simpler, ie no additional regulatory signals have been inserted before the coding sequence and the natural promoter with its regulation has not been removed. Instead, the natural regulatory sequence is mutated so that regulation stops and gene expression is increased.
• a preferred nucleic acid construct advantageously also contains one or more of the already mentioned “enhancer” sequences, functionally linked to the promoter, which allow increased expression of the nucleic acid sequence, and additional advantageous sequences can also be inserted at the 3 'end of the DNA sequences, such as further regulatory sequences Elements or Terminators
• the nucleic acids according to the invention can be present in one or more copies in the construct
• Further genes, such as antibiotic resistance or auxotrophy-complementing genes, optionally for selection on the construct, can also be contained in the construct.
• Advantageous regulatory sequences for the process according to the invention are, for example, in promoters such as cos-, tac-, trp-, tet-, trp-tet, Ipp-, lac-, Ipp-lac-, lacl " " T7-, T5-, T3 -, gal, trc, ara, rhaP (rhaP BAD ) SP6-, Iambda-P R - or contained in the Iambda-P L promoter, which are advantageously used in Gram-negative bacteria application.
• Advantageous regulatory sequences are, for example, in the gram-positive promoters amy and SPO2, in the yeast or fungal promoters ADC1, MFalpha, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH.
• the promoters of pyruvate decarboxylase and methanol oxidase e.g. - seaus Hansenula advantageous artificial regulator
• the nucleic acid construct, for expression in a host organism is advantageously inserted into a vector, such as a plasmid or a phage, which enables optimal expression of the genes in the host.
• a vector such as a plasmid or a phage
• Vectors other than plasmids and phages are also all other vectors known to those skilled in the art, ie viruses such as SV40, CMV, baculovirus and adenovirus, transposons, IS elements, phasmids, cosmids, and linear or circular DNA. These vectors can be autonomously replicated in the host organism or replicated chromosomally. These vectors represent a further embodiment of the invention. Suitable plasmids are described, for example, in E.
• nucleic acid construct for expression of the further genes contained additionally 3'- and / or 5'-terminal regulatory sequences for increasing expression, which are selected depending on the selected host organism and gene or genes for optimal expression.
• genes and protein expression are intended to allow the targeted expression of genes and protein expression. Depending on the host organism, this may mean, for example, that the gene is only expressed or overexpressed after induction, or that it is expressed and / or overexpressed immediately.
• the regulatory sequences or factors can thereby preferably influence the gene expression of the introduced genes positively and thereby increase.
• enhancement of the regulatory elements can advantageously be done at the transcriptional level by using strong transcription signals such as promoters and / or enhancers.
• an enhancement of the translation is possible by, for example, the stability of the mRNA is improved.
• the vector containing the nucleic acid construct according to the invention or the nucleic acid according to the invention can also advantageously be introduced in the form of a linear DNA into the microorganisms and integrated into the genome of the host organism via heterologous or homologous recombination.
• This linear DNA can consist of a linearized vector such as a plasmid or only of the nucleic acid construct or of the nucleic acid according to the invention.
• An expression cassette according to the invention is produced by fusion of a suitable promoter with a suitable coding nucleotide sequence and a terminator or polyadenylation signal.
• common recombination and cloning techniques are used, as described, for example, in T. Maniatis, EF Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, ColD Spring Harbor Laboratory, ColD Spring Harbor, NY (1989) and in TJ. Silhavy, ML Berman and LW Enquist, Experiments with Gene Fusions, ColD Spring Harbor Laboratory, CoId Spring Harbor, NY (1984) and in Ausubel, FM et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987).
• the recombinant nucleic acid construct or gene construct is advantageously inserted into a host-specific vector for expression in a suitable host organism, which enables optimal expression of the genes in the host.
• Vectors are well known to those skilled in the art and can be found, for example, in "Cloning Vectors" (Pouwels P.H. et al., Eds. Elsevier, Amsterdam-New York-Oxford, 1985).
• the invention also relates to a host cell which has been stably or transiently transformed or transfected with the vector according to the invention or with the polynucleotide according to the invention or in which the polynucleotide according to the invention or a polynucleotide suitable for the method according to the invention is expressed as described above or in which such Compared to a wild type is expressed increased.
• recombinant microorganisms can be produced, which are transformed, for example, with at least one vector according to the invention and can be used to produce the polypeptides according to the invention.
• the above-described recombinant constructs according to the invention are introduced into a suitable host system and expressed.
• familiar cloning and transfection methods known to the person skilled in the art, such as, for example, co-precipitation, protoplast fusion, electroporation, retroviral transfection and the like, are used in order to express the stated nucleic acids in the respective expression system. Suitable systems are described, for example, in Current Protocols in Molecular Biology, F.
• Homologously recombined microorganisms can also be produced according to the invention.
• a vector is prepared which contains at least a portion of a gene or a coding sequence according to the invention, wherein optionally at least one amino acid deletion, addition or substitution has been introduced in order to modify the sequence according to the invention, eg functionally to disrupt ("Knockouf
• the introduced sequence may, for example, also be a homologue from a related microorganism or be derived from a mammalian, yeast or insect source
• the vector used for homologous recombination may be designed to mutate the endogenous gene upon homologous recombination or otherwise modified, but the functional protein is still encoded (eg the upstream regulatory region may be altered in such a way that is altered by the expression of the endogenous protein).
• the altered portion of the gene of the invention is in the homologous recombination vector.
• suitable vectors for homologous recombination is described, for example,
• prokaryotic or eukaryotic organisms are suitable as recombinant host organisms for the nucleic acid or nucleic acid construct according to the invention.
• microorganisms such as bacteria, fungi or yeast are used as host organisms.
• gram-positive or gram-negative bacteria preferably bacteria of the families Enterobacteriaceae,
• Pseudomonadaceae, Rhizobiaceae, Streptomycetaceae or Nocardiaceae particularly preferably bacteria of the genera Escherichia, Pseudomonas, Streptomyces, Nocardia, Burkholderia, Salmonella, Agrobacterium or Rhodococcus used. Very particularly preferred is the genus and species Escherichia coli. Further advantageous bacteria are also found in the group of alpha-proteobacteria, beta-proteobacteria or gamma-proteobacteria.
• the host organism or the host organisms according to the invention preferably contain at least one of the nucleic acid sequences described in this invention, nucleic acid constructs or vectors which encode an enzyme with activity according to the invention for the conversion of compound I to II.
• the organisms used in the method according to the invention are grown or grown, depending on the host organism, in a manner known to those skilled in the art.
• Microorganisms are usually contained in a liquid medium containing a carbon source mostly in the form of sugars, a nitrogen source mostly in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as iron, manganese, magnesium salts and optionally vitamins, at temperatures between 0 ° C and 100 ° C, preferably between 10 ° C to 60 ° C attracted under oxygen fumigation.
• the pH of the nutrient fluid can be kept at a fixed value, that is regulated during the cultivation or not.
• the cultivation can be done batchwise, semi-batchwise or continuously.
• Nutrients can be presented at the beginning of the fermentation or fed semi-continuously or continuously.
• the ketone can be given directly for cultivation or advantageously after cultivation.
• the enzymes may be isolated from the organisms by the method described in the Examples or used as crude extract for the reaction.
• the invention furthermore relates to processes for the recombinant production of polypeptides according to the invention or functional, biologically active fragments thereof, which comprises cultivating a polypeptide-producing microorganism, optionally inducing the expression of the polypeptides and isolating them from the culture.
• the Po Lypeptides can thus also be produced on an industrial scale, if desired.
• the recombinant microorganism can be cultured and fermented by known methods. Bacteria can be propagated, for example, in TB or LB medium and at a temperature of 20 to 40 0 C and a pH of 6 to 9. Specifically, suitable culturing conditions are described, for example, in T. Maniatis, EF Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Colard Spring Harbor Laboratory, ColD Spring Harbor, NY (1989).
• the cells are then disrupted if the polypeptides are not secreted into the culture medium and the product recovered from the lysate by known protein isolation techniques.
• the cells may optionally be treated by high frequency ultrasound, high pressure, e.g. in a French pressure cell, by osmolysis, by the action of detergents, lytic enzymes or organic solvents, by homogenizers or by combining several of the listed methods.
• polypeptides can be achieved by known chromatographic methods, such as molecular sieve chromatography (gel filtration), such as
• Sepharose chromatography Sepharose chromatography, ion exchange chromatography and hydrophobic chromatography, as well as other conventional methods such as ultrafiltration, crystallization, salting out, dialysis and native gel electrophoresis. Suitable methods are described, for example, in Cooper, F.G., Biochemische Harvey Methoden, Verlag Walter de Gruyter, Berlin, New York or in Scopes, R., Protein Purification, Springer Verlag, New York, Heidelberg, Berlin.
• anchors such as anchors.
• hexa-histidine anchors which can be recognized as antigens of antibodies (described, for example, in Harlow, E. and Lane, D., 1988, Antibody: A Laboratory Manual, ColD Spring Harbor (NY ) Press).
• anchors may be used to attach the proteins to a solid support, e.g. a polymer matrix, which may for example be filled in a chromatography column, or used on a microtiter plate or on another carrier.
• these anchors can also be used to detect the proteins.
• conventional markers such as fluorescent dyes, enzyme markers, which, after reaction with a substrate, have a detectable reactivity. or radioactive markers, alone or in combination with the anchors used to derivatize the proteins.
• organisms in particular microorganisms, which have an increased acetonitrilase activity or in which the activity of the polypeptide according to the invention is increased in comparison to the wild type.
• such an increase may be achieved by incorporation of a corresponding nucleic acid construct, e.g. the nucleic acid construct or vector according to the invention or by targeted or untargeted mutagenesis of the organism
• the selected microorganisms are mutagenized according to the invention.
• mutagenized is meant that into the genetic information, i. targeted or untargeted mutations are introduced into the genome of microorganisms.
• Targeted or untargeted mutations alter one or more genetic information, i. the microorganisms are genetically modified. As a rule, this change leads to the fact that the affected genes are not or incorrectly expressed, so that the activity of the gene product is reduced or inhibited.
• Targeted mutations mutate a particular gene or inhibit, reduce or alter its activity.
• untargeted mutations one or more genes are randomly mutated or its activity inhibited, reduced or altered.
• a population may be e.g. with a DNA population or bank which is suitable for the inhibition of various, as many as possible, in the optimal case of all genes, are transformed so that, statistically, a DNA fragment, preferably identifiable, is integrated into each gene of the microorganism.
• the switched-off gene can be identified.
• former mutagens that differ in their mode of action: e.g. Base analogs, e.g. 5-bromouracil, 2-aminopurine; Chemicals that react with DNA, e.g. nitrous acid, hydroxylamine; or alkylating compounds, such as monofunctional (eg ethyl methanesulfonate, dimethyl sulfate, methyl methanesulfonate), bifunctional (eg nitrogen mustard gas, mitomycin, nitrosoguanidines - dialkylnitrosamines, N-nitrosourea derivatives, N-alkyl-N-nitro-N-nitroso-guanidine-), intercalating dyes (eg acridines, ethidium bromide).
• Base analogs e.g. 5-bromouracil, 2-aminopurine
• Chemicals that react with DNA e.g. nitrous acid, hydroxylamine
• alkylating compounds such as monofunctional (eg e
• Physical mutagenization is e.g. about irradiation of the organisms.
• Several forms of radiation are highly mutagenic.
• Mutations can also be induced by biological processes.
• the standard procedure is transposon mutagenesis, in which insertion or transposition of a transposable element within or around a gene causes alteration, usually loss of gene activity. By identifying the insertion site of the transposon, the gene whose activity has been altered can be isolated.
• Mutagenesis can alter the cellular activity of one or more gene products.
• the cellular activity of the arylacetonitrilase described herein is increased, most preferably the polypeptide described herein.
• the non-transgenic organisms according to the invention in particular
• Microorganisms, plants and plant cells which are characterized by a modulation of the expression and / or the binding behavior of endogenous arylacetonitrilase and have a permanent or transient pathogen resistance, by the so-called "TILLING" approach (Targeting Induced Local Lesion in genomes) manufactured become.
• TILLING Targeting Induced Local Lesion in genomes
• This method is described in detail in Colbert et al. (2001, Plant Physiology, 126, 480-484), McCallum et al. (2000, Nat. Biotechnol., 18, 455-457) and McCallum et al. (2000, Plant Physiology, 123, 439-442).
• the aforementioned references are explicitly incorporated herein by way of disclosure with respect to the "TILLING" method.
• the TILLING procedure is a strategy of so-called reverse genetics that allows the production of high levels of point mutations in mutagenized microorganisms. or plant collections, eg, by chemical mutagenesis with ethylmethanesulphonate (EMS), combined with rapid systematic identification of mutations in target sequences.
• EMS ethylmethanesulphonate
• the target sequence is amplified by PCR in DNA pools of mutagenized M2 populations.
• Denaturing and annealing reactions of the heteroallelic PCR products allow the formation of heteroduplexes in which one strand of DNA originates from the mutated and the other from the "wild-type" PCR product, at the point of point mutation, a so-called mismatch, either denaturing HPLC (DHPLC, McCallum et al., 2000, Plant Physiol., 123, 439-442) or with the cel mismatch detection system (Oleykowsky et al., 1998, Nucl. Acids Res.
• mismatch either denaturing HPLC (DHPLC, McCallum et al., 2000, Plant Physiol., 123, 439-442) or with the cel mismatch detection system (Oleykowsky et al., 1998, Nucl. Acids Res.
• Cel ⁇ is an endonuclease that recognizes mismatches in heteroduplex DNA and specifically cleaves the DNA at these sites The cleavage products can then be separated and detected by automated sequencing gel electrophoresis (Colbert et al., 2001, vide supra) Identification of target gene-specific mutations in a pool is analyzed according to individual DNA samples to isolate the microorganism or plant with the mutation In the case of the microorganisms, plants and plant cells according to the invention, after the production of the mutagenized populations by the use of primer sequences directed against arylacetonitrilase, the identification of the mutagenized plant cells or plants is carried out.
• the TILLING method is generally applicable to all microorganisms and plants and plant cells.
• the invention also relates to a composition containing substantially R- and / or S-5-norbornene-2-endo-carbonitrile and compositions comprising: R- and / or S-5-norbornene-2-endo-carboxylic acid more than 60%, 70%, 80%, 90%, 95%, 99%; and / or containing R and / or S-5-norbornene-2-exo-carboxylic acid ratio less than 40%, 30%, 20%, 10%, 5%, 1%.
• a composition has not hitherto been made in the prior art.
• norbornene acid always resulted in an enantiomeric mixture of 5-norbornene-2-endo-carboxylic acid to 5-norbornene-2-exo-carboxylic acid ratio of about 0.6: about 0.4.
• the present invention also relates to a composition
• a composition comprising substantially R- and / or S-5-norbornene-2-exo-carbonitrile and a composition containing R- and / or S-5-norbornene-2-endo-carboxylic acid to form R- and / or S-5-norbornene-2-exo-carbonitrile. and / or S-5-norbornene-2-exo-carboxylic acid in a ratio of less than 0.6 to more than 0.4.
• Such a composition has not hitherto been made in the prior art.
• norbornene acid always resulted in an enantiomeric mixture of 5-norbornene-2-endo-carboxylic acid to 5-norbornene-2-exo-carboxylic acid ratio of about 0.6: about 0.4
• the invention also relates to a composition which can be prepared by the process according to the invention.
• the invention relates to a composition prepared by the method according to the invention.
• the invention relates to the use of an enzyme, in particular a nitrilase, preferably an arylacetonitrilase, more preferably a polypeptide according to the invention having the sequence shown in SEQ ID no. 2 or 4 or a homolog or a functional fragment thereof for the enrichment of an isomer of the compound II from a mixture of isomers of the compound I.
• an enzyme in particular a nitrilase, preferably an arylacetonitrilase, more preferably a polypeptide according to the invention having the sequence shown in SEQ ID no. 2 or 4 or a homolog or a functional fragment thereof for the enrichment of an isomer of the compound II from a mixture of isomers of the compound I.
• the invention relates to the use of an enzyme, in particular a nitrilase, preferably an arylacetonitrilase, particularly preferably a polypeptide according to the invention having the sequence shown in SEQ ID no. 2 or 4 or a homologue or a functional fragment thereof for the enrichment of R- and / or S-5-norbornene-2-endo-carboxylic acid from a mixture containing R- and / or S-5-norbornene-2-endo-carbonitrile and R- and / or S-5-norbornene-2-exo-carbonitrile.
• an enzyme in particular a nitrilase, preferably an arylacetonitrilase, particularly preferably a polypeptide according to the invention having the sequence shown in SEQ ID no. 2 or 4 or a homologue or a functional fragment thereof for the enrichment of R- and / or S-5-norbornene-2-endo-carboxylic acid from a mixture containing R- and / or S
• the invention relates to the use of an arylacetonitrilase for reacting R- and / or S-5-norbornene-2-endo-carbonitrile and / or R- and / or S-5-norbornene-2-exo-carbonitrile to R- and / or S-norbornene-2-endo-carboxylic acid and / or R- and / or S-norbornene-2-exo-carboxylic acid.
• the invention also relates to the use of an arylacetonitrilase for reacting R- and / or S-5-norbornene-2-endo-carbonitrile and / or R- and / or S-5-norbornene-2-exo-carbonitrile to the R- and / or S-endo and / or R and / or S-norbornene-2-exo-carboxylic acid.
• the invention relates to the use of an enzyme, in particular a nitrilase, preferably an arylacetonitrilase, particularly preferably a polypeptide according to the invention having the sequence shown in SEQ ID no. 2 or 4 or a homolog or a functional fragment thereof for the reaction of R- and / or S-5-norbornene-2-endo-carbonitrile to isomerically pure R and / or S-5-norbornene-2-endo-carboxylic acid high substrate concentration.
• an enzyme in particular a nitrilase, preferably an arylacetonitrilase, particularly preferably a polypeptide according to the invention having the sequence shown in SEQ ID no. 2 or 4 or a homolog or a functional fragment thereof for the reaction of R- and / or S-5-norbornene-2-endo-carbonitrile to isomerically pure R and / or S-5-norbornene-2-endo-carboxylic acid high substrate concentration.
• the invention relates to the use of an enzyme, in particular a nitrilase, preferably an arylacetonitrilase, particularly preferably a polypeptide according to the invention, characterized in that a protein lypeptid is used, that is encoded by a nucleic acid molecule comprising a nucleic acid molecule selected from the group consisting of:
• nucleic acid molecule comprising at least the polynucleotide of the coding sequence of Seq. ID No .: 1 or 3;
• nucleic acid molecule whose sequence can be deduced from a polypeptide sequence encoded by a nucleic acid molecule according to (a) or (b) due to the degeneracy of the genetic code;
• nucleic acid molecule encoding a polypeptide whose sequence has an identity of at least 60% to the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) or (b);
• nucleic acid molecule encoding a polypeptide derived from an aryl acetonitrile se- crase polypeptide, wherein up to 25% of the amino acid residues are opposite
• SEQ ID NO: 2 or 4 are altered by deletion, insertion, substitution or a combination thereof and which still has at least 30% of the enzymatic activity of SEQ ID NO: 2 or 4;
• nucleic acid molecule encoding a fragment or epitope of an arylaceto-nitrilase encoded by any of the nucleic acid molecules of (a) to (c);
• the polypeptide does not have the sequence according to Seq ID No .: 2 or 4. In one embodiment, the polypeptide also does not have the sequence described in Eur. J. Biochem. 182, 349-156, 1989 called nitrilase. In one embodiment, the polypeptide also does not have the sequence of database entry AY885240.
• the invention relates to the use of a polypeptide for the production of a compound of formula II by enzymatic reaction of a compound of formula I, wherein the polypeptide is characterized in that it is encoded by a nucleic acid molecule comprising nucleic acid molecule selected from the group consisting of:
• nucleic acid molecule comprising at least the polynucleotide of the coding sequence of Seq. ID No .: 1 or 3;
• nucleic acid molecule whose sequence can be deduced from a polypeptide sequence encoded by a nucleic acid molecule according to (a) or (b) due to the degeneracy of the genetic code;
• nucleic acid molecule encoding a polypeptide whose sequence has an identity of at least 60% to the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) or (b);
• nucleic acid molecule encoding a polypeptide derived from an aryl acetonitrile polypeptide wherein up to 25% of the amino acid residues are altered from SEQ ID NO: 2 or 4 by deletion, insertion, substitution or a combination thereof has at least 30% of the enzymatic activity of SEQ ID NO: 2 or 4;
• nucleic acid molecule encoding a fragment or epitope of an arylaceto-nitrilase encoded by any of the nucleic acid molecules of (a) to (c);
• the polypeptide does not have the sequence of Seq ID No .: 2 or 4. In one embodiment, the polypeptide also does not have the sequence described in Eur. J. Biochem. 182, 349-156, 1989 called nitrilase. In one embodiment, the polypeptide also does not have the sequence of database entry AY885240.
• FIG. 1 shows enzymes with activity according to the invention.
• a high activity was observed.
• High activity was also observed at a high nitrile concentration.
• Nitrilases from Biocatalytics were used as BTM at 2 mg / ml
• the BASF nitrilases were used as recombinant whole cell biocatalysts (E. coli TG1 OpDHE system with GroELS chaperones, see PCT / EP 03 / 13367) and overnight in 30 ml LB with ampicillin (100 ⁇ g / ml), spectinomycin (100 ⁇ g / ml), chloramphenicol (20 ⁇ g / ml), IPTG (0.1 mM) and rhamnose monohydrate (0, 5 g / L) in 100 ml Erlenmeyer at 37 ° C.
• the cells were washed 1x in 30 ml of 10 mM Pipes pH 7.0 and taken up in 3 ml buffer and optionally stored at -20 ° C.
• nitrile was the Isomer mixture from the company Aldrich used.
• the PCR was carried out according to Stratagene standard protocol with Pfu ultra-polymerase (Stratagene) and the following temperature program: 95 ° C for 5 minutes; 30 cycles at 95 ° C for 45 sec, 50 ° C for 45 sec, and 72 ° C for 1 min 30 sec; 72 ° C for 10 min .; 10 ° C until use.
• the PCR product (1.2 kb) was isolated by agarose gel electrophoresis (1.2% E-gel, Invitrogen) and column chromatography (GFX kit, Amersham) and then digested with Ndel / HindIII and incubated in appropriate digested pDHE19.2 vector (a pJOE derivative, DE19848129) cloned.
• E. coli TG10 pAgro4 pHSG575 TG10: a RhaA " derivative of E. coli TG1 (Stratagene); pAgro4: Takeshita, S; Sato, M; Toba, M; Masahashi, W; Hashimoto). Gotoh, T (1987) Gene 61, 63-74, pHSG575: T. Tomoyasu et al (2001) Mol. Microbiol.
• Rhodococcus rhodochrous J1 (FERM BP-1478) was grown as described in the literature (Nagasawa et al., Arch. Microbiol 1988: 150, 89-94) and harvested. The cells were tested for their benzonitrilase activity as in Example 4 and showed full turnover after 15 min. The BASF nitrilase strains and E. coli TG10 + pDHE9632J1 (Example 4) were grown and harvested as in Example 1. Subsequently, the bio-dry masses were determined (R. rhodochrous J1: 3.5 g / L, E. coli strains: 0.8 g / L).
• Nitrilase Nit Polypeptide Sequence from ADG93882 (WO2003097810-A2 Seq. ID349)

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