Nitrilase from rhodococcus rhodochrous ncimb 11216

FIELD: biotechnology.

SUBSTANCE: invention relates to isolated nucleic acid sequence encoding of polypeptide with nitrilase activity, wherein nitriles are converted to carboxylic acids in presence of said nitrilase.

EFFECT: method for production of chiral carboxylic acids with high effectiveness and low cost.

10 cl, 4 dwg, 2 tbl, 1 ex

 

The invention relates to sequences of nucleic acids that encode a polypeptide with the activity nitrilase, to nucleic acid constructs containing nucleic acid sequences, as well as vectors containing nucleic acid sequences or nucleic acid constructs. Further, the invention relates to an amino acid sequence that is encoded by the sequences of nucleic acids, and micro-organisms that contain nucleic acid sequences, or nucleic acid constructs or vectors containing nucleic acid sequences or nucleic acid constructs.

In addition, the invention relates to an enzymatic process for the preparation of carboxylic acids from the corresponding NITRILES.

Aliphatic, aromatic and heteroaromatic carboxylic acids are important compounds for organic chemistry synthesis. They are the original products for a variety of pharmaceutical active substances or active substances for plant protection.

From the literature there are a number of different enzymatic processes of synthesis for the preparation of achiral or chiral carboxylic acids. For example, the enzymatic method are optically active amino acids. The disadvantage to this is what I for each amino acid must develop its own way. In order to provide a wide range of different compounds, applied by chemical or enzymatic methods. Chemical methods the disadvantage is that stereocenter should be built, as a rule, multi-stage, not widely used, a complex synthesis.

Enzymatic synthesis of chiral carboxylic acids are described in several patent applications and patents. International application WO 92/05275 describes the synthesis of enantiomeric α-hydroxy-α-alkyl - or α-alkylcarboxylic acids in the presence of optically active biological material. Other syntheses of optically active α-substituted acids of microorganisms are described in EP-B-0348901, ER-IN-0332379, EP-A-0348901 or its equivalent, US 5283193, EP-A-0449648, ER-IN-0473328, ER-IN-0527553 or its equivalent, US 5296373, EP-A-0610048, EP-A-0610049, EP-A-0666320 or WO 97/32030.

Biotechnological synthesis of achiral carboxylic acids by microorganisms are described, for example, in EP-A-0187680, EP-A-0229042, WO 89/00193, JP 08173152, JP 06153968, FR 2694571, EP-A-0502476, EP-A-0444640 or EP-A-0319344.

The disadvantage of these processes is that they often lead to products with a low optical purity and/or that are low out of time and space. This leads to economically uninteresting processes. In addition, the lack of t is aetsa, what enzymes are used for the synthesis of achiral or chiral carboxylic acids by microorganisms, usually have only a limited range of substrates, i.e. microorganisms become only certain aliphatic, aromatic or heteroaromatic NITRILES. Especially aromatic and heteroaromatic NITRILES, such as, for example, cyanothiophene or benzonitrile, is converted by microorganisms or poorly, or not at all converted into the corresponding carboxylic acid.

The invention therefore is to develop enzymatic methods of obtaining achiral and/or chiral carboxylic acids that do not have the above disadvantages and is particularly available for achiral or chiral carboxylic acids from the corresponding NITRILES.

This problem is solved allocated according to the invention the sequence of the nucleic acid that encodes a polypeptide with the activity nitrilase, and selected from the group including

a) the sequence of nucleic acids represented in SEQ ID NO:1 sequence,

b) nucleic acid sequences which, as a result of degenerated genetic code diverted from that presented in SEQ ID NO:1 nucleic acid sequences,

C) derivatives presented in SEQ ID NO:1 is the follower of the spine of nucleic acids, encoding the polypeptide presented in SEQ ID NO: 2 amino acid sequence having at least 95% homology at the amino acid level, without significant reduction of the enzymatic activity of the polypeptides.

Under the homology of the nucleic acid sequences according to the invention with the sequence SEQ ID NO:1 should be understood, for example, allelic variants, which have 95%homology at the designated amino acid level, preferably at least 97% homology, particularly preferably at least 98%, and very preferably at least 99% homology over the entire sequence region. Partial plot of sequence homology may be preferably higher. Abstracted from SEQ ID NO:1 amino acid sequence presented in SEQ ID NO:2. Allelic variants encompass in particular functional variants which are obtained by deletion, insertion or substitution of nucleotides listed in SEQ ID NO:1 sequence, and the enzymatic activity allocated synthesized proteins should be retained, and slightly lower for introducing one or more genes in the organism. Under slightly reduced activity, you should understand the enzymatic activity, which is preferably at least 10%, preferably 30%, especially preferably 50%, and particularly preferably 70% of the enzymatic activity is presented in SEQ ID NO:2 enzyme. The invention relates, therefore, to amino acid sequences that are encoded by the above groups of sequences of nucleic acids. Preferably the invention relates to amino acid sequences that are encoded by the sequence SEQ ID NO:1.

Further, under the homology sequence of SEQ ID NO:1 should be understood, for example, fungal or bacterial homologues, abbreviated sequence, single-stranded DNA or RNA of the coding and noncoding DNA sequence. Homologues of the sequence SEQ ID NO:1 have at the level of DNA homology of at least 60%, preferably at least 70%, particularly preferably at least 80%, particularly preferably at least 90% on all listed in SEQ ID NO:1 section of DNA.

In addition, under the homology SEQ ID NO:1 should be understood derivatives, such as, for example, variants of the promoters. The promoters that are included before the given nucleotide acids, can be modified by one or more nucleotide substitutions, through inertia (inertia), and/or deletions (deletions), without negatively affecting the functionality of, respectively, the efficiency of the promoters. Further, the promoters may b the th increased in their activity, change their sequence or may be completely replaced by more active promoters and organisms of other species.

Derivatives should also understand the options, the nucleotide sequence of which is changed in the range from -1 to -200 base pairs before the start codon and from 0 to 1000 base pairs after stopcodon in such a way that changes the expression of genes and/or expression of the protein is preferably increased.

Preferably the sequence of SEQ ID NO:1 or its homologues can be isolated from bacteria, preferably gram-positive bacteria, preferably of the species Nocardia, Rhodococcus, Streptomyces, Mycobacterium, Corynebacterium, Micrococcus, Proactinomyces or Bacillus, particularly preferably from bacteria of the species Rhodococcus, Mycobacterium or Nocardia, extremely preferably from bacteria of the species Rhodococcus sp., Rhodococcus rhodochrous, Nocardia rhodochrous or Mycobacterium rhodochrous known to the person skilled in the art methods.

Sequence SEQ ID No:1 or its homologs or parts of these sequences can be selected, for example, conventional methods of hybridization or PCR techniques from other fungi or bacteria. These DNA sequences hybridizing under standard conditions with sequences according to the invention. For hybridization are used preferably short oligonucleotides canned sections, for example, from the active center, which can be determined through comparison with other nitrilase and nitrilgidrataznoi known to the expert clicks the zoom. However, for hybridization can be used longer nucleic acid fragments according to the invention or the full sequence. Depending on the nucleic acid, oligonucleotide, longer fragment or complete sequence and depending on what type of nucleic acid, DNA or RNA used for hybridization vary these standard conditions. For example, the melting temperatures for DNA: DNA hybrids on approx. on 10°With lower than DNA: RNA hybrids of the same length.

Under standard conditions should be understood as depending on the nucleic acid temperatures between 42 and 58°in aqueous buffer solution with a concentration between 0.1 to 5 × SSC (where 1 × SSC = 0.15 M NaCl, 15 mm sodium citrate, pH of 7.2) or additionally in the presence of 50% formamide, for example, 42°in 5 × SSC, 50% formamide. Preferably, the hybridization conditions are for DNA:DNA-hydride 0,1 × SSC and temperatures between 20°and 45°C, preferably between 30°and 45°C. For DNA:DNA-hydride hybridization conditions, preferably at 0.1 × SSC and temperatures between 30°and 55°C, preferably between 45°and 55°C. These temperatures for hybridization are, for example, the calculated values of the melting point for nucleic acids with a length of approx. 100 nucleotides and a G + C-content is the use of 50% in the presence of formamide. The experimental conditions for DNA hybridization are described in well-known textbooks on genetics, as for example in Sambrook and others, "Molecular Cloning", Cold Spring Harbor Laboratory, 1989, and can be calculated by well-known specialist formulas, for example, depending on the length of the nucleic acid species hybrids or the G + C-content. Further information on hybridization professional may find the following references: Ausubel et al. (eds), 1985, Current Protocols in Molecular Biology, John Wiley & Sons, New York; Hames and Higgins (eds), 1985, Nucleic Acids Hybridization: A Practical Approach, IRL Press at Oxford University Press, Oxford; Brown (ed), 1991, Essential Molecular Biology: A Practical Approach, IRL Press at Oxford University Press, Oxford.

Under the nucleic acid construct according to the invention should be understood genes nitrilase sequence SEQ ID NO:1 and its homologues, which are functionally linked to one or more regulatory signals, preferably, to enhance gene expression. When these regulatory sequences are talking about, for example, sequences that are associated with inductors or repressor substances and thus regulate the expression of nucleic acids. In addition to these regulatory sequences may be natural regulation of these sequences to their own structural genes and, if necessary, it can be genetically modified so that the natural regulation of excluded and Express what I genes increased. The nucleic acid construct may, however, be built easier, i.e. additional regulatory signals before the sequence SEQ ID NO:1 or its homologs not inserious and the natural promoter with its regulation has not been deleted. Instead, the natural regulation mutate in such a way that no longer happens regulation and expression of genes increases. The nucleic acid construct can also contain one or more so-called enhancer sequences that are functionally associated with the promoter, which allow increased expression of nucleic acid sequences. Also on 3′-end DNA sequence can be further serirovani sequences, such as further regulatory elements or terminators. Nucleic acids according to the invention may contain one or more copies of the construct. The construct may contain other markers, such as resistant to antibiotics or complementaria auxotrophic genes, if necessary, for breeding on the construct.

Preferred regulatory sequences for the method according to the invention contains, for example, such promoters as cos-, tac-, trp-, tet-, trp-tet-, lpp-, lac-, lpp-lac-, laclq-T7-, T5-, T3-, gal-, trc-, ara-, SP6-, λ-PR-or λ-PL-the promoter, which applies who are preferably in gram-negative bacteria. Other regulatory sequences are, for example, in the gram-positive promoters amy and SPO2, in the yeast and mushroom promoters ADC1, MFa, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH. In this regard, the preferred and also the promoters of piruvatcarboksilazy and methanococcales, for example, from Hansenula. For regulation can also be applied artificial promoters.

The nucleic acid construct for expression in the organism-host interisuetsya, preferably in a vector, such as plasmid, phage or other DNA, which provides optimal gene expression. These vectors represent a further object of the invention. Suitable vectors include, for example, in E. coli pLG338, pACYC184, pBR322, pUC18, pUC19, RXT, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III113-BI lgt11 or pBdCI, streptomycete lJ101, plJ364, plJ702 or plJ361, in Bacillus pUB110, pC194 or pBD214, in Corynebacterium pSA77 or pAJ667, in fungi pALS1, plL2 or rwib, in yeast 2μM, pAG-1, YEp6, YEp13 or pEMBLYe23 or in plants pLGV23, pGHIac+, pBIN19, pAK2004 or pDH151. The above plasmids represent a small selection of the possible plasmids. Further plasmids are known to the person skilled in the art and can be found, for example, in the book of Cloning Vectors (Eds. Pouwels R.N. and other Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018).

Preferably the nucleic acid construct for the expression of other genes contains an additional 3′ and/or 5′-reg ends the Torno sequences to enhance expression, which, depending on the host body and the gene or genes are selected for optimal expression.

These regulatory sequences should allow targeted gene expression and protein expression. This may mean, depending on the host body, that gene exprimarea or sverkhekspressiya only after induction or that he exprimarea and/or sverkhekspressiya immediately.

Regulatory sequences are, respectively, the factors can preferably have a positive impact on gene expression of the introduced genes and thereby improve it. The strengthening of the regulatory elements can preferably be carried out on the temperature level, applying a strong transcription signals and/or enhacer". Along with this it is also possible and the strengthening of the broadcast due to the fact that, for example, improving the stability of the mRNA.

In another form of execution of the vector containing the nucleic acid construct according to the invention or a nucleic acid according to the invention the construct must be entered in the body of the host in the form of linear DNA and through heterologous or homologous recombination to integrate into the genome of the host body. Linear DNA may consist of a linearized vector such as a plasmid, or only from the nucleic acid construct or isoclinally acid.

For optimal expression of heterologous genes in the body should preferably modify the nucleic acid sequences in accordance with the use of the organism-specific "codon usage". This "codon usage" can easily be determined using a computer comparison of other known genes of the corresponding organism.

As organisms-owners for the nucleic acids according to the invention or the nucleic acid construct suitable, in principle, all prokaryotic or eukaryotic organisms. Preferably as organisms hosts use such microorganisms as bacteria, fungi or yeast. Preferably used are gram-positive or gram-negative bacteria of the family Enterobacteriaceae, Pseudomonadaceae, Streptomycetaceae, Mycobacteriaceae or Nocardiaceae, especially practicelink bacteria of the species Escherichia, Pseudomonas, Nocardia, Mycobacterium, Streptomyces, or Rhodococcus. In particularly preferred genera Escherichia coli, Rhodococcus rhodochrous, Nocardia rhodochrous, Mycobacterium rhodochrous or Streptomyces lividans.

Master-organism according to the invention contains as a matter of priority at least one proteiny agent for laying synthesized them polypeptides and, in particular, described in the present invention sequences nukleinovykh acids with activity nitrilase and/or encoding the genes agent, and this agent is available in quantity, the cat is PoE more than the number that corresponds to the primary number of the considered organism. Encoding this agent contains genes in the chromosome or extrachromosomal elements such as plasmids.

Another object of the invention is a method of obtaining chiral or achiral carboxylic acids, characterized in that the NITRILES in the presence of the encoded nucleic acids according to the invention the amino acid sequence and growing, resting or open the above microorganism (= organism-host)that contains or sequence of nucleic acids according to the invention, or the nucleic acid construct according to the invention, which holds the nucleic acid according to the invention associated with one or more regulatory signals, or the vector according to the invention, is converted into chiral or achiral carboxylic acid.

The preferred form of execution of the method according to the invention is the transformation of a chiral or achiral aliphatic NITRILES to the corresponding carboxylic acid.

Another form of execution of the method of obtaining chiral or achiral carboxylic acids differs in that the NITRILES of General formula I

transform in the presence of the encoded nucleic acid according to the invention the amino acid placenta is successive or growing, resting or open the above microorganism that contains or sequence of nucleic acids according to the invention, or the nucleic acid construct according to the invention, which holds the nucleic acid according to the invention associated with one or more regulatory signals, or the vector according to the invention, carboxylic acids of General formula II

moreover, the substituents in formulas I and II have the following meanings:

n = 0 or 1

m = 0, 1, 2 or 3, and when m>2 between two adjacent carbon atoms does not necessarily have a double bond,

p = 0 or 1

A, b, D, and E denote, independently of one another CH, N or CR3

N = mean O, S, NR4, CH or CR3if n=0 or CH, N or CR3if n=1,

moreover, two adjacent remnant of a, b, D, E or N together may form a different substituted or unsubstituted aromatic, saturated or unsaturated ring containing from 5 to 8 atoms in the ring which may contain one or more heteroatoms such as O, N or S, and not more than three residues a, b, D, E or N represent a heteroatom,

R1means hydrogen, substituted or unsubstituted, branched or unbranched C1-C1-alkiline C1-C10alkoxy, substituted or unsubstituted aryl or guéthary is-, hydroxy-, halogen-, C1-C10-alkylamino - or amino-,

R2means hydrogen, substituted or unsubstituted, branched or unbranched C1-C10alkyl or C1-C10alkoxy, substituted or unsubstituted aryl - or hetaryl-, hydroxy-, C1-C10alkylamino - or amino-,

R3means hydrogen, substituted or unsubstituted, branched or unbranched C1-C10alkyl-, or C1-C10alkoxy substituted or unsubstituted aryl-, hetaryl-, hydroxy-, halogen-, C1-C10alkylamino - or amino-,

R4means hydrogen, substituted or unsubstituted, branched or unbranched C1-C10alkyl-.

R1means in the compounds of formulas I and II is hydrogen, substituted or unsubstituted, branched or unbranched C1-C10alkyl or C1-C10-alkoxy, substituted or unsubstituted aryl - or hetaryl-, hydroxy-, halogen - as fluorine, chlorine or bromine, C1-C10alkylamino - or amino.

As alkyl residues should be substituted or unsubstituted, branched or unbranched C1-C10alkyl residues such as methyl, ethyl, n-propyl, 1-methylethyl, n-butyl, 1-methylpropyl-, 2-methylpropyl, 1,1-dimethylethyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethy is propyl, 1-ethylpropyl, n-hexyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl-1-methyl-propyl, 1-ethyl-2-methylpropyl, n-heptyl, n-octyl, n-nonyl or n-decyl. The preferred methyl, ethyl, n-propyl, n-butyl,-propyl or-butyl.

As alkoxylated should be substituted or unsubstituted, branched or unbranched C1-C10alkoxy-chain, such as, for example, methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy, 1 methylpropoxy, 2-methylpropoxy, 1,1-dimethylmethoxy, pentox, 1 methylbutoxy, 2-methylbutoxy, 3 methylbutoxy, 1,1-DIMETHYLPROPANE, 1,2-DIMETHYLPROPANE, 2,2-DIMETHYLPROPANE, 1 ethylpropoxy, hexose, 1 methylphenoxy, 2-methylphenoxy, 3 methylphenoxy, 4-methylphenoxy, 1,1-Dimethylbutane, 1,2-Dimethylbutane, 1,3-Dimethylbutane, 2,2-Dimethylbutane, 2,3-Dimethylbutane, 3,3-Dimethylbutane, 1 ethylbutane, 2-ethylbutane, 1,1,2-trimethylpropyl, 1,2,2-trimethyl-propoxy, 1-ethyl-1-methylpropoxy, 1-ethyl-2-methylpropoxy, hexyloxy, heptyloxy, oxylate, nonyloxy or decyloxy and other homologues branched chain.

As aryl - should be a substituted or unsubstituted aryl residues, is the quiet contain from 6 to 20 carbon atoms. While we are talking about condensed to each other aromatic rings or aromatic rings that are linked by bridges with alkyl, alkylcarboxylic, alkenyl or alkenylboronic chains, carbonyl, oxygen or nitrogen. Aryl residues can be connected with the main skeleton after another With1-C10alkyl, C3-C8alkenylphenol,3-C6alkylamino or3-C8-cycloalkyl chain. Preferred phenyl or naphthyl.

As hetaryl should be substituted or unsubstituted, branched or unbranched ring system with one or more heteroaromatic 3 - to 7-membered rings which may contain one or more heteroatoms, such as N, O or S and, if necessary, can be linked through a1-C10-alkyl, C3-C8-alkeline or3-C8-cycloalkenyl chain skeleton. Examples of such getirilmek residues are pyrazole, imidazole, oxazole, isooctanol, thiazole, triazole, pyridine, quinoline, isoquinoline, acridine, pyrimidine, pyridazine, pyrazin, fenesin, purine or pteridine. Getaline residues can be linked through a heteroatom, or through different carbon atoms in the ring or ring system or through deputies with a skeleton. Preferred n is ridin, kepsel, pyrimidine, purine, pyrazin or quinoline.

As alkylaminocarbonyl should be substituted or unsubstituted, branched or unbranched C1-C10alkylamine, such as, for example, methylamino, ethylamino, n-propylamino, 1 methylethylamine, n-butylamino, 1 methylpropylamine-, 2-methylpropylamine, 1,1-dimethylethylamine, n-pentylamine, 1 methylbutylamine, 2-methylbutylamine, 3 methylbutylamine, 2,2-dimethylpropylene, 1 ethylpropylamine, n-hexylamino, 1,1-dimethylbutylamino, 1,2-dimethylpropylene, 1 methylpentylamino, 2-methylpentylamino, 3 methylpentylamino, 4-methylpentylamino, 1,1-dimethylbutylamino, 1,2-dimethylbutylamino, 1,3-dimethylbutylamino, 2,2-dimethylbutylamino, 2,3-dimethylbutylamino, 3,3-dimethylbutylamino, 1 ethylbutylamine, 2-ethylbutylamine, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl-1-methylpropylamine, 1-ethyl-2-methylpropylamine, n-heptylamine, n-octylamine, n-nonylamine or n-decylamine. Preferred methylamino, ethylamino, n-propylamino, n-butylamino, and propylamino or-butylamino.

As Vice-described residues for R1suitable, for example, one or more such substituents as halogen, such as fluorine, chlorine or bromine, thio, cyano, nitro, amino, hydroxy, alkyl, alkoxy, alkenyl, alkenylacyl, quinil or other aromatic or friend who e saturated or unsaturated non-aromatic ring or ring system. Preferred alkyl residues, such as C1-C6-alkyl, for example methyl, ethyl, propyl or butyl, aryl, e.g. phenyl, halogen, for example chlorine, fluorine or bromine, hydroxy or amino.

R2means in the compounds of formulas I and II is hydrogen, substituted or unsubstituted, branched or unbranched C1-C10-alkyl or C1-C10-alkoxy, substituted or unsubstituted aryl - or hetaryl-, hydroxy-, C1-C10-alkylamino amino.

As alkyl residues should be substituted or unsubstituted, branched or unbranched C1-C10alkyl chain, such as methyl, ethyl, n-propyl, 1-methylethyl, n-butyl, 1-methylpropyl-, 2-methylpropyl, 1,1-dimethylethyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, 1-ethylpropyl, n-hexyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl-1-methylpropyl, 1-ethyl-2-methylpropyl, n-heptyl, n-octyl, n-nonyl or n-decyl. Preference is given to stands, ethyl, n-propylene, n-butile, and the propylene-butile.

As alkoxylated should be substituted or unsubstituted, branched sludge is non-branched C 1-C10allocsize, as for example, methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy, 1 methylpropoxy, 2-methylpropoxy, 1,1-dimethylmethoxy, pentox, 1 methylbutoxy, 2-methylbutoxy, 3 methylbutoxy, 1,1-DIMETHYLPROPANE, 1,2-DIMETHYLPROPANE, 2,2-DIMETHYLPROPANE, 1 ethylpropoxy, hexose, 1 methylphenoxy, 2-methylphenoxy, 3 methylphenoxy, 4-methylphenoxy, 1,1-Dimethylbutane, 1,2-Dimethylbutane, 1,3-Dimethylbutane, 2,2-Dimethylbutane, 2,3-Dimethylbutane, 3,3-Dimethylbutane 1 ethylbutane, 2-ethylbutane, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl-1-methylpropoxy, 1-ethyl-2-methylpropoxy, hexyloxy, heptyloxy, octyloxy, nonyloxy or decyloxy their homologues branched chain.

As aryl - should be substituted or unsubstituted, aryl residues, which contain from 6 to 20 carbon atoms in the ring or ring system. It can be condensed to each other aromatic rings or aromatic rings that are linked by a bridge across alkyl, acylcarnitine, alkeline or alkenylboronic chains, carbonyl, oxygen or nitrogen. Aryl residues can, if necessary, to be linked through C1-C10-alkyl, C3-C8-alkenylphenol,3-C8-alkylamino or3-C8-cycloalkyl chain skeleton. Preferably the phenyl or naphthyl.

As hetaryl - should be substituted or unsubstituted, simple or condensed aromatic ring system with one or more heteroaromatic 3 - to 7-membered rings which may contain one or more heteroatoms such as N, O or S, and, if necessary, can be linked to the skeleton via C1-C10-alkyl, C3-C8-alkenylphenol or3-C8-cycloalkyl chain. Examples of such getirilmek residues are pyrazole, imidazole, oxazole, isoxazol, thiazole, triazole, pyridine, quinoline, isoquinoline, acidin, pyrimidine, pyridazine, pyrazin, fenesin, purine or pteridine. Getaline residues can be linked to the skeleton via heteroatoms or through different carbon atoms in the ring or ring system or through deputies. Preferred pyridine, imidazole, pyrimidine, purine, pyrazin or quinoline.

As alkylaminocarbonyl should be substituted or unsubstituted, branched or unbranched1-C10alkylamine, such as methylamine, ethylamine, n-Propylamine, 1 methylethylamine, n-butylamino, 1 methylpropylamine-, 2-methylpropylamine, 1,1-dimethylethylamine, n-pentylamine, 1 methylbutylamine, 2-methylbutylamine, 3 methylbutylamine, 2,2-dimethylpropylene, 1 ethylpropylamine, n-hexylamino, 1,1-is metilpropanalya, 1,2-dimethylpropylene, 1 methylpentylamino, 2-methylpentylamino, 3 methylpentylamino, 4-methylpentylamino, 1,1-dimethylbutylamino, 1,2-dimethylbutylamino, 1,3-dimethylbutylamino, 2,2-dimethylbutylamino, 2,3-dimethylbutylamino, 3,3-dimethylbutylamino, 1 ethylbutylamine, 2-ethylbutylamine, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl-1-methylpropylamine, 1-ethyl-2-methylpropylamine, n-heptylamine, n-octylamine, n-nonylamine or n-decylamine. Preferred methylamino, ethylamino, n-propylamino, n-butylamino, and propylamino or-butylamino.

As Vice-described residues for R2suitable, for example, one or more such substituents as halogen, for example fluorine, chlorine or bromine, thio, nitro, amino, hydroxy, alkyl, alkoxy, alkenyl, alkenylacyl, quinil or other aromatic or other saturated or unsaturated non-aromatic ring or ring system. Preferred such alkyl residues, as C1-C6alkyl, for example methyl, ethyl, propyl or butyl, aryl, e.g. phenyl, halogen, for example chlorine, fluorine or bromine, hydroxy or amino.

R3means in the compounds of formulas I and II is hydrogen, substituted or unsubstituted, branched or unbranched C1-C10alkyl or C1-C10-alkoxy, substituted or unsubstituted aryl - or hetaryl-, hydroxy-,halogen, such as fluorine, chlorine or bromine, C1-C10alkylamino amino.

As alkyl residues should be substituted or unsubstituted, branched or unbranched C1-C10alkyl chain, such as methyl, ethyl, n-propyl, 1-methylethyl, n-butyl, 1-methylpropyl-, 2-methylpropyl, 1,1-dimethylethyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, 1-ethylpropyl, n-hexyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl-1-methylpropyl, 1-ethyl-2-methylpropyl, n-heptyl, n-axtel, n-nonyl or n-decyl. The preferred methyl, ethyl, n-propyl, n-butyl,-propyl or-butyl.

As alkoxylated should be substituted or unsubstituted, branched or unbranched1-C10-allocsize, for example, methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy, 1 methylpropoxy, 2-methylpropoxy, 1,1-dimethylmethoxy, pentox, 1 methylbutoxy, 2-methylbutoxy, 3 methylbutoxy, 1,1-DIMETHYLPROPANE, 1,2-DIMETHYLPROPANE, 2,2-DIMETHYLPROPANE, 1 ethylpropoxy, hexose, 1 methylphenoxy, 2-methylphenoxy, 3 methylphenoxy, 4-methylphenoxy, 1,1-Dimethylbutane, 1,2-Dimethylbutane, 1,3-d is methylbutoxy, 2,2-Dimethylbutane, 2,3-Dimethylbutane, 3,3-Dimethylbutane, 1 ethylbutane, 2-ethylbutane, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl-1-methylpropoxy, 1-ethyl-2-methylpropoxy, hexyloxy, heptyloxy, octyloxy, nonyloxy or decyloxy and homologues branched chain.

As aryl - should be substituted or unsubstituted, aryl residues, which contain from 6 to 20 carbon atoms in the ring or ring system. It can be condensed to each other aromatic rings or aromatic rings, through which the alkyl, acylcarnitine, alkeline or alkenylboronic chains, carbonyl, oxygen or nitrogen linked by bridges. Aryl residues, if necessary, can be linked to the skeleton via C1-C10alkyl, C3-C8alkenylphenol,3-C8alkylamino or3-C8cycloalkyl chain. Preferred phenyl or naphthyl.

As hetaryl - should be substituted or unsubstituted, simple or condensed aromatic ring system with one or more heteroaromatic 3 - to 7-membered rings which may contain one or more heteroatoms, such as N, O or S and, if necessary, through the C1-C10alkyl, C3-C8alkenylphenol or3 -C8-cycloalkyl chain can be associated with the skeleton. Examples of such getirilmek residues are pyrazole, imidazole, oxazole, isooctanol, thiazole, triazole, pyridine, quinoline, isoquinoline, acridine, pyrimidine, pyridazine, pyrazin, fenesin, purine or pteridine. Getaline residues can be linked to the skeleton via heteroatoms or through different carbon atoms in the ring or ring system or through deputies. Preferred pyridine, inidazole, pyrimidine, purine, pyrazin or quinoline.

As alkylaminocarbonyl should be called substituted or unsubstituted, branched or unbranched C1-C10-alkylamine, such as, for example, methylamino, ethylamino, n-propylamino, 1 methylethylamine, n-butylamino, 1 methylpropylamine-, 2-methylpropylamine, 1,1-dimethylethylamine, n-pentylamine, 1 methylbutylamine, 2-methylbutylamine, 3 methylbutylamine, 2,2-dimethylpropylene, 1 ethylpropylamine, n-hexylamino, 1,1-dimethylbutylamino, 1,2-dimethylpropylene, 1 methylpentylamino, 2-methylpentylamino, 3 methylpentylamino, 4-methylpentylamino, 1,1-dimethylbutylamino, 1,2-dimethylbutylamino, 1,3-dimethylbutylamino, 2,2-dimethylbutylamino, 2,3-dimethylbutylamino, 3,3-dimethylbutylamino, 1 ethylbutylamine, 2-ethylbutylamine, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl-1-methylpropylamine, 1-ethyl-2-METI is propylamino, n-heptylamine, n-octylamine, n-nonylamine or n-decylamine. Preferred methylamino, ethylamino, n-propylamino, n-butylamino, and propylamino or-butylamino.

As Vice-described residues for R3suitable, for example, one or more such substituents as halogen, for example fluorine, chlorine or bromine, thio, nitro, amino, hydroxy, alkyl, alkoxy, alkenyl, alkenylacyl, quinil or other aromatic or other saturated or unsaturated non-aromatic ring or ring system. Preferred such alkyl residues, as C1-C6-alkyl, such as methyl, ethyl, propyl or butyl, aryl, such as phenyl, halogen, such as fluorine, chlorine, or bromine, hydroxy or amino.

R4means in the compounds of formulas I and II is hydrogen or substituted or unsubstituted, branched or unbranched C1-C10alkyl-.

As alkyl residues should be substituted or unsubstituted, branched or unbranched C1-C10alkyl chain, such as methyl, ethyl, n-propyl, 1-methylethyl, n-butyl, 1-methylpropyl-, 2-methylpropyl, 1,1-dimethylethyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, 1-ethylpropyl, n-hexyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl 1,3-dimethylbutyl, 2.2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl-1-methylpropyl, 1-ethyl-2-methylpropyl, n-heptyl, n-octyl, n-nonyl or n-decyl. The preferred methyl, ethyl, n-propyl, n-butyl,-propyl or-butyl.

As Vice-described residues for R4suitable, for example, one or more such substituents as halogen, such as fluorine, chlorine or bromine, thio, nitro, amino, hydroxy, alkyl, alkoxy, alkenyl, alkenylacyl, quinil or other aromatic or other saturated or unsaturated non-aromatic ring or ring system. Preferred such alkyl residues, as1-C6alkyl, such as methyl, ethyl, propyl or butyl, aryl, such as phenyl, halogen, such as chlorine, fluorine or bromine, hydroxy or amino.

Also aliphatic, saturated or unsaturated dinitrile can be used in the method according to the invention.

The method according to the invention is carried out predominantly in the pH value from 4 to 11, preferably from 4 to 9.

Next, the method is used is preferably from 0.01 to 10 wt.%, preferably from 0.1 to 10 wt.%, particularly preferably from 0.5 to 5 wt. nitrile. Depending on the type of nitrile can apply different amounts of the nitrile in the reaction. The least amount of nitrile applied NITRILES (C is the anhydrides) (equal amounts of between 0.01 to 5 wt.%), which are in equilibrium with the corresponding aldehyde and hydrocyanic acid, due to the fact that aldehydes, as a rule, are toxic to microorganisms or enzymes. The NITRILES, which are volatile, are used preferably in amounts of between 0.01 to 5 wt.%. At higher amounts of cyanhydrin, respectively nitrile, the reaction takes place with the slowdown. When the NITRILES, which have small or too small or almost no solvent properties, or NITRILES, which dissolve only in very small quantities in aqueous media, can be used in higher amounts than the above. To improve the conversion and yield of the reaction is preferably carried out at constant addition of nitrile. The product after the reaction to be allocated or may be continuously disposed in the bypass (shunt).

The method according to the invention is carried out at a temperature of from 0°C to 80°C, preferably from 10°C to 60°S, especially preferably from 15°C to 50°C.

Preferably in the method according to the invention are applied aromatic or heteroaromatic NITRILES, such as 2-phenyl-propionitrile, nitrile 2-hydroxyphenylacetic acid, 2-amino-2-phenyl-acetonitrile, benzonitrile, phenylacetonitrile, nitrile, TRANS.-cinnamic acid, 3-Tiantian or 3-cyanomethylation.

Under chiral NITRILES in the method according to the invention should be understood NITRILES, which consist of a mixture of 50:50 pure enantiomer or of any other mixture enrichment of one of the two enantiomers in the mixture. Examples of such NITRILES are 2-phenyl-propionitrile, nitrile 2-hydroxyphenylacetic acid, 2-amino-2-phenyl-atsetatnymi, 2-chloropropionitrile or 2-hydroxypropionitrile.

Under chiral carboxylic acids in the method according to the invention should be understood such that show enrichment of enantiomers. Preferably the method is achieved purity of the enantiomers at least 90%, preferably min 95%, particularly preferably min 98%, particularly preferably min 99%.

The method according to the invention allows the conversion of a large number of chiral or achiral NITRILES into the corresponding chiral or achiral carboxylic acids. In the way that you can convert at least 25 mmol of nitrile/h × 1 mg protein or at least 25 mol of nitrile/h × 1 g dry weight of the microorganism, preferably at least 30 mmol of nitrile/h × 1 mg protein or at least 30 mmol of nitrile/h × 1 g dry weight, preferably at least 40 mmol of nitrile/h × 1 mg protein or at least 40 mmol of nitrile/h × 1 g dry weight. Especially prepost is positive at least 50 mmol of nitrile/h × 1 mg of protein, or at least 50 mmol of nitrile/h × 1 g dry weight of the microorganism.

For the method according to the invention can be applied to the growing cells which contain the nucleic acid according to the invention, the nucleic acid constructs according to the invention or vectors. Can also be used and resting or open cells. Under the open cells should be understood, for example, such cells are processed, for example, the solvent is made permeable, or cells, which are enzymatic treatment, mechanical processing (e.g., pressure or ultrasound) or by other methods. Thus obtained crude extracts suitable for the method according to the invention. Also purified enzymes can be used for the method according to the invention. Also suitable immobilized microorganisms or enzymes, which preferably can be used in the reaction.

Obtained by the method according to the invention, a chiral or achiral carboxylic acids can be isolated from the aqueous reaction solution by extraction or by crystallization or extraction and crystallization. To this aqueous reaction solution is acidified with acid, for example, mineral acid (HCl or H2SO4or organic acid to pH, preferably below 2, and then extracted with organic is Kim solvent. Extraction for higher output can be repeated several times. As the organic solvent can be used in principle all solvents which, if necessary, after the addition salts form a phase with water. Preferred solvents are solvents such as toluene, benzene, hexane, tert-butyl methyl ether or ethyl acetate. Also the purification of the product may occur preferably by binding to the ion exchanger and subsequent elution of mineral acid or a carboxylic acid as HCL, H2SO4, formic acid or acetic acid.

After concentration of the aqueous or organic phase products, as a rule, can be allocated with good chemical purity, i.e. more than 90%. After extraction the organic phase with a product can only partially concentrated and the product to crystallize. For this purpose, the solution is cooled to a temperature of from 0°to 10°C. the Crystallization can be performed directly from the organic solution. Bicrystalline product can be loaded into the same or another solvent for re-crystallization. By submitting at least one crystallization purity of the enantiomers of the product may be increased depending on the position of the eutectic.

Chiral or Ahi is real carboxylic acid, however, immediately after acidification with acid at pH value below 2 can be bicrystalline from the aqueous reaction solution. To do this, mainly aquatic environment concentrated by heating and reduce its volume by 10 to 90%, preferably 20 to 80%, especially preferably 30 to 70%. Crystallization was carried out under cooling. For the preferred crystallization temperature from 0°to 10°C. for reasons of cost-saving preference for direct crystallization from aqueous solution. Also preferred treatment of chiral carboxylic acids by extraction and, if necessary, subsequent crystallization.

In the preferred process, the product obtained by the method according to the invention, isolated with a yield of from 60 to 100%, preferably from 80 to 100%, particularly preferably from 90 to 100% in terms used in the reaction of the nitrile. The isolated product has a high chemical purity >90%, preferably >95%, particularly preferably >98%. Next product in the chiral NITRILES, respectively chiral carboxylic acids has a high purity of the enantiomers, which can be even increased by crystallization.

Thus obtained product is suitable as starting material for organic synthesis in obtaining pharmaceuticals, AGR the chemicals or splitting of the racemate.

Examples

Isolation and heterologous expression of the nitA gene from Rhodococcus rhodochrous NCIMB 11216

Example 1: allocation of the nitA gene from Rhodococcus rhodochrous NCIMB 11216

To highlight the nitA gene from Rhodococcus rhodochrous NCIMB 11216 allocate cellular DNA, form a phage genomic Bank and verify it using oligonucleotide probe.

1.1 isolation of DNA from R.rhodochrous NCIMB 11216

For isolation of genomic DNA from Rhodococcus rhodochrous NCIMB 11216 according to the authors Sambrook and others, 1989, centrifuged 2×100 ml culture, which allow to stand one night (in the environment dYT, Sambrook, J., Fritsch, E.F. and Maniatis, T., 1989, Molecular cloning: a laboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press. Cold Spring Harbor, New York.) and granules resuspended in 8ml of 25 mm Tris/HCl, 25 mm add, 10% sucrose (weight/volume), pH 8.0. After handling treated crops lysozyme for 15 min at 37°With (additive 2 ml lysozyme, 100 mg/ml in 10 mm Tris/HCI, 0.1 mm add, pH 8.0) add 2 ml 10% (V/o) Na-lauroylsarcosinate and incubated with repeated stirring for 15 minutes at 65°C. thereafter serves CsCl with a final concentration of 1 g/ml, dissolved in 65°and after the addition of ethidium bromide to a final concentration of 0.4 mg/ml spending ultracentrifugation in the rotor (Sorvall T1270, 83500 g, 48 h, 17°). The chromosomal DNA band is removed under UV light, deleteroute for 2 hours in THE 10.1 (10 mm Tris/HCl, 1 mm add, pH 8.0) and extracted 3 times with a solution of phenol (saturated e is tion of 10 mm Tris/HCl, pH 8). After that, the DNA is still 3 times deleteroute in THOSE 10.01 (10 mm Tris/HCl, 0.1 mm add, pH 8.0) and stored at 4°C. Thus receive Probl,5 ml of DNA solution with a concentration of approx. 500 mcg/ml

1.2 Obtaining phage gene Bank of DNA from R.rhodochrous NCIMB 11216

As a vector for genomic Bank applied the phage I-RESIII: This vector substitution contains lux-operon as part of homogentisate that allows visual monitoring of the background by bioluminescence and integrated res("resolution") - the site of TP and replication functions n pTW601-1, so the vector can be transformed into a strain with the corresponding transposase in Autonomous replicating plasmid (Altenbuchner, 1993, A new I RES vector with a built-in Tn1721-encoded, excision system, Gene 123, 63-68).

1.2.1 Selection 1-RESIII-DNA (according to the authors Sambrook and others, 1989)

From prodelannoj culture night .coliTAP90 (LB0, Sambrook and others, 1989, 10 mm MgSO4, and 0.2% maltose (weight/vol.)) centrifuged 1010cells and granular resuspended in 3 ml of SM-phage buffer (50 mm Tris/HCl, 100 mm NaCl, 8 mm MgSO4, 0,01% (weight/vol.) gelatin). After exposure through a 1.5×108a 1.5×109plaque-forming units (pfu) I RESIII-phage lysate for 20 min at 37°the reaction mixture is poured into a two-liter flask of Erlenmayer to 500 ml LB0, 10 mm MgSO4, and 0.2% maltose. In General, 4 the reaction mixture is stirred for from 9 to 2 hours at 37° With up until lysis of the cells do not become prominent, and full of lysis in each flask serves 10 ml of chloroform, and stirred for further 30 minutes at 37°C. Cellular nucleic acid digesting medium supply DN-ASE and the PH is basic (1 μg/ml) for 30 minutes at room temperature. Then to each reaction mixture serves 29,2 g NaCI, dissolve, centrifuged 10 minutes at 8300 g and the supernatant is mixed with 10% PEG 6000. For subsequent spooling phage reaction mixture is stirred over night at 4°and then centrifuged for 15 minutes at 14000 g. After drying, the granulate it loads in 5 ml of SM buffer, mixed with 5 ml of chloroform and centrifuged for 15 minutes at 3000 g. The aqueous phase with phages are collected, mixed with 0.75 g/ml CsCl and after complete dissolution centrifuged within 24 hours (Sorvall rotor T1270, 98400 g, 48 h, 17°). The visible band of the phage isolate and deleteroute two times in 50 mm Tris/HCl, 10 mm NaCl, 10 mm MgCl2, pH 8.0. After the addition of 20 mm etc, 50 µg/ml proteinase K and 0.5%sodium dodecyl sulfate occurs incubation for 1 hour at 65°C. Then the reaction mixture was extracted once with phenol (saturated with 10 mm Tris/HCl, pH 8), once with phenol (saturated with 10 mm Tris/HCl, pH 8) / chloroform (50/50 vol./about.) and once with chloroform. After that, the DNA deleteroute 3 times THE 10.1 and once THOSE 10.01 determine is in the title E.coliTAP 90 (see 1.2.3) and store I-RESIII-DNA at 4°C.

1.2.2 DNA Cloning in I-RESIII-vectors

For cloning genomic R.rhodochrous NCIMB 11216 DNA fragment first dissect the I-RESIII fragments, while I-RESIII-DNA in a volume of 100 μl of 2 μg digested with 20 U of BamHI for 5 hours at 37°C. After extraction with phenol (saturated with 10 mm Tris/HCI, pH 8) / chloroform (50/50 vol./vol.), precipitation with isopropanol and washing by 70% ACC. 100% ethanol (pre-cooled to 20° (C) DNA was dissolved in TE 10.01 and processed with 20 units Sall (5 hours at 37°). Again make extraction with phenol/chloroform, precipitation with isopropanol, washing and dissolving in THOSE 10.01.

To obtain genomic DNA fragment after starting the kinetics of time for the party enzymes - 10 µg genomic DNA partially digested in 100 μl of the mixture for 5 minutes using a 0.5 .Su3l. After electrophoretic separation by 0.8%agarose gel with a low melting point allocate slice sections from 8 to 14 type and according to Parker & Seed (1980) out of the gel. Genomic DNA fragment are ligated overnight at 16°using I-RESIII-shoulders.

After that ligation mixture packaged in vitro fagbemi extracts that dissect according to the "packaging extract donor" E.coli BHB 2688 ("freeze thaw lysate", FTL, Sambrook et al., 1989) and according to the "prehead donor" E.coli BHB 269 ("sonicated extract", SE, Sambrook et al., 1989). Packaging 5 ál ligation mix mix 7 ál of buffer A (20 mm Tris/HCl, 3 mm MgCl2, 1 mm etc, 0,05% β-mercaptoethanol, pH 8.0), 7 μl of buffer M1 (6,mm Tris/HCl, 33 mm spermidine, 100 mm putrescine, 17,8 mm ATP, and 0.2% β-mercaptoethanol, 20 mm MgCl2, pH 8), 15 ál of SE and 10 ál of FTL and incubated for 1 hour at room temperature. Then add 500 μl of SM buffer and 1 drop of chloroform, the reaction mixture was stirred, centrifuged and stored at 4°C.

To determine the titer of the obtained phage gene Bank infect a strain of E. coli TAR 90 (Patterson & Dean, 1987). To do this, logarithmically growing cells (grown in LB0, 10 mm MgCl2and 0.5% maltose) incubated by 100 μl of various dilutions of phage or packing lysate in SM-buffer for 30 minutes at 37°C. the Reaction mixture is mixed then with 3 ml heated to 42°C top agar (0.8% bacto-agar, mm MgCl2and 0.5% maltose). Briefly stirred, and placed on LB0-analnye plates with 10 mm MgCl2(preheated to 37°). After 12-16 hours of incubation at 37°count plaques to determine the titer. The titer obtained Bank contains about 4×105plaque-forming units/ml

1.2.3 Transformation of recombinant I-RESIII phage to plasmid

The obtained recombinant I-RESIII phage transformed into the E. coli strain HB 101 F′[::is n1739l], who is transposon TP with genome resolvase under the control of the fac-promoter (Altenbuchner, 1993, see above), in the Autonomous replicating plasmid. This strain for infection is grown in 5 ml LB0with 10 mm MgCl2and 0.5% maltose to OD600from 0.6 to 0.8 and 100 µl infect him with the appropriate number of phage lysate for 30 minutes at room temperature. The mixture is placed in a 5 ml pre-heated dYT, 1 mm isopropyl-β-thiogalactopyranoside for 1 hour at 37°centrifuged, resuspended in the reverse flow and the cells placed on the agrarian plate dYT with 100 μg/ml kanamycin and incubated overnight at 37°C.

For visualization of cells nepravishta I-RESIII-molecule still contains the initial fragment of homogentisate with lux-operon and together with it does not contain genomic insert (von bantayev), LPS induce for 3 hours at 30°and bioluminescense cell count in the dark. Background gene Bank contains the fraction of luminous cells in 13%.

1.3. Screening of the gene nitrilase nitA from R.rhodochrous NCIMB 11216

Recombinant I-RESIII-phages that contain chromosomal DNA fragments from the genome nitrilase of R.rhodochrous NCIMB 11216, identified by hybridization of phage plaques oligonucleotide probes

"nit1lower", with the sequence: 5′-TGGAA(AG)TG(CT)TCCCA(AG)CA-3′, Kobayashi, M., Komeda, H. Yanaka, N., Nagasawa, T. and Yamada, H. (1992) Nitrilase from Rhodococcus rhodochrous J1. Kobayashi, M., Izui, H., Nagasawa, T. and Yamada, H. (1993) Nitrilase in biosynthesis of the plant hormone indole-3-acetic acid from indole-3-acetonitrile: Cloning of the Alcaligenes gene and site-directed mutagenesis of cysteine residues.

The sequence of the oligonucleotide identify from canned stretch of amino acid sequences with putative catalytic residue (Kobayashi and others, J. Biol. Chem. 267, 1992, 20746-20751 und Proc. Natl. Acad. Sci. USA, 90, 1993, 247-251). This motif can be found in the known DNA sequences of the gene nitrilase of strains of Rhodococcus rhodochrous J1 (GenBank ACE. # D11425) R.rhodochrous K22 (GenBank ACE. #D12583).

1.3.1 transfer of DNA and hybridization

5 agrarian plates in total 2500 plaques, which receive as described for determination of titer in section 1.2.3, 1 minute impose nalanie round membrane. The membrane is placed on 2×5 min side plaques up on the filter paper with denaturing solution (1.5 M NaCl, 0.5 M NaOH) and then 2×5 min on filter paper with denaturing solution (0.5 M Tris/HCl, 1.5 M NaCl, pH 7.5). Then briefly washed in 50 mm NaCl, dried, and the DNA is fixed for 30 minutes at 120°C.

For hybridization membranes pre-incubated by 50 ml hybridizing buffer for 2 hours at 37°and then during the night hybridizer in 12 ml hybridizing buffer at 37°using 10 pmol32 R-bulleted oligonucleotide. Oligonucleotide labeled in 30 μl mixture with 80 MX (g32P)-ATP by 10 units of T4-polynucleotide kinase and then separated from the excess (g32P)-ATP by gel-filtration using Sephadex G-25.

After hybridization nalanie membrane was washed with 1×5 minutes at room temperature by 0.5 g/l NaCl, 8.8 g/l of sodium citrate (2 × SSC), 0.1% sodium dodecyl sulfate and 2×15 minutes at 32°by 0.125 g / 1 l of NaCl, 2.2 g/l sodium citrate (0,5×SSC), 0.1% sodium dodecyl sulfate and exhibit for 5 days in a film cassette with the reinforcing film with x-ray film.

1.3.2 Identification and sekvenirovanie nitA gene

In total, identify 3 positive clones, two of which have full nitA gene fragment and one has a full nitA gene. Positive plaques secrete, incubated for 2 hours at room temperature in 0.5 ml of SM buffer and after addition of 2 drops of chloroform stored at 4°C. Resultwise after transformation of recombinant I-RESIII-phage full nitA-gene plasmids (see 1.2.3) denote pDHE 6 (figure 1 shows pDHE 6 to 12 thousand poganovo fragment Rhodococcus rhodochrous NCIMB 11216 ) and restriction Carteret region nitA gene using Southern-gibridizatzii oligonucleotide probe "nit1lower". Pvul fragment with 1.5 thousand BP with a total nitA-genome handle fragment maple and subcloning about botany in EcoRV pBluescriptSK + (pDHE 7 Fvul-fragment of 1.5 thousand BP from genomic fragment of genomic Bank 12 thousand BP from Rhodococcus rhodochrous NCIMB 11216 in pDHE6, figure 2). After further sublimirovanny overlapping fragments pDHE 7 HindIII (vector) IEcoRI, Kpnl/Xhol, EcoRV/BamHl and Apal/EcoRI (Vektor), appropriately digested pBluescriptSK+ PvuI fragment serverinput method authors Sanger and others (Proc. Natl. Acad. Sci. USA 74, 1977, 5463-5467) using a double-strand sequence. The reaction sekvestirovanija is carried out using a commercially available kit sekvestirovanija also available on the market seed "universal"and "reverse" (Vieira &Messing, Gene, 19, 1982: 259-268). Defined for Pvul fragment 1.5 thousand BP DNA sequence presented in SEQ ID NO:1. Allocated amino acid sequence presented in SEQ ID NO:2.

2 Heterologous expression of the nitA gene from R.rhodochrous NCIMB 11216 in E. coli and purification of recombinant protein nitrilase

For cloning in the expression vector amplified nitA gene from R.rhodochrous NCIMB 11216 from codon translational the start codon translational stop. For this purpose apply seed

"nit Ndel" (upper) with the sequence

5′-TATATATCATATGGTCGAATACACAAACA-3′

and

"nit Hindlll" (lower) with the sequence:

5′-TAATTAAGCTTCAGAGGGTGGCTGTCGC-3′

in which on 5′-nitA-the end of the above perehlestyvajushchy with translational start Ndel-the cutting location and on 3′-nitA-end, perehlestyvajushchy with a stop codon Hindlll-IU is the cutting. This pair of amplified seed nitA gene from pDHE 7 with Pwo polymerase in 40 μl of reaction mixture of 8 nmol seed, 100 g pDHE 7-Template and 2.5 units of Pwo in 10 mm Tris-HCl, pH 8.85, 25 mm KCl, 5 mm (NH4)SO4, 2 mm MgSO4, 0.2 mm dATP, 0.2 mm dTTP, 0.2 mm dGTP, 0.2 mm dCTP under the following conditions:

Denaturing for 3′ 94°C;

25 cycles with denaturing for 1′ 93°With, storage of seed for 1′ 30′′ 48°and polymerization for 1′ 30′′ 72°C;

The final polymerization for 5′ 72°C.

Received nit-PCR fragment was then purified, digested with Ndel/HindIII, integrate into similarly digested vector molecules pJOE 2702 (Volff and others, Mol. Environ., 21, 1996: 1037-1047) and resultwise plasmids indicate pDHE 17 (2: pDHE 17c nitA in adulruna L-ramnose the vector expressi pJOE 2702). Due to integration through Ndel/HindIII nitA gene is plasmid pDHE 17 under the transcriptional control obtained in pJOE 2702 promoter rhapthat comes from operon L-ramnose rhaBAD in E. coli (Egan & Schleif, Mol. Biol. 243, 1994: 821 - 829). Transcription termination nitA gene and translational initiation of transcription occurs through the vector sequence (see Volff and others, 1996). After transformation pDHE 17 in E. coli JM 109 nitA gene from R.rhodochrous NCIMB 11216 can be induced by additive L-ramnose.

For purification of recombinant protein NITR the manholes using imidazole affinity chromatography nitA gene, in addition, merge with 3′sequence for C-terminal His6-motif, and for amplifying the nitA gene, which is carried out under the above conditions, along with the 5′-seed "nitNdel" (upper) used a modified 3′-seed without stop codon with the sequence 5′-CGAGGGTGGCTGTCGCCCG-3′ and the resulting PCR fragment is integrated into the modified pJOE 2702-vector, which contains behind BamHI-designated cutting sequence [SAT]6Tja. After BamHI-digestion, processing of maple and Ndel-digestion of the vector Ndel-cut nitA-Pwo-amplificat merge legatia 3′-end by "blunt ends" in the reading frames with sequence His6-the motive and the resulting plasmids indicate pDHE 18.

For heterologous expression in laboratory scale sow JM 109 (pDHE 17) of the culture temperature of 37°1:200 in 50 ml of dYT environment (see Sambrook and others, 1989) with the help of 0.2% L-ramnose and culture for 8 hours at 30°cultivated in vibrating water bath during incubation. After the cells are washed once in 50 mm Tris/HCl, pH 7.5, in accordance with OD60010 resuspending in the same buffer and by ultrasonic treatment reveals. Over JM 109 (pDHE 18) is produced by the same procedure. The protein obtained by ultrasonic treatment, clarified by centrifugation of the crude extract compared to Nein zirovanii control determines SDS-polyacrylonitriles. Under these conditions, the induction of the share nitrilase in the total protein is for JM 109 (pDHE 17) and JM 109 (pDHE 18) for each 30%.

For cleaning nitrilase with His6the motive of JM 109 (pDHE 18) cells washed in 50 mm Tris/HCl, pH 7.5, resuspended respectively pribl OD600/ml and receive extracts press French Press (2 × 20000 kg/cm2). After purification of the extract by centrifugation for 30 minutes at 15000 g do the cleaning using the QIAexpress-Ni2+-NTA (QIAGEN). To 1 ml of crude extract using 1 ml of the matrix, which result in equilibrium with 20 mm Tris/HCl, pH 7.5. After loading the column is washed with 5 column volumes of 20 mm Tris/HCl, 300 mm NaCl, 40 mm imidazole, pH 7.0 and elute with 20 mm Tris/HCl, 300 mm NaCl, 300 mm imidazole, pH 7.5. Purity determined by gel-electrophoretic method, the thus obtained protein nitrilase is >90%. After two dialysis in 50 mm Tris/HCl, 0.1 mm DTT, 0.5 M (NH4)2SO4, pH 7.5 purified nitrilase can be stored at -20°C.

For crude extracts for the conversion of 2-benzonitrile in benzoic acid define 2 units/mg and purified by QIAexpress-Ni2+-NTA NITRILES with His6-motive when the concentration of the enzyme in 50 lhs/ml 11 units/mg One unit corresponds to the production of 1 µmol of benzoic acid when the initial concentration is AI benzonitrile 10 mm, 30°C and pH 7.5. The transformation of 2-benzonitrile in benzoic acid by crude extract nitrilase occurs in 50 mm Tris/HCl, pH 7.5, transformation with purified nitrilase in 50 mm Tris/HCl, pH 7.5, 0.1 mm of dithiothreitol. The formation of benzoic acid is determined using VSGH (RP18 column, 250×4, the solvent 47% methanol, 0.3% of N3PO4,).

Similar to the above example is subjected to the interaction of a number of NITRILES and determine the conversion.

Strains of E. coli JM 109 (pDHE 17, respectively pDHE 18) convert various NITRILES. To do this, cells are grown in 250 ml LB/Amp-medium + 2 g/l ramnose at 30°C and 200 rpm for 9 hours. Harvest cells removed by centrifugation (20 minutes, 4°C, 5000 rpm). Cells resuspended in 10 mm phosphate buffer with a pH of 7.2, so that the concentration of biomass is roughly 2 g biomass/l 150 µl of cell suspension is placed in a pipette in deepening microtitre plate the plate is then centrifuged. The supernatant is sucked off and the cell granules washed twice with Na2HPO4(1.42 g/l in Finacle, pH of 7.2). After another stage of centrifugation of the cell granulates resuspending in substrate solution (150 μl). Every 12 a number of microtitre plates mixed with the substrate. As a control, we take the number of substrate solution without cells. Microtitre the new plates incubated at 30° C and 200 rpm for 1 hour in vibration incubator. After that, the cells centrifuged and the supernatant determine the amount of NH4-ions using the Biomek instrument. The measurement is carried out at 620 nm against the reference curve, which was constructed with different solutions of NH4OH. In experiment 1 (see Fig. 3, table 1) as the substrate, apply the following substrates: benzonitrile (=1), 3-hydroxypropionitrile (=2), 2-methylglutaronitrile (=3), 4-chloro-3-hydroxybutyrate (=4), malonodinitrile (=5), crotononitrile (=6), germanytel (=7), dinitrile octandiol acid (=8), mevalonate (=9), aminocaproate (=10), 3,4-dihydroxybenzoate (=11), 3,5-dibromo-4-hydroxybenzonitrile (=12), 3-cyanopyridine (=13), 4-bromobenzylcyanide (=14), 4-chloraniline (=15), 2-phenylbutyramide (=16), 2-chloraniline (=17), 2-pyridylacetonitrile (=18), 4-forbindelsen (=19), 4-methylbenzonitrile (=20), benzylcyanide (=21). In experiment 2 (see figure 4, table 2), which is carried out similarly to experiment 1, use the following substrates: 2-phenylpropionitrile (=1), mandelonitrile (=2), 2-amino-2-phenylacetonitrile (=3), 2-hydroxypropionitrile (=4), 3,3-dimethoxypropionate (=5), 3-Tiantian (=6), 3-cyanomethylation (=7), benzonitrile (=8), propionitrile (=9), nitrile, TRANS-cinnamic acid (=10), 2-hydroxy-4-phenylbutyramide (=11), dinitrile 3-phenylglutaric acid (=12), fumaronitrile (=13), gluta onitrile (=14) valeronitrile (=15).

Table 1
1benzonitrile0,4051
23-hydroxypropionitrile0,1785
32-methylglutaronitrile0,4758
44-chloro-3-hydroxybutyrate0,1208
5malonodinitrile0,1208
6crotononitrile0,4946
7germanytel0,1517
8dinitrile octandiol acid0,4548
9pilonidal0,1569
10aminocaproate0,1236
113,4-dihydroxybenzoate0,1569
123,5-dibromo-4-hydroxybenzonitrile0,1624
133-cyanopyridine0,2393
144-bromobenzylcyanide0,5213
154-chloraniline0,4830
162-phenylbutyramide0,1376
172-chloraniline0,4530
182-pyridylacetonitrile0,1222
194-forbindelsen0,2361
204-methylbenzonitrile0,4326
21benzylcyanide0,2755

Table 2
12-phenylpropionitrile0,0000
2mandelonitrile0,0000
32-amino-2-phenylacetonitrile0,0000
42-hydroxypropionitrile0,0000
53,3-dimethoxypropionate0,1466
63-cyanothiophene1,9038
73-cyanomethylation0,9949
8benzonitrile1,9518
9propionitrile0,4135
10nitrile TRANS-cinnamic acid2,2509
112-hydroxy-4-phenylbutyramide0,0000
12dinitrile 3-phenylglutaric acid0,0000
13fumaronitrile the 2,2510
14glutaronitrile2,0809
15valeronitrile1,9218

1. The sequence of nucleic acids encoding a polypeptide with the activity nitrilase selected from the group including

a) the sequence of nucleic acids represented in SEQ ID N0:1 sequence,

b) nucleic acid sequences which, as a result of degenerated genetic code, are shown in SEQ ID NO:1 nucleic acid sequences.

2. A protein with the amino acid sequence encoded by the sequence of nucleic acid according to claim 1 and having the activity nitrilase.

3. The protein according to claim 2 with the amino acid sequence encoded by represented in SEQ ID NO:1 sequence.

4. The construct of nucleic acids for gene expression nitrilase sequence SEQ ID NO:1 containing the sequence of the nucleic acid according to claim 1, and a sequence of nucleic acids associated with one or more regulatory signals.

5. The expression vector containing the series is here nucleic acid according to claim 1 or the nucleic acid construct according to claim 4.

6. The method of obtaining chiral carboxylic acids, characterized in that the NITRILES of the formula I

in the presence of the amino acid sequence according to claim 2 or 3, or growing, resting or open recombinant microorganism, preferably selected from the group comprising bacteria of the species Escherichia, Rhodococcus, Nocardia, Streptomyces, or Mycobacterium containing the sequence of the nucleic acid according to claim 1, the nucleic acid construct according to claim 4 or a vector according to claim 5, converted into carboxylic acids of General formula II

and convert at least 25 mmol of nitrile/h·mg protein or at least 25 mmol of nitrile/h·mg dry weight of the microorganism and the substituents and variables in formulas I and II have the following meanings:

n is 0 or 1;

m is 1, 2 or 3, and when m>2 between two adjacent aromani carbon may have one double bond;

p is 1;

A, b, D, and E denote, independently of one another CH, N or CR3;

N means O, S, NR4, CH or CR3if n=0 or CH, N or CR3if n=1;

moreover, two adjacent remnant of a, b, D, E or N together may form a different substituted or unsubstituted aromatic, saturated or partially saturated ring containing o is 5 to 8 atoms in the ring, which may contain one or more heteroatoms such as O, N or S, with no more than three residues a, b, D, E or N represent a heteroatom;

R1means hydrogen, substituted or unsubstituted, branched or unbranched1-C1alkyl, C1-C10alkoxy, aryl, hetaryl, or C1-C10alkylamino, hydroxyl, halogen or amino;

R2means substituted or unsubstituted, branched or unbranched1-C1alkyl, C1-C10alkoxy, aryl, hetaryl, or C1-C10alkylamino, hydroxyl, amino;

R3means hydrogen, substituted or unsubstituted, branched or unbranched C1-C10alkyl, C1-C10alkoxy, aryl, hetaryl or1-C10alkylamino, hydroxyl, halogen or amino;

R4means hydrogen, substituted or unsubstituted, branched or unbranched1-C10alkyl.

7. The method according to claim 6, characterized in that the process is conducted in an aqueous reaction solution at a pH value between 4 and 11.

8. The method according to PP or 7, characterized in that the nitrile turn in an amount of from 0.01 to 10 wt.%.

9. The method according to one of p-8, characterized in that the process is carried out at a temperature from 0 to 80°C.

10. The method according to one of p-9, characterized in that the chiral carboxylic acid is obtained by extraction or by crystallization or extraction and crystallization from the reaction solution with the release of from 60 to 100%.



 

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