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WO2004067738A2 - Nitrile hydratases provenant de rhodococcus erythropolis et leur application - Google Patents

Nitrile hydratases provenant de rhodococcus erythropolis et leur application Download PDF

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Publication number
WO2004067738A2
WO2004067738A2 PCT/EP2004/000338 EP2004000338W WO2004067738A2 WO 2004067738 A2 WO2004067738 A2 WO 2004067738A2 EP 2004000338 W EP2004000338 W EP 2004000338W WO 2004067738 A2 WO2004067738 A2 WO 2004067738A2
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nucleic acid
nitrile
date
alkyl
seq
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WO2004067738A3 (fr
Inventor
Christoph Syldatk
Pedro Brandao
Alan T Bull
Stefan Verseck
Jeannette Erfurth
Stefan Buchholz
Steffen Osswald
Karlheinz Drauz
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Evonik Operations GmbH
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Degussa GmbH
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    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
    • 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
    • C12N9/88—Lyases (4.)

Definitions

  • the present invention is directed to polypeptides exhibiting nitrile hydratase activity and the respective encoding nucleic acids. Furthermore, micoorganisms, plasmids and vectors comprising the polypetides are also embraced by this invention.
  • amino acid amides and (proteogenic or non-proteogenic) amino acids are key intermediates for the synthesis of pharmaceutical and agrochemical products.
  • amino acids can also serve as feed additives.
  • ⁇ -Amino nitriles are precursors for appreciated products which can easily be synthesized by Strecker chemistry leading to a racemic mixture of these compounds .
  • the nitriles can be converted subsequently into the corresponding amides or carboxylic acids by chemical saponification or with the help of enzymes.
  • the amides can be synthesized e.g. via a non- enantioselective nitrile hydratase and then converted to an amino acid by an enantioselective or non-enantioselective a idase.
  • nitriles into amides and/or carboxylic acids catalyzed by the action of enzymes or whole cell catalysts helps to avoid the formation of large amounts of salts, which are produced during the neutralization step necessary for chemical saponification of nitriles.
  • nitrile hydratases Enzymes converting nitriles to amides are called nitrile hydratases and belong to the group E.G. 4.2.1.84. They consist of ⁇ , ⁇ -subunits and may exist as multimeric polypeptides with up to 20 separable units (Bunch A. W. (1998), Nitriles, in: Biotechnology, Volume 8a,
  • the object of the present invention was to provide further polypeptides exhibiting nitrile hydratase activity, in particular those which were less prone to CN- inhibition compared to the ones already state of the art.
  • the enzymes should accept a broad range of nitriles and ⁇ -amino nitriles, preferably such with sterically demanding ⁇ -radicals.
  • Claims 1 to 3 protect nucleic acids encoding either the ⁇ - or ⁇ -subunits of a newly found nitrile hydratase which are subject to claims 4 to 6.
  • Microorganisms and plasmids or vectors are covered by claims 7 and 8, primers are referred to in claim 9 and claims 10 to 12 are directed to a method of producing amides from nitriles by enzymatic conversion.
  • Claim 13 provides a whole cell catalyst for the transformation of nitriles to acids.
  • One embodiment of the present invention relates to isolated nucleic acid selected from the group consisting of: i) at least one nucleic acid sequence selected from the group containing odd SEQ. ID. NO.s 1 - 36 or a fragment thereof; ii) a nucleic acid that has at least 70% homology to the sequences under i) or a fragment thereof; iii) a nucleic acid that hybridyses to a sequence under i) or ii) or its complementary sequences under stringent conditions comprise washing in 5X SSC at a temperature ranging from 50°C to 68 °C; iv) a nucleic acid obtained by: - a) mutagenizing a nucleic acid of i) , ii) or iii) ,
  • nucleic acid which encodes the protein detected in (e) , wherein said nucleic acid of (i) , (ii) , (iii) or (iv) encodes a polypeptide being part of an enzyme having nitrile hydratase activity.
  • the details of the nucleic acids advantageously provide access to substances which make it possible to assure an adequate amount of the enzymes necessary for an enzyme- based industrial process, as mentioned at the outset, for the production of e.g. amino acids or carboxylic acids or amides. Via known recombinant techniques (see below) it is possible, with the nucleic acids according to the invention, to recover high yields of the enzymes from fast- growing host organisms.
  • the gene sequences according to the invention can be used to produce mutants which may exhibit further improvements.
  • Said recombinant techniques with which those skilled in the art are sufficiently familiar (see below) , provide access to organisms which are capable of providing the enzyme in question in an amount adequate for an industrial process.
  • the rec-enzymes according to the invention are prepared by genetic engineering methods known to those skilled in the art (Sambrook et al. 1989, Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press; Balbas P. & Bolivar F. 1990, Design and construction of expression plasmid vectors in E. coli, Methods Enzymology 185, 14-37; Vectors: A Survey of Molecular Cloning Vectors and Their Uses. R.L.
  • Procedure for improving the enzymes according to the invention by mutagenesis methods are state of the art like e.g. saturation mutagenesis (A.R. Oliphant, A.L. Nussbaum, K. Struhl (1986) Cloning of random sequence oligonucleotides, Gene 44, 177-183) , random mutagenesis (R.C. Caldwell, G.F. Joyce (1992) Randomization of genes by PCR mutagenesis, PCR Methods Appl. 2, 28-33), recombination methods such as shuffling (W.P. Stemmer (1994) DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution, Proc. Natl. Acad.
  • nucleic acid sequence comprising a nucleic acid according to the invention and optionally: a) a polynucleotide encoding one or more recombinant protein (s) in heterologous expression systems (Hashimoto, Yoshihiro; Nishiyama, Makoto; Yu, Fujio; Watanabe, Ichiro; Horinouchi, Sueharu; Beppu, Teruhiko. Development of a host-vector system in a Rhodococcus strain and its use for expression of the cloned nitrile hydratase gene cluster. Journal of General Microbiology (1992), 138(5), 1003-10; Mizunashi, W.; Nishiyama, M.
  • nucleic acid fragments that comprises at least 70% of the total nucleic acid sequence of the nucleic acids according to the invention.
  • fragments comprise more than 75%, 80%, 85%, 9.0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99,5% of the total nucleic acid sequence of the nucleic acids according to the invention.
  • the present invention is directed to a polypeptide, encoded by a nucleic acid of the invention, being part of an enzyme having nitrile hydratase activity.
  • the sequences of these peptides are depicted in even SEQ. ID NO: 1 - 36. These sequences are either the ⁇ - (even Seq. 1 - 18) or the ⁇ -subunits (even Seq. 19 - 36) of nitrile hydratses.
  • an enzyme consisting of an ⁇ -unit and/or a ⁇ - unit encoded by a nucleic acid sequences selected from the group consisting of odd SEQ. ID. NOs. 1 to 18 and 19 to 36, respectively, and having nitrile hydratse activity is an object of the present invention.
  • Preferred are enzymes comprising an ⁇ - and a ⁇ -subunit combination encoded by nucleic acid sequences selected from the group consisting of 1/19, 3/21, 5/23, 7/25, 9/27, 11/29, 13/31, 15/33, 17/35, respectively, and having nitrile hydratase activity.
  • all combinations between the disclosed ⁇ - and ⁇ -subunits can be selected as long as a nitrile hydratase activity can be achieved.
  • One further embodiment of the ' instant invention is directed to a microorganism comprising one or more nucleic acids of the invention.
  • Embraced are either native or rec- microorganis s .
  • the respective native microorganisms are desposited according to the Budapest Treaty at the Deutsche Sammlung fur Mikroorgansimen und Zellkulturenj, Mascheroder Weg 4, 38124 Braunschweig.
  • the following table 1 depicts the microorganisms, their origins and deposit numbers
  • the rec-microorganisms into which the nucleic acids may be cloned are used for increasing and recovering a sufficient amount of the recombinant enzyme or polypeptide.
  • the relevant processes are well known to those skilled in the art (Sambrook et al. 1989, Molecular cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Balbas P. & Bolivar F. 1990, Design and construction of expression plasmid vectors in E. coli, Methods Enzymology 185, 14-37; and above-cited bibliography relating to recombinant techniques) .
  • the microorganisms used can be any of the organisms used for this purpose by those skilled in the art, e.g.
  • E. coli strains for this purpose, the following being very particularly preferred: E. coli NM 522, XL1 Blue, JM101, JM109, JM105, RR1, DH5 ⁇ , TOP 10 " or HB101.
  • the invention further relates to plasmids or vectors containing one or more of the nucleic acids according to the invention.
  • suitable plasmids or vectors are any of the variants available for this purpose to those skilled in the art.
  • Such plasmids and vectors can be found in Studier et al., Methods Enzymol. 1990, 185, 61-69, or in the brochures issued by Roche Biochemicals, Invitrogen, Novagen, Promega, New England Biolabs, Clontech or Gibco BRL.
  • Particularly preferred plasmids and vectors can be found in DNA cloning: a practical approach, Volume I-III, edited by D.M. Glover, IRL Press Ltd., Oxford, Washington DC, 1985, 1987;
  • Denhardt, D.T. and Colasanti, J. A survey of vectors for regulating expression of cloned DNA in E. coli.
  • Rodriguez, R.L. and Denhardt, D.T. (eds) Vectors, Butterworth, Stoneham, MA, 1987, pp. 179-204; Gene expression technology.
  • Goeddel, D.V. (eds) Methods in Enzymology, Volume 185, Academic Press, Inc., San Diego, 1990; Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989.
  • Molecular cloning a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
  • Plasmids with which the gene construct containing the nucleic acid according to the invention can very particularly preferably be cloned into the host organism are: pKK-177-3H (Roche Biochemicals), pBTac (Roche Biochemicals) , pKK-233-3 (Amersham Pharmacia Biotech) , pLex (Invitrogen) or the vectors of the pET (Novagen) or pUC- series like pUC18 or pUC19, respectively.
  • Modified plasmid pUC18/19 (Fig. 1 - plasmid map of pUC18/19 and the orientation of the nitrile hydratase ⁇ -, ⁇ - subunits, and orfP47K regarding the lac promoter) is exceedingly preferred.
  • a further feature of the invention concerns primers for the preparation of the gene sequences according to the invention by means of all kinds of PCR selected from the group comprising: 5'-GCC CGC ATA AGA AAA GGT GAA (SEQ. ID NO. 37); 5'-TCA AAC GGT CTG GTC GGT ATA (SEQ. ID NO. 38); 5'-TCT ACG ACA CCA CCG CCG AAA CT (SEQ. ID NO. 39); 5'-GCA TGA CGT ACC TCT CGT AGT ACG (SEQ. ID NO. 40); 5'-GAC CAT GAT TTC CAG TGT CC (SEQ. ID NO. 41);
  • They include the sense and antisense primers coding for the corresponding amino acid sequences.
  • primers can be obtained by methods known to those skilled in the art.
  • the primers according to the invention are identified by comparison with known DNA sequences or by translation of the contemplated amino acid sequences into the codon of the organism in question (e.g. for Streptomyces: Wright et al., Gene 1992, 113, 55-65). Common characteristics in the amino acid sequence of proteins of so-called superfamilies are also useful for this purpose (Firestine et al., Chemistry & Biology 1996, 3, 779-783) . Further information on this subject can be found in Oligonucleotide synthesis: a practical approach, edited by M.J.
  • the present invention concerns a method for preparing an amide comprising converting a nitrile into an amide using a polypeptide of the invention, preferrably in enantiomerically enriched form.
  • nitrile is an ⁇ -amino nitrile.
  • ⁇ -amino nitrile is a compound of formula (I)
  • R 1 is the ⁇ -radical of a proteinogenic or non-proteinogenic amino acid
  • R 2 is H, (C ⁇ -C 8 ) -alkyl, (C 2 -C 8 ) -alkenyl, (C 2 -C 8 ) -alkinyl, (C ⁇ -Cis) -aryl, (C 7 -C ⁇ 9 ) -aralkyl, (C 3 -C ⁇ 8 ) -heteroaryl, (C 4 -C 19 ) -heteroaralkyl, (C ⁇ -C 8 ) -alkyl- (C 6 -C ⁇ 8 ) -aryl,
  • phthaloyl or (C 7 -Ci9) -aralkyl if R 3 is H than R 4 can be OH, OR 2 , NH 2 , NHR 2 , NR 2 R 2 , CONHNH 2 or SO 2 R 1 with R 2 not being H.
  • Further groups can be depicted from Houben-Weyl, Synthese von Peptiden, Band 15/1, S. 46ff, 1974, Thiele.
  • the invention also provides a whole cell catalyst comprising a cloned gene for a nitrile hydratase and a cloned gene for an amidase, wherein both enzymes are tuned according to their turnover rates, using preferably a rec- amidase from Variovorax or Rhizobium huatlense and a nitrile hydratase of the invention (DE 10230756 or DE 10037115.9 and bibliography cited therein).
  • a rec- amidase from Variovorax or Rhizobium huatlense and a nitrile hydratase of the invention
  • Those skilled in the art are familiar with the preparation of such an organism (PCT/EP00/08473; PCT/US00/08159; see below for bibliography) .
  • the DNA fragments with the nitrile hydratase genes from different R. erythropolis strains were obtained by aplifying the ⁇ - and ⁇ -subunits and the open reading frame P47K.
  • the orfP47K is presumably responsible for a active expression of the NHase.
  • Used primer pairs were derived from sequences of R. erythropolis N-774 (Duran, R, Nishiyama, M, Horinouchi and S, Beppu, T. (1993) Characterization of nitrile hydratase genes cloned by DNA screening from Rhodococcus erythropolis.
  • Nitrile hydratase gene from Rhodococcus sp. N-774 requirement for its downstream region for efficient expression. Bioscience, Biotechnology, and Biochemistry 58(10) 1859-65).
  • the amplified DNA fragments were ligated in pUC18 or 19 plasmids, transformed in suitable microorganisms and subsequently sequenced.
  • Nitrile hydratases with an in-frame orientation regarding the lac promoter were used in heterologous expression in E. coli .
  • the enzymes in question can be used in free form as homogeneously purified compounds.
  • the enzyme can also be used as a constituent of an intact host organism or in conjunction with the digested host organism cell mass purified to any desired degree. It is also possible to use the enzymes in an immobilized, form (Bhavender P. Sharma, Lorraine F. Bailey and Ralph A. Messing, "Immobilinstrumente Biomaterialien -techniken und füren", Angew. Chem. 1982, 94, 836-852) .
  • the immobilization is advantageously effected by lyophilization (Dordick et al., J. Am. Chem. Soc. 1994, 116, 5009-5010; Okahata et al., Tetrahedron Lett.
  • a stringent hybridisation is provided if washing for 1 h with 1 x SSC (150 mM sodium chloride, 15 M sodium citrate, pH 7.0) and 0,1 % SDS (sodium dodecylsulfate) at 50 °C, preferably at 55 °C, and more preferably at 62 °C and most preferably at 68 °C and further preferably for 1 h with 0,2 x SSC and 0,1 % SDS at 50 °C, more preferably at 55 °C, most preferably at 62 °C and utmostly preferably at 68 °C leads to a ⁇ positive hybridisation signal can be monitored.
  • 1 x SSC 150 mM sodium chloride, 15 M sodium citrate, pH 7.0
  • SDS sodium dodecylsulfate
  • optically enriched (enantiomer-enriched, enantiomerically enriched) compounds are understood as indicating the presence of one optical antipode in a mixture with the other in a proportion of >50 mol%.
  • Proteinogenic amino acid is an amino acid as described in Beyer-Walter, Lehrbuch der organischen Chemie, S. Hirzel Verlag Stuttgart, 22nd edition, 1991, p. 822 et seq. However, corresponding non-proteinogenic ⁇ -amino acids, such as those listed e.g. in DE 19903268.8, are also embraced.
  • ⁇ nucleic acids' encompasses both single stranded or double stranded DNA and RNA.
  • Improved rec-enzymes are understood according to the Claims as meaning particularly rec-enzymes which are more active and/or more selective (in respect of the reaction) and/or more stable under the reaction conditions used.
  • the claimed protein sequences and the nucleic acid sequences also include sequences which have a homology (exclusive of natural degeneracy) greater than 80%, preferably greater than 90%, 91%, 92%, 93% or 94%, particularly preferably greater than 95% or 96% and very particularly preferably greater than 97%, 98% or 99% to one of these sequences, provided the mode of action or the purpose of such a sequence is preserved.
  • H denotes homology
  • X is the total number of nucleic acids/amino acids in the reference sequence
  • V is the number of different nucleic acids/amino acids in the sequence in question relative to the reference sequence.
  • the expression ⁇ nucleic acids coding for amino acid sequences' includes all sequences which appear possible according to the degeneracy of the genetic code.
  • a (C ⁇ -C 8 ) -alkyl radical in the context of the invention is understood as meaning a radical having 1 to 8 saturated C atoms, which can have any desired branchings.
  • the radicals methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec- butyl, tert-butyl, pentyl, hexyl etc. can be subsumed, in particular, under this group. This group may be substituted one ore more times by Hal, especially fluorine.
  • (C ⁇ -C 8 )-Alkoxy is a (C ⁇ -C 8 ) -alkyl group which is linked to the molecule in question via a oxygen.
  • (C ⁇ -C 8 )-Acyl is a residue embracing an (C ⁇ -C 8 ) -alkyl group which linked to the molecule in question via a carbonyl function.
  • (C ⁇ -C 8 ) -Alkyloxycarbonyl represents an (C ⁇ -C 8 )- alkoxy radical linked to the molecule in question via a carbonyl function.
  • a (C 2 -C 8 ) -alkenyl radical has the features mentioned for, the (C ⁇ C 8 ) -alkyl radical, where at least one double bond must be present within the radical.
  • a (C 2 -C 8 ) -alkinyl radical has the features mentioned for the (C ⁇ -C 8 ) -alkyl radical, where at least one triple bond must be present within the radical.
  • a (C 6 -C ⁇ 8 ) -aryl radical is understood as meaning an aromatic radical having 6 to 18 C atoms. This includes, in particular, compounds such as phenyl, naphthyl, anthryl, phenanthryl and biphenyl radicals. These radicals may be substituted one or more times by Hal, (C ⁇ -C 8 ) -alkoxy, (C ⁇ C 8 ) -alkyl.
  • a (C7-C 19 ) -aralkyl radical is a (C 6 -C ⁇ 8 ) -aryl radical bonded to the molecule via a (C ⁇ -C 8 ) -alkyl radical, like benzyl or Ph 2 CH, naphthylmethyl, fluorenylmethyl etc..
  • a (C 3 -C ⁇ 8 ) -heteroaryl radical designates a five-, six- or seven-membered aromatic ring system of 3 to 18 C atoms which contains heteroatoms such as e. g. nitrogen, oxygen or sulfur in the ring.
  • Radicals such as 1-, 2- or 3-furyl, such as 1-, 2- or 3-pyrrolyl, l-,2- or 3-thienyl, 2-, 3- or 4-pyridyl, 2-, 3-, 4-, 5-, 6- or 7-indolyl, 3-, 4- or 5-pyrazolyl, 2-,4- or 5-imidazolyl, acridinyl, quinolinyl, phenanthridinyl or 2-, 4-, 5- or 6-pyrimidinyl, in particular, are regarded as such heteroaromatics.
  • a (C-C ⁇ 9 ) -heteroaralkyl is understood as meaning a heteroaromatic system corresponding to the (C 7 -C ⁇ g) -aralkyl radical.
  • a (C 3 -C 8 ) -cycloalkyl radical consequently designates a radical of the group of cyclic alkyl radicals having 3 to 8 C atoms and optionally any desired branching.
  • the radicals cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl in particular are to be subsumed under this group.
  • a (C 3 -C 8 ) -cycloalkenyl radical is a (C 3 -C 8 ) -cycloalkyl radical with one or more double bonds in the ring system.
  • a (C ⁇ -C 8 ) -cycloalkinyl accordingly is a (C6-C 8 ) -cycloalkyl radical with a triple bond in the ring system.
  • Hal halogen
  • the medium used for the enrichment procedures and growth was chelate mineral medium (CMM; Heald SC, Brandao PFB,
  • Vitamin stock solution and trace element solution were added at 1 ml and 0.1 ml I -1 , respectively.
  • the vitamin stock solution contained (mg l "1 ) : 25, thiamine hydrochloride; 250, calcium pantothenate; 250, nicotinic acid; 0.5, biotin; 250, riboflavin; 5, p-aminobenzoic acid; 12.5, cyanocobalamin (B ⁇ 2 ) ; 500, folic acid; 500, pyridoxine hydrochloride (Be) •
  • the mixture was filter sterilised (0.2 ⁇ filter, Millipore, Bedford, USA) and stored at 4°C in the dark.
  • the trace element solution contained (mg per 100 ml -1 ): 158, Na 2 EDTA.2H 2 0; 4.7, NaMo0 4 -2H 2 0; 70, ZnSO"7H 2 0; 18, MnSO 4 * 4H 2 0; 16, FeSO 4 -7H 2 0; 4.7, CuSO 4 -5H 2 0; 5.2, CoCl 2 "6H 2 0.
  • the solution was stored at 4°C in the dark.
  • Solid media were prepared by adding 1.5% (w/v) agar (Agar No.l, Lab M, England) to liquid media before autoclaving. Isolations were routinely made at 30°C (Colquhoun et al. 1998) .
  • GYE glucose yeast extract
  • CMM CMM supplemented with 0.1% (v/v) of the relevant nitrile compound.
  • GYE contained (g l -1 ) : 10, glucose; 10, yeast extract; 15, agar.
  • Thiamine hydrochloride was added to autoclaved GYE to a final concentration of 4 mg l "1 from a filter sterilized stock (0.2 ⁇ m filter, Millipore) .
  • Nitrile-utilising organisms were recovered by directly plating deep-sea sediments, diluted in potassium phosphate buffer (0.1 M, pH 7.0) ⁇ onto CMM agar supplemented with the relevant nitrile compound.
  • Acetonitrile and benzonitrile were supplied at a final concentration of 0.1% (v/v) unless stated otherwise.
  • Batch enrichments were established by adding 0.1-0.5 g wet weight of sediment to 50 ml liquid medium containing a nitrile compound and incubating at the desired temperature with agitation (150 rpm) .
  • the primary enrichment culture (5 ml) was sequentially subcultured into 50 ml identical fresh medium at seven day intervals, at which time 0.1-ml aliquots were spread plated onto CMM agar containing the enrichment substrate.
  • a dispersion and differential centrifugation technique also was used to isolate nitrile-degrading organisms from deep-sea sediments.
  • Rhodococcus The following validated species of the genus Rhodococcus (Goodfellow M, Alderson G and Chun J (1998) Rhodococcal systematics: problems and developments. Antonie van
  • Leeuwenhoek 74: 3-20) were included as references in the taxonomic analysis: R. rhodochrous (type species) R. coprophiluS f R. egui, R . erythropolis R. fascians, R. globeruluS f R. marinonascens , R. - opacus, R. rhodnii F R. ruber; R. wratislavensis; together with R. luteus f R. percolatus and R. zopfii .
  • Genomic DNA from the bacterial strains was extracted and purified according to Pitcher et al. (Pitcher, D: G.; Saunders, N. A.; Owen, R. J. (1989) Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Letters in Applied Microbiology, 8(4), 151-6 ), followed by a phenol-chloroform extraction to obtain DNA of sufficient purity for amplification by the polymerase chain reaction (PCR) .
  • PCR polymerase chain reaction
  • PCR amplification was performed using a DNA Thermal Cycler model 9600 (Perkin-Elmer Cetus, Norwalk, CT, USA) under the following conditions: 100 ng template DNA, 10 ⁇ l reaction buffer (100 mM Tris-HCl, 15 mM MgCl 2 , 500 mM KC1, pH 8.3 at 20°C, 2.5 U Taq DNA polymerase (Roche, Mannheim, Germany) , 1 nM upstream primer, 1 nM downstream primer, 200 nM dATP, 200 nM dCTP, 200 nM dGTP, 200 nM dTTP (dNTPs supplied as a 10 mM stock mix, Promega, Madison, WI, USA) , and PCR water (Sigma) combined in a total volume of 100 ⁇ l.
  • reaction buffer 100 mM Tris-HCl, 15 mM MgCl 2 , 500 mM KC1, pH 8.3 at 20°C, 2.5 U Taq DNA polymerase (Roche
  • JR. erythropolis 870-AN019 cells were pre-cultivated in 50 ml CMM medium (Heald SC, Brandao PFB, Hardicre R and Bull AT (2001) .
  • CMM medium Heald SC, Brandao PFB, Hardicre R and Bull AT (2001) .
  • CMM "N ammonium sulphate addition
  • AN acetonitrile
  • cell centrifugation was made at 4000 rpm at 4°C.
  • cell cultures were prepared by inoculating 200 ml of CMM ⁇ N supplemented with 10 mM acetyl- valine-nitrile (Ac-ValCN) , lOmM glucose and 50 mg/L FeS04.7H20 in a 1000 ml Erlenmeyer flask, to a final optical density at 600 nm wavelength (OD 6 oo) of approximately 0,1 with sterile washed cells. Incubation was made at 30°C and 110 rpm for 28 hours.
  • nitrile hydratase activity of the cells was determined using both valine nitrile (ValCN) and Mipkan as substrates. Under these conditions the specific activity of the nitrile hydratase in strain 870-AN019 towards ValCN and Mipkan (methyl isobutyl amino nitrile) was respectively 1,6 and 0,6 ⁇ mol amide formed/min/mg cell dry weight (units/mg cdw) .
  • the DNA fragments of the different nitrile hydratase genes were amplified by PCR using chromosomal DNA from the corresponding bacterial strains (Tab. 1) . All primers (Tab. 2) were synthesized by MWG-Biotech (Ebersberg, Germany) .
  • Each reaction mixture contained 1 U Herculase DNA polymerase (Stratagene, Germany) , 10 nmol of each dNTP, 50 pmol of each primer and approx. 0.1-0.5 (g of genomic DNA in a final volume of 50 (1 with the appropriate buffer.
  • PCR amplification was performed using a programmable thermocyler (Master Cycler; Eppendorf, Germany) . Amplifications proceeds with selected primer pairs (see Tab. 2) derived from the DNA sequence of R. erythropolis (Duran, R, Nishiyama, M, Horinouchi and S, Beppu, T. (1993) 5 Characterization of nitrile hydratase genes cloned by DNA screening from Rhodococcus erythropolis.
  • PCR DNA polymerase chain reaction
  • the concentration of cyanide in biotransformation reactions was. measured -by__a_spectropho.tometric_ assay ⁇ using _a_ commercially available kit (Spectroquant® Cyanide, Merck) .
  • MeatiScation reference givisn by the DEPOSITOH Accession number given by the INTERNATIONAL DEPOSITARY AUTHORITY:
  • the microorganism identified under I. above was accompanied by;
  • the microorganism identified under L above was accompanied by.
  • the microorganism identified under I. above was accompanied by:
  • Ad ieEs M ⁇ scheroder eg l D-38124 Braunsohweig
  • the microorganism identified under L above was accompanied by.
  • microorganism identified under I above woo received by this ⁇ ntematioid Depository Authority ea (date of o iginDl depssit) and o request to convert (he originnl deposit to a deposit under die Budapest Tieaty "tra received by it ea (date of eseipt of request fer ⁇ oavESSoa .
  • the microorganism identified under L above was accompanied by:
  • microorganism identified under I above was received by t sa International Depository Authority ⁇ a (slate of original depssit) and a request to convert the original deposit to a deposit under the Budapest Treat ws recer/cd by It oa (date of receipt of re ⁇ e ⁇ for coaf eroka).
  • the microorganism identified under L above was accompanied by:
  • the microorganism identified under L above was accompanied by.
  • the microorganism identified under L above was accompanied by:
  • JDpgujsaAG • Accession number given by the
  • Ad ⁇ teGB Ro enbach ⁇ r Ctou ⁇ ss 4
  • the microorganism identified under L above was accompanied by;

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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Abstract

L'invention concerne des polypeptides présentant une activité nitrile hydratase et les acides nucléiques de codage respectifs provenant de Rhodococcus erythropolis. L'invention concerne également des micro-organismes, des plasmides et des vecteurs comprenant lesdits polypeptides.
PCT/EP2004/000338 2003-01-27 2004-01-17 Nitrile hydratases provenant de rhodococcus erythropolis et leur application Ceased WO2004067738A2 (fr)

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EP03001715 2003-01-27
EP03001715.6 2003-01-27

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WO2004067738A3 WO2004067738A3 (fr) 2004-12-16

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005090577A1 (fr) * 2004-03-20 2005-09-29 Brain Biotechnology Research And Information Network Ag Expression de nitrile hydratases dans un systeme d'expression a deux vecteurs
WO2005090394A3 (fr) * 2004-03-20 2005-12-22 Degussa Nitrilhydratases tolerantes au cyanure
US8889358B2 (en) 2009-11-03 2014-11-18 Genetic Analysis As Methods of amplifying a target sequence of a 16S rRNA or 16S rDNA in a prokaryotic species
WO2018012011A1 (fr) * 2016-07-11 2018-01-18 三菱ケミカル株式会社 Procédé d'examen de l'intérieur de la cavité buccale
US11421214B2 (en) * 2019-01-31 2022-08-23 Dalian University Of Technology System based on a new nitrile hydratase for highly efficient catalytic hydration reaction of aliphatic dinitriles

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2840253B2 (ja) * 1988-07-06 1998-12-24 輝彦 別府 ニトリルヒドラターゼ活性を有するポリペプチドをコードする遺伝子ｄｎａ、これを含有する形質転換体によるニトリル類からアミド類の製造法
US5648256A (en) * 1990-02-28 1997-07-15 Nitto Chemical Industry Co., Ltd. Gene encoding a polypeptide having nitrile hydratase activity, a transformant containing the gene and a process for the production of amides using the transformant
AU627648B2 (en) * 1990-02-28 1992-08-27 Teruhiko Beppu Dna fragment encoding a polypeptide having nitrile hydratase activity, a transformant containing the gene and a process for the production of amides using the transformant
JPH099973A (ja) * 1995-06-27 1997-01-14 Chisso Corp ロードコッカス属細菌由来のニトリルヒドラターゼ遺伝子およびアミダーゼ遺伝子
FR2770214B1 (fr) * 1997-10-24 1999-12-31 Rhodia Chimie Sa Procede de preparation d'un benzamide halogene
US6294328B1 (en) * 1998-06-24 2001-09-25 The Institute For Genomic Research DNA sequences for strain analysis in Mycobacterium tuberculosis

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005090577A1 (fr) * 2004-03-20 2005-09-29 Brain Biotechnology Research And Information Network Ag Expression de nitrile hydratases dans un systeme d'expression a deux vecteurs
WO2005090394A3 (fr) * 2004-03-20 2005-12-22 Degussa Nitrilhydratases tolerantes au cyanure
JP2007537726A (ja) * 2004-03-20 2007-12-27 デグサ ゲーエムベーハー シアン耐性ニトリルヒドラターゼ
JP4868533B2 (ja) * 2004-03-20 2012-02-01 エボニック デグサ ゲーエムベーハー シアン耐性ニトリルヒドラターゼ
US8889358B2 (en) 2009-11-03 2014-11-18 Genetic Analysis As Methods of amplifying a target sequence of a 16S rRNA or 16S rDNA in a prokaryotic species
WO2018012011A1 (fr) * 2016-07-11 2018-01-18 三菱ケミカル株式会社 Procédé d'examen de l'intérieur de la cavité buccale
US11421214B2 (en) * 2019-01-31 2022-08-23 Dalian University Of Technology System based on a new nitrile hydratase for highly efficient catalytic hydration reaction of aliphatic dinitriles

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