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WO2005026338A1 - Alcool-dehydrogenases dotees d'une stabilite amelioree aux solvants et a la temperature - Google Patents

Alcool-dehydrogenases dotees d'une stabilite amelioree aux solvants et a la temperature Download PDF

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Publication number
WO2005026338A1
WO2005026338A1 PCT/EP2004/052095 EP2004052095W WO2005026338A1 WO 2005026338 A1 WO2005026338 A1 WO 2005026338A1 EP 2004052095 W EP2004052095 W EP 2004052095W WO 2005026338 A1 WO2005026338 A1 WO 2005026338A1
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Prior art keywords
seq
sequence
alcohol dehydrogenase
oxidation
enzyme
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PCT/EP2004/052095
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Inventor
Wolfgang Stampfer
Birgit Kosjek
Wolfgang Kroutil
Kurt Faber
Frank Niehaus
Jürgen Eck
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BASF Schweiz AG
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Ciba Spezialitaetenchemie Holding AG
Ciba SC Holding AG
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Priority to CA002535710A priority Critical patent/CA2535710A1/fr
Priority to EP04766745A priority patent/EP1664286A1/fr
Publication of WO2005026338A1 publication Critical patent/WO2005026338A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/002—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by oxidation/reduction reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • C12P7/26—Ketones

Definitions

  • the invention relates to biocatalysts showing alcohol dehydrogenase activity, their preparation, their use in the oxidation of secondary alcohols and/or the reduction of ketones, as well as nucleic acids coding for these alcohol dehydogenases and microorganisms transformed with nucleic acids coding for these biocatalysts and their use for producing the biocatalyst and for the oxidation of secondary alcohols and/or the reduction of ketones.
  • a disadvantage is that the strongly basic reaction conditions lead to undesired aldol-type side reactions (see K. G. Akamanchi, B. A. Chaudhari, Tetrahedron Lett. 38, 6925-8 (1997)). Substrates sensitive to basic conditions cannot be reacted without decomposition (see T. Ooi, Y. Itagaki, T. Miura, K. Maruoka, Tetrahedron. Lett. 40, 2137-8 (1999)). Asymmetric variants of the MPV-Red that employ chiral transition metal catalysts for enantioselective hydrid transfer have been tested only on few model substrates and resulted in preparatively inacceptably low stereoselectivities (see F. Touchard, M. Bernard, F.
  • biocatalytic methods have the advantage that they can be led under mild conditions, e.g. at room temperature and approximately neutral pH in aqueous media (see K. Faber, Biotransformations in Organic Chemistry 4 th Ed., Springer Verlag, , Heidelberg 2000; ISBN 3-540-61688-8).
  • An additional valuable property of biocatalysts is their normally high intrinsic stereoselectivity.
  • the desired reaction usually takes place without side reactions.
  • Biocatalytic redox processes on the basis of isolated alcohol dehydrogenases require the presence of expensive cofactors, such as NAD + /NADH or NADP + /NADPH. The recycling of these substrates is difficulty and expensive (see W. Hummel, Adv. Biochem. Eng.
  • the substrate concentrations are below 0.15 mol/l and the co-substrate concentrations below 3 % (v/v) (see K. Nakamura, Y. Inoue, T. Matsuda, I. Misawa, J. Chem. Soc. Perkin Trans. 1 1999, 2397-2402; and A. Goswami, R. L. Bezbaruah, J. Goswami, N. Borthakur, D. Dey, A. K. Hzarika, Tetrahedron: Asymmetry 2000., 11, 3701-9).
  • the invention relates to a biocatalyst, especially an enzyme, preferably in (at least partially) purified form, which biocatalyst has alcohol dehydrogenase activity and which can be obtained from Rhodococcus ruber.
  • this enzyme has unexpected and unique properties in comparison to other, known enzymes having the same type of activity.
  • the novel enzyme has high temperature stability and in addition is capable of maintaining its activity in the presence of high concentrations of organic solvents (such as aromatic or aliphatic hydrocarbons, e.g. toluene, hexane) other than the co-substrates in up to 95, preferably up to 98 % concentration (v/v).
  • organic solvents such as aromatic or aliphatic hydrocarbons, e.g. toluene, hexane
  • This especially allows for leading the oxidation or reduction reactions under conditions of high temperature and especially in the presence of high co-substrate concentrations (in the case of oxidation of alcohols, the presence of high ketone concentrations; in the case of reduction of ketones, the presence of high alcohol concentrations).
  • Fig. 1 Native gel analysis at different steps of the purification protocol of sec-alcohol dehydrogenase A. Lanes: Lane 1 - crude cell extract; lane 2: batch DEAE cellulose; lane 3: Phenyl Sepharose; lane 4: UNO Q6; lane 5: Blue Sepharose; lane 6: Superdex 200.
  • Fig. 2 SDS-PAGE analysis at different steps of the purification protocol of sec-alcohol dehydrogenase A.
  • Lanes Lane 1 - crude cell extract; lane 2: batch DEAE cellulose; lane 3: Phenyl Sepharose; lane 4: UNO Q6; lane 5: Blue Sepharose; lane 6: Superdex 200; lane 7: low molecular weight standard.
  • the invention especially relates to an biocatalyst with alcohol dehydrogenase activity, especially an enzyme, preferably in (at least partially) purified form, which has alcohol dehydrogenase activity, especially stereospecific alcohol dehydrogenase activity in the oxidation of secondary alcohols or the reduction of ketones; and which can be obtained from a naturally occuring microorganism, especially Rhodococcus, especially Rhodococcus ruber DSM 44541 -called Rhodococcus ruber DSM 14855 hereafter, in accordance with the number of the Budapest Treaty deposit (see below), see WO 03/078615 which is enclosed here by reference especially regarding the deposit of said microorganism.
  • biocatalyst (or "enzyme") according to the invention is preferably present or used in purified form.
  • the invention also relates to a corresponding biocatalyst with alcohol dehydrogenase activity, obtained by recombinant technology (recombinant biocatalyst).
  • a further embodiment of the invention relates to the use of a biocatalyst with alcohol dehydrogenase activity according to the invention in the oxidation of secondary alcohols and/or the reduction of ketones (process of or according to the invention, hereinafter).
  • the process of the invention can be used especially for separating mixtures of stereo- isomers with respect to a center of chirality by kinetic resolution, if one stereo-isomer of an alcohol is specifically oxidized, or for the stereo-specific production of secondary alcohols having a specific chiral form from ketones.
  • Still a further embodiment relates to nucleic acids coding for such a biocatalyst with alcohol dehydrogenase activity, especially recombinant nucleic acids.
  • Another embodiment relates to microorganisms transformed with a nucleic acid coding for such a biocatalyst with alcohol dehydrogenase activity.
  • the invention relates to the use of the mentioned microorganisms, especially host cells, in the production of said biocatalyst with alcohol dehydrogenase activity, as well as their use in the catalysis of the oxidation of secondary alcohols or the reduction of ketones, especially as shown in reaction scheme (A) below.
  • a further aspect of the invention relates to a (preferably isolated) polynucleotide, namely a DNA , or a (preferably isolated) recombinant polynucleotide comprising a DNA, each coding for a biocatalyst according to the invention, namely an enzyme, showing alcohol dehydrogenase activity, preferably as described above and below and in the examples as preferred, according to the invention which is characterized by the following sequence (SEQ ID NO: 47)
  • versions of the polynucleotide or recombinant polynucleotide are comprised where conservative nucleic acid replacements that lead to no change in the resulting amino acid sequence are present and/or such changes in the base sequence that lead to amino acid replacements in the amino acid sequence resulting from translation thereof for 5 % or less, preferably 3 % or less of the total number of amino acids coded by and resulting from translation of the sequence of SEQ ID NO: 47, for example the replacement of up to 3 amino acids, provided that the resulting polypeptides still display an alcohol dehydrogenase activity as the biocatalyst according to the invention (especially the substrate specificities, ph-optimum, temperature optimum, temperature stability, activity in the presence of EDTA or solvent stability properties mentioned below, or any combination of two or more of these properties, preferably all these properties), especially with at least the high solvent and temperature stability mentioned below.
  • exactly the DNA of SEQ ID NO: 47 is comprised, in a broader embodiment also variants thereof with up to 30, e.g. up to 15 nucleic acid replacements.
  • corresponding polynucleotides or recombinant polynucleotides comprising the mentioned DNA sequence with insertions and/or deletions (either in addition to or alternatively to nucleic acid replacements), e.g. with a total of up to 300, preferably up to 90, for example up to 30 nucleic acids, preferably the insertions and deletions not affecting the reading frame, that is, resulting from addition or removal of one or more triplets, preferably in accordance with the mentioned boundaries, in each case provided that the resulting polypeptides after translation still display an alcohol dehydrogenase activity as the biocatalyst according to the invention.
  • the invention also relates to microorganisms transformed with a polynucleotide, especially a recombinant polynucleotide, each as mentioned above coding for such a biocatalyst with alcohol dehydrogenase activity - preferably to microorganisms appropriate for expressing the gene, but also those that comprise the nucleic acid for pure conservation or replication purposes.
  • the invention also relates to the use of and a method of using these microorganisms (either living or killed, in complete or in at least partially digested form) especially in the oxidation or reduction reactions described above and below.
  • the invention also relates to the complementary nucleic acid for such a nucleic acid sequence as well as to double strands with both the complementary and the coding sequence.
  • a corresponding RNA (with U instead of T) is also comprised.
  • the invention also relates to the primers given in the examples under the SEQ ID Nos starting from SEQ ID NO 7 up to SEQ ID NO 46 and to the Rhodococcus rubber DSM 14855 DNA library mentioned in the examples.
  • the invention also relates to a polypeptide showing alhohol dehydrogenase activity according to the invention (especially the substrate specificities, ph-optimum, temperature optimum, temperature stability and solvent stability properties mentioned below, or any combination of two or more of these properties, preferably all these properties), especially with at least the high solvent and temperature stability mentioned below, of the sequence with SEQ ID NO: 48
  • the invention also relates to the use of or a method of using such a polypeptide (in purified or at least partially purified form especially in the oxidation or reduction reactions described above and below.
  • the invention especially relates to a biocatalyst with alcohol dehydrogenase activity, especially an enzyme, preferably in purified form, which has alcohol dehydrogenase activity and which can be obtained from Rhodococcus ruber, especially Rhodococcus ruber DSM 14855. That the biocatalyst has alcohol dehydrogenase activity, is not intended to mean that other activities (be it of enzymatic, regulatory or any other kind) are excluded within the present disclosure.
  • biocatalyst of the invention or “enzyme of the invention” or “enzyme (or polypeptide) showing alhohol dehydrogenase activity”
  • biocatalyst having alcohol dehydrogenase activity especially an enzyme with said activity, most especially alcohol dehydrogenase "ADH-A”, as described below.
  • alcohol dehydrogenase ADH-A
  • all these terms include not only the naturally occurring, “authentic” sequence of a polypeptide of the invention, which are the preferred embodiments of the invention, but also all mutants, variants and fragments thereof which exhibit the alcohol dehydrogenase activity, preferably with the same stereoselective activity as the natural enzyme.
  • An enzyme of the invention preferably has one, more preferably two, most preferably three or more of the following properties:
  • Stability meaning alcohol dehydrogenase activity also in the presence of up to 50, preferably up to 80 percent by volume of isopropanol.
  • Stability meaning alcohol dehydrogenase activity also in the presence of up to 20, preferably up to 50 percent by volume of acetone.
  • [L/l] is leucine or isoleucine and X stands for an unidentified amino acid.
  • TD in the beginning at the amino terminus or with DT in the beginning) within the total sequence of at least one polypeptide forming the whole or part of the enzyme where the sequence in brackets is selected from the two alternatives mentioned therein and [L l] is isoleucine or leucine.
  • the term "within the total sequence of at least one polypeptide forming the whole or part of the enzyme” refers to the possibility that the biocatalyst/enzyme according to the invention may consist of one subunit (then it is formed from one polypeptide) or more than one (identical or different) subunits (then it is formed from the corresponding number of polypeptides).
  • an enzyme of the invention wherein one of the partial sequences mentioned under (xii) to (xvii) is present or wherein one of the amino acids mentioned in any of these sequences is exchanged against a different amino acid, preferably by a conservative replacement, e.g. of lipophilic against lipophilic, basic against basic, acidic against acidic, polar against polar amino acids, or the like.
  • an enzyme the peptide sequence or, if more than one different subunits are present, sequences of which comprise a partial sequence selected from two, more preferably 3, even more preferably 4, still most preferably 5, most preferably 6 of the sequences mentioned above under (xii) to (xvii).
  • the enzyme of the invention is isolated (purified) from the microorganism by methods that are, perse, well known in the art, in particular by the methods described in the examples or methods analogous thereto, the whole purification method also forming an embodiment of the invention.
  • the Alcohol dehydrogenase activity is preferably determined by oxidation of 1 -phenylethanol (6.6 ⁇ M) and addition of 10 mM NAD + (testing conditions: 30 °C, 10 ⁇ M Tris-buffer, pH 7.5, 10 min, conversion by GC-analysis), or as described in the Examples.
  • a biocatalyst of the invention is obtainable from a naturally occurring microorganism fermentable by a process comprising inoculating a selection medium with natural samples such as soil, water, or plant silage, preferably a hydrocarbon, e.g. hexane.
  • a selection medium also contains all essential ingredients necessary for allowing growth of microorganisms, such as mineral salts, N-sources, and trace elements.
  • a naturally occurring microorganism having alcohol dehydrogenase activity can be obtained by a process known in the art, for example by isolation from a natural source, such as Rhine water.
  • the microorganism having alcohol dehydrogenase activity is cultivated in an aqueous nutrient medium, e.g. comprising yeast extract, peptone, glucose and mineral salts (e.g. 10 g/1 yeast extract 10 g/l peptone,2 g/1 NaCl, 0.15 g/l MgSO 4 • 7H 2 O, 1.3 g/l NaH 2 P0 4 , 4.4 g/l K 2 HP0 4 ) for some, e.g. three, days.
  • Cell growth is followed by determination of the optical density via absorption, e.g. at 546 nm.
  • the cells are disrupted, the biomass is removed and the cell-free extract is obtained.
  • the fermentation time is so selected that optimum titers with respect to alcohol dehydrogenase activity are achieved.
  • the cultivation is discontinued.
  • the culture broth is separated off in known manner, e.g. by centrifugation, and the sedimented cells are broken down in customary manner, e.g. by shaking with fine glass beads, by ultrasound treatment, or using a French press.
  • Insoluble cell components and, if used, glass beads are removed, e.g., by centrifugation, and the residue is used as the enzyme source (crude extract).
  • the residue as an alcohol dehydrogenase activity-containing crude extract, can be used directly in the process according to the invention.
  • in order to remove nucleic acids (viscous solutions) and other impurities or interfering components e.g.
  • the crude extract is subjected to further purification in order to obtain the enzyme of the invention in purified form.
  • the crude cell extract is subjected to one or more purification steps that, as such, are known in the art in order to remove interfering components from the extract.
  • a preferred method makes use of batch pretreatment with a cation exchanger, e.g. DEAE cellulose, and subsequent chromatographic (especially FPLC) separation first by Hydro- phobic Interaction Chromatography, e.g. over Phenyl Sepharose, advantageously after removing precipitations formed in the presence of (NH ) 2 S0 4 .
  • a cation exchanger e.g. DEAE cellulose
  • FPLC e.g. over Phenyl Sepharose
  • enzyme activity is eluted.
  • the active fractions are further separated by anion exchange chromatography, preferably on an UNO Q column (BioRad). Elution by eluents with increasing sodium chloride content yields again active fractions with alcohol dehydrogenase activity.
  • the interesting fractions are then further purified on a separation matrix comprising binding places for adenylyl-comprising cofactors, e.g. carrying the dye Cibacron Blue F3G-A or other moieties allowing for such affinity chromatography, especially using Blue Sepharose CI-6B, this innovative material allowing a surprisingly good further purification, although surprisingly no binding takes place.
  • the next step is size exclusion chromatography, e.g. using a Superdex 200 column.
  • the enzyme of the invention elutes in fractions corresponding to a molecular weight between 55 to 69 kDa, with an average of about 62 kDa.
  • purified means preferably “in at least partially purified form” or “in enriched form” or, more preferably, purified in the stricter sense, that is, in practically isolated form (especially with more than 50, most especially more than 95 % purity by weight compared to other peptides present).
  • a corresponding biocatalyst with alcohol dehydrogenase activity obtained by recombinant technology, then called a recombinant biocatalyst of the invention, (or also a biocatalyst from natural sources other than Rhodococcus ruber DSM 14855), preferably is defined as follows.
  • the sequence of said biocatalyst may comprise deletions, insertions, terminal additions or exchanges (especially conservative exchanges, e.g.
  • amino acids preferably of up to 20, in case of terminal additions up to 1000; more preferably of up to 5, in case of terminal additions of up to 200 amino acids, respectively, or any combination of such changes, when compared to the sequence of the enzyme as purified in the examples or possible different subunits thereof, as long as the basic activity (alcohol dehydrogenase activity, especially with the substrates (preferably 1 -phenylethanol or acetophenone) and co- substrates mentioned in the examples) is still present, especially in connection with one or more of the additional advantageous properties mentioned for a biocatalyst according to the invention.
  • modified amino acids (with different structures than the 20 amino acids directly derivable from the genetic code) which may be modified during translation or post- translationally, may be present, e.g. 1 to 20, more preferably 1 to 5 such amino acids.
  • a biocatalyst with alcohol dehydrogenase activity in the oxidation of secondary alcohols or the reduction of ketones is the use in catalyzing the following reactions (reaction scheme (A)):
  • Ri and R 2 are two different moieties from the group consisting of unsubstituted or substituted alkyl, unsubstituted or substituted alkenyl, unsub- stituted or substituted alkinyl, unsubstituted or substituted cycloalkyl, unsubstituted or substituted aryl and unsubsituted or substituted heterocyclyl, or Ri and R 2 together form an unsubstituted or substituted bridge; and in formula III and formula IV, R 3 and R 4 are two different or preferably two identical lower alkyl or aryl moieties, or together form a bridge.
  • lower defines a moiety with up to and including maximally 7, especially up to and including maximally 4, carbon atoms, said moiety being branched or straight-chained.
  • Lower alkyl for example, is ethyl, n-propyl, sec-propyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pen- tyl, n-hexyl or n-heptyl or most preferably methyl.
  • Substituted wherever used for a moiety, means that one or more hydrogen atoms in the respective molecule, especially up to 5, more especially up to three, of the hydrogen atoms are replaced by the corresponding number of substituents which preferably are independently selected from the group consisting of alkyl, especially lower alkyl, for example methyl, ethyl or propyl, fluoro-lower alkyl, for example trifluoromethyl, C 6 -C ⁇ 6 -aryl, especially phenyl or naphthyl (where C 6 -C ⁇ 6 -aryl, especially phenyl or naphthyl, is unsubstituted or substituted by one or more, especially up to three moieties selected from halogen, carboxy, lower alkoxy- carbonyl, hydroxy, lower alkoxy, phenyl-lower alkoxy, lower alkanoyloxy, lower alkanoyl, ami- no, N-lower alkylamino, N,N-d
  • C 3 -C ⁇ 0 -cycloalkyl that is unsubstituted or substituted by one or more, especially up to three moieties selected from halogen, carboxy, lower alkoxycarbonyl, hydroxy, lower alkoxy, phenyl-lower alkoxy, lower alkanoyloxy, lower alkanoyl, amino, N-lower alkylamino, N,N-di- lower alkylamino, N-phenyl-lower alkylamino, N,N-bis(phenyl-lower alkyl)-amino, lower al- kanoylamino, fluoro-lower alkyl, e.g.
  • heterocyclyl that is unsaturated, saturated or partially saturated, is mono-, bi- or tri-cyclic and has 4 to 16 ring atoms, where instead of one or more, especially one to four, carbon ring atoms the corresponding number of heteroatoms are present (within the chemically possible limits) selected from nitrogen, oxygen and sulfur (with said heterocyclyl unsubstituted or substituted by one or more, especially up to three moieties selected from halogen, carboxy, lower alko- xycarbonyl, hydroxy, lower alkoxy, phenyl-lower alkoxy, lower alkanoyloxy, lower alkanoyl, amino, N-lower alkylamino, N,N-di-lower alkylamino, N-phenyl-lower alkylamino, N,N-bis- (phenyl-lower alkyl)-amino, lower alkanoylamino, fluoro-lower
  • hydroxy, lower alkoxy for example methoxy, phenyl-lower alkoxy, lower alkanoyloxy, amino, N-lower alkylamino, N,N-di-lower alkylamino, N-phenyl-lower alkylamino, N,N-bis- (phenyl-lower alkyl)-amino, lower alkanoylamino, carbarnoyl-lower alkoxy, N-lower alkyl- carbamoyl-lower alkoxy or N,N-di-lower alkylcarbamoyl-lower alkoxy, amino, mono- or di- lower alkylamino, lower alkanoylamino, carboxy, lower alkoxycarbonyl, phenyl-, naphthyl- or fluorenyl-lower alkoxycarbonyl, such as benzyloxycarbonyl, lower alkanoyl
  • substitutents are only at positions where they are chemically possible, the person skilled in the art being able to decide (either experimentally or theoretically) without inappropriate effort which substitutions are possible and which are not. Where more than one substituent is present, the substituents, if not indicated otherwise, are selected independently from each other.
  • Alkyl preferably has up to 24, more preferably up to 12 carbon atoms and, if possible in view of the number of carbon atoms, is linear or branched one or more times; preferred is lower alkyl, especially C ⁇ -C 4 -alkyl.
  • Alkyl can be substituted or unsubstituted, especially by one or more, more especially up to 3, of the substituents mentioned above under "substituted”. Unsubstituted alkyl, especially lower alkyl, is one preferred embodiment.
  • Alkenyl is preferably a moiety with one or more double bonds and preferably has 2 to 20, more preferably up to 12, carbon atoms; it is linear or branched one or more times (as far as possible in view of the number of carbon atoms). Preferred is C 2 -C 7 -alkenyl, especially C 3 -C 4 - alkenyl, such as allyl or crotyl. Alkenyl can be unsubstituted or substituted, especially by one or more, more especially up to three, of the substituents mentioned above under lightningsubstituted".
  • Substituents such as amino or hydroxy (with free dissociable hydrogen) preferably are not bound to carbon atoms that participate at a double bond, and also other subtituents that are not sufficiently stable are preferably excluded.
  • Alkinyl is preferably a moiety with one or more triple bonds and preferably has 2 to 20, more preferably up to 12, carbon atoms; it is linear of branched one or more times (as far as possible in view of the number of carbon atoms). Preferred is C 2 -C 7 -alkinyl, especially C 3 -C - alkinyl, such as ethinyl or propin-2-yl. Alkinyl can be unsubstituted or substituted, especially by one or more, more especially up to three, of the substituents mentioned above under ..substituted".
  • Substituents such as amino or hydroxy (with free dissociable hydrogen) preferably are not bound to carbon atoms that participate at a triple bond, and also other subtituents that are not sufficiently stable are preferably excluded. Unsubstituted alkinyl, in particular C 2 -C 7 -aIkinyl, is especially preferred.
  • Aryl preferably has a ring system with not more than 20 carbon atoms, especially not more than 14 carbon atoms; is preferably mono-, bi- or tricyclic; and is unsubstituted or substituted by one or more, especially up to three, substitu tents, preferably as defined above under "substituted".
  • aryl is selected from the group consisting of phenyl, naphthyl, indenyl, azulenyl and anthryl, each of which is unsubstituted or substituted; preferably from phenyl or 1- or 2-naphthyl, each unsubstituted or substituted by one or more, preferably up to 5, substituents as defined above under "substituted”.
  • Heterocyclyl is preferably a heterocyclic radical that is unsaturated, saturated or partially saturated, and is preferably a monocyclic or, in a broader aspect of the invention, bi- or tricyclic moiety; it has preferably 3 to 24, especially 4 to 16 carbon atoms, where instead of one or more, especially one to four, carbon ring atoms the corresponding number of heteroatoms are present, especially (within the chemically possible limits) selected from nitrogen, oxygen and sulfur; and where heterocyclyl is unsubstituted or substituted by one or more, especially up to three, substituents as defined above under "substituents".
  • heterocyclyl is selected from the group consisting of oxiranyl, azirinyl, 1,2-oxathiolanyl, imidazolyl, thienyl, furyl, tetrahydrofuryl, pyranyl, thiopyranyl, thianthrenyl, isobenzofuranyl, benzofuranyl, chromenyl, 2r -pyrrolyl, pyrrolyl, pyrrolinyl, pyrrolidinyl, imidazolyl, imidazolidinyl, benzimidazolyl, pyrazolyl, pyrazinyl, pyrazolidinyl, pyranyol, thiazolyl, isothiazolyl, dithiazolyl, oxazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, piperidyl, piperazinyl, pyridazinyl, morph
  • Cycloalkyl preferably has 3 to 12, more preferably 3 to 8 carbon atoms and is, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl; it is unsubstituted or substituted with one or more, especially up to 3, substituents, preferably as defined above under "substituted".
  • An unsubstituted or substituted bridge formed from Ri and R 2 in formula I, la, lb or II is preferably a bridge formed by 2 to 12 carbon atoms that, together with the binding atom in formula I, la, lb or II forms a ring; where the bridge may contain one or more double and/or triple bonds at places other than the binding carbon atom (then the bridge has at least three carbon atoms) or is preferably saturated.
  • the substituents are preferably chosen from 1 or more, especially up to three, substituents as defined under "substituents".
  • C 2 -C 7 -alkylen chain such as ethylene, propylene, n-butylene, n-hexylene or n-heptylene, each of which is substituted by one or more, especially 1 or 2, of the moieties defined above under "substituted", or preferably is unsubstituted.
  • saltforming groups are especially basic groups, such as amino groups, or acidic groups, such as carboxy groups.
  • acidic groups such as alkaline metal salts, e.g.
  • sodium or potassium salts, or alkaline-earth metal salts, such as calcium salts, or salts with nitrogen bases, such as ammonium-, tri-lower alkylammonium, pyridinium salts or the like, can be present; in the case of basic groups, the corresponding acid addition salts may be present, e.g. with inorganic acids, such as sulphuric acid or hydrogen halides, such as HCI or HBr, or with organic acids, e.g. carboxylic acids, such as acetic acid, or sulfonic acids, e.g. methane sulfonic acid.
  • inorganic acids such as sulphuric acid or hydrogen halides, such as HCI or HBr
  • organic acids e.g. carboxylic acids, such as acetic acid, or sulfonic acids, e.g. methane sulfonic acid.
  • Examples for preferred secondary alcohols are hydroxy group carrying isoprenoids, such as mono-, di- or tri-terpenes, e.g. geraniol, isoborneol, ipsenol, menthol (especially ( ⁇ )-menthol), nerolidol, hemandulcin, taxol or lanosterol, or steroids, such as cholestan-3-ol, cholesterol, ergosterin, stigmasterin, cholic acids, vitamin D 2 , vitamin D 3 , androsterone, testosterone, estrone, 17 ⁇ -estradiol, estriol, cortisol, corticosterone, aldosterone, triamincolone, digitoxigenin, strophanthidine, ouabagenine, scillaridine or bufotalin; di-lower alkyl- or lower alkyl-lower alkenyl methanols, such as isopropanol, butane-2-ol, n-hexane-2
  • Preferred cosubstrates for the reduction are secondary alcohols, such as isopropanol, 4-me- thyl-2-pentanol or further other C,C-di-(lower alkyl)-methanols. These are preferably present in the reaction mixture in an excess, for example compared to the ketone to be reduced, for example making up 50 % (related to the volume of the reaction mixture, v/v).
  • the concentration of the ketone to be reduced preferably lies below an upper limit of 3 mol/l, preferably up to 2,6 mol/l or lower.
  • Ketones to be reduced are especially free ketones or ketals thereof (such as especially di- (loweralkyl ketals or (cydic) (unsubstituted or substituted, for example by lower alkyl, such as methyl) lower alkylene ketals, such as ethylene ketals (dioxolan derivatives).
  • ketones or ketals thereof such as especially di- (loweralkyl ketals or (cydic) (unsubstituted or substituted, for example by lower alkyl, such as methyl) lower alkylene ketals, such as ethylene ketals (dioxolan derivatives).
  • oxo-carrying isoprene derivatives such as mono-, di- or triterpenes, e.g.
  • menthone pulegone, carvone, carone, verbenone, camphor, dihydrocarvone, dihydrocarvone hydrobromide, carvenone, hernandulcine or taxol or lanosterol, or steroids, such as androsterone, testosterone, estrone, cortisol, corticosterone, aldosterone or prednisone; di-(lower alkyl)- or lower alkyl-lower alkenyl-ketones, such as acetone, butane- one, n-hexan-2-one, n-heptan-2-one, n-octan-2-one, n-nonan-2-one, 3-octanone, 5-methyl-2- heptanone, 3-octen-2-one, 3-penten-2-one, 6-methyl-hex-5-en-2-one or 6-methyl-hept-5-en- 2-one; hydroxyacetone; or n-decan-2-one; acetophen
  • the preferred pH range for the oxidation of alcohols in the presence of ketones as co- substrates is kept in the area from pH 6 to pH 12, more preferably from pH 8 to pH 11.
  • the preferred pH range lies between pH 5 and pH 9, more preferably between pH 6 and pH 8.
  • the pH value is controlled by standard buffers, for example phosphate buffers with alkaline metal, especially potassium or sodium phosphate buffer, boronic acid/HCI/sodium hydroxide buffers, or other buffers, such as Tris/HCI, HEPES buffers or the like; and/or by automated titration with an acid, such as HCI, to keep the pH from rising, or a base, such as NaOH, to keep the pH from sinking.
  • the enzyme of the invention can also be used in the presence of surface active substances (surfactants, detergents).
  • surfactants for example, sodium dodecyl alcohol, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite
  • anionic tensides that usually include long chain fatty acids as anionic, hydrophobic component, e.g. sulfates of long chain (especially C 8 -C ⁇ 8 ) alcohols, such as alkaline metal C 8 -C ⁇ 8 alkanoylsulfates, especially sodium dodecylsulfate or sodium decylsulfate;
  • hydrophilic groups with a positive charge e.g. quaternary ammonium
  • - amphoteric detergents such as mono- or dicarboxylated imidazolines of fatty acids, such as sodium lauryldicarboxyimidazoline or sodium; or
  • - non-ionic tensides such as ethoxylated sugar esters of higher fatty acids, such as polyoxyethylene-sorbitan-monolaurate, -palmitate, -stearate or -tristearate.
  • the temperatures for the use of the enzyme of the invention in the oxidation or reduction reactions preferably lies in the range customary for biocatalytic reactions or above, preferably in the range from 10 to 65, more preferably from 40 to 65 °C.
  • the oxidation of or reduction to the S-enantiomer is especially preferred in this reaction.
  • Yet another preferred embodiment of the invention relates to the use of/process using the enzyme of the invention for the (mild) chemoselective (especially stereoselective) oxidation of secondary alcohols, where only the hydroxy group with the appropriate steric form is oxidised to the corresponding oxo group (while other oxidable hydroxy groups in the same or other molecules remain intact).
  • This can especially be used for the separation of isomers where from mixtures of alcohols only the reactive ones are oxidised, so that either the desired alcohols remain as such or the resulting oxo compounds are, in a subsequent step, again transformed into the desired alcohols by reversal of the reaction (reduction).
  • An especially preferred variant of this use relates to the use of an enzyme according to the invention for the (mild) enantioselective oxidation of only one isomer of mixtures of enantiomers (or diastereomers), especially racemates, of secondary alcohols, especially of alcohols of the formula I wherein R 1 and R 2 are two different moieties, especially as defined above, or form an asymmetric (especially asymmetrically substituted) bridge.
  • the remaining alcohol can be obtained in isomerically pure, especially enantiomerically pure, form, e.g. with 75 % or more, especially 95 % or more, more especially with 98 % or more enantiomeric excess regarding the carbon atom carrying Ri and R 2 .
  • the process of the invention can thus be used especially for separating mixtures of stereoisomers with respect to a center of chirality by kinetic resolution, if one stereoisomer of an alcohol is specifically oxidized, or for the stereospecific production of secondary alcohols representing a specific chiral form from ketones.
  • This can also be used for separation processes in that from complex mixtures of ketones only the reactive ones are taking part in the reaction while either the desired oxo compounds remain in the reaction mixtures or the resulting alcohols are, in a subsequent inverse step (oxidation), converted back into the desired keto compounds.
  • reaction prochiral oxo-substituted carbon atoms are transformed into the corresponding asymmetrically substituted hydroxy-carrying carbon atoms
  • this is also appropriate for obtaining the isomerically, especially enantiomerically, pure alcohols (especially with a purity as defined above).
  • the process of the invention can be performed with free or immobilized biocatalyst according to the invention, which can be used in enriched or preferably in purified form.
  • a recombinant microorganism especially a host cell which is present in suspension or immobilized and is expressing an enzyme of the invention is used for performing the reaction, i.e. the enzyme is in cell-bound form.
  • an enzyme of the invention for use in a process of the invention may be immobilized.
  • the immobilization of said enzyme can be carried out analogously to processes known perse, e.g. coupling to a solid support or enclosing in an enzyme membrane reactor.
  • the process of the invention may also be lead in the presence of a (free or immobilized) microorganism transformed by genetic engineering techniques with a gene coding for an enzyme of the invention to be able to produce the desired enzyme of the invention, especially in higher amounts than it would be present in the original microorganism, e.g. Rhodococcus ruber, especially Rhodococcus ruber DSM 14855.
  • a (free or immobilized) microorganism transformed by genetic engineering techniques with a gene coding for an enzyme of the invention to be able to produce the desired enzyme of the invention, especially in higher amounts than it would be present in the original microorganism, e.g. Rhodococcus ruber, especially Rhodococcus ruber DSM 14855.
  • the reaction of an alcohol or ketone substrate with an enzyme of the invention or a microbial cell extract is preferably carried out in homogeneous aqueous solution at pH 5 to 10.5, more preferably at pH 6 to 9.5.
  • the reaction is carried out in a manner known perse in buffered solution or using a pH-stat.
  • the reaction temperature is approximately from 10 to 65 °C, more preferably from 20 to 50 °C, even more preferably from 20 to 30 °C.
  • the substrate is used preferably in a concentration of 1 mM to 2 M, more preferably 50mM to 500 mM. However, if the substrate is less soluble, it is also possible to use a substrate suspension.
  • the process according to the invention can be carried out either as a batch process or continuously in an enzyme membrane reactor (EMR).
  • EMR enzyme membrane reactor
  • the enzyme membrane reactor is preferably fitted with an ultrafiltration membrane having a separation limit of less than approximately 30000 Da, so that the enzymes contained in the reaction mixture are held back whilst the low-molecular-weight products and unreacted rearfants pass through the membrane and the product can be isolated from the outflow.
  • the reactor is preferably sterilized before use so that the addition of antibacterial substances can be dispensed with.
  • the reactions are carried out in a manner analogous to that described above.
  • the process according to the invention can also be carried out by percolating the solution containing the alcohol or ketone substrate, which has been adjusted to a suitable pH value, through a solid carrier on which the enzyme of the invention has been immobilized (the matrix-bound enzyme preparation is obtainable, for example, by percolation of the crude microbial extract through CNBr-activated Sepharose, Eupergit or the like).
  • reaction mixture can be clarified by filtration or, preferably, centrifugation, and then the enzyme can be separated by ultrafiltration (membrane with separation limit of ⁇ 30 kDa) and the remaining product can be washed out of the retentate by diafiltration.
  • ultrafiltration membrane with separation limit of ⁇ 30 kDa
  • Salts of educts or products can be converted into the free compounds, free compounds into the salts using standard methods, respectively.
  • Nucleic acids are preferably DNA or RNA (in general, oligo- or polynucleotides).
  • Isolated nucleic acids coding for a biocatalyst according to the invention with alcohol dehydrogenase activity are preferably obtained and defined as follows:
  • a nucleic acid, especially a gene, coding for a biocatalyst of the invention can, for example, be obtained by identifying at least a part of the sequence of an isolated enzyme of the invention, deducing DNA sequences coding for the partial protein sequence, preparing an oligonucleotide or a mixture of oligonucleotides (taking into consideration the degeneracy of the genetic code) as probe(s), probing a DNA library derived from the microbial strain naturally expressing the activity of the biocatalyst (the term DNA library also including a "cDNA-library”), isolating the gene, and cloning it into a suitable vector for transformation of the microorganism to be genetically modified.
  • the partial sequencing of an enzyme of the invention is, for example, made using selective endoproteases for selective digestion, e.g. endo-protease Lys-C, endoprotease Glu-C, chymotrypsin, thermolysin or preferably trypsin (cleaving C-terminally from the basic amino acids arginine or lysine) and, after separation, e.g. electrophoretically on a gel or by chromatography (e.g .HPLC), determining the terminal sequences of the resulting peptides, e.g. by exopeptidases, e.g. carboxypeptidases, such as carboxypeptidase A , B or P).
  • Preferred is tryptic digestion, then MS/MS analysis (TOF).
  • DNA libraries can also be obtained by PCR methods.
  • a cDNA library (obtainable e.g. after extraction of the mRNA from the cells, transformation into DNA using reverse transcriptase, introduction of sticky ends, introduction into a cloning vector, and introduction of that vedor into an appropriate host cell, e.g. a plasmid vector into a bacterium, such as a bacteriophage ⁇ vector or a cosmid into E. coli, a yeast artificial into a yeast, such as Saccaromyces cerevisiae, a Pichia-pastoris vector into Pichia pastoris, or the like) or a DNA library (e.g. obtainable from selective digests of isolated DNA with restriction endonucleases, especially of type II, e.g.
  • Hybridization is done using standard procedures, if necessary removing possible disturbing non-coding sequences, e.g. by PCR amplifying only the desired sequence parts or by endonuclease digestion, e.g. using dot blots of colonies of microorganisms from the DNA library. The positive clones can then be isolated.
  • the sequencing is done using standard procedures, e.g. the Maxam-Gilbert or the Sanger method.
  • the complete sequence coding for an enzyme of the invention (or one subunit thereof, if more than one polypeptide form the complete enzyme) can be determined.
  • the corresponding amino acid sequence of the enzyme is (or, if more than one polypeptide forms it, the subunits thereof, the amino acid sequences are) easily determined, using the genetic code.
  • the full amino acid sequence of the isolated biocatalyst according to the invention can be determined (e.g. by different endopeptidase digests and matching of overlapping sequenced partial peptides) and a DNA coding the protein can be produced synthetically. It is also very easily possible to screen a suitable DNA library in a host, e.g. E. coli, for expression of temperature resistant alcohol dehydrogenase activity to obtain a transformed clone expressing the biocatalyst.
  • Still another method makes use of antibodies against an enzyme of the invention that can be obtained using standard procedures (up to and including the production of monoclonal antibodies obtained from myelomas obtained according to standard procedures) in order to isolate the ribosomes carrying the mRNA coding for the enzyme, transforming it into the corresponding DNA (e.g. with reverse transcriptase) and sequencing or genetically engineering the resulting enzyme.
  • the nucleic acid according to the invention is preferably present in isolated form or in recombinant form (then also in a microorganism, see below).
  • nucleic acids according to the invention also comprise modified (especially recombinant, but also naturally occurring) nucleic acids where, when compared with the form sequenced as described above, one or more nucleic acids are deleted, inserted, exchanged or added terminally, as long as the polypeptide or polypeptides for which they code still display alcohol dehydrogenase activity, especially according to the test method with 1-phenylethanol or acetophenone as described in the Examples.
  • Terminal additions may comprise the addition of sequences for vectors or host nucleic acids into which the coding sequence may be combined.
  • the modified nucleic acids are modified such as to code for a biocatalyst with alcohol dehydrogenase activity, obtained by recombinant technology (resulting in a recombinant nucleic acid) or alternatively from natural sources, where the amino acid sequence of the biocatalyst comprises deletions, insertions, terminal additions or exchanges (especially conservative exchanges, e.g.
  • the modified nucleic acids contain 1 to 50, more preferably 1 to 12, additional nucleotides by insertion (especially additions yielding no frame shift), 1 to 50, more preferably 1 to 12 changes in nucleic acids, preferably resulting in conservative amino acid changes, and/or 1 to 50, more preferably 1 to 12, deletions of nucleotides, especially without frame shift.
  • the invention also relates to probes, especially in radiolabelled or fluorescence labelled form, that are hybridizable under stringent conditions to genomic or cDNA or other nucleic acids coding and that code for the sequences of the six partial amino acid sequences given as SEQ ID NO: 1 to SEQ ID NO: 6, or for parts thereof, said probes preferably having a length of 6 to 24, more preferably of 12 to 21 nucleotides.
  • the embodiment of the invention relating to microorganisms transformed with a nucleic acid coding for such a biocatalyst with alcohol dehydrogenase activity preferably relates to microorganisms appropriate for expressing the gene, but also those that comprise the nucleic acid for pure conservation or replication purposes.
  • Appropriate microorganisms are especially viruses, bacteriophages or especially host cells, for example, bacteria, e.g. E. coli, single cell fungi, such as yeasts, e.g. Pichia pastoris, Schizosaccharomyces pombe or Saccharomyces cerevisiae, or plant cells.
  • bacteria e.g. E. coli
  • single cell fungi such as yeasts, e.g. Pichia pastoris, Schizosaccharomyces pombe or Saccharomyces cerevisiae, or plant cells.
  • microorganisms can be transformed with nucleic acids as such that code for an enzyme of the invention; however, usually they are transformed with suitable vectors, e.g. plasmids, cosmids, yeast artificial chromosome or the like, which may comprise partial sequences (e.g. useful in sequence determination) or total sequences for the enzyme (e.g. useful in the expression of the enzyme).
  • suitable vectors e.g. plasmids, cosmids, yeast artificial chromosome or the like, which may comprise partial sequences (e.g. useful in sequence determination) or total sequences for the enzyme (e.g. useful in the expression of the enzyme).
  • Transformation of host cells is made according to standard procedures known in the art and appropriate for the respective host cells, e.g. according to the calcium chloride method, by electroporation, transformation after spheroblast formation into fungi, transformation with polyethylene glycol, transformation with lithium chloride, or the like.
  • Virus or the like are modified by introduction of the sequences comprising the coding sequences for the enzyme of the invention.
  • the invention relates to the use of the microorganisms, especially host cells, in the production of said biocatalyst with alcohol dehydrogenase activity.
  • Expression systems suitable for production of an enzyme of the invention are especially phage-based expression systems in bacteria, e.g. bacteriophage ⁇ or cosmids for E. coli as host, yeast artificial chromosomes for expression in Saccharromyces cerevisiae, the Pichia pastoris expression system used for expression in Pichia pastoris, expression systems in Schizosaccaromyces pombe, the baculovirus expression system or the like.
  • the nucleotide sequences coding for an enzyme of the invention can be expressed, either as such or with additional N- or C-terminal sequences, e.g. such that allow for direct export of the resulting polypeptide outside the expressing cells. These extra sequences, if disturbing the activity or otherwise not desired, can then be cleaved off using appropriate endoproteases known in the art.
  • the invention relates in particular to the use of/process using the enzyme of the invention, and especially to the enzyme described in the Examples.
  • Rhodococcus ruber DSM 14855 is grown under aerobic conditions in baffled Erlenmeyer flasks at 30 °C and 130 rpm using a complex medium containing yeast extract, peptone, glucose and mineral salts (1O g/l yeast extract (OXOID CM129, OXOID Ltd., Hampshire, England), 10 g/l peptone, 2 g/l NaCl, 0.15 g/l MgSO 4 • 7H 0, 1.3 g/l NaH 2 P0 4 , 4.4 g/1 K 2 HPO 4 ) for three days. Cell growth is followed by determination of the optical density via absorption at 546 nm, see Table 1. After a centrifugation (2000 g, 20 min), the pellet is taken up in Tris-HCI buffer pH 7.5 (50 mM), shock-frozen in liquid nitrogen and lyophilised.
  • Tris-HCI buffer pH 7.5 50 mM
  • the cells produce several NAD + /NADH-dependent sec-alcohol dehydrogenases without any induction.
  • the highest sec-alcohol dehydrogenase activity is displayed during the late exponential and early stationary phase of the growth curve (see table 1).
  • the activity is measured as the ability of the cells to oxidise 1-phenylethanol or the ability to reduce acetophenone.
  • Rhodococcus ruber DSM 14855 (20 mg) are rehydrated in phosphate buffer (0.5 ml, 50 mM, pH 7.5) for 30 min. Activity is measured by adding acetophenone (0.27 mmol) and 2-propanol (0.4 ml). The mixture is shaken at 24 °C, 130 rpm in Eppendorf vials for 2.5 h. The reaction is quenched by addition of ethyl acetate (1 ml) and centrifugation. The conversion is determined by GC (Varian 3800, FID) on an achiral column (HP-1301 , 30 m x 0.25 mm x 0.25 ⁇ m; N 2 ).
  • Temperature program for rac-1- phenylethanol/acetophenone start temperature 80 °C - hold 2 min - 10 "C/rni ⁇ - until 130 °C - hold 2 min. The conversion is calculated from calibration curves.
  • 1- phenylethanol is oxidised in the presence of acetone as co-substrate.
  • Example 2 Purification of a sec-Alcohol dehydrogenase 'ADH-A' from Rhodococcus ruber DSM 14855 a) Cell Disruption
  • the sec-alcohol dehydrogenase might be a mem rane-associated or even membrane-bound protein
  • cell disruption using a Vibrogen cell mill glass beads, diameter 0.25 mm, Vibrogen Zellmuhle , E. Buhler, Typ VI-4; Braun Biotech Int., Melsoder, Germany
  • optimised in order to obtain the majority of the activity in the cell-free lysate and not in the cell debris fraction This results in an exceptionally long and rough procedure of 7 shaking cycles of 2 min agitation/ 5 min cooling each.
  • the enzyme shows high stability against this strong mechanical treatment.
  • the buffer used for cell disruption is 10 mM Tris-HCI buffer, pH 7.5.
  • FPLC Fast Protein Liquid Chromatography
  • the first protein purification step is a Hydrophobic Interaction Chromatography using Phenyl Sepharose High Performance material ((Amersham Pharmacia Biotech AB, Uppsala, Sweden).
  • sample preparation precipitation occurs by adding (NH 4 ) 2 SO 4 ), and solids are removed since they do not contain any sec-alcohol dehydrogenase activity.
  • (NH 4 ) 2 S0 4 as salting-out medium to the equilibration buffer and a stepwise gradient, the different hydrophobicities of proteins enable a first separation of enzymes.
  • Activity of Bi is found between 70 and 118 ml.
  • Activity of seo'ADH-A' is found between 220 and 236 ml elution volume. The latter (3 x 16 ml after 3 runs, a total of 48 ml) is used for further purification.
  • step (ii) Details of step (ii) .Column length 53 mm, diameter 12 mm.
  • Elution buffer A 10 mM Tris-HCI-buffer pH 7.5; B: A + 1.5 M NaCl ("100 % NaCl).
  • Activity of B 3 is found between 68 and 82 ml.
  • Activity of sec-'ADH-A' is found between 131 and 137 ml elution volume. The latter (6 ml) is used for further purification.
  • step (iii) Affinity Chromatography
  • the third step in protein purification of the resulting 6 ml from step (iii) is carried out on an innovative material, Blue Sepharose CI-6B [see Shaw et al., Biochem. J. 187, 181 (1996)], containing a dye, Cibacron Blue F3G-A, specific for enzymes requiring adenylyl-containing cofactors (such as NAD + /NADP + ) (Amersham Pharmacia Biotech AB, Uppsala, Sweden).
  • step (iii) Column length 60 mm, diameter 16 mm.
  • Buffer A 10 mM Bis-tris-HCI buffer pH 6.0
  • Buffer B A + 1 M NaCl.
  • Activity of alcohol dehydrogenase B 3 is found between 44 and 68 ml.
  • Activity of sec-'ADH-A' is found between 7 and 23 ml elution volume. The latter is concentrated up on Centriplus YM-10, cut-off 10 kDa (regenerated cellulose with 10 kDa exclusion size) from Millipore lipore GmbH, Vienna, AT)/Amicon to a final volume of 2 ml.
  • the molecular weight of the native enzyme is determined as approximately 62 kDa.
  • step (iv) Column length 310 mm, diameter 10 mm.
  • the sec-alcohol dehydrogenase 'ADH-A' is purified reproducibly.
  • the small overall recovery is explained by a major loss of activity during desalting procedures and buffer exchange via dialysis and during concentration to a small volume before applying it to the size-exclusion column. For this reason, the majority of biochemical characterizations are carried out with sem/-pure enzyme after the dye chromatography, where no other sec- alcohol dehydrogenase activity is left.
  • the "low" yield is attributable to the fact that in the cell extract at the beginning other alcohol dehydrogenases that are removed during the later procedure (e.g. Bi, B 2 and B 3 ) contribute to the dehydrogenase activity.
  • the activity is determined by oxidation of 1-phenylethanol (6.6 ⁇ M) and addition of 1O mM NAD + (testing conditions: 30 °C, 10 ⁇ M Tris-buffer, pH 7.5, 10 min, conversion by GC- analysis). Protein amounts are measured by the method of Bradford (Coomassie Blue Protein Assay) at 595 nm using a BioRad Protein Assay:
  • Example 3 Purification control and molecular weight determination by gel electrophoresis: The progress of the protein purification is controlled by native as well as by SDS- polyacrylamide gel electrophoresis (PAGE). For both methods, the protein fractions are subjected to a Laemmli SDS-PAGE system using a MINI-PROTEAN II dual slab cell (BioRad).
  • Active fractions are treated with non-denaturing sample buffer and loaded on a polyacrylamide gel (12%) without SDS.
  • the gel is run at 4 °C at 150 V with running buffer containing 15 g/l Tris and 72 g/l Glycine.
  • the method for visualization of sec-alcohol dehydrogenases in polyacrylamide gels was first reported by Grell et al. [see Science 149, 80 (1965)] and is based on a staining solution [see Dodgson et al., Biochem. J.
  • sec-alcohol dehydrogenases present in the microorganism, of which sec-alcohol dehydrogenase 'ADH-A' can be separated properly during the purification protocol.
  • sample buffer SDS reducing buffer, BioRad
  • SDS-polyacrylamide gel (12%) subsequently run at 200V at room temperature in the same running buffer with addition of 3 g/1 SDS. After the run, staining is carried out with Coomassie Brilliant Blue.
  • the molecular weight is determined in comparison to a low molecular weight range SigmaMarker as protein standard containing albumin (66 kDa), ovalbumin (45 kDa), glyceraldehyde-3- phosphate dehydrogenase (36 kDa), carbonic anhydrase (29 kDa), trypsinogen (24 kDa), trypsin inhibitor (20 kDa), ⁇ -lactalbumin (14.2 kDa) and aprotinin (6.5 kDa).
  • SigmaMarker as protein standard containing albumin (66 kDa), ovalbumin (45 kDa), glyceraldehyde-3- phosphate dehydrogenase (36 kDa), carbonic anhydrase (29 kDa), trypsinogen (24 kDa), trypsin inhibitor (20 kDa), ⁇ -lactalbumin (14.2 kDa) and aprotinin (6.5
  • Example 1 Example 4: Characterisation of sec-alcohol dehydrogenase "ADH-A" from Rhodococcus ruber DSM 14855 a) Determination of Molecular Weight: The molecular weight is determined by running SDS-polyacrylamide gel electrophoresis and in parallel by a size exclusion column calibrated with standard proteins. The elution of the purified protein on Superdex 200 indicates a molecular mass for the native enzyme of about 62 kDa. In contrast, the SDS-PAGE procedure results in a single band at 38 kDa.
  • the present enzyme displays activity over a broad range of pH values (see Table 4).
  • Presence of Zinc ICP-MS analysis shows the presence of Zn 2+ in the purified enzyme preparation (see Hemmers, B., et al., J. Biol. Chem.275, 35786-35791 (2000), for the method). Whether this is required for catalytic activity and forms a true component of the enzyme is not determined at present. As the enzyme is both active in the presence of complex forming agents such as EDTA and in their absence, either the zinc is bound very firmly or it might not form part of the enzyme or its active site.
  • ADH-A is ideally suited for this simple protocol, which is fully commensurate with the requirements of industrial applications.
  • the purified enzyme is exceptionally stable towards acetone or 2- propanol in up to 10% v/v concentration for cofactor-recy ing in oxidation and reduction, respectively.
  • excellent storage stability no loss of activity after 14 days at +4 °C, or after several months at -80 °C, or in lyophilised form
  • more lipophilic substrates are rapidly transformed due to their enhanced solubility in aqueous/organic systems (as compared to pure aqueous systems).
  • Table 7 Reduction Of 2-methyl-2-hepten-6-one by partially purified enzyme (after Hydrophobic Interaction Chromatography), using 2-propanol as co-substrate (10 ⁇ M substrate, 10 mM NADH, 30 °C; 100 % relative activity. 044 ⁇ mol conversion/mg protein):
  • E is the Enantiomeric ratio (see Chen., C.-S., et al., J. Am. Chem. Soc. 104, 7294-99 (1982) (quotient of the reaction velocities of the isomers) Analogously, the following reactions can take place:
  • Oxidation of: rac-(CH 3 ) 2 C €H-(CH 2 ) 2 -CHOH-CH 3 , rac-n-C 6 H 13 -CHOH-CH 3 , rac-4-phenyl-2- butanol, rac-(E)3-octen-2-on, cyclopentanol, rac-(1-(2-naphthyl)ethanol, rac-1-phenyl-1- ethanol.
  • tryptic peptides are sequenced completely (SEQ ID NO: 1-4), one of them also being found as part of a somewhat larger peptide (SEQ ID NO: 5) with possibly modified N-terminus, and a tryptic peptide with an ambiguity in the N-terminal sequence is found (SEQ ID NO: 6), where [L/l] is leucine or isoleucine and X is an unidentified amino acid:
  • Example 7 Obtaining of nucleotide sequences corresponding to the enzyme "ADH-A" Taking one of the peptide sequences given above in Example 6 or two or more thereof, especially SEQ ID NO: 1, the corresponding (fully degenerate, then preferably only the nucleotides corresponding to 3 to 6 of the amino acids given are used; partially degenerate, then also longer nucleotides can be synthesized, with non-specific nucleotides at positions of ambiguity; or "guessmers” that have only one specific sequence) nucleotide sequence corresponding to (sense sequence) or complementary (antisense) to the nucleotide sequence of the corresponding mRNA, based on the standard genetic code, is synthesized (e.g.
  • oligonucleotide synthesizers for determination of useful oligonucleotides, their purification and use and the preparation of radio-labelled probes, the methods described by Sambrook et al., Molecular Cloning - a Laboratory Manual, Cold Spring Harbor Laboratory Press, pages 11.1 to 11.44 are be used. The labelled oligonucleotide or oligonucleotides are then hybridised (for conditions see Sambrook et al., loc.
  • polymerase chain reaction is used for the preparation of the cDNA (see Lee, C.C., et al., Science 239(4845), 1288-91 (1988)).
  • sequences between a start and a termination codon include the sequence(s) coding for the peptide backbone(s) of a part or the whole enzyme "ADH-A" or homologues displaying similar activity.
  • Example 8 Isolation and characterization of the polynucleotide coding for ADH-A and determination of the corresponding amino acid sequence.
  • a Rhodococcus ruber DSM 44541 DSM 14855 preculture is cultivated in 500 ml standard complex medium (10 g Yeast Extract, Oxoid L21; 10 g Bacteriological Peptone, Oxoid L 37; 10g glucose, Fluka 49150; 2g NaCl, Roth 9265.1; 0,15g MgS0 4 2O, Fluka 63140; 18g Agar, Oxoid L11; 1,3g NaH 2 P0 4 , Fluka 71496; 4,4g K 2 HP0 4 , Merck 5101; all amounts per I itre) overnight at 30 °C and 130 rpm.
  • 500 ml standard complex medium (10 g Yeast Extract, Oxoid L21; 10 g Bacteriological Peptone, Oxoid L 37; 10g glucose, Fluka 49150; 2g NaCl, Roth 9265.1; 0,15g MgS0 4 2O, Fluka 63140; 18g Agar, Oxoid L11; 1,
  • the cell suspension is then centrifuged at 3900xg, washed with 50ml 20% glycerol and pelleted at further time. 1g of the cell pellet is resuspended with 3ml TE25S-Puffer (25mM Tris pH8; 25mM EDTA;0,3M saccharose) and incubated with addition of lysozyme (Sigma L7651 , final concentration 2mg/ml) for 3 h at 37°C.
  • 3ml TE25S-Puffer 25mM Tris pH8; 25mM EDTA;0,3M saccharose
  • lysozyme Sigma L7651 , final concentration 2mg/ml
  • the DNA is incubated with addition of 40 ⁇ g/ml RNase A (Applichem, A3832) 1 h at 37°C and, after that, further incubated with addition of 500 ⁇ g/ml proteinase K (Merck KGaA, #1.24568) 1 h at 3 ⁇ C. After another addition of phenol (see above) and precipitation with propan-2-ol, the purified DNA is dissolved in an appropriate volume of H 2 0.
  • RNase A Applichem, A3832
  • 500 ⁇ g/ml proteinase K Merck KGaA, #1.24568
  • VXAVDXDDDR SEQ ID NO: 49
  • EVGADAAVKSGAGAAGAXR SEQ ID NO: 50
  • XFEWAXAR SEQ ID NO: 51
  • XMEWAXAR SEQ ID NO: 6
  • X in each case stands for leucine or isoleucine and which are identical to the peptides mentioned above and/or deduced from them and published sequences using a data bank, alcohol dehydrogenase sequences are identified that provide partial similarity to these peptides.
  • the sequences taken into consideration for this purpose have the accession numbers NP_631415, ZP_00058234, BAA35108 and CAD36475.1 in or accessible via the NCBI databank (www.ncbi.nlm.nih.gov/).
  • degenerate oligonucleotide primers are deduced from highly conserved regions identified in these sequences.
  • the oligonucleotide primers are then used for a PCR screening using the isolated chromosomal DNA from R. ruber DSM 14855 (obtained as described under A) above).
  • degenerate primers are used for a PCR screening based on the peptide partial sequences described in B1).
  • PepB1-F2 GARGTNGGNGCNGAYGCNGC SEQ ID NO: 36 pepB1-R1 GCSGCGTCSGCGCCNACRTC SEQ ID NO: 37 pepB1-R2 GCNGCRTCNGCNCCNACYTC SEQ ID NO: 38 pepB2-R1 GATSGCGTCSGCSGCNCCSGCNGG SEQ ID NO: 39 pepC-R1 CGSGCSAGGTCSACSACGTCCAT SEQ ID NO: 40 pepC1-F GGNGCNGGNGCNGCNGAYGC " SEQ ID NO: 41 pepC1-R1 GCRTCNGCNGCNCCNGCNCC SEQ ID NO: 42 pepC1-R2 GCCGCCCCGGCGCC SEQ ID NO: 43 pepD-R "CKNGCNARIGCIACIAC SEQ ID NO: 44
  • HotStar-Taq Master-Mix (Qiagen, Hilden, Germany) is used in accordance with the instructions provided by the manufacturer.
  • the Expand l-cosmid vector with an insert capacity of 9-16 kb (Roche Diagnostics GmbH, Mannheim, Germany) is used.
  • the chromosomal DNA from R. ruber DSM 14855 described under A) is restriction digested with Alul (NEB Inc., Beverly, USA) as follows:
  • composition of the incubation mixture is a composition of the incubation mixture:
  • the in vitro packaging and transformation of Escherichia coli DH5 is performed in accordance with the manufadurer's instrudions (Expand-Cloning Kit; Roche Diagnostics GmbH, Mannheim).
  • a total of about 2500 clones is generated and archived in a 384 hole microtiter plate (MTP) format in a total of 6 MTPs in LB-liquid medium.
  • MTP microtiter plate
  • a grid PCR screening employing the nested primer mentioned using the "nested primers'' mentioned under B2), RS1_F1 und Asb_030730_R is performed.
  • the clones of the 6 MTPs obtained under C) are replicated on LB solid medium and the cosmid DNA of the cells of each single MTP is isolated (Cosmid-Midiprap Kit, Qiagen, Langen, Germany).
  • the PCR- analysis with the "nested primers” results in a DNA of MTP2with the expected size.
  • the dones of the horizontal 24 rows are each pooled and analysed by means of colony PCR.
  • a small amount of cell material is applied to the PCR incubation.
  • PCR is performed using the nested primers RS1_F1 (SEQ ID NO:45) and Asb_030730_R (SEQ ID NO:46) under the conditions described below.
  • the clones of the positive rows 15 and 16 are screened one by one by means of colony PCR (see above). By this procedure, the clones of the coordinates H15 and H16 are identified as positive. The cosmid DNA of these clones is isolated subsequently and used for sequencing.
  • sequences responsible for the ADH-A activity can be obtained both on the polynucleotide as well as peptide level.
  • Example 9 Whole cell catalysts with recombinant ADH-A activity
  • nucleotide sequence of ADH-A is cloned in an expression vector in bacterial cells.
  • clones derived from DSM 44541 Rhodococcus ruber are selected that show high ADH-A activity.
  • the activity of clones as whole cell catalysts shows up to 100 fold increase in activity compared to the wildtype.
  • the activity can be improved by a further factor of approximately two if the oxidation reaction takes place in the presence of 1 mM NAD + or the reduction reaction takes place in the presence of 1 mM NADH under otherwise the same reaction conditions as described in the preceding examples for the enzyme assay.
  • Rhodococcus ruber DSM 44541 now Rhodococcus ruber DSM 14855.
  • the strain has the following properties:
  • Morphology elementary ramification rod/coccus growth cycle.
  • Biochemical properties fatty acids: 5-30 % 16:0; 5-15 % 16:1; 5-15 % 18:0; 15-30 % 18:1;
  • Rhodococcus ruber DSM 14855 is isolated from the lower Rhine on hexane as sole carbon source.
  • the strains can be kept on a complex culture medium as described above at 30 °C and 130 rpm in L-shaking flasks with flow spoiler. After centrifugation, the pellet can be taken up in Tris-HCI buffer (pH 7.5, 50 mM), shock-frozen in liquid nitrogen and lyophilised, where desired and appropriate.

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Abstract

La présente invention se rapporte à des biocatalyseurs déployant une activité d'alcool-déhydrogénases, que l'on obtient à partir de Rhodococcus ruber, à leur préparation, à leur utilisation dans l'oxydation des alcools secondaires et/ou la réduction des cétones, et à des acides nucléiques codant pour lesdites alcool-déhydrogénases et à des micro-organismes transformés par les acides nucléiques codant pour les biocatalyseurs précités, et à l'utilisation de ces derniers dans la production du biocatalyseur ou dans l'oxydation des alcools secondaires et/ou la réduction des cétones.
PCT/EP2004/052095 2003-09-18 2004-09-08 Alcool-dehydrogenases dotees d'une stabilite amelioree aux solvants et a la temperature Ceased WO2005026338A1 (fr)

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DE102006009743A1 (de) * 2006-03-02 2007-09-06 Wacker Chemie Ag Verfahren zur Oxidation von sekundären Alkoholen durch Enzyme
WO2009131040A1 (fr) * 2008-04-25 2009-10-29 財団法人地球環境産業技術研究機構 Bactéries corynéformes génétiquement modifiées capables de produire de l’isopropanol
JP2011205921A (ja) * 2010-03-29 2011-10-20 Mitsubishi Rayon Co Ltd ロドコッカス（Ｒｈｏｄｏｃｏｃｃｕｓ）属細菌組換体及びそれを用いた光学活性（Ｒ）−３−キヌクリジノールの製造方法
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US8765433B2 (en) 2009-12-29 2014-07-01 Butamax Advanced Biofuels Llc Alcohol dehydrogenases (ADH) useful for fermentive production of lower alkyl alcohols
US8865439B2 (en) 2008-05-01 2014-10-21 Genomatica, Inc. Microorganisms for the production of methacrylic acid
US8889399B2 (en) 2007-03-16 2014-11-18 Genomatica, Inc. Compositions and methods for the biosynthesis of 1,4-butanediol and its precursors
US8993285B2 (en) 2009-04-30 2015-03-31 Genomatica, Inc. Organisms for the production of isopropanol, n-butanol, and isobutanol
US9017983B2 (en) 2009-04-30 2015-04-28 Genomatica, Inc. Organisms for the production of 1,3-butanediol
US9023636B2 (en) 2010-04-30 2015-05-05 Genomatica, Inc. Microorganisms and methods for the biosynthesis of propylene
US9175297B2 (en) 2008-09-10 2015-11-03 Genomatica, Inc. Microorganisms for the production of 1,4-Butanediol
US9222113B2 (en) 2009-11-25 2015-12-29 Genomatica, Inc. Microorganisms and methods for the coproduction 1,4-butanediol and gamma-butyrolactone
US9260729B2 (en) 2008-03-05 2016-02-16 Genomatica, Inc. Primary alcohol producing organisms
US9434964B2 (en) 2009-06-04 2016-09-06 Genomatica, Inc. Microorganisms for the production of 1,4-butanediol and related methods
US9562241B2 (en) 2009-08-05 2017-02-07 Genomatica, Inc. Semi-synthetic terephthalic acid via microorganisms that produce muconic acid
US9677045B2 (en) 2012-06-04 2017-06-13 Genomatica, Inc. Microorganisms and methods for production of 4-hydroxybutyrate, 1,4-butanediol and related compounds
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