WO2003031625A1 - Esterase esta issue de rhodococcus ruber - Google Patents

Esterase esta issue de rhodococcus ruber Download PDF

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WO2003031625A1
WO2003031625A1 PCT/EP2002/011213 EP0211213W WO03031625A1 WO 2003031625 A1 WO2003031625 A1 WO 2003031625A1 EP 0211213 W EP0211213 W EP 0211213W WO 03031625 A1 WO03031625 A1 WO 03031625A1
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polypeptide
esterase
nucleic acid
seq
enzyme
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PCT/EP2002/011213
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German (de)
English (en)
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Helmut Schwab
Andreas Lidauer
Marinka Gudelj-Wyletal
Michaela Pressnig
Kurt Faber
Mateja Pogorevc
Harald Trauthwein
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Degussa Ag
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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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C12P7/625—Polyesters of hydroxy carboxylic acids
    • 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
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6418—Fatty acids by hydrolysis of fatty acid esters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/003—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions
    • C12P41/004—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions by esterification of alcohol- or thiol groups in the enantiomers or the inverse reaction
    • 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
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • 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
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12P7/62—Carboxylic acid esters
    • 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
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6458—Glycerides by transesterification, e.g. interesterification, ester interchange, alcoholysis or acidolysis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to an esterase from Rhodococcus ruber, proteins homologous to it, nucleic acids coding for these proteins, antibodies against these proteins and the production and use of these proteins, nucleic acids and antibodies.
  • Hydrolases are a large and diverse group of enzymes that can hydrolyze peptides, amides and halides in addition to esters.
  • the lipolytic and esterolytic enzymes represent a special class, whereby it is difficult to differentiate the esterases (EC 3.1.1) from the lipases, since the definition of these enzymes is not based on the structure of their substrates, but rather on their physical state.
  • the classification of the esterases as opposed to the lipases is usually based on the length of the acyl chain of their ester substrates. Esterases preferably show activity in relation to water-soluble, short-chain fatty acid esters, while lipases show higher activity in relation to water-insoluble, emulsified long-chain fatty acid substrates [1, 2].
  • the family of alpha / beta hydrolase-folded enzymes includes a group of proteins that share a three-dimensional core structure, although no significant similarity was found in the primary structure. While the catalytic specificities of the members of the alpha / beta hydrolase-folded enzymes are completely different from one another, their enzymatic mechanism appears to be similar [12]. All have a catalytic triad with the nucleophilic acid histidine configuration consecutive in the amino acid sequence. These three amino acids are located at similar topological locations in the correctly folded protein, although they are separated by a variable number of amino acids in the primary structure of each enzyme.
  • nucleophile In all known family members of these hydrolases, the nucleophile, usually a serine residue, is localized in a conserved sequence, the “nucleophilic elbow”, with a proposed consensus region Sm-X-Nu-X-Sm-Sm, where Sm is a small amino acid , usually glycine, X stands for any amino acid and Nu for the nucleophile, which is a structural commonality of the alpha / beta hydrolase-folded enzymes [12].
  • Esterases have become increasingly important in biotechnology [13] and in organic chemistry [14, 15]. Their characteristic properties, such as substrate specificity, regioselectivity and enantioselectivity, allow a wide range of applications for these enzymes. They are very common in nature and can be found in animals, plants and microorganisms. Because of their industrial applications, intensive research interest has recently focused on microbial esterases.
  • linaloyl acetate or linalyl acetate an ester of an allylic tertiary alcohol.
  • the esterified alcohol, linalool is one of the most important terpene alcohols in the taste and smell industry and is available in different enantiomeric forms. The (r?
  • the object of the present invention was to provide a new enzyme with hydrolytic activity, in particular a new esterase and / or lipase, for which there is an urgent need in view of the possible uses of these enzymes, in particular in biotechnology and organic chemistry, and / or to provide a method by means of which new hydrolytic enzymes, especially esterases and / or lipases, can be obtained.
  • the object of the present invention was in particular to provide a hydrolytic enzyme, in particular an esterase and / or lipase, which has a different substrate specificity and / or a distinguishable degree of selectivity and / or a distinguishable reaction rate and compared to the esterases and lipases described so far / or has a distinguishable structure and / or a distinguishable reaction mechanism.
  • Another object of the present invention was to provide esterases and / or lipases which are able to cleave bulky esters and / or sterically difficult to access ester linkages, in particular the sterically difficult to access ester bond in linaloyl acetate.
  • polynucleotides, polypeptides and antibodies according to the invention in particular by polynucleotides according to SEQ ID No. 1 and SEQ ID No. 3, polypeptides according to SEQ ID No. 2 and SEQ ID No. 4 and by against polypeptides according to SEQ ID No. 2 and SEQ ID No. 4 targeted antibodies.
  • nucleic acids according to SEQ ID No. 1 and SEQ ID No. 3 or starting from polypeptides according to SEQ ID No. 2 and SEQ ID No. 4 nucleic acids and polypeptides of similar structure and function or can be obtained.
  • enzymes with improved properties can also be obtained, for example by evolution experiments.
  • the present invention therefore relates to a polynucleotide selected from the group consisting of: a) polynucleotide with a nucleic acid sequence according to SEQ ID NO. 1 or according to SEQ ID NO. 3, b) Polynucleotides coding for a polypeptide with an amino acid sequence according to SEQ ID NO. 2 or according to SEQ ID NO.
  • polynucleotides with a sequence homology or identity of at least 50%, preferably at least 60% or 70%, particularly preferably at least 80%, very particularly preferably at least 90% or 95%, in particular at least 98%, in relation to a polynucleotide according to (a), (b),
  • polynucleotides consisting of at least 10 or 15, preferably at least 20 or 25, particularly preferably at least 30 or 40, very particularly preferably 50 or 80, in particular at least 100 or 120 successive nucleotides of a polynucleotide according to ( a), (b), (c),
  • the nucleic acids according to the invention comprise one or more non-coding sequences, the non-coding sequences being, for example, naturally occurring intron sequences or regulatory sequences such as promoter or enhancer sequences, in particular those for controlling expression hydrolytic enzymes, especially esterases or lipases.
  • the nucleic acids according to the invention are preferably ribonucleic acids (RNAs) or deoxyribonucleic acids (DNAs), the nucleic acids preferably being double-stranded nucleic acids.
  • RNAs ribonucleic acids
  • DNAs deoxyribonucleic acids
  • the nucleic acids according to the invention are preferably nucleic acids which code for a protein with hydrolase activity or parts thereof.
  • the protein with hydrolase activity is preferably a lipase or esterase and / or an enzyme which is capable of ester, Cleave thioester, amide, halide or peptide bonds, particularly preferably bulky and / or sterically difficult to access ester, thioester, amide, peptide or halide bonds.
  • the compounds are, for example, esters, thioesters, peptides, amides or halides with a short acyl chain, the short acyl chain preferably referring to the acyl radical of acetic acid, propionic acid, butyric acid or valeric acid. It can very particularly preferably be an ester of a tertiary alcohol, especially an allylic tertiary alcohol, especially the ester of linalool or licareol or coriandrol, especially linaloyl acetate.
  • cleavage means above all hydrolytic cleavage, that is to say cleavage with liberation of the carboxylic acid and the corresponding alcohol, thiol, amine or hydrogen halide.
  • cleavage can also take place with the formation of a new ester, thioester, amide, peptide or halide bond, in particular if, for example, an ester bond is converted into a new ester bond, it can be a transesterification reaction act.
  • the esterase is very particularly preferably a serine esterase and / or an enzyme which is capable of cleaving regioselective or enantioselective bonds, the value for the enantiomeric excess (ee) preferably being greater than or equal to 20% , is particularly preferably greater than or equal to 40%, especially greater than or equal to 60%, in particular greater than or equal to 80%.
  • the esterase is very particularly preferably a hydrolase with an ⁇ / ⁇ -hydrolase fold and / or a serine esterase and / or a carboxylesterase, in particular one which belongs to the GDXG family of lipolytic enzymes or is homologous to these enzymes.
  • the nucleic acids according to the invention are preferably nucleic acids which are here for an esterase, particularly preferably for a microbial esterase, particularly preferably bacterial esterase, in particular for an esterase from Rhodococcus, in particular for an esterase from Rhodococcus ruber especially for the esterase EstA according to SEQ ID NO. 2, for the esterase according to SEQ ID NO. 4 and / or one of the polypeptides according to the invention described below.
  • the present invention furthermore relates to the use of the nucleic acids according to the invention, on the one hand for the production or isolation of nucleic acids according to the invention, on the other hand for the production or isolation of new nucleic acids which are homologous to the nucleic acids according to the invention, in particular those having structural and the same in relation to the nucleic acids according to the invention , similar and / or improved functional properties, with functional properties in particular hydrolytic activity, especially lipase and / or esterase activity, and improved functional properties, for example higher specificity and / or higher conversion and / or higher regio- or enantioselectivity understand is.
  • nucleic acids according to the invention can thus be used, for example, as probes for the identification and / or isolation of homologous nucleic acids from an artificial, a cDNA or genomic gene bank, preferably for the identification of nucleic acids for hydrolases, especially esterases or lipases, and / or parts thereof code, or as antisense nucleic acids or as primers in the polymerase chain reaction (PCR), in particular for the amplification of nucleic acids comprising nucleic acids coding for enzymes with hydrolytic activity, in particular esterases and / or lipases, or parts thereof.
  • PCR polymerase chain reaction
  • nucleic acids according to the invention or homologous to the nucleic acids according to the invention can also be obtained by random mutagenesis or targeted mutagenesis in a manner known to the person skilled in the art.
  • the nucleic acids according to the invention can be used, for example, for the targeted production of individual domains or epitopes of the protein according to the invention or of fusion proteins which comprise the polypeptides according to the invention.
  • the present invention therefore also relates to a process for obtaining a nucleic acid which codes for a hydrolytic enzyme, in particular an esterase and / or lipase, comprising the following steps: a) a nucleic acid library is contacted with a nucleic acid according to the invention, b) one Nucleic acid that hybridizes with a nucleic acid according to the invention is identified, c) the nucleic acid identified in step (b) is sequenced.
  • the present invention therefore also relates to a method for isolating a nucleic acid coding for a hydrolytic enzyme, in particular for an esterase and / or lipase, comprising the following steps: a) primers are prepared starting from a nucleic acid according to the invention, b) the primers according to (a) are used to amplify nucleic acids, especially cDNAs, of unknown nucleic acid sequences in the PCR, c) the nucleic acids obtained according to (b) are sequenced.
  • the present invention therefore also further relates to a method for isolating a nucleic acid coding for a hydrolytic enzyme, in particular an esterase and / or lipase, comprising the following steps: a) Nucleic acids from a library are incorporated into suitable vectors and these are preferably incorporated into suitable host organisms Bacteria, in particular E.
  • nucleic acids from the clones identified in step (d) are sequenced.
  • the present invention therefore furthermore also relates to a method for isolating a nucleic acid coding for a hydrolytic enzyme, in particular an esterase and / or lipase, comprising the following steps: a) a nucleic acid according to the invention is subjected to mutagenesis experiments, b) the mutants obtained incorporated and expressed in a suitable vector, c) the expression products are examined for hydrolytic activity, in particular esterase and / or lipase activity, d) the nucleic acids whose expression products show hydrolytic activity in step (c) are optionally sequenced.
  • the polymerase chain reaction can be used to carry out the mutagenesis experiments.
  • the PCR experiments can be carried out, for example, in particular when using a Taq polymerase, in such a way that reaction parameters such as the Mg 2+ concentration, the pH value, the reaction temperature or the substrate concentrations are varied, or error can be used -prone-PCR techniques, which are based, for example, on the addition of Mn 2+ or on the addition of unequal nucleotide concentrations.
  • the nucleic acid library is preferably a cDNA, genomic or artificial library, particularly preferably a microbial, especially bacterial library, in particular one from Rhodococcus, particularly preferably from Rhodococcus ruber.
  • the invention further relates to a nucleic acid which can be obtained by one of the aforementioned methods.
  • Another object of the present invention is a method for producing a nucleic acid according to the invention, characterized in that the nucleic acid is chemically synthesized.
  • the nucleic acids according to the invention can be chemically described, for example, in 1 or SEQ ID No. 3 specified nucleic acid sequences or using the in SEQ ID No. 2 or SEQ ID No 4 specified amino acid sequences can be synthesized based on the genetic code, for example according to the phosphotriester method.
  • the present invention furthermore relates to a vector, in particular a cloning and / or expression vector, comprising one of the aforementioned nucleic acids.
  • the expression vector can be, for example, a prokaryotic or eukaryotic expression vector.
  • the prokaryotic vectors for incorporating the nucleic acids according to the invention are, for example, the plasmids pBSII, pGEM-5Zf (+/-), pSK, pBluescript SKII (-) or pMS470 ⁇ 8 or another high copy number plasmid.
  • the available expression vectors for expression in E. coli are, for example, the vectors pBSG25, pBSS12 or pMSRul for expressing a protein according to SEQ ID No. Second
  • the expression vectors for expression in E. coli can, for example, also be other commercially available vectors, such as the T7 expression vector pGM10 or pGEX-4T-1 GST (Pharmacia Biotech), which are used for an N-terminal Met- Coding Ala-His6-Tag, which enables the purification of the expressed protein on a Ni 2+ -NTA column.
  • suitable eukaryotic expression vectors for expression in Saccharomyces cerevisiae are vectors p426Met25 or p426GAL1, for expression in insect cells, for example baculovirus vectors as disclosed in EP-B1-0127839 or EP-B1-0549721, and for expression in mammalian cells, for example SV40 vectors ,
  • the nucleic acids according to the invention can be incorporated into a vector with flanking nucleic acids in such a way that when the vector is expressed, the polypeptides encoded by the nucleic acids according to the invention are present as fusion proteins or carry a tag, a labeling amino acid sequence, which, for example, purifies or detects the Can lighten polypeptides.
  • the day can be, for example, the strep, flag, myc or his day.
  • the expression vectors preferably contain the regulatory sequences suitable for the host cell, in this case preferably the lac or tac promoter for expression in E. coli, the ADH-2, GAL1 or AOX promoter for expression in yeast, the baculovirus -Polyhedrin promoter for expression in insect cells or the early SV40 promoter or LTR promoters for expression in mammalian cells.
  • the regulatory sequences suitable for the host cell in this case preferably the lac or tac promoter for expression in E. coli, the ADH-2, GAL1 or AOX promoter for expression in yeast, the baculovirus -Polyhedrin promoter for expression in insect cells or the early SV40 promoter or LTR promoters for expression in mammalian cells.
  • the present invention furthermore relates to a host cell comprising a vector according to the invention, the host cell preferably being Rhodococcus, in particular Rhodococcus ruber, or E. coli, in particular the E. co // ' strain BL21 (DE3).
  • the host cell preferably being Rhodococcus, in particular Rhodococcus ruber, or E. coli, in particular the E. co // ' strain BL21 (DE3).
  • nucleic acids according to the invention are preferably introduced into the host cells after incorporation into a suitable vector by the methods of transfection, transformation or electroporation known to the person skilled in the art.
  • the present invention furthermore relates to a polypeptide selected from the group consisting of: a) polypeptide with an amino acid sequence according to SEQ ID NO. 2 or according to SEQ ID NO. 4, b) naturally occurring mutants, polymorphic forms or alleles of a polypeptide according to (a), in particular those which have up to ten, preferably up to five, especially exactly one, two or three, point mutations with respect to a polypeptide according to (a) possess, c) polypeptides which have a sequence homology or identity of at least 50%, preferably at least 60% or 70%, very particularly preferably at least 80 or 90%, in particular at least 95% or 98% in relation to a polypeptide according to (a) or (b) have d) polypeptides which are encoded by the aforementioned nucleic acids according to the invention, e) polypeptides consisting of at least 5 or 6, preferably at least 10 or 15, particularly preferably at least 20 or 30, in particular at least 40, 50 or 60 consecutive amino acids of a
  • polypeptides which catalyze a hydrolysis reaction, in particular the hydrolysis of ester, thioester, amide, peptide or halide bonds, or parts, in particular epitopes or domains, of such polypeptides.
  • enzymes which have a catalytic triad comprising a nucleophile, an aspartate or glutamate and a histidine, or a catalytic diad in their reactive center.
  • esterases or lipases are preferably esterases or lipases, particularly preferably a serine esterase and / or a carboxyl esterase which belongs to the GDXG family of lipolytic enzymes or has strong homology to the enzymes of this family, the serine being active Center (S *) is preferably found in the amino acid sequence IVLGGDS * AGGNLA (SEQ ID NO 5), and / or it is an enzyme which has alpha / beta hydrolase folding.
  • the compounds which can be cleaved by the polypeptides according to the invention are, for example, esters, thioesters, peptides, amides or halides, preferably those with a short acyl chain, the acyl radical in particular having a short acyl chain Acetic acid, propionic acid, butyric acid or valeric acid is meant.
  • the cleavable compound is very particularly preferably the ester of a tertiary alcohol, especially an allylic tertiary alcohol and / or a tertiary alcohol, the tertiary carbon atom of which carries an aromatic or unsaturated hydrocarbon group, especially a phenyl radical.
  • the polypeptides according to the invention are particularly preferably esterases, preferably microbial, in particular bacterial, esterases. It is very particularly preferably an esterase from Rhodococcus ruber, in particular the esterase EstA according to SEQ ID No. 2 or a mutant of this esterase according to SEQ ID No. 4th
  • the polypeptide according to the invention is a water-soluble esterase.
  • the present invention furthermore relates to mixtures and preparations, in particular bacterial preparations, which can be carried out in a manner known to the person skilled in the art, comprising a polypeptide according to the invention.
  • Another object of the invention is the use of a polypeptide according to the invention for hydrolytic cleavage and / or for the formation of ester, thioester, amide, peptide or halide bonds.
  • the compounds to be cleaved or prepared are those with a short acyl chain, very particularly preferably the ester of a tertiary alcohol, especially an allylic tertiary alcohol and / or a tertiary alcohol, the tertiary carbon atom of which is one unsaturated or aromatic hydrocarbon group, especially a phenyl group.
  • ester of linalool or licareol or coriandrol in particular linaloyl acetate, or an ester of 2-phenyl-2-butanol, 2-phenyl-2-hexanol or 2-phenyl-2-octanol, especially 2-phenyl-2-butyl acetate, 2-phenyl-2-hexyl acetate or 2-phenyl-2-octyl acetate.
  • “Short acyl chain” according to the invention is preferably a carboxylic acid residue with no more than 6 carbon atoms, particularly preferably with no more than 4 carbon atoms, in particular an acetate, propionate, butyrate or valerate residue, the hydrogen atoms of the The rest can also be substituted, in particular by organic groups or halogen atoms,
  • the alcohol residue of the ester according to the invention is, for example, naphthol, glycerol, p- and o-nitrophenol or a naphthol AS substrate.
  • Substrates or compounds to be prepared according to the invention are generally compounds of the formula R 1 -C (0) -R 2 , where R 2 for one of the halogens F, Cl, Br, I, for an amino group NR 3 R 4 , for a thiol SR 3 or stands for an alcohol OR 3 and R 1 is preferably a hydrocarbon chain, which can preferably have up to 5, particularly preferably up to 3 carbon atoms, especially exactly 1 carbon atom, the hydrocarbon chain preferably being unbranched, however can also be branched or unsaturated and at least one of the hydrogen atoms can also be substituted by other groups, such as, for example, halogens, hydrocarbon radicals or ether groups.
  • the alcohol, thiol or amine is preferably a bulky group.
  • R 3 can thus be, for example, a group CR 5 R 6 R 7 , at least one, in particular exactly one, of the radicals R 5 , R 6 and R 7 being a bulky hydrocarbon skeleton, for example an aromatic group, which also can be substituted.
  • the aromatic group can also be a heterocycle.
  • the remaining radicals, which are not a bulky group and / or aromatics, are preferably a hydrogen atom or a short-chain hydrocarbon chain with up to 8, particularly preferably up to 6, carbon atoms, at least one the C atoms can also carry a substituent, for example a halogen atom.
  • the hydrocarbon chain can be unbranched, branched, saturated or unsaturated.
  • R 3 itself can also be an aromatic, especially one heterocyclic aromatics act, the hydrogens can also be substituted.
  • Substrates or compounds to be prepared according to the invention are furthermore compounds of the formula R 1 -C (0) -R 2 , where R 1 is a sterically demanding group, especially a quaternary carbon atom, which is preferably a center of chroma.
  • R 1 is a sterically demanding group, especially a quaternary carbon atom, which is preferably a center of chroma.
  • This can be, for example, the alpha-C atom of a substituted alpha-amino acid or a carbon atom as part of a hexane ring, the hexane ring preferably carrying at least one further substituent, for example in the form of a hydrocarbon chain or a halogen atom.
  • the quaternary carbon atom can carry, for example, a hydrocarbon chain, in particular a methyl or an ethyl group.
  • R 2 is preferably a simple group, for example an amine or an alcohol residue, which comprises up to 6, especially up to 4, carbon atoms, the alcohol residue preferably being unbranched and unsaturated , but can also be branched or unsaturated.
  • the substrate or the compound to be produced is a glycerol derivative, in a preferred embodiment all three hydroxyl groups, preferably with short-chain acids, are esterified.
  • the substrate is triacetin.
  • the substrates to be cleaved or prepared by means of the polypeptides according to the invention are those of the general formula R 1 -C (0) -R 2 , where R 1 is for (C 1 -C 6 ) -alkyl, in particular ( CrC 3 ) -alkyl, which may optionally be mono- or polysubstituted, in particular by radicals from the group consisting of (-CC 6 ) -alkyl, in particular (-C-C 3 ) -alkyl, (-C-C 6 ) Alkoxy, in particular (CrC 3 ) alkoxy, halogen, in particular fluorine, chlorine, bromine or iodine, and where R 2 is halogen, in particular fluorine, chlorine, bromine or iodine, or for a radical selected from the group OR 3 , SR 3 and NR 3 stands, wherein R 3 is (C 6 -C 14) -aryl, in particular pheny
  • R 3 is particularly preferably an alkyl or alkenyl radical which bears two substituents in the 1 position or one substituent in the 1 position and one substituent in the 2 position, at least one of the substituents preferably being one Alkenyl radical or an aryl radical and the other substituent is preferably an alkyl radical.
  • the hydrolysis and / or bond formation can be carried out, for example, in the presence of BSA and / or in the presence of a detergent, in particular a nonionic, such as e.g. Octyl glucoside.
  • a detergent in particular a nonionic, such as e.g. Octyl glucoside.
  • the enzymes according to the invention are used to cleave or form enantioselectively bonds, the enantiomeric excess (ee) of the reaction in question preferably being greater than or equal to 20%, particularly preferably greater than or equal to 40%, especially greater than or equal to 60, in particular is greater than or equal to 80%.
  • the polypeptides according to the invention can also be used to isolate or enrich one of the two enantiomers starting from racemic mixtures.
  • the knotting reaction can be, for example, a transesterification reaction.
  • Possible industrial applications of the polypeptides according to the invention are, for example, the production or selective enrichment of flavor components in the food industry, detergents in the detergent industry, fine chemicals in the chemical industry or therapeutically or diagnostically usable substances in the pharmaceutical industry.
  • the present invention also relates to a method for cleaving the aforementioned ester, thioester, amide, peptide or halide bonds, characterized in that molecules comprising the corresponding bonds are incubated with at least one polypeptide according to the invention.
  • the present invention furthermore also relates to a process for forming the aforementioned ester, thioester, amide, peptide or halide bonds, characterized in that carboxylic acids and / or carboxylic acid esters, thioesters, amides or halides with a Alcohol, thiol, amine or hydrogen halide can be incubated in the presence of a polypeptide according to the invention.
  • polypeptides according to the invention can furthermore be used, for example, as epitopes for the production of mono- or polyclonal antibodies by coupling them to a carrier, for example bovine serum albumin, and then using a mammal, preferably a mouse, rabbit or rabbit Epitope, preferably using adjuvants, is immunized.
  • a carrier for example bovine serum albumin
  • Polypeptides with a length of 6-12, in particular 8, amino acids are preferably suitable for this.
  • Polypeptides with a length of more than 60, in particular more than 75, amino acids can also be used without a carrier for the production of antibodies.
  • the resulting antibodies can then optionally be isolated, and antibody fragments, for example Fab or scFv fragments, can optionally be prepared starting from the antibodies or the nucleic acids coding for them.
  • Peptides that bind to a polypeptide according to the invention can alternatively also be produced by an in vitro method known to the person skilled in the art, such as phage Display, yeast display, bacterial display or the so-called Fusagen technology are obtained, in which the nucleic acid and the polypeptide encoded by it are covalently linked to one another via a puromycin.
  • the antisera, antibodies and antibody fragments obtainable by immunization with the polypeptides according to the invention and the peptides obtainable by one of the in vitro methods mentioned are suitable, for example, for examining gene expression banks in order to make proteins homologous to the polypeptides according to the invention, especially those with hydrolytic activity, especially esterolytic and / or lipolytic activity.
  • the present invention therefore also relates to antisera, antibodies and antibody fragments against a polypeptide according to the invention and other peptides which bind to a peptide according to the invention, in particular obtainable by one of the aforementioned methods.
  • Antiserum, antibodies and antibody fragments as well as other peptides that bind to a peptide according to the invention are called “antibodies” below for the sake of simplicity.
  • the present invention further relates to a method for producing a polypeptide according to the invention, characterized in that a nucleic acid according to the invention in a suitable host cell, in particular in E. coli, especially in E. coli BL21 (DE3), or in Rhodococcus, especially Rhodococcus ruber, is expressed. This can then be followed by purification, especially chromatographic purification of the protein.
  • the method can be carried out, for example, in the following manner: a) a nucleic acid according to the invention is incorporated into a suitable vector and E. coli is transformed with the vector, b) after the expression of the protein, the cells are harvested and disrupted, c) the protein becomes optionally purified from the supernatant thus obtained.
  • E. coli is preferably cultivated between 15 and 40 ° C, particularly preferably between 20 and 37 ° C, especially at about 30 or about 37 ° C. If induction of the expression of the nucleic acids according to the invention takes place, for example, by adding IPTG or lactose, the cells are preferably allowed to grow for between 3 and 15 hours, particularly preferably for between 5 and 10 hours, after induction of the expression. Alternatively, induction of expression can also be dispensed with.
  • the harvested cells are disrupted by a method known to the person skilled in the art, for example French press, ultrasound, ball mill, but particularly preferably by using a sonifier.
  • the cells can also be chemically permeabilized, for example by EDTA or polymyxin B.
  • the protein to be purified is preferably chromatographically. Chromatographic purification of the protein can include, for example, cation and / or anion exchange chromatography, hydrophobic interaction chromatography and / or gel filtration.
  • short-chain polypeptides according to the invention can also be synthesized using classic peptide synthesis (Merrifield technique).
  • the invention furthermore relates to a test system for identifying substrates or functional interactors containing at least one nucleic acid according to the invention and / or at least one polypeptide according to the invention and / or at least one antibody according to the invention and optionally suitable auxiliaries and / or additives.
  • one or more nucleotides of the nucleic acids according to the invention or one or more amino acids of the polypeptides according to the invention or the antibodies according to the invention can also be modified.
  • the modification can be a radioactive, fluorogenic or chromogenic group or a post-translational modification.
  • 1 schematically shows substrate types for lipases and esterases.
  • the chirality center is marked with the star.
  • FIG. 5 shows a schematic overview of the production of the plasmid pMSRul.
  • FIG. 7 shows an agarose gel with a restriction analysis of the esterase-positive clones.
  • the clones were digested with ßamHI.
  • tracks 1 and 21 was H / ndlll standard plotted, in clues 2-20 and 22-27 the clones of the genomic library (G2-G20, G22-G27).
  • Figure 8 shows a native polyacrylamide gel from esterase positive clones.
  • Supernatant from the Rhodococcus ruber preparation (RR1) was applied in lane 1, supernatant (XL1 S ) and pellet (XL1 P ) from E. coli XU Blue cells with the plasmid pBS SKII as negative control in lanes 2 and 3, in the others Supernatants from the clones of the genomic library were recorded.
  • the native gel was stained using alpha-naphthyl acetate / Fast Blue.
  • FIG. 10 shows a native polyacrylamide gel for the detection of esterase activity in the subclones.
  • Raw lysates of E. coli XL1 Blue, the subclones and Rhodococcus ruber were applied. Linalyl acetate was used as the substrate.
  • FIG. 12 shows part of the sequence of the insert pMSS12 (adjacent to the T3 region of the vector; SEQ ID NO. 6) and the protein encoded by it (SEQ ID NO. 7/8).
  • Position 1-244 codes for the C-terminus of an aldehyde dehydrogenase (SEQ ID NO. 7), the stop codon is at position 245.
  • At position 325 there is a start codon ATG with an upstream Shine-Dalgarno sequence (AGGA), a stop codon follows at position 760.
  • FIG. 13 shows part of the sequence of the insert pMSS12 (adjacent to the T7 region of the vector; SEQ ID NO. 9) and the protein encoded by it (SEQ ID NO. 2). The start and stop codons of the esfcA gene and the Shine-Dalgarno sequence are marked.
  • FIG. 14 shows the sequence of the ORF of the estA gene and the associated amino acid sequence of the esterase EstA (SEQ ID NO. 2).
  • the plasmid pMSRul which comprises the estA gene, is shown schematically in FIG.
  • FIG. 17 shows growth curves of XL1 blue cells containing the vector pMSRul at 37 ° C., one induction using 0.1 mM IPTG (squares) and the other no induction (circles).
  • FIG. 18 An SDS polyacrylamide gel is shown in FIG. 18, and a native gel of supernatant (S) and pellet (P) fractions from XL1 blue cells containing the plasmid pMSRul is shown in FIG. 19. Fermentation took place at 37 ° C. "1" means that there was induction, "2" that there was no induction. Total lysate from XL1 blue cells with the vector pBluescript without insert was used as negative control “n.c.”. The native gel was stained using alpha-naphthyl acetate / Fast Blue.
  • FIG. 20 shows the results for the investigation of a possible influence of sonification on the state of the protein, the investigation in FIG. 21 using an SDS gel and in FIG. 22 using a native gel.
  • the protein was renatured and then stained using ⁇ -naphthyl acetate and Coomassie Brilliant Blue.
  • the bacteria were fermented at 30 ° C (LMW: low molecular weight standard, S: supernatant, P: pellet; 1: pMS470 ⁇ 8 E.coli XL1 Blue, 2: pMS470 ⁇ 8 induced, 3: pMSRul E.coli XL1 Blue, induced and standard sonification, 4: induced and twice the sonification time, 5: no induction and standard sonification, 6: not induced and twice the sonification time).
  • LMW low molecular weight standard
  • S supernatant
  • P pellet
  • 1 pMS470 ⁇ 8 E.coli XL1 Blue
  • 2 pMS470 ⁇ 8 induced
  • 3 pMSRul E.coli XL1 Blue
  • induced and standard sonification 4: induced and twice the sonification time
  • 5 no induction and standard sonification
  • 6 not induced and twice the sonification time
  • 25 shows the different expression levels of the clone pMSRul at different growth times using an SDS gel: (LMW: low molecular weight standard, S: supernatant, P: pellet; induction times: 1: 4.5 h, 2: 8.5 h, 3 : 19.5 h, 4: no induction)
  • LMW low molecular weight standard
  • S supernatant
  • P pellet
  • FIG. 28 shows the results for the investigation of the specific activity of supernatant (S), pellet (P) and lysate (L) fractions from fermentations of the clone pMSRul. o-Nitrophenylacetate was used as the substrate.
  • FIG. 29 shows the results for the investigation of the specific activity of the clone pMSRul in comparison to the activity of the mutant F3, using fractions in which the expression had been induced by IPTG (induced) or in which no addition of IPTG took place.
  • the supernatants from ultracentrifuged lysates were analyzed.
  • FIG. 30 shows the results for the investigation of the specific activity of the esterase EstA, which had been produced at 25 ° C., 700 rpm.
  • S stands for substrate
  • LA for linaloyl acetate, ind. means that protein expression had been induced by adding IPTG.
  • FIG. 31 shows the results for the investigation of the specific activity on the one hand of digested cells and on the other hand of non-digested cells.
  • rac-linal acetate (LA) was used as the substrate.
  • UV lamp 254 nm for analytical agarose gels, 302 nm for preparative gels; Perkin Eimer Lambda Bio UV ⁇ / IS spectrometer; Beckman DU-50 spectrophotometer; Branson Sonifier 250; Perkin Elmer Thermal Cycler, Gene Amp PCR System 2400; BIO-RAD Mini Protean II TM Cell; MWG-BIOTECH electrophoresis chamber; HT INFORS AG incubator; Eppendorf Thermomixer comfort, 1.5 ml; refrigerated centrifuges: Hettich MIKRO RAPID / K and JOUAN BR4; cooled super speed centrifuge: Sorvall ® RC-5B (GSA rotor); Sorvall ® Ultra Centrifuge, Combi; ORGANIC BIKE SLAB DRIER MODEL 483; Applied Biosystems 392 DNA / RNA synthesizer and 373A DNA sequencer
  • E. coli SURE ® Stratagene (La Jolla, CA, USA) e14- (mcrA), ⁇ (mcrCB-hsdSMR-mrr) 171, sbcC, recB, recJ, umuC :: Tn ⁇ (kan r ), uvrC, supE44 , lac, gyrA96, relA1, thi-1, endA1, [F proAB, lac q Z ⁇ M15, Tn10, (tef)]
  • E. coli XL1 Blue Stratagene (La Jolla, CA, USA) recA1, endA1, gyrA96, thi-1, hsdR17, supE44, relA1, lac, [F proAB, lac q Z ⁇ M15, Tn 10, (tef)]
  • E. coli BL21 (DE3): Promega (Madison, Wl, USA) F “ , ompT, / 7sdS B (rB " m B “ ), gal, the
  • pMS470 ⁇ 8 3992 bp + 1360 bp placeholder fragment (stuffer insert) (Balzer et ai, 1992): The expression can be regulated by an inducible fac promoter.
  • This plasmid also contains an ampicillin resistance conferring gene and the Shine-Dalgarno sequence of gene 10 of bacteriophage T7. ⁇ / del and Sph restriction sites allow the cloning of a target gene into this vector system (Fig. 3).
  • TAE buffer (0.04 M Tris acetate, 0.001 M EDTA, pH 8.0); Stop solution / sample buffer, diluted from 5x solution (5x: 4 M urea, 50% (w / v) sucrose in water, 0.1
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside: 100 mM (BTS-Bio Tech Trades GmbH),
  • Tris-Glycine electrophoresis buffer 25mM Tris; 250mM Glycine (pH 8.3); 0.1%
  • E.co// growth experiments were preferably carried out in 300 ml and 11 Erlenmeyer flasks (baffled flasks) which contained 100 ml and 250 ml LB medium with 100 ⁇ g / ml ampicillin.
  • OD optical density
  • Rhodococcus ruber (DSM 43338) showed good growth on LB plates and in LB liquid culture at a temperature of 30 ° C. Cultures seeded with single colonies (grown on fresh LB plates overnight) were grown for 24 hours (250 ml LB, 120 rpm, 30 ° C).
  • the cells were harvested by centrifugation at 4 ° C (GSA rotor, 6000 rpm, 15 min), resuspended in 2-8 ml 0.1 M Tris (pH 7.0) and frozen at -20 ° C.
  • the resuspended pellet was subjected to ultrasonification (6 x 20 seconds, on ice, Branson Sonifier: Output: micro tip limit, duty cycle: 60). This was followed by ultracentrifugation (30,000 rpm (approx. 78,000 g), 30 min, 4 ° C).
  • the resulting supernatant fractions and the resuspended pellet fractions (3 ml 0.1 M Tris-HCl, pH 7.0) were stored at -20 ° C.
  • the concentration of the agarose gels was varied between 0.7 and 1.5% depending on the size of the fragment.
  • the 1% agarose gel normally used was sufficient for most applications.
  • the required amount of agarose was dissolved in 1 x TAE buffer by heating the solution in the microwave. After cooling to 60 ° C, ethidium bromide (1-3 ⁇ l) added and the gel was poured into the intended container. Between 1/3 and% volume of DNA gel loading buffer had to be added to all DNA samples that were added to the gel.
  • the voltage applied for electrophoresis was 60 V for preparative gels and 80 V for control gels. After electrophoresis, the bands were visualized in UV light.
  • Weight standards used (size in kDa: 94, 67, 43, 30, 21, 14).
  • the samples for the native gels were placed directly in the gel pockets after mixing with the loading buffer.
  • the electrophoresis was carried out at 150 V.
  • the native gels could be rinsed directly with water and on a
  • Vacuum dryer can be dried (80 ° C, 45 min).
  • the SDS gels on the other hand, were first decolorized before rinsing and drying until the background became colorless.
  • Plasmid DNA isolated by Wizard mini-preparations was digested for 2 hours at 37 ° C using commonly available enzymes such as BamYM, HincM and EcoRI.
  • a reaction volume of 10 ul was composed of the following components: 2 ul DNA, 1 ul enzyme (10 U / ul), 1 ul 10x buffer, 6 ul bidist. H 2 0. Before the samples were placed on a 0.7-1% agarose gel, the reaction was stopped by adding 5 ⁇ l loading buffer.
  • the competent cells were thawed on ice, the DNA to be transformed was added to 200 ⁇ l of the competent cells and the cells were then left on the ice for 30 minutes. There was a heat shock at 42 ° C. for 30 seconds. The tube was then placed on the ice again for 2 minutes. After adding 800 ⁇ l SOC medium, the mixture was shaken at 37 ° C. for one hour and then 100-200 ⁇ l of the suspension obtained in this way were plated onto LB-Amp plates.
  • the purity of the DNA from the Wizard mini preps was sufficient for sequencing.
  • the sequencing reaction was performed according to the dideoxy-mediated chain termination method (Sanger et ai, 1977) with the DNA sequencing kit (Big Dye TM Terminator Cycle Sequencing Ready Reaction with AmpliTaq ® DNA Polymerase, FS by PE Applied Biosystems). The sequences were analyzed with the Applied Biosystems Sequence Editor software (SeqEd TM Version 1.0.3).
  • Carrying (-) vector was centrifuged to harvest the cells.
  • the plasmid DNA was isolated using the JETSTAR Plasmid Isolation Kit. After the elution step, the DNA was precipitated with 0.7 volume of isopropanol, washed with 70% ethanol and bidistilled in 80 ⁇ l. Water dissolved.
  • H 2 0 was made up to 100 ul.
  • the plasmid was digested at 37 ° C for 2 hours. Then more
  • Alkaline phosphatase (1 U / ⁇ l; Boehringer). This was followed by incubation for 30 min at 37 ° C. Then a further 2 ⁇ l of alkaline phosphatase were added and, after further incubation for 30 minutes at 37 ° C., the
  • the sample was loaded onto a 1% preparative agarose gel. After the sample was loaded onto a 1% preparative agarose gel. After the sample was loaded onto a 1% preparative agarose gel. After the sample was loaded onto a 1% preparative agarose gel. After the sample was loaded onto a 1% preparative agarose gel. After the sample was loaded onto a 1% preparative agarose gel. After the sample was loaded onto a 1% preparative agarose gel. After
  • the 3 kb band was cut out of the gel by electrophoresis at 60 V.
  • DNA extraction was performed using Qiagen's Qiaex Agarose Gel Extraction Kit. 1 ⁇ l of the diluted was used to estimate the DNA concentration
  • Chromosomal DNA was isolated from cells grown in 250 ml LB medium (34 ° C, 120 rpm, 24 h). This experiment was carried out according to the Qiagen Isolation Protocol. An isopropanol precipitation (at -20 ° C., overnight) followed. For the following partial digestion, the DNA concentrations were estimated on the basis of the band strength in the agarose gel.
  • the optimal conditions for partial digestion with Sau3AI had to be determined.
  • the desired size of the fragments to be cloned was 2-5 kb.
  • the process was carried out in the following manner (Sambrook et a /., 1989): A reaction mixture with 10 ⁇ g chromosomal DNA and buffer for restriction digestion with a final volume of 150 ⁇ l was introduced (tube 9). Mixing was carried out by inverting several times. 30 ⁇ l of the mixture were placed in an Eppendorf tube (tube 1) and 15 ⁇ l each in tubes 2-8, so that 15 ⁇ l remained in tube 9. It was then cooled on ice. Now 4 units of restriction enzyme (Sa ⁇ / 3AI) were added to tube 1 and mixed well.
  • the concentration of the enzyme was thus 2 units per ⁇ g DNA. 15 ⁇ l of this reaction mixture was added to tube 2, so that the enzyme concentration in tube 2 is reduced to one unit of enzyme per ⁇ g of DNA. After mixing again, the dilution was continued in a corresponding manner up to tube 8. Tubes 1 to 8 were then incubated at 37 ° C for one hour. Then 5 ⁇ l of sample buffer was added to each sample and gel electrophoresis was carried out for sample analysis. To carry out a preparation on a large scale (750 ⁇ l total volume), 50 ⁇ g DNA with a correspondingly larger amount of restriction enzyme were used.
  • reaction tubes were frozen in liquid N 2 and stored at -70 ° C.
  • precipitation was carried out with 0.7 volumes of isopropanol and subsequent dissolution of the DNA in 100 ⁇ l bidist. H 2 0. Then 20 ⁇ l of sample buffer were added and the sample was applied to a preparative 1% low melting agarose gel.
  • the DNA fragments which had a size of 3-5 kb, were cut out and the DNA was extracted using the Qiaex II Agarose Gel Extraction Kit (Qiagen, Hilden, Germany).
  • Ratio vector fragment of 1: 1 incubated. 500 ng vector,
  • E.coli SURE ® and XL1 Blue cells were transformed according to the SEM protocol.
  • LB-Amp plates with IPTG 50 ⁇ l / plate, 100 mM
  • X-gal 100 ⁇ l / plate, 2%) were used to determine the amount of religated vector by blue / white selection.
  • the optimal dilution (400-500 colonies per plate) for the screening was determined.
  • a sufficient number of clones are screened to increase the likelihood of finding the gene sought.
  • filter papers (Whatman, 0 70 mm) were first soaked with ⁇ -naphthylacetate staining solution (see material: staining solution for native gels), then the filter papers were placed on the colonies to determine whether immediate color formation occurred (esterase - positive clones show red color, the background is pink). The positive clones are now selected and plated out for further screening. A single colony was spread out on LB-Amp plates after overnight incubation with this cell material and a mini-plasmid DNA preparation was carried out. Restriction analysis (SamHI and Hindi) is then carried out in order to eliminate identical clones based on the restriction pattern.
  • the clones showing esterase activity are now grown in 100 ml LB-Amp overnight cultures. After cell disruption, the supernatant of the cell lysate is used in selectivity tests with (S) -linalyl acetate. The cell lysates are then analyzed on SDS and native polyacrylamide gels.
  • rac-linalyl acetate (10 ⁇ l) was added to 1 ml of cell lysate and the mixture was incubated at room temperature for 16 hours at 180 rpm. The reaction was stopped with 1 ml of ethyl acetate and the organic phase was extracted with 16 ml of ethyl acetate. After drying the extract with Na 2 S0 4 , the organic phase was separated off by a rotary evaporator.
  • the purpose of subcloning was to reduce the insert size of the genomic library clones (average insert size 6 kb) to 3 kb.
  • 4 shows the experimental approach for carrying out this experiment.
  • An (S) -linalyl acetate selective clone from the library (clone pBSG25) was partially digested with Sau3AI as previously described. DNA fragments with a size of 1 to 3 kb were cut out of the preparative gel and the DNA was extracted with the QIAEX II kit from Qiagen.
  • the fragments obtained were then cloned into the vector pBluescript SK II (-) at a vector: fragment ratio of 1: 1. After the ligation at room temperature for 3 hours, 10 ⁇ l of the ligation mixture were transformed into E. coli XL1 blue cells (SEM transformation).
  • Clones with esterase activity were selected by screening with ⁇ -naphthyl acetate (as previously described) and identical clones were eliminated by restriction analysis (SamHI, EcoRI, HincW).
  • the 3.5 kb insert of the subclone pBSS12 was sequenced with the primers given in Table 1. The sequences were edited on a Macintosh computer using Seq Ed TM software (Applied Biosystems, Version 1.0.3).
  • Pirher jt - 5'-3 sequence ⁇
  • Primer 1 5 '- GAGAT ⁇ A ⁇ ATGlG C C CT C GATTC -3' (SEQ ID NO. 28)
  • Primer 2 3 '-CTACTC CTG ⁇ GTAC GJG C CATAC -5' (SEQ ID NO. 29)
  • esterase gene (estA) from the pBSS12 construct was amplified by PCR in the following way:
  • Template DNA (30 ng, isolated with the Nucleobond plasmid midiprep kit), primer 1 (93 ng), primer 2 (87 ng), dNTPs (10 mM; 1 ⁇ l), incubation mix (10x; 5 ⁇ l) and dist. H 2 0 were mixed together to adjust a total volume of 50 ⁇ l.
  • "Hot start” was achieved by adding 0.5 ⁇ l Taq polymerase (5 U / ⁇ l).
  • the following thermal cycler program was used: 98 ° C 3 min // 30 x: 98 ° C 45 sec / 62 ° C 45 sec / 72 ° C 1 min // 72 ° C 2 min.
  • the entire reaction volume of 50 ⁇ l was loaded onto a preparative agarose gel. After gel electrophoresis at 60 V, the DNA band was cut out (1 kb) and the DNA was isolated and purified using the Qiaex Gel Extraction Kit.
  • the plasmid pMS470 ⁇ 8 was digested with ⁇ / del and Sphl and the stuffer fragment was removed by separation on an agarose gel and subsequent isolation and purification with the Qiaex Gel Extraction Kit.
  • the PCR fragment prepared as described above was ligated into the vector prepared as described above at room temperature. The reaction was carried out for three hours. The ratio vector: fragment was 1: 1 (100 ng vector 4 kb, 25 ng fragment 1 kb).
  • To transform competent E.coli XU Blue cells they were incubated with 10 ⁇ l of the ligation mixture. 100 ul of these cells were then plated on 2 agar plates. A series of transformants were tested for esterase activity. Positive clones were examined by restriction analysis and a suitable clone was selected. The clone obtained in this way was called pMSRul.
  • the insert of the plasmid pMSRul was sequenced.
  • the plasmid DNA was first isolated using the Nucleobond AX-100 kit.
  • the primers Tac stop, S12b1, S12b2, S12b4 and pMSRu1b4a were used for the sequencing (Table 1).
  • the plasmid pMSRul was then also transformed into E. coli BL21 cells. Both pMSRul clones (XL1 Blue and BL21) were preferably cultured overnight in 3 ml LB-Amp medium, frozen with liquid nitrogen (300 ⁇ l culture + 100 ⁇ l 80% glycerol) and stored at -70 ° C. n) Expression studies with the clone pMS Ru1
  • the cells were harvested and disrupted as previously described.
  • the cell lysates were analyzed on SDS and native gels and examined for (S) -linalyl acetate activity.
  • the substrate indicator solution 50 ⁇ l of the substrate indicator solution were mixed with 10-50 ⁇ l cell lysate or cell suspension. The color change could be observed at room temperature after 10-15 minutes.
  • Whatman filter paper was placed on the plates and dried for 15 minutes. It was then soaked in the substrate litmus solution and the color changed from blue to red in the presence of a positive clone within 10-15 minutes.
  • Enzyme activities and specific activities were determined by hydrolysis of o-nitrophenylacetate (SIGMA ® ). The formation of nitrophenol could be measured with a spectrophotometer at 405 nm.
  • substrate stock solution (84 ⁇ g o-nitrophenyl acetate, dissolved in 916 ⁇ l DMSO), 980 ⁇ l 0.1 M Tris-HCl buffer (pH 7.0), 10 ⁇ l substrate and 10 ⁇ l enzyme (diluted in 0.1 M Tris buffer pH 7.0) first mixed in an Eppendorf tube and then added to a cuvette.
  • the zero point (Autozero) was determined with a solution of 10 ⁇ l substrate and 990 ⁇ l buffer.
  • the reaction curves were recorded at 25 ° C for 10 minutes (at 30 sec intervals).
  • the slope ( ⁇ E / min) was calculated using the spectrophotometer software (Perkin Elmer, Lambda Bio).
  • the absorption curve of the reference solution was subtracted from the slope of the absorption curves of the reaction solutions.
  • the enzyme activity was calculated using the following formula:
  • the extinction coefficient ⁇ (self-determined) for o-nitrophenol is 2.4 [l-mmol "
  • Enzyme was the value for the activity [U / ml] by the protein concentration
  • the reactions for the GC / HPLC screening were carried out in 5 ml of 0.1 M NaH 2 PO (pH 7.3). 250 ⁇ l enzyme and then 100 ⁇ l substrate were added to the buffer. 2-phenyl-2-hexyl acetate (acetic acid (2-phenyl-2-butyl ester)) and 2-phenyl-2-butyl acetate (acetic acid (2-phenyl-2-hexyl ester) were used as substrates )) used. Buffer with substrate and buffer with enzyme were used as a negative control. The reaction batches were incubated at 25 ° C. at 150 rpm. Samples were taken after 2, 5 and approx. 24 hours.
  • a ChiraCel OJ column was used for the HPLC. A flow rate of 1 ml / min was chosen. The absorption was measured at 254 nm, a temperature of 25 ° C. was specified. A mixture of heptane and isopropanol was used as the eluent. First, heptane: isopropanol was given in a ratio of 95: 5. After 5 minutes a ratio of heptane: isopropanol 85:15 was specified, this was kept constant for 20 minutes and then a ratio of 95: 5 was set again.
  • the total protein concentration was determined by the Bradford (1976) method using the Bio-Rad Protein Assay.
  • the task of the mutagenesis experiment was to improve the enantioselectivity and the stability of the enzyme EstA, which was produced by the clone pMSRul.
  • the theoretical background of this method was described by Leung et al., 1989.
  • the standard PCR conditions for gene amplification were described under (m).
  • Table 2 shows the conditions for the modified PCR. The same temperature program was used as before
  • Incubation mix (10x; 1x contains 1.5 mM MgCI 2 ), MgCI 2 and MnCI 2 as indicated in Table 2 and 0.7 ⁇ l Taq polymerase (5 U / ⁇ l; hot start) was used.
  • NEB nucleic acids
  • Ratio of 1 100 ng vector, 25 ng fragment; the vector pMS470 ⁇ 8 was previously cut with ⁇ / del and Sphl). 2 ⁇ l of the ligation mix were used
  • a total of 23 selected clones were tested in a further step for activity in hiblick on rac-linalyl acetate and (R) -linalyl acetate by growing the cells in 5 ml LB-Amp medium at 37 ° C. overnight. 200 ⁇ l of this culture were centrifuged in a microtiter plate at 2500 rpm for 10 minutes in order to sediment the cells. After decanting the medium, 50 ⁇ l litmus substrate solution were added. The color change from blue to red was observed every 10 minutes and recorded on a photo.
  • mutants B2, D1, D4, F3, F5; according to the PCR conditions B, D or F
  • 250 ml LB-Amp cultures for 11 hours (30 ° C, 120 rpm) either with induction or grown without induction of expression (induction with IPTG after 5 hours).
  • the cells were harvested, disrupted and ultracentrifuged as previously described. Aliquots (1 ml) of each fraction were stored at -20 ° C for further activity tests. The supernatant fractions from all five clones were analyzed by SDS-PAGE. Whole cells, lysates, supernatant and pellet fractions after ultracentrifugation were tested for activity and selectivity in microtiter plate assays by mixing the samples (50 ⁇ l each) with 50 ⁇ l litmus substrate solution.
  • the aim of this experiment was to find out whether the mutant F3 shows a higher specific activity than the wild type.
  • the protruding Fractions of the fermentations 250 ml LB-Amp cultures induced or not induced used in the test for enzymatic activity.
  • Enzyme stability over time at various reaction conditions The enzyme reaction of the rac-linalyl acetate hydrolysis reaction was carried out as previously described, using 1 ml of supernatant fractions, crude lysate and undigested cells (induced or not induced) from pMSRul (E. coli XU Blue ) and supernatant fractions of mutant F3 (induced) were mixed with 10 ⁇ l rac-linalyl acetate and incubated at 25 ° C. on an Eppendorf thermomixer at 700 rpm for 24 hours. As a control, supernatant fractions without a substrate were incubated under the same conditions in order to observe influences of the substrate on the enzyme stability. 10 ⁇ l samples were taken from each tube after 0, 3, 5, 10, 15 and 24 hours, diluted and analyzed by o-nitrophenylacetate activity tests.
  • Freshly cultivated cells 250 ml LB-Amp cultures, induced) of the clone pMSRul (E. coli XU Blue) were harvested, resuspended in 8 ml 0.1 M Tris buffer pH 7.0 and the suspension was separated into two equal parts. One half was subjected to the normally used sonification for cell disruption. The digested and undigested cells were then examined for enzyme activity.
  • Example 1 Preparation of a Rhodococcus ruber genomic library
  • the optimal conditions for the partial digestion of the chromosomal DNA had to be found. It has been suggested that the optimal enzyme concentration is around 3-5 kb fragments obtained, is 0.03 U / ⁇ g DNA (Fig. 6). For large-scale preparation, 1.56 U SauZAl was added to 50 ⁇ g DNA.
  • Genome size of -4000 kb for Rh. Ruber (DOGS- Database of Genome Sizes) resulted in an N value of -3700 clones (99% probability).
  • N " ln (l -f)
  • clone G16 showed a very strong band at the position of one of the E.co// bands, the specific linaloyl acetate esterase band not being found in the lower region of the gel.
  • the activity staining of native gels with ⁇ -naphthyl acetate made it possible to visualize esterases contained in the lysate.
  • a GC selectivity assay found that the strong band in the lower region of the gel is due to the selective linalyl acetate esterase.
  • the selective clones G11 and G25 produced comparatively low amounts of protein.
  • the amount of enzyme produced by the clones could be that
  • the very selective subclone S12 (pBSS12) was selected for further investigations.
  • the 3.5 kb insert was sequenced in order to obtain information about the insert and in particular about the esterase gene.
  • the primers used for sequencing are listed in Table 1.
  • the results of the sequencing are shown in Figures 12-14.
  • the nucleotide sequences obtained and the peptide sequences derived therefrom were analyzed using the GCG software package (Genetics Computer Group, Wisconsin, USA).
  • the region of the insert located towards the T3 region was found to contain a partial sequence of an aldehyde dehydrogenase gene.
  • Figure 12 shows the 773 base unit long DNA sequence of this region.
  • the Sau3AI restriction site at which the insert was inserted into the vector is particularly highlighted in the front sequence area.
  • the esterase EstA (estA) gene was located in the region of the 3.5 kb insert adjacent to the T7 region of the vector (Fig. 11). Sequence information about this area is shown in FIG. 13. Reading frame 2 of this sequence was translated into the amino acid sequence. A Shine-Dalgarno sequence at bp position 529, which was followed by a start ATG at bp position 530 and a stop codon at bp position 1475 (marked in FIG. 13) resulted in an open reading frame (ORF) of 948 bp.
  • ORF open reading frame
  • the Saw3AI cloning site which was supposed to be in the 3 'direction from TGA (1475 bp), could not be found here, since the sequence which was obtained using the primer T7 was only readable from bp position 1494. However, this area was not further processed because it was of no further interest.
  • the esf gene sequence obtained from the 3'5 'strand of the insert was confirmed by sequencing the complementary strand and then used in the database for homology search (comparison of amino acid sequences). All reverse primers used are listed in Table 1. Homologies to other bacterial and eukaryotic esterase sequences were found (Table 5). The 1.2 kb gap between the aldehyde dehydrogenase gene and the estA gene could not be fully sequenced, although different primers and annealing temperatures were tried. A possible explanation for this could be the secondary structure of the insert in this region.
  • esterase gene (estA)
  • construct pBSS12 as template DNA and primers 1 and 2 (see methods (m))
  • the vector pMS470 ⁇ 8 was digested with ⁇ / del and Sphl to cut out the 1.4 kb stuffer insert.
  • the resulting 4 kb vector fragment was cut out of the gel and extracted with the Qiaex kit (Qiagen, Hilden).
  • the DNA concentrations were 10 ng / ⁇ l for the PCR fragment and 100 ng / ⁇ l for the vector.
  • plating 100 ul cells resulted in -20 colonies per plate.
  • the E.coli XU Blue clone pMSRul was grown in 250 ml LB-Amp medium. In one case, IPTG was added 3 hours after inoculation (in the logarithmic phase). Expression was not induced in the second flask. This led to different expression of the protein, like that
  • the uninduced culture grew to an optical density (OD) of 2.9, while with the addition of IPTG there was a decrease in the slope in the
  • the normal sonification time (6 x 20 sec) was increased by up to two times.
  • the patterns of the SDS gel and native gel bands were similar for both sonification conditions.
  • the pellet fractions were inhomogeneous suspensions of cell residues. The amount of protein added to the gels was therefore not the same in all traces.
  • FIG. 22 The result of renaturing an SDS gel followed by activity staining is shown in FIG. 22.
  • the denaturation of the protein was reversible.
  • the renatured, activity-Coomassie stained gel showed a strong esterase overexpression band (33 kDa).
  • Very weak bands (the top two bands) also showed activity in lanes P3 and P4 during activity staining. These bands are likely due to protein remaining in the pellet fraction.
  • the BL21 type grew to a higher optical density in the same predetermined time of 10 hours.
  • temperature effects on cell growth could be observed (FIG. 23).
  • Cells that grew at 37 ° C. grew much faster in the exponential phase, but the final OD was one unit lower than in 30 ° C. cultures.
  • Higher temperatures increased the level of overexpression of the esterase, which could have a negative impact on cell growth.
  • E. coli BL21 allowed efficient overexpression of the EstA protein (Fig. 24) at 30 ° C as well as 37 ° C. Influences of the temperature on the esterase expression could not be determined on the basis of the band pattern of the SDS gel.
  • the strategy chosen to improve the selectivity and stability of the esterase EstA was random mutagenesis by modified PCR (Arnold et al., 1999).
  • the PCR amplification of the esfcA gene was carried out at concentrations of 5 mM MgCl 2 and 0 to 3.0 mM MnCl 2 .
  • PCR conditions B, C, D and F (Table 2) were chosen for the further experiments.
  • the number of transformants obtained after transformation of the ligation batches in E. coli XU Blue was> 20,000 under all four PCR conditions.
  • the religation rate was around 10%.
  • the respective mutant library cell suspension was suitably diluted and 100 ⁇ l in each case were plated on LB-Amp plates (-300 colonies per plate).
  • Microtiter plate test to check the selectivity of the mutants
  • rac- and (R) -linalyl acetate were used as substrates to determine the selectivity.
  • the 23 selected mutants were used in this test in the form of whole cells, using pMS470 ⁇ 8 as a negative control. Almost all of the mutants examined showed a substrate turnover comparable to that of the wild type.
  • the mutant F3 showed a comparatively high overall activity and high (S) preference in the tests and was therefore selected for further studies. In this clone, the color change from blue to red could be observed only a few seconds after the litmus substrate solution was added to the cells.
  • the 1 kb insert of this clone F3 was sequenced using the primers pMSRul b4a, S12b1 and Tac stop (Table 1). It contained a total of 3 mutations, one of which was a silent mutation (Table 12).
  • Tab. 12 Sequencing results of mutant F3 (mutated position: gray background)
  • Esterase activity was determined in lysates of the clone pMSRul (in E.coli XU Blue) with the substrate o-nitrophenylacetate using the photometric assay. As shown in Fig. 28, the fractions of the induced fermentations showed a specific activity many times higher than that of the non-induced samples. Induction increased the enzyme yield by at least a factor of ten. It was thus possible to confirm with these quantitative analyzes that the supernatant fractions contained the major part of the enzyme, but enzyme activity was also detectable in the pellet fraction. However, the activity was only about a fifth of the activity of the supernatant fraction.
  • the mutant F3 showed an approximately ten times higher specific activity compared to the wild type, both in the induced and in the non-induced fermentation.
  • the previously described expression studies showed that the wild-type clone pMSRul produced smaller amounts of enzyme during this fermentation, which makes a comparison of the enzyme activities difficult.
  • the results shown in FIG. 29 confirmed that the induction also leads to an increase in the enzyme activity in the mutant F3 in approximately the same order of magnitude as in the wild-type clone.
  • the cell disruption showed no significant influence on the specific activity of the enzyme. In both cases the observed activity of the enzyme was between 50 and 60 U / mg protein. Although the lysate samples became warm during the sonification process, this treatment did not appear to have a demonstrable deleterious effect on the enzyme.
  • Example 10 Screening for mutants with increased activity on the substrate 2-phenyl-2-butyl acetate using a phenol red filter assay
  • Colonies obtained after transforming a mutant library into E. coli Top10F ' were transferred to a filter paper (Whatman) by means of an impression technique. The filter paper was then incubated in a screening mix and colonies where the color changed rapidly from red to yellow were selected. Table 13: Composition of the screening mix
  • the screening mix was filtered through a 0.2 ⁇ m membrane filter and then
  • the mutants were isolated, purified and the DNA sequence of the insert was determined. The sequencing results are shown in the table below.
  • McKay AM Microbial carboxylic ester hydrolases (EC 3.1.1.) In food biotechnology. Lett Appl Microbiol 1993; 16: 1 -6.

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Abstract

La présente invention concerne une estérase issue de Rhodococcus ruber, des protéines homologues à celle-ci, des acides nucléiques codant ces protéines, des anticorps luttant contre ces protéines, ainsi que la production et l'utilisation de ces protéines, de ces acides nucléiques et de ces anticorps.
PCT/EP2002/011213 2001-10-09 2002-10-07 Esterase esta issue de rhodococcus ruber WO2003031625A1 (fr)

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CN111727235A (zh) * 2019-01-15 2020-09-29 辽宁格瑞仕特生物制药有限公司 赤红球菌产品及其制药用途

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Title
DATABASE EMBL [online] 16 July 1999 (1999-07-16), "Petroleum-degrading bacterium HD-1 gene for esterase HDE, complete cds", XP002232124, Database accession no. AB029896 *
DATABASE GENBANK [online] 8 October 2001 (2001-10-08), XP002232125, retrieved from NCBI *
POGOREVC M ET AL: "Novel carboxyl esterase preparations for the resolution of linalyl acetate", MONATSHEFTE FUR CHEMIE, (JUN 2000) VOL. 131, NO. 6, PP. 639-644. PUBLISHER: SPRINGER-VERLAG WIEN, SACHSENPLATZ 4-6, PO BOX 89, A-1201 VIENNA, AUSTRIA. ISSN: 0026-9247., GRAZ UNIV, INST ORGAN CHEM, HEINRICHSTR 28, A-8010 GRAZ, AUSTRIA (Reprint);GRAZ UNIV, INST ORGAN CHEM, A-8010 GRAZ, AUSTRIA; GRAZ UNIV, INST BIOCHEM, A-8010 GRAZ, AUSTRIA, XP001145715 *
SCHLACHER A ET AL: "Detection of a new enzyme for steroselective hydrolysis of linalyl acetate using simple plate assays for the characterization of cloned esterases from Burkholderia gladioli", JOURNAL OF BIOTECHNOLOGY, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 62, no. 1, 11 June 1998 (1998-06-11), pages 47 - 54, XP004127152, ISSN: 0168-1656 *
SCHLACHER A ET AL: "Recombinant esterases for organic syntheses;", MEDED.FAC.LANDBOUWWET.RIJKSUNIV.GENT;(1996) 61, 4A, 1391-94 CODEN: MFLRA3 ISSN: 0368-9697 APPLIED BIOTECHNOLOGY, 10TH FORUM, GHENT, BELGIUM, 26-27 SEPTEMBER, 1996., Univ.Graz-Tech.Inst.Biotechnol.;Univ.Graz-Tech.Inst.Org.Chem., XP009006334 *

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Publication number Priority date Publication date Assignee Title
CN111727235A (zh) * 2019-01-15 2020-09-29 辽宁格瑞仕特生物制药有限公司 赤红球菌产品及其制药用途
CN111727235B (zh) * 2019-01-15 2024-03-15 辽宁天安生物制药股份有限公司 赤红球菌产品及其制药用途

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