+

WO2003018789A1 - Esterase est4b1 issue de bacillus subtilis - Google Patents

Esterase est4b1 issue de bacillus subtilis Download PDF

Info

Publication number
WO2003018789A1
WO2003018789A1 PCT/EP2002/009289 EP0209289W WO03018789A1 WO 2003018789 A1 WO2003018789 A1 WO 2003018789A1 EP 0209289 W EP0209289 W EP 0209289W WO 03018789 A1 WO03018789 A1 WO 03018789A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
nucleic acid
polynucleotide
enzyme
nucleic acids
Prior art date
Application number
PCT/EP2002/009289
Other languages
German (de)
English (en)
Inventor
Helmut Schwab
Anton Glieder
Michaela Pressnig
Norbert Klempier
Andrea Schmidt
Original Assignee
Degussa Ag
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Degussa Ag filed Critical Degussa Ag
Publication of WO2003018789A1 publication Critical patent/WO2003018789A1/fr

Links

Classifications

    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • 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)

Definitions

  • the present invention relates to an esterase from Bacillus subtilis, proteins homologous to it, nucleic acids coding for these proteins and antibodies against these proteins, and the production and use of these proteins,
  • Hydrolytic enzymes play an important role in the field of biocatalysis, especially for the production of enantiomerically pure substances [1, 2].
  • the enzymatic hydrolysis and synthesis of esters takes place in technical
  • Lipases in particular have been successfully used industrially for various reasons [3]. The reasons for this are that lipases can be provided in large quantities, that they are very stable under the reaction conditions and that very high enantioselectivity can be achieved in the hydrolysis and transesterification [4].
  • Enzymes in particular esterolytic or lipolytic enzymes, for industrial use, in particular for separating racemic mixtures into their components.
  • the object of the present invention was therefore to provide a new enzyme with hydrolytic activity, in particular esterolytic and / or lipolytic activity, for which there is an urgent need in view of the possible uses of such enzymes, in particular in biotechnology and organic chemistry, and / or to provide a method with which new hydrolytic enzymes, especially esterolytic and / or lipolytic enzymes, can be obtained.
  • the object of the present invention was, in particular, to provide a hydrolytic enzyme, in particular an esterolytic and / or iipolytic enzyme, which has a different substrate specificity than the esterolytic and lipolytic enzymes described hitherto, a distinguishable degree of selectivity, a distinguishable reaction rate and a distinguishable structure and / or has a distinguishable reaction mechanism.
  • a hydrolytic enzyme in particular an esterolytic and / or iipolytic enzyme, which has a different substrate specificity than the esterolytic and lipolytic enzymes described hitherto, a distinguishable degree of selectivity, a distinguishable reaction rate and a distinguishable structure and / or has a distinguishable reaction mechanism.
  • Another object of the present invention was to provide esterolytic and / or lipolytic enzymes which are capable of cleaving ester and / or thioester bonds.
  • polypeptides and antibodies according to the invention, in particular by a polynucleotide according to SEQ ID No. 1, a polypeptide according to SEQ ID No. 2 and one against a polypeptide according to SEQ ID No. 2 directed antibodies.
  • nucleic acid according to SEQ ID No. 1 or a polypeptide according to SEQ ID No. 2 nucleic acids and polypeptides of similar structure and the same or similar function can be obtained.
  • enzymes with improved properties can also be obtained, for example through evolution experiments [10, 11].
  • the present invention therefore relates to a polynucleotide selected from the group consisting of: a) polynucleotide with a nucleic acid sequence from positions 556 to 1287 according to SEQ ID No. 1, b) Polynucleotides coding for a polypeptide with an amino acid sequence according to SEQ ID No.
  • Polynucleotides e) polynucleotides hybridizing under stringent conditions with a polynucleotide according to (a), (b), (c) or (d), stringent conditions being understood to mean the conditions known to the person skilled in the art for hybridization experiments, preferably incubation at 60 ° C.
  • 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), (c), ( d) or (e), g) polynucleotides consisting of at least 10 or 15, preferably at least 20 or 25, particularly preferably at least 30 or 40, very particularly preferably at least 50 or 80, in particular at least 100 or 120, in succession nucleic acids of a polynucleotide according to (a), (b), (c),
  • polynucleotides with one or more deletions and / or insertions of up to 50 or 40, in particular up to 30, 20 or 10 nucleic acids with respect to a polynucleotide according to (a), (b), (c ), (d) or (e), i) polynucleotides comprising at least one of those mentioned under (a) to (h)
  • 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, in particular esterolytic or lipolytic enzymes.
  • the nucleic acids according to the invention are preferably ribonucleic acids (RNAs) or deoxyribonucleic acids (DNAs), the
  • Nucleic acids are preferably present as double-stranded nucleic acids.
  • the nucleic acids according to the invention are nucleic acids which code for a protein with hydrolase activity or parts thereof.
  • the protein with hydrolase activity is, for example, a halogen peroxidase, haloalkane dehalogenase, epoxy hydrolase, thioesterase, lipase or esterase and / or an enzyme which is capable of haloalkane, ester, thioester, amide - cleave, halide, epoxy or peptide bonds, particularly preferably ester or thioester bonds. It is very particularly preferably an enzyme which is able to cleave bonds regioselectively and / or enantioselectively.
  • 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, however, occur in particular
  • Embodiments also take place with formation of a new ester, thioester, amide, peptide or halide bond, in particular it can be a transesterification reaction, for example, if an ester bond is converted into a new ester bond.
  • Cleavage also includes those catalyzed by halogen peroxidases and haloalkane dehalogenases
  • the enzyme according to the invention is a hydrolase with an ⁇ / ⁇ hydrolase fold and / or an independent esterase or thioesterase as a component of non-ribosomal peptide synthetases (Schneider et al., Arch. Microbiol. (1998) 169: 404-410).
  • the nucleic acids according to the invention are preferably nucleic acids which are suitable for a microbial enzyme, particularly preferably a bacterial enzyme, in particular for an enzyme from Bacillus, especially for an enzyme from Bacillus subtilis, in this case in particular for the esterase Est4B1 according to SEQ ID No. 2 and / or one of the polypeptides according to the invention described below.
  • a microbial enzyme particularly preferably a bacterial enzyme, in particular for an enzyme from Bacillus, especially for an enzyme from Bacillus subtilis, in this case in particular for the esterase Est4B1 according to SEQ ID No. 2 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, functional properties in particular hydrolytic activity, especially lipolytic and / or esterolytic activity, and improved functional properties, for example, higher specificity, higher conversion and / or higher regio- or enantioselectivity.
  • the nucleic acids according to the invention can thus be used, for example, as probes for identifying and / or isolating homologous nucleic acids from an artificial, cDNA or genomic gene bank, preferably for identifying nucleic acids that are used for esterolytic enzymes, lipolytic enzymes and / or for one of the polypeptides according to the invention encode or parts thereof, 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 esterolytic and / or lipolytic activity, or parts thereof.
  • the 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.
  • Nucleic acids are used, for example, for the targeted production of individual domains or epitopes of a polypeptide according to the invention or of fusion proteins which comprise the polypeptides according to the invention.
  • the present invention therefore also relates to a method for
  • nucleic acid which codes for a hydrolytic enzyme in particular for an esterolytic and / or iipolytic enzyme, comprising the following steps: a) a nucleic acid library is contacted with a nucleic acid according to the invention, b) a nucleic acid which hybridizes with a nucleic acid according to the invention 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 esterolytic and / or iipolytic enzyme, comprising the following steps: a) primers are prepared starting from a nucleic acid according to the invention, b) the primers (a) are used to amplify nucleic acids, especially cDNAs, of unknown nucleic acid sequences in the PCR, c) the nucleic acids obtained in (b) are sequenced.
  • Another object of the present invention is therefore also a method for isolating a nucleic acid coding for a hydrolytic enzyme, in particular for an esterolytic and / or iipolytic enzyme, comprising the following steps: a) nucleic acids from a library are incorporated into suitable vectors and these are introduced into suitable host organisms, preferably bacteria, in particular E.
  • the host organisms obtained according to (a) are on a solid support, preferably an agar plate, plated out and incubated, c) an assay, for example a color reaction, is carried out on the solid support, which involves the reaction of a hydrolase substrate, for example naphthylacetate, d) hydrolase-positive clones are identified, for example by visual observation, by automated image analysis or by means of photometric measurement, and purified, e) the nucleic acids from the clones identified in step (d) are sequenced.
  • the present invention therefore also relates to a method for isolating a nucleic acid coding for a hydrolytic enzyme, in particular for an esterolytic and / or iipolytic enzyme, comprising the following steps: a) a nucleic acid according to the invention is subjected to mutagenesis experiments, b) the results obtained Mutants are incorporated into a suitable vector and expressed using a suitable host organism, c) the expression products are examined for hydrolytic activity, in particular esterase and / or lipase activity, d) the nucleic acids, the expression products of which in step (c) are hydrolytic
  • 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 come that for example, based 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, in particular a microbial, especially bacterial library, in particular one from Bacillus, particularly preferably from Bacillus subtilis.
  • 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 SEQ ID no. 1 specified
  • the present invention also relates to a vector, in particular a
  • Cloning and / or expression vector comprising one of the aforementioned nucleic acids.
  • the 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 pGEM-5Zf (+/-), pBluescript SK (-) or pMS470 or another high copy number plasmid.
  • the available expression vectors for expression in E. coli are, for example, the vectors pTB3 or pMS470BS1 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- Encode Ala-His6 tag, which is the purification of the expressed protein on a Ni 2+ -NTA column.
  • suitable eukaryotic expression vectors for expression in Saccharomyces cerevisiae are the vectors p426Met25 or p426GALT, 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 polypeptides can ease.
  • 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 an expression vector according to the invention, the host cell preferably being Bacillus, in particular Bacillus subtilis, or E coli, in particular the E. co // strain BL21 (DE3) or the strain E coli SURE.
  • Bacillus in particular Bacillus subtilis
  • E coli in particular the E. co // strain BL21 (DE3) or the strain E coli SURE.
  • 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.
  • nucleic acids in particular those which code for enzymes with hydrolytic activity
  • nucleic acids according to the invention is completely or partially by a portion of one of the Nucleic acids according to the invention replaced in order to improve the expression of these enzymes, in particular the expression in E. coli, by producing these heterologous constructs.
  • nucleic acid from position 556 to 1287 according to SEQ ID No. 1, and above all the range of the first 150 nucleic acids is characterized by a codon use which makes this nucleic acid particularly suitable for expression in E. coli, since it has a very low GC content and therefore after transcription there are less difficult melting RNA structures.
  • the 5'-terminal partial area can therefore primarily by a nucleic acid from position 556 to position 706 according to SEQ ID No. 1 or at least a part thereof, the at least one part preferably comprising at least 10 nucleic acids, particularly preferably at least 20 nucleic acids, in particular comprising at least 50 nucleic acids.
  • 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, b) naturally occurring or artificially generated mutants, polymorphic forms or alleles of a polypeptide according to (a), especially those which have up to five, in particular one, two or three, point mutations with respect to a polypeptide according to (a), 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 8 or 10, particularly preferably at least 12 or 15, in particular at least 20, 25 or 40 consecutive
  • the polypeptide is preferably a hydrolytic enzyme, particularly preferably an esterolytic or Iipolytic enzyme, or parts thereof.
  • a hydrolytic enzyme particularly preferably an esterolytic or Iipolytic enzyme, or parts thereof.
  • enzymes that can catalyze a hydrolysis reaction such as the hydrolysis of haloalkane, ester, thioester,
  • the hydrolytic enzyme can be an esterase, lipase, thioesterase, haloalkane dehalogenase, halogen peroxidase or epoxy hydrolase and / or an enzyme which has alpha / beta-hydroIase folding and / or an enzyme which according to
  • the cleavage reaction which is catalyzed by a polypeptide according to the invention can also be a reaction analogous to this, such as the formation, in particular new formation, of an ester, thioester or amide bond.
  • the polypeptide according to the invention is a microbial, in particular bacterial, enzyme. It is very particularly preferably an enzyme from Bacillus subtilis, in particular the esterase Est4B1 according to SEQ ID No. Second
  • the polypeptide according to the invention is particularly preferably a water-soluble enzyme.
  • the polypeptide according to the invention is an esterase or thioesterase which is or can be contained in a non-ribosomal peptide synthetase. This can in particular be a thioesterase according to the publication by Schneider et al. (Arch. Microbiol. 1998, 169: 404-410), here in particular a distinct thioesterase like protein.
  • Another object of the invention is the use of a polypeptide according to the invention for the cleavage of haloalkane, ester, thioester, peptide, amide, epoxy or halide bonds, the cleavage reaction being both a hydrolysis reaction with liberation of the corresponding carboxylic acid or the corresponding alcohol or alkane, as well as the new formation of an ester, thioester, peptide, amide or halide bond or the new formation of a haloalkane bond.
  • Substrates or compounds to be prepared according to the invention are, in particular, compounds of the formula R 1 -C (O) -OR 2 , where R 1 is an alkyl radical having preferably up to 7, especially up to 5, carbon atoms, in particular methyl, ethyl, propyl or Butyl, or a cycloalkyl or heterocycloalkyl radical preferably having 5 ring atoms, the
  • Heterocycloalkyl radical preferably carries exactly one hetero atom, particularly preferably oxygen
  • the alkyl radical can also comprise an epoxy group
  • the heterocycloalkyl radical preferably exactly one hetero atom, particularly preferably carries oxygen
  • the radicals mentioned for R 1 and R 2 independently of one another, can also carry at least one, particularly preferably one or two substituents known to the person skilled in the art, for example selected from the group consisting from halogen atoms, especially Cl or Br, N0 2 , NH 2 , S0 3 H, S0 2 H, phthalimido,
  • the enzymes according to the invention are able to split carboxylic acid esters in which the alcohol component is selected from the group consisting of 0- and p-nitrophenol, naphthol, phthalimidobutanol, methanol, ethanol, propanol, butanol, glycerol, glycidol and Hydroxy-tetrahydrofuran and the carboxylic acid component is selected from the group consisting of 3-hydroxybutyric acid, tetrahydrofuran-2-carboxylic acid, butyric acid, valeric acid, acetic acid, propionic acid, caproic acid and 2-
  • Chloropropionic acid where the individual components also, independently of one another, bear substituents or can be modified in some other way.
  • the cleavage reaction is carried out at a pH greater than or equal to 6.5, preferably at a pH between 7.0 and 10.0, particularly preferably at a pH between 7.5 and 8.5, especially at a pH of around 8.0.
  • polypeptides according to the invention are used as a constituent of non-ribosomal peptide synthetases, in particular for the production of antibiotics, the reaction which is catalyzed by the polypeptide according to the invention, for example the cyclization of cyclic peptide antibiotics in the form of an ester, Can be thioester or amide bond.
  • the enzymes according to the invention are used in order to isolate or enrich one of the two enantiomers starting from racemic mixtures.
  • Production or selective enrichment for example of taste components in the food industry, of detergents in the Detergent industry, fine chemicals in the chemical industry or therapeutically or diagnostically usable substances in the pharmaceutical industry.
  • 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 5-12, in particular 8, amino acids are preferably suitable for this.
  • 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 binding to a polypeptide according to the invention can alternatively also be obtained by an in vitro method known to the person skilled in the art, such as, for example, phage display, yeast display, bacterial display or the so-called Fusagen technology, in which the nucleic acid and the coding thereof
  • Polypeptide 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 binding to a polypeptide according to the invention are called “antibodies” for the sake of simplicity.
  • the present invention furthermore 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 Bacillus, in particular Bacillus subtilis, or in E. coli, in particular E. coli BL21 (DE3) , is expressed.
  • a nucleic acid according to the invention in a suitable host cell, in particular in Bacillus, in particular Bacillus subtilis, or in E. coli, in particular E. coli BL21 (DE3) , is expressed.
  • 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 is optionally purified from the supernatant thus obtained.
  • E. coli is preferably cultivated between 15 and 40 ° C., particularly preferably at about 37 ° C. Expression is preferably induced in the logarithmic growth phase.
  • expression can be induced by adding lactose, for example by setting a final concentration between 1 and 30 mM, in particular between 5 and 20 mM, in particular of about 10 mM, and / or by adding IPTG, for example by setting a final concentration between 0.01 and 10 mM, especially between 1 and 5 mM.
  • the cells are preferably allowed to grow for a further 8-18 hours, in particular a further 12-14 hours.
  • induction of expression can also be dispensed with.
  • the harvested cells can be disrupted by a method known to the person skilled in the art, for example French press, ultrasound, ball mill or by using a sonifier and / or enzymatically.
  • 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, anion or cation exchange chromatography, hydrophobic interaction chromatography and / or gel filtration.
  • the protein is purified using anion exchange chromatography and a subsequent gel filtration step.
  • the protein Est4B1 according to SEQ ID No. 2 can be cleaned in an advantageous manner.
  • the anion exchange column is preferably a QFF column (Amersham Pharmacia, Uppsala, Sweden), and the gel filtration column is a Pharmacia Sephacryl S-100 HR in XK 26/70 column.
  • Application to and elution from the anion exchange column is preferably carried out in the neutral to slightly alkaline range, preferably between pH 6.5 and pH 8.5, very particularly preferably at about pH 7.5.
  • a 10 mM Tris-HCl buffer is preferably used for this.
  • Elution of the protein is preferably achieved by slowly increasing the ionic strength of the elution buffer. This can be achieved, for example, by adding NaCl in a step gradient.
  • Elution of the protein is preferably carried out at a NaCl concentration between 150 mM and 300 mM or when using a different salt with the same ionic strength.
  • the active fractions are preferably combined and concentrated.
  • the application to the gel filtration column is preferably carried out under the same conditions as the application to the anion exchange column.
  • the protein is eluted, for example, with a flow rate between 2 and 6 ml min "1 , in particular with a flow rate of about 4 ml min " 1 .
  • the short-chain polypeptides according to the invention can also be synthesized using classic peptide synthesis (Merrifield technique).
  • the present invention furthermore relates to a process for the production of crystals containing at least one of the polypeptides according to the invention, and to the crystals obtainable by this process.
  • the crystallization is preferably carried out at a pH between 6.0 and 9.0, particularly preferably between 7.0 and 8.0, especially at a pH of about 7.5 and / or using PEG, especially PEG 4000, as a precipitant, the proportion of PEG preferably being between 10 and 50%, particularly preferably between 20 and 36%, especially about 28%, and / or in the presence of sodium acetate, the concentration of sodium acetate preferably being between 0.1 and 0.3 M, especially about 0.2 M.
  • the invention further relates to a test system for identifying substrates or functional interactors containing a nucleic acid according to the invention, a polypeptide according to the invention and / or an antibody according to the invention and, if appropriate, suitable auxiliaries and additives.
  • Bacillus subtilis is known to ward off plant pathogens and to treat symptoms of plant pests such as Erwinia, relieves. This positive effect is due not least to enzymes with hydrolytic activity, especially esterases and thioesterases.
  • the present invention therefore furthermore relates to the use of at least one polynucleotide according to the invention and / or of at least one polypeptide according to the invention and / or of at least one antibody according to the invention for defense against plant pathogens and / or plant pests and a composition for the treatment of plant pathogens and / or plant pests, comprising at least one polynucleotide according to the invention and / or at least one polypeptide according to the invention and / or at least one antibody according to the invention.
  • a polynucleotide according to the invention can also be introduced into a plant in a manner known to the person skilled in the art in order to produce a polypeptide according to the invention in the plant and thus resistance to it To confer plant pathogens.
  • the polynucleotide according to the invention can in particular be introduced into a single plant cell and a plant can be obtained therefrom by multiplication and differentiation, which comprises a polynucleotide and / or polypeptide according to the invention.
  • the present invention therefore also relates to a method for introducing a polynucleotide according to the invention into a plant cell and a plant and / or plant cell, comprising at least one polynucleotide according to the invention and / or at least one polypeptide according to the invention.
  • 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.
  • FIG. 1 shows a nucleic acid sequence according to the invention of genomic DNA from Bacillus subtilis.
  • This B. subtilis genomic DNA fragment comprises a full open reading frame (ORF) (esWßl) and two partial ORFs (ot 1 and ot ⁇ 3).
  • ORF full open reading frame
  • ot 1 and ot ⁇ 3 The putative Shine-Dalgamo sequence of the est4B gene is underlined. Sequences of the cloning vector pBluescript SK (-) are identified by bold italics.
  • FIG. 2 shows the cloned DNA fragment from FIG. 1 again schematically.
  • the ORF for Est4B1 (BS1)
  • the two partial ORFs (ORF1, ORF3)
  • some restriction interfaces were identified.
  • the Amino acid sequence shows great similarity to putative proteins from ß. subtilis A13.
  • BL21 (pMS470BS1) shown at 37 ° C. Expression was induced by adding lactose to a final concentration of 10 mM at different times: (A) induction took place immediately after inoculation, (B) 4 hours, (C) 6 hours, (D) 8 hours, (E ) 10 hours, (F) 12 hours after inoculation. (G) E. coli BL21 (pBluescript SK-) was used as a negative control. Protein crude lysates were applied.
  • Example 1 Isolation and characterization of the Est4 bacterial strain
  • Ultrathurax homogenizer used, which had previously been used for the homogenization of plant material. A large halo bacterial culture was isolated and replated several times to ensure that there was a single colony. Halo formation was observed at room temperature as well as at 30, 37 and 50 ° C. The strain thus obtained was
  • Est4A produces lecithinase but not Est4B.
  • the ß. subW / s strain Est4A was cultivated on LB agar plates, several morphological variants again being observed. It seems so that these variants represent different morphological forms of one and the same strain, and that these are not contaminating strains. Under the light microscope, the different variants showed the same shape, but different mobility. It has also been shown that Est4A, B, C and D have different esterase and lipase profiles. Esterase activity could be found in all 4 variants, but the variants Est4A and C additionally showed lipase activities with different stereo preferences. Est4B was subsequently used to clone the esterase.
  • Example 2 Bacterial strains and growth media
  • the Bacillus subtilis strains Est4A, Est4B, Est4C and Est4D were isolated from tributyrin emulsion agar plates and then cultivated on Luria Broth (LB) agar plates.
  • E. coli SURE and the cloning vector pBluescript SK (-) from Stratagene (La Jolla, CA, USA) were used for the standard cloning experiments.
  • E. coli BL21 (DE3) (Stratagene) was used as a host for enzyme production.
  • the vector pMS470 ⁇ 8 used for heterologous enzyme expression was a gift from E. Lanka (18).
  • E. co / 7 cells were normally grown at 37 ° C in LB medium containing 100 mg / l ampicillin.
  • tributyrin emulsion agar plates 5-7 g / l tributyrin was added to the melted agar and then emulsified by sonification with an ultrathurax homogenizer before the agar was poured into the dishes.
  • Genomic DNA from ß. subtilis Est4B was isolated using the Qiagen (Qiagen, Hilden, Germany) Genomic Kit (Tip 500) from cells that had grown overnight in LB medium at 37 ° C. 10 ⁇ g of the genomic DNA was partially digested with Sau3AI (Röche, Mannheim, Germany). The fragments were separated by size using agarose gel electrophoresis and
  • Example 4 Sequence analysis of the ORF (open readinq frames) of pTB3 plasmid DNA was purified using a plasmid DNA isolation kit (Qiagen) and the insert was analyzed using an automated sequencer (ABI 373A Automated Sequencer) and using the Dye Deoxy Terminator Sequencing kits sequenced by Perkin Elmer Biosystems (California, USA). DNA and protein sequences were analyzed using the GCG Program Package (Genetics Computer Group, Madison, Wisconsin, USA).
  • the sequence analysis showed that the Bacillus subtilis DNA fragment contained in the vector pTB3 completely encoded one protein and two proteins partially (FIG. 2).
  • the partially contained ORF1 which codes for the C-terminus of a protein, shows great amino acid sequence homology to integrated thioesterases from surfactin synthetase gene clusters. 98% amino acid sequence homology was found for the putative surfactin synthetase protein SrfC from Bacillus subtilis A13.
  • the gene sequence of this highly homologous protein has Accession No. in the NCBI database. AF233756.
  • the ORF2 encoded a protein with 243 amino acids and was named Est4B1.
  • This protein is homologous to a putative independent and non-C-terminally fused thioesterase from Bacillus subtilis A13 (Accession No. AAF87217) and to subunit 4 of a surfactin synthetase from Bacillus subtilis ATCC 21332, which is also known as cold shock protein CSI16 (Accession No. Q08788 ) is known.
  • the ORF2 encoded a protein with 243 amino acids and was named Est4B1.
  • This protein is homologous to a putative independent and non-C-terminally fused thioesterase from Bacillus subtilis A13 (Accession No. AAF87217) and to subunit 4 of a surfactin synthetase from Bacillus subtilis ATCC 21332, which is also known as cold shock protein CSI16 (Accession No. Q08788 ) is known.
  • Haloperoxidase L Streptomyces lividans
  • Chlorperoxidase F Pseudomonas fluorescens
  • Bromperoxidase A2 Streptomyces aureofaciens
  • Proline-Iminopeptidase Seratia marescens
  • Haloalkane-Dehalogenase Xanthobacter Autotrophic
  • B3ADstart 5'GAACACACATATGGTCCAGCTC3 '.
  • B3ADend 5 ⁇ GCGCATGCTGTCTGTCATATC3'.
  • the reaction was carried out in a total volume of 50 ul, using 10 ng template DNA (pTB3), 5 ul dNTPs (1 mM each), 200 ng of each primer, 5 ul PCR buffer (10 x Dynazyme) and 2 Units Dynazyme DNA polymerase (Finnzyme, Espoo, Finland).
  • the construct thus obtained was named pMS470BS1.
  • E. coli was transformed with the plasmid thus obtained. Clones showing esterase activity were identified by the agar plate filter paper assay described above. The plasmid DNA of a positive clone was isolated after isolation sequenced to determine if the sequence matches the native sequence.
  • Example 6 Preparation of the esterase Est4B1
  • E. coli BL21 pMS470BS1
  • pMS470BS1 E. coli BL21
  • pMS470BS1 E. coli BL21
  • This overnight culture was used to inoculate 100 ml LB medium in a 300 ml Erlenmeyer flask.
  • the cells were grown at 37 ° C until the culture was in the middle of the logarithmic growth phase.
  • the main culture 300 ml in 1 l flask was inoculated with 10 ml of the second preculture.
  • esterase Est4B1 could be obtained in very high yield.
  • the expression of the esterase was very strong compared to the expression of other esterases that were produced under the same conditions. This indicated that the codon use in this Bacillus subtilis gene is very well suited for expression in E. coli.
  • the first 150 codons in particular have a lower GC content compared to the corresponding codons from esterases from other organisms, so that there are fewer melting RNA structures and thus the expression in E. coli is facilitated.
  • the protein was purified by a two step process.
  • the supernatant was placed on a 15 ml QFF column (Amersham Pharmacia, Uppsala, Sweden) and using a step gradient, starting with buffer A (10 mM Tris-HCl, pH 7.5) and increasing the proportion of buffer B (10 mM Tris-HCl, pH 7.5, 2M NaCl).
  • buffer A 10 mM Tris-HCl, pH 7.5
  • buffer B 10 mM Tris-HCl, pH 7.5, 2M NaCl
  • the majority of Est4B1 was eluted by passing 15 ml of buffer containing 8.5% of buffer B over the column and then passing a further 30 ml of buffer over the column, with a flat one
  • Est4B1 has esterase activity.
  • Crude lysates from E. coli BL21 (DE3) or purified enzyme were used for further analysis of the esterase activity of this enzyme.
  • Photometric assay Esterase activity was measured photometrically in Tris-HCl buffer (100 mM, pH 7.0) using ortho- and para-nitrophenyl butyrate (4 mM in DMSO) as substrates. The amount of nitrophenol released in the enzymatic reaction was determined photometrically at 405 nm and room temperature. An activity unit is defined as the amount of enzyme which releases 1 ⁇ mol alcohol min ⁇ 1 under the assay conditions.
  • Enzyme preparations that had been purified by anion exchange chromatography were used in the alkali titration.
  • Esterase activity of Est4B1 with respect to tributyrin, ethyl 3-hydroxybutyrate and methyl 2-chloropropionate was determined by alkali titration using the Mettler DL 21 autotitrator from Mettler Toledo (Switzerland). For this purpose, 50 ml of 20 mM NaH 2 PO 4 solution with 100 ⁇ l of substrate and 0.75 ml of BS1 lysate (corresponding to 56 U esterase, determined using para-nitrophenyl butyrate) at pH 7.5 and 30 ° C. were used. An activity of Est4B1 with regard to methyl 2-chloropropionate of 0.5 U / mg protein was determined. As the optimum pH for the Hydrolysis was found to be 8.
  • the activity was greatly reduced at a pH of less than 6.5.
  • the protein already precipitated in the slightly acidic range.
  • the hydrolytic activity for 3-hydroxybutyric acid ethyl ester was less than 0.01 U / mg. No activity was observed with tributyrin as the substrate.
  • the thin-layer chromatographic (TLC) experiments were carried out by suspending 100 ⁇ l of crude bacteria lysate from the cultures overexpressing Est4B1 in 0.5 ml of buffer (0.1 NaH 2 PO, pH 7.3). Crude lysate from cells without a vector was used as a negative control. After the addition of the substrate (20 ⁇ l), the mixture was shaken on a rotary shaker (150 rpm) at room temperature (25 ° C.). Samples were taken after 2 and 4 hours and after 1 day (20-24 hours) and analyzed directly by TLC using Merck Silicagel 60 F 254 . As far as technically feasible, the substrates specified in Table 4 were used in the TLC.
  • composition of the eluent (cyclohexane / ethyl acetate) and the visualization method (spraying with vanillin / H 2 SO conc. Or cerammonium molybdate as well as heat treatment and / or UV detection) were selected depending on the substrate.
  • a mixture of petrol / ethyl acetate (3: 1) was used for the substrates octanyl-2-acetate and 2-chloropropionic acid-2-naphthyl ester for elution and the products were made visible by vanilla / H 2 SO 4 conc . were sprayed.
  • a mixture of petrol / ethyl acetate (1: 1) was used for the elution for the substrates mandelic acid ethyl ester and 3-hydroxybutanoic acid ethyl ester.
  • the resulting compounds were made visible by UV detection in the case of the mandelic acid ethyl ester and by spraying with cerammonium molybdate in the case of the hydroxybutanoic acid ethyl ester.
  • the substrates mentioned were obtained from Sigma (St. Louis, MO, USA), with the exception of the 2-chloropropionic acid-2-naphthyl ester and the 2-octanylacetate, which were given by Michael Schmidt from the Institute of Organic Chemistry (Graz University of Technology) were obtained.
  • the hydrolytic activity of the esterase with respect to the substrates could be determined by the conversion rate of the acetate into the corresponding alcohol or of the ester into the corresponding acid.
  • the conversion rate was determined in the TLC on the basis of the color intensities of the starting material and the products.
  • Tab. 2 gives an overview of the results of the TLC.
  • esterase Est4B1 (56% ee, 63% conversion) showed selectivity with respect to the alcohol (Tab. 2). The enantiomeric purity of the corresponding acetate has not yet been determined. Interestingly, the esterase Est4B1 showed an opposite enantio preference compared to three other esterases tested.
  • Est4B1 showed almost no activity and selectivity with regard to the acetate of (R, SJ-3-butyn-2-ol (2), tetrahydrofuran-3-acetate (4), tetrahydrofuran-2-methyl ester in GC / HPLC (9), 3-hydroxybutyric acid ethyl ester (13) and 4-hydroxyisobutyric acid methyl ester (14).
  • the carboxylic acid (10 mmol) was carried out with a catalytic amount of H 2 SO 4 (conc.) And a 10-fold excess of methanol in the reflux process. All esters were purified using silica gel chromatography or Kugelrohr distillation.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

La présente invention concerne une thioestérase issue de Bacillus subtilis, des protéines homologues de celle-ci, des acides nucléiques codant ces protéines et des anticorps luttant contre ces protéines. La présente invention concerne également la production et l'utilisation desdites protéines, desdits acides nucléiques et desdits anticorps.
PCT/EP2002/009289 2001-08-24 2002-08-20 Esterase est4b1 issue de bacillus subtilis WO2003018789A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE10140703.3 2001-08-24
DE2001140703 DE10140703A1 (de) 2001-08-24 2001-08-24 Esterase Est4B1 aus Bacillus subtilis

Publications (1)

Publication Number Publication Date
WO2003018789A1 true WO2003018789A1 (fr) 2003-03-06

Family

ID=7695976

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2002/009289 WO2003018789A1 (fr) 2001-08-24 2002-08-20 Esterase est4b1 issue de bacillus subtilis

Country Status (2)

Country Link
DE (1) DE10140703A1 (fr)
WO (1) WO2003018789A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102286441A (zh) * 2011-07-24 2011-12-21 国家海洋局第二海洋研究所 一种低温酯酶及其编码基因与应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL [online] 24 July 2000 (2000-07-24), CONRAD ET AL.: "Putative thioesterase (srfD)", XP002222535, retrieved from EBI Database accession no. AF233756 *
PODILE A R ET AL: "SEED BACTERIZATION WITH BACILLUS SUBTILIS AF 1 INCREASES PHENYLALANINE AMMONIALYASE AND REDUCES THE INCIDENCE OF FUSARIAL WILT IN PIGEONPEA", JOURNAL OF PHYTOPATHOLOGY - PHYTOPATHOLOGISCHE ZEITSCHRIFT, BLACKWELL, BERLIN, DE, vol. 146, no. 5/6, 1998, pages 255 - 259, XP000919127, ISSN: 0931-1785 *
SCHNEIDER A ET AL: "GENETIC EVIDENCE FOR A ROLE OF THIOESTERASE DOMAINS, INTEGRATED IN OR ASSOCIATED WITH PEPTIDE SYNTHETASES, IN NON-RIBOSOMAL PEPTIDE BIOSYNTHESIS IN BACILLUS SUBTILIS", ARCHIVES OF MICROBIOLOGY, BERLIN, DE, vol. 169, no. 5, 1998, pages 404 - 410, XP000925848, ISSN: 0302-8933 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102286441A (zh) * 2011-07-24 2011-12-21 国家海洋局第二海洋研究所 一种低温酯酶及其编码基因与应用
CN102286441B (zh) * 2011-07-24 2012-12-12 国家海洋局第二海洋研究所 一种低温酯酶及其编码基因与应用

Also Published As

Publication number Publication date
DE10140703A1 (de) 2003-03-13

Similar Documents

Publication Publication Date Title
EP0334841B1 (fr) Micro-organismes et plasmides pour la formation de mono-oxygenase d'acide acetique de 2,4-dichlorophenoxy (2,4-d), et procede pour la fabrication de ces plasmides et souches
DE19610984A1 (de) Alkohol-Dehydrogenase und deren Verwendung zur enzymatischen Herstellung chiraler Hydroxyverbindungen
EP2145904A1 (fr) Procédé d'hydrolyse catalysée par enzymes d'esters d'acide polyacrylique et estérases utilisées à cet effet
WO2001007574A2 (fr) Monooxygenases a cytochrome p450 modifiees
EP2126065B1 (fr) Isoformes de l'estérase de foie de porc
DE69834130T2 (de) Epoxid-hydrolase
DE68928038T2 (de) Molekulare Klonierung und Expression von Genen, die für lipolytische Enzyme kodieren
EP1343902B1 (fr) Esterases recombinees de foie de porc, leur utilisation et procede permettant de les produire
EP1313860B1 (fr) Butinol i esterase
WO2003018789A1 (fr) Esterase est4b1 issue de bacillus subtilis
DE69736420T2 (de) Esterase Gen und dessen Verwendung
DE69920470T2 (de) Neuartiges gen und transformant, welcher dieses beinhaltet
DE19536506B4 (de) Neues Creatin-Amidinohydrolase-Gen, neue rekombinante DNA und Verfahren zur Herstellung von Creatin-Amidinohydrolase
DE10149715B4 (de) Esterase EstA aus Rhodococcus ruber
DE10100913A1 (de) Verfahren zur Herstellung von L-Menthol
WO2003031609A1 (fr) Esterase esta de rhodococcus sp.
DE102013104418B4 (de) Biokatalytisches Verfahren für die Herstellung von (R)-3-Chinuclidinol
DE69428478T2 (de) Norbornan-Ester-Hydrolase
WO2004013321A1 (fr) Esterase esta (xc4a) de xanthomonas vesicatoria
JP3855329B2 (ja) エステラーゼ遺伝子及びその利用
AT399886B (de) Esterase aus pseudomonas marginata
US6472189B1 (en) Esterase gene and its use
DE19960271A1 (de) Sarcosinoxidase und Verfahren zu deren Herstellung
DE10130169A1 (de) Aktivierte rec-D-Hydantoinasen
WO2003093467A1 (fr) Proteines a activite de (r)-hydroxynitrile lyase extraites de prunus laurocerasus et de sorbus aucuparia

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BY BZ CA CH CN CO CR CU CZ DE DM DZ EC EE ES FI GB GD GE GH HR HU ID IL IN IS JP KE KG KP KR LC LK LR LS LT LU LV MA MD MG MN MW MX MZ NO NZ OM PH PL PT RU SD SE SG SI SK SL TJ TM TN TR TZ UA UG US UZ VC VN YU ZA ZM

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR IE IT LU MC NL PT SE SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ UG ZM ZW AM AZ BY KG KZ RU TJ TM AT BE BG CH CY CZ DK EE ES FI FR GB GR IE IT LU MC PT SE SK TR BF BJ CF CG CI GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载