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WO2004013321A1 - Esterase esta (xc4a) de xanthomonas vesicatoria - Google Patents

Esterase esta (xc4a) de xanthomonas vesicatoria Download PDF

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Publication number
WO2004013321A1
WO2004013321A1 PCT/EP2003/007932 EP0307932W WO2004013321A1 WO 2004013321 A1 WO2004013321 A1 WO 2004013321A1 EP 0307932 W EP0307932 W EP 0307932W WO 2004013321 A1 WO2004013321 A1 WO 2004013321A1
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Prior art keywords
polypeptide
nucleic acid
esterase
seq
vector
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
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PCT/EP2003/007932
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German (de)
English (en)
Inventor
Helmut Schwab
Zarko Herzenjak
Apichat Upaichit
Michaela Pressnig
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Evonik Operations GmbH
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Degussa GmbH
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Priority to AU2003281854A priority Critical patent/AU2003281854A1/en
Publication of WO2004013321A1 publication Critical patent/WO2004013321A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/8281—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 for bacterial 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)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)

Definitions

  • the present invention relates to an esterase from Xanthomonas vesicatoria, proteins homologous to it, its expression, purification and use and nucleic acids coding for these proteins and host cells containing these nucleic acids
  • Xanthomonas vesicatoria is a gram-negative bacterium that is widespread in nature.
  • the phylogenetic relationships of all species of Xanthomonas described so far were analyzed by sequencing and comparing the 16S ribosomal DNAs (Hauben, L, 1997).
  • Members of the Xanthomonas genus are known to be plant pathogens. Plant pathogenicity involves overcoming and destroying the cell wall, which is probably the most important protection of the plant against invading microorganisms (Williamson, G., 1998, Jones, J.B., 2000).
  • the lipases are known to be activated by the lipid / water interface before they hydrolyze water-insoluble substrates with long chain fatty acid esters.
  • Knowledge of bacterial lipolytic enzymes is increasing rapidly. An extensive classification of the bacterial esterases are those described by Arpigny et al. (1999).
  • lipolytic enzymes Three different classes of lipolytic enzymes, namely (i) carboxylesterases (EC 3.1.1.1), (ii) true lipases (EC 3.1.1.3) and (iii) different types of phospholipases (Titball, RW, 1998 and Songer , JG, 1997) are described here.
  • esterolytic enzymes are characterized by the fact that they have conserved regions that contain the catalytic triad (Drablos, F., 1997) and their ability to catalyze a wide range of reactions.
  • Each hydrolase has a specific stereo preference with respect to a given substrate under certain reaction conditions, which can be referred to as its characteristic fingerprint (Rogalska, E., 1993).
  • Esterases can be used for the enzymatic hydrolysis of racemic carboxylic esters into their corresponding carboxylic acids and alcohols. Furthermore, they can be used for transesterifications and for the synthesis of esters. The ability of the esterases to be active in both aqueous and non-aqueous systems makes them important tools for organic synthesis (Sheldon, R.A., 1993). Esterases are of particular interest for the synthesis of enantiomerically pure products (Turner, N.J., 1994).
  • esterases The physiological functions of many esterases are still unclear. Some of these enzymes are known to be involved in metabolic pathways, such as esterases, which break down the cell wall and act as virulent factors (McQueen, DA, 1987). Virulent factors often play an important role in car transporter systems. Fenselau et al. (1992) characterized genes from X. vesicatoria, which are necessary for the induction of the hypersensitive response in resistant plants, the so-called hrp genes. They suspected that these genes are involved in the secretion of molecules that are essential for the Interaction of Xc pv. Vesicatoria with the plant.
  • the virulent factors can be categorized into families, the categorization either being based on their function or on the basis of the export mechanism through which they reach the bacterial surface (Finlay, BB, 1997).
  • the object of the present invention was to provide new enzymes with hydrolytic activity, in particular new esterases and / or lipases, 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 a method to provide, with which new hydrolytic enzymes, in particular esterases and / or lipases, can be obtained.
  • the object of the present invention was in particular to provide new hydrolytic enzymes, in particular esterases and / or lipases, which have a different structure and / or different substrate specificity and / or a distinguishable degree of selectivity compared to the enzymes with hydrolytic activity described hitherto, in particular regio- or enantioselectivity, and / or have a distinguishable reaction rate and / or a distinguishable reaction mechanism, preferably at least one of the distinguishable properties being an improved property compared to the prior art.
  • Another object of the present invention was to find hydrolytic enzymes, in particular esterases or lipases, which have phytopathogenic effects and / or can act as virulent factors.
  • polypeptides according to the invention polypeptides and by substances which bind to polypeptides 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 Polypeptides according to SEQ ID NO: 2 and SEQ ID NO: 4 binding substances, in particular antibodies.
  • polynucleotides according to SEQ ID NO: 1 and SEQ ID NO: 3 polypeptides according to SEQ ID NO: 2 and SEQ ID NO: 4 binding substances, in particular antibodies.
  • 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) polynucleotide with a nucleic acid sequence from positions 586 to 1641 or from position 697 to 1641 according to SEQ ID NO: 1, c) polynucleotides coding for a polypeptide with an amino acid sequence according to SEQ ID NO: 2 or according to SEQ ID NO: 4 or from position 38 to position 351 according to SEQ ID NO: 2, d) naturally occurring or artificially generated mutants or polymorphic forms or alleles of a polynucleotide according to (a), (b) or (c), in particular those which have up to ten, in particular five, especially one, two or three, point mutations in relation to a polynucleotide (a), (b) or (c) possess, e) polynucleo
  • 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 for parts thereof, the parts in particular being domains or sequences which can be used as epitopes and which can be used, for example, for antibody production.
  • the protein with hydrolase activity is preferably a lipase or esterase and / or an enzyme which is able to cleave ester, thioester, amide, halide and / or peptide bonds.
  • cleavage means above all hydrolytic cleavage, that is cleavage with liberation of the carboxylic acid and the corresponding alcohol, thiol, amine or hydrogen halide.
  • Cleavage can be special However, embodiments also take place with the formation of an ester, thioester, amide, peptide or halide bond; in particular, if an ester bond is converted into a new ester bond, this can be a transesterification reaction.
  • the enzyme with hydrolytic activity is very particularly preferably an esterase, especially a carboxylesterase, and / or an enzyme which is capable of cleaving regioselective or enantioselective bonds, the value for the enantiomeric excess (ee) is preferably greater than or equal to 20%, 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, in particular a serine esterase which belongs to the GXSXG family or is homologous to these enzymes and / or an enzyme belonging to the family of carboxyl ester hydrolases (EC 3.1.1), in particular to the family of carboxyl esterases (EC 3.1.1.3).
  • the nucleic acids according to the invention are preferably nucleic acids which are suitable for an esterase, particularly preferably for a microbial esterase, especially a bacterial esterase, in particular for an esterase from Xanthomonas, especially for an esterase from Xanthomonas vesicatoria, in this case in particular for the esterase EstA according to SEQ ID No. 2 or from position 38 to position 351 according to SEQ ID NO: 2.
  • 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 lipase and / or esterase activity and with improved functional properties for example, higher specificity and / or higher conversion and / or higher regio- or enantioselectivity is to be understood.
  • 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, a cDNA or genomic library, preferably for identifying nucleic acids which are used for hydrolases, especially esterases or lipases and / or for 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 hydrolases, in particular for esterases and / or lipases or for 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.
  • 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 for the production of fusion proteins which comprise the polypeptides according to the invention.
  • the present invention therefore also relates to a method for obtaining a nucleic acid which codes for an enzyme with hydrolytic activity, in particular for 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 ) a nucleic acid that hybridizes with the nucleic acid according to the invention according to (a) 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 by amplification according to (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 esterase and / or lipase, comprising the following steps: a) nucleic acids of a library are incorporated into suitable vectors and these into suitable host organisms, preferably bacteria, in particular E.
  • nucleic acids from the in step (d) identified clones 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 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 are incorporated into a suitable vector and expressed in 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) show hydrolytic activity, are sequenced.
  • the mutagenesis experiments can be carried out, for example, using the polymerase chain reaction (PCR).
  • the experiments can be carried out, for example, especially 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-prone can be used -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 to be used according to the invention can be, for example, a cDNA, a genomic or an artificial library. It is preferably a microbial, particularly preferably a bacterial library, especially one from Xanthomonas, in particular from Xanthomonas vesicatoria.
  • the invention further relates to a nucleic acid which can be obtained by one of the methods mentioned above.
  • the present invention furthermore relates to 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 determined using the nucleic acid sequence given in SEQ ID NO: 1 or the SEQ ID NO: 3 or using the amino acid sequence given in SEQ ID NO: 2 or the SEQ ID NO: 4 based on the genetic code the phosphotriester method or in another manner known to the person skilled in the art.
  • the present invention furthermore relates to vectors, in particular cloning and / or expression vectors, comprising one of the aforementioned nucleic acids according to the invention.
  • the vector can be, for example, a prokaryotic or eukaryotic vector.
  • the prokaryotic vectors for incorporating the nucleic acids according to the invention are, for example, the plasmids pBSII, pBS SK-, pGEM3Zf (+/-), pGEM-5Zf (+/-) or pMS470 ⁇ 8 or another high copy number plasmid.
  • the available expression vectors for expression in E. coli are, for example, the vectors pBLXC4 ⁇ Accl, pMSXC4a, pBLXC4 and pMStacXC4a described below for the expression of a protein according to SEQ ID No. 2 or around the vector pMStacXv-86 for expression of a protein according to SEQ ID NO: 4.
  • 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- Code 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 such 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 and / or detects that can facilitate polypeptides.
  • the day can be, for example, the strep, flag, myc or his tag.
  • the expression vectors preferably contain the regulatory sequences suitable for the host cell, preferably the lac or the 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 an LTR promoter 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 Xanthomonas, in particular Xanthomonas vesicatoria, or E. coli, in particular the E. co / Z strain BL21 (DE3).
  • Xanthomonas in particular Xanthomonas vesicatoria
  • E. coli in particular the E. co / Z 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, particularly preferably according to the SEM method.
  • the present invention furthermore relates to a polypeptide selected from the group consisting of: a) polypeptide having an amino acid sequence according to SEQ ID NO: 2 or from positions 38 to 351 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), especially those which have up to five, in particular one, two or three, point mutations with respect to a polypeptide according to (a) or (b), 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), 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 15 or 20, in particular at least 30, 40
  • polypeptides which catalyze a hydrolysis reaction, in particular the hydrolysis of ester, amide, peptide and / 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 carboxyl ester hydrolase (EC 3.1.1), in particular a carboxyl esterase (EC 3.1.1.1) or a protein with significant homology to carboxyl esterases and / or an esterase belonging to the GXSXG family of serine hydrolases and / or an enzyme which has alpha / beta hydrolase folding.
  • a carboxyl ester hydrolase EC 3.1.1
  • carboxyl esterase EC 3.1.1.1
  • a protein with significant homology to carboxyl esterases and / or an esterase belonging to the GXSXG family of serine hydrolases and / or an enzyme which has alpha / beta hydrolase folding are preferably a carboxyl ester hydrolase (EC 3.1.1), in particular a carboxyl esterase (EC 3.1.1.1) or a protein with significant homology to carboxyl esterases and / or an esterase belonging to the GXSXG family of serine
  • the compounds which can be cleaved by the polypeptides according to the invention are in particular esters, thioesters, peptides, amides or halides, preferably esters, thioesters, peptides, amides and halides, especially esters, short-chain carboxylic acids.
  • the polypeptides according to the invention are particularly preferably esterases, preferably microbial, in particular bacterial, esterases. It is very particularly preferably an esterase from Xanthomonas vesicatoria, in particular the esterase EstA according to SEQ ID NO: 2.
  • the polypeptide according to the invention is a water-soluble esterase, in particular a periplasmic esterase, that is to say an esterase which is naturally conveyed into the periplasmic space through a corresponding signal sequence.
  • polypeptide according to the invention is an esterase according to SEQ ID NO: 4, in which the natural signal sequence has been removed by deleting the amino acids from sequence positions 3 to 37 according to SEQ ID NO: 2.
  • polypeptide according to the invention is a virulent factor and / or a plant pathogen.
  • the present invention furthermore relates to mixtures and preparations, in particular bacterial preparations, which contain the nucleic acids and / or polypeptides according to the invention.
  • the mixtures or preparations can be prepared in a manner known to those skilled in the art.
  • the invention furthermore relates to 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.
  • Substrates or compounds to be prepared according to the invention are generally compounds of the formula R 1 -C (0) -R 2 .
  • R 1 is a hydrocarbon chain, which can preferably comprise up to 5, particularly preferably up to 3 carbon atoms, in particular exactly 1 carbon atom, the hydrocarbon chain preferably being unbranched and saturated, but also branched and / or can be unsaturated and at least one of the hydrogen atoms of the hydrocarbon chain can also be substituted by other groups, preferably selected from the group consisting of halogens, hydrocarbon radicals, in particular comprising 1 to 3 carbon atoms, S0 3 , N0 2 and other functional groups known to the person skilled in the art.
  • Preferred examples of R 1 are the methyl, ethyl, propyl and chloro-ethyl radical.
  • R 1 is a cyclic compound, in particular a heterocyclic compound, especially one having 5 ring atoms.
  • at least one of the hydrogen atoms of the cyclic compound can be substituted by another group, as mentioned above.
  • An example of such a compound is tetrahydrofuran.
  • R 2 represents one of the halogens F, Cl, Br, I, an amino group NR 3 R 4 , a thiol group SR 3 and particularly preferably an alcohol group OR 3 .
  • R 3 and / or R 4 can be an aryl radical with 6 to 14 C atoms or a heteroaryl radical with 5 to 13 C atoms, at least one hydrogen atom of the aryl or heteroaryl radical may also be substituted by a group preferably selected from hydrocarbon radicals, especially comprising from 1 to 6 carbon atoms, halogen atoms and known to those skilled functional groups such as N0 2, NR 2, S0 3, OR.
  • the aryl radical can be, for example, a phenyl, o- or p-nitrophenyl radical or a naphthyl radical, in particular a -naphthyl radical.
  • R 3 and / or R 4 can also be a bridge ring system, in particular a norbornane radical, the bridge ring system also being unsaturated and bearing substituents, as mentioned above.
  • R 3 and / or R 4 can furthermore be an alkyl group or a heteroalkyl group, preferably one with up to 10 C atoms, particularly preferably with up to 6 C atoms, the alkyl group or heteroalkyl group also be unsaturated and can carry at least one substituent, for example the phthalimido group or one of the aforementioned substituents.
  • the group can also be epoxidized.
  • the group can also be a cycloalkyl or a cycloheteroalkyl group, particularly preferably with 6 Ring atoms. Examples of such groups are the glycidyl group, the cis-1, 2-dihydroxymethyl-cyclohexenyl group and the 2-phthalimido-butyl group.
  • R 3 and / or R 4 can furthermore be, for example, a group CR 5 R 6 R 7 , the radicals R 5 , R 6 and R 7 independently of one another for H or a hydrocarbon radical with preferably up to 6 C atoms , particularly preferably up to 4 carbon atoms, where the hydrocarbon radical can be saturated or unsaturated, branched or unbranched and at least one of the H atoms of at least one of the hydrocarbon radicals can also be substituted by one of the aforementioned substituents.
  • R 5 is an H atom
  • R 6 is an at least monounsaturated hydrocarbon chain
  • R 7 is a saturated hydrocarbon chain.
  • R 6 and R 7 each comprise, independently of one another, preferably up to 6 C atoms, particularly preferably up to 4 C atoms, especially up to 2 C atoms. This can be branched or unbranched residues.
  • R 6 very particularly preferably comprises at least one, especially exactly one triple bond, the triple bond being particularly preferably arranged in the 2 position.
  • R 6 is a prop-2-ynyl radical and R 7 is a methyl radical.
  • At least one of the radicals R 5 , R ⁇ and R 7 is a multifunctional alcohol, in particular glycerol, it being possible for at least one further OH group of the glycerol to be esterified.
  • glycerol a multifunctional alcohol
  • An example of this is triacetin.
  • the substrate is a component of the plant cell wall.
  • the enzymes according to the invention are used here to cleave or form enantioselectively bonds, wherein the enantiomeric excess (ee) of the reaction in question is preferably greater than or equal to 20%, particularly preferably greater than or equal to 40%, especially greater than or equal to 60%, in particular 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 present invention also relates to a process for cleaving the aforementioned esters, thioesters, amides, peptides or halides, characterized in that these compounds are incubated with at least one polypeptide according to the invention.
  • the present invention also relates to a process for the preparation of the aforementioned esters, thioesters, amides, peptides or halides, characterized in that carboxylic acids and / or carboxylic acid esters, thioesters, amides or halides with an alcohol, thiol, amine or Hydrogen halide can be incubated in the presence of at least one polypeptide according to the invention.
  • polypeptides according to the invention are, for example, the production or selective enrichment of flavor components in the food industry, of detergents in the detergent industry, of fine chemicals in the chemical industry or of 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.
  • 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 isolated, and starting from the antibodies or the nucleic acids coding for them, antibody fragments, for example Fab or scFv fragments, can optionally be produced.
  • 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, for example, the so-called Fusagen technology, in which the nucleic acid and the polypeptide encoded by it are obtained a puromycin are covalently linked.
  • 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, in particular 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 binding to a peptide according to the invention are referred to below as "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, particularly preferably in E. coli, in particular in E. coli BL21 (DE3) or E. coli SURE TM, or in Xanthomonas, in particular in Xanthomonas vesicatoria, is expressed.
  • a suitable host cell particularly preferably in E. coli, in particular in E. coli BL21 (DE3) or E. coli SURE TM, or in Xanthomonas, in particular in Xanthomonas vesicatoria.
  • a protein purification can then optionally be carried out.
  • E. coli is preferably cultivated between 20 and 37 ° C, particularly preferably at about 30 ° C or at about 37 ° C.
  • Harvest and digestion of the Cells are made by methods known to those skilled in the art. The digestion can thus be carried out, for example, by 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.
  • the chromatographic purification of the protein can include, for example, cation and / or anion exchange chromatography, hydrophobic interaction chromatography and / or gel filtration.
  • the method can be carried out, for example, in the following way: 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) anion exchange chromatography is carried out with the supernatant, d) after the elution, the active fractions are optionally combined and e) hydrophobic interaction chromatography is carried out, then f) the active fractions are optionally combined again and then concentrated.
  • the anion exchange chromatography is preferably carried out using an anion exchange column, in particular using a Q-Sepharose column, for example using the Q-Sepharose column FF from Pharmacia.
  • Hydrophobic interaction chromatography is preferably carried out with a hydrophobic interaction chromatography column, in particular with a butyl-Sepharose column, for example with the HIC-Butyl-Sepharose column from Merck (Darmstadt, Germany).
  • the application to and elution from the anion exchange column is preferably carried out between pH 6.0 and 8.0, particularly preferably between pH 6.5 and 7.5, in particular approximately at pH 7.0.
  • a Tris-HCI is used for this Buffer with a concentration of, for example, 100 mM or another buffer with a similar ionic strength.
  • hydrophobic interaction column is preferably carried out at a pH between 7.0 and 9.0, particularly preferably between pH 7.5 and 8.5, in particular approximately at pH 8.0, and at a (NH 4 ) 2 SO 4 concentration between 0.4 and 0, 8 M, in particular about 0.6 M or using another salt with the setting of a corresponding ionic strength.
  • the pH is preferably kept constant by applying a decreasing salt gradient.
  • the elution of the polypeptides according to the invention can thus be achieved, for example, at a concentration of about 0.1 M (NH 4 ) 2 SO 4 or when using another salt at a corresponding ionic strength.
  • short-chain polypeptides according to the invention can also be synthesized using classic peptide synthesis (Merrifield technique).
  • the present invention also relates to a method for controlling plant pathogenic factors, characterized in that a plant is treated with a nucleic acid according to the invention, a polypeptide according to the invention or an antibody according to the invention becomes.
  • the present invention therefore also relates to a composition for the treatment of plants, comprising a nucleic acid according to the invention, a polypeptide according to the invention and / or an anti-body according to the invention.
  • the present invention furthermore relates to plant cells and transgenic plants, comprising a nucleic acid according to the invention and / or a polypeptide according to the invention and / or an antibody according to the invention, and a method for producing such transgenic plants, which consists in that a nucleic acid according to the invention or a for a polypeptide according to the invention or nucleic acid coding for an antibody according to the invention in is introduced into a plant cell in a manner known to the person skilled in the art and, if appropriate, a transgenic plant is grown from the transgenic cells, which preferably carries plant pathogens and / or factors which are directed against plant pathogens.
  • the plant cells or the transgenic plant are preferably those which can be infected by Xanthomonas, in particular Xanthomonas vesicatoria, and / or related phytopathogenic bacteria.
  • the invention furthermore 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.
  • the present invention furthermore relates to the substrates and functional interactors obtainable with the aid of the test system according to the invention, which can have a modulating, inhibiting or activating effect on a polypeptide according to the invention.
  • one or more nucleotides of the nucleic acid or one or more amino acids of the polypeptides or antibodies can also be modified.
  • the modification can be, for example, a radioactive, fluorogenic or chromogenic group or a post-translational modification.
  • Xanthomonas vesicatoria DSM 50071 was used as a source for the esterase genes and cultivated at 30 ° C. in LB medium.
  • E. coli HB101 (Boyer and Roulland-Dussoix, 1969): hsd S20 (rB “ , mB “ ), supE44, ara14, ⁇ ; galK2, / acY1, proA2, rspL20, xyl-5, mf / -1, recA13 (ATCC 33649) and E.
  • coli SURE hsdR, merk, mer B, mrr, endf ⁇ , sup AA, thi-, ⁇ " , gyrA96, right / A1, lac, recB, recJ, sbcC, umuC, uvrC, [F ⁇ proAB, / aqlqZ ⁇ M15, Tn10, (tet r )] were used as hosts for the work with the recombinant DNA.
  • Recombinant E. coli HB 101 and recombinant E. coli SURE strains were normally incubated at 37 ° C. on LB petri dishes with LB medium and 100 ⁇ g / ml ampicillin overnight.
  • Constructs for overexpression were incubated at 30 ° C. in 100 ml liquid LB medium with 100 ⁇ g / ml ampicillin overnight to avoid possible misfolding of the overexpression product.
  • plasmid constructs pBluescript® SK- (Stratagene Cloning Systems, La Jolla, USA); AmpR, lacZ, f1 ori; and pMS470 ⁇ 8 (Balzer et al., 1994; Amp R , laclQ, Ptac, SD-T7) were used as cloning vectors for standard techniques with the recombinant DNA and for the production of the expression constructs.
  • pMS4K the insertion-less form of pMS470 ⁇ 8 was used for control purposes.
  • PBLXC4 is the esterase-positive clone of the primary gene library.
  • pBL XC4 ⁇ Accl was prepared starting from pBLXC4 by deleting a 2 kbp yAccI fragment.
  • PMS XC4a is an expression construct for the expression of EstA, in which the sequence of the native promoter, as shown in FIG. 1, is still present between the strong tac promoter of the expression vector and the structural gene for the esterase EstA. In this construct, expression can also be controlled by the native promoter still present.
  • the vector pMS470 ⁇ 8 was cut with Nde I and Hind III.
  • the vector pBLXC4 ⁇ Accl containing the entire estA ORF (open reading frame) was cut with Dralll and Hind III (Dralll cuts within the 5 'region of the esM-ORF). The missing estA 5 'end was obtained using the following 43 bp linker fragment:
  • Ndel Dr ⁇ III The Nde I and Dra III restriction sites are in bold. An ATG codon that may encode an extended polypeptide is underlined.
  • the linearized vector pMS470 ⁇ 8, the partial estA-ORF, and the DNA linker were ligated together in a single reaction.
  • PMStacEstA is a construct for the expression of EstA under the sole control of the tec promoter present on the expression vector. To produce this expression construct, the vector pMSEstA was cut with Nde I and Accl, releasing the esM-ORF.
  • the vector pBLXC4 was cut with Pvul and Accl, resulting in a 5 'deleted esfc4 ORF lacking the native estA promoter.
  • the missing estA 5 'end was obtained from the following 16 bp linker fragment:
  • the Nde I and Dra III restriction sites are in bold.
  • the ATG start codon is underlined.
  • the linearized vector pMS470 ⁇ 8, the partial esM-ORF and the DNA linker were ligated together in a single reaction. Plasmid DNA was isolated and purified using Promega Wizard TM MiniPreps.
  • DNA modification and transformation restriction endonucleases and modifying enzymes were obtained from New England BioLabs (Beverly, USA), Röche Diagnostics GmbH (Vienna, Austria), and Promega Corporation (Madison, USA). Restriction digestion and DNA modifications were carried out as recommended by the manufacturer. The E. co / 7 strains were transformed by the SEM method (Inoue et al., 1990).
  • Genomic DNA from Xanthomonas vesicatoria DSM 50071 was prepared using the Qiagen Genomic Kit (Qiagen GmbH, Hilden, Germany). After partial digestion with Sau3AI, the genomic DNA fragments with a size of 3-5 kbp were cloned into the vector pBluescript TM SK according to the "partial fill-in" technique, which had previously been linearized by digestion with Xho ⁇ The ligation products were used to transform E. coli SURE cells using the SEM method, and the transformants were plated onto LB-Amp plates containing IPTG / X-Gal. A total of 13,800 clones were obtained. E) Search for active clones
  • the Dye-Deoxy Terminator Sequencing Kit (Applied Biosystems Inc., Faster City, CA, USA) and the AmplyTaq DNA polymerase (Perkin-Elmer Corporation, Norwalk, USA) were used for the automated sequencing of double-stranded DNA on an ABI 373A sequencing device. In addition, some subclones and gene-specific primers were used to obtain complete sequence information for both strands of DNA. Sequence analysis and homolog search was performed using the Wisconsin Sequence Analysis Package (Devereux et al., 1984) and the Blast program (Altschul et al., 1990). The expasy proteomics tools server (http://expasy.hcuqe.ch/www/tools.htmn) was used for detailed protein sequence analysis.
  • esterase gene estA was expressed in E. coli SURE in 250 ml LB medium containing 100 ⁇ g / ml ampicillin and 0.1 mM IPTG.
  • a 100 ml preculture was inoculated with a single colony of the corresponding expression clone and allowed to grow overnight at 37 ° C.
  • a second pre-culture was inoculated with 10 ml of this pre-culture, which was grown until the exponential growth phase.
  • the 250 ml main culture was inoculated with 10 ml of the second preculture.
  • the cells were grown at 30 ° C. to an OD595 of 0.2, after which enzyme expression was induced by adding IPTG to a final concentration between 0.005 and 0.5 mM.
  • the cells were harvested by centrifugation at 10,000 g for 10 minutes.
  • the pellets were resuspended in 10 ml of 100 mM Tris-HCl, pH 7.0.
  • After freezing the cells at -20 ° C. they were thawed, mixed by vortexing and disrupted by sonification with the Branson Sonifier 250 (3x30 seconds on ice; duty cycle 70%).
  • the supernatant fractions were collected and the pellets were resuspended in 100 mM Tris-HCl, pH 7.0, adjusting the previous volume.
  • the crude lysate, the supernatant and the pellet fraction were examined for esterase activity.
  • the supernatant fraction was placed on an anion exchange chromatography (IEX) column (Pharmacia QFF) and the protein was eluted with the flow-through fractions.
  • IEX anion exchange chromatography
  • a hydrophobic interaction chromatography (HIC) column butyl Sepharose) from Merck (Darmstadt, Germany) was used for the second purification step.
  • the protein fractions from the lEX step with esterase activity were combined and a concentration of 0.1 M sodium phosphate (pH 8.0) and 0.6 M (NH 4 ) 2 SO 4 was established. Under these conditions the protein bound to the butyl sepharose column. A decreasing salt gradient was then created.
  • the active protein was eluted from the column at a concentration of 0.1 M (NH 4 ) S0 4 .
  • the samples were separated from one another by means of denaturing SDS gels and testing for activity was carried out after renaturation of the esterase, which was achieved by incubating the gels in water for 60 minutes.
  • the detection of the esterase activity for the protein bands on the gel was carried out by incubating the gel in a solution of 10 ml of 0.1 M phosphate buffer (pH 7.0), 1 ml of substrate stock solution ( ⁇ -naphthylacetate, 1% [w / v] in acetone) and 250 ⁇ l Fast Blue B stock solution (2% [w / v] in H 2 0). The reaction was stopped by incubating the gels with 10% acetic acid. Bands with esterase activity were detectable as dark red bands.
  • Esterase activity was measured with o- and p-nitrophenyl fatty acid esters (Sigma) as substrates.
  • the photometric assay was carried out at room temperature in 100 mM Tris-HCl, pH 7.0, with substrate in a concentration of 4 mM.
  • Substrate stock solutions were made up with DMSO as a solvent.
  • the release of o- and p-nitrophenol was monitored spectrophotometrically at 405 nm, the activity being calculated on the basis of the specific absorption coefficients of 2.4 for o-nitrophenol and 9.6 for p-nitrophenol.
  • the absorption coefficients were calculated by linear regression analysis of a standard curve of p- or o-nitrophenol at 405 nm.
  • a unit of enzyme is defined as the amount of enzyme that releases 1 ⁇ mol of o- or p-nitrophenol per minute.
  • Ester hydrolysis releases the ester components alcohol and fatty acid.
  • the release of the acid component was examined using a pH indicator.
  • the enzymatic hydrolysis was carried out at room temperature in 100 mM Tris-HCl, pH 7.0, containing 4 mM substrate.
  • the esterase activity was determined directly with cell crude lysates or after separation of the cell crude lysate with a native polyacrylamide gel.
  • the assays were carried out at room temperature in 100 mM Tris-HCl, pH 7.0. Substrate stock solutions prepared using DMSO as a solvent. The fluorescence was excited using a transiluminator at 315 nm and evaluated visually.
  • esterase activity By removing a 1.3 kb fragment, the esterase activity could be assigned to a 2 kb subfragment (FIG. 1). By sequencing this fragment, an ORF (open reading frame) could be determined that codes for a 352 amino acid polypeptide called EstA.
  • EstA A possible ribosome binding region, a putative promoter consensus sequence from the E. coli ⁇ . The o-type and a Shine-Dalgamo sequence (SD) could be found in the 5 'direction from the start codon (FIG. 1).
  • Ruminococcus flavefaciens Another ruminant bacterium, Ruminococcus flavefaciens, is known to have an enzyme complex that degrades the plant cell wall and also includes a xylanase (XynB) with a C-terminal esterase domain (Kirby et al., 1998).
  • XynB xylanase
  • EstA expression should be inducible using the vector pMS470 ⁇ 8 (by adding IPTG), no significant increase in EstA activity could be observed under induced conditions. This suggests that the native promoter still present in this expression construct controls the expression and not the inducible tac promoter present on the vector.
  • the second expression construct pMStacXC4a was produced by removing the insert sequence in the 5 'direction from the ORF of the esM gene and thus placing the gene directly behind the inducible tac promoter and the highly regulatable SD sequence from T7.
  • E. coli SURE pMStacXC4a
  • a further increase in the expression and activity of the esterase EstA could be achieved by replacing the native promoter from Xanthomonas vesicatoria with the inducible tac promoter, which is located on the vector pMS470 ⁇ 8 (FIG. 4).
  • the activity could be increased tenfold again when induction of the E. co / Z cultures was carried out with 0.1 mM IPTG (Table 1).
  • esterase EstA was accomplished by anion exchange chromatography followed by hydrophobic interaction chromatography. A 2.5-fold purification of the enzyme was achieved by placing the soluble fraction of the crude cell lysate on an anion exchange column (QFF from Pharmacia). In a solution of 0.1 M Tris-HCl and pH 7.0, the esterase EstA does not bind to the column and could thus be collected with the first flow-through fractions. The protein pre-purified in this way was analyzed by SDS-PAGE (Fig. 5).
  • the flow-through fractions were combined and buffered while adjusting 0.1 M sodium phosphate, pH 8.0, 0.6 M (NH 4 ) 2 SO and placed on a butyl-Sepharose hydrophobic interaction column (Merck, Darmstadt, Germany). In this way, a further 15-fold purification of the enzyme could be achieved.
  • the purified protein could be visualized in the SDS-PAGE as a 33 kDa band (FIG. 5). These purification steps resulted in an enzyme with a purity of approximately 70%. Isoelectric focusing of the purified protein confirmed as p1 of the unprocessed peptide a value of 8.1, as previously calculated based on the sequence.
  • the clone E. coli SURE pMStacXC4a was used to determine the hydrolytic activity of EstA in relation to various substrates.
  • p- and o-nitrophenol fatty acid esters of different chain lengths were used as substrates, whereby a clear preference of the enzyme for ortho-substituted nitrophenols and short-chain fatty acids could be determined (Tab. 2).
  • no hydrolytic activity could be determined for the purified enzyme with nitrophenol fatty acid esters that have a longer fatty acid chain, such as valerate, caproat, caprylate, nonaoate and palmitate.
  • Esterase EstA was also assayed for hydrolytic activity for triacetin and tributyrin using a plate assay. Here, only by using triacetin activity could be determined by forming a plaque.
  • the gene coding for the esterase was modified in such a way that the nucleotide sequences coding for the amino acids 3 to 37 were removed. This removed a large part of the naturally occurring signal peptide.
  • EstA polypeptide corresponds, amplified.
  • the two amino acids Met and Gin additionally introduced at the N-terminus compared to the native processed EstA are contained in the primer for the N-terminal end (marked in bold)
  • the PCR reaction batches were carried out under standard conditions, as recommended by the manufacturer for the TaqHotStar DNA polymerase used, using "Q-solution” (Qiagen, Germany).
  • the reaction conditions for the PCR were 95 ° C.
  • the bacterial strains were grown under aerobic conditions on Luria-Bertani (LB) medium (1% bactotrypton, 0.5% yeast extract, 0.5% NaCl). Ampicillin (100 mg I "1 ) was added if plasmid selection should be performed.
  • the bacterial cells were grown on LB agar plates (15 g I " 1 Bactoagar) at 37 ° C and in liquid LB medium at 30 ° C , For enzyme production, 5 ml LB medium were inoculated with bacterial cells and grown overnight at 30 ° C. The cultures were placed in 100 ml of LB medium and the cultures thus obtained were allowed to grow to the middle exponential phase.
  • the pellets were disrupted by ice three times for 30 seconds each on ice (Braun Labsonic 2000, Branson Sonifier 250; duty cycle: 50%; Output control: 5).
  • the supernatant obtained by subsequent ultracentrifugation (30,000 g; 60 min) was used for biocatalytic enzyme studies.
  • the anion exchange chromatography with a QSepharose R Fast Flow Matrix column the column was first equilibrated with a buffer containing 20 mM Tris-HCl, 50 mM NaCl, pH 7.5. After the sample application, washing was carried out with the same buffer, the enzyme being in the flow-through fraction.
  • the hydrophobic interaction chromatography with a Pharmacia glass column containing Fractogel TSK Butyl 650 (Merck, Darmstadt, Germany) as the column material the column was firstly treated with a buffer containing 20 mM Tris-HCl, 1.5 M NaCI, pH 7.5 equilibrated. After the sample application, washing was carried out with the same buffer. Protein elution takes place through a buffer containing 20 mM Tris-HCl, 0.1 M NaCl, pH 7.5.
  • Example 6 Reaction of the enzyme with 1-methylprop-2-ynyl acetate
  • But-3-yn-2-ol is a very important chiral building block for many materials, for example for the 5-lipoxygenase inhibitor "Fenleuton".
  • But -3-in-2-ol is an intermediate in the production of the sex pheromone of the male mountain beetle, Dendroctonus brevicomis, and the pharmaceutical and biological activity of these compounds is due to the steric configuration of the C atom carrying the hydroxyl group.
  • the hydrolysis reactions were carried out by introducing 5 ml of 0.1 M sodium phosphate buffer (pH 7.3) and then adding 250 ⁇ l of crude enzyme preparation and 100 l of substrate. The reaction solution was stirred at 150 revolutions per minute at room temperature. Samples were taken after 2 and 5 hours and after one day. After extraction with CH 2 CI 2 , centrifugation was carried out and then the organic phase was dried over Na 2 SO 4 .
  • the reaction was monitored using a Shimadzu GC-14A gas chromatograph, a C-R5A Chromatopac integrator and an FID detector at 250 ° C.
  • the separation of the enantiomeric acetates and alcohols was carried out using a Chirasil-Dex-CB fused silica capillary column (2,3,6-tri-O-methyl - ⁇ - cyclodextrin column from Chrompak, 25 mx 0.32 mm, 0, 25 ⁇ m coating).
  • the samples Prior to use in the chiral GC, the samples were filtered (filters Needle, 1, 1 mm x 40 mm, 19 G1 1/2 TW Becton Dickinson).
  • the retention times determined by means of reversed phase HPLC were 9.6 minutes for 1-methylprop-2-ynyl acetate and 2.3 minutes for but-3-yn-2-ol (FIG. 8).
  • the conversion rates determined were 75% (at 204 nm) and 76% (at 210 nm) after 2 Hours response time. With longer reaction times, yields of up to 85% could be achieved.
  • this process is the first described process for the preparation of enantiomerically enriched 1-methylprop-2-ynyl acetate and but-3-yn-2-ol by an enzymatically catalyzed hydrolysis reaction.
  • Example 7 Determination of the Kinetic Properties of the Esterase EstA To determine the kinetic parameters of the enzyme against p- / o-nitrophenyl acetate, measurements with the substrate in 100 mM Tris-HCl, pH 7.0 at 25 ° C. were used. One unit (U) is defined as 1 / mol of alcohol released per minute. The values determined for KM were 0.45 (o) and 0.46 (p) mM, for Vmax 188 (o) and 384 (p) mol min “1 mg " 1 and for k ca t 110 (o) and 224 (p) s "1.
  • Example 8 Production of mutants with improved activity in aqueous solution
  • random mutagenesis experiments were carried out by means of PCR.
  • reaction solutions were prepared to prepare the library: 30 ng plasmid; 0.4 mM dNTP; 3 mM MgCl2; 0.15 mM MnCl2, 3.5 unit DNA polymerase (Appligene).
  • the following temperature program was used to carry out the PCR: 95 ° C / 5 min; 25 cycles with 98 ° C / 30s - 62 ° C / 60s - 72 ° C / 90s each; 72 ° C / 10 min; 4 ° C.
  • a mutation rate of 0.51% could be set.
  • the DNA fragments obtained in this way were digested with ⁇ / ctel and Hind ⁇ and cloned into the correspondingly linearized vector pMS470 ⁇ 8.
  • the ligated vectors were then electroporated into E. coli SURE cells.
  • the cells thus obtained were plated on LB / ampicillin agar plates and grown overnight at 37 ° C. The colonies were then transferred to a filter paper.
  • the information on the position in the protein relates to the modified esterase in the expression plasmid pMStacXv-86
  • the cells were cultivated as described above and, after harvesting and disrupting the cells, the protein was purified by anion exchange chromatography (see FIG. 9).
  • the activity of the mutants against 1-methylprop-2-ynyl acetate and also against ortho-nitrophenylacetate was then determined in comparison to the wild type.
  • a concentration of 4 mM substrate was specified.
  • the determined specific activities of the mutants against 1-methylprop-2-ynyl acetate were above the activity of the wild-type enzyme, in the case of G1, A1 and A6 even by almost up to 100%.
  • the mutants showed approximately the same activity as the wild type (FIG. 11).
  • Example 7 The library described in Example 7 was used to screen for improved activity and stability. To screen for activity, 1-methylprop-2-ynyl acetate in a solution of 37.5 ml of 1% (w / v) litmus and 12.5 ml of 25% DMF was used. The following mutants could be identified
  • the mutant 02 determined in this way is identical to the mutant A5 and the mutation of the mutant DM14 has already been found in the double mutant A1.
  • FIG. 1 shows the structure of the 2 kbp construct pBLXC4 ⁇ accl, comprising the ORF for EstA from Xanthmonas vesicatoria.
  • the insertion is limited by a 5 'X / .o / restriction interface, which is a partial fill-in cloning strategy the gene bank has been changed and is therefore no longer intact.
  • the 3 'limit is an Acc / restriction interface.
  • the estA gene is identified by a black bar and is located at positions 590 to 1640 of the cloned fragment.
  • the ORF is preceded by a putative Shine Dalgamo (SD) sequence (underlined).
  • SD putative Shine Dalgamo
  • FIG. 2 shows part of the DNA sequence of the fragment cloned into the vector pBLXC4 ⁇ accl and the predicted protein sequence from EstA.
  • the underlined areas show the conserved promoter boxes and the double underlined sequences show the Shine Dalgarno sequence.
  • the suspected leader peptide is underlined.
  • the carboxylesterase profile is characterized by a gray background.
  • Fig. 3 Amino acid alignment of the EstA sequence (top) and the C-terminus of xylanase B from Butyrivibrio fibrisolvens. Identical amino acids are highlighted in black. Areas with a high level of homology are identified by a gray background.
  • FIG. 4 shows the functional map and the restriction map of pMSXC4a and pMStac-XC4a.
  • A Protein staining using Coomassie Brillant Blue.
  • Lane 1 LMW marker
  • Lane 2 E. coli SURE (pMS4K)
  • Lane 3 E. coli SURE
  • FIG. 7 shows a chromatogram of the chiral GC of the reaction shown in FIG. 6 and catalyzed by EstA. The assignment of the peaks to the starting materials and products is shown in the figure.
  • FIG. 8 shows a reversed phase HPLC chromatogram for acetic acid-1-methyl-prop-2-ynyl and the corresponding but-3-yn-2-ol after a reaction time of one day. The assignment of the peaks to the starting materials and products is shown in the figure.
  • FIG. 9 shows the expression of some mutants of EstA using an SDS-polyacrylamide gel stained with Coomassie Brillant Blue. WT identifies the wild type EstA.
  • Figure 10 shows a comparison of the activities of some mutants of EstA and the activity of the wild-type enzyme with respect to 1-methylprop-2-ynyl acetate.
  • Figure 11 shows a comparison of the activities of some mutants of EstA and the activity of the wild-type enzyme with respect to ortho-nitrophenyl acetate.

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Abstract

La présente invention concerne une estérase de Xanthomonas vesicatoria, des protéines homologues à cette dernière, son expression, sa purification et son utilisation, des acides nucléiques codant pour ces protéines, des anticorps contre ces protéines ainsi que des cellules hôtes et plantes transgéniques, contenant des acides nucléiques et/ou protéines selon ladite invention.
PCT/EP2003/007932 2002-07-26 2003-07-21 Esterase esta (xc4a) de xanthomonas vesicatoria Ceased WO2004013321A1 (fr)

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CN104694558A (zh) * 2014-12-16 2015-06-10 华南农业大学 一种酯酶基因estZ及其编码的蛋白质和应用

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WO1997035980A1 (fr) * 1996-03-25 1997-10-02 University Of Florida Anticorps diriges contre les proteines d'avirulence ou de pathogenicite des agents pathogenes des vegetaux
WO2000009698A2 (fr) * 1998-08-14 2000-02-24 Kansas State University Research Foundation Composes s'utilisant pour rendre des plantes resistantes et procedes afferents

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WO2000009698A2 (fr) * 1998-08-14 2000-02-24 Kansas State University Research Foundation Composes s'utilisant pour rendre des plantes resistantes et procedes afferents

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104694558A (zh) * 2014-12-16 2015-06-10 华南农业大学 一种酯酶基因estZ及其编码的蛋白质和应用
CN104694558B (zh) * 2014-12-16 2017-10-20 华南农业大学 一种酯酶基因estZ及其编码的蛋白质和应用

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