DE10140703A1 - Esterase Est4B1 from Bacillus subtilis - Google Patents

Abstract

The invention relates to a thioesterase obtained from <i>Bacillus subtilis</i>, to homologous proteins of said thioesterase, to nucleic acids that code for the proteins and to antibodies against said proteins. The invention also relates to the production and use of said proteins, nucleic acids and antibodies.

Description

The present invention relates to an esterase from Bacillus subtilis homologous proteins, nucleic acids coding for these proteins and antibodies against these proteins and the production and use of these proteins, Nucleic acids and antibodies.

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 Applications mainly through three groups of enzymes: lipases, Esterases and proteases.

Lipases in particular are industrially successful for various reasons have been applied [3]. The reasons for this are that lipases are ready in large quantities can be made that they are very stable under the reaction conditions and that in hydrolysis and transesterification very high enantioselectivity can be achieved [4].

However, resolving racemates is usually difficult if it is there is no clear steric difference between the two stereoisomers. In addition, many substrates are known, which are hardly or not at all can be reacted slowly using esterases [5-9].

For the reasons mentioned, there is an urgent need for hydrolytic Enzymes, especially esterolytic or lipolytic enzymes, for the industrial Use, especially for the separation of racemic mixtures in their Components.

The object of the present invention was therefore to use a new enzyme hydrolytic activity, in particular esterolytic and / or lipolytic activity, to make available, why in view of the application possibilities of such Enzymes, especially in biotechnology and organic chemistry there is an urgent need and / or to provide a method by which new hydrolytic enzymes, especially esterolytic and / or lipolytic enzymes, can be obtained.

The object of the present invention was, in particular, a hydrolytic enzyme, in particular an esterolytic and / or lipolytic enzyme represent the one compared to the esterolytic and lipolytic enzymes different substrate specificity, a distinguishable degree in selectivity, a distinct reaction rate, a distinguishable structure and / or a distinguishable reaction mechanism has.

The object of the present invention was furthermore esterolytic and / or to provide lipolytic enzymes that are capable of ester- and / or cleave thioester bonds.

This object is achieved by the polynucleotides according to the invention, Polypeptides and antibodies, 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.

It is obvious to the person skilled in the art how he starts from a 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 function or function can. In particular, for example, through evolution experiments, too Enzymes with improved properties can be obtained [10, 11].

• a) Polynukleotid mit einer Nukleinsäuresequenz von Position 556 bis 1287 gemäß SEQ ID No. 1,
• b) Polynukleotide kodierend für ein Polypeptid mit einer Aminosäuresequenz gemäß SEQ ID No. 2,
• c) natürlicherweise vorkommende Mutanten oder polymorphe Formen oder Allele eines Polynukleotids gemäß (a) oder (b),
• d) zu einem Polynukleotid gemäß (a), (b) oder (c) komplementäre Polynukleotide,
• e) unter stringenten Bedingungen mit einem Polynukleotid gemäß (a), (b), (c) oder (d) hybridisierende Polynukleotide, wobei unter stringenten Bedingungen die dem Fachmann bekannten Bedingungen für Hybridisierungsexperimente zu verstehen sind, vorzugsweise Inkubation bei 60°C in 0,1× SSC und 0,1% Natriumdodecylsulfat (SDS), wobei 20× SSC eine Lösung enthaltend 3 M NaCl und 0,3 M Natriumcitrat (pH 7,0) ist,
• f) Polynukleotide mit einer Sequenzhomologie oder -identität von mindestens 50%, bevorzugt mindestens 60% oder 70%, besonders bevorzugt mindestens 80%, ganz besonders bevorzugt mindestens 90%, insbesondere mindestens 95% in Bezug auf ein Polynukleotid gemäß (a), (b), (c), (d) oder (e),
• g) Polynukleotide, die aus mindestens 10 oder 15, bevorzugt mindestens 20 oder 25, besonders bevorzugt mindestens 30 oder 40, ganz besonders bevorzugt mindestens 50 oder 80, insbesondere mindestens 100 oder 120 aufeinanderfolgenden Nukleinsäuren eines Polynukleotids gemäß (a), (b), (c), (d) oder (e) bestehen,
• h) Polynukleotide mit Deletionen oder Insertionen von bis zu 50 oder 40, insbesondere bis zu 30, 20 oder 10 Nukleinsäuren gegenüber einem Polynukleotid gemäß (a), (b), (c), (d) oder (e),
• i) Polynukleotide, umfassend mindestens eines der unter (a) bis (h) genannten Polynukleotide.

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. 2,
• c) naturally occurring mutants or polymorphic forms or alleles of a polynucleotide according to (a) or (b),
• d) polynucleotides complementary to a polynucleotide according to (a), (b) or (c),
• 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. in 0 , 1 × SSC and 0.1% sodium dodecyl sulfate (SDS), 20 × SSC being a solution containing 3 M NaCl and 0.3 M sodium citrate (pH 7.0),
• f) 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%, in particular at least 95% 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 successive nucleic acids of a polynucleotide according to (a), (b), (c), (d) or (e) exist,
• h) polynucleotides with deletions 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 the polynucleotides mentioned under (a) to (h).

In a further preferred embodiment, the invention comprise Nucleic acids one or more non-coding sequences, where it is the non-coding sequences for example by natural occurring intron sequences or regulatory sequences, such as promoter or Enhancer sequences, in particular those for controlling the expression of hydrolytic enzymes, especially esterolytic or lipolytic enzymes, is.

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.

In a preferred embodiment, the inventive ones Nucleic acids are nucleic acids that are responsible for a protein with hydrolase activity or Encode parts of it. 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, halide, epoxy or Cleave peptide bonds, particularly preferably ester or thioester Bonds. It is very particularly preferably an enzyme that is able to cleave bonds regioselectively and / or enantioselectively.

According to the invention, cleavage is primarily hydrolytic cleavage, ie cleavage with release of the carboxylic acid and the corresponding alcohol, thiols, amine or hydrogen halide. However, cleavage can occur in particular Embodiments also with formation of a new ester, thioester, amide, Peptide or halide binding take place, in particular this can be for example, if an ester bond is converted to a new ester bond will be a transesterification reaction. With division are also those catalyzed by halogen peroxidases and haloalkane dehalogenases Cleavage reactions of haloalkanes meant to release the appropriate alcohols and alkanes.

In a preferred embodiment, it is the one according to the invention Enzyme around a hydrolase with α / β-hydrolase folding and / or around a 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 for a microbial enzyme, particularly preferably a bacterial one Enzyme, especially for an enzyme from Bacillus, especially for an enzyme from Bacillus subtilis, in particular for the esterase Est4B1 according to SEQ ID No. 2 and / or one of the polypeptides according to the invention described below encode.

The present invention also relates to the use of 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 production or isolation newer nucleic acids homologous to the nucleic acids according to the invention, in particular those with regard to the nucleic acids according to the invention similar structural and same, similar and / or improved functional Properties, with functional properties in particular hydrolytic Activity, especially lipolytic and / or esterolytic activity, and below improved functional properties, for example higher specificity, higher Sales and / or higher regio- or enantioselectivity is to be understood.

The nucleic acids according to the invention can thus, for example, as probes for Identification and / or isolation of homologous nucleic acids from a artificial, a cDNA or genomic library, preferably for Identification of nucleic acids required for esterolytic enzymes, lipolytic Enzymes and / or for one of the polypeptides according to the invention or parts thereof encode, or as an antisense nucleic acid or as a primer in the Polymerase chain reaction (PCR), in particular for the amplification of Nucleic acids comprising nucleic acids coding for enzymes with hydrolytic Activity, especially esterolytic and / or lipolytic activity, or parts of which are used.

The nucleic acids according to the invention or homologous to the nucleic acids according to the invention Nucleic acids can also by random mutagenesis or targeted Mutagenesis can be obtained in a manner known to the person skilled in the art.

After incorporation into an expression vector, the inventive Nucleic acids for example for the targeted production of individual domains or Epitopes of the protein according to the invention or of fusion proteins which comprise polypeptides according to the invention can be used.

• a) eine Nukleinsäurebibliothek wird mit einer erfindungsgemäßen Nukleinsäure kontaktiert,
• b) eine Nukleinsäure, die mit einer erfindungsgemäßen Nukleinsäure hybridisiert, wird identifiziert,
• c) die in Schritt (b) identifizierte Nukleinsäure wird sequenziert.

The present invention therefore also relates to a method for obtaining a nucleic acid which codes for a hydrolytic enzyme, in particular for an esterolytic and / or lipolytic 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 is identified,
• c) the nucleic acid identified in step (b) is sequenced.

• a) ausgehend von einer erfindungsgemäßen Nukleinsäure werden Primer hergestellt,
• b) die Primer gemäß (a) werden verwendet, um in der PCR Nukleinsäuren, vor allem cDNAs, unbekannter Nukleinsäuresequenz zu amplifizieren,
• c) die gemäß (b) erhaltenen Nukleinsäuren werden sequenziert.

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 lipolytic enzyme, 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 in (b) are sequenced.

• a) eine erfindungsgemäße Nukleinsäure wird Mutagenese-Experimenten unterworfen,
• b) die erhaltenen Mutanten werden in einen geeigneten Vektor eingebaut und exprimiert,
• c) die Expressionsprodukte werden auf hydrolytische Aktivität, insbesondere Esterase- und/oder Lipase-Aktivität hin untersucht.

Finally, the present invention also relates to a method for isolating a nucleic acid coding for a hydrolytic enzyme, in particular for an esterolytic and / or lipolytic enzyme, 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,
• c) the expression products are examined for hydrolytic activity, in particular esterase and / or lipase activity.

For example, the polymerase chain reaction (PCR) can be used to carry out the mutagenesis experiments. The PCR experiments can be carried out, for example, in particular when using a Taq polymerase, in such a way that reaction parameters such as the Mg ²⁺ concentration, the pH value, the reaction temperature or the substrate concentrations are varied, or error can be used -prone-PCR techniques, which are based, for example, on the addition of Mn ²⁺ or on the addition of unequal nucleotide concentrations.

The nucleic acid library is preferably a cDNA, genomic or artificial library, especially a microbial one, especially bacterial library, especially one from Bacillus, particularly preferably from Bacillus subtilis.

The invention further relates to a nucleic acid according to one of the preceding mentioned method is available.

Another object of the present invention is a method for manufacturing a nucleic acid according to the invention, characterized in that the Nucleic acid is chemically synthesized. The nucleic acids according to the invention can, for example, be chemically based on the 1 specified Nucleic acid sequence or using the in SEQ ID No. 2 specified Amino acid sequence based on the genetic code e.g. B. after the Phosphotriester method can be synthesized.

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. In the prokaryotic vectors for Incorporation of the nucleic acids according to the invention is, for example the plasmids pGEM-5Zf (+/-), pBluescript SK (-) or pMS470 or any other high copy number plasmid. With the available expression vectors for the Expression in E. coli is, for example, the vectors pTB3 or pMS470BS1 for the expression of 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- Code Ala-His6 tag, which enables the purification of the expressed protein on a Ni ²⁺ -NTA column. As eukaryotic expression vectors for expression in Saccharomyces cerevisiae are, for. B. the vectors p426Met25 or p426GAL1, for expression in insect cells z. B. baculovirus vectors as disclosed in EP-B1-0127839 or EP-B1-0549721, and for expression in mammalian cells e.g. B. SV40 vectors.

In one embodiment, the nucleic acids according to the invention can also be used flanking nucleic acids can be built into a vector that when expressed of the vector, the polypeptides encoded by the nucleic acids according to the invention present as fusion proteins or a day, a labeling Amino acid sequence, carry, for example, the purification or detection of Can facilitate polypeptides. For example, the day may be trade strep, flag, myc or his day.

The expression vectors preferably contain those suitable for the host cell regulatory sequences, preferably the lac or tac promoter for expression in E. coli, the ADH-2, GAL1 or AOX promoter for the Expression in yeast, the baculovirus polyhedrin promoter for expression in Insect cells or the early SV40 promoter or LTR promoters for the Expression in mammalian cells.

The present invention further relates to a host cell an expression vector according to the invention, it being the host cell preferably around Bacillus, in particular Bacillus subtilis, or E. coli, in particular around the E. coli strain BL21 (DE3) or the strain E. coli SURE.

The nucleic acids according to the invention are preferred after incorporation into a suitable vector by the methods of transfection known to the person skilled in the art, Transformation or electroporation introduced into the host cells.

In a further preferred embodiment, the 5'-terminal portion of for enzymes with hydrolytic activity coding nucleic acids completely or partly through a portion of one of the nucleic acids of the invention replaced to hereby the expression of these enzymes, in particular the expression in E. coli to improve. Especially the nucleic acid from position 556 to 1287 according to SEQ ID No. 1, and especially the area of the first 150 nucleic acids, is characterized by a codon use that this nucleic acid for Expression in E. coli is particularly suitable because it has a very low GC Contains content and therefore less difficult to melt after transcription RNA structures are present. The 5'-terminal section can therefore mainly through a nucleic acid from position 556 to position 706 according to SEQ ID No. 1 or at least a part thereof, the at least a part preferably at least 10 nucleic acids, particularly preferably at least 20 nucleic acids, especially comprises at least 50 nucleic acids.

• a) Polypeptid mit einer Aminosäuresequenz gemäß SEQ ID No. 2,
• b) natürlicherweise vorkommende Mutanten, polymorphe Formen oder Allele eines Polypeptids gemäß (a),
• c) Polypeptide, die eine Sequenzhomologie oder -identität von mindestens 50%, bevorzugt mindestens 60% oder 70%, ganz besonders bevorzugt mindestens 80 oder 90%, insbesondere mindestens 95% in Bezug auf ein Polypeptid gemäß (a) oder (b) besitzen,
• d) Polypeptide, die von den zuvor genannten erfindungsgemäßen Nukleinsäuren kodiert werden,
• e) Polypeptide bestehend aus mindestens 5 oder 6, bevorzugt mindestens 8 oder 10, besonders bevorzugt mindestens 12 oder 15, insbesondere mindestens 20, 25 oder 40 aufeinanderfolgenden Aminosäuren eines Polypeptids gemäß (a) oder (b),
• f) Polypeptide mit Insertionen und/oder Deletionen von bis zu 60, 50 oder 40, insbesondere bis zu 30, 20 oder 10 Aminosäuren in Bezug auf ein Polypeptid gemäß (a), (b) oder (c),
• g) Polypeptide umfassend mindestens eines der unter (a) bis (f) genannten Polypeptide.

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 mutants, polymorphic forms or alleles of 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%, 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 12 or 15, in particular at least 20, 25 or 40 consecutive amino acids of a polypeptide according to (a) or (b),
• f) polypeptides with insertions and / or deletions of up to 60, 50 or 40, in particular up to 30, 20 or 10, amino acids with respect to a polypeptide according to (a), (b) or (c),
• g) polypeptides comprising at least one of the polypeptides mentioned under (a) to (f).

The polypeptide is preferably a hydrolytic enzyme, particularly preferably an esterolytic or lipolytic enzyme, or parts from that. By this we mean enzymes that catalyze a hydrolysis reaction can, such as the hydrolysis of haloalkane, ester, thioester, Amide, epoxy, peptide or halide bonds, or parts, in particular epitopes or Domains of such polypeptides. In particular, it can hydrolytic enzyme around an esterase, lipase, thioesterase, haloalkane Dehalogenase, halogen peroxidase or epoxy hydrolase and / or an enzyme, which has alpha / beta hydrolase folding and / or an enzyme which according to the Classification of Arpigny is assigned to the V family, act.

In a preferred embodiment, these are enzymes that have a catalytic triad comprising a nucleophile, an aspartate or glutamate and a Histidine, or have a catalytic diad in its reactive center.

In the cleavage reaction which is catalyzed by the enzyme according to the invention, In addition to a hydrolysis reaction, it can also be an analogous one Reaction, such as the formation, especially new formation, of an ester, Act thioester or amide bond.

In a preferred embodiment, it is microbial, in particular bacterial enzymes. Enzymes are very particularly preferred Bacillus subtilis, especially around the esterase Est4B1 according to SEQ ID No. Second

The polypeptide according to the invention is particularly preferably a water soluble enzyme.

In a further preferred embodiment, the polypeptide according to the invention to an esterase or thioesterase, which in a non-ribosomal peptide synthetase is included. It can in particular for a thioesterase according to the publication by Schneider et al. (Arch. Microbiol. 1998, 169: 404-410), above all a distinct thioesterase like protein, act.

Another object of the invention is the use of an inventive Polypeptide for cleaving haloalkane, ester, thioester, peptide, amide, Epoxy or halide bonds, the cleavage reaction being both a hydrolysis reaction with release of the corresponding carboxylic acid or of the corresponding alcohol or alkane can act, as well as the New formation of an ester, thioester, peptide, amide or halide bond or the New formation of a haloalkane bond.

In a preferred embodiment, the enzymes according to the invention are in able to split a carboxylic acid ester, especially one in which the alcohol component is selected from the group consisting of o- 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, the individual Components also carry, independently of one another, substituents can otherwise be modified.

In a preferred embodiment, 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 preferred at a pH between 7.5 and 8.5, especially at one pH of about 8.0.

In a further preferred embodiment, the invention Polypeptides use as a component of non-ribosomal peptide synthetases, here in particular for the production of antibiotics, the reaction being caused by the polypeptide according to the invention is catalyzed, for example the ring closure of cyclic peptide antibiotics in the form of an ester, thioester or Amide bond can be.

In a further preferred embodiment, the inventive Enzymes are used to isolate from racemic mixtures or enrichment of one of the two enantiomers.

Possible applications of the polypeptides according to the invention are, for example Production or selective enrichment of, for example Flavor components in the food industry, from detergents in the Detergent industry, fine chemicals in the chemical industry or therapeutically or diagnostically usable substances in the pharmaceutical industry.

The polypeptides according to the invention can furthermore, for example, as epitopes can be used for the production of mono- or polyclonal antibodies by they are coupled to a carrier, for example bovine serum albumin, and then a mammal, preferably a mouse, rabbit or rabbit the epitope, preferably using adjuvants. For this are preferably polypeptides with a length of 5-12, in particular 8, Amino acids. Polypeptides with a length of more than 60, in particular more than 75 amino acids can also be used without a carrier to produce antibodies be used. The resulting antibodies can then optionally isolated, and starting from the antibodies or for them coding nucleic acids can optionally be antibody fragments, for example, Fab or scFv fragments.

Peptides binding to a polypeptide according to the invention can alternatively also by an in vitro method known to the person skilled in the art, such as phage Display, Yeast Display, Bacterial Display or the so-called Fusagen- Technology are obtained in which the nucleic acid and the encoded by it Polypeptide are covalently linked to one another via a puromycin.

Those obtainable by immunization with the polypeptides according to the invention Antisera, antibodies and antibody fragments as well as those of one of the named Peptides obtainable in vitro are suitable, for example, for the investigation from gene expression banks in order to be homologous to the polypeptides according to the invention Proteins, 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 others on Polypeptide-binding peptides according to the invention, in particular obtainable from one of the previously mentioned procedures. Antiserum, Antibody and Antibody Fragment and other peptides binding to a polypeptide according to the invention are described in hereinafter called "antibodies" for simplicity.

The present invention further relates to a method for the production of 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, especially Bacillus subtilis, or in E. coli, especially E. coli BL21 (DE3), is expressed.

• a) eine erfindungsgemäße Nukleinsäure wird in einen geeigneten Vektor eingebaut und E. coli wird mit dem Vektor transformiert,
• b) nach der Expression des Proteins werden die Zellen geerntet und aufgeschlossen.
• c) das Protein wird aus dem so erhaltenen Überstand gegebenenfalls aufgereinigt.

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) the protein is optionally purified from the supernatant thus obtained.

Cultivation of E. coli is preferably carried out between 15 and 40 ° C, particularly preferred at about 37 ° C. Expression is preferably induced in the logarithmic growth phase. When using, for example, a pMS470 vector which encodes a peptide according to the invention Nucleic acid can include induction of expression by adding lactose take place, for example by setting a final concentration between 1 and 30 mM, especially 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. After induction of the Expression, the cells are preferably a further 8-18 hours, in particular grown another 12-14 hours. Alternatively, you can also click on the Induction of expression can be dispensed with.

The harvested cells can be disrupted by a method known to the person skilled in the art Method, for example French press, ultrasound, ball mill or by the Use of a sonifier and / or enzymatically. Alternatively, the Cells are also chemically permeabilized, for example by EDTA or Polymyxin B. become.

The protein is optionally purified chromatography. The chromatographic purification of the protein can for example anion or cation exchange chromatography include hydrophobic interaction chromatography and / or gel filtration.

In a special embodiment, the protein is purified under Using anion exchange chromatography and a subsequent one Gel filtration step. In particular the protein Est4B1 according to SEQ ID No. 2 can cleaned in an advantageous manner.

The anion exchange column is preferably a QFF Column (Amersham Pharmacia, Uppsala, Sweden) around the gel filtration column a Pharmacia Sephacryl S-100 HR in XK 26/70 column.

Application to and elution from the anion exchange column is preferably carried out here in the neutral to slightly alkaline range, preferably between pH 6.5 and pH 8.5, very particularly preferably at about pH 7.5. This is preferably a 10 mM Tris-HCl buffer was used. Elution of the protein is preferred thereby achieves that the ionic strength of the elution buffer is slowly increased. This can can be achieved, for example, by adding NaCl in a step gradient. The protein is preferably eluted at a NaCl concentration between 150 mM and 300 mM or when using another salt below Specification of the same ionic strength.

After elution of the protein, 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 .

In particular, the short-chain polypeptides according to the invention can also be used classical peptide synthesis (Merrifield technique) can be synthesized.

The present invention also relates to a method for producing Crystals containing at least one of the polypeptides according to the invention, and the crystals obtainable by this method. The crystallization takes place here preferably at a pH between 6.0 and 9.0, particularly preferred between 7.0 and 8.0, especially at a pH of around 7.5 and / or below Use of PEG, especially PEG 4000, as a precipitant, the Amount of PEG preferably between 10 and 50%, particularly preferred between 20 and 36%, especially about 28%, and / or in the presence of Sodium acetate, the concentration of sodium acetate preferably between 0.1 and 0.3 M, especially about 0.2 M.

Another object of the invention is a test system for the identification of Substrates or functional interactors containing an inventive Nucleic acid, a polypeptide according to the invention and / or one Antibodies according to the invention and, if appropriate, suitable auxiliaries and Additives.

Bacillus subtilis is known to and can ward off plant pathogens Symptoms of plant pests, such as B. Erwinia. This positive effect is not least due to enzymes with hydrolytic activity, especially esterases and Attributed to thioesterases. Another object of the present invention is therefore 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 according to the invention Antibody.

In addition, a polynucleotide according to the invention can also be used in a Those skilled in the art can be introduced into a plant in order to do so to produce the polypeptide according to the invention and thus resistance to it To confer plant pathogens. The polynucleotide according to the invention can in particular be introduced into and out of a single plant cell this can be obtained by multiplying and differentiating a plant that a comprises 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 according to the invention 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 modified polypeptides according to the invention or the antibodies according to the invention his. The modification can be a radioactive, fluorogenic one or chromogenic group or a post-translational modification.

In the following, certain embodiments of the invention are exemplary explained using illustrations and exemplary embodiments.

pictures

In Fig. 1, a nucleic acid sequence of genomic DNA from Bacillus subtilis according to the invention is shown. This B. subtilis genomic DNA fragment comprises a complete open reading frame (ORF) (est4B1) and two partial ORFs (orf1 and orf3). The putative Shine-Dalgarno sequence of the est4B1 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. In addition to the ORF for Est4B1 (BS1) and the two partial ORFs (ORF1, ORF3), some restriction interfaces were identified. The amino acid sequence shows great similarity to putative proteins from B. subtilis A13.

In FIG. 3, results are shown from studies on the expression of Est4B1 means of E. coli BL21 (pMS470BS1) at 37 ° C. Expression was induced by adding lactose with a final concentration of 10 mM at various 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.

In FIG. 4, the crystals obtained in the invention of Est4B1 are shown.

embodiments example 1 Isolation and characterization of the Est4 bacterial strain

Large halos were found on contaminated tributyrin emulsion agar plates observed. An insufficiently cleaned one was used in the production of the agar plates Ultrathurax homogenizer used previously for the homogenization of Plant material had been used. A bacterial culture that's a big one Halo was isolated and replated several times to ensure that there is a single colony. Halo formation could occur both at room temperature as well as at 30, 37 and 50 ° C can be observed. The strain thus obtained was Called Est4 and for characterization to the Deutsche Stammsammlung für Microorganisms and cell cultures (DSMZ) in Braunschweig (Germany) cleverly. There 4 different variants of this strain could be identified, which were named Est4A, Est4B, Est4C and Est4D. Est4A and Est4B were created in further characterized the following. Both strains are B. subtilis Strains and the only difference between the two is that Est4A Lecithinase produces, but not Est4B. After receiving the separate strains of the DSMZ, the B. subtilis strain Est4A was cultivated on LB agar plates, whereby again several morphological variants could be observed. It seems so that these variants have different morphological forms one and the same Represent tribe, and that these are not contaminating strains is. The different variants showed the same under the light microscope 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 also 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 derived from tributyrin Emulsion agar plates isolated and then on Luria Broth (LB) agar plates cultured. 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 pMS470A8, which was used for the heterologous enzyme expression, was a gift from E. Lanka (18).

E. coli cells were normally at 37 ° C in LB medium containing 100 mg / l Ampicillin, grown. For the preparation of the tributyrin emulsion agar plates, 5-7 g / l tributyrin was added to the molten agar and then emulsification by sonification with an ultrathurax homogenizer, before the agar was poured into the dishes.

Example 3 Generation and screening of a B. subtilis genomic library Est4B

B. subtilis Est4B genomic DNA was obtained using Qiagen (Qiagen, Hilden, Germany) Genomic Kit (Tip 500) isolated from cells over Grown overnight in LB medium at 37 ° C. 10 µg of the genomic DNA was partially digested with Sau3Al (Roche, Mannheim, Germany). The fragments were size-separated by agarose gel electrophoresis and Fragments between 4-8 kb in size were cut out of the gel and purified using the Qiaex DNA Elution Kit (Qiagen). The genomic DNA fragments were inserted into the BamHI restriction site of the Vector pBluescript SK (-) ligated. The transformation of E. coli SURE cells was completed performed directly with this ligation approach, without prior amplification of the Library.

Active clones were identified using an agar plate filter paper esterase assay [13, 14]. 2-Chloropropionic acid-2-naphthyl ester was used as the substrate for screening the library. A clone called E. coli (pTB3) was isolated and the insert of the plasmid pTB3 was sequenced. The sequence is shown in Fig. 1. It shows that the insert is unusually ligated to the Bluescript vector SK (-) at one end.

Example 4 Sequence analysis of the ORF (open reading frames) of pTB3

Plasmid DNA was isolated using a plasmid DNA isolation kit (Qiagen) cleaned 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).

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 shows strong homology to a putative independent and not C-terminally fused thioesterase from Bacillus subtilis A13 (Accession No. AAF87217) and to subunit 4 of a surfactin synthetase from Bacillus subtilis ATCC 21332, also known as cold shock protein CSI16 (Accession No. Q08788) is known. The function of this protein has so far not been clarified. However there is Guesses that it is in the termination of peptide synthesis or in the release incorrectly charged molecules is involved [28].

Interestingly, the predicted structure of Est4B1 was stronger Homology to other enzymes in the family of alpha / beta-folded hydrolases than about the esterases and lipases of this family. The strongest homology was found for the following enzymes: Haloperoxidase L (Streptomyces lividans), chloroperoxidase F (Pseudomonas fluorescens), bromoperoxidase A2 (Streptomyces aureofaciens), proline iminopeptidase (Seratia marescens), Haloalkane dehalogenase (Xanthobacter autotrophicus) and hydroxynitrile lyase (Hevea brasiliensis). For this reason it can be assumed that also halogenated ester compounds and esters with nitrogen-containing groups of Est4B1 can be implemented.

Example 5 Production of the expression clone pMS470BS1

PCR primers with restriction sites for Ndel and Sphl were produced
(B3ADstart: 5'GAACACA CATATG GTCCAGCTC3 ', B3ADend: 5'AGC GCATGC TGTCTGTCATATC3'). These primers were prepared in order to be able to introduce suitable restriction sites at the 5 'and 3' ends of the gene coding for the protein Est4B1 by means of PCR. The reaction was carried out in a total volume of 50 μl, using 10 ng template DNA (pTB3), 5 μl dNTPs (1 mM each), 200 ng of each primer, 5 μl PCR buffer (10 × Dynazyme) and 2 Units Dynazyme DNA polymerase (Finnzyme, Espoo, Finland). 6 cycles of 94 ° C (1 min), 38 ° C (2 min) and 72 ° C (2 min) were performed, followed by 23 cycles of 94 ° C (1 min), 65 ° C (2 min) and 72 ° C (2 min). The PCR product was purified using the Qiaquick PCR Purification Kit (Qiagen) and then digested with Ndel and Sphl. This was followed by ligation into the expression plasmid pMS470, replacing the insert of the vector pMS470Δ8. This vector contains the heavily regulated tac promoter and enables the gene to be inserted into an ATG start codon in the 3 'direction from a highly efficient Shine Dalgarno sequence which is derived from the T7 gene 10. The construct thus obtained was named pMS470BS1.

E. coli was transformed with the plasmid thus obtained. Clones, the esterase Showed activity by the previously described agar plate filter paper Assay identified. The plasmid DNA of a positive clone was isolated after isolation sequenced to determine whether the sequence matches the native sequence matches.

Example 6 Preparation of the esterase Est4B1

A preculture of E. coli BL21 (pMS470BS1) was made by culturing of cells in LB medium with 100 mg / l ampicillin overnight at 37 ° C below Shake. This overnight culture was used to inject 100 ml of LB medium inoculate a 300 ml Erlenmeyer flask. The cells were shaken while shaking Let 37 ° C grow until the culture is in the middle of the logarithmic Growth phase. Finally the main culture (300 ml in 1 liter flask) inoculated with 10 ml of the second preculture. For the induction of expression a 1M lactose stock solution was added immediately after inoculation to set a final concentration of 10 mM. The cells were used for further Cultivated at 37 ° C for 12-14 hours and then harvested by centrifugation. The Cell sediment was resuspended in 20 ml Tris-HCl buffer (0.1 M, pH 7.5) and after Add 1 unit of frozen benzonase per flask. The cell suspension was thawed and lysed by sonification (Branson Sonifier 250, pulsed Sonification for 6 min). The cell residues were removed by centrifugation at 100,000 g for 1 Hour separated. The supernatant with the soluble Est4B1 protein was aliquoted and frozen at -18 ° C.

In this way, the esterase Est4B1 could be obtained in very high yield become. The expression of the esterase was very strong compared to others Esterases that were produced under the same conditions. This indicated suggests that codon use in this Bacillus subtilis gene is very good for that Expression in E. coli is suitable. In particular, the first 150 codons in Comparison to the corresponding codons from esterases from other organisms have a lower GC content, so that less difficult-melting RNA Structures are present and thus the expression in E. coli is facilitated.

Example 7 Purification of the esterase Est4B1

The protein was purified by a two step process. For this purpose, 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) while increasing the proportion of 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% buffer B over the column and then passing a further 30 ml of buffer over the column, applying a flat gradient so that the fraction was increased on buffer B increased from 8.5% to 13%. The fractions with the enzyme were then combined and concentrated to 5-8 ml by ultrafiltration with the Centricon YM-10 centrifugation columns (Millipore, Bedford, USA). For further purification, gel filtration was carried out with a Pharmacia Sephacryl S-100 HR in an XK 26/70 column at a flow rate of 4 ml min ^-1 . In this way, Est4B1 was obtained in a very clean form.

Using MALDI, the molecular weight of the enzyme was 27445 Da certainly. However, the calculated average molecular weight was 27760 Da.

This small difference may be due to one post-translational N-terminal modification of the first amino acids or Inaccuracies in mass spectrometry.

Example 8 Crystallization of the Est4B1 protein

For the crystallization experiments, an enzyme solution with a Protein concentration of 6.5 mg / ml used. The vapor pressure Diffusion method of hanging drop (hangig drop) applied. For this siliconized glass platelets and 24-well Linbro culture plates are used. The glass plates with the Drops were applied to the wells and then followed Sealing using high vacuum grease. To make the drops 5 .mu.l protein solution and 5 .mu.l of the precipitant solution containing the 750 µl reservoir applied to the well was removed mixed. The experiments were preferably carried out at 20 ° C. As Hampton's Coarse Matrix Crystallization Screen was used as standard. Plate-shaped crystals were observed under the following conditions: 28% PEG 4000, 0.2 M NaAc, 0.1 M Tris-HCl, pH 7.5. Place the crystals obtained an important step towards solving the 3D structure of this class of hydrolytic Enzymes.

Example 9 Enzyme assays to determine the hydrolytic activity of Est4B1 Agar plates filter paper assay

Positive signals with the agar plate filter paper assay regarding hydrolysis of 2-chloropropionic acid-2-naphthyl ester showed that Est4B1 esterase activity has. Crude lysates of E. coli BL21 (DE3) or purified enzyme were used for the further analysis of the esterase activity of this enzyme was used.

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 that releases 1 µmol alcohol min ^-1 under the assay conditions. Under the given conditions, the extinction coefficients of ortho- and para-nitrophenol are 2.42 and 9.6 ml µmol / l cm. A specific activity of 0.3 U / mg was found in the photometric assay with 4-nitrophenyl butyrate, while a value of less than 0.01 U / mg was determined with 2-nitrophenyl butyrate as substrate. The results of the photometric and alkalimetric assay are summarized in Tab. 1.

Enzyme preparations purified by anion exchange chromatography were used in thioesterase assays and in alkali titration used.

Thioesterase assay

A was used to determine the free SH groups after hydrolysis of the CoA esters photometric assay performed as described by Cho and Cronan [19] becomes. With the tested fatty acid thioesterase substrates (propionyl-CoA, DL-β- Hydroxybutyryl-CoA, DL-β-hydroxyglutaryl-CoA, n-decanoyl-CoA and myristoyl- However, no activity was found here. However, this was not surprisingly, since thioesterases with respect to their thioester substrate in are usually very selective, so that was not necessarily to be expected to find feasible substrate.

Alkalimetric titration

Esterase activity of Est4B1 for tributyrin, 3-hydroxybutyric acid ethyl ester and 2-chloropropionic acid methyl ester 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 μl substrate and 0.75 ml 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 could be determined. A value of 8 was determined as the optimum pH for the hydrolysis. 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 with regard to ethyl 3-hydroxybutyrate was less than 0.01 U / mg. No activity was observed with tributyrin as the substrate. Table 1 Substrates which were examined for hydrolysis by Est4B1 photometrically or alkalimetrically

Thin layer chromatography (TLC)

The thin-layer chromatography (TLC) experiments were carried out by suspending 100 μl crude bacterial lysate of the cultures overexpressing Est4B1 in 0.5 ml buffer (0.1 NaH ₂ PO ₄ , pH 7.3). Crude lysate from cells without a vector was used as a negative control. After the addition of the substrate (20 μl), 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 silica gel 60 F ₂₅₄ . As far as technically feasible, the substrates listed in Table 4 were used in the TLC.

The composition of the eluent (cyclohexane / ethyl acetate) and the visualization method (spraying with vanillin / H ₂ SO ₄ conc. Or cerammonium molybdate as well as heat treatment and / or UV detection) were selected depending on the substrate.

For example, for the substrates octanyl-2-acetate and 2-chloropropionic acid-2-naphthyl ester, a mixture of petrol / ethyl acetate (3: 1) was used for elution and the products were made visible by adding vanillin / 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 in the case of the mandelic acid ethyl ester by UV detection, in the case of the hydroxybutanoic acid ethyl ester by spraying with cerammonium molybdate. 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. Tab. 2 Results of the TLC analysis (+ weak activity (<20% conversion); ++ high activity (~ 20-50% conversion); - no detectable activity; for numbering the substrates see Table 4)

It was observed that after 20 hours the majority of the 2- Chloropropionic acid 2-naphthyl ester has been hydrolyzed by Est4B1 while hydrolysis of ethyl mandelate (15, 16) or octanoyl-2-acetate is not could be observed. A total of 5 substrates were found that are sufficient in terms of industrial applications by the Esterase Est4B1 can be converted (marked with ++ in Tab. 2) and the could then be examined for selectivity of sales. All Substrates in which there was up to about 50% conversion were used in further Studies used to increase the enantioselectivity of sales investigate.

The activity of the esterase with respect to the compounds 2 and 12 had to be Gas chromatography to be investigated since compound 12 was not by TLC can be visualized and connection 2 is very volatile.

Furthermore, the acid (R, S) - formed during the reaction of compound 14 3-hydroxy-2-methyl-propionic acid is not a stable product, so that also Investigation of this reaction by TLC was not possible.

Chiral gas chromatography (GC) and high pressure liquid chromatography (HPLC)

The activity analysis was carried out in aqueous sodium phosphate buffer (0.1 M; pH 7.3). Usually 5 ml of buffer were mixed with 250 µl enzyme (raw bacterial lysate) and 100 µl substrate was added. The mixture thus obtained was shaken on a stirred shaker at 150 rpm at room temperature (approx. 25 ° C.). Samples were taken after 2, 5, and about 20 hours and the reaction was stopped by adding 0.5 ml CH ₂ Cl ₂ . After extraction, centrifugation and separation of the aqueous phase, the organic phase was dried over sodium sulfate. A 1 µl aliquot was used for the GC after being filtered through a filter membrane (nylon, ∅ 3 mm, 0.2 µm, Roth). The enantiomeric purity of the product was determined on a 2,3,6-tri-O-methyl-β-cyclodextrin column (Chrompack Chirasil-Dex-CB, 25 m × 0.32 mm, 0.25 µm film, H ₂ ) certainly.

2-chloropropionic acid 2-naphthyl ester 11 and 1-acetoxy-2-phthalimido-butane 6 were subjected to HPLC analysis. In the first case, however, only the Esters can be observed by UV, while with respect to compound 6 only the corresponding alcohol on a Chiralcel OD-H column (Daicel 25 cm × 0.46 cm) could be determined.

The following reaction conditions were specified: Analysis parameters for the 6-alcohol: heptane: isopropanol = 90: 10; Flow rate 0.5 ml min ^-1 , 10 ° C, UV detection at 230 nm. Analysis parameters for compound 11: heptane: isopropanol = 95: 5, flow rate 0.5 ml min ^-1 , 10 ° C, UV- Detection at 254 nm. Table 3 Results of chiral gas chromatography and HPLC for substrate 6 (values after 2, 5 and 24 hours)

Although the B. subtilis Est4B library was examined for cloning, the (R, S) - Hydrolyze 2-chloropropionic acid 11 and the results of TLC Analysis in relation to compound 11 gave no hope (Table 1) enantioselective hydrolysis of this compound by the esterase Est4B1 to be watched.

With respect to the substrate 6 (±) -N-phthaloyl-2-amino-1-butyl acetate showed the Esterase Est4B1 (56% e.e., 63% conversion) selectivity in relation to the alcohol (Tab. 2). The enantiomeric purity of the corresponding acetate has so far been unsuccessful cannot be determined. Interestingly, the esterase showed Est4B1 an opposite to three other esterases tested Enantiopreference.

Est4B1 showed almost no activity and selectivity for this in GC / HPLC Acetate of (R, S) -3-butyn-2-ol (2), tetrahydrofuran-3-acetate (4), tetrahydrofuran-2- methyl ester (9), 3-hydroxybutyric acid ethyl ester (13) and 4-Hydroxyisobutyric acid methyl ester (14).

Manufacture of substrates

Acetates 1, 2, 4, and 7 and butyrate 3, as well as methyl esters 8 and 9, were prepared according to the following protocol:
A solution of alcohol (10 mmol) in CH ₂ Cl ₂ (10 ml) was treated with acid anhydride (20 mmol) at room temperature, then pyridine (20 mmol) and a catalytic amount of 4- (dimethylamino) pyridine (Merck- Schuchardt) added. All compounds were purified using silica gel chromatography or Kugelrohr distillation.

The carboxylic acid (10 mmol) was refluxed with a catalytic amount of H ₂ SO ₄ (conc.) And a 10-fold excess of methanol. All esters were purified using silica gel chromatography or Kugelrohr distillation.

Substrate 6

The amino group (±) -2-phthalimido-1-butanol (6) (Kumar et al. 1997) was protected by standard protective group methods:
Phthalic anhydride (50 mmol; 7.4 g) and 2-amino-1-butanol (45.5 mmol; 4.05 g) were melted at a temperature of 140 ° C. The reaction mixture was diluted with 200 ml CH ₂ Cl ₂ and washed with NaHCO ₃ (4 × 100 ml) and water (4 × 500 ml). After drying over Na ₂ SO ₄ , the crude product was acetylated and then purified.

The acetate was made by the following procedure:
A solution of alcohol (28.5 mmol; 6.26 g) in CH ₂ Cl ₂ (10 ml) was treated with acid anhydride (57.1 mmol; 4.52 g), then with pyridine (57.1 mmol; 4 , 52 g) and with a catalytic amount of 4- (dimethylamino) pyridine (Merck-Schuchardt) at room temperature for 5 hours. In addition, a stoichiometric amount of methanol was added to destroy the excess of acid anhydride, and the mixture thus obtained was stirred for 4 hours. The reaction mixture was washed with HCl 0.5N (2 x 40 ml), NaHCO ₃ (2 x 50 ml) and water (1 x 50 ml). After drying over Na ₂ SO ₄ , the acetate obtained was purified by means of silica gel chromatography (gasoline: ethyl acetate = 2: 1). Table 4 Overview of some of the substrates tested



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SEQUENCE LISTING

Claims (33)

1. 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. 2, c) naturally occurring mutants or polymorphic forms or alleles of a polynucleotide according to (a) or (b), d) polynucleotides complementary to a polynucleotide according to (a), (b) or (c), e) polynucleotides which hybridize under stringent conditions with a polynucleotide according to (a), (b), (c) or (d), f) polynucleotides with a sequence homology or identity of at least 50% with respect to a polynucleotide according to (a), (b), (c), (d) or (e), g) polynucleotides which consist of at least 10 successive nucleic acids of a nucleic acid according to (a), (b), (c), (d) or (e), h) polynucleotides with deletions or insertions of up to 50 nucleic acids compared to a nucleic acid according to (a), (b), (c), (d) or (e) i) polynucleotides which comprise at least one of the nucleic acids mentioned under (a) to (h). 2. Polynucleotide according to claim 1, coding for an enzyme with hydrolytic Activity. 3. Polynucleotide according to claim 2, coding for an enzyme with esterolytic or lipolytic activity. 4. The polynucleotide of claim 1, wherein the enzyme is a Enzyme with alpha / beta hydrolase folding. 5. The polynucleotide of claim 1, wherein the enzyme is a Esterase, thioesterase, dehalogenase, halogen peroxidase, epoxy hydrolase or lipase. 6. The polynucleotide of claim 1, wherein the enzyme is the Esterase Est4B1 according to SEQ ID No. 2 acts. 7. The nucleic acid of claim 1, wherein the nucleic acid is one or more includes non-coding sequences. 8. A method for producing and / or identifying a nucleic acid according to one of the preceding claims, characterized in that the Nucleic acid chemically synthesized using a probe from a gene bank isolated, by polymerase chain reaction and / or by mutagenesis Experiments is obtained. 9. The method according to claim 8, comprising the following steps: a) a nucleic acid library is contacted with a nucleic acid according to claim 1, b) a nucleic acid which hybridizes with a nucleic acid according to claim 1 is identified, c) the nucleic acid identified in step (b) is sequenced. 10. The method according to claim 8, comprising the following steps: a) starting from a nucleic acid according to SEQ ID No. 1 primers are produced, b) the primers according to (a) are used to amplify nucleic acids of unknown nucleic acid sequence in the PCR, c) the nucleic acids obtained in (b) are sequenced. 11. The method according to claim 8, comprising the following steps: a) a nucleic acid according to claim 1 is subjected to mutagenesis experiments, b) the mutants obtained are sequenced, incorporated into a suitable vector and expressed, c) the expression products according to (b) are examined for hydrolytic activity. 12. Vector comprising a nucleic acid according to claim 1. 13. Host cell comprising a vector according to claim 12. 14. The host cell of claim 13, wherein the host cell is Bacillus subtilis or E. coli. 15. Polypeptide selected from the group consisting of: a) polypeptide with an amino acid sequence according to SEQ ID No. 2, b) naturally occurring mutants, polymorphic forms or alleles of a polypeptide according to (a), c) polypeptides which have a sequence homology or identity of at least 50% with respect to a polypeptide according to (a) or (b), d) polypeptides which are encoded by nucleic acids according to claim 1, e) polypeptides consisting of at least 5 consecutive amino acids of a polypeptide according to (a) or (b), f) polypeptides with insertions and / or deletions of up to 60 amino acids with respect to a polypeptide according to (a), (b) or (c), g) polypeptides which comprise at least one of the polypeptides mentioned under (a) to (f). 16. The polypeptide of claim 15, wherein the polypeptide is a Protein with hydrolytic activity. 17. The polypeptide of claim 16, wherein the polypeptide is one lipolytic or esterolytic enzyme. 18. The polypeptide of claim 16, wherein the polypeptide is a Esterase, thioesterase, dehalogenase, halogen peroxidase, epoxy hydrolase or lipase. 19. The polypeptide of claim 15, wherein the polypeptide is one Enzyme with alpha / beta hydrolase folding. 20. The polypeptide of claim 15, wherein the polypeptide is the Esterase Est4B1 according to SEQ ID No. 2 acts. 21. The polypeptide of claim 15, which is a water-soluble protein is. 22. A method for producing a polypeptide according to claim 15, characterized characterized in that a nucleic acid according to claim 1 in a suitable host cell is expressed. 23. The method of claim 22, wherein the host cell is Bacillus subtilis or E. coli. 24. The method of claim 23, comprising the following steps: 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 purified from the supernatant thus obtained. 25. Antibody against a polypeptide according to claim 15. 26. A method for producing an antibody according to claim 25, characterized characterized in that a mammal with a polypeptide according to claim 15 is immunized and the resulting antibodies isolated if necessary become. 27. Test system for the identification of substrates or functional interactors containing a nucleic acid according to claim 1, a polypeptide according to Claim 15 and / or an antibody according to Claim 25 and if appropriate, suitable auxiliaries and additives. 28. Use of a polypeptide according to claim 15 for the cleavage of Substrates with haloalkane, ester, thioester, amide, halide or peptide Bonds. 29. Use according to claim 28, wherein the cleavage is the Hydrolysis or to form an amide, ester, thioester or Acyl halogen bond. 30. Use of a polynucleotide according to claim 1, a polypeptide according to claim 15 and / or an antibody according to claim 25 for Control of plant pathogens and / or plant pests. 31. Composition for combating plant pathogens and / or Plant pests containing at least one polynucleotide according to Claim 1 and / or at least one polypeptide according to Claim 15 and / or at least one antibody according to claim 25 and suitable Auxiliaries and / or additives. 32. Plant and / or plant cell containing at least one polynucleotide according to claim 1 and / or at least one polypeptide according to claim 15 and / or at least one antibody according to claim 25. 33. Crystals containing at least one polypeptide according to claim 15.