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WO2005040408A1 - Procede d'identification d'enzymes presentant des proprietes voulues, par ancrage des produits de reaction sur la surface d'organismes presentant des enzymes - Google Patents

Procede d'identification d'enzymes presentant des proprietes voulues, par ancrage des produits de reaction sur la surface d'organismes presentant des enzymes Download PDF

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WO2005040408A1
WO2005040408A1 PCT/EP2004/012017 EP2004012017W WO2005040408A1 WO 2005040408 A1 WO2005040408 A1 WO 2005040408A1 EP 2004012017 W EP2004012017 W EP 2004012017W WO 2005040408 A1 WO2005040408 A1 WO 2005040408A1
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enzyme
substrate
host
cells
host organism
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Harald Kolmar
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Nascacell IP GmbH
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Nascacell IP GmbH
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase

Definitions

  • the present invention relates to a method for the identification of enzymes with a sought activity by the random generation of a large collection of enzyme variants, the synthesis of these variants in host organisms, their presentation on the surface of the organisms and the isolation of enzyme variants with the desired properties by detection the covalent deposition of the reaction product on the surface of the host organism.
  • the present invention relates in particular to a method for identifying hydrolases with desired properties, wherein hydrolase variants are presented on the surface of organisms which are decorated with a second enzyme which acts as an auxiliary enzyme.
  • the product released by the hydrolase is in turn substrate for the auxiliary enzyme which activates the product so that it is covalently fixed on the surface of the host organism by coupling to functional groups.
  • the host organism which has the desired enzyme activity is labeled and can then be isolated from a large collection of organisms which express different hydrolase variants.
  • This method is particularly suitable for screening large libraries and allows several million enzyme variants to be tested in a short time and thus enzyme variants with the desired properties can be obtained more quickly and more specifically than previously possible.
  • Hydrolytic enzymes and especially esterases and lipases, represent a class of enzymes that have become indispensable aids for a variety of applications in organic chemistry and biotechnology. Their potential is based on their ability, not just hydrolysis, but also that To catalyze the synthesis of many different esters, which usually takes place with high specificity and selectivity.
  • Lipases (EC 3.1.1.3) are carboxylesterases that have the ability to hydrolyze long-chain acylglyceryl esters (> C ⁇ 0 ), while esterases (EC 3.1.1.1) hydrolyze ester substrates of shorter-chain fatty acids ( ⁇ C 10 ).
  • Hydrolases especially lipases and esterases, are used in biotechnology primarily for the catalysis of stereoselective conversions of a large number of amines, as well as primary and secondary alcohols (Rogalska et al., Biochem. Soc. Trans. 25 (1997), 161- 164; Jaeger et al., FEMS Microbiol. Rev. 15 (1994), 29-63).
  • a correspondingly selective hydrolase is not known for every desired reaction. Therefore, new approaches were taken to isolate hydrolase mutants from natural sources or to modify known hydrolases in such a way that they meet the requirements for selectivity and specificity for commercial use.
  • Beta-lactam antibiotics which are normally poor substrates for beta-lactamase, have been isolated (Venkatachalam et al., J. Biol. Chem. 269 (1994), 23444- 23450).
  • the variant obtained can be characterized with regard to its deviations from the base sequence of the gene originally used.
  • this strategy e.g. Moore and Arnold (Nature Biotechnol. 14 (1996), 458-467) isolate a variant of the p-nitrobenzyl esterase, which had a 16-fold higher activity in 30% DMF than the starting enzyme.
  • the enzyme variant obtained from a single round of mutagenesis and screening shows slight improvements in its enzymatic properties in the direction of the desired one, but does not yet fully express the desired property. Therefore, mostly starting from this Enzyme variant obtained generates a new set of gene variants, and with these the process of gene expression in microorganisms, determination of the enzymatic properties of individual clones and identification of variants improved with regard to the desired properties run again until the desired property is obtained.
  • this method has a major disadvantage: it requires physical separation and cultivation in spatially separated culture vessels (e.g. microtiter plates or test tubes) of the individual microbial clones, which carry randomly modified genes for the enzyme and thus also produce different enzyme variants.
  • the enzymatic property of the enzyme variant produced by the respective microbial clone must then be measured in each of the separate culture vessels.
  • This method is very complex in terms of the separate handling of the microbial clones required and the separate determination of the enzymatic properties of each individual microbial clone. For reasons of logistics and costs, only a subset of the gene variants actually created can be screened. Usually several hundred to several thousand clones are examined as described above with regard to the property sought.
  • the ompT variants operatively linked to these were expressed and the respective variant enzymatic protein domain on the surface of the respective, which produce bacterial cells exposed.
  • the bacterial cells were incubated with a synthetic peptide, which carries two fluorophores in the vicinity. Hydrolytic cleavage - caused by an OmpT variant with the desired substrate specificity - results in the separation of the fluorophores and thus a change in the fluorescence properties of the product compared to the substrate, which then shows an increased green fluorescence.
  • the released product has a positive net charge, so that it remains bound on the (negatively charged) surface of the cell which has proteolysis activity against the substrate.
  • the binding of the product to the cell surface is only maintained under low salt conditions and the method is only applicable if the product obtained has a charge and if this charge is contrary to the charge on the surface of the host cell. In addition, it is only applicable to those microorganisms that have a charged surface. Furthermore, the method cannot be used if the charge on the substrate or product molecule has an unfavorable effect on the enzyme reaction.
  • the present invention is therefore based on the object of making such methods available.
  • the present invention relates to a method for identifying an enzyme with a desired substrate-cleaving activity, a library which encodes a large number of different candidate polypeptides being expressed by suitable host organisms in such a way that the candidate polypeptides are presented on the surface of the host organisms, and that Host organisms are brought into contact with the substrate to be cleaved, characterized in that (a) an auxiliary enzyme is provided on the surface of the host organism which enables a covalent bond to be formed between the surface of the organism and a product resulting from the substrate cleavage reaction catalyzed by a candidate polypeptide, and
  • the method according to the invention is particularly advantageous since it enables the covalent binding of a product of the reaction catalyzed by the candidate polypeptide on the surface of the host organism. This drastically increases the reliability with which host organisms that express the desired enzyme activity can be identified.
  • the covalent fixation of the reaction product on the surface of the host organism allows a better correlation between the detection of the desired enzyme activity and the host organism which expresses this enzyme activity.
  • the disadvantages of the methods described in the prior art, in which the reaction product is bound to the cell surface of a host cell via ionic interactions, are avoided.
  • the method of the invention is also very widely applicable, i. H. on all possible enzyme activities and substrates, since no consideration has to be given to the charge ratios of the substrate or the host organism used.
  • enzyme in the context of the present invention means a polypeptide which is able to catalyze a biochemical reaction.
  • the method according to the present invention can be used for the identification of enzymes with substrate-cleaving activity.
  • substrate cleaving means that the enzyme catalyzes a reaction by which a given substrate (educt) is cleaved into at least two products. Reactions which lead to the cleavage of a starting material by an enzyme into at least two products are, for example, hydrolysis, phosphorolysis or elimination. Phosphorolysis is the cleavage of a bond by orthophosphate.
  • Hydrolysis refers to a reaction in which a bond is cleaved by water, an OH group being incorporated into one product of the cleavage reaction and a hydrogen atom into the other product.
  • Called elimination a reaction in which two substituents of a pair of adjacent atoms in a molecule are removed without being replaced by other atoms or groups.
  • the enzyme with substrate-splitting activity therefore has hydrolase activity, ie it is able to catalyze a hydrolysis reaction.
  • hydrolase activity examples include esterases, lipases, phosphatases, glucosidases, acylases or amidases.
  • Phosphatases are hydrolases through which phosphonic acid monoesters e.g. B. from sugar phosphates, nucleoside monophosphates or as a terminal phosphate group of nucleic acids hydrolytically. Depending on the pH in which these enzymes develop their optimal effectiveness, a distinction is made between acid phosphatases and alkaline phosphatases.
  • Glucosidases are hydrolases that release glycosidically bound glucose. Amidases catalyze the hydrolytic cleavage of amides. Acylases are enzymes that are able to catalyze the removal of acyl groups from a molecule. These include e.g. B. deacylases, but also lipases. Lipases (EC 3.1.1.3) are carboxylesterases that have the ability to hydrolyze long-chain fatty acid esters (> C 10 ). Esterases (EC 3.1.1.1), on the other hand, have the ability to hydrolyze ester substrates of shorter-chain fatty acids ( ⁇ C 10 ).
  • lipases and esterases include, for example, biotechnologically important lipases and esterases, such as phospholipases (leather processing), acylases from B. megaterium and E. coli (chemical synthesis), lipases from Aspergillus sp. (Prostaglandin synthesis), LipA from B. subtilis (cephalosporin synthesis), lipase from Candida sp. (Pyrolidinion synthesis), lipase from C. rugosa (synthesis of ibuprofen), from Chromobacterium (vitamin D synthesis), from M. miehei (synthesis of ketoprofen), from P. cepacia (rapamycin synthesis), from P. fluorescens (synthesis of hydantoins) and from Streptomyces sp. (Synthesis of penicillins).
  • biotechnologically important lipases and esterases such as phospholipases (leather processing), acylases from
  • the enzyme with esterase activity is preferably derived from an esterase from a prokaryotic organism, preferably a bacterium, particularly preferably a bacterium of the genus Pseudomonas and very particularly preferably from the species Pseudomonas aeruginosa.
  • the esterase is particularly preferably derived from the esterase EstA from Pseudomonas aeruginosa. This enzyme is described for example in Wilhelm et al. (J. Bacteriol. 181 (1999), 6977-6986).
  • the nucleotide and amino acid sequence of the EstA esterase are shown in Figure 4.
  • the enzyme with lipase activity is derived from the lipase LipA from Bacillus subtilis (Eggert et al., Eur. J. Biochem. 267 (2000), 6459-6469; Van Pouderoyen et al., J. Mol. Biol . 309 (2001), 215-216; Eggert et al., FEBS Lett. 502 (2001), 89-92; Eggert et al., FEMS Microbiol. Lett. 225 (2003), 319-324).
  • the substrate which is used in the process according to the invention and for which an enzyme with appropriate substrate-splitting activity is sought can in principle be any substrate which can be cleaved by an enzyme.
  • the substrate is selected such that it is cleaved by the desired enzyme activity to be identified.
  • the substrate serves only as the substrate for the desired enzyme activity to be identified, but not for the auxiliary enzyme which is simultaneously provided on the surface of the host organism.
  • It is preferably a substrate which can be hydrolytically cleaved by an enzyme-catalyzed reaction, particularly preferably the substrate is an ester.
  • the substrate is preferably an ester of shorter-chain fatty acids ( ⁇ C 10 ) or a derivative of such an ester or a long-chain fatty acid (> C 10 ) or a derivative of such an ester.
  • derivatives are e.g. B. halogenated fatty acids, branched chain fatty acids, amino acids or hydroxy acids and many others.
  • the enzyme has esterase or lipase activity and the substrate is a phenol derivative of an ester of any carboxylic acids, ie a phenol ester.
  • the substrate preferably has a constituent which, after cleavage by the desired enzyme activity, leads to a product which can be converted into an activated form by the auxiliary enzyme, which then forms a covalent bond with groups on the surface of the host organism.
  • auxiliary enzyme which then forms a covalent bond with groups on the surface of the host organism.
  • Such components are known to the person skilled in the art, e.g. U.S. Patent 5,196,306, and include, for example, tyramine and p-hydroxyphenylpropionylbiocytin.
  • an ester e.g. B. the alcohol component can be converted into a radical by the auxiliary enzyme after cleavage by an esterase.
  • the substrate to be used carries a component which allows the detection of a product of the substrate cleavage reaction.
  • a component which allows detection of a product of the substrate cleavage reaction.
  • markers routinely used in molecular biology which allow detection, such as. B. fluorescent markers, chemiluminescent markers, radioactive markers, biotin, avidin, streptavidin, antigens for antibodies, magnetic particles or an enzyme that leads to a detectable dye on contact with a chromogenic substance.
  • markers and their uses are known to the person skilled in the art and are also described in connection with the identification of new desired enzyme activities in US patent application 20030036092.
  • the component which allows the detection of a product of the substrate cleavage reaction is located on the substrate on a component, which is covalently fixed on the surface of the host organism after the cleavage reaction by the auxiliary enzyme.
  • a component which is covalently fixed on the surface of the host organism after the cleavage reaction by the auxiliary enzyme.
  • the alcohol function in the phenolic ester can be linked to a detectable signaling molecule (in the example attached, biotin).
  • the tyramide released by hydrolysis of the ester, which carries the signaling molecule is activated by the peroxidase fixed on the surface of the host organism in the presence of H 2 0 2 .
  • the phenol radical reacts with aromatic residues on the surface of the host organism and is thereby covalently fixed.
  • the presence of the product labeled with the signal molecule on the surface of the host organism can then be detected using detection methods known to the person skilled in the art.
  • the detection can e.g. B. on the formation of biotin / streptavidin conjugates.
  • the streptavidin can, for example, with a fluorescent substance, e.g. B. R-phycoerythrin, which allows the detection of biotin via the formation of the biotin / streptavidin conjugates.
  • Cells which have corresponding fluorescence-labeled conjugates on their surface can be determined by flow cytometry, e.g. B. by fluorescence-activated cell sorting, identified and isolated.
  • the substrate itself can also be linked with a fluorescent dye, so that the product fixed on the cell surface by the auxiliary enzyme is fluorescently labeled.
  • the fluorescence-labeled cells by flow cytometry, e.g. B. fluorescence-activated cell sorting, identified and isolated.
  • an "auxiliary enzyme" is provided on the surface of the host organism, which presents a candidate molecule on its surface.
  • This auxiliary enzyme is able to catalyze a reaction which enables a product of the cleavage reaction to be covalently bound to the surface of the host organism.
  • the auxiliary enzyme is an enzyme capable of converting a product released by the substrate cleavage reaction into an activated form capable of covalently binding with groups present on the surface of the host organism are known to those skilled in the art and preferably include those described in US Pat. No. 5,196,306, the disclosure of which is hereby incorporated by reference into the present application.
  • auxiliary enzymes are thus peroxidases, ligases, oxidoreductases, transferases and isomerases.
  • O xidases e.g., amino oxidases, and transferases.
  • the auxiliary enzyme is preferably a peroxidase.
  • peroxidase generally refers to enzymes that catalyze the oxidation of a compound with peroxide as the oxidizing agent.
  • any peroxidase can be used in the process according to the invention.
  • Examples are myeoloperoxidase (McCormick et al., J. Biol. Chem. 273 (1998), 32030-32037; myeoloperoxidase from human leukocytes is sold by Fluka, Sigma-Aldrich), lactoperoxidase (Heinecke, Toxicology 177 (2002), 11- 22; Ostdal et al., J. Agric. Food Chem.
  • HRP horseradish peroxidase
  • the auxiliary enzyme is preferably capable of converting a product released by the substrate cleavage reaction into an activated form which is capable of covalently bonding with groups, which are localized on the surface of the host organism.
  • groups on the host organism include in particular aromatic residues, such as. B. the side chains of tyrosine, tryptophan or histidine residues.
  • An “activated form” is preferably understood to mean a highly reactive, short-lived reaction product.
  • An activated form can, for example, be a radical. Radicals have the advantage that they are deactivated very quickly by water molecules if they do not immediately have molecules on the surface Other activated forms are those described in US Pat. No. 5,196,306.
  • an aminooxidase as an auxiliary enzyme if the product released by the cleavage reaction carries a free amino group. The aminooxidase then sets the free amino group to form an aldehyde, which then reacts with primary amines on the surface of the organism via a Schiff base formation and can form a covalent bond.
  • oxidases can also be used as auxiliary enzymes, which convert a product released in the cleavage reaction into an aldehyde, e.g. B. galactose oxidase, which converts released galactose to the corresponding aldehyde.
  • the expression “provided on the surface of the host organism” means that the auxiliary enzyme is present on the surface of the host organism.
  • the auxiliary enzyme can be provided on the surface according to known methods. For example, it is possible to irreversibly or reversibly immobilize the auxiliary enzyme On the surface of the host organism An irreversible immobilization can be achieved, for example, by covalent bonding of the auxiliary enzyme to groups which are present on the surface of the host organism, so that it is possible to add an auxiliary enzyme, in particular peroxidase, to the surface of the host organism to immobilize that an oxidation of the sugar side chains of the protein with sodium periodate and Schiff base reaction of the sugar aldehydes thus produced is carried out with primary amino groups present on the surface of the host organism.
  • auxiliary enzyme it is also possible for the auxiliary enzyme to bind to the surface of the host organism in the form of a conjugate of auxiliary enzyme and receptor, the receptor being able to bind a molecule which occurs on the surface of the host organism.
  • a receptor is an antibody that has a structure, e.g. B. recognizes a protein on the surface of the host organism.
  • This can e.g. B. a conjugate of the auxiliary enzyme and an anti-E. coli antibody that recognizes lipopolysaccharides on the cell surface.
  • conjugates are commercially available, e.g. B. at Maine Biotechnology Services Inc ..
  • Another possibility is a conjugate of a sugar-binding lectin and an auxiliary enzyme (see, for example, Appukuttan et al., Biochem. Biophys. 37 (2000), 77-80).
  • Another possibility of making the auxiliary enzyme available on the surface of the host organism is that it is expressed by the host organism in such a way that it is presented on the surface of the host organism. Methods by which this can be achieved are known to the person skilled in the art and are described in detail below in connection with the expression of the candidate polypeptides.
  • the enzyme to be identified is an esterase
  • the auxiliary enzyme is a peroxidase
  • the substrate is a phenol derivative of an ester of any carboxylic acids, ie a phenol ester.
  • phenol esters carry a functional group which enables the detection of the deposition of the product of the esterase activity on the surface of the host organism.
  • Use is made of the known fact that derivatives of phenol are activated by peroxidase in the presence of H 2 0 2 by the formation of a phenolic radical which is linked to electron-rich groups of other molecules such as e.g. B. tyrosine or tryptophan residues, form covalent adducts.
  • the phenol ester itself is not a substrate for the peroxidase. Thus, the phenol ester cannot be activated by the peroxidase and deposited on the surface of the host organism.
  • a covalent bond of the phenolic component, ie a reaction product of the enzyme activity, on the surface of the host organism is only possible if the acid activity is split off by the enzyme activity presented on the surface of the host organism and the free phenolic component is present.
  • the substrate in particular the part which is fixed as a product by the auxiliary enzyme on the surface of the host organism after the cleavage reaction
  • the phenol ester can be coupled with biotin in such a way that the biotin is attached to the phenol residue.
  • the biotin-coupled phenol component fixed on the surface of the host organism can then be detected using detection methods for biotin known to the person skilled in the art.
  • flow cytometry comes here, e.g. B. fluorescence-activated cell sorting, in question.
  • phages are used as host organisms, their detection or enrichment and isolation, e.g. B. achieved by adsorption on surfaces coated with a corresponding receptor molecule.
  • plastic surfaces e.g. microtiter plates
  • magnetic particles come into consideration as surfaces.
  • a common method is detection via biotin or digoxigenin, whereby the strong binding of biotin to strepavidin or of digoxigenin to a dioxigenin antibody is used.
  • the medium in which the host organisms are present at the time of Bringing into contact with the substrate H 2 0 2 , which is necessary for the enzymatic reaction of the peroxidase.
  • the concentration of H 2 0 2 is adjusted so that it is not toxic to the host organism. Suitable concentrations are preferably in the range from 0.00005% (v / v) to 0.005% (v / v), preferably in the range from 0.000075% (v / v) to 0.004% (v / v), particularly preferably in Range from 0.00009% (v / v) to 0.003% (v / v).
  • the concentration is very particularly preferably in the range from 0.0001% (v / v) to 0.001% (v / v).
  • H2O2-mediated covalent deposition of phenolic components have hitherto only been used for the immunohistochemical staining of fixed cell material, but not with living cells or other organisms, since H 2 0 2 is a strong cell poison and it was to be expected that this would be for a peroxidase Reaction used H 2 0 2 concentration is not compatible with the survival of the cells. Surprisingly, it was found that the concentration of H 2 0 2 can be adjusted to such an extent that on the one hand it does not lead to cell death, but on the other hand it still allows efficient deposition of the phenolic alcohol on the cell surface.
  • this method can be used successfully to selectively label living cells with the desired esterase activity through the simultaneous cell surface exposure of enzyme (esterase) and auxiliary enzyme (peroxidase) and the use of phenol esters as substrates and through iterative rounds of isolation labeled cells, proliferation of the isolated labeled cells and re-labeling and isolation, isolating cells with the desired enzyme activity from a population of cells without enzyme activity.
  • esterase enzyme
  • peroxidase auxiliary enzyme
  • the host organisms used in the method according to the invention can be any type of host organism which is suitable for the presentation of the candidate polypeptides on their surface.
  • any organism that is capable of carrying, expressing and replicating genetic information can be considered as the host organism.
  • the term “organism” encompasses any type of cells, but also viruses and phages.
  • the host organisms are cells or phages. If the host organisms are cells, they can be eukaryotic or prokaryotic cells Procaryotic host cells Organisms, particularly preferably bacteria. Gram-negative bacteria are preferred, and host cells of the type E. coli are particularly preferred.
  • host organisms which express the candidate polypeptides in such a way that the host organisms present them on their surface.
  • a variety of methods are known to those skilled in the art to achieve surface exposure of a protein to a host organism. Such methods are e.g. B. known under the terms “phage display” and “microbial display”. Exposure on the surface is mostly based on the fact that by using suitable genetic engineering methods, an enzyme variant is provided on the surface of the host organism in a covalent link with a component of the surface.
  • this is achieved, for example, by operatively linking the gene which codes for the enzyme which is to be optimized in terms of its properties to the coding sequence for a protein of the outer membrane of a microbial producer, so that a fusion protein is formed which is anchored in the outer membrane of the bacterium and exposes the linked protein domain on the outside of the outer membrane.
  • a fragment of the E. coli OmpA protein (Francisco et al., Proc. Natl. Acad. Sci. USA 90 (1993), 10444-10448), an Escherichia coli adhesin (Maurer et al., J. Bacteriol. 179 (1997), 794-804) or the intimin from enteropathogenic E.
  • phage display The presentation of polypeptides on the surface of phages, the so-called phage display, has already been described in detail and is widely used (see, for example, Miyakubo et al. (Nucleic Acids Symp. Ser. 44 (2000), 165-166), Widersten et al. (Meth. Enzymol. 328 (2000), 389-404), Widersten and Mannervik (J. Mol. Biol. 250 (1995), 115-122), Korn et al. (Biol. Chem. 381 ( 2000), 179-181) and Droge et al. (J. Biotechnol. 101 (2003), 19-28)).
  • the method according to the invention serves to identify enzymes with a desired substrate-splitting activity. It builds up methods known in the art in which enzymes with desired activities are identified by expressing a large number of different candidate polypeptides in host organisms and by identifying the host organisms that express the desired enzyme activity. Such host organisms are generally produced by providing DNA libraries which code for a large number of polypeptides and introducing them into corresponding host organisms.
  • the DNA libraries can e.g. B. are produced by the in vitro mutagenesis of a starting gene which codes for a specific enzyme. Mutagenesis results in variants of the enzyme generated, which can then be tested or selected for expression in the host organisms for their enzymatic properties.
  • Suitable methods for in vitro mutagenesis and for the production of suitable host organisms are known to the person skilled in the art and are known, for. B. described in detail in US patent application US 20030036092. They include e.g. B. chemical mutagenesis, especially of isolated DNA, gene amplification by "error prone" PCR and oligonucleotide mutagenesis. Other possibilities are the ligation of randomized gene segments (cassette mutagenesis), gene shuffling, in vivo mutagenesis with mutagenic agents and the use of E. According to the invention, a library is thus expressed in the host organisms, this library encoding a large number of candidate polypeptides.
  • step (b) of the method can be carried out by methods known to the person skilled in the art, such as, for. B. are described in US patent application 20030036092. If the product produced by the substrate cleavage reaction is inherently a product that is amenable to direct or indirect detection, the use of a marker molecule is not absolutely necessary. As explained above, however, a substrate is preferably used in the method according to the invention which is coupled to a marker molecule which allows detection of the product bound to the surface of the host organism. Examples of such markers are mentioned above and include biotin or fluorescent dyes.
  • such markers can also allow the isolation of these organisms.
  • the insulation is preferably carried out as described in US patent application 20030036092.
  • cells which carry such a labeled product on their surface can be determined by flow cytometry, e.g. B. fluorescence-activated cell sorting.
  • flow cytometry e.g. B. fluorescence-activated cell sorting.
  • the label coupled to the product is conjugated to another substance that fluoresces itself or is linked to a fluorescent label.
  • biotin as a marker on the product and to detect the biotin by streptavidin, to which a fluorescent marker is coupled.
  • the use of phages z. B. insulation over surfaces in question, the one Wear receptor for the marker molecule used, as already described above.
  • Another way to isolate the host organisms that have (labeled) product bound to their surface is by magnetic (cell) sorting.
  • the host organisms are brought into contact with magnetic particles which carry a molecule on their surface which binds the product fixed on the surface of the host organism or the marker coupled to it. So z. B. such magnetic particles carry a biotin-binding molecule on their surface (e.g. streptavidin) and would then bind organisms that carry a biotin-coupled product on their surface.
  • step (b) the method according to the invention leads to the identification of host organisms in which the product of the substrate-cleaving activity is fixed on the surface and which therefore are likely to express the desired enzyme activity.
  • the method according to the invention can be used repeatedly.
  • the DNA sequence which encodes the enzyme activity and which is contained in the identified host organisms is used as the starting point for the generation of a new library which encodes a large number of candidate polypeptides. This can be done by mutagenesis methods known to the person skilled in the art, as already mentioned above. Host organisms that express this library are then used in turn in the method according to the invention.
  • the present invention also relates to host organisms which express a candidate polypeptide (enzyme) in such a way that it is presented on the surface of the host organism and which at the same time carry on their surface an auxiliary enzyme which is able to catalyze a reaction which inhibits the formation covalent bonding allows a surface cleavage reaction between the surface of the host organism and a product catalyzed by the candidate polypeptide.
  • a candidate polypeptide enzyme
  • FIG. 1 Schematic representation of the inventive method using the example of the isolation of cells with esterase activity by covalent deposition of the hydrolysis product on the cell surface.
  • Clockwise Use a library of E. coli cells that carry randomly varied esterase genes. After induction of gene expression, the esterase is presented on the cell surface. The auxiliary enzyme peroxidase is then fixed on the surface of the bacteria. Unbound enzyme is removed by centrifuging the bacteria and discarding the supernatant. Then an ester substrate is added, which is a phenolic ester in which the alcohol function is linked to a detectable signal molecule (here biotin). The biotin pyramid released by hydrolysis of the ester (indicated schematically by a triangle in FIG.
  • the esterase activity of the cell is thus coupled to a detectable cell surface signal, which enables the isolation of an esterase-active cell from a population of cells, for example by using flow cytometry or magnetic cell sorting.
  • FIG. 2 TSA reaction (Tyramid Signal Amplification). Peroxidase-mediated covalent coupling of biotin tyramide with a tyrosyl residue of a protein. On Biotin molecule, which is linked to a phenol derivative, is activated by peroxidase in the presence of H 2 O 2 by formation of a phenyl radical. As it is very short-lived, this radical reacts with aromatic residues near where it originates. A covalent fixation of the detectable signal - here biotin - is achieved.
  • FIG. 3 Structure of LC-LC-biotin-tyramide octanoic acid ester. This ester can be used as a substrate molecule for an esterase. The released biotin pyramid can then be activated by peroxidase in the presence of H 2 O 2 .
  • Figure 4 Nucleotide sequence and deduced amino acid sequence of the esterase gene estA from Pseudomonas aeruginosa (from Wilhelm et al., J. Bacteriol. 181 (1999), 6977-6986).
  • the coding amino acid sequence begins at base 206.
  • the putative signal sequence is highlighted by an arrow.
  • Figure 5 Line surface presentation by EstA.
  • EstA induced E. coli cells, which express the estA gene under / ac promoter control, were incubated with anti-EstA antibody and stained with a biotinylated second antibody and streptavidin, phycoerythrin conjugate. The immunofluorescence of the cells was analyzed in a Zeiss axioscope (filter 15). Left: fluorescence; right: transmitted light image.
  • FIG. 6 Esterase-mediated deposition of biotin on the surface of E. coli cells.
  • Induced E. coli cells that carry both peroxidase and EstA on their surface (E. coli pBBX +) and control cells treated the same that do not contain an estA gene (pBBRI MCS) were treated with the substrate octanoic acid-biotin-LC-LC - Tyramide ester (Figure 3) incubated in the presence of 0.001% H 2 O 2 .
  • Cells of the same E. coli strain that do not carry an estA gene served as a control. After 15 minutes of incubation, the cells were washed in PBS buffer and stained with streptavidin, phycoerythrin conjugate. The immunofluorescence of the cells was analyzed in a Zeiss axioscope (filter 15). Left: fluorescence microscopy; right: transmitted light microscopy.
  • EstA is an esterase from Pseudomonas aeruginosa, which consists of an amino-terminal exposed cell surface-exposed enzyme domain and a membrane anchor domain, which is located in the outer membrane and mediates a translocation of the amino-terminal domain through the outer membrane.
  • the nucleotide and amino acid sequence of EstA is shown in Figure 4.
  • the coding sequence of EstA (FIG. 4) was introduced into the vector PBBX + (Wilhelm et al., Loc. Cit.) And thereby brought under the control of the lac promoter.
  • PBBX + Wild-Beet al., Loc. Cit.
  • E. coli cells were obtained which were able to hydrolyze the esterase substrate octanoic acid p-nitrophenyl ester (Wilhelm et al., Loc. Cit.).
  • the cell surface presentation of EstA was detected by immunofluorescence staining of the cells by successive incubation with anti-EstA antibody (from rabbits), a biotinylated anti-rabbit antibody and streptavidin, phycoerythrin conjugate. Cells treated in this way showed red fluorescence when analyzed in a fluorescence microscope (Zeiss Axioskop, Filter 15) (FIG. 5).
  • HRP Horseradish Peroxidase
  • the EstA substrate (FIG. 3) was made from LC-LC-biotin tyramide by reacting 0.2 mg (1.17 ⁇ mol) octanoyl chloride with 0.34 mg (0.57 ⁇ mol) LC-LC-biotin tyramide (Pierce ) made in 0.2 ml of pyridine. After incubation for 60 minutes at room temperature, the reaction mixture was lyophilized and taken up in 0.1 ml of dimethylformamide.
  • E. coli JM109 cells carrying the plasmid pBBX + were added at an optical density of 0.4 with IPTG (final concentration 1mM) to induce the expression of the estA gene and incubated at 37 ° C. for 60 minutes.
  • E. coli JM109 cells which do not contain an esfA gene were used as controls.
  • 500 ⁇ l of the cultures of both cell types were centrifuged off, coupled with oxidized peroxidase, then washed three times with 500 ⁇ l PBS each and in 500 ⁇ l 100 mM potassium phosphate buffer containing 0.0029 ⁇ mol biotin-LC-LC-tyramide-octanoic acid ester and 0.001% H 2 O. 2 contained incubated for 15 min.
  • the cells were centrifuged and resuspended in 10 ⁇ l PBS, which contained 1 ⁇ l streptavidin, R-phycoerythrin conjugate (Molecular Probes). After 5 minutes of incubation, the cells were spun down and the supernatant was discarded. The cells were washed in 500 ul PBS buffer. An aliquot was then analyzed in a fluorescence microscope (Zeiss Axioskop, Filter 15). Deposition of biotin tyramide was only detectable in the cells with EstA activity (FIG. 6).
  • E. coli JM109 cells induced with IPTG which contain the plasmid pBBRI MCS, which does not comprise an esf> A gene
  • JM109 cells containing the plasmid pBBX + which contains an estA gene, mixed in a ratio of 10 6 : 1.
  • the 1:10 6 mixture was then labeled with the biotin-LC-LC-tyramide-octanoic acid ester as described above.
  • Another 0.0029 ⁇ mol substrate in 500 ⁇ l potassium phosphate buffer was used.
  • the incubation time was again 15 minutes.
  • the cells were then centrifuged off and washed three times with 500 ⁇ l of PBS buffer. 20 ⁇ l streptavidin-coated paramagnetic beads (Miltenyi Biotech, Bergisch Gladbach) were then added to 200 ⁇ l of the cell suspension. After 15 minutes of incubation, the cells were centrifuged and washed three times with 500 ⁇ l PBS each. The cells were resuspended in 500 ul PBS. The suspension was then passed through a column filled with iron balls and placed in a strong magnetic field (MidiMacs, Mitenyi Biotech Bergisch Gladbach). The column was washed three times with 2 ml PBS buffer each. Cells retained in the column were eluted with 2 ml of PBS buffer after removal from the magnetic field. The cells were plated on selective plates and grown overnight. The next day the cells were washed away and the process repeated.
  • 20 ⁇ l streptavidin-coated paramagnetic beads (Miltenyi Biotech, Berg
  • the sorted cells were harvested from the plates with 2 ml of dYT and re-grown. For this, 50 ⁇ l of harvested cells were inoculated in 50 ml of dYT-Cm 25 . After induction of the culture, the next round of labeling and sorting took place. Three rounds of selection were carried out in this way
  • induced cells from each round of selection were checked for their esterase activity.
  • P-Nitrophenylcaprylate served as the hydrolyzable standard substrate. While the starting mixture shows no esterase activity, a slightly increased substrate hydrolysis can already be seen after the first round of selection. After the second round of selection, this is almost at the level of a corresponding sample of induced pBBX + transformants from E. coli JM109.

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Abstract

L'invention concerne un procédé permettant d'identifier des enzymes présentant une activité voulue, par production aléatoire d'une collection importante de variantes d'enzymes, la synthèse de ces variantes dans des organismes hôtes, leur présentation à la surface desdits organismes et l'isolement de variantes d'enzymes aux propriétés voulues, par mise en évidence du dépôt covalent du produit de réaction à la surface de l'organisme hôte.
PCT/EP2004/012017 2003-10-22 2004-10-22 Procede d'identification d'enzymes presentant des proprietes voulues, par ancrage des produits de reaction sur la surface d'organismes presentant des enzymes Ceased WO2005040408A1 (fr)

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EP04790808A EP1675958A1 (fr) 2003-10-22 2004-10-22 Procede d'identification d'enzymes presentant des proprietes voulues, par ancrage des produits de reaction sur la surface d'organismes presentant des enzymes

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008002472A3 (fr) * 2006-06-23 2008-03-13 Danisco Us Inc Genencor Div Évaluation systématique de relations entre séquence et activité à l'aide de bibliothèques d'évaluation de sites pour l'ingénierie de protéines multiples
CN111733102A (zh) * 2020-06-30 2020-10-02 东南大学 基于酪氨酸酶催化的革兰氏阳性菌表面修饰方法及其应用
CN119614505A (zh) * 2023-09-12 2025-03-14 珠海圣美生物诊断技术有限公司 稀有细胞捕获试剂组合及方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002034906A2 (fr) * 2000-10-26 2002-05-02 Georg-August-Universität Göttingen Procede pour l'exposition de peptides et de polypeptides sur la surface cellulaire de bacteries

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002034906A2 (fr) * 2000-10-26 2002-05-02 Georg-August-Universität Göttingen Procede pour l'exposition de peptides et de polypeptides sur la surface cellulaire de bacteries

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
BECKER S. ET AL.: "Ultra-high-throughput screening based on cell-surface display and fluorescence-activated cell sorting for the identification of novel biocatalysts.", CURRENT OPINION IN BIOTECHNOLOGY, vol. 15, no. 4, August 2004 (2004-08-01), pages 323 - 329, XP002321737, ISSN: 0958-1669 *
KLIS F. M.: "REVIEW: CELL WALL ASSEMBLY IN YEAST", YEAST, CHICHESTER, SUSSEX, GB, vol. 10, 1994, pages 851 - 869, XP000196331, ISSN: 0749-503X *
MOORE J.C. ET AL.: "DIRECTED EVOLUTION OF A PARA-NITROBENZYL ESTERASE FOR AQUEOUS-ORGANIC SOLVENTS", BIO/TECHNOLOGY, NATURE PUBLISHING CO. NEW YORK, US, vol. 14, no. 4, April 1996 (1996-04-01), pages 458 - 467, XP001148071, ISSN: 0733-222X *
OLSEN M.J. ET AL.: "FUNCTION-BASED ISOLATION OF NOVEL ENZYMES FROM A LARGE LIBRARY", NATURE BIOTECHNOLOGY, NATURE PUBLISHING, US, vol. 18, no. 10, October 2000 (2000-10-01), pages 1071 - 1074, XP001147401, ISSN: 1087-0156 *
OLSEN MARK ET AL: "High-throughput screening of enzyme libraries", CURRENT OPINION IN BIOTECHNOLOGY, LONDON, GB, vol. 11, no. 4, August 2000 (2000-08-01), pages 331 - 337, XP002207011, ISSN: 0958-1669 *
SAMUELSON P. ET AL.: "Display of proteins on bacteria", JOURNAL OF BIOTECHNOLOGY, vol. 96, no. 2, 26 June 2002 (2002-06-26), pages 129 - 154, XP002321735, ISSN: 0168-1656 *
SCHREUDER M.P. ET AL.: "Immobilizing proteins on the surface of yeast cells", TRENDS IN BIOTECHNOLOGY, ELSEVIER PUBLICATIONS, CAMBRIDGE, GB, vol. 14, no. 4, April 1996 (1996-04-01), pages 115 - 120, XP004035795, ISSN: 0167-7799 *
WILHELM S. ET AL.: "A novel lipolytic enzyme located in the outer membrane of Pseudomonas aeruginosa", JOURNAL OF BACTERIOLOGY, vol. 181, no. 22, November 1999 (1999-11-01), pages 6977 - 6986, XP002321736, ISSN: 0021-9193 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008002472A3 (fr) * 2006-06-23 2008-03-13 Danisco Us Inc Genencor Div Évaluation systématique de relations entre séquence et activité à l'aide de bibliothèques d'évaluation de sites pour l'ingénierie de protéines multiples
US8648015B2 (en) 2006-06-23 2014-02-11 Danisco Us Inc. Systematic evaluation of sequence and activity relationships using site evaluation libraries for engineering multiple properties
CN111733102A (zh) * 2020-06-30 2020-10-02 东南大学 基于酪氨酸酶催化的革兰氏阳性菌表面修饰方法及其应用
CN111733102B (zh) * 2020-06-30 2023-08-18 东南大学 基于酪氨酸酶催化的革兰氏阳性菌表面修饰方法及其应用
CN119614505A (zh) * 2023-09-12 2025-03-14 珠海圣美生物诊断技术有限公司 稀有细胞捕获试剂组合及方法

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