WO2001060986A2 - Esterase enzymes having selective activity - Google Patents

Abstract

This application relates to methods for identifying enzymes having selective activity and the enzymes identified thereby. In particular, the present application relates to novel hydrolases. Methods for identifying and using the enzymes are also provided.

Description

HYDROLASE ENZYMES HAVING SELECTIVE ACTIVITY FIELD OF THE INVENTION

This application relates to methods for identifying enzymes having selective activity and the enzymes identified thereby. In particular, the present application relates to novel hydrolases.

BACKGROUND OF THE INVENTION

There is an increasingly important need to develop new biocatalysis processes rapidly and inexpensively, especially for the development of novel pharmaceuticals where time is extremely valuable. The use of a powerful, analytical screening strategy is often the key to speeding up development time at several different levels of the process. In the discovery of novel enzymes, screening plays an important role in identifying which subset of candidates contain an enzyme of interest from a collection of organisms, clone banks, or enzyme libraries. Directed evolution approaches to engineer custom biocatalysts require powerful screening strategies to sift through large mutant pools to find enzymes with properties that have often been only slightly altered against a high activity background. Finally, process optimization and development can often take an excessive amount of time, especially to perform a comprehensive analysis of different reaction conditions including temperature, pH, cosolvent, reaction time, and other parameters, both individually and in combination. This type of analysis requires the implementation of a rapid, high-throughput assay which is amenable to automation and use in a hierarchical screening strategy. Hydrolases are well established as valuable tools for the food, pharmaceuticals and fine chemicals industry. (Gerhartz W. Ed. Enzymes in Industry VCH: Weinheim, 1990). The importance of biocatalysis has led to the search for novel enzymes with singular activities. Recently, extremophilic microorganisms have been investigated as a source of these novel activities. (Kristjansson, J. K. TIBTECH 1989, 7, 349; Adams,et al. Bio/Technology , 1995, 13, 662; Govardhan, et al. Chem. Ind. 1995, 17, 689- 93; Newell, J., Chemistry in Britain 1995, 31; Vieille, et al. TIBTECH , 1996, 14, 183). Because of their properties, enzymes are important in the selective biotransformation of many molecules. Enzymes are often desired for their chemoselectivity, regioselectivity, enantioselectivity properties or for substrate selectivity. For example, synthesis of enantiomerically pure compounds (EPC) with one or several chiral centers is one of the most challenging tasks in modern organic chemistry. Enzymes are able to contribute significantly to this challenge and have been increasingly considered as a useful class of catalysts for organic synthesis. (Davies, G. et al. Biotransformations in Preparative Organic Chemistry; Academic Press: London, 1989; Wong, et al. Enzymes in Synthetic Organic Chemistry; Pergamon: Oxford, 1994; Faber, K. Biotransformations in Preparative Organic Chemistry , Springer- Verlag: Berlin-Heidelberg, 1995; Drauz, K; Waldmann, H Eds. Enzyme Catalysis in Organic Synthesis Vol 1 & 2VCH: Weinheim, 1995). Scientists at ThermoGen, Inc. (Chicago, IL) have developed a set of tools to obtain libraries of thermophilic enzymes by genetic engineering (see, for example, U.S. Pat. Ser. No. 08/694,078). ThermoCat® consists of a set of twenty stable esterases, capable of working well either at room or high temperature and also in organic solvents. It is of high interest to study the selectivity of each enzyme in the library, especially their enantiodiscrimination when exposed to racemic substrates. Time is the limiting factor in carrying out the work when screening a library of enzymatic activities against an array of substrates for either enzyme discovery, enzyme engineering (such as directed evolution) or process optimization experiments. The analytical methods typically employed for this purpose include high- pressure liquid chromatography (HPLC), thin-layer chromatography (TLC), and gas chromatography (GC), which are often not amenable to high- throughput assays.

There is a need in the industry for new methods for the identification of new biocatalysts requires rapid screening assays combined with hierarchical screening strategies. The approach works by eliminating the weakest candidates as one of the earliest steps in the bioprocess development timeline, rendering a streamlined process-viability study. (See, for example, Demirjian, et al. Top. Curr. Chem., 1998, 200.)

One of the most convenient ways to assay an enzyme is through a method that allows the development of color and thus can be used in qualitative as well as quantitative measurements. A number of colorimetric methods to measure enzymatic activity have been described. (Michal, et al. in Methods of Enzymatic Analysis; Bergmeyer, H. U., Ed.; Verlag Chemie: Weinheim, 1983; Vol. I; pp 197; Demirjian, et al. Top. Curr. Chem., 1998, 200.) Hydrolytic enzymes can be rapidly screened with chromogenic (nitrophenyl), fluorogenic (4-methylumbelliferone) or indigogenic (indoxyl) substrates that yield colored products upon hydrolysis. The main limitation of this approach is the presence of the latent colorimetric functionality within the substrate, whose introduction is at least time-consuming and yields a structure for analysis essentially different from the actual target (usually a methyl or ethyl ester).

Several types of pH-dependent assays have been described including enzyme-catalyzed processes with hexokinase. (Wajzer, J. Hebd. Seances Acad. Sci., 1949, 229, 1270; Darrow, et al. Methods in Enzymology, 1962; Vol. V, 226; Crane, et al. Methods in Enzymology, 1960; Vol. I, 277) and cholinesterase (Lowry, et al. J. Biol. Chem. 1954, 207, 19) and in enzyme- free studies of carbon dioxide hydration. (Gibbons, et al. J. Biol. Chem. 1963, 238, 3502). Since the 1970's, such strategies have been used in kinetic analysis of enzyme reactions. Examples of this include human carbonic anhydrase (Khallifah, R. G. J. Biol. Chem., 1971, 246, 2561), amino acid decarboxylases (Rosenberg, et al. Anal. Biochem. 1989, 181, 59) and serine proteases (Whittaker, et al. Anal. Biochem. 1994, 220, 238). The progress of the hydrolysis is monitored by visual inspection of the solution color after the enzyme has been added or by using a microplate reader to get a quantitative reading. Whittaker et al. (Anal. Biochem. 1994, 220, 238-243) measured the esterase activity of proteases in 96-well microplates using a pH-dependent assay. However, the Whittaker assay requires additional experiments beyond those required in practicing the methodologies provided herein in that Whittaker does not use an indicator-buffer pair with the same pK, values and does not reliably measure the true rates of enzyme-catalyzed hydrolysis.

Recently, the use of pH indicators has been extended to monitor the directed evolution of an esterase on a plate assay using a whole cell system, rather than the isolated enzyme. (Bomscheuer, et al. Biotechnol. Bioeng. 1998, 58, 554). However, a pH-dependent assay has not been utilized to determine the enatioselectivity of an enzyme. Kazlauskas has partially solved the problem of competition by using a reference non-chiral additive in classical chromogenic substrate assays. (Janes, et al. J. Org. Chem., 1997, 62, 4560.) The same author developed a quantitative method for the evaluation of the enantioselectivity (without considering the competition factor) for actual substrates based on a pH indicator/buffer system (/?-nitrophenol / BES) with equal pK_, so the linearity of the color transition allows the quantitation of the enantioselectivity. (Janes, et al. Chem. Eur. J. ,1998, 4, 2317). Certain of these assay systems have proven useful for the identification of novel esterases, as shown below. Provided herein are several novel esterases having enantioselective activity. These new enzymes are useful the carrying out reactions in many different fields of research and industry.

SUMMARY OF THE INVENTION

The present invention provides esterases isolated from microorganisms and having particular biochemical fingerprints that allow for the enzymes to be distinguished from one another. In one embodiment, the present invention provides an esterase selected from the group consisting of NE01, NE02, NE03, NE04, NE05, NE06, NE07, NE08, NE09, NE10, NE11, NE12, NE13, NE14, NE15, NE16, NE17, NE18, NE19, NE20, NE21 and NE22, the biochemical fingerprint being represented by the reactivity and enantioselectivity profile to substrates 1-26 illustrated in Figure 1, and the PNP priopionate profile of the enzyme.

In another embodiment, the present invention provides an esterase having the biochemical fingerprint of the esterase NE01, the biochemical fingerprint being represented by the reactivity and enantioselectivity profile to substrates 1-26 illustrated in Figure 1, the PNP priopionate profile, a relative molecular weight of 48 kDa, and the N-terminal amino acid sequence TEXQYIVALD.

In another embodiment, the present invention provides an esterase having the biochemical fingerprint of the esterase NE02, the biochemical fingerprint being represented by the reactivity and enantioselectivity profile to substrates 1-26 as illustrated in Figure 1, the PNP priopionate profile, and a relative molecular weight of 36 kDa.

In another embodiment, the present invention provides an esterase having the biochemical fingerprint of the esterase NE03, said biochemical fingerprint being represented by the reactivity and enantioselectivity profile to substrates 1-26 as illustrated in Figure 1, the PNP priopionate profile, a relative molecular weight of 43 kDa, and the N-terminal amino acid sequence XQXPYDMPLE. In another embodiment, the present invention provides an esterase having the biochemical fingerprint of the esterase NE04A, the biochemical fingerprint being represented by the reactivity and enantioselectivity profile to substrates 1-26 illustrated in Figure 1, the PNP priopionate profile, a relative molecular weight of 42 kDa, and the N-terminal amino acid sequence RPMGFXGAXX.

In another embodiment, the present invention provides an esterase having the biochemical fingerprint of the esterase NE04B, the biochemical fingerprint being represented by the reactivity and enantioselectivity profile to substrates 1-26 illustrated in Figure 1, the PNP priopionate profile, a relative molecular weight of 31 kDa, and the N-terminal amino acid sequence XLDPVI(Q/X)QVL.

In another embodiment, the present invention provides an esterase having the biochemical fingerprint of the esterase NE05, said biochemical fingerprint being represented by the reactivity and enantioselectivity profile to substrates 1-26 illustrated in Figure 1, the PNP priopionate profile, a relative molecular weight of 46 kDa, and the N-terminal amino acid sequence MENFKHLPEP. In another embodiment, the present invention provides an esterase having the biochemical fingerprint of the esterase NE06, the biochemical fingerprint being represented by the reactivity and enantioselectivity profile to substrates 1-26 illustrated in Figure 1, PNP priopionate profile, and relative molecular weight of 50 kDa.

In another embodiment, the present invention provides an esterase having the biochemical fingerprint of the esterase NE09, the biochemical fingerprint being represented by the reactivity and enantioselectivity profile to substrates 1-26 illustrated in Figure 1, the PNP priopionate profile, and a relative molecular weight of 81 kDa.

In another embodiment, the present invention provides an esterase having the biochemical fingerprint of the esterase NE10, said biochemical fingerprint being represented by the reactivity and enantioselectivity profile to substrates 1-26 illustrated in Figure 1, the PNP priopionate profile, a relative molecular weight of 48 kDa, and the N-terminal amino acid sequence MEVE(K/T)HLPE(P/L).

In another embodiment, the present invention provides a method for identifying esterases using a biochemical fingerprint consisting of any combination of characterisitics including but not limited to reactivity and enantioselectivity profiles, PNP priopionate profiles, relative molecular weight, and N-terminal, internal, or C-terminal amino acid sequence.

In another embodiment, a method for performing selective hydrolysis on a substrate using an esterase having a biochemical fingerprint substantially similar to that of NE01, NE02, NE03, NE04, NE05, NE06, NE07, NE08, NE09, NE10, NE11, NE12, NE13, NE14, NE15, NE16, NE17, NE18, NE19, NE20, NE21 and NE22. The biochemical fingerprint may be represented by the reactivity and enantioselectivity profile to substrates 1-26 illustrated in Figure 1 , and the PNP priopionate profile of an esterase, for instance. In a preferred embodiment, the substrate is selected from compounds 1-26, as illustrated in Figure 1.

In another embodiment, the present invention provides a method for mutating an esterase identified herein in order to identify esterases having a desired activity that is more preferred than that of the originally isolated esterase. As such, the present invention provides for mutation of a DNA molecule encoding an esterase by directed mutagenesis, PCR, error prone PCR, DNA shuffling, protein domain shuffling (see, for example US 5,981,177) or similar technique and selection of esterases possessing enhanced activities, such as increased enantioselectivity. As such, the present invention provides bacterial clones comprising DNA molecules encoding the esterases of the invention, and the DNA molecules encoding the esterases may serve as templates for modification of the coding sequences. The present invention further encompasses a method for performing multiple cycles of mutation and selection in order to select enzymes having even more desired characteristics, such as enhanced selectivity. Many methods for mutating a DNA molecule are well known within the art and are contemplated by this invention, as applied to the esterases and coding sequences therefor described in this application.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the library of substrates employed to obtain the activity profiles.

Figures 2A and 2B show the kinetic profiles obtained by running the assay in 96-well microplates and reading absorbance decrease at 405nm.

Figures 3A-D reduce the data after analyzing the kinetic plots. The most preferred enzymes are listed in Figures 4 A and 4B.

Figures 4A-B illustrate the PNP propionate profiles for particular esterases.

DETAILED DESCRIPTION

Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references including: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, CA), PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, CA), and Gene Transfer and Expression Protocols, pp. 109-128, ed. E.J. Murray, The Humana Press Inc., Clifton, N.J.). All references cited in this application are hereby incorporated by reference.

Methods for the production of enzymes having selective activities are much desired in the art. The selective activities may relate to chemoselectivity, regioselectivity, enantioselectivity or substrate selection. A particularly challenging task for modern chemists is the synthesis of enantiomerically pure compounds (EPC) with one or several chiral centers. Those of skill in the art understand that enzymes are a useful class of biocatalysts for organic synthesis. Among these biocatalysts, hydrolases are particularly valuable tools for the food, pharmaceuticals and fine chemicals industry. The importance of biocatalysis has led to the search of novel enzymes with singular activities.

In one embodiment, the present invention provides an esterase having a biochemical fingerprint substantially similar to that of NE01, NE02, NE03, NE04, NE05, NE06, NE07, NE08, NE09, NE10, NE11, NE12, NE13, NE14, NE15, NE16, NE17, NE18, NE19, NE20, NE21 and NE22. In another embodiment, the esterase is formulated as a composition in a suitable buffer such that the enzyme may be utilized in a chemical or other reaction. In one embodiment, the present invention provides a method for performing selective hydrolysis on a substrate. In a preferred embodiment, the substrate is one of compounds 1-26 illustrated in Figure 1.

Many different enzymes may be isolated, screened, characterized, and/or modified using the techniques described herein. For example, the techniques may be applicable to Upases or esterases useful for enantioselective hydrolysis of esters (lipids)/thioesters (i.e., resolution of racemic mixtures and / or synthesis of optically active acids or alcohols from meso-diesters; see, for example U.S. Pat. No. 5,969,121), selective syntheses (i.e., regiospecific hydrolysis of carbohydrate esters, selective hydrolysis of cyclic secondary alcohols), synthesis of optically active esters, lactones, acids, alcohols (i.e., transesterification of activated/nonactivated esters, interesterifϊcation, optically active lactones from hydroxyesters, regio- and enantioselective ring opening of anhydrides), detergents, fat/oil conversion, and cheese ripening); protease (useful for ester/amide synthesis, peptide synthesis, resolution of racemic mixtures of amino acid esters, synthesis of non-natural amino acids, and detergents/protein hydrolysis, for example); glycosidase/glycosyl transferases (for example, useful for sugar/polymer synthesis, cleavage of glycosidic linkages to form mono, di- and oligosaccharides, synthesis of complex oligosaccharides, glycoside synthesis using UDP-galactosyl transferase, transglycosylation of disaccharides, glycosyl fluorides, aryl galactosides, glycosyl transfer in oligosaccharide synthesis, diastereoselective cleavage of β-glucosylsulfoxides, asymmetric glycosylations, food processing, and paper processing); phosphatase/kinases (useful for synthesis/hydrolysis of phosphate esters (i.e., regio-, enantioselective phosphorylation, introduction of phosphate esters, synthesize phospholipid precursors, controlled polynucleotide synthesis), activating a biological molecule, and selective phosphate bond formation without protecting groups, for example); mono/dioxygenases (useful for direct oxyfunctionalization of unactivated organic substrates, hydroxylation of alkane, aromatics, steroids, epoxidation of alkenes, enantioselective sulphoxidation, and regio- and stereoselective Bayer-Villiger oxidations); haloperoxidases (for oxidative addition of halide ion to nucleophilic sites, addition of hypohalous acids to olefinic bonds, ring cleavage of cyclopropanes, conversion of activated aromatic substrates to ortho and para derivatives, coversion of 1,3 diketones to 2-halo-derivatives, oxidation of heteroatom of sulfur and nitrogen containing substrates, oxidation of enol acetates, alkynes and activated aromatic rings, for example); lignin peroxidase/diarylpropane peroxidases (useful for oxidative cleavage of C— C bonds, oxidation of benzylic alcohols to aldehydes, hydroxylation of benzylic carbons, phenol dimerization, hydroxylation of double bonds to form diols, and cleavage of lignin aldehydes, for example); epoxide hydrolase (useful for synthesis of enantiomerically pure bioactive compounds, regio- and enantioselective hydrolysis of epoxide, epoxidation of aromatic and olef inic by monooxygenases to form epoxides, resolution of racemic epoxides, and hydrolysis of steroid epoxides, for example); nitrile hydratase/nitrilases (for hydrolysis of aliphatic nitriles to carboxamides, hydrolysis of aromatic, heterocyclic, unsaturated aliphatic nitriles to corresponding acids, hydrolysis of acrylonitrile, production of aromatic and carboxamides, carboxylic acids (nicotinamide, picolinamide, isonicotinamide), regioselective hydrolysis of acrylic dinitrile, production of alpha.-amino acids from . alpha. -hydroxynitriles, for example); transaminases (i.e., for transferring amino groups into oxo-acids); and / or amidases / acylases (i.e., hydrolysis of amides, amidines, and other C--N bonds, resolution and synthesis of non-natural amino acids). Many other such enzymes are known in the art, and could be modified and assayed as described herein.

In order to produce large amounts of enzyme or conduct mutational analysis or enhancement of enzymes, it is useful to isolate a DNA molecule encoding the enzyme. Cloning of esterases using these systems is described within U.S. Pat. No. 5.969,121, for example. Using phage- or plasmid-based system, a library including sequences encoding esterases is prepared in phage, bacteria such as E. coli are infected with phage, and the infected bacteria are screened for esterase activity. A similar approach may be taken using a plasmid-based system.

It may also be desireable to use a DNA library generated from random, "normalized", cultivated, uncultivated, isolated or mixed microorganisms (see, for example, U.S. 5,969,121; U.S. Pat. No. 5,763,329; U.S. Pat. No. 6,001,574; U.S. Pat. No. 5,958,672 or US. Pat. No. 5,939,250; each of which being hereby incorporated by reference in their entirety) that may be screened to identify DNA molecules encoding enzymes having a particular activity or biochemical profile. Any of the enzymes isolated from such libraries may also be modified using the techniques described above, or other suitable techniques as are known in the art.

Another method that may be used to identify clones containing DNA molecules encoding enzymes to prepare "selective libraries". As described herein, organism and/or polynucleotide libraries from mixed populations of organisms having maximal diversity may be prepared. It is important to have the ability to prepare diverse libraries of organisms to either utlize directly or for the preparation of polynucleotide libraries, such as genomic DNA or cDNA libraries. Diversity of the library is important to ensure that the most information possible can be derived from that library. It has been previously demonstrated that DNA libraries can be prepared from mixed populations of organisms (see, for example, Schmidt, et al. Analysis of a Marine Picoplankton Community by 16S rRNA Gene Cloning and Sequencing, Bacteriology. July 1991, pp. 4371-4378). However, previous studies have not provided a method for optimizing the diversity of the libraries resulting in the possibility of over- or under-representation of library members. Provided herein is a solution to this difficulty where a preliminary screen is performed to eliminate redundant species or library members, thus maximizing the diversity of the resultant library.

To enhance the quality of a DNA library to be used in identifying esterases, an organism collection may be fingerprinted using the RNA typing, 16S ribosomal typing, and / or Rapid PCR techniques. Organisms are first separated, such as by culturing. This technique is adaptable for either mixed, non-related populations of organisms where the DNA sequence variations may be substantial or related populations, where the DNA sequence variations may be relatively minor. It is conceivable that uncultured organisms could be separated by techniques other than culturing, such as by flow cytometry or other methods. The skilled artisan would understand that there are many methods available for the separation of organisms other than culturing.

DNA is then isolated from each of the organisms and amplified by polymerase chain reaction (PCR). Random primers are utilized to amplify the DNA of the organisms in the population under standard conditions for PCR. The various organisms are then fingerprinted based on the resultant PCR amplification pattern. The fingerprints are compared, and organisms sharing identical fingerprints are eliminated from the population. In this manner, duplicate or redundant organisms are eliminated from the population. This provides a unique strain collection. By eliminating redundant organisms prior to the library preparation step, representation of a diverse group of organisms ensured. A problem associated with mixed population library preparation is that one or more species of the population may be over- or under-represented in the library. This is because one or more representative members of that species may be present in the initial sample, thus causing a bias for that species during library preparation. Preferably, the resultant library is equally representative of each of the members of the mixed population. This ensures that the signal from less populous members of the population is not "drowned out" by the signal from more populous members of the population.

Following preparation of suitable expression libraries, the libraries are screened to identify clones containing DNA molecules encoding particular enzymes. Many assays for screening are well-known and widely available in the art. For example, Assays for the identification of selective enzymes having are demonstrated in U.S. Pat. No. 5,969,121, U.S. Ser. No. 08/694,078, U.S. Ser. No. 09/348,976, PCT/US99/15400, and PCT/US99/14448, for example. The present application provides methods for identifying novel enzymes having selective activities.

The assay systems described herein are useful for generating a biochemical fingerprint for an enzyme, especially where the DNA sequence is not yet known. This fingerprint can be used by the skilled artisan to differentiate various enzymes from one another. For instance, as shown herein, esterases have been identified and distinguished from one another using a pH assay to determine reactivity and enantioselectivity, and by determining the PNP propionate profile.

The reactivity and enantioselectivity profiles may be generated using compounds such as compounds 1-26 as shown in Figure 1. These compounds include methyl 2,2-dimethyl-l,3-dioxolane-4-carboxylate (1), methyl l-methyl-2-oxo-cyclohexane propionate (2), methyl 2- chloropropionate (3), methyl lactate (4), glycidyl butyrate (5), tryptophan methyl ester (6), methyl mandelate (7), methyl 3-hydroxy-2-methylpropionate (8), methyl 3-hydroxybutyrate (9), ethyl 4-chloro-3-hydroxybutyrate (10), oxabicyclo[3.3.0]oct-6-en-3-one (11), menthyl acetate (12), neomenthyl acetate (13), 3-hydroxy-3-methyl-4,4,4-trichlorobutyric-β-lactone (14), dimethyl malate (15), dimethyl 2,3-O-isopropylidenetartrate (16), methyl mandelate acetate (17), methyl 3-hydroxy-3-phenylpropionate (18), indanol acetate (19), phenethyl alcohol acetate (20), O-acetyl mandelonitrile (21), acetyl α-hydroxy-γ-butyro-lactone (22), glycidyl 4-nitrobenzoate (23), 2- methylglycidyl 4-nitrobenzoate (24), α-methyl-1-naphthalene-methanol acetate (25), and α-methyl-2-naphthalene-methanol acetate (26). As described above, another embodiment of the present invention is a method for performing selective hydrolysis of a compound, preferably one of compounds 1-26 as illustrated in Figure 1.

In addition, for certain enzymes, the relative molecular weight (MW_r), and N-terminal amino acid sequence has been determined. In describing the N-terminal amino acid sequence of an enzyme, the following abbreviations may be used:

TABLE 2

Where an ambiguity is identified within a sequence a "/" is placed between the two possible amino acid residues and is typically highlighted by placing the residues within parentheses. Where a particular residue is unknown, a "?" is inserted.

In considering the amino acid sequence component of a biochemical fingerprint, two sequences are identical where 60-100% of the amino acid residues are the same between two proteins. Preferably, two sequences are identical where 70-100% of the amino acid residues are the same. More preferably, two sequences are identical where 80-100% of the amino acid residues are the same. Even more preferably, the sequence is identical where 90-100%) of the residues are the same. Even more preferably, two sequences are identical where 95-100%> of the amino acid residues are the same. And most preferably, two sequences are the same where 100% of the amino acid residues are the same. As shown in the Examples section herein, these biochemical parameters which together, in some combination, for a biochemical fingerprint with which at least 10 distinct enzyme activities (i.e., at least 10 different DNA molecules encoding the enzyme) have been identified and isolated from a genomic DNA library. The biochemical fingerprint may comprise any combination of biochemical characteristics that is useful for distinguishing enzymes. Two enzymes are distinct where the biochemical fingerprint of the two enzymes are not substantially similar. Two enzymes are indistinct where the biochemical fingerprint of the two enzymes are substantially similar. In characterizing enzymes, the reactivity, enantioselectivity and

PNP propionate profile may be known and in some instances this will be sufficient to distinguish the enzymes. In other instances, MW_r and / or amino acid sequence may also be known. Other biochemical characteristics, as would be known by one of skill in the art, may also be helpful and may be form additional features of a biochemical fingerprint for an enzyme. Accordingly, any suitable combination of such characteristics may be combined to generate the biochemical fingerprint of an enzyme.

Following isolation of DNA molecules encoding an enzyme of interest, the skilled artisan may desire to modify, improve, or otherwise alter one or more characteristics of the enzyme. This may be accomplished using any of the known methods in the art. For example, directed evolution can provide rapid access to an enzyme with the desired properties. This process utilizes a variety of methods such as sequential random mutagenesis, error- prone mutagenesis (i.e., error-prone PCR) or gene shuffling in combination with high-throughput screening or selection to identify libraries of potential biocatalysts. Directed evolution does not necessarily require any prior knowledge of the structure-function relationship.

The major steps of directed evolution include the selection of the gene, creation of the variant library, insertion of the library into an expression vector, expression of the gene library to product mutant enzyme libraries, screening of the mutant enzymes for the property of interest, and the isolation of the gene corresponding to the improved variant properties so that the cycle can be repeated as desired. The generation and screening of mutants with improved performance is carried out in iterative steps. After several cycles, the performance of mutant proteins should be optimal under the application- specific conditions. One exemplary system is provided by PCT US98/09627 (WO98/51802), incorporated herein by reference in its entirety. Many suitable systems for performing such development cycles are available to one of skill in the art. Certain non-limiting examples of such systems are reviewed below.

In one aspect, evolution of an enzyme may be accomplished by random domain shuffling, as described in U.S. Pat. No. 5,981,177, which is hereby incorporated by reference in its entirety. As described therein, a transposon may be utilized to randomly shuffle protein domains, thus providing enzymes having improved or unique activities. The instantly described screening methodology is useful for identifying the desired enzymes expressed from a library of enzymes created by randomly shuffling the various domains of the enyzmes, for instance. Other methods for recombining DNA sequences to generate novel enzymes have also been described in the art. For instance, U.S. Pat. No. 5,605,793 (incorporated herein by reference in its entirety) describes the random fragmentation of a template DNA sequence and re-assembly in the presence of a partially random oligonucleotide having overlapping sequence with the template. In this manner, novel libraries of DNA sequences encoding enzymes are produced. The high-throughput screening methodology set forth herein is useful for screening such libraries for enzymes having particular characteristics. Another method for recombining sequences is described in U.S.

5,811,238; U.S. Pat. No. 5,830,721; and, 5,837,458 (each of which being incorporated herein by reference in their entirety) wherein a DNA sequence is randomly fragmented, "shuffled" to randomly recombine the sequences, and screened to identify DNA sequences encoding proteins having particular characteristics. This cycle is then repeated to further select for enzymes having even more evolved characteristics. The present assay system is particularly useful for identifying enzymes having the desired activity from a very large population of variants.

Yet another method for generating large numbers of randomly associated DNA sequences encoding enzymes is provided by U.S. Pat. No. 5,965,408 (incorporated herein by reference in its entirety). As shown therein, "sexual" PCR is performed using a template DNA and random oligonucleotide primers and interrupting synthesis of the template DNA molecule. In this way, novel DNA sequences are provided that encode enzymes having unique characteristics. The high-throughput assay system provided herein is useful for identifying those DNA sequences that encode unique enzymes.

It is also possible to generate functional polypeptides by randomly assembling small oligonucleotides, such as described by U.S. Pat. No. 5,723,323; U.S. Pat. No. 5,814,476; U.S. Pat. No. 5,817,483; U.S. Pat. No. 5,824,514; U.S. Pat. No. 5,976,862, each of which being hereby incorporated by reference in their entirety. As shown therein, polypeptides may be generated using randomly generated 7-mer and / or 8-mer oligonucleotides, for example, to generate larger "random" DNA sequences. These sequences may then be cloned into expression vectors, which are then transformed into the appropriate host cell. The host cells are then screened for expression of particular enzymatic activities and the DNA encoding the responsible enzymes are isolated. Following isolation of such DNA molecules, the enzyme of interest may be studied further, and potentially further manipulated using the techniques described herein.

There are a multitude of suitable vectors for use in cloning enzymes that are available to one of skill in the art. One such vector is described in U.S. Pat. No. 5,786,174, incorporated herein by reference in its entirety, and describes a novel cloning vector for use in thermophiles, such as Thermus. The vector allows for identification of organisms containing cloned sequences by selection in antibiotic, such as kanamycin. Many other suitable vectors also available for cloning in thermophilic or non-thermophilic organisms, as is known by those of skill in the art.

Multiple methods for detecting enzyme activity are also available to the skilled artisan. For instance, U.S. Pat. No. 5,969,121 demonstrates multiple esterase screening techniques for identification of esterase-producing clones from DNA expression libraries. Similarly, U.S. Pat. No. 6,004,788 describes screening techniques for identifying enzyme activity. PCT US99/14448 and PCT/US99/11540 (both of which being hereby incorporated by reference in their entirety) describe pH-dependent assays for enzymes that may be utilized in the instantly described high-throughput assay. Assays are also described in PCT/US98/22607 and PCT/US98/09627 (both of which being hereby incorporated by reference in their entirety) that are useful for identifying alcohol dehydrogenases, for example. These methods are useful for identifying enzymes with a particular activity or characteristic. Many other suitable methods are known by those of skill in the art and are encompassed by the instant invention. The Examples provided herein are for illustrative purposes only and are not intended, nor should they be construed as limiting the invention in any manner. Those skilled in the art will appreciate that variations and modifications can be made without violating the spirit or scope of the invention.

EXAMPLES Example 1 Preparation of DNA libraries for Identification of Esterases

As described above, libraries may be prepared either from known cultured organisms, unknown cultured organisms or uncultured organisms. For example, the known strain Thermus sp. T351 (ATCC 31674) is available from the American Type Culture Collection (ATCC). Isolated strains and cultures are grown on TT medium, which consists of (per liter): BBL Polypeptone (8 gm), Difco Yeast Extract (4 gm), and NaCl (2 gm). Small scale cultures for screening are grown at 65°C at 250-300 rpm with 1 liter of medium in a 2 liter flask. Larger scale production of cells for enzyme purification are grown in 17 liter fermentors (LH Fermentation, Model 2000 series 1). The fermentors have a working volume of 15 liters and cultures were grown in TT broth, 250 rpm, 0.3 to 0.5 vvm (volumes air/volume media per minute) at 65°C. Temperature is maintained by circulating 65°C water from a 28 liter 65°C water reservoir through hollow baffles within the stirred jars. E. coli strains are grown under standard conditions. To isolate unknown organisms, multiple stream sediments, composting organic materials, and soil samples may be collected. For these experiments, samples were collected from numerous geographic sites, including Florida, Montana, and Maryland. Samples (~1 gm) were resuspended in 2 ml of TT broth and 50-100 μl of these samples were plated onto TT agar plates containing twice the usual amount of agar (3%). Agar was added to a final concentration of 1.5% for solid media to prevent highly motile microorganisms from overcrowding the plate at the expense of other microbes. Plates were incubated at 55°C or 65°C for one to two days and isolates then purified by numerous restreaks onto fresh plates for single colony isolation. The initial basis for differentiation was color, colony morphology, microscopic examination, temperature of growth, and lipase and esterase activities. Genomic DNA was then isolated from the organisms, digested with Sau3A using standard techniques, and inserted into a lambda phage (lambda ZAP) to prepare a DNA library representative of various types of organisms. Esterases 23-1, 23-4, 23-7, 23-31, 14-2 were isolated in pBluescript. The following esterases were cloned in lambda zap: 69-10, 4AE-1, 4AE-2, 72-6, 84-5, 84-7, 84-12, 84-13, 84-16, 81-7, 82-26, 9-19, 9-20, 32-81, 32-82, 48-28, 62-10, 82-81. Methods for preparing libraries were performed as is standard in the art.

The libraries were then be screened for enzyme activity. In a preferred embodiment, the library is screened for esterase activity essentially as described in U.S. Pat. No. 5,969,121. The skilled artisan has available many methods for screening libraries for either activity or antibody binding.

In this manner, unique organisms and proteins, for example, may be isolated.

Example 2 Identification of Novel Esterases

To identify novel esterases in the libraries, a hierarchical screening approach was taken in which a broad screen was first performed, followed by a selective screen, and then a specific screen. For example, a broad screen that serves to identify enzymes having a broadly-defined desired activity or being members of a particular family may be performed by selecting for activity against substrate analogs. Clones expressing enzymes identified as having the particular activity may then be screened using actual substrates (a selective screen). Clones expressing the desired activity following this screen are then screened under actual reaction conditions, providing the highest level of accuracy. These methods are utilized below to isolate novel esterases having selective activity.

Native (non denaturing) 10% polyacrylamide gels were run on crude extracts of E. coli containing either phage- or plasmid-based vectors clones from the library. After electrophoresis, the gels were equilibrated in pH 7.6 Trizma buffer and then stained for activity in either 0.15% X- acetate. The gels are then incubated at 55°C for up to 30 minutes. These gels were then be stained with an esterase activity stain containing 5-bromo-4-chloro-3-indolyl acetate (X-acetate) (5-bromo-4-chloro-3-indolyl butyrate (X-butyrate) or 5- bromo-4-chloro-3 -indolyl caprylate (X-caprylate) will also work), producing indigo precipitates. The clones were then further screened using the pNP priopionate screening method. Esterase activity was measured by monitoring the hydrolysis of p-nitrophenylproprionate (pNP) (MUB may also be used). Each substrate is dissolved in acetonitrile and added to the reaction mixture (100 μM final concentration) which contain 50 mM Tris HC1 pH 8.5 adjusted for temperature dependent pH variation. Reactions are thermally equilibrated at 37°C for 5 minutes prior to initiation of the reaction by addition of 10 μL of enzyme sample, while control reactions substituted equivalent amounts of

BSA. The reaction is monitored spectrophotometrically at 405 run ε=17mM^~ icπr¹ for pNP and 360 n ε=7.9 mM-ϊcn ¹ for MUB. The rates of enzyme catalyzed hydrolysis was corrected for the spontaneous hydrolysis of the substrate. Protein concentrations were determined by either the absorbance at 280 nm or by Lowery assay. Crude activity was determined by a colorimetric assay based on the hydrolysis of 5- bromo-4-chloro-3-indoyl esters suspended in a 0.7% agar matrix on microtiter plates. A 0.1 mg/ml solution of the indolyl derivative was dissolved in a minimal volume of acetonitrile and added to a warm solution of 0.7% agar containing 0.1M phosphate buffer pH 7.5. 10 μL of this solution was distributed to microtiter plates which, when cooled, could be used with as much as 100 μL of enzyme sample and incubated at temperatures from ambient to >65°C.

As described below, twenty-two esterase clones were identified using the X-acetate screening method primarily, followed by secondary screening using pNP propionate using these methods. In addition, the coding sequences of the DNA encoding the esterases within the different clones are unique from one another as determined by DNA restriction map analysis.

Using a pH assay developed by Kazlauskas, et al. (as described in U.S. Ser. No. 09/348,976 and PCT/US99/15400, for example), the activity profile of these esterases was determined by reaction with a library of chiral esters, such that a bioctalytic fingerprint can be generated to rule out duplicate activities. Using these profiles, several novel enzymes having unique activities have been identified and are provided herein.

Figure 1 illustrates the library of substrates employed to obtain the activity profiles. These are 26 pairs of chiral esters bearing the asymmetric center either in the acid or alcohol moiety. In this particular case, substrate 22 was not used for the assay. A diverse library was utilized in order to identify enzymes having duplicate activities using the pH assay.

Figures 2A-B illustrate the kinetic profiles of the novel esterases NE01, NE02, NE03, NE04, NE05, NE06, NE07, NE08, NE09, NE10, NE11, NE12, NE13, NE14, NE15, NE16, NE17, NE18, NE19, NE20, NE21 and NE22 obtained by running the assay in 96-well microplates and reading absorbance decrease at 405nm. lOOμL total volume are split as follows: 95 μL of enzyme solution (equivalent to 1 unit of activity measured as hydrolysis of pNP propionate) in 5mM BES buffer containing 0.45mM of p-nitrophenol and 5μL of a 250mM substrate solution in acetonitrile. For substrates 23-26, the buffer contained 0.5% of triton X-100 to help solubility. If the substrate and enzyme combination react, a negative slope will show, otherwise a flat line is expected (no change in the absorbance). For example, NE20 shows a flat line for IS and a negative sloping line for IR. This indicates that NE20 is selective for IR. As can be derived from Figures 2 A and 2B, several of the enzymes exhibit significant enantioselective activity.

Figures 3A-D provides a summary or reduced version of the data after further analysis of the kinetic plots. As can be derived from this data, 10 of the 22 initially identified esterases show different profiles, while the others can be considered as duplicate activities (at least for this library of substrates).

Figures 4A-B illustrates the PNP propionate profiles for the ten distinct enzymes identified above. As can be seen, each enzyme has a distinct activity profile. Table 2 summarizes the activity results for the 10 preferred enzymes identified in the library. It is shown whether the enzyme was reactive or reactive and enantioselective at the same time. An empty space means that no activity was detected under the conditions mentioned above. Table 3 illustrates the relative molecular weight (MW_r) and N- terminal amino acid sequence for each of the ten enzymes.

Table 3 Additional Biochemical Properties of Esterases

The numbers in parenthesis represent internal designations for these enzymes.

²Numerical values represent kDa.

³A major and a minor band were observed in SDS-PAGE analysis of enzyme 23-31. The relative molecular weight and N-terminal amino acid sequence is reported here for both the major and the minor species of NE04.

As can be seen from this data, these various features may be combined to generate a biochemical fingeφrint with which these enzymes can be distinguished from one another. The enzymes may then be further processed or otherwise utilized.

Example 3 Selection of Esterases Having Enhanced Enantioselectivity

As described above, multiple clones are provided that each contain a recombinant polynucleotide encoding enantioselective esterases. To select for an esterase having enhanced enantioselectivity, a polynucleotide encoding an esterase selected from the group consisting of NE01, NE02, NE03, NE04,

NE05, NE06, NE07, NE08, NE09, NE10, NE11, NE12, NE13, NE14, NE15, NE16, NE17, NE18, NE19, NE20, NE21 and NE22 is isolated from said clone and subjected to mutation or otherwise modifed as indicated herein.

The resultant mutated polynucleotide is then cloned into an expression vector and transformed into E. coli or other suitable organism. The primary X- acetate screening method and the secondary pNP screening method are performed as described above to identify those clones encoding functional esterases. The clones encoding esterases are then selected and the esterases further assayed for enantioselectivity as described above. The activity of the newly isolated esterase encoded by the mutated polynucleotide are then compared to the activity of the parental esterase. Those DNA molecules encoding esterases having increased enantioselectivity over that of the parental esterase are selected for further analysis. The selected DNA molecules may be subjected to further rounds of mutation and selection until an esterase having the desired activity is obtained.

While a preferred form of the invention has been shown in the drawings and described, since variations in the preferred form will be apparent to those skilled in the art, the invention should not be construed as limited to the specific form shown and described, but instead is as set forth in the claims.

Claims

What is claimed is:

I . An esterase having the biochemical fingeφrint of an esterase selected from the group consisting of NE01, NE02, NE03, NE04, NE05, NE06, NE07, NE08, NE09, NE10, NE11, NE12, NE13, NE14, NE15,

NE16, NE17, NE18, NE19, NE20, NE21 and NE22, said biochemical fingeφrint being represented by the reactivity and enantioselectivity profile to substrates 1-26 illustrated in Figure 1, and the PNP priopionate profile.

2. An esterase of claim 1 wherein said esterase is selected from the group consisting of NE01, NE02, NE04A/B, NE06, NE08, and NE22.

3. An esterase of claim 1 wherein said esterase is NE05.

4. An esterase of claim 1 wherein said esterase is NE09.

5. An esterase of claim 1 wherein said esterase is selected from the group consisting of NE11, NE12, NE14 and NE15.

6. An esterase of claim 1 wherein said esterase is NE13.

7. An esterase of claim 1 wherein said esterase is NE16.

8. An esterase of claim 1 wherein said esterase is selected from the group consisting of NE17 and NE20.

9. An esterase of claim 1 wherein said esterase is NE18.

10. An esterase of claim 1 wherein said esterase is NE19.

I I. An esterase having the biochemical fingeφrint of the esterase NE01, said biochemical fingeφrint being represented by the reactivity and enantioselectivity profile to substrates 1-26 illustrated in Figure 1, PNP priopionate profile, relative molecular weight of 48 kDa, and N- terminal amino acid sequence TEXQYIVALD.

12. An esterase having the biochemical fingeφrint of the esterase NE02, said biochemical fingeφrint being represented by the reactivity and enantioselectivity profile to substrates 1-26 illustrated in Figure 1, PNP priopionate profile, and relative molecular weight of 36 kDa.

13. An esterase having the biochemical fingeφrint of the esterase NE03, said biochemical fingeφrint being represented by the reactivity and enantioselectivity profile to substrates 1-26 illustrated in Figure 1, PNP priopionate profile, relative molecular weight of 43 kDa, and N- terminal amino acid sequence XQXPYDMPLE.

14. An esterase having the biochemical fingeφrint of the esterase NE04A, said biochemical fingeφrint being represented by the reactivity and enantioselectivity profile to substrates 1-26 illustrated in Figure 1,

PNP priopionate profile, relative molecular weight of 42 kDa, and N- terminal amino acid sequence RPMGFXGAXX.

15. An esterase having the biochemical fingeφrint of the esterase NE04B, said biochemical fingeφrint being represented by the reactivity and enantioselectivity profile to substrates 1-26 illustrated in Figure 1,

PNP priopionate profile, relative molecular weight of 31 kDa, and N- terminal amino acid sequence XLDPVI(Q/X)QVL.

16. An esterase having the biochemical fingeφrint of the esterase NE05, said biochemical fingeφrint being represented by the reactivity and enantioselectivity profile to substrates 1-26 illustrated in Figure 1,

PNP priopionate profile, relative molecular weight of 46 kDa, and N- terminal amino acid sequence MENFKHLPEP.

17. An esterase having the biochemical fingeφrint of the esterase NE06, said biochemical fingeφrint being represented by the reactivity and enantioselectivity profile to substrates 1-26 illustrated in Figure 1,

PNP priopionate profile, and relative molecular weight of 50 kDa.

18. An esterase having the biochemical fingeφrint of the esterase NE09, said biochemical fingeφrint being represented by the reactivity and enantioselectivity profile to substrates 1-26 illustrated in Figure 1, PNP priopionate profile, and relative molecular weight of 81 kDa.

19. An esterase having the biochemical fingeφrint of the esterase NE10, said biochemical fmgeφrint being represented by the reactivity and enantioselectivity profile to substrates 1-26 illustrated in Figure 1, PNP priopionate profile, relative molecular weight of 48 kDa, and N- terminal amino acid sequence MEVE(K/T)HLPE(P/L).

20. A method for performing selective hydrolysis on a substrate using an esterase having a biochemical fingeφrint substantially similar to that of NE01, NE02, NE03, NE04, NE05, NE06, NE07, NE08, NE09, NE10, NE11, NE12, NE13, NE14, NE15, NE16, NE17, NE18, NE19,

NE20, NE21 and NE22, said biochemical fingeφrint being represented by the reactivity and enantioselectivity profile to substrates 1-26 illustrated in Figure 1 and the PNP priopionate profile.

21. A method of claim 20, wherein said substrate is selected from the group consisting of compounds 1-26, as illustrated in Figure 1.

22. A method of claim 20, wherein said substrate is selected from the group consisting of methyl 2,2-dimethyl-l,3-dioxolane-4-carboxylate, methyl l-methyl-2-oxo-cyclohexane propionate, methyl 2- chloropropionate, methyl lactate, glycidyl butyrate, tryptophan methyl ester, methyl mandelate, methyl 3-hydroxy-2-methylpropionate, methyl 3-hydroxybutyrate, ethyl 4-chloro-3-hydroxybutyrate, oxabicyclo[3.3.0]oct-6-en-3-one, menthyl acetate, neomenthyl acetate, 3-hydroxy-3-methyl-4,4,4-trichlorobutyric-β-lactone, dimethyl malate, dimethyl 2,3-O-isopropylidenetartrate, methyl mandelate acetate, methyl 3-hydroxy-3-phenylpropionate, indanol acetate, phenethyl alcohol acetate, O-acetyl mandelonitrile, acetyl α-hydroxy- γ-butyro-lactone, glycidyl 4-nitrobenzoate, 2-methylglycidyl 4- nitrobenzoate, α-methyl-1-naphthalene-methanol acetate, and α- methyl-2-naphthalene-methanol acetate.