Carboxylesterase Biocatalysts

Abstract

To provide a carboxyesterase polypeptide, and to provide a method for producing amide using the carboxyesterase polypeptide.SOLUTION: Provided are, as one aspect, a carboxyesterase polypeptide comprising an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to a specific amino acid sequence, or a functional fragment thereof, the carboxyesterase polypeptide amino acid sequence comprising the feature that the residue corresponding to the 198th in the specific amino acid sequence is chosen from a non-polar residue, an aromatic residue, and an aliphatic residue, and a method for producing amide using the carboxyesterase polypeptide.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to improved carboxylesterase biocatalysts and methods for producing amides using the biocatalysts.

[0002] Amide bond formation is one of the most common reactions in organic synthesis, and amides are commonly found in active pharmaceutical ingredients, bioactive molecules, synthetic polymers, peptides, and proteins. A study by the Novartis Institute for BioMedical Research found that amide bond formation and related acylation chemistry accounted for 21.3% of all chemical reactions performed in pharmaceutical synthesis over the last 40 years (Schneider, et al., J. Med. Chem. 59, 4385-4402, 2016). Traditional amide synthesis methods use carboxylic acid and amine substrates and require stoichiometric coupling reagents. Because the reaction proceeds through highly reactive activated intermediates, unwanted side reactions can occur, resulting in the formation of undesirable by-products such as ureas. The poor atom economy and the significant amount of (metal-containing and often toxic) waste generated make the amide formation method costly. Other drawbacks of conventional amide synthesis methods include the lack of enantio- and chemoselectivity, the use of volatile or toxic coupling reagents, and the need to protect other functional groups present in the reactants.

[0003] Chemical catalytic approaches have been developed that eliminate the need for stoichiometric coupling reagents, thus improving atom economy and generating less waste (reviewed in Pattabiraman and Bode, Nature, 480, 471-479, 2011 and de Figueiredo, et al., Chemical Reviews, 12029-12122, 2016). Boronic acid catalysis is the oldest approach to chemical amidation, where temporary activation of carboxylic acids with aryl boronic acids allows catalytic amide formation. However, these methods suffer from limited substrate scope and often require high temperatures, as well as very low solvent tolerance, limiting their wider use. More recent work includes the use of metal-catalyzed amidation, where metal salts are used as Lewis acids for temporary activation of carboxylic acids to aid in amidation. To date, these work suffer from many of the same drawbacks as boronic acid catalysis, requiring high temperatures, catalyst addition, and limited solvent scope and substrate tolerance. Redox-based methods have also been explored using either N-heterocyclic carbenes (NHCs) or metal catalysts that allow the oxidative conversion of alcohols, aldehydes, ketones, or nitriles to their corresponding amides. Unfortunately, both metal and NHC catalysts are expensive, are highly toxic themselves, often require harmful co-solvents, and generally suffer from poor functional group tolerance.

[0004] Lipases have been used as biocatalysts to form amide bonds in organic solvents by direct activation of ester starting materials followed by coupling with amines. Advantageously, these enzymes are generally highly enantioselective and thermostable (for a review, see Gotor, Bioorg Med Chem, 7, 2189-2197, 1999). However, most of the lipases currently studied appear to have narrow substrate specificity and must be used in dry organic solvents to prevent unwanted hydrolysis. This specificity problem is particularly pronounced in the synthesis of tertiary amides, where very few enzymes have been shown to be even marginally active (reviewed in van Pelt, Green, Chem. 13, 1791-1798, 2011).

[0005] To overcome such limitations, directed evolution methods are commonly used in which enzyme variants are expressed and studied in a high-throughput manner. However, these enzymes are often derived from Pseudomonas or Bacillus strains and cannot be easily expressed in laboratory strains, such as E. coli or S. cerevisiae, for which robust genetic engineering techniques exist. Furthermore, the need for drying solvents and molecular sieves makes directed evolution methods extremely challenging, both due to the high water content of cell lysates and the technical difficulty of drying hundreds of reactions in parallel. [Brief description of the drawings]

[0006] [Figure 1]FIG. 1 is a schematic representation of a polynucleotide sequence encoding an E. coli codon optimized for the wild-type carboxylesterase enzyme A. acidocaldarius esterase 2 (SEQ ID NO: 1), which is referred to in Table 3 below and is included in the following sequence listing (SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 125, 127, 129, 130, 131, 132, 133, 134, 135, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, and 125), all of which are 95-99% identical to each other. [Diagram 2] FIG. 2 is a sequence diagram of a polypeptide sequence derived from the wild-type carboxylesterase enzyme A. acidocaldarius esterase 2 (SEQ ID NO: 2), which is referred to in Table 3 below and which includes the following sequence listings (SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 15 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, and 126), all of which are 95-99% identical to each other. Summary of the Invention

[0007] In view of the limitations of the prior art, the present inventors have developed a series of mutants from the wild-type carboxylesterase enzyme A. acidocaldarius esterase 2 (SEQ ID NO:2) that are highly thermotolerant, and these mutant enzymes have improved amidation activity that is more than 785,000-fold higher than the wild-type enzyme. Due to this dramatically altered activity, these mutant enzymes have substantial water and alcohol tolerance, enabling the large-scale direct synthesis of amides from simple ester and amine precursors. This direct synthesis strategy shortens the amide synthesis by one or two chemical steps, reduces the use of organic solvents, and eliminates the use of stoichiometric activators, and overall represents a dramatic improvement to the state of the art in chemistry.

[0008] The disclosure provides, inter alia, polypeptides, polynucleotides encoding the polypeptides, and methods of using the polypeptides, for the biocatalytic conversion of ethyl oxazole-5-carboxylate (the "ester substrate") to (4-isopropylpiperazin-1-yl)(oxazol-5-yl)methanone (the "product") in the presence of 1-isopropylpiperazine (the "amine substrate"). The disclosure also provides, inter alia, polypeptides, polynucleotides encoding the polypeptides, and methods of using the polypeptides, for the biocatalytic conversion of ethyl oxazole-5-carboxylate (the "ester substrate") ester substrate to ((2S,6R)-2,6-dimethylmorpholino)(oxazol-5-yl)methanone (the other product) in the presence of the amine substrate cis-2,6-dimethylmorpholine. These products are starting materials for pharmaceuticals of interest in development for the treatment of chronic obstructive pulmonary disease (COPD). In particular, these products can be used to synthesize phosphoinositide 3-kinase delta inhibitors (PI3K delta inhibitors), a class of drugs used to treat inflammation, autoimmune diseases, and cancer. The compositions of the invention can also be used as starting materials for other pharmaceutical classes as well.

[0009] As shown in Table 3, the wild-type polypeptide, carboxylesterase enzyme A. acidocaldarius esterase 2 (SEQ ID NO: 2), acts on ester substrates with very low efficiency (less than 1% of the substrate converted to product), whereas the engineered carboxylesterases (EC 3.1.1) of the present disclosure are capable of readily converting ester substrates to products in the presence of amine substrates. Thus, in one aspect, the present disclosure relates to improved carboxylesterases capable of converting the ester substrate, ethyl oxazole-5-carboxylate, in the presence of 1-isopropylpiperazine "amine substrate," to the product, (4-isopropylpiperazin-1-yl)(oxazol-5-yl)methanone, at levels measurable by analytical techniques such as HPLC-UV absorbance, down to about 0.1% conversion.

[0010] In some embodiments, the improved carboxylesterase polypeptide comprises an amino acid sequence at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical or greater to the amino acid sequence set forth in SEQ ID NO:4, or a functional fragment thereof, and the improved carboxylesterase amino acid sequence includes the characteristic that the residue corresponding to X198 is selected from a non-polar residue, an aromatic residue, and an aliphatic residue. Guidelines for the selection of various amino acid residues that may be present at the designated residue positions are provided in the detailed description below as well as in the claims.

[0011] In some embodiments, the improved carboxylesterase polypeptide comprises an amino acid sequence corresponding to the amino acid sequence set forth in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, or 126.

[0012] In some embodiments, the improved carboxylesterase polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, or 126.

[0013] In some embodiments, the disclosure provides a carboxylesterase polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 122. In another embodiment, the disclosure provides a polynucleotide sequence encoding the carboxylesterase polypeptide sequence set forth in SEQ ID NO: 122. In yet another embodiment, the disclosure provides a polynucleotide encoding a carboxylesterase polypeptide, the polynucleotide comprising the polynucleotide sequence set forth in SEQ ID NO: 121. In yet another embodiment, the polynucleotide encodes a carboxylesterase polypeptide consisting of the polynucleotide sequence set forth in SEQ ID NO: 121.

[0014] In another aspect, the improved carboxylesterase polypeptides can be used in a method for producing an amide, comprising: (a) R1-COOR2, where R1 is a sp 3 and R2 is selected from a methyl group; an ethyl group; and a 1-6 carbon alkyl chain; (b) an amine substrate; (c) an improved carboxylesterase polypeptide comprising an amino acid sequence at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical or greater to the amino acid sequence set forth in SEQ ID NO:4, or a functional fragment thereof, the carboxylesterase polypeptide amino acid sequence including the characteristic that the residue corresponding to X198 in SEQ ID NO:4 is selected from a non-polar residue, an aromatic residue, and an aliphatic residue; and (d) a component comprising a solvent. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0015] The present disclosure provides a highly efficient biocatalyst that can mediate transformations involving the amidation of certain amide group acceptors, e.g., the synthesis of a compound of formula III. The biocatalyst is an engineered amidating polypeptide that can convert a substrate of formula I, ethyl oxazole-5-carboxylate (the "ester substrate"), to a product of formula III, (4-isopropylpiperazin-1-yl)(oxazol-5-yl)methanone (the "product"), in the presence of an amine substrate of formula II, 1-isopropylpiperazine, as follows: [ka]

[0016] In certain embodiments, the engineered carboxylesterase is derived from a native carboxylesterase from A. acidocaldarius esterase 2, a carboxylesterase enzyme that catalyzes the hydrolysis of esters via the formation and decomposition of an acyl-enzyme intermediate amine. The carboxylesterase of SEQ ID NO:4 differs from the native enzyme from wild-type carboxylesterase A. acidocaldarius esterase 2 (SEQ ID NO:2) in that it has a substitution of leucine (L) for glutamate (E) at residue position X198 and has measurable activity towards the ester substrate ethyl oxazole-5-carboxylate (Formula I). ​​The carboxylesterase of SEQ ID NO:4 has been engineered to mediate the efficient conversion of an ester substrate of Formula I to a product of Formula III in the presence of an amine substrate such as 1-isopropylpiperazine (Formula II). This conversion can be carried out under mild conditions (30° C., high conversion %), making the method applicable to the large-scale production of amides of Formula III and Formula V.

[0017] Abbreviations and Definitions In this specification, the abbreviations used for genetically encoded amino acids are conventional and are as shown in Table 1.

[0018] [Table 1]

[0019] When three-letter abbreviations are used, the amino acid may be in either the L- or D-form about the α-carbon (Cα), unless specifically preceded by "L" or "D" or unless it is clear from the context in which the abbreviation is used. For example, "Ala" represents alanine without specifying the configuration about the α-carbon, while "D-Ala" and "L-Ala" represent D-alanine and L-alanine, respectively. When one-letter abbreviations are used, capital letters represent amino acids in the L-form about the α-carbon, and lower case letters represent amino acids in the D-form about the α-carbon. For example, "A" represents L-alanine and "a" represents D-alanine. Peptide sequences are represented as a series of one-letter or three-letter abbreviations (or mixtures thereof), and the sequences are shown in the N→C direction according to convention.

[0020] Technical and scientific terms used herein shall have the meanings commonly understood by one of ordinary skill in the art unless specifically defined otherwise. Accordingly, the following terms are intended to have the following meanings: All U.S. patents and published U.S. patent applications cited herein, including all sequences disclosed within such patents and patent applications, are expressly incorporated herein by reference.

[0021] "Acid by-product" or "hydrolysis by-product" refers to a carboxylic acid resulting from the reaction of an ester substrate with water as a result of a carboxylesterase enzyme. The acid by-product is a molecule of general formula (3) where R3 is -H. R1 is defined above. [ka]

[0022] "Acidic amino acid or residue" refers to a hydrophilic amino acid or residue having a side chain that exhibits a pK value of less than about 6 when the amino acid is included in a peptide or polypeptide. Acidic amino acids generally have a side chain that is negatively charged at physiological pH due to loss of a hydrogen ion. Commonly encoded acidic amino acids include L-Glu (E) and L-Asp (D).

[0023] "Alkyl" is intended to include alkyl groups of the specified length in either a straight or branched configuration. Exemplary alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, isohexyl, and the like. Alkyl groups may be unsubstituted or substituted with halogen, hydroxy, carboxy, aminocarbonyl, amino, C l-4 Alkoxy, and C l-4 Substituted with 1 to 3 groups independently selected from alkylthio.

[0024] "Amide" is intended to mean a functional group containing a carbonyl group linked to a nitrogen atom. Amide also refers to any compound containing an amide functional group. Amides are derived from a carboxylic acid and an amine.

[0025] "Amidate" or "amidation" is intended to mean the formation of an amide functional group from a carbonyl-containing compound, generally resulting from the reaction of a carboxylic acid with an amine functional group, but also formed herein from an ester with an amine functional group.

[0026] "Amine" is intended to mean a functional group that contains an sp3 hybridized nitrogen atom. Amine also means any compound that contains an amine function.

[0027] "Amine substrate" refers to an amino compound that can displace the alcohol side chain of an ester substrate under the action of a carboxylesterase, thereby yielding an amide product. An amine substrate is a molecule of general formula (5), where each of R3 and R4, when taken independently, is an alkyl or aryl group that is unsubstituted or substituted with at least one non-enzymatically inhibiting group. The groups R3 and R4, when taken together, may form a ring that is unsubstituted, substituted, or fused to another ring. Exemplary amine substrates that can be used with the present invention include, but are not limited to, cyclic piperazinyl or morpholino moieties, as well as primary or aromatic amines. For the purposes of this disclosure, amine substrates include, among others, 1-isopropylpiperazine, a compound of formula II, and cis-2,6-dimethylmorpholine, a compound of formula IV. [ka]

[0028] "Amino acid" or "residue," when used in reference to the polypeptides disclosed herein, refers to a particular monomer at a certain sequence position (e.g., P5 indicates that the "amino acid" or "residue" at position 5 of SEQ ID NO:2 is proline).

[0029] An "amino acid difference" or "residue difference" refers to a change in a residue at a specified position in a polypeptide sequence as compared to a reference sequence. The position in a polypeptide sequence at which a particular amino acid or amino acid change (a "residue difference") occurs may be referred to herein as "Xn" or "position n," where n refers to the residue position relative to the reference sequence.

[0030] For example, a residue difference at position X8, where the reference sequence has a serine, refers to a change of the residue at position X8 to any residue other than serine. As disclosed herein, an enzyme can contain one or more residue differences relative to a reference sequence, where multiple residue differences are generally indicated by a list of designated positions at which there is a change relative to the reference sequence (e.g., "one or more residue differences at the following residue positions: X27, X30, X35, X37, X57, X75, X103, X185, X207, X208, X271, X286, or X296, as compared to SEQ ID NO:4").

[0031] Specific substitution mutations, which are replacements of a particular residue in a reference sequence with a specified different residue, can be represented by the conventional notation "X(number)Y," where X is the single-letter identifier of the residue in the reference sequence, "number" is the residue position in the reference sequence, and Y is the single-letter identifier of the residue substitution in the engineered sequence.

[0032] "Aliphatic amino acid or residue" refers to a hydrophobic amino acid or residue having an aliphatic hydrocarbon side chain. Genetically encoded aliphatic amino acids include L-Ala (A), L-Val (V), L-Leu (L), and L-Ile (I).

[0033] "Aromatic amino acid or residue" refers to a hydrophilic or hydrophobic amino acid or residue having a side chain containing at least one aromatic or heteroaromatic ring. Genetically encoded aromatic amino acids include L-Phe (F), L-Tyr (Y) and L-Trp (W). Histidine is classified herein as a hydrophilic residue or as a "constrained residue" (see below), although L-His (H) may be classified as a basic residue because of the pKa of its heteroaromatic nitrogen atom, or as an aromatic residue because its side chain contains a heteroaromatic ring.

[0034] "Aryl" is intended to mean an aromatic group, including phenyl and naphthyl. "Aryl" is unsubstituted or substituted with fluoro, hydroxy, trifluoromethyl, amino, Cl-4 Alkyl, and C l-4 Substituted with 1 to 5 substituents independently selected from alkoxy.

[0035] "Basic amino acid or residue" refers to a hydrophilic amino acid or residue having a side chain that exhibits a pKa value greater than about 6 when the amino acid is included in a peptide or polypeptide. Basic amino acids generally have a side chain that is positively charged at physiological pH due to association with a hydronium ion. Genetically encoded basic amino acids include L-Arg (R) and L-Lys (K).

[0036] The term "carboxylesterase" is used to refer to a polypeptide having the enzymatic activity of interconverting the side chain of an ester substrate (1) with the side chain of a donor compound (2), converting the ester substrate (1) to its corresponding ester product (3) and the free alcohol form of the ester side chain (4). [ka]

[0037] "Codon optimized" refers to changing the codons of a polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is efficiently expressed in that organism. Although the genetic code is degenerate in that most amino acids are represented by a few codons, called "synonyms" or "synonymous" codons, it is well known that the frequency of codon usage by a particular organism is not random, but is biased toward certain codon triplets. This bias in codon usage appears to be higher for a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and aggregated protein-coding regions of an organism's genome. In some embodiments, a polynucleotide encoding a carboxylesterase enzyme can be codon optimized for optimal production from the host organism selected for expression.

[0038] A "comparison window" refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acid residues, where a sequence can be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids, and the sequence positions within the comparison window may contain no more than 20 percent additions or deletions (i.e., gaps) compared to the reference sequence (no additions or deletions) for optimal alignment of the two sequences. The comparison window may be longer than 20 contiguous residues, and may optionally include a window of 30, 40, 50, 100, or more.

[0039] "Conservative" amino acid substitution or mutation refers to the interchangeability of residues with similar side chains, and therefore generally includes the substitution of the amino acid of the polypeptide with the same or similar amino acid within a defined class of amino acids.However, as used herein, in some embodiments, conservative mutation may be the substitution of aliphatic residue to aliphatic residue, non-polar residue to non-polar residue, polar residue to polar residue, acidic residue to acidic residue, basic residue to basic residue, aromatic residue to aromatic residue, or constrained residue to constrained residue, but does not include the substitution of otherwise hydrophilic residue to hydrophilic residue, hydrophobic residue to hydrophobic residue, hydroxyl-containing residue to hydroxyl-containing residue, or small residue to small residue.Furthermore, as used herein, A, V, L, or I can be conservatively mutated to another aliphatic residue or another non-polar residue.Table 2 below shows examples of conservative substitution.

[0040] [Table 2]

[0041] "Constrained amino acid or residue" refers to an amino acid or residue that has a constrained geometry. As used herein, constrained residues include L-Pro (P) and L-His (H). Histidine has a constrained geometry because it has a relatively small imidazole ring. Proline also has a constrained geometry because it has a five-membered ring.

[0042] As used herein, "control sequences" are defined to include all components necessary or advantageous for the expression of a polynucleotide and / or polypeptide of the present disclosure. Each control sequence may be native or foreign to the subject polynucleotide. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator.

[0043] "Conversion" refers to the enzymatic conversion of a substrate to the corresponding product.

[0044] "Corresponding to," "referring to," or "relative to," when used in reference to a number of a given amino acid sequence or polynucleotide sequence, refers to the residue number of the stated reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is given with respect to the reference sequence, not according to the actual number position of the residue in the given amino acid or polynucleotide sequence that is not the reference sequence. For example, a given amino acid sequence, such as the amino acid sequence of an engineered carboxylesterase, can be aligned to a reference sequence by introducing gaps to optimize residue matching between the two sequences. In these cases, although gaps are present, the residues in the given amino acid sequence or polynucleotide sequence are numbered with respect to the reference sequence to which it is aligned.

[0045] "Cysteine" or L-Cys(C) is unique in that it can form disulfide bridges with other L-Cys(C) amino acids or other sulfanyl- or sulfhydryl-containing amino acids. "Cysteine-like residues" include cysteine ​​and other amino acids that contain sulfhydryl moieties available for disulfide bridge formation. The ability of L-Cys(C) (and other amino acids with -SH-containing side chains) to exist within a peptide in either the reduced free -SH or oxidized disulfide bridged form affects whether L-Cys(C) imparts net hydrophobicity or hydrophilicity to the peptide. L-Cys(C) exhibits a hydrophobicity of 0.29 according to Eisenberg's normalized consensus scale (Eisenberg et al., 1984, supra), but for purposes of this disclosure, L-Cys(C) should be understood to be classified in its own group.

[0046] "Deletion" refers to the modification of a polypeptide by removing one or more amino acids from a reference polypeptide. Deletions can comprise the removal of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, up to 10% of the total number of amino acids, up to 20% of the total number of amino acids, or up to 30% of the total number of amino acids that make up the polypeptide, while retaining enzymatic activity and / or retaining the improved properties of the engineered carboxylesterase enzyme. Deletions can be to internal and / or terminal portions of the polypeptide. In various embodiments, deletions can comprise a contiguous segment or can be discontinuous.

[0047] "Derived from," as used herein with respect to an engineered enzyme, identifies the source enzyme and / or the gene encoding the enzyme on which the engineering was based. For example, the engineered carboxylesterase enzyme of SEQ ID NO:4 was obtained by mutagenesis of the carboxylesterase of SEQ ID NO:2. Thus, the engineered carboxylesterase enzyme of SEQ ID NO:4 is "derived from" the polypeptide of SEQ ID NO:2.

[0048] "Engineered carboxylesterase," as used herein, refers to a carboxylesterase-type protein that has been systematically modified through the insertion of new amino acids into its reference sequence, the deletion of amino acids present in its reference sequence, or the mutation of amino acids in its reference sequence to other amino acids, either by random mutagenesis followed by selection of mutants with particular properties, or by the deliberate introduction of particular amino acids into the protein sequence.

[0049] "Ester" is intended to mean a functional group containing a carbonyl group linked to an oxygen atom which is then linked to a carbon atom. Ester also refers to any compound containing an ester functional group. Esters are derived from carboxylic acids and alcohols.

[0050] "Ester substrate" refers specifically to a compound of formula (1) that contains an ester that reacts with an engineered carboxylesterase. For the purposes of this disclosure, ester substrates include, among others, the compound of formula I, ethyl oxazole-5-carboxylate. [ka]

[0051] "Fragment," as used herein, refers to a polypeptide having an amino- and / or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions of the sequence. Fragments can be at least 14 amino acids long, at least 20 amino acids long, at least 50 or more amino acids long, and up to 70%, 80%, 90%, 95%, 98%, and 99% or more of a full-length carboxylesterase polypeptide, e.g., the polypeptide of SEQ ID NO:4.

[0052] A "functional fragment" or "biologically active fragment," as used interchangeably herein, refers to a polypeptide that has an amino-terminal and / or a carboxy-terminal and / or an internal deletion, but in which the remaining amino acid sequence is identical to the corresponding positions in a sequence to which it is compared (e.g., a full-length engineered T. fusca enzyme of the invention) and which retains substantially all of the activity of the full-length polypeptide.

[0053] "Halogen" is intended to include the halogen atoms fluorine, chlorine, bromine, and iodine.

[0054] A "heterologous" polynucleotide refers to any polynucleotide that is introduced into a host cell by laboratory techniques, and includes polynucleotides that have been removed from a host cell, subjected to laboratory manipulation, and then reintroduced into the host cell.

[0055] "Hybridization stringency" refers to hybridization conditions, such as washing conditions, in the hybridization of nucleic acids. Generally, hybridization reactions are performed under conditions of lower stringency, followed by washing of various, but higher stringency. "Moderate stringency hybridization" refers to conditions under which target DNA can bind to complementary nucleic acids that have about 60% identity with the target DNA, preferably about 75% identity, about 85% identity, or more than about 90% identity with the target polynucleotide. Exemplary moderate stringency conditions are those that correspond to hybridization in 50% formamide, 5x Denhardt's solution, 5x saline-sodium phosphate-EDTA (SSPE), 0.2% sodium dodecyl sulfate (SDS) at 42°C, followed by washing in 0.2x SSPE, 0.2% SDS at 42°C. "High stringency hybridization" generally refers to hybridization that is performed using a method that achieves a thermal melting point (T) of a given polynucleotide sequence under solution conditions. mHigh stringency conditions refer to conditions that are approximately 10°C lower than 0.018M NaCl. In some embodiments, high stringency conditions refer to conditions that allow hybridization only for nucleic acid sequences that form stable hybrids at 65°C in 0.018M NaCl (i.e., if a hybrid is not stable at 65°C in 0.018M NaCl, it is not stable under high stringency conditions as contemplated herein). High stringency conditions can be provided, for example, by hybridization at 42°C in conditions equivalent to 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS, followed by washing at 65°C in 0.1x SSPE and 0.1% SDS. Another high stringency condition is hybridization at 65°C in 5x SSC containing 0.1% (w:v) SDS, and washing at 65°C in 0.1x SSC containing 0.1% SDS. Other highly stringent hybridization conditions, as well as moderately stringent conditions, are described in the references cited above.

[0056] "Hydrophilic amino acid or residue" refers to an amino acid or residue having a side chain that exhibits a hydrophobicity of less than 0 according to the normalized consensus hydrophobicity scale of Eisenberg et al., 1984, J. Mol. Biol. 179:125-142. Genetically encoded hydrophilic amino acids include L-Thr (T), L-Ser (S), L-His (H), L-Glu (E), L-Asn (N), L-Gln (Q), L-Asp (D), L-Lys (K) and L-Arg (R). "Hydrophobic amino acid or residue" refers to an amino acid or residue having a side chain that exhibits a hydrophobicity of greater than 0 according to the normalized consensus hydrophobicity scale of Eisenberg et al., 1984, J. Mol. Biol. 179:125-142. Genetically encoded hydrophobic amino acids include L-Pro (P), L-Ile (I), L-Phe (F), L-Val (V), L-Leu (L), L-Trp (W), L-Met (M), L-Ala (A) and L-Tyr (Y).

[0057] "Hydroxyl-containing amino acid or residue" refers to an amino acid that contains a hydroxyl (-OH) moiety. Genetically encoded hydroxyl-containing amino acids include L-Ser (S), L-Thr (T), and L-Tyr (Y).

[0058] "Improved enzyme properties" refers to any enzyme property that is better or more desirable for a particular purpose as compared to the properties found in a reference enzyme. For the engineered carboxylesterase polypeptides described herein, a comparison is generally made to a wild-type carboxylesterase enzyme, although in some embodiments, the reference carboxylesterase can be another improved, engineered carboxylesterase. Enzyme properties for which improvements can be made include, but are not limited to, enzyme activity (which can be expressed as % conversion of substrate over a period of time), thermostability, solvent stability, pH activity profile, coenzyme requirement, resistance to inhibitors (e.g., product inhibition), stereospecificity, and suppression of acid by-product production.

[0059] "Increased enzyme activity" or "increased activity" refers to an improved property of an engineered enzyme that can be expressed by an increase in specific activity (e.g., product produced / time / weight protein) or an increase in the % conversion of substrate to product (e.g., % conversion of starting amount of substrate to product in a specified time period using a specified amount of carboxylesterase) when compared to a reference enzyme. Exemplary methods for measuring enzyme activity are shown in the Examples. K that can lead to increased enzyme activity m , V max or k catThe improved enzyme activity can affect any property related to enzyme activity, including classical enzyme properties of the corresponding wild-type or engineered enzyme. The improved enzyme activity can be from about 1.5-fold that of the corresponding wild-type or engineered enzyme to as much as 2-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, 1000-fold, 10,000-fold, 100,000-fold, 500,000-fold, 785,000-fold or more than the enzyme activity of the native enzyme (e.g., carboxylesterase) or another engineered enzyme from which the enzyme exhibiting the increased activity is derived. In certain embodiments, the engineered carboxylesterase enzymes of the disclosure exhibit improved enzyme activity in the range of 1.5-50-fold, 1.5-100-fold or more than the enzyme activity of the parent carboxylesterase enzyme (i.e., the wild-type or engineered carboxylesterase from which they are derived). One of skill in the art will appreciate that the activity of any enzyme is diffusion limited such that it cannot exceed the diffusion rate of the substrate, including any coenzyme for which a catalytic turnover rate is required. The theoretical maximum value of the diffusion limit is generally about 10 8 ~10 9 (M -1 s -1 ). Thus, any improvement in the enzyme activity of a carboxylesterase has an upper limit with respect to the diffusion rate of the substrate on which the carboxylesterase enzyme acts. Carboxylesterase activity can be measured by any of the standard assays used to measure carboxylesterases, such as changing the substrate or product concentration, or changing the amine substrate concentration. Comparisons of enzyme activity are made using a defined enzyme preparation, a defined assay under a set of conditions, and one or more defined substrates, as described in more detail herein. In general, when enzymes in cell lysates are compared, the same expression system and the same host cells are used, and the number of cells and the amount of protein assayed are determined to minimize variation in the amount of enzyme produced by the host cells and present in the lysate.

[0060] "Insertion" refers to the modification of a polypeptide by the addition of one or more amino acids to a reference polypeptide. In some embodiments, improved, engineered carboxylesterase enzymes comprise an insertion of one or more amino acids into a native carboxylesterase polypeptide as well as an insertion of one or more amino acids into other improved carboxylesterase polypeptides. The insertion may be in an internal portion of the polypeptide or to the carboxy or amino terminus. Insertions, as used herein, include fusion proteins as known in the art. The insertion may be a contiguous amino acid segment or may be separated by one or more of the amino acids of the native polypeptide.

[0061] An "isolated polypeptide" refers to a polypeptide that is substantially separated from other contaminants that naturally accompany it, such as proteins, lipids, and polynucleotides. The term encompasses polypeptides that have been removed or purified from their natural environment or expression system (e.g., a host cell or in vitro synthesis). The improved carboxylesterase enzymes may be present intracellularly, in cell media, or prepared in a variety of forms, such as a lysate or isolated preparation. Thus, in some embodiments, the improved carboxylesterase enzyme may be an isolated polypeptide.

[0062] "Non-conservative substitution" refers to the substitution or mutation of an amino acid in a polypeptide with an amino acid that has significantly different side chain properties. Non-conservative substitutions may use amino acids between the groups listed above, rather than within the defined groups. In one embodiment, non-conservative mutations affect (a) the replacement area of ​​the peptide backbone structure (e.g., proline instead of glycine); (b) charge or hydrophobicity; or (c) the bulk of the side chain.

[0063] "Nonpolar amino acid" or "nonpolar residue" refers to a hydrophobic amino acid or residue having a side chain with a bond that is uncharged at physiological pH and in which the electron pair shared by the two atoms is generally held equally by each of the two atoms (i.e., the side chain is nonpolar). Genetically encoded nonpolar amino acids include L-Gly (G), L-Leu (L), L-Val (V), L-Ile (I), L-Met (M), and L-Ala (A).

[0064] "Percent conversion" refers to the percent of a substrate that is converted to a product within a period of time under specified conditions. Thus, for example, the "enzyme activity" or "activity" of a carboxylesterase polypeptide can be expressed as the "percent conversion" of substrate to product.

[0065] "Percentage of sequence identity," "percent identity," and "percent identical" are used herein to refer to a comparison between polynucleotide or polypeptide sequences and are determined by comparing two optimally aligned sequences in a comparison window, where the portion of the polynucleotide or polypeptide sequence within the comparison window may contain additions or deletions (i.e., gaps) when compared to a reference sequence for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where identical nucleobases or amino acid residues are found in both sequences, or the nucleobases or amino acid residues are aligned with gaps to determine the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying by 100 to obtain the percentage of sequence identity. Optimal alignment and determination of percent sequence identity are performed using the BLAST and BLAST 2.0 algorithms (see, e.g., Altschul, et al., 1990, J. Mol. Biol. 215: 403-410 and Altschul, et al., 1977, Nucleic Acids Res. 3389-3402). Software for performing BLAST analyses is publicly available at the website of the National Center for Biotechnology Information.

[0066] Briefly, BLAST analysis involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that, when aligned with words of the same length in the database sequences, either match or meet some positive threshold score T. T is referred to as the neighborhood word score threshold (Altschul, et al, supra). These initial neighborhood word hits act as seeds to initiate searches to find longer HSPs containing them. Each word hit is extended in both directions along the sequence for as long as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is terminated when the cumulative alignment score falls by an amount X from its maximum achieved value; when the accumulation of one or more negative scoring residue alignments causes the cumulative score to fall below zero; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, and an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).

[0067] Many other algorithms are available which function similarly to BLAST in obtaining the percent identity of two sequences. Optimal alignment of sequences for comparison can be determined, for example, by the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, by the similarity search method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin software package), or by visual inspection (generally as described in Current Protocols in Molecular Biology, FM Ausubel, et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (See, Ausubel). Additionally, for sequence alignment and determination of percent sequence identity, the BESTFIT or GAP programs of the GCG Wisconsin software package (Accelrys, Madison WI) can be used with the default parameters provided.

[0068] "pH stable" refers to a polypeptide that maintains equivalent activity (e.g., greater than 60%-80%) compared to the intact enzyme after exposure to low or high pH (e.g., 4.5-6 or 8-12) for a period of time (e.g., 0.5-24 hours).

[0069] "Polar amino acid or residue" refers to a hydrophilic amino acid or residue having a side chain that is uncharged at physiological pH but has at least one bond in which the electron pair shared by two atoms is held more closely by one of the atoms. Genetically encoded polar amino acids include L-Asn (N), L-Gln (Q), L-Ser (S) and L-Thr (T).

[0070] "Preferred, optimal, high, codon usage bias codons" refer interchangeably to codons that are used more frequently in protein coding regions than codons that code for the same amino acid. Preferred codons can be determined with respect to codon usage in a single gene, codon usage in a set of genes of common function or origin, codon usage in highly expressed genes, codon frequency in aggregated protein coding regions across organisms, codon frequency in aggregated protein coding regions of related organisms, or combinations thereof. Codons whose frequency increases with the level of gene expression are generally optimal codons for expression. A variety of methods are known for determining codon frequency (e.g., codon usage, relative synonym codon usage) and codon preference in a particular organism, including, for example, cluster or correspondence analysis, and multivariate analyses using the effective number of codons used in a gene (see, e.g., GCG CodonPreference, Genetics Computer Group Wisconsin Package; CodonW, John Peden, University of Nottingham; McInerney, J. O, 1998, Bioinformatics 14:372-73; Stenico, et al., 1994, Nucleic Acids Res. 222437-46; Wright, F., 1990, Gene 87:23-29). Codon usage tables are becoming available for an increasing number of organisms (see, e.g., Wada, et al., 1992, Nucleic Acids Res. 20:2111-2118; Nakamura, et al., 2000, Nucl. Acids Res. 28:292; Duret, et al., supra; Henaut and Danchin, "Escherichia coli and Salmonella," 1996, Neidhardt, et al., eds., ASM Press, Washington DC, pp. 2047-2066).The data source for obtaining codon usage can rely on any available nucleotide sequence that can code for a protein. These data sets include nucleic acid sequences that are actually known to code for expressed proteins (e.g., complete protein coding sequences-CDS), expressed sequence tags (ESTs), or putative coding regions of genomic sequences (see, e.g., Mount, D., Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001; Uberbacher, EC, 1996, Methods Enzymol. 266:259-281; ​​Tiwari, et al., 1997, Comput. Appl. Biosci. 13:263-270).

[0071] "Protein," "polypeptide," and "peptide" are used interchangeably herein to refer to polymers of at least two amino acids covalently joined by amide bonds, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristoylation, ubiquitination, etc.). Included within this definition are D- and L-amino acids, and mixtures of D- and L-amino acids.

[0072] "Reference sequence" refers to a defined sequence to which another (e.g., altered) sequence is compared. A reference sequence may be a subset of a larger sequence, such as a segment of a full-length gene sequence or a full-length polypeptide sequence. Generally, a reference sequence is at least 20 nucleotides or amino acid residues long, at least 25 residues long, at least 50 residues long, or the entire length of a nucleic acid or polypeptide. Since two polynucleotides or polypeptides each comprise a similar sequence between the two sequences (i.e., a portion of the complete sequence), and (2) may further comprise a sequence that differs between the two sequences, sequence comparison between two (or more) polynucleotides or polypeptides is generally performed by comparing the sequences of the two polynucleotides in a comparison window to identify and compare local regions of sequence similarity.

[0073] The term "reference sequence" is not intended to be limited to wild-type sequences, and may include sequences that have been manipulated or modified. For example, in some embodiments, a "reference sequence" may be an amino acid sequence that has been previously manipulated or modified. For example, a "reference sequence based on SEQ ID NO:2 with a glycine residue at position X12" refers to a reference sequence that corresponds to SEQ ID NO:2 with a glycine residue at X12 (the unmodified version of SEQ ID NO:2 has an aspartate at X12).

[0074] "Small amino acid" or "small residue" refers to an amino acid or residue having a side chain consisting of a total of three or fewer carbon and / or heteroatoms (excluding the α-carbon and hydrogen). Small amino acids or residues may be further classified as aliphatic, non-polar, polar or acidic small amino acids or residues according to the above definitions. Genetically encoded small amino acids include L-Ala (A), L-Val (V), L-Cys (C), L-Asn (N), L-Ser (S), L-Thr (T) and L-Asp (D).

[0075] "Solvent stable" or "solvent stable" refers to a polypeptide that maintains equivalent (e.g., greater than 60%-80%) activity after exposure to various concentrations (e.g., 5-99%) of solvents (e.g., isopropyl alcohol, dimethyl sulfoxide, tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyl tert-butyl ether, acetonitrile, etc.) for a period of time (e.g., 0.5-24 hours) compared to the intact enzyme.

[0076] "Substantially identical" refers to a polynucleotide or polypeptide sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 91, 93, 94, 95, 96, 97, 98, 99 or more percent sequence identity when compared to a reference sequence in a comparison window of at least 20 residue positions, and often in a window of at least 30-50 residues. The percentage of sequence identity is calculated by comparing the reference sequence to a sequence that contains a total of 20 percent or less deletions or additions of the reference sequence in the comparison window. In certain embodiments applied to polypeptides, the term "substantially identical" means that two polypeptide sequences, when optimally aligned, have at least 80 percent sequence identity, preferably at least 89 percent sequence identity, at least 95 percent sequence identity or more (e.g., 99 percent sequence identity), such as by the GAP or BESTFIT programs using default gap weights. Preferably, residue positions that are not identical differ by conservative amino acid substitutions.

[0077] A "substantially pure polypeptide" refers to a composition in which the polypeptide species is the predominant species present (i.e., it is more abundant than any other individual macromolecular species in the composition, on a molar or weight basis), and is generally a substantially purified composition when the species of interest constitutes at least about 50 percent of the macromolecular species present, on a molar or weight percent basis. Generally, a substantially pure carboxylesterase composition constitutes about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more, on a molar or weight percent basis, of all macromolecular species present in the composition. In some embodiments, the species of interest is purified to essential homogeneity (i.e., contaminants cannot be detected in the composition by conventional detection methods), and the composition consists essentially of a single macromolecular species. Solvent species and elemental ion species are not considered to be macromolecular species. In some embodiments, the isolated improved carboxylesterase polypeptide is a substantially pure polypeptide composition.

[0078] "Substrate," as used herein, refers to a carboxylesterase-reactive compound consisting of a compound containing an ester (1), an amine (5), or an alcohol (2). In the context of this disclosure, substrates for carboxylesterases include, inter alia, compounds of formula I and compounds of formula II, as further described herein.

[0079] "Thermostable" or "thermostable" are used interchangeably to refer to a polypeptide that is resistant to inactivation when exposed to a set of temperature conditions (e.g., 40-80°C) for a period of time (e.g., 0.5-24 hours) compared to the intact enzyme, and thus retains a level of residual activity (e.g., greater than 60%-80%) after exposure to elevated temperatures.

[0080] As used herein, "solvent stable" refers to the ability of a polypeptide to maintain equivalent activity (e.g., greater than 60%-80%) after exposure to various concentrations (e.g., 5-99%) of solvents (e.g., isopropyl alcohol, tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.) for a period of time (e.g., 0.5-24 hours) compared to the intact enzyme.

[0081] DETAILED DESCRIPTION OF EMBODIMENTS OF THEINVENTION In embodiments herein, the engineered carboxylesterases have improved ability to convert the ester substrate ethyl oxazole-5-carboxylate to the product (4-isopropylpiperazin-1-yl)(oxazol-5-yl)methanone in the presence of the amine substrate 1-isopropylpiperazine, as compared to the wild-type carboxylesterase enzyme A. acidocaldarius esterase 2 (SEQ ID NO: 2). Carboxylesterases, including those described herein, are free-standing enzymes that lack chemical cofactors and are water-soluble enzymes that may be formulated as soluble enzymes, as enzymes immobilized on a resin, or as lyophilized powders in the presence of one or more salts.

[0082] In some embodiments, the improved enzyme activity is with respect to another engineered carboxylesterase, such as the polypeptide of SEQ ID NO: 4. The improved activity on an ester substrate can be represented by an increase in the amount of substrate converted to product by the engineered enzyme (e.g., % conversion) compared to a reference enzyme (e.g., wild type, SEQ ID NO: 2) under defined conditions. The improved activity can include an increase in the rate of product formation resulting in an increase in the conversion of the ester substrate to product under defined conditions and in the presence of an amine substrate for a defined period of time. The increased activity (e.g., % conversion and / or increased conversion rate) can also be characterized by the conversion of the substrate to the same amount of product with a smaller amount of enzyme. The amount of product can be assessed by various techniques, such as separation of the reaction mixture (e.g., by chromatography) and detection of the separated products by UV absorbance or tandem mass spectrometry (MS / MS) (see, e.g., Example 4). Examples of prescribed reaction conditions for comparison with the activity of SEQ ID NO:2 include approximately 40 g / L of ethyl oxazole-5-carboxylate (ester substrate), 44 g / L of 1-isopropylpiperazine (amine substrate), and 20 g / L of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 138, 140, 141, 142, 143, 144, 145, 146, 6, 88, 92, or 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, or 126, which enzyme is produced in the presence of sodium sulfate and operates in the presence of about 10 g / L to about 20 g / L water in methyl isobutyl ketone (MIBK) as described below in the description of reaction conditions for the carboxylesterases listed in Table 3. Defined reaction conditions for comparison with specific engineered carboxylesterases are also provided in the description of the carboxylesterases listed in Table 3 and in the corresponding description in Example 7.In some embodiments, the engineered carboxylesterase has at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500, 1000, 1,500, 2,000, 10,000, 100,000, 500,000, 785,000 or more times the activity of the polypeptide of SEQ ID NO:2 under defined reaction conditions.

[0083] In some embodiments, the improved enzyme activity is also associated with other improvements in enzyme properties, hi some embodiments, the improved enzyme properties relate to thermostability, such as above 60°C.

[0084] In some embodiments, improved enzyme activity is associated with improved solvent stability, such as when performed at 98% (vol / vol) in methyl isobutyl ketone (MIBK) or tert-butyl methyl ether (TBME).

[0085] In some embodiments, an engineered carboxylesterase polypeptide of the disclosure is capable of converting an ester substrate, ethyl oxazole-5-carboxylate, to the product, (4-isopropylpiperazin-1-yl)(oxazol-5-yl)methanone, in the presence of an amine substrate, e.g., 1-isopropylpiperazine, with an activity greater than the activity of the polypeptide of SEQ ID NO:2, and comprises an amino acid sequence at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical or greater to the reference sequence of SEQ ID NO:2, or a functional fragment thereof.

[0086] In some embodiments, the engineered carboxylesterase polypeptides of the disclosure can convert the ester substrate ethyl oxazole-5-carboxylate to the product (4-isopropylpiperazin-1-yl)(oxazol-5-yl)methanone with greater activity in the presence of an amine substrate, such as 1-isopropylpiperazine, than the polypeptide of SEQ ID NO:2, and can be synthesized using the reference sequences listed in Table 3, e.g., SEQ ID NOs:4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 11 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, or 126, or a functional fragment thereof.

[0087] In some embodiments, an engineered carboxylesterase polypeptide comprises an amino acid sequence having one or more residue differences when compared to a carboxylesterase reference sequence. The residue differences can be non-conservative substitutions, conservative substitutions, or a combination of non-conservative and conservative substitutions. With respect to the description of residue differences and residue positions, the carboxylesterases provided herein can be described with reference to the amino acid sequence of a native carboxylesterase of A. acidocaldarius esterase 2 (SEQ ID NO:2), the carboxylesterase of SEQ ID NO:2, or an engineered carboxylesterase, such as the polypeptide of SEQ ID NO:4. With respect to the description herein, the positions of amino acid residues within the reference sequence are determined starting from the initial methionine (M) residue in the carboxylesterase (i.e., M indicates residue position 1), however, one of skill in the art will understand that this initial methionine residue can be removed by biological mechanisms, such as a host cell or an in vitro translation system, to generate a mature protein lacking the initial methionine residue.

[0088] In some embodiments, the residue differences are at the following residue positions: X2, X7, X9, X10, X19, X20, X22, X27, X28, X29, X30, X33, X34, X35, X36, X37, X38, X46, X48, X54, X57, X66, X75, X85, X86, X87, X96, X103, X139 , X160, X176, X181, X183, X185, X188, X190, X197, X198, X205, X207, X208, X212, X216, X248, X249, X255, X263, X266, X270, X271, X278, X280, X286, X290 and X296. In some embodiments, the residue differences or combinations thereof are associated with improved enzyme properties. In some embodiments, the carboxylesterase polypeptide can further have 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 1-18, 1-19, 1-20, 1-22, 1-24, 1-26, 1-28, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, 1-60, or 1-62 residue differences at residue positions other than the specific positions designated "Xn" above. In some embodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 55, 60, or 62 residue differences at other amino acid residue positions. In some embodiments, the residue differences at other residue positions comprise substitutions with conservative amino acid residues.

[0089] In an embodiment herein, the residue differences at residue positions that affect substrate binding to carboxylesterases compared to SEQ ID NO: 2 allow for the accommodation of ester substrates of structural formula (I), in particular the ester substrate ethyl oxazole-5-carboxylate, as further described below. Without being bound by theory, at least two regions, namely the first substrate binding region and the second substrate binding region, interact with different structural elements of the ester substrate. The first binding region comprises residues X85, X185, X214, X215 and X254, and the second binding region comprises residue positions X30, X33, X34, X37, X82, X205, X210, X283, X286 and X287, while the positions of X83, X84, X155, X156, X206, X214 and X282 overlap at these two sites. These residues were determined by X-ray crystallography. Thus, the carboxylesterase polypeptides herein have one or more residue differences at residue positions including X30, X33, X34, X37, X85, X185, X205, and X286. In some embodiments, the carboxylesterase polypeptides herein have at least two or more, or three or more residue differences at the designated residue positions involved in substrate binding.

[0090] In other embodiments, the improved carboxylesterase polypeptide comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO:4, wherein the improved carboxylesterase amino acid sequence has the characteristic that the residue corresponding to X198 is selected from a non-polar residue, an aromatic residue, and an aliphatic residue. In yet other embodiments, the improved carboxylesterase polypeptide comprises the characteristic that X198 is selected from F, L, I, Y, and M. In some embodiments, the improved carboxylesterase polypeptide may comprise an amino acid sequence comprising one or more residue differences at residue positions corresponding to X27, X30, X35, X37, X57, X66, X75, X103, X207, X208, X271 X286, and X296 when compared to the sequence of SEQ ID NO:4. Guidance regarding the selection of various amino acid residues that may be present at the specified residue positions is provided in the detailed description below.

[0091] In other embodiments, the improved carboxylesterase polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO:4, wherein the amino acid sequence is such that the residue corresponding to X27 is a constrained residue; the residue corresponding to X30 is an aliphatic residue; the residue corresponding to X35 is selected from basic and polar residues; the residue corresponding to X37 is selected from aliphatic and polar residues; the residue corresponding to X57 is a non-polar residue; the residue corresponding to X75 is selected from basic and polar residues; and the residue corresponding to X103 is a non-polar residue. the residue corresponding to X185 is selected from an aliphatic, non-polar, and aromatic residue; the residue corresponding to X207 is selected from an acidic and polar residue; the residue corresponding to X208 is selected from an aliphatic, basic, and polar residue; the residue corresponding to X271 is selected from an acidic and polar residue; the residue corresponding to X286 is selected from an aliphatic, polar and small residue; and the residue corresponding to X296 is selected from an aliphatic and basic residue.

[0092] In still other embodiments, the improved carboxylesterase polypeptides comprise an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO:4, which comprises at least one feature selected from: X27 is P; X30 is selected from I, L, and V; X35 is H; X37 is selected from I, L, T, and V; X57 is M; X75 is R; X103 is selected from F, M, and W; X185 is selected from F, I, and M; X207 is E; X208 is selected from R, L, and H; X271 is D; X286 is selected from M, V, and G; and X296 is selected from V, L, and R. In some embodiments, the improved carboxylesterase polypeptides comprise an amino acid sequence comprising the following features: X35 is selected from a basic residue and a polar residue; and X185 is selected from a polar residue and an aliphatic residue. In another embodiment, the improved carboxylesterase polypeptide comprises an amino acid sequence including the following features: X35 is H; and X185 is selected from F, I, and M.

[0093] In some embodiments, the improved carboxylesterase polypeptide comprises a residue difference at at least one residue position selected from X9, X19, X34, X35, X37, X46, X48, X66, X87, X103, X139, X190, X207, X216, X263, X271, X278, and X296 when compared to the amino acid sequence set forth in SEQ ID NO: 24. In other embodiments, the improved carboxylesterase polypeptide has the following characteristics: the residue corresponding to X9 is an aromatic residue; the residue corresponding to X19 is selected from a basic residue and a polar residue; the residue corresponding to X34 is selected from a constrained residue, an acidic residue, and a polar residue; the residue corresponding to X35 is selected from a polar residue; the residue corresponding to X46 is an aliphatic residue; the residue corresponding to X48 is an aliphatic residue; the residue corresponding to X66 is an aliphatic residue; the residue corresponding to X87 is selected from an aliphatic residue and a small residue; and the residue corresponding to X103 is selected from an aromatic residue. the residue corresponding to X207 is a basic residue; the residue corresponding to X216 is selected from aromatic, basic, and polar residues; the residue corresponding to X263 is selected from aliphatic and polar residues; the residue corresponding to X271 is selected from acidic and polar residues; the residue corresponding to X278 is selected from aliphatic and aromatic residues, and the residue corresponding to X296 is selected from aliphatic and basic residues.

[0094] In yet other embodiments, the improved carboxylesterase polypeptides comprise an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO:24, wherein the amino acid sequence comprises at least one feature selected from: X9 is Y; X19 is R; X34 is selected from E, N or P; X35 is S, X37 is T; X46 is selected from I, L or V; X48 is L; X66 is V; X87 is A; X103 is selected from W or F; X139 is R; X190 is Y; X207 is E; X216 is selected from N and W; X263 is selected from T and A; X271 is D; X278 is selected from W and L; and X296 is selected from V, L, and R. In yet other embodiments, the improved carboxylesterase polypeptides comprise an amino acid sequence comprising the following features: X9 is an aromatic residue, and X87 is an aliphatic residue. In another embodiment, the improved carboxylesterase polypeptides comprise an amino acid sequence comprising the following features: X9 is Y; and X87 is A.

[0095] In other embodiments, the improved carboxylesterase polypeptides comprise an amino acid sequence that comprises a residue different from the amino acid sequence set forth in SEQ ID NO: 54 at at least one residue position selected from X20, X28, X29, X30, X33, X34, X188, X216, and X286. In another embodiment, the improved carboxylesterase polypeptides comprise an amino acid sequence that comprises at least one feature selected from: X20 is selected from an aliphatic residue and a basic residue; the residue corresponding to X28 is selected from an acidic residue, a polar residue, and a constrained residue; the residue corresponding to X29 is selected from an acidic residue and a polar residue; the residue corresponding to X30 is an aliphatic residue; the residue corresponding to X33 is an aromatic residue; the residue corresponding to X34 is a small residue; the residue corresponding to X188 is selected from a small residue and an aromatic residue; the residue corresponding to X216 is a polar residue, and the residue corresponding to X286 is selected from an aliphatic residue, a small residue, a non-polar residue, and a polar residue.

[0096] In some embodiments, the improved carboxylesterase polypeptide comprises the amino acid sequence set forth in SEQ ID NO:54, which contains at least one specific mutation selected from: X20 selected from I and R; X28 selected from D, P, and S; X29 is D; X30 is V; X33 is W; X34 is G; X188 is selected from G and F; X216 is N, and X286 is selected from S, M, V, G, and A. In another embodiment, the improved carboxylesterase polypeptide comprises an amino acid sequence comprising the characteristic that X216 is a polar residue. In yet another embodiment, the improved carboxylesterase polypeptide comprises an amino acid sequence comprising the characteristic that X216 is N.

[0097] In other embodiments, the improved carboxylesterase polypeptides comprise an amino acid sequence that comprises a residue different from the amino acid sequence set forth in SEQ ID NO:68 at one residue position selected from X10, X20, X22, X28, X30, X33, X36, X37, X46, X66, X75, X103, X197, X263, X266, X280, and X290. In another embodiment, the improved carboxylesterase polypeptide comprises an amino acid sequence comprising at least one feature selected from: the residue corresponding to X10 is an aliphatic residue; the residue corresponding to X20 is selected from an aliphatic and a basic residue; the residue corresponding to X22 is an aromatic residue; the residue corresponding to X28 is selected from an acidic, a polar, and a constrained residue; the residue corresponding to X30 is an aliphatic residue; the residue corresponding to X33 is an aromatic residue; the residue corresponding to X36 is an aliphatic or aromatic residue; the residue corresponding to X37 is an aromatic or small residue; the residue corresponding to X46 is a basic residue; the residue corresponding to X66 is a polar residue; the residue corresponding to X75 is a basic residue; the residue corresponding to X103 is an aromatic residue; the residue corresponding to X197 is an aliphatic residue; the residue corresponding to X263 is a basic residue; the residue corresponding to X266 is a polar residue; the residue corresponding to X280 is selected from an aliphatic and a polar residue; and the residue corresponding to X290 is selected from an aliphatic and an aromatic residue.

[0098] In yet other embodiments, the improved carboxylesterase polypeptides comprise an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO:68, which comprises at least one feature selected from: X10 is selected from L and M; X20 is selected from I and R; X22 is W; X28 is selected from D, P, and S; X30 is V; X33 is W; X36 is selected from F, I, and M; X37 is selected from G and Y; X46 is R; X66 is T; X75 is R; X103 is W; X197 is L; X263 is R; X266 is T; X280 is selected from M and T; and X290 is selected from W and I. In another embodiment, the improved carboxylesterase polypeptides comprise an amino acid sequence comprising at least one feature selected from: X30 is an aliphatic residue, X33 is an aromatic residue, X75 is a basic residue, and X103 is an aromatic residue. In yet another embodiment, the improved carboxylesterase polypeptides comprise an amino acid sequence comprising the following features: X30 is V; X33 is W; X75 is R; and X103 is W.

[0099] In other embodiments, the improved carboxylesterase polypeptides comprise an amino acid sequence that comprises a residue different from the amino acid sequence set forth in SEQ ID NO:68 at one residue position selected from X28, X38, X46, X54, X66, X75, X85, X86, X96, X160, X176, X183, X188, X205, X212, X248, X249, X255, X270, and X286. In another embodiment, the improved carboxylesterase polypeptide has a residue corresponding to X28 selected from an acidic residue, a polar residue, a small residue, and a constrained residue; a residue corresponding to X38 selected from an aliphatic residue and a basic residue; a residue corresponding to X46 selected from an acidic residue and a basic residue; a residue corresponding to X54 selected from an acidic residue and a polar residue; a residue corresponding to X66 is a polar residue; a residue corresponding to X75 is a basic residue; a residue corresponding to X85 is selected from an aromatic residue or a basic residue and a small residue; a residue corresponding to X86 is a polar residue; a residue corresponding to X96 is selected from a non-polar residue and an aliphatic residue; a residue corresponding to X160 is a polar residue and a constrained residue. the residue corresponding to X176 is selected from an aliphatic, aromatic or basic residue and a non-polar residue; the residue corresponding to X183 is a non-polar residue; the residue corresponding to X188 is selected from an aromatic residue and a small residue; the residue corresponding to X205 is an aromatic residue; the residue corresponding to X212 is an acidic residue; the residue corresponding to X248 is an aliphatic residue; the residue corresponding to X249 is an aromatic residue; the residue corresponding to X255 is a polar residue; the residue corresponding to X270 is selected from an aliphatic residue and a polar residue; and the residue corresponding to X286 is selected from an aliphatic residue, a non-polar residue, a small residue and a polar residue.

[0100] In yet another embodiment, the improved carboxylesterase polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO:68, wherein the amino acid sequence is selected from: X28 is selected from C, D, S, H, P, G, and R; X38 is selected from E and L; X46 is selected from K, R and Q; X54 is selected from R, Q, and S; X66 is selected from L, T, and V; X75 is R; X85 is selected from G and H; X and X286 is selected from M, V, G, N and S. In another embodiment, the improved carboxylesterase polypeptide comprises an amino acid sequence comprising at least one feature selected from: X28 is a polar residue, X38 is a basic residue, and X85 is a small residue. In yet another embodiment, the improved carboxylesterase polypeptide comprises an amino acid sequence comprising the following features: X28 is C; X38 is E; and X85 is G.

[0101] In other embodiments, the improved carboxylesterase polypeptides comprise an amino acid sequence that comprises a residue at one residue position selected from X7, X22, X36, X38, X46, X54, X66, and X75 that differs from the amino acid sequence set forth in SEQ ID NO: 100. In another embodiment, the improved carboxylesterase polypeptides comprise an amino acid sequence that includes at least one feature selected from: the residue corresponding to X7 is an aliphatic residue; the residue corresponding to X22 is selected from an aliphatic residue and an aromatic residue; the residue corresponding to X36 is selected from a polar residue and a non-polar residue; the residue corresponding to X38 is an aromatic residue; the residue corresponding to X46 is selected from a polar residue and a basic residue; the residue corresponding to X54 is selected from a polar residue and a basic residue; the residue corresponding to X66 is a polar residue; and the residue corresponding to X75 is selected from a basic residue and a non-polar residue.

[0102] In yet another embodiment, the improved carboxylesterase polypeptide comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 100, which comprises at least one feature selected from: X7 is L; X22 is selected from W and L; X36 is selected from T and M; X38 is W; X46 is selected from K and Q; X54 is selected from S, Q, and K; X66 is selected from G and T; and X75 is selected from M and R. In another embodiment, the improved carboxylesterase polypeptide comprises an amino acid sequence comprising at least one feature selected from: X36 is a polar residue, X38 is an aromatic residue, and X75 is a basic residue. In yet another embodiment, the improved carboxylesterase polypeptide comprises an amino acid sequence comprising the following features: X36 is T; X38 is W; and X75 is R.

[0103] In other embodiments, the improved carboxylesterase polypeptides comprise an amino acid sequence that comprises a residue at one residue position selected from X2, X181, and X286 that differs from the amino acid sequence set forth in SEQ ID NO: 114. In another embodiment, the improved carboxylesterase polypeptides comprise an amino acid sequence that comprises at least one feature selected from: the residue corresponding to X2 is selected from an aliphatic residue, a basic residue, a polar residue, and an aromatic residue; the residue corresponding to X181 is a basic residue; and the residue corresponding to X286 is selected from a polar residue and a non-polar residue.

[0104] In yet another embodiment, the improved carboxylesterase polypeptide comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 114, which comprises at least one feature selected from: X2 selected from L, Q, R, and H; X181 is Q; and X286 is selected from C and S. In another embodiment, the improved carboxylesterase polypeptide comprises an amino acid sequence comprising at least one feature selected from: X286 is a non-polar residue. In yet another embodiment, the improved carboxylesterase polypeptide comprises an amino acid sequence comprising the feature: X286 is C.

[0105] In some embodiments, the improved carboxylesterase polypeptide comprises an amino acid sequence corresponding to the amino acid sequence set forth in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, or 126.

[0106] In a further aspect, the disclosure provides polynucleotides encoding each of the above-described improved engineered carboxylesterase polypeptides, hi some embodiments, these polynucleotides can be part of an expression vector having one or more regulatory sequences for expression of the carboxylesterase polypeptide. In another embodiment, the polynucleotide corresponds to any one of the nucleotide sequences set forth in SEQ ID NO:3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, or 125.

[0107] In another aspect, the disclosure provides a host cell comprising a polynucleotide encoding an engineered carboxylesterase or an expression vector capable of expressing the engineered carboxylesterase. In some embodiments, the host cell can be a bacterial host cell, such as E. coli. These host cells can be used for expression and isolation of the engineered carboxylesterase enzymes described herein, or the host cells can be used directly for conversion of ester substrates to products. In some embodiments, the engineered amides can be used in the form of whole cells, crude extracts, isolated polypeptides, or purified polypeptides, individually or as a combination of various engineered amides.

[0108] Those skilled in the art will recognize that post-translational modifications may occur during the production of enzymes, particularly depending on the cell line used and the specific amino acid sequence of the enzyme. For example, such post-translational modifications may include cleavage of specific leader sequences, addition of various sugar moieties in various glycosylation and phosphorylation patterns, deamidation, oxidation, disulfide bond scrambling, isomerization, C-terminal lysine clipping, and N-terminal glutamine cyclization. The present invention encompasses the use of engineered carboxylesterase enzymes that have or may have undergone one or more post-translational modifications. Thus, the engineered carboxylesterases of the present invention may include those that have undergone post-translational modifications such as those described herein.

[0109] Deamidation is an enzymatic reaction that primarily converts asparagine (N) to isoaspartic acid (isoaspartate) and aspartic acid (aspartate) (D) in an approximately 3:1 ratio. This deamidation reaction is therefore associated with the isomerization of aspartate (D) to isoaspartate. Both the deamidation of asparagine and the isomerization of aspartate involve the intermediate succinimide. Deamidation can occur at glutamine residues as well, although to a much lesser extent.

[0110] Oxidation can occur during production and storage (i.e., in the presence of oxidizing conditions) and results in the covalent modification of proteins induced directly by reactive oxygen species or indirectly by reaction with secondary by-products of oxidative stress. Oxidation occurs primarily at methionine residues, but can also occur at tryptophan and free cysteine ​​residues.

[0111] Disulfide bond scrambling can occur during production and basic storage conditions. Under certain circumstances, disulfide bonds can break or form improperly, resulting in unpaired cysteine ​​residues (-SH). These free (unpaired) sulfhydryls (-SH) can facilitate shuffling.

[0112] The N-terminal glutamine (Q) and glutamate (E) in engineered carboxylesterases can form pyroglutamate (pGlu) via cyclization. Most formation of pGlu occurs during production, but it can also form nonenzymatically depending on the pH and temperature of processing and storage conditions.

[0113] C-terminal lysine clipping is an enzymatic reaction catalyzed by carboxypeptidases and is commonly found in enzymes. Variations of this process include the removal of lysine from enzymes derived from recombinant host cells.

[0114] In the present invention, the above post-translational modifications and changes in the primary amino acid sequence are not known to result in significant changes in the activity of the engineered carboxylesterase enzymes.

[0115] Table 3 below provides examples of engineered carboxylesterase polypeptides, with two SEQ ID NOs in each row, where odd numbers represent nucleotide sequences that encode the amino acid sequences represented by the even numbers. Residue differences are based on comparison to the reference sequence of SEQ ID NO:2, which is a carboxylesterase that corresponds to the wild-type A. acidocaldarius esterase 2 shown in Example 6.In the activity column, the level of activity enhancement (i.e., "+", "++", "+++", etc.) was defined as follows: "-" indicates less than 1% substrate to product conversion, with a conversion rate of 0.9% or less (175 μL lysis solution, 100 mM ester, 100 mM isopropyl piperazine, 2% water); "+" indicates at least 1.1-80 times the activity of SEQ ID NO:2, but not more than the activity of SEQ ID NO:4 (175 μL lysis solution, 100 mM ester, 100 mM isopropyl piperazine, 2% water in TBME). piperazine, 2% water); "++" indicates at least 1.1-11 times the activity of SEQ ID NO:4 but not more than the activity of SEQ ID NO:18 (150 μL lysis solution, 200 mM ester, 200 mM isopropyl piperazine, 2% water); "+++" indicates at least 1.1-5 times the activity of SEQ ID NO:18 but not more than the activity of SEQ ID NO:54 (120 μL lysis solution, 300 mM ester, 300 mM isopropyl piperazine, 2% water in MIBK); "++++" indicates at least 1.1-5 times the activity of SEQ ID NO:54 (120 μL lysis solution, 300 mM ester, 300 mM isopropyl piperazine, 2% water in MIBK); "++++" indicates at least 1.1-2.0 times the activity of SEQ ID NO:68 (50 μL of solution, 300 mM ester, 300 mM isopropylpiperazine, 2% water in MIBK); "$" indicates at least 1.1-2 times the activity of SEQ ID NO:68 but not more than the activity of SEQ ID NO:100 (50 μL of solution, 300 mM ester, 300 mM isopropylpiperazine, 2% water in MIBK). solution in MIBK, 354 mM ester, 425 mM isopropylpiperazine, 2% water); "$$" indicates at least 1.1-5 times the activity of SEQ ID NO:100 but not more than the activity of SEQ ID NO:114 (50 μL solution in MIBK, 354 mM ester, 425 mM isopropylpiperazine, 2% water); "$$$" indicates at least 1.1-2 times the activity of SEQ ID NO:114 (50 μL solution in MIBK, 354 mM ester, 354 mM isopropylpiperazine, 2% water).In each case, activity was determined using various amounts of lysate, which was added to a multi-well lyophilized and activity screen as described in Example 5, and then reacted with the indicated concentrations of substrate in the indicated solvent system in a volume of 200 μL for 16 hours.

[0116] [Table 3] TIFF0007663635000009.tif196150TIFF0007663635000010.tif203150TIFF0007663635000011.tif50150

[0117] As described above, in some embodiments, the improved carboxylesterase polypeptides are selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 15 In one embodiment, the amino acid sequence is at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical or more to a reference sequence of 0, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, or 126. In some embodiments, the improved carboxylesterase polypeptides can have 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 1-18, 1-19, 1-20, 1-22, 1-24, 1-26, 1-28, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, 1-60, or 1-62 residue differences when compared to the carboxylesterase set forth in SEQ ID NO:2. In some embodiments, the number of residue differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 55, 60, or 62 differences when compared to SEQ ID NO:2.

[0118] In some embodiments, the improved carboxylesterase polypeptide is selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, or 126, wherein the improved carboxylesterase amino acid sequence comprises an amino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 8%, or 99% identical to a reference sequence based on SEQ ID NO:116, 118, 120, 122, 124, or 126, provided that the improved carboxylesterase amino acid sequence comprises any one of a set of residue differences contained in any one of the polypeptide sequences listed in Table 3 when compared to SEQ ID NO:2. In some embodiments, the improved carboxylesterase polypeptides can further have 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, 1-60, or 1-62 residue differences at other amino acid residue positions when compared to a reference sequence. In some embodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or 62 residue differences at other residue positions. In some embodiments, residue differences at other residue positions comprise substitutions with conservative amino acid residues.

[0119] In some embodiments, the improved carboxylesterase polypeptides capable of converting an ester substrate, ethyl oxazole-5-carboxylate, in the presence of an amine substrate in water-saturated MIBK to product levels detectable at 230 nm by HPLC-UV are selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 118, 120, 122, 124, 126, 6, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, or 126.

[0120] In some embodiments, the engineered carboxylesterase polypeptides are capable of converting ester substrates to products with 2-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, 1000-fold, 10,000-fold, 100,000-fold, 500,000-fold, 785,000-fold or more activity than the polypeptide of SEQ ID NO:2. In some embodiments, the engineered carboxylesterase polypeptide is capable of converting ester substrates to products with 50-100 fold or more activity than the polypeptide of SEQ ID NO:2 and comprises an amino acid sequence corresponding to SEQ ID NO:16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, or 126.

[0121] In some embodiments, the engineered carboxylesterase polypeptide is capable of converting an ester substrate to a product with about 1.1-5 times or more activity than the polypeptide of SEQ ID NO: 24. In some embodiments, the engineered carboxylesterase polypeptide is capable of converting an ester substrate to a product with about 1.1-5 times or more activity than the polypeptide of SEQ ID NO: 24 and comprises an amino acid sequence corresponding to the sequence of SEQ ID NO: 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, or 126.

[0122] In some embodiments, the engineered carboxylesterase polypeptide is capable of converting an ester substrate to a product with about 1.1-5-fold or greater activity than the polypeptide of SEQ ID NO: 54. In some embodiments, the engineered carboxylesterase polypeptide is capable of converting an ester substrate to a product with about 1.1-5-fold or greater activity than the polypeptide of SEQ ID NO: 54 and comprises a sequence corresponding to the sequence of SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, or 126.

[0123] In some embodiments, the engineered carboxylesterase polypeptide is capable of converting ester substrates to products with about 1.1-6 fold or greater activity than the polypeptide of SEQ ID NO: 68. In some embodiments, the engineered carboxylesterase polypeptide is capable of converting ester substrates to products with about 1.1-5 fold or greater activity than the polypeptide of SEQ ID NO: 68 and comprises an amino acid sequence corresponding to the sequence of SEQ ID NO: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, or 126.

[0124] In some embodiments, the engineered carboxylesterase polypeptide is capable of converting ester substrates to products with about 1.1-5 fold or greater activity than the polypeptide of SEQ ID NO: 100. In some embodiments, the engineered carboxylesterase polypeptide is capable of converting ester substrates to products with about 1.7 fold or greater activity than the polypeptide of SEQ ID NO: 100 and comprises an amino acid sequence corresponding to the sequence of SEQ ID NO: 108, 110, 112, 114, 116, 118, 120, 122, 124, or 126.

[0125] In some embodiments, the engineered carboxylesterase polypeptide is capable of converting ester substrates to products with about 1.1-2 fold or greater activity than the polypeptide of SEQ ID NO: 114. In some embodiments, the engineered carboxylesterase polypeptide is capable of converting ester substrates to products with about 1.1-5 fold or greater activity than the polypeptide of SEQ ID NO: 114 and comprises an amino acid sequence corresponding to the sequence of SEQ ID NO: 122, 124, or 126.

[0126] In some embodiments, the improved engineered carboxylesterase polypeptides can comprise deletions at specific amino acid residues of the engineered carboxylesterase polypeptides described herein. Thus, for each embodiment of the carboxylesterase polypeptides of the disclosure, the deletions can comprise 1 or more amino acids, 2 or more amino acids, 3 or more amino acids, 4 or more amino acids, 5 or more amino acids, 6 or more amino acids, 8 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 5% of the total number of amino acids, up to 10% of the total number of amino acids, up to 20% of the total number of amino acids, or up to 30% of the total number of amino acids of the carboxylesterase polypeptide, so long as the functional activity of the carboxylesterase activity is maintained. In some embodiments, the deletions may comprise 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 14, 1 to 15, 1 to 16, 1 to 18, 1 to 20, 1 to 22, 1 to 24, 1 to 26, 1 to 30, 1 to 35, 1 to 40, 1 to 45, 1 to 50, 1 to 55, 1 to 60, or up to 1 to 62 amino acid residues. In some embodiments, the number of deletions may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, 60, or up to 62 amino acids. As described herein, the carboxylesterase polypeptides of the disclosure can be in the form of a fusion polypeptide in which the carboxylesterase polypeptide is fused to other polypeptides, such as, by way of example and not limitation, an antibody tag (e.g., a myc epitope), a purification sequence (e.g., a His tag for binding to metals), and a cellular localization signal (e.g., a secretion signal). Thus, the carboxylesterase polypeptides can be used with or without being fused to other polypeptides.

[0127] The polypeptides described herein are not limited to genetically encoded amino acids. In addition to genetically encoded amino acids, the polypeptides described herein may be composed entirely or partially of natural and / or synthetic non-encoded amino acids. Certain commonly encountered non-encoded amino acids that may comprise the polypeptides described herein include, but are not limited to, the D-stereoisomers of the genetically encoded amino acids; 2,3-diaminopropionic acid (Dpr); α-aminoisobutyric acid (Aib); ε-aminohexanoic acid (Aha); δ-aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly or Sar); ornithine (Orn); citrulline (Cit); t-butylalanine (Bua); t-butylglycine (Bug); N-methylisoleucine (MeIle); phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle); naphthylalanine (Nal); 2-chlorophenylalanine (Ocf); 3-chlorophenylalanine (Mcf); 4-chlorophenylalanine (Pcf); 2-fluorophenylalanine (Off); 3-fluorophenylalanine (Mff); 4-fluorophenylalanine (Pff); 2-bromophenylalanine (Obf );3-Bromophenylalanine (Mbf);4-Bromophenylalanine (Pbf);2-Methylphenylalanine (Omf);3-Methylphenylalanine (Mmf);4-Methylphenylalanine (Pmf);2-Nitrophenylalanine (Onf);3-Nitrophenylalanine (Mnf);4-Nitrophenylalanine (Pnf);2-Cyanophenylalanine (Ocf);3-Cyanophenylalanine (Mcf);4-Cyanophenylalanine (Pcf);2-Trifluoromethylphenylalanine (Mbf); 3-trifluoromethylphenylalanine (Otf); 3-trifluoromethylphenylalanine (Mtf); 4-trifluoromethylphenylalanine (Ptf); 4-aminophenylalanine (Paf); 4-iodophenylalanine (Pif); 4-aminomethylphenylalanine (Pamf); 2,4-dichlorophenylalanine (Opef); 3,4-dichlorophenylalanine (Mpcf); 2,4-difluorophenylalanine (Opff); 3,4-difluorophenylalanine (Mpff);Pyrid-2-ylalanine (2pAla); Pyrid-3-ylalanine (3pAla); Pyrid-4-ylalanine (4pAla); Naphth-1-ylalanine (1nAla); Naphth-2-ylalanine (2nAla); Thiazolylalanine (taAla); Benzothienylalanine (bAla); Thienylalanine (tAla); Furylalanine (fAla); Homophenylalanine (hPhe); Homotyrosine (hTyr); Homotryptophan (hTrp); Pentafluorophenylalanine (5ff); Styrylkalanine (sAla); Anthrylalanine (aAla); 3,3-Diphenylalanine (Dfa); 3-Amino-5-phenylpentanoic acid (phenypentanoic acid)(Afp);penicillamine (Pen);1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic);β-2-thienylalanine (Thi);methionine sulfoxide (Mso);N(w)-nitroarginine (nArg);homolysine (hLys);phosphonomethylphenylalanine (pmPhe);phosphoserine (pSer);phosphothreonine (pThr);homoaspartic acid (hAsp);homoglutamic acid (hGlu);1-aminocyclopent-(2 or 3)-ene-4-carboxylic acid;pipecolic acid (P A), azetidine-3-carboxylic acid (ACA); 1-aminocyclopentane-3-carboxylic acid; allylglycine (aOly); propargylglycine (pgGly); homoalanine (hAla); norvaline (nVal); homoleucine (hLeu), homovaline (hVal); homoisoleucine (hIle); homoarginine (hArg); N-acetyl-lysine (AcLys); 2,4-diaminobutyric acid (Dbu); 2,3-diaminobutyric acid (Dab); N-methylvaline (MeVal); homocysteine ​​(hCys); homoserine (hSer);These include hydroxyproline (Hyp) and homoproline (hPro). Additional non-encoded amino acids that may comprise the polypeptides described herein will be apparent to those of skill in the art (see, e.g., the various amino acids set out in Fasman, 1989, CRC Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Boca Raton, FL, at pp. 3-70 and the references cited therein). These amino acids may be in the L- or D-form;

[0128] Those skilled in the art will recognize that amino acids or residues with side chain protecting groups can also constitute the polypeptides described herein.Non-limiting examples (protecting groups are listed in parentheses) of such protected amino acids (in this case belonging to the aromatic category) include, but are not limited to, Arg(tos), Cys(methylbenzyl), Cys(nitropyridine sulfenyl), Glu(δ-benzyl ester), Gln(xanthyl), Asn(N-δ-xanthyl), His(bom), His(benzyl), His(tos), Lys(fmoc), Lys(tos), Ser(O-benzyl), Thr(O-benzyl) and Tyr(O-benzyl).

[0129] Conformationally constrained non-coded amino acids that may be comprised in the polypeptides described herein include, but are not limited to, N-methyl amino acids (L-form); 1-aminocyclopent-(2 or 3)-ene-4-carboxylic acid; pipecolic acid; azetidine-3-carboxylic acid; homoproline (hPro); and 1-aminocyclopentane-3-carboxylic acid.

[0130] As noted above, various modifications introduced into a native polypeptide to create an engineered carboxylesterase enzyme may be aimed at affecting particular properties of the enzyme, such as activity, specificity for its substrates, and thermostability.

[0131] In another aspect, the disclosure provides polynucleotides encoding improved carboxylesterase polypeptides. These polynucleotides may be operably linked to one or more heterologous regulatory sequences that control gene expression to generate recombinant polynucleotides capable of expressing the carboxylesterase polypeptides. Expression constructs containing heterologous polynucleotides encoding engineered carboxylesterases can be introduced into suitable host cells to express the corresponding carboxylesterase polypeptides.

[0132] Because of the knowledge of the codons corresponding to the various amino acids, the availability of a protein sequence provides a description of all polynucleotides that can code for the subject. The degeneracy of the genetic code, in which the same amino acids are coded for by alternative or synonymous codons, allows for the creation of a very large number of nucleic acids, all of which code for the improved carboxylesterase polypeptides disclosed herein. Thus, once a particular amino acid sequence has been identified, one of skill in the art can create any number of different nucleic acids by simply modifying the sequence of one or more codons without changing the amino acid sequence of the protein. In this regard, the present disclosure specifically contemplates each possible variant of a polynucleotide that can be made by selecting combinations based on possible codon choices, and all such variants should be considered specifically disclosed with respect to any polypeptide disclosed herein, including the amino acid sequences shown in Table 3.

[0133] In some embodiments, these polynucleotides can be selected and / or engineered to comprise codons selected to be compatible with the host cell in which the protein is produced. For example, preferred codons used in bacteria are used for expression of genes in bacteria; preferred codons used in yeast are used for expression in yeast; preferred codons used in mammals are used for expression in mammalian cells. Because it is not necessary to replace all codons to optimize the codon usage of a carboxylesterase (e.g., because the native sequence may have preferred codons and because preferred codon usage is not required for all amino acid residues), a codon-optimized polynucleotide encoding a carboxylesterase polypeptide can include preferred codons at more than about 40%, 50%, 60%, 70%, 80%, or 90% of the codon positions of the full-length coding region.

[0134] In some embodiments, the polynucleotide encodes a carboxylesterase polypeptide comprising an amino acid sequence at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical or greater to the reference sequence of SEQ ID NO:4, or a functional fragment thereof, which is capable of converting an ester substrate in the presence of an amine substrate with an activity that is improved compared to the activity of the carboxylesterase of SEQ ID NO:2 from A. acidocaldarius esterase 2.

[0135] In some embodiments, the polynucleotide is at least about identical to a polypeptide comprising an amino acid sequence corresponding to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, or 126. The polypeptides encode carboxylesterase polypeptides comprising an amino acid sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity, or a functional fragment thereof, which have at least one improved property in converting an ester substrate, ethyl oxazole-5-carboxylate, to the product, (4-isopropylpiperazin-1-yl)(oxazol-5-yl)methanone, in the presence of an amine substrate, 1-isopropylpiperazine. In some embodiments, the encoded carboxylesterase polypeptide has activity equal to or greater than the activity of the polypeptide of SEQ ID NO:2.

[0136] In some embodiments, the polynucleotide is selected from the group consisting of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, or 126. or a functional fragment thereof, wherein the improved carboxylesterase amino acid sequence comprises any one of a set of residue differences included in any one of the polypeptide sequences listed in Table 3 when compared to SEQ ID NO:2.

[0137] In some embodiments, the polynucleotide encoding the improved carboxylesterase polypeptide is selected from SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, or 125.

[0138] In some embodiments, the polynucleotide is selected from the group consisting of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 300, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 349, 351, 353, 354, 355 09, 111, 113, 115, 117, 119, 121, 123, or 125, or their complements, and polynucleotides that hybridize under high stringency conditions encode carboxylesterase polypeptides that can convert an amine substrate to a product with activity equal to or greater than that of the polypeptide of SEQ ID NO:2 in the presence of an amine substrate.

[0139] In some embodiments, these polynucleotides encode a polypeptide as described herein that has about 80% or greater sequence identity, about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater sequence identity at the nucleotide level to a reference polynucleotide encoding an engineered carboxylesterase as described herein. In some embodiments, the reference polynucleotide is selected from SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, or 125.

[0140] In some embodiments, the carboxylesterase polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 122. In another embodiment, the disclosure provides a polynucleotide sequence encoding the carboxylesterase polypeptide sequence set forth in SEQ ID NO: 122. In yet another embodiment, the disclosure provides a polynucleotide encoding the carboxylesterase polypeptide, the polynucleotide comprising the polynucleotide sequence set forth in SEQ ID NO: 121. In yet another embodiment, the carboxylesterase polypeptide consists of the polypeptide sequence set forth in SEQ ID NO: 122. In another embodiment, the carboxylesterase polypeptide consists of residues 2-310 of SEQ ID NO: 122.

[0141] Improved carboxylesterases and polynucleotides encoding such polypeptides can be produced using methods familiar to those skilled in the art. As mentioned above, the native amino acid sequence encoding the wild-type carboxylesterase enzyme A. acidocaldarius esterase 2 from which the parent sequence SEQ ID NO:2 was derived and the corresponding polynucleotides are available in WO02 / 057411 (see SEQ ID NO:10). In some embodiments, the parent polynucleotide sequence is codon-optimized to enhance expression of the carboxylesterase in a specified host cell. Engineered carboxylesterases can be obtained by subjecting polynucleotides encoding native carboxylesterases to mutagenesis and / or directed evolution methods. Examples of directed evolution techniques are mutagenesis and / or DNA shuffling, as described in Stemmer, 1994, Proc Natl Acad Sci USA 91:10747-10751; WO95 / 22625; WO97 / 0078; WO97 / 35966; WO98 / 27230; WO00 / 42651; WO01 / 75767; and U.S. Patent No. 6,537,746.

[0142] Other directed evolution methods that can be used include, among others, the staggered extension process (StEP), in vitro recombination (Zhao, et al., 1998, Nat. Biotechnol. 16:258-261), mutagenic PCR (Caldwell, et al., 1994, PCR Methods Appl. 3:S136-S140), and cassette mutagenesis (Black, et al., 1996, Proc Natl Acad Sci USA 93:3525-3529). Mutagenesis and directed evolution techniques useful for purposes herein are also described in the following references: Ling, et al., 1997, "Approaches to DNA mutagenesis: an overview," Anal. Biochem. 254(2):157-78; Dale, et al., 1996, "Oligonucleotide-directed random mutagenesis using the phosphorothioate method," Methods Mol. Biol. 57:369-74; Smith, 1985, "In vitro mutagenesis," Ann. Rev. Genet. 19:423-462; Botstein, et al., 1985, "Strategies and applications of in vitro mutagenesis," Science 229:1193-1201; Carter, 1986, "Site-directed mutagenesis," Biochem. J. 237:1-7; Kramer, et al., 1984, “Point Mismatch Repair,” Cell 38:879-887; Wells, et al., 1985, “Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites,” Gene 34:315-323; Minshull, et al., 1999, “Pro tein evolution by molecular breeding,” Curr Opin Chem Biol 3:284-290; Christians, et al., 1999, “Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling,” Nature Biotech 17:259-264; Crameri, et al., 1998, “DNA shuffling of a family of genes from diverse species accelerates directed evolution,” Nature 391:288-291; Crameri, et al., 1997, “Molecular evolution of an arsenate detoxification pathway by DNA shuffling,” Nature Biotech 15:436-438; Zhang, et al., 1997, “Directed evolution of an effective fructosidase from a galactosidase by DNA shuffling and screening,” Proc Natl Acad Sci USA 94:45-4-4509; Crameri, et al., 1996, “Improved green fluorescent protein by molecular evolution using DNA shuffling,’ Nature Biotech 14:315-319; and Stemmer, 1994, “Rapid evolution of a protein in vitro by DNA shuffling,” Nature 370:389-391。

[0143] In some embodiments, the clones obtained after the mutagenesis treatment are screened for carboxylesterases with the desired improved enzymatic properties. Measurement of carboxylesterase enzyme activity from an expression library can be performed using standard techniques, such as separation of products (e.g., by HPLC) and detection of the products by measuring UV absorbance of the separated substrates and products and / or detection using tandem mass spectrometry (e.g., MS / MS). An exemplary assay is described in Example 4 below. The rate of increase of the desired product per unit time indicates the relative (enzymatic) activity of the carboxylesterase polypeptide in a fixed amount of lysate (or lyophilized powder made therefrom). If the desired improvement in the enzymatic properties is thermostability, the enzymatic activity can be measured by exposing the enzyme preparation to a defined temperature and then measuring the amount of enzymatic activity remaining after heat treatment. Clones containing polynucleotides encoding the desired carboxylesterases are then isolated, sequenced to identify changes in the nucleotide sequence (if any), and used to express the enzyme in host cells.

[0144] In other embodiments known in the art, enzymes may be genetically diversified while maintaining their target activity, such as by the technique of neutral drift.

[0145] When the sequence of the engineered polypeptide is known, the polynucleotide encoding the enzyme can be prepared by standard solid-phase methods according to known synthesis methods.In some embodiments, fragments of up to about 100 bases can be synthesized individually and then linked (e.g., by enzymatic or chemical litigation methods, or polymerase-mediated methods) to form any desired continuous sequence.For example, the polynucleotides and oligonucleotides of the present invention can be prepared by chemical synthesis, for example, using the classical phosphoramidite method described by Beaucage, et al., 1981, Tet Lett 22:1859-69 or the method described by Matthes, et al., 1984, EMBO J. 3:801-05, as is usually carried out in automated synthesis methods.According to the phosphoramidite method, oligonucleotides are synthesized, purified, annealed, ligated, and cloned into suitable vectors, for example, in an automatic DNA synthesizer. Additionally, essentially any nucleic acid can be obtained from any of a variety of commercial sources, including The Great American Gene Company, Ramona, Calif., ExpressGen Inc, Chicago, Ill., Operon Technologies Inc, Alameda, Calif., and many others.

[0146] The engineered carboxylesterase enzyme expressed in the host cells can be recovered from the cells and / or culture medium using any one or more of the well-known techniques for protein purification, including lysozyme treatment, sonication, filtration, salting out, ultracentrifugation, and chromatography, among others. A solution suitable for lysis and highly efficient extraction of proteins from bacteria such as E. coli is commercially available from Sigma-Aldrich of St. Louis, MO under the trademark CelLytic BTM.

[0147] Chromatographic techniques for isolation of carboxylesterase polypeptides include, among others, reverse phase chromatography, high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, and affinity chromatography. Conditions for purifying a particular enzyme will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, and the like, and will be apparent to one of skill in the art. In some embodiments, the engineered carboxylesterase can be expressed as a fusion protein with a purification tag, such as a His tag, which has affinity for metals, or an antibody tag for binding with an antibody, e.g., a myc epitope tag.

[0148] In some embodiments, affinity techniques can be used to purify the improved carboxylesterase enzyme. For affinity chromatography purification, any antibody that specifically binds to the carboxylesterase polypeptide can be used. For production of antibodies, various host animals, including but not limited to rabbits, mice, rats, etc., can be immunized by injection with the engineered polypeptide. The polypeptide can be attached to a suitable carrier, such as BSA, by a side chain functional group or a linker that is attached to a side chain functional group. Depending on the host species, various adjuvants can be used to enhance the immune response, including but not limited to Freund's (complete and incomplete), inorganic gels such as aluminum hydroxide, lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, surfactants such as dinitrophenol, and potentially useful human adjuvants such as bacilli Calmette Guerin (BCG) and Corynebacterium parvum.

[0149] In a further aspect, the improved carboxylesterase polypeptides described herein can be used in methods for amidating specific amide group acceptors (eg, ester substrates) in the presence of an amine substrate.

[0150] In some embodiments, the improved carboxylesterase comprises (a) R1-COOR2 [R1 is sp 3 and an aromatic ring, and R2 is selected from a methyl group; an ethyl group; and an alkyl chain of 1 to 6 carbon atoms; (b) amine substrates; (c) an improved carboxylesterase polypeptide; and (d) Solvent The compound can be used in a process for producing an amide by combining components containing

[0151] In one embodiment, the solvent is an organic solvent containing up to 3 molar equivalents of water relative to the ester substrate in an amount of about 0.5% (vol / vol) to about 3% (vol / vol).

[0152] In some embodiments of this method, the improved carboxylesterase is selected from SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, and 126.

[0153] In other embodiments, the improved carboxylesterase polypeptides include (a) R1-COOR2, where R1 has 0-3 alkyl substituents, and 3The method can be used in a method for making an amide molecule, comprising combining components comprising an ester substrate of the structure: (a) an amine substrate; (b) an amine substrate; (c) an improved carboxylesterase polypeptide as described above; and (d) a solvent. In another embodiment of the method, an organic solvent is used. In one embodiment, the organic solvent is selected from toluene; 2-methyltetrahydrofuran; tetrahydrofuran; dimethylacetamide; methylisobutylketone (MIBK); dichloromethane; tert-butylmethylether; cyclopentylmethylether; methylcyclohexane; dichloromethane; acetonitrile; methylethylketone; isopropylacetate; ethanol; isopropanol; ethylacetate; heptane; xathane; and 2-methyltetrahydrofuran (2-Me-THF); and water. In yet another embodiment, the organic solvent contains up to 3 molar equivalents of water relative to the ester substrate in an amount of about 0.5% (vol / vol) to about 3% (vol / vol). In another embodiment of this method, the carboxylesterase polypeptide in step (c) is prepared in the presence of a salt to stabilize its physical form during the reaction. In yet another embodiment, a salt is added as an additional reaction component. In one embodiment of this method, the ester has the formula: [ka] and the amine substrate is ethyl oxazole-5-carboxylate having the formula: [ka] and the amide is 1-isopropylpiperazine having the formula: [ka] The compound is (4-isopropylpiperazin-1-yl)(oxazol-5-yl)methanone having the formula:

[0154] In yet another embodiment of this method, the ester has the formula: [ka] and the amine substrate is an ethyl oxazole-5-carboxylate having the formula: [ka] and the amide is cis-2,6-dimethylmorpholine having the formula: [ka] The compound is ((2S,6R)-2,6-dimethylmorpholino)(oxazol-5-yl)methanone having the formula:

[0155] In another embodiment, the reactants in this method comprise about 50 g / L of ethyl oxazole-5-carboxylate, 44 g / L of 1-isopropylpiperazine, and about 25 g / L of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74 , 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, or 126 amino acid sequences, which are produced in the presence of sodium sulfate and function in the presence of about 10 g / L to about 20 g / L water in MIBK.

[0156] In some embodiments, the invention is an amide made by these methods using the improved carboxylesterase. In another embodiment, the invention is an amide, of the formula: [ka] and the amide of formula III is prepared by the methods described above.

[0157] In another embodiment, the present invention provides an amide, of the formula: [ka] and the amide of formula V is prepared by the method described above.

[0158] Compounds of formula I (Astatech, AS-23210), formula II (Oakwood Products Inc, OAK-008910), and formula IV (Oakwood Products Inc, OAK-091224) were obtained from commercial suppliers.

[0159] In some embodiments, the method comprises contacting or incubating an ester substrate, ethyl oxazole-5-carboxylate, with an improved carboxylesterase in the presence of an amine substrate under suitable reaction conditions to convert the ester substrate to the product (4-isopropylpiperazin-1-yl)(oxazol-5-yl)methanone at about 50 to about 785,000-fold or greater conversion and / or activity relative to SEQ ID NO:2. Exemplary polypeptides comprise an amino acid sequence corresponding to SEQ ID NO: 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, or 126.

[0160] In some embodiments of the above process, the reaction solvent for carrying out the process is selected from methyl isobutyl ketone (MIBK), toluene, tert-butyl methyl ether (TBME) or 2-methyltetrahydrofuran (2-Me-THF).

[0161] In some embodiments of the above methods, the enzyme preparation for the reaction includes a salt selected from one of potassium phosphate (KPi), potassium sulfate, or sodium sulfate.

[0162] In some embodiments, the reaction conditions for carrying out the method may comprise a temperature of about 15°C to about 30°C. In one embodiment, the amine substrate used in the method may be a chiral amine or an achiral amine. Achiral amine substrates have the advantage that they are not limited to a particular stereoisomer in the reaction, and therefore less amine substrate is required. A variety of suitable amine substrates may be used, including, but not limited to, 1-isopropylpiperazine and cis-2,6-dimethylmorpholine.In some embodiments, other amine substrates can be used, including, among others, α-phenethylamine (also called 1-phenylethanamine) and its enantiomers (S)-1-phenylethanamine and (R)-1-phenylethanamine, 2-amino-4-phenylbutane, glycine, L-glutamic acid, L-glutamate, monosodium glutamate, L-aspartic acid, L-lysine, L-ornithine, β-alanine, taurine, n-octylamine, cyclohexane, cyclohexane-1, cyclohexane-1, cyclohexane-1, cyclohexane-1, cyclohexane-1 Hexylamine, 1,4-butanediamine, 1,6-hexanediamine, 6-aminohexanoic acid, 4-aminobutyric acid, tyramine, and benzylamine, 2-aminobutane, 2-amino-1-butanol, 1-amino-1-phenylethane, l-amino-1-(2-methoxy-5-fluorophenyl)ethane, 1-amino-1-phenylpropane, 1-amino-1-(4-hydroxyphenyl)propane, 1-amino-1-(4-bromophenyl)propane, 1-amino-1- (4-nitrophenyl)propane, l-phenyl-2-aminopropane, 1-(3-trifluoromethylphenyl)-2-aminopropane, 2-aminopropanol, l-amino-l-phenylbutane, l-phenyl-2-aminobutane, 1-(2,5-dimethoxy-4-methylphenyl)-2-aminobutane, l-phenyl-3-aminobutane, 1-(4-hydroxyphenyl)-3-aminobutane, 1-amino-2-methylcyclopentane, l-amino-3-methylcyclopentane Examples of suitable isomers include 1-aminopentane, 1-amino-2-methylcyclohexane, 1-amino-1-(2-naphthyl)ethane, 3-methylcyclopentylamine, 2-methylcyclopentylamine, 2-ethylcyclopentylamine, 2-methylcyclohexylamine, 3-methylcyclohexylamine, 1-aminotetralin, 2-aminotetralin, 2-amino-5-methoxytetralin, and 1-aminoindan, including both the (R) and (S) single isomers where possible.

[0163] In some embodiments, a method for converting an ester substrate, ethyl oxazole-5-carboxylate, comprises contacting about 36 mL / L of the ester substrate with about 20 g / L of a carboxylesterase described herein in MIBK in the presence of 43 mL / L of 1-isopropylpiperazine at a temperature of about 30° C., where at least 80%, 85%, 90%, 92%, 94%, 96%, or 98% or more of the ester substrate is converted to product in 24 hours. In some embodiments, a carboxylesterase polypeptide capable of carrying out the above reaction comprises an amino acid sequence corresponding to SEQ ID NO:122.

[0164] In some embodiments, the above method may further comprise the step of isolating the compound of formula III or the compound of formula V from the reaction solvent.

[0165] Also provided herein are compositions of carboxylesterases and substrates / products. In some embodiments, these compositions can comprise a compound of Formula III or Compound V and an improved carboxylesterase of the present disclosure.

[0166] Any one or more of the improved engineered carboxylesterases can be part of the composition. EXAMPLES

[0167] Various features and embodiments of the present disclosure are illustrated in the following representative examples, which are illustrative and not intended to be limiting.

[0168] Example 1: Obtaining A. acidocaldarius esterase 2 wild-type carboxylesterase gene and constructing expression vector A carboxylesterase (CE)-encoding gene was designed for expression in E. coli based on the reported amino acid sequence of the carboxylesterase A. acidocaldariu esterase 2 (SEQ ID NO:2) and the codon optimization algorithm described in Example 1 of patent application US2008 / 0248539. The oligonucleotides were synthesized separately and then ligated using an oligonucleotide generally consisting of 42 nucleotides. The gene was then cloned under the control of the lac promoter in the expression vector pCK110900 (shown in FIG. 3 of US application 2006 / 195947, both of which are incorporated herein by reference in their entirety for all purposes). This expression vector also contains a P15a origin of replication and a chloramphenicol resistance gene. The resulting plasmid was transformed into E. coli W3110 using standard methods. The codon-optimized gene and the encoded polypeptide are shown in Table 3 and in the sequence listing below as SEQ ID NOs:1 and 2, respectively.

[0169] Similarly, genes encoding the engineered carboxylesterases of the present disclosure listed in Table 3 (SEQ ID NOs: 3-94) were cloned into the vector pCK110900 for expression in E. coli W3110.

[0170] Example 2: Production of Carboxylesterase Powder - Shake Flask Method A single microbial colony of E. coli containing a plasmid encoding the carboxylesterase of interest was inoculated into 50 mL of Luria Bertoni broth containing 30 μg / mL chloramphenicol and 1% glucose. The cells were grown overnight (at least 16 hours) in an incubator at 30°C with shaking at 250 rpm. The culture was diluted with 1000 mL of Terrific broth containing 30 μg / mL chloramphenicol to an OD600 of approximately 0.2 and grown at 30°C with shaking at 250 rpm. Expression of the carboxylesterase gene was induced by adding isopropyl βD-thiogalactoside (IPTG) to a final concentration of 1 mM when the culture had an OD600 of 0.6-0.8, after which incubation was continued overnight (at least 16 hours). Cells were harvested by centrifugation (3738 RCF, 20 min, 4°C) and the supernatant was discarded. The pellet was frozen at -80°C for 2 hours. The pellet was then thawed and resuspended in 3 mL of sodium sulfate buffer (consisting of 15 g / L anhydrous sodium sulfate in water) per gram of final pellet mass (e.g., 10 g of frozen pellet is suspended in 30 mL of sodium sulfate buffer). Cell debris was removed by centrifugation (15,777 RCF, 40 min, 4°C). The supernatants of the clear lysates were collected, pooled, and lyophilized to obtain a dry powder of crude carboxylesterase enzyme.

[0171] Example 3: Production of Carboxylesterase Powder - Fermentation Method An aliquot of frozen working stock (E. coli containing a plasmid with the carboxylesterase gene of interest) was removed from the freezer and allowed to thaw at room temperature. 300 μL of this working stock was inoculated into a primary seed stage of 250 ml of M9YE medium (1.0 g / L ammonium chloride, 0.5 g / L sodium chloride, 6.0 g / L disodium monohydrogen phosphate, 3.0 g / L potassium dihydrogen phosphate, 2.0 g / L Tastone-154 yeast extract, 1 L / L deionized water) containing 30 μg / ml chloramphenicol and 1% glucose in a 1 L flask. Grow at 26 °C with shaking at 220 pm. The flask was removed from the incubator when the culture had an OD600 of 0.5-1.0 and used immediately to inoculate a secondary seed stage.

[0172] The secondary seed stage was performed in a bench-scale 5 L fermenter using 4 L of growth medium (0.88 g / L ammonium sulfate, 0.98 g / L sodium citrate; 12.5 g / L dipotassium hydrogen phosphate trihydrate, 6.25 g / L potassium dihydrogen phosphate, 3.3 g / L Springer 0251 yeast extract, 0.083 g / L ferric ammonium citrate, 0.5 mL / L antifoam, and 8.3 ml / L of trace element solution containing 2 g / L calcium chloride dihydrate, 2.2 g / L zinc sulfate heptahydrate, 0.5 g / L manganese sulfate monohydrate, 1 g / L cuprous sulfate heptahydrate, 0.1 g / L ammonium molybdate tetrahydrate, and 0.02 g / L sodium tetraborate) sterilized at 120° C. for 40 minutes. The fermenter was inoculated with 2 ml of primary seed at OD 0.5-1.0 and incubated at 30°C, 300 rpm and 0.5 vvm aeration. When the culture reached an OD600 of 0.5-1.0 OD600, the secondary seed was immediately transferred to the final stage of fermentation.

[0173] Final stage fermentation was performed at bench scale in a 10 L fermenter sterilized at 121°C for 40 min and then sterilized in 6 L of growth medium (0.88 g / L ammonium sulfate, 0.98 g / L sodium citrate; 12.5 g / L dipotassium hydrogen phosphate trihydrate, 6.25 g / L potassium dihydrogen phosphate, 3.3 g / L Springer Buffered Soluble Hydrogen ... The fermentation was carried out in 0251 yeast extract, 0.083 g / L ferric ammonium citrate, 0.5 mL / L antifoam, and 8.3 ml / L trace element solution containing 2 g / L calcium chloride dihydrate, 2.2 g / L zinc sulfate heptahydrate, 0.5 g / L manganese sulfate monohydrate, 1 g / L cuprous sulfate heptahydrate, 0.1 g / L ammonium molybdate tetrahydrate, and 0.02 g / L sodium tetraborate. The fermenter was inoculated with 500 ml of secondary seed at OD600 0.5-1.0 and incubated at 30 C and 1.6 vvm aeration. Dissolved oxygen was controlled at 30% by variable shaking speed from 300-950 rpm. pH was maintained at 7.0 by addition of 20% v / v ammonium hydroxide. Growth of the culture was maintained by adding a feed solution containing 500 g / L glucose monohydrate, 12 g / L ammonium chloride and 5.1 g / L magnesium sulfate heptahydrate.

[0174] After the culture reached an OD600 of 80+ / -10, expression of the carboxylesterase was induced by adding isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 1 mM, and fermentation was continued for an additional 24 hours. The culture was then cooled to 8°C and maintained at this temperature until harvest. Cells were harvested by centrifugation at 5000G for 40 minutes at 4°C in a Sorvall RC12BP centrifuge. The harvested cell pellet was then frozen at -80°C and stored until downstream processing and regeneration as described below.

[0175] The pellet was frozen at -80°C for 2 hours. The pellet was then thawed and resuspended in 3 mL of sodium sulfate buffer (consisting of 15 g / L anhydrous sodium sulfate in water) per gram of final pellet mass (e.g., 10 g of frozen pellet suspended in 30 mL of sodium sulfate buffer). After resuspension, the cells were filtered through a 200 um mesh and then passed twice through a microfluidizer at 12000 psig. Cell debris was removed by centrifugation (15,777 RCF, 40 min, 4°C). The supernatant of the clear lysate was collected, pooled, and lyophilized to obtain a dry powder of crude carboxylesterase enzyme. The carboxylesterase powder was stored at -80°C.

[0176] Example 4: High-throughput assay for the identification of mutants of A. acidocaldarius esterase 2 capable of converting ester substrates to amides under aqueous conditions A UPLC method for determining the conversion of ester substrate I to amide III The enzymatic conversion of ester substrates of formula I (commercially available, CAS number 118994-89-1) to amides of formula III was determined using an Agilent 1290 UPLC equipped with an Agilent Zorbax RRHD Eclipse Plus Phenyl-Hexyl column (3.0×50 mm, 1.8 μm) using a gradient of 5 mM NH4Ac in water (mobile phase A) and acetonitrile (mobile phase B) at a flow rate of 2 mL / min and a column temperature of 60° C. Starting with a 99.9:0.1 ratio of A:B, the method followed a 0.25 min hold, followed by a 0.05 min gradient to 80:20 A:B, then a 0.5 min gradient to 60:40 A:B, then a 0.1 min purge gradient to 0:100 A:B, a 0.2 min hold at 0:100 A:B, and a 0.1 min gradient to 99.9:0.1 A:B, and finally a 0.3 min hold at 99.9:0.1 A:B. Elution of the compounds was monitored at 210 nm and 230 nm, with the ester eluting at 0.56 min, the amide eluting at 0.52 min, and the acid by-product of the reaction eluting as a narrow peak near the solvent front at 0.14 min.

[0177] UPLC method for determining the conversion of ester substrates of formula I to amides of formula III:The enzymatic conversion of ester substrates of formula I to amides of formula III was determined using an Agilent 1290 UPLC equipped with an Agilent Zorbax SB-C18 RRHD column (3.0×50 mm, 1.8 μm) using a gradient of 0.05% TFA in water (mobile phase A) and 0.05% TFA in acetonitrile (mobile phase B) at a flow rate of 2 mL / min and a column temperature of 60° C. Starting with a 99.9:0.10 ratio of A:B, the method followed a 0.25 min hold, followed by a 0.0.25 min gradient to 80:20 A:B, followed by a 0.1 min gradient to 100:0 A:B, followed by a 0.1 min hold, followed by a 0.1 min gradient to 99.9:0.1 A:B, followed by a 0.2 min hold. Elution of the compounds was monitored at 210 nm and 230 nm with the ester eluting at 0.53 min, the amide eluting at 0.23 min and the acid by-product of the reaction eluting as a narrow peak near the solvent front at 0.2 min with an injection volume of 1 uL.

[0178] UPLC method for determining the conversion of ester substrates of formula I to amides of formula V: The enzymatic conversion of the ester substrate of formula I to the amide of formula V was determined using an Agilent 1290 UPLC equipped with an Agilent Zorbax SB-C18 column (3.0×50 mm, 1.8 μm) using a gradient of 0.05% TFA in water (mobile phase A) and 0.05% TFA in acetonitrile (mobile phase B) at a flow rate of 1.5 mL / min and a column temperature of 60° C. Starting with an 80:20 ratio of A:B, the method followed a 0.9 min hold, followed by a 0.1 min gradient to 0:100 A:B, followed by a 0.1 min gradient to 80:20 A:B, followed by a 0.4 min hold. Compound elution was monitored at 210 nm and 230 nm, with the ester eluting at 0.5 min, the amide eluting at 0.39 min, and the acid by-product of the reaction eluting as a narrow peak near the solvent front at 0.14 min.

[0179] Example 5: High-throughput screening for the identification of mutants of A. acidocaldarius esterase 2 capable of converting ester substrates to amides The gene encoding A. acidocaldarius esterase 2 (SEQ ID NO:2), constructed as described in Example 1, was mutagenized using the methods described below and the population of altered DNA molecules was used to transform a suitable E. coli host strain. Antibiotic resistant transformants were selected and processed to identify those expressing carboxylesterases with improved ability to convert ester substrates of formula I to compounds of formulas (III) and (V) in the presence of amine substrates of either formula (II) or (IV), respectively. Cell selection, growth, induction of expression of the carboxylesterase mutant enzymes and collection of cell pellets are described below.

[0180] Recombinant E. coli colonies carrying genes encoding carboxylesterases were picked using a Q-PIX molecular devices robotic colony picker (Genetix USA, Inc., Boston, MA) into 96-well shallow-bottom microtiter plates containing 180 μL of LB broth, 1% glucose, and 30 μg / mL chloramphenicol (CAM) per well. The cells were grown overnight at 30 °C with shaking at 200 rpm. A 20 μL aliquot of this culture was then transferred to a 96-well deep-bottom plate containing 380 μL of TB broth and 30 μg / mL CAM. After incubating these deep-bottom plates for 2-3 h at 30 °C with shaking at 250 rpm, expression of the recombinant genes in the cultured cells was induced by adding IPTG to a final concentration of 1 mM. The plates were then incubated for 18 h at 30 °C with shaking at 250 rpm.

[0181] Cells were pelleted by centrifugation (3738RCF, 10 min, 4°C), resuspended in 200 μL lysis buffer, and lysed by shaking at room temperature for 2 h. For lyophilized screening conditions, the lysis buffer contained 100 mM sodium sulfate (14.2 g / L), 1 mg / mL lysozyme, 500 μg / mL polymyxin B sulfate (PMBS), and 12.5 U / mL benzonase. For aqueous screening conditions, the lysis buffer contained 10 mM potassium phosphate, pH 7.0, 1 mg / mL lysozyme, and 500 μg / mL PMBS. After sealing the plates with breathable nylon seals, they were shaken vigorously at room temperature for 2 h. Cell debris was pelleted by centrifugation (3738RCF, 10 min, 4°C), and the clear supernatant was assayed directly or stored at 4°C until use.

[0182] For screening in semi-aqueous conditions with early stage engineered carboxylesterases, 120 μL aliquots of substrate solution (720 mL / L DMSO, 90 mL / L 200-proof ethanol, 19.67 mL / L isopropyl-piperazine, 23.5 mL / L ester substrate, and 8.5 mL / L 6N HCl) were added to each well of a Costar deep-well plate, followed by 80 μL of collected lysate supernatant using a Biomek FX robotic instrument (Beckman Coulter, Fullerton, CA). A solution of 100 mM ester substrate, 100 mM isopropyl-piperazine, 45% DMSO, 5% EtOH with a final pH of 9.0 was obtained. The plates were heat sealed with aluminum / polypropylene laminate heat seal tape at 165° C. for 4 seconds, and then shaken at 50° C. overnight (at least 16 hours). Reactions were quenched by adding 200 μL of acetonitrile using a Biomex FX. Plates were resealed and shaken for 5 minutes before being centrifuged at 3738 RCF for 10 minutes. 20 μL of substrate samples were transferred to shallow-bottom polypropylene plates (Costar #3365) containing 180 μL of 75% acetonitrile in water, sealed, shaken for 10 minutes, and then analyzed as described in Example 4.

[0183] For early-stage organic screening with engineered carboxylesterases, 150 μL aliquots of harvested lysate supernatants were added to aluminum 96-well racks (F158359, Unchained Labs, Pleasanton, CA) containing 1 mL glass vial inserts (S11168, Unchained Labs, Pleasanton, CA). The apparatus was then lyophilized, gently warmed to room temperature, and 4 μL of distilled water was added using a Multidrop Combi Reagent Dispenser (Thermo Scientific, Waltham, MA), followed by 200 μL of organic substrate solution (10.93 mL / L isopropyl-piperazine, 23.5 mL / L ester substrate) in tert-butyl methyl ether (tBME). The plates were then sealed by use of a Teflon sheet (S11690-2, Unchained Labs, Pleasanton, CA) laminated under two rubber gaskets (S13086, Unchained Labs, Pleasanton, CA) and a metal rack lid (F158424, Unchained Labs, Pleasanton, CA) attached by seven screws (C151943-050, Unchained Labs, Pleasanton, CA). The constructs were then incubated overnight (at least 16 hours) at 50°C with shaking. The reactions were quenched by adding 200 μL of isopropyl alcohol by a Biomek FX before being sealed, shaken for 10 minutes, and centrifuged at 235 RCF for 2 minutes to settle any remaining solids. 200 μL of sample was then transferred to a Costar deep-bottom plate containing 200 μL of isopropanol per well, heat sealed with aluminum / polypropylene laminate heat seal tape at 165° C. for 4 seconds, shaken for 10 minutes, and centrifuged at 3738 RCF for 10 minutes. 40 μL of substrate sample was transferred to a shallow-bottom polypropylene plate (Costar #3365) containing 160 μL of isopropanol, sealed, shaken for 10 minutes, and then analyzed as described in Example 4.

[0184] For organic screening with late-stage engineered carboxylesterases, 120 μL aliquots of the harvested lysate supernatant were added to an aluminum 96-well rack (F158359, Unchained Labs, Pleasanton, CA) containing a 1 mL glass vial insert (S11168, Unchained Labs, Pleasanton, CA). The device was then lyophilized, gently warmed to room temperature, and 200 μL of organic substrate solution (53.2 mL / L isopropyl-piperazine, 43 mL / L ester substrate) in methyl isobutyl ester (MIBK) was added, followed by 4 μL of distilled water using a Multidrop Combi Reagent Dispenser (Thermo Scientific, Waltham, MA). The plates were then sealed by use of a Teflon sheet (S11690-2, Unchained Labs, Pleasanton, CA) laminated under one rubber gasket (S13086, Unchained Labs, Pleasanton, CA) and a metal rack lid (F158424, Unchained Labs, Pleasanton, CA) attached by five screws (C151943-050, Unchained Labs, Pleasanton, CA). The constructs were then incubated overnight (at least 16 hours) at 15°C with shaking. The reactions were removed from the incubator and centrifuged at 235 RCF. 20 μL of supernatant samples were transferred to shallow-bottom polypropylene plates (Costar #3365) containing 180 μL of isopropanol (containing 5 g / L naphthalene) per well, heat sealed with aluminum / polypropylene laminate heat seal tape at 165° C. for 4 seconds, shaken for 10 minutes, and then centrifuged at 3738 RCF for 10 minutes. 10 μL of substrate samples were transferred to shallow-bottom polypropylene plates (Costar #3365) containing 190 μL of isopropanol, sealed, shaken for 10 minutes, and then analyzed as described in Example 4.

[0185] Example 6: Amidation of ester substrates of formula I and amine substrates of formula II in methyl isobutyl esterase (MIBK) by engineered carboxylesterase derived from A. acidocaldarius esterase 2 The improved late-stage carboxylesterases described in Table 3 were evaluated at a preparative scale in MIBK as follows: 10 mg of lyophilized enzyme powder was added to 1.5 mL HPLC vials with 15 μL of distilled water. 485 μL of substrate solution (42.93 mL isopropylpiperazine / L MIBK, 36.4 mL ester / L MIBK) was then added and the vials were sealed. The reactions were shaken at 50° C. and 850 rpm on an Eppendorf Thermomixer C heated vial shaker for 16 hours. The reactions were quenched by the addition of 500 μL isopropanol. 50 μL of sample was then transferred to a shallow-bottom polypropylene plate (Costar #3365) containing 150 μL isopropanol and shaken for 10 minutes before centrifuging at 3738 RCF for 10 minutes. 10 μL of substrate samples were transferred to shallow-bottom polypropylene plates (Costar #3365) containing 190 μL of isopropanol, sealed, shaken for 10 minutes, and then analyzed as described in Example 4.

[0186] The improved late-stage carboxylesterases described in Table 3 were evaluated on a preparative scale in MIBK as follows: 11.25 mg of lyophilized enzyme powder was added to 1.5 mL HPLC vials, then 750 μL of substrate solution (53.2 mL isopropylpiperazine / L MIBK, 43 mL ester / L MIBK) was added along with 15 μL of distilled water, and the vials were sealed. The reactions were shaken at 15° C. and 850 rpm on an Eppendorf Thermomixer C heated vial shaker for 16 hours. The reactions were quenched by removing 20 uL as described in Example 5 for late-stage engineered carboxylesterase screening. Table 3 shows the sequence numbers corresponding to the carboxylesterase variants tested in this method as well as the number of amino acid residue differences from the A. acidocaldarius esterase 2 wild-type carboxylesterase (SEQ ID NO: 2).

[0187] Example 7: Amidation of ester substrate I and amine substrate II in methyl isobutyl esterase (MIBK) by engineered carboxylesterase derived from A. acidocaldarius esterase The following examples describe a gram-scale method used to increase the conversion of the ester substrate, ethyl oxazole-2-carboxylate, compound of formula I, and the amine substrate, 1-isopropylpiperazine, compound of formula II. The method utilizes improved liquid mixing at a large scale to increase the conversion of substrates to products. Related monitoring indicates that capture and isolation of over 70% of the total product yield is possible.

[0188] The large scale reaction process contains the following reaction components: [Table 4]

[0189] method To a 2L CLR reactor equipped with an overhead stirrer, 340mL of MIBK was added and set to agitation at ambient temperature. Then, 11mL of water was added and then gently heated to 30°C. 14g of carboxylesterase was added followed by 20mL of MIBK wash. Next, 23.62g of 1-isopropylpiperazine was added followed by 20mL of MIBK wash. Finally, 20g of ethyl oxazole 2-carboxylate was added followed by a final 20mL of MIBK wash. After 16 hours, the conversion was over 90%.

[0190] The remaining solids were filtered and washed with 80 mL of MIBK to isolate the adsorbed material. The washes were then pooled and washed with 20 mL of 10% w / w sodium chloride, stirred and separated. The organic phase was collected and concentrated under vacuum to 60 mL and then cooled to 15° C., at which point crystallization began to occur. 70 mL of n-heptane was added over 10 minutes and crystallization was allowed to proceed to completion over 2 hours, after which the crystalline material was filtered off. The crystalline product was washed twice with 80 mL of 1:4 MIBK:n-heptane and dried under vacuum at 50° C. This method gave an overall yield (w / w) of 66% as assessed by the mass of the product.

[0191] Example 8: Amidation of ester substrate I and amine substrate IV in methyl isobutyl esterase (MIBK) by engineered carboxylesterase derived from A. acidocaldarius esterase Vial-scale reactions were performed to produce amides of formula V using the engineered carboxylesterases. 25 mg of lyophilized enzyme powder was added to 1.5 mL HPLC vials along with 10 μL of distilled water. 490 μL of substrate solution (26.72 mL of (2S,6R)-2,6-dimethylmorpholine / L MIBK, 24.27 mL of ester / L MIBK) was then added and the vials were sealed. The reactions were shaken at 50° C. and 800 rpm on a heated vial shaker for 16 hours. A 100 μL aliquot of each reaction solution was added to a shallow-bottom polypropylene plate (Costar #3365) containing 100 μL of isopropanol, sealed, shaken for 10 minutes, and then centrifuged at 3738 RCF for 10 minutes. A 10 μL aliquot of the supernatant was then diluted in 190 μL of acetonitrile containing 25% water in a shallow-bottom polypropylene plate (Costar #3365), sealed, and shaken at 850 rpm for 5 minutes. The reactions were analyzed as described in Example 4. In all cases observed, the enzyme activities closely matched those of the reactions described in Example 6 and Table 3.

[0192] While various specific embodiments have been illustrated and described, it will be understood that various changes can be made without departing from the spirit and scope of the invention.

[0193] [Table 5] TIFF0007663635000022.tif224150 TIFF0007663635000023.tif221150 TIFF0007663635000024.tif222150 TIFF0007663635000025.tif216150 TIFF0007663635000026.tif221150 TIFF0007663635000027.tif221150 TIFF0007663635000028.tif221150 TIFF0007663635000029.tif222150 TIFF0007663635000030.tif213150 TIFF0007663635000031.tif225150 TIFF0007663635000032.tif222150 TIFF0007663635000033.tif221150 TIFF0007663635000034.tif221150 TIFF0007663635000035.tif223150 TIFF0007663635000036.tif221150 TIFF0007663635000037.tif222150 TIFF0007663635000038.tif219150 TIFF0007663635000039.tif221150 TIFF0007663635000040.tif218150 TIFF0007663635000041.tif222150 TIFF0007663635000042.tif220150 TIFF0007663635000043.tif220150 TIFF0007663635000044.tif219150 TIFF0007663635000045.tif218150 TIFF0007663635000046.tif218150 TIFF0007663635000047.tif221150 TIFF0007663635000048.tif219150 TIFF0007663635000049.tif218150 TIFF0007663635000050.tif217150 TIFF0007663635000051.tif219150

Claims

1. A carboxylesterase polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:4, or a functional fragment thereof, wherein the amino acid sequence of the carboxylesterase polypeptide comprises the characteristic that the residue corresponding to X198 in SEQ ID NO:4 is selected from F, I, Y, and M.

2. 2. The carboxylesterase polypeptide of claim 1, wherein the amino acid sequence of the carboxylesterase polypeptide comprises a residue which differs from the amino acid sequence set forth in SEQ ID NO:4 at at least one residue position selected from X27, X30, X35, X37, X57, X75, X103, X185, X207, X208, X271, X286, and X296.

3. The amino acid sequence of the carboxylesterase polypeptide is 3. The carboxylesterase polypeptide of claim 1 or 2, comprising at least one characteristic in which the residue corresponding to X103 is selected from an aliphatic residue and an aromatic residue.

4. The carboxylesterase polypeptide of claim 3, wherein X103 is an aromatic residue.

5. The carboxylesterase polypeptide of claim 4, wherein X103 is W.

6. 6. The carboxylesterase polypeptide of any one of claims 1 to 5, wherein the amino acid sequence of the carboxylesterase polypeptide comprises the characteristic that the residue corresponding to X185 is selected from a non-polar residue, an aliphatic residue, and an aromatic residue.

7. The carboxylesterase polypeptide of claim 6, wherein X185 is an aromatic residue.

8. The carboxylesterase polypeptide of claim 7, wherein X185 is F.

9. 4. The carboxylesterase polypeptide of any one of claims 1 to 3, wherein the amino acid sequence of the carboxylesterase polypeptide comprises at least one feature selected from: X27 is P; X30 is selected from I, L, and V; X35 is H; X37 is selected from I, L, T, and V; X57 is M; X75 is R; X103 is selected from F, M, and W; X185 is selected from F, I, and M; X207 is E; X208 is selected from R, L, and H; X271 is D; X286 is selected from M, V, and G; and X296 is selected from V, L, and R.

10. A polynucleotide encoding the polypeptide according to any one of claims 1 to 9.

11. A vector comprising the polynucleotide of claim 10.

12. A host cell comprising the vector of claim 11.

13. A composition comprising at least one engineered carboxylesterase according to any one of claims 1 to 9.

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