EA043748B1 - PENICILLIN G ACYLASES - Google Patents

Description

This application claims benefit to U.S. Patent Application Serial Nos. 62/442810 and 62/472055, filed January 5, 2017 and March 16, 2017, respectively, both of which are hereby incorporated by reference in their entirety for all purposes. .

Field of invention

The present invention provides engineered penicillin G acylase (PGA) enzymes, polynucleotides encoding the enzymes, compositions containing the enzymes, and methods of using the engineered PGA enzymes.

Link to sequence list, table, or computer program

An official copy of the sequence listing is submitted along with the description as an ASCII text file via EFS-Web with the file name Sequence Listings^, creation date June 5, 2019, and size 967 KB. The sequence listing provided via EFS-Web is part of the specification and is incorporated in its entirety by reference into this specification.

BACKGROUND OF THE INVENTION

Penicillin G acylase (PGA) (penicillin amidase, EC 3.5.1.11) catalyzes the cleavage of the amide bond of the penicillin G (benzylpenicillin) side chain. The enzyme is used commercially in the preparation of 6-amino-penicillanic acid (6-APA) and phenylacetic acid (PAA). 6-APA is a key compound in the industrial production of semisynthetic β-lactam antibiotics such as amoxicillin, ampicillin and cephalexin. The naturally occurring PGA enzyme exhibits instability in commercial processes, requiring immobilization on solid supports for commercial applications. PGA is covalently linked to various backbones, and systems with immobilized PGA have been described as effective tools for the synthesis of pure optical isomers. However, attachment to solid surfaces leads to impairment of enzyme properties, such as reduced activity and/or selectivity, and limited access of solutes. In addition, although attachment to solid supports allows enzymes to be captured and reused in additional processing cycles, the stability of the enzyme is such that such applications may be limited. The enzymatic catalysis of penicillin G by PGA to 6APA is regiospecific (no cleavage of the lactam-amide bond occurs) and stereospecific. The production of 6-APA is perhaps the greatest benefit from enzymatic catalysis in the production of pharmaceuticals. The enzymatic activity of PGA, associated with the phenacetyl moiety, allows stereospecific hydrolysis of a wide range of phenacetyl derivatives of primary amines, as well as alcohols.

The essence of the invention

The present invention provides engineered penicillin G acylase (PGA) enzymes, polynucleotides encoding the enzymes, compositions containing the enzymes, and methods of using the engineered PGA enzymes. The present invention provides engineered penicillin G acylases capable of acylating insulin, wherein the polypeptide sequence of said penicillin G acylase is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% or greater identical to SEQ ID Nos. 2, 4, 12, 24, 40, 56, 70, 82, 100, 108, 110, 116, 136, 142, 154, and 160. In some embodiments, penicillin acylase G contains SEQ ID No. 4, 12, 24, 40, 56, 70, 82, 108, 110, 116, 136, 142, 154, or 160. In some additional embodiments, the engineered penicillin acylase G contains a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to at least one sequence shown in table. 5.1, 5.1, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 7.1, 8.1, 9.1, 11.1, 12.1, 13.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1 and/or table. 20.1. In some further embodiments, the engineered penicillin G acylase comprises the sequence comprising the sequence shown in Table. 5.1, 5.1, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 7.1, 8.1, 9.1, 11.1, 12.1, 13.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1 and/or table. 20.1. In some other additional embodiments, the engineered penicillin G acylase contains a histidine tag. In some embodiments, a histidine tag is present at the C-terminus of the engineered penicillin G acylase. In some further embodiments, the engineered penicillin G acylase comprises a polypeptide sequence selected from SEQ ID NO: 110 and SEQ ID NO: 142.

The present invention also provides engineered polynucleotide sequences encoding the engineered penicillin G acylases provided herein. In some embodiments, the engineered polynucleotide sequence comprises a polynucleotide sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identical to SEQ ID Nos. 1, 11, 23, 39, 55, 69, 81, 99, 107, 109, 115, 135, 141, 153 and/or 159.

The present invention also relates to vectors containing the designed polynucleotide sequences provided herein. In some embodiments, the vectors contain at least one engineered polynucleotide sequence comprising polynucleotide sequence(s) of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% or more identical to SEQ ID Nos. 2, 11, 23, 39, 55, 69, 81, 99,

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107, 109, 115, 135, 141, 153 and/or 159. In some further embodiments, the vectors comprise at least one escape sequence.

The present invention also relates to host cells containing at least one vector as provided herein. In some embodiments, the host cell vectors comprise at least one engineered polynucleotide sequence comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide sequence(s) 96%, 97%, 98%, 99% or greater identical to SEQ ID Nos. 1, 11, 23, 39, 55, 69, 81, 99, 107, 109, 115, 135, 141, 153 and/or 159. In some further embodiments, the vectors comprise at least one escape sequence.

The present invention also relates to methods for producing acylated insulin, which include providing: at least one engineered penicillin G acylase according to any one of claims 1 to 7 and insulin; and exposing the engineered penicillin G acylase and insulin under conditions such that the engineered penicillin G acylase acylates insulin, thereby producing acylated insulin. In some embodiments, the acylation is carried out in the presence of methyl phenylacetate. In some further embodiments, the acylation occurs at any of the A1, B1 and/or B29 positions of said insulin. In some further embodiments, the acylation occurs at the A1 position of the insulin, while in some alternative embodiments, the acylation occurs at the B1 position of the insulin, and in other embodiments, the acylation occurs at the B29 position of the insulin. In some embodiments, acylation occurs at positions A1, B1 and/or B29 of said insulin. In some additional embodiments, acylation occurs at positions A1, B1, and B29 of insulin. In some further embodiments of the methods, the engineered penicillin G acylase produces greater than 90% more acylated insulin compared to producing acylated insulin using the polypeptide of SEQ ID NO: 2, 4, 12, 24, 40, 56, 70, 82, 100, 108, 110, 116, 136, 142, 154 and/or 160.

The present invention also relates to acylated insulin compositions prepared using at least one engineered penicillin G acylase provided herein. In some further embodiments, the present invention provides compositions comprising acylated insulin produced using at least one method provided herein.

Brief description of the figures

In fig. 1 shows a chromatogram for the analytical method described in table. 21.5, which is used to quantify insulin and the elution order of acylated products.

In fig. 2 shows the results of the experiments described in example 10.

In fig. 3 shows the results of the experiments described in example 16.

Description of the invention

The present invention provides engineered penicillin G acylases (PGAs) that are capable of degrading penicillin to phenylacetic acid and 6-aminopenicillanic acid (6-APA), which is a key intermediate in the synthesis of a wide range of β-lactam antibiotics. In particular, the present invention provides engineered PGAs that are capable of producing monoprotected or diprotected phenylacetate insulin by adding a protecting group to the A1, B1 or B29 positions of free insulin or removing protecting groups from A1/B1/B29 of triprotected insulin or removing A1/B1/B29 of triphenylacetate protective groups in order to release free insulin.

In general, naturally occurring PGAs are heterodimeric enzymes consisting of an alpha subunit and a beta subunit. In nature, wild-type PGA is synthesized as a prepro-PGA polypeptide containing an N-terminal signal peptide that mediates translocation into the periplasm and a linker region connecting the C-terminus of the alpha subunit to the N-terminus of the beta subunit. Proteolytic processing leads to a mature heterodimeric enzyme. The intermolecular linker region may also serve the function of facilitating proper folding of the enzyme. The PGAs provided herein are based on PGAs from Kluyvera citrophila, into which various modifications have been introduced in order to create improved enzymatic properties, as described in detail below.

For the purposes of the descriptions provided herein, the use of the singular form includes the plural (and vice versa) unless specifically stated otherwise. For example, singular forms include the plural unless the context clearly indicates otherwise. Likewise, contain, contains, containing, include, includes, and including are interchangeable and not intended to be limiting. It should also be understood that when the term comprising is used in the descriptions of various embodiments, those skilled in the art will appreciate that in some particular cases, an embodiment may alternatively be described using the language consisting essentially of or consisting of.

Both the foregoing general description, including the drawings, and the following detailed description are exemplary and explanatory only and are not intended to limit this disclosure. In addition, section headings used in this specification are for organizational purposes only and should not be construed as limiting the subject matter described.

Definitions

As used herein, the following terms are given the following meanings.

With respect to the present disclosure, technical and scientific terms used in the descriptions herein shall have the meanings in which they are commonly understood by one skilled in the art, unless specifically defined otherwise. Accordingly, the following terms are given the following meanings. All patents and publications, including all sequences disclosed in such patents and publications mentioned herein, are expressly incorporated by reference. Unless otherwise indicated, the practice of the present invention includes conventional methods widely used in molecular biology, fermentation, microbiology and related fields that are known to those skilled in the art. Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention relates. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, preferred methods and materials are described. Indeed, the present invention is not intended to be limited to the specific methods, protocols and reagents described herein, as they may vary depending on the context in which they are used. The headings provided herein are not intended to limit the various aspects or embodiments of the present invention.

However, to facilitate understanding of the present invention, many terms are defined below. Numeric ranges include numbers that define a range. Thus, each numerical range disclosed herein is intended to cover each narrower numerical range that falls within such narrower numerical range as if all such narrower numerical ranges were expressly set forth herein. It is also provided that each maximum (or minimum) numerical limitation disclosed herein includes each lesser (or greater) numerical limitation as if such lesser (or greater) numerical limitations were expressly set forth herein.

As used herein, the term containing and its derivatives are used in their inclusive meaning (ie, equivalent to the term including and its corresponding derivatives).

As used herein and in the appended claims, the singular form includes the plural unless the context clearly indicates otherwise. Thus, for example, reference to a host cell includes a plurality of such host cells.

Unless otherwise noted, nucleic acids are written from left to right in a 5' to 3' orientation and amino acid sequences are written from left to right in an amino to carboxy orientation, respectively.

The headings provided herein are not intended to limit the various aspects or embodiments of the invention, which may be obtained by reference to the specification as a whole. Accordingly, the terms that are defined below are more fully defined by reference to the description as a whole.

As used herein, the terms protein, polypeptide, and peptide are used interchangeably herein to refer to a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modifications (e.g., glycosylation, phosphorylation, lipidation, myristylation, ubiquitinylation, and etc.). Included in this definition are D- and L-amino acids and mixtures of D- and L-amino acids.

As used herein, polynucleotide and nucleic acid refer to two or more nucleosides that are covalently linked together. A polynucleotide may be composed entirely of ribonucleosides (i.e., RNA), composed entirely of 2'-deoxyribonucleotides (i.e., DNA), or mixtures of ribo- and 2'-deoxyribonucleosides. Although nucleosides are usually linked together through standard phosphodiester linkages, polynucleotides may contain one or more non-standard linkages. The polynucleotide may be single-stranded or double-stranded, or may contain both single-stranded regions and double-stranded regions. In addition, although a polynucleotide typically consists of naturally occurring coding nucleic acid bases (i.e., adenine, guanine, uracil, thymine, and cytosine), it may contain one or more modified and/or synthetic nucleic acid bases (e.g., inosine, xanthine, hypoxanthine, etc.). Preferably, such modified or synthetic nucleic acid bases will be coding nucleic acid bases.

As used herein, the degree of hybridization stringency refers to the hybridization conditions, such as washing conditions, when hybridizing nucleic acids. Overall, the reactions

- 3 043748 hybridizations are carried out under lower stringency conditions, followed by variable but higher stringency washes. The term moderately stringent hybridization refers to conditions that allow the binding of target DNA to a complementary nucleic acid that has about 60% identity, preferably about 75% identity, about 85% identity to the target DNA; with greater than approximately 90% identity to the target polynucleotide. Exemplary moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5x Denhart's solution, 5x SSPE, 0.2% SDS at 42°C, followed by a wash in 0.2x SSPE, 0.2% SDS at 42°C WITH. High stringency hybridization generally refers to conditions that are approximately 10° C. or less of the thermal melting temperature T _m as determined under solution conditions for a particular polynucleotide sequence. In some embodiments, high stringency conditions refer to conditions that allow hybridization of only those nucleic acid sequences that form stable hybrids in 0.018 M NaCl at 65°C (i.e., if the hybrid is not stable in 0.018 M NaCl at 65°C , it will not be stable under high stringency conditions as provided herein). High stringency conditions can be achieved, for example, by hybridization under conditions equivalent to 50% formamide, 5x Denhart's solution, 5x SSPE, 0.2% SDS at 42°C, followed by a wash in 0.1x SSPE and 0.1% SDS at 65°C. Other high stringency conditions are hybridization under conditions equivalent to hybridization in 5x SSC containing 0.1% (w:v) SDS at 65°C and washing in 0.1x SSC containing 0.1% SDS at 65°C. Other high stringency hybridization conditions, as well as moderate stringency conditions, are known to those skilled in the art.

As used herein, a coding sequence refers to that portion of a nucleic acid (eg, a gene) that encodes the amino acid sequence of a protein.

As used herein, codon optimization refers to changes in the codons of a polynucleotide encoding a protein to those preferentially used by a particular organism, such that the encoded protein is efficiently expressed in the organism of interest. In some embodiments, polynucleotides encoding PGA enzymes may be codon optimized for optimal production from the host selected for expression. Although the genetic code is degenerate in that most amino acids are represented by multiple codons, called synonyms or synonymous codons, it is well known that codon usage by specific organisms is non-random and biased towards specific codon triplets. This codon preference may be higher for a given gene, genes with common function or descent from a common ancestor, highly expressed versus low copy number proteins, and aggregate protein coding regions in an organism's genome. In some embodiments, polynucleotides encoding PGA enzymes may be codon optimized for optimal production from the host selected for expression.

As used herein, preferred, optimal, and high codon preference codons refer interchangeably to codons that are used at a higher frequency in protein coding regions than other codons that code for the same amino acid. Preferred codons can be determined with respect to codon usage in a single gene, a set of genes with a common function or origin, highly expressed genes, the frequency of codons in aggregate protein-coding regions of an entire organism, the frequency of codons in aggregate protein-coding regions of related organisms, or combinations thereof. Codons whose frequency increases with the level of gene expression generally represent the optimal codons for expression. Various methods are known to determine codon frequency (e.g., codon usage, relative synonymous codon usage) and codon preferences in specific organisms, including multivariate analysis, e.g., using cluster or correspondence analysis, and the effective number of codons used in a gene (see eg GCG CodonPreference, Genetics Computer Group Wisconsin Package; CodonW, John Peden, University of Nottingham; McInerney, Bioinform., 14:372-73 [1998]; Stenico et al., Nucleic Acids Res., 222:437-46 [1994]; and Wright, Gene 87:23-29 [1990]). Codon usage tables are available for a growing list of organisms (see, for example, Wada et al., Nucleic Acids Res., 20:2111-2118 [1992]; Nakamura et al., Nucl. Acids Res., 28:292 [2000] ; Duret, et al., supra; Henaut and Danchin, Escherichia coli and Salmonella, Neidhardt, et al. (eds.), ASM Press, Washington D.C., [1996], pp. 2047-2066. Source of data for obtaining codon usage can rely on any available nucleotide sequence capable of encoding a protein. These data sets include nucleic acid sequences that are known to actually encode expressed proteins (e.g. complete protein coding sequences - CDS), expressed sequence tags (ESTS) or predicted coding regions of genomic sequences (see, for example, Uberbacher, Meth. Enzymol., 266:259-281 [1996]; Tiwari et al., Comput. Appl. Biosci., 13:263-270 [1997]).

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As used herein, a control sequence is defined herein to include all components that are necessary or beneficial for expression of the polynucleotide and/or polypeptide of the present invention, each control sequence being native or foreign to the polynucleotide of interest. Such control sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, and a transcription terminator.

As used herein, operably linked is defined herein as a configuration in which a control sequence is suitably placed (i.e., operably linked) at a position relative to the polynucleotide of interest such that the control sequence controls or regulates expression of the polynucleotide and/or or a polypeptide of interest.

As used herein, a promoter sequence refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The control sequence may comprise a suitable promoter sequence. The promoter sequence contains transcriptional control sequences that mediate expression of the polynucleotide of interest. A promoter can be any nucleic acid sequence that exhibits transcriptional activity in a host cell, preferably including mutant, truncated, and hybrid promoters, and that can be derived from genes encoding extracellular or intracellular polypeptides homologous or heterologous to the host cell.

As used herein, naturally occurring or wild type refers to the form that is found in nature. For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be derived from a source in nature and that has not been purposefully modified by human manipulation.

As used herein, non-naturally occurring, engineered, and recombinant, when used in this disclosure with reference to (e.g., a cell, nucleic acid, or polypeptide), refers to a material, or material corresponding to a natural or native form of the material, that is modified in a way that would not otherwise exist in nature. In some embodiments, the material is identical to a naturally occurring material, but is produced or created from synthetic materials and/or through manipulation using recombinant methods. Non-limiting examples include, but are not limited to, recombinant cells expressing genes that are not found in the native (non-recombinant) form of the cell, or expressing native genes that are otherwise expressed at a different level.

As used herein, percent sequence identity, percent identity, and percent identical refer to a comparison between polynucleotide sequences or polypeptide sequences and are determined by comparing two optimally aligned sequences in a comparison window, wherein a portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., omissions) compared to a reference sequence to optimally align two sequences. The percentage is calculated by determining the number of positions at which identical nucleic acid bases or amino acid residues occur in both sequences, or aligning the nucleic acid base or amino acid residue with a gap to obtain the number of matching positions, and dividing the number of matching positions by the total number of positions in the window comparisons and multiply the result by 100 to obtain the percentage sequence identity. Determination of optimal alignment and percent sequence identity is performed using the BLAST and BLAST 2.0 algorithms (see, for example, Altschul et al., J. Mol. Biol. 215:403-410 [1990]; and Altschul et al., Nucl. Acids Res. 3389-3402 [1977]). Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information website.

Briefly, BLAST analysis involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or meet a certain score threshold with a positive T value when aligned with a word of the same length in the sequence Database. T is designated as the threshold for judging adjacent words (Altschul et al., supra). These initial matches of neighboring words act as seeds to initiate searches to find longer HSPs containing them. Word matches are then expanded in both directions along each sequence as long as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, parameters M (reward per matching pair

- 5 043748 giving remainders; always >0) and N (penalty for mismatched balances; always <0). For amino acid sequences, a substitution matrix is used to calculate a cumulative score. Expansion of word matches in each direction is stopped when: the cumulative alignment score is reduced by an amount X from its maximum value achieved; the cumulative score is reduced to zero or lower due to the accumulation of one or more residual alignments with negative scores; or reach the end of any of the sequences. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of alignment. The BLASTN program (for nucleotide sequences) uses a default word length (W) of 11, expectation (E) of 10, M=5, N=4, and comparison of both strands. For amino acid sequences, BLASTP defaults to a word length (W) of 3, an expectation (E) of 10, and a BLOSUM62 substitution matrix (see, for example, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 [1989 ]).

Many other algorithms are available and known in the art that function similarly to BLAST by providing percent identity between two sequences. Optimal alignment of sequences for comparison can be performed using any suitable method known in the art (eg, using the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 [1981]; using the homology alignment algorithm of Needleman and Wunsch , J. Mol. Biol. 48:443 [1970]; using the similarity search method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 [1988]; and/or using computer implementations of these algorithms [GAP , BESTFIT, FASTA and TFASTA in the GCG Wisconsin Software Package]), or by visual inspection using methods well known in the art. Additionally, the BESTFIT or GAP programs in the GCG Wisconsin Software Package (Accelrys, Madison WI) can be used to determine sequence alignment and percent sequence identity using the default parameters provided.

As used herein, substantial identity refers to a polynucleotide or polypeptide sequence that has at least 80% sequence identity, at least 85% sequence identity and 89-95% sequence identity, more typically at least 99% sequence identity compared to with the reference sequence in a comparison window of at least 20 residue positions, often in a window of at least 30-50 residues, where the percentage of sequence identity is calculated by comparing the reference sequence with a sequence that contains deletions or additions that are 20% or less from the reference sequence in the comparison window. In specific embodiments, as applied to polypeptides, the term substantial identity means that two polypeptide sequences, when optimally aligned, e.g., using the GAP or BESTFIT programs using default gap weights, have at least 80% sequence identity, preferably at least at least 89% sequence identity, at least 95% sequence identity, or greater (eg, 99% sequence identity). In some preferred embodiments, residue positions that are not identical are distinguished by conservative amino acid substitutions.

As used herein, a reference sequence refers to a specific sequence to which another sequence is compared. The reference sequence may be a subset of a larger sequence, such as a segment of the full-length sequence of a gene or polypeptide. In general, the reference sequence is at least 20 nucleotide or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, or the entire length of a nucleic acid or polypeptide. Because each of two polynucleotides or polypeptides may (1) contain a sequence (i.e., part of the complete sequence) that is similar between the two sequences, and (2) additionally contain a sequence that is divergent between the two sequences, comparison of the sequences between the two (or more ) polynucleotides or polypeptides are typically performed by comparing the sequences of two polynucleotides in a comparison window in order to identify and compare local regions of sequence similarity. The term reference sequence is not intended to be limited to wild-type sequences, and may include engineered or altered sequences. For example, in some embodiments, the reference sequence may be a pre-designed or modified amino acid sequence.

As used herein, a comparison window refers to a speculative segment of at least about 20 contiguous nucleotide positions or amino acid residues in which the sequence can be compared to a reference sequence of at least 20 contiguous nucleotides or amino acid residues and where a portion of the sequence in the comparison window can contain additions or deletions (i.e., gaps) of 20% or less compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The comparison window may be longer than 20 continuous residues and may not

- 6 043748 optionally includes 30, 40, 50, 100 or longer windows.

As used herein, corresponding, in relation to, and relative to, when used in the context of numbering a given amino acid or polynucleotide sequence, refers to the numbering of residues of a particular reference sequence when a given amino acid or polynucleotide sequence is compared to a reference sequence. In other words, the residue number or position of a residue in a given polymer is designated relative to a reference sequence and not by the actual numbering position of the residue in a given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as that of a designed PGA, can be aligned to a reference sequence by introducing gaps in order to optimize residue matches between the two sequences. In these cases, although gaps are present, the residue numbering in a given amino acid or polynucleotide sequence is relative to the reference sequence to which it is aligned. As used herein, reference to a residue position such as Xn, as further described below, should be interpreted as indicating the corresponding residue unless specifically stated otherwise. Thus, for example, X94 refers to any amino acid at position 94 in the polypeptide sequence.

As used herein, an improved enzyme property refers to a PGA that exhibits an improvement in any enzyme property compared to a reference PGA. For the engineered PGA polypeptides described herein, comparisons are generally made with the wild-type PGA enzyme, although in some embodiments the reference PGA may be another improved engineered PGA. Enzyme properties for which improvement is desirable include, but are not limited to, enzymatic activity (which can be expressed in units of percent substrate conversion over a specific reaction time using a specific amount of PGA), chemoselectivity, thermal stability, solvent stability, pH profile of activity, cofactor requirements, inhibitor resistance (e.g., product inhibition), stereospecificity, and stereoselectivity (including enantioselectivity).

As used herein, increased enzymatic activity refers to an improved property of engineered PGA polypeptides, which may represent an increase in specific activity (e.g., product produced/time/weight of protein) or an increase in the percentage of substrate converted to product (e.g., the percentage of initial amount of substrate converted to product over a specific period of time using a specific amount of PGA) compared to a reference PGA enzyme. Exemplary methods for determining enzyme activity are given in the examples. Any property related to enzyme activity can be influenced, including the classical enzyme properties Km, V _max or kc _at , changes in which can lead to increased enzymatic activity. The improvement in enzyme activity can range from approximately 1.5 times the enzymatic activity of the corresponding wild-type PGA enzyme, up to 2 times, 5 times, 10 times, 20 times, 25 times, 50 times, 75 times, 100 times or more enzyme activity relative to that found in the nature of PGA or other engineered PGA from which PGA polypeptides were derived. In specific embodiments, the engineered PGA enzyme exhibits improved enzymatic activity ranging from 1.5 to 50 times, 1.5 to 100 times greater than that of the parent PGA enzyme. One skilled in the art will appreciate that the activity of any enzyme is diffusion limited such that the rate of catalytic turnover cannot exceed the rate of diffusion of the substrate, including any required cofactors. The theoretical maximum diffusion limit, or kcat/Km, is generally approximately 10 ⁸ to 109 (M ^-1 s ^-1 ). Thus, any improvements in PGA enzyme activity will have an upper limit related to the rate of diffusion of the substrates on which the PGA enzyme acts. PGA activity can be measured using any one of the standard assays used to measure the release of phenylacetic acid from the breakdown of penicillin G, for example, by titration (see, for example, Simons and Gibson, Biotechnol. Tech., 13:365-367 [1999] ). In some embodiments, PGA activity can be measured using 6-nitrophenylacetamidobenzoic acid (NIPAB), the cleavage product of which 5-amino-2-nitrobenzoic acid is detectable spectrophotometrically (λ _max =405 nm). Comparisons of enzyme activities are performed using a specific enzyme preparation, a specific assay under specific conditions, and one or more specific substrates, as further described in detail herein. In general, when lysates are compared, the number of cells and the amount of protein analyzed are determined, as is the use of identical expression systems and identical host cells in order to minimize variations in the amount of enzyme produced by the host cells and present in the lysates.

As used herein, increased enzyme activity and increased activity refer to an improved property of the engineered enzyme, which may be represented by an increase in specific activity (e.g., product/time/weight of protein

- 7 043748 ka) or an increase in the percentage conversion of substrate to product (eg, the percentage conversion of an initial amount of substrate to product over a specific period of time using a specific amount of PGA) compared to a reference enzyme as disclosed herein. Any property related to enzyme activity can be influenced, including the classical enzyme properties Km, V _max or k _cat , changes in which can lead to increased enzymatic activity. In some embodiments, PGA enzymes provided herein release insulin by removing triphenylacetate protecting groups from specific residues in insulin. Comparisons of enzyme activities are performed using a specific enzyme preparation, a specific assay under specific conditions, and one or more specific substrates, as further described in detail herein. In general, when enzymes in cell lysates are compared, the number of cells and the amount of protein analyzed are determined, as is the use of identical expression systems and identical host cells to minimize variation in the amount of enzyme produced by the host cells and present in the lysates.

As used herein, conversion refers to the enzymatic conversion of a substrate to the corresponding product.

As used herein, percentage conversion refers to the percentage of substrate that is converted to product over a given period of time under specific conditions. Thus, for example, the enzymatic activity or activity of a PGA polypeptide can be expressed as the percentage of substrate to product conversion.

As used herein, chemoselectivity refers to the preferential formation of one product relative to others in a chemical or enzymatic reaction.

As used herein, thermostable is used to refer to a polypeptide that is resistant to inactivation when exposed to a set of temperature conditions (eg, 40-80°C) over a specified period of time (eg, 0.5-24 hours), versus with untreated enzyme, thus maintaining a certain level of residual activity (eg, more than 60-80%) after exposure to elevated temperatures.

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

As used herein, pH stable refers to a PGA polypeptide that retains similar activity (e.g., greater than 60-80%) after exposure to high or low pH (e.g., 4.5-6 or 8-12) for a specified period time (eg 0.5-24 hours) compared to untreated enzyme.

As used herein, heat-stable and solvent-stable refers to a PGA polypeptide that is both heat-stable and solvent-stable.

As used herein, a hydrophilic amino acid or residue refers to an amino acid or residue having a side chain exhibiting hydrophobicity less than zero according to the normalized consensus hydrophobicity scale of Eisenberg et al., (Eisenberg et al., J. Mol. Biol., 179:125-142 [1984]). 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).

As used herein, an acidic amino acid or residue refers to a hydrophilic amino acid or residue having a side chain exhibiting a pK value of less than about 6 when the amino acid is included in a peptide or polypeptide. Acidic amino acids typically have negatively charged side chains at physiological pH due to the loss of a hydrogen ion. Genetically encoded acidic amino acids include L-Glu (E) and L-Asp (D).

As used herein, a basic amino acid or residue refers to a hydrophilic amino acid or residue having a side chain exhibiting a pK value greater than about 6 when the amino acid is included in a peptide or polypeptide. Basic amino acids typically have positively charged side chains at physiological pH due to association with the hydronium ion. Genetically encoded essential amino acids include L-Arg (R) and L-Lys (K).

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

As used herein, a hydrophobic amino acid or residue refers to an amino acid or residue having a side chain exhibiting hydrophobicity greater than zero according to the normalized consensus hydrophobicity scale of Eisenberg et al., (Eisenberg et al., J. Mol. Biol.,

- 8 043748

179:125-142 [1984]). 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).

As used herein, an aromatic amino acid or residue refers to a hydrophilic or hydrophobic amino acid or residue having a side chain that contains at least one aromatic or heteroaromatic ring. Genetically encoded aromatic amino acids include L-Phe (F), L-Tyr (Y), and L-Trp (W). Although due to the pKa of its heteroaromatic nitrogen atom, L-His(H) is sometimes classified as a basic residue, or as an aromatic residue because its side chain contains a heteroaromatic ring, herein histidine is classified as a hydrophilic residue or a conformationally hindered residue (see below).

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

As used herein, a nonpolar amino acid or residue refers to a hydrophobic amino acid or residue having a side chain that is uncharged at physiological pH and that has bonds in which a pair of electrons typically shared by two atoms is generally held equally by each of two atoms (i.e. the side chain is not polar). 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).

As used herein, an 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).

It should be noted that cysteine (or L-Cys or [C]) is unusual 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 that are available for the formation of disulfide bridges. The ability of L-Cys(C) (and other amino acids with SH-containing side chains) to exist in the peptide, either in a reduced form with free SH or an oxidized form with a disulfide bridge, influences whether L-Cys(C) contributes net hydrophobic or hydrophilic character in the peptide. Although L-Cys (C) exhibits a hydrophobicity of 0.29 according to the Eisenberg normalized consensus scale (Eisenberg et al., 1984, supra), it should be understood that for purposes of the present disclosure, L-Cys (C) is referred to as its own unique group.

As used herein, a small amino acid or residue refers to an amino acid or residue having a side chain that consists of only three or fewer carbon atoms and/or heteroatoms (not including α-carbon and hydrogens). Small amino acids or residues can further be referred to as aliphatic, non-polar, polar or acidic small amino acids or residues, in accordance with 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).

As used herein, a hydroxyl-containing amino acid or residue refers to an amino acid containing a hydroxyl (-OH) moiety.

Genetically encoded hydroxyl-containing amino acids include L-Ser (S), L-Thr (T), and LTyr (Y).

As used herein, amino acid difference and residue difference refer to the difference of an amino acid residue at a particular position in a polypeptide sequence relative to an amino acid residue at a corresponding position in a reference sequence. The positions of the various amino acids are generally referred to herein as Xn, where n refers to the corresponding position in the reference sequence on which the residue difference is based. For example, the difference in residues at position X40 compared to SEQ ID No. 2 refers to the difference in amino acid residue at position of the polypeptide corresponding to position 40 in SEQ ID No. 2. Thus, if the reference polypeptide from SEQ ID No. 2 has a histidine at position 40, then a residue difference at position X40 compared to SEQ ID No. 2 refers to the replacement of an amino acid with any residue other than histidine at the position of the polypeptide corresponding to position 40 in SEQ ID No. 2. In most cases herein, a specific difference in amino acid residues at a particular position is indicated as XnY, where Xn is the specific corresponding position as described above, and Y is the one-letter identifier of the amino acid occurring in the designed polypeptide (ie, a different residue than in the reference polypeptide). In some cases, the present disclosure also makes specific distinctions

- 9,043,748 amino acids, designated by the standard notation AnB, where A is the one-letter residue identifier in the reference sequence, n is the residue position number in the reference sequence, and B is the one-letter residue substitution identifier in the sequence of the designed polypeptide. In some cases, a polypeptide of the present disclosure may contain one or more amino acid residue differences relative to a reference sequence, which is indicated by a list of specific positions where residue differences relative to the reference sequence are present. In some embodiments, where more than one amino acid can be used at a particular residue position of the polypeptide, the different amino acid residues that can be used are separated by / (eg, X192A/G). The present disclosure includes engineered polypeptide sequences containing one or more amino acid differences that include conservative and/or non-conservative amino acid substitutions. The amino acid sequences of specific recombinant carbonic anhydrase polypeptides included in the sequence listing of the present disclosure contain an initial methionine residue (M) (ie, M represents the position of residue 1). However, one skilled in the art will appreciate that this initial methionine residue can be removed by biological processing mechanisms, such as in a host cell translation system or in vitro, to create a mature protein that does not contain the initial methionine residue but otherwise retains the properties of the enzyme. Therefore, the term amino acid residue difference relative to SEQ ID No. 2 at position Xn, as used herein, may refer to position Xn or a corresponding position (eg, position (X-1)n) in a reference sequence that is processed so that the initial methionine was absent.

As used herein, the phrase conservative amino acid substitutions refers to the interchangeability of residues having similar side chains, and thus generally includes the replacement of an amino acid in a polypeptide with amino acids in the same or similarly defined class of amino acids. By way of example and not limitation, in some embodiments, an amino acid with an aliphatic side chain is replaced with another aliphatic amino acid (eg, alanine, valine, leucine, and isoleucine); an amino acid with a hydroxyl side chain is replaced with another amino acid with a hydroxyl side chain (eg, serine and threonine); amino acids having aromatic side chains are replaced with another amino acid having an aromatic side chain (eg, phenylalanine, tyrosine, tryptophan and histidine); an amino acid with a basic side chain is replaced with another amino acid with a basic side chain (eg, lysine and arginine); an amino acid with an acidic side chain is replaced with another amino acid with an acidic side chain (for example, aspartic acid or glutamic acid); and/or a hydrophobic or hydrophilic amino acid is replaced by another hydrophobic or hydrophilic amino acid, respectively. Exemplary conservative substitutions are given in table. 1.

Table 1. Exemplary conservative amino acid substitutions

Remainder Possible conservative substitutions A, L, V, I Other aliphatic (A, L, V, I) Other non-polar (A, L, V, I, G, M) G, M Other non-polar (A, L, V, I, G, M) D, E Other sour (D, E) K, R Other main (K, R) N, Q, S, T Another polar Η, Y, W, F Other aromatic (Η, Y, W, F) S, R Non-polar

As used herein, the phrase non-conservative substitution refers to the replacement of an amino acid in a polypeptide with an amino acid with significantly different side chain properties. Non-conservative substitutions allow the use of amino acids between certain groups rather than within them, and affect (a) the structure of the peptide backbone at the site of the substitution (e.g. proline instead of glycine) (b) charge or hydrophobicity or (c) side chain volume. By way of example and not limitation, an exemplary non-conservative substitution may be an acidic amino acid replaced by a basic or aliphatic amino acid; an aromatic amino acid replaced by a small amino acid; and a hydrophilic amino acid replaced by a hydrophobic amino acid.

As used herein, deletion refers to modification of a polypeptide by removing one or more amino acids from a reference polypeptide. Deletions may include the deletion of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total amino acids, or up to 20% of the total amino acids forming the polypeptide, while maintaining enzymatic activity and/or maintaining the improved properties of the constructed enzyme. Deletions may be directed to the internal portions and/or terminal portions of the polypeptide. In various embodiments, the deletion may comprise a continuous segment or may be discontinuous.

As used herein, insertion refers to modifying a polypeptide by adding one or more amino acids to a reference polypeptide. In some embodiments, the improved engineered PGA enzymes comprise insertions of one or more amino acids into a naturally occurring PGA polypeptide, as well as insertions of one or more amino acids into engineered PGA polypeptides. Insertions can be located in the internal parts of the polypeptide or at the carboxy or amino terminus. Insertions, as used herein, include fusion proteins as known in the art. The insertion may be a contiguous segment of amino acids or may be separated by one or more amino acids in a naturally occurring polypeptide.

The term amino acid substitution set or substitution set refers to a group of amino acid substitutions in a polypeptide sequence compared to a reference sequence. A set of substitutions may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions. In some embodiments, a substitution set refers to a set of amino acid substitutions that are present in any of the PGA variants listed in the tables provided in the examples.

As used herein, a fragment refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence. The fragments typically have about 80%, about 90%, about 95%, about 98%, or about 99% of the full-length PGA polypeptide, such as the polypeptide of SEQ ID NO: 2. In some embodiments, the fragment is biologically active (i.e., it exhibits same enzymatic activity as the full-length sequence).

As used herein, an isolated polypeptide refers to a polypeptide that is substantially separated from other contaminants that naturally accompany it, such as protein, lipids, and polynucleotides. The term covers polypeptides that are removed from or purified from the naturally occurring environment or expression system (eg, host cell or in vitro synthesis). The improved PGA enzymes may be present in the cell, present in the cellular environment, or may be obtained in various forms, such as lysates or isolated preparations. As such, in some embodiments, the engineered PGA polypeptides of the present disclosure may be an isolated polypeptide.

As used herein, a substantially pure polypeptide refers to a composition in which the polypeptide particles are the predominant particles present (i.e., on a molar or mass basis, it is more numerous than any other individual macromolecular particles in the composition), and generally represents a substantially purified composition when the particles in question constitute at least about 50% of the macromolecular particles present by mole or % by weight. In general, the substantially pure engineered PGA polypeptide composition contains about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% of all macromolecular particles by moles or % by weight present in the composition. Solvent particles, low molecular weight compounds (<500 Da) and elemental ion particles are not considered macromolecular particles. In some embodiments, the isolated improved PGA polypeptide is a substantially pure polypeptide composition.

As used herein, when used in relation to a nucleic acid or polypeptide, the term heterologous refers to a sequence that an organism (eg, a wild-type organism) does not normally express or secrete. In some embodiments, the term covers a sequence that contains two or more subsequences that do not occur in the same association with each other as they are typically found in nature, or are recombinantly engineered such that its expression level or physical association with other nucleic acids or other molecules in a cell or structure not usually found in nature. For example, a heterologous nucleic acid is typically produced recombinantly, having two or more sequences from unrelated genes that are arranged in a manner not found in nature (e.g., the open reading frame (ORF) of a nucleic acid of the invention is operably linked to a promoter sequence inserted into an expression cassette such as vector). In some embodiments, a heterologous polynucleotide refers to any polynucleotide that is introduced into a host cell by laboratory techniques, and which includes polynucleotides that are removed from a host cell subjected to laboratory manipulation and then reintroduced into the host cell.

- 11 043748

As used herein, suitable reaction conditions refer to those conditions in the biocatalytic reaction solution (e.g., ranges of enzyme loading, substrate loading, cofactor loading, temperature, pH, buffers, cosolvents, etc.) under which the PGA polypeptide is presently opening is capable of releasing free insulin by removing triphenylacetate protecting groups. Exemplary suitable reaction conditions are provided in the present disclosure and are illustrated by examples.

As used herein, loading, for example in compound loading, enzyme loading, or cofactor loading, refers to the concentration or amount of a component in the reaction mixture at the start of the reaction.

As used herein, substrate, in the context of a biocatalyst-mediated process, refers to the compound or molecule that is acted upon by the biocatalyst.

As used herein, product, in the context of a biocatalyst-mediated process, refers to a compound or molecule resulting from the action of a biocatalyst.

As used herein, equilibration, as used herein, refers to the process leading to an equilibrium concentration of chemical species in a chemical or enzymatic reaction (e.g., the interconversion of two species A and B), including the interconversion of stereoisomers, as determined by a direct constant rate and inverse rate constant of a chemical or enzymatic reaction.

As used herein, acylase and acyltransferases are used interchangeably to refer to enzymes that are capable of transferring an acyl group from a donor to an acceptor to form esters or amides. The acylase-mediated reverse reaction leads to hydrolysis of the ester or amide.

As used herein, penicillin G and benzylpenicillin refer to the antibiotic also known as (2S,5R,6R)-3,3-dimethyl-7-oxo-6-(2-phenylacetamido)-4-thia-1-azabicyl [3.2.0]heptane-2-carboxylic acid (C ₁₆ H ₁₈ N ₂ O ₄ S). It is primarily effective against gram-positive organisms, although some gram-negative organisms are also susceptible to it.

As used herein, penicillin G acylase and PGA are used interchangeably to refer to an enzyme having the ability to mediate the cleavage of penicillin G (benzylpenicillin) to phenylacetic acid (PHA) and 6-aminopenicillanic acid (6APA). In some embodiments, PGA activity may be based on the cleavage of model substrates, for example, the cleavage of 6-nitro-3-(phenylacetamide)benzoic acid to phenylacetic acid and 5-amino-2-nitrobenzoic acid. PGAs are also capable of performing the reverse reaction of acyl group transfer from an acyl donor to an acyl acceptor. PGAs, as referred to herein, include naturally occurring PGAs (wild type) as well as non-naturally occurring PGA enzymes comprising one or more engineered polypeptides created by human manipulation. The wild-type PGA gene is a heterodimer consisting of an alpha subunit (23.8 kDa) and a beta subunit (62.2 kDa) linked by a 54 amino acid spacer region. Due to the presence of a spacer region, an autoprocessing step is required in order to form the active protein.

As used herein, an acyl donor refers to that portion of an acylase substrate that donates an acyl group to an acyl acceptor to form esters or amides.

As used herein, an acyl acceptor refers to that part of an acylase substrate that accepts an acyl group from an acyl donor in order to form esters or amides.

As used herein, an α-chain sequence refers to an amino acid sequence that corresponds to (e.g., has at least 85% identity to) residues at positions 27 to 235 in SEQ ID No. 2. As used herein, a single chain polypeptide may contain the release sequence and additional sequence(s).

As used herein, a β chain sequence refers to an amino acid sequence that corresponds to (e.g., has at least 85% identity to) residues at positions 290 to 846 in SEQ ID No. 2. As used herein, a single chain polypeptide may contain a β-strand sequence and additional sequence(s).

As used herein, derived from, when used in the context of engineered PGA enzymes, identifies the parent PGA enzyme and/or the gene encoding such PGA enzyme on which the design is based. For example, the single-chain engineered PGA enzyme of SEQ ID NO: 60 was produced by artificially evolving, over several generations, the gene encoding PGA from K. citrophila. Thus, in some embodiments, engineered PGA enzymes are derived from naturally occurring or wild type PGA with

- 12 043748

SEQ ID NO: 2, although in some additional embodiments, engineered PGA enzymes are derived from other engineered PGA enzymes. In some embodiments, the engineered PGA comprises an α chain sequence and a β chain sequence, which may be present as separate polypeptides in the mature enzyme or may be present as part of a single chain polypeptide. In some embodiments, when present in single-chain form, the engineered PGA polypeptide may comprise, from amino to carboxy terminus, a B-L-A structure in which B is a β-strand sequence (or B unit); A represents an α-chain sequence (or A unit); and L is a linker connecting the α-chain and β-chain sequences. In some embodiments, the spacer or linker L comprises a spacer or linker of sufficient length and flexibility to allow proper folding and interaction of the A and B units to form a functional PGA enzyme. An exemplary linker/spacer contains the amino acid sequence Gln-Leu-Asp-Gln.

Whether in the form of individual polypeptides or as a single-chain polypeptide, the α- and β-chain sequences may have one or more residue differences compared to the naturally occurring α- and β-chain sequences from the K. citrophila PGA.

As used herein, insulin refers to a polypeptide hormone produced by pancreatic beta cells in normal individuals. Insulin is necessary to regulate carbohydrate metabolism by lowering blood glucose levels. A systematic lack of insulin leads to diabetes. Insulin consists of 51 amino acids and has a molecular weight of approximately 5800 Da. Insulin consists of two peptide chains (designated A and B) containing one intrasubunit and two intersubunit disulfide bonds. Chain A consists of 21 amino acids and chain B consists of 30 amino acids. The two chains form a highly ordered structure, with several alpha-helical regions in both chains A and B. Isolated chains are inactive. In solution, insulin is a monomer, dimer or hexamer. It is a hexamer in highly concentrated preparations used for subcutaneous injection, but becomes a monomer as it is diluted by body fluids. The definition is intended to include proinsulin and any purified isolated polypeptide having part or all of the primary structural conformation and at least one of the biological properties of naturally occurring insulin. It is also intended to cover natural and synthetically produced insulin, including glycoforms, as well as analogues (eg, polypeptides having deletions, insertions and/or substitutions).

Insulin contains three nucleophilic amines that can potentially react with the phenylacetate donor and be protected by PGA. These residues include Lys on the B chain at position 29 (B29) and two N-terminal free amines, Gly on the A chain at position 1 (A1) and Phe on the B chain at position 1 (B1). Triprotected insulin (phenylacetate chemically attached to the A1, B1, B29 residues in human insulin) is provided herein. It was previously reported that PGA catalyzes the hydrolysis of N-phenylacetate-protected peptides and insulin with exceptional selectivity for the phenylacetate amide bond, leaving the remaining peptide bonds of the protein intact (Brtnik et al., Coll. Czech. Chem. Commun., 46 (8), 1983 -1989 [1981] and Wang et al. Biopolym. 25 (Suppl.), S109-S114 [1986]).

As used herein, a triphenylacetate protecting group refers to an insulin molecule that has three primary amines at positions B1, B29 and A1, which are protected by a phenylacetate group.

As used herein, a diphenylacetate protecting group refers to an insulin molecule that has two primary amines at positions B1, B29 and/or A1 that are protected by a phenylacetate group.

As used herein, a diphenylacetate protecting group refers to an insulin molecule that has one primary amine at the B1, B29 or A1 positions that is protected by a phenylacetate group.

Penicillin G acylases

Penicillin acylase was first described from Penicillium chrysogenum Wise. Q176 by Sakaguchi and Murao (Sakaguchi and Murao, J. Agr. Chem. Soc. Jpn., 23:411 [1950]). Penicillin G acylase is a hydrolytic enzyme that acts on the side chains of penicillin G, cephalosporin G, and related antibiotics to produce the β-lactam antibiotic intermediates 6-aminopenicillanic acid and 7-aminodesacetoxycephalosporanic acid, with phenylacetic acid as a common byproduct. These antibiotic intermediates are potential building blocks for semisynthetic antibiotics such as ampicillin, amoxicillin, cloxacillin, cephalexin, and cefatoxime.

As stated above, penicillin G acylases (PGAs) are distinguished by their ability to catalyze the hydrolytic cleavage of penicillin G, with its conjugate base of structural formula (I), to 6-aminopenicillanic acid, with its conjugate base of structural formula (II), and phenylacetic acid.

- 13 043748 acid of structural formula (III), as shown in scheme 1:

Scheme 1

W (P) (tp)

Without being limited by theory, substrate specificity appears to be associated with the recognition of a hydrophobic phenyl group, while the nucleophile, which in some PGAs is a serine residue at the N-terminus of the beta chain, functions as an acceptor for the beta-lactam and various other groups such as beta-amino acids. PGAs may also differ in their ability to cleave model substrates similar to penicillin G, such as the cleavage of 6-nitro-3-(phenylacetamido)benzoic acid (NIPAB) of structural formula (IV), as shown in Scheme 2:

Scheme 2

^(TV) (V) (HI) to phenylacetic acid of structural formula (III) and 5-amino-2-nitrobenzoic acid of structural formula (V) (see, for example, Alkema et al., Anal. Biochem., 275:47 -53 [1999]). Since 5-amino2-nitrobenzoic acid is chromogenic, the substrate of formula (IV) provides a convenient method for measuring PGA activity. In addition to the above reactions, PGA can also be used to kinetically resolve DL-tert-leucine to produce optically pure tert-leucine (see, for example, Liu et al., Prep. Biochem. Biotechnol., 36:235-41 [2006 ]).

The PGAs of the present invention are based on an enzyme obtained from the organism Kluyvera citrophila (K. citrophila). As with PGA from other organisms, PGA from K. citrophila is a heterodimeric enzyme consisting of an alpha subunit and a beta subunit, which arise through proteolytic processing of the PGA prepropolypeptide. Removal of the signal peptide and spacer peptide produces a mature heterodimer (see, eg, Barbero et al., Gene 49:69-80 [1986]). The amino acid sequence of the naturally occurring PGA prepropolypeptide from K. citrophila is publicly available (see, e.g., GenBank accession number P07941, [gi:129551]) and is provided herein as SEQ ID No. 2. Naturally occurring PGA K alpha chain sequence. citrophila corresponds to residues 27 to 235 of SEQ ID No. 2. The beta chain sequence of the naturally occurring K. citrophila PGA corresponds to residues 290 to 846 of SEQ ID No. 2.

Residues 1 to 26 of SEQ ID No. 2 correspond to the signal peptide and residues 236 to 289 of SEQ ID No. 2 correspond to the binding propeptide, both of which are removed to create the naturally occurring mature PGA enzyme, which is a heterodimer containing an α chain subunit and a β-chain subunit. In some embodiments, the engineered PGA comprises an α chain sequence and a β chain sequence, which may be present as separate polypeptides in the mature enzyme or may be present as part of a single chain polypeptide. In some embodiments, which is present in single-chain form, the engineered PGA polypeptide may comprise, from amino to carboxy terminus, a B-L-A structure in which B is a β-strand sequence (or B unit); A represents an α-chain sequence (or A unit); and L is a linker connecting the α chain and β chain sequences. In some embodiments, the spacer or linker L comprises a spacer or linker of sufficient length and flexibility to allow proper folding and interaction of the A and B units to form a functional PGA enzyme. An exemplary linker/spacer contains the amino acid sequence Gln-Leu-Asp-Gln.

Whether in the form of individual polypeptides or as a single-chain polypeptide, the α- and β-chain sequences may have one or more residue differences compared to the naturally occurring α- and β-chain sequences from the K. citrophila PGA.

In some embodiments, the present invention provides engineered PGA polypeptides with amino acid sequences that have at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater sequence identity to SEQ ID Nos. 12, 24, 40, 56, 70, 82, 100, 108, 110, 116, 136, 154 and/or 160 .

The present invention provides insulin-specific acylation biocatalysts suitable for use on a commercial scale. Directed evolution has been used to develop efficient acylase variants that are capable of adding a phenylacetate protecting group to insulin at positions A1, B1 and/or B29. The PGA variants provided herein are capable of accepting a wide range of acyl groups, exhibiting increased stability in

- 14 043748 solvent and improved thermal stability compared to wild type PGA. The PGA variants provided herein do not contain a spacer region. Thus, an autoprocessing step is not required in order to obtain active enzymes. The present invention also relates to polynucleotides encoding engineered PGA polypeptides. In some embodiments, the polynucleotides are operably linked to one or more heterologous regulatory sequences that control gene expression to create a recombinant polynucleotide capable of expressing the polypeptide.

Expression constructs containing a heterologous polynucleotide encoding the engineered PGA polypeptides can be introduced into suitable host cells to express the corresponding PGA polypeptide. Because the codons corresponding to different amino acids are known, the availability of a protein sequence provides a description of all polynucleotides capable of encoding the one in question. The degeneracy of the genetic code, where the same amino acids are encoded by alternative or synonymous codons, allows for the creation of an extremely large number of nucleic acids, all of which encode the improved PGA enzymes disclosed herein. Thus, once a specific amino acid sequence has been identified, those skilled in the art can create any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not change the amino acid sequence of the protein. In this regard, the present disclosure specifically provides for each and every possible variation of polynucleotides that can be obtained by selecting combinations based on the selection of possible codons, and all such variations are to be considered as specifically disclosed for any polypeptide disclosed herein, including amino acid sequences presented in the tables in the examples.

In various embodiments, codons are preferably selected to suit the host cell in which the protein is produced. For example, preferred codons used in bacteria are used to express a gene in bacteria; preferred codons used in yeast are used for expression in yeast; and preferred codons used in mammals are used for expression in mammalian cells.

In certain embodiments, all codons do not need to be replaced in order to optimize codon usage for PGA polypeptides because the natural sequence contains preferred codons and because the use of preferred codons may not be required for all amino acid residues. Therefore, codon-optimized polynucleotides encoding PGA enzymes may contain preferred codons at approximately 40%, 50%, 60%, 70%, 80%, or greater than 90% of the codon positions of the full-length coding region.

In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a PGA polypeptide with an amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, or 99% or greater sequence identity to any alpha chain and/or beta chain of the reference engineered PGA polypeptides described herein. Accordingly, in some embodiments, the polynucleotide encodes an amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identical to the alpha and beta chain reference sequences based on SEQ ID Nos. 12, 24, 40, 56, 70, 82, 100, 108, 110, 116, 136, 154 and/or 160. In some embodiments, the polynucleotide encodes the alpha and/or beta chain amino acid sequence of SEQ ID NO: 12, 24, 40, 56, 70, 82, 100, 108, 110, 116, 136, 154 and/ or 160.

In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a PGA polypeptide with an amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, or 99% or greater sequence identity to SEQ ID No. 12, 24, 40, 56, 70, 82, 100, 108, 110, 116, 136, 154 and/or 160. Accordingly, in some embodiments, the polynucleotide encodes an amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99% or greater is identical to SEQ ID No. 12, 24, 40, 56, 70, 82, 100, 108, 110, 116, 136, 154 and/or 160.

In some embodiments, an isolated polynucleotide encoding an improved PGA polypeptide has been manipulated in various ways to provide improved activity and/or expression of the polypeptide. Manipulation of the isolated polynucleotide before its insertion into a vector may be desirable or necessary, depending on the expression vector. Techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA techniques are well known in the art.

For example, mutagenesis and directed evolution techniques can be readily applied to polynucleotides to create expressible library variants, screen them, and analyze them. Mutagenesis and directed evolution techniques are well known in the art.

- 15 043748 (see, for example, US patents No. 5605793, 5830721, 6132970, 6420175, 6277638, 6365408, 6602986,

7288375, 6287861, 6297053, 6576467, 6444468, 5811238, 6117679, 6165793, 6180406, 6291242, 6995017,

6395547, 6506602, 6519065, 6506603, 6413774, 6573098, 6323030, 6344356, 6372497, 7868138, 5834252,

5928905, 6489146, 6096548, 6387702, 6391552, 6358742, 6482647, 6335160, 6653072, 6355484, 603344,

6319713, 6613514, 6455253, 6579678, 6586182, 6406855, 6946296, 7534564, 7776598, 5837458, 6391640,

6309883, 7105297, 7795030, 6326204, 6251674, 6716631, 6528311, 6287862, 6335198, 6352859, 6379964,

7148054, 7629170, 7620500, 6365377, 6358740, 6406910, 6413745, 6436675, 6961664, 7430477, 7873499,

7702464, 7783428, 7747391, 7747393, 7751986, 6376246, 6426224, 6423542, 6479652, 6319714, 6521453,

6368861, 7421347, 7058515, 7024312, 7620502, 7853410, 7957912, 7904249 and all related non-US equivalents; Ling et al., Anal. Biochem., 254(2):157-78 [1997]; Dale et al., Meth. Mol. Biol., 57:369-74 [1996]; Smith, Ann. Rev. Genet., 19:423-462 [1985]; Botstein et al., Science, 229:1193-1201 [1985]; Carter, Biochem. J., 237:1-7 [1986]; Kramer et al., Cell, 38:879-887 [1984]; Wells et al., Gene 34:315-323 [1985]; Minshull et al., Curr. Op. Chem. Biol., 3:284-290 [1999]; Christians et al., Nat. Biotechnol., 17:259-264 [1999]; Crameri et al., Nature, 391:288-291 [1998]; Crameri, et al., Nat. Biotechnol., 15:436-438 [1997]; Zhang et al., Proc. Nat. Acad. Sci. U.S.A. 94:4504-4509 [1997]; Crameri et al., Nat. Biotechnol., 14:315-319 [1996]; Stemmer, Nature, 370:389-391 [1994]; Stemmer, Proc. Nat. Acad. Sci. USA, 91:10747-10751 [1994]; WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651; WO 01/75767 and WO 2009/152336, all of which are incorporated herein by reference).

In some embodiments, the PGA acylase variants of the present invention further comprise additional sequences that do not alter the encoded enzyme activity. For example, in some embodiments, PGA acylase variants are linked to an epitope tag or other sequence that can be used in purification.

In some embodiments, the PGA acylase variant polypeptides of the present invention are secreted by the host cell in which they are expressed (e.g., a yeast or filamentous fungus host cell) and expressed as a preprotein containing a signal peptide (i.e., an amino acid sequence that is linked with the amino terminus of the polypeptide and which directs the encoded polypeptide along the cellular secretory pathway).

In some embodiments, the signal peptide is an endogenous K. citrophila PGA acylase signal peptide. In some other embodiments, signal peptides from other secreted proteins of K. citrophila are used.

In some embodiments, other signal peptides are used, depending on the host cell and other factors. Effective signal peptide-encoding regions for filamentous fungal host cells include, but are not limited to, signal peptide-encoding regions derived from Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, Humicola lanuginosa lipase and T. reesei cellobiohydrolase II. Signal peptide coding regions for bacterial host cells include, but are not limited to, signal peptide coding regions derived from the Bacillus NClB 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis β-lactamase, neutral protease (nprT) genes , nprS, nprM) Bacillus stearothermophilus and prsA Bacillus subtilis. In some additional embodiments, other signal peptides find use in the present invention (see, for example, Simonen and Palva, Microbiol. Rev., 57: 109-137 [1993], incorporated herein by reference). Additional effective signal peptides for yeast host cells include those of the Saccharomyces cerevisiae alpha factor, SUC2 invertase genes (see, e.g., Taussig and Carlson, Nucl. Acids Res., 11:1943-54 [1983]; SwissProt accession no. P00724; and Romanos et al., Yeast 8:423-488 [1992]). In some embodiments, variants of these signal peptides and other signal peptides are used. In fact, the present invention is not intended to be limited to any particular signal peptide, as any suitable signal peptide known in the art finds use in the present invention.

In some embodiments, the present invention provides polynucleotides encoding PGA acylase variant polypeptides and/or biologically active fragments thereof, as disclosed herein. In some embodiments, the polynucleotide is operably linked to one or more heterologous regulatory or control sequences that control gene expression to create a recombinant polynucleotide capable of expressing the polypeptide. In some embodiments, expression constructs comprising a heterologous polynucleotide encoding a PGA acylase variant are introduced into suitable host cells to express the PGA acylase variant.

Those of ordinary skill in the art will appreciate that, due to the degeneracy of the genetic code, there are many nucleotide sequences encoding the polypeptides of the PGA acylase variants of the present invention. For example, codons AGA, AGG, CGA, CGC, CGG and CGU all code for the amino acid arginine. Thus, at each position in the nucleic acids of the invention,

- 16 043748 where arginine is specified by a codon, the codon can be changed to any of the corresponding codons described above without changing the encoded polypeptide. It is clear that the U in the RNA sequence corresponds to the T in the DNA sequence. The invention relates to and provides for each and every possible variation of the nucleic acid sequence encoding a polypeptide of the invention, which can be accomplished by selecting combinations based on possible codon alternatives.

As stated above, it is also possible to design the DNA sequence encoding PGA with high codon preference codons (codons that are used at a higher frequency in protein coding regions than other codons that code for the same amino acid). Preferred codons can be determined with respect to codon usage in a single gene, a set of genes with a common function or origin, highly expressed genes, the frequency of codons in aggregate protein-coding regions of a whole organism, the frequency of codons in aggregate protein-coding regions of related organisms, or combinations thereof. A codon whose frequency increases with the level of gene expression is usually the optimal codon for expression. In particular, the DNA sequence can be optimized for expression in a particular host organism. Various methods are well known in the art for determining codon frequency (e.g., codon usage, relative usage of synonymous codons) and codon preferences in specific organisms, including multivariate analysis (e.g., using cluster analysis or correspondence analysis) and the effective number of codons used in gene The data source may rely on any available nucleotide sequence capable of encoding a protein to obtain codon usage.

These data sets include nucleic acid sequences that accurately encode expressed proteins (eg, complete protein coding sequences -CDS), expressed sequence tags (ESTs), or predicted coding regions of genomic sequences, as is well known in the art. Polynucleotides encoding PGA variants can be prepared using any suitable methods known in the art. Typically, oligonucleotides are synthesized individually, then joined (eg, by enzymatic or chemical ligation or polymerase-mediated methods) to form essentially any desired contiguous sequence. In some embodiments, the polynucleotides of the present invention are produced by chemical synthesis using any suitable methods known in the art, including, but not limited to, automated synthesis methods. For example, in the phosphoramidite method, oligonucleotides are synthesized (eg, in an automated DNA synthesizer), purified, annealed, ligated, and cloned into suitable vectors. In some embodiments, double-stranded DNA fragments are then produced either by synthesizing the complementary strand and annealing the strands together under suitable conditions or by adding the complementary strand using DNA polymerase and a suitable primer sequence. There are many general and standard texts that provide methods that can be used in the present invention and that are well known to those skilled in the art.

Engineered PGAs can be produced by subjecting a polynucleotide encoding a naturally occurring PGA to mutagenesis and/or directed evolution methods, as discussed above. Mutagenesis can be carried out in accordance with any of the techniques known in the art, including random and site-specific mutagenesis. Directed evolution can be carried out using any of the techniques known in the art for screening improved variants, including shuffling. Other directed evolution procedures that are used include, but are not limited to, stepwise extension, in vitro recombination, mutagenic PCR, cassette mutagenesis, splicing by overlap extension (SOE), ProSAR™ directed evolution techniques, etc. ., as well as any other suitable methods.

Clones obtained after mutagenetic treatment are screened for engineered PGAs having the desired improved enzyme property. Measurement of enzyme activity from expression libraries can be accomplished using standard biochemical techniques for monitoring the rate of product formation. When the desired improved property of an enzyme is thermal stability, enzyme activity can be measured after exposing enzyme preparations to specific temperatures and measuring the amount of enzyme activity remaining after heat treatment. Clones containing the polynucleotide encoding PGA are then isolated, sequenced to identify changes in the nucleotide sequence (if appropriate), and used to express the enzyme in a host cell.

Once the sequence of the designed polypeptide is known, polynucleotides encoding the enzyme can be prepared by standard solid phase methods, in accordance with known synthetic methods. In some embodiments, fragments of up to about 100 bases can be synthesized individually and then joined (eg, by enzymatic or chemical ligation or polymerase-mediated methods) to form any desired contiguous sequence. For example, polynucleotides and oligonucleotides

- 17 043748 of the invention can be obtained by chemical synthesis (for example, using the classical phosphoramidite method described in Beaucage et al., Tet. Lett., 22:1859-69 [1981], or the method described in Matthes et al., EMBO J., 3:801-05 [1984], as commonly practiced in automated synthesis methods). In the phosphoramidite method, oligonucleotides are synthesized (eg in an automated DNA synthesizer), purified, annealed, ligated and cloned into suitable vectors. In addition, essentially any nucleic acid can be obtained from any of a variety of commercial sources (e.g., Midland Certified Reagent Company, Midland, TX, The Great American Gene Company, Ramona, CA, ExpressGen Inc. Chicago, IL, Operon Technologies Inc., Alameda, CA and many others).

The present invention also provides recombinant constructs containing a sequence encoding at least one PGA variant as provided herein. In some embodiments, the present invention provides an expression vector comprising a PGA variant polynucleotide operably linked to a heterologous promoter. In some embodiments, the expression vectors of the present invention are used to transform suitable host cells to allow the host cells to express a PGA variant protein. Methods for recombinant expression of proteins in fungi and other organisms are well known in the art, and many expression vectors are available or can be constructed using standard methods. In some embodiments, the nucleic acid constructs of the present invention comprise a vector, such as a plasmid, cosmid, phage, virus, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), and the like, into which a nucleic acid sequence is inserted at invention. In some embodiments, the polynucleotides of the present invention are inserted into any one of various expression vectors suitable for expressing the PGA variant polypeptide(s). Suitable vectors include, but are not limited to, chromosomal, non-chromosomal and synthetic DNA sequences (e.g. SV40 derivatives), as well as bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, viral DNA, e.g. vaccinia virus, adenovirus, fowlpox virus, pseudorabies, adenovirus, adeno-associated virus, retroviruses and many others. Any suitable vector that transduces genetic material into a cell and, if replication is desired, that is replicable and viable in the relevant host finds use in the present invention.

In some embodiments, the construct further comprises regulatory sequences, including, but not limited to, a promoter, operably linked to a protein coding sequence. A large number of suitable vectors and promoters are known to those skilled in the art. In fact, in some embodiments, in order to achieve high levels of expression in a particular host, it is often useful to express the PGA variants of the present invention under the control of a heterologous promoter. In some embodiments, the promoter sequence is operably linked to the 5' region of the PGA variant coding sequence using any suitable method known in the art. Examples of effective promoters for the expression of PGA variants include, but are not limited to, fungal promoters. In some embodiments, a promoter sequence is used that directs the expression of a gene other than the PGA gene in a fungal strain. As a non-limiting example, a fungal promoter from a gene encoding an endoglucanase can be used. In some embodiments, a promoter sequence is used that directs expression of the PGA gene in a fungal strain other than the fungal strain from which the PGA was derived. Examples of other suitable promoters that can be used to drive transcription of the nucleotide constructs of the present invention in filamentous fungal host cells include, but are not limited to, promoters derived from the Aspergillus oryzae TAKA amylase genes, Rhizomucor miehei aspartic proteinase, neutral alpha-amylase Aspergillus niger, Aspergillus niger acidic stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase, Aspergillus nidulans acetamidase and Fusarium oxysporum trypsin-like protease (see for example WO 96 /00787, incorporated herein by reference), as well as the NA2-tpi promoter (a hybrid of promoters from the Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase genes), promoters such as cbh1, cbh2, egl1, egl2, pepA, hfb1, hfb2, xyn1, amy and glaA (see, for example, Nunberg et al., Mol. Cell Biol., 4:2306-2315 [1984]; Boel et al., EMBO J., 3:1581-85 [1984]; and European Patent Application 137280, all of which are incorporated herein by reference), and their mutant, truncated and hybrid promoters.

In yeast host cells, effective promoters include, but are not limited to, those of the Saccharomyces cerevisiae enolase (eno-1), Saccharomyces cerevisiae galactokinase (gal1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP) and 3phosphoglycerate kinases from S. cerevisiae. Additional effective promoters that can be used for yeast host cells are known in the art (see, for example, Romanos et al., Yeast

8:423-488 [1992], incorporated herein by reference). In addition, promoters associated with chitinase production in fungi find use in the present invention (see, for example, Blaiseau and Lafay, Gene 120243-248 [1992]; and Limon et al., Curr. Genet., 28:478- 83 [1995], both of which are incorporated herein by reference).

For bacterial host cells, suitable promoters for driving transcription of the nucleic acid constructs of the present disclosure include, but are not limited to, promoters derived from the E. coli lac operon, E. coli trp operon, bacteriophage A, Streptomyces coelicolor agarase gene (dagA ), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), xylA and xylB of Bacillus subtilis and the prokaryotic beta-lactamase gene (see, e.g., Villa-Kamaroff et al., Proc. Natl. Acad. Sci. USA 75: 3727-3731 [1978]), as well as the tac promoter (see, e.g. , DeBoer et al., Proc. Natl. Acad. Sci. USA 80: 21-25 [1983]).

In some embodiments, the cloned PGA variants of the present invention also have a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is operably linked to the 3' end of the nucleic acid sequence encoding the polypeptide. Any terminator that is functional in a host cell preferably finds use in the present invention. Exemplary transcription terminators for filamentous fungal host cells include, but are not limited to, those derived from the Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease genes (see also patent US No. 7,399,627, incorporated herein by reference). In some embodiments, exemplary terminators for yeast host cells include those derived from the Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYCl) and Saccharomyces cerevisiae glyceraldehyde-3phosphate dehydrogenase genes. Other effective terminators for yeast host cells are well known to those skilled in the art (see, eg, Romanos et al., Yeast 8:423-88 [1992]).

In some embodiments, a suitable leader sequence that is part of the PGA variant sequence to be cloned is an untranslated region of the mRNA that is important for translation in the host cell. The leader sequence is operably linked to the 5' end of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in a host cell preferably finds use in the present invention. Exemplary leaders for filamentous fungal host cells include, but are not limited to, those derived from the Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase genes. Suitable leaders for yeast host cells include, but are not limited to, those derived from the Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3phosphoglycerate kinase, Saccharomyces cerevisiae alpha factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2) genes. /GAP).

In some embodiments, the sequences of the present invention also contain a polyadenylation sequence, which is a sequence operably linked to the 3' end of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to the transcribed mRNA. Any polyadenylation sequence that is functional in a host cell preferably finds use in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to, those derived from the Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase genes. Efficient polyadenylation sequences for yeast host cells are known in the art (see, for example, Guo and Sherman, Mol. Cell. Biol., 15:5983-5990 [1995]).

In some embodiments, the control sequence comprises a signal peptide coding region encoding an amino acid sequence that binds to the amino terminus of the polypeptide and directs the encoded polypeptide to the secretory pathway of the cell. The 5' end of the coding sequence in the nucleic acid sequence may inherently contain a signal peptide coding region, naturally associated with conservation of translational reading frame with a segment of the coding region that encodes the secreted polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding region that is foreign to the coding sequence. When the coding sequence does not naturally contain a signal peptide coding region, a foreign signal peptide coding region may be required.

Alternatively, the foreign signal peptide coding region may simply replace the native signal peptide coding region in order to enhance secretion of the polypeptide.

However, any signal peptide coding region that directs the expressed polypeptide to the secretory pathway of a host cell can preferably be used in the present invention.

Effective signal peptide coding regions for bacterial host cells include, but are not limited to, signal peptide coding regions derived from the Bacillus NC1B 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus neutral protease genes stearothermophilus (nprT, nprS, nprM) and prsA Bacillus subtilis.

Additional signal peptides are known in the art (see, for example, Simonen and Palva, Microbiol. Rev., 57: 109-137 [1993]).

Effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to, signal peptide coding regions derived from the Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and lipase genes Humicola lanuginosa.

Effective signal peptides for yeast host cells include, but are not limited to, the Saccharomyces cerevisiae alpha factor and Saccharomyces cerevisiae invertase genes. Other effective signal peptide coding regions are known in the art (see, eg, Romanos et al., [1992], supra).

In some embodiments, the control sequence comprises a propeptide coding region, which encodes an amino acid sequence located at the amino terminus of the polypeptide. The resulting polypeptide is known as a proenzyme or propolypeptide (or zymogen, in some cases). The propolypeptide is generally inactive and can be converted to the mature active PGA polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region can be derived from the Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha factor, Rhizomucor miehei aspartic proteinase and Myceliophthora thermophila lactase genes (see, for example, WO 95/33836).

When both signal peptide and propeptide regions are present at the amino terminus of the polypeptide, the propeptide region is located adjacent to the amino terminus of the polypeptide and the signal peptide region is located adjacent to the amino terminus of the propeptide region.

In some embodiments, regulatory sequences are also used to allow the expression of the polypeptide to be regulated relative to the growth of the host cell. Examples of regulatory systems are those that provide expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. In prokaryotic host cells, suitable regulatory sequences include, but are not limited to, the lac, tac and trp operator systems. In yeast host cells, suitable regulatory systems include, for example, the ADH2 system or the GAL1 system. In filamentous fungi, suitable regulatory sequences include the TAKA alpha-amylase promoter, the Aspergillus niger glucoamylase promoter, and the Aspergillus oryzae glucoamylase promoter.

Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, these include the dihydrofolate reductase gene, which is amplified in the presence of methotrexate, and the metallothionein genes, which are amplified in the presence of heavy metals. In these cases, the nucleic acid sequence encoding the PGA polypeptide of the present invention should be operably linked to a regulatory sequence.

Thus, in additional embodiments, the present invention provides recombinant expression vectors comprising a polynucleotide encoding an engineered PGA polypeptide or variant thereof, and one or more expression control regions, such as a promoter and terminator, an origin of replication, etc., depending on the type of hosts into which they should be introduced. In some embodiments, the various nucleic acid sequences and control sequences described above are linked together to produce a recombinant expression vector, which may contain one or more convenient restriction sites to allow the insertion or replacement of a polypeptide-encoding nucleic acid sequence in such areas. Alternatively, in some embodiments, nucleic acid sequences are expressed by inserting the nucleic acid sequence or a nucleic acid construct containing the sequence into a suitable expression vector. When creating an expression vector, a coding sequence is positioned in the vector so as to operably link the coding sequence to suitable expression control sequences.

A recombinant expression vector includes any suitable vector (eg, plasmid or virus) that can be conveniently manipulated with recombinant DNA and can provide expression of a polynucleotide sequence. The choice of vector generally depends on the compatibility of the vector with the host cell into which the vector is to be introduced. In some embodiments, the vectors are linear or closed circular plasmids.

In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid, extrachromosomal element, minichromosome, or artificial chromosome). In some embodiments, the vector contains any means to enable self-replication. Alternatively, in some other embodiments, when introduced into a host cell, the vector is inserted into the genome and replicated along with the chromosome(s) into which it is inserted. In addition, in additional embodiments, one vector or plasmid or two or more vectors or plasmids that together contain the entire DNA to be introduced into the host cell genome, or transposon, are used.

In some embodiments, the expression vector of the present invention contains one or more selectable markers that allow simple selection of transformed cells. A selectable marker is a gene whose product provides resistance to a biocide or virus, resistance to antimicrobials or heavy metals, prototrophy for auxotrophs, etc. Any suitable selectable markers for use in filamentous fungal host cells find use in the present invention, including, but not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinotricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine 5'phosphate decarboxylase), sC (sulfate adenyl transferase) and trpC (anthranilate synthase), as well as their equivalents. Additional markers that can be used in host cells such as Aspergillus include, but are not limited to, the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae, and the bar gene of Streptomyces hygroscopicus. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1 and URA3. Examples of bacterial selectable markers include, but are not limited to, dal genes from Bacillus subtilis or Bacillus licheniformis or markers that confer antibiotic resistance, such as resistance to ampicillin, kanamycin, chloramphenicol and/or tetracycline.

In some embodiments, the expression vectors of the present invention contain element(s) that allow the vector to be inserted into the genome of a host cell or to replicate autonomously in the cell independently of the genome. In some embodiments involving insertion into the genome of a host cell, the vectors are based on a nucleic acid sequence encoding a polypeptide or any other vector element for insertion of the vector into the genome via homologous or non-homologous recombination.

In some alternative embodiments, expression vectors contain additional nucleic acid sequences to direct integration through homologous recombination into the host cell genome. Additional nucleic acid sequences enable the vector to be inserted into the host cell genome at precise location(s) on the chromosome(s). To increase the likelihood of insertion at a precise location, the integration elements preferably contain a sufficient number of nucleotides, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 base pairs, that are highly homologous to the corresponding target sequence, to increase the likelihood of homologous recombination. Integration elements can be any sequence that is homologous to a target sequence in the host cell genome. In addition, integration elements can be non-coding or coding nucleic acid sequences. Alternatively, the vector can be inserted into the host cell genome through non-homologous recombination.

For autonomous replication, the vector may further comprise an origin of replication that allows autonomous replication of the vector in the host cell in question. Examples of bacterial origins of replication are ori P15A or origins of replication of plasmids pBR322, pUC19, pACYC177 (this plasmid has ori P15A) or pACYC184, which are permissible for replication in E. coli, and pUB110, pE194, pTA1060 or pAMe1, which are permissible for replication in Bacillus. Examples of origins of replication for use in yeast host cells are the 2 μm origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6. An origin of replication may be one that has a mutation that makes its operation temperature sensitive in the host cell (see, for example, Ehrlich, Proc. Natl. Acad. Sci. USA 75:1433 [1978]).

In some embodiments, more than one copy of a nucleic acid sequence of the present invention is inserted into a host cell to increase production of a gene product. An increase in the number of copies of a nucleic acid sequence can be obtained by inserting at least one additional copy of the sequence into the genome of a host cell or by incorporating an amplified selectable marker gene with the nucleic acid sequence, wherein cells containing amplified copies of the selectable marker gene and thereby additional copies of the nucleic acid sequence can be selected by culturing the cells in the presence of a suitable selection agent.

Many expression vectors for use in the present invention are commercially available. Suitable commercial expression vectors include, but are not limited to, p3xFLAGTMTM expression vectors (Sigma-Aldrich Chemicals), which contain the CMV promoter and hGH polyadenylation site for expression in mammalian host cells and the pBR322 origin of replication and ampicillin resistance markers for amplification in E .coli. Other suitable expression vectors include, but are not limited to, pBluescriptII SK(-) and pBK-CMV (Stratagene), and plasmids derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4 (Invitrogen) or pPoly ( see, for example, Lathe et al., Gene 57:193-201 [1987]).

Thus, in some embodiments, a vector containing a sequence encoding at least one PGA variant is transformed into a host cell to allow propagation of the vector and expression of the PGA variant(s). In some embodiments, PGA variants are post-translationally modified to remove a signal peptide, and in some cases may be cleaved after secretion. In some embodiments, the transformed host cell described above is cultured in a suitable growth medium under conditions allowing expression of the PGA variant(s). Any suitable medium that can be used to culture host cells finds use in the present invention, including, but not limited to, minimal or complex media containing suitable additives. In some embodiments, host cells are grown in HTP media. Suitable media are available from various commercial suppliers or can be obtained from published recipes (eg, American Type Culture Collection catalogs).

In another aspect, the present invention provides host cells containing a polynucleotide encoding an improved PGA polypeptide as provided herein, the polynucleotide being operably linked to one or more control sequences for expression of a PGA enzyme in the host cell. Host cells for use in the expression of PGA polypeptides encoded by the expression vectors of the present invention are well known in the art and include, but are not limited to, bacterial cells such as E. coli, Bacillus megaterium, Lactobacillus kefir, Streptomyces and Salmonella typhimurium cells ; fungal cells, such as yeast cells (for example, Saccharomyces cerevisiae or Pichia pastoris (Accession No. ATCC 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293 and Bowes melanoma cells; and plant cells. Suitable culture media and growth conditions for the host cells described above are well known in the art.

PGA expression polynucleotides can be introduced into cells using a variety of methods known in the art. Techniques include electroporation, biolistic particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion, among others. Various methods for introducing polynucleotides into cells are known to those skilled in the art.

In some embodiments, the host cell is a eukaryotic cell. Suitable eukaryotic host cells include, but are not limited to, fungal cells, algal cells, insect cells and plant cells. Suitable fungal host cells include, but are not limited to, Ascomycota, Basidiomycota, Deuteromycota, Zygomycota, Fungi imperfecti. In some embodiments, the fungal host cells are yeast cells and filamentous fungal cells. The filamentous fungal host cells of the present invention include all filamentous forms of the subdivision Eumycotina and Oomycota. Filamentous fungi are distinguished by vegetative mycelium with a cell wall consisting of chitin, cellulose and other complex polysaccharides. The host cells of the filamentous fungi of the present invention are morphologically distinct from yeast.

In some embodiments of the present invention, the filamentous fungal host cells are any suitable genus and species, including, but not limited to, Achlya, Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus, Corynascus, Cryphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia, Endothis, Fusarium, Gibberlla, Gliocladium, Humicola, Hypocrea, Myceliophthora, Mucor, Neurospora, Penicillium, Podospora, Phlebia, PIROMYCES, PIROMYCES, PIROMYCES, PIROMYCES, PIROMYCES, PIROMYCES, PIROMYCES, PIROMYCES Yricularia, Rhizomucor, Rhizopus, Schizophyllum, Scytalidium, Sporotrichum, Talaromyces, Thermoascus, Thielavia, Trametes, Tolypocladium, Trichoderma, Verticillium and/or Volvariella and/or teleomorphs or anamorphs, as well as their synonyms, basionyms or taxonomic equivalents.

In some embodiments of the present invention, the host cell is a yeast cell, including but not limited to Candida, Hansenula, Saccharomyces, Schizosaccharomyces, Pichia, Kluyveromyces, or Yarrowia species. In some embodiments of the present invention, the yeast cell is Hansenula polymorpha, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces diastaticus, Saccharomyces norbensis, Saccharomyces

- 22 043748 kluyveri, Schizosaccharomyces pombe, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia kodamae,

Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia quercuum, Pichia pijperi, Pichia stipitis, Pichia methanolica, Pichia angusta, Kluyveromyces lactis, Candida albicans or Yarrowia lipolytica.

In some embodiments, the host cell is an algal cell, such as Chlamydomonas (eg, C. reinhardtii) and Phormidium (P. sp. ATCC29409).

In some other embodiments, the host cell is a prokaryotic cell. Suitable prokaryotic cells include, but are not limited to, gram-positive, gram-negative and gram-variable bacterial cells. Any suitable bacterial organism finds use in the present invention, including but not limited to Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Acinetobacter, Acidothermus, Arthrobacter, Azobacter, Bacillus, Bifidobacterium, Brevibacterium, Butyrivibrio, Buchnera, Campestris, Camplyobacter, Clostridium, Corynebacterium, Chromatium , Coprococcus, Escherichia, Enterococcus, Enterobacter, Erwinia, Fusobacterium, Faecalibacterium, Francisella, Flavobacterium, Geobacillus, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Lactococcus, Ilyobacter, Micrococcus, Microbacterium, Mesorhizobium, Methylobacterium, Methylobacterium, Mycobacterium, Neisseria, Pantoea, Pseudomonas , Prochlorococcus, Rhodobacter, Rhodopseudomonas, Rhodopseudomonas, Roseburia, Rhodospirillum, Rhodococcus, Scenedesmus, Streptomyces, Streptococcus, Synecoccus, Saccharomonospora, Staphylococcus, Serratia, Salmonella, Shigella, Thermoanaerobacterium, Tropheryma, Tularensis, Temecula, Therm osynechococcus, Thermococcus, Ureaplasma, Xanthomonas, Xylella , Yersinia and Zymomonas. In some embodiments, the host cell is a species of Agrobacterium, Acinetobacter, Azobacter, Bacillus, Bifidobacterium, Buchnera, Geobacillus, Campylobacter, Clostridium, Corynebacterium, Escherichia, Enterococcus, Erwinia, Flavobacterium, Lactobacillus, Lactococcus, Pantoea, Pseudomonas, Staphylococcus, Salmonella, Streptococcus, Streptomyces or Zymomonas. In some embodiments, the host bacterial strain is not pathogenic to humans. In some embodiments, the host bacterial strain is a commercial strain. Many commercial bacterial strains are known and are suitable for the present invention. In some embodiments of the present invention, the bacterial host is an Agrobacterium species (eg, A. radiobacter, A. rhizogenes, and A. rubi). In some embodiments of the present invention, the bacterial host cell is an Arthrobacter species (e.g., A. aurescens, A. citreus, A. globiformis, A. hydrocarboglutamicus, A. mysorens, A. nicotianae, A. paraffineus, A. protophonniae, A. roseoparqffinus, A. sulfureus and A. ureafaciens). In some embodiments of the present invention, the bacterial host cell is a Bacillus species (e.g., B. thuringensis, B. anthracis, B. megaterium, B. subtilis, B. lentus, B. circulans, B. pumilus, B. lautus, B. .coagulans, B. brevis, B. firmus, B. alkaophius, B. licheniformis, B. clausii, B. stearothermophilus, B. halodurans and B. amyloliquefaciens). In some embodiments, the host cell is a commercial strain of Bacillus, including but not limited to B. subtilis, B. pumilus, B. licheniformis, B. megaterium, B. clausii, B. stearothermophilus, or B. amyloliquefaciens. In some embodiments, the Bacillus host cells are B. subtilis, B. licheniformis, B. megaterium, B. stearothermophilus, and/or B. amyloliquefaciens. In some embodiments, the bacterial host cell is a Clostridium species (eg, C. acetobutylicum, C. tetani E88, C. lituseburense, C. saccharobutylicum, C. perfringens, and C. beijerinckii). In some embodiments, the bacterial host cell is a Corynebacterium species (eg, C. glutamicum and C. acetoacidophilum). In some embodiments, the bacterial host cell is an Escherichia species (eg, E. coli). In some embodiments, the bacterial host cell is an Erwinia species (eg, E. uredovora, E. carotovora, E. ananas, E. herbicola, E. punctata, and E. terreus). In some embodiments, the bacterial host cell is a Pantoea species (eg, P. Citrea and P. agglomerans). In some embodiments, the bacterial host cell is a Pseudomonas species (eg, P. putida, P. aeruginosa, P. mevalonii, and P. sp. D-01 10). In some embodiments, the bacterial host cell is a Streptococcus species (eg, S. equisimiles, S. Pyogenes, and S. uberis). In some embodiments, the bacterial host cell is a Streptomyces species (e.g., S. ambofaciens, S. achromogenes, S. avermitilis, S. coelicolor, S. aureofaciens, S. aureus, S. fungicidicus, S. griseus, and S. lividans ). In some embodiments, the bacterial host cell is a Zymomonas species (eg, Z. mobilis and Z. lipolytica).

The model host cell is Escherichia coli W3110. An expression vector was created by operably linking a polynucleotide encoding an improved PGA into plasmid pCK110900, operably linked to the lac promoter under the control of the lad repressor. The expression vector also contained the P15a origin of replication and the chloramphenicol resistance gene. Cells containing the polynucleotide in question in Escherichia coli W3110 were isolated through cell selection using chloramphenicol.

Many prokaryotic and eukaryotic strains that find use in the present invention are readily available to the public in many culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Cen- 23 043748 traalbureau Voor Schimmelcultures (CBS) and Agricultural Research Service Patent Culture Collection, Northern

Regional Research Center (NRRL).

In some embodiments, host cells are genetically modified to have characteristics that improve protein secretion, protein stability, and/or other properties desired for protein expression and/or secretion. Genetic modification can be carried out using genetic engineering techniques and/or classical microbiological techniques (for example, chemical or UV mutagenesis and subsequent selection). In fact, in some embodiments, combinations of recombinant modification and classical selection techniques are used to obtain host cells. Using recombinant technology, nucleic acid molecules can be introduced, removed, inhibited or modified in a manner that leads to increased yield of PGA variant(s) in the host cell and/or culture medium. For example, knockout of Alp1 function leads to a cell with protease deficiency, and knockout of pyr5 function leads to a cell with a pyrimidine deficiency phenotype. In one genetic engineering approach, homologous recombination is used to induce modifications of a target gene by specifically targeting the gene in vivo to inhibit expression of the encoded protein. Alternative approaches include the use of siRNA, antisense and/or ribozyme technology to inhibit gene expression. Various methods are known in the art to reduce protein expression in cells, including, but not limited to, deletion of all or part of the gene encoding the protein and site-directed mutagenesis to disrupt the expression or activity of the gene product. (See, for example, Chaveroche et al., Nucl. Acids Res., 28:22 e97 [2000]; Cho et al., Molec. Plant Microbe Interact., 19:7-15 [2006]; Maruyama and Kitamoto, Biotechnol Lett., 30:1811-1817 [2008]; Takahashi et al., Mol. Gen. Genom., 272: 344-352 [2004]; and You et al., Arch. Micriobiol., 191:615-622 [2009], all of which are incorporated by reference herein). Random mutagenesis followed by screening for desired mutations is also used (see, for example, Combier et al., FEMS Microbiol. Lett., 220:141-8 [2003]; and Firon et al., Eukary. Cell 2:247 -55 [2003], both included by reference).

Introduction of a vector or DNA construct into a host cell can be accomplished using any suitable method known in the art, including, but not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, PEG-mediated transformation, electroporation, or other conventional techniques known in the art. .

In some embodiments, engineered host cells (ie, recombinant host cells) of the present invention are cultured in standard culture media modified as appropriate for promoter activation, transformant selection, or PGA polynucleotide amplification. Culture conditions such as temperature, pH, etc. are those previously used for the host cell selected for expression and are well known to those skilled in the art. As noted, many standard sources and texts are available for the culture and production of many cells, including cells derived from bacteria, plants, animals (particularly mammals), and archaebacteria.

In some embodiments, cells expressing PGA variant polypeptides of the invention are grown under batch or continuous fermentation conditions. Classic batch fermentation is a closed system in which the composition of the medium is set at the beginning of fermentation and is not subject to artificial changes during fermentation. A variation of the batch system is fed-batch fermentation, which also finds use in the present invention. In this variation, the substrate is added in portions as fermentation progresses. Fed-batch systems can be used when catabolite repression is likely to inhibit cell metabolism and when it is desirable to have limited amounts of substrate in the medium. Batch fermentation and fed-batch fermentation are common and well known in the art. Continuous fermentation is an open system where a specified fermentation medium is continuously added to the bioreactor and simultaneously an equal amount of conditioned medium is removed for processing. Continuous fermentation generally maintains cultures at a constant high density, where cells are primarily in logarithmic growth phase. Continuous fermentation systems strive to maintain sustainable growth conditions. Techniques with modulating nutrients and growth factors for a continuous fermentation process, as well as techniques for maximizing the rate of product formation, are well known in the field of industrial microbiology.

In some embodiments of the present invention, cell-free transcription/translation systems are used in the production of PGA variant(s). Several systems are commercially available and the methods are well known to those skilled in the art.

The present invention provides methods for producing PGA variant polypeptides or biologically active fragments thereof. In some embodiments, the method includes: providing a host cell transformed with a polynucleotide encoding an amino acid sequence that has at least about 70% (or at least about 75%, at least about 80%, about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) sequence identity to SEQ ID No. 12, 24, 40, 56, 70, 82, 100, 108, 110, 116, 136, 154 and/or 160, and contains at least one mutation as provided herein; culturing the transformed host cell in a culture medium under conditions in which the host cell expresses the encoded PGA variant polypeptide; and optionally recovering or isolating the expressed PGA variant polypeptide and/or recovering or isolating a culture medium containing the expressed PGA variant polypeptide. In some embodiments, the methods further comprise optionally lysing transformed host cells following expression of the encoded PGA polypeptide and optionally extracting and/or isolating the expressed PGA variant polypeptide from the cell lysate. The present invention further provides methods for producing a PGA variant polypeptide, comprising culturing a host cell transformed with a PGA variant polypeptide under conditions suitable for producing a PGA variant polypeptide, and recovering the PGA variant polypeptide. Typically, recovery or isolation of the PGA polypeptide occurs from the culture medium of the host cell, the host cell, or both, using protein recovery techniques that are well known in the art, including those described herein. In some embodiments, host cells are collected by centrifugation, disrupted by physical or chemical means, and the resulting crude extract is stored for further purification. Microbial cells used in the expression of proteins can be disrupted by any convenient method, including, but not limited to, freeze-thaw cycling, sonication, mechanical disruption, and/or the use of cell lysis agents, as well as many other suitable methods well known to those skilled in the art. areas.

The engineered PGA enzymes expressed in the host cell can be recovered from the cells and/or culture medium using any one or more conventional protein purification techniques including, but not limited to, lysozyme treatment, sonication, filtration, salting out, ultracentrifugation, and chromatography . Suitable solutions for lysing and highly efficient extraction of proteins from bacteria such as E. coli are commercially available under the trade name CelLytic B™ (Sigma-Aldrich).

Thus, in some embodiments, the resulting polypeptide is recovered/isolated and optionally purified by any of many methods known in the art. For example, in some embodiments, the polypeptide is isolated from the culture medium using standard procedures, including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation, chromatography (e.g., ion exchange, affinity, hydrophobic interaction, chromatofocusing and size exclusion) or precipitation. In some embodiments, protein refolding steps optionally utilize the mature protein configuration upon completion. Additionally, in some embodiments, high performance liquid chromatography (HPLC) is used in the final purification steps. For example, in some embodiments, methods known in the art are used in the present invention (see, for example, Parry et al., Biochem. J., 353:117 [2001]; and Hong et al., Appl. Microbiol. Biotechnol ., 73:1331 [2007], both incorporated herein by reference). In fact, any suitable purification methods known in the art find use in the present invention.

Chromatographic techniques for isolating PGA polypeptide include, but are not limited to, reverse phase chromatography, high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, and affinity chromatography. The conditions for purifying a particular enzyme depend in part on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., which are known to those skilled in the art.

In some embodiments, affinity techniques are used in the isolation of improved PGA enzymes. Any antibody that specifically binds a PGA polypeptide can be used for affinity chromatography purification. To obtain antibodies, various animal hosts, including but not limited to rabbits, mice, rats, etc., can be immunized by injection of PGA. The PGA polypeptide can be attached to a suitable carrier, such as BSA, through a side chain functional group or linkers attached to a side chain functional group. Various adjuvants can be used to enhance the immunological response, depending on the host species, including, but not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, polyols Pluronic, polyanions, peptides, oil emulsions, limpet hemocyanin, dinitrophenol and potentially effective human adjuvants such as BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum.

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In some embodiments, PGA variants are prepared and used in the form of enzyme-expressing cells, as crude extracts, or as isolated or purified preparations. In some embodiments, PGA variants are prepared as lyophilisates, in powder form (eg, acetone powders), or are prepared as enzyme solutions. In some embodiments, the PGA variants are in the form of substantially pure preparations.

In some embodiments, the PGA polypeptides are attached to any suitable solid support. Solid supports include, but are not limited to, a solid phase, a surface and/or a membrane. Solid carriers include, but are not limited to, organic polymers such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy and polyacrylamide, as well as copolymers and graft polymers thereof. The solid support may also be inorganic, such as glass, silica, controlled pore glass (CPG), reverse phase silica, or a metal such as gold or platinum. The substrate configuration can be in the form of beads, spheres, particles, granules, gel, membrane or surface. The surfaces may be planar, substantially planar, or non-planar. Solid carriers may be porous or non-porous and may have swelling or non-swelling characteristics. The solid carrier may be in the form of a well, depression, or other container, vessel, feature, or location. The plurality of supports may be arranged in an array at different locations, reversible for robotic delivery of reagents or by sensing methods and/or tools.

In some embodiments, immunological methods are used to purify PGA variants. In one approach, an antibody raised against a PGA variant polypeptide (e.g., against a polypeptide comprising any of SEQ ID NOs: 2, 4, 12, 24, 40, 56, 70, 82, 100, 108, 110, 116, 136, 154 and/or 160 and/or an immunogenic fragment thereof), using standard methods, are immobilized on beads, mixed with cell culture media under conditions in which the PGA variant is bound, and precipitated. A related approach finds application in immunochromatography.

In some embodiments, PGA variants are expressed as a fusion protein containing a non-enzymatic portion. In some embodiments, the PGA variant sequence is fused to a purification-facilitating domain. As used herein, the term purification facilitating domain refers to a domain that mediates the purification of the polypeptide to which it is fused. Suitable domains for purification include, but are not limited to, metal-chelating peptides, histidine-tryptophan modules that allow purification on immobilized metals, a sequence that binds glutathione (e.g., GST), a hemagglutinin tag (HA) (corresponding to an epitope derived from influenza hemagglutinin protein; see, for example, Wilson et al., Cell 37:767 [1984]), maltose-binding protein sequences, the FLAG epitope used in the FLAGS extension/affinity purification system (for example, the system available from Immunex Corp), and etc. One expression vector contemplated for use in the compositions and methods described herein provides expression of a fusion protein comprising a polypeptide of the invention fused to a polyhistidine region separated by an enterokinase cleavage site. Histidine residues facilitate IMIAC (immobilized metal ion affinity chromatography; see, e.g., Porath et al., Prot. Exp. Purif., 3:263-281 [1992]) purification, while the enterokinase cleavage site provides a means for polypeptide separation PGA variant from the fusion protein. pGEX vectors (Promega) can also be used to express foreign polypeptides as glutathione S-transferase (GST) fusion proteins. In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption onto ligand-agarose beads (eg, glutathione-agarose in the case of a GST fusion), followed by elution in the presence of free ligand.

experimental part

Various features and embodiments of the disclosure are illustrated in the following representative examples, which are provided by way of illustration and not limitation.

Further, in the disclosure of experiments, the following abbreviations are used: ppm. (ppm); M (molar); mM (millimolar), µM (micromolar); nM (nanomolar); mole(s); g (gram); mg (milligram); mcg (microgram); l (liter); ml (milliliter); cm (centimeter); mm (millimetre); µm (micrometer); s(second); min (minute); h (hour); Unit (unit); MW (molecular weight); rpm (revolutions per minute); °C (degrees Celsius); RT (room temperature); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); a.k. (amino acid); TV (TV medium; 12 g/L bactotryptone, 24 g/L yeast extract, 4 ml/L glycerol, 65 mM potassium phosphate, pH 7.0, 1 mM MgSO ₄ ); CAM (chloramphenicol); PMBS (polymyxin B sulfate); IPTG (isopropylthiogalactoside); TFA (trifluoroacetic acid); CHES (2-cyclohexylamino)ethanesulfonic acid; HPLC (high performance liquid chromatography); FIOPC (fold improvement relative to positive control); HTR (high throughput); LB (Luria broth); Codexis (Codexis, Inc., Redwood City, CA); Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO); Millipore (Millipore, Corp., Billerica MA); Difco (Difco Laboratories, BD Diagnostic Systems, Detroit, MI); Daicel (Daicel, West

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Chester, PA); Genetix (Genetix USA, Inc., Beaverton, OR); Molecular Devices (Molecular Devices, LLC,

Sunnyvale, CA); Applied Biosystems (Applied Biosystems, part of Life Technologies, Corp., Grand Island, NY),

Agilent (Agilent Technologies, Inc., Santa Clara, CA); Thermo Scientific (part of Thermo Fisher Scientific, Waltham, MA); (Infors; Infors-HT, Bottmingen/Basel, Switzerland); Corning (Corning, Inc., Palo Alto, CA); and BioRad (Bio-Rad Laboratories, Hercules, CA); Microfluidics (Microfluidics Corp., Newton, MA).

Example 1. E. coli expression hosts containing recombinant PGA genes.

The parent PGA enzymes used to make the variants of the present invention were obtained from either the Codex® acylase panel (PGA panel plate; Codexis) or the variants disclosed in co-owned US Patent Application Publication No. 2016/0326508. The PGA panel plate contains a collection of engineered PGA polypeptides that have improved properties compared to wild-type Kluyvera citrophila PGA. The wild-type PGA gene is a heterodimer consisting of an alpha subunit (23.8 kDa) and a beta subunit (62.2 kDa), which are linked by a 54-aa spacer region. Due to the presence of a spacer region, an autoprocessing step is required in order to form the active protein. During the development of the present invention, the wild type gene was modified to eliminate the spacer region, thereby eliminating the autoprocessing step. The PGA panel plate (Codexis) contains PGA variants that do not contain a spacer region (see, for example, US patent application publication 2010/0143968 A1). The PGA encoding genes were cloned into the expression vector pCK110900 (see Fig. 3 in US Patent Application Publication No. 2006/0195947), operably linked to the lac promoter under the control of the lacl repressor. The expression vector also contains the P15a origin of replication and the chloramphenicol resistance gene. The resulting plasmids were transformed into E. coli W3110 using standard methods known in the art. Transformants were isolated by subjecting cells to selection with chloramphenicol, as is known in the art (see, for example, US patent No. 8383346 and WO2010/144103).

Example 2. Preparation of NTP PGA-containing wet cell sediments

E. coli cells containing recombinant PGA-encoding genes from monoclonal colonies were inoculated in 180 μl of LB containing 1% glucose and 30 μg/ml chloramphenicol in the wells of 96-well microtiter plates with fine wells. The plates were sealed with O ₂ permeable seals and cultures were grown overnight at 30°C, 200 rpm and 85% humidity. Then, 10 μl of each cell culture was transferred into the wells of 96-well deep well plates containing 390 ml TB and 30 μg/ml CAM. Deep well plates were sealed with O ₂ -permeable seals and incubated at 30°C, 250 rpm and 85% humidity until OD6000.6-0.8 was reached. Cell cultures were then induced using IPTG to a final concentration of 1 mM and incubated overnight under the same conditions as originally used. Cells were then pelleted using centrifugation at 4000 rpm for 10 min. Supernatants were discarded and pellets were frozen at −80°C before lysis.

Example 3. Preparation of NTP PGA-containing cell lysates

First, 200 μl of lysis buffer containing 10 mM Tris-HCl buffer, pH 7.5, 1 mg/ml lysozyme and 0.5 mg/ml PMBS was added to the cell mass in each well prepared as described in example 2. Cells lysed at room temperature for 2 h with shaking on a tabletop shaker. The plate was then centrifuged for 15 min at 4000 rpm and 4°C. The clear supernatants were then used in biocatalytic reactions to determine their activity levels.

Example 4: Preparation of lyophilized lysates from shake flask (SF) cultures.

Selected NTP cultures, which were grown as described above, were plated on LB agar plates with 1% glucose and 30 μg/ml CAM and grown overnight at 37°C. One colony from each culture was transferred to 6 ml LB with 1% glucose and 30 μg/ml CAM.

Cultures were grown for 18 h at 30°C, 250 rpm and subcultured approximately 1:50 in 250 ml TB containing 30 μg/ml CAM to a final OD600 of 0.05. Cultures were grown for approximately 195 minutes at 30°C, 250 rpm, to an OD600 of 0.6-0.8 and induced using 1 mM IPTG. The cultures were then grown for 20 h at 30°C, 250 rpm. Cultures were centrifuged at 4000 rpm for 20 min. The supernatant was discarded and the pellets were resuspended in 30 ml of 20 mM TRIS-HCl, pH 7.5. Cells were pelleted (4000 rpm x 20 min) and frozen at -80°C for 120 min. Frozen pellets were resuspended in 30 ml of 20 mM TRIS-HCl, pH 7.5 and lysed using a Microfluidizer® Processing System (Microfluidics) at ^18,000 psi. Lysates were pelleted (10,000 rpm x 60 min) and supernatants were frozen and lyophilized to generate shake flask (SF) enzymes.

Example 5 Improvements in Insulin Acylation at Positions A1, B1 and B29 relative to SEQ ID No. 4

SEQ ID No. 4 was selected as the parent enzyme based on the results of screening variants disclosed in joint US Patent Application Publication No. 2016/0326508 to obtain the B29 deacylated product. Libraries of engineered genes were prepared using

- 27 043748 well techniques (eg, saturation mutagenesis and recombination of previously identified beneficial mutations).

The polypeptides encoded by each gene were prepared in the NTR as described in Example 2, and soluble lysates were created as described in Example 3.

Each reaction well contained 200 μl of 0.1 M CHES, pH 10, 10 g/l insulin, 17 g/l methylphenylacetate, and 20 μl of HTP supernatant. NTP plates were incubated in a Thermotron® shaker (3 mm amplitude, model no. AJ185, Infors) at 30°C, 300 rpm, for 20 hours. Reactions were quenched using 200 μl of acetonitrile and mixed for 5 minutes using a benchtop shaker The plates were then centrifuged at 4000 rpm for 5 min and loaded onto HPLC for analysis.

The activity relative to SEQ ID No. 4 (FIOP activity) was calculated as the percent conversion of the product formed by the variant relative to the percent conversion provided by SEQ ID No. 4. The results are shown in table. 5.1. The percent conversion was calculated by dividing the peak area of the product by the sum of the peak areas of the substrate, product, and impurities/by-product as observed by HPLC analysis. In table 5.2 shows the results showing the selectivity of the variants relative to SEQ ID No. 4.

Table 5.1. Activity of variants relative to SEQ ID No. 4

No. option SEQ ID No. (n./a.k.) Differences amino acids (relative to SEQ ID no. 4) Percentage of acylation conversion of FIOP ¹ at the indicated sites (relative to SEQ ID No. 4) Al B29 Bl A1/B2 9 Al/B 1 B1/B2 9 4 11/12 F71G;G74D +++ +++ ++++ ++++ ++++ ++++ 5 17/18 F2 4Y;V2 8A;F71C;F7 01W + ++++

¹ Levels of increased activity were set relative to the reference polypeptide of SEQ ID No. 4 and were determined as follows: + > 1.2-fold but less than 2.5-fold increased activity; ++ > more than 2.5 times, but less than 5 times increased activity; +++ > more than 5 times increased activity, but less than 10 times; ++++ > than 10 times.

Table 5.2. Selectivity of variants relative to SEQ ID No. 4

No. varia nta SEQ ID No. (n./a.k.) Amino Acid Differences (relative to SEQ ID No. 4) Acylation Selectivity Percentage (FIOP) ¹ at designated regions (relative to SEQ ID No. 4) Al B29 Bl Al/B 29 Al/B 1 Bl/B 29 3 85/86 F71R + 4 11/12 F71G;G74D + +++ + + + + + + + + + + + + + 5 17/18 F24Y;V28A;F71C;F 701W + + + +

¹ Levels of increased selectivity were set relative to the reference polypeptide of SEQ ID No. 4 and were determined as follows: + > 1.2-fold but less than 2.5-fold increased selectivity; + + > more than 2.5 times, but less than 5 times increased selectivity; +++ > more than 5 times increased selectivity, but less than 10 times; ++++ > than 10 times.

Example 6: Improvement of Insulin Acylation at Positions A1, B1 and B29 Compared to SEQ ID No. 12 in a High Throughput Screen

SEQ ID No. 12 was selected as the next parent enzyme based on the results described in Example 5 (ie, the best enzyme identified for acylation of insulin at position B29). Libraries of engineered genes were prepared using well techniques (eg, saturation mutagenesis and recombination of previously identified beneficial mutations).

The polypeptides encoded by each gene were prepared in the NTR as described in Example 2, and soluble lysates were created as described in Example 3.

Screening of each variant was carried out in a 200 μl reaction consisting of 10 g/l insulin, 0.1 M TRIS buffer pH 9.25, 20% acetonitrile, 17 g/l methylphenylacetate and 10 μl clarified lysate for 5 hours at 30° WITH. 96-well plates were heat sealed and incubated in a shaker

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Thermotron® at 100 rpm. Reactions were quenched using 200 μl of acetonitrile and mixed for 5 min using a benchtop shaker. The plates were then centrifuged at 4000 rpm for 5 min and loaded onto HPLC for analysis.

The percentage conversion relative to SEQ ID No. 12 (FIOP conversion percentage) was calculated as the percentage conversion of the product formed by the variant relative to the percentage conversion provided by SEQ ID No. 12. These results are presented in table. 6.1, 6.2, 6.3, 6.4, 6.5, 6.6 and 6.7. The percent conversion was calculated by dividing the peak area of the product by the sum of the peak areas of the substrate, product, and impurities/by-product as observed by HPLC analysis.

The percentage selectivity relative to SEQ ID No. 12 (FIOP selectivity percentage) was calculated as the percentage selectivity of the product formed by the variant relative to the percentage selectivity provided by SEQ ID No. 12. The results are presented in table. 6.1, 6.2, 6.3, 6.4, 6.5, 6.6 and 6.7. The percent selectivity was calculated by dividing the peak area of the product by the sum of the peak areas of the product and the impurities/by-product as observed by HPLC analysis.

Table 6.1. Activity and selectivity of variants acylating at the A1 site relative to SEQ ID No. 12

No. Variation SEQ ID No. (n./a.k.) Amino Acid Differences (relative to SEQ ID No. 12) Acylation Conversion Percentage (FIOP) ¹ at Site A1 (relative to SEQ ID NO. 12) Acylation selectivity percentage (FIOP) ¹ FOR region A1 (relative to SEQ ID No. 12) 6 Y27T;A255G;W3701; + + + 7 D623N; + 8 T384R; + + + 9 L253S; + + 10 T705S; + eleven A373Y; + + 12 Y27T;F254W;A470V; + + 13 Y27T;L253V;A255G;N348R; + 14 Y27T;D74S;F254W;A255G;N34 8R;KZ69C;TZ84P; + + 15 Y27T;D74N;L253V;F254W;N34 + + + 8R;KZ69C;TZ84P; 16 L253M; + + 17 N457T; + + 18 R317S;Q380P; + + 19 69/70 K128W; + +

¹ Levels of increased activity or selectivity were determined relative to the reference polypeptide of SEQ ID No. 12 and were defined as follows: + > 1.0-fold but less than 1.5-fold increased; ++ > more than 1.5 times, but less than 2.0 times; +++ > than 2.0 times.

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Table 6.2. Activity and selectivity of variants acylating at the B29 site relative to SEQ ID No. 12

No. varia nta SEQ ID No. (n./a.k.) Amino acid differences (relative to SEQ ID No. 12) Acylation conversion percentage (FIOP) ¹ at site B2 9 (relative to SEQ ID NO. 12) Acylation selectivity percentage (FIOP) ² for site B2 9 (relative to SEQ ID NO. 12) 20 Y27T;N348R;D381K; + + 21 Y27T;D74S;A255G;N348R; K369C;D381K; + 22 15/16 Y271;D74S;A255G;N348R; D381K;T384P; + + 23 Y2 7Т;N34 8R;КЗ 6 9С;W37 01; D381K;T384P; + 24 Y27T;D74G;F254W;A255G; N348R;K369S;W370I; D381K; + 25 Y27T;F254W;A255G;N348R; W370I;D381K; + 26 13/14 Y27T;A255G;N348R;W3701; D381K;T384P; + 27 D381F; + + 28 Q134M; + +

- 30 043748

29 D623W; ++ + thirty L253R; + + 31 N627M; + + 32 N627R; + + 33 D623N; ++ + 34 K615V; + + 35 D381L; + + 36 D381R; + ++ 37 A132G; + + 38 A467S; + + 39 F256Y; + + 40 D623V; + + 41 K615N; + 42 D623A; + + 43 D381Q; + + 44 K615S; + 45 T384R; + + 46 F256H; + + 47 T453S; + 48 D381V; + 49 D381K; + + 50 D381F;Q672K; + 51 D623Y; + + 53 D623R; +++ ++ 54 D623F; + + 55 D623K; +++ ++ 56 D381I; + 57 AZ73K; +++ ++ 58 S706K; + ++ 59 N348K;A467T; + + 60 D709G; + + 61 D709A; + + 62 F620R; + + 63 D709N; + ++ 64 D709H; + + 65 E377A; + 66 F620K; + + 67 S706R; + 68 21/22 D709R; + ++++ 69 N20S;D709Q; + 70 D709S; + + 71 V618C; + + 72 A6 9M; + 73 F2 5 4K; + + 74 A8 4V; + 75 F701H; + 76 P383K; + 77 Ab 9L; ++ 78 I708V; + 79 A255K; ++ ++ 80 A255R; ++ ++ 81 A6 9V; + 82 P383R; ++++

¹ Levels of increased activity were set relative to the reference polypeptide from SEQ ID No. 12 and were determined as follows: + > 2.0-fold but less than 4.0-fold increased; ++ > more than 4.0 times, but less than 6.0 times; +++ > than 6.0 times.

² Levels of increased selectivity were set relative to the reference polypeptide of SEQ ID No. 12 and were determined as follows: + > greater than 2.0-fold but less than 5.0-fold increased

- 31 043748 us; ++ > more than 5.0 times, but less than 10.0 times; +++ > than 10.0 times, but less than 15 times;

++++ > than 15 times.

Table 6.3. Activity and selectivity of variants acylating at sites A1 and B29 relative to SEQ ID No. 12

No. option a SEQ ID No. (n./a.k.) Amino acid differences (relative to SEQ ID No. 12) Percentage of conversion of IL by acylation (FIOP) ¹ in regions A1 and B29 (relative to SEQ ID No. 12) Acylation selectivity percentage (FIOP) ² FOR sites A1 and B29 (relative to SEQ ID No. 12) 198 Y271; D74S; F254W; N348R; D381W; + + 199 Y27T;A255G;N348R;W370I; + 200 Y27T;N348R;T384P; + + 201 Y27T;D74G;F254W;A255G;N348R; D381W; + + 202 Y27T;D74S;N348R; + 203 Y27T;A255G;N348R; + + 204 Y27T;L253V;N348R;D38IF;T384P r + + 205 Y27T;L253V;F254W;N348R;1384P r + 206 Y27I;D74P;F254W;A255G;N348R; D381K;T384P; + + 207 Y27T;N348R; + + 208 Y27T;F254W;A255G;N348R;W370I ;D381W;1384P; + 22 15/16 Y27T;D74S;A255G;N348R;D381K; 1384P; + + 209 Y27T;D74G;F254W;N348R;D381W; 1384P; + + 210 Y27T;F254W;A255G;D38IK;T384P r + + 211 Y2 7T;D7 4 S;F2 5 4W;N3 4 8R;D3 81F; + + 212 Y27T;A255G;N348R;D381W;T384P + + 213 Y27T;F254W;N348R;D381W;T384P r + + 214 Y27T;D74G;N348R; + + 181 Y27T;F254W;A255G;T384P; ++ + 182 Y27T;F254W;A255G; ++ + 215 Y27T;D74S;L253V;N348R; +++ ++ 183 Y27T;L253V;F254W;A255G;N348R ;D381F;T384P; + + 184 Y27T;F254W;A255G;N348R; + + 185 Y27T;D74N;F254W;T384P; +++ +

- 32 043748

186 Y27T;L253V;N348R; + 187 Y27T;D74G;A255G;N348R; ++ ++ 12 Y27T;F254W;A470V; + 13 Y27T;L253V;A255G;N348R; + + 189 Y27T;D74G;L253 V;A255G;N348R; D381F; ++ + 6 Y27T;A255G;W3701; ++ + 190 Y27T;D74S;F254W;A255G;N348R; +++ ++ 216 Y27T;D74N;L253V;F254W; + 191 9/10 Y27T;D74S;L253V;F254W;N348R; D381W;T384P; ++ ++ 192 Y27T;D74N;F254W;A255G;N348R; +++ ++ 217 5/6 Y27T;D74G;L253V;F254W;A255G; N348R; +++ + 194 Y27T;L253V;N348R;W3701;D38IF ;T384P; + + 26 13/14 Y27T;A255G;N348R;W3701;D38IK ;T384P; + + 195 Y2 7T;D7 4 S;A2 55G;W37 01; ++ + 29 D623W; + + thirty L253R; + + 42 D623A; + + 218 D623N; + + 46 F256H; + + 219 A616R; + 220 D623L; + + 51 D623Y; + + 54 D623F; + + 55 D623K; + + 221 D381Q; + + 222 T384R; ++ ++ 223 19/20 D623R; ++ +++ 57 A373K; + + 224 H472R; + 225 F620R; + + 197 A255P; + 73 F2 5 4K; + + 80 A255R; + +

¹ Levels of increased activity were set relative to the reference polypeptide from SEQ ID No. 12 and were determined as follows: + > 2.0-fold but less than 5.0-fold increased; ++ > more than 5.0 times, but less than 10.0 times; +++ > than 10.0 times.

Levels of increased selectivity were set relative to the reference polypeptide of SEQ ID No. 12 and were determined as follows: + > 2.0-fold but less than 5.0-fold increased; ++ > more than 5.0 times, but less than 7.0 times; +++ > than 7.0 times.

- 33 043748

Table 6.4. Activity and selectivity of variants acylating at sites A1 and B1 relative to SEQ ID No. 12

No. option SEQ ID No. (n./ A. To. ) Amino Acid Differences (relative to SEQ ID No. 12) Acylation conversion percentage (FIOP) ¹ at Al and Bl sites (relative to SEQ ID No. 12) Acylation Selectivity Percentage (FIOP) ² for Regions A1 and B1 (relative to SEQ ID NO. 12) 226 Y2 7T;L253V;A255G;N348R ;T384P; + + 83 Y27T;L253V; + + 84 Y27T;D74G;L253V;F254W; + + 85 Y27T;D74G;L253V;A255G; N348R;W370I;TZ84R; + + 86 Y27T;D74N;L253V;F254W; N348R;W3701;D38 IK;TZ84 P; + + 87 Y27T;D74N;L253V;F254W; A255G;N348R;W370I; + + 199 Y27T;A255G;N348R;W370I + +

- 34 043748

227 Y27T;D74P;L253V;F254W; A255G;N348R;K369C;W370 I; + + 88 Y27T;D74G;L253V;N348R; KZ 6 9C;W3 7 01;D3 81F;T3 8 4 P; + + 89 Y27T;L253V;F254W;N348R + + 90 Y27T;D74P;L253V;F254W; A255G;N348R; + + 228 Y27T; + 229 Y27T;D74G;L253V;N348R; K369C;W370I; + + 91 Y27T;D74G;L253V;F254W; A255G;W370I; + + + 92 Y27T;L253V;F254W;A255G + + 93 Y27T;D74G;L253V;F254W; T384P; + + 94 Y27T;D74N;F254W;N348R; W370I; + + 95 Y27T;D74S;L253V;N348R; W3701;D38 IK;T384P; + + 96 Y27T;L253V;A255G;W370I + + 97 Y27T;D74G;F254W;A255G; D381F; + + 98 Y27T;L253V;KZ69C;W370I + + 230 Y27T;D74P;F254W;A255G; N348R; + + 99 Y27T;L253V;F254W;N348R ;D381F; + + 101 Y27T;D74S;L253V;A255G; T384P; + +

- 35 043748

231 Y27T;D74G;A255G;W370I; + + 232 Y27T;D74S;N348R; + 102 Y27T;D74N;L253V;N348R; W370I;D381W;T384P; + + 233 Y27T;D74G;L253V;F254W; N348R; + + 103 Y27T;D7 4P;L253V;N348R; + + 234 Y27T;L253V;F254W;A255G ;N348R; + + 235 Y27T;D74S;L253V;F254W; A255G;N348R; + + 104 Y27T;D74G;F254W;A255G; N348R;KZ69C;W3701;D381 F; + + 105 Y27T;D74P;F254W;A255G; N348R;KZ69C;W3701; + + 106 65/6 6 Y27T;D74N;L253V;F254W; W370I;D381K; + + + 236 Y27T;D74G;F254W;KZ69C; W370I; + + 237 Y27T;D74G;L253V;N348R; W370I; + + 107 Y27T;D74G;L253V;T384P; + + 108 Y27T;D74S;N348R;W370I; + + 109 Y27T;D74S;L253V;F254W; A255G;KZ69C;W3701; + + 238 Y27T;D74G;F254W;A255G; N348R; + + 239 Y27T;L253V;N348R;W370I ;T384P; + + 111 Y27T;D74N;L253V;F254W; A255G; + + 240 Y27T;L253V;F254W; + + 241 Y27T;D74G;KZ69C;W370I; + + 242 Y27T;F254W;A449V; + +

- 36 043748

114 Y27T;D74G;L253V;A255G; N348R;D381W; + + 115 Υ27Τ;D74Ρ;L253V;W370I; + + 117 Υ27Τ;D74G;L253V;F254W; Α2 55G;W3701;D38 IK;Τ384 Ρ; + 118 Y27T;D74N;F254W;A255G; Ν348R;W3701;D38 IK; + + 119 Y27T;D74S;L253V;N348R; KZ 69C;W3701;TZ84P; + + 243 Y2 7 T;D7 4N;L2 5 3V;N3 4 8R; + + 120 Y27Τ;D74Ν;L253V;F254W; Ν348R;W370I;D381F; + + 121 Y27T;D74S;F254W;A255G; N348R;W370I;D381F;T384 P; + + 122 Y27T;D74G;L253V;F254W; A255G;D381K; + + 123 Y27T;D74S;F254W;K369L; W370I; + + 124 Y27T;L253V;F254W;D381F ;T384P; + + 125 Y27T;D74G;L253V;F254W; A255G;KZ69C;W370I; + + 126 Y27T;D74G;F254W;A255G; N348R;W370I; + + 127 Y27T;D74G;L253V;F254W; A2 55G;N348R;W3701;T384 P; + + 128 L253V;N348R;W370I; + + 244 Y27T;F254W;N348R;W370I r + + 129 Y27T;D74N;L253V;F254W; K369C; + + 131 Y27T;D74S;L253V;N348R; + +

- 37 043748

D381W; 245 Y27T;D74S;A255G; + + 134 Y27T;D74S;L253V;A255G; N348R;W3701;D38 IK; + + 136 Y27T;D74N;L253V;F254W; A2 55G;N348R;W3701;T384 P; + + 137 Y27T;D74S;F254W;A255G; W370I; + + + 138 Y27T;F254W;A255G;KZ69C ;W3701;D381F;T384P; + + 139 Y27T;L253V;F254W;D381F r + + 140 Y27T;D74N;F254W;N348R; + + 141 67/6 8 Y27T;F254W;A255G;W370I r + + + 142 Y27T;D74N;A107V;A255G; N348R;KZ69C;W370I; + + 143 Y27T;F254W;A255G;N348R ;W370I; + + 144 Y27T;D74N;F254W; + + 246 Y27T;D74S;F254W;K369C; T384P; + + 145 Y27T;D74G;L253V;D381F; T384P; + + 146 Y27T;D7 4P;L253V;A255G; + + 247 Y27T;D74G;A255G;N348R; K369C;W370I; + + 148 Y27T;D74S;F254W;A255G; N348R;KZ69C;W370I; + + 248 Y27T;F254W;A255G;N348R ;KZ69C;W3701; + + 149 Y27T;D74P;W3701; + + 249 Y27T;D74P;L253V;F254W; N348R;KZ69C;W370I; + +

- 38 043748

150 Y27T;L253V;F254W;T384P + + 205 Y27T;L253V;F254W;N348R ;T384P; + + 151 Y27T;D74N;L253V;F254W; A255G;W370I; + + 250 Y27T;F254W;A255G;N348R f + + 251 Y27T;D74S;F254W;N348R; + + 153 Y27T;F254W;A255G;N348R ;W370I;T384P; + + 252 D74N;L253V;F254W;K369C ;W370I; + + 253 Y27T;D74S;KZ69C;W3701; D381K;T384P; + + 155 Y27T;L253V;A255G;W370I ;D381F;T384P; + + + 157 Y27T;L253V;F254W;N348R ;W370I;T384P; + + 158 Y27T;D74N;F254W;N348R; W370I;D381K; + + 159 Y27T;D74N;L253V;A255G; N348R;KZ69C;W370I; + + 160 Y27T;L253V;F254W;N348R ;D381W;T384P; + + 161 Y27T;L253V;F254W;A255G ;G260C;N348R;D38IF;T38 4P; + 162 Y27T;D74P;L253V;F254W; N348R;D381F;T384P; + + 165 Y27T;D74G;L253V;A255G; N348R;T384P; + + 166 Y27T;D74N;L253V;A255G; W370I; + + 167 Y27T;D74N;A255G;N348R; + +

- 39 043748

W370I; 208 Y27T;F254W;A255G;N348R ;W3701;D381W;TZ84P; + + 254 Y2 7T;L253V;A255G;N348R ;KZ69C;W3701; + + 169 Y2 7T;L253V;D381F;T384P r + + 170 Y27T;D74P;L253V;F254W; N348R;W370I;D381W;T384 P; + + 171 Y27T;L253V;F254W;A255G ;N348R;W3701;T384P; + + 255 A255G;N348R;W370I; + + 209 Y27T;D74G;F254W;N348R; D381W;T384P; + 210 Y27T;F254W;A255G;D381K ;T384P; + + 256 Y27T;D74N;F254W;A255G; K369C; + + 257 Y27T;D74P;L253V;F254W; N348R; + + 173 Y27T;L253V;F254W;N348R ;W370I;D381F; + + 174 Y2 7T;D74G;L253V;A255G; + + 175 Y27T;D74N;L253V;F254W; N348R;W370I; + + 177 Y27T;D74G;L253V;F254W; A255G;N348R;KZ69C;W370 I;D381F; + + 178 Y27T;D74G;L253V;F254W; N348R;KZ69C;W370I; + + 258 Y27T;L253V;F254W;A255G ;N348R;W370I; + + + 179 Y27T;D74N;L253V; + + 181 Y27T;F254W;A255G;T384P + + + +

- 40 043748

G 182 Y27T;F254W;A255G; + + ++ 215 Y27T;D74S;L253V;N348R; + + + + 183 Y27T;L253V;F254W;A255G ;N348R;D381F;T384P; + + 185 Y27T;D74N;F254W;T384P; + + + + + 186 Y27T;L253V;N348R; + + 187 Y27T;D74G;A255G;N348R; + + 12 Y27T;F254W;A470V; + + 13 Y27T;L253V;A255G;N348R r + + 189 Y27T;D74G;L253V;A255G; N348R;D381F; + + 6 Y27T;A255G;W3701; + + + + 190 Y27T;D74S;F254W;A255G; N348R; + + + 216 Y27T;D74N;L253V;F254W; ++++ + + 191 9/10 Y27T;D74S;L253V;F254W; N348R;D381W;T384P; + + + + 192 Y27T;D74N;F254W;A255G; N348R; + + + 193 Y27T;D74P;L253V;F254W; A255G; + + + + + 217 5/6 Y27T;D74G;L253V;F254W; A255G;N348R; + + + + 14 Y27T;D74S;F254W;A255G; N348R;KZ69C;T384P; + 15 Y27T;D74N;L253V;F254W; N348R;KZ69C;T384P; + + 194 Y27T;L253V;N348R;W370I ;D381F;T384P; + + 195 Y27T;D74S;A255G;W3701; +++ ++ 27 D381F; + + 259 A132S; + + 28 Q134M; + + 29 D623W; + + 260 T131L; + + 35 D381L; + + 37 A132G; + + 262 W370V; + + 263 D381R; + + 264 T384R; + + 265 D623Y; + + 7 D623N; + + 266 D623R; + + 267 S619I; + 268 L253V; + + 269 T133K; + 197 A255P; + + 270 I708M; + 271 F254T; + + 272 T705S; +

- 41 043748 ¹ Levels of increased activity were established relative to the reference polypeptide from SEQ ID No. 12 and were determined as follows: + > more than 2.0-fold, but less than 10.0-fold increased; ++ > more than 10.0 times, but less than 50.0 times; +++ > more than 50.0 times, but less than 100 times; ++++ >

than 100.

² Levels of increased selectivity were set relative to the reference polypeptide of SEQ ID No. 12 and were determined as follows: + > 2.0-fold but less than 10.0-fold increased; ++ > more than 10.0 times, but less than 50.0 times; +++ > more than 50.0 times, but less than 100 times; ++++ > than 100.

Table 6.5. Activity and selectivity of variants acylating at sites A1 and B29 relative to SEQ ID No. 12

No. varia nta SEQ ID No. (n./a. Amino acid differences (relative to SEQ ID No. 12) Conversion percentage acylated Acyl selectivity percentage

To. ) FIOP ¹ no to Al and B29 sections (relative to SEQ ID No. 12) FIOP ² FOR Al and B29 areas (relative to SEQ ID No. 12) 85 Y27Т;D74G;L253V;А255G;N348R ;W3701;ТЗ84Р; + + 86 Y27Т;D74N;L253V;F254W;N348R ;W370I;D381К;ТЗ84Р; + + 87 Y27T;D74N;L253V;F254W;A255G ;N348R;W370I; + + 91 Y27T;D74G;L253V;F254W;A255G ;W370I; + 93 Y27T;D74G;L253V;F254W;T384P + 95 Y27T;D74S;L253V;N348R;W370I ;D381K;T384P; + + 102 Y27T;D74N;L253V;N348R;W370I ;D381W;T384P; + + + 233 Y27T;D74G;L253V;F254W;N348R + 273 Y27T;L253V;F254W;A255G;N348 R;KZ69C;W3701;D381W;T384P; + + 235 Y27T;D74S;L253V;F254W;A255G ;N348R; + + 106 65/6 6 Y27T;D74N;L253V;F254W;W370I ;D381K; + + 108 Y27T;D74S;N348R;W370I; + + 111 Y27T;D74N;L253V;F254W;A255G r + 113 Y27T;D74G;A255G;W370I; + + 114 Y27T;D74G;L253V;A255G;N348R + +

- 42 043748

;D381W; 115 Y27T;D74P;L253V;W370I; + + 117 Y27T;D74G;L253V;F254W;A255G ;W3701;D38IK;T384P; + + + 274 Y27T;L253V;F254W;A255G;N348 R;K369C;T384P; + + 118 Y27T;D74N;F254W;A255G;N348R ;W370I;D38IK; + + 120 Y27T;D74N;L253V;F254W;N348R ;W370I;D381F; + + 121 Y27T;D74S;F254W;A255G;N348R ;W3701;D381F;T384P; + + 122 Y27T;D74G;L253V;F254W;A255G ;D381K; + + 126 Y27T;D74G;F254W;A255G;N348R ;W370I; + + 127 Y27T;D74G;L253V;F254W;A255G ;N348R;W370I;T384P; + + 131 Y27T;D74S;L253V;N348R;D381W r + + 245 Y27T;D74S;A255G; + 134 Y27T;D74S;L253V;A255G;N348R ;W370I;D38IK; + + 136 Y27T;D74N;L253V;F254W;A255G ;N348R;W370I;T384P; + + 137 Y27T;D74S;F254W;A255G;W370I r + + 140 Y27T;D74N;F254W;N348R; + 141 67/6 8 Y27T;F254W;A255G;W370I; + 143 Y27T;F254W;A255G;N348R;W370 i; + 145 Y27T;D74G;L253V;D381F;T384P + + 149 Y27T;D74P;W370I; +

- 43 043748

151 Y27Τ;D74Ν;L253V;F254W;A255G ;W370I; + + 152 Y27T;D74G;L253V;Ν348R;W370I G + + 157 Y27T;L253V;F254W;N348R;W370 I;T384P; + + 160 Y27T;L253V;F254W;N348R;D381 W;T384P; + + 161 Y27T;L253V;F254W;A255G;G260 C;N348R;D381F;T384P; + + 162 Y27T;D74P;L253V;F254W;N348R ;D381F;T384P; + + 275 Y27T;D74G;L253V;F254W; + + 164 Y27T;D74N;L253V;N348R; + + 165 Y27T;D74G;L253V;A255G;N348R ;T384P; + + 166 Y27T;D74N;L253V;A255G;W370I r + + 167 Y27T;D74N;A255G;N348R;W370I r + + 276 Y27T;D74P;F254W;A255G;N348R ;D381K;T384P; + + 208 Y27T;F254W;A255G;N348R;W370 I;D381W;T384P; + + 254 Y27T;L253V;A255G;N348R;KZ 69 C;W370I; + 22 15/1 6 Y27T;D74S;A255G;N348R;D38 IK ;T384P; + + 170 Y27T;D74P;L253V;F254W;N348R ;W3 7 01;D3 81W;T 3 8 4 P; + + 171 Y27T;L253V;F254W;A255G;N348 R;W3701;T384P; + + 209 Y27T;D74G;F254W;N348R;D381W ;T384P; + + 277 Y27T;D74N;F254W;N348R;W370I + +

- 44 043748

278 Y27T;D74P;L253V;F254W;N348R ;K369S; + + 257 Y27T;D74P;L253V;F254W;N348R + 279 Y27T;F254W;KZ69S;D381F;TZ84 R; + + 173 Y27T;L253V;F254W;N348R;W370 I;D381F; + + 175 Y27T;D74N;L253V;F254W;N348R ;W370I; + + 280 Y27T;F254W;N348R;KZ69C;W370 I;D381N;T384P; + + 180 Y27T;D74G;L253V;F254W;A255G ;N348R; + 281 D623N; + + 264 T384R; + + 220 D623L; + + 62 F620R; ++ + + + 63 D709N; ++ + +++ 19 69/7 0 K128W; + + ++ 282 T705E; ++ ++ 283 A255E; ++ + + 284 F254T; ++ ++

¹ Levels of increased activity were set relative to the reference polypeptide of SEQ ID No. 12 and were determined as follows: + > 2.0-fold but less than 10.0-fold increased; ++ > more than 10.0 times, but less than 50.0 times; +++ > more than 50.0 times, but less than 100 times; ++++ > than 100.

² Levels of increased selectivity were set relative to the reference polypeptide of SEQ ID No. 12 and were determined as follows: + > 2.0-fold but less than 10.0-fold increased; ++ > more than 10.0 times, but less than 50.0 times; +++ > more than 50.0 times, but less than 100 times; ++++ > _Than 100.

- 45 043748

Table 6.6. Activity and selectivity of variants acylating at sites A1, B1 and B29, relative to SEQ ID No. 12

No. Variation SEQ ID No. (n./ A. To. ) Amino acid differences (relative to SEQ ID No. 12) Acylation conversion percentage (FIOP) ¹ at Al, B1 and B29 sites (relative to SEQ ID No. 12) Acylation Selectivity Percentage (FIOP) ² FOR Al, B1, and B29 Sites (relative to SEQ ID No. 12) 226 Y2 7T;L253V;A255G;N348R;T384P + + 83 Y27T;L253V; + + 84 Y27T;D74G;L253V;F254W; + + 198 Y27T;D74S;F254W;N348R;D381W; + + 285 Y27T;D74G;A255G;N348R;KZ69C; D381F;T384P; + + 85 Y27T;D74G;L253V;A255G;N348R; W370I;T384P; + + 86 Y27T;D74N;L253V;F254W;N348R; W3701;D38 IK;T384P; + + + + 87 Y27T;D74N;L253V;F254W;A255G; N348R;W370I; + + 199 Y27T;A255G;N348R;W370I; + + + 200 Y27T;N348R;T384P; + + 88 Y27T;D74G;L253V;N348R;KZ69C; W370I;D381F;T384P; + +

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90 Y27T;D7 4P;L253V;F254W;A255G; N348R; + + 228 Y27T; + + 91 Y27T;D74G;L253V;F254W;A255G; W370I; + + 92 Y27T;L253V;F254W;A255G; + + 93 Y27T;D74G;L253V;F254W;T384P; + + 94 Y27T;D74N;F254W;N348R;W370I; + + 95 Y27T;D74S;L253V;N348R;W370I; D381K;T384P; + + + + 286 Y27T;L253V;N348R; + + 96 Y27T;L253V;A255G;W370I; + + 97 Y27T;D74G;F254W;A255G;D381F; + + 287 Y27T;F254W;N348R;T384P; + 201 Y27T;D74G;F254W;A255G;N348R; D381W; + + 288 D74N;F254W;A255G;N348R;T384P + + 289 Y27T;F254W;A255G; + + 202 Y27T;D74S;N348R; + + 230 Y27T;D74P;F254W;A255G;N348R; + + 99 Y27T;L253V;F254W;N348R;D381F r + + 101 Y27T;D74S;L253V;A255G;T384P; + + 231 Y27T;D74G;A255G;W370I; + + + 102 Y27T;D74N;L253V;N348R;W370I; D381W;T384P; + + + + 233 Y27T;D74G;L253V;F254W;N348R; + + + 103 Y27T;D74P;L253V;N348R; + + 234 Y27T;L253V;F254W;A255G;N348R r + + 235 Y27T;D74S;L253V;F254W;A255G; N348R; + + 104 Y27T;D74G;F254W;A255G;N348R; KZ69C;W370I;D381F; + +

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105 Y27T;D74P;F254W;A255G;N348R; K369C;W370I; + + 106 65/6 6 Y27T;D74N;L253V;F254W;W370I; D381K; + + + + 236 Y27T;D74G;F254W;KZ69C;W370I; + + 237 Y27T;D74G;L253V;N348R;W370I; + + + 107 Y27T;D74G;L253V;T384P; + + 108 Y27T;D74S;N348R;W370I; + + + 290 Y27T;D74G;A255G;N348R; + + 238 Y27T;D74G;F254W;A255G;N348R; + + 239 Y27T;L253V;N348R;W370I;T384P + + + 111 Y27T;D74N;L253V;F254W;A255G; + + 242 Y27T;F254W;A449V; + + 114 Y27T;D74G;L253V;A255G;N348R; D381W; + + 115 Y27T; D74P;L253V;W3701; + + 117 Y27T;D74G;L253V;F254W;A255G; W3701;D38 IK;T384P; + + + + 118 Y27T;D74N;F254W;A255G;N348R; W370I;D381K; + + 120 Y27T;D74N;L253V;F254W;N348R; W370I;D381F; + + + + 121 Y2 7 T;D7 4 S;F2 5 4W;A2 5 5G;N3 4 8R; W370I;D381F;T384P; + + + + 122 Y27T;D74G;L253V;F254W;A255G; D381K; + + + 123 Y27T;D74S;F254W;KZ69L;W370I; + + 291 Y27T;D74N;F254W;A255G;N348R; K369C;D381F; + + 124 Y27T;L253V;F254W;D381F;T384P + + + 126 Y27T;D74G;F254W;A255G;N348R; W370I; + + + + 127 Y27T;D74G;L253V;F254W;A255G; + +

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N348R;W3701;TZ84P; 128 L253V;N348R;W3701; + + 244 Y27T;F254W;N348R;W370I; + + 131 Y27T;D74S;L253V;N348R;D381W; + + + + 132 Y27T;D74S;L253V;N348R; + + 245 Y27T;D74S;A255G; + + 292 Y27T;L253V;A255G;N348R; + + 134 Y27T;D74S;L253V;A255G;N348R; W370I;D381K; + + 136 Y27T;D74N;L253V;F254W;A255G; N348R;W370I;T384P; + + 137 Y27T;D74S;F254W;A255G;W370I; + + + 138 Y27T;F254W;A255G;KZ69C;W370I ;D381F;T384P; + + 203 Y27T;A255G;N348R; + + 139 Y27T;L253V;F254W;D381F; + + 140 Y27T;D74N;F254W;N348R; + + 141 67/6 8 Y27T;F254W;A255G;W370I; + + + + 204 Y27T;L253V;N348R;D381F;T384P Λ + + 143 Y27T;F254W;A255G;N348R;W370I + + + + 293 Y27T;L253V;F254W;N348R; + + 144 Y27T;D74N;F254W; + + 294 Y27T;D74N;F254W;N348R;KZ69C; D381F;T384P; + + 145 Y27T;D74G;L253V;D381F;T384P; + + + + 146 Y27T;D7 4P;L253V;A255G; + + 148 Y2 7 T;D7 4 S;F2 5 4W;A2 5 5G;N3 4 8R; K369C;W370I; + + 149 Y27T;D74P;W370I; + + + 150 Y27T;L253V;F254W;T384P; + + 295 Y27T;D74N;N348R; + + 205 Y27T;L253V;F254W;N348R;T384P + +

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G 151 Y27T;D74N;L253V;F254W;A255G; W370I; + + 250 Y27T;F254W;A255G;N348R; + + 251 Y27T;D74S;F254W;N348R; + + 206 Y27T;D74P;F254W;A255G;N348R; D381K;T384P; + + 153 Y27T;F254W;A255G;N348R;W370I ;T384P; + + + + 296 7/8 Y27T;D74N;N348R;T384P; + + 253 Y27T;D74S;KZ69C;W3701;D38IK; T384P; + + 155 Y27T;L253V;A255G;W370I;D381F ;T384P; + + + + 157 Y27T;L253V;F254W;N348R;W370I ;T384P; + + + 158 Y27T;D74N;F254W;N348R;W370I; D381K; + + 160 Y2 7T;L2 53V;F2 54W;N34 8R; D3 81W ;T384P; + + + 297 Y27T;L253V;F254W; + + 161 Y27T;L253V;F254W;A255G;G260C ;N348R;D381F;T384P; + + 162 Y27T;D74P;L253V;F254W;N348R; D381F;T384P; + + + + 164 Y2 7 T;D7 4N;L2 5 3V;N3 4 8R; + + 165 Y27T;D74G;L253V;A255G;N348R; T384P; + + 166 Y27T;D74N;L253V;A255G;W370I; + + 167 Y27T;D74N;A255G;N348R;W370I; + + 207 Y27T;N348R; + + 208 Y27T;F254W;A255G;N348R;W370I ;D381W;T384P; + + + + 169 Y2 7T;L2 53V;D3 81F;T3 8 4P; + + 22 15/1 Y27T;D74S;A255G;N348R;D38 IK; + +

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6 T384R; 170 Y27T;D74P;L253V;F254W;N348R; W370I;D381W;TZ84R; + + + + 171 Y27T;L253V;F254W;A255G;N348R ;W370I;TZ84R; + + + 255 A255G;N348R;W370I; + + 209 Y27T;D74G;F254W;N348R;D381W; T384R; + + + + 210 Y27T;F254W;A255G;D381K;T384P Λ + + + + 211 Y27T;D74S;F254W;N348R;D381F; + + 257 Y27T;D74P;L253V;F254W;N348R; + + 173 Y27T;L253V;F254W;N348R;W3701 ;D381F; + + 174 Y27T;D74G;L253V;A255G; + + 175 Y27T;D74N;L253V;F254W;N348R; W370I; + + 177 Y27T;D74G;L253V;F254W;A255G; N348R;KZ69C;W3701;D38IF; + + 178 Y27T;D74G;L253V;F254W;N348R; K369C;W370I; + + 212 Y27T;A255G;N348R;D381W;T384P r + + 258 Y27T;L253V;F254W;A255G;N348R ;W370I; + + + 213 Y27T;F254W;N348R;D381W;T384P r + + 214 Y27T;D74G;N348R; + + 179 Y27T;D74N;L253V; + + 180 Y27T;D74G;L253V;F254W;A255G; N348R; + + 29 D623W; + + 40 D623V; + + 42 D623A; + + 43 D381Q; + + 220 D623L; + + 298 D623N; + + 8 T384R; + + + 57 A373K; + + 299 F620R; + + 300 F254T; + + 197 A255P; + +

¹ Levels of increased activity were set relative to the reference polypeptide of SEQ ID No. 12 and were determined as follows: + > 2.0-fold but less than 10.0-fold increased; ++ > more than 10.0 times, but less than 50.0 times; +++ > more than 50.0 times, but less than 100 times; ++++ > than 100.

Levels of increased selectivity were set relative to the reference polypeptide of SEQ ID No. 12 and were determined as follows: + > 2.0-fold but less than 10.0-fold increased; ++ > more than 10.0 times, but less than 50.0 times; +++ > more than 50.0 times, but less than 100 times; ++++ > than 100.

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Table 6.7. Activity and selectivity of variants acylating at the B1 site relative to SEQ ID No. 12

No. varia nta SEQ ID No. (n./ A. To. ) Amino Acid Differences (relative to SEQ ID No. 12) Acylation Conversion Percentage (FIOP) ¹ at Site B1 (relative to SEQ ID NO. 12) Acylation Selectivity Percentage (FIOP) ² for Site B1 (relative to SEQ ID NO: 12) 83 Y27T;L253V; + 84 Y27T;D74G;L253V;F254W; + + + 85 Y27T;D74G;L253V;A255G;N34 8R;W370I;TZ84Ρ; + + + 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 Υ27Τ;D74Ν;L253V;F254W;N34 8R;W37 01;D3 81K;TZ 8 4Ρ; Υ27Τ;D74Ν;L253V;F254W;A25 5G;Ν348R;W370I; Υ27Τ;D74G;L253V;Ν348R;KZ 6 9С;W3 7 01;D3 81F;ТЗ 8 4 P; Y27T;L253V;F254W;N348R; Y27T;D74P;L253V;F254W;A25 5G;N348R; Y27T;D74G;L253V;F254W;A25 5G;W370I; Y27T;L253V;F254W;A255G; Y27T;D74G;L253V;F254W;T38 4P; Y27T;D74N;F254W;N348R;W37 01; Y27T;D74S;L253V;N348R;W37 01;D381K;T384P; Y27T;L253V;A255G;W370I; Y27T;D74G;F254W;A255G;D38 IF; Y27T;L253V;KZ69C;W370I; Y27T;L253V;F254W;N348R;D3 8 IF; Y27T;L253V;F254W;A255G;N3 48R;W370I; Y27T;D74S;L253V;A255G;T38 4P; Y27T;D74N;L253V;N348R;W37 01;D381W;T384P; + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 103 Y27T;D74P;L253V;N348R; + + 104 Y27T;D74G;F254W;A255G;N34 8R;KZ69C;W370I;D381F; + + 105 Y27T;D74P;F254W;A255G;N34 8R;KZ69C;W3701; + +

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106 65/6 6 Y27T;D74Ν;L253V;F254W;W37 0I;D381K; + + + + 107 Y27T;D7 4G;L253V;T384P; + + 108 Y27T;D74S;N348R;W370I; + + 109 Y27T;D74S;L253V;F254W;A25 5G;KZ69C;W3701; + + 110 Y27T;D74G;L253V;N348R;KZ 6 9C;W370I; + 111 Y27T;D74N;L253V;F254W;A25 5G; + + + 112 Y27T;D74P;L253V;F254W;N34 8R; + + 113 Y27T;D74G;A255G;W3701; + + 114 Y27T;D74G;L253V;A255G;N34 8R;D381W; + + 115 Y27T;D74P;L253V;W370I; + + + 116 Y27T;L253V;N348R;W3701; T3 84P; + 117 Y27T;D74G;L253V;F254W;A25 5G;W3701;D38 IK;T384P; + + + 118 Y27T;D74N;F254W;A255G;N34 8R;W3701;D38 IK; + + 119 Y27T;D74S;L253V;N348R;SC 6 9C;W370I;T384P; + + 120 Y27T;D74N;L253V;F254W;N34 8R;W370I;D381F; + + + + 121 Y27T;D74S;F254W;A255G;N34 8R;W37 01;D3 81F;T3 8 4P; + + 122 Y27T;D74G;L253V;F254W;A25 5G;D381K; + + + + 123 Y27T;D74S;F254W;KZ69L;W37 01; + + 124 Y27T;L253V;F254W;D381F;T3 84P; + + 125 Y27T;D74G;L253V;F254W;A25 +

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5G;KZ69C;W3701; 126 Y27T;D74G;F254W;A255G;N34 8R;W37OI; + + + 127 Y27T;D74G;L253V;F254W;A25 5G;N348R;W370I;T384P; + + + + 128 L253V;N348R;W370I; + 129 Y27T;D74N;L253V;F254W;K36 9C; + + 130 Y27T;D74G;F254W;A255G;N34 8R; + 131 Y27T;D74S;L253V;N348R;D38 1W; + + 132 Y27T;D74S;L253V;N348R; + + 133 Y27T;L253V;F254W;A255G;N3 4 8R; + 134 Y27T;D74S;L253V;A255G;N34 8R;W3701;D38 IK; + + + + 135 Y27T;F254W;A255G;N348R;KZ 69C;W370I; + + 136 Y27T;D74N;L253V;F254W;A25 5G;N348R;W370I;T384P; + + + + + 137 Y27T;D74S;F254W;A255G;W37 01; + + + + 138 Y27T;F254W;A255G;KZ69C;W3 701;D38IF;T384P; + 139 Y27T;L253V;F254W;D381F; + + + 140 Y27T;D74N;F254W;N348R; + 141 67/6 8 Y27T;F254W;A255G;W370I; + 142 Y27T;D74N;A107V;A255G;N34 8R;KZ69C;W3701; + 143 Y27T;F254W;A255G;N348R;W3 701; + 144 Y27T;D74N;F254W; + + 145 Y27T;D74G;L253V;D381F;T38 + +

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4P; 146 Y27T;D74P;L253V;A255G; + + 147 Y27T;D74S;L253V;F254W;A25 5G;N348R; + + 148 Y27T;D74S;F254W;A255G;N34 8R;KZ69C;W3701; + 149 Y27T;D74P;W370I; + 150 Y27T;L253V;F254W;T384P; + + 151 Y27T;D74N;L253V;F254W;A25 5G;W370I; + + + + + + 152 Y27T;D74G;L253V;N348R;W37 01; + + + 153 Y27T;F254W;A255G;N348R;W3 70I;T384P; + 154 Y27T;D74G;L253V;F254W;N34 8R; + + 155 Y27T;L253V;A255G;W370I;D3 81F;T384P; + 156 Y27T;L253V;A255G;N348R;D3 8 IK; + + 157 Y27T;L253V;F254W;N348R;W3 70I;T384P; + + 158 Y27T;D74N;F254W;N348R;W37 0I;D381K; + + 159 Y27T;D74N;L253V;A255G;N34 8R;KZ69C;W3701; + + 160 Y27T;L253V;F254W;N348R;D3 81W;T384P; + + 161 Y27T;L253V;F254W;A255G;G2 60C;N348R;D381F;T384P; + + 162 Y27T;D74P;L253V;F254W;N34 8R;D381F;T384P; + + 163 Y2 7T;D7 4G;L2 53V;N348R;SC 6 9C;W370I;D381F; + + 164 Y27T;D74N;L253V;N348R; + +

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165 Y27Τ;D74G;L253V;Α255G;Ν34 8R;T384P; + + 166 Υ27Τ;D74N;L253V;A255G;W37 01; + + + + 167 Y27T;D74Ν;Α255G;Ν348R;W37 01; + + 168 Υ2 7Τ;L2 53V;N34 8R;D3 81F; TZ 84R; + 169 Y2 7T;L2 53V;D3 81F;TZ 8 4P; + 170 Y27T;D74P;L253V;F254W;N34 8R;W370I;D381W;TZ84P; + + + 171 Y27T;L253V;F254W;A255G;N3 48R;W370I;T384P; + 172 Y27T;L253V;F254W; + + 173 Y27T;L253V;F254W;N348R;W3 70I;D381F; + + + + 174 Y27T;D74G;L253V;A255G; + + 175 Y27T;D74N;L253V;F254W;N34 8R;W370I; + + + + + + 176 Y27T;D74N;L253V;F254W;N34 8R;KZ69C;W3701;D38 IK; + + 177 Y27T;D74G;L253V;F254W;A25 5G;N348R;KZ69C;W370I;D381 F; + + 178 Y27T;D74G;L253V;F254W;N34 8R;KZ69C;W3701; + + 179 Y27T;D74N;L253V; + + 180 Y27T;D74G;L253V;F254W;A25 5G;N348R; + + 181 Y27T;F254W;A255G;T384P; + 182 Y27T;F254W;A255G; + + + 183 Y27T;L253V;F254W;A255G;N3 48R;D381F;T384P; + + 184 Y27T;F254W;A255G;N348R; + + 185 Y27T;D74N;F254W;T384P; +

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186 Y27T;L253V;N348R; + + 187 Y27T;D74G;A255G;N348R; + 12 Y27T;F254W;A470V; + 13 Y27T;L253V;A255G;N348R; + 188 Y27Т;D74N;L253V;КЗ69С;D38 1К;Т384Р; + + 189 Y27T;D74G;L253V;A255G;N34 8R;D381F; + + + 6 Y27Τ;Α255G;W370I; + 190 Y27T;D74S;F254W;A255G;N34 8R; + 191 9/10 Υ27Τ;D74S;L253V;F254W;N34 8R;D381W;T384P; + + + + + 192 Y27T;D74N;F254W;A255G;N34 8R; + 193 Y27T;D74P;L253V;F254W;A25 5G; + + + 194 Y27T;L253V;N348R;W370I;D3 81F;T384P; + + + + 195 Y27T;D74S;A255G;W370I; + 196 N388E; + + + 197 A255P; +

¹ Levels of increased activity were set relative to the reference polypeptide from SEQ ID No. 12 and were determined as follows: + > 2.0-fold but less than 5.0-fold increased; ++ > more than 5.0 times, but less than 10.0 times; +++ > than 10.0 times.

² Levels of increased selectivity were set relative to the reference polypeptide of SEQ ID No. 12 and were determined as follows: + > 2.0-fold but less than 5.0-fold increased; ++ > more than 5.0 times, but less than 7.0 times; +++ > than 7.0 times.

Example 7: Improvement in Insulin Acylation at Position B29 Compared to SEQ ID No. 12

Acylation at B29 with four options listed in table. 7.1 was tested on a shake flask scale. Shake flask powders were prepared as described in Example 4. Reactions were carried out in 96-well deep well plates, each containing 200 μl of 0.2 M TRIS, pH 9.25, 20% acetonitrile, 10 g/l insulin, 17 g/l l methylphenylacetate and 0.9 g/l lyophilized enzyme powder reconstituted in 10 mM TRIS, pH 7.5. NTR plates were heat sealed and incubated in Thermotron® shakers (3 mm amplitude, model no. AJ185, Infors) at 30°C, 100 rpm, for 5 h. Reactions were quenched using 200 μl of acetonitrile and mixed for 5 min. using a table shaker. The plates were then centrifuged at 4000 rpm for 5 min and loaded onto HPLC for analysis.

The percentage conversion relative to SEQ ID No. 12 (FIOP conversion percentage) was calculated as the percentage conversion of the product formed by the variant relative to the percentage conversion provided by SEQ ID No. 12. The results are presented in table. 7.1. The percent conversion was quantified by dividing the peak area of the product by the sum of the peak areas of the substrate, product, and impurities/by-product as observed by HPLC analysis.

The percentage selectivity relative to SEQ ID No. 12 (FIOP selectivity percentage) was calculated as the percentage selectivity of the product produced by the variant relative to the percentage selectivity provided by SEQ ID No. 12. The percentage selectivity was calculated by dividing the peak area of the product by the sum of the peak areas of the product and the impurities/by-product product as observed by HPLC analysis.

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Table 7.1. Activity and selectivity of variants acylating at the B29 site relative to SEQ ID No. 12

Y27Т;V28А;G71Н;D74G;К103

E;W119Y;L253Y;F256R;N348

H;T352K;A373R;S374T;S390

K;G444N;A451K;N494D;Q547

TO; A616Y;S646D;

Amino Acid Differences (relative to SEQ ID No. 12)

D709R;

D709K;

Conversion percentage acylated

Percentage of acylation selectivity

we eat I (FIOP) ² (FIOP) ¹ to for the site site B29 B29 (relative (relative but SEQ ID no. Exactly SEQ ID 12) No. 12)

¹ Levels of increased activity were set relative to the reference polypeptide of SEQ ID No. 12 and were determined as follows: + > 10.0-fold but less than 50.0-fold increased; ++ > more than 50.0 times, but less than 100.0 times; +++ > than 100.0 times.

² Levels of increased selectivity were set relative to the reference polypeptide of SEQ ID No. 12 and were determined as follows: + > 10.0-fold but less than 50.0-fold increased; ++ > more than 50.0 times, but less than 100.0 times; +++ > than 100.0 times.

Example 8: Improvement of Insulin Acylation at Position B29 Compared to SEQ ID No. 108 in a High Throughput Screen

SEQ ID No. 108 was selected as the next parent enzyme based on the results described in Example 7. Engineered gene libraries were generated using well techniques (eg, saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were prepared in the NTR as described in Example 2, and soluble lysates were created as described in Example 3.

HTR reactions were carried out in 96-well plates with deep wells. Each reaction well contained 200 μl of 0.1 M TRIS, pH 9.25, 20% acetonitrile, 25 g/l insulin, 17 g/l methylphenylacetate, and 10 μl of NTR supernatant. NTP plates were incubated in Thermotron® shakers (amplitude 3 mm, model no. AJ185, Infors) at 30°C, 100 rpm, for 3 hours. Reactions were quenched using 200 μl of acetonitrile and mixed for 5 minutes using a benchtop shaker. Then, 400 μL of water was added and mixed for 5 min using a table shaker. The plates were then centrifuged at 4000 rpm for 5 min and loaded onto HPLC for analysis.

The percentage conversion relative to SEQ ID No. 108 (FIOP conversion percentage) was calculated as the percentage conversion of the product formed by the variant relative to the percentage conversion provided by SEQ ID No. 108. The results are presented in table. 8.1. The percent conversion was quantified by dividing the peak area of the product by the sum of the peak areas of the substrate, product, and impurities/by-product as observed by HPLC analysis.

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Table 8.1. Activity of variants acylating at the B29 site relative to SEQ ID No. 108

No. option SEQ ID No. (n./a.k.) Amino Acid Differences (relative to SEQ ID No. 108) Acylation Conversion Percentage (FIOP) ¹ at Al Site (relative to SEQ ID NO: 108) 301 25/26 R256V; + + + + 302 23/24 N71M; + + + + 303 27/28 K390A; + + + + 304 33/34 H71L; + + + + 305 31/32 R256L; + + + 306 35/36 R256K; + + + 307 29/30 KZ9OR; + + + 308 K390L; + + + 309 K390R; + + 310 KZ 9OS; + + 311 H71Q; + + 312 H71D; + + 313 KZ 9ON; + + 314 E707K; + + 315 D623W; + 316 F50V;S619W; + 317 D252C; + 318 T705R; + 319 K369S; + 320 K369R;S619M; + 321 N185D;G415Н;N444L;К723D G + 322 N185D;N444L;K723D;R748G + 323 N9D;G415H;N444L;N457Q;K 723D;R748E; + 324 N185D;K723D;R748E; + 325 K128H;K369R; + 326 N9D;T443D;N444K;K723D;R 748G;A764E; + 327 S619F; + 328 N9D;N185D;G415H;K723D;R 748E; + 329 S619M; + 330 N185D;R256H;T560G;R748E r + 331 N9D;K723D;R748S; +

- 59 043748

332 K436G; + 333 N185D;N444K;N457Q;Kb68E ;K723D;R748G; + 334 K128N; + 335 K723D; + 336 N185D;G415H;T443D;N444R ;K723D;R748S; + 337 N444S;K723D;R748D; + 338 N185D;T560G;K723D; + 339 K128Q; + 340 N185D;R748D; + 341 N9D;N185D; + 342 T129W;N185D;N444L;K723D ;R748E; + 343 N185D;N444K;R748S; + 344 N185D;G415H;N444S;R748E r + 345 A467V; + 346 F620L; + 347 N185D;T443D;N444S;K723D ;R748E; + 348 N9D;G415H;T443D;N444L;K 723D;R748E; + 349 Q626E; + 350 K723D;R748E; + 351 Q626M; + 352 N9D;T443D;N444S;K723D;R 748E; + 353 D709E; + 354 K369W; + 355 G415H;N444S;T560G;K723D ;R748S; + 356 G415H;K723D; + 357 T384E; + 358 N185D;N444K;K723D; +

- 60 043748

359 N444L;K723D; + 360 K369R;T384Y;S619M; + 361 N9D;N185D;N444L;K723D;R 748D; + 362 N457Q;K723D;R748D; + 363 K369R;H546P; + 364 N9D;N185D;G415H;N444L;R 748E; + 365 K369Q; + 366 D623H; + 367 N9D;G415H;T443D;N444L;K 723D; + 368 F254W; + 369 K369R; + 370 N185D;G415H;T443D;N444S ;N457Q;R748E; + 371 N185D;G415H;N444S;K723D ;R748S; + 372 N9D;N185D;N444S;K723D;R 748E; + 373 N9D;N185D;T560G;K723D;R 748E; + 374 K723D;R748D; + 375 T379N; + 376 N18 5D;G415H;N444L; T560G ;K723D; + 377 N444K;K723D;R748E; + 378 N18 5D;T4 4 3D;N4 57Q; T5 60G ;R748S; + 379 N185D;N457Q;R748S; + 380 N9D;N185D;K723D;R748S; + 381 N9D;G415H;T443D;N444L;R 748S; + 382 N9D;N185D;G415H;T443D; +

¹ Levels of increased activity were set relative to the reference polypeptide from SEQ ID No. 108 and were determined as follows: + > 1.2-fold but less than 2.0-fold increased; ++ > more than 2.0 times, but less than 4.0 times; +++ > than 4.0 times, but less than 8.0 times; ++++ > than 8.0 times.

Example 9: Improvement of Insulin Acylation at Position B29 Compared to SEQ ID No. 24 in a High Throughput Screen

SEQ ID No. 24 was selected as the next parent enzyme based on the results described in Example 8. Engineered gene libraries were generated using well techniques (eg, saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were prepared in the NTR as described in Example 2, and soluble lysates were created as described in Example 3.

HTR reactions were carried out in 96-well plates with deep wells. Each reaction well contained 200 μl of 0.2 M TRIS, 20% acetonitrile, 25 g/l insulin, 17 g/l methylphenylacetate, and 10 μl of HTR supernatant (initial pH before addition of lysate was 9.4). NTP plates were incubated in Thermotron® shakers (amplitude 3 mm, model no. AJ185, Infors) at 30°C, 100 rpm, for 3 hours. Reactions were quenched using 200 μl of acetonitrile and mixed for 5 minutes using a benchtop shaker. Then, 400 μL of water was added and mixed for 5 min using a table shaker. The plates were then centrifuged at 4000 rpm for 5 minutes and loaded onto HPLC for analysis.

The percentage conversion relative to SEQ ID No. 24 (FIOP conversion percentage) was calculated as the percentage conversion of the product formed by the variant relative to the percentage conversion provided by SEQ ID No. 24. The results are presented in table. 9.1. The percentage of conversion was quantified by dividing the peak area of the product by the sum of the peak areas of the substrate, product, and

- 61 043748 impurities/by-product as observed by HPLC analysis.

Table 9.1. Activity of variants acylating at the B29 site relative to SEQ ID No. 24

No. option SEQ ID No. (n./a. To. ) Amino acid differences (relative to SEQ ID No. 24) Acylation Conversion Percentage (FIOP) ¹ at Site A1 (relative to SEQ ID NO: 24) 383 71/72 S67A;M7IQ;N185D;R256L;T443D;Q 62 6M; + + + 384 S67A;Nl85D;KZ90A;N444S; + + + 385 81/82 S67A;M7IQ;R256L;T443D;S619W; +++ 386 S67A;Nl85D;K390A;T443D;Q626E; +++ 387 83/84 S67A;M7IQ;Nl85D;R256L;S619W; + + + 388 75/76 S67A;M7IQ;Nl85D;R256L;T443D;E 707K;K723D; +++ 389 73/74 S67A;M7IQ;R256L;K436G;T443D;S 619W;Q626M; + + + 390 79/80 S67A; KZ90A;K436G;T443D;S619W; Q626E; +++ 391 77/78 M71Q;N185D;R256L;T443D; +++ 392 S67A;M7IQ;Nl85D;R256L; +++ 393 S67A;M71F;KZ90A;S619W; + + 394 S67A;KZ90A;N444S;S619F; + + 395 S67A;K390A; ++ 396 M7IQ;Nl85D;R256L;T443D;Q626E; K723D; + + 397 S67A;M71F;Nl85D;KZ90A;S619W; + + 398 Nl85D;KZ90A;T443D; ++ 399 S67A;M7IQ;Nl85D;R256K; + + 400 M71Q;R256L;T443D; + +

- 62 043748

401 M7IQ;N185D;R256L;T443D;Q626M; E707K; + + 402 S67A;M71F;KZ90A;T443D;S619F;Q 62 6M; + + 403 S67A;M7IQ;N185D;R256L;Q626M;K 723D; + + 404 S67A; KZ90A;T443D;S619W;Q626E; + + 405 M7IQ;N185D;R256L;T443D;Q626E; E707K; ++ 406 M7IL;VI84Q;D252C;KZ69S;KZ90P; G415H;R748E; + + 407 S67A;KZ90A;S619W; + + 408 M71F;KZ90A;S619W; + + 409 M71Q;R256L;K436G;E707K; + + 410 S67A;K390A;K723D; ++ 411 S67A;M7IQ;N185D;R256K;T384E;N 444S;S619W; + + 412 M7IQ;N185D;R256K;S619F; ++ 413 S67A;M7IQ; R256L;K436G;Q626M; ++ 414 K3 90A; ++ 415 N185D; KZ90A;K436G;Q626M; + + 416 KZ90A;N444S;K723D; + + 417 N185D;KZ90A;S619F; + + 418 K369S;G415H;F620L;T705P; + + 419 N185D;KZ90A;Q626M; ++ 420 M7IQ; R256L;T443D;S619W;Q626E; ++ 421 M7IQ;R256L;S619W;E707K; + + 422 VI84Q;D252C;KZ69S;KZ90P;R748E G ++ 423 M7IQ;N185D;R256L;T384E; + + 424 M71L;F254W;A638Q;D709E;R748S; + + 425 S67A;M7IL;D252C;KZ69S;KZ90P; ++ 426 K390A;T443D;S619W; + + 427 M7IQ;R256K;T443D;Q626M; ++ 428 KZ90A;N444S;S619W; + +

- 63 043748

429 S67A;M7IQ;Nl85D;KZ90A;N444S;S 619W; + + 430 Nl85D;KZ90A;S619W; + + 431 S67A;M7IQ;Nl85D;KZ90A;S619W;E 707K; + + 432 Nl85D;KZ90A;Q626M;K723D; + + 433 K128Q;KZ69S;T705P;R748E; + + 434 M7IQ;R256L;Q626M;E707K; ++ 435 KZ90A;K436G;S619W;K723D; + + 436 K390A;K723D; ++ 437 K128Q;D252S;KZ69S;KZ90R; + + 438 M7IQ;N185D;R256L;TZ84E;S619W; ++ 439 M71Q;N18 5D;R2 5 6K;KZ 9 0A;K4 3 6G; E707K; ++ 440 S67A;M71F;N185D;N444S;S619W;K 723D; ++ 441 Nl85D;K436G;T443D;S619W;Q626M ;E707K;K723D; + + 442 M71Q;R256K; ++ 443 M7IQ;R256L;KZ90L;T443D; + + 444 S67A;M7IF;K390L;T443D;Q626M; + 445 М7IQ;R256L;КЗ90L;Т443D;Q626М; + 446 M7IQ;Nl85D;KZ90A;T443D;S619W; Q626E; + 447 K128Q;KZ69S;G415N; + 448 K128N;F620L;T705P;R748E; + 449 M7IL;T379N;D709E;R748G; + 450 TZ84E;P418G;N444S;A638Q;D709E;R748S; + 451 M71Q; Nl8 5D;KZ 9 OA;N4 4 4 S; + 452 M71F; Nl85D;KZ90L;N444S;S619W; K723D; + 453 M71L;D709E; + 454 N185D;R256K;S619W;E707K; + 455 K369S;K390P;F620L; +

- 64 043748

456 M7IQ;R256L;Q626E;E707K; + 457 S67A;M71F;N185D;N444S;S619W; + 458 К128Q;VI84Q;D252C;КЗ69S;G415Н + 459 F254W;TZ84E;A638Q;D709E;R748S G + 460 S67A;M7IQ;Q134H;R256L;K390L;T 443D;Q626M;E707K; + 461 K128Q;T705P; + 462 М7IQ;N185D;R256L;КЗ90L; + 463 S67A;T443D;S619R; + 464 M7IQ;R256K;KZ90A;E707K; + 465 S67A;M71Q;TZ84E;KZ90A;N444S;S 619W; + 466 M71L;F254W;N309T;N444S; + 467 M71L;P418G;R748S; + 468 М7IQ;N185D;R256L;КЗ90L;Q626М; + 469 M71L; + 470 S67A;M7IQ;N185D;R256K;KZ90L;S 619W; + 471 S67A;M71F;Nl85D;KZ90L;S619W; + 472 M71Q;N185D;K390A;K436G;T443D; Q626M; + 473 G415H;F620L; + 474 М7IL;VI84Q;КЗ69S;G415Н;Т705Р; +

Levels of increased activity were set relative to the reference polypeptide of SEQ ID No. 24 and were determined as follows: + > 1.2-fold but less than 2.0-fold increased; ++ > more than 2.0 times, but less than 4.0 times; +++ > than 4.0 times.

Example 10 Effect of Adding a Histidine Tag to SEQ ID NO: 82

The B29 acylation of SEQ ID No. 82, as described in Example 9, and SEQ ID No. 110, which contains a six-histidine tag at the C-terminus, were compared on a shake flask scale. Shake flask powders were prepared as described in Example 4. Reactions were carried out in 96-well deep well plates, each containing 200 μl, consisting of 0.2 M TRIS, pH 9.25, 20% acetonitrile, 25 g/l insulin, 17 g/l methyl phenylacetate and 0.3-10 g/l lyophilized enzyme powder reconstituted in 10 mM TRIS, pH 7.5. NTR plates were heat sealed and incubated in Thermotron® shakers (3 mm amplitude, model no. AJ185, Infors) at 30°C, 100 rpm, for 3 h. Reactions were quenched using 200 μl of acetonitrile and mixed for 5 min. using a table shaker. Then, 400 μL of water was added and mixed for 5 min using a table shaker. The plates were then centrifuged at 4000 rpm for 5 min and loaded onto HPLC for analysis. In fig. Figure 2 is a graph showing the results. As shown, the addition of a histidine tag had minimal effect on the enzyme relative to the version without the histidine tag.

Example 11: Improvement of Insulin Acylation at Position B29 Compared to SEQ ID No. 110 in a High Throughput Screen

SEQ ID No. 110 was selected as the parent enzyme after the histidine tag was shown to have minimal effect on the activity of SEQ ID No. 82. Libraries of engineered genes were generated using well techniques (eg, saturation mutagenesis and recombination of previously identified beneficial mutations).

The polypeptides encoded by each gene were prepared in the NTP as described in Example 2, and soluble lysates were created as described in Example 3, but using 400 μl of lysis buffer instead of 200 μl.

HTR reactions were carried out in 96-well plates with deep wells. Each reaction well contained 200 μl of 0.2 M TRIS, 20% acetonitrile, 25 g/l insulin, 17 g/l methylphenylacetate, and 10 μl of HTR supernatant (initial pH before addition of lysate was 9.4). NTP plates were incubated in Thermotron® shakers (amplitude 3 mm, model no. AJ185, Infors) at 30°C, 100 rpm, for 2 hours. Reactions were quenched using 200 μl of acetonitrile and mixed for 5 min with

- 65 043748 using a table shaker. Then, 400 μL of water was added and mixed for 5 min using a table shaker. The plates were then centrifuged at 4000 rpm for 5 min and loaded onto HPLC for analysis.

The percentage conversion relative to SEQ ID No. 110 (FIOP conversion percentage) was calculated as the percentage conversion of the product formed by the variant relative to the percentage conversion provided by SEQ ID No. 110. The results are presented in table. 9.1. The percent conversion was quantified by dividing the peak area of the product by the sum of the peak areas of the substrate, product, and impurities/by-product as observed by HPLC analysis.

Table 11.1. Activity of variants acylating at the B29 site relative to SEQ ID No. 110

No. option SEQ ID No. (n./a.k.) Amino Acid Differences (relative to SEQ ID No. 110) Acylation Conversion Percentage (FIOP) ¹ at Site A1 (relative to SEQ ID NO: 110) 475 E482Q; + + 476 E482M; + + 477 E482Y; + + 478 37/38 E482I; + + 479 E482T; + + 480 G54A; + 481 39/40 S386G; + 482 D484T; + 483 E482L; + 484 T32V; + 485 N444S;D709E;R748S; +

- 66 043748

486 G54S; + 487 G415H;D709E; + 488 Q556G; + 489 Y616D; + 490 K128Q;D252C;G415H;N444S ;D709E;R748S; + 491 N185D;D709E; + 492 Y616N; + 493 Y616G; + 494 L557P; + 495 S704T; + 496 Y616A; + 497 47/48 L557S; + 498 43/44 P496A; + 499 P496T; + 500 45/46 L557Q; + 501 L557R; + 502 S639G; + 503 49/50 S704A; + 504 Q233A; + 505 D334P; + 506 Q233D; + 507 P496R; + 508 S639A; + 509 T131D; + 510 T131E; + 511 Q112D; + 512 S639E; + 513 L557V; + 514 S639D; + 515 P496N; + 516 S740A; + 517 L225T; + 518 T131N; + 519 L557M; + 520 D484L; +

¹ Levels of increased activity were set relative to the reference polypeptide from SEQ ID No. 110 and were determined as follows: + > 1.0-fold but less than 2.0-fold increased; ++ > more than 2.0 times, but less than 4.0 times; +++ > than 4.0 times, but less than 8.0 times; ++++ > than 8.0 times.

Example ^: Improvement of insulin acylation at position B29 compared to SEQ ID No. 40 in a high-throughput screen

SEQ ID No. 40 was selected as the next parent enzyme based on the results described in Example 11. Engineered gene libraries were generated using well techniques (eg, saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were prepared in the NTP as described in Example 2, and soluble lysates were created as described in Example 3, but using 400 μl of lysis buffer instead of 200 μl.

HTR reactions were carried out in 96-well plates with deep wells. Each reaction well contained 200 μl of 0.2 M TRIS, 20% acetonitrile, 25 g/l insulin, 17 g/l methylphenylacetate, and 80 μl of HTR supernatant (initial pH before addition of lysate was 9.4). NTP plates were incubated in Thermotron® shakers (amplitude 3 mm, model no. AJ185, Infors) at 30°C, 100 rpm, for 2 hours. Reactions were quenched using 200 μl of acetonitrile and mixed for 5 min with

- 67 043748 using a table shaker. Then, 400 μL of water was added and mixed for 5 min using a table shaker. The plates were then centrifuged at 4000 rpm for 5 min and loaded onto HPLC for analysis.

The conversion percentage relative to SEQ ID No. 40 (FIOP) was calculated as the percentage conversion of the product formed by the variant relative to the percentage conversion provided by SEQ ID No. 40. The results are presented in table. 12.1. The percent conversion was quantified by dividing the peak area of the product by the sum of the peak areas of the substrate, product, and impurities/by-product as observed by HPLC analysis.

The percentage selectivity relative to SEQ ID No. 40 (FIOP selectivity percentage) was calculated as the percentage selectivity of the product produced by the variant relative to the percentage selectivity provided by SEQ ID No. 40. The percentage selectivity was calculated by dividing the peak area of the product by the sum of the peak areas of the product and the impurities/by-product product as observed by HPLC analysis.

Table 12.1. Activity and selectivity of variants acylating at the B29 site relative to SEQ ID No. 40

No. Variation SEQ ID No. (n./a.k.) Amino acid differences (relative to SEQ ID No. 40) Percentage converted by acylation (FIOP) ¹ at site B29 (relative to SEQ ID no. 40) Acylation selectivity percentage (FIOP) ² FOR region B29 (relative to SEQ ID No. 40) 524 53/54 W370K; + + 525 57/58 D623V; + + 526 51/52 A2 8V;Y52L;L55I;T131E;L175H ;Q233R;G415Н;N444G;Р513S; + + 527 55/56 D623L; + + 528 59/60 P366G; + + 529 A28V;G74D;T374S;S704A; + + 530 A28V;L175H;P513S;W619S; + + 531 P366S; + + 532 LI75Н;G415Н;К723D; + + 533 H472A; + + 534 Н348N;N444G;S704А; + + 535 T374S;Y616A;S704A; + + 536 T27Y;A28V;G74D;N185D; T374S ;N444G;S704A; + + 537 A2 8R; + + 538 N185D;T374S; + + 539 Y52L;L55I;QI12D;TZ74S;N444 G; + 540 H348N; + 541 Q380F; + 542 S150A; + 543 A362R; + 544 T374S;G415H;K547Q; +

¹ Levels of increased activity were set relative to the reference polypeptide from SEQ ID No. 40 and were determined as follows: + > 1.0-fold but less than 2.0-fold increased; ++ > more than 2.0 times, but less than 4.0 times; +++ > than 4.0 times, but less than 8.0 times; ++++ > than 8.0 times.

² Levels of increased selectivity were established relative to the reference polypeptide from SEQ ID No. 40 and were determined as follows: + > 1.0-fold but less than 2.0-fold increased; ++ > more than 2.0 times, but less than 4.0 times; +++ > than 4.0 times, but less than 8.0 times;

++++ > than 8.0 times.

Example ^: Improvement of insulin acylation at position B29 compared to SEQ ID No. 56 in a high-throughput screen

SEQ ID No. 56 was selected as the next parent enzyme based on the results described in Example 12. Engineered gene libraries were generated using well techniques (eg, saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were prepared in the NTP as described in Example 2, and soluble lysates were created as described in Example 3, but using 400 μl of lysis buffer instead of 200 μl.

HTR reactions were carried out in 96-well plates with deep wells. Each reaction well contained 200 μl of 0.2 M TRIS, 10% acetonitrile, 50 g/l insulin, 17 g/l methylphenylacetate, and 10 μl of HTR supernatant (initial pH before addition of lysate was 9.4). NTP plates were incubated in Thermotron® shakers (amplitude 3 mm, model no. AJ185, Infors) at 30°C, 100 rpm, for 3 hours. Reactions were quenched using 200 μl of acetonitrile and mixed for 5 minutes using a benchtop shaker Then, 400 μL of water was added and mixed for 5 min using a table shaker. The plates were then centrifuged at 4000 rpm for 5 min and diluted 2 more times in water before loading into HPLC for analysis.

The percent conversion relative to SEQ ID No. 56 (FIOP conversion percent) was calculated as the percent conversion of the product formed by the variant relative to the percent conversion provided by SEQ ID No. 56 and is presented in the table below. The percent conversion was quantified by dividing the peak area of the product by the sum of the peak areas of the substrate, product, and impurities/by-product as observed by HPLC analysis.

The percentage selectivity relative to SEQ ID No. 56 (FIOP selectivity percentage) was calculated as the percentage selectivity of the product formed by the variant relative to the percentage selectivity provided by SEQ ID No. 56. The results are presented in table. 13.1. The percent selectivity was calculated by dividing the peak area of the product by the sum of the peak areas of the product and the impurities/by-product as observed by HPLC analysis.

- 69 043748

Table 13.1. Activity and selectivity of variants acylating at the B29 site relative to SEQ ID No. 56

No. option SEQ ID No. (n./a.k.) Amino acid differences (relative to SEQ ID No. 56) Acylation conversion percentage (FIOP) ¹ at site B29 (relative to SEQ ID No. 56) Acylation selectivity percentage (FIOP) ² FOR region B29 (relative to SEQ ID No. 56) 561 Q112D;W619S; + 562 87/88 RZ66S;D484T;W619S;1708V; + 563 RZ66Q;D484T;K547N;W619S; + 564 W619S; + 565 РЗ66G;D484Т;W619S;F620L; + 566 95/96 K390Q; + 567 K390A; + 568 K390S; + 569 W619S;S740A; + 570 E103K;Q112D;A361G;P366G;D48 4T;W619S; + 571 Q157S;K723D; + 572 E103K;W619S; + 573 A361G;РЗ66Q;D484T;W619S;F62 0L; + 574 QI57S;K436G;L623V;K723D; + 575 P366G;W619S;S740A; + 576 D484T;K547H;W619S;F620L;S74 0A; + 577 D484T;I708V; + 578 P366Q;Q556H;W619S;S740A; + 579 P366S;D484T;W619S;F620L;170 +

- 70 043748

8V; 580 ElOZK;QI12L;РЗ66Q;D484Т;W61 9S;I708V;S740A; + 581 D130E;QI57S;L62ZT;Q626M; + 582 T131E;S704A;K723D; + 583 D130E;R317A;L623T;K723D; + 584 E561P; + 585 W37OK;D484T;W619S; + 586 K436G;L623T; + 587 РЗ66S;D484Т;S740А; + 588 T13ID;L557V;S639T;K723D; + 589 K369D; + 590 R317A;L62ZT;Q626M;K723D; + 591 D130E;QI57S;K436G;K723D; + 592 Y616T; + 593 K128D;D130E;QI57S;K436G;L62 ZT;Q626M;K723D; + 594 L557P; + 595 91/92 A255G; + 596 P366S;D484T; + 597 QI12L;РЗ66G;D484Т;К547Н;W61 9S;I708V; + 598 T131D;T491S;L557V;D709E;K72 3D; + 599 Q71G; + 600 D484T;I708V;S740A; + 601 S675E; + 602 РЗ66S;W37OK;D484Т;Q556H;W61 9S; + 603 D130E;L623T; + 604 A255D; + 605 T131D;L557V; + 606 L225T;L557V;S704A;D709E;K72 3D; +

607 E1OZK;W37OK;D484T;W619S;170 8V; + 608 D484T; + 609 QI12D;РЗ66S;D484Т; + 610 Q112L;D484T;I708V; 611 97/98 F57H; + 612 99/10 0 F57C; + 614 89/90 A67S; +

¹ Levels of increased activity were set relative to the reference polypeptide from SEQ ID No. 56 and were determined as follows: + > 1.2-fold but less than 2.0-fold increased; ++ > more than 2.0 times, but less than 4.0 times; +++ > than 4.0 times, but less than 8.0 times; ++++ > than 8.0 times.

² Levels of increased selectivity were set relative to the reference polypeptide of SEQ ID No. 56 and were determined as follows: + > 1.2-fold but less than 2.0-fold increased; ++ > more than 2.0 times, but less than 4.0 times; +++ > than 4.0 times, but less than 8.0 times; ++++ > than 8.0 times.

- 71 043748

Example 14: Improvement of Insulin Acylation at Position A1 Compared to SEQ ID No. 70 in a High Throughput Screen

SEQ ID No. 70 was selected as the additional parent enzyme based on the results described in Example 7. Engineered gene libraries were generated using well techniques (eg, saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were prepared in the NTR as described in Example 2, and soluble lysates were created as described in Example 3, but using 400 μl of lysis buffer instead of 200 μl.

NTR reactions were carried out in 96-well deep well plates containing 200 μl of 0.1 M Tris-HCl, pH 9.25, 20% acetonitrile, 20 g/l insulin, 17 g/l methylphenylacetate and 10 μl of HTR lysate. NTP plates were incubated in Thermotron® shakers (amplitude 3 mm, model no. AJ185, Infors) at 30°C, 100 rpm, for 5 hours. Reactions were quenched using 200 μl of acetonitrile and mixed for 5 minutes using a benchtop shaker. The plates were then centrifuged at 4000 rpm for 5 min, diluted 2 times in water and loaded into HPLC for analysis.

The percentage conversion relative to SEQ ID No. 70 (FIOP conversion percentage) was calculated as the percentage conversion of the product formed by the variant relative to the percentage conversion provided by SEQ ID No. 70. The results are presented in table. 14.1. The percent conversion was quantified by dividing the peak area of the product by the sum of the peak areas of the substrate, product, and impurities/by-product as observed by HPLC analysis.

Table 14.1. Activity of variants acylating at the A1 site relative to SEQ ID No. 70

No. option SEQ ID No. (n./a.k.) Amino acid differences (relative to SEQ ID No. 70) Acylation Conversion Percentage (FIOP) ¹ at Al Site (relative to SEQ ID No. 70) 642 121/1 22 R317S;Q380Р;G415Н;Т443D;А517 K;T560G;F701W; + + + 643 119/1 20 А255Р;Q380P;G444K;N457T;A517 K;T560G; +++ 644 123/1 24 A2 55Р;R317S;Q3 8 OP;G415Н;Т4 4 3 D;G4 4 4 S;A517K;T5 6 0G;F7 01W; + + + 645 113/1 14 A2 55P;R317S;Q3 8 OP;G415H;T4 4 3 D;G444S;F454W;N457T;A517K;T5 60G;F701W; 646 115/1 16 L55I;A255P;R317S;Q38 OP;T443D ;G444S;F454W;H472P;A517K;T56 + + +

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0G;F701W; 647 111/1 12 A2 55P; R317S ; Q3 8 OP; G415H; G4 4 4 S;N457T;A517K;T560G;F701W; + + + 648 117/1 18 A2 55P;R317S;Q3 8 OP;G415H;T4 4 3 D;F454W;N457T;A517K;T560G;F7 01W; 649 A2 55P;R317S;Q3 8 OP;T4 4 3D;G4 4 4 S;F454W;A517K;T560G;F701W; + + 650 A2 55P;R317S;Q3 8 OP;T4 4 3D;G4 4 4 S;N457T;T560G;F701W; + + 651 A255P;R317S;Q38 OP;G444K;F4 54 W;A517K;T560G; + + 652 L253S; + + 653 A255P;R317S;G415H;T443D;G444 S;F454W;N457T;A517K;F701W; + + 654 A2 55P;R317S;Q3 8 OP;G415H;T4 4 3 D;F454W;N457T;A517K;F701W; + + 655 A255P;R317S;Q38 OP;G444K;A517 K;T560G;F701W; + + 656 R317S;Q38 OP;T443D;G444K;F4 54 W;A517K;T560G;F701W; + + 657 Q556G; + + 658 A255P;R317S;A373K;G415H;T443 D;F454W;N457T;A517K;T560G;F7 01W; + + 659 N185E; + + 660 W370L; + + 661 A2 55P;R317S;Q3 8 OP;G4 4 4S; N4 57 T;A517K;T560G;F701W; + + 662 A2 55P; R317S ; A37 3K; G415H; G4 4 4 K;N457T;A517K;T560G;F701W; + + 663 A2 55P;R317S;Q38 OP;G444K;F4 54 W;N457T;A517K;T560G;F701W; + + 664 N348D; + 665 N348H; +

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666 А255Р;R317S;А37ЗК;G415Н;G444 S;F454W;N457T;A517K;T560G;F7 01W; + 667 А255Р;А37ЗК;G415Н;G444S;N457 Т;А517К;T560G;F701W; + 668 L557P; + 669 A362V; + 670 A255P;R317S;Q380P;T443D;F454 W;T560G;F701W; + 671 E707A; + 672 A255P;R317S;Q380P;G444S;A517 K;T560G;F701W; + 673 A255P;R317S;Q380P;G444K;N457 T;A517K;T560G;F701W; + 674 T560G; + 675 А255Р;R317S;G415Н;G444S;F454 W;A517K;T560G;F701W; + 676 R317S;A37ЗК;G415Н;Т443D;F454 W;A517K;T560G;F701W; + 677 А255Р;R317S;КЗ69Т;G415Н;Т443 D;N457T;A517K;F701W; + 678 А255Р;R317S;А37ЗК;G415Н;G444 K;N457T;A517K;S530D;F701W; + 679 L557G; + 680 А255Р;R317S;G415Н;F454W;N457 T;A517K;T560G;F701W; + 681 А255Р;R317S;А37ЗК;G415Н;G444 K;F454W;A517K;T560G;F701W; + 682 A255P;R317S;Q380P;G444Q;F454 W;A517K;T560G;F701W; + 683 L253R; + 684 A2 55P;R317S;Q3 8 0P;G4 4 4S;F4 54 W;A517K;S530D;T560G;F701W; + 685 R317S;A37ЗК;G415Н;G444К;F454 W;A517K;T560G;F701W; + 686 A255P; + 687 L557S; + 688 A255P;R317S;Q38OP;G444K;F454 W;N457T;A517K;F701W; + 689 S386G; + 690 L557E; + 691 F254G; + 692 A451K; + 693 A2 55P;R317S;Q3 8 0P;G4 4 4S;F4 54 W;A517K;T560G; F701W; + 694 A255P;R317S;Q380P;G444K; F4 54 W;T560G;F701W; + 695 F756P; + 696 R317S;Q380P;T443D;N457T;A517 K;F701W; +

¹ Levels of increased activity were set relative to the reference polypeptide from SEQ ID No. 70 and were determined as follows: + > 1.2-fold but less than 1.5-fold increased; ++ > more than 1.5 times, but less than 2.0 times; +++ > than 2.0 times.

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Example 15 Improvement in insulin acylation at position A1 compared to SEQ ID No. 116 in a high throughput screen

SEQ ID No. 116 was selected as the next parent enzyme based on the results described in Example 14. Engineered gene libraries were generated using well techniques (eg, saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were prepared in the NTP as described in Example 2, and soluble lysates were created as described in Example 3, but using 400 μl of lysis buffer instead of 200 μl.

HTP reactions were carried out in 96-well deep well plates containing 200 μl of 0.25 M Tris-HCl, pH 9.25, 20% acetonitrile, 50 g/L insulin, 17 g/L methylphenylacetate, and 10 μL HTP lysate. NTP plates were incubated in Thermotron® shakers (3 mm amplitude, model no. AJ185, Infors) at 30°C, 100 rpm for 4 h. Reactions were quenched using 200 μl of acetonitrile and mixed for 5 min using a tabletop shaker . The plates were then centrifuged at 4000 rpm for 5 min, diluted 24x in water and loaded onto HPLC for analysis.

The percentage conversion relative to SEQ ID No. 116 (FIOP conversion percentage) was calculated as the percentage conversion of the product formed by the variant relative to the percentage conversion provided by SEQ ID No. 116. The results are presented in table. 15.1. The percent conversion was quantified by dividing the peak area of the product by the sum of the peak areas of the substrate, product, and impurities/by-product as observed by HPLC analysis.

Table 15.1. Activity of variants acylating at the A1 site relative to SEQ ID No. 116

No. option a SEQ ID No. (on the. To. ) Amino acid differences (relative to SEQ ID NO: 116) Acylation conversion percentage (FIOP) ¹ at site A1 (relative to SEQ ID No. 116) 697 125/12 6 L253S;N348D;G415Н;N457Т;Q556G;L557P G + + + + 698 131/13 2 N185E;L253S;N348Н;G415Н;N457Т;Q556G ;L557G;К72ЗЭ; ++++ 699 L2 53S;S317R;N348Н;AZ 62V;G415Н;N4 57T ;Q556G;L557G;K723E; + + + + 700 133/13 N185E;L253S;S317R;N348D;AZ62V; + + + +

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4 701 N185E;L253S;S317R;N348H;A3 62V;Q556G ;L557G; + + + + 702 L253S;N348D;W370L;N457T;Q556G;L557G r ++++ 703 N185D;L253S;S317R;N348H;A3 62V;G415H ;N457T; ++++ 704 N185E;L253S;S317R;G415H;N457T;L557P ;K723E; + + + + 705 129/13 0 N185D;L253S;N348H;A3 62V; ++++ 706 L253S;S317R;N348D;A3 62V;G415H;N457T ++++ 707 L253S;N348D;A3 62V;L557G;K723E; + + + + 708 L253S;N348D;A3 62V;N457T;L557G;K723E ++++ 709 L253S;A3 62V;N457T;L557G; + + + + 710 L253S;N348D;A3 62V;G415H;L557G; ++++ 711 L253S;S317R;A3 62V;G415H;L557G; ++++ 712 L253S;S317R;N348H;A3 62V;W37OL;Q556G ;L557P; + + + + 713 L253S;S317R;N348D;A3 62V;G415H;Q556G ;L557G;K723E; ++++ 714 L253S;N348D;L557P; + + + + 715 L253S;N348D;A3 62V;W37OL;N457T;Q556G ;L557G;K723E; ++++ 716 N185D;L253S;N348H;G415H;L557G; ++++ 717 N185E;L253S;N348H;A3 62V;K723E; + + + + 718 N185D;L253Q;G415H;N457T;L557G; ++++ 719 L253S;S317R;N348D;W37OL;N457T;K723E r + + + + 720 L253S;S317R;N348D;G415H;L557G; ++++ 721 N185D;L253S;S317R;W37OL;G415H;Q556G ;L557G; + + + + 722 N185E;L253S;S317R;N348H;A3 62V;L557G ++++

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;K723E; 723 N185D;L253S;S317R;N348H;L557G;R602C + + + + 724 N185E;L253S;N348D;A3 62V;N457T;Q556G ;L557P; ++++ 725 N185D;L253S;S317R;A3 62V; ++++ 726 L253S;S317R;N348D;A3 62V;N457T;Q556G ;L557G;K723E; + + + + 727 N185D;L253S;N348H;A3 62V;G415H; ++++ 728 N185E;L253S;N348H;A3 62V;W370L;G415H ;Q556G;L557G; ++++ 729 L253S;S317R;N348D;A362V;Q556G;L557P + + + + 730 L253S;S317R;A362V;N457T;Q556G;L557G ;K723E; + + + + 731 N185E;L253S;S317R;N348D;A3 62V;G415H ;L557G;K723E; ++++ 732 L253S;S317R;N348D;L557P; ++++ 733 N18 5E;L2 53S;S317R;N348H;A3 62V;G415H ;N457T;K723E; + + + + 734 137/13 8 N18 5D;L2 53S;S317R;N348H;A3 62V;G415H ++++ 735 N18 5D;L2 53S;A3 62V;L557G; ++++ 736 N185D;L253Q;S317R;N348D;A362V;G415H ;N457T;K723E; + + + 737 N185E;L253S;S317R;A3 62V;W37OL;L557G ;K723E; + + + 738 N185E;L253S;G415H;L557G; +++ 739 L253Q;N348D;N457T;Q556G;L557P; +++ 740 N185E;L253S;N348H;A3 62V;N457T;L557G + + + 741 N185D;L253S;S317R;A3 62V;G415H;N457T +++ 742 N185D;L253Q;S317R;N348D;W37OL;N457T + + +

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743 N185D;L253Q;S317R;A3 62V;G415H;K72ЗЭ + + + 744 N185D;L253S;S317R;G415Н;L557G; + + + 745 L253S;N348D;AZ 62V;Q556G;L557G; + + + 746 L253S;S317R;A362V; + + + 747 L253S;S317R;G415Н;N457Т; + + + 748 L253S;S317R;N348D;AZ 62V;N457T;Q556G + + + 749 N185E;L253S;N348H;AZ 62V;L557G; +++ 750 L253S;N348N;AZ 62V;K72ZE; +++ 751 L253Q;N348D;A362V;N457T;L557P; +++ 752 L253S;S317R;N348D;AZ 62V; +++ 753 N185E;L253S;N457T;L557P; +++ 754 N185E;L253Q;N348D;Q556G;L557G; +++ 755 L253S;N348D;AZ 62V;W370L;L557P;K72ZE + + + 756 L253Q;S317R;N348D;L557G; +++ 757 L253Q;AZ 62V;W37OL;N457T;Q556G;L557G + + + 758 L253S;N348D;L557G;K72ЗЭ; +++ 759 L253S;N348D;L557G; +++ 760 N185E;L253S;L557G;K72ЗЭ; +++ 761 N185D;L253Q;S317R;Q556G;L557G; +++ 762 L2 53Q;S317R;A3 62V;N4 57T;Q55 6G; L557P + + + 763 L253S;A3 62V;L557G; +++ 764 N185D;L253S;N348D;A3 62V;G415H;Q556G ;K723E; +++ 765 L253S;S317R;N348H;N457T;Q556G;K72ЗЭ + + + 766 L253S;S317R;A3 62V;L557G;K72ЗЭ; +++ 767 N185D;L253S;N457T;L557G; +++ 768 127/12 8 L253S;N348H;G415H; +++ 769 N185E;S317R;N348N;AZ 62V;N457T;L557G +++

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G 770 N185E;L253Q;N348H;A362V;N457T; + + + 771 L253S;S317R;N348D;K72ZE; +++ 772 L253S;AZ 62V;G415N; + + + 773 N348D;AZ 62V;N457T;Q556G;L557G;K72ZE G +++ 774 N185E;L253S;S317R;N348N;AZ 62V;K72ZE G + + + 775 N185E;L253S;AZ 62V;Q556G;K72ZE; + + + 776 L253S;S317R;AZ 62V;Q556G;L557G; + + + 777 L253Q;N348D;A362V;Q556G;L557G; +++ 778 N185E;S317R;N348D;A362V;Q556G;L557P G + + + 779 N185E;L253S;S317R;N457T; +++ 780 N185E;L253S;S317R;N348H;G415H;L557G ;K723E; + + + 781 T105N;N185D;L253Q;S317R;N348D;L557G + + + 782 N185D;S317R;N348D;A362V;N457T; +++ 783 L253S;G415H;L557G; + + + 784 N18 5E;N348D;A3 62V;G415H;K72 3E; +++ 785 N185D;L253Q;N348D;G415H;N457T;L557G ;K723E; + + + 786 N185E;L253Q;W370L;G415H;Q556G;L557G r + + + 787 N185D;A3 62V;G415H;Q556G;L557G;K723E r +++ 788 N185D;L253Q;S317R;N348D;N457T; +++ 789 L253Q;N348D;A362V;L557G; + + + 790 N185D;L253S;N348D;N457T;Q556G;L557G ;K723E; + + + 791 N185E;S317R;N348H;N457T;Q556G;L557G r +++ 792 L253S;N348D;A3 62V;G415H;Q556G;L557P r +++

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793 N348D;A362V;G415H;L557G; + + + 794 N18 5D;L2 53S;A3 62V; + + + 795 N185D;A3 62V;W37OL;Q556G;L557G;W701F ;K723E; + + + 796 L253S;G415H;N457T;L557G; + + + 797 N185E;S317R;G415H;N457T;L557G; + + + 798 L253Q;S317R;N348D;A362V;G415H; + + + 799 N18 5E;L2 53S;A3 62V; + + + 800 L253S;L557P; + + + 801 L253Q;S317R;N348H;A362V;G415H;L557G r + + + 802 N185E;L253Q;S317R;N348D; + + + 803 L253S;S317R;N348H;W37OL;G415D;N457T ;K723E; + + + 804 L253Q;S317R;N348D;A362V;N457T;Q556G ;K723E; +++ 805 N185D;S317R;N348D;G415H;L557G; + + + 806 L253Q;S317R;N348D;N457T;Q556G;L557G ;K723E; + + + 807 N185D;L253S;N348H; +++ 808 N185E;S317R;N348H;W701F;K723E; +++ 809 N185E;N348D;A3 62V;W37OL;G415H;K723E r +++ 810 S317R;A3 62V;N457T;L557G;K723E; +++ 811 N185D;N348H;A3 62V;Q556G;L557G;K723E + + + 812 L253S;S317R;A3 62V;K723E; +++ 813 N185E;N348D;N457T;Q556G;K723E; + + + 814 L253S;N348H;A3 62V; + + + 815 L253S;N348D; + + + 816 S317R;N348D;A362V;G415H; +++ 817 L253Q;N348D;A3 62V;L557G;K723E; +++ 818 L253Q;N348D;G415H;L557G;K723E; + + + 819 L253Q;N348D;A362V;G415H;L557P; +++ 820 135/13 L253S;S317R;N348D; +++

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6 821 N185D;L253Q;S317R;N348D;L557G;K72ЗЭ + + + 822 L253S;S317R;L557G;K72ЗЭ; + + + 823 N185E;S317R;N348D;A362V;G415H;L557P + + + 824 L253S;Q556G;L557G;K723E; + + + 825 N185E;L253S;S317R; + + + 826 N185E;L253Q;S317R;N348D;A3 62V;K72ЗЭ f + + + 827 L2 53Q;S317R;N348D;A3 62V;Q55 6G;K72 3E f + + + 828 N185E;L253S;N457T;L557P;R602C; + + + 829 L253Q;A3 62V;Q556G;L557G;K72ЗЭ; + + + 830 N185D;N348D;A362V; + + + 831 N185E;L253S;S317R;A3 62V; + + + 832 L253Q;S317R;A362V;L557G; + + + 833 N185D;N348D;L557G; + + + 834 N185D;L253Q;S317R;N457T; + + + 835 S317R;N348D;A362V;N457T;L557G; + + + 836 L253Q;S317R;G415H;L557P;K723E; + + + 837 N185D;N348D;W370L;G415H;Q556G;L557P ;K723E; + + + 838 KZ04E;S317R;N348H;A3 62V;L557G;W701F f + + + 839 N185D;L253S; + + + 840 N185E;G415H;N457T;Q556G;K723E; + + + 841 G415H;Q556G; + + + 842 L253S;N457T;L557P; + + + 843 S317R;N348D;G415H;N457T; + + + 844 L253Q;G415H;L557G; + + + 845 L253Q;S317R;N348D; + + + 846 N185D;N348H;A362V;L557P; + + + 847 N185D;L253S;N348D;A3 62V;W37OL;G415H ;D428G;Q556G;K723E; + + +

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848 N348D;Q556G;L557G; + + + 849 N185D;N348D;N457T;K72ЗЭ; + + + 850 N185E;L253Q;N348D;L557G; + + + 851 N185D;A362V;G415H;L557P; + + + 852 N185E;S317R;A3 62V;N457T;L557G;K723E r + + + 853 L253Q;S317R;G415H;Q556G;L557G;K723E r +++ 854 L253Q;N348H;A362V;L557G; +++ 855 N185E;N348D;A362V;L557P; +++ 856 L253Q;A3 62V;W701F;K723E; + + + 857 S317R;N348D;A3 62 V;Q556G;L557P;K723E r + + + 858 N185E;L253S;K723E; + + + 859 N185D;S317R;N348D; +++ 860 N185D;S317R;N348H;A362V; +++ 861 N185E;S317R;N457T;Q556G;L557G; +++ 862 N185E;N348D;A362V; +++ 863 S317R;A362V;L557G; +++ 864 N185E;A362V;N457T; +++ 865 N185D;L253Q;S317R;N348H;K723E; +++ 866 N348H;A3 62V;N457T;W701F;K723E; + + + 867 N348D;A362V;N457T; +++ 868 N185D;S317R;N348H;N457T; +++ 869 N185E;N457T;Q556G; + + + 870 N185E;S317R;N348D;G415H;L557P;W701F r +++ 871 L253S;N348H;N457T; + + + 872 N18 5E;L2 5 3Q;N3 4 8 D;G415H;K7 2 3E; + + + 873 L253S;Q556G;L557G; +++ 874 N18 5E;L2 53Q;W7 01F;K7 2 3E; +++ 875 N185D;S317R;A362V;Q556G; + + + 876 L253S;N348H;K723E; +++ 877 N185E;L253Q;S317R;N457T; +++

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878 Nl85D;G415H;N457T;K72ЗЭ; + + + 879 N185E;N348D;G415H; + + + 880 L253Q;N348Н;AZ 62V;W37OL;L557P; + + + 881 N185D;G415H;Q556G; + + + 882 Nl85E;N348D;A3 62V;N457T;K72ЗЭ; + + + 883 N185D;S317R;L557P; + + + 884 L253S;N348H;L557G; + + + 885 Nl85D;S317R;W37OL;N457T;L557G; + + + 886 N185D;S317R;N348D;A362V;K723E; + + + 887 S317R;A3 62V;W37OL;L557G; + + + 888 Nl 85D;S317R;W701F; + + + 889 L253Q;G415H;N457T; + + + 890 L253S;A362V; + + + 891 N348H;G415H;Q556G;L557P; + + + 892 N185D;N348D;A362V;K723E; + + + 893 N185D;N348H;A362V; + + + 894 N185E;L253Q;S317R;A362V; + + + 895 N185E;L253Q;N348H;L557G; + + + 896 N185D;W701F; + + + 897 L253Q;S317R;A362V;G415H; + + + 898 L253Q;S317R;W37OL;N457T;L557G;K723E r + + + 899 N185E;S317R;A362V; + + + 900 L253S;S317R; + + + 901 L253Q;N348H;L557G; + + + 902 Nl85D;L557G;K723E; + + + 903 N185E;G415H;L557G; + + + 904 N185E;W701F; + + + 905 S317R;N348D;L557G; + + + 906 L253S;N348H;N457T;Q556G;L557G;Y580N ;K723E; + + + 907 N185E;A362V;N457T;K723E; + + + 908 N348D;A3 62V;L557G;K72 3E; + + + 909 L253Q;S317R;A3 62V;G415H;K723E; + + +

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910 L253Q;N457T;Q556G;L557G; + + + 911 G415H;N457T;Q556G;L557G; + + + 912 L253Q;S317R;L557G;K72ЗЭ; + + + 913 S317R;N457T;Q556G;L557G; + + + 914 N348H;G415H;N457T; + + + 915 N185D;L253Q;N348H;L557P;K723E; + + + 916 L253Q;A3 62V;N457T;K723E; + + + 917 N185D;L253Q;Q556G;L557P;K723E; + + + 918 N185D;G415H;K723E; + + + 919 S317R;A362V;N457T; + + + 920 L253Q;N348D;Q556G;K723E; + + + 921 S317R;L557G;K723E; + + + 922 N185E;Q556G;L557G;K723E; + + + 923 N185D;A362V;L557G; + + + 924 N185E;N348D;K723E; + + + 925 L253Q;A362V;N457T; + + + 926 L253S;Q556G; + + + 927 N185D;N348D; + + + 928 L253Q;S317R;N457T; + + + 929 N185E;L253Q; + + + 930 L253S;L557G; + + + 931 N185E;N348D; + + + 932 L253Q;N348D;A3 62V;K723E; + + + 933 N185D;A3 62 V;G415H;K723E; + + + 934 N185E;A362V;Q556G; + + + 935 L253Q;L557G;K723E; + + + 936 S317R;N348H;L557G; + + + 937 S317R;A362V;G415H; + + + 938 N185E;L253Q;S317R; + + + 939 N185D;L253Q;L557G; + + + 940 G415H;L557G; + + + 941 S317R;N457T;L557G; + + + 942 A3 62V;L557G;K723E; + + + 943 L253Q;S317R;Q556G; + + +

- 84 043748

944 N348D;G415H; + + + 945 N18 5D;L2 53Q;N348H;A3 62V;K72 3E; + + + 946 N348H;Q556G; + + + 947 A362V;N457T;L557G; + + + 948 L253Q;S317R;N348H; + + + 949 L253Q;N348D;W370L;Q556G;L557G;K723E r + + + 950 N185E;Q556G;L557P; +++ 951 A3 62V;W37OL;L557G;K723E; +++ 952 L253Q;Q556G;L557G; + + + 953 L253S;N457T; + + + 954 N185E;S317R;N457T;K723E; + + + 955 S317R;A362V;Q556G;L557G; +++ 956 N185D;N348H;N457T; +++ 957 A362V;N457T;Q556G;L557P; +++ 958 G415H;L557P; +++ 959 L253S; + + + 960 N185E;L253Q;N457T; + + + 961 L253S;A642V; +++ 962 N348D;A362V; +++ 963 N185E;S317R;W37OL;Q556G; + + + 964 L253Q;A362V; +++ 965 L253Q;N348H;A362V; +++ 966 N457T;Q556G;L557P; +++ 967 Al 60S;N18 5E;S317R;A3 62V;L557G; +++ 968 S317R;W37OL;L557G; + + + 969 S317R;W37OL;N457T; +++ 970 N457T;L557P; +++ 971 S317R;Q556G; + + + 972 A362V;N457T; + + + 973 S317R;A362V; + + + 974 S317R;Q556G;L557G;K723E; +++ 975 S317R;G415H; + + + 976 N185D;L253S;V360I;G415H;L557G; + + +

- 85 043748

977 N348D; + + + 978 N348D;AZ 62V;K72ZE; + + + 979 N185E;N348H; + + + 980 AZ 62V;Q556G;L557P; + + + 981 N185D;N457T; + + + 982 N185D;A362V; + + + 983 L253Q;S317R; + + + 984 N348H;A362V; + + + 985 A362V;Q556G;K723E; + + + 986 N185E;A362V; + + + 987 A362V;L557G; + + + 988 S317R;Q556G;L557G; + + 989 L253Q;K723E; + + 990 N185D;S317R; + + 991 S317R;N457T; + + 992 S317R;L557G; + + 993 G415H; + + 994 A3 62V;N457T;K723E; + + 995 N185D;K723E; + + 996 Q556G;L557G; + + 997 A3 62 V; + + 998 N185D; + + 999 L253Q;S317R; + + 1000 S317R;K723E; + + 1001 Q556G;K723E; + + 1002 L253Q; + + 1003 L557G; + + 1004 N185E; + + 1005 S317R; + + 1006 Al40E;N185E;L253Q;G415H;N457T;L557G r + + 1007 N457T; + 1008 L2 53S;S317R;A3 62V;N457T;K723E;S750R r + 1009 N185D;A362V;E707A; + 1010 L557P; + 1011 L253Q;F277L;L557P; + 1012 N185D;A3 62V;L557G;E707A; + 1013 N348H; + 1014 K723E; + 1015 N185D;S317R;A362V;E707A; +

¹ Levels of increased activity were set relative to the reference polypeptide from SEQ ID No. 116 and were determined as follows: + > 1.2-fold but less than 1.5-fold increased; ++ > more than 1.5 times, but less than 2.0 times; +++ > more than 2.0 times, but less than 5.0 times; ++++ > than 5.0 times.

Example 16: Effect of adding a histidine tag on SEQ ID NO: 136

The A1 acylation of SEQ ID No. 136 (described in Example 15) and SEQ ID No. 142, which contains a six-histidine tag at the C-terminus of SEQ ID No. 136, were compared on a shake flask scale. Shake flask powders were prepared as described in Example 4. Reactions were carried out in 96-well deep well plates containing 200 μl of 0.25 M Tris-HCl, pH 9.25, 20% acetonitrile, 50 g/l insulin, 17 g /l methylphenylacetate and 0.05-0.5 g/l lyophilized enzyme powder. The plates were incubated in Thermotron® shakers (3 mm amplitude, model no. AJ185, Infors) at 30°C, 100 rpm, for 4 hours. Reactions were quenched using 200 μl of acetonitrile and mixed in

- 86 043748 for 5 minutes using a table shaker. The plates were then centrifuged at 4000 rpm for 5 min, diluted 20x in water and loaded onto HPLC for analysis. The results are presented in Fig. 3. As shown in this FIG. , the addition of a histidine tag had minimal effect on the enzyme relative to the version without the histidine tag.

Example 17: Improvement of Insulin Acylation at Positions A1 and B29 Compared to SEQ ID No. 40 in a High Throughput Screen

SEQ ID No. 40 was selected as the additional parent enzyme based on the results described in Example 11. Engineered gene libraries were generated using well techniques (eg, saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were prepared in the NTR as described in Example 2, and soluble lysates were created as described in Example 3.

NTR reactions were carried out in 96-well deep well plates, each containing 200 μl containing 0.2 M TRIS, pH 9.25, 20% acetonitrile, 50 g/l insulin, 17 g/l methylphenylacetate and 10 μl NTR supernatant. NTP plates were incubated in Thermotron® shakers (3 mm amplitude, model no. AJ185, Infors) at 30°C, 100 rpm for 2 h. Reactions were quenched using 200 μl of acetonitrile and mixed for 5 min using a tabletop shaker . The plates were then centrifuged at 4000 rpm for 5 min and loaded onto HPLC for analysis.

The percentage conversion relative to SEQ ID No. 40 (FIOP conversion percentage) was calculated as the percentage conversion of the product formed by the variant relative to the percentage conversion provided by SEQ ID No. 40. The results are presented in table. 17.1. The percent conversion was quantified by dividing the peak area of the product by the sum of the peak areas of the substrate, product, and impurities/by-product as observed by HPLC analysis.

The percentage selectivity relative to SEQ ID No. 40 (FIOP selectivity percentage) was calculated as the percentage selectivity of the product formed by the variant relative to the percentage selectivity provided by SEQ ID No. 40. The results are presented in table. 17.1. The percent selectivity was calculated by dividing the peak area of the product by the sum of the peak areas of the product and the impurities/by-product as observed by HPLC analysis.

- 87 043748

Table 17.1. The activity and selectivity of variants acylating at the B29 site ________________ relative to SEQ ID No. 40________________

No. Variation SEQ ID No. (n./a.k.) Amino Acid Differences (relative to SEQ ID No. 40) Acylation conversion percentage (FIOP) ¹ at sites A1 and B29 (relative to SEQ ID NO. 40) Acylation selectivity percentage (FIOP) ² FOR sites A1 and B2 9 (relative to SEQ ID No. 40) 1018 143/1 44 L55I;Q71G;A255P;N444S ; T56 0G; + + + + + + + 1030 L55I;Q7IG;K128W;Y253S;T56 0G; + + + + + + + + 1020 147/1 48 L55I;Q7IG;G74D;N444S; + + + + + + + 1031 L256Y;N457M;G461R; ++++ ++++ 1032 L55I;Y253S;H348D; ++++ ++++ 1019 145/1 46 L55I;Q7IG;K128W;N4 4 4S; ++++ +++ 1033 L55I;Q7IG;G74D; ++++ +++ 1034 L55I;Q71G; ++++ +++ 1035 H348N;K352T;T384P; ++++ ++++ 1036 L55I;H348D; ++++ ++++ 1037 H348N;K352T;T384P; Q559H; ++++ ++++ 1038 L55I;Q7IG;Y253S;A255P; ++++ +++ 1039 L55I;G74D;H348D;Q38 OP;N44 4S;T560G; + + + + + + + 1040 H348N;K352T;P364L;Y616R; ++++ ++++ 1041 L55I;Q7IG;G74D;Y253S;A255 P; + + + + + + + 1042 L55I;Q7IG;G74D;A255P;H348 ++++ +++

- 88 043748

D; 1043 L55I;Q7IG;G74D;A517K; + + + + +++ 1044 L55I;Q7IG;G74D;N444S;F454 W;A517K; + + + + + + + + 1023 153/1 54 L55I;Q7IF;G74D;K390L; + + + ++ 1045 H348N;K352T;T384R;N457M;Y 61 6R; + + + +++ 1046 L55I;Q7IG;G74D;H348D;N444 S;T560G; + + + +++ 1047 Q7IM;G74S;N185D;KZ90A;Q62 6M; + + + + + 1048 L55I;Y253S;Q38OP;T560G; +++ ++ 1022 151/1 52 L55I;Q7IM;G74S;Q38 OP; + + + + + 1021 149/1 50 L55I;Q7IM;Q38 OP;K436G;W61 9F; +++ ++ 1049 H348N;K352T; +++ +++ 1050 K128W;Y253S;F254W;A362V;N 457T;Q556G;L557G; +++ +++ 1051 L55I;Q71F;W619F; +++ ++ 1052 L55I;Q71G;H348D;F701W; +++ +++ 1053 L55I;Q7IM;G74D;N185D; +++ + 1054 L55I;Q7IM;N185D;K436G;W61 9F; + + + + 1055 L55I;Y253S;N444S; +++ ++ 1056 H348N;K352T;T384R; +++ +++ 1057 L55I;Q71F; +++ ++ 552 L55I;H348D;Q38OP; +++ + 1058 T12 9W;H34 8N;KZ52T; +++ +++ 1059 L55I;Q71M;Q380P; +++ + 1060 T129W;G202A;F254K;K352T;R 373K;T384R;N457M; +++ +++ 1061 N185E;KZ69C;N457T;Q556G;L 557G;D709E;K723E; + + + + + 1062 L55I;N444S; +++ + 1063 N185E;G415H;N457T;K723E; +++ + 1064 A67S;Q71F;A255P;L256K;T38 4E; +++ ++++ 1065 Q71F;L256K;N444S;T560G; +++ ++ 1066 L256Y;H348N;T384R; +++ +++ 1067 Q71F;N444K;Y616E; +++ ++ 1068 H546L; + +++ 1069 N440L; + + 1070 D518R; + +++ 1071 A279P; + + 1072 A349E; + + 1073 N440Y; + + 1074 K682A; + + 1075 N333S; + + 1076 K682G; + + 1077 N333A; + +

¹ Levels of increased activity were set relative to the reference polypeptide from SEQ ID No. 40 and were determined as follows: + > 1.2-fold but less than 5.0-fold increased; ++ >

- 89 043748 than 5.0 times, but less than 10.0 times; +++ > than 10.0 times, but less than 20.0 times; ++++ >

than 20.0 times.

² Levels of increased selectivity were set relative to the reference polypeptide of SEQ ID No. 40 and were determined as follows: + > 1.2-fold but less than 5.0-fold increased; ++ > more than 5.0 times, but less than 10.0 times; +++ > than 10.0 times, but less than 20.0 times; ++++ > than 20.0 times.

Example 18: Improvement of Insulin Acylation at Positions A1 and B29 Compared to SEQ ID No. 154 in a High Throughput Screen

SEQ ID No. 154 was selected as the next parent enzyme based on the results described in Example 17. Engineered gene libraries were generated using well techniques (eg, saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were prepared in the NTR as described in Example 2, and soluble lysates were created as described in Example 3.

NTR reactions were carried out in 96-well deep well plates, each containing 200 μl containing 0.2 M TRIS, pH 9.25, 20% acetonitrile, 50 g/l insulin, 17 g/l methylphenylacetate and 10 μl NTR supernatant. NTR plates were incubated in Thermotron® shakers (3 mm amplitude, model no. AJ185, Infors) at 30°C, 100 rpm, for 2 hours. Reactions were quenched using 200 μl of acetonitrile and mixed for 5 minutes using a benchtop shaker. The plates were then centrifuged at 4000 rpm for 5 min and loaded onto HPLC for analysis.

The percentage conversion relative to SEQ ID No. 154 (FIOP conversion percentage) was calculated as the percentage conversion of the product formed by the variant relative to the percentage conversion provided by SEQ ID No. 154. The results are presented in table. 18.1. The percent conversion was quantified by dividing the peak area of the product by the sum of the peak areas of the substrate, product, and impurities/by-product as observed by HPLC analysis.

The percentage selectivity relative to SEQ ID No. 154 (FIOP selectivity percentage) was calculated as the percentage selectivity of the product formed by the variant relative to the percentage selectivity provided by SEQ ID No. 154. The results are presented in table. 18.1. The percent selectivity was calculated by dividing the peak area of the product by the sum of the peak areas of the product and the impurities/by-product as observed by HPLC analysis.

- 90 043748

Table 18.1. Activity and selectivity of variants acylating at the B29 site relative to SEQ ID No. 154

No. varia SEQ ID No. Amino acid differences (relative to SEQ ID no. Conversion percentage Percent voter nta (on the. To. ) 154) acylated (FIOP) ¹ at sites A1 and B29 (relative to SEQ ID No. 154) acylation properties (FIOP) ² FOR sites A1 and B2 9 (relative to SEQ ID No. 154) 1026 159/16 0 N185E;H348D;K352T;L390A; N444S; + + + + + 1027 163/16 4 F71G;F254W;Н348D;ТЗ84Р;L 390A;N444S; + + + + + 1078 Y253S;Н348D;ТЗ84Р;L390А; N444S;N457T; +++ + + 1079 F71G;N185E;Y253S;F254W;H 348D;K352T;TZ84P;L390A;N 444S; 1028 161/16 2 N185E;Y253S;F254W;H348N; K352T;L390A;T560G; + + + + + 1029 165/16 6 F71G;Y253S;H348D;K352T;T 384P;L390A;T560G; + + + + 1080 H348D;K352T;L390A;N444S; +++ + 1081 F254W;H348N;TZ84P;L390A; + + + + + 1082 K352T;L390A;N444S; + + + + 1083 H348D;K352T;L390A; + + + 1084 N185E;F254W;H348N;K352T; T384P;L390A;N444S; + + + 1085 Y253S;F254W;K352T;T384P; L390A; + + + 1086 F254W;T384P;L390A; ++ + 1087 F71G;E165G;N185E;Y253S;F 254W;H348D;K352T;L390A;N 444S; + 1088 F71G;K352T;L390A; + + +

- 91 043748

1089 F71G;TZ84P;L390A;N444S;T 560G; + + + 1090 F71G;N185E;F254W;H348D;K 352T;L390A;N444S; + + + 1091 F71G;F254W;H348N;K352T;T 384P;L390A;N444S; + + + 1092 G202A;F254W;H348D;T384P; L390A;N444S; + + + 1093 Y253S;H348D;T384P;L390A; N444S; + + + 1094 F71G;A466M; ++ + 1095 F7IG;Y253S;K352T;T384P;L 390A;T560G; + + + 1096 H348D;L390A;N444S; ++ + 1097 F7IG;H348N;T384P;L390A;T 560G; + + + 1098 F254W;K352T;L390A; ++ + 1099 Y253S;F254W;T384P;L390A; + + + 1100 L390A;N444S; + + + 1024 155/15 6 D7 4S;A3 62V;Q55 6G; ++ + 1101 F71G;Y253S;F254W;H348N;K 352T;L390A; + + + 1102 D74S;A362V; + + + 1103 D74S;A3 62V;T384R;L557G; + + + 1104 F7IM;S251C;A279P;N333A;N 444S; + + + 1105 H348D;K352T;A3 62V;N444S; K723E; + + + 1106 F71G;F254W;H348D;K352T;T 384P;L390A;N444S; + + + 1107 A279P;N44 OY;N444S; ++ + 1108 F7IG;A250S;A279P;N44OL;N 444S;A466M;A642E; ++ + 1109 F254W;H348N;L390A; + + +

- 92 043748

1110 D74S;AZ 62V;N444K; + + + 1025 157/15 8 D74G;N185E;K352T;Q380P;K 436G;Y616R; + + + 1111 D74S; + + + 1112 D7 4S;AZ 62V;N4 4 4S;Q55 6G; + + + 1113 K352T;T384P;L390A;T560G; + + + 1114 F7IM;D74S;AZ 62V;L390A;G4 15H;N444K; + + + 1115 F71G;Н348D;ТЗ84Р;L390А; + + 1116 D74G;H348D;Q380P;K436G;K 723E; + + 1117 F71G;P164S;Y253S;H348D;T 384P;L390A;N444S;I637M; + + 1118 F71G;D74S;L390A;G415Н;W6 19F; + + 1119 N185E;TZ84P;L390A;N472P; + + 1120 F254W;K352T;AZ 62V;K723D; + + 1121 D74G;H348D;T560G;K72ZE; + + 1122 A67S;Y253S;F254W;H348D;K 352Т;G415Н;К723D; + + 1123 F71G;Н348D;К352Т;ТЗ84Р;L 390A;N444S; + + 1124 F71G;H348D;L390A; + + 1125 F71G;N185E;H348N;L390A; + + 1126 N185D;H348D;K352T;K436G; T560G;K723D; + + 1127 F71G;F254W;T384P;L390A;N 444S;T560G; + + 1128 D74S;A3 62V;T384P; + + 1129 A279P;N333A;A466M; + + ISO F71G;Y253S;K352T;L390A; + + 1131 D74S;L557G; + +

¹ Levels of increased activity were set relative to the reference polypeptide from SEQ ID No. 40 and were determined as follows: + > 1.2-fold but less than 2.0-fold increased; ++ > more than 2.0 times, but less than 3.0 times; +++ > than 3.0 times, but less than 5.0 times; ++++ > than 5.0 times.

Levels of increased selectivity were set relative to the reference polypeptide of SEQ ID No. 40 and were determined as follows: + > 1.2-fold but less than 2.0-fold increased; ++ > more than 2.0 times, but less than 3.0 times; +++ > than 3.0 times, but less than 5.0 times; ++++ > than 5.0 times.

Example 19: Improvement of Insulin Acylation at Positions A1 and B1 Compared to SEQ ID No. 12 in a High Throughput Screen

SEQ ID No. 12 was selected as the additional parent enzyme based on the results described in Example 7. Engineered gene libraries were generated using well techniques (eg, saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were prepared in the NTR as described in Example 2, and soluble lysates were created as described in Example 3.

NTR reactions were carried out in 96-well deep well plates containing 200 μl of 0.1 M TRIS, pH 9.25, 20% acetonitrile, 10 g/l insulin, 17 g/l methylphenylacetate and 10 μl of NTR supernatant. NTR plates were incubated in Thermotron® shakers (amplitude 3 mm, model no. AJ185, Infors) at 30°C, 100 rpm for 5 hours. Reactions were quenched using 200 μl of acetonitrile or dimethylacetamide and mixed for 5 min using table shaker. The plates were then centrifuged at 4000 rpm for 5 min and loaded onto HPLC for analysis.

The percent conversion relative to SEQ ID No. 12 (FIOP conversion percent) was calculated as the percent conversion of the product formed by the variant relative to the percent conversion provided by SEQ ID No. 12. The results are presented in Table 1. 19.1. The percent conversion was quantified by dividing the peak area of the product by the sum of the peak areas of the substrate, product, and impurities/by-product as observed by HPLC analysis.

Table 19.1. Activity and selectivity of variants acylating at the B29 site relative to SEQ ID No. 12

No. Variation SEQ ID No. (n./ A. To. ) Amino acid differences (relative to SEQ ID No. 12) Percentage of ion converted by acylation (FIOP) ¹ in regions A1 and B1 (relative to SEQ ID No. 12) Acylation selectivity percentage (FIOP) ¹ FOR sites A1 and B1 (relative to SEQ ID No. 12) 106 65/6 6 Y27T;D7 4N;L253V;F254W;W370I; D381K; + + + + + + + 141 67/6 8 Y27T;F254W;A255G;W370I; + + + + + + + 117 Y27T;D7 4G;L253V;F254W;A255G; W3701;D38 IK;TZ84P; ++++ +++ 120 Y27T;D7 4N;L253V;F254W;N348R; W370I;D381F; + + + + + + + 1132 Y27T;L253V;F254W;N348R;W370I +++ + + 157 Y27T;L253V;F254W;N348R;W370I ;T384P; + + + + + 1133 Y27T;D7 4N;L253V;F254W;A255G; N348R;W370I;D381W;T384P; +++ + + 86 Y27T;D7 4N;L253V;F254W;N348R; W3701;D38IK;T384P; + + + + + 137 Y27T;D74S;F254W;A255G;W370I; +++ ++ 170 Y27T;D74P;L253V;F254W;N348R; W370I;D381W;T384P; +++ ++

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91 Y27T;D74G;L253V;F254W;A255G; W370I; + + + + + 155 Y27T;L253V;A255G;W370I;D381F ;T384P; + + + + + 1134 Y27T;L253V;F254S;A255G;N348R ;W370I; +++ + + 258 Y27T;L253V;F254W;A255G;N348R ;W370I; + + + + + 1135 Y27T;L253V;F254W;A255G;N348R ;W370I; +++ + + 126 Y27T;D74G;F254W;A255G;N348R; W370I; + + + + + 143 Y27T;F254W;A255G;N348R;W370I +++ + + 124 Y27T;L253V;F254W;D381F;T384P +++ + + 240 Y27T;L253V;F254W; +++ ++ 171 Y27T;L253V;F254L;A255G;N348R ;W370I;T384P; +++ + + 275 Y27T;D74G;L253V;F254W; +++ ++ 84 Y27T;D74G;L253V;F254W; +++ ++ 96 Y27T;L253V;A255G;W370I; ++ ++ 115 Y27T;D74P;L253V;W370I; + + ++ 136 Y27T;D74N;L253V;F254W;A255G; N348R;W370I;T384P; + + + + 153 Y27T;F254W;A255G;N348R;W370I ;T384P; + + + + 102 Y27T;D74N;L253V;N348R;W370I; D381R;T384P; ++ + + 95 Y27T;D74S;L253V;N348R;W370I; D381K; + + + + 1136 Y27T;D74G;F254W;W370I; ++ ++ 122 Y27T;D74G;L253V;F254W;A255G; D381K; ++ + + 145 Y27T;D74G;L253V;D381F;T384P; + + ++ 116 Y27T;L253V;N348R;W370I;T384P + + + + 154 Y27T;D74G;L253V;F254W;N348R; ++ ++ 1137 Y27T;L253V;N348R;W370I;T384P + + + + 152 Y27T;D74G;L253V;N348R;W370I; ++ ++ 162 Y27T;D74P;L253V;F254W;N348R; D381F;T384P; + + + + 237 Y27T;D74G;L253V;N348R;W370I; ++ ++ 113 Y27T;D74G;A255G;W370I; ++ ++ 1138 Y27T;F254W;A255G;N348R;W370I ;D381F;T384P; + + + 208 Y27T;F254W;A255G;N348R;W370I ;D381F;T384P; + + + 643 119/ 120 K128W;A255P;Q380P;G444K;N457 T;A517K;T560G; + +

¹ Levels of increased activity or selectivity were determined relative to the reference polypeptide of SEQ ID No. 12 and were defined as follows: “+” > 2.0-fold but less than 5-fold increased; ++ > more than 5 times, but less than 10 times; +++ > than 10 times, but less than 15 times: ”++++” > than 15 times.

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Example 20: Improvement of Insulin Acylation at Position B29 in the Presence of Phenylacetic Acid Compared to High Throughput SEQ ID No. 56

SEQ ID No. 56 was selected as the additional parent enzyme based on the results described in Example 12. Engineered gene libraries were generated using well techniques (eg, saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were prepared in the NTP as described in Example 2, and soluble lysates were created as described in Example 3, but using 400 μl of lysis buffer instead of 200 μl.

HTR reactions were carried out in 96-well plates with deep wells. Each reaction well contained 200 μl of 0.2 M TRIS, 10% acetonitrile, 50 g/l insulin, 17 g/l methylphenylacetate, 12.5 or 15 g/l phenylacetic acid and 10 μl of HTR supernatant (initial pH before addition of lysate was 9.4). NTP plates were incubated in Thermotron® shakers (3 mm amplitude, model no. AJ185, Infors) at 30°C, 100 rpm for 3 h. Reactions were quenched using 200 μl of acetonitrile and mixed for 5 min using a tabletop shaker . Then, 400 μL of water was added and mixed for 5 min using a table shaker. Then the plates were centrifuged at 4000 rpm. /min for 5 min and diluted another 2-fold in water before loading into HPLC for analysis.

The percentage conversion relative to SEQ ID No. 56 (FIOP conversion percentage) was calculated as the percentage conversion of the product formed by the variant relative to the percentage conversion provided by SEQ ID No. 56. The results are presented in table. 20.1. The percent conversion was quantified by dividing the peak area of the product by the sum of the peak areas of the substrate, product, and impurities/by-product as observed by HPLC analysis.

Table 20.1. The activity and selectivity of variants acylating at the B29 site are __________________ relative to SEQ ID No. 56__________________

No. option a SEQ ID No. (n./a. To. ) Differences amino acids (relative to SEQ ID No. 56) Acylation Conversion Percentage (FIOP) ¹ at B29 (relative to SEQ ID No. 56) 615 T27S; + + 611 97/98 F57H; + 614 89/90 A67S; + 616 Al 6OS; + 612 99/100 F57C; + 617 F57V; + 618 S704A; + 619 R373I; + 620 T379S; +

¹ Levels of increased activity were set relative to the reference polypeptide from SEQ ID No. 56 and were determined as follows: + > 1.2-fold but less than 2.0-fold increased; ++ > than 2.0 times.

Example 21. Analytical detection of insulin and its acylated products.

The data described in examples 5-18 were collected using the analytical methods in table. 21.1, 21.2, 21.3, 21.4 and 21.5. All methods provided in the present description find use in the analysis of variants obtained using the present invention. However, it is not intended that the methods described herein are exclusive methods as applied to the analysis of the variants provided herein and/or obtained using the methods provided herein. The results presented in Fig. 1 correspond to the elution order of the compounds for these methods.

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Table. 21.1. Analytical method

Device Agilent HPLC 1200 Series Column Ascentis Express C18, 4.6 x 100 or 150 mm, 2.7 µM Mobile phase Gradient I (A: 0.05% TEA in water; B: 0.05% TEA in MeCN) Time min) %A 0, 0 95 0, 1 70 8, 8.5 or 9 50 8.1, 8, 6 or 9.1 5 8.2, 8, 7, or 9.2 95 9, 9, 2 or 9.5 95 Time ( 0 7 or 8 7, 1 or 9 or 1 Flow rate Transit time Product elution order Column temperature Gradient II ((A: 0.05% TEA in water; B: 0.05% TEA in MeCN) min) %A 70 50 8.1 70 0 70 1.0 ml/min ~10 min Insulin; Al-acylated insulin; B2 9- acylated insulin; Bl-acylated insulin; di-Al,B29-acylated insulin; di-A1,Bl- acylated insulin; di-B1,B29-acylated insulin; tri-A1, Bl, B29-acylated insulin 40°C Injection volume 5 µl Detection UV 218 nm and 280 nm Detector: MWD (Agilent 1200 series) Gap=4 nm

Table 21.2. Analytical method

Device Agilent HPLC 1290 Series Column Waters Cortecs UPLC C18 2.1 x 50 mm, 1.6 µM Mobile phase Gradient MeCN) for (A: 0.05% samples TEA in water; IN: 0.05 %TEA in Time ( min) %A 0, 0 72 2.5 50 2.51-2, 7 10 2.71 72 3 72 Gradient MeCN) for (A: 0.05% TEA rinsing in water; IN: 0.05 %TEA in Time ( min) Isa

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0, 0 72 1.7 0 1.71-2 72 Flow rate 0.9 ml/min Time passing 3 min Product elution order Insulin; A1-acylated insulin; B2 9acylated insulin; B1-acylated insulin; di-Al,B29-acylated insulin; di-A1,Blacylated insulin; di-B1,B29-acylated insulin; tri-A1, Bl, B29-acylated insulin Column temperature 40°C Injection volume 0.5 µl Detection UV 218 nm and 280 nm Detector: MWD (Agilent 1290 series); gap=4 nm; peak width > 0.1 min; Standard=360; BW=8

Table 21.3. Analytical method

Device Agilent HPLC 1200 Series Column Ascentis Express C18, 4.6 x 100 or 150 mm, 2.7 µM Mobile phase Flow rate 1.0 ml/min Time passing ~10 min Product elution order Insulin; Al-acylated insulin; B2 9acylated insulin; Bl-acylated insulin; di-A1,B29-acylated insulin; di-A1,Blacylated insulin; di-B1,B29-acylated insulin; tri-A1,Bl,B29-acylated insulin Temperature 40°C speakers Injection volume 5 µl Detection UV 218 nm and 280 nm Detector: MWD (Agilent 1200 series); gap=4 nm; peak width > 0.1 min; Standard=360; BW=8

Table 21.4. Analytical method

Device Agilent HPLC 1290 Series Column Waters Cortecs UPLC C18 2.1 χ 50 mm, 1.6 µM Mobile phase Flow rate 0.9 ml/min Time passing 3 min Product elution order Insulin; Al-acylated insulin; B2 9acylated insulin; Bl-acylated insulin; di-A1,B29-acylated insulin; di-A1,Blacylated insulin; di-B1,B29-acylated insulin; tri-A1, Bl, B29-acylated insulin Temperature speakers 40°C Injection volume 0.5 µl Detection UV 218 nm and 280 Detector: MWD peak width > 0 nm (Agilent, 1 min; series 1290); gap=4 nm; standard=360; BW=8

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Table 21.5. Analytical method

Device An Agilent UPLC 1290 system equipped with a four-component pump and DAD UV detector, or a Thermo Vanquish UPLC system equipped with a DAD UV detector. Column Column Waters Cortecs UPLC C18 (50 χ 2.1 mm, 1.6μ) Mobile phase Flow rate 1.0 ml/min Time passing 3.25 min Product elution order Phenylacetic acid, 0.32 minutes; insulin, 0.78 minutes; A1-monoacylinsulin, 0.91 minutes; B29-monoacylinsulin, 0.95 minutes; Blmono acylinsulin, 1.02 minutes; A1,B29 diacylinsulin, 1.09 minutes; A1, B1-diacillinsulin, 1.14 minutes; Bl,B29-diacylinsulin, 1.21 minutes; Al, Bl, B29-triacylinsulin, 1.3 8 minutes Column temperature 40°C Injection volume 1.0 µl Detection UV detection (λ=218 nm, slit=4 nm, peak width >0.1 min, reference=360 nm)

Example 22: Improvement of Insulin Acylation at A1 and B2 Positions 9 Compared to SEQ ID No. 160 in a High Throughput Screen

SEQ ID No. 160 was selected as the additional parent enzyme based on the results described in Example 18. Engineered gene libraries were generated using well techniques (eg, saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were prepared in the NTR as described in Example 2, and soluble lysates were created as described in Example 3.

NTR reactions were carried out in 96-well deep well plates, each containing 200 μl, consisting of 0.5 M TRIS, pH 10.0, 20% acetonitrile, 50 g/l insulin, 17 g/l methylphenylacetate and 5-10 μl NTR of the supernatant. NTR plates were incubated in Thermotron® shakers (3 mm amplitude, model no. AJ185, Infors) at 30°C, 100 rpm, for 2 hours. Reactions were quenched using 200 μl of acetonitrile and mixed for 5 minutes using a benchtop shaker. The plates were then centrifuged at 4000 rpm for 5 min and loaded onto HPLC for analysis.

The percentage conversion relative to SEQ ID No. 160 (FIOP conversion percentage) was calculated as the percentage conversion of the product formed by the variant relative to the percentage conversion provided by SEQ ID No. 160. The results are presented in table. 22.1. The percent conversion was quantified by dividing the peak area of the product by the sum of the peak areas of the substrate, product, and impurities/by-product as observed by HPLC analysis.

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Table 22.1. Activity and selectivity of variants acylating at sites A1 and B29 relative to SEQ ID No. 160

No. option SEQ ID No. (on the. To. ) Amino Acid Differences (relative to SEQ ID No. 160) Acylation conversion percentage (FIOP) ¹ at sites A1 and B2 9 (relative to SEQ ID No. 160) Acylation selectivity percentage (FIOP) ² for sites A1 and B29 (relative to SEQ ID No. 160) 1139 S251G; + 1140 N44S; + + 1141 N44R; + 1142 N4 4M; + + 1143 F254H; + 1144 N44Q; + + 1145 S251A; + + 1146 R317A; + + 1147 D348H; + + 1148 F254V; + + 1149 F254L; + + 115 S444A; + + 1151 I55V; + 1152 S444K; + 1153 S444R; + + 1154 N44D; + + 1155 N4 4E; + + 1156 R317W; + + 1157 N44T; + + 1158 F254C; + + 1159 S444L; + + 116 N4 4H; + + 1161 N44I; + + 1162 A3 9T; + + 1163 F254T; + + 1164 F254G; + + 1165 167/16 8 K172R; D484T + + 1166 F254Y; + + 1167 S444T; + + 1168 F254S; ++ + 1169 169/17 S251T; ++ + 117 171/17 2 A466M;A47T;K723 E; + + + 1171 173/17 4 El 85D;A279P;Q38 P;A466M; + + + 1172 175/17 6 F254W; + + + + + 1173 177/17 8 F2 54A; + + + + + 1174 179/18 A3 9S; +++ ++

¹ Levels of increased activity were set relative to the reference polypeptide from SEQ ID No. 160 and were determined as follows: + > 1.2-fold but less than 2.0-fold increased; ++ > more than 2.0 times, but less than 3.0 times; +++ > than 3.0 times, but less than 5.0 times; ++++ > than 5.0 times.

² Levels of increased selectivity were set relative to the reference polypeptide of SEQ ID No. 160 and were determined as follows: + > 1.2-fold but less than 2.0-fold increased; ++ > more than 2.0 times, but less than 3.0 times; +++ > than 3.0 times, but less than 5.0 times; ++++ > than 5.0 times.

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Example 23: Improvement of Insulin Acylation at Position B29 Compared to SEQ ID No. 100 in a High Throughput Screen

SEQ ID No. 100 was selected as the next parent enzyme based on the results described in Example 13. Engineered gene libraries were generated using well techniques (eg, saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were prepared in the NTP as described in Example 2, and soluble lysates were created as described in Example 3, but using 400 μl of lysis buffer instead of 200 μl.

HTR reactions were carried out in 96-well plates with deep wells. Each reaction well contained 200 μl of 0.5 M TRIS, 20% acetonitrile, 50 g/l insulin, 17 g/l methylphenylacetate and 40 μl of HTR supernatant (initial pH before addition of lysate=10). NTP plates were incubated in Thermotron® shakers (amplitude 3 mm, model no. AJ185, Infors) at 30°C, 100 rpm, for 5 hours. Reactions were quenched using 200 μl of acetonitrile and mixed for 5 minutes using a benchtop shaker. 400 μl of water was added and the plates were mixed again for 5 min using a table shaker. The plates were then centrifuged at 4000 rpm for 5 minutes and diluted an additional 2-fold in water before loading into HPLC for analysis.

The percent conversion relative to SEQ ID No. 100 (FIOP conversion percent) was calculated as the percent conversion of the product formed by the variant relative to the percent conversion provided by SEQ ID No. 100 and is shown below in the table. The percent conversion was quantified by dividing the peak area of the product by the sum of the peak areas of the substrate, product, and impurities/by-product as observed by HPLC analysis.

Table 23.1. The activity and selectivity of variants acylating at site B29 are _____________ relative to SEQ ID No. 100________________

No. option SEQ ID No. (on the. To. ) Amino Acid Differences (relative to SEQ ID No. 100) Acylation Conversion Percentage (FIOP) ¹ at B29 (relative to SEQ ID NO: 100) 1175 195/196 N444S;N457T;A466M;D709E; + + 1176 201/202 N333A;AZ 62V;N457T;Y616T; + + 1177 193/194 A255G;K390Q;Y616T;S675E; + + 1178 199/200 N333A;N444S;N457T;Y616T;D 709E; + + 1179 A255G;L557G;Y616T; + + 1180 181/182 A255G;L557G;E561P;Y616T;S 67 5E; + + 1181 F254H;A255G;L557G;Y616T; + + 1182 A255G;R373N;Y616T; + + 1183 R373N;L557G;E561P;Y616T;S 67 5E; + + 1184 Al60T;A255G;L557G;Y616T; + + 1185 L557G;Y616T;S675E; + + 1186 Al60T;A255G;KZ90Q;L557G;S 67 5E; + + 1187 K390Q;Y616T; + + 1188 C57H;A255G;KZ69D;KZ90Q; + + 1189 N444S;N457T;D709E; + + 1190 L557G;Y616T; + 1191 K390Q;L557G; + 1192 N333A;A362V;T384E;Y616T; + 1193 F254H;A255G;L557G;S675E; + 1194 C57H;A255G;L557G;S675E; + 1195 QI57S;R316H;A3 62V;KZ90A; N 444S;T560G; + 1196 A2 8 V;P2 4 9A;T5 6 0G;D7 0 9E; + 1197 K390Q; +

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1198 C57H;A255G;L557G;E561P;S6 7 5E; + 1199 Al 60T;L557G;Y616T; + 1200 L256Y;N333A;N444S;N457T;T 560G; + 1201 A255G;R373N; + 1202 Y616T; + 1203 R373N;E561P; + 1204 KZ69D;L557G;E561P; + 1205 L557G;S675E; + 1206 C57H;A255G;L557G; + 1207 А160Т;F254H;K390Q;L557G;Y 61 6Т; + 1208 A255G;KZ69D;R373Ν;S675E; + 1209 V56L;A255G;Y616T; + 1210 N333A;N444S;T560G; + 1211 C57H;Y616T; + 1212 F254H;A255G;KZ69D;KZ90Q; + 1213 L557G; + 1214 KZ69D;E561P;S675E; + 1215 C57H;A255G; + 1216 N333A;N444S;T560G;D709E; + 1217 V56L;L557G;Y616T; + 1218 V5 6L;C57H;A255G;L557G;E56 1P; + 1219 L256Y;N333A;Y616T;D709E; +

¹ Levels of increased activity were established relative to the reference polypeptide from SEQ ID No. 100, which showed a conversion of 33.9 ± 1.4%. Efficiency improvements were defined as: + > greater than 1.2 times but less than 2.0 times increased; ++ > than 2.

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Table 23.2

Activity and selectivity of variants acylating at the B29 site relative to SEQ ID No. 100

No. option SEQ ID No. (n./a.k.) Amino acid differences (relative to SEQ ID No. 100) Conversion percentage acylation of m (FIOP) ¹ at site B29 (relative to SEQ ID No. 100) 1220 189/190 A28T;L557V;K723D; + 1221 187/188 С57Н;A255G;L557G;Y616T;S675 E; + 1222 197/198 AZ 62V;N444S;N457T; + 1223 185/186 C57H;A255G;Y616T;S675E; + 1224 183/184 С57Н;F254H;A255G;L557G;Y616 T; + 1225 191/192 A28T;C57V;K723D; + 1226 E561P;Y616T; + 1227 A255G;S675E; + 1228 N333A;H348N;AZ 62V;N444S;T56 0G;Y616T; + 1229 V56L;C57H;F254H;A255G;R373N ;Y616T;S675E; + 1230 F254H;A255G;L557G;S675E; + 1231 Y616T;S675E; + 1232 V56L;C57H;E561P;Y616T;S675E + 1233 N333A;A3 62 V;N444S;D709E; + 1234 A28T;Q112D; + 1235 L557V;W619S;K723D; + 1236 L557G;Y616T; + 1237 C57H;F254H;A255G;S675E; + 1238 C57V;QI12D;QI57S;Al 60C;L557 V;L623T;K723D; + 1239 C57H;L557G;Y616T; + 1240 A2 8T;C57V;L557V; + 1241 A362V;N444S;D709E; + 1242 Y616T; +

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Claims (6)

1243 L557V;W619S;L62ЗТ; + 1244 Q112D;A160C;W619S; + 1245 A255G; + 1246 P249A;AZ 62V;N444S;A466M;D70 9E; + 1247 D484T;S704A; + 1248 F254Н;КЗ90Q;Y616Т; + 1249 QI12D;QI57S;L557V;W619S;L62 ST; + 1250 D484Т;L557V;L62ЗТ; + 1251 QI12D;QI57S;L62ЗТ; + 1252 С57Н;Al60Т;А255G;Y616T;S675 E; + 1253 L557V;L62ЗТ;К723D; + 1254 C57H;A255G;L557G; + 1255 L623T; + 1256 D484T;L623T; + 1257 QI12D;Al60С;D484Т;L557V;W61 9S;L623T;K723D; + 1258 AZ 62 V; + 1259 C57H;F254H;A255G; + 1260 С57Н;R373N;D484Т;L557G;S675 E; + 1261 C57H;R373N;L557G;S675E; + 1262 C57H;A255G;R373N; + 1263 P249A;R316H;A362V;N444S;A46 6M;Y616T; + 1264 L557V;K723D; + ¹ Levels of increased activity were established relative to the reference polypeptide from SEQ ID NO: 100, which showed a conversion of 52.1 ± 2.2%. Efficiency improvements were defined as: + > greater than 1.2 times but less than 2.0 times increased; ++ > than 2. All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated for inclusion by reference for all purposes. While specific embodiments have been illustrated and described, it should be appreciated that various modifications can be made without departing from the spirit and scope of the invention(s). CLAIM 1. An engineered penicillin G acylase capable of acylating insulin at position B29, wherein the polypeptide sequence of said penicillin G acylase is at least 97, 98, 99% or more identical to SEQ ID No. 100 and wherein the engineered penicillin G acylase has an enzymatic activity of at least 1.5 times the enzymatic activity of the engineered penicillin G acylase of SEQ ID NO: 4. 2. The engineered penicillin G acylase according to claim 1, wherein said penicillin G acylase contains SEQ ID NO: 100, 24, 40, 56, 82, 108, 110, 154 or 160. 3. The engineered penicillin G acylase of claim 1 or 2, wherein said penicillin G acylase has an enzymatic activity that is 2, 5, 10, 20, 25, 50, 75, 100 or more times the enzymatic activity of the engineered penicillin G acylase of SEQ ID No. 4. 4. The engineered penicillin G acylase according to any one of claims 1 to 3, wherein said engineered penicillin G acylase contains a histidine tag. 5. The engineered penicillin G acylase of claim 4, wherein said histidine tag is present at the C-terminus of said engineered penicillin G acylase, optionally wherein said penicillin G acylase comprises a polypeptide sequence selected from SEQ ID NO: 100 and SEQ ID NO: 110. 6. An engineered nucleic acid molecule encoding an engineered penicillin G acylase according to any one of claims 1 to 5. - 104 -