Polypeptides having deamidase inhibitor activity

Abstract

The present invention relates to polypeptides having deamidase inhibitor activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells containing the polynucleotides, as well as methods of producing and using the polypeptides.

Description

[Technical Field]

[0001] Sequence Listing Reference This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

[0002] The present invention relates to polypeptides having deamidase inhibitor activity, polynucleotides encoding the polypeptides, nucleic acid constructs, vectors, and host cells comprising the polynucleotides, and methods of making and using the polypeptides. [Background technology]

[0003] Deamidase enzymes are produced by microbial cells in an inactive proform, which contains a propeptide domain tightly linked to a deamidase domain. The proform has little deamidase activity to protect host cell viability. By nature, the proform is post-processed to remove the propeptide and release the active deamidase outside the host cell. However, in recombinant expression systems, the propeptide is not naturally removed, and the inactive deamidase proform is secreted outside the host cell.

[0004] The propeptide domain cannot be separated from the deamidase domain by simple cleavage due to the high binding affinity of the propeptide for the deamidase polypeptide. To facilitate separation of the propeptide and active deamidase, binding affinity can be engineered by mutagenesis, but this results in a partially activated intracellular deamidase, reducing host cell viability.

[0005] It is an object of the present invention to provide novel deamidase inhibitors that can be co-expressed with deamidases to improve recombinant expression of the deamidase by mitigating the negative effects of active intracellular deamidase on host cell viability. Summary of the Invention [Means for solving the problem]

[0006] The present invention provides polypeptides that can reversibly bind to deamidases and inhibit deamidase activity by interacting with the deamidase active site.

[0007] Thus, in a first aspect, the present invention provides a method for producing a medicament for the treatment of a medicament comprising: (a) a polypeptide having at least 60% amino acid sequence identity to SEQ ID NO:2; (b) a polypeptide derived from SEQ ID NO: 2 by having 1 to 30 modifications (e.g., substitutions, deletions, and / or insertions), for example 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23, or 24, or 25, or 26, or 27, or 28, or 29, or 30 modifications, particularly substitutions, at one or more positions; (c) a polypeptide derived from the polypeptide of (a) or (b), wherein the N-terminus and / or C-terminus are extended by the addition of one or more amino acids; and (d) a fragment of the polypeptide of (a), (b), or (c). The present invention relates to a polypeptide having deamidase inhibitor activity selected from the group consisting of:

[0008] In further aspects, the invention relates to polynucleotides encoding the polypeptides of the invention, nucleic acid constructs comprising the polynucleotides, recombinant expression vectors, recombinant host cells, and methods for producing the polypeptides.

[0009] Other aspects and embodiments of the invention will become apparent from the description and examples.

[0010] array SEQ ID NO: 1: Polynucleotide encoding a deamidase inhibitor from Chryseobacterium sp.-62563. SEQ ID NO:2: Amino acid sequence of the deamidase inhibitor encoded by SEQ ID NO:1. SEQ ID NO: 3: Polynucleotide encoding an active deamidase from Chryseobacterium sp.-62563. SEQ ID NO: 4: Amino acid sequence of the deamidase encoded by SEQ ID NO: 3. SEQ ID NO: 5: Amino acid sequence motif of deamidase inhibitor. SEQ ID NO: 6: Amino acid sequence motif of deamidase inhibitor. SEQ ID NO: 7: Amino acid sequence motif of deamidase inhibitor. SEQ ID NO: 8: Amino acid sequence motif of deamidase inhibitor. SEQ ID NO: 9: Amino acid sequence motif of deamidase inhibitor. SEQ ID NO: 10: Amino acid sequence motif of deamidase inhibitor. SEQ ID NO: 11: Amino acid sequence motif of deamidase inhibitor. SEQ ID NO: 12: Amino acid sequence motif of deamidase inhibitor. SEQ ID NO: 13: Amino acid sequence motif of deamidase inhibitor. SEQ ID NO: 14: Amino acid sequence motif of the deamidase active site. SEQ ID NO: 15: Amino acid sequence motif of the deamidase active site. SEQ ID NO: 16: Amino acid sequence motif of the deamidase active site.

[0011] definition In accordance with this detailed description, the following definitions apply: Note that the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise.

[0012] Unless otherwise defined or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0013] Deamidase activity: The term "deamidase activity" refers to protein-glutamine glutaminase (also known as glutaminylpeptide glutaminase) activity as described in EC 3.5.1.44, which catalyzes the hydrolysis of gamma-amides of glutamine substituted at the carboxyl position or at both the alpha-amino and carboxyl positions (e.g., L-glutaminylglycine and L-phenylalanyl-L-glutaminylglycine). Polypeptides having deamidase activity are also commonly referred to as deamidases. Thus, deamidases can deamidate glutamine residues in proteins to glutamic acid residues and are also referred to as protein glutamine deamidases. The deamidase comprises a Cys-His-Asp catalytic triad (e.g., Cys-156, His-197, and Asp-217 as shown in Hashizume et al., "Crystal structures of protein glutaminase and its pro forms converted into enzyme-substrate complex," Journal of Biological Chemistry, vol. 286, no. 44, pp. 38691-38702) and belongs to InterPro entry IPR041325. In a preferred embodiment, the deamidase of the present invention belongs to the PFAM domain PF18626. The deamidase amino acid sequence may include the amino acid sequence motif DGCYARAH (SEQ ID NO: 14), which corresponds to amino acid residues 40-47 of SEQ ID NO: 4, and / or the amino acid sequence motif CYARAH[R / K / Q] (SEQ ID NO: 15), which corresponds to amino acid residues 42-48 of SEQ ID NO: 4, and / or the amino acid sequence motif HVA[L / V / I]LVS (SEQ ID NO: 16), which corresponds to amino acid residues 83-89 of SEQ ID NO: 4. These motifs overlap the deamidase active site. Preferably, the deamidase activity is that exhibited by the polypeptide set forth as SEQ ID NO: 4.

[0014] Deamidase activity was measured by using a fluorescent substrate containing a glutamine residue and a fluorescence quenching group as described in Example 1. The glutamine residue is converted to a glutamic acid residue by the deamidase activity, and the substrate is then cleaved by glutamyl endopeptidase to remove the fluorescence quenching group.

[0015] Deamidase activity may also be measured by deamidating a glutamine substrate (e.g., Cbz-Gln-Gly), generating ammonia in the process. Ammonia, in combination with α-ketoglutarate, is used as a substrate for glutamate dehydrogenase to generate glutamate. This latter enzymatic reaction requires NADH as a coenzyme. NADH depletion can be followed by kinetic absorbance measurements at 340 nm and is directly proportional to deamidase activity. The reaction is carried out at pH 7 and 37°C.

[0016] Deamidase inhibitor: The term "deamidase inhibitor" refers to a sequence of amino acids that interact with amino acid residues in a deamidase active site. Thus, deamidase inhibitor activity reduces or inhibits deamidase activity, preferably the deamidase activity exhibited by the polypeptide set forth as SEQ ID NO: 4. For example, in the presence of a deamidase inhibitor, the deamidase activity can be reduced to less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, or less than 40% (compared to the deamidase activity in the absence of the deamidase inhibitor). Polypeptides having deamidase inhibitor activity are also commonly referred to as deamidase inhibitors. Deamidase inhibitors can comprise an amino acid sequence motif selected from the group consisting of F[F / Y][I / L / V][F / Q / S][E / K / R], L[I,T]WY[D,H,K,N], G[I,M]S[A,P,Q]Q, [D,H,K,N,S][I,L][G,V][I,V][D,E], [N,H][I,L,M,V,Q][I,V][K,R,Q][E,I,Q], [D / N][P / S][D / E][H / K / N / Q / R][A / P / S], and combinations thereof. Deamidase inhibitors can also comprise an amino acid sequence motif selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and combinations thereof.

[0017] cDNA: The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from a spliced mature mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before emerging as a spliced mature mRNA.

[0018] Coding sequence: The term "coding sequence" refers to a polynucleotide that directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0019] Control sequence: The term "control sequence" refers to a nucleic acid sequence involved in regulating the expression of a polynucleotide in a particular organism or in vitro. Each control sequence may be native (i.e., derived from the same gene) or heterologous (i.e., derived from different genes) to the polynucleotide encoding a polypeptide, and may be native or heterologous to each other. Such control sequences include, but are not limited to, leader, polyadenylation, prepropeptide, propeptide, signal peptide, promoter, terminator, enhancer, and transcriptional or translational initiator and terminator sequences. At a minimum, control sequences include a promoter and transcriptional and translational stop signals. Control sequences may be provided with linkers to introduce specific restriction sites facilitating ligation of the control sequences with the coding region of a polynucleotide encoding a polypeptide.

[0020] Expression: The term "expression" refers to any step involved in producing a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0021] Expression vector: An "expression vector" refers to a linear or circular DNA construct containing a DNA sequence encoding a polypeptide, wherein the coding sequence is operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, and enhancers and sequences that control the termination of transcription and translation.

[0022] Extension: The term "extension" refers to the addition of one or more amino acids to the amino and / or carboxyl terminus of a polypeptide, wherein the "extended" polypeptide has deamidase inhibitor activity.

[0023] Fragment: The term "fragment" refers to a polypeptide having one or more amino acids not present at the amino and / or carboxyl terminus of the mature polypeptide, which fragment has deamidase inhibitor activity.

[0024] Fusion polypeptide: The term "fusion polypeptide" refers to a polypeptide in which one polypeptide of the present invention is fused at the N-terminus and / or C-terminus of another polypeptide of the present invention. A fusion polypeptide is produced by fusing two or more polynucleotides that together encode a polypeptide of the present invention. Techniques for producing fusion polypeptides are known in the art and include ligating coding sequences encoding the polypeptides so that they are in frame and expression of the fusion polypeptide is under the control of the same promoter and terminator. Fusion polypeptides can also be constructed using intein technology, in which the fusion polypeptide is generated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266:776-779). A fusion polypeptide can further include a cleavage site between the two polypeptides. Thus, a fusion polypeptide can include, for example, a cleavage site for a site-specific endopeptidase within 20 amino acids, preferably within 10 amino acids, of the C-terminus of the first polypeptide. Examples of well-known site-specific endopeptidases include glutamyl endopeptidases (e.g., EC 3.4.21.19 or EC 3.4.21.82), trypsin- and chymotrypsin-like endopeptidases (including enteropeptidase).Many other examples of cleavage sites and corresponding endopeptidases include Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3:568-576; Svetina et al., 2000, J. Biotechnol. 76:245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63:3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9:378-381; Eaton et al., 1986, Biochemistry 25:505-512; Collins-Racie et al., 1995, Biotechnology 13:982-987; Carter et al. al., 1989, Proteins: Structure, Function, and Genetics 6:240-248; and Stevens, 2003, Drug Discovery World 4:35-48.

[0025] Heterologous: The term "heterologous" with respect to a host cell means that the polypeptide or nucleic acid does not naturally occur in the host cell. The term "heterologous" with respect to a polypeptide or nucleic acid means that the regulatory sequences of the polypeptide or nucleic acid, e.g., a promoter, are not naturally associated with the polypeptide or nucleic acid, i.e., the regulatory sequences are derived from a gene other than the gene encoding the mature polypeptide.

[0026] Host strain or host cell: A "host strain" or "host cell" is an organism into which an expression vector, phage, virus, or other DNA construct containing a polynucleotide encoding a polypeptide of interest (e.g., an amylase) has been introduced. Exemplary host strains are microbial cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing a polypeptide of interest and / or fermenting sugars. The term "host cell" includes protoplasts made from cells.

[0027] Introduced: The term "introduced" in the context of inserting a nucleic acid sequence into a cell means "transfection," "transformation," or "transduction," as known in the art.

[0028] Isolated: The term "isolated" refers to a polypeptide, nucleic acid, cell, or other specified material or component that is separated from at least one other material or component, including, but not limited to, other proteins, nucleic acids, cells, etc. An isolated polypeptide, nucleic acid, cell, or other material is therefore in a form that is not found in nature. Isolated polypeptides include, but are not limited to, culture broths containing the polypeptide expressed and secreted in a host cell.

[0029] Mature polypeptide: The term "mature polypeptide" refers to the mature form of a polypeptide after N-terminal and / or C-terminal processing (e.g., removal of a signal peptide). In one aspect, the mature polypeptide is SEQ ID NO:2.

[0030] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" refers to a polynucleotide that encodes a mature polypeptide having deamidase inhibitor activity. In one embodiment, the mature polypeptide coding sequence is SEQ ID NO: 1.

[0031] Native: The term "native" refers to a nucleic acid or polypeptide that is naturally present in a host cell.

[0032] Nucleic Acid: The term "nucleic acid" includes DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids may be single- or double-stranded and may be chemically modified. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode specific amino acid sequences. Unless otherwise indicated, nucleic acid sequences are presented in the 5' to 3' direction.

[0033] Nucleic Acid Construct: The term "nucleic acid construct" refers to a nucleic acid molecule, either single-stranded or double-stranded, that is isolated from a naturally occurring gene or that has been modified to contain a segment of nucleic acid in a manner not normally found in nature, or that is synthetic, and that includes one or more regulatory sequences operably linked to the nucleic acid sequence.

[0034] Operably linked: The term "operably linked" means that the specified components are in a relationship, including but not limited to, a juxtaposition, permitting them to function in their intended manner. For example, a regulatory sequence is operably linked to a coding sequence so that expression of the coding sequence is under the control of the regulatory sequence.

[0035] Purified: The term "purified" refers to a nucleic acid, polypeptide, or cell that is substantially free of other components as determined by analytical techniques well known in the art (e.g., a purified polypeptide or nucleic acid may form a discrete band in an electrophoretic gel, a chromatographic eluate, and / or a medium subjected to density gradient centrifugation). A purified nucleic acid or polypeptide is at least about 50% pure, and typically at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or more pure (e.g., percent by weight or molar basis). In a related sense, a composition is enriched with respect to a molecule if there is a substantial increase in the concentration of that molecule after application of a purification or concentration technique. The term "enriched" refers to a compound, polypeptide, cell, nucleic acid, amino acid, or other specified material or component that is present in a composition at a higher relative or absolute concentration compared to the starting composition.

[0036] In one aspect, the term "purified," as used herein, refers to a polypeptide or cell that is essentially free from components, particularly insoluble components, from the producing organism. In another aspect, the term "purified" refers to a polypeptide that is essentially free from insoluble components, particularly insoluble components, from the natural organism from which the polypeptide is obtained. In one aspect, the polypeptide has been separated from a portion of the soluble components of the organism and culture medium from which the polypeptide is recovered. The polypeptide may be purified (i.e., separated) by one or more of the unit operations filtration, precipitation, or chromatography.

[0037] Thus, a polypeptide can be purified such that only trace amounts of other proteins (particularly other polypeptides) are present. The term "purified," as used herein, can refer to the removal of other components (particularly other proteins, most particularly other enzymes) present in the cell from which the polypeptide originates. A polypeptide can also be "substantially pure," i.e., free from other components from the organism that produces the polypeptide (e.g., the host organism in the case of a recombinantly produced polypeptide). In one aspect, the polypeptide is at least 40% pure by weight of total polypeptide material present in a preparation. In one aspect, the polypeptide is at least 50%, 60%, 70%, 80%, or 90% pure by weight of total polypeptide material present in a preparation. As used herein, a "substantially pure polypeptide" can mean a polypeptide preparation that contains up to 10%, preferably up to 8%, more preferably up to 6%, more preferably up to 5%, more preferably up to 4%, more preferably up to 3%, even more preferably up to 2%, most preferably up to 1%, and most preferably up to 0.5%, by weight of other polypeptide material with which the polypeptide is naturally or recombinantly associated.

[0038] Thus, a substantially pure polypeptide is preferably at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99% pure, and most preferably at least 99.5% pure, by weight of total polypeptide material present in the preparation. The polypeptides of the invention are preferably in substantially pure form (i.e., the preparation is essentially free of other polypeptide material with which it is naturally or recombinantly associated). This can be accomplished, for example, by preparing the polypeptide by well-known recombinant or classical purification methods.

[0039] Recombinant: The term "recombinant" is used in its conventional sense to refer to the manipulation, e.g., cutting and recombining, of nucleic acid sequences to form constellations that differ from those found in nature. The term recombinant refers to a cell, nucleic acid, polypeptide, or vector that has been modified from its natural state. Thus, for example, a recombinant cell expresses genes that are not found within the native form of the cell (non-recombinant), or expresses native genes at levels or under conditions that are different from those found in nature. The term "recombinant" is synonymous with "genetically modified" and "transgenic."

[0040] Recover: The term "recover" or "recovery" refers to the removal of a polypeptide from at least one fermentation broth component selected from the list of cells, nucleic acids, or other specified materials, e.g., recovery of a polypeptide from a whole fermentation broth or from a cell-free fermentation broth, by collecting polypeptide crystals, by filtration, e.g., depth filtration (using filter aids or packed filter media, chamber filter fabric filtration, rotary drum filtration, drum filtration, rotary vacuum drum filters, candle filters, horizontal leaf filters, or similar, with seed or pad filtration in a framework or modular setup), or membrane filtration (using sheet filtration, module filtration, candle filtration, microfiltration, ultrafiltration, in either cross-flow, dynamic cross-flow, or dead-end operation), or by centrifugation (using a decanter centrifuge, disc centrifuge, hydrocyclone, or similar), or by precipitating the polypeptide and using particle size classification separation using an appropriate solid-liquid separation method. Recovery encompasses isolation and / or purification of the polypeptide.

[0041] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity."

[0042] For the purposes of the present invention, sequence identity between two amino acid sequences is determined as the "longest identity" output using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), preferably version 6.6.0 or later. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order for the Needle program to report the longest identity, the -nobrief option must be specified on the command line. The Needle output labeled "longest identity" is calculated as follows: (identical residues × 100) / (length of alignment−total number of gaps in the alignment).

[0043] For purposes of the present invention, sequence identity between two polynucleotide sequences is preferably determined as the "longest identity" output using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS; The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), version 6.6.0 or later. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and an EDNAFULL (the EMBOSS version in NCBI NUC4.4) substitution matrix. The nobrief option must be specified on the command line to cause the Needle program to report the longest identity. The Needle output labeled "longest identity" is calculated as follows: (identical nucleotides × 100) / (length of alignment−total number of gaps in the alignment).

[0044] Signal peptide: A "signal peptide" is a sequence of amino acids attached to the N-terminal portion of a protein that facilitates secretion of the protein outside the cell. The mature form of the extracellular protein lacks the signal peptide, which is cleaved during the secretion process.

[0045] Subsequence: The term "subsequence" refers to a polynucleotide having one or more nucleotides missing at the 5' and / or 3' end of the mature polypeptide coding sequence, wherein the subsequence encodes a fragment that has deamidase inhibitor activity.

[0046] Thermal denaturation temperature: "melting temperature", "T m The term "thermal denaturation temperature," also referred to as "midpoint temperature," or "denaturation temperature," refers to the temperature at which approximately 50% of a protein unfolds. Typically, the percentage of folded protein is predominant (>99.999%) at room temperature, and the temperature at which it unfolds is greater than the melting temperature, T m It decreases as the m At this rate, approximately 50% of the molecule is in a folded state and approximately 50% is in an unfolded state. m At temperatures above 100°C, the unfolded state becomes the predominant species (>50%). A preferred method for determining the terminal denaturation temperature of a deamidase / inhibitor complex using an inhibitor of the present invention is nanoDSF. Using nanoDSF, T m The determination of T can be performed at 330 nm, 350 nm, and / or the ratio 330 nm / 350 nm. m is equal to the temperature of the inflection point (IP) of the first derivative. At the inflection point, the first derivative reaches a local maximum or minimum and the second derivative has an isolated zero. m Alternatively, the thermal denaturation temperature is the temperature of the inflection point (IP) of the first derivative determined by nanoDSF at 330 nm as described in Example 2.

[0047] Other methods for determining the thermal denaturation temperature are thermal shift assays as described in Current Protocols in Protein Science: "Analysis of protein stability and ligand interactions by thermal shift assay" (K. Huynh and C.L. Partch, 2015) or the method described in Encyclopedia of Industrial Biotechnology: "Proteins: Thermal Unfolding" (R. Lonescu and L. Shi, 2009).

[0048] Thermal denaturation is an important tool for assessing protein stability. To assess a protein's preference for maintaining its folded (active) conformation, the protein is exposed to high levels of denaturing stress (temperature), and protein stability is determined based on the stress level required to produce a significant proportion of unfolded protein. Factors that affect thermal stability include, but are not limited to, protein structure, the presence of ligands or excipients, the binding strength between inhibitors and enzymes, and solvent conditions.

[0049] Variant: The term "variant" refers to a polypeptide having deamidase inhibitor activity that contains an artificial mutation (i.e., a substitution, an insertion (including an extension), and / or a deletion (e.g., a truncation)) at one or more positions. A substitution refers to the replacement of an amino acid occupying a position with a different amino acid; a deletion refers to the removal of an amino acid occupying a position; and an insertion refers to the addition of 1 to 5 amino acids (e.g., 1 to 3 amino acids, particularly 1 amino acid) adjacent to and immediately following the amino acid occupying a position.

[0050] Wild-type: The term "wild-type" with respect to an amino acid sequence or a nucleic acid sequence means that the amino acid sequence or nucleic acid sequence is a native or naturally occurring sequence. As used herein, the term "naturally occurring" refers to something that is found in nature (e.g., a protein, amino acid, or nucleic acid sequence). Conversely, the term "non-naturally occurring" refers to something that is not found in nature (e.g., recombinant nucleic acid and protein sequences produced in the laboratory, or modifications of a wild-type sequence). DETAILED DESCRIPTION OF THE INVENTION

[0051] Polypeptides having deamidase inhibitor activity The present invention provides (a) a polypeptide having at least 60% amino acid sequence identity to SEQ ID NO:2; (b) a polypeptide derived from SEQ ID NO: 2 by having 1 to 30 modifications (e.g., substitutions, deletions, and / or insertions), for example 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23, or 24, or 25, or 26, or 27, or 28, or 29, or 30 modifications, particularly substitutions, at one or more positions; (c) a polypeptide derived from the polypeptide of (a) or (b), wherein the N-terminus and / or C-terminus are extended by the addition of one or more amino acids; and (d) a fragment of the polypeptide of (a), (b), or (c). The present invention relates to a polypeptide having deamidase inhibitor activity selected from the group consisting of:

[0052] In one embodiment, a polypeptide having deamidase inhibitor activity has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to SEQ ID NO:2.

[0053] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:2.

[0054] The polypeptide may have an N-terminal and / or C-terminal extension of one or more amino acids, for example, 1 to 20 amino acids, 1 to 10 amino acids, 1 to 5 amino acids.

[0055] In another aspect, the polypeptide comprises an amino acid sequence motif selected from the group consisting of F[F / Y][I / L / V][F / Q / S][E / K / R], L[I,T]WY[D,H,K,N], G[I,M]S[A,P,Q]Q, [D,H,K,N,S][I,L][G,V][I,V][D,E], [N,H][I,L,M,V,Q][I,V][K,R,Q][E,I,Q], [D / N][P / S][D / E][H / K / N / Q / R][A / P / S], and combinations thereof.

[0056] In another embodiment, the polypeptide also comprises an amino acid sequence motif selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and combinations thereof.

[0057] In another aspect, the polypeptide is derived from SEQ ID NO: 2 by substitution, deletion, or addition of one or more amino acids. In some embodiments, the polypeptide is a variant of SEQ ID NO: 2 comprising substitutions, deletions, and / or insertions at one or more positions. In one embodiment, the number of amino acid substitutions, deletions, and / or insertions introduced into a polypeptide of SEQ ID NO: 2 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes can be minor in nature, such as conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity, small deletions, typically of 1-30 amino acids, small amino- or carboxyl-terminal extensions such as an amino-terminal methionine residue, small linker peptides of up to 20-25 residues, or small extensions that facilitate purification by altering net charge or another function, such as a polyhistidine tract, antigenic epitope, or binding module.

[0058] Essential amino acids in a polypeptide can be identified using art-known procedures such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations are introduced at every residue in a molecule to identify those critical to the molecule's activity, and the resulting molecules are tested for deamidase inhibitor activity. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. Enzyme active sites or other biological interactions can also be determined by physical analysis of structures measured by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, combined with mutations of putative contact site amino acids. See, e.g., de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. Identification of essential amino acids can also be inferred from alignments with related polypeptides and / or from sequence homology and conserved catalytic mechanisms with related polypeptides, or with polypeptides / proteins (typically with similar three-dimensional structure, function, and significant sequence similarity) derived from a common ancestor within a polypeptide or protein family. Additionally or alternatively, protein structure prediction tools can be used for protein structure modeling to identify essential amino acids and / or the active site of a polypeptide. See, e.g., Jumper et al., 2021, "Highly accurate protein structure prediction with AlphaFold," Nature 596:583-589.

[0059] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known mutagenesis, recombination, and / or shuffling methods followed by associated screening procedures such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86:2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, CRISPR gene editing, phage display (e.g., Lowman et al., 1991, Biochemistry 30:10832-10837; U.S. Pat. No. 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0060] Mutagenesis / shuffling methods can be combined with high-throughput automated screening methods to detect the activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17;893-896). Mutagenized DNA molecules encoding active polypeptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues within a polypeptide.

[0061] The polypeptide may be a fusion polypeptide.

[0062] In one aspect, the polypeptide is isolated.

[0063] In another embodiment, the polypeptide is purified.

[0064] In another aspect, the present invention provides a method for:

[0065] In yet another aspect, the present invention provides a composition comprising a deamidase according to the present invention and a deamidase inhibitor.

[0066] Sources of wild-type deamidase inhibitor polypeptides The wild-type deamidase inhibitor polypeptide may be obtained from any genus of microorganism (donor strain). Preferably, the wild-type deamidase inhibitor polypeptide is obtained from a Chryseobacterium species.

[0067] For purposes of the present invention, the term "obtained from," when used herein with reference to a given source, shall mean that the polypeptide encoded by the polynucleotide is produced by the source or by the strain into which the polynucleotide of the invention has been inserted. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.

[0068] In one embodiment, the wild-type deamidase inhibitor polypeptide is obtained from a Chryseobacterium species.

[0069] Wild-type deamidase inhibitor polypeptides can be identified and obtained from sources including microorganisms isolated from nature (e.g., soil, compost, water, etc.) or DNA samples obtained directly from natural sources (e.g., soil, compost, water, etc.). Techniques for directly isolating microorganisms and DNA from natural habitats are well known in the art. Polynucleotides encoding the polypeptides can then be obtained by similarly screening genomic DNA or cDNA libraries of other microorganisms or mixed DNA samples. After the polynucleotide encoding the polypeptide is detected by a probe, the polynucleotide can be isolated or cloned using techniques known to those skilled in the art (see, for example, Davis et al., 2012, Basic Methods in Molecular Biology, Elsevier).

[0070] Polynucleotides The present invention also relates to polynucleotides encoding the polypeptides of the invention as described herein.

[0071] The polynucleotide may be modified by the introduction of nucleotide substitutions that do not result in a change in the amino acid sequence of the polypeptide but correspond to the codon usage of the host organism in which the enzyme is intended to be produced, or that may result in a different amino acid sequence. For a general description of nucleotide substitutions, see, e.g., Ford et al., 1991, Protein Expression and Purification 2:95-107.

[0072] In one aspect, the polynucleotide is isolated.

[0073] In another embodiment, the polynucleotide is purified.

[0074] Nucleic Acid Constructs The present invention also relates to nucleic acid constructs comprising a polynucleotide of the invention operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0075] Polynucleotides can be manipulated in a variety of ways to provide for expression of a polypeptide. It may be desirable or necessary to manipulate the polynucleotide depending on the expression vector prior to insertion into the vector. Techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

[0076] promoter The control sequence may be a promoter, which is a polynucleotide recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the invention. The promoter contains transcriptional control sequences that mediate expression of the polypeptide. The promoter may be any polynucleotide that exhibits transcriptional activity in the host cell, including mutant, truncated, and hybrid promoters, and may be derived from a gene encoding an extracellular or intracellular polypeptide either homologous or heterologous to the host.

[0077] Examples of suitable promoters for directing transcription of the polynucleotides of the invention in bacterial host cells are described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab., NY; Davis et al., 2012, supra; and Song et al., 2016, PLOS One 11(7):e0158447.

[0078] Examples of suitable promoters for directing transcription of the polynucleotides of the invention in filamentous fungal host cells are promoters obtained from Aspergillus, Fusarium, Rhizomucor, and Trichoderma cells, such as the promoters described in Mukherjee et al., 2013, "Trichoderma: Biology and Applications," and Schmoll and Dattenboeck, 2016, "Gene Expression Systems in Fungi: Advancements and Applications," Fungal Biology.

[0079] For expression in yeast hosts, examples of useful promoters are described by Smolke et al., 2018, "Synthetic Biology: Parts, Devices and Applications" (Chapter 6: Constitutive and Regulated Promoters in Yeast: How to Design and Make Use of Promoters in S. cerevisiae), and Schmoll and Dattenboeck, 2016, "Gene Expression Systems in Fungi: Advancements and Applications", Fungal Biology.

[0080] Terminator The control sequence may also be a transcription terminator recognized by the host cell to terminate transcription. The terminator is operably linked to the 3' end of the polynucleotide encoding the polypeptide. Any terminator functional in the host cell may be used in the present invention.

[0081] Preferred terminators for bacterial host cells can be obtained from the genes for alkaline protease of Bacillus clausii (aprH), alpha-amylase of Bacillus licheniformis (amyL), and ribosomal RNA of Escherichia coli (rrnB).

[0082] Preferred terminators for filamentous fungal host cells can be obtained from Aspergillus or Trichoderma species, such as those obtained from the genes for Aspergillus niger glucoamylase, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, and Trichoderma reesei endoglucanase I, such as the terminators described in Mukherjee et al., 2013, "Trichoderma: Biology and Applications," and Schmoll and Dattenboeck, 2016, "Gene Expression Systems in Fungi: Advancements and Applications," Fungal Biology.

[0083] Preferred terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, Yeast 8:423-488.

[0084] mRNA stabilizers A regulatory sequence can also be an mRNA stabilizing region downstream of a promoter and upstream of the coding sequence of a gene that increases expression of the gene.

[0085] Examples of suitable mRNA stabilization regions are obtained from the cryIIIA gene of Bacillus thuringiensis (WO 94 / 25612) and the Bacillus subtilis SP82 gene (Hue et al., 1995, J. Bacteriol. 177:3465-3471).

[0086] Examples of mRNA stabilization regions for fungal cells are described in Geisberg et al., 2014, Cell 156(4):812-824, and Morozov et al., 2006, Eukaryotic Cell 5(11):1838-1846.

[0087] Leader sequence The control sequence may also be a leader, untranslated region of an mRNA that is important for translation by the host cell. The leader is operably linked to the 5' terminus of the polynucleotide encoding the polypeptide. Any leader that is functional in the host cell may be used.

[0088] Suitable leaders for bacterial host cells are described in Hambraeus et al., 2000, Microbiology 146(12):3051-3059, and Kaberdin and Blaesi, 2006, FEMS Microbiol. Rev. 30(6):967-979.

[0089] Preferred leaders for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0090] Suitable leaders for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0091] Polyadenylation sequence The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3' end of the polynucleotide, which, when transcribed, is recognized by a host cell as a signal for the addition of polyadenosine residues to the transcribed mRNA. Any polyadenylation sequence functional in the host cell may be used.

[0092] Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0093] Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15:5983-5990.

[0094] signal peptide The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a polypeptide and directs the polypeptide into the secretory pathway of a cell. The 5' end of the coding sequence of a polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence encoding the polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding sequence heterologous to the coding sequence. A heterologous signal peptide coding sequence may be required if the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, a heterologous signal peptide coding sequence may simply replace the native signal peptide coding sequence to enhance secretion of the polypeptide. Any signal peptide coding sequence that directs an expressed polypeptide into the secretory pathway of a host cell may be used.

[0095] Effective signal peptide coding sequences for bacterial host cells include those obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alpha-amylase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prsA. Additional signal peptides are described by Freudl, 2018, Microbial Cell Factories 17:52.

[0096] Effective signal peptide coding sequences for filamentous fungal host cells are those obtained from genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase, such as the signal peptides described by Xu et al., 2018, Biotechnology Letters 40:949-955.

[0097] Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, supra.

[0098] Regulatory sequences It may also be desirable to add regulatory sequences that regulate polypeptide expression relative to the growth of the host cell. Examples of regulatory sequences are those that turn gene expression on or off in response to chemical or physical stimuli, including the presence of a regulatory compound. Regulatory sequences in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 or GAL1 system may be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, the Aspergillus oryzae TAKA alpha-amylase promoter, the Aspergillus oryzae glucoamylase promoter, the Trichoderma reesei cellobiohydrolase I promoter, and the Trichoderma reesei cellobiohydrolase II promoter may be used. Other examples of regulatory sequences are those that allow for gene amplification. In fungal systems, these regulatory sequences include dihydrofolate reductase, which is amplified in the presence of methotrexate, and metallothionein genes, which are amplified with heavy metals.

[0099] transcription factors The regulatory sequence may also be a transcription factor, which is a polynucleotide encoding a polynucleotide-specific DNA-binding polypeptide that controls the rate of transcription of genetic information from DNA to mRNA by binding to a specific polynucleotide sequence. Transcription factors can function alone and / or with one or more other polypeptides or transcription factors in a complex by promoting or blocking the recruitment of RNA polymerase. Transcription factors are characterized by containing at least one DNA-binding domain, which often binds to a specific DNA sequence adjacent to the genetic element regulated by the transcription factor. Transcription factors can regulate the expression of a protein of interest directly, i.e., by activating the transcription of a gene encoding the protein of interest by binding to its promoter, or indirectly, i.e., by activating the transcription of an additional transcription factor that regulates the transcription of the gene encoding the protein of interest, such as by binding to the promoter of the additional transcription factor. Suitable transcription factors for fungal host cells are described in WO 2017 / 144177. Suitable transcription factors for prokaryotic host cells are described in Seshasayee et al., 2011, Subcellular Biochemistry 52:7-23, and Balleza et al., 2009, FEMS Microbiol. Rev. 33(1):133-151.

[0100] Expression vector The present invention also relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals. Various nucleotide and control sequences can be ligated together to generate a recombinant expression vector, which may contain one or more convenient restriction sites to allow for insertion or substitution of a polynucleotide encoding a polypeptide at such sites. Alternatively, a polynucleotide can be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In forming an expression vector, a coding sequence is placed in the vector such that the coding sequence is operably linked to appropriate control sequences for expression.

[0101] The recombinant expression vector can be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of a polynucleotide. The choice of vector will usually depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear or closed circular plasmid.

[0102] The vector may be, for example, a self-replicating vector, i.e., a vector that exists as an extrachromosomal entity and whose replication is independent of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may include any means for ensuring self-replication. Alternatively, the vector may be one that is integrated into the genome when introduced into a host cell and replicated together with the chromosome or chromosomes into which it is integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids, or transposons, that together contain the total DNA to be introduced into the genome of the host cell, may be used.

[0103] Vectors preferably contain one or more selectable markers which facilitate the selection of cells that have been transformed, transfected, transduced, etc. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc.

[0104] The vector preferably contains at least one element that allows for integration of the vector into the genome of the host cell or autonomous replication of the vector within the cell independent of the genome.

[0105] For integration into the host cell genome, the vector may rely on the sequence of the polynucleotide encoding the polypeptide or any other element of the vector for integration into the genome by homologous recombination, such as homology directed repair (HDR), or non-homologous recombination, such as non-homologous end joining (NHEJ).

[0106] For autonomous replication, the vector may further comprise an origin of replication that allows the vector to replicate autonomously in a host cell of interest. The origin of replication may be any plasmid replicator that mediates autonomous replication that functions within the cell. The term "origin of replication" or "plasmid replicator" refers to a polynucleotide that allows a plasmid or vector to replicate in vivo.

[0107] Two or more copies of the polynucleotide of the present invention can be inserted into a host cell to increase the production of the polypeptide. For example, two, three, four, five or more copies can be inserted into the host cell. Increasing the copy number of the polynucleotide can be achieved by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene in the polynucleotide, and by culturing the cells in the presence of an appropriate selection agent, cells containing an amplified copy of the selectable marker gene, and therefore cells containing additional copies of the polynucleotide, can be selected.

[0108] host cell The present invention also relates to recombinant host cells containing a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of a polypeptide of the present invention.

[0109] The construct or vector containing the polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extrachromosomal vector as described above. The choice of host cell will largely depend on the gene encoding the polypeptide and its source. The polypeptide may be native or heterologous to the recombinant host cell. Also, at least one of the one or more control sequences may be heterologous to the polynucleotide encoding the polypeptide. The recombinant host cell may contain a single copy of the polynucleotide of the invention, or at least two copies, e.g., three, four, five, or more copies.

[0110] The host cell can be any microbial cell, such as a prokaryotic or fungal cell, useful for the recombinant production of the polypeptides of the invention.

[0111] Prokaryotic host cells can be any gram-positive or gram-negative bacterium, including, but not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0112] Bacterial host cells include, but are not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus spp. ... The Bacillus cell can be any Bacillus cell, including Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells. In one embodiment, the Bacillus cell is a Bacillus amyloliquefaciens, Bacillus licheniformis, or Bacillus subtilis cell.

[0113] For purposes of the present invention, the class / genus / species of Bacillus shall be defined as set forth in Patel and Gupta, 2020, Int. J. Syst. Evol. Microbiol. 70:406-438.

[0114] A bacterial host cell can also be any Streptococcus cell, including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

[0115] A bacterial host cell can also be any Streptomyces cell, including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

[0116] Methods for introducing DNA into prokaryotic host cells are well known in the art and any suitable method can be used, including but not limited to protoplast transformation, competent cell transformation, electroporation, conjugation, transduction, where the DNA is introduced as a linear polynucleotide or as a circular polynucleotide. One skilled in the art will readily be able to identify a suitable method for introducing DNA into a given prokaryotic cell, depending, for example, on the genus. Methods for introducing DNA into prokaryotic host cells are described, for example, in Heinze et al., 2018, BMC Microbiology 18:56, Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98:6289-6294, Choi et al., 2006, J. Microbiol. Methods 64:391-397, and Donald et al., 2013, J. Bacteriol. 195(11):2612-2620.

[0117] The host cell may be a fungal cell. As used herein, "fungi" includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota, as well as Oomycota and all vegetative spore-forming fungi (as defined by Hawksworth et al., in Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

[0118] Fungal cells may be transformed by processes including protoplast-mediated transformation, Agrobacterium-mediated transformation, electroporation, biolistic methods, and shock wave-mediated transformation, as reviewed by Li et al., 2017, Microbial Cell Factories 16:168, and procedures described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, Christensen et al., 1988, Bio / Technology 6:1419-1422, and Lubertozzi and Keasling, 2009, Biotechn. Advances 27:53-75. However, any method known in the art for introducing DNA into fungal host cells may be used, and the DNA may be introduced as a linear or circular polynucleotide.

[0119] The fungal host cell may be a yeast cell. As used herein, "yeast" includes ascosporogenous yeasts (Endomycetales), basidiomycete yeasts, and yeasts belonging to the Fungi Imperfecti (Blastomycetes). For purposes of the present invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0120] Yeast host cells may be selected from the group consisting of Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, and Yarrowia lipolytica. The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Pichia lipolytica cell. In a preferred embodiment, the yeast host cell is a Pichia or Komagataella cell, such as a Pichia pastoris cell (Komagataella phaffii).

[0121] The fungal host cell can be a filamentous fungal cell. "Filamentous fungi" includes all filamentous forms of the subdivisions Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). Filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation, and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus, and carbon catabolism can be fermentative.

[0122] Filamentous fungal host cells include Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, The host cell may be a Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell. In a preferred embodiment, the filamentous fungal host cell is an Aspergillus, Trichoderma, or Fusarium cell. In a further preferred embodiment, the filamentous fungal host cell is an Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, or Fusarium venenatum cell.

[0123] For example, filamentous fungal host cells may be selected from the group consisting of Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, and Ceriporiopsis girvescens. gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenstrandicum queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bacteridioidesbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochromium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Talaromyces emersonii, Thielavia terrestrisThe cell may be a Trichoderma terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

[0124] In one embodiment, the host cell is isolated.

[0125] In another embodiment, the host cells are purified.

[0126] In yet another aspect, the host cell further comprises a co-expressed polypeptide that exhibits deamidase activity. In a related aspect, the invention also relates to a method for producing a polypeptide having deamidase activity, comprising culturing a recombinant host cell (comprising a co-expressed polypeptide that exhibits deamidase activity) under conditions conducive to the production of the polypeptide having deamidase activity, and preferably further comprising recovering the polypeptide having deamidase activity. Such recovery of the polypeptide having deamidase activity may comprise a diafiltration step.

[0127] Generation method The present invention also relates to methods for producing a polypeptide of the present invention, comprising (a) culturing a recombinant host cell of the present invention under conditions conducive to production of the polypeptide, and optionally (b) recovering the polypeptide.

[0128] The host cells are cultured in a nutrient medium suitable for production of the polypeptide using methods known in the art. For example, the cells can be cultured in shake flask cultures or by small- or large-scale fermentation (including continuous, batch, fed-batch, or solid-state and / or microcarrier-based fermentation) in laboratory or industrial fermentors in a suitable medium and under conditions that allow for expression and / or isolation of the polypeptide. Suitable media are available from commercial suppliers or can be prepared according to published compositions (e.g., in catalogs of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, the polypeptide can be recovered from cell lysates.

[0129] Polypeptides can be detected using methods known in the art that are specific for the polypeptides, including, but not limited to, the use of specific antibodies, formation of an enzyme product, disappearance of an enzyme substrate, or assays that determine the relative or specific activity of the polypeptide.

[0130] The polypeptide may be recovered from the culture medium using methods known in the art, including, but not limited to, harvesting, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. In one aspect, the whole fermentation broth containing the polypeptide is recovered. In another aspect, the cell-free fermentation broth containing the polypeptide is recovered.

[0131] Polypeptides can be purified by various procedures known in the art to obtain substantially pure polypeptides and / or polypeptide fragments (see, e.g., Wingfield, 2015, Current Protocols in Protein Science; 80(1):6.1.1-6.1.35; Labrou, 2014, Protein Downstream Processing, 1129:3-10).

[0132] In an alternative embodiment, the polypeptide is not recovered.

[0133] solid formulation The present invention also relates to enzyme granules / particles comprising the polypeptides of the present invention. In one embodiment, the granules comprise a core and optionally one or more coatings (outer layers) surrounding the core.

[0134] The core may have a diameter, measured as an equivalent spherical diameter (average particle size by volume), of 20 to 2000 μm, in particular 50 to 1500 μm, 100 to 1500 μm, or 250 to 1200 μm. The core diameter, measured as an equivalent spherical diameter, may be determined using laser diffraction, such as using a Malvern Mastersizer and / or the method described under ISO 13320 (2020).

[0135] In one embodiment, the core comprises a polypeptide having deamidase inhibitor activity of the present invention.

[0136] The core may contain additional materials such as fillers, fibrous materials (cellulose or synthetic), stabilizers, solubilizers, suspending agents, viscosity modifiers, light spheres, plasticizers, salts, lubricants, and fragrances.

[0137] The core may include a binder such as a synthetic polymer, a wax, a fat, or a carbohydrate.

[0138] The core may include salts of multivalent cations, reducing agents, antioxidants, peroxide decomposition catalysts and / or acidic buffer components, typically as a homogeneous blend.

[0139] The core may comprise an inert particle into which the polypeptide is absorbed, or onto which the enzyme is applied, for example, by fluidized bed coating.

[0140] The core may have a diameter of 20 to 2000 μm, in particular 50 to 1500 μm, 100 to 1500 μm or 250 to 1200 μm.

[0141] The core may be surrounded by at least one coating, for example to improve storage stability, reduce dust formation during handling, or to color the granules. Optional coatings may include salt coatings or other suitable coating materials such as polyethylene glycol (PEG), methylhydroxypropylcellulose (MHPC), and polyvinyl alcohol (PVA).

[0142] The coating may be applied in an amount of at least 0.1% by weight of the core, such as at least 0.5%, at least 1%, at least 5%, at least 10%, or at least 15% by weight, which may be up to 100%, 70%, 50%, 40%, or 30%.

[0143] The coating is preferably at least 0.1 μm thick, particularly at least 0.5 μm, at least 1 μm, or at least 5 μm thick. In some embodiments, the coating thickness is less than 100 μm, for example less than 60 μm, or less than 40 μm.

[0144] The coating must encapsulate the core unit by forming a substantially continuous layer, which is to be understood as a coating with few or no holes, so that there are few or no uncovered areas of the core unit. The layer or coating must in particular be uniform in thickness.

[0145] The coating may further contain other materials known in the art, such as fillers, anti-adherents, pigments, dyes, plasticizers and / or binders, such as titanium dioxide, kaolin, calcium carbonate or talc.

[0146] The salt coating can comprise at least 60% by weight salt, e.g., at least 65% by weight, at least 70% by weight, at least 75% by weight, at least 80% by weight, at least 85% by weight, at least 90% by weight, at least 95% by weight, or at least 99% by weight salt.

[0147] To provide acceptable protection, the salt coating is preferably at least 0.1 μm thick, e.g., at least 0.5 μm, at least 1 μm, at least 2 μm, at least 4 μm, at least 5 μm, or at least 8 μm thick, hi certain embodiments, the salt coating is less than 100 μm thick, e.g., less than 60 μm or less than 40 μm thick.

[0148] The salt may be added from a salt solution in which the salt is completely dissolved or from a salt suspension in which the fine particles are less than 50 μm, such as less than 10 μm or less than 5 μm.

[0149] The salt coating may comprise a single salt or a mixture of two or more salts. The salt may be water-soluble, in particular having a solubility of at least 0.1 g in 100 g of water at 20° C., preferably at least 0.5 g per 100 g of water, such as at least 1 g per 100 g of water, for example at least 5 g per 100 g of water.

[0150] The salts may be inorganic salts, such as sulfates, sulfites, phosphates, phosphonates, nitrates, chlorides, or carbonates, or salts of simple organic acids (having less than 10 carbon atoms, e.g., 6 or fewer carbon atoms), such as citrates, malonates, or acetates. Examples of cations in these salts include alkali or earth alkali metal ions, ammonium ions, or first transition series metal ions, such as sodium, potassium, magnesium, calcium, zinc, or aluminum. Examples of anions include chloride, bromide, iodide, sulfate, sulfite, bisulfite, thiosulfate, phosphate, monobasic phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, metasilicate, citrate, malate, maleate, malonate, succinate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate, or gluconate. In particular, alkali or earth alkali metal sulfates, sulfites, phosphates, phosphonates, nitrates, chlorides or carbonates or salts of simple organic acids such as citrates, malonates or acetates may be used.

[0151] The salt of the coating may have a constant moisture content of more than 60%, in particular more than 70%, 80% or more than 85% at 20° C., or may be another hydrated form of such a salt (e.g., anhydrous). The salt coating may be as described in WO 00 / 01793 or WO 2006 / 034710.

[0152] Specific examples of suitable salts include NaCl(CH 20℃ = 76%), Na2CO3(CH 20℃ = 92%), NaNO3(CH 20℃ = 73%), Na2HPO4(CH 20℃ = 95%), Na3PO4(CH 25℃ = 92%), NH4Cl(CH 20℃ =79.5%), (NH4)2HPO4(CH 20℃ =93.0%), NH4H2PO4(CH 20℃ = 93.1%), (NH4)2SO4(CH20℃ = 81.1%), KCl(CH 20℃ = 85%), K2HPO4(CH 20℃ = 92%), KH2PO4(CH 20℃ =96.5%), KNO3(CH 20℃ = 93.5%), Na2SO4(CH 20℃ = 93%), K2SO4(CH 20℃ = 98%), KHSO4(CH 20℃ = 86%), MgSO4(CH 20℃ = 90%), ZnSO4(CH 20℃ = 90%) and sodium citrate (CH 25℃ =86%). Other examples include NaH2PO4, (NH4)H2PO4, CuSO4, Mg(NO3)2, and magnesium acetate.

[0153] The salts may be in anhydrous form or may be hydrated salts, i.e., crystalline salt hydrates containing bound water of crystallization, such as those described in WO 99 / 32595. Specific examples include anhydrous sodium sulfate (NaSO), anhydrous magnesium sulfate (MgSO), magnesium sulfate heptahydrate (MgSO·7H2O), zinc sulfate heptahydrate (ZnSO·7H2O), dibasic sodium phosphate heptahydrate (NaHPO·7H2O), magnesium nitrate hexahydrate (Mg(NO3)2(6H2O)), sodium citrate dihydrate, and magnesium acetate tetrahydrate.

[0154] Preferably the salt is applied as a solution of the salt, for example using a fluidized bed.

[0155] The coating material can be a wax coating material or a film-forming coating material. Examples of wax coating materials are poly(ethylene oxide) products (polyethylene glycol, PEG) with an average molecular weight of 1,000 to 20,000, ethoxylated nonylphenols with 16 to 50 ethylene oxide units, ethoxylated fatty alcohols in which the alcohol contains 12 to 20 carbon atoms and there are 15 to 80 ethylene oxide units, fatty alcohols, fatty acids, and mono-, di-, and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluidized bed technology are given in GB 1,483,591.

[0156] The granules may optionally have one or more additional coatings. Examples of suitable coating materials include polyethylene glycol (PEG), methylhydroxypropylcellulose (MHPC) and polyvinyl alcohol (PVA). Examples of multi-coated enzyme granules are described in WO 93 / 07263 and WO 97 / 23606.

[0157] The cores can be prepared by granulating a blend of ingredients by methods including granulation techniques such as, for example, crystallization, precipitation, pan coating, fluid bed coating, fluid bed agglomeration, rotary spraying, extrusion, prilling, spheronization, size reduction methods, drum granulation and / or high shear granulation.

[0158] Methods for preparing the cores can be found in Handbook of Powder Technology; Particle size enlargement by CECapes; Vol. 1; 1980; Elsevier. Preparation methods include known feeding and granule blending techniques, such as: (a) Spray-dried products, in which a liquid polypeptide-containing solution is atomized in a spray-drying tower to form droplets that are dried during passage through the drying tower to form a polypeptide-containing particulate material, thus producing microparticles (Michael S. Showell (editor); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker). (b) Layered products, in which a polypeptide is coated as a layer surrounding a preformed inert core particle, a polypeptide-containing solution is atomized, typically in a fluidized bed apparatus, the preformed core particle is fluidized, the polypeptide-containing solution adheres to the core particle, and the core particle is completely dried, leaving a layer of dried polypeptide on the surface of the core particle. If a useful core particle of the desired particle size can be found, particles of the desired particle size can be obtained in this way. This type of product is described, for example, in WO 97 / 23606. (c) Adsorbed core particles, in which the polypeptide is adsorbed onto and / or into the surface of the core, rather than being coated as a layer around the core. Such a process is described in WO 97 / 39116. (d) Extruded or pelletized products, in which a polypeptide-containing paste is pressed into pellets or extruded under pressure through small orifices and cut into particles, which are then dried. Such particles usually have a significant size because the material from which the extrusion orifices are made (usually a perforated plate) limits the allowable pressure drop across the orifice. When small orifices are used, very high extrusion pressures also increase heat generation within the polypeptide paste, which is detrimental to the polypeptide (Michael S. Showell (editor); Powdered Detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker). (e) A granulated product in which a polypeptide-containing powder is suspended in molten wax and the suspension is sprayed, for example, with a rotating disk atomizer, into a cooled chamber where the droplets rapidly solidify (Michael S. Showell (editor); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker). The resulting product is one in which the polypeptide is uniformly distributed throughout the inert material instead of being concentrated on its surface. U.S. Pat. Nos. 4,016,040 and 4,713,245 describe this technique. (f) Mixer-granulated products in which a polypeptide-containing liquid is added to a dry powder composition of conventional granulation components. The liquid and powder are mixed in the appropriate proportions, and as the moisture from the liquid is absorbed into the dry powder, the dry powder components begin to adhere and aggregate, resulting in particle deposition and the formation of granules containing the polypeptide. Such processes are described in U.S. Pat. No. 4,106,991, EP 170360, EP 304332, EP 304331, WO 90 / 09440, and WO 90 / 09428. In certain products of this process, various high-shear mixers can be used as granulators. Granules consisting of polypeptide, fillers, and binders are mixed with cellulose fibers to reinforce the particles, producing so-called T-granules. The reinforced particles are more robust and less likely to release enzyme dust. (g) Size reduction, in which cores are produced by grinding or crushing larger particles, pellets, tablets, briquettes, etc., containing the polypeptide. The desired core particle fraction is obtained by sieving the ground or crushed product. The large and small particles can be recycled. Size reduction is described in Martin Rhodes (editor); Principles of Powder Technology; 1990; Chapter 10; John Wiley & Sons. (h) Fluidized Bed Granulation. Fluidized bed granulation involves suspending fine particles in an air stream and spraying a liquid through a nozzle onto the fluidized particles. Particles hit by the spray droplets become wet and sticky. The sticky particles collide with other particles, causing them to adhere to each other, forming granules. (i) The cores may be subjected to drying, such as in a fluidized bed dryer. Other known methods for drying granules in the feed or enzyme industries may be used by those skilled in the art. Drying is preferably carried out at a product temperature of 25 to 90°C. For some polypeptides, it is important that the core containing the polypeptide contains a small amount of water before coating with salt. If a water-sensitive polypeptide is coated with salt before removing excess water, the excess water may become trapped within the core, which may adversely affect the activity of the polypeptide. After drying, the cores preferably contain 0.1 to 10% water by weight.

[0159] Non-shattering granules may be produced, for example, as disclosed in US Pat. Nos. 4,106,991 and 4,661,452, and may optionally be coated by methods known in the art.

[0160] The granules may further comprise one or more additional enzymes, such as hydrolases, isomerases, ligases, lyases, oxidoreductases, and transferases. The one or more additional enzymes are preferably selected from the group consisting of acetyl xylan esterase, acylglycerol lipase, amylase, alpha-amylase, beta-amylase, arabinofuranosidase, cellobiohydrolase, cellulase, feruloyl esterase, galactanase, alpha-galactosidase, beta-galactosidase, beta-glucanase, beta-glucosidase, lysophospholipase, lysozyme, alpha-mannosidase, beta-mannosidase (mannanase), phytase, phospholipase A1, phospholipase A2, phospholipase D, protease, pullulanase, pectinesterase, triacylglycerol lipase, xylanase, beta-xylosidase, or any combination thereof. Each enzyme will then be present in more granules, ensuring a more uniform enzyme distribution and also reducing the physical separation of the various enzymes due to different particle sizes. A method for making multi-enzyme co-granules is disclosed in ip.com disclosure IPCOM000200739D.

[0161] Another example of the formulation of polypeptides through the use of co-granules is disclosed in WO 2013 / 188331.

[0162] The present invention also relates to protected polypeptides prepared according to the methods disclosed in EP 238216.

[0163] liquid formulation The present invention also relates to liquid compositions comprising the polypeptides of the present invention. The compositions may contain enzyme stabilizers (examples include polyols such as propylene glycol or glycerol, sugars or sugar alcohols, lactic acid, reversible protease inhibitors, boric acid or boric acid derivatives, such as aromatic borate esters, or phenylboronic acid derivatives, such as 4-formylphenylboronic acid).

[0164] In some embodiments, fillers or carrier materials are included to increase the volume of such compositions. Suitable fillers or carrier materials include, but are not limited to, various salts of sulfates, carbonates, and silicates, as well as talc, clay, and the like. Suitable fillers or carrier materials for liquid compositions include, but are not limited to, water or low molecular weight primary and secondary alcohols, including polyols and diols. Examples of such alcohols include, but are not limited to, methanol, ethanol, propanol, and isopropanol. In some embodiments, the compositions contain from about 5% to about 90% of such materials.

[0165] In one aspect, the liquid formulation comprises 20-80% by weight of a polyol. In one embodiment, the liquid formulation comprises 0.001-2% by weight of a preservative.

[0166] In another embodiment, the present invention provides (a) 0.001 to 25% by weight of the polypeptide of the present invention having deamidase inhibitor activity; (b) 20 to 80 wt. % of a polyol; (c) optionally, 0.001 to 2% by weight of a preservative; and (d) water The present invention relates to a liquid formulation comprising:

[0167] In another embodiment, the present invention provides (a) 0.001 to 25% by weight of the polypeptide of the present invention having deamidase inhibitor activity; (b) 0.001 to 2% by weight of a preservative; (c) optionally, 20 to 80 wt. % of a polyol, and (d) water The present invention relates to a liquid formulation comprising:

[0168] Preferably, the liquid compositions of the above two embodiments further contain a polypeptide having deamidase activity, for example, in an amount of 0.001 to 25% by weight.

[0169] In another embodiment, the liquid formulation comprises one or more formulating agents selected from the group consisting of polyols, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, PVA, acetates, and phosphates, preferably sodium sulfate, dextrin, cellulose, sodium thiosulfate, kaolin, and calcium carbonate. In one embodiment, the polyol is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propylene glycol or 1,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight of less than about 600, and polypropylene glycol (PPG) having an average molecular weight of less than about 600, more preferably glycerol, sorbitol, and propylene glycol (MPG), or any combination thereof.

[0170] In another embodiment, the liquid formulation comprises 20-80% polyol (i.e., total amount of polyol), e.g., 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40-60% polyol. In one embodiment, the liquid formulation comprises 20-80% polyol, e.g., 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40-60% polyol, where the polyol is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propylene glycol or 1,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight of less than about 600, and polypropylene glycol (PPG) having an average molecular weight of less than about 600. In one embodiment, the liquid formulation comprises 20-80% polyol (i.e., total amount of polyol), for example 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40-60% polyol, wherein the polyol is selected from the group consisting of glycerol, sorbitol, and propylene glycol (MPG).

[0171] In another embodiment, the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate, and potassium benzoate, or any combination thereof. In one embodiment, the liquid formulation comprises 0.02-1.5 wt.% preservative, e.g., 0.05-1 wt.% preservative, or 0.1-0.5 wt.% preservative. In one embodiment, the liquid formulation comprises 0.001-2 wt.% preservative (i.e., total amount of preservative), e.g., 0.02-1.5 wt.% preservative, 0.05-1 wt.% preservative, or 0.1-0.5 wt.% preservative, wherein the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate, and potassium benzoate, or any combination thereof.

[0172] In another embodiment, the liquid formulation further comprises one or more additional enzymes, such as hydrolases, isomerases, ligases, lyases, oxidoreductases, and transferases. Preferably, the one or more additional enzymes are selected from the group consisting of acetyl xylan esterase, acylglycerol lipase, amylase, alpha-amylase, beta-amylase, arabinofuranosidase, cellobiohydrolase, cellulase, feruloyl esterase, galactanase, alpha-galactosidase, beta-galactosidase, beta-glucanase, beta-glucosidase, lysophospholipase, lysozyme, alpha-mannosidase, beta-mannosidase (mannanase), phytase, phospholipase A1, phospholipase A2, phospholipase D, protease, pullulanase, pectinesterase, triacylglycerol lipase, xylanase, beta-xylosidase, or any combination thereof.

[0173] Fermentation Broth Composition or Cell Composition The present invention also relates to fermentation broth formulations or cell compositions comprising a polypeptide of the invention. The fermentation broth formulation or cell composition further comprises additional components used in the fermentation process, such as, for example, cells (including host cells containing a gene encoding a polypeptide of the invention used to produce the polypeptide of interest), cell debris, biomass, fermentation medium, and / or fermentation products. In some embodiments, the composition is a killed whole broth containing organic acids, killed cells and / or cell debris, and culture medium.

[0174] The term "fermentation broth," as used herein, refers to a preparation produced by cell fermentation with no or minimal recovery and / or purification. For example, a fermentation broth is produced when a microbial culture is grown to saturation, incubated under carbon-limiting conditions, and proteins are synthesized (e.g., enzymes expressed by the host cells) and secreted into the cell culture medium. Fermentation broth can contain the unfractionated or fractionated contents of the fermentation material obtained at the end of fermentation. Typically, fermentation broth is unfractionated and includes spent culture medium and cellular debris present after the microbial cells (e.g., filamentous fungal cells) have been removed, for example, by centrifugation. In some embodiments, the fermentation broth contains spent cell culture medium, extracellular enzymes, and viable and / or non-viable microbial cells.

[0175] In some embodiments, the fermentation broth formulation or cell composition comprises a first organic acid component and / or salt thereof comprising an organic acid of at least 1-5 carbons and a second organic acid component and / or salt thereof comprising an organic acid of at least 1-6 or more carbons. In some embodiments, the first organic acid component is acetic acid, formic acid, propionic acid, a salt thereof, or a mixture of two or more of the foregoing, and the second organic acid component is benzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid, phenylacetic acid, a salt thereof, or a mixture of two or more of the foregoing.

[0176] In one aspect, the composition contains an organic acid and optionally further contains dead cells and / or cell debris, hi some embodiments, the dead cells and / or cell debris are removed from the cell-killed whole broth to obtain a composition free of these components.

[0177] The fermentation broth formulation or cell composition may further comprise preservatives and / or antimicrobial (e.g., bacteriostatic) agents, including, but not limited to, sorbitol, sodium chloride, potassium sorbate, and others known in the art.

[0178] The cell-killed whole broth or cell composition may contain the unfractionated contents of the fermentation material obtained at the end of fermentation. Typically, the cell-killed whole broth or cell composition contains spent culture medium and cell debris present after microbial cells (e.g., filamentous fungal cells) have been grown to saturation and incubated under carbon-limited conditions that allow protein synthesis. In some embodiments, the cell-killed whole broth or cell composition contains spent cell culture medium, extracellular enzymes, and killed filamentous fungal cells. In some embodiments, the microbial cells present in the cell-killed whole broth or composition can be permeabilized and / or lysed using methods known in the art.

[0179] Whole broth or cell compositions as described herein are typically liquid but may contain insoluble components such as dead cells, cell debris, culture medium components, and / or one or more insoluble enzymes. In some embodiments, the insoluble components may be removed to obtain a clarified liquid composition.

[0180] The whole broth formulations and cell compositions of the present invention may be produced by the methods described in WO 90 / 15861 or WO 2010 / 096673.

[0181] The present invention is further defined by the following numbered embodiments:

[0182] Embodiment 1. (a) a polypeptide having at least 60% amino acid sequence identity to SEQ ID NO:2; (b) a polypeptide derived from SEQ ID NO: 2 by having 1 to 30 alterations (e.g., substitutions, deletions, and / or insertions) at one or more positions; (c) a polypeptide derived from the polypeptide of (a) or (b), wherein the N-terminus and / or C-terminus are extended by the addition of one or more amino acids; and (d) a fragment of the polypeptide of (a), (b), or (c). A polypeptide having deamidase inhibitor activity selected from the group consisting of:

[0183] Embodiment 2. The polypeptide of embodiment 1, which is derived from SEQ ID NO: 2 by having 1 to 25 modifications.

[0184] Embodiment 3. The polypeptide of embodiment 1, which is derived from SEQ ID NO: 2 by having 1 to 20 modifications.

[0185] Embodiment 4. The polypeptide of embodiment 1, which is derived from SEQ ID NO: 2 by having 1 to 15 modifications.

[0186] Embodiment 5. The polypeptide of embodiment 1, which is derived from SEQ ID NO: 2 by having 1 to 10 modifications.

[0187] Embodiment 6. The polypeptide of embodiment 1, which is derived from SEQ ID NO: 2 by having 1 to 5 modifications.

[0188] Embodiment 7. The polypeptide of any one of embodiments 1 to 6, wherein the modification is a substitution, deletion, and / or insertion, preferably a substitution.

[0189] Embodiment 8. The polypeptide of any one of embodiments 1 to 7, wherein the modification is a substitution.

[0190] Embodiment 9. The polypeptide of embodiment 1, having at least 70% amino acid sequence identity to SEQ ID NO:2.

[0191] Embodiment 10. The polypeptide of embodiment 1, having at least 80% amino acid sequence identity to SEQ ID NO:2.

[0192] Embodiment 11. The polypeptide of embodiment 1, having at least 90% amino acid sequence identity to SEQ ID NO:2.

[0193] Embodiment 12. The polypeptide of embodiment 1, having at least 95% amino acid sequence identity to SEQ ID NO:2.

[0194] Embodiment 13. The polypeptide of embodiment 1, having at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO:2.

[0195] Embodiment 14. The polypeptide of any one of embodiments 1 to 13, wherein the N-terminus and / or C-terminus is extended by the addition of 1 to 20 amino acids.

[0196] Embodiment 15. The polypeptide of any one of embodiments 1 to 14, wherein the N-terminus and / or C-terminus is extended by the addition of 1 to 10 amino acids.

[0197] Embodiment 16. The polypeptide of any one of embodiments 1 to 15, wherein the N-terminus and / or C-terminus is extended by the addition of 1 to 5 amino acids.

[0198] Embodiment 17. The polypeptide of any one of embodiments 1 to 16, comprising the amino acid sequence motif F[F / Y][I / L / V][F / Q / S][E / K / R].

[0199] Embodiment 18. The polypeptide of any one of embodiments 1 to 17, comprising the amino acid sequence motif L[I,T]WY[D,H,K,N].

[0200] Embodiment 19. The polypeptide of any one of embodiments 1 to 18, comprising the amino acid sequence motif G[I,M]S[A,P,Q]Q.

[0201] Embodiment 20. The polypeptide of any one of embodiments 1 to 19, comprising the amino acid sequence motif [D,H,K,N,S][I,L][G,V][I,V][D,E].

[0202] Embodiment 21. The polypeptide of any one of embodiments 1 to 20, comprising the amino acid sequence motif [N,H][I,L,M,V,Q][I,V][K,R,Q][E,I,Q].

[0203] Embodiment 22. The polypeptide of any one of embodiments 1 to 21, comprising the amino acid sequence motif [D / N][P / S][D / E][H / K / N / Q / R][A / P / S].

[0204] Embodiment 23. The polypeptide of any one of embodiments 1 to 22, comprising an amino acid sequence motif selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and combinations thereof.

[0205] Embodiment 24 The polypeptide of any one of embodiments 1 to 23, wherein the deamidase inhibitor activity reduces the deamidase activity to less than 90% activity.

[0206] Embodiment 25 The polypeptide of any one of embodiments 1 to 24, wherein the deamidase inhibitor activity reduces the deamidase activity to less than 80% activity.

[0207] Embodiment 26 The polypeptide of any one of embodiments 1 to 25, wherein the deamidase inhibitor activity reduces the deamidase activity to less than 70% activity.

[0208] Embodiment 27 The polypeptide of any one of embodiments 1 to 26, wherein the deamidase inhibitor activity reduces the deamidase activity to less than 60% activity.

[0209] Embodiment 28 The polypeptide of any one of embodiments 1 to 27, wherein the deamidase inhibitor activity reduces the deamidase activity to less than 50% activity.

[0210] Embodiment 29 The polypeptide of any one of embodiments 1 to 28, wherein the deamidase inhibitor activity reduces the deamidase activity to less than 40% activity.

[0211] Embodiment 30. The polypeptide of any one of embodiments 1 to 29, wherein the deamidase activity is derived from a deamidase comprising an amino acid sequence motif selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and combinations thereof.

[0212] Embodiment 31. The polypeptide of any one of embodiments 1 to 30, wherein the deamidase activity is derived from a polypeptide having deamidase activity derived from a Chryseobacterium species.

[0213] Embodiment 32 The polypeptide of any one of embodiments 1 to 31, wherein the deamidase activity is derived from the polypeptide set forth as SEQ ID NO:4.

[0214] Embodiment 33. A polynucleotide encoding the polypeptide of any one of embodiments 1 to 32.

[0215] Embodiment 34. The polynucleotide of embodiment 33, which is isolated and / or purified.

[0216] Embodiment 35. A nucleic acid construct or expression vector comprising the polynucleotide of embodiment 33, wherein the polynucleotide is operably linked to one or more control sequences that direct the production of the polypeptide in an expression host.

[0217] Embodiment 36. A recombinant host cell comprising the nucleic acid construct or expression vector of embodiment 35.

[0218] Embodiment 37. The recombinant host cell of embodiment 36, wherein the polynucleotide is heterologous to the recombinant host cell.

[0219] Embodiment 38. The recombinant host cell of embodiment 36 or 37, wherein at least one of the one or more regulatory sequences is heterologous to the polynucleotide encoding the polypeptide.

[0220] Embodiment 39. The recombinant host cell of any one of embodiments 36 to 38, comprising at least two copies, for example, three, four, or five or more copies, of the polynucleotide of any one of embodiments 33 to 35.

[0221] Embodiment 40. A yeast recombinant host cell, for example a cell of the genus Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces or Yarrowia, for example Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri. 40. The recombinant host cell of any one of embodiments 36 to 39, which is a cell of Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis or Yarrowia lipolytica.

[0222] Embodiment 41. Filamentous fungal recombinant host cells, such as Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocalima. Cells of the genera Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes or Trichoderma, in particular Aspergillus awamori awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirinaaneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium meldarium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulantumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Talaromyces emersonii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum 40. The recombinant host cell of any one of embodiments 36-39, which is a Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

[0223] Embodiment 42. A prokaryotic recombinant host cell, for example a Gram-positive cell selected from the group consisting of a Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus or Streptomyces cell, or a Campylobacter, Escherichia coli (E.Gram-negative bacteria selected from the group consisting of Bacillus coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma cells, such as Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus arginense ... firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis uberis) and Streptococcus equi subsp. zooepidemicus (Streptococcus equi subsp.40. The recombinant host cell of any one of embodiments 36 to 39, which is a Streptomyces zooepidemicus, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cell.

[0224] Embodiment 43. The recombinant host cell of any one of embodiments 36 to 39, which is a Bacillus licheniformis cell.

[0225] Embodiment 44. The recombinant host cell of any one of embodiments 36 to 43, which is isolated.

[0226] Embodiment 45. The recombinant host cell of any one of embodiments 36 to 44, which is purified.

[0227] Embodiment 46. The recombinant host cell of any one of embodiments 36 to 45, further comprising a co-expressed polypeptide that exhibits deamidase activity, preferably wherein the co-expressed polypeptide that exhibits deamidase activity is the polypeptide set forth as SEQ ID NO: 4 or a polypeptide having at least 80%, 90%, or 95% amino acid sequence identity to SEQ ID NO: 4.

[0228] Embodiment 47. A method for producing the polypeptide of any one of embodiments 1 to 32, comprising culturing a recombinant host cell of any one of embodiments 36 to 45 under conditions conducive to production of the polypeptide.

[0229] Embodiment 48. The method of embodiment 47, further comprising recovering the polypeptide.

[0230] Embodiment 49. (a) 0.001 to 25% by weight of the polypeptide of any one of embodiments 1 to 32; (b) a polyol, preferably 20 to 80 wt. % of a polyol, and (c) water A liquid composition comprising:

[0231] Embodiment 50 The liquid composition of embodiment 49, further comprising a polypeptide exhibiting deamidase activity.

[0232] Embodiment 51. The liquid composition of embodiment 49, further comprising 0.001 to 25% by weight of a polypeptide exhibiting deamidase activity.

[0233] Embodiment 52. The liquid composition of embodiment 50 or 51, wherein the polypeptide exhibiting deamidase activity has at least 80%, 90%, or 95% amino acid sequence identity to the polypeptide set forth as SEQ ID NO:4.

[0234] Embodiment 53. The liquid composition of any one of embodiments 50 to 52, wherein the polypeptide exhibiting deamidase activity is the polypeptide set forth as SEQ ID NO: 4.

[0235] Embodiment 54. A method for producing a polypeptide having deamidase activity, comprising culturing a recombinant host cell of embodiment 46 under conditions conducive to production of a polypeptide having deamidase activity.

[0236] Embodiment 55 The method of embodiment 54, further comprising recovering the polypeptide having deamidase activity.

[0237] Embodiment 56 The method of embodiment 55, wherein recovering the polypeptide having deamidase activity comprises diafiltration.

[0238] The present invention is further described by the following examples, which should not be construed as limiting the scope of the invention. [Example]

[0239] KK Chryseobacterium sp.-62563 was isolated from a soil sample collected in September 2013 in Sibhult, Sweden.

[0240] Example 1 Deamidase activity material Glutamyl endopeptidase from Bacillus licheniformis FITC-PP-Dnp (FITC-Ahx-His-His-Gln-Ser-Ser-ED-Dnp) is a custom synthesized substrate molecule from TAG Copenhagen, Kong Georgs Vej 12, DK-2000 Frederiksberg (www.tagc.com). FITC is a fluorescein with excitation and emission maxima at approximately 490 nm and 520 nm; Ahx is aminohexanoic acid ED is ethylenediamine, and Dnp is 2,4-dinitrophenyl (fluorescein fluorescence quencher).

[0241] Deamidase activity assay The assay measures the (relative) activity of the active deamidase.

[0242] 50 μL of the deamidase sample was transferred to a standard black 96-well plate. 20μL of 0.25μg / mL FITC-PP-Dnp 50 μL of 50 μg / mL glutamyl endopeptidase 130 μL of 100 mM HEPES buffer pH 7.0, and 0.01% v / v Triton X detergent was added.

[0243] The fluorescence signal (RFU) was measured using a Biotek Synergy H1 fluorescent plate reader for 30 min using excitation / emission wavelengths of 485 nm / 525 nm.

[0244] Data were analyzed for initial velocity using variable time intervals depending on the shape of the curve (signal vs. time). The initial velocity for each sample was normalized to the initial velocity of a fully active deamidase reference enzyme (produced by a wild-type donor strain). Activity was measured as "% initial velocity," i.e., the initial velocity of a sample molecule relative to the initial velocity of the % active deamidase reference enzyme.

[0245] When a deamidase inhibitor of the present invention (SEQ ID NO: 2) was added to a sample containing active deamidase (SEQ ID NO: 4) at approximately a 1:1 ratio (deamidase:inhibitor), the deamidase activity (% initial rate) was significantly reduced.

[0246] Example 2 Nano Differential Scanning Fluorescence (nanoDSF) - Thermal Denaturation Temperature Nanodifferential scanning fluorimetry (nanoDSF) was used to assess the conformational stability of deamidase / inhibitor complexes using deamidase inhibitors of the present invention. Molecules were exposed to a temperature gradient as shown below. The resulting conformational changes are reflected by changes in fluorescence intensity, providing a measure of thermal stability. Binding of the propeptide domain to the deamidase inhibitor contributes to the stability of the molecule; therefore, nanoDSF thermal denaturation temperatures also provide information on deamidase / inhibitor binding affinity.

[0247] His-tag purified samples were received in elution buffer from an IMAC (immobilized metal affinity chromatography) column; 20 mM sodium phosphate; 500 mM sodium chloride; 500 mM imidazole; pH 7.4.

[0248] 60 μL of sample was transferred in duplicate to a black bottom 384-well plate and the plate was briefly centrifuged to remove potential air bubbles.

[0249] The thermal denaturation temperatures of the samples were analyzed using a Prometheus NT.Plex system from NanoTemper Technologies GmbH with the following settings: (i) temperature scan rate: 3.3°C per minute; and (ii) Temperature scan interval: 20–95°C.

[0250] During the analysis, samples were loaded into capillaries (Prometheus NT.Plex-Capillary Chips, Standard, Cat#PR-AC002) and then subjected to a temperature gradient. The generated data was analyzed by PR.Stability analysis v.1.0.1 software. The denaturation midpoint temperature (T m , °C) were annotated based on the first derivative of the trace at 330 nm. In some cases, two or more T m In these cases, the main peak in the first derivative trace at 330 nm corresponds to the T m was selected as.

[0251] Example 3 Thermal denaturation temperature of the deamidase / inhibitor complex The nanoDSF procedure was used to measure the thermal denaturation temperatures of the active deamidase and the corresponding deamidase / inhibitor complexes, as described in Example 2.

[0252] [Table 1]

Claims

1. (a) a polypeptide having at least 60% amino acid sequence identity to SEQ ID NO:2; (b) a polypeptide derived from SEQ ID NO: 2 by having 1 to 30 alterations (e.g., substitutions, deletions, and / or insertions), for example 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23, or 24, or 25, or 26, or 27, or 28, or 29, or 30 alterations, particularly substitutions, at one or more positions; (c) a polypeptide derived from the polypeptide of (a) or (b), wherein the N-terminus and / or C-terminus is extended by the addition of one or more amino acids; and (d) a fragment of the polypeptide of (a), (b), or (c). A polypeptide having deamidase inhibitor activity selected from the group consisting of:

2. 2. The polypeptide of claim 1, comprising an amino acid sequence motif selected from the group consisting of F[F / Y][I / L / V][F / Q / S][E / K / R], L[I,T]WY[D,H,K,N], G[I,M]S[A,P,Q]Q, [D,H,K,N,S][I,L][G,V][I,V][D,E], [N,H][I,L,M,V,Q][I,V][K,R,Q][E,I,Q], [D / N][P / S][D / E][H / K / N / Q / R][A / P / S], and combinations thereof.

3. 2. The polypeptide of claim 1, which also comprises an amino acid sequence motif selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and combinations thereof.

4. 2. The polypeptide of claim 1, comprising, consisting essentially of, or consisting of SEQ ID NO:

2.

5. 5. The polypeptide of any one of claims 1 to 4, wherein the deamidase inhibitor activity reduces the deamidase activity to less than 90% of the activity, preferably, the deamidase inhibitor activity reduces the deamidase activity of the polypeptide set forth as SEQ ID NO: 4 to less than 90%.

6. A polynucleotide encoding the polypeptide of any one of claims 1 to 5.

7. 7. A nucleic acid construct or expression vector comprising the polynucleotide of claim 6, wherein the polynucleotide is operably linked to one or more control sequences that direct the production of the polypeptide in an expression host.

8. A recombinant host cell comprising the nucleic acid construct or expression vector of claim 7.

9. A method for producing a polypeptide according to any one of claims 1 to 5, comprising culturing a recombinant host cell according to claim 8 under conditions conducive to the production of said polypeptide.

10. (a) 0.001 to 25% by weight of the polypeptide according to any one of claims 1 to 5; (b) a polyol, and (c) water A liquid composition comprising:

11. The liquid composition according to claim 10, further comprising a polypeptide exhibiting deamidase activity, preferably in an amount of 0.001 to 25% by weight.

12. 12. The liquid composition of claim 11, wherein the polypeptide exhibiting deamidase activity is a polypeptide set forth as SEQ ID NO: 4 or a polypeptide having at least 80%, 90%, or 95% amino acid sequence identity to SEQ ID NO:

4.

13. 9. The recombinant host cell of claim 8, further comprising a co-expressed polypeptide exhibiting deamidase activity, preferably wherein the co-expressed polypeptide exhibiting deamidase activity is the polypeptide set forth as SEQ ID NO:4 or a polypeptide having at least 80%, 90%, or 95% amino acid sequence identity to SEQ ID NO:

4.

14. 14. A method for producing a polypeptide having deamidase activity, comprising culturing the recombinant host cell of claim 13 under conditions that promote the production of said polypeptide having deamidase activity.

15. 15. The method of claim 14, further comprising recovering the polypeptide having deamidase activity.

16. 16. The method of claim 15, wherein said recovering said polypeptide having deamidase activity comprises diafiltration.

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