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 comprising the polynucleotides as well as methods of producing and using the polypeptides.

Description

[0001] References to sequence listings

[0002] This application contains a sequence list in computer-readable form, which is incorporated herein by reference. Technical Field

[0003] This invention relates to polypeptides with deamidase inhibitor activity, polynucleotides encoding these polypeptides, nucleic acid constructs containing these polynucleotides, vectors, and host cells, along with methods for producing and using these polypeptides. Background Art

[0004] Deamidases are produced by microbial cells in an inactive proform, which contains a propeptide domain tightly bound to the deamidase domain. This proform possesses almost no deamidase activity to protect the host cell's viability. Essentially, this 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 proform of the deamidase is secreted extracellularly.

[0005] Due to the high binding affinity of the propeptide to the deamidase peptide, the propeptide domain cannot be separated from the deamidase domain by simple cleavage. This binding affinity can be manipulated through mutagenesis to make the propeptide and active deamidase more easily separated, but this leads to partial activation of the intracellular deamidase, reducing the viability of the host cell.

[0006] The purpose of this invention is to provide a novel deamidase inhibitor that can be co-expressed with deamidases to improve the recombinant expression of deamidases by mitigating the negative impact of active intracellular deamidases on host cell viability. Summary of the Invention

[0007] This invention provides a polypeptide capable of inhibiting deamidase activity by reversibly binding to and interacting with the active site of deamidase.

[0008] Therefore, in a first aspect, the present invention relates to polypeptides having deamidase inhibitor activity, the polypeptides being selected from the group consisting of:

[0009] (a) A polypeptide having at least 60% amino acid sequence identity with SEQ ID NO: 2;

[0010] (b) A polypeptide derived from SEQ ID NO: 2 by having 1 to 30 alterations (e.g., substitution, deletion and / or insertion) at one or more locations, such as 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;

[0011] (c) A polypeptide derived from (a) or (b) wherein the N- and / or C-terminus has been extended by adding one or more amino acids; and

[0012] Fragments of the polypeptides in (d), (a), (b), or (c).

[0013] In another aspect, the present invention relates to polynucleotides encoding the polypeptides of the present invention; nucleic acid constructs comprising these polynucleotides, recombinant expression vectors, recombinant host cells; and methods for producing these polypeptides.

[0014] Other aspects and embodiments of the invention will be apparent from the description and examples.

[0015] sequence

[0016] SEQ ID NO: 1: Encoding a polynucleotide of a deamidase inhibitor from species-62563 of the genus *Cyclocarya*.

[0017] SEQ ID NO: 2: The amino acid sequence of the deamidase inhibitor encoded by SEQ ID NO: 1.

[0018] SEQ ID NO: 3: Encoding a polynucleotide of an active deamidase from species-62563 of the genus *Citrus*.

[0019] SEQ ID NO: 4: The amino acid sequence of the deamidase encoded by SEQ ID NO: 3.

[0020] SEQ ID NO: 5: Amino acid sequence motif of a deamidase inhibitor.

[0021] SEQ ID NO: 6: Amino acid sequence motif of a deamidase inhibitor.

[0022] SEQ ID NO: 7: Amino acid sequence motif of a deamidase inhibitor.

[0023] SEQ ID NO: 8: Amino acid sequence motif of a deamidase inhibitor.

[0024] SEQ ID NO: 9: Amino acid sequence motif of a deamidase inhibitor.

[0025] SEQ ID NO: 10: Amino acid sequence motif of a deamidase inhibitor.

[0026] SEQ ID NO: 11: Amino acid sequence motif of a deamidase inhibitor.

[0027] SEQ ID NO: 12: Amino acid sequence motif of a deamidase inhibitor.

[0028] SEQ ID NO: 13: Amino acid sequence motif of a deamidase inhibitor.

[0029] SEQ ID NO: 14: Amino acid sequence motif of the deamidase active site.

[0030] SEQ ID NO: 15: Amino acid sequence motif of the deamidase active site.

[0031] SEQ ID NO: 16: Amino acid sequence motif of the deamidase active site.

[0032] definition

[0033] Based on this detailed description, the following definitions apply. Note that the singular forms “a / an” and “the” include plural indicators unless the context explicitly indicates otherwise.

[0034] Unless otherwise defined or explicitly indicated by the 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 pertains.

[0035] Deamidase activity: The term "deamidase activity" refers to the activity of protein-glutamylglutaminase (also known as glutamylpeptide glutaminase), as described in EC 3.5.1.44, which catalyzes the hydrolysis of the γ-amide of glutamine substituted at the carboxyl position or both the α-amino and carboxyl positions, such as L-glutamylglycine and L-phenylalanyl-L-glutamylglycine. Peptides exhibiting deamidase activity are generally also called deamidases. Therefore, deamidases can deamidate glutamine residues in proteins to glutamate residues, and deamidases are also referred to as protein glutamine deamidases. Deamidases include the 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 proforms converted into enzyme-substrate complex,” Journal of Biological Chemistry, Vol. 286, No. 44, pp. 38691-38702) and belong 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) corresponding 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) corresponding 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) corresponding to amino acid residues 83-89 of SEQ ID NO: 4. These motifs overlap with the deamidase active site. Preferably, the deamidase activity is the activity exhibited by the polypeptide as shown in SEQ ID NO: 4.

[0036] As described in Example 1, deamidase activity was measured using a fluorescent substrate containing glutamine residues and a fluorescence quencher group. The glutamine residues were converted to glutamate residues by deamidase activity, and then the substrate was cleaved by glutamyl endopeptidase to remove the fluorescence quencher group.

[0037] Deamidase activity can also be measured by deamidating glutamine substrates (e.g., Cbz-Gln-Gly), in which ammonia is generated. Ammonia is used as a substrate for glutamate dehydrogenase in combination with α-ketoglutarate to produce glutamate. This latter enzymatic reaction requires NADH as a coenzyme. NADH consumption can be tracked by measuring the kinetic absorbance at 340 nm and is directly proportional to deamidase activity. The reaction is carried out at pH 7 and 37°C.

[0038] Deamidase Inhibitor: The term "deamidase inhibitor" refers to an amino acid sequence that interacts with the amino acid residues at the active site of a deamidase. Therefore, deamidase inhibitor activity reduces or inhibits deamidase activity, preferably the deamidase activity exhibited by a polypeptide as shown in SEQ ID NO: 4. For example, in the presence of a deamidase inhibitor, 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 deamidase activity in the absence of a deamidase inhibitor). Polypeptides exhibiting deamidase inhibitor activity are generally also referred to as deamidase inhibitors. Deamidase inhibitors may contain amino acid sequence motifs selected from the following groups: 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 may also contain amino acid sequence motifs 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.

[0039] cDNA: The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from mature, spliced ​​mRNA molecules obtained from eukaryotic or prokaryotic cells. cDNA lacks intron sequences that can be present in the corresponding genomic DNA. The initial primary RNA transcript is the precursor of mRNA, which is processed through a series of steps (including splicing) to become mature, spliced ​​mRNA.

[0040] Coding sequence: The term "coding sequence" refers to a polynucleotide that directly specifies the amino acid sequence of a polypeptide. The boundaries of a coding sequence are typically defined by an open reading frame (OPG), which begins with a start codon (such as ATG, GTG, or TTG) and ends with a stop codon (such as TAA, TAG, or TGA). Coding sequences can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0041] Control Sequences: The term "control sequence" refers to a nucleic acid sequence involved in regulating the expression of polynucleotides in a particular organism, either in vivo or in vitro. Each control sequence can be native (i.e., from the same gene) or heterologous (i.e., from different genes) for the polynucleotide encoding a polypeptide, and is native or heterologous to each other. Such control sequences include, but are not limited to, leader sequences, polyadenylated sequences, propeptides, propeptides, signal peptides, promoters, terminators, enhancers, and transcription or translation initiator and terminator sequences. At a minimum, these control sequences include promoters and transcription and translation termination signals. These control sequences may be provided with multiple linkers for the purpose of introducing specific restriction sites that facilitate the linking of control sequences to the coding regions of polynucleotides encoding polypeptides.

[0042] Expression: The term “expression” refers to any step involved in polypeptide production, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0043] Expression vector: An expression vector is a linear or circular DNA construct containing a DNA sequence encoding a polypeptide, which is operatively linked to a suitable control sequence capable of influencing DNA expression in a suitable host. Such control sequences may include promoters that influence transcription, optional operon sequences that control transcription, sequences encoding suitable ribosome binding sites on mRNA, enhancers, and sequences that control the termination of transcription and translation.

[0044] Extension: The term "extension" refers to the addition of one or more amino acids to the amino and / or carboxyl termini of a polypeptide, wherein the "extended" polypeptide has deamidase inhibitor activity.

[0045] Fragment: The term "fragment" refers to a polypeptide that has one or more amino acids missing from the amino and / or carboxyl termini of a mature polypeptide, wherein the fragment has deamidase inhibitory activity.

[0046] Fusion polypeptide: The term "fusion polypeptide" is a polypeptide in which one polypeptide of the present invention is fused to the N-terminus and / or C-terminus of another polypeptide of the present invention. Fusion polypeptides are generated by fusing together two or more polynucleotides encoding the polypeptide of the present invention. Techniques for generating fusion polypeptides are known in the art and include linking the coding sequences of the polypeptides such that they conform to reading frames, and that the expression of the fusion polypeptide is under the control of one or more identical promoters and terminators. Fusion polypeptides can also be constructed using intronomer techniques in which fusion polypeptides are generated post-translational (Cooper et al., 1993, EMBO J. [European Journal of Molecular Biology] 12: 2575-2583; Dawson et al., 1994, Science [Science] 266: 776-779). Fusion polypeptides may further include a cleavage site between the two polypeptides. Thus, these fusion polypeptides may include a site-specific endopeptidase cleavage site, for example, within 20 amino acids, preferably within 10 amino acids, at the C-terminus of the first polypeptide. Examples of well-known site-specific endopeptidases include gamma-glutamyl endopeptidases (e.g., EC 3.4.21.19 or EC 3.4.21.82), trypsin-like endopeptidases, and chymotrypsin-like endopeptidases (including intestinal peptidases). Many other examples of cleavage sites and corresponding endopeptidases include, but are not limited to, those disclosed in: Martin et al., 2003, J. Ind. Microbiol. Biotechnol. [Journal of Industrial Microbiology] 3: 568-576; Svetina et al., 2000, J. Biotechnol. [Journal of Biotechnology] 76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. [Applied and Environmental Microbiology] 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., 1989, Proteins: Structure, Function, and Genetics 6:240-248; and Stevens, 2003, Drug Discovery World 4:35-48.

[0047] Heterogeneous: For host cells, the term "heterogeneous" means that the polypeptide or nucleic acid is not naturally present in the host cell. For polypeptides or nucleic acids, the term "heterogeneous" means that the control sequence (e.g., the promoter of the polypeptide or nucleic acid) is not naturally associated with that polypeptide or nucleic acid; that is, the control sequence comes from a gene other than the gene encoding the mature polypeptide.

[0048] Host strain or host cell: A "host strain" or "host cell" is an organism in which an expression vector, bacteriophage, virus, or other DNA construct (including a polynucleotide encoding a target polypeptide, such as an amylase) has been introduced. Exemplary host strains are microbial cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing a target polypeptide and / or fermenting sugars. The term "host cell" includes protoplasts produced by cells.

[0049] Introduction: In the context of inserting a nucleic acid sequence into a cell, the term “introduction” means “transfection,” “conversion,” or “transduction,” as is known in the art.

[0050] Isolated: The term "isolated" means a polypeptide, nucleic acid, cell, or other specific material or component that has been separated from at least one other material or component (including, but not limited to, other proteins, nucleic acids, cells, etc.). Therefore, the isolated polypeptide, nucleic acid, cell, or other material exists in a form not found in nature. Isolated polypeptides include, but are not limited to, culture media containing secreted polypeptides expressed in host cells.

[0051] Mature polypeptide: The term "mature polypeptide" refers to a polypeptide that has undergone processing at its N-terminus and / or C-terminus (e.g., removal of the signal peptide) to obtain its mature form. In one respect, this mature polypeptide is SEQ ID NO: 2.

[0052] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" refers to a polynucleotide encoding a mature polypeptide with deamidase inhibitor activity. In one respect, this mature polypeptide coding sequence is SEQ ID NO: 1.

[0053] Natural: The term "natural" refers to nucleic acids or polypeptides that are naturally present in host cells.

[0054] Nucleic acid: The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding polypeptides. Nucleic acids can be single-stranded or double-stranded and can be chemically modified. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon can be used to encode a specific amino acid, and the compositions and methods of the present invention cover nucleotide sequences encoding specific amino acid sequences. Unless otherwise stated, nucleic acid sequences are presented in a 5′ to 3′ orientation.

[0055] Nucleic acid constructs: "Nucleic acid constructs" refer to single-stranded or double-stranded nucleic acid molecules that are isolated from naturally occurring genes or modified in a way that does not originally exist in nature to contain segments of nucleic acid or are synthesized and contain one or more control sequences that are operatively linked to the nucleic acid sequence.

[0056] Operationally linked: "Operationally linked" means that the specified components are in a relationship that allows them to function in the intended manner (including but not limited to juxtaposition). For example, a regulatory sequence is operationally linked to a coding sequence such that the expression of the coding sequence is under the control of the regulatory sequence.

[0057] Purified: The term "purified" means nucleic acids, peptides, or cells that are substantially free of other components, as determined by analytical techniques well known in the art (e.g., in electrophoretic gels, chromatographic eluates, and / or media subjected to density gradient centrifugation, where purified peptides or nucleic acids form discrete bands). Purified nucleic acids or peptides are at least about 50% pure, and typically at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or more pure (e.g., by weight or molar percentage). In a relevant sense, a composition is enriched with the molecule when the concentration of the molecule increases significantly after the application of purification or enrichment techniques. The term “enrichment” refers to the presence of compounds, peptides, cells, nucleic acids, amino acids, or other specified materials or components in a composition at a relative or absolute concentration higher than that of the starting composition.

[0058] In one respect, the term "purified," as used herein, means that the polypeptide or cell is substantially free of components (especially insoluble components) from the producing organism. In other respects, the term "purified" means that the polypeptide is substantially free of insoluble components (especially insoluble components) from the natural organism from which it was obtained. In one respect, the polypeptide is separated from some soluble components of the organism from which it was recovered and the culture medium. The polypeptide can be purified (i.e., separated) by one or more of the following methods: unit operation filtration, precipitation, or chromatography.

[0059] Accordingly, the polypeptide can be purified such that only small amounts of other proteins, particularly other polypeptides, are present. The term "purified" as used herein can mean the removal of other components, particularly other proteins and most particularly other enzymes, present in the cells from which the polypeptide originates. The polypeptide can be "substantially pure," meaning it is free from other components from the organism that produced it (e.g., the host organism used to recombinantly produce the polypeptide). In one aspect, the polypeptide is at least 40% pure by weight of the total polypeptide material present in the formulation. In another aspect, the polypeptide is at least 50%, 60%, 70%, 80%, or 90% pure by weight of the total polypeptide material present in the formulation. As used herein, "substantially pure polypeptide" can mean a polypeptide formulation containing, by weight, at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0.5% of the polypeptide and other polypeptide materials associated with it naturally or recombinantly.

[0060] Therefore, it is preferred that the substantially pure polypeptide, based on the weight of the total polypeptide material present in the formulation, is 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. The polypeptides of the present invention are preferably in a substantially pure form (i.e., the formulation is substantially free of other polypeptide materials associated with it, either naturally or recombinantly). This can be achieved, for example, by preparing the polypeptide using well-known recombinant methods or classical purification methods.

[0061] Recombination: The term "recombination," used in its conventional sense, refers to the manipulation (e.g., cutting and rejoining) of nucleic acid sequences to form a sequence group different from that found in nature. The term recombination refers to cells, nucleic acids, polypeptides, or vectors that have been modified from their natural state. Thus, for example, recombinant cells express genes not found in their natural (non-recombinant) forms, or express natural genes at different levels or under different conditions compared to those found in nature. The term "recombination" is synonymous with "genetically modified" and "transgenic."

[0062] Recovery: The term "recovery" refers to the removal of peptides from at least one fermentation broth component selected from a list of cells, nucleic acids, or other specified materials, for example, by means of: harvesting peptides from whole or cell-free fermentation broths by means of: peptide crystallization; filtration (e.g., deep filtration (using filter aids or packed filter media, cloth filtration in a box filter, rotary drum filtration, drum filtration, rotary vacuum drum filtration, candle filter, horizontal leaf filter, or the like, sheet or pad filtration in a frame or modular apparatus) or membrane filtration (using plate filtration, modular filtration, candle filtration, microfiltration, crossflow, dynamic crossflow, or ultrafiltration in dead-end operation)); or centrifugation (using a horizontal centrifuge, disc stack centrifuge, hyrdo cyclone, or the like); or by precipitation of peptides and using relevant solid-liquid separation methods to harvest peptides from broth media by means of particle size fractionation. Recovery encompasses the isolation and / or purification of peptides.

[0063] Sequence identity: The degree of association between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity".

[0064] For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. [Journal of Molecular Biology] 48: 443-453) is used to determine the sequence identity between two amino acid sequences as the output of "longest identity". This algorithm is implemented in the Needle program using the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. [Trends in Genetics] 16: 276-277) (preferably version 6.6.0 or later). The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. For the Needle program to report the longest identity, the -nobrief option must be specified in the command line. The output of "longest identity" marked by Needle is calculated as follows:

[0065] (identical residues × 100) / (alignment length - total number of vacancies in the alignment)

[0066] For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, ibid.) is used to determine the sequence identity between two polynucleotide sequences as the output of "longest identity," as implemented by the Needle program in the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, ibid.) (preferably version 6.6.0 or later). The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. For the Needle program to report the longest identity, the nobrief option must be specified on the command line. The Needle-marked "longest identity" output is calculated as follows:

[0067] (identical nucleotides x 100) / (alignment length - total number of gaps in alignment)

[0068] Signal peptide: A signal peptide is an amino acid sequence that attaches to the N-terminal portion of a protein and promotes its secretion outside the cell. The mature form of extracellular proteins lacks the signal peptide, which is cleaved during secretion.

[0069] Subsequence: The term “subsequence” refers to a polynucleotide in which one or more nucleotides are deleted from the 5′ and / or 3′ end of the coding sequence of a mature polypeptide; wherein the subsequence encodes a fragment having deamidase inhibitor activity.

[0070] Pyrolysis folding temperature: The term "pyrolysis folding temperature" is also known as "chain breaking temperature" or "T". m The term "midpoint unfolding temperature" or "middle point unfolding temperature" refers to the temperature at which approximately 50% of a protein is unfolded. Typically, the fraction of folded proteins is dominant at room temperature (>99.999%), and this fraction decreases as the temperature approaches the unfolding temperature (T0). m And thus decrease. In T m At this point, approximately 50% of the molecules are in a folded state, and approximately 50% are in an unfolded state. At temperatures above T... m At this time, the unfolded state becomes the dominant species (>50%). A preferred method for determining the pyrolysis folding temperature of the deamidase / inhibitor complex containing the inhibitor of the present invention is nanoDSF. Using nanoDSF, pyrolysis folding can be performed at 330 nm, 350 nm, and / or at a 330 nm / 350 nm ratio. m The determination of T, where T mThe temperature is equal to 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. For this invention, T m Alternatively, the pyrolysis folding temperature is the temperature at which the inflection point (IP) of the first derivative is determined by nanoDSF at 330 nm, as described in Example 2.

[0071] Other methods for determining the pyrolysis folding temperature include 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 methods described in Encyclopedia of Industrial Biotechnology: “Proteins: Thermal Unfolding” (R. Lonescu and L. Shi, 2009).

[0072] Thermal defolding represents an important tool for assessing protein stability. To evaluate a protein's preference for maintaining its folded (active) conformation, the protein is exposed to increased levels of denaturing stress (temperature), and protein stability is determined based on the stress level required to produce significant fractions of unfolded protein. Factors affecting 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.

[0073] Variant: The term "variant" refers to a polypeptide with deamidase inhibitor activity that contains artificial mutations (i.e., substitutions, insertions (including extensions), and / or deletions (e.g., truncations)) at one or more positions. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and insertion means adding 1-5 amino acids (e.g., 1-3 amino acids, especially 1 amino acid) adjacent to and immediately following an amino acid occupying a position.

[0074] Wild-type: When referring to an amino acid or nucleic acid sequence, the term "wild-type" means that the amino acid or nucleic acid sequence is a naturally occurring or naturally occurring sequence. As used herein, the term "naturally occurring" refers to any substance found in nature (e.g., a protein, amino acid, or nucleic acid sequence). Conversely, the term "non-naturally occurring" refers to any substance not found in nature (e.g., recombinant nucleic acid and protein sequences produced in a laboratory, or modifications of wild-type sequences). Detailed Implementation

[0075] Peptides with deamidase inhibitor activity

[0076] This invention relates to polypeptides having deamidase inhibitor activity, wherein the polypeptides are selected from the group consisting of:

[0077] (a) A polypeptide having at least 60% amino acid sequence identity with SEQ ID NO: 2;

[0078] (b) A polypeptide derived from SEQ ID NO: 2 by having 1 to 30 alterations (e.g., substitution, deletion and / or insertion) at one or more locations, such as 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;

[0079] (c) A polypeptide derived from (a) or (b) wherein the N- and / or C-terminus has been extended by adding one or more amino acids; and

[0080] Fragments of the polypeptides in (d), (a), (b), or (c).

[0081] In one aspect, the polypeptide having deamidase inhibitor activity has at least 60%, for example, 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 with SEQ ID NO: 2.

[0082] The polypeptide preferably comprises, is substantially composed of, or is composed of the amino acid sequence of SEQ ID NO: 2.

[0083] A polypeptide may have an N-terminus and / or C-terminus of one or more amino acids, for example, 1-20 amino acids, 1-10 amino acids, or 1-5 amino acids.

[0084] In another aspect, the polypeptide contains 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.

[0085] In another respect, the polypeptide 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.

[0086] 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 containing substitutions, deletions, and / or insertions at one or more positions. In one aspect, the number of amino acid substitutions, deletions, and / or insertions introduced into the polypeptide of SEQ ID NO: 2 is up to 15, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. Amino acid changes can be minor, i.e., conserved amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; typically small deletions of 1-30 amino acids; short amino or carboxyl terminal extensions, such as methionine residues at the amino terminus; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering net charge or another function (such as polyhistidine fragments, antigenic epitopes, or binding modules).

[0087] Essential amino acids in peptides can be identified using procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, a single alanine mutation is introduced at each residue in the molecule, and the deamidase inhibitory activity of the resulting molecule is tested to identify amino acid residues critical to the activity of the molecule. See also Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active sites of enzymes or other biological interactions can also be determined by physical analysis of the structure, such as by techniques like nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, along with mutation of the amino acid at the putative contact site. See, for example, 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. The identity of essential amino acids can also be inferred from alignments with related peptides, and / or from sequence homology and conserved catalytic mechanisms with related peptides or peptide / protein families from a common ancestor (which typically have similar three-dimensional structures, functions, and significant sequence similarities). Alternatively or additionally, protein structure prediction tools can be used for protein structure modeling to identify essential amino acids and / or active sites of peptides. See, for example, Jumper et al., 2021, “Highly accurate protein structure prediction with AlphaFold”, Nature 596: 583-589.

[0088] Using known mutagenesis, recombination, and / or tampering methods, followed by relevant screening procedures, one or more amino acid substitutions, deletions, and / or insertions can be made and tested, 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; US 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

[0089] Mutagenesis / reorganization methods can be combined with high-throughput automated screening methods to detect the activity of cloned mutagenic peptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenic DNA molecules encoding active peptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues within the peptide.

[0090] This polypeptide can be a fusion polypeptide.

[0091] In one respect, the polypeptide is isolated.

[0092] On the other hand, the polypeptide is purified.

[0093] In another aspect, the present invention provides a method for...

[0094] In yet another aspect, the present invention provides a composition comprising a deamidase and a deamidase inhibitor according to the invention.

[0095] Sources of wild-type deamidase inhibitor peptides

[0096] Wild-type deamidase inhibitor peptides can be obtained from microorganisms of any genus (donor strains). Preferably, wild-type deamidase inhibitor peptides are obtained from species of the genus *Chlorella*.

[0097] For the purposes of this invention, the term "obtained from," as used herein in conjunction with a given source, should mean that the polypeptide encoded by the polynucleotide is produced by that source or by a strain that has inserted the polynucleotide of this invention. In one aspect, polypeptides obtained from a given source are secreted extracellularly.

[0098] In one embodiment, the wild-type deamidase inhibitor polypeptide is obtained from a species of the genus *Chlorella*.

[0099] Wild-type deamidase inhibitor peptides 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 materials (e.g., soil, compost, water, etc.). Techniques for directly isolating microorganisms and DNA from their natural habitats are well known in the art. The polynucleotide encoding the peptide can then be obtained by similarly screening a library of genomic DNA or cDNA from another microorganism or a mixed DNA sample. Once the polynucleotide encoding the peptide has been detected with one or more probes, it 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).

[0100] polynucleotides

[0101] This invention also relates to polynucleotides encoding the polypeptides of this invention, as described herein.

[0102] This polynucleotide can be mutated by introducing a nucleotide substitution that does not cause a change in the amino acid sequence of the polypeptide but corresponds to the codon used by the host organism intended to produce the enzyme, or by introducing a nucleotide substitution that may produce a different amino acid sequence. For a general description of nucleotide substitutions, see, for example, Ford et al., 1991, Protein Expression and Purification 2: 95-107.

[0103] In one respect, the polynucleotide is isolated.

[0104] On the other hand, the polynucleotide is purified.

[0105] Nucleic acid constructs

[0106] The present invention also relates to nucleic acid constructs comprising the polynucleotide of the present invention, wherein the polynucleotide is operatively linked to one or more control sequences, which, under conditions compatible with the control sequences, direct the expression of the coding sequence in a suitable host cell.

[0107] Polynucleotides can be manipulated in various ways to provide polypeptide expression. Depending on the expression vector, manipulation of the polynucleotide prior to insertion into the vector may be desirable or necessary. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0108] promoter

[0109] The control sequence may be a promoter, i.e., a polynucleotide recognized by the host cell for expressing the polypeptide encoding the present invention. The promoter contains a transcriptional control sequence that mediates polypeptide expression. The promoter can be any polynucleotide exhibiting transcriptional activity in the host cell, including mutant promoters, truncated promoters, and heterozygous promoters, and can be a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to that of the host cell.

[0110] Examples of suitable promoters for guiding the transcription of polynucleotides 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, ibid., and Song et al., 2016, PLOS One 11(7): e0158447.

[0111] Examples of suitable promoters for guiding the transcription of polynucleotides in filamentous fungal host cells in this invention are promoters obtained from Aspergillus, Fusarium, Rhizomucor, and Trichoderma cells, such as those described in: Mukherjee et al., 2013, “Trichoderma: Biology and Applications” and Schmoll and 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.

[0112] 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 et al. 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.

[0113] terminator

[0114] The control sequence can also be a transcription terminator that is recognized by the host cell to terminate transcription. The terminator is operatively linked to the 3' end of a polynucleotide encoding a polypeptide. Any terminator that is functional in the host cell can be used in this invention.

[0115] Preferred terminators for bacterial host cells can be obtained from the following genes: Bacillus clausti alkaline protease (aprH), Bacillus licheniformis α-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

[0116] Preferred terminators for filamentous fungal host cells can be obtained from *Aspergillus* or *Trichoderma* species, such as genes obtained from *Aspergillus niger* glucosidase, *Trichoderma reesei* β-glucosidase, *Trichoderma reesei* cellobiase I, and *Trichoderma reesei* endoglucanase I, such as terminators described in: Mukherjee et al., 2013, “Trichoderma: Biology and Applications”, and Schmoll and 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.

[0117] Preferred terminators for yeast host cells are derived from the following genes: *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.

[0118] mRNA stabilizers

[0119] Control sequences can also be mRNA stabilizing regions downstream of the promoter and upstream of the gene's coding sequence, which enhance the expression of the gene.

[0120] Examples of suitable mRNA stabilizer regions were obtained from the Bacillus thuringiensis cryIIIA gene (WO 94 / 25612) and the Bacillus subtilis SP82 gene (Hue et al., 1995, J. Bacteriol. [Journal of Bacteriology] 177: 3465-3471).

[0121] Examples of mRNA stabilizer regions in 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.

[0122] Leader sequence

[0123] The control sequence can also be a leader sequence, which is a non-translated region of mRNA that is important for translation in the host cell. The leader sequence is operatively linked to the 5' end of a polynucleotide encoding a polypeptide. Any leader sequence that is functional in the host cell can be used.

[0124] The appropriate leader sequence for bacterial host cells was determined by Hambraeus et al., 2000, Microbiology, 146(12): 3051-3059, and by Kaberdin and... Description in 2006, FEMS Microbiol.Rev. [FEMS Microbiology Review] 30(6): 967-979.

[0125] Preferred leader sequences for filamentous fungal host cells can be obtained from the following genes: Aspergillus oryzae TAKA amylase and Aspergillus nidulans triphosphate isomerase.

[0126] Suitable leader sequences for yeast host cells can be obtained from the following genes: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0127] polyadenylation sequence

[0128] The control sequence can also be a polyadenylation sequence, which is a sequence operatively linked to the 3' end of a polynucleotide that is recognized by the host cell during transcription as a signal to add polyadenylation residues to the transcribed mRNA. Any polyadenylation sequence that is functional in the host cell can be used.

[0129] Preferred polyadenylation of filamentous fungal host cells is obtained from the following genes: Aspergillus nidulans o-aminobenzoate synthase, Aspergillus niger glucosidase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0130] Useful polyadenylation sequences in yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. [Molecular Cell Biology] 15: 5983-5990.

[0131] signal peptide

[0132] The control sequence can also be a signal peptide coding region encoding a signal peptide that is linked to the N-terminus of the polypeptide and guides the polypeptide into the secretory pathway of the cell. The 5' end of the polynucleotide coding sequence itself may contain a signal peptide coding sequence naturally linked to the coding sequence segment of the polypeptide within the translation read frame. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding sequence that is heterologous to the coding sequence. In cases where the coding sequence does not naturally contain a signal peptide coding sequence, a heterologous signal peptide coding sequence may be required. Alternatively, a heterologous signal peptide coding sequence may simply replace the natural signal peptide coding sequence to enhance polypeptide secretion. Any signal peptide coding sequence that guides the expressed polypeptide into the host cell's secretory pathway can be used.

[0133] The effective signal peptide coding sequences for bacterial host cells are obtained from the gene sequences of *Bacillus NCIB 11837* maltose amylase, *Bacillus licheniformis* subtilis protease, *Bacillus licheniformis* β-lactamase, *Bacillus thermophilus* α-amylase, *Bacillus thermophilus* neutral proteases (nprT, nprS, nprM), and *Bacillus subtilis* prsA. Additional signal peptides are described in Freudl, 2018, *Microbial Cell Factories*, 17:52.

[0134] The effective signal peptide coding sequences for filamentous fungal host cells are obtained from the signal peptide coding sequences of the following genes: Aspergillus niger neutral amylase, Aspergillus niger glucosylase, Aspergillus oryzae TAKA amylase, Specific Humus cellulase, Specific Humus endoglucanase V, Specific Humus lipase, and Rhizopus oryzae aspartic protease, such as the signal peptide described by Xu et al., 2018, Biotechnology Letters 40: 949-955.

[0135] Useful signal peptides in yeast host cells are derived from the genes for *Saccharomyces cerevisiae* α-factor and *Saccharomyces cerevisiae* invertase. Sequences encoding other useful signal peptides are described by Romanos et al., 1992, ibid.

[0136] Regulation sequence

[0137] It is also desirable to add regulatory sequences that regulate the expression of host cell growth-related peptides. Examples of regulatory sequences are those that cause gene expression to turn on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. Regulatory sequences in prokaryotic systems include the lac, tac, and trp operon systems. In yeast, the ADH2 or GAL1 systems can be used. In filamentous fungi, the *Aspergillus niger* glucosylamylase promoter, the *Aspergillus oryzae* TAKA α-amylase promoter and *Aspergillus oryzae* glucosylamylase promoter, the *Trichoderma reesei* cellobiose hydrolase I promoter, and the *Trichoderma reesei* cellobiose hydrolase II promoter can be used. Other examples of regulatory sequences are those that allow gene amplification. In fungal systems, these regulatory sequences include dihydrofolate reductase genes amplified in the presence of methotrexate and metallothionein genes amplified with heavy metals.

[0138] transcription factors

[0139] Control sequences can also be transcription factors, which are polynucleotides encoding polynucleotide-specific DNA-binding polypeptides that control the rate of transcription of genetic information from DNA to mRNA by binding to specific polynucleotide sequences. Transcription factors can function alone and / or together 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 is typically attached to a specific DNA sequence adjacent to the genetic element regulated by the transcription factor. Transcription factors can regulate the expression of a target protein directly (i.e., by binding to their promoter to activate the transcription of a gene encoding the target protein) or indirectly (i.e., by binding to the promoter of another transcription factor that regulates the transcription of a gene encoding the target protein) or indirectly (e.g., by binding to the promoter of another transcription factor that regulates the transcription of a gene encoding the target protein). 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.

[0140] expression carrier

[0141] The present invention also relates to recombinant expression vectors comprising the polynucleotide of the present invention, a promoter, and transcription and translation termination signals. Multiple nucleotides and control sequences can be linked together to produce a recombinant expression vector, which may include one or more convenient restriction sites to allow the insertion or substitution of the polynucleotide encoding the polypeptide at such sites. Alternatively, the polynucleotide can be expressed by inserting the polynucleotide or a nucleic acid construct containing the polynucleotide into a suitable vector for expression. In producing the expression vector, the coding sequence is located in the vector such that the coding sequence is operatively linked to a suitable control sequence for expression.

[0142] Recombinant expression vectors can be any vector (e.g., plasmids or viruses) that can readily undergo recombinant DNA procedures and induce expression of the polynucleotide. The choice of vector will typically depend on its compatibility with the host cell to which it will be introduced. Vectors can be linear or closed circular plasmids.

[0143] The vector can be a self-replicating vector, that is, a vector that exists as an extrachromosomal entity and replicates independently of chromosome replication, such as a plasmid, extrachromosomal element, microchromosome, or artificial chromosome. The vector can contain any means to ensure self-replication. Alternatively, the vector can be one that integrates into the genome when introduced into a host cell and replicates along with one or more chromosomes in which it has already been integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids collectively containing the total DNA of the host cell genome to be introduced, or transposons can be used.

[0144] The vector preferably contains one or more selective markers that allow for convenient selection of cells such as transformed cells, transfected cells, and transduced cells. A selective marker is a gene whose product provides resistance to biocides or viruses, resistance to heavy metals, or prototrophic auxotrophic traits, etc.

[0145] The vector preferably contains at least one element that allows the vector to integrate into the genome of the host cell or to replicate autonomously in the cell independently of the genome.

[0146] In order to integrate into the host cell genome, the vector may depend on a polynucleotide sequence encoding a polypeptide or any other element of the vector used for integration into the genome via homologous recombination (such as homologous directed repair (HDR)) or non-homologous recombination (such as non-homologous end joining (NHEJ)).

[0147] For autonomous replication, the vector may further include an origin of replication, which enables the vector to replicate autonomously in the host cell discussed. The origin of replication can be any plasmid replicon that mediates autonomous replication and functions within the cell. The terms "origin of replication" or "plasmid replicon" refer to the polynucleotide that enables a plasmid or vector to replicate in vivo.

[0148] More than one copy of the polynucleotide of the present invention can be inserted into host cells to improve polypeptide production. For example, two, three, four, five, or more copies can be inserted into host cells. An increased copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene along with the polynucleotide, wherein cells containing the amplified copy of the selectable marker gene and thus additional copies of the polynucleotide can be selected by culturing cells in the presence of a suitable selectivity reagent.

[0149] host cells

[0150] The present invention also relates to recombinant host cells containing polynucleotides of the present invention operably linked to one or more control sequences that direct the production of polypeptides of the present invention.

[0151] A construct or vector containing a polynucleotide is introduced into a host cell, such that the construct or vector is maintained as a chromosomal integrase or as an autonomously replicating extrachromosomal vector, as previously described. The choice of host cell will depend largely on the gene encoding the polypeptide and its origin. The polypeptide may be native or heterologous for the recombinant host cell. Furthermore, at least one of the one or more control sequences may be heterologous for the polynucleotide encoding the polypeptide. The recombinant host cell may contain a single copy or at least two copies, such as three, four, five or more copies, of the polynucleotide of the invention.

[0152] The host cell can be any microbial cell that can be used to recombinantly generate the polypeptides of the present invention, such as prokaryotic cells or fungal cells.

[0153] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0154] The bacterial host cell can be any Bacillus genus cell, including but not limited to Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus croceae, Bacillus coagulans, Bacillus sclerotiorum, Bacillus brevicornuate, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus thermophilus, Bacillus subtilis, and Bacillus thuringiensis cells. In one embodiment, the Bacillus genus cell is a Bacillus amyloliquefaciens, Bacillus licheniformis, or Bacillus subtilis cell.

[0155] For the purposes of this invention, Bacillus species should be defined as described in Patel and Gupta, 2020, Int. J. Syst. Evol. Microbiol. [International Journal of Systematic and Evolutionary Microbiology] 70: 406-438.

[0156] The bacterial host cell can also be any streptococcus cell, including but not limited to Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equisubsp. Zooepidemicus.

[0157] The bacterial host cell can also be any Streptomyces cell, including but not limited to non-chromogenic Streptomyces, insecticidal Streptomyces, sky blue Streptomyces, gray Streptomyces, and light blue Streptomyces cells.

[0158] 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, and transduction, wherein the DNA is introduced as a linearized or circular polynucleotide. Those skilled in the art will be able to readily determine, for example, a suitable method for introducing DNA into a given prokaryotic cell based 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.

[0159] The host cell can be a fungal cell. As used herein, “fungus” includes Ascomycota, Basidiomycota, Chytridiomycota, Zygomycota, Oomycota, and all mitotic 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).

[0160] Fungal cells can be transformed via processes involving protoplast-mediated transformation, Agrobacterium-mediated transformation, electroporation, gene gun methods, and shock wave-mediated transformation (as reviewed in 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 can be used, and the DNA can be introduced as linearized or circular polynucleotides.

[0161] The host cell for fungi can be a yeast cell. As used herein, “yeast” includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeasts belonging to the class Fungi Imperfecti (Blastomycetes). For the purposes of this invention, yeast should be defined as described in *Biology and Activities of Yeast* (edited by Skinner, Passmore, and Davenport, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0162] Yeast host cells can be cells from the genera *Candida*, *Hansenula*, *Kluyveromyces*, *Pichia*, *Saccharomyces*, *Schizosaccharomyces*, or *Yarrowia*, such as *Kluyveromyces lactis*, *Saccharomyces carlsbergensis*, *Saccharomyces diastaticus*, *Saccharomyces douglasii*, *Saccharomyces kluyveri*, *Saccharomyces norbensis*, *Saccharomyces oviformis*, or *Yarrowia lipolytica*. In a preferred embodiment, the yeast host cell is a Pichia or Komagataella cell, such as a Pichia pastoris cell (Komagataella phaffii).

[0163] Fungal host cells can be filamentous fungal cells. "Filamentous fungi" includes all filamentous forms of the phylum Eumycota and subphyla of Oomycetes (as defined by Hawksworth et al., 1995, see above). Filamentous fungi are generally characterized by a hyphal wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth occurs through hyphal extension, and carbon metabolism is obligate aerobic. In contrast, yeast (such as Saccharomyces cerevisiae) undergoes vegetative growth through budding of single-celled cells, and carbon metabolism can be fermentation.

[0164] The host cells of filamentous fungi can be *Acremonium*, *Aspergillus*, *Aureobasidium*, *Bjerkandera*, *Ceriporiopsis*, *Chrysosporium*, *Coprinus*, *Coriolus*, *Cryptococcus*, *Filibasidium*, *Fusarium*, *Humicola*, *Magnaporthe*, *Mucor*, and *Myceliophthora*. Cells of the genera *Neocallimastix*, *Neurospora*, *Paecilomyces*, *Penicillium*, *Phanerochaete*, *Phlebia*, *Piromyces*, *Pleurotus*, *Schizophyllum*, *Talaromyces*, *Thermoascus*, *Thielavia*, *Tolypocladium*, *Trametes*, or *Trichoderma* are used. In a preferred embodiment, the filamentous fungal host cell is a *Aspergillus*, *Trichoderma*, or *Fusarium* cell. In a further preferred embodiment, the filamentous fungal host cell is a *Aspergillus niger*, *Aspergillus oryzae*, *Fusarium moniliforme*, or *Trichoderma reesei* cell.

[0165] For example, the host cells of filamentous fungi can be *Aspergillus bubomori*, *Aspergillus stolonifer*, *Aspergillus fumigatus*, *Aspergillus japonicus*, *Aspergillus nidus*, *Aspergillus oryzae*, *Bjerkandera adusta*, *Ceriporiopsis saneirina*, *Ceriporiopsis caregiea*, *Ceriporiopsis gilvescens*, *Ceriporiopsis pannocinta*, *Ceriporiopsis rivulosa*, and *Ceriporiopsis microerythros*. subrufa), Ceriporiopsis subvermispora, Chrysosporium inopsinae, Chrysosporium aucubin, Lukenowens aucubin, Fecal aucubin, Felt aucubin, Queensland aucubin, Tropical aucubin, Striped aucubin, Coprinus comatus, Coriolus (hirsutus), Fusarium moniliforme, Fusarium graminearum, Fusarium kuwai, Fusarium scabra, Fusarium graminearum, Fusarium graminearum, Fusarium moniliforme ...

[0166] In one respect, the host cell is isolated.

[0167] On the other hand, the host cell is purified.

[0168] In another aspect, the host cell further comprises a co-expressed polypeptide exhibiting deamidase activity. In a related aspect, the invention also relates to a method for producing a polypeptide with deamidase activity, the method comprising culturing recombinant host cells (containing a co-expressed polypeptide exhibiting deamidase activity) under conditions favorable for producing the polypeptide with deamidase activity; preferably, the method further comprises recovering the polypeptide with deamidase activity. Such recovery of the polypeptide with deamidase activity may include a percolation step.

[0169] Generation method

[0170] The present invention also relates to methods for producing the polypeptides of the invention, the methods comprising (a) culturing the recombinant host cells of the invention under conditions conducive to the production of the polypeptide; and optionally (b) recovering the polypeptide.

[0171] The host cells are cultured in a nutrient medium suitable for producing peptides using methods known in the art. For example, cells can be cultured in a suitable medium and under conditions that allow for peptide expression and / or isolation by shake-flask culture or by small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid-state and / or microcarrier-based fermentation) in a laboratory or industrial fermenter. Suitable media are available from commercial suppliers or can be prepared according to publicly available compositions (e.g., in the catalogue of the American Type Culture Collection). If the peptide is secreted into the nutrient medium, it can be recovered directly from that medium. If the peptide is not secreted, it can be recovered from cell lysates.

[0172] Peptides can be detected using methods known in the art that are specific to peptides, including but not limited to the use of specific antibodies, enzyme product formation, enzyme substrate disappearance, or determination of the relative or specific activity of the peptide.

[0173] Peptides can be recovered from culture media using methods known in the art, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. In one aspect, whole fermentation broth containing peptides is recovered. In another aspect, cell-free fermentation broth containing peptides is recovered.

[0174] Peptides can be purified using a variety of procedures known in the art to obtain substantially pure peptides and / or peptide fragments (see, for example, Wingfield, 2015, Current Protocols in Protein Science; 80(1): 6.1.1-6.1.35; Labrou, 2014, Protein Downstream Processing, 1129: 3-10).

[0175] In terms of alternatives, peptides are not recycled.

[0176] solid preparations

[0177] The present invention also relates to enzyme particles / granules comprising the polypeptides of the present invention. In one embodiment, the particle comprises a core and optionally one or more coatings (outer layers) surrounding the core.

[0178] The core diameter (measured as equivalent sphere diameter (volume-average particle size)) can be 20–2000 μm, particularly 50–1500 μm, 100–1500 μm, or 250–1200 μm. The core diameter, as measured as equivalent sphere diameter, can be determined using laser diffraction methods such as those using the Malvern Mastersizer and / or those described under ISO 13320 (2020).

[0179] In one embodiment, the core comprises a polypeptide having the deamidase inhibitor activity of the present invention.

[0180] The core may include additional materials such as fillers, fibrous materials (cellulose or synthetic fibers), stabilizers, solubilizers, suspending agents, viscosity modifiers, light spheres, plasticizers, salts, lubricants, and fragrances.

[0181] The core may include adhesives such as synthetic polymers, waxes, fats, or carbohydrates.

[0182] The core, typically a homogeneous blend, may include salts of polyvalent cations, reducing agents, antioxidants, peroxide decomposition catalysts, and / or acidic buffer components.

[0183] The core may contain inert particles into which peptides are adsorbed or applied (e.g., by fluidized bed coating) to the surface of the inert particles.

[0184] The diameter of the core can be 20-2000μm, especially 50-1500μm, 100-1500μm or 250-1200μm.

[0185] The core can be coated with at least one coating, for example, to improve storage stability, to reduce dust formation during processing, or to color the particles. Optionally, one or more coatings may include salt coatings or other suitable coating materials such as polyethylene glycol (PEG), methyl hydroxypropyl cellulose (MHPC), and polyvinyl alcohol (PVA).

[0186] The coating may be applied at a rate of at least 0.1% (e.g., at least 0.5%, at least 1%, at least 5%, at least 10%, or at least 15%) based on the weight of the core. This amount may be at most 100%, 70%, 50%, 40%, or 30%.

[0187] 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, such as less than 60 μm or less than 40 μm.

[0188] The coating should seal the core unit by forming a substantially continuous layer. A substantially continuous layer should be understood as a coating with very few or no pores, such that the core unit has very few or no uncoated areas. The layer or coating should in particular be uniform in thickness.

[0189] The coating may further contain other materials known in the art, such as fillers, anti-sticking agents, pigments, dyes, plasticizers and / or adhesives, such as titanium dioxide, kaolin, calcium carbonate or talc.

[0190] Salt coatings may contain at least 60% salt by weight, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% salt by weight.

[0191] To provide acceptable protection, the salt coating is preferably at least 0.1 μm thick, such as 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. In one specific embodiment, the thickness of the salt coating is less than 100 μm, such as less than 60 μm or less than 40 μm.

[0192] Salt can be added from a salt solution (where the salt is completely dissolved) or from a salt suspension (where the fine particles are less than 50 μm, for example less than 10 μm or less than 5 μm).

[0193] Salt coatings may contain a single salt or a mixture of two or more salts. The salt may be water-soluble, particularly having a solubility of at least 0.1 g in 100 g of water at 20°C, preferably at least 0.5 g / 100 g of water, for example at least 1 g / 100 g of water, or at least 5 g / 100 g of water.

[0194] The salt can be an inorganic salt, such as a sulfate, sulfite, phosphate, phosphonate, nitrate, chloride, or carbonate, or a salt of a simple organic acid (less than 10 carbon atoms, such as 6 or fewer carbon atoms), such as a citrate, malonate, or acetate. Examples of cations in these salts are alkali or alkaline earth metal ions, ammonium ions, or first transition metal ions, such as sodium, potassium, magnesium, calcium, zinc, or aluminum. Examples of anions include chloride, bromine, iodine, sulfate, sulfite, bisulfite, thiosulfate, phosphate, dihydrogen phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, silicate, citrate, malate, maleate, malonic acid, succinate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate, or gluconate. In particular, alkali or alkaline earth metal salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate, or salts of simple organic acids such as citrate, malonate or acetate can be used.

[0195] The salt in the coating may have a constant humidity of more than 60% at 20°C, particularly more than 70%, more than 80% or more than 85%, or it may be another hydrated form of the salt (e.g., anhydrous). Salt coating may be as described in WO00 / 01793 or WO 2006 / 034710.

[0196] A specific example of a suitable salt is NaCl (CH3) 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(CH 20 ℃=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.

[0197] The salt can be in anhydrous form, or it can be a hydrated salt, i.e., a crystalline salt hydrate with one or more bound water crystals, as described, for example, in WO 99 / 32595. Specific examples include anhydrous sodium sulfate (Na₂SO₄), anhydrous magnesium sulfate (MgSO₄), magnesium sulfate heptahydrate (MgSO₄·7H₂O), zinc sulfate heptahydrate (ZnSO₄·7H₂O), disodium hydrogen phosphate heptahydrate (Na₂HPO₄·7H₂O), magnesium nitrate hexahydrate (Mg(NO₃)₂(6H₂O)), sodium citrate dihydrate, and magnesium acetate tetrahydrate.

[0198] Preferably, the salt is applied as a salt solution, for example using a fluidized bed.

[0199] Coating materials can be waxy coating materials and film-forming coating materials. Examples of waxy coating materials are poly(ethylene oxide) products (polyethylene glycol, PEG) with an average molecular weight of 1,000 to 20,000; ethoxylated nonylphenol having 16 to 50 ethylene oxide units; ethoxylated fatty alcohols containing 12 to 20 carbon atoms and having 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and monoglycerides, diglycerides, and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application via fluidized bed technology are given in GB 1483591.

[0200] The particles may optionally have one or more additional coatings. Examples of suitable coating materials are polyethylene glycol (PEG), methyl hydroxypropyl cellulose (MHPC), and polyvinyl alcohol (PVA). Examples of enzyme particles with multiple coatings are described in WO 93 / 07263 and WO 97 / 23606.

[0201] The core can be prepared by blends of granulated components, for example by methods including granulation techniques such as crystallization, precipitation, pan-coating, fluidized bed coating, fluidized bed agglomeration, rotary atomization, extrusion, granulation, spheronization, particle size reduction, drum granulation, and / or high-shear granulation.

[0202] Methods for preparing the core can be found in *Handbook of Powder Technology*; *CECapes: Particle Size Enlargement*; Volume 1; 1980; Elsevier. Preparation methods include known feed and pellet formulation techniques, for example...

[0203] (a) Spray-dried products, wherein a liquid polypeptide solution is atomized in a spray drying tower to form small droplets, which are dried as they descend along the tower to form polypeptide-containing particulate material. This method can produce very small particles (Michael S. Showell; Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker).

[0204] (b) Layered products, wherein the polypeptide is coated in layers around a pre-formed inert core particle, wherein the polypeptide-containing solution is typically atomized in a fluidized bed apparatus, in which the pre-formed core particle is fluidized and the polypeptide-containing solution adheres to the core particle and is dried until a dry polypeptide layer remains on the surface of the core particle. If useful core particles of a desired size can be found, particles of the desired size can be obtained in this manner. This type of product is described, for example, in WO 97 / 23606.

[0205] (c) Absorbed core particles, wherein instead of coating the polypeptide in layers around the core, the polypeptide is absorbed onto and / or into the surface of the core. Such a method is described in WO 97 / 39116.

[0206] (d) Extruded or pelletized products, wherein a peptide-containing paste is compressed into pellets or extruded and cut into particles under pressure through small openings, followed by drying of these pellets. Such particles are typically of considerable size because the material with the extrusion openings (usually a flat plate with perforations) limits the pressure drop permissible through the extrusion openings. Furthermore, when using small openings, the very high extrusion pressure increases the heat generation in the peptide paste, which is detrimental to the peptide (Michael S. Showell; Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker).

[0207] (e) Spray-granulated products, wherein a peptide-containing powder is suspended in molten wax, and the suspension is sprayed (e.g., via a rotary sprayer) into a cooling chamber where the droplets rapidly solidify (Michael S. Showell; Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker). The resulting product is one in which the peptide is uniformly distributed throughout the inert material rather than concentrated on its surface. This technique is described in US 4,016,040 and US 4,713,245.

[0208] (f) A mixer-granulated product in which a polypeptide-containing liquid is added to a dry powder composition of conventional granulation components. The liquid and powder are mixed in a suitable ratio, and as the moisture from the liquid is absorbed into the dry powder, the components of the dry powder begin to adhere and aggregate, and the particles accumulate to form granules containing the polypeptide. Such methods are described in US4,106,991, EP 170360, EP 304332, EP 304331, WO 90 / 09440, and WO 90 / 09428. In certain aspects of this process, various high-shear mixers can be used as granulators. Granules consisting of polypeptides, fillers, and binders are mixed with cellulose fibers to reinforce the granules, thereby producing so-called T-granules. The reinforced granules are more robust and release less enzyme dust.

[0209] (g) Particle size reduction, in which a core is generated by grinding or crushing larger particles, pellets, flat sheets, briquettes, etc., containing polypeptides. The desired core particle fraction is obtained by sieving the ground or crushed product. Oversized and undersized particles can be recovered. Particle size reduction is described in Martin Rhodes (ed.); Principles of Powder Technology; 1990; Chapter 10; John Wiley & Sons.

[0210] (h) Fluidized bed granulation. Fluidized bed granulation involves suspending microparticles in an airflow and spraying liquid through a nozzle onto the fluidized particles. The particles hit by the sprayed droplets become wetted and sticky. The sticky particles collide with and adhere to other particles to form granules.

[0211] (i) These cores can be dried, for example, in a fluidized bed dryer. Those skilled in the art can use other known methods for drying particles in the feed or enzyme industries. Drying is preferably carried out at a product temperature from 25°C to 90°C. For some peptides, it is important that the core containing the peptide contains a small amount of water before salt coating. If water-sensitive peptides are salt-coated before removing excess water, the excess water will be trapped in the core and may negatively affect the activity of the peptide. After drying, these cores preferably contain 0.1% w / w to 10% w / w of water.

[0212] Dust-free particles can be produced, for example, as disclosed in US 4,106,991 and US 4,661,452, and can optionally be coated by methods known in the art.

[0213] The particles may further contain one or more additional enzymes, such as hydrolases, isomerases, ligases, lyases, oxidoreductases, and transferases. One or more additional enzymes are preferably selected from the group consisting of: acetylxylan esterase, acylglycerol lipase, amylase, α-amylase, β-amylase, arabinofuranylase, cellobiase, cellulase, ferulic acid esterase, galactanase, α-galactosidase, β-galactosidase, β-glucanase, β-glucosidase, lysophospholipase, lysozyme, α-mannosidase, β-mannosidase (mannanase), phytase, phospholipase A1, phospholipase A2, phospholipase D, protease, pullulanase, pectin esterase, triacylglycerol lipase, xylanase, β-xylosidase, or any combination thereof. Each enzyme will then be present in more particles, ensuring a more uniform distribution of the enzymes and also reducing the physical separation of different enzymes due to the varying particle sizes. The method for generating multi-enzyme coparticles is disclosed in IP.com disclosure IPCOM000200739D.

[0214] Another example of peptide formulation using coparticles is disclosed in WO 2013 / 188331.

[0215] The present invention also relates to protected polypeptides prepared according to the method disclosed in EP 238216.

[0216] Liquid preparations

[0217] The present invention also relates to liquid compositions comprising the polypeptides of the present invention. The composition may comprise an enzyme stabilizer (examples of enzyme stabilizers include polyols (such as propylene glycol or glycerol), sugars or sugar alcohols, lactic acid, reversible protease inhibitors, boric acid or boric acid derivatives (e.g., aromatic borate esters, or phenyl boric acid derivatives (such as 4-formylphenylboronic acid))).

[0218] In some embodiments, one or more fillers or one or more 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, etc. 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, these compositions contain about 5% to about 90% of such materials.

[0219] In one aspect, the liquid formulation contains 20% w / w to 80% w / w of a polyol. In one embodiment, the liquid formulation contains 0.001% w / w to 2% w / w of a preservative.

[0220] In another embodiment, the present invention relates to a liquid preparation comprising:

[0221] (a) 0.001% w / w to 25% w / w polypeptides having the deamidase inhibitor activity of the present invention;

[0222] (b) Polyols ranging from 20% w / w to 80% w / w;

[0223] (c) Optionally 0.001% w / w to 2% w / w preservative; and

[0224] (d) Water.

[0225] In another embodiment, the present invention relates to a liquid preparation comprising:

[0226] (a) 0.001% w / w to 25% w / w polypeptides having the deamidase inhibitor activity of the present invention;

[0227] (b) 0.001% w / w - 2% w / w preservative;

[0228] (c) optionally 20% w / w to 80% w / w polyols; and

[0229] (d) Water.

[0230] Preferably, the liquid composition of the two embodiments described above further comprises a polypeptide having deamidase activity, for example, in an amount of 0.001% w / w to 25% w / w.

[0231] In another embodiment, the liquid formulation comprises one or more formulations, such as formulations 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, acetate, and phosphate, preferably selected from the group consisting of: sodium sulfate, dextrin, cellulose, sodium thiosulfate, kaolin, and calcium carbonate. In one embodiment, the polyols are selected from the group consisting of: glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propanediol or 1,3-propanediol, dipropylene glycol, polyethylene glycol (PEG) with an average molecular weight of less than about 600, and polypropylene glycol (PPG) with an average molecular weight of less than about 600, more preferably selected from the group consisting of: glycerol, sorbitol, and propylene glycol (MPG), or any combination thereof.

[0232] In another embodiment, the liquid formulation comprises 20%-80% polyol (i.e., the total amount of polyol), for example, 25%-75% polyol, 30%-70% polyol, 35%-65% polyol, or 40%-60% polyol. In one embodiment, the liquid formulation comprises 20%-80% 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, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propanediol or 1,3-propanediol, dipropylene glycol, polyethylene glycol (PEG) with an average molecular weight of less than about 600, and polypropylene glycol (PPG) with an average molecular weight of less than about 600. In one embodiment, the liquid formulation comprises 20%-80% polyol (i.e., the 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).

[0233] 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 contains 0.02% w / w to 1.5% w / w preservative, such as 0.05% w / w to 1% w / w preservative or 0.1% w / w to 0.5% w / w preservative. In one embodiment, the liquid formulation contains 0.001% w / w to 2% w / w preservative (i.e., the total amount of preservative), such as 0.02% w / w to 1.5% w / w preservative, 0.05% w / w to 1% w / w preservative, or 0.1% w / w to 0.5% w / w preservative, wherein the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate, and potassium benzoate, or any combination thereof.

[0234] In another embodiment, the liquid formulation further comprises 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: acetylxylan esterase, acylglycerol lipase, amylase, α-amylase, β-amylase, arabinofuranylase, cellobiase, cellulase, ferulic acid esterase, galactanase, α-galactosidase, β-galactosidase, β-glucanase, β-glucosidase, lysophospholipase, lysozyme, α-mannosidase, β-mannosidase (mannanase), phytase, phospholipase A1, phospholipase A2, phospholipase D, protease, pullulanase, pectin esterase, triacylglycerol lipase, xylanase, β-xylosidase, or any combination thereof.

[0235] Fermentation broth preparations or cell compositions

[0236] The present invention also relates to fermentation broth formulations or cell compositions comprising the polypeptides of the present invention. The fermentation broth formulations or cell compositions further comprise additional components used in the fermentation process, such as cells (including host cells containing genes encoding the polypeptides of the present invention, these host cells being used to produce the target polypeptide), cell debris, biomass, fermentation medium, and / or fermentation products. In some embodiments, the composition is a cell-killing whole culture medium containing one or more organic acids, killed cells and / or cell debris, and a culture medium.

[0237] As used herein, the term "fermentation broth" refers to a preparation produced by cell fermentation that undergoes little or no recovery and / or purification. For example, fermentation broth is produced when a microbial culture is incubated to saturation under carbon-limited conditions that allow protein synthesis (e.g., expression of enzymes by the host cell) and secretion of proteins into the cell culture medium. Fermentation broth may contain unfractionated or fractionated contents of fermentation material derived at the end of fermentation. Typically, fermentation broth is unfractionated and contains used culture medium and cell debris remaining after, for example, removal of microbial cells (e.g., filamentous fungal cells) by centrifugation. In some embodiments, the fermentation broth contains used cell culture medium, extracellular enzymes, and viable and / or non-viable microbial cells.

[0238] In some embodiments, the fermentation broth formulation or cell composition comprises a first organic acid component (an organic acid containing at least one to five carbon atoms and / or its salt) and a second organic acid component (an organic acid containing at least six or more carbon atoms and / or its salt). In some embodiments, the first organic acid component is acetic acid, formic acid, propionic acid, its salt, 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, its salt, or a mixture of two or more of the foregoing.

[0239] In one aspect, the composition contains one or more organic acids and optionally further contains killed cells and / or cell debris. In some embodiments, these killed cells and / or cell debris are removed from the cell-killing whole culture medium to provide a composition free of these components.

[0240] Fermentation broth formulations or cell compositions may further contain preservatives and / or antimicrobial agents (e.g., bacteriostatic agents), including but not limited to sorbitol, sodium chloride, potassium sorbate, and other preservatives and / or antimicrobial agents known in the art.

[0241] The cell-killing whole culture or cell composition may contain ungraded contents of fermentation material derived at the end of fermentation. Typically, the cell-killing whole culture or cell composition contains used 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 to allow protein synthesis. In some embodiments, the cell-killing whole culture or cell composition contains used cell culture medium, extracellular enzymes, and killed filamentous fungal cells. In some embodiments, methods known in the art can be used to permeate and / or lyse the microbial cells present in the cell-killing whole culture or composition.

[0242] The whole culture medium or cell composition described herein is typically a liquid, but may contain insoluble components, such as killed cells, cell debris, culture medium components, and / or one or more insoluble enzymes. In some embodiments, insoluble components may be removed to provide a clear liquid composition.

[0243] The whole culture medium formulations and cell compositions of the present invention can be produced by the methods described in WO 90 / 15861 or WO 2010 / 096673.

[0244] The invention is further defined by the following numbered embodiments:

[0245] Example 1. A polypeptide with deamidase inhibitor activity, the polypeptide being selected from the group consisting of:

[0246] (a) A polypeptide having at least 60% amino acid sequence identity with SEQ ID NO: 2;

[0247] (b) A polypeptide derived from SEQ ID NO: 2 by having 1-30 alterations (e.g., substitution, deletion and / or insertion) at one or more sites;

[0248] (c) A polypeptide derived from (a) or (b) wherein the N- and / or C-terminus has been extended by adding one or more amino acids; and

[0249] Fragments of the polypeptide in (d), (a), (b), or (c).

[0250] Example 2. A polypeptide as described in Example 1, which is derived from SEQ ID NO: 2 by having 1-25 variations.

[0251] Example 3. A polypeptide as described in Example 1, which is derived from SEQ ID NO: 2 by having 1-20 variations.

[0252] Example 4. A polypeptide as described in Example 1, which is derived from SEQ ID NO: 2 by having 1-15 variations.

[0253] Example 5. A polypeptide as described in Example 1, which is derived from SEQ ID NO; 2 by having 1-10 modifications.

[0254] Example 6. A polypeptide as described in Example 1, which is derived from SEQ ID NO: 2 by having 1-5 modifications.

[0255] Example 7. A polypeptide as described in any of the preceding examples, wherein the changes are substitution, deletion and / or insertion; preferably substitution.

[0256] Example 8. A polypeptide as described in any of the preceding examples, wherein these changes are substitutions.

[0257] Example 9. A polypeptide as described in Example 1, which has at least 70% amino acid sequence identity with SEQ ID NO: 2.

[0258] Example 10. A polypeptide as described in Example 1, which has at least 80% amino acid sequence identity with SEQ ID NO: 2.

[0259] Example 11. A polypeptide as described in Example 1, which has at least 90% amino acid sequence identity with SEQ ID NO: 2.

[0260] Example 12. A polypeptide as described in Example 1, which has at least 95% amino acid sequence identity with SEQ ID NO: 2.

[0261] Example 13. A polypeptide as described in Example 1, which has at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with SEQ ID NO: 2.

[0262] Example 14. A polypeptide as described in any of the preceding examples, wherein the N- and / or C-terminus has been extended by adding 1-20 amino acids.

[0263] Example 15. A polypeptide as described in any of the preceding examples, wherein the N- and / or C-terminus has been extended by adding 1-10 amino acids.

[0264] Example 16. A polypeptide as described in any of the preceding examples, wherein the N- and / or C-terminus has been extended by adding 1-5 amino acids.

[0265] Example 17. A polypeptide as described in any of the preceding examples, the polypeptide comprising the amino acid sequence motif F[F / Y][I / L / V][F / Q / S][E / K / R].

[0266] Example 18. A polypeptide as described in any of the preceding examples, the polypeptide comprising the amino acid sequence motif L[I,T]WY[D,H,K,N].

[0267] Example 19. A polypeptide as described in any of the preceding examples, the polypeptide comprising the amino acid sequence motif G[I,M]S[A,P,Q]Q.

[0268] Example 20. A polypeptide as described in any of the preceding examples, the polypeptide comprising the amino acid sequence motifs [D,H,K,N,S][I,L][G,V][I,V][D,E].

[0269] Example 21. A polypeptide as described in any of the preceding examples, the polypeptide comprising the amino acid sequence motifs [N,H][I,L,M,V,Q][I,V][K,R,Q][E,I,Q].

[0270] Example 22. A polypeptide as described in any of the preceding examples, the polypeptide comprising the amino acid sequence motif [D / N][P / S][D / E][H / K / N / Q / R][A / P / S].

[0271] Example 23. A polypeptide as described in any of the preceding examples, the polypeptide 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.

[0272] Example 24. A polypeptide as described in any of the preceding examples, wherein the deamidase inhibitor activity reduces the deamidase activity by at least 90%.

[0273] Example 25. A polypeptide as described in any of the preceding examples, wherein the deamidase inhibitor activity reduces the deamidase activity by at least 80%.

[0274] Example 26. A polypeptide as described in any of the preceding examples, wherein the deamidase inhibitor activity reduces the deamidase activity by at least 70%.

[0275] Example 27. A polypeptide as described in any of the preceding examples, wherein the deamidase inhibitor activity reduces the deamidase activity by at least 60%.

[0276] Example 28. A polypeptide as described in any of the preceding examples, wherein the deamidase inhibitor activity reduces the deamidase activity by at least 50%.

[0277] Example 29. A polypeptide as described in any of the preceding examples, wherein the deamidase inhibitor activity reduces the deamidase activity by at least 40%.

[0278] Example 30. A polypeptide as described in any of the preceding examples, 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.

[0279] Example 31. A polypeptide as described in any of the preceding examples, wherein the deamidase activity is derived from a polypeptide having deamidase activity derived from a species of the genus Chlorella.

[0280] Example 32. A polypeptide as described in any of the preceding examples, wherein the deamidase activity is derived from the polypeptide shown in SEQ ID NO: 4.

[0281] Example 33. A polynucleotide encoding a polypeptide as described in any of the preceding examples.

[0282] Example 34. A polynucleotide as described in Example 33, wherein the polynucleotide is isolated and / or purified.

[0283] Example 35. A nucleic acid construct or expression vector comprising a polynucleotide as described in Example 33, wherein the polynucleotide is operatively linked to one or more control sequences that direct the production of the polypeptide in an expression host.

[0284] Example 36. A recombinant host cell comprising a nucleic acid construct or expression vector as described in Example 35.

[0285] Example 37. The recombinant host cell as described in Example 36, wherein the polypeptide is heterologous to the recombinant host cell.

[0286] Example 38. A recombinant host cell as described in Example 36 or 37, wherein at least one of the one or more control sequences is heterologous to the polynucleotide encoding the polypeptide.

[0287] Example 39. A recombinant host cell as described in any one of Examples 36-38, wherein the recombinant host cell comprises at least two copies, such as three, four, or five or more copies, of the polynucleotide as described in any one of Examples 33-35.

[0288] Example 40. The recombinant host cell as described in any one of Examples 36-39, wherein the recombinant host cell is a yeast recombinant host cell, for example, Candida, Hansenula, Kluyveromyces, Pichia, Yeast, Fissionomyces or Yersinia cells, such as Kluyveromyces lactis, Kelvin, Saccharomyces cerevisiae, Saccharomyces sacchariformis, Douglas yeast, Kluyveromyces rubiginii, Nodi yeast, Ovoyces or Yersinia lipolytica cells.

[0289] Example 41. The recombinant host cell as described in any one of Examples 36-39, wherein the recombinant host cell is a filamentous fungal recombinant host cell, such as *Cladosporium*, *Aspergillus*, *Briefomus*, *Cirsium*, *Pseudomonas*, *Aureospora*, *Coprinus*, *Cladosporium*, *Cryptococcus*, *Ustilago*, *Fusarium*, *Pyrophyllus*, *Pyrophyllus*, *Mucor*, *Pyrophyllus*, *Nematophytes*, *Penicillium*, *Penicillium*. Cells of the genera *Penicillium*, *Pleurotus*, *Gnaphalium*, *Ruminocytophyte*, *Pleurotus*, *Schizophyllum*, *Basilaria*, *Thermophilic Ascomycetes*, *Fusporium*, *Cyclophorus*, *Pterygomyces*, or *Trichoderma*, especially *Aspergillus amblycera*, *Aspergillus scabra*, *Aspergillus fumigatus*, *Aspergillus japonicus*, *Aspergillus nidus*, *Aspergillus oryzae*, *Aspergillus niger*, *Aspergillus oryzae*, *Pterygomyces nidus*, *Pterygomyces carnegieae*, *Pterygomyces paeonii*, *Pterygomyces pannohita*, *Pterygomyces circumferentiale*, and *Pterygomyces purpureus*. *Pseudomonas cereus*, *Pseudomonas tinctoria*, *Aureobasidium stenoptera*, *Aureobasidium keratophilum*, *Aureobasidium ruthenicum*, *Aureobasidium foetida*, *Aureobasidium quinquefolium*, *Aureobasidium stenoptera*, *Aureobasidium glomeratum*, *Aureobasidium triflorum*, *Fusarium moniliforme*, *Fusarium graminearum ... Fusarium, Fusarium scabiosifolium, Fusarium pseudocranioides, Fusarium sulfideum, Fusarium scabiosifoli ...

[0290] Example 42. The recombinant host cell as described in any one of Examples 36-39, wherein the recombinant host cell is a prokaryotic recombinant host cell, for example, Gram-positive cells selected from the group consisting of: Bacillus spp., Clostridium spp., Enterococcus spp., Bacillus terrestris spp., Lactobacillus spp., Lactococcus spp., Bacillus spp., Staphylococcus spp., Streptococcus spp., or Streptomyces spp. cells, or Gram-negative bacteria selected from the group consisting of: Campylobacter spp., Escherichia coli, Flavobacterium spp., Fusobacterium spp., Helicobacter spp., Coptis spp., Neisseria spp., Pseudomonas spp., Salmonella spp. and Ureaplasma spp. cells, such as Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus croceus, Bacillus coagulans, Bacillus sclerotiorum, Bacillus splenium, Bacillus tarda, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus thermophilus, Bacillus subtilis, Bacillus thuringiensis, Streptococcus equi, Streptococcus pyogenes, Streptococcus lactis and Streptococcus equi subsp. vesicant, Streptococcus nonchromogenic, Streptococcus pyogenes, Streptococcus aureus, Streptococcus gray, and Streptococcus purpureus cells.

[0291] Example 43. The recombinant host cell as described in any one of Examples 36-39, wherein the recombinant host cell is a Bacillus licheniformis cell.

[0292] Example 44. The recombinant host cell as described in any one of Examples 36-43, wherein the recombinant host cell is isolated.

[0293] Example 45. The recombinant host cell as described in any one of Examples 36-44, wherein the recombinant host cell is purified.

[0294] Example 46. A recombinant host cell as described in any one of Examples 36-45, the recombinant host cell further comprising a co-expressed polypeptide exhibiting deamidase activity; preferably, the co-expressed polypeptide exhibiting deamidase activity is a polypeptide as shown in SEQ ID NO: 4, or a polypeptide having at least 80%, 90%, or 95% amino acid sequence identity with SEQ ID NO: 4.

[0295] Example 47. A method for producing a polypeptide as described in any one of Examples 1-32, the method comprising culturing recombinant host cells as described in any one of Examples 36-45 under conditions favorable for producing the polypeptide.

[0296] Example 48. The method as described in Example 47, further comprising recovering the polypeptide.

[0297] Example 49. A liquid composition comprising

[0298] (a) 0.001% w / w to 25% w / w of a polypeptide as described in any one of Examples 1-32,

[0299] (b) a polyol, preferably a polyol of 20% w / w to 80% w / w, and

[0300] (c) Water.

[0301] Example 50. A liquid composition as described in Example 49, the liquid composition further comprising a polypeptide exhibiting deamidase activity.

[0302] Example 51. A liquid composition as described in Example 49, the liquid composition further comprising 0.001% w / w to 25% w / w of a polypeptide exhibiting deamidase activity.

[0303] Example 52. A liquid composition as described in Example 50 or 51, wherein the polypeptide exhibiting deamidase activity has at least 80%, 90%, or 95% amino acid sequence identity with the polypeptide shown in SEQ ID NO: 4.

[0304] Example 53. A liquid composition as described in any one of Examples 50-52, wherein the polypeptide exhibiting deamidase activity is the polypeptide shown in SEQ ID NO: 4.

[0305] Example 54. A method for producing a polypeptide with deamidase activity, the method comprising culturing recombinant host cells as described in Example 46 under conditions conducive to the production of the polypeptide with deamidase activity.

[0306] Example 55. The method as described in Example 54, further comprising recovering the polypeptide having deamidase activity.

[0307] Example 56. The method as described in Example 55, wherein the recovery of the polypeptide having deamidase activity includes percolation.

[0308] The invention is further described by the following examples, which should not be construed as limiting the scope of the invention.

[0309] Examples

[0310] strains

[0311] A strain of *Aureobacterium* species-62563 was isolated from soil samples collected in Sibhult, Sweden in September 2013.

[0312] Example 1

[0313] Deamidase activity

[0314] Material

[0315] Glutamine endopeptidase from Bacillus licheniformis

[0316] 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), in which:

[0317] FITC is a fluorescein with excitation and emission maxima at approximately 490 nm and 520 nm.

[0318] Ahx is aminocaproic acid.

[0319] ED is ethylenediamine. And...

[0320] Dnp is 2,4-dinitrophenyl (fluorescein fluorescence quencher).

[0321] Deamidase activity assay

[0322] This assay measures the (relative) activity of active deamidases.

[0323] Transfer 50 μL of deamidase sample to a standard black 96-well plate and add:

[0324] 20μL of 0.25μg / mL FITC-PP-Dnp

[0325] 50 μL of 50 μg / mL gamma-glutamyl endopeptidase

[0326] 130 μL of 100 mM HEPES buffer (pH 7.0), and

[0327] 0.01% v / v Triton X detergent.

[0328] Fluorescence signal (RFU) was measured for 30 min using a Biotek Synergy H1 fluorescent plate reader at emission / excitation wavelengths of 485 nm / 525 nm.

[0329] The initial rate of the data was analyzed using variable time intervals that depended on the curve shape (signal versus time). The initial rate of each sample was normalized relative to the initial rate of a fully active deamidase reference enzyme (produced by a wild-type donor strain). Activity was measured as “initial rate %”, which is the initial rate of the sample molecules relative to the initial rate of the active deamidase reference enzyme.

[0330] When the deamidase inhibitor (SEQ ID NO: 2) of the present invention was added to a sample containing an active deamidase (SEQ ID NO: 4) at a ratio of about 1:1 (deamidase: inhibitor), the deamidase activity (initiation rate %) was significantly reduced.

[0331] Example 2

[0332] Nano differential scanning fluorometry (nanoDSF) - pyrolysis folding temperature

[0333] The conformational stability of the deamidase / inhibitor complexes (using the deamidase inhibitor of this invention) was assessed using nano-differential scanning fluorescence (nanoDSF). These molecules were exposed to a temperature gradient, as shown below. The resulting structural changes are reflected in the changes in fluorescence intensity and provide a measure of temperature stability. The binding of the deamidase inhibitor to the deamidase contributes to molecular stability; therefore, the nanoDSF pyrolysis folding temperature also provides information about the deamidase / inhibitor binding affinity.

[0334] The His-Tag purified sample was received in an elution buffer consisting of an IMAC (immobilized metal affinity chromatography) column, 20 mM sodium phosphate, 500 mM sodium chloride, 500 mM imidazole, and pH 7.4.

[0335] 60 μL of sample was repeatedly transferred to a black-backed 384-well plate, and the plate was briefly centrifuged to remove potential air bubbles.

[0336] The pyrolysis folding temperature of the samples was analyzed using a Prometheus NT.Plex system from NanoTemper Technologies GmbH with the following settings:

[0337] (i) Temperature scan rate: 3.3 °C / min; and

[0338] (ii) Temperature scan interval: 20℃-95℃.

[0339] During operation, the sample was loaded into a capillary (Prometheus NT.Plex - capillary action sheet, standard, catalog number PR-AC002) before being subjected to a temperature gradient. Data generated were analyzed using PR.Stability Analysis v.1.0.1 software. The first derivative of the 330 nm trace was used to align with the midpoint defolding temperature (T). m Annotate (℃). In some cases, more than one T can be observed. m In these cases, the main peak in the first derivative trace at 330 nm is selected as the Ts of the sample. m .

[0340] Example 3

[0341] Thermolytic folding temperature of deamidase / inhibitor complex

[0342] The pyrolysis folding temperatures of the active deamidase and the corresponding deamidase / inhibitor complex were measured using the nanoDSF procedure as described in Example 2.

[0343] Table 1. NanoDSF pyrolysis folding temperatures of peptides.

[0344]

Claims

1. A polypeptide having deamidase inhibitor activity, the polypeptide being selected from the group consisting of: (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 1-30 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- and / or C-terminus has been extended by the addition of one or more amino acids; and (d) A fragment of the polypeptide of (a), (b) or (c).

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. The polypeptide of claim 1, 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.

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

2.

5. A polypeptide as described in any of the preceding claims, wherein the deamidase inhibitor activity reduces the deamidase activity to less than 90% activity; preferably, the deamidase inhibitor activity reduces the deamidase activity of the polypeptide as shown in SEQ ID NO: 4 to less than 90%.

6. A polynucleotide encoding the polypeptide of any preceding claim.

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 according to claim 7.

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

10. A liquid composition comprising (a) 0.001% w / w-25% w / w of a polypeptide according to any one of claims 1 to 5, (b) polyols, and (c) Water.

11. The liquid composition of claim 10, further comprising a polypeptide exhibiting deamidase activity; preferably, present in an amount of 0.001% w / w-25% w / w.

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

4.

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

4.

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

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

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

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