Deamidase inhibitors and fusion polypeptides having a deamidase domain

Abstract

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

Description

[Technical field]

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

[0002] The present invention relates to fusion polypeptides comprising a propeptide and a polypeptide with deamidase activity, as well as separate propeptides and polypeptides with deamidase activity; polynucleotides encoding the polypeptides, nucleic acid constructs, vectors, and host cells comprising the polynucleotides and methods of making and using the polypeptides. [Background technology]

[0003] Deamidase enzymes are produced by microbial cells in an inactive proform, which contains a propeptide domain tightly bound to a deamidase domain. The proform is expressed as a fusion protein and has reduced deamidase activity that protects the viability of the host cell. By nature, the fusion protein is post-processed to remove the propeptide and release the active deamidase outside the host cell. However, in recombinant expression systems, the fusion protein is secreted outside the host cell as an inactive proform containing the propeptide. For many deamidase proforms, the propeptide domain cannot be separated from the deamidase domain by simple cleavage due to the high binding affinity of the propeptide to the deamidase polypeptide. For other deamidase proforms produced in recombinant expression systems, the binding affinity between the propeptide and the deamidase polypeptide is too weak, resulting in an activated deamidase that reduces the viability of the recombinant host cell and makes commercial production impossible.

[0004] The object of the present invention is to provide novel proforms of deamidase enzymes (fusion polypeptides) in which the binding affinity of the propeptide domain to the mature deamidase domain is tailored to allow recombinant expression and secretion without reducing host cell viability, and at the same time allowing extracellular separation of the propeptide and the active mature deamidase enzyme. Summary of the Invention [Means for solving the problem]

[0005] The invention provides fusion polypeptides comprising a propeptide and a deamidase polypeptide, as well as separate propeptide and deamidase polypeptides, and polynucleotides encoding the peptides and polypeptides.

[0006] Thus, in a first aspect, the present invention provides a method for producing a method for treating a cancer cell comprising the steps of: (a) a first polypeptide comprising a deamidase inhibitor domain, and (b) a recombinant fusion polypeptide comprising or consisting of a second polypeptide having deamidase activity, The fusion polypeptide has a thermal denaturation temperature in the range of 65-82° C.; and the fusion polypeptide has a deamidase activity that is less than 50% compared to the deamidase activity of the second polypeptide.

[0007] In another aspect, the invention provides a method for producing a polypeptide having deamidase activity, the method comprising contacting a fusion polypeptide of the invention with an endopeptidase to separate the first polypeptide from the second polypeptide.

[0008] In yet another aspect, the present invention provides a composition that exhibits deamidase activity, comprising isolated first and second polypeptides of a fusion polypeptide of the invention.

[0009] The present invention also relates to polynucleotides encoding the polypeptides of the invention; nucleic acid constructs; recombinant expression vectors; recombinant host cells containing the polynucleotides; and methods for producing the polypeptides.

[0010] Other aspects and embodiments of the invention will become apparent from the description and examples.

[0011] array SEQ ID NO: 1: Polynucleotide encoding the propeptide from Chryseobacterium sp-62563. SEQ ID NO:2: Amino acid sequence of the propeptide encoded by SEQ ID NO:1. SEQ ID NO: 3: Polynucleotide encoding a deamidase polypeptide from Chryseobacterium sp-62563. SEQ ID NO:4: Amino acid sequence of the deamidase polypeptide encoded by SEQ ID NO:3. SEQ ID NO:5: Amino acid sequence of a fusion polypeptide comprising SEQ ID NOs:2 and 4. SEQ ID NO: 6: Polynucleotide encoding the propeptide derived from Chryseobacterium gambrini. SEQ ID NO:7: Amino acid sequence of the propeptide encoded by SEQ ID NO:6. SEQ ID NO: 8: Polynucleotide encoding a deamidase polypeptide derived from Chryseobacterium gambrini. SEQ ID NO:9: Amino acid sequence of the deamidase polypeptide encoded by SEQ ID NO:8. SEQ ID NO:10: Amino acid sequence of a fusion polypeptide comprising SEQ ID NOs:7 and 9. SEQ ID NO:11: Polynucleotide encoding the propeptide derived from Chryseobacterium culicis. SEQ ID NO:12: Amino acid sequence of the propeptide encoded by SEQ ID NO:11. SEQ ID NO: 13: A polynucleotide encoding a deamidase polypeptide derived from Chryseobacterium culicis. SEQ ID NO:14: Amino acid sequence of the deamidase polypeptide encoded by SEQ ID NO:13. SEQ ID NO:15: Amino acid sequence of a fusion polypeptide comprising SEQ ID NOs:12 and 14. SEQ ID NO: 16: Polynucleotide encoding the propeptide derived from Chryseobacterium defluvii. SEQ ID NO:17: Amino acid sequence of the propeptide encoded by SEQ ID NO:16. SEQ ID NO: 18: A polynucleotide encoding a deamidase polypeptide derived from Chryseobacterium defluvii. SEQ ID NO:19: Amino acid sequence of the deamidase polypeptide encoded by SEQ ID NO:18. SEQ ID NO:20: Amino acid sequence of a fusion polypeptide comprising SEQ ID NOs:17 and 19. SEQ ID NO: 21: Amino acid sequence motif overlapping the deamidase active site. SEQ ID NO: 22: Amino acid sequence motif overlapping the deamidase active site. SEQ ID NO: 23: Amino acid sequence motif overlapping the deamidase active site. SEQ ID NO: 24: Amino acid sequence of SEQ ID NO: 9 without the His-tag. SEQ ID NO: 25: Amino acid sequence of SEQ ID NO: 10 without the His-tag. SEQ ID NO: 26: Amino acid sequence of SEQ ID NO: 14 without the His-tag. SEQ ID NO: 27: Amino acid sequence of SEQ ID NO: 15 without the His-tag. SEQ ID NO: 28: Amino acid sequence of SEQ ID NO: 19 without the His-tag. SEQ ID NO: 29: Amino acid sequence of SEQ ID NO: 20 without the His-tag. SEQ ID NO: 30: Polynucleotide encoding the propeptide from Chryseobacterium proteolyticum. SEQ ID NO:31: Amino acid sequence of the propeptide encoded by SEQ ID NO:30. SEQ ID NO: 32: Polynucleotide encoding a deamidase polypeptide from Chryseobacterium proteolyticum. SEQ ID NO:33: Amino acid sequence of the deamidase polypeptide encoded by SEQ ID NO:32. SEQ ID NO:34: Amino acid sequence of a fusion polypeptide comprising SEQ ID NOs:31 and 33. SEQ ID NO: 35: Amino acid sequence of SEQ ID NO: 33 without the His-tag. SEQ ID NO: 36: Amino acid sequence of SEQ ID NO: 34 without the His-tag.

[0012] definition In accordance with this Detailed Description, the following definitions apply: It should be noted that the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise.

[0013] Unless otherwise defined or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0014] Deamidase: The term "deamidase" refers to a protein-glutamine glutaminase (also known as glutaminylpeptide glutaminase) activity as described in EC 3.5.1.44 that catalyzes the hydrolysis of gamma-amides of glutamine substituted at the carboxyl position or at both the alpha-amino and carboxyl positions (e.g., L-glutaminylglycine and L-phenylalanyl-L-glutaminylglycine). Thus, deamidases can deamidate glutamine residues in proteins to glutamic acid residues and are also referred to as protein glutamine deamidases. The deamidase comprises a Cys-His-Asp catalytic triad (e.g., Cys-156, His-197, and Asp-217 as shown in Hashizume et al. "Crystal structures of protein glutaminase and its pro forms converted into enzyme-substrate complex", Journal of Biological Chemistry, vol. 286, no. 44, pp. 38691-38702) and belongs to InterPro entry IPR041325. In a preferred embodiment, the deamidase of the invention belongs to the PFAM domain PF18626. The deamidase amino acid sequence may comprise the active site overlapping amino acid motifs DGCYARAH (SEQ ID NO: 21), CYARAH[R / K / Q] (SEQ ID NO: 22), and / or HVA[L / V / I]LVS (SEQ ID NO: 23).

[0015] Deamidase activity: Deamidase activity was measured by using a fluorescent substrate containing a glutamine residue and a fluorescence quenching group as described in Example 1. The glutamine residue is converted to a glutamic acid residue by the deamidase activity, and the substrate is subsequently cleaved by glutamyl endopeptidase to remove the fluorescence quenching group.

[0016] Deamidase activity may also be measured by deamidating a glutamine substrate (e.g., Cbz-Gln-Gly), generating ammonia in the process. Ammonia is used as a substrate for glutamate dehydrogenase in combination with α-ketoglutarate to generate glutamate. This latter enzymatic reaction requires NADH as a coenzyme. NADH depletion can be followed by kinetic absorbance measurements at 340 nm and is directly proportional to the deamidase activity. The reaction is carried out at pH 7 and 37°C.

[0017] Deamidase inhibitory domain: The term "deamidase inhibitory domain" refers to a sequence of amino acids that interact with amino acid residues in the deamidase active site and inhibit or reduce the deamidase activity. For example, the deamidase activity may be reduced by less than 50%, preferably less than 40%, in the presence of the deamidase inhibitory domain (compared to the deamidase activity without the presence of the deamidase inhibitory activity). The deamidase inhibitory domain may comprise the amino acid sequence motif [I / M][L / I / V][S / T]AQ corresponding to amino acids 35-43 of SEQ ID NO:5, or amino acids 39-43 of SEQ ID NO:10, amino acids 39-43 of SEQ ID NO:15, or amino acids 41-45 of SEQ ID NO:20. The deamidase inhibitory domain may comprise the amino acid sequence motif [K / R][V / I / L][S / A / N]X[I / M][L / I / V][S / T]AQ corresponding to amino acids 35 to 43 of SEQ ID NO:5, or amino acids 35 to 43 of SEQ ID NO:10, amino acids 35 to 43 of SEQ ID NO:15, or amino acids 37 to 45 of SEQ ID NO:20.

[0018] cDNA: The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from a spliced ​​mature mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before it occurs as a spliced ​​mature mRNA.

[0019] Coding sequence: The term "coding sequence" refers to a polynucleotide that directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which generally begins with a start codon, such as ATG, GTG, or TTG, and ends with a stop codon, such as TAA, TAG, or TGA. The coding sequence can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0020] Control sequence: The term "control sequence" refers to a nucleic acid sequence involved in regulating the expression of a polynucleotide in a particular organism or in vitro. Each control sequence may be native (i.e., from the same gene) or heterologous (i.e., from different genes) to the polynucleotide encoding the polypeptide, and may be native or heterologous to each other. Such control sequences include, but are not limited to, leader, polyadenylation, prepropeptide, propeptide, signal peptide, promoter, terminator, enhancer, and transcriptional or translational initiator and terminator sequences. At a minimum, control sequences include promoters and transcriptional and translational stop signals. Control sequences may be provided with linkers to introduce specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding the polypeptide.

[0021] Expression: The term "expression" refers to any step involved in the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0022] Expression vector: An "expression vector" refers to a linear or circular DNA construct containing a DNA sequence encoding a polypeptide, where the coding sequence is operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers and sequences that control the termination of transcription and translation.

[0023] 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 activity.

[0024] Fragment: The term "fragment" refers to a polypeptide having one or more amino acids not present at the amino and / or carboxyl terminus of the mature polypeptide, which fragment has deamidase activity.

[0025] Fusion polypeptide: The term "fusion polypeptide" refers to a polypeptide in which one polypeptide of the invention is fused at the N-terminus and / or C-terminus of another polypeptide of the invention. A fusion polypeptide is generated by fusing two or more polynucleotides that together code for a polypeptide of the invention. Techniques for generating fusion polypeptides are known in the art and include linking coding sequences encoding the polypeptides such that they are in frame and the expression of the fusion polypeptide is under the control of the same promoter and terminator. Fusion polypeptides can also be constructed using intein technology, in which the fusion polypeptide is generated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266:776-779). A fusion polypeptide can further comprise a cleavage site between the two polypeptides. Thus, a fusion polypeptide can, for example, comprise a cleavage site for a site-specific endopeptidase within 20 amino acids, preferably within 10 amino acids, of the C-terminus of the first polypeptide. Examples of well-known site-specific endopeptidases include glutamyl endopeptidases (eg, EC 3.4.21.19 or EC 3.4.21.82), trypsin- and chymotrypsin-like endopeptidases (including enteropeptidase).Many other examples of cleavage sites and corresponding endopeptidases include Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3:568-576; Svetina et al., 2000, J. Biotechnol. 76:245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63:3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9:378-381; Eaton et al., 1986, Biochemistry 25:505-512; Collins-Racie et al., 1995, Biotechnology 13:982-987; Carter et al. al., 1989, Proteins: Structure, Function, and Genetics 6:240-248; and Stevens, 2003, Drug Discovery World 4:35-48.

[0026] Heterologous: The term "heterologous" with respect to a host cell means that a polypeptide or nucleic acid does not naturally occur in the host cell. The term "heterologous" with respect to a polypeptide or nucleic acid means that a regulatory sequence of the polypeptide or nucleic acid, e.g., a promoter, is not naturally associated with the polypeptide or nucleic acid, i.e., the regulatory sequence is derived from a gene other than the gene encoding the mature polypeptide.

[0027] Host strain or host cell: A "host strain" or "host cell" is an organism into which an expression vector, phage, virus, or other DNA construct containing a polynucleotide encoding a polypeptide of interest (e.g., an amylase) has been introduced. Exemplary host strains are microbial cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing a polypeptide of interest and / or fermenting sugars. The term "host cell" includes protoplasts produced from cells.

[0028] Introduced: The term "introduced" in the context of inserting a nucleic acid sequence into a cell means "transfection," "transformation," or "transduction" as known in the art.

[0029] Isolated: The term "isolated" refers to a polypeptide, nucleic acid, cell, or other specified material or component that is separated from at least one other material or component, including but not limited to other proteins, nucleic acids, cells, etc. Thus, an isolated polypeptide, nucleic acid, cell, or other material is in a form that is not found in nature. Isolated polypeptides include, but are not limited to, culture broths containing the polypeptide expressed and secreted in a host cell.

[0030] Mature Polypeptide: The term "mature polypeptide" refers to the mature form of a polypeptide following N-terminal and / or C-terminal processing (e.g., removal of a signal peptide). In one aspect, the mature polypeptide is SEQ ID NO:4.

[0031] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" refers to a polynucleotide that encodes a mature polypeptide having deamidase activity. In one embodiment, the mature polypeptide coding sequence is SEQ ID NO:3.

[0032] Native: The term "native" refers to a nucleic acid or polypeptide that is naturally present in a host cell.

[0033] Nucleic Acid: The term "nucleic acid" includes DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids may be single- or double-stranded and may be chemically modified. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to code for a particular amino acid, and the present compositions and methods encompass nucleotide sequences that code for a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in the 5' to 3' direction.

[0034] Nucleic Acid Construct: The term "nucleic acid construct" refers to a nucleic acid molecule, either single-stranded or double-stranded, that is isolated from a naturally occurring gene, or that is modified or synthetic to contain a segment of nucleic acid in a manner that would not otherwise occur in nature, and that includes one or more regulatory sequences operably linked to the nucleic acid sequence.

[0035] Operably linked: The term "operably linked" means that the specified components are in a relationship (including, but not limited to, a proximal relationship) that permits them to function in their intended manner. For example, a regulatory sequence is operably linked to a coding sequence such that expression of the coding sequence is under the control of the regulatory sequence.

[0036] Purified: The term "purified" refers to a nucleic acid, polypeptide, or cell that is substantially free of other components as determined by analytical techniques well known in the art (e.g., a purified polypeptide or nucleic acid may form a discrete band in an electrophoretic gel, a chromatographic eluate, and / or a medium subjected to density gradient centrifugation). A purified nucleic acid or polypeptide is at least about 50% pure, and typically is 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., percent on a weight or molar basis). In a related sense, a composition is enriched with respect to a molecule if there is a substantial increase in the concentration of that molecule following application of a purification or concentration technique. The term "enriched" refers to a compound, polypeptide, cell, nucleic acid, amino acid, or other specified material or component that is present in a composition at a higher relative or absolute concentration as compared to the starting composition.

[0037] In one aspect, the term "purified" as used herein refers to a polypeptide or cell that is essentially free of components, particularly insoluble components, from the producing organism. In another aspect, the term "purified" refers to a polypeptide that is essentially free of insoluble components, particularly insoluble components, from the natural organism from which the polypeptide is obtained. In one aspect, the polypeptide is separated from the organism from which it is recovered and from a portion of the soluble components of the culture medium. The polypeptide may be purified (i.e., separated) by one or more of the unit operations filtration, precipitation, or chromatography.

[0038] Thus, a polypeptide may be purified such that only trace amounts of other proteins (particularly other polypeptides) are present. The term "purified" as used herein may refer to the removal of other components (particularly other proteins, most particularly other enzymes) present in the cell of origin of the polypeptide. A polypeptide may be "substantially pure", i.e., free of other components from the organism that produces the polypeptide (e.g., the host organism in the case of recombinantly produced polypeptides). In one aspect, the polypeptide is at least 40% pure by weight of the total polypeptide material present in a preparation. In one aspect, the polypeptide is at least 50%, 60%, 70%, 80%, or 90% pure by weight of the total polypeptide material present in a preparation. As used herein, a "substantially pure polypeptide" can mean a polypeptide preparation that contains up to 10% by weight of other polypeptide material with which the polypeptide is naturally or recombinantly associated, preferably up to 8%, more preferably up to 6%, more preferably up to 5%, more preferably up to 4%, more preferably up to 3%, even more preferably up to 2%, most preferably up to 1%, and most preferably up to 0.5%.

[0039] Thus, a substantially pure polypeptide is preferably at least 92% pure, more preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99% pure, and most preferably at least 99.5% pure, by weight of the total polypeptide material present in the preparation. The polypeptides of the invention are preferably in substantially pure form (i.e., the preparation is essentially free of other polypeptide material with which it is naturally or recombinantly associated). This can be accomplished, for example, by preparing the polypeptide by well-known recombinant or classical purification methods.

[0040] Recombinant: The term "recombinant" is used in its conventional sense to refer to the manipulation, e.g., cutting and rejoining, of nucleic acid sequences to form a constellation different from that found in nature. The term recombinant refers to a cell, nucleic acid, polypeptide, or vector that has been altered from its natural state. Thus, for example, a recombinant cell expresses genes not found within the native form of the cell (non-recombinant), or expresses native genes at levels or under different conditions than found in nature. The term "recombinant" is synonymous with "genetically modified" and "transgenic."

[0041] Recover: The term "recover" or "recovery" refers to the removal of the polypeptide from at least one fermentation broth component selected from the list of cells, nucleic acids or other specified materials, for example, recovery of the polypeptide from the whole fermentation broth or from the cell-free fermentation broth, which is carried out by collecting the polypeptide from the broth medium by filtration, for example, depth filtration (by using filter aids or packed filter materials, fabric filtration in chamber filters, rotary drum filtration, drum filtration, rotary vacuum drum filters, candle filters, horizontal leaf filters or similar, with seed or pad filtration in a framework or modular setup) or membrane filtration (using sheet filtration, module filtration, candle filtration, microfiltration, ultrafiltration, either in cross-flow, dynamic cross-flow or dead-end operation), or by centrifugation (using decanter centrifuges, disc centrifuges, hydrocyclones or similar), or by precipitating the polypeptide and using particle size classification separation using suitable solid-liquid separation methods. Recovery encompasses isolation and / or purification of the polypeptide.

[0042] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity."

[0043] For the purposes of the present invention, sequence identity between two amino acid sequences is determined as the "longest identity" output using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48; 443-453), preferably as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), version 6.6.0 or later. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order for the Needle program to report the longest identity, the -nobrief option must be specified on the command line. The output of Needle labeled "longest identity" is calculated as follows: (identical residues × 100) / (length of alignment−total number of gaps in the alignment).

[0044] For the purposes of the present invention, sequence identity between two polynucleotide sequences is preferably determined as the "longest identity" output using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, see above) as implemented in the Needle program of the EMBOSS package (EMBOSS; The European Molecular Biology Open Software Suite, Rice et al., 2000, see above), version 6.6.0 or later. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and an EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix. In order for the Needle program to report the longest identity, the nobrief option must be specified on the command line. The output of Needle labeled "longest identity" is calculated as follows: (identical nucleotides × 100) / (length of alignment−total number of gaps in the alignment).

[0045] Signal peptide: A "signal peptide" is a sequence of amino acids attached to the N-terminal portion of a protein that facilitates secretion of the protein outside of the cell. The mature form of the extracellular protein lacks the signal peptide, which is cleaved off during the secretion process.

[0046] Subsequence: The term "subsequence" refers to a polynucleotide having one or more nucleotides missing at the 5' and / or 3' end of a mature polypeptide coding sequence, wherein the subsequence encodes a fragment that has deamidase activity.

[0047] Thermal denaturation temperature: "melting temperature", "T m The term "thermal denaturation temperature", also referred to as "thermal melting temperature" or "denaturation midpoint temperature", refers to the temperature at which approximately 50% of the fusion protein is unfolded. Usually, the percentage of folded protein is predominant (>99.999%) at room temperature and the melting temperature, T m It decreases as it approaches T m At this rate, approximately 50% of the molecules are in a folded state and approximately 50% are in an unfolded state. m At temperatures above 100° C., the unfolded state becomes the predominant species (>50%). A preferred method for determining the terminal unfolding temperature of a fusion protein of the invention is nanoDSF. Using nanoDSF, T m The determination of T can be performed at 330 nm, 350 nm, and / or the ratio 330 nm / 350 nm. m is equal to the temperature of the inflection point (IP) of the first derivative, where the first derivative reaches a local maximum or minimum and the second derivative has an isolated zero. m Or the thermal denaturation temperature is the temperature of the inflection point (IP) of the first derivative determined by nanoDSF at 330 nm as described in Example 2.

[0048] Other methods for determining the thermal denaturation temperature are the thermal shift assay as described in Current Protocols in Protein Science: “Analysis of protein stability and ligand interactions by thermal shift assay” (K. Huynh and CL Partch, 2015) or the method described in Encyclopedia of Industrial Biotechnology: “Proteins: Thermal Unfolding” (R. Lonescu and L. Shi, 2009).

[0049] Thermal denaturation is an important tool for evaluating protein stability. To evaluate the protein preference to maintain its folded (active) conformation, the protein is exposed to high levels of denaturation stress (temperature), and protein stability is determined based on the stress level required to generate a significant proportion of unfolded protein. Factors that affect thermal stability include, but are not limited to, protein structure, the presence of ligands or excipients, binding strength between inhibitors and enzymes, and solvent conditions.

[0050] Variant: The term "variant" refers to a polypeptide having deamidase activity and which contains an artificial mutation (i.e., a substitution, an insertion (including an extension), and / or a deletion (e.g., a truncation)) at one or more positions. A substitution refers to the replacement of an amino acid occupying a position with a different amino acid; a deletion refers to the removal of an amino acid occupying a position; and an insertion refers to the addition of 1 to 5 amino acids (e.g., 1 to 3 amino acids, specifically 1 amino acid) adjacent to and immediately following the amino acid occupying a position.

[0051] Wild-type: The term "wild-type" with respect to an amino acid sequence or a nucleic acid sequence means that the amino acid sequence or nucleic acid sequence is a natural or naturally occurring sequence. As used herein, the term "naturally occurring" refers to something that is found in nature (e.g., a protein, amino acid, or nucleic acid sequence). Conversely, the term "non-naturally occurring" refers to something that is not found in nature (e.g., recombinant nucleic acids and modifications of protein sequences or wild-type sequences produced in the laboratory).

[0052] Rules for naming variants: For the purposes of the present invention, the polypeptide disclosed in SEQ ID NO: 5 is used to determine the corresponding amino acid residue position in another propeptide and / or deamidase. The amino acid sequence of another propeptide and / or deamidase is aligned with the polypeptide disclosed in SEQ ID NO: 5, and based on this alignment, the amino acid position number corresponding to any amino acid residue in the polypeptide disclosed in SEQ ID NO: 5 is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48; 443-453), preferably as implemented in the Needle program of the EMBOSS package (EMBOSS; The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16; 276-277), version 5.0.0 or later. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

[0053] In describing the variants of the present invention, the nomenclature described below is adapted for ease of reference: Recognized IUPAC one-letter or three-letter amino acid abbreviations are employed.

[0054] Substitutions. In the case of amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Thus, a substitution of threonine with alanine at position 226 is designated "Thr226Ala" or "T226A". Multiple mutations are separated by additional marks ("+"), e.g., "Gly205Arg+Ser411Phe" or "G205R+S411F" represent the substitution of serine (S) with glycine (G) and phenylalanine (F) with arginine (R) at positions 205 and 411, respectively.

[0055] Deletions. In the case of amino acid deletions, the following nomenclature is used: original amino acid, position, * Therefore, the deletion of glycine at position 195 is called "Gly195 * " or "G195 * " Multiple deletions are separated by additional marks ("+"), e.g., "Gly195 * +Ser411 * " or "G195 * +S411 * "

[0056] Insertions. In the case of amino acid insertions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Thus, an insertion of a lysine after the glycine at position 195 would be designated as "Gly195GlyLys" or "G195GK". Insertions of multiple amino acids are designated as [original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2, etc.]. For example, an insertion of a lysine and an alanine after the glycine at position 195 would be designated as "Gly195GlyLysAla" or "G195GKA".

[0057] In such cases, the inserted amino acid residue is numbered by adding a lower case letter to the position number of the amino acid residue preceding it. In the above example, the sequence would be:

[0058] [Table 1]

[0059] Multiple modifications. Variants containing multiple modifications are separated by additional marks ("+"), for example, "Arg170Tyr+Gly195Glu" or "R170Y+Gly195E" represent the substitution of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively.

[0060] Different modifications. When different modifications can be introduced at a position, the different modifications are separated by commas, for example, "Arg170Tyr,Glu" or "R170Y,E" represents the substitution of arginine with tyrosine or glutamic acid at position 170. Thus, "Tyr167Gly,Ala+Arg170Gly,Ala" or "Y167G,A+R170G,A" designate the following variants: "Tyr167Gly+Arg170Gly", "Tyr167Gly+Arg170Ala", "Tyr167Ala+Arg170Gly", and "Tyr167Ala+Arg170Ala". DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061] Recombinant deamidase fusion polypeptides The present invention relates to a recombinant deamidase fusion polypeptide (pro-form). In one aspect, the present invention relates to a recombinant deamidase fusion polypeptide (pro-form), (a) a first polypeptide comprising a deamidase inhibitor domain, and (b) a recombinant fusion polypeptide comprising or consisting of a second polypeptide having deamidase activity, The fusion polypeptide has a thermal denaturation temperature in the range of 65-82° C.; and the fusion polypeptide has a deamidase activity that is less than 50% compared to the deamidase activity of the second polypeptide.

[0062] In one embodiment, the thermal denaturation temperature is in the range of 65 to 80° C., preferably 67 to 80° C., and more preferably 70 to 80° C. The thermal denaturation temperature can be measured as the nanoDSF thermal denaturation temperature, for example, as described in Example 2.

[0063] In certain embodiments, the C-terminus of the first polypeptide precedes the N-terminus of the second polypeptide.

[0064] In one embodiment, the first polypeptide is located near the N-terminus of the fusion polypeptide; preferably within 30 amino acids, e.g., within 20 amino acids or within 10 amino acids, of the N-terminus of the fusion polypeptide. Similarly, the second polypeptide may be located near the C-terminus of the fusion polypeptide; preferably within 30 amino acids, e.g., within 20 amino acids or within 10 amino acids, of the C-terminus of the fusion polypeptide.

[0065] In certain embodiments, the fusion polypeptide has less than 40% deamidase activity, preferably less than 35% deamidase activity, compared to the second polypeptide (leakage activity).

[0066] In one embodiment, the first polypeptide comprises the amino acid sequence motif [I / M][L / I / V][S / T]AQ, and / or the amino acid sequence motif [K / R][V / I / L][S / A / N]X[I / M][L / I / V][S / T]AQ.

[0067] In certain embodiments, the second polypeptide comprises an amino acid sequence motif selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and combinations thereof.

[0068] In one embodiment, the first polypeptide comprises an amino acid change at a position corresponding to a position of SEQ ID NO:2 selected from the group consisting of 23, 38, 39, 43, 45, 67, 69, 88, 91, 92, 94, 95, 96, 98, 99, 100, and 101.

[0069] Preferably, the first polypeptide comprises an amino acid change at a position corresponding to a position of SEQ ID NO:2 selected from the group consisting of V23, F38, M39, Q43, Y45, E67, P69, T88, D91, I92, Y94, F95, K96, F98, F99, T100, and K101.

[0070] More preferably, the first polypeptide is selected from the group consisting of V23G,D,Y,S; F38A,C,D,G,N,T,V; M39D,E,F,G,H,K,N,P,Q,R,S,W,Y; Q43D,E,F,G,I,K,M,R,Y; Y45A,C,G,I,K,M,N,Q,R,S,T,V; E67D,K,N,P,W; P69D,F,G,H,K,L,M,Q,R,S,T,W,Y; T88F,I,K,L,P,R,V,W,Y; D91F,G,H,K,L,M,N,P,Q,R,S,Y; I92G,N,P ,Q,S,T; Y94A,E,I,K,P,Q,R,T; F95A,D,E,G,H,I,K,L,M,N,R,S,T,V; K96C,F,I,P,V,Y; F98A,C,D,E,G,H,K,N,P,Q,R,S,T,W,Y; F99A,C,D,G,H,K,P,Q,R,V,W,Y; T100E,P,W; and K101E,P (shown in the left column of Table 1, "Heat denaturation temperature in the range of 65 to 82°C").

[0071] More preferably, the first polypeptide is selected from the group consisting of V23G,D,Y,S; F38A,C,D,G,N,T,V; M39D,E,F,G,H,K,N,P,Q,R,S,Y; Q43D,E,I,K,R; Y45A,C,G,I,K,M,N,Q,R,S,T,V; E67D,N,P; P69D,F,G,H,K,M,Q,R,S,T,W,Y; T88I,P,W; D91G,H,K,N,P,Q,R, The amino acid changes are at positions corresponding to positions of SEQ ID NO:2 selected from the group consisting of S; I92G,P,S; Y94A,E,I,P,Q,R,T; F95A,D,E,G,H,K,N,R,S,T,V; K96P; F98D,E,G,N,P,Q,S; F99A,C,D,G,H,K,P,Q,R; and T100P,W (shown in the left column of Table 2, "Heat denaturation temperature in the range of 65 to 80°C").

[0072] Even more preferably, the first polypeptide comprises an amino acid change at a position corresponding to a position of SEQ ID NO:2 selected from the group consisting of V23S, F38C, Y45G, Y45T, Y45I, Y45M, Y45R, P69D, P69G, P69L, P69M, P69Q, P69S, P69T, Y94P, F99A, F99G, and F99K.

[0073] Table 1 shows examples of amino acid substitutions in the Chryseobacterium sp-62563 propeptide (first polypeptide) of SEQ ID NO: 2, with thermal denaturation temperatures in the range of 65 to 82° C. The data was generated according to the procedure in Example 4.

[0074] [Table 2]

[0075] Table 2 shows examples of amino acid substitutions in the Chryseobacterium sp-62563 propeptide (first polypeptide) of SEQ ID NO: 2, and the thermal denaturation temperature is in the range of 65 to 80°C.

[0076] [Table 3]

[0077] In one embodiment, the second polypeptide comprises an amino acid change at a position corresponding to a position of SEQ ID NO:5 selected from the group consisting of 190, 252, 254, 255, 256, 258, 259, 260, 268, and 285.

[0078] Preferably, the second polypeptide comprises an amino acid change at a position corresponding to a position of SEQ ID NO:5 selected from the group consisting of V190, Y252, S254, P255, S256, S258, L259, L260, T268, and P285.

[0079] More preferably, the second polypeptide comprises an amino acid change at a position corresponding to a position of SEQ ID NO:5 selected from the group consisting of V190A, V190D, V190F, V190G, V190K, V190M, V190P, V190Q, V190Y, Y252S, S254K, P255D, S256D, S258E, L259P, L260E, L260K, T268I, and P285D.

[0080] In one embodiment, the fusion polypeptide has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:29.

[0081] In one embodiment, the fusion polypeptide has 1 to 30 modifications (e.g., substitutions, deletions and / or insertions), preferably 1 to 20 modifications, 1 to 10 modifications, or 1 to 5 modifications, in particular substitutions, compared to an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:29.

[0082] In one embodiment, the first polypeptide has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:12, and SEQ ID NO:17.

[0083] In one embodiment, the second polypeptide has 1 to 30 modifications (e.g., substitutions, deletions and / or insertions), preferably 1 to 20 modifications, 1 to 10 modifications, or 1 to 5 modifications, particularly substitutions, compared to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:12, and SEQ ID NO:17.

[0084] In one embodiment, the second polypeptide has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:26, and SEQ ID NO:28.

[0085] In one embodiment, the second polypeptide has up to 30 modifications (e.g., substitutions, deletions and / or insertions), preferably up to 20 modifications, up to 10 modifications, or up to 5 modifications, particularly substitutions, compared to an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:26, and SEQ ID NO:28.

[0086] In certain embodiments, the first polypeptide comprises a cleavage site for a site-specific endopeptidase, such as glutamyl endopeptidase or trypsin. Preferably, the cleavage site is located within 20 amino acids, within 15 amino acids, or within 10 amino acids of the C-terminus.

[0087] In another aspect, the invention provides a method for producing a polypeptide having deamidase activity, the method comprising contacting a fusion polypeptide of the invention with a site-specific endopeptidase, such as glutamyl endopeptidase, trypsin-like endopeptidase, or chymotrypsin-like endopeptidase, to separate the first polypeptide from the second polypeptide.

[0088] In yet another aspect, the present invention provides a composition exhibiting deamidase activity, comprising a first polypeptide and a second polypeptide of a fusion polypeptide, wherein the first and second polypeptides are not covalently linked. Preferably, the composition further comprises a site-specific endopeptidase.

[0089] In one embodiment, the composition comprises: (a) 0.001 to 25% w / w of a first polypeptide; (b) 0.001 to 25% w / w of a second polypeptide; (c) a polyol, and (d) water A liquid composition comprising:

[0090] In yet another embodiment, the present invention provides a method for modifying a protein comprising contacting the protein with a composition exhibiting deamidase activity of the present invention. Preferably, the modification is deamidation of a glutamine residue (conversion of glutamine to glutamic acid).

[0091] The positions to be mutated can be identified by visual inspection of the 3D structure of the fusion polypeptide and selecting all those positions that are in or near the interaction interface between the first polypeptide (the propeptide domain) and the second polypeptide (the deamidase domain).

[0092] Amino acid modifications may be of a minor nature, being conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein, such as small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, such as a polyhistidine tract, an antigenic epitope or a binding module, as described above.

[0093] Essential amino acids in a polypeptide can be identified according to 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, single alanine mutations are introduced at every residue in the molecule to identify amino acid residues critical to the activity of the molecule, and the resulting molecules are tested for deamidase activity. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. Enzyme active sites or other biological interactions can also be determined by physical analysis of the structure, as measured by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in combination with mutations of putative contact site amino acids. See, e.g., de Vos et al., 1992, Science 255; 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. Identification of essential amino acids can also be inferred from alignment with related polypeptides and / or from sequence homology and conserved catalytic mechanism with related polypeptides or with polypeptides / proteins (typically with similar three-dimensional structure, function and significant sequence similarity) derived from a common ancestor within a polypeptide or protein family. Additionally or alternatively, protein structure prediction tools can be used for protein structure modeling to identify essential amino acids and / or active sites of polypeptides. See, e.g., Jumper et al., 2021, "Highly accurate protein structure prediction with AlphaFold", Nature 596: 583-589.

[0094] Single or multiple amino acid substitutions, deletions and / or insertions can be made and tested using known mutagenesis, recombination, and / or shuffling methods followed by associated screening procedures such as those disclosed in Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86; 2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, CRISPR gene editing, phage display (e.g., Lowman et al., 1991, Biochemistry 30:10832-10837; U.S. Pat. No. 5,223,409; WO 92 / 06204), and region-specific mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0095] Mutagenesis / shuffling methods can be combined with high-throughput automated screening methods to detect the activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17;893-896). Mutagenized DNA molecules that code for active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues within a polypeptide.

[0096] In certain aspects, the polypeptide is isolated.

[0097] In another embodiment, the polypeptide is purified.

[0098] In another aspect, the invention provides a method for producing a polypeptide having deamidase activity, the method comprising contacting a fusion polypeptide of the invention with an endopeptidase to separate the first polypeptide from the second polypeptide.

[0099] In yet another aspect, the present invention provides a composition that exhibits deamidase activity, comprising isolated first and second polypeptides of a fusion polypeptide of the invention.

[0100] Source of wild-type deamidase fusion polypeptide Wild-type deamidase fusion polypeptides (pro-forms) that can be used to generate recombinant fusion polypeptides of the invention may be obtained from any genus of microorganism (donor strain). The wild-type fusion polypeptide may then be modified in the propeptide (first polypeptide) to reduce the thermal denaturation temperature of the fusion polypeptide according to the invention. Some examples of wild-type fusion polypeptides obtained from Chryseobacterium species are shown in Example 5. For the purposes of the present invention, the term "obtained from", when used herein in relation to a given source, shall mean that the polypeptide encoded by the polynucleotide is produced by the source or produced by a strain into which the polynucleotide of the invention has been inserted. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.

[0101] In certain embodiments, the wild-type fusion polypeptide is obtained from a Chryseobacterium species.

[0102] Wild-type fusion polypeptides may 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.). Methods for directly isolating microorganisms and DNA from natural habitats are well known in the art. Polynucleotides encoding the polypeptides may then be obtained by similarly screening genomic DNA or cDNA libraries of other microorganisms or mixed DNA samples. After the polynucleotides encoding the polypeptides are detected by the probe, the polynucleotides may be isolated or cloned by using techniques known to those skilled in the art (see, for example, Davis et al., 2012, Basic Methods in Molecular Biology, Elsevier).

[0103] Polynucleotides The present invention also relates to polynucleotides encoding the polypeptides of the present invention as described herein.

[0104] The polynucleotide may be mutated by the introduction of nucleotide substitutions that do not result in a change in the amino acid sequence of the polypeptide, but that correspond to the codon usage of the host organism in which the enzyme is intended to be produced, or that may result in a different amino acid sequence. For a general description of nucleotide substitutions, see, e.g., Ford et al., 1991, Protein Expression and Purification 2:95-107.

[0105] In certain aspects, the polynucleotide is isolated.

[0106] In another embodiment, the polynucleotide is purified.

[0107] Nucleic Acid Constructs The present invention also relates to a nucleic acid construct comprising a polynucleotide of the invention operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0108] Polynucleotides can be manipulated in a variety of ways to provide for expression of a polypeptide. It may be desirable or necessary to manipulate the polynucleotide depending on the expression vector prior to insertion into the vector. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0109] promoter The control sequence may be a promoter polynucleotide that is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention. The promoter contains transcriptional control sequences that mediate expression of the polypeptide. The promoter may be any polynucleotide that exhibits transcriptional activity in a host cell, including mutant, truncated and hybrid promoters, and may be derived from a gene encoding an extracellular or intracellular polypeptide, either homologous or heterologous to the host.

[0110] Examples of suitable promoters for directing transcription of the polynucleotides of the invention in bacterial host cells are described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab., NY, Davis et al., 2012, supra, and Song et al., 2016, PLOS One 11(7):e0158447.

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

[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 and Dattenboeck, 2016, "Gene Expression Systems in Fungi: Advancements and Applications", Fungal Biology.

[0113] Terminator The control sequence may also be a transcription terminator recognized by a host cell to terminate transcription. The terminator is operably linked to the 3' end of the polynucleotide encoding the polypeptide. Any terminator functional in the host cell may be used in the present invention.

[0114] Preferred terminators for bacterial host cells may be obtained from the genes for alkaline protease from Bacillus clausii (aprH), alpha-amylase from Bacillus licheniformis (amyL), and ribosomal RNA from Escherichia coli (rrnB).

[0115] Preferred terminators for filamentous fungal host cells may be obtained from Aspergillus or Trichoderma species, such as those obtained from the genes for Aspergillus niger glucoamylase, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, and Trichoderma reesei endoglucanase I, such as the terminators described in Mukherjee et al., 2013, "Trichoderma: Biology and Applications", and Schmoll and Dattenboeck, 2016, "Gene Expression Systems in Fungi: Advancements and Applications", Fungal Biology.

[0116] Preferred terminators for yeast host cells may be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1) and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, Yeast 8:423-488.

[0117] mRNA Stabilizing Agents A regulatory sequence can also be an mRNA stabilizing region downstream of a promoter and upstream of the coding sequence of a gene that increases expression of the gene.

[0118] Examples of suitable mRNA stabilization regions are obtained from the cryIIIA gene of Bacillus thuringiensis (WO 94 / 25612) and the Bacillus subtilis SP82 gene (Hue et al., 1995, J. Bacteriol. 177:3465-3471).

[0119] Examples of mRNA stabilization regions for fungal cells are described in Geisberg et al., 2014, Cell 156(4):812-824, and Morozov et al., 2006, Eukaryotic Cell 5(11):1838-1846.

[0120] Leader sequence The control sequence may also be a leader, untranslated region of an mRNA that is important for translation by the host cell. The leader is operably linked to the 5' end of the polynucleotide encoding the polypeptide. Any leader that is functional in the host cell may be used.

[0121] Suitable leaders for bacterial host cells are described in Hambraeus et al., 2000, Microbiology 146(12):3051-3059, and Kaberdin and Blaesi, 2006, FEMS Microbiol. Rev. 30(6):967-979.

[0122] Preferred leaders for filamentous fungal host cells may be obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0123] Suitable leaders for yeast host cells may be obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0124] Polyadenylation sequence The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3' end of a polynucleotide which, when transcribed, is recognized by a host cell as a signal to add polyadenosine residues to the transcribed mRNA. Any polyadenylation sequence functional in the host cell may be used.

[0125] Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0126] Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15:5983-5990.

[0127] Signal peptide The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a polypeptide and directs the polypeptide into the secretory pathway of a cell. The 5' end of the coding sequence of a polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding sequence that is heterologous to the coding sequence. A heterologous signal peptide coding sequence may be required if the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, the heterologous signal peptide coding sequence may simply replace the native signal peptide coding sequence to enhance secretion of the polypeptide. Any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of the host cell may be used.

[0128] Effective signal peptide coding sequences for bacterial host cells are those obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alpha-amylase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prsA. Additional signal peptides are described by Freudl, 2018, Microbial Cell Factories 17:52.

[0129] Effective signal peptide coding sequences for filamentous fungal host cells are signal peptide coding sequences obtained from genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase, such as the signal peptides described by Xu et al., 2018, Biotechnology Letters 40:949-955.

[0130] Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., supra, 1992.

[0131] Regulatory Sequences It may also be desirable to add regulatory sequences that regulate the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory sequences are those that turn expression of the gene on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory sequences in prokaryotic systems include the lac, tac and trp operator systems. In yeast, the ADH2 or GAL1 system may be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, the Aspergillus oryzae TAKA alpha-amylase promoter and the Aspergillus oryzae glucoamylase promoter, the Trichoderma reesei cellobiohydrolase I promoter and the Trichoderma reesei cellobiohydrolase II promoter may be used. Other examples of regulatory sequences are those that allow for gene amplification. In fungal systems, these regulatory sequences include dihydrofolate reductase, which is amplified in the presence of methotrexate, and metallothionein genes, which are amplified with heavy metals.

[0132] Transcription factors The control sequence may also be a transcription factor, which is a polynucleotide that encodes a polynucleotide-specific DNA-binding polypeptide that controls the rate of transcription of genetic information from DNA to mRNA by binding to a specific polynucleotide sequence. Transcription factors may 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 that often binds to a specific DNA sequence adjacent to the genetic element regulated by the transcription factor. Transcription factors may regulate the expression of a protein of interest directly, i.e., by activating the transcription of a gene encoding the protein of interest by binding to its promoter, or indirectly, i.e., by activating the transcription of a further transcription factor that regulates the transcription of the gene encoding the protein of interest, such as by binding to the promoter of the further transcription factor. Suitable transcription factors for fungal host cells are described in WO 2017 / 144177. Suitable transcription factors for prokaryotic host cells are described in Seshasayee et al., 2011, Subcellular Biochemistry 52:7-23, and Balleza et al., 2009, FEMS Microbiol. Rev. 33(1):133-151.

[0133] Expression vector The present invention also relates to recombinant expression vectors comprising the polynucleotide of the present invention, a promoter, and transcription and translation termination signals. Various nucleotides and control sequences can be linked together to generate recombinant expression vectors that may contain one or more convenient restriction sites to allow for the insertion or replacement of a polynucleotide encoding a polypeptide at such site. Alternatively, a polynucleotide can be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into a vector suitable for expression. In forming an expression vector, a coding sequence is placed in the vector such that the coding sequence is operably linked to a control sequence suitable for expression.

[0134] The recombinant expression vector can be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA techniques and can bring about the expression of a polynucleotide. The choice of vector will usually depend on the compatibility of the vector with the host cell into which it is to be introduced. The vector can be a linear or closed circular plasmid.

[0135] The vector may be a self-replicating vector, i.e. a vector that exists as an extrachromosomal entity and does not depend on chromosomal replication, such as, for example, a plasmid, an extrachromosomal element, a minichromosome or an artificial chromosome. The vector may contain any means for ensuring self-replication. Alternatively, the vector may be integrated into the genome when introduced into a host cell and replicated together with the chromosome or chromosomes that it is integrated into. Furthermore, a single vector or plasmid, or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.

[0136] Vectors preferably contain one or more selectable markers which facilitate the selection of cells that have been transformed, transfected, transduced, etc. A selectable marker is a gene the product of which provides biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.

[0137] The vector preferably contains at least one element that allows for integration of the vector into the genome of the host cell or for autonomous replication of the vector within the cell independent of the genome.

[0138] For integration into the host cell genome, the vector may rely on the sequence of the polynucleotide encoding the polypeptide or any other element of the vector for integration into the genome by homologous recombination, such as homology directed repair (HDR), or non-homologous recombination, such as non-homologous end joining (NHEJ).

[0139] For autonomous replication, the vector may further comprise an origin of replication that allows the vector to replicate autonomously in a host cell of interest. The origin of replication may be any plasmid replicator that mediates autonomous replication in a cell. The term "origin of replication" or "plasmid replicator" refers to a polynucleotide that allows a plasmid or vector to replicate in vivo.

[0140] Two or more copies of the polynucleotide of the present invention can be inserted into host cell to increase the production of polypeptide.For example, two or three or four or five or more copies are inserted into host cell.Increasing the copy number of polynucleotide can be achieved by integrating at least one additional copy of sequence into host cell genome or by including an amplifiable selectable marker gene in polynucleotide, and by culturing cells in the presence of a suitable selection agent, the cells that contain the amplified copy of the selectable marker gene, and therefore the cells that contain the additional copy of polynucleotide, can be selected.

[0141] host cell The present invention also relates to recombinant host cells containing a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of a polypeptide of the present invention.

[0142] The construct or vector containing the polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extrachromosomal vector as described above. The choice of host cell will depend largely on the gene encoding the polypeptide and its source. The polypeptide may be native or heterologous to the recombinant host cell. Also, at least one of the one or more control sequences may be heterologous to the polynucleotide encoding the polypeptide. The recombinant host cell may contain a single copy or at least two copies, for example, three, four, five or more copies, of the polynucleotide of the invention.

[0143] The host cell can be any microbial cell useful for the recombinant production of the polypeptides of the invention, such as a prokaryotic or fungal cell.

[0144] Prokaryotic host cells can be any gram-positive or gram-negative bacteria, including, but not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0145] Bacterial host cells include, but are not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus The Bacillus cell may be any Bacillus cell, including Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells. In some embodiments, the Bacillus cell is a Bacillus amyloliquefaciens, Bacillus licheniformis, and Bacillus subtilis cell.

[0146] For purposes of this invention, the Bacillus class / genus / species shall be defined as set forth in Patel and Gupta, 2020, Int. J. Syst. Evol. Microbiol. 70:406-438.

[0147] A bacterial host cell may also be any Streptococcus cell, including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

[0148] A bacterial host cell can also be any Streptomyces cell, including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

[0149] Methods for introducing DNA into prokaryotic host cells are well known in the art and any suitable method can be used, including but not limited to protoplast transformation, competent cell transformation, electroporation, conjugation, transduction, where the DNA is introduced as a linear or circular polynucleotide. A person skilled in the art will be able to readily identify a suitable method for introducing DNA into a given prokaryotic cell, for example depending 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.

[0150] The host cell may be a fungal cell. As used herein, "fungi" includes Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota, as well as Oomycota and all vegetative spore-forming fungi (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

[0151] Fungal cells may be transformed by processes including protoplast-mediated transformation, Agrobacterium-mediated transformation, electroporation, gene gun methods and shock wave-mediated transformation, which are reviewed by Li et al., 2017, Microbial Cell Factories 16:168 and procedures described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, Christensen et al., 1988, Bio / Technology 6:1419-1422, and Lubertozzi and Keasling, 2009, Biotechn. Advances 27:53-75. However, any method known in the art for introducing DNA into fungal host cells may be used, and DNA may be introduced as a linear or circular polynucleotide.

[0152] The fungal host cell may be a yeast cell. As used herein, "yeast" includes ascosporogenous yeasts (Endomycetales), basidiomycete yeasts, and yeasts belonging to the fungi imperfecti (Blastomycetes). For purposes of the present invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0153] Yeast host cells may be selected from the group consisting of Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, and Yarrowia lipolytica. The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Candida lipolytica cell. In a preferred embodiment, the yeast host cell is a Pichia or Komagataella cell, such as a Pichia pastoris cell (Komagataella phaffii).

[0154] The fungal host cell may be a filamentous fungal cell. "Filamentous fungi" includes all filamentous forms of the subdivisions Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). Filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.

[0155] Filamentous fungal host cells include those of the genera Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, The host cell may be a cell of the genus Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma. In a preferred embodiment, the filamentous fungal host cell is an Aspergillus, Trichoderma, or Fusarium cell. In a further preferred embodiment, the filamentous fungal host cell is an Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, or Fusarium venenatum cell.

[0156] For example, filamentous fungal host cells include Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis girvescens, and the like. gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bacteridioidesbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochromium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Talaromyces emersonii, Thielavia terrestrisThe cell may be a Trichoderma terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei or Trichoderma viride cell.

[0157] In some embodiments, the host cell is isolated.

[0158] In another embodiment, the host cells are purified.

[0159] How to generate The present invention also relates to a method of producing a polypeptide of the present invention comprising: (a) culturing a recombinant host cell of the present invention under conditions conducive to the production of the polypeptide; and, optionally, (b) recovering the polypeptide.

[0160] The host cells are cultured in a nutrient medium suitable for the production of the polypeptide using methods known in the art. For example, the cells can be cultured in a suitable medium and under conditions that allow expression and / or isolation of the polypeptide by shake flask culture or by small- or large-scale fermentation in laboratory or industrial fermenters (including continuous, batch, fed-batch, or solid-state and / or microcarrier-based fermentation). Suitable media are available from commercial suppliers or can be prepared according to published compositions (e.g., in catalogs of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, the polypeptide can be recovered from cell lysates.

[0161] Polypeptides may be detected using methods known in the art that are specific for the polypeptides, including, but not limited to, the use of specific antibodies, formation of an enzyme product, disappearance of an enzyme substrate, or assays that determine the relative or specific activity of the polypeptide.

[0162] The polypeptide may be recovered from the medium using methods known in the art, including, but not limited to, harvesting, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. In one aspect, the whole fermentation broth containing the polypeptide is recovered. In another aspect, the cell-free fermentation broth containing the polypeptide is recovered.

[0163] Polypeptides can be purified by various procedures known in the art to obtain substantially pure polypeptides and / or polypeptide fragments (see, e.g., Wingfield, 2015, Current Protocols in Protein Science; 80(1):6.1.1-6.1.35; Labrou, 2014, Protein Downstream Processing, 1129:3-10).

[0164] In an alternative embodiment, the polypeptide is not recovered.

[0165] Deamidase Granules The present invention also relates to enzyme granules / particles comprising the polypeptides of the present invention. In one embodiment, the granules comprise a core and optionally one or more coatings (outer layers) surrounding the core.

[0166] The core may have a diameter, measured as an equivalent spherical diameter (average particle size by volume), of 20 to 2000 μm, in particular 50 to 1500 μm, 100 to 1500 μm or 250 to 1200 μm. The core diameter, measured as an equivalent spherical diameter, may be determined using laser diffraction, such as using a Malvern Mastersizer and / or the method described under ISO 13320 (2020).

[0167] In certain embodiments, the core comprises a polypeptide having deamidase activity of the present invention.

[0168] The core may contain additional materials such as fillers, fibrous materials (cellulose or synthetic), stabilizers, solubilizers, suspending agents, viscosity modifiers, light spheres, plasticizers, salts, lubricants and fragrances.

[0169] The core may include a binder such as a synthetic polymer, a wax, a fat, or a carbohydrate.

[0170] The core may include salts of multivalent cations, reducing agents, antioxidants, peroxide decomposition catalysts and / or acidic buffer components, typically as a homogeneous blend.

[0171] The core may comprise an inert particle into which the polypeptide is absorbed or onto which the enzyme is applied, for example, by fluidized bed coating.

[0172] The core may have a diameter of 20 to 2000 μm, in particular 50 to 1500 μm, 100 to 1500 μm or 250 to 1200 μm.

[0173] The core may be surrounded by at least one coating, for example to improve storage stability, reduce dust formation during handling, or to color the granules. Optional coatings may include salt coatings or other suitable coating materials such as polyethylene glycol (PEG), methylhydroxy-propyl cellulose (MHPC), and polyvinyl alcohol (PVA).

[0174] The coating may be applied in an amount of at least 0.1% by weight of the core, such as at least 0.5%, at least 1%, at least 5%, at least 10% or at least 15% by weight, which may be up to 100%, 70%, 50%, 40% or 30%.

[0175] The coating preferably has a thickness of at least 0.1 μm, particularly at least 0.5 μm, at least 1 μm or at least 5 μm, hi some embodiments the coating has a thickness of less than 100 μm, for example less than 60 μm or less than 40 μm.

[0176] The coating must encapsulate the core unit by forming a substantially continuous layer, which is to be understood as a coating with few or no holes, so that there are few or no uncovered areas of the core unit. The layer or coating must in particular be uniform in thickness.

[0177] The coating may further contain other materials known in the art, such as fillers, anti-adherents, pigments, dyes, plasticizers, and / or binders, such as titanium dioxide, kaolin, calcium carbonate, or talc.

[0178] The salt coating may comprise at least 60% by weight salt, such as at least 65% by weight, at least 70% by weight, at least 75% by weight, at least 80% by weight, at least 85% by weight, at least 90% by weight, at least 95% by weight or at least 99% by weight.

[0179] To provide acceptable protection, the salt coating is preferably at least 0.1 μm, 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 thick. In certain embodiments, the salt coating is less than 100 μm thick, such as less than 60 μm or less than 40 μm thick.

[0180] The salt may 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, such as less than 10 μm or less than 5 μm.

[0181] The salt coating may comprise a single salt or a mixture of two or more salts. The salt may be water soluble, in particular having a solubility of at least 0.1 g in 100 g water at 20° C., preferably at least 0.5 g per 100 g water, such as at least 1 g per 100 g water, for example at least 5 g per 100 g water.

[0182] The salts may be inorganic salts, such as sulfates, sulfites, phosphates, phosphonates, nitrates, chlorides or carbonates, or salts of simple organic acids (having less than 10 carbon atoms, e.g., 6 or less carbon atoms), such as citrates, malonates or acetates. Examples of cations in these salts include alkali or earth alkali metal ions, ammonium ions or metal ions of the first transition series, such as sodium, potassium, magnesium, calcium, zinc or aluminium. Examples of anions include chloride, bromide, iodide, sulfate, sulfite, bisulfite, thiosulfate, phosphate, monobasic phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, metasilicate, citrate, malate, maleate, malonate, succinate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate or gluconate. In particular, alkali or earth alkali metal sulfates, sulfites, phosphates, phosphonates, nitrates, chlorides or carbonates or salts of simple organic acids such as citrates, malonates or acetates may be used.

[0183] The salt of the coating may have a constant moisture content of more than 60%, in particular more than 70%, 80% or more than 85% at 20° C., or may be another hydrated form of such salt (e.g. anhydrous). The salt coating may be as described in WO 00 / 01793 or WO 2006 / 034710.

[0184] Specific examples of suitable salts include NaCl(CH 20℃ = 76%), Na2CO3(CH 20℃ = 92%), NaNO3(CH 20℃= 73%), Na2HPO4(CH 20℃ = 95%), Na3PO4(CH 25℃ = 92%), NH4Cl(CH 20℃ = 79.5%), (NH4)2HPO4(CH 20℃ = 93.0%), NH4H2PO4(CH 20℃ = 93.1%), (NH4)2SO4(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.

[0185] The salts may be in anhydrous form or may be hydrated salts, i.e., crystalline salt hydrates containing bound water of crystallization, such as those described in WO 99 / 32595. Specific examples include anhydrous sodium sulfate (Na2SO4), anhydrous magnesium sulfate (MgSO4), magnesium sulfate heptahydrate (MgSO4·7H2O), zinc sulfate heptahydrate (ZnSO4·7H2O), dibasic sodium phosphate heptahydrate (Na2HPO4·7H2O), magnesium nitrate hexahydrate (Mg(NO3)2(6H2O)), sodium citrate dihydrate, and magnesium acetate tetrahydrate.

[0186] Preferably the salt is applied as a solution of the salt, for example using a fluidized bed.

[0187] The coating materials can be wax coating materials and film-forming coating materials. Examples of wax coating materials are poly(ethylene oxide) products (polyethylene glycol, PEG) with an average molecular weight of 1000 to 20000; ethoxylated nonylphenols with 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains 12 to 20 carbon atoms and there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by the fluidized bed technique are given in GB 1483591.

[0188] The granules may optionally have one or more additional coatings. Examples of suitable coating materials include polyethylene glycol (PEG), methylhydroxy-propyl cellulose (MHPC) and polyvinyl alcohol (PVA). Examples of multi-coated enzyme granules are described in WO 93 / 07263 and WO 97 / 23606.

[0189] The cores can be prepared by granulating a blend of the ingredients by methods including granulation techniques such as, for example, crystallization, precipitation, pan coating, fluid bed coating, fluid bed agglomeration, rotary spraying, extrusion, prilling, spheronization, size reduction methods, drum granulation and / or high shear granulation.

[0190] Methods for preparing the cores can be found in Handbook of Powder Technology; Particle size enlargement by CECapes; Vol. 1; 1980; Elsevier. Preparation methods include known feeding and granule formulation techniques, such as: (a) A spray-dried product, in which a liquid polypeptide-containing solution is atomized in a spray-drying tower to form droplets which are dried during passage through the drying tower to form a polypeptide-containing particulate material, thus producing microparticles (Michael S. Showell (editor); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker). (b) Layered products, in which the polypeptide is coated as a layer surrounding a preformed inert core particle, the polypeptide-containing solution is atomized, typically in a fluidized bed apparatus, the preformed core particle is fluidized, the polypeptide-containing solution adheres to the core particle, and is completely dried, leaving a layer of dry polypeptide on the surface of the core particle. In this way, particles of the desired particle size can be obtained, if useful core particles of the desired particle size can be found. This type of product is described, for example, in WO 97 / 23606. (c) Absorbed core particles, in which the polypeptide is absorbed onto and / or into the surface of the core, rather than being coated as a layer around the core. Such a process is described in WO 97 / 39116. (d) Extruded or pelletized products, in which the polypeptide-containing paste is pressed into pellets or extruded under pressure through small openings and cut into particles, which are then dried. Such particles usually have a significant size, since the material from which the extrusion openings are made (usually a plate with perforations) limits the allowable pressure drop across the extrusion openings. When small openings are used, the very high extrusion pressures also increase the heat generation in the polypeptide paste, which is detrimental to the polypeptide (Michael S. Showell (editor); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker). (e) A granulated product in which a polypeptide-containing powder is suspended in molten wax and the suspension is sprayed, for example with a rotating disk atomizer, into a cooling chamber where the droplets rapidly solidify (Michael S. Showell (editor); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker). The resulting product is one in which the polypeptide is uniformly distributed throughout the inert material instead of being concentrated on its surface. U.S. Pat. Nos. 4,016,040 and 4,713,245 describe this technology. (f) Mixer granulation products, where a polypeptide-containing liquid is added to a dry powder composition of conventional granulation components. The liquid and powder in the appropriate ratio are mixed, and as the moisture of the liquid is absorbed into the dry powder, the dry powder components start to adhere and aggregate, and the particles accumulate to form granules containing the polypeptide. Such processes are described in US Pat. No. 4,106,991, EP 170360, EP 304332, EP 304331, WO 90 / 09440 and WO 90 / 09428. In certain products of this process, various high shear mixers can be used as granulators. Granules consisting of polypeptide, fillers and binders etc. are mixed with cellulose fibres to reinforce the particles, producing so-called T-granules. The reinforced particles are more robust and less prone to release enzyme dust. (g) Size reduction, where cores are produced by grinding or crushing larger particles, pellets, tablets, briquettes, etc., containing the polypeptide. The desired core particle fraction is obtained by sieving the ground or crushed product. The large and small particles can be recycled. Size reduction is described in Martin Rhodes (editor); Principles of Powder Technology; 1990; Chapter 10; John Wiley & Sons. (h) Fluidized Bed Granulation. Fluidized bed granulation involves suspending fine particles in an air stream and spraying a liquid through a nozzle onto the fluidized particles. Particles hit by the spray droplets become wet and sticky. The sticky particles collide with other particles causing them to adhere to each other, forming granules. (i) The core may be subjected to drying, such as in a fluid bed dryer. Other known methods for drying granules in the feed or enzyme industry may be used by the skilled person. It is preferred to carry out the drying at a product temperature of 25-90°C. For some polypeptides, it is important that the core containing the polypeptide contains a small amount of water before coating with salt. If a water sensitive polypeptide is coated with salt before removing the excess water, the excess water will become trapped in the core, which may adversely affect the activity of the polypeptide. After drying, it is preferred that the core contains 0.1-10% w / w water.

[0191] Non-shattering granules may be prepared, for example, as disclosed in US Pat. Nos. 4,106,991 and 4,661,452, and may optionally be coated by methods known in the art.

[0192] The granules may further comprise one or more additional enzymes, such as hydrolases, isomerases, ligases, lyases, oxidoreductases, and transferases. The one or more additional enzymes are preferably selected from the group consisting of acetyl xylan esterase, acylglycerol lipase, amylase, alpha-amylase, beta-amylase, arabinofuranosidase, cellobiohydrolase, cellulase, feruloyl esterase, galactanase, alpha-galactosidase, beta-galactosidase, beta-glucanase, beta-glucosidase, lysophospholipase, lysozyme, alpha-mannosidase, beta-mannosidase (mannanase), phytase, phospholipase A1, phospholipase A2, phospholipase D, protease, pullulanase, pectin esterase, triacylglycerol lipase, xylanase, beta-xylosidase, or any combination thereof. In this way, each enzyme is present in more granules, ensuring a more uniform enzyme distribution and reducing physical separation of the various enzymes due to different particle sizes. A method for making multi-enzyme co-granules is disclosed in ip.com disclosure IPCOM000200739D.

[0193] Another example of the formulation of polypeptides by the use of co-granules is disclosed in WO 2013 / 188331.

[0194] The present invention also relates to protected polypeptides produced according to the methods disclosed in EP238216.

[0195] liquid formulation The present invention also relates to liquid compositions comprising the polypeptides of the present invention. The compositions may include enzyme stabilizers (examples include polyols such as propylene glycol or glycerol, sugars or sugar alcohols, lactic acid, reversible protease inhibitors, boric acid or boric acid derivatives, such as aromatic borate esters or phenylboronic acid derivatives, such as 4-formylphenylboronic acid).

[0196] In some embodiments, fillers or carrier materials are included to increase the volume of such compositions. Suitable fillers or carrier materials include, but are not limited to, various salts of sulfates, carbonates, and silicic acids, as well as talc, clay, and the like. Suitable fillers or carrier materials for liquid compositions include, but are not limited to, water or low molecular weight primary and secondary alcohols, including polyols and diols. Examples of such alcohols include, but are not limited to, methanol, ethanol, propanol, and isopropanol. In some embodiments, the compositions contain about 5% to about 90% of such materials.

[0197] In one aspect, the liquid formulation comprises 20-80% w / w polyol. In one embodiment, the liquid formulation comprises 0.001-2% w / w preservative.

[0198] In another embodiment, the present invention provides (a) 0.001 to 25% w / w of the polypeptide of the present invention having deamidase activity; (b) 20-80% w / w of a polyol; (c) optionally, 0.001 to 2% w / w of a preservative; and (d) water The present invention relates to a liquid formulation comprising:

[0199] In another embodiment, the present invention provides (a) 0.001 to 25% w / w of the polypeptide of the present invention having deamidase activity; (b) 0.001-2% w / w of a preservative; (c) optionally, 20-80% w / w of a polyol; and (d) water The present invention relates to a liquid formulation comprising:

[0200] In another embodiment, the liquid formulation comprises one or more formulation agents such as a formulation agent selected from the group consisting of polyols, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, PVA, acetates and phosphates, preferably selected from the group consisting of sodium sulfate, dextrin, cellulose, sodium thiosulfate, kaolin and calcium carbonate. In one embodiment, the polyol is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propylene glycol or 1,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight less than about 600 and polypropylene glycol (PPG) having an average molecular weight less than about 600, more preferably selected from the group consisting of glycerol, sorbitol and propylene glycol (MPG) or any combination thereof.

[0201] In another embodiment, the liquid formulation comprises 20-80% polyol (i.e., total amount of polyol), e.g., 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40-60% polyol. In one embodiment, the liquid formulation comprises 20-80% polyol, e.g., 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40-60% polyol, where the polyol is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propylene glycol or 1,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight of less than about 600, and polypropylene glycol (PPG) having an average molecular weight of less than about 600. In one embodiment, the liquid formulation comprises 20-80% polyol (i.e., total amount of polyols), such as 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40-60% polyol, where the polyol is selected from the group consisting of glycerol, sorbitol, and propylene glycol (MPG).

[0202] In another embodiment, the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof. In one embodiment, the liquid formulation comprises 0.02-1.5% w / w of the preservative, e.g., 0.05-1% w / w of the preservative or 0.1-0.5% w / w of the preservative. In one embodiment, the liquid formulation comprises 0.001-2% w / w of the preservative (i.e., total amount of preservative), e.g., 0.02-1.5% w / w of the preservative, 0.05-1% w / w of the preservative, or 0.1-0.5% w / w of the preservative, wherein the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof.

[0203] 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 acetyl xylan esterase, acylglycerol lipase, amylase, alpha-amylase, beta-amylase, arabinofuranosidase, cellobiohydrolase, cellulase, feruloyl esterase, galactanase, alpha-galactosidase, beta-galactosidase, beta-glucanase, beta-glucosidase, lysophospholipase, lysozyme, alpha-mannosidase, beta-mannosidase (mannanase), phytase, phospholipase A1, phospholipase A2, phospholipase D, protease, pullulanase, pectin esterase, triacylglycerol lipase, xylanase, beta-xylosidase or any combination thereof.

[0204] use Protein deamidase can be applied to almost all kinds of proteins (plant proteins, animal proteins, fermentation proteins, etc.), the enzyme will lower the isoelectric point of the protein, and when the protein is applied at a pH above the isoelectric point, it will improve solubility, electrostatic repulsion, improve different kinds of functionalities such as foaming, emulsification, hydration, etc., change affinity for flavor and off-flavor, gelling properties, improve thermal stability, etc. The enzymatically modified proteins can be applied as ingredients in various foods and beverages or protein deamidase can be directly applied in food production processes such as yogurt fermentation.

[0205] Vegetable proteins often have low solubility and poor functional properties. Deamidation is known to improve the solubility of vegetable proteins, some of which results in improved functional properties including foaming activity, foaming stability, emulsifying activity and emulsion stability. This has been observed on the hybridization of several vegetable protein substrates, including cereal proteins such as oat, wheat, corn proteins, and legume proteins such as soybean and pea proteins, coconut proteins, etc. Negative attributes associated with partially insoluble proteins, such as grittiness and roughness, are mitigated by enzymatic deamidation.

[0206] For example, enzymatically partially amidated oat proteins become essentially completely soluble at neutral pH and also result in significantly improved emulsifying properties. (Zl Jiang at al, J Cereal Science (2015): 64: 126-132). One practical relevance is the use of protein deamidases in processes for oat milk production resulting in oat milk with increased protein content, improved foaming properties, and good stable emulsions well suited to meet the requirements for the barista segment (WO 2014 / 123466). Similarly, emulsifying and foaming properties are improved when soy protein isolates are enzymatically deamidated (I Suppavorasatit et al J. Agric. Food Chem (2011) 59: 11621-11628). Improved solubility, uniformity, dispersibility, and suspendability as well as reduced off-flavors, harshness, and lumps have been observed for enzymatically deamidated pea protein isolates (L Fang et al J, Agric. Food Chem. (2020) 68:1691-1697). Even highly insoluble corn protein (zein) becomes soluble at pH 5 and 7 and has significantly improved emulsifying properties (YH Yong et al. J. Agric Food Chem. (2006):54:6034-6040).

[0207] The improved functional properties provided by enzymatic deamidation make protein deamidases well suited for a variety of food applications, such as vegetable protein-containing milk analogs with increased protein content, reduced graininess and roughness, improved mouthfeel, and barista properties. Similarly, solutions for the yogurt analog segment have improved mouthfeel, texture, and hydrocolloid replacements. Protein deamidases have also been suggested to improve the texture of plant-based meat analogs and plant-based eggs (X Liu et al Foods (2022) 11:440).

[0208] Deamidation of plant proteins also has a positive effect on the flavor of the protein. Plant proteins are associated with various hydrophobic off-flavors, such as lipid oxidation products with, for example, off-flavors or saponins, phenols, and flavonoids that give bitter off-flavors. Enzymatic deamidation of plant proteins reduces the hydrophobicity of the protein and therefore reduces its affinity for hydrophobic off-flavors. Thus, protein deamidases can be applied to improve the flavor of plant proteins by inclusion of the enzyme in the process for recovery of protein concentrates or isolates, or by processing of recovered proteins such as protein isolates (X Liu et al Foods (2022) 11: 440). Flavor improvement is demonstrated, for example, for soybeans (I Suppavorasatit et al J.Agric.Food Chem (2012) 60: 7817-7823).

[0209] The application of protein deamidase in protein recovery processes, such as pea protein recovery processes, leads to improved protein recovery yields, as when applied in recovery processes resulting in legume protein concentrates and isolates (WO2021049591).

[0210] Protein deamidase also has some applications for dairy proteins and dairy-based foods. Deamidation of whey results in better electrostatic repulsion of proteins, giving better thermal stability when the protein solution is heat treated, and avoiding undesirable aggregation in whey protein solutions (e.g., beverages fortified with added protein) (N Miwa et al J.Agric.Food Chem (2013) 61:2205-2212). Enzymatic deamidation in skim milk results in much improved solubility, viscosity, and provides a translucent milk beverage (N Miwa et al International dairy journal (2010) 20:393-399). Application of protein deamidase in yogurt process results in improved stabilization and can be applied, for example, to replace pectin and other hydrocolloids in drinking yogurt.

[0211] Protein (glutaminase) deamidase can be applied together with other enzymes, including other enzymes that modify or degrade proteins. The combination between protein glutamine deamidase and protein asparagine deamidase can provide a higher degree of protein deamidation, thereby providing better application performance. Protein deamidase can be applied together with protein cross-linking enzymes, such as transglutaminase, to modify protein cross-linking, in part because transglutaminase will prevent reaction with glutamine converted to glutamic acid by deamidase. When a combination of transglutaminase and protein deamidase is used in yogurt process, a textural effect is obtained, which can be applied in place of added dairy proteins or hydrocolloids, providing yogurt with a smooth texture and avoiding the lumpy texture seen when transglutaminase is used alone. Similar effects are observed when this enzyme combination is used for the production of plant-based yogurt analogs. Furthermore, protein deamidases can be applied together with proteases, and the resulting protein hydrolysates will have improved solubility and taste as well as altered functional properties.

[0212] Fermentation broth blend or cell composition The present invention also relates to fermentation broth formulations or cell compositions comprising the polypeptides of the invention. The fermentation broth formulations or cell compositions further comprise additional components used in the fermentation process, such as, for example, cells (including host cells containing genes encoding the polypeptides of the invention used to produce the polypeptide of interest), cell debris, biomass, fermentation medium and / or fermentation products. In some embodiments, the composition is a cell-killed whole broth containing organic acids, killed cells and / or cell debris, and culture medium.

[0213] The term "fermentation broth" as used herein refers to a preparation produced by cell fermentation with no or minimal recovery and / or purification. For example, a fermentation broth is produced when a microbial culture is grown to saturation, incubated under carbon-limited conditions, and proteins are synthesized (e.g., expression of enzymes by the host cells) and secreted into the cell culture medium. The fermentation broth may contain unfractionated or fractionated contents of the fermentation material obtained at the end of fermentation. Typically, the fermentation broth is not fractionated and includes spent culture medium and cell debris present after the microbial cells (e.g., filamentous fungal cells) have been removed, for example, by centrifugation. In some embodiments, the fermentation broth contains spent cell culture medium, extracellular enzymes, and viable and / or non-viable microbial cells.

[0214] In some embodiments, the fermentation broth formulation or cell composition comprises a first organic acid component comprising an organic acid of at least 1-5 carbons and / or a salt thereof and a second organic acid component comprising an organic acid of at least 1-6 or more carbons and / or a salt thereof. In some embodiments, the first organic acid component is acetic acid, formic acid, propionic acid, a salt thereof, or a mixture of two or more of the foregoing, and the second organic acid component is benzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid, phenylacetic acid, a salt thereof, or a mixture of two or more of the foregoing.

[0215] In one aspect, the composition contains an organic acid and optionally further contains dead cells and / or cell debris, hi some embodiments, the dead cells and / or cell debris are removed from the cell-killed whole broth to obtain a composition free of these components.

[0216] The fermentation broth formulation or cell composition may further include preservatives and / or antimicrobial (e.g., bacteriostatic) agents, including, but not limited to, sorbitol, sodium chloride, potassium sorbate, and others known in the art.

[0217] The cell-killed whole broth or cell composition may contain the unfractionated contents of the fermentation material obtained at the end of fermentation. Typically, the cell-killed whole broth or cell composition contains spent culture medium and cell debris present after microbial cells (e.g., filamentous fungal cells) have been grown to saturation and incubated under carbon-limited conditions that allow protein synthesis. In some embodiments, the cell-killed whole broth or cell composition contains spent cell culture medium, extracellular enzymes, and killed filamentous fungal cells. In some embodiments, the microbial cells present in the cell-killed whole broth or composition can be permeabilized and / or lysed using methods known in the art.

[0218] Whole broth or cell compositions as described herein are typically liquid, but may contain insoluble components such as dead cells, cell debris, culture medium components, and / or one or more insoluble enzymes, etc. In some embodiments, the insoluble components may be removed to obtain a clarified liquid composition.

[0219] The whole broth formulations and cell compositions of the present invention may be made by the methods described in WO 90 / 15861 or WO 2010 / 096673.

[0220] The present invention is further defined by the following numbered embodiments: Embodiment 1. (a) a first polypeptide comprising a deamidase inhibitor domain, and (b) a recombinant fusion polypeptide comprising or consisting of a second polypeptide having deamidase activity, A recombinant fusion polypeptide, wherein the fusion polypeptide has a thermal denaturation temperature in the range of 65-82°C; and the fusion polypeptide has less than 50% of the deamidase activity compared to a second polypeptide. Embodiment 2. The fusion polypeptide of embodiment 1, wherein the thermal denaturation temperature is in the range of 65 to 80°C. Embodiment 3. The fusion polypeptide of embodiment 1, having a thermal denaturation temperature in the range of 67 to 80°C. Embodiment 4. The fusion polypeptide of embodiment 1, wherein the thermal denaturation temperature is in the range of 70 to 80°C. Embodiment 5. The fusion polypeptide of any of the preceding embodiments, wherein the thermal denaturation temperature is the nanoDSF thermal denaturation temperature. Embodiment 6 The fusion polypeptide of any of the preceding embodiments, wherein the thermal denaturation temperature is the nanoDSF thermal denaturation temperature, as described in Example 2. Embodiment 7 The fusion polypeptide of any of the preceding embodiments, wherein the first polypeptide is located near the N-terminus of the fusion polypeptide. Embodiment 8. The fusion polypeptide of any of the preceding embodiments, wherein the N-terminus of the first polypeptide is located within 30 amino acids, such as within 20 amino acids or within 10 amino acids, of the N-terminus of the fusion polypeptide. Embodiment 9. The fusion polypeptide of any of the preceding embodiments, wherein the second polypeptide is located near the C-terminus of the fusion polypeptide. Embodiment 10. The fusion polypeptide of any of the preceding embodiments, wherein the C-terminus of the second polypeptide is located within 30 amino acids, such as within 20 amino acids or within 10 amino acids, of the C-terminus of the fusion polypeptide. Embodiment 11 The fusion polypeptide of any of the preceding embodiments, wherein the C-terminus of the first polypeptide is located before the N-terminus of the second polypeptide. Embodiment 12. The fusion polypeptide of any of the preceding embodiments, having less than 40% deamidase activity compared to the second polypeptide (leakage activity). Embodiment 13. The fusion polypeptide of any of the preceding embodiments, having less than 35% deamidase activity compared to the second polypeptide (leakage activity). Embodiment 14 The fusion polypeptide of any of the preceding embodiments, wherein the first polypeptide comprises the amino acid sequence motif [I / M][L / I / V][S / T]AQ. Embodiment 15. The fusion polypeptide of any of the preceding embodiments, wherein the first polypeptide comprises the amino acid sequence motif [K / R][V / I / L][S / A / N]X[I / M][L / I / V][S / T]AQ. Embodiment 16 The fusion polypeptide of any of the preceding embodiments, wherein the second polypeptide comprises an amino acid sequence motif selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and combinations thereof. Embodiment 17. The fusion polypeptide of any of the preceding embodiments, wherein the first polypeptide comprises an amino acid change at a position corresponding to a position selected from the group consisting of 23, 38, 39, 43, 45, 67, 69, 88, 91, 92, 94, 95, 96, 98, 99, 100, and 101 of SEQ ID NO:2. Embodiment 18. The fusion polypeptide of any of the preceding embodiments, wherein the second polypeptide comprises an amino acid change at a position corresponding to a position selected from the group consisting of 190, 252, 254, 255, 256, 258, 259, 260, 268, and 285 of SEQ ID NO:5. Embodiment 19. The fusion polypeptide of any of the preceding embodiments, wherein the first polypeptide comprises an amino acid change at a position corresponding to a position selected from the group consisting of V23, F38, M39, Q43, Y45, E67, P69, T88, D91, I92, Y94, F95, K96, F98, F99, T100, and K101 of SEQ ID NO:2. Embodiment 20. The fusion polypeptide of any of the preceding embodiments, wherein the second polypeptide comprises an amino acid change at a position corresponding to a position selected from the group consisting of V190, Y252, S254, P255, S256, S258, L259, L260, T268, and P285 of SEQ ID NO:5. Embodiment 21 The fusion polypeptide of any of the preceding embodiments, wherein the second polypeptide comprises an amino acid change at a position corresponding to a position selected from the group consisting of V190, Y252, S258, L259, L260, T268, and P285 of SEQ ID NO:5. Embodiment 22. The fusion polypeptide of any of the preceding embodiments, wherein the first polypeptide comprises an amino acid change at a position corresponding to a position selected from the group consisting of V23S, F38C, Y45G, Y45T, Y94P, F99A, F99G, and F99K in SEQ ID NO:2. Embodiment 23. The first polypeptide is selected from the group consisting of V23G,D,Y,S; F38A,C,D,G,N,T,V; M39D,E,F,G,H,K,N,P,Q,R,S,W,Y; Q43D,E,F,G,I,K,M,R,Y; Y45A,C,G,I,K,M,N,Q,R,S,T,V; E67D,K,N,P,W; P69D,F,G,H,K,L,M,Q,R,S,T,W,Y; T88F,I,K,L,P,R,V,W,Y; D91F,G,H,K,L,M,N,P,Q,R,S of SEQ ID NO:2. ,Y; I92G,N,P,Q,S,T; Y94A,E,I,K,P,Q,R,T; F95A,D,E,G,H,I,K,L,M,N,R,S,T,V; K96C,F,I,P,V,Y; F98A,C,D,E,G,H,K,N,P,Q,R,S,T,W,Y; F99A,C,D,G,H,K,P,Q,R,V,W,Y; T100E,P,W; and K101E,P. Embodiment 23a. The fusion polypeptide of any of the preceding embodiments, wherein the second polypeptide comprises an amino acid change at a position corresponding to a position selected from the group consisting of V190A, V190D, V190F, V190G, V190K, V190M, V190P, V190Q, V190Y, Y252S, S254K, P255D, S256D, S258E, L259P, L260E, L260K, T268I, and P285D in SEQ ID NO:5. Embodiment 24. The first polypeptide is selected from the group consisting of V23G,D,Y,S; F38A,C,D,G,N,T,V; M39D,E,F,G,H,K,N,P,Q,R,S,Y; Q43D,E,I,K,R; Y45A,C,G,I,K,M,N,Q,R,S,T,V; E67D,N,P; P69D,F,G,H,K,M,Q,R,S,T,W,Y; T88I,P,W; D91G,H ,K,N,P,Q,R,S; I92G,P,S; Y94A,E,I,P,Q,R,T; F95A,D,E,G,H,K,N,R,S,T,V; K96P; F98D,E,G,N,P,Q,S; F99A,C,D,G,H,K,P,Q,R; and T100P,W. Embodiment 24a. The fusion polypeptide of any of the preceding embodiments, wherein the first polypeptide comprises an amino acid change at a position corresponding to a position selected from the group consisting of V190A, V190D, V190F, V190G, V190K, V190P, V190Q, Y252S, S258E, L259P, L260E, L260K, T268I, and P285D in SEQ ID NO:5. Embodiment 25. The fusion polypeptide of any of the preceding embodiments, having at least 60% but less than 100% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:15, and SEQ ID NO:20. Embodiment 26. The fusion polypeptide of any of the preceding embodiments, having at least 60% but less than 100% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:34, and SEQ ID NO:36. Embodiment 27. The fusion polypeptide of any of the preceding embodiments, having at least 70% but less than 100% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:15, and SEQ ID NO:20. Embodiment 28. The fusion polypeptide of any of the preceding embodiments, having at least 70% but less than 100% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:34, and SEQ ID NO:36. Embodiment 29. The fusion polypeptide of any of the preceding embodiments, having at least 80% but less than 100% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:15, and SEQ ID NO:20. Embodiment 30. The fusion polypeptide of any of the preceding embodiments, having at least 80% but less than 100% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:34, and SEQ ID NO:36. Embodiment 31 The fusion polypeptide of any of the preceding embodiments, having at least 90% but less than 100% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:15, and SEQ ID NO:20. Embodiment 32. The fusion polypeptide of any of the preceding embodiments, having at least 90% but less than 100% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:34, and SEQ ID NO:36. Embodiment 33. The fusion polypeptide of any of the preceding embodiments, having at least 95% but less than 100% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:15, and SEQ ID NO:20. Embodiment 34. The fusion polypeptide of any of the preceding embodiments, having at least 95% but less than 100% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:34, and SEQ ID NO:36. Embodiment 35. The fusion polypeptide of any of the preceding embodiments, having at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:15, and SEQ ID NO:20. Embodiment 36. The fusion polypeptide of any of the preceding embodiments, having at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:34, and SEQ ID NO:36. Embodiment 37. The fusion polypeptide of any of the preceding embodiments, having 1 to 30 modifications (e.g., substitutions, deletions and / or insertions), preferably 1 to 20 modifications, 1 to 10 modifications, or 1 to 5 modifications, in particular substitutions, compared to an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:15, and SEQ ID NO:20. Embodiment 38. The fusion polypeptide of any of the preceding embodiments, having 1 to 30 modifications (e.g. substitutions, deletions and / or insertions), preferably 1 to 20 modifications, 1 to 10 modifications, or 1 to 5 modifications, in particular substitutions, compared to an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:34, and SEQ ID NO:36. Embodiment 39. The fusion polypeptide of any of the preceding embodiments, wherein the first polypeptide has at least 60% but less than 100% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:12, SEQ ID NO:17, and SEQ ID NO:31. Embodiment 40. The fusion polypeptide of any of the preceding embodiments, wherein the first polypeptide has at least 70% but less than 100% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:12, SEQ ID NO:17, and SEQ ID NO:31. Embodiment 41. The fusion polypeptide of any of the preceding embodiments, wherein the first polypeptide has at least 80% but less than 100% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:12, SEQ ID NO:17, and SEQ ID NO:31. Embodiment 42. The fusion polypeptide of any of the preceding embodiments, wherein the first polypeptide has at least 90% but less than 100% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:12, SEQ ID NO:17, and SEQ ID NO:31. Embodiment 43. The fusion polypeptide of any of the preceding embodiments, wherein the first polypeptide has at least 95% but less than 100% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:12, SEQ ID NO:17, and SEQ ID NO:31. Embodiment 44. The fusion polypeptide of any of the preceding embodiments, wherein the first polypeptide has at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:12, SEQ ID NO:17, and SEQ ID NO:31. Embodiment 45. The fusion polypeptide of any of the preceding embodiments, wherein the first polypeptide has 1 to 30 modifications (e.g., substitutions, deletions and / or insertions), preferably 1 to 20 modifications, 1 to 10 modifications, or 1 to 5 modifications, in particular substitutions, compared to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:12, SEQ ID NO:17, and SEQ ID NO:31. Embodiment 46 The fusion polypeptide of any of the preceding embodiments, wherein the second polypeptide has at least 60% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:14, and SEQ ID NO:19. Embodiment 47. The fusion polypeptide of any of the preceding embodiments, wherein the second polypeptide has at least 60% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:33, and SEQ ID NO:35. Embodiment 48. The fusion polypeptide of any of the preceding embodiments, wherein the second polypeptide has at least 70% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:14, and SEQ ID NO:19. Embodiment 49. The fusion polypeptide of any of the preceding embodiments, wherein the second polypeptide has at least 70% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:33, and SEQ ID NO:35. Embodiment 50. The fusion polypeptide of any of the preceding embodiments, wherein the second polypeptide has at least 80% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:14, and SEQ ID NO:19. Embodiment 51. The fusion polypeptide of any of the preceding embodiments, wherein the second polypeptide has at least 80% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:33, and SEQ ID NO:35. Embodiment 52. The fusion polypeptide of any of the preceding embodiments, wherein the second polypeptide has at least 90% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:14, and SEQ ID NO:19. Embodiment 53. The fusion polypeptide of any of the preceding embodiments, wherein the second polypeptide has at least 90% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:33, and SEQ ID NO:35. Embodiment 54. The fusion polypeptide of any of the preceding embodiments, wherein the second polypeptide has at least 95% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:14, and SEQ ID NO:19. Embodiment 55. The fusion polypeptide of any of the preceding embodiments, wherein the second polypeptide has at least 95% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:33, and SEQ ID NO:35. Embodiment 56. The fusion polypeptide of any of the preceding embodiments, wherein the second polypeptide has at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:14, and SEQ ID NO:19. Embodiment 57. The fusion polypeptide of any of the preceding embodiments, wherein the second polypeptide has at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:33, and SEQ ID NO:35. Embodiment 58. The fusion polypeptide of any of the preceding embodiments, wherein the second polypeptide has up to 30 modifications (e.g., substitutions, deletions and / or insertions), preferably up to 20 modifications, up to 10 modifications, or up to 5 modifications, in particular substitutions, compared to an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:14, and SEQ ID NO:19. Embodiment 59. The fusion polypeptide of any of the preceding embodiments, wherein the second polypeptide has up to 30 modifications (e.g. substitutions, deletions and / or insertions), preferably up to 20 modifications, up to 10 modifications, or up to 5 modifications, in particular substitutions, compared to an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:33, and SEQ ID NO:35. Embodiment 60 The fusion polypeptide of any of the preceding embodiments, wherein the first polypeptide comprises a cleavage site for a site-specific endopeptidase, such as glutamyl endopeptidase or trypsin. Embodiment 61. The fusion polypeptide of any of the preceding embodiments, wherein the first polypeptide comprises a cleavage site for a site-specific endopeptidase, such as glutamyl endopeptidase or trypsin, within 20 amino acids of the C-terminus. Embodiment 62. The fusion polypeptide of any of the preceding embodiments, wherein the first polypeptide comprises a cleavage site for a site-specific endopeptidase, such as glutamyl endopeptidase or trypsin, within 15 amino acids of the C-terminus. Embodiment 63. The fusion polypeptide of any of the preceding embodiments, wherein the first polypeptide comprises a cleavage site for a site-specific endopeptidase, such as glutamyl endopeptidase or trypsin, within 10 amino acids of the C-terminus. Embodiment 64 A polynucleotide encoding the fusion polypeptide of any of the preceding embodiments. Embodiment 65. The polynucleotide of embodiment 64, which is isolated and / or purified. Embodiment 66. A nucleic acid construct or expression vector comprising the polynucleotide of embodiment 64, wherein the polynucleotide is operably linked to one or more control sequences that direct the production of a fusion polypeptide in an expression host. Embodiment 67. A recombinant host cell comprising the nucleic acid construct or expression vector of embodiment 66. Embodiment 68 The recombinant host cell of embodiment 67, wherein the fusion polypeptide is heterologous to the recombinant host cell. Embodiment 69. The recombinant host cell of any of embodiments 67-68, wherein at least one of the one or more control sequences is heterologous to the polynucleotide encoding the fusion polypeptide. Embodiment 70. The recombinant host cell of any of embodiments 67 to 69, comprising at least two copies, such as three, four, or five or more copies, of the polynucleotide of embodiment 64. Embodiment 71. Yeast recombinant host cells, for example cells of the genera Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces or Yarrowia, for example Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri. 71. The recombinant host cell of any of embodiments 67 to 70, which is a cell of Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis or Yarrowia lipolytica. Embodiment 72. Filamentous fungal recombinant host cells, such as Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocalima. Cells of the genera Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes or Trichoderma, in particular Aspergillus awamori. awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirinaaneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium meldarium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulantumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Talaromyces emersonii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma 71. The recombinant host cell of any of embodiments 67 to 70, which is a Trichoderma longibrachiatum, Trichoderma reesei or Trichoderma viride cell. Embodiment 73. A prokaryotic recombinant host cell, such as a Gram-positive cell selected from the group consisting of a Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus or Streptomyces cell, or a Campylobacter, E. coli or Escherichia coli cell.Gram-negative bacteria selected from the group consisting of Bacillus coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella and Ureaplasma cells, such as Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus argin ... firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis uberis and Streptococcus equi subsp.71. The recombinant host cell of any of embodiments 67-70, which is a Streptomyces zooepidemicus, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cell. Embodiment 74. The recombinant host cell of any of embodiments 67 to 70, which is a Bacillus licheniformis cell. Embodiment 75. A recombinant host cell of any of embodiments 67 to 74, which is isolated. Embodiment 76. The recombinant host cell of any of embodiments 67 to 75, which is purified. Embodiment 77. A method for producing a fusion polypeptide of any of embodiments 1 to 63, comprising culturing a recombinant host cell of any of embodiments 67 to 76 under conditions conducive to the production of the fusion polypeptide. Embodiment 78 The method of embodiment 77, further comprising recovering the fusion polypeptide. Embodiment 79. A method for producing a polypeptide having deamidase activity, comprising contacting a fusion polypeptide of any of embodiments 1 to 63 with a site-specific endopeptidase to separate the first polypeptide from the second polypeptide. Embodiment 80 The method of embodiment 79, wherein the site-specific endopeptidase is selected from the group consisting of glutamyl endopeptidase, trypsin-like endopeptidase, and chymotrypsin-like endopeptidase. Embodiment 81. A composition exhibiting deamidase activity, comprising a first polypeptide and a second polypeptide of a fusion polypeptide of any of embodiments 1 to 63, wherein the first and second polypeptides are not covalently linked. Embodiment 82 The composition of embodiment 81, further comprising a site-specific endopeptidase. 83. (a) 0.001 to 25% w / w of a first polypeptide; (b) 0.001 to 25% w / w of a second polypeptide; (c) a polyol, and (d) water 83. The composition of embodiment 81 or 82, which is a liquid composition comprising: Embodiment 84. A method for modifying a protein, comprising contacting the protein with a composition of any of embodiments 81 to 83. Embodiment 85. The method of embodiment 84, wherein the modification is deamidation of a glutamine residue.

[0221] The present invention is further described by the following examples, which should not be construed as limiting the scope of the invention. EXAMPLES

[0222] KK Chryseobacterium sp-62563 strain was isolated from a soil sample collected in September 2013 in Sibhult, Sweden.

[0223] Example 1 Deamidase activity material Glutamyl endopeptidase from Bacillus licheniformis FITC-PP-Dnp (FITC-Ahx-His-His-Gln-Ser-Ser-ED-Dnp) is a custom synthesized substrate molecule from TAG Copenhagen, Kong Georgs Vej 12, DK-2000 Frederiksberg. FITC is a fluorescein with excitation and emission maxima at approximately 490 nm and 520 nm; Ahx is aminohexanoic acid, ED is ethylenediamine, and Dnp is 2,4-dinitrophenyl (a fluorescein fluorescence quencher).

[0224] Deamidase maturation assay The assay describes maturation of the deamidase pro-form (fusion polypeptide) by cleavage of the propeptide (first polypeptide) and deamidase (second polypeptide) domains and measuring the (relative) activity of the active deamidase ("activated sample") after treatment with a site-specific endopeptidase. The site-specific endopeptidase used in the assay is glutamyl endopeptidase from Bacillus licheniformis.

[0225] After purification and quantification, deamidase pro-form (fusion polypeptide) samples were standardized to 50 nM deamidase in 100 mM HEPES buffer, pH 7.0, 0.01% v / v Triton X detergent containing 10 μg / mL glutamyl endopeptidase and incubated at room temperature for 30 minutes to allow "activation" (cleavage of the propeptide and deamidase domains), as described in Examples 6 and 7. After incubation, 50 μL of sample was transferred to a standard black 96-well plate and incubated for 30 minutes at room temperature to allow "activation" (cleavage of the propeptide and deamidase domains). 20μL of 0.25μg / mL FITC-PP-Dnp 50 μL of 50 μg / mL glutamyl endopeptidase 130 μL of 100 mM HEPES buffer pH 7.0, and 0.01% v / v Triton X detergent Added.

[0226] Fluorescence signal (RFU) was measured using a Biotek Synergy H1 fluorescent plate reader for 30 min using excitation / emission wavelengths of 485 nm / 525 nm.

[0227] Data was analyzed for initial velocity using variable time intervals depending on the shape of the curve (signal vs. time). The initial velocity for each sample was normalized to the initial velocity of the fully activated deamidase reference enzyme (produced by the wild-type donor strain). Activity was measured as "% initial velocity", i.e., the initial velocity of the sample molecules relative to the initial velocity of the % mature deamidase reference enzyme.

[0228] Deamidase (leakage activity) of fusion polypeptide The assay measures the activity of the deamidase pro-form (fusion polypeptide), designated "native", relative to the mature form, designated "activated". This ratio is also referred to as "leakage activity". Maturation was performed by pretreatment with glutamyl endopeptidase from Bacillus licheniformis.

[0229] After purification and quantification, as described in Examples 6 and 7, the deamidase (pro-form) samples were split in half and one half ("activated") was standardized to 10 nM deamidase in 100 mM HEPES buffer, pH 7.0, 0.01% v / v Triton X detergent, and 10 μg / mL glutamyl endopeptidase was added.

[0230] The other half ("native") was standardized to a concentration of 100 nM deamidase in 100 mM HEPES buffer, pH 7.0, 0.01% v / v Triton X detergent.

[0231] Both the "native" and "activated" samples were reacted with 0.2 μg / mL FITC-PP-Dnp for 90 minutes at 30° C. under gentle shaking. After reaction, 100 μL of each of the two samples was heated to 95° C. for 5 minutes in a PCR thermal cycler to inactivate the deamidase activity. After inactivation, 50 μL of the heat-treated samples were transferred to a standard black 96-well plate and 150 μL of 13 μg / mL glutamyl endopeptidase was added to hydrolyze a fraction of the FITC-PP-Dnp, and glutamine was converted to glutamic acid by the deamidase activity.

[0232] The endpoint signal was measured in a standard plate reader using excitation / emission wavelengths of 485 nm / 525 nm in a Biotek Synergy H1 fluorescent plate reader.

[0233] Leakage activity was defined as the activity of the deamidase pro-form ("native") relative to the activity of the mature deamidase ("activated") in percentage (RFU / nM deamidase).

[0234] Example 2 Nano Differential Scanning Fluorometry (nanoDSF)-Thermal Denaturation Temperature Nano differential scanning fluorimetry (nanoDSF) was used to evaluate the conformational stability of the fusion polypeptide of the present invention. The molecules were exposed to a temperature gradient as shown below. The resulting conformational changes are reflected in the changes in fluorescence intensity, which provide a measure of thermal stability. The binding of the propeptide domain to the deamidase domain contributes to the stability of the molecule, and therefore the nanoDSF thermal denaturation temperature also provides information on the propeptide-deamidase binding affinity.

[0235] The His-tag purified sample was received in elution buffer from an IMAC (immobilized metal affinity chromatography) column; 20 mM sodium phosphate; 500 mM sodium chloride; 500 mM imidazole; pH 7.4.

[0236] 60 μL of sample was transferred in duplicate to a black bottom 384-well plate and the plate was briefly centrifuged to remove potential air bubbles.

[0237] The thermal denaturation temperatures of the samples were analyzed using a Prometheus NT.Plex system from NanoTemper Technologies GmbH with the following settings: (i) temperature scan rate: 3.3° C. per minute; and (ii) Temperature scan interval: 20–95°C.

[0238] During the analysis, samples were loaded into a capillary (Prometheus NT.Plex-Capillary Chips, Standard, Cat#PR-AC002) and then subjected to a temperature gradient. The generated data was analyzed by PR.Stability analysis v.1.0.1 software. The denaturation midpoint temperature (T m , °C) were annotated based on the first derivative of the trace at 330 nm. In some cases, more than one T m In these cases, the main peak in the first derivative trace at 330 nm corresponds to the T m was selected as.

[0239] Example 3 Thermal denaturation temperature of wild-type fusion polypeptide Using the nanoDSF procedure, we measured the thermal denaturation temperatures of several recombinant wild-type fusion polypeptides containing a deamidase inhibitor domain and a deamidase domain, as described in Example 2. They all exhibit very low deamidase activity and cannot be activated / matured by proteolytic cleavage / separation of the two domains. The sequence identities of the exemplified polypeptides indicate that they exhibit high sequence diversity.

[0240] [Table 4]

[0241] The data in Table 3 show that the wild-type fusion polypeptides with completely different amino acid sequences have high thermal denaturation temperatures (as determined by nanoDSF), indicating high stability and high binding affinity between the propeptide and deamidase domains.

[0242] Example 4 Thermal denaturation temperature of deamidase variant fusion polypeptide Using the nanoDSF procedure, we measured the thermal denaturation temperatures of several recombinant fusion polypeptides as described in Example 2. All recombinant fusion polypeptides contain a variant deamidase propeptide (first polypeptide) or a variant deamidase (second polypeptide) derived from a donor organism / sequence as shown in Table 4.

[0243] All recombinant fusion polypeptides exhibited sufficiently low deamidase activity to allow recombinant expression / production (see Example 8) and could also be matured by proteolytic cleavage and separation of the fusion polypeptides (see Example 9). The variant deamidase propeptides contained mutations (substitutions, deletions, or insertions) as shown in Table 4, and the nanoDSF thermal denaturation temperatures of the resulting fusion polypeptides are also shown.

[0244] The fusion polypeptide of SEQ ID NO:5 from Chryseobacterium sp-62563 is shown as the donor sequence when the mutated amino acid position is higher than 109, because SEQ ID NO:2 contains only the first 109 amino acids of SEQ ID NO:5. The amino acid numbering is the same as in SEQ ID NO:2 and SEQ ID NO:5.

[0245] [Table 5]

[0246] [Table 6]

[0247] [Table 7]

[0248] The data in Table 4 show that by introducing mutations in the deamidase inhibitor domain, the binding affinity / stability of a fusion polypeptide comprising a deamidase propeptide (and deamidase inhibitor domain) and a deamidase is sufficiently reduced to allow maturation by cleavage / separation of the two domains, and at the same time, the variant fusion polypeptide exhibits sufficiently low deamidase activity to allow recombinant expression without compromising viability of the recombinant host cell.

[0249] Example 5 Identification and cloning of the protein glutamine deamidase Identification of protein glutamine deamidase gene from Chryseobacterium sp-62563 The DNA sequence (SEQ ID NO: 1 fused to SEQ ID NO: 3) encoding the protein glutamine deamidase was isolated from a bacterial strain collected in Sweden as described above. Taxonomy of the donor strain was established by 16S ribosomal DNA sequencing. Blast homology analysis using the 16S ribosomal sequence returned 99.1% homology with the 16S ribosomal sequence from EMBL:KC108937 (Chryseobacterium sp. UA-JF4202) and 99.1% with the 16S DNA sequence from EMBL:AM988898 (Chryseobacterium sp. AKB-2008-HE85). The strain was therefore given the new name Chryseobacterium sp-62563.

[0250] Chryseobacterium sp-62563 genome sequencing was performed on pure genomic DNA using next generation sequencing Illumina technology, sequence reads were assembled by ibda v1.1.1 (Bioinformatics. 2012 Jun 1;28(11):1420-8.) and open reading frames were annotated using GeneFinder Prodigal 2.50 (BMC Bioinformatics. 2010;11:119.). Protein glutamine deamidase DNA sequence (SEQ ID NO:1 fused to SEQ ID NO:3) was identified within this assembly. This gene encodes the protein glutamine deamidase pro-form (SEQ ID NO:5) consisting of a propeptide domain (SEQ ID NO:2) and a deamidase domain (SEQ ID NO:4).

[0251] The gene encoding the protein glutamine deamidase pro-form (SEQ ID NO: 5) was PCR amplified from genomic DNA of Chryseobacterium sp-62563 and the native N-terminal region encompassing the native lipoprotein signal peptide (Teufel et al., “SignalP 6.0 predicts all five types of signal peptides using protein language models”, Nature Biotechnology (2022)) was replaced with the Bacillus licheniformis alpha-amylase secretion signal (amyL signal peptide as described in WO 2014 / 206806).

[0252] A 6His tag was added at the C-terminus of the CDS to facilitate enzyme recovery. This engineered sequence was fused to a transcription promoter from sequence GENESEQN:BGM50663 (WO 2015 / 004013) and a transcription terminator from sequence GENESEQN:BET98406 (WO 2018 / 009520) by linear overlap PCR using the SOE-PCR fusion strategy (Horton, RM, et al. (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension Gene 77(1)61-68). The SOE PCR method is also described in more detail in patent application WO 2003 / 095658. Further additional 5' and 3' DNA regions corresponding to the L-arabinose (ara) insertion locus were added during the SOE-PCR reaction.

[0253] Bacillus subtilis in a low protease background derived from B. subtilis A164 (ATCC 6051A) was used for expression of the Chryseobacterium sp-62563 protein glutamine deamidase. Integration of the SOE PCR product into the expression host genome was achieved by homologous recombination into the Bacillus subtilis chromosomal region of the L-arabinose (ara) operon and selection driven by the erm (erythromycin) marker on agar plates.

[0254] Strategy for generating deamidase variants Point mutation variants were generated using the giga-primer strategy described in WO2016066756 with non-degenerate primers. In the first PCR (PCR1), a C-terminal fragment was generated using a mutagenic forward primer and a reverse primer complementary to a sequence required for homologous integration in the Bacillus genome. In the second PCR, the C-terminal fragment from PCR1 was used as a giga-primer and a second primer complementary to a sequence required for homologous integration into the Bacillus genome was used. The resulting variants were spread on agar plates with erythromycin and single colonies were picked for sequence verification before fermentation. The polymerase used for the PCR reaction was Phusion DNA polymerase (Finnzymes Oy (ThermoFisher Scientific)).

[0255] Cloning and expression of deamidases from Chryseobacterium gambrini, Chryseobacterium culitis, and Chryseobacterium defluvii The published sequences TREMBL:A0A2S9CPY3 from Chryseobacterium culicis, TREMBL:A0A1N7P918 from Chryseobacterium gambrini and TREMBL:A0A495SBZ2 from Chryseobacterium defluvii were engineered to ensure a similar expression strategy as for the Chryseobacterium sp-62563 deamidase SEQ ID NO:5. The N-terminal regions of the wild-type sequences (TREMBL: A0A2S9CPY3 from Chryseobacterium culicis, TREMBL: A0A1N7P918 from Chryseobacterium gambrini, and TREMBL: A0A495SBZ2 from Chryseobacterium defluvii) including their lipoprotein signal peptides were replaced by the Bacillus licheniformis alpha-amylase secretion signal as described above. The linker region (defined by sequence alignment with SEQ ID NO:5) located at the C-terminus of the propeptide region (SEQ ID NO:2) and the N-terminus of the deamidase peptide (SEQ ID NO:4) was replaced by the linker region of Chryseobacterium sp-62563 and a 6-histidine tag was added to the C-terminus to ensure a process for expression maturation and purification similar to that for Chryseobacterium sp-62563 deamidase (SEQ ID NO:5). SEQ ID NO:10 for Chryseobacterium culitis deamidase (encoded by the propeptide domain SEQ ID NO:6 and the deamidase domain SEQ ID NO:8), SEQ ID NO:15 for Chryseobacterium gambrini deamidase (encoded by the propeptide domain SEQ ID NO:11 and the deamidase domain SEQ ID NO:13), and The engineered sequence corresponding to SEQ ID NO:20 for the Chryseobacterium defluvii deamidase (encoded by the propeptide domain SEQ ID NO:16 and the deamidase domain SEQ ID NO:18) is It was purchased as synthetic DNA. 5' and 3' overlaps corresponding to an amyl (3A) signal peptide on the 5' end and a His tag / stop / terminator at the 3' end were also included to allow for the generation of constructs for transformation by linear overlap PCR using the SOE-PCR fusion strategy (Horton, RM, et al. (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension Gene 77 (1) 61-68). The SOE PCR method is also described in more detail in patent application WO2003095658.

[0256] Bacillus subtilis in a low protease background derived from B. subtilis A164 (ATCC 6051A) was used for expression of three Chryseobacterium protein glutamine deamidase pro-forms: SEQ ID NO: 10, SEQ ID NO: 15, and SEQ ID NO: 20. Integration of the SOE PCR products into the expression host genome was achieved by homologous recombination into the Bacillus subtilis chromosomal region of the L-arabinose (ara) operon and erm (erythromycin) marker-driven selection on agar plates.

[0257] Example 6 Fermentation and purification of protein glutamine deamidase Sequence verified Bacillus subtilis transformed with constructs encompassing SEQ ID NO:1 fused to SEQ ID NO:3, SEQ ID NO:6 fused to SEQ ID NO:8, SEQ ID NO:11 fused to SEQ ID NO:13, SEQ ID NO:16 fused to SEQ ID NO:18, or their corresponding point mutation variants, were seeded into 96-well plates pre-filled with 1 mL of Cal18-2 medium (Cal18-2 medium composition is described in EP 1187925 B1) supplemented with erythromycin, and fermentation was carried out at 30°C under 700 rpm agitation. After 4 days, plates were centrifuged to pellet Bacillus cells, and 0.4 mL of supernatant was transferred to a 96-well His MultiTrap purification filter (GE Healthcare 11-0036-62 AB). His-tag purification was performed according to the manufacturer's protocol. Elution of protein glutamine deamidase SEQ ID NO: 5, 10, 15, 20 and protein glutamine deamidase variants was done in 0.2 mL of buffer composed of 20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole pH 7.4. For activation assays, His-purified samples were buffer exchanged using a 96-well desalting PD MultiTrap G-25 (Cytiva) and elution was performed in 50 mM Hepes, 100 mM NaCl pH 7 buffer.

[0258] Example 7 Expression analysis and quantification of the protein glutamine deamidase His-tag purified samples were analyzed by SDS PAGE electrophoresis and separated on 26-well NuPAGE™ gels (Invitrogen ref.WG1403Bx10). 10 μL of purified enzyme was mixed with 10 μL of sample buffer mixture (containing loading buffer and reducing agent) that was heat treated at 99° C. for 2 min, and 10 μL was loaded onto the gel. Electrophoresis was performed using 1×MOPS buffer, and after electrophoresis the gel was stained overnight in Ready Blue™ reagent (Sigma Aldrich). Gels were scanned on a BioRad Criterion Stain Free™ image system.

[0259] Tiff-file format scans from SDS PAGE gels were used for densitometric analysis performed with ImageJ software v1.52a. The band intensity of the deamidase pro-form (approximately 32 kDa) was measured and normalized across the gel using the wild-type deamidase pro-form as a standard. Densitometric measurements were used to evaluate the effect of the mutations on the expression levels of the protein glutamine deamidase.

[0260] Example 8 Leakage activity of recombinant fusion polypeptides Using the leakage activity procedure, we measured the activity of several recombinant fusion polypeptides in comparison with the corresponding mature / active deamidases, as described in Example 1. The leakage activity measures the activity of the deamidase proform before maturation (fusion polypeptide). It also indicates the ability of the fusion polypeptide to separate the propeptide domain (first polypeptide) and the deamidase domain (second polypeptide) to release the active deamidase.

[0261] The recombinant fusion polypeptides are derived from Chryseobacterium sp-62563 (SEQ ID NO: 5) and contain a mutated propeptide domain (first polypeptide) or a mutated deamidase domain (second polypeptide). The mutations and measured leakage activity of the resulting recombinant fusion polypeptides are shown in Table 5.

[0262] The fusion polypeptide of SEQ ID NO:5 from Chryseobacterium sp-62563 is shown as the donor sequence when the mutated amino acid positions are higher than 109, because SEQ ID NO:2 contains only the first 109 amino acids of SEQ ID NO:5.

[0263] [Table 8]

[0264] [Table 9]

[0265] [Table 10]

[0266] The data in Table 5 show that all recombinant fusion polypeptides exhibited less than 26% deamidase activity compared to the mature deamidase (leakage activity).

[0267] Example 9 Maturation of deamidase from recombinant fusion polypeptide Using the deamidase maturation procedure, we measured the activity of several mature / active deamidases, as described in Example 1. The recombinant fusion polypeptide is derived from Chryseobacterium sp-62563 (SEQ ID NO:5) and contains a mutated propeptide domain (first polypeptide, SEQ ID NO:2). The deamidase domain (second polypeptide) is the same for all recombinant fusion polypeptides, which is also derived from Chryseobacterium sp-62563 (SEQ ID NO:4).

[0268] The fusion polypeptide of SEQ ID NO:5 is shown as the donor sequence when the mutated amino acid positions are higher than 109, because SEQ ID NO:2 contains only the first 109 amino acids of SEQ ID NO:5.

[0269] All recombinant fusion polypeptides exhibit sufficiently low deamidase activity (leakage activity, see Example 8) to allow recombinant expression / production. The mutations and relative activities of the resulting mature deamidases are shown in Table 6.

[0270] [Table 11]

[0271] [Table 12]

[0272] [Table 13]

[0273] [Table 14]

[0274] [Table 15]

[0275] [Table 16]

[0276] [Table 17]

[0277] [Table 18]

[0278] The data in Table 6 show that the recombinant fusion polypeptide was successfully matured using a site-specific endopeptidase to release the deamidase activity.

Claims

1. (a) A first polypeptide comprising a deamidase inhibitory domain, and (b) A second polypeptide having deamidase activity A recombinant fusion polypeptide comprising or consisting thereof A recombinant fusion polypeptide wherein the fusion polypeptide has a thermal denaturation temperature in the range of 65 to 82°C; and the fusion polypeptide has less than 50% of the deamidase activity compared to the second polypeptide.

2. The recombinant fusion polypeptide according to claim 1, wherein the C-terminus of the first polypeptide is located before the N-terminus of the second polypeptide.

3. The recombinant fusion polypeptide according to any one of claims 1 to 2, wherein the first polypeptide comprises the amino acid sequence motif [I / M][L / I / V][S / T]AQ and / or the amino acid sequence motif [K / R][V / I / L][S / A / N]X[I / M][L / I / V][S / T]AQ.

4. The recombinant fusion polypeptide according to any one of claims 1 to 2, wherein the second polypeptide comprises an amino acid sequence motif selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and combinations thereof.

5. The recombinant fusion polypeptide according to any one of claims 1 to 2, wherein the fusion polypeptide has amino acid sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% with an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 34, and SEQ ID NO:

36.

6. The recombinant fusion polypeptide according to any one of claims 1 to 2, wherein the first polypeptide has amino acid sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% with an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 12, SEQ ID NO: 17, and SEQ ID NO:

31.

7. The recombinant fusion polypeptide according to any one of claims 1 to 2, wherein the second polypeptide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 9, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 33, and SEQ ID NO:

35.

8. The recombinant fusion polypeptide according to any one of claims 1 to 2, wherein the first polypeptide comprises an amino acid change at a position selected from the group consisting of 23, 38, 39, 43, 45, 67, 69, 88, 91, 92, 94, 95, 96, 98, 99, 100, and 101 of SEQ ID NO: 2; preferably, the amino acid change is a substitution.

9. The recombinant fusion polypeptide according to any one of claims 1 to 2, wherein the second polypeptide comprises an amino acid change at a position selected from the group consisting of 190, 252, 254, 255, 256, 258, 259, 260, 268, and 285 of SEQ ID NO: 5; preferably, the amino acid change is a substitution.

10. A method for producing a fusion polypeptide according to any one of claims 1 to 2, comprising culturing a recombinant host cell containing a polynucleotide encoding the recombinant fusion polypeptide under conditions that contribute to the production of the recombinant fusion polypeptide.

11. A method for producing a polypeptide having deamidase activity, comprising contacting a recombinant fusion polypeptide according to any one of claims 1 to 2 with a site-specific endopeptidase to separate the first polypeptide from the second polypeptide.

12. A composition exhibiting deamidase activity, comprising the first polypeptide and the second polypeptide of a recombinant fusion polypeptide according to any one of claims 1 to 2, wherein the first and second polypeptides are not covalently linked.

13. The composition according to claim 12, further comprising a site-specific endopeptidase.

14. (a) 0.001 to 25% w / w of the first polypeptide, (b) 0.001 to 25% w / w of the second polypeptide, (c) Polyols, and (d) Water The composition according to claim 12, which is a liquid composition containing the following:

15. A method for modifying a protein, comprising contacting the protein with the composition described in claim 12; preferably, the modification is the deamidation of a glutamine residue.