Stabilized liquid deamidase compositions

Abstract

The invention provides methods for stabilizing liquid enzyme compositions by incubating the liquid compositions at a temperature of at least 40°C for at least 24 hours, and subsequently removing precipitated solids.

Description

[0001] STABILIZED LIQUID DEAMIDASE COMPOSITIONS

[0002] Reference to a Sequence Listing

[0003] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to methods for making liquid deamidase compositions having improved physical stability after storage.

[0006] BACKGROUND

[0007] Liquid enzyme compositions are popular product formats because they are easy to handle in an industrial environment. They can be pumped directly into an industrial process, and generally do not require safety measures to avoid exposure to enzyme dust, as compared to solid enzyme compositions.

[0008] While solid enzyme compositions generally exhibit excellent physical stability, liquid enzyme compositions are much more challenging, because high concentrations of solubilized enzyme may form precipitates during storage.

[0009] Protein-glutamine glutaminase (“deamidase”) enzymes are protein modifying enzymes that can change the physical properties of proteins comprising glutamine residues. Liquid compositions comprising this class of enzymes may be prone to poor physical stability during storage. It is an object of the invention to provide liquid deamidase compositions having improved physical stability after storage.

[0010] SUMMARY OF THE INVENTION

[0011] The present invention provides, in a first aspect, a method for stabilizing a liquid enzyme composition, comprising

[0012] (a) providing a liquid composition comprising a protein-glutamine glutaminase, which is recovered from a microbial fermentation broth;

[0013] (b) incubating the liquid composition at a temperature of at least 40°C for at least 24 hours; and

[0014] (c) removing precipitated solids from the incubated liquid composition by solid-liquid separation to provide a stabilized liquid enzyme composition.

[0015] Other aspects and embodiments of the invention are apparent from the description and examples.

[0016] Unless otherwise indicated, or if it is apparent from the context that something else is meant, all percentages are percentage by weight (% w / w).

[0017] As used herein, the term "consists essentially of" (and grammatical variants thereof), as applied to the compositions and methods of the invention, means that the compositions / methods may contain additional components so long as the additional components do not materially alter the composition / method.

[0018] As used herein, the term "essentially free of' (and grammatical variants thereof), as applied to the compositions and methods of the invention, means that the compositions / methods may contain minor amounts of the specified component so long as the amount of the component does not materially alter, or provide any material effect on, the composition / method. In an embodiment, "essentially free of" means 0% w / w.

[0019] Sequences

[0020] SEQ ID NO: 1: Amino acid sequence of a deamidase from Chryseobacterium viscerum. SEQ ID NO: 2: Amino acid sequence of a deamidase propeptide from Chryseobacterium viscerum.

[0021] DETAILED DESCRIPTION

[0022] Physical stability is the ability to maintain a transparent, preferably clear, composition. This may be evaluated by visual inspection, or by centrifugation. For example, the liquid composition may centrifugated at 1200 G for 10 minutes to determine if a pellet (solid phase) is formed. Alternatively, transparency may be measured as turbidity or haziness, by using a nephelometer to measure NTU to determine light scattering.

[0023] In the context of the invention, the term “deamidase” means a protein-glutamine glutaminase (also known as glutaminylpeptide glutaminase, or protein deamidase) activity, as described in EC 3.5.1.44, which catalyzes the hydrolysis of the gamma-amide of glutamine substituted at the carboxyl position or 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 glutamate residues and are also referred to as protein glutamine deamidase. Deamidases comprise 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 belong to the InterPro entry IPR041325. In a preferred embodiment, the deamidases of the present invention belong to PFAM domain PF18626.

[0024] Deamidases are catalytic proteins (enzymes), and the term “active (deamidase) enzyme protein” is defined herein as the amount of catalytic protein(s), which exhibits deamidase activity. This can be determined using an activity based analytical enzyme assay. This technique is well-known in the art.

[0025] Deamidase activity was measured using the assay described in Example 1. The activity assay consists of two separate de-coupled parts: (1) an enzymatic step wherein ammonia is formed by the catalytic action of the protein deamidase; and (2) a non-enzymatic detection step, wherein the ammonia formed in step (1) is derivatized to a blue indophenol compound with an absorption maximum at 630 nm. The amount of enzyme producing 1 pmol ammonia per minute at 37°C is defined as 1 unit (given in Indophenol Assay Unit: IPA(U)). The activity may be determined relative to a standard of declared strength.

[0026] For purposes of the present invention, the sequence identity between two amino acid sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order for the Needle program to report the longest identity, the -nobrief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:

[0027] (Identical Residues x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0028] Stabilized liquid enzyme composition

[0029] We have found that the physical stability of liquid deamidase enzyme compositions produced by microbial fermentation can be significantly improved by incubation at high temperatures for an extended time. During incubation precipitates will form, which are subsequently removed by solid-liquid separation, such as filtration, sedimentation / decanting or centrifugation.

[0030] Contrary to expectations, we found that the precipitate formed during incubation was not a mix of unspecific host proteins, but predominantly consisted of inactivated deamidase protein. The reason for this partial instability of deamidase protein is unknown, but without being bound by theory it could be misfolding, modification in posttranslational processing, or other modifications after secretion into the fermentation broth.

[0031] The incubation is carried out for at least 24 hours at a temperature of at least 40°C, but higher temperatures and extended incubation time further improves the physical stability of the resulting deamidase enzyme solution. Due care must be taken not to thermally inactivate the deamidase enzyme, but a small loss of up to 5% enzyme activity, or even up to 10, 15 or 20%, may be acceptable if the resulting liquid deamidase enzyme product has significantly improved physical stability during storage.

[0032] Accordingly, we provide a method for stabilizing a liquid enzyme composition, comprising

[0033] (a) providing a liquid composition comprising a protein-glutamine glutaminase, which is recovered from a microbial fermentation broth;

[0034] (b) incubating the liquid composition at a temperature of at least 40°C for at least 24 hours; and

[0035] (c) removing precipitated solids from the incubated liquid composition by solid / liquid separation to provide a stabilized liquid enzyme composition. In an embodiment, the liquid enzyme composition is incubated at a temperature of at least 45°C, such as at least 50°C, or at least 55°C. Depending on the thermostability of the deamidase, the upper limit of the incubation temperature may be at most 70°C, such as at most 65°C, or at most 60°C.

[0036] In an embodiment, the liquid enzyme composition is incubated for at least 48 hours, such as at least 60 hours, or at least 72 hours.

[0037] The incubation temperature and time may be interrelated, so that a low temperature and long incubation time, or a high temperature and a short incubation time, both provide satisfactory results. Examples of such treatments could be incubation at a temperature of 40- 50°C for 48-96 hours, and incubation at a temperature of 50-60°C for 24-60 hours.

[0038] In an embodiment, the liquid enzyme composition has a pH of at least pH 3.5, preferably at least pH 4.0. Preferably the pH is in the range of pH 3.5-10, more preferably in the range of pH 4-9 or in the range of pH 4-8. In an embodiment, the pH of the composition is at most pH 8.

[0039] The composition may further comprise at least 10% w / w of water.

[0040] In an embodiment, the composition comprises the deamidase (protein-glutamine glutaminase) in an amount of 20-10000 IPA(ll) per gram; preferably in an amount of 40-8000 IPA(ll) per gram, 60-6000 IPA(ll) per gram, 80-5000 IPA(ll) per gram, or in an amount of 100- 4000 IPA(ll) per gram. The amount of deamidase may also be expressed in “active (deamidase) enzyme protein”; thus, the liquid enzyme composition may comprise the deamidase (protein-glutamine glutaminase) in an amount of 0.01-15% w / w of active deamidase enzyme protein; preferably in an amount of 0.05-10% w / w, in an amount of 0.1-5% w / w, or in an amount of 0.1-3% w / w of active deamidase enzyme protein.

[0041] As described below, the liquid enzyme composition may further comprise polyol(s) and / or salt(s) to provide additional stability to the deamidase enzyme.

[0042] The liquid enzyme composition may further comprise a reducing agent to avoid oxidation of the cysteine residue in the active site and maintain the deamidase enzymatic activity during storage. The reducing agent may, for example, be a salt of sulfite, metabisulfite, thiosulphate, or ascorbate. The liquid enzyme composition may comprise at least 0.1% w / w of the reducing agent; such as 0.1-5% w / w or 0.1-2% w / w of the reducing agent.

[0043] Polyols

[0044] The liquid enzyme composition used in the method of the invention may comprise one or more polyols. Polyols (or polyhydric alcohols) according to the invention are alcohols with two or more hydroxyl groups. The polyols may have a molecular weight lower than 500 g / mol.

[0045] Polyols include non-sugar polyols, such as glycerol, ethylene glycol, diethylene glycol, triethylene glycol, propylene glycol, dipropylene glycol, tripropylene glycol, polyethylene glycol (PEG), sugar alcohols, and combinations thereof. The polyethylene glycol may have an average molecular weight at or below about 500. Examples of sugar alcohols are sorbitol, mannitol, erythritol, dulcitol, inositol, xylitol, adonitol, isomalt, and maltitol.

[0046] Polyols also include sugar polyols, such as mono- and disaccharides, like glucose, fructose, galactose, sucrose, lactose, maltose, trehalose, cellobiose, and combinations thereof.

[0047] The polyols may be combinations of non-sugar polyols and sugar polyols.

[0048] The liquid enzyme composition may comprise the polyol(s) in an amount of 10-80% w / w, such as 10-75% w / w, 10-70% w / w, 10-65% w / w, or 10-60% w / w. In an embodiment, the liquid composition comprises at least 10% w / w, preferably at least 15% w / w, at least 20% w / w, at least 25% w / w, at least 30% w / w, or at least 35% w / w of polyol(s).

[0049] Salts

[0050] The liquid enzyme composition used in the method of the invention may further comprise at least 2% w / w, preferably at least 4% w / w, at least 6% w / w, or at least 8% w / w of salt(s). The amount of salt(s) is calculated as unhydrated salt, thus excluding any complexed water (crystal water). The skilled person will recognize that the upper limit of a salt in the liquid enzyme composition is determined by the solubility of the salt. In an embodiment, the liquid enzyme composition may comprise at most 20% w / w of salt, preferably at most 15% w / w of salt or at most 10% w / w of salt.

[0051] The salt(s) may be selected from the group consisting of alkali metal sulfates, carbonates, nitrates, phosphates, halides, formates, acetates, and citrates; alkaline earth metal sulfates, carbonates, nitrates, phosphates, halides, formates, acetates, and citrates; transition metal sulfates, carbonates, nitrates, phosphates, halides, formates, acetates, and citrates; and ammonium sulfates, carbonates, nitrates, halides, formates, acetates, and citrates.

[0052] Examples of such salts include sodium sulfate, potassium sulfate, ammonium sulfate, magnesium sulfate, zinc sulfate, sodium carbonate, potassium carbonate, ammonium carbonate, sodium bicarbonate, potassium bicarbonate, ammonium bicarbonate, sodium nitrate, potassium nitrate, ammonium nitrate, magnesium nitrate, zinc nitrate, calcium nitrate, sodium phosphate, potassium phosphate, ammonium phosphate, magnesium phosphate, zinc phosphate, calcium phosphate, sodium chloride, potassium chloride, ammonium chloride, magnesium chloride, zinc chloride, calcium chloride, sodium formate, potassium formate, ammonium formate, magnesium formate, zinc formate, calcium formate, sodium acetate, potassium acetate, ammonium acetate, magnesium acetate, zinc acetate, calcium acetate, sodium citrate, potassium citrate, ammonium citrate, and magnesium citrate. Included are also the hydrates thereof.

[0053] Preferred salts are sodium, potassium, ammonium, magnesium, zinc, and calcium salts of formate, acetate, citrate, and chloride. More preferred salts are sodium and potassium salts of formate, acetate, citrate, and chloride. Deamidase enzymes

[0054] The deamidase enzyme comprised in the liquid enzyme composition is produced by a microbial host cell fermentation and subsequent recovery process. The subsequent recovery process may comprise a maturation / activation step, where an inhibitory propeptide is separated from a deamidase pro-form to produce an active (or more active) deamidase. Thus, the liquid enzyme composition of the invention may further comprise a deamidase inhibitory propeptide, which is not covalently linked to the deamidase. Deamidase inhibitory propeptides are, for example, described in WO 2023 / 170177. An exemplary propeptide is shown in SEQ ID NO: 2, and others can be identified using protein structure prediction tools (see, for example, Jumper et al., 2021, “Highly accurate protein structure prediction with AlphaFold”, Nature 596: 583-589). Such propeptides may be cleaved / separated from the deamidase by the (recombinant) microbial expression organism, or extracellularly by using a suitable site-specific protease. Preferred expression organisms are Chryseobacterium and Bacillus species.

[0055] The fermentation liquid / broth may be subjected to a flocculation / precipitation step to provide a purified deamidase supernatant, and subsequently the purified deamidase supernatant may be subjected to a membrane filtration to provide a concentrated deamidase solution. Preferably, the membrane filtration comprises an ultra-filtration. The concentrated deamidase solution may subsequently be subjected to the incubation step of the method of the invention.

[0056] Depending on the desired product concentration, the fermentation broth (from the fermentation), the deamidase supernatant (from the flocculation), or the concentrated deamidase solution (from the membrane filtration) may be subjected to spray-drying (or freeze drying) to provide a deamidase powder. The deamidase powder may subsequently be used to produce a liquid composition in a process that comprises mixing the deamidase powder with water and polyol. The resulting liquid composition may subsequently be subjected to the incubation step of the method of the invention.

[0057] The deamidase comprised in the liquid enzyme composition of the invention may have an amino acid sequence identity of at least 60%, preferably at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%, as compared to the amino acid sequence of SEQ ID NO: 1.

[0058] Alternatively, the deamidase may have up to 30 alterations (e.g., substitutions, deletions and / or insertions), preferably up to 25, up to 20, up to 15, up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 alteration(s), in particular substitutions, as compared to the amino acid sequence of SEQ ID NO: 1.

[0059] Amino acid alterations of both the deamidase and propeptide, as described above, may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding module.

[0060] 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, and the resultant molecules are tested for enzyme activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899- 904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related polypeptide, and / or be inferred from sequence homology and conserved catalytic machinery with a related polypeptide or within a polypeptide or protein family with polypeptides / proteins descending from a common ancestor, typically having similar three-dimensional structures, functions, and significant sequence similarity.

[0061] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known methods of mutagenesis, recombination, and / or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, CRISPR gene editing, phage display (e.g., Lowman et al., 1991, Biochemistry 30: 10832-10837; US 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et a / ., 1988, DNA 7: 127).

[0062] Microbial host cell

[0063] The microbial host cell may be of any genus. The deamidase enzyme may be homologous or heterologous to the host cell, which is capable of producing the deamidase.

[0064] The term “homologous protein” or “native protein” means a protein encoded by a gene that is derived from the host cell in which it is produced.

[0065] The term “heterologous protein” means a protein encoded by a gene which is foreign to the host cell in which it is produced.

[0066] The term "recombinant host cell", as used herein, means a host cell which harbors gene(s) encoding the deamidase and is capable of expressing said gene(s) to produce the deamidase. The deamidase coding gene(s) may be transformed, transfected, transduced, or the like, into the recombinant host cell using techniques well known in the art.

[0067] When the deamidase is a heterologous protein, the recombinant host cell capable of producing the deamidase is preferably of fungal or bacterial origin. The choice of recombinant host cell will to a large extent depend upon the gene coding for the deamidase and the source organism from which it originates.

[0068] The term "wild-type host cell", as used herein, refers to a host cell that natively harbors gene(s) coding for the deamidase and is capable of expressing said gene(s).

[0069] A mutant thereof may be a wild-type host cell in which one or more genes have been deleted, e.g., in order to enrich the desired deamidase preparation.

[0070] In a preferred embodiment, the recombinant or wild-type microbial host cell is a bacterium or a fungus.

[0071] The microbial host cell may be a yeast cell such as a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia strain. In another aspect, the strain is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis strain.

[0072] The microbial host cell may be a filamentous fungal strain such as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meri pilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria strain.

[0073] In another aspect, the strain is an Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa, Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride strain.

[0074] In one aspect, the fungal host cell is a strain selected from the group consisting of Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, Yarrowia, Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium, and Trichoderma.

[0075] In a more preferred embodiment, the filamentous fungal host cell is selected from the group consisting of Trichoderma and Aspergillus host cells, in particular a strain of Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma viride\, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae, especially a strain of Trichoderma reesei.

[0076] In another preferred embodiment, the recombinant or wild-type microbial host cell is a bacterium.

[0077] The recombinant host cell may comprise a single copy, or at least two copies, e.g., three, four, five, or more copies of the polynucleotide encoding the deamidase.

[0078] The host cell may be any Gram-positive or Gram-negative bacterium. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0079] The host cell may be any Bacillus cell including, but 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 stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells. In an embodiment, the Bacillus cell is a Bacillus amyloliquefaciens, Bacillus licheniformis or Bacillus subtilis cell.

[0080] In one embodiment, the Bacillus cell is a Bacillus subtilis cell.

[0081] In another embodiment, the Bacillus cell is a Bacillus licheniformis cell.

[0082] For purposes of this invention, Bacillus classes / genera / species shall be defined as described in Patel and Gupta, 2020, Int. J. Syst. Evol. Microbiol. 70: 406-438.

[0083] The 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. The bacterial host cell may also be any Streptomyces cell including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

[0084] 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, with DNA introduced as linearized or as circular polynucleotide. Persons skilled in the art will be readily able to identify a suitable method for introducing DNA into a given prokaryotic cell depending, e.g., on the genus. Methods for introducing DNA into prokaryotic host cells are for example described in Heinze et at., 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.

[0085] Recovery from fermentation broth

[0086] Fermentation broth

[0087] The present invention may be useful for any fermentation in industrial scale, e.g., for any fermentation having culture media of at least 50 liters, preferably at least 500 liters, more preferably at least 5,000 liters, even more preferably at least 50,000 liters.

[0088] The microorganism producing the deamidase may be fermented by any method known in the art. The fermentation medium may be a minimal medium as described in, e.g., WO 98 / 37179, or the fermentation medium may be a complex medium comprising complex nitrogen and carbon sources, wherein the complex nitrogen source may be partially hydrolyzed as described in WO 2004 / 003216.

[0089] The fermentation may be performed as a batch, a repeated batch, a fed-batch, a repeated fed-batch or a continuous fermentation process.

[0090] In a fed-batch process, either none or part of the compounds comprising one or more nutrient(s) is added to the medium before the start of the fermentation and either all or the remaining part, respectively, of the compounds comprising one or more nutrients are fed during the fermentation process. The compounds which are selected for feeding can be fed together or separately to the fermentation process.

[0091] In a repeated fed-batch or a continuous fermentation process, the complete start medium is additionally fed during fermentation. The start medium can be fed together with or separately from the structural element feed(s). In a repeated fed-batch process, part of the fermentation broth comprising the biomass is removed at regular time intervals, whereas in a continuous process, the removal of part of the fermentation broth occurs continuously. The fermentation process is thereby replenished with a portion of fresh medium corresponding to the amount of withdrawn fermentation broth. In a preferred embodiment of the invention, a fermentation broth from a fed-batch fermentation process is preferred.

[0092] In one embodiment, the fermentation broth is provided after a cultivation time of at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours.

[0093] In a particular embodiment, the fermentation broth is provided after at least 120 hours of cultivation time.

[0094] According to the present invention, the fermentation broth may be diluted up to 2000% (w / w) with water; preferably the fermentation broth may be diluted 10-2000% (w / w) with water; more preferably the fermentation broth may be diluted 100-1500% (w / w) with water; more preferably the fermentation broth may be diluted 100-1000% (w / w) with water; more preferably the fermentation broth may be diluted 200-800% (w / w) with water.

[0095] Dilution with water means, according to the present invention, that the dilution medium may be water, or it may be an ultra-filtration permeate from the production of the deamidase, or it may be a recycle of water from the production of the deamidase, or it may be a condensate from a heater, or it may be any combination of the above mentioned, e.g., a mixture of water and an ultra-filtration permeate.

[0096] The fermentation broth comprises host cells (including, the host cells containing the gene encoding the deamidase which are used to produce the deamidase), cell debris, biomass, recombinant DNA from the bacterial host cells, fermentation media and / or fermentation products. In some embodiments, the composition is a cell-killed whole broth containing organic acid(s), killed cells and / or cell debris, and culture medium.

[0097] For example, fermentation broths are produced when microbial cultures are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis (e.g., expression of enzymes by host cells) and secretion into cell culture medium. The fermentation broth can contain unfractionated or fractionated contents of the fermentation materials derived at the end of the fermentation. Typically, the fermentation broth is unfractionated and comprises the spent culture medium and cell debris present after the microbial cells (e.g., Bacillus cells) are removed, e.g., by centrifugation. In some embodiments, the fermentation broth contains spent cell culture medium, extracellular enzymes, and viable and / or nonviable microbial cells.

[0098] The cell-killed whole broth or cell composition may contain the unfractionated contents of the fermentation materials derived at the end of the fermentation. Typically, the cell-killed whole broth or cell composition contains the spent culture medium and cell debris present after the microbial cells (e.g., Bacillus cells) are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis. In some embodiments, the cell-killed whole broth or cell composition contains the spent cell culture medium, recombinant DNA from the microbial host cells, extracellular enzymes, and killed microbial 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. A whole broth or cell composition as described herein is typically a liquid, but may contain insoluble components, such as killed cells, recombinant DNA from the microbial host cells, cell debris, culture media components, and / or insoluble enzyme(s). In some embodiments, insoluble components may be removed to provide a clarified liquid composition.

[0099] The whole broth formulations and cell compositions of the present invention may be produced by a method described in WO 90 / 15861 or WO 2010 / 096673.

[0100] Flocculation / precipitation

[0101] In order to flocculate the fermentation broth a divalent salt may be added to the fermentation broth, in particular a calcium salt and / or a magnesium salt, e.g., calcium chloride or magnesium chloride. A preferred embodiment is a calcium salt, in particular calcium chloride.

[0102] The salt may be added to the fermentation broth in a concentration of 0.01-10% (w / w) per kg fermentation broth (un-diluted); preferably 0.5-10% (w / w) per kg fermentation broth (undiluted); more preferably 1-9% (w / w) per kg fermentation broth (un-diluted); in particular 2-8% (w / w) per kg fermentation broth (un-diluted).

[0103] Filtration and other downstream operations

[0104] The flocculated cell debris may be removed by methods known in the art such as, but not limited to, filtration, e.g., drum filtration, membrane filtration, filter-press dead end filtration, cross-flow filtration, or centrifugation.

[0105] The resulting fermentation supernatant may then be further processed or refined by methods known in the art. For example, the protein may be recovered by conventional procedures including, but not limited to, further filtration such as ultra-filtration and dia-filtration, extraction, spray-drying, evaporation, precipitation or crystallization.

[0106] The terms "recover" or “recovery” means the removal of a polypeptide from at least one fermentation broth component selected from the list of a cell, a nucleic acid, or other specified material, e.g., recovery of the polypeptide from the whole fermentation broth, or from the cell- free fermentation broth, by polypeptide crystal harvest, by filtration, e.g. depth filtration (by use of filter aids or packed filter medias, cloth filtration in chamber filters, rotary-drum filtration, drum filtration, rotary vacuum-drum filters, candle filters, horizontal leaf filters or similar, using sheed or pad filtration in framed or modular setups) or membrane filtration (using sheet filtration, module filtration, candle filtration, microfiltration, ultrafiltration in either cross flow, dynamic cross flow or dead end operation), or by centrifugation (using decanter centrifuges, disc stack centrifuges, hydro cyclones or similar), or by precipitating the polypeptide and using relevant solid-liquid separation methods to harvest the polypeptide from the broth media by use of classification separation by particle sizes. Recovery encompasses isolation and / or purification of the polypeptide. The isolated protein may then be further purified and / or modified by a variety of procedures known in the art including, but not limited to, chromatography e.g. ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion and / or electrophoretic procedures e.g. preparative isoelectric focusing and / or differential solubility e.g., ammonium sulfate precipitation and / or extraction.

[0107] Microfiltration may involve a membrane having a size exclusion limit of more than 1000 kDa, more than 500 kDa, more than 100 kDa, or more than 50 kDa; and / or more than 5 pm, more than 1 pm, more than 0.5 pm, more than 0.4 pm, more than 0.3 pm, more than 0.2 pm, or more than 0.1 pm; or other filters with equivalent molecular weight exclusion properties; wherein the fermentation product is a filtrate of the microfiltration.

[0108] Subsequent to the microfiltration, or in a combined process, the fermentation product may be subjected to an ultra-filtration step, which involves a membrane having a size exclusion limit of more than 100 kDa, more than 80 kDa, more than 60 kDa, more than 50 kDa, more than 40 kDa, more than 30 kDa, more than 20 kDa, more than 15 kDa, more than 10 kDa, more than 5 kDa, or more than 1 kDa; or another filter with equivalent molecular weight exclusion properties.

[0109] Uses

[0110] Protein deamidase can be applied on almost all types of proteins (plant proteins, animal protein, fermented proteins etc) where the enzyme will lower the isoelectric point of the proteins, and will, when the proteins are applied at a pH above the isoelectric point, improve solubility, electrostatic repulsion, improve different types of functionalities like foaming, emulsification, water binding etc., change affinity to flavours and off-flavours, gelling properties, improve thermostability etc. The enzymatically modified proteins can be applied as ingredients in various foods and beverages or the protein deamidase can be applied directly into a food production process like in a yogurt fermentation.

[0111] Plant proteins often have a low solubility and low functional properties. Deamidation is known to improve the solubility of plants proteins and partly of consequence hereof improve the functional properties including foaming activity, foaming stability, emulsification activity and emulsification stability. This has been observed on cross of several plant protein substrates including cereals protein like oat, wheat, corn protein and legume proteins like soy and pea protein, coconut protein etc. Negative attributes associated with the partly insoluble proteins like sandiness and grittiness are being mitigated by the enzymatic deamidation.

[0112] For example, enzymatically partly deamidated oat protein becomes essentially fully soluble at neutral pH also leading to significantly improved emulsification properties. (Z-l Jiang et al, J Cereal Science (2015): 64: 126-132). One practical implication is the use of protein deamidase in the process for oat milk production, leading to an oat milk with increased protein content, well suited to meet the requirement for the barista segment (WO 2014 / 123466). Similarly, emulsification and foaming properties are improved when soy protein isolate is enzymatically deamidated (I Suppavorasatit et al. J. Agric. Food Chem (2011) 59: 11621- 11628). For enzymatically deamidated pea protein isolate improved solubility, homogeneity, dispersibility, and suspendability and reduced beany flavour, grittiness and lumpiness have been observed (L Fang et al. J, Agric. Food Chem. (2020) 68: 1691-1697). Even the highly insoluble corn protein (zein) becomes soluble at pH 5 and 7 and with significantly improved emulsification properties (YH Yong et al. J. Agric Food Chem. (2006): 54: 6034-6040).

[0113] The improved functional properties provided by enzymatic deamidation makes the protein deamidase well suited for a variety of food applications of plant protein containing food products like milk analogues with increased protein content, reduced graininess & grittiness, improved mouthfeel, and barista properties, Similarly solutions for the yogurt analogue segments with improved mouthfeel, texture and hydrocolloid replacement. Protein deamidase has also been suggested to improve the texture of plant-based meat analogues and plant-based eggs (X Liu et al. Foods (2022) 11: 440).

[0114] Deamidation of plant proteins also have a positive impact on the flavour of proteins. Plant proteins are associated with various hydrophobic off-flavours like lipid oxidation products, e.g., having a beany off-flavour, or saponins, phenolics, and flavonoids giving a bitter off-flavour. Enzymatic deamidation of plant proteins reduces the hydrophobicity of the proteins, which therefore reduces the affinity for the hydrophobic off-flavours. Protein deamidase can therefore be applied to improve the flavour of plant proteins by inclusion of the enzyme in the process for recovery of protein concentrates or isolates, or by treatment of recovered proteins like protein isolates (X Liu et al. Foods (2022) 11: 440). Flavour improvement is, e.g., demonstrated for soy (I Suppavorasatit et al. J. Agric. Food Chem (2012) 60: 7817-7823).

[0115] Application of protein deamidase in protein recovery process like the pea protein recovery process leads to improved recovery yield of the proteins, like when applied in the recovery process leading to legume protein concentrate and isolates (WO 2021049591).

[0116] Protein deamidase also have several applications on dairy proteins and dairy based foods. Deamidation of whey lead to a better electrostatic repulsion of the proteins, giving a better thermostability, avoiding undesirable aggregation in whey protein solutions (e.g., in protein fortified beverages) when the protein solution is heat-treated (N Miwa et al. J. Agric. Food Chem (2013) 61 : 2205-2212). Enzymatic deamidation in skim milk leads to much improved solubility, viscosity and provides a translucent milk drink (N Miwa et al. International dairy journal (2010) 20: 393-399). Application of protein deamidase in the yogurt process leads to an improved stabilization, which can e.g. be applied to replace pectin and other hydrocolloids in drinking yogurt.

[0117] Protein (glutaminase) deamidase can be applied together with other enzymes including other enzymes modifying or degrading protein. Combinations between protein glutamine deamidase and protein asparagine deamidase can provide a higher degree of deamidation of the proteins and thereby an even better applicational performance. Protein deamidase can be applied together with protein crosslinking enzymes like transglutaminase, where the crosslinking of the protein is modified, partly as the transglutaminase will be prevented from reacting with the glutamines which have been converted to glutamic acid by the deamidase. When a combination of transglutaminase and protein deamidase is used in the yogurt process, a texturing effect is obtained, applicable to replace added dairy proteins or hydrocolloids, providing a yogurt with a smooth texture and avoiding the lumpy texture which is seen when transglutaminase is used alone. A similar effect is observed when this enzyme combination is used for production of plant-based yogurt analogues. Furthermore, the protein deamidase can be applied together with proteases where the resulting protein hydrolysate will have improved solubility and taste and changed functional properties.

[0118] Further embodiments of the invention include:

[0119] Embodiment 1. A method for stabilizing a liquid enzyme composition, comprising

[0120] (a) providing a liquid composition comprising a protein-glutamine glutaminase, which is recovered from a microbial fermentation broth;

[0121] (b) incubating the liquid composition at a temperature of at least 40°C for at least 24 hours; and

[0122] (c) removing precipitated solids from the incubated liquid composition by solid-liquid separation to provide a stabilized liquid enzyme composition.

[0123] Embodiment 2. The method of the preceding embodiment, wherein the liquid composition is incubated at a temperature of at least 45°C.

[0124] Embodiment 3. The method of any of the preceding embodiments, wherein the liquid composition is incubated at a temperature of at least 50°C.

[0125] Embodiment 4. The method of any of the preceding embodiments, wherein the liquid composition is incubated at a temperature of at least 55°C.

[0126] Embodiment 5. The method of any of the preceding embodiments, wherein the liquid composition is incubated at a temperature of at least 60°C.

[0127] Embodiment 6. The method of any of the preceding embodiments, wherein the liquid composition is incubated for at least 48 hours.

[0128] Embodiment 7. The method of any of the preceding embodiments, wherein the liquid composition is incubated for at least 60 hours.

[0129] Embodiment 8. The method of any of the preceding embodiments, wherein the liquid composition is incubated for at least 72 hours.

[0130] Embodiment 9. The method of any of the preceding embodiments, wherein the liquid composition is incubated at a temperature of 40-50°C for 48-96 hours, or at a temperature of 50-60°C for 24-60 hours. Embodiment 10. The method of any of the preceding embodiments, wherein the liquid composition comprises the protein-glutamine glutaminase in an amount of 20-10000 IPA(ll) per gram.

[0131] Embodiment 11. The method of any of the preceding embodiments, wherein the liquid composition comprises the protein-glutamine glutaminase in an amount of 40-8000 IPA(ll) per gram.

[0132] Embodiment 12. The method of any of the preceding embodiments, wherein the liquid composition comprises the protein-glutamine glutaminase in an amount of 60-6000 IPA(ll) per gram.

[0133] Embodiment 13. The method of any of the preceding embodiments, wherein the liquid composition comprises the protein-glutamine glutaminase in an amount of 80-5000 IPA(ll) per gram.

[0134] Embodiment 14. The method of any of the preceding embodiments, wherein the liquid composition comprises the protein-glutamine glutaminase in an amount of 100-4000 IPA(ll) per gram.

[0135] Embodiment 15. The method of any of the preceding embodiments, wherein the liquid composition comprises the protein-glutamine glutaminase in an amount of 0.01-15% w / w of active deamidase enzyme protein.

[0136] Embodiment 16. The method of any of the preceding embodiments, wherein the liquid composition comprises the protein-glutamine glutaminase in an amount of 0.05-10% w / w of active deamidase enzyme protein.

[0137] Embodiment 17. The method of any of the preceding embodiments, wherein the liquid composition comprises the protein-glutamine glutaminase in an amount of 0.05-5% w / w of active deamidase enzyme protein.

[0138] Embodiment 18. The method of any of the preceding embodiments, wherein the liquid composition comprises the protein-glutamine glutaminase in an amount of 0.1-5% w / w of active deamidase enzyme protein.

[0139] Embodiment 19. The method of any of the preceding embodiments, wherein the liquid composition comprises the protein-glutamine glutaminase in an amount of 0.1-3% w / w of active deamidase enzyme protein.

[0140] Embodiment 20. The method of any of the preceding embodiments, wherein the liquid composition comprises the protein-glutamine glutaminase in an amount of 0.1-2% w / w of active deamidase enzyme protein.

[0141] Embodiment 21. The method of any of the preceding embodiments, wherein the protein- glutamine glutaminase belongs to EC 3.5.1.44.

[0142] Embodiment 22. The method of any of the preceding embodiments, wherein the protein- glutamine glutaminase has an amino acid sequence identity of at least 60%, preferably at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%, as compared to the amino acid sequence of SEQ ID NO: 1.

[0143] Embodiment 23. The method of any of the preceding embodiments, wherein the protein- glutamine glutaminase has up to 30 alterations (e.g., substitutions, deletions and / or insertions), preferably up to 25, up to 20, up to 15, up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 alteration(s), in particular substitutions, as compared to the amino acid sequence of SEQ ID NO: 1.

[0144] Embodiment 24. The method of any of the preceding embodiments, wherein a polyol is added to the liquid composition before incubation.

[0145] Embodiment 25. The method of any of the preceding embodiments, wherein a polyol is added to the liquid composition before incubation in an amount of at least 10% w / w of the liquid composition.

[0146] Embodiment 26. The method of any of the preceding embodiments, wherein a polyol is added to the liquid composition before incubation in an amount of 10-75% w / w of the liquid composition.

[0147] Embodiment 27. The method of any of the preceding embodiments, wherein a polyol is added to the liquid composition before incubation in an amount of 10-70% w / w of the liquid composition.

[0148] Embodiment 28. The method of any of the preceding embodiments, wherein a polyol is added to the liquid composition before incubation in an amount of 10-65% w / w of the liquid composition.

[0149] Embodiment 29. The method of any of the preceding embodiments, wherein a polyol is added to the liquid composition before incubation in an amount of 10-60% w / w of the liquid composition.

[0150] Embodiment 30. The method of any of the preceding embodiments, wherein a polyol is added to the liquid composition before incubation in an amount of at least 20% w / w of the liquid composition.

[0151] Embodiment 31. The method of any of the preceding embodiments, wherein a polyol is added to the liquid composition before incubation in an amount of at least 25% w / w of the liquid composition.

[0152] Embodiment 32. The method of any of the preceding embodiments, wherein a polyol is added to the liquid composition before incubation in an amount of at least 30% w / w of the liquid composition.

[0153] Embodiment 33. The method of any of the preceding embodiments, wherein a polyol is added to the liquid composition before incubation in an amount of at least 35% w / w of the liquid composition.

[0154] Embodiment 34. The method of any of embodiments 24-33, wherein the polyol is selected from the group consisting of glucose, fructose, galactose, sucrose, lactose, maltose, trehalose, cellobiose, glycerol, ethylene glycol, diethylene glycol, triethylene glycol, propylene glycol, dipropylene glycol, tripropylene glycol, polyethylene glycol (PEG), sugar alcohols, and combinations thereof

[0155] Embodiment 35. The method of any of the preceding embodiments, wherein the pH of the liquid composition is at least pH 3.5.

[0156] Embodiment 36. The method of any of the preceding embodiments, wherein the pH of the liquid composition is at least pH 4.0.

[0157] Embodiment 37. The method of any of the preceding embodiments, wherein the pH of the liquid composition is at most pH 8.0.

[0158] Embodiment 38. The method of any of the preceding embodiments, wherein the pH of the liquid composition is in the range of pH 3.5-10.

[0159] Embodiment 39. The method of any of the preceding embodiments, wherein the pH of the liquid composition is in the range of pH 4-9.

[0160] Embodiment 40. The method of any of the preceding embodiments, wherein the pH of the liquid composition is in the range of pH 4-8.

[0161] Embodiment 41. The method of any of the preceding embodiments, wherein the pH of the liquid composition is in the range of pH 4-7.

[0162] Embodiment 42. The method of any of the preceding embodiments, wherein the pH of the liquid composition is in the range of pH 4-6.

[0163] Embodiment 43. The method of any of the preceding embodiments, wherein the liquid composition further comprises at least 2% w / w of salt.

[0164] Embodiment 44. The method of any of the preceding embodiments, wherein the liquid composition comprises at least 4% w / w of salt.

[0165] Embodiment 45. The method of any of the preceding embodiments, wherein the liquid composition further comprises at least 6% w / w of salt.

[0166] Embodiment 46. The method of any of the preceding embodiments, wherein the liquid composition further comprises at least 8% w / w of salt.

[0167] Embodiment 47. The method of any of the preceding embodiments, wherein the liquid composition comprises at most 20% w / w of salt.

[0168] Embodiment 48. The method of any of the preceding embodiments, wherein the liquid composition comprises at most 15% w / w of salt.

[0169] Embodiment 49. The method of any of the preceding embodiments, wherein the liquid composition comprises at most 10% w / w of salt.

[0170] Embodiment 50. The method of any of embodiments 43-49, wherein the salt is selected from the group consisting of sodium sulfate, potassium sulfate, ammonium sulfate, magnesium sulfate, zinc sulfate, sodium carbonate, potassium carbonate, ammonium carbonate, sodium bicarbonate, potassium bicarbonate, ammonium bicarbonate, sodium nitrate, potassium nitrate, ammonium nitrate, magnesium nitrate, zinc nitrate, calcium nitrate, sodium phosphate, potassium phosphate, ammonium phosphate, magnesium phosphate, zinc phosphate, calcium phosphate, sodium chloride, potassium chloride, ammonium chloride, magnesium chloride, zinc chloride, calcium chloride, sodium formate, potassium formate, ammonium formate, magnesium formate, zinc formate, calcium formate, sodium acetate, potassium acetate, ammonium acetate, magnesium acetate, zinc acetate, calcium acetate, sodium citrate, potassium citrate, ammonium citrate, magnesium citrate, and hydrates thereof.

[0171] Embodiment 51. The method of the preceding embodiment, wherein the salt is selected from the group consisting of sodium chloride, potassium chloride, ammonium chloride, magnesium chloride, zinc chloride, calcium chloride, sodium formate, potassium formate, ammonium formate, magnesium formate, zinc formate, calcium formate, sodium acetate, potassium acetate, ammonium acetate, magnesium acetate, zinc acetate, calcium acetate, sodium citrate, potassium citrate, ammonium citrate, magnesium citrate, and hydrates thereof.

[0172] Embodiment 52. The method of the preceding embodiment, wherein the salt is selected from the group consisting of sodium chloride, potassium chloride, sodium formate, potassium formate, sodium acetate, potassium acetate, sodium citrate, potassium citrate, and hydrates thereof.

[0173] Embodiment 53. The method of any of the preceding embodiments, wherein the solidliquid separation is selected from the group consisting of filtration, sedimentation, decanting, and centrifugation.

[0174] Embodiment 54. The method of any of the preceding embodiments, wherein the liquid composition further comprises a reducing agent.

[0175] Embodiment 55. The method of any of the preceding embodiments, wherein the liquid composition further comprises a reducing agent in an amount of at least 0.1% w / w.

[0176] Embodiment 56. The method of any of the preceding embodiments, wherein the liquid composition further comprises a reducing agent in an amount of at least 0.1-5% w / w.

[0177] Embodiment 57. The method of any of the preceding embodiments, wherein the liquid composition further comprises a reducing agent in an amount of at least 0.1-2% w / w.

[0178] Embodiment 58. The method of any of the preceding embodiments, wherein the liquid composition further comprises a salt of sulfite, metabisulfite, thiosulphate, or ascorbate.

[0179] Embodiment 59. The method of any of the preceding embodiments, wherein the liquid composition further comprises a salt of sulfite, metabisulfite, thiosulphate, or ascorbate, in an amount of at least 0.1% w / w.

[0180] Embodiment 60. The method of any of the preceding embodiments, wherein the liquid composition further comprises a salt of sulfite, metabisulfite, thiosulphate, or ascorbate, in an amount of at least 0.1-5% w / w.

[0181] Embodiment 61. The method of any of the preceding embodiments, wherein the liquid composition further comprises a salt of sulfite, metabisulfite, thiosulphate, or ascorbate, in an amount of at least 0.1-2% w / w. Embodiment 62. The method of the preceding embodiment, wherein the microbial fermentation broth is a bacterial fermentation broth.

[0182] Embodiment 63. The method of the preceding embodiment, wherein the microbial fermentation broth is a bacterial fermentation broth comprising a Chryseobacterium or Bacillus species.

[0183] Embodiment 64. The method of any of the preceding embodiments, wherein the recovery comprises a membrane filtration, such as an ultra-filtration.

[0184] Embodiment 65. A method for deamidating a glutamine residue in a plant or milk protein, comprising contacting the plant or milk protein with the stabilized liquid enzyme composition of any of the preceding embodiments.

[0185] Embodiment 66. The method of the preceding embodiment, wherein the plant protein is derived from cereals or legumes, and the milk protein is whey protein; more preferably the plant protein is derived from oat, wheat, corn, soy, pea or almond.

[0186] EXAMPLES

[0187] Chemicals were commercial products of at least reagent grade.

[0188] The deamidase used in the examples is derived from Chryseobacterium viscerum. The amino acid sequence of the deamidase is shown as SEQ ID NO: 1.

[0189] EXAMPLE 1

[0190] Deamidase activity assay

[0191] The deamidase activity assay consists of two separate de-coupled parts:

[0192] 1) an enzymatic step wherein ammonia is formed by the catalytic action of the deamidase; and

[0193] 2) a non-enzymatic detection step wherein the ammonia formed in step (1) is derivatized to a blue indophenol compound with an absorption maximum at 630 nm.

[0194] In step (1), the ammonia is developed by the deamidating action of the deamidase. In step (2), the generated ammonia reacts with phenol to form dioxyphenylamine under alkaline conditions. The reaction is catalyzed by sodium pentacyanonitrosylferrate(lll) (sodium nitroprusside). “Color Reagent solution A” contains phenol and sodium nitroprusside. “Color Reagent Solution B” provides alkaline reaction conditions. The intermediate is then oxidized by addition of sodium hypochlorite (“Color Reagent Solution C”) to form indophenol blue. This compound absorbs visible light at 630 nm. The enzyme activity is then calculated using a standard curve.

[0195] Assay Procedure

[0196] Step (1) Enzymatic step with ammonia formation Reagents:

[0197] Assay dilution solution: 0.2 M Na-phosphate buffer, 0.01% Triton X-100, pH 6.5.

[0198] Assay buffer: Same as above. Used to prepare stock solution and diluted sample of protein deamidase (referred to in the following as “enzyme”).

[0199] Substrate solution: 30 mM Z-GIn-Gly (Merck C6154-1 G) in assay dilution solution (check pH after dissolution).

[0200] Stop solution: 0.4 M TCA.

[0201] Standard: NH4CI (Ammonium Standard for IC, Merck 59755-100ML, 1000 mg / L NH4+in water) diluted in assay dilution solution (see also “Standard curve” section).

[0202] Dissolve / dilute enzyme product in assay buffer and prepare suitable dilution resulting in a linear assay response.

[0203] Incubation:

[0204] 1. Add 10 pL of diluted enzyme samples in triplicates to the wells of a 96-well microtiter plate (MTP).

[0205] 2. Add 100 pL of substrate solution to each well.

[0206] 3. For blank samples add 100 pL 0.4 M TCA solution.

[0207] 4. Seal the plate using transparent plate sealer.

[0208] 5. Incubate the plate for 10 minutes at 37°C, 500 rpm, on a thermomixer equipped with a lid heating function.

[0209] 6. To stop the reaction, carefully add 100 pL 0.4 M TCA solution (except for the blank samples, which already contain TCA).

[0210] Total reaction volume: 210 pL

[0211] Step (2) Ammonia detection step

[0212] Reagents:

[0213] Color reagent A: 4% (w / v) Phenol, 0.015% (w / v) sodium pentacyanonitrosylferrate(lll) dihydrate (sodium nitroprusside) (Na2[Fe(CN)sNO]-2H2O).

[0214] Color reagent B: 5% (w / v) Potassium hydroxide.

[0215] Color reagent C: 28% (w / v) Potassium carbonate, 6% (v / v) sodium hypochlorite (Sigma-Aldrich 239305-25ml, < 5% available Cl2).

[0216] Incubation:

[0217] 1. Transfer 15 pL from each well from step (1) into a new 96-well MTP.

[0218] 2. Transfer 45 pL Milli-Q water to each well.

[0219] 3. To each well, add 30 pL of color reagent B (on lab table, shake gently by hand to mix). 4. To each well, add 60 pL of color reagent A (on lab table, shake gently by hand to mix).

[0220] 5. To each well, add 60 pL of color reagent C (on lab table, shake gently by hand to mix).

[0221] 6. Color development: Carefully seal the plate and leave it on lab table for 30 minutes.

[0222] 7. Carefully transfer the MTP to a plate reader and measure absorbance at 630 nm.

[0223] Total reaction volume: 210 pL

[0224] Standard curve:

[0225] Standard stock solution: 1000 mg NH4+ / L.

[0226] The standard curve is prepared by adding dilutions of the ammonium standard in the assay dilution buffer in the ammonia detection step. That is, mixing 15 pL diluted ammonia standard with 45 pL water and then add the color reagents in the order given above; B, A, and C.

[0227] The amount of enzyme producing 1 pmol ammonia per minute at 37°C is defined as 1 unit (Indophenol Assay Unit; IPA(U)): which can be shortened to in. where

[0228] • CNH4+ is the ammonia concentration in the reaction solution derived from the ammonium standard curve (i.e., taking into account the dilution of the prediluted ammonium standard solution in the ammonia derivatization step).

[0229] • 18.04 is the molecular mass of ammonium used for the standard solution.

[0230] • Vreaaion is the reaction volume in the well when ammonia is generated (210 pL).

[0231] • V enzyme is the volume of enzyme solution added to the well when ammonia is generated (10 pL).

[0232] • VNH3 detection is the reaction volume in the well when ammonia is detected (210 pL).

[0233] EXAMPLE 2

[0234] Physical stability of stabilized liquid compositions In the first study, the physical stability of several liquid deamidase compositions was evaluated after the liquid compositions were treated according to the invention by incubation at 40°C for 4 days followed by filtration.

[0235] After this treatment, the stabilized liquid compositions were stored at 10°C or 25°C for 2, 4, or 13 weeks. The treated compositions were compared with non-treated compositions (references).

[0236] All liquid compositions contained the deamidase in an amount of approx. 370 IPA(U) / g before treatment. None of the liquid compositions lost more than 5% enzyme activity after treatment.

[0237] The liquid compositions contained (about) 10% w / w NaCI and (about) 60% w / w glycerol or 50% w / w sucrose. The pH of the compositions was adjusted to pH 4, pH 4.5, or pH 5, as indicated in the tables.

[0238] The physical stability was evaluated by visual inspection on a scale from 1 to 6, where 1 = “clear”, and 6 = “strong precipitate”.

[0239] Table 1. Physical stability of liquid composition with glycerol at pH 4, evaluated visually on a scale from 1 to 6.

[0240] Table 2. Physical stability of liquid composition with glycerol at pH 5, evaluated visually on a scale from 1 to 6. Table 3. Physical stability of liquid composition with sucrose at pH 4.5, evaluated visually on a scale from 1 to 6.

[0241] Tables 1-3 show an improved physical stability of the liquid compositions after treatment according to the method of the invention.

[0242] EXAMPLE 3

[0243] Improving physical stability

[0244] In the second study, the liquid compositions were incubated at 40°C for 4 days, 50°C for 1 day, or 50°C for 2 days, followed by filtration. After the treatments, the stabilized liquid compositions were stored at 25°C for 2 weeks. The physical stability was evaluated by visual inspection using the same scale as in Example 2, where 1 = “clear”, and 6 = “strong precipitate”.

[0245] All liquid compositions contained the deamidase in an amount of approx. 1080 IPA(U) / g before treatment. None of the liquid compositions lost more than 5% enzyme activity after treatment, except the composition treated at 60°C which lost 12% enzyme activity.

[0246] The liquid compositions contained 10% w / w NaCI and 40% w / w sucrose and was adjusted to pH 4.0.

[0247] Table 4. Physical stability after storage at 25°C for 2 weeks, evaluated visually from 1 to 6.

[0248] Table 4 shows that incubation at higher temperatures for a shorter time may be more effective for improving the physical stability of the stabilized liquid compositions. The stabilized liquid composition treated at 60°C for 1 day was still clear after storage for 8 weeks.

Claims

CLAIMS1. A method for stabilizing a liquid enzyme composition, comprising(a) providing a liquid composition comprising a protein-glutamine glutaminase, which is recovered from a microbial fermentation broth;(b) incubating the liquid composition at a temperature of at least 40°C for at least 24 hours; and(c) removing precipitated solids from the incubated liquid composition by solid-liquid separation to provide a stabilized liquid enzyme composition.

2. The method of the preceding claim, wherein the liquid composition comprises the protein- glutamine glutaminase in an amount of 20-10000 IPA(U) / g3. The method of any of the preceding claims, wherein the protein-glutamine glutaminase belongs to EC 3.5.1.44.

4. The method of the preceding claim, wherein the liquid composition has a pH of at least pH 3.5, preferably at least pH 4.0, more preferably in the range of pH 3.5-10, even more preferably in the range of pH 4-9 or in the range of pH 4-8.

5. The method of the preceding claim, wherein the liquid composition is incubated at a temperature of at least 40°C, such as at least 45°C, at least 50°C, at least 55°C, or at least 60°C.

6. The method of the preceding claim, wherein the liquid composition is incubated for at least 48 hours, such as at least 60 hours, or at least 72 hours.

7. The method of any of the preceding claims, wherein the solid-liquid separation is selected from the group consisting of filtration, sedimentation, decanting, and centrifugation.

8. The method of any of the preceding claims, wherein a polyol is added to the liquid composition before incubation, preferably in an amount of at least 10% w / w of the liquid composition, such as 10-70% w / w, 20-70% w / w, or 20-60% w / w of the liquid composition.

9. The method of the preceding claim, wherein the polyol is selected from the group consisting of glucose, fructose, galactose, sucrose, lactose, maltose, trehalose, cellobiose, glycerol, ethylene glycol, diethylene glycol, triethylene glycol, propylene glycol, dipropylene glycol, tripropylene glycol, polyethylene glycol (PEG), sugar alcohols, and combinations thereof.

10. The method of any of the preceding claims, wherein a salt is added to the liquid composition before incubation, preferably in an amount of at least 2% w / w; preferably at least 4% w / w, at least 6% w / w, or at least 8% w / w, of the liquid composition.

11. The method of the preceding claim, wherein the salt is selected from the group consisting of sodium chloride, potassium chloride, ammonium chloride, magnesium chloride, zinc chloride, calcium chloride, sodium formate, potassium formate, ammonium formate, magnesium formate, zinc formate, calcium formate, sodium acetate, potassium acetate, ammonium acetate, magnesium acetate, zinc acetate, calcium acetate, sodium citrate, potassium citrate, ammonium citrate, magnesium citrate, and hydrates thereof.

12. The method of any of the preceding claims, wherein the microbial fermentation broth comprises a Chryseobacterium or Bacillus species; and / or where the recovery comprises flocculation and membrane filtration steps.

13. A method for deamidating a glutamine residue in a plant or milk protein, comprising contacting the plant or milk protein with the stabilized liquid enzyme composition of any of the preceding claims; preferably the plant protein is derived from cereals or legumes, and the milk protein is whey protein; more preferably the plant protein is derived from oat, wheat, corn, soy, pea or almond.

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