Stable liquid deamidase compositions

Abstract

The present invention provides a liquid deamidase composition comprising one or more monosaccharides or disaccharides, which liquid deamidase composition exhibits high enzymatic stability after storage.

Description

[0001] References to sequence lists This application contains a sequence list in computer-readable form, which is incorporated herein by reference. Technical Field

[0002] The present invention relates to a liquid deamidase composition comprising monosaccharides or disaccharides, which exhibits high enzymatic stability after storage. Background Technology

[0003] Liquid enzyme compositions are a desirable product form because they are easy to handle in industrial environments. Compared to solid enzyme compositions, they can be directly pumped into industrial processes and typically do not require safety measures to avoid exposure to enzyme dust.

[0004] While solid enzyme compositions typically exhibit excellent enzymatic storage stability, liquid enzyme compositions present a greater challenge because dissolved enzymes are more fragile than enzymes in solid form.

[0005] WO 2013 / 064736 discloses a liquid deamidase composition containing glycerol / sorbitol with a pH of 4.4-5.1.

[0006] WO 2022 / 118914 discloses a liquid deamidase composition containing sorbitol with a pH of 5.5 or higher. Summary of the Invention

[0007] In a first aspect, the present invention provides a liquid enzyme composition comprising... (a) Protein-glutamine glutaminase, and (b) 10-80% w / w of one or more monosaccharides or disaccharides.

[0008] Other aspects and embodiments of the invention will be apparent from the description and examples.

[0009] Unless otherwise specified, or if the context clearly indicates otherwise, all percentages are weight percentages (% w / w).

[0010] As used herein, the term “consistently of” (and its grammatical variations) applied to the compositions and methods of the present invention means that such compositions / methods may contain additional components, provided that such additional components do not substantially alter the composition / method.

[0011] As used herein, the term "substantially free of" (and its grammatical variations) applied to the compositions and methods of the present invention means that such compositions / methods may contain small amounts of the specified component, provided that the amount of the component does not substantially alter the composition / method or provide any substantial effect on it. In the embodiments, "substantially free of" means 0% w / w.

[0012] sequence SEQ ID NO: 1: From the genus *Citrus* ( Chryseobacterium The amino acid sequence of the deamidase of species-62563.

[0013] SEQ ID NO: 2: Amino acid sequence of deamidase precursor peptide from Chrysobacterium species-62563. Detailed Implementation

[0014] In the context of this invention, the term "deamidase" refers to the activity of protein-glutamine glutaminase (also known as glutamylpeptide glutaminase or protein deamidase), as described in EC 3.5.1.44, which catalyzes the hydrolysis of γ-amides of glutamines (e.g., L-glutamylglycine and L-phenylalanyl-L-glutamylglycine) substituted at the carboxyl position or both the α-amino and carboxyl positions. Thus, deamidases can deamidate glutamine residues in proteins to glutamate residues, and deamidases are also referred to as protein glutamine deamidases. Deamidases include the Cys-His-Asp catalytic triad (e.g., Cys-156, His-197, and Asp-217, as shown in Hashizume et al., “Crystal structures of protein glutaminase and its pro forms converted into enzyme-substrate complex,” Journal of Biological Chemistry, Vol. 286, No. 44, pp. 38691-38702) and belong to InterPro entry IPR041325. In a preferred embodiment, the deamidase of the present invention belongs to the PFAM domain PF18626.

[0015] Deamidases are catalytic proteins (enzymes), and the term "active (deamidase) enzyme protein" is defined herein as the amount of one or more catalytic proteins exhibiting deamidase activity. This can be determined using activity-based analytical enzyme assays. This technique is well known in the art.

[0016] The deamidase activity was measured using the assay described in Example 1. The activity assay consisted of two separate parts: (1) an enzymatic step in which ammonia was formed by the catalysis of a protein deamidase; and (2) a non-enzymatic assay step in which the ammonia formed in step (1) was derivatized into an indophenol blue compound with maximum absorption at 630 nm. The amount of enzyme that produces 1 μmol of ammonia per minute at 37 °C was defined as one unit (expressed in indophenol assay units: IPA(U)). The activity could be determined relative to known strength standards.

[0017] For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. [Journal of Molecular Biology] 48: 443-453) is used to determine the sequence identity between two amino acid sequences as the output of "longest identity". This algorithm is implemented in the Niedel program using the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. [Trends in Genetics] 16: 276-277) (preferably version 6.6.0 or later). The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. For the Niedel program to report the longest identity, the non-brief (-nobrief) option must be specified on the command line. The Niedel-marked "longest identity" output is calculated as follows: (Identical residues × 100) / (Alignment length - Total number of vacancies in the alignment)

[0018] Liquid enzyme composition We have found that when an aqueous solution of a deamidase contains one or more monosaccharides or disaccharides, the solution exhibits improved enzyme stability after storage. Glycerol and / or sorbitol are known from the prior art to be useful stabilizers in liquid deamidase compositions. However, we have found that deamidase compositions containing one or more monosaccharides or disaccharides exhibit even better storage stability. Therefore, in a first aspect, the present invention provides a liquid enzyme composition containing… (a) Protein-glutamine glutaminase, and (b) 10-80% w / w of one or more monosaccharides or disaccharides.

[0019] The one or more monosaccharides or disaccharides may be selected from the group consisting of glucose, fructose, galactose, sucrose, lactose, maltose, trehalose, cellobiose, and combinations thereof.

[0020] Preferably, the monosaccharide or disaccharide is a disaccharide, such as sucrose, lactose, maltose, trehalose, and / or cellobiose; especially sucrose.

[0021] In the embodiments, the liquid enzyme composition comprises 10-70% w / w, preferably 10-60% w / w, 20-70% w / w, or 20-60% w / w of monosaccharides or disaccharides.

[0022] In the examples, the liquid enzyme composition has a pH of at least 3.5, preferably at least 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 the examples, the pH of the composition is at most pH 8.

[0023] The composition may further contain at least 10% w / w of water.

[0024] In the examples, the composition contains 20-10000 IPA(U) / g of deamidase (protein-glutamine glutaminase); preferably 40-8000 IPA(U) / g, 60-6000 IPA(U) / g, 80-5000 IPA(U) / g of deamidase (protein-glutamine glutaminase), or 100-4000 IPA(U) / g of deamidase (protein-glutamine glutaminase). The amount of deamidase can also be expressed as "active (deamidase) enzyme protein"; therefore, the liquid enzyme composition may contain 0.01-15% w / w of active deamidase protein of deamidase (protein-glutamine-glutaminase); preferably 0.05-10% w / w, 0.1-5% w / w, or 0.1-3% w / w of active deamidase protein.

[0025] As described below, the liquid enzyme composition may further comprise one or more salts at least 2% w / w, preferably at least 4% w / w, at least 6% w / w, or at least 8% w / w.

[0026] The liquid enzyme composition may further contain a reducing agent to prevent oxidation of cysteine ​​residues at the active site and to maintain deamidation enzymatic activity during storage. The reducing agent may be, for example, a salt of sulfite, metabisulfite, thiosulfate, or ascorbate. The liquid enzyme composition may contain at least 0.1% w / w of the reducing agent; such as 0.1-5% w / w or 0.1-2% w / w.

[0027] Monosaccharides or disaccharides The liquid enzyme composition comprises 10-80% w / w one or more monosaccharides or disaccharides, such as 10-75% w / w, 10-70% w / w, 10-65% w / w, or 10-60% w / w. In embodiments, the liquid composition comprises at least 20% w / w, preferably at least 25% w / w, at least 30% w / w, or at least 35% w / w one or more monosaccharides or disaccharides.

[0028] The compositions of the present invention may further comprise a subset selected from the group consisting of glycerol, ethylene glycol, diethylene glycol, triethylene glycol, propylene glycol, dipropylene glycol, tripropylene glycol, polyethylene glycol (PEG), and sugar alcohols. The polyethylene glycol may have an average molecular weight equal to or less than about 500. Examples of sugar alcohols are sorbitol, mannitol, erythritol, galactitol, inositol, xylitol, ribitol, isomaltitol, and maltitol. The compositions may comprise amounts of such polyols less than 40% w / w, less than 30% w / w, less than 20% w / w, less than 10% w / w, or less than 5% w / w.

[0029] Salt The liquid enzyme composition 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 one or more salts. The amount of salt is calculated on an unhydrated basis, thus excluding any complexing water (water of crystallization). Those skilled in the art will recognize that the upper limit of salt in the liquid enzyme composition is determined by the solubility of the salt. In examples, the liquid enzyme composition may further comprise up to 20% w / w of salt, preferably up to 15% w / w or up to 10% w / w of salt.

[0030] The one or more salts may be selected from the group consisting of: sulfates, carbonates, nitrates, phosphates, halides, formates, acetates, and citrates of alkali metals; sulfates, carbonates, nitrates, phosphates, halides, formates, acetates, and citrates of alkaline earth metals; sulfates, carbonates, nitrates, phosphates, halides, formates, acetates, and citrates of transition metals; and sulfates, ammonium carbonates, nitrates, halides, formates, acetates, and citrates of ammonium.

[0031] 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. Their hydrates are also included.

[0032] 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.

[0033] Deamidase The deamidases contained in the liquid enzyme compositions of the present invention are typically produced via microbial fermentation and subsequent recovery processes. These recovery processes may include a maturation / activation step, wherein an inhibitory propeptide is separated from the deamidase prototype to produce an active (or more active) deamidase. Therefore, the liquid enzyme compositions of the present invention may further comprise a deamidase inhibitory propeptide that is not covalently linked to the deamidase. Deamidase inhibitory propeptides are described, for example, in PCT / EP2023 / 055936. An exemplary propeptide is shown in SEQ ID NO: 2, and other propeptides 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 can be cleaved / isolated from the deamidase by (recombinant) microbial expression organisms or cleaved / isolated extracellularly using suitable site-specific proteases. Preferred expression organisms are species of the genus *Aureobacterium* and the genus *Bacillus*. Bacillus ) species.

[0034] The fermentation liquid / broth can undergo a flocculation / precipitation step to provide a purified deamidase supernatant, and the purified deamidase supernatant can subsequently undergo membrane filtration to provide a concentrated deamidase solution. Preferably, the membrane filtration includes ultrafiltration. The concentrated deamidase solution can then be used to produce the liquid composition of the present invention in a method comprising mixing the concentrated deamidase solution with a monosaccharide or disaccharide, and optionally evaporating some water. The water in the concentrated deamidase solution may also be evaporated before adding the one or more monosaccharides or disaccharides.

[0035] Depending on the desired product concentration, the fermentation broth (from fermentation), the deamidase supernatant (from flocculation), or the concentrated deamidase solution (from membrane filtration) can be spray-dried (or freeze-dried) to provide deamidase powder. The deamidase powder can then be used to produce the liquid compositions of the present invention in a method comprising mixing the deamidase powder with water and a polyol.

[0036] Compared with the amino acid sequence of SEQ ID NO: 1, the deamidase contained in the liquid enzyme composition of the present invention may have 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% amino acid sequence identity.

[0037] Alternatively, the deamidase may have up to 30 alterations (e.g., substitutions, deletions and / or insertions) compared to the amino acid sequence of SEQ ID NO: 1, 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, particularly substitutions.

[0038] As mentioned above, the amino acid alterations of both deamidases and propeptides can be minor, i.e., conserved amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; typically small deletions of 1–30 amino acids; small N-terminal or C-terminal extensions, such as methionine residues at the N-terminus; small linker peptides of up to 20–25 residues; or small extensions that facilitate purification by altering net charge or another function (such as polyhistidine fragments, antigenic epitopes, or binding modules).

[0039] Essential amino acids in peptides can be identified using procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, a single alanine mutation is introduced at each residue in the molecule, and the enzymatic activity of the resulting molecule is tested to identify the amino acid residues critical to the molecule's activity. See also Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active sites of enzymes or other biological interactions can also be determined by physical analysis of the structure, such as by techniques like nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, along with mutagenesis of the amino acids at the putative contact sites. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from alignments with related polypeptides, and / or from sequence homology and conserved catalytic mechanisms with related polypeptides or polypeptide / protein families from a common ancestor (typically possessing similar three-dimensional structures, functions, and significant sequence similarities).

[0040] Using known mutagenesis, recombination, and / or tampering methods, followed by relevant screening procedures, one or more amino acid substitutions, deletions, and / or insertions can be made and tested, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, CRISPR gene editing, phage display (e.g., Lowman et al., 1991, Biochemistry 30: 10832-10837; US 5,223,409; WO 92 / 06204), and regional directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA7: 127).

[0041] use Protein deamidases can be applied to almost all types of proteins (plant proteins, animal proteins, fermented proteins, etc.). The enzymes lower the isoelectric point of the protein, and when applied at a pH above the isoelectric point, they improve solubility, reduce electrostatic repulsion, enhance various functions (such as foaming, emulsification, and water binding), alter flavor and off-flavor affinity, gelling properties, and improve thermal stability. Enzymatically modified proteins can be used as ingredients in various foods and beverages, or deamidated proteins can be directly applied in food production processes, such as in yogurt fermentation.

[0042] Plant proteins typically exhibit low solubility and poor functional properties. Deamidation is known to improve the solubility of plant proteins, and consequently, to some extent, enhance functional properties, including foaming activity, foaming stability, emulsifying activity, and emulsifying stability. This has been observed on cross-contaminations of several plant protein substrates, including cereal proteins such as oat, wheat, and corn proteins, as well as legume proteins such as soybean and pea proteins, coconut proteins, etc. Negative properties associated with some insoluble proteins, such as sandiness and gritty texture, are being reduced through enzymatic deamidation.

[0043] For example, enzymatic partial deamidation of oat protein makes it substantially completely soluble at neutral pH and also significantly improves emulsifying properties (Zl Jiang et al., J Cereal Science (2015): 64: 126-132). One practical application is the use of protein deamidases in oat milk production methods that produce oat milk with increased protein content, well-suited to meeting the requirements of barista (WO 2014 / 123466). Similarly, when soy protein isolates are enzymatically deamidated, emulsifying and foaming properties are improved (Isuppavorasatit et al., J Agric. Food Chem (2011) 59: 11621-11628). For enzymatically deamidated pea protein isolates, improved solubility, homogeneity, dispersibility, and suspension properties, as well as reduced beany odor, gritty texture, and clumping characteristics have been observed (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, exhibiting significantly improved emulsifying properties (YH Yong et al. J. Agric. Food Chem. (2006): 54: 6034-6040).

[0044] The improved functional properties provided by enzymatic deamidation make protein deamidases highly suitable for many food applications in food products containing plant proteins, such as food analogs with increased protein content, reduced graininess and gritty texture, improved mouthfeel, and Baresta properties, similarly for solutions of yogurt analog portions with improved mouthfeel, texture, and aqueous colloidal substitution. Protein deamidases have also been suggested for improving the texture of plant-based meat analogs and plant-based egg analogs (X Liu et al., Foods [Food] (2022) 11: 440).

[0045] Deamidation of plant proteins also has a positive impact on protein flavor. Plant proteins are associated with various hydrophobic off-flavors (such as lipid oxidation products, for example, with a beany taste), or saponins, phenols, and flavonoids with bitter off-flavors. Enzymatic deamidation of plant proteins reduces their hydrophobicity, thus reducing their affinity for hydrophobic off-flavors. Therefore, protein deamidases can be used to improve the flavor of plant proteins by incorporating enzymes during the recovery of protein concentrates or isolates, or by treating recovered proteins such as protein isolates (X Liu et al., Foods [Food] (2022) 11: 440). For example, flavor improvement has been demonstrated in soybeans (I Suppavorasatit et al., J. Agric. Food Chem [Journal of Agricultural and Food Chemistry] (2012) 60: 7817-7823).

[0046] The application of protein deamidases in protein recycling processes (such as pea protein recycling) has improved protein recycling yields, such as when applied in the recycling process that produces pea protein concentrates and isolates (WO2021049591).

[0047] Protein deamidases also have several applications in dairy proteins and dairy-based foods. Deamidation of whey improves the electrostatic repulsion of proteins, provides better thermal stability, and avoids undesirable aggregation in whey protein solutions (e.g., in protein-fortified beverages) when protein solutions are heat-treated (N Miwa et al. J. Agric. Food Chem [Journal of Agricultural and Food Chemistry] (2013) 61: 2205-2212). Enzymatic deamidation in skim milk significantly improves solubility and viscosity, and provides translucent milky beverages (N Miwa et al. International dairyjournal [International Dairy Journal] (2010) 20: 393-399). The application of protein deamidases in yogurt processing improves stability; they can be used, for example, to replace pectin and other aqueous colloids in drinkable yogurts.

[0048] Protein (glutaminase) deamidases can be used in conjunction with other enzymes, including those that modify or degrade proteins. Combinations between protein glutaminase and protein asparagine deamidase can provide a higher degree of protein deamidation, resulting in even better application performance. Protein deamidases can be used with protein cross-linking enzymes, such as transglutaminase, where the cross-linking of the protein is modified, partly to prevent transglutaminase from reacting with glutamine that has already been converted to glutamate by the deamidase. When the combination of transglutaminase and protein deamidase is used in yogurt production, a texture-enhancing effect is achieved, which can replace added dairy proteins or aqueous colloids, providing a smooth texture to yogurt and avoiding the lumpy texture seen when transglutaminase is used alone. Similar effects have been observed when this enzyme combination is used to produce plant-based yogurt analogs. Furthermore, protein deamidases can be used with proteases, where the resulting protein hydrolysates will have improved solubility, enhanced flavor, and altered functional properties.

[0049] Other embodiments of the present invention include:

[0050] Example 1. A liquid enzyme composition, the liquid enzyme composition comprising: (a) Protein-glutamine glutaminase, and (b) 10-80% w / w of one or more monosaccharides or disaccharides.

[0051] Example 2. A liquid enzyme composition as described in the preceding examples, the liquid enzyme composition comprising 20-10000 IPA(U) / g of the protein-glutamine glutaminase.

[0052] Example 3. A liquid enzyme composition as described in any of the preceding examples, the liquid enzyme composition comprising the protein-glutamine glutaminase in an amount of 40-8000 IPA(U) / g.

[0053] Example 4. A liquid enzyme composition as described in any of the preceding examples, the liquid enzyme composition comprising 60-6000 IPA(U) / g of the protein-glutamine glutaminase.

[0054] Example 5. A liquid enzyme composition as described in any of the preceding examples, the liquid enzyme composition comprising an amount of the protein-glutamine glutaminase in the form of 80-5000 IPA(U) / g.

[0055] Example 6. A liquid enzyme composition as described in any of the preceding examples, the liquid enzyme composition comprising 100-4000 IPA(U) / g of the protein-glutamine glutaminase.

[0056] Example 7. A liquid enzyme composition as described in any of the preceding examples, wherein the liquid enzyme composition comprises 0.01-15% w / w of the amount of the protein-glutamine glutaminase, an active deamidase protein.

[0057] Example 8. A liquid enzyme composition as described in any of the preceding examples, the liquid enzyme composition comprising 0.05-10% w / w of the amount of the protein-glutamine glutaminase, an active deamidase protein.

[0058] Example 9. A liquid enzyme composition as described in any of the preceding examples, wherein the liquid enzyme composition comprises 0.05-5% w / w of the amount of the protein-glutamine glutaminase, an active deamidase protein.

[0059] Example 10. A liquid enzyme composition as described in any of the preceding examples, the liquid enzyme composition comprising 0.1-5% w / w of the amount of the protein-glutamine glutaminase, an active deamidase protein.

[0060] Example 11. A liquid enzyme composition as described in any of the preceding examples, the liquid enzyme composition comprising 0.1-3% w / w of the amount of the protein-glutamine glutaminase, an active deamidase protein.

[0061] Example 12. A liquid enzyme composition as described in any of the preceding examples, the liquid enzyme composition comprising 0.1-2% w / w of the amount of the protein-glutamine glutaminase, an active deamidase protein.

[0062] Example 13. A liquid enzyme composition as described in any of the preceding examples, wherein the protein-glutamine glutaminase belongs to EC 3.5.1.44.

[0063] Example 14. A liquid enzyme composition as described in any of the preceding examples, wherein the protein-glutamine glutaminase has 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% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO: 1.

[0064] Example 15. A liquid enzyme composition as described in any of the preceding examples, wherein the protein-glutamine glutaminase has up to 30 alterations (e.g., substitutions, deletions, and / or insertions) compared to the amino acid sequence of SEQ ID NO: 1, 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, particularly substitutions.

[0065] Example 16. A liquid enzyme composition as described in any of the preceding examples, wherein the liquid enzyme composition comprises 10-75% w / w of the monosaccharide or disaccharide.

[0066] Example 17. A liquid enzyme composition as described in any of the preceding examples, wherein the liquid enzyme composition comprises 10-70% w / w of the monosaccharide or disaccharide.

[0067] Example 18. A liquid enzyme composition as described in any of the preceding examples, wherein the liquid enzyme composition comprises 10-65% w / w of the monosaccharide or disaccharide.

[0068] Example 19. A liquid enzyme composition as described in any of the preceding examples, wherein the liquid enzyme composition comprises 10-60% w / w of the monosaccharide or disaccharide.

[0069] Example 20. A liquid enzyme composition as described in any of the preceding examples, wherein the liquid enzyme composition contains at least 20% w / w of the monosaccharide or disaccharide.

[0070] Example 21. A liquid enzyme composition as described in any of the preceding examples, wherein the liquid enzyme composition contains at least 25% w / w of the monosaccharide or disaccharide.

[0071] Example 22. A liquid enzyme composition as described in any of the preceding examples, wherein the liquid enzyme composition contains at least 30% w / w of the monosaccharide or disaccharide.

[0072] Example 23. A liquid enzyme composition as described in any of the preceding examples, wherein the liquid enzyme composition contains at least 35% w / w of the monosaccharide or disaccharide.

[0073] Example 24. A liquid enzyme composition as described in any of the preceding examples, wherein the monosaccharide or disaccharide is selected from the group consisting of glucose, fructose, galactose, sucrose, lactose, maltose, trehalose, cellobiose, and combinations thereof.

[0074] Example 25. A liquid enzyme composition as described in any of the preceding examples, wherein the monosaccharide or disaccharide is a disaccharide.

[0075] Example 26. A liquid enzyme composition as described in any of the preceding examples, wherein the pH of the composition is at least pH 3.5.

[0076] Example 27. A liquid enzyme composition as described in any of the preceding examples, wherein the pH of the composition is at least pH 4.0.

[0077] Example 28. A liquid enzyme composition as described in any of the preceding examples, wherein the pH of the composition is at most pH 8.0.

[0078] Example 29. A liquid enzyme composition as described in any of the preceding examples, wherein the pH of the composition is in the range of pH 3.5-10.

[0079] Example 30. A liquid enzyme composition as described in any of the preceding examples, wherein the pH of the composition is in the range of pH 4-9.

[0080] Example 31. A liquid enzyme composition as described in any of the preceding examples, wherein the pH of the composition is in the range of pH 4-8.

[0081] Example 32. The liquid enzyme composition according to any one of the foregoing examples, the liquid enzyme composition further comprising one or more polyols selected from the group consisting of: glycerol, ethylene glycol, diethylene glycol, triethylene glycol, propylene glycol, dipropylene glycol, tripropylene glycol, polyethylene glycol (PEG) and sugar alcohols.

[0082] Example 33. A liquid enzyme composition as described in the preceding examples, wherein the liquid enzyme composition comprises an amount of one or more polyols of less than 40% w / w, less than 30% w / w, less than 20% w / w, less than 10% w / w, or less than 5% w / w.

[0083] Example 34. A liquid enzyme composition as described in any of the preceding examples, wherein the liquid enzyme composition further comprises at least 2% w / w salt.

[0084] Example 35. A liquid enzyme composition as described in any of the preceding examples, wherein the liquid enzyme composition contains at least 4% w / w salt.

[0085] Example 36. A liquid enzyme composition as described in any of the preceding examples, wherein the liquid enzyme composition further comprises at least 6% w / w of salt.

[0086] Example 37. A liquid enzyme composition as described in any of the preceding examples, wherein the liquid enzyme composition further comprises at least 8% w / w salt.

[0087] Example 38. A liquid enzyme composition as described in any of the preceding examples, wherein the liquid enzyme composition further comprises up to 20% w / w of salt.

[0088] Example 39. A liquid enzyme composition as described in any of the preceding examples, wherein the liquid enzyme composition further comprises up to 15% w / w of salt.

[0089] Example 40. A liquid enzyme composition as described in any of the preceding examples, wherein the liquid enzyme composition further comprises up to 10% w / w of salt.

[0090] Example 41. A liquid enzyme composition as described in any one of Examples 34-40, 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 their hydrates.

[0091] Example 42. A liquid enzyme composition as described in any one of Examples 34-40, 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 their hydrates.

[0092] Example 43. A liquid enzyme composition as described in any one of Examples 34-40, 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 their hydrates.

[0093] Example 44. A liquid enzyme composition as described in any of the preceding examples, wherein the liquid enzyme composition further comprises a reducing agent.

[0094] Example 45. A liquid enzyme composition as described in any of the preceding examples, wherein the liquid enzyme composition further comprises at least 0.1% w / w of a reducing agent.

[0095] Example 46. A liquid enzyme composition as described in any of the preceding examples, wherein the liquid enzyme composition further comprises at least 0.1-5% w / w of a reducing agent.

[0096] Example 47. A liquid enzyme composition as described in any of the preceding examples, wherein the liquid enzyme composition further comprises a reducing agent in an amount of at least 0.1-2% w / w.

[0097] Example 48. A liquid enzyme composition as described in any of the preceding examples, wherein the liquid enzyme composition further comprises a salt of sulfite, metabisulfite, thiosulfate or ascorbate.

[0098] Example 49. A liquid enzyme composition as described in any of the preceding examples, the liquid enzyme composition further comprising at least 0.1% w / w of a salt of sulfite, metabisulfite, thiosulfate or ascorbate.

[0099] Example 50. A liquid enzyme composition as described in any of the preceding examples, the liquid enzyme composition further comprising at least 0.1-5% w / w of a salt of sulfite, metabisulfite, thiosulfate or ascorbate.

[0100] Example 51. A liquid enzyme composition as described in any of the preceding examples, the liquid enzyme composition further comprising at least 0.1-2% w / w of a salt of sulfite, metabisulfite, thiosulfate or ascorbate.

[0101] Example 52. A method for preparing a liquid enzyme composition as described in any of the preceding examples, the method comprising recovering the protein-glutamine-glutaminase from a microbial fermentation broth; and mixing the protein-glutamine-glutaminase with the monosaccharide or disaccharide.

[0102] Example 53. The method described in the preceding examples, wherein the microbial fermentation broth is a bacterial fermentation broth.

[0103] Example 54. The method described in the preceding examples, wherein the microbial fermentation broth is a bacterial fermentation broth containing species of the genus *Aureobacterium* or *Bacillus*.

[0104] Example 55. The method as described in any of the preceding examples, wherein the recovery includes membrane filtration, such as ultrafiltration.

[0105] Example 56. A method for deamidating glutamine residues in a plant protein or a milk protein, the method comprising contacting the plant protein or the milk protein with a liquid enzyme composition as described in any of the preceding examples.

[0106] Example 57. The method as described in the preceding examples, wherein the plant protein is derived from grains or legumes, and the milk protein is whey protein; more preferably, the plant protein is derived from oats, wheat, corn, soybeans, peas, or almonds.

[0107] Example Chemicals are commodities that are at least reagent grade.

[0108] The deamidase used in this example is derived from *Citrus* species-62563. The amino acid sequence of this deamidase is shown in SEQ ID NO: 1.

[0109] Example 1 Deamidase activity assay The deamidase activity assay consists of two separate parts: 1) Enzymatic steps, in which ammonia is formed through the catalytic action of deamidases; and 2) Non-enzymatic detection step, wherein the ammonia formed in step (1) is derivatized into an indophenol blue compound with maximum absorption at 630 nm.

[0110] In step (1), ammonia is generated through deamidation by a deamidase. In step (2), the generated ammonia reacts with phenol under alkaline conditions to form dioxane. This reaction is catalyzed by sodium pentacyanonitroferro(III) ferric(III) nitrosyl ferrate (sodium nitroprusside). "Colorimetric reagent solution A" contains phenol and sodium nitroprusside. "Colorimetric reagent solution B" provides the alkaline reaction conditions. The intermediate is then oxidized by adding sodium hypochlorite ("Colorimetric reagent solution C") to form indophenol blue. This compound absorbs visible light at 630 nm. Enzyme activity is then calculated using a standard curve.

[0111] Measurement Procedure The enzymatic step involving ammonia formation (1) Reagents: Diluent for assay: 0.2 M sodium phosphate buffer, 0.01% Triton X-100, pH 6.5.

[0112] Assay buffer: Same as above. Used for preparing stock solutions and diluting samples for protein deamidases (hereinafter referred to as "enzymes").

[0113] Substrate solution: 30 mM Z-Gln-Gly (Merck C6154-1G), in the assay dilution (check pH after dissolution).

[0114] Termination solution: 0.4 M TCA.

[0115] Standard: NH4Cl diluted in the assay dilution solution (ammonium standard for IC, Merck 59755-100ML, 1000 mg / L NH4Cl). + (in water) (see also the "Standard Curve" section).

[0116] The enzyme product is dissolved / diluted in the assay buffer and a suitable dilution is prepared to produce a linear assay response.

[0117] Incubation: 1. Add 10 μL of diluted enzyme sample in triplicate to the wells of a 96-well microtiter plate (MTP).

[0118] 2. Add 100 μL of substrate solution to each well.

[0119] 3. For blank samples, add 100 μL of 0.4 M TCA solution.

[0120] 4. Seal the board with a transparent sealing film.

[0121] 5. Incubate the plate at 37°C and 500 rpm for 10 minutes on a thermostatic mixer equipped with a heated lid.

[0122] 6. To terminate the reaction, carefully add 100 μL of 0.4 M TCA solution (except for the blank sample, which already contains TCA).

[0123] Total reaction volume: 210 μL Step (2) Ammonia Detection Step Reagents: Colorimetric reagent A: 4% (w / v) phenol, 0.015% (w / v) sodium pentacyanonitrosylferro(III) ferrate dihydrate (sodium nitroprusside) (Na2[Fe(CN)5NO]∙2H2O).

[0124] Colorimetric reagent B: 5% (w / v) potassium hydroxide.

[0125] Colorimetric reagent C: 28% (w / v) potassium carbonate, 6% (v / v) sodium hypochlorite (Sigma-Aldrich 239305-25 ml, effective Cl2 <5%).

[0126] Incubation: 1. Transfer 15 μL from each well in step (1) to a new 96-well MTP.

[0127] 2. Transfer 45 μL of Milli-Q water into each well.

[0128] 3. Add 30 μL of colorimetric reagent B to each well (on the lab bench, gently shake to mix).

[0129] 4. Add 60 μL of colorimetric reagent A to each well (shake gently by hand on the lab bench to mix).

[0130] 5. Add 60 μL of colorimetric reagent C to each well (shake gently by hand on the lab bench to mix).

[0131] 6. Color development: Carefully seal the plate and place it on the lab bench for 30 minutes.

[0132] 7. Carefully transfer the MTP to the microplate reader and measure the absorbance at 630 nm.

[0133] Total reaction volume: 210 μL Standard curve: Standard stock solution: 1000 mg NH4 + / L.

[0134] In the ammonia detection procedure, a standard curve is prepared by adding ammonium standards at different dilutions to the determination dilution buffer. Specifically, 15 μL of diluted ammonia standard is mixed with 45 μL of water, and then the colorimetric reagents B, A, and C are added in the order given above.

[0135] The amount of enzyme that produces 1 μmol of ammonia per minute at 37°C is defined as 1 unit (indophenol assay unit; IPA(U)): in It can be abbreviated as in

[0136] • C NH4+ It is the ammonia concentration in the reaction solution obtained from the ammonium standard curve (i.e., taking into account the dilution of the pre-diluted ammonium standard solution in the ammonia derivatization step).

[0137] • 18.04 is the molecular weight of ammonium used in the standard solution.

[0138] • V 反应 It is the reaction volume in the pore when ammonia is generated (210 μL).

[0139] • V 酶 This is the volume (10 μL) of enzyme solution added to the well when ammonia is generated.

[0140] • V NH3 The detection volume is the reaction volume in the well when detecting ammonia (210 μL).

[0141] Example 2 Deamidase storage stability I In the first study, the enzymatic stability of several liquid deamidase compositions was evaluated after storage at 40°C for 4 days. All compositions contained approximately 1080 IPA(U) / g of deamidase.

[0142] The composition contains 10% w / w NaCl and 40% w / w glycerol (reference) or 40% w / w sucrose. Adjust the pH of the composition to pH 3.8, pH 4.2, or pH 4.5. Activity loss is defined as the loss of activity relative to sample preparation and incubation. The lower the activity loss, the better the enzyme stability.

[0143] Table 1. Composition and activity loss after incubation at 40°C for 4 days.

[0144] Example 3 Deamidase storage stability II The setup was the same as in Example 1, but the sample was adjusted to pH 4.0 and evaluated after being stored at 50°C or 60°C for 2 days.

[0145] Table 2. Composition and activity loss after 2 days of incubation at pH 4.0.

[0146] Conclusions from Examples 2 and 3 As shown in Tables 1-2, compared with glycerol, the liquid deamidase compositions stabilized with sucrose consistently improved residual deamidase activity after storage.

Claims

1. A liquid enzyme composition, said liquid enzyme composition comprising: (a) Protein-glutamine glutaminase, (b) 10-80% w / w of one or more monosaccharides or disaccharides.

2. The liquid enzyme composition as claimed in the preceding claims, wherein the liquid enzyme composition comprises the protein-glutamine glutaminase in an amount of 20-10000 IPA(U) / g.

3. The liquid enzyme composition as claimed in any of the preceding claims, wherein the protein-glutamine glutaminase belongs to EC 3.5.1.

44.

4. The liquid enzyme composition as claimed in any of the preceding claims, wherein the liquid enzyme composition further comprises at least 10% w / w water.

5. The liquid enzyme composition as claimed in any of the preceding claims, wherein the pH of the composition is 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.

6. The liquid enzyme composition as claimed in any of the preceding claims, wherein the monosaccharide or disaccharide is selected from the group consisting of glucose, fructose, galactose, sucrose, lactose, maltose, trehalose, cellobiose, and combinations thereof.

7. The liquid enzyme composition as claimed in any of the preceding claims, wherein the monosaccharide or disaccharide is a disaccharide; preferably selected from the group consisting of sucrose, lactose, maltose, trehalose, cellobiose, and combinations thereof.

8. The liquid enzyme composition as claimed in any of the preceding claims, wherein the liquid enzyme composition comprises 10-70% w / w of the monosaccharide or disaccharide, preferably 20-70% w / w or 20-60% w / w of the monosaccharide or disaccharide.

9. The liquid enzyme composition as claimed in any of the preceding claims, wherein the liquid enzyme composition further comprises at least 2% w / w salt; preferably at least 4% w / w, at least 6% w / w, or at least 8% w / w salt.

10. The liquid enzyme composition as claimed in any of the preceding claims, wherein the liquid enzyme composition comprises up to 20% w / w of salt, preferably up to 15% w / w or up to 10% w / w of salt.

11. The liquid enzyme composition as claimed in any of the preceding claims, 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 their hydrates.

12. The liquid enzyme composition as claimed in any of the preceding claims, wherein the liquid enzyme composition further comprises less than 40% w / w, preferably less than 30% w / w, less than 20% w / w, or less than 10% w / w of one or more polyols, said one or more polyols being selected from the group consisting of: glycerol, ethylene glycol, diethylene glycol, triethylene glycol, propylene glycol, dipropylene glycol, tripropylene glycol, polyethylene glycol (PEG), and sugar alcohols.

13. A method for preparing a liquid enzyme composition as described in any of the preceding claims, the method comprising recovering the protein-glutamine-glutaminase from a microbial fermentation broth; and mixing the recovered protein-glutamine-glutaminase with the monosaccharide or disaccharide.

14. The method of the preceding claims, wherein the microbial fermentation broth comprises a species of the genus *Aureobacterium* or a species of the genus *Bacillus*; and / or wherein the recovery comprises membrane filtration.

15. A method for deamidating glutamine residues in a plant protein or a milk protein, the method comprising contacting the plant protein or the milk protein with a liquid enzyme composition as described in 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 oats, wheat, corn, soybeans, peas, or almonds.

Images