Stable deamidase compositions

Abstract

The present invention provides deamidase compositions comprising a deamidase inhibitor, wherein the deamidase exhibits less self-deamidation and / or modification to other enzymes 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 deamidase compositions comprising deamidase inhibitors, wherein the deamidase exhibits less self-deamidation and / or modification of other enzymes during and / or after storage. Background Technology

[0003] Deamidases are used in industrial applications as processing aids to modify protein substrates, such as those derived from plants. However, deamidases and other co-formulated enzymes can also modify protein substrates and alter their properties during storage.

[0004] Therefore, the object of the present invention is to provide a deamidase composition that exhibits less enzymatic modification during storage. Summary of the Invention

[0005] In a first aspect, the present invention provides an enzyme composition comprising... (a) Protein-glutamine-glutaminase; and (b) Protein-glutamine-glutaminase inhibitors, which are protein-glutamine-glutaminase inhibitors (i) Has at least 50% amino acid sequence identity with SEQ ID NO: 2 and contains the amino acid sequence motif [IM][LIV][ST]AQ. (ii) Has at least 50% amino acid sequence identity with SEQ ID NO: 3, and contains the amino acid sequence motif G[IM]S[APQ]Q, and / or (iii) Having a TM-score of at least 0.80 compared to the three-dimensional structure of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3, wherein the three-dimensional structure is calculated using AlphaFold; The protein-glutamine glutaminase and the protein-glutamine glutaminase inhibitor are not covalently linked.

[0006] In a second aspect, the present invention provides a method for modifying plant proteins or milk proteins, the method comprising contacting the plant protein or milk protein with the enzyme composition of the present invention.

[0007] Other aspects and embodiments of the invention will be apparent from the description and examples.

[0008] Unless otherwise specified, or if the context clearly indicates otherwise, all percentages are weight percentages (% w / w).

[0009] 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.

[0010] 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.

[0011] sequence SEQ ID NO: 1 From the genus *Citrus* ( Chryseobacterium The amino acid sequence of the deamidase of species-62563.

[0012] SEQ ID NO: 2 : The amino acid sequence of the deamidase precursor peptide from species-62563 of the genus *Citrus*.

[0013] SEQ ID NO: 3 : Amino acid sequence of a deamidase inhibitor from species-62563 of the genus *Citrus*.

[0014] SEQ ID NO: 4 From *Gynostemma pentaphyllum* ( Chryseobacterium proteolyticum The amino acid sequence of the deamidase. Attached Figure Description

[0015] Figure 1 Two superimposed 3D protein models of a deamidase precursor inhibitor of SEQ ID NO: 2 and a deamidase inhibitor of SEQ ID NO: 3. Detailed Implementation

[0016] Deamidase In the context of this invention, the term "deamidase" refers to protein-glutamine glutaminase (also known as glutamylpeptide glutaminase) activity, as described in EC 3.5.1.44, which catalyzes the hydrolysis of the γ-amide of glutamine (e.g., L-glutamylglycine and L-phenylalanyl-L-glutamylglycine) substituted at the carboxyl position or simultaneously at 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. The deamidase active site includes 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 belongs to InterPro entry IPR041325. In a preferred embodiment, the deamidase of the present invention belongs to the PFAM domain PF18626.

[0017] 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.

[0018] 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.

[0019] Deamidase inhibitors The term "deamidase inhibitor" (or "protein-glutamine-glutaminase inhibitor") refers to an amino acid sequence that interacts with the amino acid residues at the active site of a deamidase (see above). Therefore, a deamidase inhibitor reduces or inhibits deamidase activity, preferably the deamidase activity exhibited by the deamidase shown in SEQ ID NO: 1. For example, in the presence of a deamidase inhibitor, deamidase activity can be reduced to less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, or less than 40% (compared to deamidase activity in the absence of a deamidase inhibitor).

[0020] In the embodiments, the deamidase inhibitor comprises the amino acid sequence motif [IM][LIV][ST]AQ, which is characteristic of deamidase inhibitors derived from the deamidase propeptide inhibitory domain.

[0021] In the embodiments, the deamidase inhibitors comprise the amino acid sequence motif G[IM]S[APQ]Q, which is characteristic of the deamidase inhibitors, which are part of the same operon as the deamidase but expressed separately from the deamidase.

[0022] In the embodiments, the deamidase inhibitor comprises an amino acid sequence motif selected from the group consisting of: F[FY][ILV][FQS][EKR]; L[IT]WY[DHKN]; [DHKNS][IL][GV][IV][DE]; [NH][ILMVQ][IV][KRQ][EIQ]; [DN][PS][DE][HKNQR][APS]; and their combinations.

[0023] Sequence identity 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 in 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)

[0024] AlphaFold structural prediction AlphaFold is a computational method for predicting the three-dimensional structure of peptides based on their amino acid sequences (Jumper et al., Highly accurate protein structure prediction with AlphaFold, Nature, 2021). The predicted structures of millions of peptides in the UniProt database are stored in the AlphaFold protein structure database, using the AlphaFold monomer v2.0 model (Varadi et al., AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models, Nucleic Acids Research, 2021). In the AlphaFold protein structure database, the three-dimensional structure of a peptide can be obtained by searching for its UniProt accession number.

[0025] In addition to many publicly available 3D structures, the code can be used to reproduce and predict the structures of novel peptides in source code repositories, such as notebooks / AlphaFold.ipynb at deepmind / alphafold / on Github.com using Alphafold v2.3.1 or later. It can also be found at sokrypton / ColabFold on Github.com using v1.5.2 or later (using AlphaFold2.ipynb). For technical details, see Jumper et al. (see above).

[0026] AlphaFold generates per-residue estimates of its confidence level on a scale of 0 to 100. This confidence level metric is called pLDDT and corresponds to the model's prediction score on the lDDT-Cα index. It is stored in the B-factor field of the downloadable mmCIF and PDB files (although, unlike the B-factor, a higher pLDDT score is better). Regions with pLDDT scores above 90 are expected to be modeled with high accuracy. These should be suitable for any application that benefits from high accuracy (e.g., characterization of binding sites). Regions with pLDDT scores between 70 and 90 are expected to be well-modeled, corresponding to generally good main-chain predictions.

[0027] Structural similarity The correlation between two amino acid sequences is typically described by the parameter "sequence identity" (see above). However, since the biological function of a polypeptide is defined by its three-dimensional structure rather than its amino acid sequence, a better approach to assess the functional relationship between polypeptides is to compare their three-dimensional structures. Therefore, for the purposes of this invention, the correlation between the three-dimensional structures of two polypeptides is described by the parameter "structural similarity".

[0028] The three-dimensional structure of any polypeptide can be obtained experimentally, for example by X-ray crystallography or using computational methods such as AlphaFold (see above). The structural similarity between the three-dimensional structures can then be determined by a TM-score, which is calculated using the following general formula (Zhang and Skolnick, Proteins 57:702–710, 2004): -in L N It is the length of the natural structure. L T It is the length of the residues compared to the template structure. d i It is the first i Compare the distance between the paired residues. d 0 represents the scale of the standardized matching difference. “Max” represents the maximum value after optimal spatial superposition.

[0029] For the purposes of this invention, L N Always referencing the protein length indicates the use of a fixed reference length. L To prevent artificially large TM-scores in substructure alignment: Before the TM-score can be calculated, structural alignment of the three-dimensional structures of the two peptides is necessary. This is achieved by optimizing the structural overlap algorithm, and several methods are available, such as CEalign (Shindyalov and Bourne, ProteinEng., 11, 739-747, 1998), DALI (Holm and Sander, Trends Biochem., 20, 478-480, 1995), or TM-align (Nucleic Acids Res. 33:2302-2309, 2005).

[0030] For the purposes of this invention, TM-align is applied. For convenience, the TM-score is integrated into the TM-align software, which is available from the authors' website. The version of TM-align is preferably the latest version released on August 22, 2019, or later, and the TM-score between the reference protein and the query protein is determined by running the following command: TMalign<query.pdb><reference.pdb> -L<length of reference> -in<query.pdb> It is the name of the PDB file containing the coordinates of the queried peptide.<reference.pdb> This is the name of the PDB file containing the coordinates of the reference peptide. The TM-score is calculated and reported in the output, along with several other parameters from the alignment.

[0031] Enzyme composition Deamidases (protein-glutamine-glutaminases) are protein-modifying enzymes that convert glutamine residues into glutamate. This alters the protein's charge and can lead to changes in solubility or other modified physicochemical properties.

[0032] Since deamidases and other potentially co-formulated enzymes are also proteins, deamidase activity can lead to self-deamidation and / or modification of these other enzymes. Enzymes are known to be sensitive to modifications of amino acid residues, and the deamidation of glutamine residues to glutamate can significantly alter enzyme properties (lower isoelectric point (pI) and / or altered solubility / colloidal stability / aggregation) or even lead to loss of enzyme activity. This is particularly relevant to compositions in which the concentration of deamidase is high and storage time is extended (such as industrial enzyme product compositions).

[0033] The adverse effects of high deamidase concentrations can be mitigated by reversibly inhibiting deamidases with deamidase inhibitors. When deamidase products are used in applications, according to Le Chatelier's principle, product dilution facilitates the release of the inhibitor. The separation of the deamidase and the inhibitor leads to the reactivation of the deamidase in the application.

[0034] Two groups of deamidase inhibitors have evolved that are structurally similar but have considerably different amino acid sequences. One group of inhibitors is derived from the deamidase propeptide domain (see, for example, SEQ ID NO: 2), while the other group consists of a separate set of inhibitors expressed by the same operon as the deamidase (see, for example, SEQ ID NO: 3). Each group of inhibitors contains a characteristic amino acid sequence motif that forms part of a loop that directly interacts with the active site of the mature deamidase. The unexpected structural similarity between the two groups of inhibitors can be represented using a “TM-score”.

[0035] Therefore, the present invention provides an enzyme composition comprising... (a) Protein-glutamine-glutaminase; and (b) Protein-glutamine-glutaminase inhibitors, which are protein-glutamine-glutaminase inhibitors (i) Has at least 50% amino acid sequence identity with SEQ ID NO: 2 and contains the amino acid sequence motif [IM][LIV][ST]AQ. (ii) Has at least 50% amino acid sequence identity with SEQ ID NO: 3, and contains the amino acid sequence motif G[IM]S[APQ]Q, and / or (iii) Having a TM-score of at least 0.80 compared to the three-dimensional structure of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3, wherein the three-dimensional structure is calculated using AlphaFold; The protein-glutamine glutaminase and the protein-glutamine glutaminase inhibitor are not covalently linked.

[0036] In the embodiments, the composition comprises a deamidase (protein-glutamine-glutaminase) in an amount of 10-10000 IPA(U) / g; preferably 20-8000 IPA(U) / g, 30-6000 IPA(U) / g, 40-5000 IPA(U) / g, or 50-4000 IPA(U) / g. The amount of deamidase can also be expressed as "active (deamidase) enzyme protein"; therefore, the enzyme composition may comprise 0.01-15% w / w of active deamidase protein (protein-glutamine-glutaminase); preferably 0.05-10% w / w, 0.1-5% w / w, or 0.1-3% w / w of active deamidase protein.

[0037] In embodiments, the composition further comprises a hydrolytic enzyme selected from the group consisting of: proteases, amylases, pectins, cellulases, hemicellulases, xylanases, mannanases, dextranases, and combinations thereof. The composition may also comprise non-hydrolytic enzymes, such as transglutaminases or oxidoreductases.

[0038] In the examples, the pH of a 1% w / w solution of the composition is in the range of pH 4-8.

[0039] In the embodiments, the molar ratio of protein to glutamine-glutaminase divided by protein to glutamine-glutaminase inhibitor is less than 1000:1, preferably less than 500:1 or less than 100:1. Preferably, the molar ratio of protein to glutamine-glutaminase divided by protein to glutamine-glutaminase inhibitor is greater than 1:10 or greater than 1:1.

[0040] The enzyme composition may further contain a reducing agent to prevent oxidation of cysteine ​​residues at the active site and to maintain deamidase activity during storage. The reducing agent may be, for example, a salt of sulfite, metabisulfite, thiosulfate, or ascorbate. The 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.

[0041] In the examples, the composition is a liquid composition.

[0042] The enzyme composition may contain 20-80% w / w of one or more polyols, such as 20-75% w / w, 20-70% w / w, 20-65% w / w, or 20-60% w / w of one or more polyols.

[0043] In the examples, the composition comprises at least 25% w / w, preferably at least 30% w / w, at least 35% w / w, or at least 40% w / w of one or more polyols. The composition may be substantially free of polyols other than glycerol and one or more sugar alcohols.

[0044] The polyol (polyol or polyhydric alcohol) according to the present invention is an alcohol having two or more hydroxyl groups. The polyol may have a molecular weight of less 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), and sugar alcohols. PEG can 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.

[0046] Polyols also include sugar polyols, such as monosaccharides and disaccharides, such as glucose, fructose, galactose, sucrose, lactose, maltose, and trehalose.

[0047] The present invention also provides a method for modifying plant proteins or milk proteins, the method comprising contacting the plant protein or milk protein with an enzyme composition as described above. Preferably, the plant protein is derived from cereals or legumes; more preferably, the plant protein is derived from oats, wheat, corn, soybeans, peas, or almonds. Preferably, the milk protein is whey protein.

[0048] In the examples, the modification is the deamidation of glutamine residues in plant proteins or glutamine residues in milk proteins.

[0049] Deamidase The deamidase (protein-glutamine-glutaminase) included in the enzyme composition of the present invention is typically produced through microbial fermentation and subsequent recovery processes. The subsequent 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. Such a propeptide 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 *Chlorella* and the genus *Bacillus*. Bacillus ) species.

[0050] 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 formulations of the present invention in a process comprising mixing the concentrated deamidase solution with a polyol (such as glycerol) and a salt, and optionally evaporating some water. The water in the concentrated deamidase solution can also be evaporated before adding the polyol / salt.

[0051] 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 formulations of the present invention in a process comprising mixing the deamidase powder with water and a polyol, and optionally other components such as one or more salts.

[0052] Compared with the amino acid sequence of SEQ ID NO: 1, the deamidase contained in the 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.

[0053] 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.

[0054] As described above, structural similarity can be represented using a “TM-score”. Therefore, compared to the three-dimensional structure of the amino acid sequence of SEQ ID NO: 1, the deamidase can have a TM-score of at least 0.80, preferably at least 0.81, at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, where the three-dimensional structure is calculated using AlphaFold.

[0055] Deamidase inhibitors The deamidase inhibitor (protein-glutamine-glutaminase inhibitor) included in the enzyme composition of the present invention is not covalently linked to the deamidase.

[0056] A group of known deamidase inhibitors are derived from deamidase propeptides, such as those described in PCT / EP2023 / 055936. Exemplary propeptide inhibitors are 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).

[0057] Another group of deamidase inhibitors are essentially expressed separately from the deamidases, as described, for example, in PCT / EP2023 / 068212. Exemplary inhibitors from this group are shown in SEQ ID NO: 3, 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).

[0058] Although the two groups of inhibitors mentioned above have different amino acid sequences, they are very similar in structure, such as... Figure 1 The two superimposed 3D protein models are shown (the "propeptide inhibitor" of SEQ ID NO: 2 is in blue, and the "inhibitor alone" of SEQ ID NO: 3 is in yellow). Figure 1 It was prepared using the “super” command from PyMOL, which is specifically recommended for cases with low sequence similarity but high structural similarity. It performs a structure-based stacking and reports an all-atom RMSD of RMSD = 1.178 Ångstrøm (based on 3D coordinates of 360 atoms in this case) between the “propeptide inhibitor” and the “inhibitor alone”, indicating that the structures are indeed highly similar.

[0059] Both inhibitors contain an inhibitory loop that directly interacts with the Cys-His-Asp catalytic triplet at the deamidase active site, but despite this shared function, the characteristic amino acid sequence motifs of the loops differ slightly. The "propeptide inhibitor" (exemplified by SEQ ID NO: 2) contains the amino acid sequence motif [IM][LIV][ST]AQ, while the "single inhibitor" (exemplified by SEQ ID NO: 3) contains the amino acid sequence motif G[IM]S[APQ]Q.

[0060] Therefore, the enzyme composition of the present invention comprises a deamidase inhibitor that may have at least 60% amino acid sequence identity with SEQ ID NO: 2 and contain the amino acid sequence motif [IM][LIV][ST]AQ, or may have at least 60% amino acid sequence identity with SEQ ID NO: 3 and contain the amino acid sequence motif G[IM]S[APQ]Q.

[0061] Compared with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3, the deamidase inhibitor contained in the 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.

[0062] Alternatively, the deamidase inhibitor may have up to 30 alterations (e.g., substitutions, deletions, and / or insertions) compared to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3, 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.

[0063] As described above, structural similarity can be represented using a “TM-score”. Therefore, compared to the three-dimensional structure of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3, the deamidase inhibitor can have a TM-score of at least 0.80, preferably at least 0.81, at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, where the three-dimensional structure is calculated using AlphaFold.

[0064] As mentioned above, both deamidases and deamidase inhibitors can have minor amino acid alterations, 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).

[0065] 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 comparisons 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).

[0066] 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, DNA 7:127).

[0067] use Deamidases can be applied to almost all types of proteins (plant proteins, animal proteins, yeast 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 affinity for flavor and off-flavors, improve gelling properties, and increase thermal stability. Enzymatically modified proteins can be used as ingredients in various foods and beverages, or protein deamidases can be directly applied in food production processes, such as in yogurt fermentation.

[0068] Plant proteins typically exhibit low solubility and low functional properties. Deamidation is known to improve the solubility of plant proteins, and consequently, in part, improve functional properties, including foaming activity, foaming stability, emulsifying activity, and emulsifying stability. This has been observed in cross-contaminations with several plant protein substrates, including cereal proteins such as oat, wheat, and corn proteins, legume proteins such as soybean and pea proteins, and coconut and almond proteins. Negative properties associated with some insoluble proteins, such as sandiness and gritty texture, are being reduced through enzymatic deamidation.

[0069] 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, which produces 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 and exhibits significantly improved emulsifying properties (YH Yong et al. J. Agric. Food Chem. (2006): 54: 6034-6040).

[0070] 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; and similarly for solutions in 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).

[0071] 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).

[0072] 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).

[0073] 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

[2013] 61: 2205-2212). Enzymatic deamidation in skim milk significantly improves solubility and viscosity, and provides translucent milky beverages (N Miwa et al., 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.

[0074] 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.

[0075] Other embodiments of the present invention include:

[0076] Example 1. An enzyme composition comprising... (a) Protein-glutamine-glutaminase; and (b) Protein-glutamine-glutaminase inhibitors, which are protein-glutamine-glutaminase inhibitors (i) Has at least 50% amino acid sequence identity with SEQ ID NO: 2 and contains the amino acid sequence motif [IM][LIV][ST]AQ. (ii) Has at least 50% amino acid sequence identity with SEQ ID NO: 3, and contains the amino acid sequence motif G[IM]S[APQ]Q, and / or (iii) Having a TM-score of at least 0.80 compared to the three-dimensional structure of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3, wherein the three-dimensional structure is calculated using AlphaFold; The protein-glutamine glutaminase and the protein-glutamine glutaminase inhibitor are not covalently linked.

[0077] Example 2. An enzyme composition as described in the preceding examples, comprising 10-10000 IPA(U) / g of the protein-glutamine glutaminase.

[0078] Example 3. An enzyme composition as described in any of the preceding examples, comprising the protein-glutamine glutaminase in an amount of 20-8000 IPA(U) / g.

[0079] Example 4. An enzyme composition as described in any of the preceding examples, comprising the protein-glutamine glutaminase in an amount of 30-6000 IPA(U) / g.

[0080] Example 5. An enzyme composition as described in any of the preceding examples, wherein the enzyme composition comprises 40-5000 IPA(U) / g of the protein-glutamine glutaminase.

[0081] Example 6. An enzyme composition as described in any of the preceding examples, wherein the enzyme composition comprises 50-4000 IPA(U) / g of the protein-glutamine glutaminase.

[0082] Example 7. An enzyme composition as described in any of the preceding examples, wherein the enzyme composition comprises 0.01-15% w / w of the amount of the protein-glutamine glutaminase, an active deamidase protein.

[0083] Example 8. An enzyme composition as described in any of the preceding examples, wherein the enzyme composition comprises 0.05-10% w / w of the amount of the protein-glutamine glutaminase, an active deamidase protein.

[0084] Example 9. An enzyme composition as described in any of the preceding examples, wherein the enzyme composition comprises 0.05-5% w / w of the amount of the protein-glutamine glutaminase, an active deamidase protein.

[0085] Example 10. An enzyme composition as described in any of the preceding examples, wherein the enzyme composition comprises 0.1-5% w / w of the amount of the protein-glutamine glutaminase, an active deamidase protein.

[0086] Example 11. An enzyme composition as described in any of the preceding examples, wherein the enzyme composition comprises 0.1-3% w / w of the amount of the protein-glutamine glutaminase, an active deamidase protein.

[0087] Example 12. An enzyme composition as described in any of the preceding examples, wherein the enzyme composition comprises 0.1-2% w / w of the amount of the protein-glutamine glutaminase, an active deamidase protein.

[0088] Example 13. An enzyme composition as described in any of the preceding examples, wherein the protein-glutamine glutaminase belongs to EC 3.5.1.44.

[0089] Example 14. An 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.

[0090] Example 15. An 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), 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, compared to the amino acid sequence of SEQ ID NO: 1.

[0091] Example 16. An enzyme composition as described in any of the preceding examples, wherein the protein-glutamine glutaminase has a TM-score of at least 0.80, preferably at least 0.81, at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0 compared to the three-dimensional structure of the amino acid sequence of SEQ ID NO: 1, wherein the three-dimensional structure is calculated using AlphaFold.

[0092] Example 17. An enzyme composition as described in any of the preceding examples, wherein the protein-glutamine glutaminase inhibitor 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: 2 or SEQ ID NO: 3.

[0093] Example 18. An enzyme composition as described in any of the preceding examples, wherein the protein-glutamine glutaminase inhibitor 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, particularly substitutions, compared to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3.

[0094] Example 19. An enzyme composition as described in any of the preceding examples, wherein the protein-glutamine glutaminase inhibitor has a TM-score of at least 0.80, preferably at least 0.81, at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0 compared to the three-dimensional structure of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3, wherein the three-dimensional structure is calculated using AlphaFold.

[0095] Example 20. An enzyme composition as described in any of the preceding examples, wherein the enzyme composition is a liquid composition.

[0096] Example 21. An enzyme composition as described in any of the preceding examples, wherein the enzyme composition further comprises at least 10% w / w water.

[0097] Example 22. An enzyme composition as described in any of the preceding examples, wherein the enzyme composition further comprises at least 20% w / w water.

[0098] Example 23. An enzyme composition as described in any of the preceding examples, wherein the enzyme composition further comprises at least 30% w / w water.

[0099] Example 24. An enzyme composition as described in any of the preceding examples, the enzyme composition further comprising at least 20% w / w of a polyol and at least 10% w / w of water.

[0100] Example 25. An enzyme composition as described in any of the preceding examples, wherein the enzyme composition comprises 20-80% w / w of a polyol, preferably 20-60% w / w of a polyol.

[0101] Example 26. An enzyme composition as described in any of the preceding examples, wherein the polyol is selected from the group consisting of glycerol, sugar alcohols, monosaccharides or disaccharides, and combinations thereof.

[0102] Example 27. An enzyme composition as described in any of the preceding examples, wherein the enzyme composition comprises 20-75% w / w of the polyol.

[0103] Example 28. An enzyme composition as described in any of the preceding examples, wherein the enzyme composition comprises 20-70% w / w of the polyol.

[0104] Example 29. An enzyme composition as described in any of the preceding examples, wherein the enzyme composition comprises 20-65% w / w of the polyol.

[0105] Example 30. An enzyme composition as described in any of the preceding examples, wherein the enzyme composition comprises 20-60% w / w of the polyol.

[0106] Example 31. An enzyme composition as described in any of the preceding examples, wherein the enzyme composition comprises at least 25% w / w of the polyol.

[0107] Example 32. An enzyme composition as described in any of the preceding examples, wherein the enzyme composition comprises at least 30% w / w of the polyol.

[0108] Example 33. An enzyme composition as described in any of the preceding examples, wherein the enzyme composition comprises at least 35% w / w of the polyol.

[0109] Example 34. An enzyme composition as described in any of the preceding examples, wherein the enzyme composition comprises at least 40% w / w of the polyol.

[0110] Example 35. An enzyme composition as described in any of the preceding examples, wherein the enzyme composition is substantially free of polyols other than glycerol and one or more sugar alcohols.

[0111] Example 36. An enzyme composition as described in any of the preceding examples, wherein the pH of the composition is below pH 8.

[0112] Example 37. An enzyme composition as described in any of the preceding examples, wherein the pH of the composition is below pH 7.

[0113] Example 38. An enzyme composition as described in any of the preceding examples, wherein the polyol comprises or is composed of a non-sugar polyol.

[0114] Example 39. An enzyme composition as described in any of the preceding examples, wherein the polyol comprises or is composed of a non-sugar polyol 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.

[0115] Example 40. An enzyme composition as described in any of the preceding examples, wherein the polyol comprises, or is composed of, polyethylene glycol with an average molecular weight of 500 or less.

[0116] Example 41. An enzyme composition as described in any of the preceding examples, wherein the polyol comprises or is composed of a sugar alcohol selected from the group consisting of: sorbitol, mannitol, erythritol, galactitol, inositol, xylitol, ribitol, isomaltitol, and maltitol.

[0117] Example 42. An enzyme composition as described in any of the preceding examples, wherein the polyol comprises or consists of one or more monosaccharides or disaccharides.

[0118] Example 43. An enzyme composition as described in any of the preceding examples, wherein the polyol comprises or is composed of a monosaccharide or disaccharide selected from the group consisting of glucose, fructose, galactose, sucrose, lactose, maltose, and trehalose.

[0119] Example 44. An enzyme composition as described in any of the preceding examples, wherein the pH of a 1% w / w solution of the composition is in the range of pH 4-9.

[0120] Example 45. An enzyme composition as described in any of the preceding examples, wherein the pH of a 1% w / w solution of the composition is in the range of pH 5-8.

[0121] Example 46. An enzyme composition as described in any of the preceding examples, wherein the molar ratio of the protein-glutamine glutaminase to the protein-glutamine glutaminase inhibitor is less than 1000:1.

[0122] Example 47. An enzyme composition as described in any of the preceding examples, wherein the molar ratio of the protein-glutamine glutaminase to the protein-glutamine glutaminase inhibitor is less than 500:1.

[0123] Example 48. An enzyme composition as described in any of the preceding examples, wherein the molar ratio of the protein-glutamine glutaminase to the protein-glutamine glutaminase inhibitor is less than 100:1.

[0124] Example 49. An enzyme composition as described in any of the preceding examples, wherein the molar ratio of the protein-glutamine glutaminase to the protein-glutamine glutaminase inhibitor is greater than 1:10.

[0125] Example 50. An enzyme composition as described in any of the preceding examples, wherein the molar ratio of the protein-glutamine glutaminase to the protein-glutamine glutaminase inhibitor is greater than 1:1.

[0126] Example 51. An enzyme composition as described in any of the preceding examples, the enzyme composition further comprising an enzyme selected from the group consisting of: hydrolases, oxidoreductases and transglutaminases.

[0127] Example 52. An enzyme composition as described in any of the preceding examples, the enzyme composition further comprising a hydrolase selected from the group consisting of: proteases, amylases, pectinases, cellulases, hemicellulases, xylanases, mannanases, dextranases, and combinations thereof.

[0128] Example 53. An enzyme composition as described in any of the preceding examples, wherein the enzyme composition further comprises transglutaminase.

[0129] Example 54. An enzyme composition as described in any of the preceding examples, wherein the enzyme composition further comprises a protease.

[0130] Example 55. An enzyme composition as described in any of the preceding examples, wherein the enzyme composition further comprises a reducing agent.

[0131] Example 56. An enzyme composition as described in any of the preceding examples, wherein the enzyme composition further comprises at least 0.1% w / w of a reducing agent.

[0132] Example 57. An enzyme composition as described in any of the preceding examples, wherein the enzyme composition further comprises at least 0.1-5% w / w of a reducing agent.

[0133] Example 58. An enzyme composition as described in any of the preceding examples, wherein the enzyme composition further comprises at least 0.1-2% w / w of a reducing agent.

[0134] Example 59. An enzyme composition as described in any of the preceding examples, wherein the enzyme composition further comprises a salt of sulfite, metabisulfite, thiosulfate, or ascorbate.

[0135] Example 60. An enzyme composition as described in any of the preceding examples, the enzyme composition further comprising at least 0.1% w / w of a salt of sulfite, metabisulfite, thiosulfate or ascorbate.

[0136] Example 61. An enzyme composition as described in any of the preceding examples, the enzyme composition further comprising at least 0.1-5% w / w of a salt of sulfite, metabisulfite, thiosulfate or ascorbate.

[0137] Example 62. An enzyme composition as described in any of the preceding examples, wherein the enzyme composition further comprises at least 0.1-2% w / w of a salt of sulfite, metabisulfite, thiosulfate or ascorbate.

[0138] Example 63. A method for modifying plant protein or milk protein, the method comprising contacting the plant protein or milk protein with an enzyme composition as described in any of the preceding examples.

[0139] Example 64. 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.

[0140] Example 65. The method as described in the preceding examples, wherein the plant protein is derived from oats, wheat, corn, soybeans, peas, or almonds.

[0141] Example 66. The method as described in any of the preceding examples, wherein the modification is a deamidation of glutamine residues in plant proteins or glutamine residues in milk proteins.

[0142] Example Chemicals are commodities that are at least reagent grade.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] Assay buffer: Same as above. Used for preparing stock solutions and diluting samples for protein deamidases (hereinafter referred to as "enzymes").

[0148] Substrate solution: 30 mM Z-Gln-Gly (Merck C6154-1G), in the assay dilution (check pH after dissolution).

[0149] Termination solution: 0.4 M TCA.

[0150] 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).

[0151] The enzyme product is dissolved / diluted in the assay buffer and a suitable dilution is prepared to produce a linear assay response.

[0152] Incubation:

[0153] 1. Add 10 μL of diluted enzyme sample in triplicate to the wells of a 96-well microtiter plate (MTP).

[0154] 2. Add 100 μL of substrate solution to each well.

[0155] 3. For blank samples, add 100 μL of 0.4 M TCA solution.

[0156] 4. Seal the board with a transparent sealing film.

[0157] 5. Incubate the plate at 37°C and 500 rpm for 10 minutes on a thermostatic mixer equipped with a heated lid.

[0158] 6. To terminate the reaction, carefully add 100 μL of 0.4 M TCA solution (except for the blank sample, which already contains TCA).

[0159] Total reaction volume: 210 μL

[0160] 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).

[0161] Colorimetric reagent B: 5% (w / v) potassium hydroxide.

[0162] Colorimetric reagent C: 28% (w / v) potassium carbonate, 6% (v / v) sodium hypochlorite (Sigma-Aldrich 239305-25 ml, effective Cl2 <5%).

[0163] Incubation:

[0164] 1. Transfer 15 μL from each well in step (1) to a new 96-well MTP.

[0165] 2. Transfer 45 μL of Milli-Q water into each well.

[0166] 3. Add 30 μL of colorimetric reagent B to each well (on the lab bench, gently shake to mix).

[0167] 4. Add 60 μL of colorimetric reagent A to each well (shake gently by hand on the lab bench to mix).

[0168] 5. Add 60 μL of colorimetric reagent C to each well (shake gently by hand on the lab bench to mix).

[0169] 6. Color development: Carefully seal the plate and place it on the lab bench for 30 minutes.

[0170] 7. Carefully transfer the MTP to the microplate reader and measure the absorbance at 630 nm.

[0171] Total reaction volume: 210 μL Standard curve: Standard stock solution: 1000 mg NH4 + / L.

[0172] 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.

[0173] 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

[0174] • 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).

[0175] • 18.04 is the molecular weight of ammonium used in the standard solution.

[0176] • V 反应 It is the reaction volume in the pore when ammonia is generated (210 μL).

[0177] • V 酶 This is the volume (10 μL) of enzyme solution added to the well when ammonia is generated.

[0178] • V NH3 The detection volume is the reaction volume in the well when detecting ammonia (210 μL).

[0179] Example 2 Nano differential scanning fluorometry (nanoDSF) – pyrolysis folding temperature The conformational stability of the deamidase / inhibitor complex (using the deamidase inhibitor of this invention) was assessed using nano-differential scanning fluorescence (nanoDSF). These molecules were exposed to a temperature gradient, as shown below. The resulting structural changes are reflected in the changes in fluorescence intensity, providing a measure of temperature stability. The binding of the deamidase inhibitor to the deamidase contributes to molecular stability; therefore, the nanoDSF pyrolysis folding temperature also provides information about the deamidase / inhibitor binding affinity.

[0180] The purified sample was received in a buffer solution containing: 20 mM sodium phosphate; 500 mM sodium chloride; 500 mM imidazole; pH 7.4.

[0181] 60 µL of sample was repeatedly transferred to a black-backed 384-well plate, and the plate was briefly centrifuged to remove potential air bubbles.

[0182] The pyrolysis folding temperature of the samples was analyzed using a Prometheus NT.Plex system from NanoTemper Technologies GmbH with the following settings: (i) Temperature scan rate: 3.3 °C / min; and (ii) Temperature scan interval: 20℃-95℃.

[0183] During operation, the sample was loaded into a capillary (Prometheus NT.Plex – capillary action sheet, standard, catalog number PR-AC002) before being subjected to a temperature gradient. Data generated were analyzed using PR.Stability Analysis v.1.0.1 software. The first derivative of the 330 nm trace was used to correlate with the midpoint folding temperature. T m Annotate at (℃) (alternatively, a ratio of 350 nm / 330 nm can be used). In some cases, more than one can be observed. T m In these cases, the main peak in the first derivative trace at 330 nm is selected as the sample's... T m .

[0184] Example 3 Thermolytic folding temperature of deamidase / inhibitor complex Using the nanoDSF program with minor modifications as described in Example 2, the pyrolysis folding temperatures of the active deamidase, the corresponding deamidase / inhibitor, and the deamidase / propeptide complex were measured.

[0185] As shown in Table 1, both the propeptide and the inhibitor increased the folding temperature and conformational stability. This may indicate the formation of the deamidase / inhibitor complex and a decrease in deamidase activity.

[0186] Table 1. NanoDSF pyrolysis folding temperatures of peptides.

[0187] Example 4 Inhibition of deamidase activity Two different active deamidases from *Cryptospira* species-62563 (shown as SEQ ID NO: 1) and *Cryptospira utilis* (shown as SEQ ID NO: 4) were each incubated with skim milk at 55°C for 30 minutes. The skim milk was pre-heat-treated at 90°C for 10 minutes. Deamidase activity was measured in mg ammonium / L (see also Example 1).

[0188] Each experiment was repeated twice, but this time the deamidase was supplemented with either the propeptide of SEQ ID NO: 2 or the inhibitor of SEQ ID NO: 3.

[0189] As shown in Table 2, both the propeptide and the inhibitor effectively reduced the deamidase activity of both deamidases.

[0190] Table 2. Deamidase activity measured in mg ammonium / L. .

Claims

1. An enzyme composition comprising... (a) Protein-glutamine-glutaminase; and (b) A protein-glutamine-glutaminase inhibitor, wherein the protein-glutamine-glutaminase inhibitor (i) Has at least 50% amino acid sequence identity with SEQ ID NO: 2 and contains the amino acid sequence motif [IM][LIV][ST]AQ. (ii) Has at least 50% amino acid sequence identity with SEQ ID NO: 3, and contains the amino acid sequence motif G[IM]S[APQ]Q, and / or (iii) Having a TM-score of at least 0.80 compared to the three-dimensional structure of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3, wherein the three-dimensional structure is calculated using AlphaFold; The protein-glutamine glutaminase and the protein-glutamine glutaminase inhibitor are not covalently linked.

2. The enzyme composition as claimed in the preceding claims, wherein the enzyme composition comprises the protein-glutamine glutaminase in an amount of 10-10000 IPA(U) / g.

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

44.

4. The enzyme composition according to any one of the preceding claims, wherein the protein-glutamine glutaminase has at least 80%, preferably at least 90% or at least 95% amino acid sequence identity with the amino acid sequence of SEQ ID NO:

1.

5. The enzyme composition as claimed in any of the preceding claims, wherein the protein-glutamine glutaminase inhibitor has at least 60%, preferably at least 70%, at least 80%, or at least 90% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:

3.

6. The enzyme composition as claimed in any of the preceding claims, wherein the protein-glutamine glutaminase inhibitor has a TM-score of at least 0.90, preferably at least 0.95, at least 0.98, or at least 0.99 compared to the three-dimensional structure of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3, wherein the three-dimensional structure is calculated using AlphaFold.

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

8. The enzyme composition as claimed in the preceding claims, wherein the enzyme composition further comprises at least 20% w / w of a polyol and at least 10% w / w of water.

9. The enzyme composition as claimed in the preceding claims, wherein the enzyme composition comprises 20-80% w / w of a polyol, preferably 20-60% w / w of a polyol.

10. The enzyme composition of the preceding claims, wherein the polyol is selected from the group consisting of glycerol, sugar alcohols, monosaccharides or disaccharides, and combinations thereof.

11. The enzyme composition as claimed in any of the preceding claims, wherein the pH of a 1% w / w solution of the composition is in the range of pH 4-8.

12. The enzyme composition according to any one of the preceding claims, wherein the molar ratio of the protein-glutamine glutaminase to the protein-glutamine glutaminase inhibitor is less than 1000:1; preferably less than 100:

1.

13. The enzyme composition of any one of the preceding claims, wherein the enzyme composition further comprises transglutaminase or a hydrolase selected from the group consisting of: protease, amylase, pectinase, cellulase, hemicellulase, xylanase, mannanase, glucanase, and combinations thereof.

14. A method for modifying plant protein or milk protein, the method comprising contacting the plant protein or milk protein with an 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.

15. The method of the preceding claims, wherein the modification is a deamidation of glutamine residues in plant proteins or glutamine residues in milk proteins.

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