WO2025056701A1

WO2025056701A1 - Stabilized deamidase compositions

Info

Publication number: WO2025056701A1
Application number: PCT/EP2024/075535
Authority: WO
Inventors: Allan Noergaard; Marc Dominique Morant
Original assignee: Novozymes AS
Current assignee: Novozymes AS
Priority date: 2023-09-13
Filing date: 2024-09-12
Publication date: 2025-03-20
Anticipated expiration: 2026-03-13
Also published as: AU2024339471A1

Abstract

The invention provides deamidase compositions comprising a deamidase inhibitor, where the deamidase exhibit less self-deamidation and/or modification of other enzymes after storage.

Description

STABILIZED DEAMIDASE COMPOSITIONS

Reference to a Sequence Listing

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

FIELD OF THE INVENTION

The present invention relates to deamidase compositions comprising a deamidase inhibitor, where the deamidase exhibit less self-deamidation and/or modification of other enzymes during and/or after storage.

BACKGROUND

Deamidase enzymes are used as processing aids in industrial applications for modifying protein substrates, for example from plants. However, deamidases and other co-formulated enzymes are also protein substrates that during storage may suffer from modification and a corresponding change of properties.

Thus, the object of the present invention is to provide deamidase compositions exhibiting less enzyme modification during storage.

SUMMARY OF THE INVENTION

The present invention provides, in a first aspect, an enzyme composition, comprising

(a) a protein-glutamine glutaminase; and

(b) a protein-glutamine glutaminase inhibitor, which

(i) has at least 50% amino acid sequence identity to SEQ ID NO: 2 and comprises the amino acid sequence motif [IM][LIV][ST]AQ,

(ii) has at least 50% amino acid sequence identity to SEQ ID NO: 3 and comprises the amino acid sequence motif G[IM]S[APQ]Q, and/or

(iii) has 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, where the three-dimensional structures are calculated using AlphaFold; wherein the protein-glutamine glutaminase and the protein-glutamine glutaminase inhibitor are not covalently linked.

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

Other aspects and embodiments of the invention are apparent from the description and examples. Unless otherwise indicated, or if it is apparent from the context that something else is meant, all percentages are percentage by weight (% w/w).

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

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

Sequences

SEQ ID NO: 1: Amino acid sequence of a deamidase from Chryseobacterium sp-62563. SEQ ID NO: 2: Amino acid sequence of a deamidase propeptide from Chryseobacterium sp- 62563.

SEQ ID NO: 3: Amino acid sequence of a deamidase inhibitor from Chryseobacterium sp- 62563.

SEQ ID NO: 4: Amino acid sequence of a deamidase from Chryseobacterium proteolyticum.

Brief description of the drawing

Figure 1 : Two superimposed 3D protein models of the deamidase propeptide inhibitor of SEQ ID NO: 2 and the deamidase inhibitor of SEQ ID NO: 3.

DETAILED DESCRIPTION

Deamidase

In the context of the invention, the term “deamidase” means a protein-glutamine glutaminase (also known as glutaminylpeptide glutaminase) activity, as described in EC 3.5.1.44, which catalyzes the hydrolysis of the gamma-amide of glutamine substituted at the carboxyl position or both the alpha-amino and carboxyl positions, e.g., L-glutaminylglycine and L-phenylalanyl-L-glutaminylglycine. Thus, deamidases can deamidate glutamine residues in proteins to glutamate residues and are also referred to as protein glutamine deamidase. The deamidase active site comprises a Cys-His-Asp catalytic triad (e.g., Cys-156, His-197, and Asp- 217, as shown in Hashizume et al. “Crystal structures of protein glutaminase and its pro forms converted into enzyme-substrate complex”, Journal of Biological Chemistry, vol. 286, no. 44, pp. 38691-38702) and belong to the InterPro entry IPR041325. In a preferred embodiment, the deamidases of the present invention belong to PFAM domain PF18626. Deamidases are catalytic proteins (enzymes), and the term “active (deamidase) enzyme protein’’ is defined herein as the amount of catalytic protein(s), which exhibits deamidase activity. This can be determined using an activity based analytical enzyme assay. This technique is well-known in the art.

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

Deamidase inhibitor

The term “deamidase inhibitor” (or “protein-glutamine glutaminase inhibitor”) means a sequence of amino acids that interacts with the amino acid residues of the deamidase active site (see above). Thus, deamidase inhibitors reduce or inhibit deamidase activity, preferably the deamidase activity exhibited by the deamidase shown as SEQ ID NO: 1. For example, the 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%, in the presence of the deamidase inhibitor (as compared to the deamidase activity without the presence of the deamidase inhibitor).

In an embodiment, the deamidase inhibitors comprise the amino acid sequence motif [IM][LIV][ST]AQ, which is characteristic of deamidase inhibitors derived from deamidase propeptide inhibitory domains.

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

In an embodiment, the deamidase inhibitors comprise the 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 combinations thereof.

Sequence identity

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

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

AlphaFold structure prediction

AlphaFold is a computational method for predicting the three-dimensional structure of a polypeptide from its amino acid sequence (Jumper et al., Highly accurate protein structure prediction with AlphaFold, Nature, 2021). Predicted structures for millions of polypeptides deposited in the UniProt database have been deposited 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 polypeptide can be obtained by searching for the UniProt accession number of the polypeptide.

In addition to the many three-dimensional structures that are already publicly available, code is available for reproducing and predicting structures of new polypeptides at source code repositories such as Github.com under deepmind/alphafold/, using notebooks/AlphaFold.ipynb, which uses Alphafold v2.3.1 or newer. Additionally, it can be found in Github.com under sokrypton/ColabFold using v1.5.2 or newer, using AlphaFold2.ipynb. For technical details, please see Jumper et al. (vide supra).

AlphaFold produces a per-residue estimate of its confidence on a scale from 0 to 100. This confidence measure is called pLDDT and corresponds to the model’s predicted score on the IDDT-Ca metric. It is stored in the B-factor fields of the mmCIF and PDB files available for download (although unlike a B-factor, higher pLDDT is better). Regions with pLDDT score of more than 90 are expected to be modelled to high accuracy. These should be suitable for any application that benefits from high accuracy (e.g., characterization of binding sites). Regions with a pLDDT score between 70 and 90 are expected to be modelled well, corresponding to a generally good backbone prediction.

Structural Similarity

The relatedness between two amino acid sequences has conventionally been 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 way of assessing a functional relationship between polypeptides is by comparing their three-dimensional structures. Thus, for the purposes of the present invention, the relatedness between the three-dimensional structure of two polypeptides is described by the parameter “structural similarity”.

A three-dimensional structure of any polypeptide may be obtained experimentally via, e.g., X-ray crystallography or using in silico methods such as AlphaFold (vide supra). The structural similarity between three-dimensional structures may then be determined by the TM-score, which is calculated using the following general formula (Zhang & Skolnick, Proteins 57:702-710, 2004):

TM-score

- where LN is the length of the native structure, LT is the length of the aligned residues to the template structure, di is the distance between the /th pair of aligned residues and do is a scale to normalize the match difference. ‘Max’ denotes the maximum value after optimal spatial superposition.

For the purposes of the present invention, LN is always the length of the reference protein, indicating the use of a fixed reference length L to prevent artificially large TM-scores from alignment of substructures:

A structural alignment of the three-dimensional structure of two polypeptides is necessary before the TM-score can be calculated. This is achieved via algorithms that optimize the structural overlap, and several methods are available, such as CEalign (Shindyalov and Bourne, Protein Eng., 11, 739-747, 1998), DALI (Holm and Sander, Trends Biochem. Sci., 20, 478-480, 1995), or TM-align (Nucleic Acids Res. 33:2302-2309, 2005).

For the purposes of the present invention, TM-align is applied. For convenience, TM-score is integrated in the TM-align software, which is available from the author’s website. The version of TM-align is preferably updated 2019-08-22 or later, and the TM-score between a reference and query protein is determined by running this command:

TMalign <query . pdb> <ref erence . pdb> -L <length of reference>

- where <query.pdb> is the name of the PDB file containing coordinates of the query polypeptide, <reference.pdb> is the name of the PDB file containing coordinates of the reference polypeptide. The TM-score is calculated and reported in the output, along with several other parameters from the alignment.

Enzyme composition

Deamidase enzymes (protein-glutamine glutaminases) are protein modifying enzymes, which convert glutamine residues to glutamate. This changes the charge of the protein and may result in modified solubility or other modified physicochemical properties.

Since deamidases, and other possible co-formulated enzymes, are also proteins, the deamidase activity may result in self-deamidation and/or modification of such other enzymes. It is well-known that enzymes are sensitive to modification of the amino acid residues, and deamidation of glutamine residues to glutamate may significantly change the enzyme properties (decreased isoelectric point (pl) and/or changed solubility/colloidal stability/aggregation) or even result in loss of enzymatic activity. This is particularly relevant for compositions where the deamidase concentration is high, and which are stored for an extended time, such as industrial enzyme product compositions.

The adverse effect of a high deamidase concentration can be reduced by reversibly inhibiting the deamidase with a deamidase inhibitor. When the deamidase product is applied in an application, the product dilution favors release of the inhibitor according to Le Chatelier’s principle. The separation of the deamidase and the inhibitor results in reactivation of the deamidase in the application of use.

Two groups of deamidase inhibitors have evolved which are structurally similar but have quite 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 is a group of separate inhibitors expressed from the same operon as the deamidase (see, for example, SEQ ID NO: 3). Each group of inhibitors comprises a characteristic amino acid sequence motif, which forms part of a loop that directly interacts with the active site of the mature deamidase. The surprising structural similarity of the two groups of inhibitors can be expressed using the “TM-score”.

Thus, the present invention provides an enzyme composition, comprising

(a) a protein-glutamine glutaminase; and

(b) a protein-glutamine glutaminase inhibitor, which

(ii) has at least 50% amino acid sequence identity to SEQ ID NO: 3 and comprises the amino acid sequence motif G[IM]S[APQ]Q, and/or (iii) has a TM-score of at least 0.80 as compared to the three-dimensional structure of the amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 3, where the three-dimensional structures are calculated using AlphaFold; wherein the protein-glutamine glutaminase and the protein-glutamine glutaminase inhibitor are not covalently linked.

In an embodiment, the composition comprises the deamidase (protein-glutamine glutaminase) in an amount of 10-10000 IPA(U) per gram; preferably in an amount of 20-8000 IPA(U) per gram, 30-6000 IPA(U) per gram, 40-5000 IPA(U) per gram, or in an amount of 50- 4000 IPA(U) per gram. The amount of deamidase may also be expressed in “active (deamidase) enzyme protein”; thus, the enzyme composition may comprise the deamidase (protein-glutamine glutaminase) in an amount of 0.01-15% w/w of active deamidase enzyme protein; preferably in an amount of 0.05-10% w/w, in an amount of 0.1-5% w/w, or in an amount of 0.1-3% w/w of active deamidase enzyme protein.

In an embodiment, the composition further comprises a hydrolyzing enzyme selected from the group consisting of protease, amylase, pectinase, cellulase, hemicellulase, xylanase, mannanase, glucanase, and combinations thereof. The composition may also comprise a nonhydrolyzing enzyme, like transglutaminase or oxidoreductase.

In an embodiment, the pH of a 1 % w/w solution of the composition is in the range of pH 4- 8.

In an embodiment, the molar ratio of the protein-glutamine glutaminase divided by the protein-glutamine glutaminase inhibitor is less than 1000:1 , preferably less than 500:1 or less than 100:1. Preferably, the molar ratio of the protein-glutamine glutaminase divided by the protein-glutamine glutaminase inhibitor is higher than 1 :10 or higher than 1 :1.

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

In an embodiment, the composition is a liquid composition.

The enzyme composition may comprise 20-80% w/w of polyol(s), such as 20-75% w/w, 20-70% w/w, 20-65% w/w, or 20-60% w/w of polyol(s).

In an embodiment, 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 polyol(s). The composition may be essentially free of other polyols than glycerol and sugar alcohol(s).

Polyols (or polyhydric alcohols) according to the invention are alcohols with two or more hydroxyl groups. The polyols may have a molecular weight lower than 500 g/mol. 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. The polyethylene glycol may have an average molecular weight at or below about 500. Examples of sugar alcohols are sorbitol, mannitol, erythritol, dulcitol, inositol, xylitol, adonitol, isomalt, and maltitol.

Polyols also include sugar polyols, such as mono- and disaccharides, like glucose, fructose, galactose, sucrose, lactose, maltose, and trehalose.

The present invention also provides a method for modifying a plant or milk protein, comprising contacting the plant or milk protein with the enzyme composition, as described above. Preferably, the plant protein is derived from cereals or legumes; more preferably the plant protein is derived from oat, wheat, corn, soy, pea or almond. Preferably, the milk protein is whey protein.

In an embodiment, the modification is deamidation of a plant or milk protein glutamine residue.

Deamidase enzymes

The deamidase (protein-glutamine glutaminase) comprised in the enzyme composition of the invention is usually produced by a microbial fermentation and subsequent recovery process. The subsequent recovery process may comprise a maturation/activation step, where an inhibitory propeptide is separated from a deamidase pro-form to produce an active (or more active) deamidase. Such propeptides may be cleaved/separated from the deamidase by the (recombinant) microbial expression organism, or extracellularly by using a suitable site-specific protease. Preferred expression organisms are Chryseobacterium and Bacillus species.

The fermentation liquid/broth may be subjected to a flocculation/precipitation step to provide a purified deamidase supernatant, and subsequently the purified deamidase supernatant may be subjected to a membrane filtration to provide a concentrated deamidase solution. Preferably, the membrane filtration comprises an ultra-filtration. The concentrated deamidase solution may subsequently be used to produce the formulation of the invention in a process that comprises mixing the concentrated deamidase solution with a polyol (such as glycerol) and salt, and optionally evaporating some water. Water may also be evaporated from the concentrated deamidase solution before adding the polyol/salt.

Depending on the desired product concentration, the fermentation broth (from the fermentation), the deamidase supernatant (from the flocculation), or the concentrated deamidase solution (from the membrane filtration) may be subjected to spray-drying (or freeze drying) to provide a deamidase powder. The deamidase powder may subsequently be used to produce the formulation of the invention in a process that comprises mixing the deamidase powder with water and polyol, and optionally other ingredients, such as salt(s). The deamidase comprised in the enzyme composition of the invention may have an amino acid sequence identity of at least 60%, preferably at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%, as compared to the amino acid sequence of SEQ ID NO: 1.

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

As described above, the structural similarity can be expressed using the “TM-score”.

Thus, the deamidase may 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, as compared to the three- dimensional structure of the amino acid sequence of SEQ ID NO:1 , wherein the three- dimensional structures are calculated using AlphaFold.

Deamidase inhibitors

The deamidase inhibitors (protein-glutamine glutaminase inhibitors) comprised in the enzyme composition of the invention are not covalently linked to the deamidase.

One known group of deamidase inhibitors are derived from deamidase propeptides, for example as described in PCT/EP2023/055936. An exemplary propeptide inhibitor is shown in SEQ ID NO: 2, and others can be identified using protein structure prediction tools (see, for example, Jumper et al., 2021, “Highly accurate protein structure prediction with AlphaFold”, Nature 596: 583-589).

Another group of deamidase inhibitors are in nature expressed separately from deamidases, for example as described in PCT/EP2023/068212. An exemplary inhibitor from this group is shown in SEQ ID NO: 3, and others can be identified using protein structure prediction tools (see, for example, Jumper et a/., 2021, “Highly accurate protein structure prediction with AlphaFold”, Nature 596: 583-589).

While the two groups of inhibitors described above have different amino acid sequences, they are structurally very similar, as shown in the two superimposed 3D protein models in Figure 1 (“propeptide inhibitor” of SEQ ID NO: 2 in blue, and “separate inhibitor” of SEQ ID NO: 3 in yellow). Figure 1 was prepared using the “super” command from PyMOL, which is specifically suggested to be used for cases with low sequence similarity but high structural similarity. It does a structure-based superposition and reports an all-atom RMSD between the “propeptide inhibitor” and the “separate inhibitor” of RMSD=1.178 Angstrom (based on the 3D coordinates of 360 atoms in this case), indicating that the structures are indeed highly similar.

Both inhibitors comprise an inhibitory loop that directly interacts with the Cys-His-Asp catalytic triad of the deamidase active site, but despite this common functionality, the characteristic amino acid sequence motifs of the loops are slightly different. The “propeptide inhibitors” (exemplified by SEQ ID NO: 2) comprise the amino acid sequence motif [IM][LIV][ST]AQ, while the “separate inhibitors” (exemplified by SEQ ID NO: 3) comprise the amino acid sequence motif G[IM]S[APQ]Q.

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

The deamidase inhibitor comprised in the enzyme composition of the invention may have an amino acid sequence identity of at least 60%, preferably at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%, as compared to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 3.

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

As described above, the structural similarity can be expressed using the “TM-score”. Thus, the deamidase inhibitor may 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, as 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 structures are calculated using AlphaFold.

Amino acid alterations of both the deamidase and deamidase inhibitors, as described above, may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of 1- 30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding module. Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant molecules are tested for enzyme activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271 : 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et a!., 1992, J. Mol. Biol. 224: 899- 904; Wlodaver et a!., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related polypeptide, and/or be inferred from sequence homology and conserved catalytic machinery with a related polypeptide or within a polypeptide or protein family with polypeptides/proteins descending from a common ancestor, typically having similar three-dimensional structures, functions, and significant sequence similarity.

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

Uses

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

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

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

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

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

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

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

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

Further embodiments of the invention include:

Embodiment 1. An enzyme composition, comprising

(a) a protein-glutamine glutaminase; and

(b) a protein-glutamine glutaminase inhibitor, which

(iii) has 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, where the three-dimensional structures are calculated using AlphaFold; wherein the protein-glutamine glutaminase and the protein-glutamine glutaminase inhibitor are not covalently linked. Embodiment . The enzyme composition of the preceding embodiment, which comprises the protein-glutamine glutaminase in amount of 10-10000 IPA(U)/g.

Embodiment 3. The enzyme composition of any of the preceding embodiments, which comprises the protein-glutamine glutaminase in an amount of 20-8000 IPA(U) per gram.

Embodiment 4. The enzyme composition of any of the preceding embodiments, which comprises the protein-glutamine glutaminase in an amount of 30-6000 IPA(U) per gram.

Embodiment 5. The enzyme composition of any of the preceding embodiments, which comprises the protein-glutamine glutaminase in an amount of 40-5000 IPA(U) per gram.

Embodiment 6. The enzyme composition of any of the preceding embodiments, which comprises the protein-glutamine glutaminase in an amount of 50-4000 IPA(U) per gram.

Embodiment 7. The enzyme composition of any of the preceding embodiments, which comprises the protein-glutamine glutaminase in an amount of 0.01-15% w/w of active deamidase enzyme protein.

Embodiment 8. The enzyme composition of any of the preceding embodiments, which comprises the protein-glutamine glutaminase in an amount of 0.05-10% w/w of active deamidase enzyme protein.

Embodiment 9. The enzyme composition of any of the preceding embodiments, which comprises the protein-glutamine glutaminase in an amount of 0.05-5% w/w of active deamidase enzyme protein.

Embodiment 10. The enzyme composition of any of the preceding embodiments, which comprises the protein-glutamine glutaminase in an amount of 0.1-5% w/w of active deamidase enzyme protein.

Embodiment 11. The enzyme composition of any of the preceding embodiments, which comprises the protein-glutamine glutaminase in an amount of 0.1-3% w/w of active deamidase enzyme protein.

Embodiment 12. The enzyme composition of any of the preceding embodiments, which comprises the protein-glutamine glutaminase in an amount of 0.1-2% w/w of active deamidase enzyme protein.

Embodiment 13. The enzyme composition of any of the preceding embodiments, where the protein-glutamine glutaminase belongs to EC 3.5.1.44.

Embodiment 14. The enzyme composition of any of the preceding embodiments, where the protein-glutamine glutaminase has an amino acid sequence identity of at least 60%, preferably at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%, as compared to the amino acid sequence of SEQ ID NO:1. Embodiment 15. The enzyme composition of any of the preceding embodiments, where the protein-glutamine glutaminase has up to 30 alterations (e.g., substitutions, deletions and/or insertions), preferably up to 25, up to 20, up to 15, up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 alteration(s), in particular substitutions, as compared to the amino acid sequence of SEQ ID NO: 1.

Embodiment 16. The enzyme composition of any of the preceding embodiments, where 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, as compared to the three- dimensional structure of the amino acid sequence of SEQ ID NO:1 , wherein the three- dimensional structures are calculated using AlphaFold.

Embodiment 17. The enzyme composition of any of the preceding embodiments, where the protein-glutamine glutaminase inhibitor has an amino acid sequence identity of at least 60%, preferably at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%, as compared to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 3.

Embodiment 18. The enzyme composition of any of the preceding embodiments, where 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 alterations, in particular substitutions, as compared to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3.

Embodiment 19. The enzyme composition of any of the preceding embodiments, where 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, as 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 structures are calculated using AlphaFold.

Embodiment 20. The enzyme composition of any of the preceding embodiments, which is a liquid composition.

Embodiment 21 . The enzyme composition of any of the preceding embodiments, which further comprises at least 10% w/w of water.

Embodiment 22. The enzyme composition of any of the preceding embodiments, which further comprises at least 20% w/w of water. Embodiment 23. The enzyme composition of any of the preceding embodiments, which further comprises at least 30% w/w of water.

Embodiment 24. The enzyme composition of any of the preceding embodiments, which further comprises at least 20% w/w of polyol and at least 10% w/w of water.

Embodiment 25. The enzyme composition of any of the preceding embodiments, which comprises 20-80% w/w of polyol, preferably 20-60% w/w of polyol.

Embodiment 26. The enzyme composition of any of the preceding embodiments, where the polyol is selected from the group consisting of glycerol, a sugar alcohol, a mono- or disaccharide, and combinations thereof.

Embodiment 27. The enzyme composition of any of the preceding embodiments, which comprises the polyol in an amount of 20-75% w/w.

Embodiment 28. The enzyme composition of any of the preceding embodiments, which comprises the polyol in an amount of 20-70% w/w.

Embodiment 29. The enzyme composition of any of the preceding embodiments, which comprises the polyol in an amount of 20-65% w/w.

Embodiment 30. The enzyme composition of any of the preceding embodiments, which comprises the polyol in an amount of 20-60% w/w.

Embodiment 31 . The enzyme composition of any of the preceding embodiments, which comprises the polyol in an amount of at least 25% w/w.

Embodiment 32. The enzyme composition of any of the preceding embodiments, which comprises the polyol in an amount of at least 30% w/w.

Embodiment 33. The enzyme composition of any of the preceding embodiments, which comprises the polyol in an amount of at least 35% w/w.

Embodiment 34. The enzyme composition of any of the preceding embodiments, which comprises the polyol in an amount of at least 40% w/w.

Embodiment 35. The enzyme composition of any of the preceding embodiments, which is essentially free of other polyols than glycerol and sugar alcohol(s).

Embodiment 36. The enzyme composition of any of the preceding embodiments, where the pH of the composition is below pH 8.

Embodiment 37. The enzyme composition of any of the preceding embodiments, where the pH of the composition is below pH 7.

Embodiment 38. The enzyme composition of any of the preceding embodiments, where the polyol comprises or consists of a non-sugar polyol.

Embodiment 39. The enzyme composition of any of the preceding embodiments, where the polyol comprises or consists 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. Embodiment 40. The enzyme composition of any of the preceding embodiments, where the polyol comprises or consists of a polyethylene glycol having an average molecular weight at or below 500.

Embodiment 41 . The enzyme composition of any of the preceding embodiments, where the polyol comprises or consists of a sugar alcohol selected from the group consisting of sorbitol, mannitol, erythritol, dulcitol, inositol, xylitol, adonitol, isomalt, and maltitol.

Embodiment 42. The enzyme composition of any of the preceding embodiments, where the polyol comprises or consists of mono- or disaccharide(s).

Embodiment 43. The enzyme composition of any of the preceding embodiments, where the polyol comprises or consists of a mono- or disaccharide selected from the group consisting of glucose, fructose, galactose, sucrose, lactose, maltose, and trehalose.

Embodiment 44. The enzyme composition of any of the preceding embodiments, where the pH of a 1% w/w solution of the composition is in the range of pH 4-9.

Embodiment 45. The enzyme composition of any of the preceding embodiments, where the pH of a 1% w/w solution of the composition is in the range of pH 5-8.

Embodiment 46. The enzyme composition of any of the preceding embodiments, where the molar ratio of the protein-glutamine glutaminase divided by the protein-glutamine glutaminase inhibitor is less than 1000:1 .

Embodiment 47. The enzyme composition of any of the preceding embodiments, where the molar ratio of the protein-glutamine glutaminase divided by the protein-glutamine glutaminase inhibitor is less than 500:1.

Embodiment 48. The enzyme composition of any of the preceding embodiments, where the molar ratio of the protein-glutamine glutaminase divided by the protein-glutamine glutaminase inhibitor is less than 100:1.

Embodiment 49. The enzyme composition of any of the preceding embodiments, where the molar ratio of the protein-glutamine glutaminase divided by the protein-glutamine glutaminase inhibitor is higher than 1 :10.

Embodiment 50. The enzyme composition of any of the preceding embodiments, where the molar ratio of the protein-glutamine glutaminase divided by the protein-glutamine glutaminase inhibitor is higher than 1:1.

Embodiment 51 . The enzyme composition of any of the preceding embodiments, which further comprises an enzyme selected from the group consisting of hydrolyzing enzymes, oxidoreductases, and transglutaminase.

Embodiment 52. The enzyme composition of any of the preceding embodiments, which further comprises a hydrolyzing enzyme selected from the group consisting of protease, amylase, pectinase, cellulase, hemicellulase, xylanase, mannanase, glucanase, and combinations thereof. Embodiment 53. The enzyme composition of any of the preceding embodiments, which further comprises a transglutaminase.

Embodiment 54. The enzyme composition of any of the preceding embodiments, which further comprises a protease.

Embodiment 55. The enzyme composition of any of the preceding embodiments, which further comprises a reducing agent.

Embodiment 56. The enzyme composition of any of the preceding embodiments, which further comprises a reducing agent in an amount of at least 0.1 % w/w.

Embodiment 57. The enzyme composition of any of the preceding embodiments, which further comprises a reducing agent in an amount of at least 0.1-5% w/w.

Embodiment 58. The enzyme composition of any of the preceding embodiments, which further comprises a reducing agent in an amount of at least 0.1-2% w/w.

Embodiment 59. The enzyme composition of any of the preceding embodiments, which further comprises a salt of sulfite, metabisulfite, thiosulphate, or ascorbate.

Embodiment 60. The enzyme composition of any of the preceding embodiments, which further comprises a salt of sulfite, metabisulfite, thiosulphate, or ascorbate, in an amount of at least 0.1% w/w.

Embodiment 61 . The enzyme composition of any of the preceding embodiments, which further comprises a salt of sulfite, metabisulfite, thiosulphate, or ascorbate, in an amount of at least 0.1-5% w/w.

Embodiment 62. The enzyme composition of any of the preceding embodiments, which further comprises a salt of sulfite, metabisulfite, thiosulphate, or ascorbate, in an amount of at least 0.1-2% w/w.

Embodiment 63. A method for modifying a plant or milk protein, comprising contacting the plant or milk protein with the enzyme composition of any of the preceding embodiments.

Embodiment 64. The method of the preceding embodiment, where the plant protein is derived from cereals or legumes, and the milk protein is whey protein.

Embodiment 65. The method of the preceding embodiment, where the plant protein is derived from oat, wheat, corn, soy, pea or almond.

Embodiment 66. The method of any of the preceding embodiments, where the modification is deamidation of a plant or milk protein glutamine residue.

EXAMPLES

Chemicals were commercial products of at least reagent grade.

The deamidase used in the examples is derived from Chryseobacterium sp-62563. The amino acid sequence of the deamidase is shown as SEQ ID NO: 1. EXAMPLE 1

Deamidase activity assay

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

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

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

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

Assay Procedure

Step (1) Enzymatic step with ammonia formation

Reagents:

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

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

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

Stop solution: 0.4 M TCA.

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

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

Incubation:

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

2. Add 100 pL of substrate solution to each well.

3. For blank samples add 100 pL 0.4 M TCA solution. 4. Seal the plate using transparent plate sealer.

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

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

Total reaction volume: 210 pL

Step (2) Ammonia detection step

Reagents:

Color reagent A: 4% (w/v) Phenol, 0.015% (w/v) sodium pentacyanonitrosylferrate(lll) dihydrate (sodium nitroprusside) (Na₂[Fe(CN)₅NO] 2H₂O).

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

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

Incubation:

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

2. T ransfer 45 pL Milli-Q water to each well.

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

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

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

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

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

Total reaction volume: 210 pL

Standard curve:

Standard stock solution: 1000 mg NH₄ ⁺/L.

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

The amount of enzyme producing 1 pmol ammonia per minute at 37°C is defined as 1 unit (Indophenol Assay Unit; IPA(U)):

where

which can be shortened to 21 _ >

180.04 ml • min. where

• C_NH4+ is the ammonia concentration in the reaction solution derived from the ammonium standard curve (i.e., taking into account the dilution of the prediluted ammonium standard solution in the ammonia derivatization step).

• 18.04 is the molecular mass of ammonium used for the standard solution. is the reaction volume in the well when ammonia is generated (210 pL).

• V_mzyme is the volume of enzyme solution added to the well when ammonia is generated (10 pL).

• VNHZ detection is the reaction volume in the well when ammonia is detected (210 pL).

EXAMPLE 2

Nano differential scanning fluorimetry (nanoDSF) - thermal unfolding temperature

Nano differential scanning fluorimetry (nanoDSF) was used to evaluate the conformational stability of the deamidase/inhibitor complex, using the deamidase inhibitor of the invention. The molecules were exposed to a temperature gradient, as indicated below. The resulting changes in structure is reflected in changes in fluorescence intensity and gives a measure of temperature stability. Binding of the deamidase inhibitor to the deamidase contributes to stability of the molecule, thus the nanoDSF thermal unfolding temperatures also give information on the deamidase/inhibitor binding affinity.

Purified samples were received in a buffer containing 20 mM sodium phosphate; 500 mM sodium chloride; 500 mM imidazole; pH 7.4.

60 pL sample was transferred in replicates to a black-bottomed 384 well plate, and the plate was briefly centrifugated to remove potential air bubbles.

The thermal unfolding 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 per minute; and

(ii) Temperature scan interval: 20-95°C.

During the run, samples were loaded in capillaries (Prometheus NT.Plex - Capillary Chips, Standard, Cat# PR-AC002) before being subjected to the temperature gradient. The generated data were analyzed by the PR. Stability analysis v.1 .0.1 software. Midpoint unfolding temperatures (T_m, °C) were annotated based on the first derivative of the 330 nm trace (alternatively, the ratio 350nm/330nm may be used). In some cases, more than one T_m may be observed. In these cases, the main peak in the first derivative trace at 330 nm was chosen as the Tm of the sample.

EXAMPLE 3

Thermal unfolding temperature of deamidase/inhibitor complex

Using the nanoDSF procedure, as described in Example 2 with minor modifications, the thermal unfolding temperatures of an active deamidase, the corresponding deamidase/inhibitor, and deamidase/propeptide complex were measured.

As shown in Table 1, both the propeptide and the inhibitor increase the unfolding temperature and conformational stability. This likely indicates formation of a deamidase/inhibitor complex and reduced deamidase activity.

Table 1. NanoDSF thermal unfolding temperatures of polypeptides.

EXAMPLE 4

Inhibition of deamidase activity

Two different active deamidases from Chryseobacterium sp-62563 (shown as SEQ ID NO: 1) and Chryseobacterium proteolyticum (shown as SEQ ID NO: 4) were each incubated for 30 minutes at 55°C with skimmed milk. The skimmed milk was previously heat treated for 10 minutes at 90°C. The deamidase activity was measured as mg ammonium/L (see also Example 1).

The two experiments were each repeated twice, but this time the deamidases were supplemented with the propeptide of SEQ ID NO: 2 or the inhibitor of SEQ ID NO: 3.

As shown in Table 2, both the propeptide and the inhibitor effectively reduced the deamidase activity of both deamidases. Table 2. Deamidase activity was measured in mg ammonium/L.

Claims

1. An enzyme composition, comprising

(a) a protein-glutamine glutaminase; and

(b) a protein-glutamine glutaminase inhibitor, which

2. The enzyme composition of the preceding claim, which comprises the protein-glutamine glutaminase in amount of 10-10000 IPA(U)/g

3. The enzyme composition of any of the preceding claims, where the protein-glutamine glutaminase belongs to EC 3.5.1.44.

4. The enzyme composition of any of the preceding claims, where the protein-glutamine glutaminase has an amino acid sequence identity of at least 80%, preferably at least 90%, or at least 95%, as compared to the amino acid sequence of SEQ ID NO:1.

5. The enzyme composition of any of the preceding claims, where the protein-glutamine glutaminase inhibitor has an amino acid sequence identity of at least 60%, preferably at least 70%, at least 80%, or at least 90%, as compared to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 3.

6. The enzyme composition of any of the preceding claims, where 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, as 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 structures are calculated using AlphaFold.

7. The enzyme composition of any of the preceding claims, which is a liquid composition.

8. The enzyme composition of the preceding claim, which further comprises at least 20% w/w of polyol and at least 10% w/w of water.

9. The enzyme composition of the preceding claim, which comprises 20-80% w/w of polyol, preferably 20-60% w/w of polyol.

10. The enzyme composition of the preceding claim, where the polyol is selected from the group consisting of glycerol, a sugar alcohol, a mono- or disaccharide, and combinations thereof.

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

12. The enzyme composition of any of the preceding claims, where the molar ratio of the protein-glutamine glutaminase divided by the protein-glutamine glutaminase inhibitor is less than 1000:1 ; preferably less than 100:1.

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

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

15. The method of the preceding claim, where the modification is deamidation of a plant or milk protein glutamine residue.