Protein sweeteners

Abstract

The present disclosure relates to modified proteins having an amino acid sequence comprising at least one amino acid deletion and at least one amino acid substitution as compared to a reference protein and having improved stability.

Description

Technical Field

[0001] The present disclosure relates to protein sweeteners and uses thereof. Background Art

[0002] The following is a list of references that are believed to be relevant to the background art of the presently disclosed subject matter:

[0003] -Weiffert T, Linse S. Protein stabilization with retained function ofmonellin using a split GFP system. Sci Rep. 2018Aug 24;8(1):12763.

[0004] doi:10.1038 / s41598-018-31177-z.PMID:30143736; PMCID:

[0005] PMC6109104.

[0006] -Delfi, M.; Emendato, A.; Leone, S.; Lampitella, E. A.; Porcaro, P.;

[0007] Cardinale,G.;Petraccone,L.;Picone,DA Super Stable Mutant of thePlant Protein Monellin Endowed with Enhanced Sweetness.Life 2021,

[0008] 11,236. https: / / doi.org / 10.3390 / life11030236

[0009] -Zhao,M.et al.Structure basis of the improved sweetness andthermostability of a unique double-sites single-chain sweet-tasting proteinmonellin(MNEI)mutant.Biochimie 2018,154,156-163.doi:

[0010] https: / / doi.org / 10.1016 / j.biochi.2018.08.010

[0011] -Somoza,J.R.et al.The Taste-active Regions of Monellin,a PotentlySweet Protein.Chem Senses.1995,20(1),61-68.

[0012] https: / / doi.org / 10.1093 / chemse / 20.1.61

[0013] -Yang et al.The flexible loop is a new sweetness determinant site ofthe sweet-tasting protein:Characterization of novel sweeter mutants of thesingle-chain Monellin(MNEI).Chemical Senses 2019,44(8),607-614..

[0014] doi:10.1093 / chemse / bjz057

[0015] -Liu et al.,Functional characterization of the heterodimeric sweettaste receptor T1R2 and T1R3 from a New World monkey species(squirrel monkey)and its response to sweet-tasting proteins.Biochemical and BiophysicalResearch Communications 2012,427(2),431-437.doi:

[0016] 10.1016 / j.bbrc.2012.09.083.

[0017] -Leone et al.,Sweeter and stronger:enhancing sweetness and stability of the single chain monellin MNEI through molecular design.Scientific Reports2016,6(1).doi:10.1038 / srep34045.

[0018] Acknowledgment herein of the above references should not be inferred as an intention that these references are in any way relevant to the patentability of the presently disclosed subject matter. Background Art

[0020] Sweet proteins are a class of proteins that interact with sweet taste receptors T1R2 / T1R3 and stimulate sweet taste perception. Single-chain tamoxifen (Monelli) MNEI is an example of such sweet proteins, however, its application in food and beverage products is limited due to its low stability.

[0021] Weiffert T. et al. describe functional studies showing that the S76Y substitution retains sweet taste and has potential use in the food industry.

[0022] Delfi, M. et al. describe new constructs of MNEI that have increased sweetness and stability compared to MNEI.

[0023] Zhao, M. et al. described the effects of E2N and E23A substitutions on MNEI, namely their effects on thermal stability, protein structure and sweetness.

[0024] Somoza, JR et al. describe mutagenesis studies on mebutam and show that at least four residues are involved in the taste active region of single-chain mebutam.

[0025] Yang et al. described the effects of substitutions in the flexible loop (L23) of MNEI on the sweetness of MNEI.

[0026] Liu et al. describe hybrids of human and squirrel monkey sweet taste receptor subunits. These hybrids highlight the interactions between native receptor subunits and the important role of electrostatics involved in MNEI:receptor interactions.

[0027] Leone et al. describe substitutions in MNEIs with increased sweetness and thermostability. Summary of the invention

[0028] According to some aspects, the present disclosure provides a modified protein having an amino acid sequence comprising at least one amino acid deletion and at least one amino acid substitution compared to a reference protein, wherein the reference protein is represented by the amino acid sequence shown in SEQ ID NO:1, wherein the at least one amino acid deletion is at one or more amino acids located between T46 and I56 of the reference protein, and wherein the at least one amino acid substitution is at amino acid S76 of the reference protein.

[0029] According to some other aspects, the present disclosure provides a modified protein having an amino acid sequence comprising at least one amino acid deletion and at least one amino acid substitution compared to a reference protein, wherein the reference protein is represented by the amino acid sequence shown in SEQ ID NO:1, wherein the at least one amino acid deletion is at one or more of at least amino acids E50, F52 and R53 of the reference protein, and wherein the at least one amino acid substitution is at amino acid S76 of the reference protein.

[0030] According to some additional aspects, the present disclosure provides a modified protein having an amino acid sequence comprising at least one amino acid deletion and at least one amino acid substitution compared to a reference protein, wherein the reference protein is represented by the amino acid sequence shown in SEQ ID NO: 1, wherein the at least one amino acid deletion is at one or more amino acids E50, F52 and R53 of the reference protein, and wherein the at least one amino acid substitution is at at least amino acids E2, E23, Y65, L70, S76 of the reference protein.

[0031] According to some yet further aspects, the present disclosure provides a modified protein having an amino acid sequence comprising at least one amino acid deletion and at least one amino acid substitution compared to a reference protein, wherein the reference protein is represented by the amino acid sequence shown in SEQ ID NO:1, wherein the at least one amino acid deletion is at amino acids E50, F52 and R53 of the reference protein, wherein the at least one amino acid substitution is at least E2N, E23A, Y65R, L70I and at amino acid S76 of the reference protein.

[0032] According to some aspects, the present disclosure provides a modified protein having an amino acid sequence comprising at least one amino acid deletion and at least one amino acid substitution compared to a reference protein, wherein the reference protein is represented by the amino acid sequence shown in SEQ ID NO:1, wherein the at least one amino acid deletion is at one or more amino acids between T46 and I56 of the reference protein, wherein the at least one amino acid substitution is at amino acid S76 of the reference protein, and wherein the modified protein has at least increased stability compared to the reference protein.

[0033] According to some other aspects, the present disclosure provides a modified protein having an amino acid sequence comprising at least one amino acid deletion and at least one amino acid substitution compared to a reference protein, wherein the reference protein is represented by the amino acid sequence shown in SEQ ID NO: 1, wherein the at least one amino acid deletion is at one or more of at least amino acids E50, F52 and R53 of the reference protein, wherein the at least one amino acid substitution is at amino acid S76 of the reference protein, and wherein the modified protein has at least increased stability compared to the reference protein.

[0034] According to some additional aspects, the present disclosure provides a modified protein having an amino acid sequence comprising at least one amino acid deletion and at least one amino acid substitution compared to a reference protein, wherein the reference protein is represented by the amino acid sequence shown in SEQ ID NO: 1, wherein the at least one amino acid deletion is at one or more amino acids E50, F52 and R53 of the reference protein, wherein the at least one amino acid substitution is at at least amino acids E2, E23, Y65, L70, S76 of the reference protein, and wherein the modified protein has at least increased stability compared to the reference protein.

[0035] According to some yet further aspects, the present disclosure provides a modified protein having an amino acid sequence comprising at least one amino acid deletion and at least one amino acid substitution compared to a reference protein, wherein the reference protein is represented by the amino acid sequence shown in SEQ ID NO: 1, wherein the at least one amino acid deletion is at amino acids E50, F52 and R53 of the reference protein, wherein the at least one amino acid substitution is at least E2N, E23A, Y65R, L70I and at amino acid S76 of the reference protein, and wherein the modified protein has at least increased stability compared to the reference protein.

[0036] According to some aspects, the present disclosure provides a stable modified protein as described herein.

[0037] According to some aspects, the present disclosure provides a food product comprising a modified protein as described herein.

[0038] According to some aspects, the present disclosure provides a method for increasing the stability of a reference protein, the method comprising deleting at least one amino acid of the reference protein and replacing at least one amino group of the reference protein, wherein the reference protein is represented by the amino acid sequence shown in SEQ ID NO:1, wherein at least one deleted amino acid is at one or more amino acids between T46 and I56 of the reference protein, and wherein at least one substituted amino acid is at amino acid S76 of the reference protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to better understand the subject matter disclosed herein and to illustrate how it may be implemented in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0040] Figure 1A and Figure 1B The structure of the DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y) protein is presented. Figure 1A amino acids Y76 (cyan) and E59 (yellow) close to the loop (magenta); Figure 1B Amino acids Y76 (cyan) and R84 (orange) are located close to the loop (magenta).

[0041] Figure 2 The structure of the DM652 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, E59V, S76V) protein is presented. The colored residues are V59 (yellow) and V76 (cyan). The loop is also labeled (magenta), and the modification of residues 59 and 76 to valine prevents water molecules from interacting with the backbone.

[0042] Figure 3 An anisotropic network model (ANM) analysis of the global motion of DM31 is presented; the motion of the Cα residues is shown as arrows, the length of the arrows indicates the magnitude of the motion, and the most moving parts of the protein are the β-hairpins consisting of β-strands 2 and 3 and β-strands 4 and 5. These drive the motion of the β-sheets, helices, and the C-terminal loop of the helix.

[0043] 4A to 4F Electrostatic potential contour plots representing equipotential surfaces for DM31-based variants are presented, with positive (blue) and negative (red) potentials at + / -1 kT / e. Figure 4A DM222 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, T33R, S76Y); Figure 4B DM479 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, K25R, D68N, S76Y); Figure 4C DM31; Figure 4D DM224 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, S76Y); Figure 4E DM606 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76R); Figure 4F DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y), substitutions of specific DMs, and additional residues known to be important for sweet taste are labeled and presented as spheres.

[0044] Figure 5 RMSF plots of variants DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y), DM653 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, E59I, S76V), DM656 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, E59I, S76I), DM663 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76V), DM31 and MNEI. DETAILED DESCRIPTION

[0045] The present disclosure relates to novel sweet tasting proteins characterized by high stability. The novel proteins are designed using a series of computational optimization methods. Specifically, the computational methods are based on the computational identification of specific regions and amino acid residues in the protein that can be considered to affect protein characteristics (including especially stability and sweet taste), and thus obtain novel proteins with improved characteristics as further described herein.

[0046] As shown below, the inventors have found that introducing specific modifications (such as deletions or substitutions) into the amino acid sequence of a reference protein produces novel proteins (represented herein as "modified proteins" or "designed proteins") having at least improved stability compared to the reference protein. Based on these results, it is shown that the novel proteins with improved stability can be used in food and beverage applications to prepare a variety of edible products.

[0047] Thus, in its broadest aspect, the present disclosure relates to a modified protein comprising an amino acid sequence having at least one of (i) at least one amino acid deletion, (ii) at least one amino acid substitution (replacement), (iii) at least one amino acid insertion, or (iv) any combination thereof compared to the amino acid sequence of a reference protein. As described herein, the modified protein has at least improved thermal stability compared to the reference protein.

[0048] Hereinafter, when referring to the modified protein, it is understood that reference is also made to the food product disclosed herein. Thus, whenever characteristics relating to the modified protein are provided, it is understood that the same characteristics relating to the food product are defined mutatis mutandis.

[0049] In the present disclosure, modified proteins, which are sometimes also expressed as designed proteins, can be considered as variants of reference proteins. The term "variant" as used herein refers to a sequence containing at least one amino acid modification (e.g., at least one substitution, at least one deletion, at least one insertion, or any combination thereof) compared to a reference protein. It should be noted that, as a result of the modification, a modified / variant sequence is obtained.

[0050] The present disclosure also encompasses a modified nucleic acid sequence comprising a corresponding substitution, deletion or insertion of a codon similar to the substitution, deletion or insertion in the modified protein. In other words, the same modification is applicable to the nucleic acid sequence generating the modified sequence, including the corresponding substitution, deletion or insertion of a codon.

[0051] The present disclosure is not limited to a specific number of amino acid modifications made in a reference protein that ultimately results in the formation of a modified protein.

[0052] Modification as used herein refers to a modification in one or more amino acids of a reference protein and encompasses amino acid substitutions (replacements), amino acid deletions, amino acid insertions, or any combination thereof.

[0053] In some examples, the modification may be an amino acid insertion.

[0054] In some embodiments, the modified protein comprises an amino acid sequence having at least one, at least two, at least three, at least four, at least five amino acid insertions compared to the reference protein.

[0055] In some examples, the modification may be an amino acid deletion.

[0056] In some embodiments, the modified protein comprises an amino acid sequence having at least two, at least three, at least four, at least five amino acid deletions compared to a reference protein.

[0057] In some cases, the modification may be an amino acid substitution.

[0058] In some embodiments, the modified protein comprises an amino acid sequence having at least one, at least two, at least three, at least four, at least five amino acid substitutions compared to a reference protein.

[0059] As described herein, modified proteins can be produced by amino acid modifications (substitutions or deletions) of different regions of a protein. As used herein, "protein region" refers to an amino acid sequence or structural motif that is a part of a protein sequence (amino acid sequence) or structure. Non-limiting examples of protein regions include protein surfaces, protein cores, protein loops, secondary structural elements, secondary structural capping, disulfide bonds, binding sites, linkers, hydrophobic patches, or hydrophobic regions of proteins.

[0060] As shown in the examples below, molecular dynamics is used to design new modified proteins based on the optimization of different regions of reference proteins. Specifically, using the Rosetta energy unit (REU) score for the newly designed modified proteins, it is possible to predict those proteins that will exhibit improved properties, specifically at least the stability of the modified proteins. As part of the computational optimization process, based on energy considerations, after computational, bioinformatic or structural biology analysis, the modified proteins can be selected from a large number of output populations of amino acid sequences, i.e., those with low energy.

[0061] Energy calculations can be applied to the entire amino acid sequence, or alternatively limited to specific regions or selected amino acids within the protein. In the latter (different regions or selected amino acids), the information can be integrated to measure the entire protein.

[0062] The calculation of each amino acid sequence (e.g., modified protein) in an amino acid sequence can be accomplished by combining physics-based (also referred to as biophysical methods) and statistical-based potentials (also referred to as knowledge-based potentials or informatics methods), such as by using Rosetta Energy Units (REU). Rosetta Energy Units (REU) are algorithms of Rosetta software, which is an algorithm package for computational modeling and protein structure analysis. Rosetta software has achieved significant scientific progress in computational biology, including de novo protein design, enzyme design, ligand docking, and structural prediction of biomacromolecules and macromolecular complexes. The Rosetta energy function is a combination of physical and statistical potentials that do not match any actual physical energy unit. Rosetta energy is arbitrarily scaled and is sometimes referred to as REU (standing for "Rosetta Energy Unit").

[0063] In some embodiments, the REU of the complete protein sequence comprising at least one amino acid modification can be calculated. In some other embodiments, the REU of at least one region comprising at least one amino acid modification of the complete protein sequence can be calculated. In some other embodiments, the REU of at least one amino acid modification in the complete protein sequence can be calculated.

[0064] In some embodiments, the modified protein has an energy of less than about -315 given as REU.

[0065] In some embodiments, the modified protein has an energy less than about -321 given as REU.

[0066] In some embodiments, the modified protein has an energy lower than -316 given in REU. In some embodiments, the modified protein has an energy lower than -317 given in REU. In some embodiments, the modified protein has an energy lower than -318 given in REU. In some embodiments, the modified protein has an energy lower than -319 given in REU. In some embodiments, the modified protein has an energy lower than -320 given in REU. In some embodiments, the modified protein has an energy lower than -321 given in REU. In some embodiments, the modified protein has an energy lower than -322 given in REU. In some embodiments, the modified protein has an energy lower than -323 given in REU. In some embodiments, the modified protein has an energy lower than -324 given in REU. In some embodiments, the modified protein has an energy lower than -325 given in REU. In some embodiments, the modified protein has an energy lower than -326 given in REU. In some embodiments, the modified protein has an energy below -327 given in REU. In some embodiments, the modified protein has an energy below -328 given in REU. In some embodiments, the modified protein has an energy below -329 given in REU. In some embodiments, the modified protein has an energy below -330 given in REU.

[0067] In some embodiments, the modified protein has an energy given as REU of about -315 and about -330.

[0068] In some embodiments, the modified protein comprises an amino acid sequence that is 40% to 98% identical to the amino acid sequence of the reference protein. In some embodiments, the modified protein comprises an amino acid sequence that is 90% to 98% identical to the reference amino acid sequence.

[0069] In some embodiments, the modified protein comprises an amino acid sequence that is 60% to 90% identical to a reference amino acid sequence. In some embodiments, the modified protein comprises an amino acid sequence that is 70% to 90% identical to a reference amino acid sequence.

[0070] In some embodiments, the modified protein comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to a reference amino acid sequence.

[0071] In some embodiments, the modified protein comprises an amino acid sequence that is 90% to 98% identical to a reference amino acid sequence.

[0072] When two or more sequences are compared and aligned for maximum correspondence, the % identity between two or more amino acid sequences is determined. In the context of the present disclosure, the sequences (amino acids) with % identity described herein are considered to have the same function / activity as the reference sequence for which the identity is calculated.

[0073] In some embodiments, the modified protein comprises an amino acid sequence having 40% to 98% similarity to the amino acid sequence of the reference protein. In some embodiments, the modified protein comprises an amino acid sequence having 90% to 98% similarity to the reference amino acid sequence.

[0074] In some embodiments, the modified protein comprises an amino acid sequence having 60% to 90% similarity to a reference amino acid sequence. In some embodiments, the modified protein comprises an amino acid sequence having 70% to 90% similarity to a reference amino acid sequence.

[0075] In some embodiments, the modified protein comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% similar to a reference amino acid sequence.

[0076] In some embodiments, the modified protein comprises an amino acid sequence having 90% to 98% similarity to a reference amino acid sequence.

[0077] As used herein, sequence similarity or sequence homology refers to the amount of amino acids (eg, leucine and isoleucine) that retain similar physicochemical properties.

[0078] When determining sequence identity, gaps are not calculated, and the sequence identity is relative to the shorter of the two sequences. In this regard, it should be noted that the length of the reference protein (amino acid sequence) may be the same as the modified protein (amino acid sequence), or may be different from the modified protein (amino acid sequence).

[0079] As described herein, the reference protein used as the basis for the computational analysis is a sweet protein. The reference sweet protein can be a naturally occurring protein or a synthetic protein.

[0080] In some examples, the reference protein is a synthetic protein. When referring to a synthetic protein, it is understood to be a protein that is not found in nature and is therefore considered to be a synthetic protein.

[0081] According to the present disclosure, the reference protein is MNEI or a modified protein based on MNEI.

[0082] In some embodiments, the reference protein is MNEI.

[0083] As will be appreciated, MNEI is a synthetic protein composed of a combination of chain A mebutam (GenBank Cataloging Serial No. P02881) and chain B mebutam (GenBank Cataloging Serial No. P02882).

[0084] MNEI is represented herein by SEQ ID NO: 1 and has the following amino acid sequence:

[0085] GEWEIIDIGPFTQNLGKFAVDEENKIGQYGRLTFNKVIRPCMKKTIYE NEGFREIKGYEYQLYVYASDKLFRADISEDYKTRGRKLLRFNGPVPPP

[0086] In some embodiments, the reference protein is represented herein by SEQ ID NO:1.

[0087] The modified proteins according to the present disclosure may be considered as variants of MNEI. It should be noted that a variant sequence (modified) is considered as such as long as it comprises a different sequence compared to a reference sequence (in this case MNEI), regardless of how it is synthesized.

[0088] As shown in the examples below, substitutions in the reference protein, at least substitutions in the amino acid S76 located at the beta strand exposed to the solvent, produce modified proteins with improved stability. As further shown in the examples below, substitutions in S76 combined with one or more amino acid modifications (including particularly amino acid deletions and / or amino acid substitutions) improve the stability of the modified protein.

[0089] In some embodiments, the modified protein comprises an amino acid substitution in at least amino acid S76 of SEQ ID NO:1.

[0090] Therefore, according to some aspects, the present disclosure provides a modified protein comprising an amino acid sequence having at least one amino acid substitution in at least amino acid S76 compared to a reference protein, wherein the reference protein is represented by SEQ ID NO:1.

[0091] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having at least one amino acid substitution in at least amino acid S76 compared to a reference protein, wherein the modified protein has at least improved stability compared to the reference protein, wherein the reference protein is represented by SEQ ID NO: 1.

[0092] The term stability as used herein refers to the ability of a protein to maintain a three-dimensional structure. In the context of the present disclosure, the term stability includes thermal stability, short-term and long-term functional stability (structure and / or function), long-term functional stability (shelf life stability), pH stability, salt concentration stability, ionic strength stability or stability in a fat- or protein-containing matrix, and stability in the presence of different preservatives.

[0093] In some embodiments, the stability is thermal stability.

[0094] As used herein, the term "thermal stability" refers to the ability of a modified protein to maintain its 3D structure at a temperature higher than the temperature of a reference protein. The 3D structural stability of a protein can be measured by any method known in the art, such as circular dichroism (CD) or thermal shift assays, such as differential scanning fluorimetry (DSF) or differential scanning calorimetry (DSC). The 3D structure of a protein can affect the function of the protein.

[0095] In some embodiments, the modified protein has the same or greater thermal stability relative to a reference protein.

[0096] As used herein, the term "functional stability" refers to the ability of a modified protein to retain its function after exposure to high temperature compared to a reference protein.

[0097] In some embodiments, the modified proteins herein can maintain a sweet taste effect at higher temperatures or after exposure to higher temperatures for a possibly limited time. In other words, there is a significant perceived change in sweetness or sensory characteristics after the product is exposed to temperatures above room temperature, sometimes up to 50° C., sometimes up to 100° C., or even up to 150° C. Protein function, such as sweetest, can be measured by sensory testing.

[0098] In some embodiments, the modified protein has the same or higher pH stability relative to the reference protein. pH stability refers to the stability of the modified protein over a wider pH range relative to the reference protein, i.e., the modified protein maintains 3D structure (and / or function) after the product is exposed to any pH between 3 and 8, sometimes between 4 and 8. For example, soda water such as cola has a pH of 2.3-2.5, at which pH values, some of the sweet proteins in the sweet protein are unstable and lose functionality immediately or after a time shorter than the normal shelf life of the beverage.

[0099] In some embodiments, the modified protein has a higher solubility relative to a reference protein.Solubility can be in an aqueous, partially aqueous, or non-aqueous environment, such as in a fat-containing food.

[0100] In some embodiments, the modified protein has an improved shelf life relative to a reference protein. Improved shelf life means that the product comprising the composition has no perceived change in sweetness (function) or physical deterioration (e.g., color change, phase separation, etc.) after the product is exposed to any temperature up to 45°C, sometimes to any temperature between 4°C and 32°C or 45°C.

[0101] In some embodiments, the modified protein has a PI value between 7.8 and 8.4.

[0102] In some other embodiments, the modified protein is characterized by at least one of the following being the same or improved relative to a reference protein: (1) folding kinetics, (2) a post-translational modification (e.g., glycosylation) pattern of the protein that is different from the reference protein.

[0103] In some embodiments, the modified protein has the same or higher folding kinetics relative to the reference protein. That is, the protein folding rate from the unfolded or partially folded structure is faster (as evaluated on a computer, for example, by molecular dynamics or by in vitro or in vivo experimental methods). Alternatively, faster folding kinetics also refers to slower unfolding kinetics in denaturation experiments, for example, by denaturant titration (e.g., guanidine chloride and / or high concentration urea) or other methods.

[0104] In some embodiments, the modified protein is characterized by the same or higher expression yield in the host organism being evaluated relative to a reference protein.

[0105] It is noteworthy that the shelf life and functional stability required of food and beverage products may be related to structural thermal stability and consist of different measurable components, for example pasteurization can be applied through different protocols, and thermal resistance and functional properties can be measured over very short or extended periods of time.

[0106] As described herein, in some examples, the reference protein is a MNEI protein.

[0107] In some embodiments, the amino acid substitution compared to MNEI includes amino acid S76.

[0108] In some embodiments, the modified protein is a MNEI protein having SEQ ID NO: 1, comprising a substitution in amino acid residue S76.

[0109] In some embodiments, the modified protein comprises amino acid substitutions S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A and has improved stability compared to SEQ ID NO: 1.

[0110] In some embodiments, the modified protein is represented by an amino acid represented by DM129 herein. In some embodiments, the modified protein is represented by an amino acid represented by DM147 herein. In some embodiments, the modified protein is represented by an amino acid represented by DM89 herein. In some embodiments, the modified protein is represented by an amino acid represented by DM658 herein. In some embodiments, the modified protein is represented by an amino acid represented by DM137 herein. In some embodiments, the modified protein is represented by an amino acid represented by DM114 herein. In some embodiments, the modified protein is represented by an amino acid represented by DM606 herein. In some embodiments, the modified protein is represented by an amino acid represented by DM663 herein. In some embodiments, the modified protein is represented by an amino acid represented by DM659 herein. In some embodiments, the modified protein is represented by an amino acid represented by DM690 herein.

[0111] In some embodiments, the modified protein comprises the amino acid substitution S76F or S76Y, having improved stability compared to SEQ ID NO: 1. In some embodiments, the modified protein is represented by the amino acid denoted DM129 herein. In some embodiments, the modified protein is represented by the amino acid denoted DM147 herein.

[0112] As can be seen from Tables 2 and 4, modification of amino acid S76 increases the Tm value. For example, the experimental Tm value of DM89 is 101°C, the experimental Tm value of DM137 is 97°C, the experimental Tm value of DM114 is 101.5°C, and the experimental Tm value of DM89 is 101°C. In addition, the predicted Tm value of DM658 is 96°C, the predicted Tm value of DM663 is 98°C, the predicted Tm value of DM664 is 99°C, the predicted Tm value of DM659 is 93.6°C, and the predicted Tm value of DM690 is 98.5°C.

[0113] Based on these results, it was shown that different modifications in this amino acid are highly effective as they significantly increase the stability of the protein.

[0114] As shown in Table 2, substitution of S76 and at least one additional amino acid results in a modified protein with at least improved stability.

[0115] In some embodiments, the modified protein is a MNEI protein having SEQ ID NO: 1, comprising a substitution in amino acid residue S76 and at least one amino acid substitution.

[0116] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having a substitution in at least amino acid S76 compared to SEQ ID NO: 1 and having an additional modification in at least one amino acid residue of SEQ ID NO: 1, wherein the modified protein has at least improved stability compared to SEQ ID NO: 1.

[0117] According to some aspects, the present disclosure provides a modified protein comprising an amino acid sequence having a substitution in at least amino acid S76 compared to SEQ ID NO: 1 and having an additional modification in at least one amino acid residue of SEQ ID NO: 1, wherein at least the additional modification is in at least one of (i) an alpha helix of the reference protein, (ii) the core of the reference protein, (iii) the sweet loop of the reference protein, (iv) a linker region of the reference protein, or (v) any combination thereof.

[0118] According to some aspects, the present disclosure provides a modified protein comprising an amino acid sequence having a substitution in at least amino acid S76 compared to SEQ ID NO: 1 and having an additional modification in at least one amino acid residue of SEQ ID NO: 1, wherein at least the additional modification is a modification that stabilizes the alpha helical structure and the helical ends, and wherein the modified protein has at least improved stability compared to SEQ ID NO: 1.

[0119] As used herein, the term stabilizing the alpha helical structure refers to any modification that affects the helical structure of MNEI. Such modifications include modifications in any of the amino acid residues K25, I26, Q28 of MNEI.

[0120] In some embodiments, the modified protein is an MNEI protein having SEQ ID NO: 1, comprising a substitution in amino acid residue S76 and comprising at least one additional substitution in at least one amino acid residue K25, I26, Q28, or any combination thereof.

[0121] In some embodiments, the modified protein is a MNEI protein having SEQ ID NO: 1, comprising a substitution in amino acid residue S76 and comprising at least one additional substitution in at least one amino acid residue E23.

[0122] According to some aspects, the present disclosure provides a modified protein comprising an amino acid sequence having a substitution in at least amino acid S76 compared to SEQ ID NO: 1 and having an additional modification in at least one amino acid residue of SEQ ID NO: 1, wherein at least the additional modification is a modification of a stabilizing linker region and wherein the modified protein has at least one improved food-related property compared to SEQ ID NO: 1.

[0123] The term stabilizing linker region as used herein refers to any modification affecting the linker region of MNEI.

[0124] In some embodiments, the modified protein is a MNEI protein having SEQ ID NO: 1, comprising a substitution in amino acid residue S76 and a deletion in at least one of E50, F52, R53, or any combination thereof.

[0125] According to some aspects, the present disclosure provides a modified protein comprising an amino acid sequence having a substitution in at least amino acid S76 compared to SEQ ID NO: 1 and having an additional modification in at least one amino acid residue of SEQ ID NO: 1, wherein at least the additional modification is a modification that affects aggregation and wherein the modified protein has at least improved stability compared to SEQ ID NO: 1. In some embodiments, at least one modification is a modification that reduces protein aggregation.

[0126] As used herein, the term reducing protein aggregation refers to any modification that affects protein aggregation. It has been shown that the two chains of MNEI can form a disulfide bond between two cysteine ​​residues (C41). Therefore, such modifications include modifications in amino acid residue C41.

[0127] In some embodiments, the modified protein is an MNEI protein having SEQ ID NO: 1, comprising a substitution in amino acid residue S76 and comprising at least one additional substitution in C41. Based on computational analysis, it has been shown that C41 is prone to disulfide bonds and promotes aggregation, so any modification in this residue can reduce protein aggregation.

[0128] According to some aspects, the present disclosure provides a modified protein comprising an amino acid sequence having a substitution in at least amino acid S76 compared to SEQ ID NO: 1 and having an additional modification in at least one amino acid residue of SEQ ID NO: 1, wherein at least the additional modification is a modification that affects core repack and wherein the modified protein has at least improved stability compared to SEQ ID NO: 1.

[0129] The term core reassembly as used herein refers to any modification that affects core reassembly and thus reduces accessibility to water, thereby stabilizing the modified protein. It is suggested that there are holes that make the core accessible to surface water (the protein is small, so the core can be half exposed).

[0130] The MNEI core is partially (half) exposed to the surface and is susceptible to water interference, and therefore the proposed modification stabilizes the protein. Such modifications include modifications in at least one amino acid residue T12, C41, A19, V20, V64, A73, I75, F89 or any combination thereof.

[0131] In some embodiments, the modified protein is an MNEI protein having SEQ ID NO: 1, comprising a substitution in amino acid residue S76 and comprising a substitution in at least one amino acid residue T12, C41, A19, V20, V64, A73, I75, F89, or any combination thereof.

[0132] According to some aspects, the present disclosure provides a modified protein comprising an amino acid sequence having a substitution in at least amino acid S76 compared to SEQ ID NO: 1 and having an additional modification in at least one amino acid residue of SEQ ID NO: 1, wherein at least the additional modification is a modification affecting electrostatics and hydrophobicity, and wherein the modified protein has at least improved stability compared to SEQ ID NO: 1.

[0133] The term electrostatics as used herein refers to any modification that affects electrostatics. Such modifications include modifications in at least one amino acid residue T33, E4, or any combination thereof.

[0134] In some embodiments, the modified protein is a MNEI protein having SEQ ID NO: 1, comprising a substitution in amino acid residue S76 and comprising a substitution in at least one amino acid residue T33, E4, or any combination thereof.

[0135] In some embodiments, the modified protein is a MNEI protein having SEQ ID NO: 1, comprising a substitution in amino acid residue S76 and comprising a substitution in at least one of E2, Y65, D68, or any combination thereof.

[0136] In some embodiments, the modified protein is a MNEI protein having SEQ ID NO: 1, comprising a substitution in amino acid residue S76 and comprising a substitution in at least one of R31, Q61, N35, or any combination thereof.

[0137] In some embodiments, the modified protein is a MNEI protein having SEQ ID NO: 1, comprising a substitution in amino acid residue S76 and comprising a substitution in at least R84.

[0138] In some embodiments, the modified protein is an MNEI protein having SEQ ID NO: 1, comprising a substitution in amino acid residue S76 and comprising a modification in at least one amino acid residue selected from E23, K25, I26, Q28, E50, F52, R53, C41, T12, C41, A19, V20, A73, I75, F89, T33, E4, E2, Y65, D68, R31, Q61, N35, R84, V64, or any combination thereof.

[0139] In some embodiments, the modified protein is an MNEI protein having SEQ ID NO: 1, comprising a substitution in amino acid residue S76 and at least one amino acid substitution in at least one residue selected from E23, K25, I26, Q28, T12, C41, A19, V20, V64, A73, I75, F89, E2, Y65, D68, R84, T33, E4, R31, Q61, N35, or any combination thereof.

[0140] In some embodiments, the modified protein is an MNEI protein having SEQ ID NO: 1, comprising a substitution in amino acid residue S76 and at least one amino acid substitution in E23, K25, I26, Q28, or any combination thereof.

[0141] In some embodiments, the modified protein is an MNEI protein having SEQ ID NO: 1, comprising a substitution in amino acid residue S76 and at least one amino acid substitution in T12, C41, A19, V20, V64, A73, I75, F89, or any combination thereof.

[0142] In some embodiments, the modified protein is a MNEI protein having SEQ ID NO: 1, comprising a substitution in amino acid residue S76 and at least one amino acid substitution in E2, Y65, D68, or any combination thereof.

[0143] As shown in Table 2, computational analysis indicated that substitution of S76 and at least one other amino acid in the amino acid sequence of MNEI resulted in a modified protein having at least improved stability compared to MNEI.

[0144] In some embodiments, the modified protein comprises a substitution in amino acid S76 and at least one amino acid substitution compared to MNEI, wherein the modified protein has at least improved stability compared to MNEI.

[0145] In some embodiments, the amino acid substitutions include amino acids S76 and R84. In some embodiments, the modified protein comprises an amino acid sequence having at least two amino acid substitutions in at least amino acids R84 and S76 compared to SEQ ID NO: 1.

[0146] In some embodiments, the modified protein having at least two amino acid substitutions R84L and S76Y has improved stability compared to SEQ ID NO: 1. In some embodiments, the modified protein is represented by the amino acid denoted herein as DM298.

[0147] The main difference between wild-type mebutin and MNEI is the region where the two mebutin subunits are linked into a single chain mebutin called MNEI. The amino acid sequence between amino acid T46 and amino acid 156 represents the loop region linking the two mebutin subunits.

[0148] As shown herein, CPD analysis has identified specific amino acid residues / regions in MNEI proteins that can be modified to obtain modified proteins with improved properties, including, inter alia, stability and taste.

[0149] For example, MD simulations of MNEI have shown that the β-fold of MNEI is less stable. Without being bound by theory, it is shown that the loops of β, especially the loops between 2 and 3 of β strands, do not have a defined secondary structure, and their movement reduces stability. In addition, it is shown that the main chain of β is exposed to water. Simulation analysis of MNEI at high temperature shows that the main weakness of MNEI is the interface between strands 3 and 4 and the edges of the main chain. The main chain atoms are exposed to water. The simulation also shows that water enters and interferes with hydrogen bonds in this area.

[0150] As shown in the examples below, the modification in the loop region of the modified protein increases the sweetness and / or stability of the reference protein. Specifically, the deletion of amino acid residues in the protein loop and β strand edge produces a modified protein that is stable compared to the reference protein (in some examples, MNEI).

[0151] In some embodiments, the modified protein comprises modifications in amino acids located in the linker (loop) region compared to SEQ ID NO: 1.

[0152] In some embodiments, the modified protein is a MNEI protein having SEQ ID NO: 1, comprising a substitution in amino acid residue S76 and at least one amino acid deletion.

[0153] In some embodiments, the modified protein comprises a modification in one or more amino acids located between amino acid T46 and amino acid 156 compared to SEQ ID NO: 1.

[0154] In some embodiments, the amino acid sequence of the reference protein can be modified in the linker region of the reference protein.

[0155] The amino acid sequence of the reference protein may be modified in the linker region of the reference protein such that at least one amino acid is deleted.

[0156] A modified protein comprises at least one, sometimes at least two, sometimes at least three amino acid deletions from a reference protein.

[0157] Therefore, according to some aspects, the present disclosure provides a modified protein having an amino acid sequence comprising at least one amino acid deletion and at least one amino acid substitution compared to a reference protein, wherein the reference protein is represented by the amino acid sequence shown in SEQ ID NO:1, wherein the at least one amino acid deletion is at one or more amino acids located between amino acid T46 and amino acid I56 of the reference protein, and wherein the at least one amino acid substitution is at amino acid S76 of the reference protein.

[0158] As described above, in some examples, the reference protein is MNEI represented herein by SEQ ID NO:1.

[0159] Therefore, according to some aspects, the present disclosure provides a modified protein comprising an amino acid sequence having an amino acid deletion in at least three amino acids between amino acid T46 and amino acid I56 compared to a reference protein, and wherein the reference protein has the amino acid sequence as shown in SEQ ID NO: 1.

[0160] As shown in the Examples below, which form a part of this patent application, the deletion of three amino acids E50, F52 and R53 from the amino acid sequence of MNEI (DM29, represented herein as SEQ ID NO: 33) indicates that the modified protein will have increased stability based on a REU value of -319.65 compared to a REU value of -315.35 for MNEI. The deletion of amino acids E50, F52 and R53 is sometimes referred to herein as ΔE50, ΔF52 and ΔR53.

[0161] In some embodiments, at least one amino acid is deleted at one or more of E50, F52, R53, or any combination thereof of SEQ ID NO: 1.

[0162] Therefore, according to some aspects, the present disclosure provides a modified protein having an amino acid sequence comprising at least one amino acid deletion and at least one amino acid substitution compared to a reference protein, wherein the reference protein is represented by the amino acid sequence shown in SEQ ID NO: 1, wherein the at least one amino acid deletion is at one or more of at least amino acids E50, F52 and R53 of the reference protein, and wherein the at least one amino acid substitution is at amino acid S76 of the reference protein.

[0163] As shown in Table 2 below, the combination of an amino acid substitution at S76 and an amino acid deletion in at least amino acids E50, F52, and R53 produces a modified protein with at least improved stability compared to SEQ ID NO: 1. In some embodiments, the modified protein comprises an amino acid substitution S76Y and an amino acid deletion in amino acids E50, F52, and R53 compared to SEQ ID NO: 1, and has improved stability compared to SEQ ID NO: 1. In some embodiments, the modified protein is represented by the amino acid denoted herein as DM148.

[0164] As shown in Table 2 below, substitution of at least amino acid residue S76 of the MNEI protein in combination with other amino acid substitutions in the protein and with amino acid deletions as described herein significantly increased protein stability as demonstrated by computational modeling and optimization approaches.

[0165] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R53, an amino acid substitution in S76, and at least one amino acid substitution in one or more of G1, E2, E4, T12, A19, V20, E23, K25, I26, G27, Q28, T33, N35, C41, Q61, V64, Y65, D68, L70, A73, I75, R84, F89, or any combination thereof.

[0166] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R53, an amino acid substitution in S76, and at least one amino acid substitution in one or more of K25, I26, Q28, C41, E23, D68, E2, L70, Y65, E4, G1, or any combination thereof.

[0167] In some embodiments, the modified protein comprises the amino acid substitution S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H or S76A, and has improved stability compared to SEQ ID NO: 1. In some embodiments, the modified protein comprises the amino acid substitution S76F or S76Y, and has improved stability compared to SEQ ID NO: 1.

[0168] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R53, an amino acid substitution in S76, and at least one amino acid substitution of one or more of: (i) K25R, (ii) at least one of I26W, I26V, I26T, or any combination thereof, (iii) at least one of Q28K, Q28R, Q28S, Q28E, or any combination thereof, (iv) at least one of C41A, C41V, C41T, C41S, or any combination thereof, (v) E23A or E23Q, (vi) at least one of D68N or D68T, (vii) E2M or E2N, (viii) L70I, (ix) Y65R, (x) E4Q, (xi) G1M, or (xii) any combination thereof. In some embodiments, the amino acid substitution in S76 comprises one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H or S76A. In some embodiments, the amino acid substitution in S76 is S76Y.

[0169] In some embodiments, the amino acid substitutions include deletions in amino acids S76, Q28 and E23 and amino acids E50, F52 and R53 compared to SEQ ID NO: 1. In some embodiments, the modified protein has at least three amino acid substitutions E23A, Q28K and S76Y and deletions in amino acids E50, F52 and R53 compared to SEQ ID NO: 1, and has improved stability compared to SEQ ID NO: 1. In some embodiments, the modified protein is represented by the amino acid denoted herein as DM335.

[0170] In some embodiments, the amino acid substitutions include deletions in amino acids S76, K25, D68, E2, L70, and E23 and amino acids E50, F52, and R53 compared to SEQ ID NO: 1. In some embodiments, the modified protein has at least amino acid substitutions S76Y, K25R, D68N, E2N, L70I, and E23A and deletions in amino acids E50, F52, and R53 compared to SEQ ID NO: 1, and has improved stability compared to SEQ ID NO: 1. In some embodiments, the modified protein is represented by the amino acid denoted herein as DM480.

[0171] In some embodiments, the amino acid substitutions include deletions in amino acids S76, D68, and E23 and amino acids E50, F52, and R53 compared to SEQ ID NO: 1. In some embodiments, the modified protein has at least amino acid substitutions S76Y, D68N, and E23A and deletions in amino acids E50, F52, and R53 compared to SEQ ID NO: 1, and has improved stability compared to SEQ ID NO: 1. In some embodiments, the modified protein is represented by the amino acid denoted herein as DM362.

[0172] In some embodiments, the amino acid substitutions include deletions in amino acids S76, E2 and E23 and amino acids E50, F52 and R53 compared to SEQ ID NO: 1. In some embodiments, the modified protein has at least amino acid substitutions S76Y, E2N and E23A and deletions in amino acids E50, F52 and R53 compared to SEQ ID NO: 1, and has improved stability compared to SEQ ID NO: 1. In some embodiments, the modified protein is represented by the amino acid denoted herein as DM364.

[0173] In some embodiments, the amino acid substitutions include deletions in amino acids S76, E23, C41, Y65, and amino acids E50, F52, and R53 compared to SEQ ID NO: 1. In some embodiments, the modified protein has at least amino acid substitutions S76Y, E23A, C41A, Y65R, and deletions in amino acids E50, F52, and R53 compared to SEQ ID NO: 1, and has improved stability compared to SEQ ID NO: 1. In some embodiments, the modified protein is represented by the amino acid denoted herein as DM126.

[0174] In some embodiments, the amino acid substitutions include deletions in amino acids S76, E4 and E23 and amino acids E50, F52 and R53 compared to SEQ ID NO: 1. In some embodiments, the modified protein has at least amino acid substitutions S76Y, E23A, E4Q and deletions in amino acids E50, F52 and R53 compared to SEQ ID NO: 1 and has improved stability compared to SEQ ID NO: 1. In some embodiments, the modified protein is represented by the amino acid denoted herein as DM365.

[0175] In some embodiments, the amino acid substitutions include deletions in amino acids S76, D68, E2, and E23 and amino acids E50, F52, and R53 compared to SEQ ID NO: 1. In some embodiments, the modified protein has at least amino acid substitutions S76Y, D68N, E2N, E23A and deletions in amino acids E50, F52, and R53 compared to SEQ ID NO: 1, and has improved stability compared to SEQ ID NO: 1. In some embodiments, the modified protein is represented by the amino acid denoted herein as DM397.

[0176] In some embodiments, the amino acid substitutions include deletions in amino acids S76, E23, Y65, and L70 and amino acids E50, F52, and R53 compared to SEQ ID NO: 1. In some embodiments, the modified protein has at least amino acid substitutions S76Y, E23A, Y65R, L70I and deletions in amino acids E50, F52, and R53 compared to SEQ ID NO: 1, and has improved stability compared to SEQ ID NO: 1. In some embodiments, the modified protein is represented by the amino acid denoted herein as DM122.

[0177] In some embodiments, the amino acid substitutions include deletions in amino acids S76, E2, E23, and Y65 and amino acids E50, F52, and R53 compared to SEQ ID NO: 1. In some embodiments, the modified protein has at least amino acid substitutions S76Y, E2N, E23A, Y65R and deletions in amino acids E50, F52, and R53 compared to SEQ ID NO: 1, and has improved stability compared to SEQ ID NO: 1. In some embodiments, the modified protein is represented by the amino acid denoted herein as DM124.

[0178] In some embodiments, the amino acid substitutions include deletions in amino acids S76, E2, E23, and L70 and amino acids E50, F52, and R53 compared to SEQ ID NO: 1. In some embodiments, the modified protein has at least amino acid substitutions S76Y, E2N, E23A, L70I and deletions in amino acids E50, F52, and R53 compared to SEQ ID NO: 1, and has improved stability compared to SEQ ID NO: 1. In some embodiments, the modified protein is represented by the amino acid represented herein as DM123.

[0179] In some embodiments, the amino acid substitutions include deletions in amino acids S76, D68, E2, E23, and L70 and amino acids E50, F52, and R53 compared to SEQ ID NO: 1. In some embodiments, the modified protein has at least amino acid substitutions S76Y, D68N, E2N, E23A, L70I and deletions in amino acids E50, F52, and R53 compared to SEQ ID NO: 1, and has improved stability compared to SEQ ID NO: 1. In some embodiments, the modified protein is represented by the amino acid denoted herein as DM361.

[0180] In some embodiments, the amino acid substitutions include deletions in amino acids S76, Q28, E2, E23, and L70 and amino acids E50, F52, and R53 compared to SEQ ID NO: 1. In some embodiments, the modified protein has at least amino acid substitutions S76Y, Q28K, E2N, E23A, L70I and deletions in amino acids E50, F52, and R53 compared to SEQ ID NO: 1, and has improved stability compared to SEQ ID NO: 1. In some embodiments, the modified protein is represented by the amino acid denoted herein as DM473.

[0181] In some embodiments, the amino acid substitutions include deletions in amino acids S76, Q28, D68, E2, E23, and L70 and amino acids E50, F52, and R53 compared to SEQ ID NO: 1. In some embodiments, the modified protein has at least amino acid substitutions S76Y, Q28R, D68N, E2N, E23A, L70I and deletions in amino acids E50, F52, and R53 compared to SEQ ID NO: 1, and has improved stability compared to SEQ ID NO: 1. In some embodiments, the modified protein is represented by the amino acid denoted herein as DM521.

[0182] In some embodiments, the amino acid substitutions include deletions in amino acids S76, C41, D68, E2, E23, and L70 and amino acids E50, F52, and R53 compared to SEQ ID NO: 1. In some embodiments, the modified protein has at least amino acid substitutions S76Y, C41T, D68N, E2N, E23A, L70I and deletions in amino acids E50, F52, and R53 compared to SEQ ID NO: 1, and has improved stability compared to SEQ ID NO: 1. In some embodiments, the modified protein is represented by the amino acid denoted herein as DM477.

[0183] In some embodiments, the amino acid substitutions include deletions in amino acids S76, I26, Q28, D68, E2, E23, and L70 and amino acids E50, F52, and R53 compared to SEQ ID NO: 1. In some embodiments, the modified protein has at least amino acid substitutions S76Y, I26T, Q28K, D68N, E2N, E23A, L70I and deletions in amino acids E50, F52, and R53 compared to SEQ ID NO: 1, and has improved stability compared to SEQ ID NO: 1. In some embodiments, the modified protein is represented by the amino acid denoted herein as DM486.

[0184] In some embodiments, the amino acid substitutions include amino acid S76, Q28, E23 and deletions in amino acids E50, F52 and R53 compared to SEQ ID NO: 1. In some embodiments, the modified protein has at least amino acid substitutions S76Y, Q28K, E23Q and deletions in amino acids E50, F52 and R53 compared to SEQ ID NO: 1, and has improved stability compared to SEQ ID NO: 1. In some embodiments, the modified protein is represented by the amino acid denoted herein as DM334.

[0185] In some embodiments, the amino acid substitutions include amino acid S76, D68, and deletions in amino acids E50, F52, and R53 compared to SEQ ID NO: 1. In some embodiments, the modified protein has at least amino acid substitutions S76Y, D68N, and deletions in amino acids E50, F52, and R53 compared to SEQ ID NO: 1, and has improved stability compared to SEQ ID NO: 1. In some embodiments, the modified protein is represented by the amino acid denoted herein as DM333.

[0186] In some embodiments, the amino acid substitutions include amino acid S76, E2, and deletions in amino acids E50, F52, and R53 compared to SEQ ID NO: 1. In some embodiments, the modified protein has at least amino acid substitutions S76Y, E2N, and deletions in amino acids E50, F52, and R53 compared to SEQ ID NO: 1, and has improved stability compared to SEQ ID NO: 1. In some embodiments, the modified protein is represented by the amino acid denoted herein as DM338.

[0187] In some embodiments, the amino acid substitutions include amino acid S76, L70, and deletions in amino acids E50, F52, and R53 compared to SEQ ID NO: 1. In some embodiments, the modified protein has at least amino acid substitutions S76Y, L70I, and deletions in amino acids E50, F52, and R53 compared to SEQ ID NO: 1, and has improved stability compared to SEQ ID NO: 1. In some embodiments, the modified protein is represented by the amino acid denoted herein as DM125.

[0188] In some embodiments, the amino acid substitutions include amino acid S76, E4, and deletions in amino acids E50, F52, and R53 compared to SEQ ID NO: 1. In some embodiments, the modified protein has at least amino acid substitutions S76Y, E4Q, and deletions in amino acids E50, F52, and R53 compared to SEQ ID NO: 1, and has improved stability compared to SEQ ID NO: 1. In some embodiments, the modified protein is represented by the amino acid denoted herein as DM337.

[0189] In some embodiments, the amino acid substitutions include amino acid S76, G1, E2, and deletions in amino acids E50, F52, and R53 compared to SEQ ID NO: 1. In some embodiments, the modified protein has at least amino acid substitutions S76Y, G1M, E2M, and deletions in amino acids E50, F52, and R53 compared to SEQ ID NO: 1, and has improved stability compared to SEQ ID NO: 1. In some embodiments, the modified protein is represented by the amino acid denoted herein as DM332.

[0190] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R53, an amino acid substitution in S76, and at least one amino acid substitution in one or more of E2, E23, Y65, L70.

[0191] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R53 and an amino acid substitution in at least E2, E23, Y65, L70, and S76 compared to MNEI (SEQ ID NO: 1).

[0192] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R5 and amino acid substitutions in at least E2, E23, Y65, L70, and S76 compared to MNEI (SEQ ID NO: 1), the amino acid substitution in E2 is E2M or E2N, the amino acid substitution in E23 is E23A or E23Q, the amino acid substitution in Y65 is Y65R, the amino acid substitution in L70 is L70I, and the amino acid substitution in S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A.

[0193] In some examples, the modified protein comprises a deletion of amino acids E50, F52 and R54 and an amino acid substitution in at least E2M, E23A, Y65R, L70I and S76Y compared to MNEI (SEQ ID NO: 1). In some examples, the modified protein comprises a deletion of amino acids E50, F52 and R54 and an amino acid substitution in E2M, E23A, Y65R, L70I and S76Y compared to MNEI (SEQ ID NO: 1), represented herein as DM396.

[0194] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and an amino acid substitution in E2, E23, Y65, L70, and S76, and in one or more amino acids G1, A19, V20, K25, I26, Q28, R31, T33, N35, C41, E59, D68, A73, D74, I75, E77, R84, compared to MNEI (SEQ ID NO: 1).

[0195] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and an amino acid substitution in E2, E23, Y65, L70, and S76, and in one or more amino acids G1, E2, A19, V20, K25, I26, Q28, R31, T33, N35, C41, E59, D68, A73, D74, I75, E77, R84, compared to MNEI (SEQ ID NO: 1). the amino acid substitution in E2 is E2M or E2N, the amino acid substitution in E23 is E23A or E23Q, the amino acid substitution in Y65 is Y65R, the amino acid substitution in L70 is L70I, the amino acid substitution in S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H or S76A, the amino acid substitution in G1 is G1M, the amino acid substitution in A19 is A19V, the amino acid substitution in V20 is V20I, the amino acid substitution in K25 is K25R, the amino acid substitution in I26 is one or more of I26W, I26V, I26T, the amino acid substitution in Q28 is Q28R, Q28K, Q28S, Q28 the amino acid substitution in R31 is R31T, the amino acid substitution in T33 is T33R, the amino acid substitution in N35 is N35T, the amino acid substitution in C41 is one or more of C41A, C41V, C41T, C41S, the amino acid substitution in E59 is one or more of E59T, E59V, E59I, E59Y, E59F, E59W, E59R, the amino acid substitution in D68 is D68N or D68T, the amino acid substitution in A73 is A73F or A73V, the amino acid substitution in D74 is D74V, the amino acid substitution in I75 is I75V or I75L, the amino acid substitution in E77 is E77V, and the amino acid substitution in R84 is R84L.

[0196] In some examples, compared to SEQ ID NO: 1, the modified protein comprises a deletion of amino acids E50, F52 and R54 and an amino acid substitution in at least G1, E2, E23, Y65, L70, Y76. In some examples, compared to SEQ ID NO: 1, the modified protein comprises a deletion of amino acids E50, F52 and R5 and an amino acid substitution in at least G1M, E2M, E23A, Y65R, L70I, S76Y. In some examples, compared to SEQ ID NO: 1, the modified protein comprises a deletion of amino acids E50, F52 and R5 and an amino acid substitution in at least G1M, E2M, E23A, Y65R, L70I, S76Y. In some examples, compared to SEQ ID NO: 1, the modified protein comprises a deletion of amino acids E50, F52 and R5 and an amino acid substitution in G1M, E2M, E23A, Y65R, L70I, S76Y. In some examples, the modified protein is represented by SEQ ID NO: 360.

[0197] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54 and an amino acid substitution in at least E2, E23, Y65, and L70 compared to SEQ ID NO: 1. In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R5 and an amino acid substitution in at least E2N, E23A, Y65R, and L70I compared to SEQ ID NO: 1.

[0198] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54 and an amino acid substitution in at least E2N, E23A, Y65R, L70I, and S76Y compared to MNEI (SEQ ID NO: 1).

[0199] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54 and amino acid substitutions in at least E2N, E23A, Y65R, L70I compared to SEQ ID NO: 1, represented herein as DM31 and represented herein by SEQ ID NO: 34.

[0200] According to some aspects, a modified protein is provided having an amino acid sequence comprising at least one amino acid deletion and at least one amino acid substitution compared to a reference protein, wherein the reference protein is represented by the amino acid sequence shown in SEQ ID NO:1, wherein the at least one amino acid deletion is at one or more amino acids E50, F52 and R53 of the reference protein, and wherein the at least one amino acid substitution is at at least amino acids E2, E23, Y65, L70, S76 of the reference protein.

[0201] According to some aspects, a modified protein having an amino acid sequence comprising at least one amino acid deletion and at least one amino acid substitution compared to a reference protein is provided, wherein the reference protein is represented by the amino acid sequence shown in SEQ ID NO: 1, wherein the at least one amino acid deletion is at amino acids E50, F52 and R53 of the reference protein, and wherein the at least one amino acid substitution is in amino acids E2, E23, Y65, L70, S76 of the reference protein. In some examples, the amino acid substitution in S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H or S76A.

[0202] According to some aspects, a modified protein having an amino acid sequence comprising at least one amino acid deletion and at least one amino acid substitution compared to a reference protein is provided, wherein the reference protein is represented by the amino acid sequence shown in SEQ ID NO: 1, wherein the at least one amino acid deletion is at amino acids E50, F52, and R53 of the reference protein, and wherein the at least one amino acid substitution is at least E2N, E23A, Y65R, L70I of the reference protein and at S76. In some examples, the amino acid substitution in S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A.

[0203] As shown in the Examples below, substitution of amino acid S76 in DM31 improves stability compared to DM31.

[0204] In some examples, the modified protein comprises an amino acid sequence having amino acid deletions in amino acids E50, F52 and R54 and amino acid substitutions in E2N, E23A, Y65R, L70I, S76Y compared to SEQ ID NO: 1. In some examples, the modified protein is represented herein as DM89.

[0205] In some examples, the modified protein comprises an amino acid sequence having amino acid deletions in amino acids E50, F52 and R54 and amino acid substitutions in E2N, E23A, Y65R, L70I, S76F compared to SEQ ID NO: 1. In some examples, the modified protein is represented herein as DM114.

[0206] In some examples, the modified protein comprises an amino acid sequence having amino acid deletions in amino acids E50, F52 and R54 and amino acid substitutions in E2N, E23A, Y65R, L70I, S76W compared to SEQ ID NO: 1. In some examples, the modified protein is represented herein as DM137.

[0207] In some examples, the modified protein comprises an amino acid sequence having amino acid deletions in amino acids E50, F52 and R54 and amino acid substitutions in E2N, E23A, Y65R, L70I, S76T compared to SEQ ID NO: 1. In some examples, the modified protein is represented herein as DM658.

[0208] In some examples, the modified protein comprises an amino acid sequence having amino acid deletions in amino acids E50, F52 and R54 and amino acid substitutions in E2N, E23A, Y65R, L70I, S76R compared to SEQ ID NO: 1. In some examples, the modified protein is represented herein as DM606.

[0209] In some examples, the modified protein comprises an amino acid sequence having amino acid deletions in amino acids E50, F52 and R54 and amino acid substitutions in E2N, E23A, Y65R, L70I, S76I compared to SEQ ID NO: 1. In some examples, the modified protein is represented herein as DM664.

[0210] In some examples, the modified protein comprises an amino acid sequence having amino acid deletions in amino acids E50, F52 and R54 and amino acid substitutions in E2N, E23A, Y65R, L70I, S76V compared to SEQ ID NO: 1. In some examples, the modified protein is represented herein as DM663.

[0211] In some examples, the modified protein comprises an amino acid sequence having amino acid deletions in amino acids E50, F52 and R54 and amino acid substitutions in E2N, E23A, Y65R, L70I, S76H compared to SEQ ID NO: 1. In some examples, the modified protein is represented herein as DM659.

[0212] In some examples, the modified protein comprises an amino acid sequence having amino acid deletions in amino acids E50, F52 and R54 and amino acid substitutions in E2N, E23A, Y65R, L70I, S76A compared to SEQ ID NO: 1. In some examples, the modified protein is represented herein as DM690.

[0213] In some examples, the modified protein is represented by one or more of DM89, DM114, DM137, DM658, DM606, DM664, DM663, DM659, or DM690. In some examples, the modified protein is represented by one or more of DM89, DM114, or DM137.

[0214] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and an amino acid substitution in E2N, E23A, Y65R, L70I, and S76, and in one or more amino acids G1, A19, V20, K25, I26, Q28, R31, T33, N35, C41, E59, D68, A73, D74, I75, E77, R84, compared to MNEI (SEQ ID NO: 1).

[0215] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52, and R54, and an amino acid substitution in E2N, E23A, Y65R, L70I, and S76, and in one or more amino acids G1, E2, A19, V20, K25, I26, Q28, R31, T33, N35, C41, E59, D68, A73, D74, I75, E77, R84, S76. The amino acid substitution in G1 is G1M, the amino acid substitution in A19 is A19V, the amino acid substitution in V20 is V20I, the amino acid substitution in K25 is K25R, the amino acid substitution in I26 is I26W, I26V, I26T the amino acid substitution in Q28 is one or more of Q28R, Q28K, Q28S, Q28E, the amino acid substitution in R31 is R31T, the amino acid substitution in T33 is T33R, the amino acid substitution in N35 is N35T, the amino acid substitution in C41 is one or more of C41A, C41V, C41T, C41S, the amino acid substitution in E59 is one or more of E59T, E59V, E59I, E59Y, E59F, E59W, E59R, the amino acid substitution in D68 is D68N or D68T, the amino acid substitution in A73 is A73F or A73V, the amino acid substitution in D74 is D74V, the amino acid substitution in I75 is I75V or I75L, the amino acid substitution in E77 is E77V, and the amino acid substitution in R84 is R84L.

[0216] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and one or more of amino acids G1, E2, A19, V20, K25, I26, Q28, R31, T33, N35, C41, E59, D68, A73, D74, I75, E77, R84 in S76. The amino acid substitution in S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H or S76A, the amino acid substitution in G1 is G1M, the amino acid substitution in A19 is A19V, the amino acid substitution in V20 is V20I, the amino acid substitution in K25 is K25R, the amino acid substitution in I26 is I26W, I26V, I 26T, the amino acid substitution in Q28 is one or more of Q28R, Q28K, Q28S, Q28E, the amino acid substitution in R31 is R31T, the amino acid substitution in T33 is T33R, the amino acid substitution in N35 is N35T, the amino acid substitution in C41 is one or more of C41A, C41V, C41T, C41S, the amino acid substitution in E59 is E59 T, E59V, E59I, E59Y, E59F, E59W, and E59R, the amino acid substitution in D68 is D68N or D68T, the amino acid substitution in A73 is A73F or A73V, the amino acid substitution in D74 is D74V, the amino acid substitution in I75 is I75V or I75L, the amino acid substitution in E77 is E77V, and the amino acid substitution in R84 is R84L.

[0217] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein amino acids include deletions of E50, F52, and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in S76, A19 and one or more amino acids G1, E2, A19, V20, K25, I26, Q28, R31, T33, N35, C41, E59, D68, A73, D74, I75, E77, R84. The amino acid substitution in S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H or S76A, the amino acid substitution in G1 is G1M, the amino acid substitution in A19 is A19V, the amino acid substitution in V20 is V20I, the amino acid substitution in K25 is K25R, the amino acid substitution in I26 is I26W, I26V , I26T, the amino acid substitution in Q28 is one or more of Q28R, Q28K, Q28S, Q28E, the amino acid substitution in R31 is R31T, the amino acid substitution in T33 is T33R, the amino acid substitution in N35 is N35T, the amino acid substitution in C41 is one or more of C41A, C41V, C41T, C41S, the amino acid substitution in E59 is E5 the amino acid substitution in D68 is D68N or D68T, the amino acid substitution in A73 is A73F or A73V, the amino acid substitution in D74 is D74V, the amino acid substitution in I75 is I75V or I75L, the amino acid substitution in E77 is E77V, and the amino acid substitution in R84 is R84L.

[0218] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in S76, A19 and one or more amino acids Q28, T33, N35, R84, the amino acid substitution in S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, the amino acid substitution in A19 is A19V, the amino acid substitution in Q28 is one or more of Q28R, Q28K, Q28S, Q28E, the amino acid substitution in T33 is T33R, and the amino acid substitution in R84 is R84L.

[0219] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and substitutions in S76, V20, and one or more of amino acids G1, E2, A19, V20, K25, I26, Q28, R31, T33, N35, C41, E59, D68, A73, D74, I75, E77, R84. amino acid substitution, the amino acid substitution in S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H or S76A, the amino acid substitution in G1 is G1M, the amino acid substitution in A19 is A19V, the amino acid substitution in V20 is V20I, the amino acid substitution in K25 is K25R, the amino acid substitution in I26 is I26W, I26V , I26T, the amino acid substitution in Q28 is one or more of Q28R, Q28K, Q28S, Q28E, the amino acid substitution in R31 is R31T, the amino acid substitution in T33 is T33R, the amino acid substitution in N35 is N35T, the amino acid substitution in C41 is one or more of C41A, C41V, C41T, C41S, the amino acid substitution in E59 is E5 the amino acid substitution in D68 is D68N or D68T, the amino acid substitution in A73 is A73F or A73V, the amino acid substitution in D74 is D74V, the amino acid substitution in I75 is I75V or I75L, the amino acid substitution in E77 is E77V, and the amino acid substitution in R84 is R84L.

[0220] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in S76, V20 and one or more amino acids Q28, R31, T33, N35, D68, R84, the amino acid substitution in S76 is S76Y, S76F, S76T, S76W, S76R, S76V, S76I, one or more of S76H or S76A, the amino acid substitution in V20 is V20I, the amino acid substitution in Q28 is one or more of Q28R, Q28K, Q28S, Q28E, the amino acid substitution in R31 is R31T, the amino acid substitution in T33 is T33R, the amino acid substitution in N35 is N35T, the amino acid substitution in D68 is D68N or D68T, and the amino acid substitution in R84 is R84L.

[0221] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in S76 and K25 and one or more amino acids G1, E2, A19, V20, I26, Q28, R31, T33, N35, C41, E59, D68, A73, D74, I75, E77, R84. , the amino acid substitution in S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H or S76A, the amino acid substitution in G1 is G1M, the amino acid substitution in A19 is A19V, the amino acid substitution in V20 is V20I, the amino acid substitution in K25 is K25R, the amino acid substitution in I26 is I26W, I26V, I26I the amino acid substitution in R31 is R31T, the amino acid substitution in T33 is T33R, the amino acid substitution in N35 is N35T, the amino acid substitution in C41 is one or more of C41A, C41V, C41T, C41S, the amino acid substitution in E59 is E59T , one or more of E59V, E59I, E59Y, E59F, E59W, and E59R, the amino acid substitution in D68 is D68N or D68T, the amino acid substitution in A73 is A73F or A73V, the amino acid substitution in D74 is D74V, the amino acid substitution in I75 is I75V or I75L, the amino acid substitution in E77 is E77V, and the amino acid substitution in R84 is R84L.

[0222] In some examples, the same as MNEI (SEQ ID NO:1), the modified protein comprises a deletion of amino acids E50, F52 and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in S76 and K25 and in one or more amino acids Q28, T33, C41, D68, the amino acid substitution in S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H or S76A, the amino acid substitution in K25 is K25R, the amino acid substitution in Q28 is one or more of Q28R, Q28K, Q28S, Q28E, the amino acid substitution in T33 is T33R, the amino acid substitution in C41 is one or more of C41A, C41V, C41T, C41S, and the amino acid substitution in D68 is D68N or D68T.

[0223] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acids in S76, I26 and one or more of amino acids G1, E2, A19, V20, K25, Q28, R31, T33, N35, C41, E59, D68, A73, D74, I75, E77, R84. The amino acid substitution in S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H or S76A, the amino acid substitution in G1 is G1M, the amino acid substitution in A19 is A19V, the amino acid substitution in V20 is V20I, the amino acid substitution in K25 is K25R, the amino acid substitution in I26 is I26W, I26V, I 26T, the amino acid substitution in Q28 is one or more of Q28R, Q28K, Q28S, Q28E, the amino acid substitution in R31 is R31T, the amino acid substitution in T33 is T33R, the amino acid substitution in N35 is N35T, the amino acid substitution in C41 is one or more of C41A, C41V, C41T, C41S, the amino acid substitution in E59 is E59 T, E59V, E59I, E59Y, E59F, E59W, and E59R, the amino acid substitution in D68 is D68N or D68T, the amino acid substitution in A73 is A73F or A73V, the amino acid substitution in D74 is D74V, the amino acid substitution in I75 is I75V or I75L, the amino acid substitution in E77 is E77V, and the amino acid substitution in R84 is R84L.

[0224] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52, and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in S76, I26, and Q28, the amino acid substitution in S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, the amino acid substitution in I26 is one or more of I26W, I26V, I26T, and the amino acid substitution in Q28 is one or more of Q28R, Q28K, Q28S, Q28E.

[0225] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in S76 and Q28 and one or more amino acids G1, E2, A19, V20, K25, I26, R31, T33, N35, C41, E59, D68, A73, D74, I75, E77, R84. , the amino acid substitution in S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H or S76A, the amino acid substitution in G1 is G1M, the amino acid substitution in A19 is A19V, the amino acid substitution in V20 is V20I, the amino acid substitution in K25 is K25R, the amino acid substitution in I26 is I26W, I26V, I26I the amino acid substitution in R31 is R31T, the amino acid substitution in T33 is T33R, the amino acid substitution in N35 is N35T, the amino acid substitution in C41 is one or more of C41A, C41V, C41T, C41S, the amino acid substitution in E59 is E59T , one or more of E59V, E59I, E59Y, E59F, E59W, and E59R, the amino acid substitution in D68 is D68N or D68T, the amino acid substitution in A73 is A73F or A73V, the amino acid substitution in D74 is D74V, the amino acid substitution in I75 is I75V or I75L, the amino acid substitution in E77 is E77V, and the amino acid substitution in R84 is R84L.

[0226] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in S76 and Q28 and in one or more amino acids A19, V20, K25, I26, R31, T33, C41, D68, the amino acid substitution in S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, the amino acid substitution in A19 is A19V, the amino acid substitution in V20 is The amino acid substitution in R31 is R31T, the amino acid substitution in T33 is T33R, the amino acid substitution in N35 is N35T, the amino acid substitution in C41 is one or more of C41A, C41V, C41T, C41S, and the amino acid substitution in D68 is D68N or D68T.

[0227] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in S76 and R31 and one or more amino acids G1, E2, A19, V20, K25, I26, Q28, R31, T33, N35, C41, E59, D68, A73, D74, I75, E77, R84. the amino acid substitution in S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H or S76A, the amino acid substitution in G1 is G1M, the amino acid substitution in A19 is A19V, the amino acid substitution in V20 is V20I, the amino acid substitution in K25 is K25R, the amino acid substitution in I26 is I26W, I26V, the amino acid substitution in R31 is R31T, the amino acid substitution in T33 is T33R, the amino acid substitution in N35 is N35T, the amino acid substitution in C41 is one or more of C41A, C41V, C41T, C41S, the amino acid substitution in E59 is E59 T, E59V, E59I, E59Y, E59F, E59W, and E59R, the amino acid substitution in D68 is D68N or D68T, the amino acid substitution in A73 is A73F or A73V, the amino acid substitution in D74 is D74V, the amino acid substitution in I75 is I75V or I75L, the amino acid substitution in E77 is E77V, and the amino acid substitution in R84 is R84L.

[0228] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in S76 and R31 and in one or more amino acids V20, Q28, T33, D68, the amino acid substitution in S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, the amino acid substitution in V20 is V20I, the amino acid substitution in Q28 is one or more of Q28R, Q28K, Q28S, Q28E, the amino acid substitution in R31 is R31T, the amino acid substitution in T33 is T33R, and the amino acid substitution in D68 is D68N or D68T.

[0229] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in S76 and T33 and one or more amino acids G1, E2, A19, V20, K25, I26, Q28, R31, N35, C41, E59, D68, A73, D74, I75, E77, R84. , the amino acid substitution in S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H or S76A, the amino acid substitution in G1 is G1M, the amino acid substitution in A19 is A19V, the amino acid substitution in V20 is V20I, the amino acid substitution in K25 is K25R, the amino acid substitution in I26 is I26W, I26V, I26I the amino acid substitution in R31 is R31T, the amino acid substitution in T33 is T33R, the amino acid substitution in N35 is N35T, the amino acid substitution in C41 is one or more of C41A, C41V, C41T, C41S, the amino acid substitution in E59 is E59T , one or more of E59V, E59I, E59Y, E59F, E59W, and E59R, the amino acid substitution in D68 is D68N or D68T, the amino acid substitution in A73 is A73F or A73V, the amino acid substitution in D74 is D74V, the amino acid substitution in I75 is I75V or I75L, the amino acid substitution in E77 is E77V, and the amino acid substitution in R84 is R84L.

[0230] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in S76 and T33 and one or more amino acids A19, V20, K25, Q28, D68, the amino acid substitution in S76 is S76Y, S76F, S76T, S76W, S76R, S76V, S76I ... The amino acid substitution in G1 is G1M, the amino acid substitution in A19 is A19V, the amino acid substitution in V20 is V20I, the amino acid substitution in K25 is K25R, the amino acid substitution in Q28 is one or more of Q28R, Q28K, Q28S, Q28E, the amino acid substitution in T33 is T33R, and the amino acid substitution in D68 is D68N or D68T.

[0231] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in S76 and N35 and one or more amino acids G1, E2, A19, V20, K25, I26, Q28, R31, T33, N35, C41, E59, D68, A73, D74, I75, E77, R84. the amino acid substitution in S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H or S76A, the amino acid substitution in G1 is G1M, the amino acid substitution in A19 is A19V, the amino acid substitution in V20 is V20I, the amino acid substitution in K25 is K25R, the amino acid substitution in I26 is I26W, I26V, the amino acid substitution in R31 is R31T, the amino acid substitution in T33 is T33R, the amino acid substitution in N35 is N35T, the amino acid substitution in C41 is one or more of C41A, C41V, C41T, C41S, the amino acid substitution in E59 is E59 T, E59V, E59I, E59Y, E59F, E59W, and E59R, the amino acid substitution in D68 is D68N or D68T, the amino acid substitution in A73 is A73F or A73V, the amino acid substitution in D74 is D74V, the amino acid substitution in I75 is I75V or I75L, the amino acid substitution in E77 is E77V, and the amino acid substitution in R84 is R84L.

[0232] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in S76 and N35 and in one or more amino acids A19, V20, the amino acid substitution in S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, the amino acid substitution in G1 is G1M, the amino acid substitution in A19 is A19V, the amino acid substitution in V20 is V20I, and the amino acid substitution in N35 is N35T.

[0233] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in S76 and C41 and one or more amino acids G1, E2, A19, V20, K25, I26, Q28, R31, T33, N35, C41, E59, D68, A73, D74, I75, E77, R84. the amino acid substitution in S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H or S76A, the amino acid substitution in G1 is G1M, the amino acid substitution in A19 is A19V, the amino acid substitution in V20 is V20I, the amino acid substitution in K25 is K25R, the amino acid substitution in I26 is I26W, I26V, the amino acid substitution in R31 is R31T, the amino acid substitution in T33 is T33R, the amino acid substitution in N35 is N35T, the amino acid substitution in C41 is one or more of C41A, C41V, C41T, C41S, the amino acid substitution in E59 is E59 T, E59V, E59I, E59Y, E59F, E59W, and E59R, the amino acid substitution in D68 is D68N or D68T, the amino acid substitution in A73 is A73F or A73V, the amino acid substitution in D74 is D74V, the amino acid substitution in I75 is I75V or I75L, the amino acid substitution in E77 is E77V, and the amino acid substitution in R84 is R84L.

[0234] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52, and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions of S76 and C41 and one or more amino acids K25, Q28, the amino acid substitution in S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, the amino acid substitution in K25 is K25R, the amino acid substitution in Q28 is one or more of Q28R, Q28K, Q28S, Q28E, and the amino acid substitution in C41 is one or more of C41A, C41V, C41T, C41S.

[0235] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in S76 and E59 and one or more amino acids G1, E2, A19, V20, K25, I26, Q28, R31, T33, N35, C41, D68, A73, D74, I75, E77, R84. , the amino acid substitution in S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H or S76A, the amino acid substitution in G1 is G1M, the amino acid substitution in A19 is A19V, the amino acid substitution in V20 is V20I, the amino acid substitution in K25 is K25R, the amino acid substitution in I26 is I26W, I26V, I26I the amino acid substitution in R31 is R31T, the amino acid substitution in T33 is T33R, the amino acid substitution in N35 is N35T, the amino acid substitution in C41 is one or more of C41A, C41V, C41T, C41S, the amino acid substitution in E59 is E59T , one or more of E59V, E59I, E59Y, E59F, E59W, and E59R, the amino acid substitution in D68 is D68N or D68T, the amino acid substitution in A73 is A73F or A73V, the amino acid substitution in D74 is D74V, the amino acid substitution in I75 is I75V or I75L, the amino acid substitution in E77 is E77V, and the amino acid substitution in R84 is R84L.

[0236] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in S76, E59, and D74, the amino acid substitution in S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, the amino acid substitution in E59 is one or more of E59T, E59V, E59I, E59Y, E59F, E59W, E59R, and the amino acid substitution in D74 is D74V.

[0237] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in S76 and D68 and one or more amino acids G1, E2, A19, V20, K25, I26, Q28, R31, T33, N35, C41, E59, A73, D74, I75, E77, R84. , the amino acid substitution in S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H or S76A, the amino acid substitution in G1 is G1M, the amino acid substitution in A19 is A19V, the amino acid substitution in V20 is V20I, the amino acid substitution in K25 is K25R, the amino acid substitution in I26 is I26W, I26V, I26I the amino acid substitution in R31 is R31T, the amino acid substitution in T33 is T33R, the amino acid substitution in N35 is N35T, the amino acid substitution in C41 is one or more of C41A, C41V, C41T, C41S, the amino acid substitution in E59 is E59T , one or more of E59V, E59I, E59Y, E59F, E59W, and E59R, the amino acid substitution in D68 is D68N or D68T, the amino acid substitution in A73 is A73F or A73V, the amino acid substitution in D74 is D74V, the amino acid substitution in I75 is I75V or I75L, the amino acid substitution in E77 is E77V, and the amino acid substitution in R84 is R84L.

[0238] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in S76 and D68 and in one or more amino acids V20, K25, Q28, T33, the amino acid substitution in S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, the amino acid substitution in V20 is V20I, the amino acid substitution in K25 is K25R, the amino acid substitution in Q28 is one or more of Q28R, Q28K, Q28S, Q28E, the amino acid substitution in T33 is T33R, the amino acid substitution in N35 is N35T, and the amino acid substitution in D68 is D68N or D68T.

[0239] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acids in S76, D74 and one or more of amino acids G1, E2, A19, V20, K25, I26, Q28, R31, T33, N35, C41, E59, D68, A73, I75, E77, R84. The amino acid substitution in S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H or S76A, the amino acid substitution in G1 is G1M, the amino acid substitution in A19 is A19V, the amino acid substitution in V20 is V20I, the amino acid substitution in K25 is K25R, the amino acid substitution in I26 is I26W, I26V, I 26T, the amino acid substitution in Q28 is one or more of Q28R, Q28K, Q28S, Q28E, the amino acid substitution in R31 is R31T, the amino acid substitution in T33 is T33R, the amino acid substitution in N35 is N35T, the amino acid substitution in C41 is one or more of C41A, C41V, C41T, C41S, the amino acid substitution in E59 is E59 T, E59V, E59I, E59Y, E59F, E59W, and E59R, the amino acid substitution in D68 is D68N or D68T, the amino acid substitution in A73 is A73F or A73V, the amino acid substitution in D74 is D74V, the amino acid substitution in I75 is I75V or I75L, the amino acid substitution in E77 is E77V, and the amino acid substitution in R84 is R84L.

[0240] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in S76, D74, and E59, the amino acid substitution in S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, the amino acid substitution in E59 is one or more of E59T, E59V, E59I, E59Y, E59F, E59W, E59R, and the amino acid substitution in D74 is D74V.

[0241] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acids in S76, I75 and one or more of amino acids G1, E2, A19, V20, K25, I26, Q28, R31, T33, N35, C41, E59, D68, A73, D74, E77, R84. The amino acid substitution in S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H or S76A, the amino acid substitution in G1 is G1M, the amino acid substitution in A19 is A19V, the amino acid substitution in V20 is V20I, the amino acid substitution in K25 is K25R, the amino acid substitution in I26 is I26W, I26V, I 26T, the amino acid substitution in Q28 is one or more of Q28R, Q28K, Q28S, Q28E, the amino acid substitution in R31 is R31T, the amino acid substitution in T33 is T33R, the amino acid substitution in N35 is N35T, the amino acid substitution in C41 is one or more of C41A, C41V, C41T, C41S, the amino acid substitution in E59 is E59 T, E59V, E59I, E59Y, E59F, E59W, and E59R, the amino acid substitution in D68 is D68N or D68T, the amino acid substitution in A73 is A73F or A73V, the amino acid substitution in D74 is D74V, the amino acid substitution in I75 is I75V or I75L, the amino acid substitution in E77 is E77V, and the amino acid substitution in R84 is R84L.

[0242] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acids in S76, E77 and one or more of amino acids G1, E2, A19, V20, K25, I26, Q28, R31, T33, N35, C41, E59, D68, A73, D74, I75, R84. The amino acid substitution in S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H or S76A, the amino acid substitution in G1 is G1M, the amino acid substitution in A19 is A19V, the amino acid substitution in V20 is V20I, the amino acid substitution in K25 is K25R, the amino acid substitution in I26 is I26W, I26V, I 26T, the amino acid substitution in Q28 is one or more of Q28R, Q28K, Q28S, Q28E, the amino acid substitution in R31 is R31T, the amino acid substitution in T33 is T33R, the amino acid substitution in N35 is N35T, the amino acid substitution in C41 is one or more of C41A, C41V, C41T, C41S, the amino acid substitution in E59 is E59 T, E59V, E59I, E59Y, E59F, E59W, and E59R, the amino acid substitution in D68 is D68N or D68T, the amino acid substitution in A73 is A73F or A73V, the amino acid substitution in D74 is D74V, the amino acid substitution in I75 is I75V or I75L, the amino acid substitution in E77 is E77V, and the amino acid substitution in R84 is R84L.

[0243] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in S76, I75, and E77, the amino acid substitution in S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A, the amino acid substitution in I75 is I75V or I75L, and the amino acid substitution in E77 is E77V.

[0244] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acids in S76, R84 and one or more of amino acids G1, E2, A19, V20, K25, I26, Q28, R31, T33, N35, C41, E59, D68, A73, D74, I75, E77. The amino acid substitution in S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H or S76A, the amino acid substitution in G1 is G1M, the amino acid substitution in A19 is A19V, the amino acid substitution in V20 is V20I, the amino acid substitution in K25 is K25R, the amino acid substitution in I26 is I26W, I26V, I 26T, the amino acid substitution in Q28 is one or more of Q28R, Q28K, Q28S, Q28E, the amino acid substitution in R31 is R31T, the amino acid substitution in T33 is T33R, the amino acid substitution in N35 is N35T, the amino acid substitution in C41 is one or more of C41A, C41V, C41T, C41S, the amino acid substitution in E59 is E59 T, E59V, E59I, E59Y, E59F, E59W, and E59R, the amino acid substitution in D68 is D68N or D68T, the amino acid substitution in A73 is A73F or A73V, the amino acid substitution in D74 is D74V, the amino acid substitution in I75 is I75V or I75L, the amino acid substitution in E77 is E77V, and the amino acid substitution in R84 is R84L.

[0245] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52, and R54 and an amino acid substitution E2N, E23A, Y65R, L70I, and an amino acid substitution in S76, R84 and one or more amino acids G1, E2, A19, V20, K25, I26, Q28, R31, T33, N35, C41, E59, D68, A73, D74, I75, E77. The amino acid substitution in S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H or S76A, the amino acid substitution in G1 is G1M, the amino acid substitution in A19 is A19V, the amino acid substitution in V20 is V20I, the amino acid substitution in D68 is D68N or D68T, and the amino acid substitution in R84 is R84L.

[0246] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in G1 and S76 compared to MNEI (SEQ ID NO: 1).

[0247] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54 and an amino acid substitution E2N, E23A, Y65R, L70I, G1M, and an amino acid substitution in S76 as one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A compared to MNEI (SEQ ID NO: 1).

[0248] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, G1M and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM413.

[0249] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in A19 and S76. In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises A19V and an amino acid substitution in S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A.

[0250] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, A19V and S76Y. In some embodiments, the modified protein is represented by the amino acids denoted herein as DM202.

[0251] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in V20 and S76. In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises V20I and an amino acid substitution in S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A.

[0252] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, V20I and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM206.

[0253] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, K25, and S76 compared to MNEI (SEQ ID NO: 1).

[0254] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54 and an amino acid substitution E2N, E23A, Y65R, L70I, K25R, and an amino acid substitution in S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A compared to MNEI (SEQ ID NO: 1).

[0255] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, K25R and S76W. In some embodiments, the modified protein is represented by the amino acid represented herein as DM162.

[0256] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, K25R and S76F. In some embodiments, the modified protein is represented by the amino acid represented herein as DM160.

[0257] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, K25R and S76Y. In some embodiments, the modified protein is represented by the amino acids denoted herein as DM101.

[0258] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in I26 and S76 compared to MNEI (SEQ ID NO: 1).

[0259] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54 and an amino acid substitution E2N, E23A, Y65R, L70I, I26T, and an amino acid substitution in S76 as one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A compared to MNEI (SEQ ID NO: 1).

[0260] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54 and an amino acid substitution E2N, E23A, Y65R, L70I, I26V, and an amino acid substitution in S76 as one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A compared to MNEI (SEQ ID NO: 1).

[0261] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54 and an amino acid substitution E2N, E23A, Y65R, L70I, I26W, and an amino acid substitution in S76 as one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A compared to MNEI (SEQ ID NO: 1).

[0262] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, I26T and S76F. In some embodiments, the modified protein is represented by the amino acid represented herein as DM159.

[0263] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, I26T and S76W. In some embodiments, the modified protein is represented by the amino acids denoted herein as DM161.

[0264] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, I26T and S76Y. In some embodiments, the modified protein is represented by the amino acids denoted herein as DM100.

[0265] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in Q28 and S76 compared to MNEI (SEQ ID NO: 1).

[0266] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54 and an amino acid substitution E2N, E23A, Y65R, L70I, Q28S, and an amino acid substitution in S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A compared to MNEI (SEQ ID NO: 1).

[0267] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, Q28S and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM520.

[0268] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54 and an amino acid substitution E2N, E23A, Y65R, L70I, Q28K, and an amino acid substitution in S76 as one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A compared to MNEI (SEQ ID NO: 1).

[0269] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, Q28K and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM363.

[0270] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54 and an amino acid substitution E2N, E23A, Y65R, L70I, Q28R, and an amino acid substitution in S76 as one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A compared to MNEI (SEQ ID NO: 1).

[0271] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, Q28R and S76Y. In some embodiments, the modified protein is represented by the amino acids denoted herein as DM517.

[0272] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in R31 and S76 compared to MNEI (SEQ ID NO: 1).

[0273] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54 and an amino acid substitution E2N, E23A, Y65R, L70I, R31T, and an amino acid substitution in S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A compared to MNEI (SEQ ID NO: 1).

[0274] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, R31T and S76Y. In some embodiments, the modified protein is represented by the amino acid indicated herein as DM286.

[0275] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in T33 and S76 compared to MNEI (SEQ ID NO: 1).

[0276] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54 and an amino acid substitution E2N, E23A, Y65R, L70I, T33R, and an amino acid substitution in S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A compared to MNEI (SEQ ID NO: 1).

[0277] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, T33R and S76W. In some embodiments, the modified protein is represented by the amino acid indicated herein as DM234.

[0278] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, T33R and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM222.

[0279] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, T33R and S76F. In some embodiments, the modified protein is represented by the amino acid represented herein as DM228.

[0280] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in N35 and S76 compared to MNEI (SEQ ID NO: 1).

[0281] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54 and an amino acid substitution E2N, E23A, Y65R, L70I, N35T, and an amino acid substitution in S76 as one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A compared to MNEI (SEQ ID NO: 1).

[0282] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, N35T and S76Y. In some embodiments, the modified protein is represented by the amino acid indicated herein as DM289.

[0283] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in C41 and S76 compared to MNEI (SEQ ID NO: 1).

[0284] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54 and an amino acid substitution E2N, E23A, Y65R, L70I, C41V, and an amino acid substitution in S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A compared to MNEI (SEQ ID NO: 1).

[0285] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, C41V and S76F. In some embodiments, the modified protein is represented by the amino acid indicated herein as DM249.

[0286] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, C41V and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM146.

[0287] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, C41V and S76W. In some embodiments, the modified protein is represented by the amino acid indicated herein as DM269.

[0288] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54 and an amino acid substitution E2N, E23A, Y65R, L70I, C41T, and an amino acid substitution in S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A compared to MNEI (SEQ ID NO: 1).

[0289] In some examples, the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, C41T and S76W compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid denoted herein as DM291.

[0290] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, C41T and S76F. In some embodiments, the modified protein is represented by the amino acid indicated herein as DM294.

[0291] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, C41T and S76Y. In some embodiments, the modified protein is represented by the amino acid indicated herein as DM287.

[0292] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54 and an amino acid substitution E2N, E23A, Y65R, L70I, C41S, and an amino acid substitution in S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A compared to MNEI (SEQ ID NO: 1).

[0293] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, C41S, and S76Y compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid denoted DM346 herein. [DS replaces DM211]

[0294] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54 and an amino acid substitution E2N, E23A, Y65R, L70I, C41A, and an amino acid substitution in S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A compared to MNEI (SEQ ID NO: 1).

[0295] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, C41A and S76Y. In some embodiments, the modified protein is represented by the amino acids denoted herein as DM210.

[0296] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in E59 and S76 compared to MNEI (SEQ ID NO: 1).

[0297] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54 and an amino acid substitution E2N, E23A, Y65R, L70I, E59T, and an amino acid substitution in S76 as one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A compared to MNEI (SEQ ID NO: 1).

[0298] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, E59T, and S76I compared to MNEI (SEQ ID NO: 1), and is represented by an amino acid designated herein as DM657.

[0299] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, E59T and S76V. In some embodiments, the modified protein is represented by the amino acid indicated herein as DM654.

[0300] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, E59T and S76T. In some embodiments, the modified protein is represented by the amino acid represented herein as DM651.

[0301] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54 and an amino acid substitution E2N, E23A, Y65R, L70I, E59V, and an amino acid substitution in S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A compared to MNEI (SEQ ID NO: 1).

[0302] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, E59V and S76I. In some embodiments, the modified protein is represented by the amino acid indicated herein as DM655.

[0303] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, E59V and S76V. In some embodiments, the modified protein is represented by the amino acid indicated herein as DM652.

[0304] In some examples, the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, E59V and S76T compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid represented herein as DM605.

[0305] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54 and an amino acid substitution E2N, E23A, Y65R, L70I, E59I, and an amino acid substitution in S76 as one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A compared to MNEI (SEQ ID NO: 1).

[0306] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, E59I and S76I. In some embodiments, the modified protein is represented by the amino acid indicated herein as DM656.

[0307] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, E59I and S76V. In some embodiments, the modified protein is represented by the amino acid represented herein as DM653.

[0308] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, E59I and S76T. In some embodiments, the modified protein is represented by the amino acid indicated herein as DM650.

[0309] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54 and an amino acid substitution E2N, E23A, Y65R, L70I, E59Y, and an amino acid substitution in S76 that is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A compared to MNEI (SEQ ID NO: 1).

[0310] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, E59Y and S76V. In some embodiments, the modified protein is represented by the amino acid indicated herein as DM662.

[0311] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54 and an amino acid substitution E2N, E23A, Y65R, L70I, E59F, and an amino acid substitution in S76 as one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A compared to MNEI (SEQ ID NO: 1).

[0312] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, E59F and S76M. In some embodiments, the modified protein is represented by the amino acid denoted herein as DM660.

[0313] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54 and an amino acid substitution E2N, E23A, Y65R, L70I, E59W, and an amino acid substitution in S76 as one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A compared to MNEI (SEQ ID NO: 1).

[0314] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, E59W and S76Y. In some embodiments, the modified protein is represented by the amino acid denoted herein as DM661.

[0315] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54 and an amino acid substitution E2N, E23A, Y65R, L70I, E59R, and an amino acid substitution in S76 as one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A compared to MNEI (SEQ ID NO: 1).

[0316] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, E59R and S76T. In some embodiments, the modified protein is represented by the amino acid indicated herein as DM604.

[0317] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, D68, and S76 compared to MNEI (SEQ ID NO: 1).

[0318] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54 and an amino acid substitution E2N, E23A, Y65R, L70I, D68N, and an amino acid substitution in S76 as one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A compared to MNEI (SEQ ID NO: 1).

[0319] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, D68N and S76W. In some embodiments, the modified protein is represented by the amino acid indicated herein as DM236.

[0320] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, D68N and S76F. In some embodiments, the modified protein is represented by the amino acid indicated herein as DM230.

[0321] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, D68N and S76Y. In some embodiments, the modified protein is represented by the amino acid indicated herein as DM224.

[0322] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54 and an amino acid substitution E2N, E23A, Y65R, L70I, D68T, and an amino acid substitution in S76 as one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A compared to MNEI (SEQ ID NO: 1).

[0323] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, D68T and S76Y. In some embodiments, the modified protein is represented by the amino acid indicated herein as DM649.

[0324] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, A73, and S76 compared to MNEI (SEQ ID NO: 1).

[0325] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54 and an amino acid substitution E2N, E23A, Y65R, L70I, A73V, and an amino acid substitution in S76 as one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A compared to MNEI (SEQ ID NO: 1).

[0326] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, A73V and S76F. In some embodiments, the modified protein is represented by the amino acid represented herein as DM248.

[0327] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, A73V and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM204.

[0328] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54 and an amino acid substitution E2N, E23A, Y65R, L70I, A73F, and an amino acid substitution in S76 as one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A compared to MNEI (SEQ ID NO: 1).

[0329] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, I75, and S76 as one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A compared to MNEI (SEQ ID NO: 1).

[0330] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54 and an amino acid substitution E2N, E23A, Y65R, L70I, I75V, and an amino acid substitution in S76 as one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A compared to MNEI (SEQ ID NO: 1).

[0331] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, I75V and S76Y. In some embodiments, the modified protein is represented by the amino acid indicated herein as DM600.

[0332] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, I75L, and one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A compared to MNEI (SEQ ID NO: 1).

[0333] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, E77, and S76 compared to MNEI (SEQ ID NO: 1).

[0334] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54 and an amino acid substitution E2N, E23A, Y65R, L70I, E77V, and an amino acid substitution in S76 as one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A compared to MNEI (SEQ ID NO: 1).

[0335] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, E77V and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM594.

[0336] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in R84 and S76 compared to MNEI (SEQ ID NO: 1).

[0337] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54 and an amino acid substitution E2N, E23A, Y65R, L70I, R84L, and an amino acid substitution in S76 as one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H, or S76A compared to MNEI (SEQ ID NO: 1).

[0338] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, R84L and S76Y. In some embodiments, the modified protein is represented by the amino acid denoted herein as DM90.

[0339] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in A19, Q28, and S76 compared to MNEI (SEQ ID NO: 1).

[0340] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, A19V, Q28K and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM610.

[0341] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, A19V, Q28R and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM621.

[0342] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in A19, T33, and S76 compared to MNEI (SEQ ID NO: 1).

[0343] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, A19V, T33R and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM617.

[0344] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in A19, N35, and S76 compared to MNEI (SEQ ID NO: 1).

[0345] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, A19V, N35T and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM625.

[0346] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in A19, R84, and S76 compared to MNEI (SEQ ID NO: 1).

[0347] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, A19V, R84L and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM626.

[0348] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in V20, Q28, and S76 compared to MNEI (SEQ ID NO: 1).

[0349] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, V20I, Q28R and S76F. In some embodiments, the modified protein is represented by the amino acid represented herein as DM681.

[0350] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, V20I, Q28R and S76Y. In some embodiments, the modified protein is represented by the amino acid indicated herein as DM620.

[0351] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, V20I, Q28K and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM613.

[0352] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in V20, R31, and S76 compared to MNEI (SEQ ID NO: 1).

[0353] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, V20I, R31T and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM633.

[0354] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in V20, T33, and S76 compared to MNEI (SEQ ID NO: 1).

[0355] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, V20I, T33R and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM616.

[0356] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in V20, N35, and S76 compared to MNEI (SEQ ID NO: 1).

[0357] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, V20I, N35T and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM634.

[0358] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in V20, D68, and S76 compared to MNEI (SEQ ID NO: 1).

[0359] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, V20I, D68N and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM611.

[0360] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, V20, R84, and S76 compared to MNEI (SEQ ID NO: 1).

[0361] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, V20I, R84L and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM635.

[0362] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in K25, Q28, and S76 compared to MNEI (SEQ ID NO: 1).

[0363] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70, K25R, Q28K and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM166.

[0364] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70, K25R, Q28K and S76F. In some embodiments, the modified protein is represented by the amino acid represented herein as DM168.

[0365] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, K25R, Q28R and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM165.

[0366] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70, K25R, Q28R and S76F. In some embodiments, the modified protein is represented by the amino acid represented herein as DM167.

[0367] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in K25, I26, Q28 and S76. In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, K25R, I26T, Q28K and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM288.

[0368] In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in K25, T33, and S76, compared to MNEI (SEQ ID NO: 1). In some examples, the modified protein comprises a deletion of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, K25R, T33R, and S76Y, compared to MNEI (SEQ ID NO: 1). In some embodiments, the modified protein is represented by the amino acid represented herein as DM618.

[0369] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in K25, C41 and S76. In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, K25R, C41T and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM481.

[0370] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in K25, D68 and S76. In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, K25R, D68N and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM479.

[0371] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in I26, Q28, and S76 compared to MNEI (SEQ ID NO: 1).

[0372] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, I26W, Q28K and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM128.

[0373] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, I26W, Q28E and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM127.

[0374] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, I26V, Q28K and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM455.

[0375] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, I26T, Q28K and S76W. In some embodiments, the modified protein is represented by the amino acid represented herein as DM164.

[0376] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, I26T, Q28R and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM526.

[0377] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, I26T, Q28K and S76F. In some embodiments, the modified protein is represented by the amino acid represented herein as DM163.

[0378] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, I26T, Q28K and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM158.

[0379] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, I26T, Q28S and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM456.

[0380] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in I26, Q28, C41, and S76 compared to MNEI (SEQ ID NO: 1).

[0381] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, I26T, Q28K, C41T and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM483.

[0382] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in I26, Q28, D68, and S76 compared to MNEI (SEQ ID NO: 1).

[0383] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, I26T, Q28K, D68N and S76Y. In some embodiments, the modified protein is represented by the amino acid indicated herein as DM484.

[0384] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in Q28, R31, and S76 compared to MNEI (SEQ ID NO: 1).

[0385] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, Q28R, R31T and S76Y. In some embodiments, the modified protein is represented by the amino acids represented herein as DM171.

[0386] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, Q28K, R31T and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM630.

[0387] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in Q28, T33, and S76 compared to MNEI (SEQ ID NO: 1).

[0388] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, Q28K, T33R and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM619.

[0389] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, Q28R, T33R and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM622.

[0390] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in Q28, N35, and S76 compared to MNEI (SEQ ID NO: 1).

[0391] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, Q28K, N35T and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM631.

[0392] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, Q28R, N35T and S76Y. In some embodiments, the modified protein is represented by the amino acid indicated herein as DM640.

[0393] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in Q28, C41, and S76 compared to MNEI (SEQ ID NO: 1).

[0394] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, Q28K, C41A and S76W. In some embodiments, the modified protein is represented by the amino acid indicated herein as DM682.

[0395] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, Q28K, C41A and S76F. In some embodiments, the modified protein is represented by the amino acid represented herein as DM683.

[0396] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, Q28K, C41S and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM534.

[0397] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, Q28R, C41A and S76F. In some embodiments, the modified protein is represented by the amino acid represented herein as DM685.

[0398] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, Q28R, C41A and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM532.

[0399] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, Q28R, C41T and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM528.

[0400] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, Q28R, C41S and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM530.

[0401] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, Q28K, C41A and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM536.

[0402] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, Q28K, C41T and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM478.

[0403] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in Q28, D68, and S76 compared to MNEI (SEQ ID NO: 1).

[0404] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, Q28K, D68T and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM607.

[0405] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, Q28K, D68N and S76Y. In some embodiments, the modified protein is represented by the amino acid indicated herein as DM472.

[0406] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, Q28R, D68T and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM608.

[0407] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, Q28R, D68N and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM542.

[0408] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in Q28, R84, and S76 compared to MNEI (SEQ ID NO: 1).

[0409] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, Q28K, R84L and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM632.

[0410] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, Q28R, R84L and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM641.

[0411] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in R31, T33, and S76 compared to MNEI (SEQ ID NO: 1).

[0412] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, R31T, T33R and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM636.

[0413] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in T33, N35, and S76 compared to MNEI (SEQ ID NO: 1).

[0414] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, T33R, N35T and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM637.

[0415] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in T33, D68, and S76 compared to MNEI (SEQ ID NO: 1).

[0416] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, T33R, D68N and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM614.

[0417] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in T33, R84, and S76 compared to MNEI (SEQ ID NO: 1).

[0418] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, T33R, R84L and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM638.

[0419] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in N35, D68, and S76 compared to MNEI (SEQ ID NO: 1).

[0420] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, N35T, D68T and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM643.

[0421] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, N35T, D68N and S76Y. In some embodiments, the modified protein is represented by the amino acid indicated herein as DM628.

[0422] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in E59, D74, and S76 compared to MNEI (SEQ ID NO: 1).

[0423] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, E59V, D74V and S76V. In some embodiments, the modified protein is represented by the amino acid represented herein as DM602.

[0424] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in D68, R31, and S76 compared to MNEI (SEQ ID NO: 1).

[0425] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, D68T, R31T and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM642.

[0426] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in D68, R31, and S76 compared to MNEI (SEQ ID NO: 1).

[0427] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, D68N, R31T and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM627.

[0428] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in D68, R84, and S76 compared to MNEI (SEQ ID NO: 1).

[0429] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, D68N, R84L and S76F. In some embodiments, the modified protein is represented by the amino acid represented herein as DM242.

[0430] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, D68N, R84L and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM240.

[0431] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, D68T, R84L and S76Y. In some embodiments, the modified protein is represented by the amino acid indicated herein as DM644.

[0432] In some examples, the modified protein comprises deletions of amino acids E50, F52, and R54, and amino acid substitutions E2N, E23A, Y65R, L70I, and amino acid substitutions in I75, E77, and S76 compared to MNEI (SEQ ID NO: 1).

[0433] In some examples, compared to MNEI (SEQ ID NO: 1), the modified protein comprises a deletion of amino acids E50, F52 and R54 and amino acid substitutions E2N, E23A, Y65R, L70I, I75V, E77V and S76Y. In some embodiments, the modified protein is represented by the amino acid represented herein as DM595.

[0434] In some examples, the modified protein is one or more of DM89, DM114 (Tm=101.5° C.), DM137 (Tm=97° C.), or any combination thereof.

[0435] In some examples, the modified protein is DM456 (Tm 104°C), DM517 (Tm = 100°C), DM100 (Tm = 100°C), DM165 (Tm = 99°C), DM361 (Tm = 97°C), DM224 (Tm = 95°C), DM346 (Tm = 97°C), DM101 (Tm = 96°C), DM206 (Tm = 96°C), DM209 (Tm = 96°C), DM202 (Tm = 96°C), DM479 (Tm = 97°C), or any combination thereof. As described herein, Tm can be measured by any method known in the art, such as using DSF as described in the Examples below.

[0436] As noted herein and shown in the Examples below, the predicted Tm values ​​correlated highly with the experimentally measured Tm values. In some examples, the modified protein is DM635 (predicted Tm 105.7°C), DM644 (predicted Tm 105.43°C), DM242 (predicted Tm 105.06°C), DM632 (predicted Tm 104°C), DM90 (predicted Tm 104°C), DM240 (predicted Tm 104°C), DM641 (predicted Tm 103.8°C), DM162 (predicted Tm 102°C), DM630 (predicted Tm 101.9°C), DM657 (predicted Tm 101.8°C), DM655 (predicted Tm 101.8°C), DM642 (predicted Tm 101.7°C), DM633 (predicted Tm 101.6°C), DM595 (predicted Tm 101.5°C), DM656 (predicted Tm 101.6°C). 101.3°C), DM638 (predicted Tm 101.2°C), DM335 (predicted Tm 101.1°C), DM286 (predicted Tm 101.1°C).

[0437] Interestingly, and as shown in the examples below, the substitutions are shown to be synergistic substitutions. As used herein, the term synergistic substitution refers to the simultaneous modification of multiple amino acids within a protein sequence that together enhance or modify its function. In other words, the combined effect of these substitutions is greater than the sum of their individual effects.

[0438] In some examples, the modified protein is one or more of DM479, DM472, DM346, DM165, DM361, DM456, or any combination thereof.

[0439] In some examples, the modified protein is DM240, DM641, DM633, DM627, DM171, DM480, DM362, DM236, DM396, DM479, DM167, DM165, DM397, DM679, DM230, DM681, DM361, DM224, DM611, DM620, DM473, DM517, DM472, DM685, DM608, DM628, DM521, DM542, DM477, DM640, DM532, DM528, DM621, DM530, DM360, DM73, DM614, DM526, DM622, DM94, DM486, DM484, DM457, DM209, DM540, DM333, DM One or more of M511, DM539, DM524, DM148 or any combination thereof.

[0440] In some examples, the modified protein is one or more of DM202, DM207, DM289, DM210, DM89, DM287, DM224, DM346, DM209, DM203, DM274, DM273, DM222, DM206, DM160, DM292, DM253, DM241, DM204, DM128, DM158, DM250, DM240, DM247, DM127, DM178, DM174, DM248, DM254, DM208, DM179, DM242, or any combination thereof.

[0441] Table 1 shows proteins related to the present disclosure, including modified proteins encompassed by the present disclosure and their corresponding SEQ ID NOs:.

[0442] Table 1: Sequences of modified proteins and MNEI

[0443]

[0444]

[0445]

[0446]

[0447]

[0448]

[0449]

[0450]

[0451]

[0452]

[0453]

[0454]

[0455]

[0456]

[0457]

[0458]

[0459]

[0460]

[0461]

[0462]

[0463] In some embodiments, the modified protein comprises SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:221, SEQ ID NO:108, SEQ ID NO:9, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:52, SEQ ID NO:182, SEQ ID NO:10, SEQ ID NO:69, SEQ ID NO:157, SEQ ID NO:114, SEQ ID NO:174, SEQ ID NO:162, SEQ ID NO:231, SEQ ID NO:57, SEQ ID NO:192, SEQ ID NO:68, SEQ ID NO:106, SEQ ID NO:8, SEQ ID NO:79, SEQ ID NO:76, SEQ ID NO:22, SEQ ID NO:194, SEQ ID NO:262, SEQ ID NO:84, SEQ ID NO:239, SEQ ID NO:135, SEQ ID NO:211, SEQ ID The amino acid sequence set forth in one or more of SEQ ID NO:237, SEQ ID NO:198, SEQ ID NO:179, SEQ ID NO:172, or any combination thereof.

[0464] As described herein, the modified protein has at least improved stability compared to a reference protein.

[0465] According to the present disclosure, the modified protein has at least one improved food-related property characteristic compared to a reference protein.

[0466] The term food-related properties as used herein encompasses properties that effectuate the suitability of the modified protein in food and beverage applications, such as flavor, texture, taste, sweetness threshold, sweetness level, sweetness profile, sensory profile, sweetness kinetics, stability (structural and functional), thermotolerance, suitability for food matrices, shelf life, masking and / or enhancement of other flavors, off-taste, taste onset, aftertaste, roundness of taste, or a sugar-like taste.

[0467] In some embodiments, the at least one food-related characteristic is a sensory characteristic. As used herein, the term "sensory characteristic" refers to a change in a sensory impression determined, for example, by taste. Sensory characteristics include, for example, sweetness characteristics, such as sweetness potency (sugar-like taste), sweetness dynamics (onset time, aftertaste time, taste duration), lack of off-flavors, and masking and / or enhancement of other flavors, off-flavors (e.g., metallic taste). For example, the improved characteristic is associated with increased sweetness, reduced onset time, or reduced aftertaste.

[0468] According to some embodiments in which at least one property is a property that affects the organoleptic properties, the modified protein can be considered a sugar substitute. In some embodiments, the at least one food-related property is at least one of sweetness potency, reduced onset time, or reduced aftertaste.

[0469] In some examples, the modified protein has an improved aftertaste. In some examples, the modified protein is DM114. In some examples, the modified protein is DM137. In some examples, the modified protein is DM157. In some examples, the modified protein is DM224. In some examples, the modified protein is DM157. In some examples, the modified protein is DM02.

[0470] In some examples, the modified protein is DM165.

[0471] As mentioned above, the modified protein can be used in combination with at least one additional food ingredient. In some embodiments, the at least one food-related characteristic can refer to a synergistic effect between the modified protein and at least one food ingredient. Non-limiting examples of food ingredients include artificial flavors or natural flavors, food additives, food coloring agents, preservatives, or additional sugar additives. The food ingredient can have a masking taste effect or an enhanced taste effect.

[0472] As described herein, modified proteins as described herein have improved food-related properties. The sweet taste characteristics of the protein can be determined by any taste test known in the art, such as the sweetness effectiveness (sugar-like flavor), lack of staleness, reduced onset time and reduced aftertaste of the modified protein. For example, a comparison with the sweetness of sucrose or other sweeteners can be performed by a tasting panel, and the sweetness effectiveness can be graded according to the detailed description in the examples below.

[0473] The comparison can be made by determining a threshold for the modified protein as compared to a known sweetener, such as sucrose, for example, by determining the minimum concentration required to elicit a sweet taste sensation, or a sweetness profile assessment, including characteristics such as sweetness profile, sweetness onset time, aftertaste, mouthfeel, aftertaste, off-taste, and masking of unwanted tastes.

[0474] As used herein, the term sweetening-affecting property encompasses a sweetness threshold of about 0.28 mg / L or more, a sweetness duration of about 1 to 20 seconds, sometimes 2 to 18 seconds, and sometimes 2 to 4 seconds, as determined by at least one of the following:

[0475] The modified protein, similar to the reference protein, binds to the sweet taste receptor.

[0476] In some embodiments, the modified protein has a perceived sweetness threshold of 300-16,000 higher than sugar, based on weight.

[0477] Sensory characteristics include flavor dynamics, which are typically Gaussian, showing flavor intensity over time, i.e., duration of onset (time until the flavor is perceived), flavor duration, and aftertaste time (corresponding to the tail of the Gaussian distribution). Additional characteristics include off-taste (e.g., due to binding to other receptors), mouthfeel roundness, metallic and other side flavors, synergy with other ingredients (e.g., masking and enhancing other flavors or their unwanted flavors, such as stevia), etc.

[0478] As described herein, computational analysis is performed to identify regions and / or amino acid residues based on MNEI or MNEI-based proteins.

[0479] The modified proteins described herein can be designed by a variety of methods.

[0480] In some embodiments, protein design is completed using computational tools or by expert protein design and structural biology methods, such as site-directed mutagenesis, protein engineering or directed evolution, as described in addition below. The inventors have developed computational methods based on sequence data, structural data and / or evolutionary data of other proteins with local similarity or global similarity to the reference flavor protein and in sequence and / or structural features. The computational methods developed and applied herein enable the inventors to design proteins with specific amino acid substitutions, which are advantageous to a large extent and are therefore predicted to have improved properties, such as thermal stability, salt stability, pH stability, shelf life, folding and solubility characteristics. Specifically, computational protein design (CPD) is applied to specific sites or regions within the reference protein structure and / or sequence, which are not necessary for functional binding to receptors. In addition, CPD allows the inventors to limit substitutions to predetermined amino acid groups that meet the desired improved features. The predetermined amino acid group is present both in the input data, i.e., the region where the protein undergoes CPD, and in the output data, i.e., the position and type of amino acids present in the resulting modified protein.

[0481] For example, by using CPD, it is possible to replace "non-ideal" amino acids (such as hydrophilic amino acids in the hydrophobic core or hydrophobic amino acids on the external surface region) with "ideal" amino acids (such as hydrophilic amino acids in the external surface region and hydrophobic amino acids in the hydrophobic core).

[0482] Without being bound by theory, the inventors suggest that replacing hydrophobic amino acids with hydrophilic amino acids in the external surface region will reduce non-specific binding to the oral cavity and reduce residual aftertaste.

[0483] The method developed herein includes finding "stable substitutions", for example, amino acid substitutions that will reduce the total energy of the protein structure. The total energy can be calculated by applying algorithms known in the art. Non-limiting examples of such algorithms include Rosetta, OSPREY (M. Hallen, J. Martin et al., Journal of Computational Chemistry 2018; 39 (30): 2494-2507) or EnCoM (Frappier V, Chartier M, Najmanovich RJ, Nucleic Acids Res., 2015; 43 (W1): W395-400). These CPD methods are focused and filtered by a series of orthogonal methods, such as evolutionary sequence and structure sharing, conventional and high temperature molecular dynamics (MD) and other dynamic simulations, correlation mutation analysis (CMA), surface electrostatic analysis, visual inspection, and analysis of cavities, hydrophobic patches, unsatisfied hydrogen bonds, etc.

[0484] Amino acid substitutions were based on considerations of: (a) surface electrostatic potential and (lack of) surface hydrophobic patches, (b) retention of the protein isoelectric point (pI) within a specific range, (c) analysis of the protein internal cavity, (d) dynamic stability, including correlation mutation analysis, normal mode analysis, and root mean square fluctuations (RMSF) in high temperature kinetics or room temperature kinetics, (e) entropic and / or enthalpic components of substitution energetics, (f) visualization of specific substitutions, (g) allowed amino acid types in a family of related proteins; as reflected by evolutionary conservation analysis of post-planning multiple sequence alignments (MSAs), and (h) substitution frequencies as reflected in low pseudo-energy CPD calculations.

[0485] The calculation method includes one or more of the following steps:

[0486] (1) Multiple sequence alignment (MSA) or multiple structural alignment. In this step, DNA sequences and / or protein sequences similar to the target reference protein or its fragments are queried in public databases. Based on the results obtained, a multiple sequence alignment (MSA) or multiple structural alignment is constructed, and the conservation rate is calculated. Based on the MSA results, a decision is made about the level of CPD to be performed. At non-conserved positions, all amino acids (with or without cysteine) are allowed in the CPD, while for more conserved positions, CPD is limited to residues with similar properties (charge, size, internal dynamics, etc.). This step involves limiting substitutions at each position based on biophysical knowledge and conservation data. MSA can generate a position-specific substitution matrix (PSSM), in which each position along the sequence is described in a manner related to the relative abundance of each amino acid, possibly taking into account the potential possibility of amino acid substitution or deletion or insertion.

[0487] (2) Protein function analysis and structure-function-dynamic relationship analysis. In this step, a database of substitutions with known effects (such as on activity, structure, binding, etc.) is constructed using prior knowledge. Based on prior knowledge, substitution positions and substitution-adjacent positions (e.g., 0.5 nm-1 nm distance) known to interfere with protein stability and / or function are restricted during CPD and are not substituted.

[0488] (3) CPD. This step is partially completed by designated software, such as ROSETTA, OSPREY, SCWRL, PyMol, AlphaFold, etc. Before performing deterministic CPD, the reference protein 3D structure / model is minimized in energy. CPD may include site-specific amino acid substitutions, in which one amino acid is replaced by another amino acid, or a protein region is replaced by other amino acid sequences, thereby producing proteins with different lengths. The latter can be accomplished by reconstructing regions, such as loops, or removing regions from other proteins by ab initio calculation methods, a method that can be referred to as "grafting". For each reference protein, multiple models are considered.

[0489] Another consideration in CPD is to retain the functional plasticity required for receptor binding while increasing thermal stability, which is often associated with protein hardening in nature. In order to activate the receptor, the protein must undergo some conformational changes (also known as "functional plasticity"). Therefore, the inventors focused on regions that can be hardened while retaining regions that must maintain functional plasticity.

[0490] The terms "amino acid sequence" and / or "polypeptide chain" are used to describe a protein having an amino acid sequence or polypeptide chain. Thus, the term "reference protein" is equivalent to the term "reference amino acid sequence", and the term "modified protein" is equivalent to the term "modified amino acid sequence". It should be noted that the terms "amino acid sequence" and / or "polypeptide chain" encompass sequences having a 3D structure as well as sequences without a 3D structure.

[0491] Amino acids may be referred to herein by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Amino acid substitution (replacement) as used herein refers to the change from one amino acid to a different amino acid. This is usually due to a point mutation in the DNA sequence caused by a non-synonymous missense mutation, which changes the codon sequence to encode other amino acids rather than a reference. Amino acid substitution may have an impact on the function or structure of the protein, and this generally depends on how similar or dissimilar the replaced amino acid is and their position in the sequence or structure. For example, amino acid substitution can be carried out on the basis of the similarity of polarity, charge, solubility, hydrophobicity, hydrophilicity, bulkiness (or flexibility), β-branching, aromaticity, the ability to confer specific binding interactions (hydrogen bonds, salt bridges, polar and non-polar interactions), pK, the ability to bind sugars and other post-translational modifications and / or the amphipathic nature of the residues involved.

[0492] In some embodiments, an amino acid substitution may be a conservative substitution. Such substitutions encompass the change of one amino acid to another amino acid that exhibits similar properties. A conservative amino acid substitution (also denoted as a conservative amino acid "substitution" or conservative amino acid mutation) is an amino acid substitution in a protein that changes a given amino acid to a different amino acid with similar biochemical, structural, and / or chemical properties.

[0493] For example, amino acids can be classified into six major classes based on their structure and the general chemical characteristics of their side chains (R groups).

[0494] Aliphatic: isoleucine (I), leucine (L), glycine (G), alanine (A), valine (V);

[0495] Hydroxyl or sulfur / selenium-containing: serine (S), cysteine ​​(C), threonine (T), methionine (M);

[0496] Cyclic: Proline (P)

[0497] Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W)

[0498] Alkaline: Histidine (H), Lysine (K), Arginine (R)

[0499] Acidic and its amides: aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q).

[0500] In addition, each of the following groups contains other exemplary amino acids that are conservative substitutions for one another:

[0501] 1) Very small: alanine (A), glycine (G);

[0502] 2) Negative charge: aspartic acid (D), glutamic acid (E);

[0503] 3) Polar (amidated carboxyl side chain): asparagine (N), glutamine (Q);

[0504] 4) Positively charged: arginine (R), lysine (K);

[0505] 6) Aromatic: phenylalanine (F), tyrosine (Y), tryptophan (W), and occasionally histidine (H);

[0506] 7) Small polarity: serine (S), threonine (T);

[0507] 8) Sulfur-containing: cysteine ​​(C), methionine (M)

[0508] 9) Small: Ala (A), glycine (G), serine (S).

[0509] 10) β-branched: valine (V), isoleucine (I), and occasionally threonine

[0510] (T);

[0511] 11) Polarity: Asparagine (N), Glutamine (Q), Serine (S), Threonine

[0512] (T);

[0513] However, there are a large number of amino acid clusters that give rise to a large number of amino acid indices, each of which highlights a different aspect of the characteristics of the amino acid - see, for example, the aa index database https: / / www.genome.jp / aaindex / Thus, some conservative substitutions may actually represent other features that are important for the suitability of the protein for industrial use in the food industry, such as nonspecific binding to the tongue or other sensory characteristics.

[0514] In addition, additional conservative analysis was based on the following:

[0515] - non-polar "hydrophobic" amino acids are selected from the group consisting of valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tryptophan (W), cysteine ​​(C), alanine (A), tyrosine (Y), histidine (H), threonine (T), serine (S), proline (P), glycine (G), arginine (R) and lysine (K);

[0516] - "polar" amino acids are selected from the group consisting of: arginine (R), lysine (K), aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q);

[0517] - the "positively charged" amino acid is selected from the group consisting of: arginine (R), lysine (K) and histidine (H); and

[0518] - "Acidic" amino acids are selected from the group consisting of: aspartic acid (D), asparagine (N), glutamic acid (E) and glutamine (Q).

[0519] In some embodiments, the substitution is a free radical substitution. A free radical substitution (substitution) is the exchange of an amino acid with another amino acid having different properties.

[0520] The degree of sequence similarity and / or sequence identity between the reference protein and the modified protein will generally affect the properties of the modified protein. For example, a large number of substitutions may affect binding kinetics, folding kinetics, solubility, thermal stability, salt stability, pH stability, shelf life, binding to non-aqueous particles (e.g., proteins or fats in food matrices or hydrophobic regions in the oral cavity), 3D structure, and its activity and related properties. The computational methods developed and applied herein provide a thorough understanding of the putative amino acid residues for substitution, which will produce improved modified proteins.

[0521] The modified proteins can be used as flavor modifiers or flavor enhancers.

[0522] The proteins described herein are used as oral products. In some embodiments, the product is a food product, a food supplement product or a medicine. For the preparation of the product, the proteins described herein can be combined with any food grade additive. The food product can be provided and used in any solid dry form, including but not limited to fine powders, lyophilizates, granules, tablets, etc. In some embodiments, the composition is provided in liquid form, for example, as a solute (aqueous solution) in water.

[0523] Products comprising the protein may have a variety of applications. This includes, but is not limited to (each of the following constitutes a separate embodiment of the present disclosure) in the food and beverage industry (soft drinks, ready-to-drink beverages, syrups, functional beverages, sports drinks, etc.), in the dairy industry (i.e. dairy products, yogurt and pudding), in the pharmaceutical industry, in the natural remedy industry, the nutrition industry and other health products (e.g. toothpaste, mouthwash); in the candy and chewing gum industry as a sweetener, flavoring agent, enhancer or masking agent, or any other application requiring the use of a flavor-modifying composition as an excipient or additive.

[0524] The product may include additional food ingredients. In some embodiments, the food ingredient is a sweetener, such as stevioside. As shown in the examples below, the combination of the modified protein described herein and stevioside produces a synergistic effect. Therefore, in some embodiments, the product includes at least one modified protein as described herein and stevioside.

[0525] It should be noted that, according to the present invention, modified proteins can be produced by any method known in the art, for example, the protein can be produced by recombinant DNA technology, or by protein production in a microorganism via a fermenter or in a plant or in a plant callus or other bioreactor and synthetically produced. In some embodiments, modified proteins can be produced in bacteria, such as Escherichia coli (E.Coli). In some other embodiments, modified proteins can be produced by yeast, such as Saccharomyces cerevisiae or Pichia pastoris. In some embodiments, the DNA sequence of the selected amino acid sequence is optimized at the RNA level and the DNA level. At the RNA level, this includes minimizing RNA secondary structure to ensure rapid insertion into the ribosome. At the DNA level, this includes codon optimization of the host organism (taking into account RNA level optimization). Codon usage optimization provides a preference for using the most abundant tRNA in the host organism for each expressed amino acid.

[0526] As used herein, the term "about" means that the value may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% above or below the referenced value, the deviation range including the integer values ​​constituting the continuous range, and, if applicable, non-integer values. In some embodiments, the term "about" refers to ± 10%.

[0527] As indicated herein, a modified protein has at least improved stability compared to a reference protein. As used herein, the term "improved" in relation to the stability or any other characteristic of a modified protein refers to an increase or improvement in stability of about 1% to 100%, specifically, 5% to 100%, more specifically, about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85%, about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%, 100% or more compared to the stability of a reference protein.

[0528] Unless otherwise indicated, all scientific and technical terms used herein have the meaning commonly used in the art. The definitions provided herein are to facilitate understanding of certain terms frequently used herein and are not intended to limit the scope of the present disclosure.

[0529] As used herein, the term "about" means that the value may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% above or below the referenced value, and the deviation range includes integer values ​​constituting a continuous range, and if applicable, non-integer values. Therefore, as used herein, the term "about" refers to ±10%.

[0530] It should be noted that various embodiments of the present invention can be presented in a range format. The description of a range should be considered to have specifically disclosed all possible subranges, as well as each numerical value within the range. For example, the description of a range such as from 1 to 6 or between 1 and 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, and each number within the range, such as 1, 2, 3, 4, 5 and 6.

[0531] As used herein, the forms "a," "an," and "the" include singular as well as plural references, unless the context clearly dictates otherwise.

[0532] It should be understood that certain features of the invention described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention described in the context of a single embodiment for simplicity may also be provided individually or in any suitable subcombination, or, where appropriate, in any other described embodiment of the invention. Certain features described in the context of various embodiments are not considered essential features of those embodiments, unless the embodiment cannot be implemented without those elements.

[0533] It should be noted that the various embodiments and examples described in detail in conjunction with various aspects of the present invention herein may be applicable to one or more aspects disclosed herein. It should also be noted that any embodiment described herein (e.g., relating to methods) may be applied alone or in various combinations. Various embodiments and aspects of the present invention as described above and as claimed in the claims section below are experimentally supported in the following examples. The phrase "in another embodiment" or any reference to an embodiment as used herein does not necessarily refer to a different embodiment, although it may refer to a different embodiment. Therefore, various embodiments of the present invention may be combined (from the same aspect or from different aspects) without departing from the scope of the present invention.

[0534]

[00136] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0535] It should be understood that the present invention is not limited to the specific embodiments, method steps and reactors disclosed herein, as such method steps and reactors may be slightly modified. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims and their equivalents.

[0536] The following examples represent techniques employed by the inventors in carrying out various aspects of the present invention. It should be understood that although these techniques are examples of preferred embodiments for practicing the present invention, those skilled in the art will recognize, based on this disclosure, that many modifications may be made without departing from the spirit and intended scope of the present invention.

[0537] Non-limiting Examples

[0538] Example 1: Design of MNEI-based proteins

[0539] The design of the MNEI-based protein was performed as follows: Single-chain mebutin, MNEI (SEQ ID NO: 1) is a polypeptide consisting of 96 amino acids with a molecular weight of about 11 kD and a pi of about 8.7.

[0540] method :

[0541] ANM (Anisotropic Network Model)

[0542] ANM is a computational method used to study the dynamics and motion of proteins, assuming that proteins can be approximated as a network of interacting atoms or residues.

[0543] ANM considers the interactions between neighboring atoms in a protein and calculates the force constants between them. By representing a protein as a network of nodes (representing atoms) connected by edges (representing interactions), ANM analyzes the vibrational modes and collective motions of a protein structure.

[0544] The analysis represents the structure as a network of nodes and springs. For the matrix of all interacting residues, ANM dynamics is based only on the inter-residue contact topology represented by the N×N harmonic potential of the uniform force constant γ, and the 3N×3N Hessian H of the second-order derivative of the potential for N nodes / residues. The mode spectrum of N-1 (or 3N-6) non-zero modes is obtained by the eigenvalue decomposition of H. The modes are sorted by increasing frequency so that mode 1 is the slowest and softest (most accessible) mode. Soft modes are highly cooperative. The pseudo-inverse of H is proportional to the cross-correlation between residue fluctuations, organized in an N×N (or 3N×3N) covariance matrix (Hongchun Li et al., 2017, Nucleic Acids Research 45(W1), W374-W380).

[0545] Molecular dynamics

[0546] Molecular dynamics (MD) system is established and simulated.For all simulated proteins, PROPKA (Olsson residues, 2011, Journal of Chemical Theory and Computation, 7 (22), 525-537) version 3.4 is used to appropriately set the residue protonation state at pH 7.0. All systems are placed in a dodecahedral box equipped with TIP4P water (Jorgensen et al., 1983, Chem. Phys., 79, 926-935), and its minimum distance from protein to box wall is 1.5nm. Each system is neutralized by randomly placing sodium or chlorine counterions with a concentration of 150mM NaCl. MD simulations are performed using GROMACS software (Abraham et al., 2015, SoftwareX, 1-2, 19-25) version 2022.1. System parameterization (Hornak et al., 2006, Proteins, 65, 712-725) is used.

[0547] The system was relaxed using 50,000 steps of steepest descent energy minimization and then equilibrated using the NVT ensemble. At equilibrium, the initial velocities of the atoms were randomly distributed at 300 K according to a Maxwell–Boltzmann distribution. 1000 kJ mol - 1 nm -2 Harmonic position restraints of were applied to the protein atoms, and the temperature was steadily increased to 300K over the course of 1000ps, then increased to 433K for another 1000ps using V-rescale (modified Berendsen thermostat). The Parrinello-Rahman method was then used to equilibrate to 1atm (Parrinello and Rahman, 1981, J.Appl.Phys., 52, 7182-7190), and the restraints were steadily removed in 5 steps of 1000ps each. Production simulations were performed in the NPT ensemble without position restraints, using an integration time step of 2fs, and snapshots were saved every 10ps for analysis. Independent repetitions for each system were performed for 50ns each. MD simulations were performed under periodic boundary conditions, and PME was used to calculate long-range interactions (T. Darden, D. York, L. Pedersen, Particle mesh Ewald: An N·log(N) method for Ewald sums in large systems. J. Chem. Phys. 98, 10089-10092 (1993)) with a cutoff radius of 1.5 nm.

[0548] MD analysis includes the following parts :

[0549] The average native contacts were calculated using the Best-Hummer score of the native contact function (Best et al., 2013, PNAS, 110 (44), 17874-17879), as implemented in MDTraj version 1.9.6 (McGibbon et al., 2015, Biophys J., 109 (8), 1528-32). A native contact is a contact between two amino acids that are not adjacent in the amino acid sequence but are spatially close in the native tertiary structure of the protein. These contacts are used to measure deviations in molecular dynamics. A β constant of 501 / nm, a λ constant of 1.8, and a native contact cutoff of 0.45nm were used. The scores of native contacts were averaged over the simulation time. The more contacts between the heavy atoms of the protein, the higher the stability during the simulation.

[0550] 1. Use the GROMACS hbond utility to calculate the average helical H-bonds during the simulation using intra-main-chain hydrogen bonds to detect secondary structure unfolding. Helical residues were defined as residues 10–26.

[0551] 2. The average β-sheet H-bonds during the simulation were calculated using the GROMACS hbond utility using intra-main-chain hydrogen bonds. The β-sheet residues were defined as residues 2-6, 35-37, 42-48, 54-66, 69-78, 82-90 numbered according to the sequence and structure of MNEI (2O9U).

[0552] 3. Water-β-sheet backbone H-bonds - measures the average number of water molecules surrounding the β-sheets of a protein that are stable enough to link the protein's backbone to β-stranded secondary structure segments.

[0553] 4. Fraction of typical helices - This is the fraction of i:i+4 type H bonds in the helix. There are three types of helical H bonds: i:i+3, i:i+4, and i:i+5, representing 3-10 helices, α-helices, and π-helices, respectively. α-helices with i:i+4 bonds are the most typical. This value indicates that α-helices are maintained during the simulation. The higher the fraction of typical H bonds, the more stable the helix is ​​expected to be.

[0554] 5. Use the GROMACS rms utility to calculate the helical region (residues 10-26), loops (residues 38-42, 49-53, 67-68, 79-81, 7-9, 27-34), the C-terminus of the protein (residues 90-96), and the β-sheet (residues 2-6, 35-37, 42-48, 54-66,

[0555] The sum of RMSD (root mean square deviation) of C-α atoms of proteins in 69-78, 82-90)

[0556] The average of the RMSDs was then summed to produce a single measure. The residues are numbered according to the sequence and structure of MNEI (SEQ ID NO: 8, PDB ID: 209U).

[0557] 6. RMSF (root mean square fluctuation) of the protein backbone atoms throughout the MD simulation. These values ​​were calculated using the GROMACS rmsf utility and averaged for each residue.

[0558] 7. Use the average RMSF of three MD simulation replicates for each DM to convert the RMSF

[0559] AUC (area under the curve) difference (ΔAUC) was calculated as ΔAUC = (AUC(variant) -

[0560] AUC(reference), where reference is DM31 or MNEI).

[0561] Molecular Graphics Structural analysis, measurements, pictures and movie editing were performed using either VMD (Humphrey, W., Dalke, A. and Schulten, K., VMD-Visual Molecular Dynamics, J. Molec. Graphics 1996, 14.1, 33-38.) version 1.9.4. The images were visualized using VMD or PyMol (The PyMOL Molecular Graphics System, version 2.5.2, LLC.) Renderings and videos.

[0562] Structure prediction and energy calculation

[0563] By using the template of DM31 and / or MNEI crystal structure (DM31 is determined by the crystallization unit of Weizmann Institute (Weizmanninstitute); MNEI is based on the structure from Protein Data Bank (Protein Data Bank, PDB), ID209U) the new variant of MNEI is modeled. The obtained structure is submitted to Rosetta software, and further energy minimization and energy scoring calculations are performed using Rosetta 3.8 (Schueler-Furman et al., 2005, Science, 310, 638-642; Baker, 2006, Philos Trans R Soc Lond B Biol Sci., 361, 459-63; Kaufmann et al., 2010, Biochemistry, 49, 2987-2998). Minimization and scoring (Park et al., 2016, J Chem Theory Comput, 12, 6201-6212) are performed using Rosetta's FastRelax scheme and REF2015 energy function. For each input sequence, the scheme was repeated at least 30K times to obtain a variety of structures and their Rosetta energy functions (REU) scores. For each variant, the 30K scores were classified and the 500 lowest scoring structures were used for further inspection and analysis. For MNEI and DM31, the input structure was the corresponding crystal structure (2O9U for MNEI), and the scheme was repeated 100K times. Table 2 presents the new variants selected for expression in the laboratory, and their corresponding Rosetta energy units (REU) scores. For some analyses, the lowest energy structure of each new variant was used.

[0564] Additional protein features

[0565] PyMol was used to visualize and analyze hydrogen bonds. In addition, we used the Baker-Hubbard method to check hydrogen bonds (Baker and Hubbard, 1984, Progress in Biophysics and Molecular Biology, 44.2, 97-179), as implemented in the MDTraj Python package (McGibbon et al., 2015, Biophys J., 109 (8), 1528-1532) version 1.9.6. Another hydrophobicity measure-SAP (spatial aggregation tendency) score (Lauer et al., 2012, JPharm Sci, 101 (1), 102-115) was used to calculate using Rosetta. This score measures the local hydrophobicity of surface patches. Such surface regions may form hydrophobic interactions with other patches on other proteins, thereby increasing the risk of aggregation.

[0566] Another measure is used to estimate protein structure assembly - VoroMQA (Olechnovi and Venclovas, 2017, Proteins, 85, 1131-1145). This method combines statistical potentials with the use of interatomic contact areas rather than distances. The contact areas obtained using Voronoi partitioning of the protein structure are used to describe and integrate explicit interactions between protein atoms and implicit interactions of protein atoms with the solvent. VoroMQA produces scores at the atomic, residue, and global levels, all in a fixed range from 0 to 1.

[0567] result

[0568] Table 1 presented above presents examples of proteins designed using the methods described herein. Table 1 also includes the sequence of MNEI (a known protein).

[0569] Table 2 presents the calculated Rosetta Energy Unit (REU) scores for MNEI variant y. For both minimization and calculation, Rosetta's REF2015 energy function was used. The table also presents the SAP score, which refers to hydrophobicity and indicates aggregation tendency.

[0570] The average RMSD was calculated using the backbone atoms of the protein. The average H bonds in the helical and β-sheet backbones were calculated in GROMACS (see under Methods, Molecular Dynamics) and colored by a green-yellow-red gradient (green marks a higher number of H bonds). The average RMSD of specific secondary structure elements (helices, La2 and L23) was calculated using the C-α atoms of the protein and colored by a green-yellow-red gradient (green indicates a lower RMSD).

[0571] Table 2: Molecular dynamics (MD) simulation results .

[0572]

[0573]

[0574]

[0575]

[0576]

[0577]

[0578]

[0579]

[0580]

[0581]

[0582]

[0583]

[0584]

[0585]

[0586]

[0587]

[0588]

[0589]

[0590]

[0591]

[0592]

[0593]

[0594]

[0595] Rosetta energy scores for the MNEI-based variants: DM212 (A19V, S76Y) has a score of -304.306REU. DM217 (I75L, S76Y) has a score of -308.118REU. DM299 (N35T, S76Y) has a score of -312.195REU. DM276 (A19V, S76W) has a score of -307.017REU. DM256 (A19V, S76F) has a score of -307.977REU. DM220 (C41A, S76Y) has a score of -313.722REU. DM281 (I75L, S76W) has a score of -308.084REU. DM129 (S76Y) has a score of -313.148 REU. DM297 (C41T, S76Y) has a score of -312.022 REU. DM221 (C41S, S76Y) has a score of -312.548 REU. DM227 (D68N, S76Y) has a score of -314.09 REU. DM219 (A73F, F89M, S76Y) has a score of -315.41 REU. DM213 (V64I, S76Y) has a score of -313.15 REU. DM183 (S76W, I26T) has a score of -310.896 REU. DM284 (C41A, S76W) has a score of -312.41 REU. DM283 (A73F, F89M, S76W) has a score of -312.802REU. DM225 (T33R, S76Y) has a score of -314.501REU. DM307 (N35T, S76F) has a score of -312.43REU. DM216 (V20I, S76Y) has a score of -316.389REU. DM182 (S76F, K25R) has a score of -313.12REU. DM303 (N35T, S76W) has a score of -312.963REU. DM237 (T33R, S76W) has a score of -311.444REU. DM263 (A73F, F89M, S76F) has a score of -315.016REU. DM244 (D68N, R84L, S76W) has a score of -316.843REU. DM214 (A73V, S76Y) has a score of -316.179REU. DM201 (I26W, Q28K, S76Y) has a score of -319.166REU. DM261 (I75L, S76F) has a score of -311.468REU. DM180 (Q28K, I26T, S76Y) has a score of -315.492REU. DM260 (V20I, S76F) has a score of -314.026REU.DM243 (D68N, R84L, S76Y) has a score of -317.997REU. DM285 (C41S, S76W) has a score of -311.278REU. DM257 (V64I, S76F) has a score of -313.764REU. DM280 (V20I, S76W) has a score of -313.281REU. DM239 (D68N, S76W) has a score of -313.781REU. DM200 (I26W, Q28E, S76Y) has a score of -319.278REU. DM198 (I26W, Q28K, S76W) has a score of -316.895REU. DM181 (S76F, I26T) has a score of -312.985REU. DM265 (C41S, S76F) has a score of -312.498REU. DM187 (K25R, Q28R, S76Y) has a score of -313.041REU. DM302 (R84L, S76W) has a score of -317.531REU. DM258 (A73V, S76F) has a score of -317.64REU. DM233 (D68N, S76F) has a score of -314.319REU. DM264 (C41A, S76F) has a score of -315.808REU. DM147 (S76F) has a score of -314.468REU. DM282 (F89M, S76W) has a score of -312.956 REU. DM231 (T33R, S76F) has a score of -313.647 REU. DM218 (F89M, S76Y) has a score of -317.154 REU.

[0596] Rosetta energy scores for DM31-based variants: DM202 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, A19V, S76Y) has a score of -326.104 REU. DM207 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, I75L, S76Y) has a score of -325.596 REU. DM289 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, N35T, S76Y) has a score of -327.695 REU. DM266 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, A19V, S76W) has a score of -322.122 REU. DM246 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, A19V, S76F) has a score of -322.969 REU. DM210 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41A, S76Y) has a score of -328.185 REU. DM271 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, I75L, S76W) has a score of -322.428 REU. DM287 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41T, S76Y) has a score of -325.849 REU. DM211 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41S, S76Y) has a score of -326.053 REU. DM224 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, S76Y) has a score of -327.546 REU. DM203 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, V64I, S76Y) has a score of -326.036 REU. DM161 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76W, I26T) has a score of -323.585 REU. DM274 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41A, S76W) has a score of -324.868 REU. DM273 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, A73F, F89M, S76W) has a score of -324.977 REU.DM222 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, T33R, S76Y) has a score of -326.421 REU. DM295 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, N35T, S76F) has a score of -324.151 REU. DM206 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, V20I, S76Y) has a score of -327.962 REU. DM160 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76F, K25R) has a score of -324.536 REU. DM292 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, N35T, S76W) has a score of -324.361 REU. DM234 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, T33R, S76W) has a score of -322.785 REU. DM241 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, R84L, S76W) has a score of -327.95 REU. DM204 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, A73V, S76Y) has a score of -327.016 REU. DM128 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, I26W, Q28K, S76Y) has a score of -329.989 REU. DM251 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, I75L, S76F) has a score of -322.262 REU. DM158 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28K, I26T, S76Y) has a score of -326.098 REU. DM250 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, V20I, S76F) has a score of -324.555 REU. DM240 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, R84L, S76Y) has a score of -328.51 REU. DM275 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41S, S76W) has a score of -321.76 REU.DM247 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, V64I, S76F) had a score of -324.213 REU. DM270 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, V20I, S76W) had a score of -323.622 REU. DM236 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, S76W) had a score of -324.065 REU. DM127 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, I26W, Q28E, S76Y) has a score of -329.421 REU. DM178 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, I26W, Q28K, S76W) has a score of -326.997 REU. DM159 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76F, I26T) has a score of -323.01 REU. DM255 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41S, S76F) has a score of -322.513 REU. DM165 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, K25R, Q28R, S76Y) has a score of -322.968 REU. DM174 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, R84L, S76W) has a score of -327.282 REU. DM248 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, A73V, S76F) has a score of -327.375 REU. DM230 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, S76F) has a score of -324.008 REU. DM254 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41A, S76F) has a score of -325.464 REU. DM272 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, F89M, S76W) had a score of -322.55 REU. DM228 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, T33R, S76F) had a score of -323.104 REU.DM208 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, F89M, S76Y) had a score of -326.518 REU. DM679 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, V20I, D68N, S76F) had a score of -324.319 REU. DM687 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41T, Q28K, S76F) had a score of -323.70 REU. DM688 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41T, Q28R, S76W) has a score of -320.87 REU. DM689 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41T, Q28R, S76F) has a score of -322.01 REU.

[0597] Example 2: Cloning, expression and characterization of MNEI designed proteins

[0598] Recombinant MNEI protein was produced in E. coli BL21 (DE3+) under the T7 promoter induced with isopropyl β-D-1-thiogalactopyranoside (IPTG). Using this system, MNEI protein was expressed as cytosolic protein (soluble fraction) during high-density fermentation. Designed MNEI (DM) designed proteins had up to 11% amino acid substitutions.

[0599] All DMs were produced in E. coli fermentations and purified to levels >90%.

[0600] clone

[0601] DMs were generated using site-directed mutagenesis (SDM).

[0602] Fermentation

[0603] All DM clones were produced by bacterial fermentation in flasks incubated in horizontal operation at 37° C. Protein variant production was induced by IPTG.

[0604] purification

[0605] Purify all DM samples using the following steps:

[0606] 1. Pressure homogenizer lysis.

[0607] 2. Capture the protein on the multimodal resin and elute with increasing NaCl concentrations in the same buffer.

[0608] At least one polishing step using a resin from the following groups:

[0609] 1. Ion exchange.

[0610] 2. Hydrophobic interaction.

[0611] 3. Size exclusion.

[0612] 4. Final microbial filtration (0.2um) and storage at 2℃-8℃.

[0613] Characterization

[0614] Purification level

[0615] The level of purification was assessed by SDS-page followed by Coomassie blue staining and RP-HPLC.

[0616] Sweetness evaluation

[0617] Amai Proteins uses a regular, biweekly panel for sensory evaluations. The sensory panel includes panelists calibrated with sugar solutions on a scale of 0-100 (magnitude estimate), where 0 = not sweet at all and 100 = very sweet. A linear scale for sucrose is obtained at concentrations of 2°Bx, 4°Bx, 6°Bx, and 8°Bx.

[0618] Brix (Bx) = gr / 100 ml. After calibration, tasters graded the test samples on the same scale according to a validated tasting protocol.

[0619] The newly selected DM was tested in aqueous solution at 6° Bx, equivalent to the X3500 potency.

[0620] All tested products are provided using a code number.

[0621] Before and between samples, tasters were asked to rinse their mouths with mineral water, eat an unsalted cracker and a cucumber, and then drink water again.

[0622] Sweetness after heat treatment

[0623] The sweetness intensity of the new DM was measured after heat treatment at 95 °C for 30 min.

[0624] New DMs were tested in citrate buffer (pH ~3.1) at 5° Bx equivalent to the potency of X5000.

[0625] The citrate buffer was heated to 95°C in a hot mix, then DM was added to the hot mix and the temperature was kept at 95°C for 30 minutes. The heated buffer solution containing DM was then poured into the bottle and immediately cooled. Sweetness intensity was measured at room temperature.

[0626] All tested products are provided using a code number.

[0627] Before and between samples, tasters were asked to rinse their mouths with mineral water, eat an unsalted cracker and a cucumber, and then drink water again.

[0628] Time intensity

[0629] Temporal Intensity = measurement of sweetness intensity over time.

[0630] 1. Test the newly selected DM in aqueous solution at 6° Bx equivalent to X3500 potency.

[0631] 2. The panelists first calibrate with sugar solutions (in water) on a scale of 0-100, with 0 = no sweetness at all. The calibration solutions used were 4° Bx sucrose (defined as 50 sweetness) and 8° Bx (defined as 100 sweetness rating).

[0632] 3. After calibration, tasters rate each sample in terms of time-intensity according to the following stages:

[0633] a. After the signal was given, all tasters placed the sample in their mouth, rolled it in their oral cavity for 2 seconds, and then swallowed.

[0634] b. After swallowing, the instructor measures the number of times and, at each time point, asks the taster to rate the current perceived sweetness on the same scale (0-100) until the taste disappears.

[0635] c. The time periods for inspection are: 1, 2, 5, 7, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 135, 150, 180, 210, 240, 270, and 300 seconds.

[0638] 4. All tested products are provided using code numbers.

[0639] 5. Before and between samples, tasters were asked to rinse their mouths with mineral water, eat an unsalted cracker and cucumber, and then drink water again.

[0640] DSF Analysis and DSC Analysis - Thermal Sensitivity

[0641] Nanotemper Prometheus was used to determine the relative Tm by differential scanning fluorimetry (DSF). DSF is a simple, fast, and accurate method for analyzing protein stability and aggregation. DSF detects fluorescence changes of tryptophan residues and tyrosine residues in proteins. The fluorescence of tryptophan residues and tyrosine residues strongly depends on their surroundings. Changes in protein conformation will be reflected as fluorescence changes. The first-order derivative of the fluorescence ratio (330 / 350) is used to determine the inflection point. Since no secondary reporter fluorophore is required, protein solutions can be analyzed independently of buffer components and in a concentration range of 250 mg / ml to 10 μg / ml. DM was analyzed at a concentration of 0.5 mg / ml in 10 mM phosphate buffer at pH 7.

[0642] Table 3: Experimental results

[0643]

[0644]

[0645] #Determined by sensory panel.

[0646] Δ time / maximum intensity is the ratio between Δ time and maximum sweetness intensity of DM relative to DM31. Δ time (Delta time) is the difference between the time point when sweetness equals zero and the start of the experiment. Maximum sweetness intensity is the maximum value of the sweetness of DM. For each new variant, the ratio Δ time / maximum intensity of the variant is divided by the same ratio calculated for DM31 from the same sensory panel.

[0647] Table 4 shows the predicted and experimental Tm values ​​for modified proteins with substitutions in amino acid S76.

[0648] Table 4: Comparison of predicted and experimental Tm values ​​for exemplary proteins .

[0649]

[0650] As can be seen, the prediction follows the experimental data. This shows that the predicted Tm value can be used to predict the experimental Tm. As can be seen from the table, the modification in the amino acid S76 of MNEI or compared to DM31 increases the Tm value. Based on these results, it is shown that the different modifications in this amino acid are highly effective because they significantly increase the stability of the protein.

[0651] Combining substitutions in S76 with ring remodeling:

[0652] It has been shown that the L23 loop of MNEI is close to amino acid S76, and therefore substitution at amino acid S76 is thought to improve stability.

[0653] Several modified proteins with substitutions in S76 were prepared. Table 3 shows a list of modified proteins in which amino acid S76 was substituted and the experimental results for these proteins. Some variants are based on S76 in the context of DM31.

[0654] Figure 1A DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y) is shown, where the L23 loop is magenta and S76Y is cyan. The proximity of the loop and S76 is clearly visible. This indicates the possibility of interaction and possible synergy. The results show that adding the substitution in residue S76 to DM31 does improve stability, which can be seen by the DSF results (Table 3). DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y) is as sweet or slightly sweet as DM31, with a Tm of 101°C. In addition, it has a reduced aftertaste effect compared to DM31, which is shown in the fact that DM89 has a Δ time / maximum sweetness intensity reduction of about 10% (Table 3). DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y, Tm 101°C), DM114 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76F, Tm 101.5°C), DM137 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76W, Tm 97°C) are more stable than DM31. Their sweetness is similar to that of DM31, however, all three proteins show a reduced aftertaste (reduced by more than 25% for DM114 and by 10% for DM89 and DM137, see Table 3, Δ time / maximum sweetness intensity).

[0655] Amino Acid R84 :

[0656] DM Minimization Model 89( Figure 1B ) showed that R84 was relatively close to S76. Therefore, modified proteins containing substitutions in R84 were analyzed.

[0657] DM90 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, R84L, S76Y) had more native contacts, better REU scores, and lower RMSF AUC than DM70 (ΔE50, ΔF52, ΔR53, , E2N, E23A, Y65R, L70I, R8RL), DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y), and DM31. In addition, DM90 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, R84L, S76Y) has a predicted Tm of 104°C, which is higher than DM70 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, R84L) with an experimental Tm of 94°C.

[0658] As can be seen from the table above, combinations of one or more substitutions provide greater improved stability than each substitution alone.

[0659] Table 5 shows exemplary calculations for representative modified proteins, showing the advantages of combining one or more modifications. point .

[0660]

[0661]

[0662] The following modified proteins are predicted to have advantages in the modified combination: DM240, DM641, DM633, DM627, DM171, DM480, DM362, DM236, DM396, DM479, DM167, DM165, DM397, DM679, DM230, DM681, DM361, DM224, DM611, DM620, DM473, DM517, DM472 , DM685, DM608, DM628, DM521, DM542, DM477, DM640, DM532, DM528, DM621, DM530, DM360, DM73, DM 614, DM526, DM622, DM94, DM486, DM484, DM457, DM209, DM540, DM333, DM511, DM539, DM524, DM148.

[0663] Tm predictions were calculated in comparison to DM31.

[0664] Unless otherwise stated, all DMs below are based on MNEI (SEQ ID NO: 1) with substitutions of E2N, Y65R, L70I and E23A and deletions in E50, F52 and R53.

[0665] DM635 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, V20I, R84L, S76Y), the predicted Tm for this DM is 106°C, which is higher than DM31. The global VoroMQA packing score for this DM (0.51) is higher than that of DM31 (0.49) and DM89 (0.49), and the residue-specific packing score at position 76 (0.60) is much higher than that of DM31 (0.52) and DM89 (0.53).

[0666] The predicted Tm of DM286 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, R31T, S76Y) is 101°C, which is surprisingly high compared to the predicted Tms of 95°C (DM47) and 98°C (DM89).

[0667] DM combining the substitution in S76 with substitutions in D68, T33, G1M, G2M :

[0668] Taking into account computational analysis and predictions obtained with S76, the inventors designed additional proteins with additional substitutions that were believed to have improved properties, such as improved stability.

[0669] The inventors have preliminarily demonstrated the effect of substitutions in D68. D68N was previously shown to increase sweetness (Kohmura 1992), but its effect on stability was not described.

[0670] The following modified proteins were designed to predict the effect of substitutions in D68:

[0671] Modification of DM31 (DM108) with the substitution D68T showed increased sweetness (Table 3).

[0672] As can be seen from Table 2, the Rosetta energy score for the substitution D68N+S76Y (DM227) on MNEI decreased by 1.3 REU, while the same substitution (DM224) on DM31 increased the energy score by -5.9 REU. The DM224 REU score indicates its high stability. This suggests that there is a synergistic effect when combining D68N+S76Y with the substitutions made in DM31.

[0673] These calculation conclusions were verified by experiments, and as can be seen in Table 3, DM224 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, S76Y) has higher sweetness and higher stability than DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N) (Table 3).

[0674] DM361 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, D68N, S76Y) It has a higher Tm (97°C) (Table 3) than DM224 (D68N, S76Y) (95°C), and its stability prediction is better (Table 2). The experimental Tm of DM361 is 97°C, which is 3 degrees higher than DM31. In addition, the substituted Tm contained in the following: DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, D68N, Tm 90°C), DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y, Tm 101°) and DM591 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, Tm 91°). Therefore, the combination of D68N, S76Y and R65Y (reverse) is synergistic.

[0675] DM397 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y, D68N) The predicted Tm is 99.06°C and the ΔTm is 7.74°C, showing synergistic effect because it has a ΔTm that is better than the sum of the ΔTms of the following DMs that constitute it (3.4°C): DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N) has a predicted Tm of 87.09°C, DM89 (ΔE50, ΔF52, The predicted Tm of DM142 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R) is 98.19°C, the predicted Tm of DM142 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R) is 91.52°C, and the predicted Tm of DM591 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I) is 91.89°C.

[0676] DM362 (ΔE50, ΔF52, ΔR53, E23A, S76Y, D68N) The Tm is predicted to be 100°C, which is higher than DM224 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, S76Y). It has a predicted Tm of 100.32°C and a ΔTm of 9.0°C, and exhibits synergy as it has a ΔTm that is better than the sum of the ΔTms of the following DMs that constitute it (5.44°C): DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, D68N, with a predicted Tm of 87.09°C), DM122 (ΔE50, ΔF52, ΔR53, E23A, Y65R, L70I, S76Y, with a predicted Tm of 100.23°C, DM142 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, with a predicted Tm of 91.52°C), and DM591 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, with a predicted Tm of 91.89°C).

[0677] replace

[0678] DM627 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, R31T, S76Y) The Tm is predicted to be 100.65°C and the ΔTm is 9.33°C, which exhibits synergistic effects as it has a ΔTm that is better than the sum of the ΔTms of the following DMs that constitute it (5.55°C): DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N) Tm is predicted to be 87.09°C, and DM286 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, R31T, S76Y) Tm is predicted to be 101.1°C.

[0679] DM642 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68T, R31T, S76Y) has a predicted Tm of 101°C, while DM567 (D68T, R31T) has a predicted Tm of 94°C.

[0680] DM614 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, T33R, S76Y) It has a predicted Tm of 94°C, which is higher than DM31, DM511 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, T33R), DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N) and DM57 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, T33R). Molecular dynamics analysis showed that it has better REU and more native contacts than DM31. This DM has a ΔTm of 2.6°C, which exhibits synergism because it has a ΔTm that is better than the sum of the ΔTms of the following DMs that constitute it (-0.77°C): DM57 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, T33R) Tm predicted to be 87.92°C, DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N) Tm predicted to be 87.09°C, and DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y) Tm predicted to be 98.19°C.

[0681] DM542 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, Q28R, S76Y)It is based on DM66 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28R), which has a higher sweetness. DM542 has a higher predicted Tm than DM540 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, Q28R). With a predicted Tm of 96.7°C and a ΔTm of 5.38°C, this DM exhibits synergy as it has a ΔTm that is better than the sum of the ΔTms of the following DMs that constitute it (-11.49°C): DM66 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28R) with a predicted Tm of 77.2°C, DM87 (D68N) with a predicted Tm of 87.09°C, and DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y) with a predicted Tm of 98.19°C.

[0682] DM240 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, R84L, S76Y) Tm is predicted to be 104.03°C and ΔTm is 12.71°C, which shows synergy because it has a ΔTm that is better than the sum of the ΔTms of the following DMs that constitute it (8.85°C): DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N) Tm is predicted to be 87.09°C, and DM90 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y, R84L) Tm is predicted to be 104.4°C. In addition, there is synergy in terms of Rosetta energy score. These combinations produce an improvement of 6.85REU to DM31, and a smaller improvement of only 2.65REU to MNEI, indicating that these substitutions have synergy with the modification of DM31.

[0683] DM472 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, Q28K, S76Y)It has a higher Tm (100°C) than DM31. It shows synergy because it has a ΔTm of 6°C, which is higher than the sum of the ΔTms of DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y), DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N) and DM65 (ΔE5 0, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28K), or DM224 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, S76Y) and DM65 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28K). DM479 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, K25R, D68N, S76Y) has a low RMSF, which also indicates high stability. DM479 has a Tm of 97, which is 3 degrees better than DM31. It exhibits synergy as it has a ΔTm of 3 degrees, which is better than the sum of the ΔTms of DM101 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, K25R, S76Y), DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N) or DM224 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, S76Y) with DM43 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, K25R). DM240 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, R84L, S76Y) has a high predicted Tm with an REU of -329, which is an improvement of 6.9 REU - better than the improvement of DM243 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, R84L, S76Y) relative to MNEI (2.6 REU) - indicating a synergistic effect of this combination with DM31 and a better RMSF AUC.

[0684] DM241 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, R84L, S76W)DM31 has lower REU than DM137 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76W), DM70 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, R84L), and DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N). The Rosetta score for DM241 was -327.9 REU, an improvement of 6 REU relative to DM31, which was higher when compared to the -1.50 improvement of DM244 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, R84L, S76W on MNEI) on MNEI - these data suggest that the combination with DM31 is synergistic.

[0685] DM236 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, S76W) The Rosetta energy scores for these substitutions on MNEI (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, DM239) were reduced by 1.6 REU compared to MNEI, while the same substitutions on DM31 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, S76W) increased the energy score by -2.4 REU compared to DM31. This suggests that there is a synergistic effect when combining the modifications of substitutions D68N, S76W (of DM236) and DM31. This DM has a Tm of 99.75 and a ΔTm of 8.43, exhibiting synergistic effects as it has a ΔTm that is better than the sum of the ΔTms of the following DMs that constitute it (5.21): DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N) with a Tm of 87.09 and DM137 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76W) with a Tm of 97.

[0686] DM230 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, S76F)The Rosetta energy score for these substitutions on MNEI (DM233) was reduced by 1.0 REU compared to MNEI, while the same substitutions on DM31 (D68N, S76F) increased the energy score by -2.3 REU. This suggests that there is a synergistic effect when combining the substitutions of DM230 (D68N, S76F) and the modifications of DM31. This DM has a Tm of 98.95 and a ΔTm of 7.63, exhibiting synergistic effects as it has a ΔTm that is better than the sum of the ΔTms of the following DMs that constitute it (3.83): DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N) with a Tm of 87.09 and DM114 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76F) with a Tm of 99.39.

[0687] DM608 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68T, Q28R, S76Y) It has a higher Tm prediction than DM108 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68T) and DM66 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28R). The ΔTm is 5.74°C, which exhibits synergistic effects as it has a ΔTm that is better than the sum of the ΔTms of the following DMs that constitute it (-7.05°C): DM66 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28R) Tm is predicted to be 77.2°C, and DM649 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68T, S76Y) Tm is predicted to be 98.4°C.

[0688] DM644 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68T, R84L, S76Y) With a Tm prediction of 105. RMSF AUC -0.56, similar to DM31. And the assembly (using VoroMQA) at position 76 was very good.

[0689] G1M, E2M is a combination known to increase sweetness (Somoza et al. 1995). When these substitutions are combined with S76Y, DM360 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, G1M, E2M, S76Y) has lower β-sheet RMSD and lower REU than DM413 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, G1M, S76Y). DM360 also has a Tm of 94.48°C and a ΔTm of 3.16°C, which exhibits synergism because it has a ΔTm that is better than the sum of the ΔTms of the following DMs that constitute it (1.09°C): DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y) Tm is predicted to be 98.19°C, DM395 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, E2M) Tm is predicted to be 90.63°C, and DM412 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, G1M) Tm is predicted to be 86.23°C.

[0690] DM396 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, E2M, S76Y) Contains E2M for sweetening mentioned by Zhao et al. 2018. Its sweetness is 3 times higher than that of MNEI, however, its Tm is 4 degrees lower than that of MNEI (see Zhao et al.), indicating a significant decrease in stability. Based on the prediction model, we predicted an increase of 9°C for the combination of E2M and S76Y on the DM31 template. DM396 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, E2M, S76Y) is predicted to have a Tm of 99.6°C. It exhibits synergism as it has a ΔTm that is better than the sum of the ΔTms of the following DMs that constitute it (6.18°C): DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y) Tm predicted to be 98.19°C, DM395 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, E2M) Tm predicted to be 90.63°C.

[0691] DM222 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, T33R, S76Y)Tm prediction (98.19°C) is higher than DM57 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, T33R) (87.92°C). RMSF is similar to DM57 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, T33R) and DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y). The Rosetta energy score for these substitutions (DM225) on MNEI was reduced by 0.8REU, while the same substitutions on DM31 increased the energy score by -4.8REU. This indicates that there is a synergistic effect between the substitutions of DM222 and the modification of DM31.

[0692] DM638 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, T33R, R84L, S76Y) With a predicted Tm of 101.2.

[0693] DM622 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, T33R, Q28R, S76Y) Tm is predicted to be higher than DM539 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, T33R, Q28R), DM57 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, T33R) and DM65 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28K). Both Q28R and T33R have been shown to increase sweetness (Table 3). With a predicted Tm of 93.72°C and a ΔTm of 2.4°C, this DM exhibits synergism as it has a ΔTm that is better than the sum of the ΔTms of the following DMs that constitute it (-10.65°C): DM57 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, T33R) with a predicted Tm of 87.92°C, DM66 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28R) with a predicted Tm of 77.2°C, and DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y) with a predicted Tm of 98.19°C.

[0694] DM242 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, R84L, S76F)The Tm for this DM is predicted to be 105°C, which is higher than DM70 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, R84L), DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N) and DM114 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76F). It has an REU of -328, which is 6.6 units different from DM31, which is an improvement over DM245 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, R84L, S76F on MNEI, which improved by 3.8 REU), meaning that the combination has a synergistic effect on DM31.

[0695] DM628 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N, N35T, S76Y) The predicted Tm is 96.88°C and the ΔTm is 5.56°C, which exhibits synergism as it has a ΔTm that is better than the sum of the ΔTms of the following DMs that constitute it (3.04°C): DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N) Tm is 87.09°C and DM289 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, N35T, S76Y) Tm is 98.6°C.

[0696] Figure 4 shows the electrostatic potential of: DM31( Figure 4C ), DM89( Figure 4F )(ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y), DM222( Figure 4A )(ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, T33R, S76Y), DM224( Figure 4D )(D68N, S76Y), DM479( Figure 4B )(ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, K25R, D68N, S76Y), DM606( Figure 4E)(ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76R). Residues known to be important for sweet taste are labeled and presented as spheres. R88 and R72, R39, K36 and D7 are known to be important for receptor binding and sweet taste (Somoza et al. 1995, Yang et al. 2020, Liu et al. 2012). Substitutions of specific DMs are labeled and presented as spheres. S76Y does not interfere with the positive electrostatic surface of R88, R72, R39. T33R and D68N increase the positive surface imbalance and therefore have a positive effect on sweet taste.

[0697] Core substitution in combination with S76Y

[0698] Additional modified proteins were designed in which the MNEI core region was replaced .

[0699] DM247 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, V64I, S76F) The Rosetta energy score for these substitutions on MNEI (DM257) was 1.6 REU worse, while the same substitutions on DM31 improved the energy score by -2.6 REU. This suggests that the substitutions in DM247 and the modifications in DM31 have a synergistic effect.

[0700] DM202 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, A19V, S76Y) With an experimental Tm of 96°C, higher than DM31. . The RMSF AUC for this variant also indicates stability. In addition, the global VoroMQA assembly score for this variant (0.52) is better than that of DM31 (0.49) and DM89 (0.49). It is worth noting that DM202 has a better REU than DM31, reducing it by 4 points (-326.1 for DM202 and -321.66 for DM31), while the substitution A19V, S76Y (DM212) on MNEI increases the REU by 11 points (-304.31 for DM212 and -315 for MNEI). This means that the substitution and deletion of A19V, S76Y with DM31 have a synergistic effect. DM276 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, A19V, S76W) and DM256 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, A19V, S76F) decreased REU by 8 points compared to MNEI. However, the same substitutions on DM31, i.e., DM266 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, A19V, S76W) and DM246 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, A19V, S76F) increased REU by 1 point. In conclusion, the interactions of residues 19 and 76 are synergistic with other substitutions in DM31.

[0701] DM204 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, A73V, S76Y) This DM has a predicted Tm of 93°C, indicating increased stability. The global VoroMQA assembly score (0.51) is higher than the scores of its precursors DM31 (0.49), DM89 (0.49) and DM85 (0.5) (A73V on DM31). The Rosetta energy score (REU) for this variant is similar to the score of DM89, and is higher than the scores of DM31 and DM85. Its Rosetta score is -327REU, an improvement of 5REU relative to DM31, and when compared with DM214 (A73V, S76Y on MNEI) without any improvement relative to MNEI, the improvement is greater-this shows that the combination with DM31 works synergistically.

[0702] DM206 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, V20I, S76Y) The experimental Tm was shown to be 96°C, which is higher than DM31. The Rosetta energy score (REU) for this variant was -6REU better than DM31, and also better than DM89 and DM96 (V20I on DM31). The same substitution on MNEI (DM216) increased the REU by -1, which indicates a synergistic effect between the substitution of DM206 and the modification of DM31.

[0703] DM250 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, V20I, S76F) The REU decreased by 3 points relative to DM31. However, DM260 (V20I, S76F on MNEI) increased the REU by 1 point relative to MNEI. This indicates a synergistic effect between the substitution of DM250 and the modification of DM31.

[0704] DM209 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, A73F, F89M, S76Y) Its experimental Tm is 96°C, which is higher than DM31. This combination of substitutions improved REU by 8 points (-328.41 for DM209 and -321.66 for D31). However, the same combination on MNEI (DM219) did not improve REU (-315 in both MNEI and DM219). This means that A73F, F89M, S76Y are synergistic with DM31 substitutions and deletions.

[0705] DM253 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, A73F, F89M, S76F)Improved the REU by 6 points (-326.13 for DM253 and -321.66 for DM31). However, the same combination on MNEI (DM263) did not improve the REU (-315 in both MNEI and DM263). This means that the A73F, F89M, S76F substitutions are synergistic with DM31. This DM has a ΔREU of -4.47, so it exhibits synergy because it has a ΔREU that is better than the sum of the ΔREUs of the following DMs that constitute it (-0.86): DM131 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, A73F) has an REU of -320.83 and DM252 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, F89M, S76F) has an REU of -323.36. Furthermore, it exhibits synergistic effects as it has a ΔREU that is superior to the sum of the ΔREUs of the DMs that constitute it (0.06): DM104 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, F89M) REU is -319.99, DM114 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, S76F) REU is -324.1, and DM131 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, A73F) REU is -320.83.

[0706] DM273 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, A73F, F89M, S76W) Improves REU by 3.3 points, however, the same combination on MNEI (DM283) increases REU by 2.5 points. This means that the A73F, F89M, S76W substitutions are synergistic with DM31. This DM has a ΔREU of -3.32, showing synergy as it has a ΔREU that is better than the sum of the ΔREUs of the following DMs that compose it (-0.06): DM131 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, A73F) has an REU of -320.83 and DM272 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, F89M, S76W) has an REU of -322.55. Furthermore, it exhibits synergistic effects as it has a ΔREU that is superior to the sum of the ΔREUs of the DMs that constitute it (0.21): DM104 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, F89M) REU is -319.99, DM131 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, A73F) REU is -320.83, and DM137 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, S76W) REU is -323.95.

[0707] DM248 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, A73V, S76F) The predicted Tm for this DM is 95°C, indicating good stability. The Rosetta energy score (REU) for this DM is almost the same as that of DM89, and is greater than the sum of the effects of the substitutions of individual A73V (DM85) and S76F (DM114). Its Rosetta score is -327REU, an improvement of 5.7REU relative to DM31, which is greater when compared with DM258 (A73V, S76F on MNEI) with only 2.3REU improvement relative to MNEI-this indicates that the combination with DM31 works synergistically. The average native contact for this DM is higher than the average native contact of DM31. The A73V substitution (DM85) had a higher global VoroMQA assembly score (0.5) than DM31 and DM89 (0.49), and this effect was carried over into DM248 and was also observed in DM114 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I). 、 S76F), where a reduction in residue-specific assembly was observed at positions 73 and 76. Substitution of phenylalanine at position 76 was found in a structural homolog with a similar fold to this stem and loop structure.

[0708] DM210 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41A, S76Y) The Tm of this DM is predicted to be 96°C, higher than DM31. The Rosetta energy score for this DM is superior to that of DM31 - a Rosetta score of 328REU is much better - an improvement of 6.5 REU compared to DM31. The same combination on MNEI (DM220) yields a poorer Rosetta score of 1.6REU relative to MNEI, indicating synergy of this combination with DM31.

[0709] DM208 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41A, S76F) Compared with DM31, REU was reduced by 4 points, while DM264 (C41A, S76F on MNEI) had the same REU as MNEI, which means that C41A, S76F and DM31 substitutions and deletions have synergistic effects.

[0710] DM274 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41A, S76W (decreased REU by 3 points compared to DM31. However, DM284 (C41A, S76W on MNEI) increased REU of MNEI by 2.9 points. This suggests synergy of C41A+S76W with DM31 substitutions and deletions.

[0711] DM346 (ΔE5, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41S, S76Y)The Tm for this DM measured by DSF was 97°C, which is higher than DM31 and DM151 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, C41S). The RMSF AUC for this DM is better than DM31, DM89 and DM151. The average native contact is also better than DM31. The Rosetta score is better than DM31 and DM151. The residue-specific VoroMQA assembly score (0.54) at position 76 is better than that of DM31 (0.52) and DM89 (0.53). DM221 (C41S, S76Y on MNEI) increased the REU of MNEI by 2.8 points. However, DM346 reduced the REU by 4.3 points. This means synergy between C41S, S76Y and DM31 substitutions and deletions.

[0712] DM287 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41T, S76Y) The predicted Tm for this DM is 96°C, which is higher than DM31. The VoroMQA global assembly score (0.5) for this DM is better than DM31 (0.49), DM89 (0.49) and DM46 (C41T on DM31) with the same score. Surprisingly, the RMSF AUC for this DM is significantly inferior to DM31, DM89 and DM46, wherein the values ​​of DM89 and DM46 are similar and better than DM31. Despite the predicted instability, the Tm is higher than DM31. DM287 reduces the REU of DM31 by 4 points. However, the same substitution (DM297) on MNEI increases REU by 3 points. This means the synergistic effect between C41T, S76Y and DM31 substitutions and deletions.

[0713] DM477 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, C41T, D68N, S76Y) The predicted Tm for this DM is 97°C, which is higher than DM31, and the ΔTm is 5.3°C, which exhibits synergistic effects because it has a ΔTm that is better than the sum of the ΔTms of the following DMs that constitute it (1.38°C): DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, D68N), with a predicted Tm of 87.09°C, DM287 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, C41T, S76Y), with a predicted Tm of 96.37°C, and DM591 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I) with a predicted Tm of 91.89°C.

[0714] DM203 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, V64I, S76Y) The Rosetta energy score for these substitutions on MNEI (DM213) was 2.2 REU worse, while the same substitutions on DM31 improved the energy score by -4.4 REU. This suggests that the substitutions in DM203 and DM31 have a synergistic effect.

[0715] DM207 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, I75L, S76Y) With an REU of -325.6, it improves DM31 by 3 points. However, DM217 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, I75L, S76Y on MNEI) has an REU of -308.12, which increases the MNEI REU (-315) by 11 points. This implies synergy between I75L, S76Y and DM31 substitutions and deletions.

[0716] DM595( ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I , I75V, S76Y, E77V) The predicted Tm for this DM is 102°C, which is higher than all the DMs that make it up: DM31, DM89, DM593, DM594, DM600, DM601. The average β-sheet RMSD is also lower than that of the DMs that make it up, indicating that the β-sheet is stabilized by these substitutions.

[0717] DM611( ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, V20I, D68N, S76Y) The predicted Tm for this DM is 98°C, which is higher than DM31. The ΔTm is 6.78°C, which shows synergy because it has a ΔTm that is better than the sum of the ΔTms of the following DMs that constitute it (4.08°C): DM87 (D68N) Tm predicted 87.09°C, DM89 (S76Y) Tm predicted 98.19°C, DM96 (V20I) Tm predicted 92.76°C.

[0718] DM679( ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, V20I, D68N, S76F) The Tm is predicted to be 98.98°C and the ΔTm is 7.66°C, which exhibits synergism because it has a ΔTm that is better than the sum of the ΔTms of the following DMs that constitute it (5.28°C): DM87 (D68N) Tm is predicted to be 87.09°C, DM96 (V20I) Tm is predicted to be 92.76°C, and DM114 (S76F) Tm is predicted to be 99.39°C.

[0719] DM620( ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, V20I, Q28R, S76Y)The predicted Tm for this DM is 98°C, which is higher than DM31. Both V20I and Q28R showed increased sweetness (Table 3). This DM has a ΔTm of 6.58°C, which shows synergy because it has a ΔTm that is better than the sum of the ΔTms of the following DMs that constitute it (-5.81°C): DM66 (Q28R) Tm predicted 77.2°C, DM89 (S76Y) Tm predicted 98.19°C, DM96 (V20I) Tm predicted 92.76°C.

[0720] DM681( ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, V20I, Q28R, S76F) The Tm is predicted to be 98.64°C and the ΔTm is 7.32°C, which shows synergy because it has a ΔTm that is better than the sum of the ΔTms of the following DMs that constitute it (-4.61°C): DM66 (Q28R) Tm is predicted to be 77.2°C, DM96 (V20I) Tm is predicted to be 92.76°C, and DM114 (S76F) Tm is predicted to be 99.39°C.

[0721] DM621( ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, A19V, Q28R, S76Y) The predicted Tm for this DM is 95°C, which is higher than DM31. Both A19V and Q28R showed increased sweetness (Table 3). The ΔTm is 3.93°C, which shows synergy because it has a ΔTm that is better than the sum of the ΔTms of the following DMs that constitute it (-9.92°C): DM66 (Q28R) Tm predicted 77.2°C, DM202 (A19V, S76Y) Tm predicted 95.52°C.

[0722] DM289 ( ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I 、 N35T, S76Y) has a Tm prediction of 98°C. RMSF_AUC is better than DM31. It also improves REU by 6 points. However, DM299 (N35T, S76Y on MNEI) increases REU by 3 points, which means a synergistic effect between N35T, S76Y and DM31 substitutions and deletions.

[0723] DM292( ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I , N35T, S76W)This combination of substitutions on DM31 improved the Rosetta energy score by 2.7 REU, while these substitutions on MNEI resulted in a worse Rosetta score of 2.38 REU. These results suggest that there is a synergistic effect between these substitutions and the modification of DM31 relative to MNEI.

[0724] DM626 ΔE50, ΔF52, (( ΔE50, ΔF52ΔR53, E2N, E23A, Y65R, L70I 、A19V、R84L、 S76Y) The predicted Tm for this DM is 101°C, which is higher than DM31 and equal to DM89. The Rosetta energy score for this DM is better than that for DM31 (approximately -4 ΔREU).

[0725] DM633( ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, (( ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I , V20I, R31T, S76Y) The predicted Tm for this DM is 102°C, which is higher than DM31 and DM89 and all its precursors (DM96, DM47, DM286, DM206). The ΔTm is 10.36°C, which shows synergy because it has a ΔTm that is better than the sum of the ΔTms of the following DMs that constitute it (8.13°C): DM89 (S76Y) Tm is predicted to be 98.19°C, DM555 ( ΔE50, ΔF52,

[0726] ( ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I 、 V20I, R31T) Tm predicted to be 92.58°C. The Rosetta energy score for this DM is higher than DM31 and DM89. The average native contact for this DM is better than all its precursors except DM89.

[0727] DM600 ΔE50, ΔF52, (( ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I , I75V, S76Y) Has a predicted Tm (99.6°C) that is surprisingly and synergistically greater than the sum of the substitutions that make it up (DM601 and DM89). The RMSF AUC for this DM is superior to DM31. The I75V substitution is associated with S76Y compared to spatially similar stem and loop structures found in other proteins. It also has a better global VoroMQA assembly score (0.5) than its precursors: DM31 (0.49), DM89 (0.49) and DM601.

[0728] DM208 ΔE50, ΔF52 (( ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I , F89M, S76Y) Rosetta score of -326 REU, an improvement of 4.8 REU over DM31, is significantly lower when compared to DM218 which has only a 1.8 REU improvement over MNEI ( F89M、S76Y ) - indicating a synergistic effect of the combination with DM31.

[0729] Spiral Correlation

[0730] S76Y was also combined with two substitutions of helix capping, DM100 and DM101. DM100 was very stable - its experimental Tm was 100°C, 6 degrees greater than DM31. DM101 was very stable - its experimental Tm was 96°C, 2 degrees greater than DM31. Among these DMs with DM31 changes (including ring remodeling, S76Y and helix capping substitutions), it was shown that they had the following synergistic effects:

[0731] ○ Stabilize β by hardening the ring

[0732] ○ Use S76Y to protect β from water

[0733] ○ Use spiral capping instead to stabilize the spiral

[0734] ○ The natural contact of DM100 and DM101 is higher than that of DM31 and DM89 (but not higher than that of DM130).

[0735] DM100 shows a higher number of backbone H-bonds within β-sheets and within helices than MNEI, DM31, and DM130. These H-bonds maintain the secondary structure of these segments during simulations. The fraction of typical helices in DM100 is also higher in DM100 compared to these references.

[0736] Combining β-sheet stabilization with α-helices for synergistic stability enhancement:

[0737] ANM analysis Figure 3 ) showed that the global movement of MNEI is mainly driven by two flexible regions in the protein: 1-β-hairpin, which consists of β-strands 2 and 3 and β-strands 4 and 5 that drive the movement of the β-sheet; 2-helix and its C-terminal loop. The combined hardening of these two key regions is predicted to achieve a synergistic increase in stability. This is accomplished by adding the S76Y modification to the modifications in the C-terminus of the helix: Q28K, Q28R, Q28S, K25R.

[0738] Q28K :

[0739] This substitution was originally discovered by Leone et al. (Leone 2016) to enhance sweetness. Q28K was found to enhance the helical end by improving the assembly of this region (ref. PCT March). We have found that Q28K enhances the helical end by improving the assembly of this region (ref. PCT March). 、 The two substitutions are combined in DM89 (S76Y), whose MD simulation shows that it is better in maintaining native contacts and it has a RMSD that is somewhat better than that of DM89 (S76Y) (Table 2, RMSD some and). This confirms the hypothesis that the β-sheet and helix are related.

[0740] DM632 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I 、 Q28K, R84L, S76Y) has a predicted Tm of 104.5, which is higher than DM31.

[0741] DM335 (ΔE50, ΔF52, ΔR53, E23A 、 Q28K, S76Y) (which are Q28K, S76Y on MNEI) are also better in terms of average native contacts and have greatly improved RMSD (Table 2).

[0742] DM630 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I 、 Q28K, R31T, S76Y relative to DM552 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I) 、 Q28K, R31T) will be added later.

[0743] DM158 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I 、 Q28K, I26T, S76Y on MNEI) has a Tm prediction of 93.98. Its Rosetta score is -326REU, an improvement of 4.4REU over DM31, which is greater when compared to DM180 (Q28K, I26T, S76Y on MNEI) which has no improvement over MNEI - indicating a synergistic effect in combination with DM31.

[0744] DM486( ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, I26T, Q28K, D68N, S76Y)With a similar Tm prediction to DM31, Tm prediction of 93.48°C and ΔTm of 2.16°C, it exhibits synergy as it has a ΔTm that is better than the sum of the ΔTms of the following DMs that make it up (-1.0°C): DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I 、 D68N) Tm is predicted to be 87.09℃, DM158 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I 、 Q28K, I26T, S76Y) Tm is predicted to be 93.98°C, and DM591 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I) Tm is predicted to be 91.89°C.

[0745] DM456( ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28S, I26T, S76Y) . It has a better native contact (Table 2). Its experimental Tm is 104°C, which is 10 degrees more than DM31. It exhibits synergy because it has a better ΔTm than the sum of the ΔTms of: DM100 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I 、 I26T, S76Y) Tm is 100, and DM330 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I 、 Q28S)Tm is 94.1. DM455 (ΔE50, ΔF52ΔR53 E2N, E23A, Y65R, L70I 、 Q28K, I26V, S76Y) has a better Tm prediction than DM31, and it has a better canonical helix score than DM89, DM31 and DM65.

[0746] DM473( ΔE50, ΔF52, ΔR53, E2N, E23A, L70I , D68N, Q28K, S76Y) With a predicted Tm of 97.75 and a ΔTm of 6.43°C, it exhibits synergy as it has a ΔTm that is better than the sum of the ΔTms of the following DMs that constitute it (2.85°C): DM87 (ΔE50, ΔF52,

[0747] ΔR53, E2N, E23A, Y65R, L70I 、 D68N) Tm is predicted to be 87.09℃, DM363 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I 、Q28K, S76Y) Tm is predicted to be 97.83°C, and DM591 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I) Tm is predicted to be 91.89°C.

[0748] Helix and core combination of S76Y :

[0749] Since the region connecting the beta sheets and the alpha helices is the core of the protein, we added core modifications to the above combination to improve the interaction between these regions, thereby combining the effects of the two stabilizing modifications.

[0750] Residue C41 is in the core of the protein, and a C41S substitution was previously shown to increase sweetness (Liu 2015, Zhao 2019). C41A was predicted to have no effect on stability and increase sweetness (Tang 2020).

[0751] Q28R

[0752] We proposed Q28R in March 2023, which follows the activation pattern of Q28K and shows a stronger positive effect on assembly.

[0753] DM517( ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28R, S76Y) Its experimental Tm is 100°C, which is 6 degrees more than DM31. Its sweetness intensity is similar to DM31. The sweetness after heat treatment is similar to DM31. MD simulations indicate better H-bonding of β-sheets and better water-proof protection of H-bonds (Table 2).

[0754] DM641( ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I , Q28R, R84L, S76Y) With a Tm prediction of 103.8, this is better than the Tm of DM31. With a ΔTm of 12.52°C, it exhibits synergy as it has a ΔTm that is better than the sum of the ΔTms of the following DMs that make it up (-1.04°C): DM66 ( ΔE50, ΔF52,

[0755] ΔR53, E2N, E23A, Y65R, L70I 、 Q28R) Tm is predicted to be 77.2℃, DM90 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I 、 S76Y, R84L) Tm is predicted to be 104.4℃.

[0756] DM640( ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28R, N35T, S76Y) The Tm is predicted to be 96.57°C and the ΔTm is 5.25°C, which shows synergy as it has a ΔTm that is better than the sum of the ΔTms of the following DMs that constitute it (-6.84°C): DM66 ( ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I 、 Q28R) Tm is 77.2℃, DM289 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I 、 N35T, S76Y) Tm is 98.6℃.

[0757] To further improve protein stability and improve RMSF parameters via modified cores, we have tested DM517 in combination with C41A, C41S, and C41T. All of these DMs have Tm predictions that are better than DM31 and similar to DM89. ( ΔE50, ΔF52,

[0758] ΔR53, E2N, E23A, Y65R, L70I 、 C41A, Q28R, S76Y) has a predicted Tm of 96.43°C and a ΔTm of 5.11°C, which exhibits synergy as it has a ΔTm that is better than the sum of the ΔTms of the following DMs that constitute it (-9.79°C): DM66 ( ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I 、 Q28R) Tm is predicted to be 77.2°C, DM210 (C41A, S76Y) Tm is predicted to be 95.65°C. DM528 ( ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I 、 (C41T, Q28R, S76Y) and DM530 ( ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I 、 C41S, Q28R, S76Y) had a superior RMSF AUC compared to DM66, DM31 and DM DM89 (Table 2). DM530 also had a superior RMSD than DM66, DM31 and DM89. Both criteria indicated an increase in stability (Table 2). DM530 had a Tm of 94.79°C and a ΔTm of 3.47°C, which exhibited synergy because it had a ΔTm that was superior to the sum of the ΔTms of the following DMs that constitute it (-9.65°C): DM66 ( ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I 、Q28R) Tm is predicted to be 77.2℃, DM89 (S76Y) Tm is predicted to be 98.19℃, and DM702 (C41S) Tm is predicted to be 88.93℃.

[0759] DM685( ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41A, Q28R, S76F) The predicted Tm is 97.31°C and the ΔTm is 5.99°C, which shows synergy because it has a ΔTm that is better than the sum of the ΔTms of the following DMs that constitute it (-6.47°C): DM66 ( ( ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I 、 Q28R) Tm is predicted to be 77.2℃, DM114 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I 、 S76F) Tm is predicted to be 99.39℃, DM150 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I 、 C41A) Tm predicted to be 90.9°C.

[0760] DM528( ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I , C41T, Q28R, S76Y) The predicted Tm is 96.31°C and the ΔTm is 4.99°C, which shows synergy as it has a ΔTm that is better than the sum of the ΔTms of the following DMs that constitute it (-9.07°C): DM66 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I 、 Q28R) Tm is predicted to be 77.2℃, DM287 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I 、 C41T, S76Y) Tm is predicted to be 96.37℃.

[0761] DM165( ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I , K25R, Q28R, S76Y) It has a better Tm prediction than DM31 and DM89. Its Tm is experimentally evaluated (99°C). This Tm is similar to DM101 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I 、 K25R, S76Y) and DM66 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I) 、The combination of DM165 and DM31 (Q28R) was synergistic because DM165 had a Δ of 5 degrees relative to DM31, while DM66 and DM101 had a sum of only 3 degrees. DM165 also showed synergy in terms of sweetness intensity because it was higher than DM101. ( ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I 、 K25R, S76Y) and DM66 ( ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I 、 The combination of K25R, Q28K and S76Y with DM31 (DM187) can increase sweetness. It has better RMSD than DM66, DM31 and DM89, and it better maintains the native contacts of the starting structure (Table 2, average native contacts). DM165 has a Rosetta energy score of 1REU lower than DM31 (-322.97 relative to -321.66), while the same substitution on MNEI (DM187) increases REU (-313.04 for DM187 and -315 for MNEI). This may mean the synergy of K25R, Q28K and S76Y substitutions with DM31.

[0762] DM167( ΔE50, ΔF52 (( ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I 、K25R、Q28R、 S76F) The predicted Tm is 99.37°C and the ΔTm is 8.05°C, which shows synergy because it has a ΔTm that is better than the sum of the ΔTms of the following DMs that constitute it (-5.1°C): DM66 (Q28R) Tm is predicted to be 77.2°C, DM160 ( ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I 、 S76F, K25R) Tm is predicted to be 100.34℃.

[0763] DM171( ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I , Q28R, R31T, S76Y) It has a better Tm prediction than DM31 and DM89. It has a Tm prediction of 100.46°C and a ΔTm of 9.14°C, showing synergy because it has a ΔTm that is better than the sum of the ΔTms of the following DMs that constitute it (-4.34°C): DM66 (ΔE50, ΔF52,

[0764] ΔR53, E2N, E23A, Y65R, L70I, Q28R) has a predicted Tm of 77.2°C, and DM286 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, R31T, S76Y) has a predicted Tm of 101.1°C. It has a better RMSFAUC than DM31.

[0765] DM521 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, D68N, Q28R, S76Y) has a Tm prediction better than DM31 and similar to DM89, and a ΔTm of 5.41°C, which exhibits synergy because it has a ΔTm that is better than the sum of the ΔTms of the following DMs that constitute it (-10.7°C): DM66 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28R) Tm is predicted to be 77.2°C, DM87 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N) Tm is predicted to be 87.09°C, and DM123 (S76Y, R65Y (reverse)) Tm is predicted to be 98.98°C.

[0766] DM526 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28R, I26T, S76Y) has a similar Tm prediction to DM31, with a Tm prediction of 93.89°C and a ΔTm of 2.57°C, which exhibits synergy as it has a ΔTm that is better than the sum of the ΔTms of the following DMs that constitute it (-9.87°C): DM66 (ΔE50, ΔF52,

[0767] The predicted Tm of ΔR53, E2N, E23A, Y65R, L70I, Q28R) is 77.2°C, and the predicted Tm of DM100 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y, I26T) is 95.57°C.

[0768] K25R

[0769] We submitted the K25R substitution as part of the PCT in March 2023. This substitution provides stronger H-bonding and protection of the backbone between positions 21 and 25, resulting in increased stability. The complex of K25R and S76Y (DM101: (ΔE50, ΔF52,

[0770] ΔR53, E2N, E23A, Y65R, L70I, K25R, S76Y) produced better RMSF than DM31, DM43 (K25R) and DM89.

[0771] DM160 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, K25R, S76F) has a Tm prediction of 100°C. Its REU is better than DM31 (-324.54 vs. -321.66). However, K25R, S76F on MNEI (DM182) has a poorer REU than MNEI (-313.12 for DM182 vs. -315 for MNEI). This shows a synergistic effect between the K25R, S76F and DM31 substitutions.

[0772] DM480 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I, K25R, D68N, S76Y) has a better Tm than DM31 and DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y) with a ΔTm of 9.01°C, which exhibits synergistic effects because it has a ΔTm that is better than the sum of the ΔTms of the following DMs that constitute it (5.32°C): DM 87 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, D68N) Tm was predicted to be 87.09 ° C, DM101 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y, K25R) Tm was predicted to be 100.3 ° C, and DM591 (ΔE50, ΔF52, ΔR53, E2N, E23A, L70I) Tm was predicted to be 91.89 ° C. Compared with DM31, it has a better RMSF AUC (Table 2) and has a better RMSD than DM31, DM43 (K25R) and DM89, indicating a stronger increase in stability.

[0773] DM687 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41T, Q28K, S76F) with an REU of -323.7 and a ΔREU of -2.04 exhibits synergy as it has a ΔREU that is superior to the sum of the ΔREUs of the DMs that constitute it (-0.01): DM65 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28K) with an REU of -320.54 and DM294 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41T, S76F) with an REU of -322.79. Furthermore, it exhibits synergistic effects as it has a ΔREU that is superior to the sum of the ΔREUs of the following DMs that constitute it (0.07): DM46 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, C41T) with an REU of -320.28, DM65 (ΔE50, ΔF52,

[0774] The REU of ΔR53, E2N, E23A, Y65R, L70I, Q28K) is -320.54, and the REU of DM114 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76F) is -324.1.

[0775] DM127 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, I26W, Q28E, S76Y) and DM128 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, I26W, Q28K, S76Y) had Tm predictions as good as DM89, with better protected H bonds (water-β-sheet backbone H bonds, Table 2) and favorable minimum REUs of -329.421 (ΔREU -7.76) and -329.989 (ΔREU -1.06). U-8.33), the improvement in REU of this combination relative to DM31 was greater than that of the same combinations on MNEI: DM200 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, I26W, Q28E, S76Y) (ΔREU of -3.93) and DM201 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, I26W, Q28K, S76Y) (ΔREU of -3.82) - indicating synergy of these combinations with DM31. DM127 and DM128 are superior to DM31, DM89, DM115 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, I26W, Q28E) and DM116 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, I26W, Q28K).

[0776] DM162 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76W, K25R) has better Tm prediction than DM31 and DM89. Its average native contacts are better than DM31 and similar to DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y). Its helices are more typical. Its RMSF AUC is better than DM43 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, K25R).

[0777] DM159 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76F, I26T) has a Tm prediction of 96 and has a better RMSF AUC compared to DM31 and DM89. (Table 2)

[0778] DM178 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, I26W, Q28K, S76W) had a Rosetta score of -327 REU, an improvement of 5 REU over DM31, which was greater when compared to DM198 (I26W, Q28K, S76W over MNEI) which had only a 1.5 REU improvement over MNEI - suggesting that the combination with DM31 is synergistic. Furthermore, this DM has a ΔREU of -5.34 and exhibits synergistic effects as it has a ΔREU that is superior to the sum of the ΔREUs of the following DMs that constitute it (-2.98): DM65 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, Q28K) with an REU of 320.54, DM137 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76W) with an REU of -323.95, and DM144 (I26W) with an REU of -323.48.

[0779] DM179 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, I26W, Q28E, S76W) had a Rosetta score of -326 REU, an improvement of 4.4 REU over DM31, which was greater when compared to DM199 (I26W, Q28E, S76W over MNEI) which had only a 1.5 REU improvement over MNEI - suggesting that the combination with DM31 is synergistic.

[0780] DM100 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y, I26T) has an experimental Tm of 100°C, which is 10 degrees more than DM31. It shows a higher number of backbone H bonds as intra-β-sheet and intra-helical H bonds than MNEI, DM31, and DM130. These H bonds maintain the secondary structure of these segments during the simulation. The fraction of typical helices is also higher in DM100 when compared to the reference design described above.

[0781] DM174 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, R84L, S76W) had a Rosetta score of -327 REU, an improvement of 5.6 REU over DM31, which was greater when compared to DM302 (R84L, S76W on MNEI) which had only a 2.2 REU improvement over MNEI - suggesting that the combination with DM31 is synergistic.

[0782] Alternatives to S76Y prompt new title: β-sheet stabilization via protection of main-chain h-bonds (59aa-76aa)

[0783] To stabilize the β-sheet of the protein, we added modifications that protect the H-bonds of the main chain between β-strands 3 and 4. This was primarily achieved by substituting residues in positions 59 and 76 with β-branched or bulky residues. Since position 59 is close to position 76, substitutions at this position can protect hydrogen bonds within the β-sheet ( Figure 1A ). For example, DM652 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, E59V, S76Y) has β-branched substitutions, and structural analysis shows that the backbone is not exposed to solvent ( Figure 2 ).

[0784] DM652 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, E59V, S76V) has a predicted Tm of 101°C for this DM, similar to DM89 and much higher than DM31. The Rosetta energy score is better than DM31 (about -1.5 ΔREU). The average native contacts for this DM are much higher than those for DM31 and DM89. In the homologous structures of this stem-loop, the substitution of E59V is associated with S76V, and this pair together dominates in these homologous protein substructures.

[0785] DM653 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, E59I, S76V) has a predicted Tm of 100°C for this DM, similar to DM89 and much higher than DM31. The Rosetta energy score is better than DM31 (approximately -2ΔREU). The RMSF AUC for this DM is better than DM31 ( Figure 5 ) and DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y). The average native contact for this DM is higher than DM31 and slightly lower than DM89. In the homologous structure of this stem-loop, the substitution of E59I is associated with S76V.

[0786] The predicted Tm of DM654 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, E59T, S76V) for this DM is 101°C, which is similar to DM89 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76Y) and much higher than DM31. In the homologous structure of this stem-loop, the substitution of E59T is associated with S76V.

[0787] DM655 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, E59V, S76I) has a predicted Tm of 102°C for this DM, which is higher than DM89 and DM31. The Rosetta energy score for this DM is better than DM31 (about -2ΔREU), but worse than DM89. The global VoroMQA assembly score (0.55) is better than DM31 (0.49) and DM89 (0.49), and the residue assembly score at position 76 (0.66) is higher than DM31 (0.52) and DM89 (0.53). The RMSF AUC is lower than its precursor DM664 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76I). The Tm values ​​obtained here are surprising since the RMSF AUC values ​​are much worse than those for DM31 and DM89, also for the average native contacts.

[0788] The predicted Tm of DM656 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, E59I, S76I) for this DM is 101°C, similar to DM89 and much higher than DM31. The Rosetta energy score is better than DM31 (about -3ΔREU). The RMSF AUC is better than DM31 and DM89 ( Figure 5 ). The average RMSD of the β-sheet is lower than that of DM31 and DM89, indicating a stable fold. The VoroMQA scores for residues at positions 59 (-0.58) and 76 (-0.55) are better than those of DM31 (0.57 and 0.52), with the value for position 76 also being better than that of DM89 (0.53). In the homologous structure of the stem-loop, the substitution of E59I is associated with S76I.

[0789] DM657 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, E59T, S76I) has a predicted Tm of 102°C for this DM, which is higher than DM89 and DM31. The RMSF AUC is better than DM31 and DM89. The average RMSD of the β-sheet is lower than that of DM31 and DM89, indicating a stable fold. The average native contacts are lower than those of DM31 and DM89. The Rosetta energy score is similar to DM31. Surprisingly, despite the lower average native contacts and no improvement in the Rosetta energy score compared to DM31, the predicted Tm is very high.

[0790] DM662 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, E59Y, S76V) has a predicted Tm of 100°C for this DM, which is higher than DM31. The Rosetta energy score for this DM is better than DM31 by about -2REU. The average native contacts for this DM are better than those of DM31, DM89, and DM663 (S76V). The global VoroMQA assembly score for this DM (0.51) is also better than DM31 (0.49) and DM89 (0.49), and is similar to DM661.

[0791] The predicted Tm of DM663 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76V) for this DM is 98°C, which is higher than DM31. The Rosetta score for this DM is better than DM31. The RMSF AUC for this DM is better than DM31 and DM89 ( Figure 5 ). Homologous structures of the stem-loop structure in this region typically contain valine at position 76.

[0792] DM664 (ΔE50, ΔF52, ΔR53, E2N, E23A, Y65R, L70I, S76I) has a predicted Tm of 100°C for this DM, much higher than DM31 and similar to DM89. The average native contact for this DM is higher than DM31 and DM89. The Rosetta energy score for this DM is better than DM31, but worse than DM89. Homologous structures of the stem-loop structure in this region generally contain isoleucine at position 76.

[0793] Example 3: Energy bar formulation containing designer MNEI (DM) protein

[0794] The energy bar formula containing DM protein is presented in Table 6.

[0795] Table 6: DM-based energy bar formulation (40% reduction in added sugar compared to energy bars without DM)

[0796] Element Energy Bars Whole oats 21g-26g Whole Crispy Rice 17g-22g Pecan 8g-12g almond 8g-12g Glucose syrup 17g-22g Palm Oil 6g-9g glycerin 1g-3g Soy lecithin 0.2g-1g Salt 0.1g-0.5g Maltodextrin 2g-5g DM protein 7mg-10mg

[0797] Example 4: Almond protein formulation containing designer MNEI (DM) protein

[0798] Marzipan is a confectionery made mainly from ground almonds and sugar. It is widely used in the baking industry and for preparing different kinds of desserts.

[0799] The marzipan recipe containing DM protein is presented in Table 7.

[0800] Table 7: DM-based marzipan recipe (70% less added sugar) compared to a DM-free bar

[0801] Element percentage White almonds 28％-48％ Maltodextrin 20％-40％ Powdered sugar 3％-23％ water 1％-20％ fiber 1％-10％ glucose 1％-5％ lemon juice 0.1％-0.6％ Almond Extract 0.1％-0.6％ DM 0.01％-0.05％

[0802] Preparation Instructions :

[0803] Grind the almonds, gradually add all the powder to the mixer and grind thoroughly. Add all the liquid ingredients as well as the sweetening protein to the mass and continue grinding until a crystalline dough is obtained. Add 0.01% - 0.05% protein by weight to the recipe. The DM potency is 1000-3000 and can be equivalent to 25-45 Brix.

[0804] Example 5: Non-dairy milk prototype with designer DM protein

[0805] Non-dairy milk formulas containing DM protein are presented in Table 8.

[0806] Table 8: DM-based non-dairy milk formulas with 50% less added sugars compared to non-dairy milk formulas with 50% less added sugars Milk formula

[0807] formula :

[0808] Element Oat milk Oat milk with no added sugar 98g-99.4g sugar 0.6g-2g AMAI sweet protein DM-31 0.3mg-0.7mg

[0809] Element Almond milk Almond milk with no added sugar 98g-99.4g sugar 0.6g-2g AMAI sweet protein DM-31 0.6mg-1.4mg

[0810] Element Soy milk Soy milk with no added sugar 97.7g-99.3g sugar 0.7g-2.3g AMAI Sweet Protein Dm-31 0.8mg-1.6mg

[0811] Preparation Instructions :

[0812] Sugar and AMAI sweetened protein DM were added to unsweetened non-dairy milk and mixed into a homogenous solution.

[0813] Example 6: Granola Preparation

[0814] The formulation of granola containing DM protein is presented in Table 9.

[0815] Table 9: DM-based granola

[0816]

[0817]

[0818] Preparation Instructions

[0819] Toast the oats, crispy rice, pecans and almonds at 160°C for 15 minutes.

[0820] Heat the glucose syrup, powdered sugar, sunflower oil, glycerine, lecithin and salt into a homogeneous syrup.

[0821] Crush the roasted pecans and almonds into small pieces. Add the dry ingredients (oats, crispy rice, pecans and almonds) to the syrup and mix. Add the maltodextrin and DM 31 last.

[0822] Pour mixture into baking dish.

[0823] Bake the granola mixture at 90°C for 10 minutes, then remove, cool and store in a cool place in an airtight container.

[0824] Example 7: Peanut Butter Spread

[0825] Peanut butter spread formulations for various filling options containing DM protein are presented in Table 10.

[0826] Table 10: DM-based peanut butter spreads

[0827] Material percentage peanut 56％-76％ Polydextrose 5％-15％ sugar 3％-14％ Maltodextrin 2％-13％ fiber 1％-11％ Palm Oil 1％-5％ Salt 0.1％-0.5％ DM 0.1％-0.5％

[0828] Preparation Instructions :

[0829] 1. Grind the peanuts.

[0830] 2. Gradually add ingredients 2-7 to the mixer and grind well until a homogeneous dough is obtained.

[0831] 3. Add the sweetened protein DM to the mass and continue grinding at a slow rate until fully assimilated.

Claims

1. A modified protein, the modified protein having an amino acid sequence comprising at least one amino acid deletion and at least one amino acid substitution compared to a reference protein, wherein the reference protein is represented by the amino acid sequence shown in SEQ ID NO: 1, wherein the at least one amino acid deletion is at one or more amino acids between T46 and I56 of the reference protein, and wherein the at least one amino acid substitution is at amino acid S76 of the reference protein.

2. The modified protein of claim 1, wherein the at least one amino acid is deleted at one or more of amino acids E50, F52, R53, or any combination thereof of the reference protein.

3. The modified protein of claim 1, wherein the at least one amino acid deletion comprises a deletion of at least three amino acids between amino acid T46 and amino acid I56.

4. The modified protein according to any one of claims 1 to 3, wherein the at least one amino acid deletion comprises a deletion of amino acids E50, F52 and R53.

5. A modified protein according to any one of the preceding claims, wherein the at least one amino acid substitution comprises at least one amino acid substitution in addition to the amino acid substitution at amino acid S76 of the reference protein.

6. A modified protein according to any one of the preceding claims, wherein the at least one amino acid substitution comprises at least two amino acid substitutions in addition to the amino acid substitution at amino acid S76 of the reference protein.

7. A modified protein according to any one of the preceding claims, wherein the at least one amino acid substitution comprises at least three amino acid substitutions in addition to the amino acid substitution at amino acid S76 of the reference protein.

8. The modified protein of any one of the preceding claims, wherein the at least one amino acid substitution comprises an amino acid substitution at one or more of amino acids G1, E2, E4, T12, A19, V20, E23, K25, I26, G27, Q28, T33, N35, C41, Q61, V64, Y65, D68, L70, A73, I75, R84, F89, or any combination thereof.

9. The modified protein according to any one of the preceding claims, wherein the at least one amino acid substitution comprises one or more of the amino acid substitutions (i) G1M, (ii) A19V, (iii) V20I, (iv) K25R, (v) one or more of I26W, I26V or I26T, (vi) one or more of Q28R, Q28K, Q28S, Q28E, (vii) R31T, (viii) T33R, (ix) N35T, (x) one or more of C41A, C41V, C41T or C41S, (xi) one or more of E59T, E59V, E59I, E59Y, E59F, E59W or E59R, (xii) D68N or D68T, (xiii) A73F or A73V, (xiv) D74V, (xv) I75V or I75L, (xvi) E77V, (xvii) R84L.

10. The modified protein of any one of the preceding claims, wherein the at least one amino acid substitution comprises an amino acid substitution at one or more of amino acids E2, E23, Y65, L70, or any combination thereof.

11. The modified protein of any one of the preceding claims, wherein the at least one amino acid substitution comprises an amino acid substitution at one or more of amino acids E2, E23, Y65, L70.

12. The modified protein of any one of the preceding claims, wherein the amino acid substitution at amino acid S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H or S76A.

13. The modified protein of any one of the preceding claims, wherein the at least one amino acid substitution comprises an amino acid substitution at one or more of amino acids E2N, E23A, Y65R, L70I, or any combination thereof.

14. The modified protein of any one of the preceding claims, wherein the at least one amino acid substitution comprises an amino acid substitution at one or more of amino acids E2M, E23A, Y65R, L70I, or any combination thereof.

15. A modified protein according to any one of the preceding claims, wherein the at least one amino acid substitution comprises one or more of the following amino acid substitutions: (i) E2N, E23A, Y65R, L70I, (ii) E2M, E23A, Y65R, L70I, (iii) E2N, E23Q, Y65R, L70I, or (iv) E2M, E23Q, Y65R, L70I.

16. A modified protein according to any one of the preceding claims, wherein the at least one amino acid substitution comprises one or more of the amino acid substitutions (i) G1M, (ii) A19V, (iii) V20I, (iv) K25R, (v) one or more of I26W, I26V or I26T, (vi) one or more of Q28R, Q28K, Q28S, Q28E, (vii) R31T, (viii) T33R , (ix) N35T, (x) one or more of C41A, C41V, C41T or C41S, (xi) one or more of E59T, E59V, E59I, E59Y, E59F, E59W or E59R, (xii) D68N or D68T, (xiii) A73F or A73V, (xiv) D74V, (xv) I75V or I75L, (xvi) E77V, (xvii) R84L.

17. A modified protein according to any one of the preceding claims, comprising SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:221, SEQ ID NO:108, SEQ ID NO:9, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:52, SEQ ID NO:182, SEQ ID NO:10, SEQ ID NO:69, SEQ ID NO:157, SEQ ID NO:114, SEQ ID NO:174, SEQ ID NO:162, SEQ ID NO:231, SEQ ID NO:57, SEQ ID NO:192, SEQ ID NO:68, SEQ ID NO:106, SEQ ID NO:8, SEQ ID NO:79, SEQ ID NO:76, SEQ ID NO:22, SEQ ID NO:194, SEQ ID NO:262, SEQ ID NO:84, SEQ ID NO:239, SEQ ID NO:135, SEQ ID NO: The amino acid sequence set forth in one or more of SEQ ID NO:211, SEQ ID NO:237, SEQ ID NO:198, SEQ ID NO:179, SEQ ID NO:172, or any combination thereof.

18. The modified protein according to any one of the preceding claims, having increased stability compared to the reference protein.

19. The modified protein of claim 18, wherein the stability is one or more of thermal stability, chemical stability (over a range of pH values, at different hydrophobicity levels, in different food and beverage matrices and at different levels of protein, lipid and preservatives in the matrix), functional stability (in terms of sensory characteristics, short-term or long-term, and included in shelf life studies).

20. The modified protein according to any one of the preceding claims for use in the preparation of a product for oral delivery.

21. The modified protein according to any one of the preceding claims for use as a flavor improving agent, flavor enhancing agent or flavor masking agent.

22. The modified protein according to any one of the preceding claims for use as a sweetener.

23. The modified protein according to any one of the preceding claims for use as a stabilizer.

24. A food product comprising the modified protein according to any one of the preceding claims.

25. A method for increasing the stability of a reference protein, the method comprising deleting at least one amino acid of the reference protein and replacing at least one amino group of the reference protein, wherein the reference protein is represented by the amino acid sequence shown in SEQ ID NO:1, wherein the at least one deleted amino acid is at one or more amino acids between T46 and I56 of the reference protein, and wherein the at least one substituted amino acid is at amino acid S76 of the reference protein.

26. The method of claim 25, wherein the at least one amino acid is deleted at one or more of amino acids E50, F52, R53, or any combination thereof of the reference protein.

27. The method according to claim 25, wherein the at least one amino acid deletion comprises a deletion of at least three amino acids between amino acid T46 and amino acid I56.

28. The method according to any one of the preceding claims, wherein the at least one amino acid deletion comprises deletion of amino acids E50, F52 and R53.

29. The method of any of the preceding claims, wherein the at least one amino acid substitution comprises at least one amino acid substitution in addition to the amino acid substitution at amino acid S76 of the reference protein.

30. The method of any of the preceding claims, wherein the at least one amino acid substitution comprises at least two amino acid substitutions in addition to the amino acid substitution at amino acid S76 of the reference protein.

31. The method of any of the preceding claims, wherein the at least one amino acid substitution comprises at least three amino acid substitutions in addition to the amino acid substitution at amino acid S76 of the reference protein.

32. The method of any of the preceding claims, wherein the at least one amino acid substitution comprises an amino acid substitution at one or more of amino acids G1, E2, E4, T12, A19, V20, E23, K25, I26, G27, Q28, T33, N35, C41, Q61, V64, Y65, D68, L70, A73, I75, R84, F89, or any combination thereof.

33. The method of any one of the preceding claims, wherein the at least one amino acid substitution comprises one or more of the amino acid substitutions (i) G1M, (ii) A19V, (iii) V20I, (iv) K25R, (v) one or more of I26W, I26V, or I26T, (vi) one or more of Q28R, Q28K, Q28S, Q28E, (vii) R31T, (viii) T33R, (ix) N35T, (x) one or more of C41A, C41V, C41T or C41S, (xi) one or more of E59T, E59V, E59I, E59Y, E59F, E59W or E59R, (xii) D68N or D68T, (xiii) A73F or A73V, (xiv) D74V, (xv) I75V or I75L, (xvi) E77V, (xvii) R84L.

34. The method of any one of the preceding claims, wherein the at least one amino acid substitution comprises an amino acid substitution at one or more of amino acids E2, E23, Y65, L70, or any combination thereof.

35. The method of any one of the preceding claims, wherein the at least one amino acid substitution comprises an amino acid substitution at one or more of amino acids E2, E23, Y65, L70.

36. The method of any one of the preceding claims, wherein the amino acid substitution at amino acid S76 is one or more of S76Y, S76F, S76T, S76W, S76R, S76V, S76I, S76H or S76A.

37. The method of any one of the preceding claims, wherein the at least one amino acid substitution comprises an amino acid substitution at one or more of amino acids E2N, E23A, Y65R, L70I, or any combination thereof.

38. The method of any one of the preceding claims, wherein the at least one amino acid substitution comprises an amino acid substitution at one or more of amino acids E2M, E23A, Y65R, L70I, or any combination thereof.

39. The method of any of the preceding claims, wherein the at least one amino acid substitution comprises one or more of the following amino acid substitutions: (i) E2N, E23A, Y65R, L70I, (ii) E2M, E23A, Y65R, L70I, (iii) E2N, E23Q, Y65R, L70I, or (iv) E2M, E23Q, Y65R, L70I.

40. The method of any one of the preceding claims, wherein the at least one amino acid substitution comprises one or more of the amino acid substitutions (i) G1M, (ii) A19V, (iii) V20I, (iv) K25R, (v) one or more of I26W, I26V, or I26T, (vi) one or more of Q28R, Q28K, Q28S, Q28E, (vii) R31T, (viii) T33R, (ix) N35T, (x) one or more of C41A, C41V, C41T or C41S, (xi) one or more of E59T, E59V, E59I, E59Y, E59F, E59W or E59R, (xii) D68N or D68T, (xiii) A73F or A73V, (xiv) D74V, (xv) I75V or I75L, (xvi) E77V, (xvii) R84L.

41. The method of any one of the preceding claims, wherein the modified protein comprises SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 221, SEQ ID NO: 108, SEQ ID NO: 9, SEQ ID NO: 102, SEQ ID NO: 105, SEQ ID NO: 52, SEQ ID NO: 182, SEQ ID NO: 10, SEQ ID NO: 69, SEQ ID NO: 157, SEQ ID NO: 114, SEQ ID NO: 174, SEQ ID NO: 162, SEQ ID NO: 231, SEQ ID NO: 57, SEQ ID NO: 192, SEQ ID NO: 68, SEQ ID NO: 106, SEQ ID NO: 8, SEQ ID NO: 79, SEQ ID NO: 76, SEQ ID NO: 22, SEQ ID NO: 194, SEQ ID NO: 262, SEQ ID NO: 84, SEQ ID NO: 239, SEQ ID NO: 135, SEQ ID NO: 211, SEQ ID NO: The amino acid sequence set forth in one or more of SEQ ID NO:237, SEQ ID NO:198, SEQ ID NO:179, SEQ ID NO:172, or any combination thereof.

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