Novel carbohydrate-binding polypeptides

Abstract

The present invention relates to novel carbohydrate-binding polypeptides and their use in the preparation of hydrogels.

Description

[Technical field]

[0001] The present invention relates to novel carbohydrate-binding polypeptides and their use in the preparation of hydrogels. [Background technology]

[0002] Hydrogels are a big and fast-growing business, with applications in many areas including drug delivery, tissue scaffolds, life science research, the cosmetics industry, bio-based agriculture, and industrial lubrication. The majority of hydrogels are still made from fossil-derived polymers that are assembled into 3D networks by chemical modification and cross-linking. This production method is not sustainable and has not been well received by consumers, so there is an urgent need to start using biological polymers for gel formation.

[0003] Hydrogels can already be produced from several natural polymers, such as cellulose and other polysaccharides from plants or microorganisms, as well as mucins from animals, but these still require chemical modification to enable crosslinking, resulting in a high chemical waste footprint and potentially containing compounds that can irritate the skin or interfere with other biological systems depending on the application.

[0004] There is a strong push in industry to develop new methods to produce hydrogels from sustainably sourced polysaccharides such as cellulose and scleroglucan. Scleroglucan is secreted by industrial fungi such as Sclerotium rolfsii and can be obtained in reasonably high yields and purity (Schmid et al. 2011). In current practice, it is common to dissolve scleroglucan in DMSO to separate the triple helices of the scleroglucan structure prior to gel formation (Palleschi et al. 2005). This is then oxidized using sodium periodate to generate the scleroaldehyde derivative (Maeda et al. 2001). In some cases, further oxidation catalyzed by sodium chlorite to generate carboxyl groups leads to a product called sclerox (Crescenzi et al. 1983). This oxidized form can be crosslinked with 1,6-hexanedibromide to form gel-forming networks, with different sucralox:dihalide ratios resulting in significantly different mechanical and rheological properties (Coviello et al. 1999; Coviello et al. 2001). More recently, an efficient alternative method has been developed using boric acid (borax) to directly crosslink polysaccharides through 4,6-gluco-borate bonds and physical associations to create physical gels (Coviello et al. 2005; Palleschi et al. 2006). However, borax is a regulated Substance of Very High Concern (SVHC) (European Chemicals Agency).

[0005] Therefore, there is an unmet need to improve the sustainability of hydrogel production. Summary of the Invention

[0006] Herein, we present a completely new method for cross-linking polysaccharides that does not require modification of the starting polymer and does not use chemical cross-linking. The cross-linker is actually a polypeptide that belongs to a novel carbohydrate-binding module (CBM) family. Each polypeptide in the novel CBM family disclosed herein, called CBM92, contains three distinct repeating regions that are binding sites for monosaccharides, oligosaccharides, and polysaccharides. Polypeptides of this novel CBM family have binding specificity and gelation tendency for high molecular weight polysaccharides such as scleroglucan. We have demonstrated that proteins of this novel CBM family are highly advantageous as cross-linkers in hydrogel formation.

[0007] One aspect of the present disclosure is a polypeptide comprising: [ka] Repeat region 1 (R1) comprising or consisting of the amino acid sequence or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO: 8 in that the amino acid sequence of the variant comprises 1 to 6 distinct amino acid substitutions. [ka] Repeat region 2 (R2) comprising or consisting of the amino acid sequence or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO: 10 in that the amino acid sequence of the variant comprises 1 to 6 distinct amino acid substitutions. [ka] Repeat region 3 (R3) comprising or consisting of the amino acid sequence or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO: 12 in that the amino acid sequence of the variant contains 1 to 6 distinct amino acid substitutions. The present invention relates to a polypeptide comprising at least two repeat regions selected from the group consisting of:

[0008] Another aspect of the disclosure is a polypeptide comprising: a. SEQ ID NO:220: X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 X 31 X 32 X 33 X 34 WEX 37 F Repeat region 1 (R1) comprising or consisting of the amino acid sequence [In the formula, X1 is isoleucine (I) or valine (V); X2 is any amino acid, such as tryptophan (W), serine (S), threonine (T) or alanine (A); X3 is leucine (L) or isoleucine (I); X4 is lysine (K) or arginine (R) or is absent; X5 is glutamine (Q), arginine (R), alanine (A), lysine (K), or is absent; X6 is glycine (G), serine (S), tyrosine (Y), cysteine ​​(C), leucine (L) or threonine (T); X7 is any amino acid, such as phenylalanine (F) or serine (S); X8 is glycine (G) or is absent; X9 is asparagine (N), histidine (H), aspartic acid (D) or threonine (T); X 10 is asparagine (N), serine (S), threonine (T), glutamine (Q), lysine (K) or glycine (G); X 11 is lysine (K), glutamine (Q), asparagine (N) or leucine (L); X 12 is tyrosine (Y), leucine (L) or phenylalanine (F), X 13 is valine (V), leucine (L) or alanine (A), X 14 is serine (S), threonine (T), cysteine ​​(C) or asparagine (N), X 15 is alanine (A), serine (S) or glycine (G), X 16 is glutamic acid (E), lysine (K), arginine (R), glutamine (Q) or aspartic acid (D), X 17 is asparagine (N), glycine (G), glutamine (Q) or proline (P), X 18 is glycine (G), asparagine (N), threonine (T) or aspartic acid (D); X 19 is leucine (L) or is absent, X 20 is glycine (G) or alanine (A) or is absent, X 21 is alanine (A) or asparagine (N) or is absent, X 22 is any amino acid, such as threonine (T), or is absent, X 23 is any amino acid, such as glycine (G), X 24 is proline (P), alanine (A), glutamine (Q) or arginine (R), X 25 is leucine (L), methionine (M), alanine (A) or isoleucine (I), X 26 is any amino acid, such as threonine (T), X 27 is alanine (A), cysteine ​​(C) or tryptophan (W), X 28 is asparagine (N), aspartic acid (D), glutamic acid (E) or arginine (R); X 29 is arginine (R), alanine (A) or serine (S), X 30 is threonine (T), aspartic acid (D), proline (P), isoleucine (I), asparagine (N) or alanine (A); X 31 is threonine (T), alanine (A), valine (V), glycine (G), lysine (K) or isoleucine (I); X 32 is alanine (A), valine (V), proline (P) or leucine (L); X 33 is glycine (G), glutamine (Q), serine (S) or aspartic acid (D), X 34 is any amino acid, such as glycine (G), X 37 is any amino acid, such as glutamine (Q) or lysine (K)]; or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO: 220 in that the amino acid sequence of the variant comprises 1 to 6 distinct amino acid substitutions. b. SEQ ID NO:221: X1X2X3X4X5X6X7X8X9X 10 X 11 X 12X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 X 31 WEX 34 F Repeat region 2 (R2) comprising or consisting of the amino acid sequence [In the formula, X1 is isoleucine (I) or valine (V); X2 is alanine (A), asparagine (N), tyrosine (Y) or threonine (T); X3 is leucine (L) or isoleucine (I); X4 is glutamine (Q), arginine (R), leucine (L) or lysine (K); X5 is glycine (G), alanine (A), asparagine (N) or serine (S); X6 is any amino acid, such as alanine (A), or is absent; X7 is glycine (G) or serine (S) or is absent; X8 is asparagine (N), methionine (M), glutamine (Q), histidine (H) or serine (S); X9 is glycine (G) or alanine (A); X 10 is lysine (K), leucine (L), methionine (M), asparagine (N) or serine (S); X 11 is tyrosine (Y), X 12 is valine (V) or phenylalanine (F), X 13is serine (S), cysteine ​​(C), lysine (K) or glutamine (Q), X 14 is alanine (A), valine (V) or serine (S), X 15 is glutamic acid (E), aspartic acid (D), glutamine (Q) or glycine (G), X 16 is asparagine (N), proline (P), glycine (G), threonine (T) or aspartic acid (D), X 17 is glycine (G), aspartic acid (D), isoleucine (I), asparagine (N), or is absent; X 18 is valine (V) or glycine (G) or is absent; X 19 is glycine (G), threonine (T), leucine (L), alanine (A), or glutamic acid (E), or is absent; X 20 is any amino acid, such as glutamine (Q), or is absent, X 21 is proline (P), alanine (A), glutamine (Q) or asparagine (N), X 22 is leucine (L), isoleucine (I), methionine (M) or valine (V); X 23 is threonine (T), asparagine (N), phenylalanine (F) or arginine (R); X 24 is alanine (A), cysteine ​​(C) or arginine (R), X 25 is asparagine (N), alanine (A), threonine (T) or glutamine (Q), X 26 is alanine (A), glycine (G) or arginine (R), X 27is threonine (T), alanine (A), lysine (K), proline (P) or leucine (L); X 28 is any amino acid, such as alanine (A), X 29 is isoleucine (I), valine (V), proline (P), alanine (A), lysine (K), tyrosine (Y) or leucine (L); X 30 is glycine (G), alanine (A), glutamic acid (E), threonine (T), serine (S), glutamine (Q) or aspartic acid (D); X 31 is glycine (G), alanine (A), aspartic acid (D), serine (S), proline (P) or lysine (K); X 34 is any amino acid, such as lysine (K)], or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO: 221 in that the amino acid sequence of the variant comprises 1 to 6 distinct amino acid substitutions. c. SEQ ID NO:222: X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 X 31 X 32 X 33 X 34 X 35 WEX 38 F Repeat region 3 (R3) comprising or consisting of the amino acid sequence [In the formula, X1 is valine (V), isoleucine (I), tyrosine (Y) or phenylalanine (F); X2 is alanine (A), serine (S), cysteine ​​(C), threonine (T) or glycine (G); X3 is leucine (L), phenylalanine (F) or isoleucine (I); X4 is arginine (R), lysine (K) or glutamine (Q); X5 is glycine (G), alanine (A) or serine (S); X6 is arginine (R) or valine (V) or is absent; X7 is any amino acid, such as asparagine (N); X8 is asparagine (N), histidine (H), alanine (A), lysine (K) or absent; X9 is glycine (G), tryptophan (W) or asparagine (N); X 10 is lysine (K), leucine (L), methionine (M), serine (S), alanine (A) or glutamine (Q), X 11 is tyrosine (Y), tryptophan (W) or phenylalanine (F), X 12 is valine (V), leucine (L), methionine (M) or isoleucine (I), X 13 is serine (S) or glutamine (Q), X 14 is alanine (A), serine (S), histidine (H) or glycine (G), X 15 is glutamic acid (E), aspartic acid (D), glutamine (Q), serine (S) or asparagine (N); X 16is asparagine (N), leucine (L), tryptophan (W), methionine (M) or aspartic acid (D); X 17 is glycine (G), serine (S) or aspartic acid (D), X 18 is any amino acid, such as serine (S) or threonine (T), X 19 is asparagine (N), valine (V), or aspartic acid (D), or is absent; X 20 is alanine (A) or is absent, X 21 is serine (S) or is absent, X 22 is any amino acid, such as alanine (A), or is absent, X 23 is proline (P), alanine (A), arginine (R), threonine (T) or glycine (G), X 24 is leucine (L), methionine (M), valine (V) or isoleucine (I), X 25 is threonine (T), tyrosine (Y), asparagine (N), leucine (L) or glutamine (Q), X 26 is alanine (A), cysteine ​​(C), phenylalanine (F) or tryptophan (W), X 27 is asparagine (N), alanine (A), threonine (T) or serine (S), X 28 is arginine (R) or lysine (K), X 29 is aspartic acid (D) or is absent, X 30 is threonine (T), alanine (A), proline (P), serine (S) or glycine (G); X 31is any amino acid, such as alanine (A), X 32 is isoleucine (I), glutamic acid (E), proline (P), alanine (A) or valine (V), X 33 is glycine (G), lysine (K), or aspartic acid (D), or is absent; X 34 is glycine (G), serine (S), lysine (K), aspartic acid (D) or glutamine (Q), X 35 is glycine (G), alanine (A), aspartic acid (D) or cysteine ​​(C), X 38 is any amino acid, such as lysine (K)], or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO: 222 in that the amino acid sequence of the variant contains 1 to 6 distinct amino acid substitutions. The present invention relates to a polypeptide comprising at least two repeat regions selected from the group consisting of:

[0009] A further aspect of the present disclosure relates to an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4, or any of the sequences SEQ ID NO: 20 to SEQ ID NO: 33, or any of the sequences SEQ ID NO: 71 to SEQ ID NO: 219, wherein the polypeptide is capable of binding one or more saccharide units.

[0010] A further aspect of the present disclosure relates to a polypeptide comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:20 to SEQ ID NO:33, wherein the polypeptide is capable of linking one or more saccharide units.

[0011] A further aspect of the present disclosure pertains to polynucleotides that encode the polypeptides disclosed herein.

[0012] Another aspect of the present disclosure pertains to recombinant vectors comprising a polynucleotide disclosed herein, or a nucleic acid sequence encoding a carbohydrate binding polypeptide disclosed herein.

[0013] Another aspect of the present disclosure pertains to host cells expressing the recombinant vectors disclosed herein.

[0014] Another aspect of the present disclosure relates to the use of at least one polypeptide disclosed herein to cross-link two or more polysaccharides.

[0015] A further aspect of the present disclosure relates to a hydrogel comprising at least one polypeptide disclosed herein and two or more polysaccharides, the hydrogel having a crosslinked structure.

[0016] A further aspect of the present disclosure relates to a cross-linking agent for cross-linking polysaccharides, the cross-linking agent being a polypeptide as disclosed herein.

[0017] Another aspect of the present disclosure is a method of making a hydrogel, comprising: a. preparing a solution containing a polysaccharide; b. providing a solution comprising a polypeptide of the present disclosure; c. mixing the solution at room temperature with continuous stirring; The present invention relates to a method for obtaining a hydrogel through cross-linking of the polysaccharide. [Brief description of the drawings]

[0018] [Figure 1] The repeating structural unit of scleroglucan polysaccharides. [Diagram 2] Schematic depiction of the superimposed crystal structures of Cp-F1 and Cp-F2, showing glucose monosaccharide ligands in two of the binding sites. The upper arrow indicates the glucose in the alpha binding site, and the lower arrow indicates the glucose in the beta binding site. [Diagram 3]Schematic depiction of the crystal structure of Cp-F1 showing a close-up of the Cp-F2 binding site bound to the ligand glucose, with the relevant amino acids indicated. [Figure 4] Depiction of solid gel formation. Top: scleroglucan only. Bottom: a mixture of scleroglucan and Cp-F1 protein. [Diagram 5] Varying the polysaccharide:protein ratio (9:1, 8:1, and 2:1) alters gel properties such as solidity and transparency. These hydrogels were made by mixing a stock solution of scleroglucan with a concentration of 5 g / L with a stock solution of protein (Cp-F1) with a concentration of 4 g / L. These ratios are therefore based on the volumes of the stock solutions mixed. [Figure 6A] Example of rheological evaluation. All analyses shown concern hydrogels containing a final concentration of 4 g / L scleroglucan. The final protein concentrations in the hydrogels, from lowest to highest, are 0.08 g / L, 0.2 g / L, 0.4 g / L, 0.8 g / L, 1.2 g / L, 1.6 g / L, and 2 g / L. [Figure 6B] Example of rheological evaluation. All analyses shown concern hydrogels containing a final concentration of 4 g / L scleroglucan. The final protein concentrations in the hydrogels, from lowest to highest, are 0.08 g / L, 0.2 g / L, 0.4 g / L, 0.8 g / L, 1.2 g / L, 1.6 g / L, and 2 g / L. [Figure 7] Grayscale heatmap of polysaccharide binding data for 12 proteins and five variants of the Cp-F1 protein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] definition As used herein, the term "polypeptide" refers to a single polypeptide chain that may or may not be modified by the addition of non-amino acid groups. The term does not refer to a specific length of the product, and thus peptides, oligopeptides, and proteins are included in the definition of a polypeptide. In one embodiment, the term "polypeptide" as used herein also includes variants, mutants, modifications, analogs, and / or derivatives of the disclosed polypeptides described herein. Amino acid sequence variants of the disclosed polypeptides can be prepared by introducing appropriate nucleotide changes into the nucleic acids of the invention or by in vitro synthesis of the desired polypeptide. Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acid sequence. A combination of deletions, insertions, and substitutions can be made to arrive at the final construct, so long as the final polypeptide product has the desired properties.

[0020] An "amino acid residue" may be a naturally occurring or unnatural amino acid residue linked by a peptide bond or a bond other than a peptide bond. The amino acid residue may be in the D or L configuration. An amino acid residue comprises an amino terminal portion (NH2) and a carboxyl terminal portion (COOH) separated by a central portion comprising a carbon atom or a chain of carbon atoms, at least one of which comprises at least one side chain or functional group. NH2 refers to the amino group present at the amino terminus of an amino acid or peptide, and COOH refers to the carboxyl group present at the carboxyl terminus of an amino acid or peptide. The collective term amino acid includes both natural and unnatural amino acids. Natural amino acids according to standard nomenclature as described in J. Biol. Chem., 243:3552-59 (1969) and adopted in 37 CFR section 1.822(b)(2) belong to the group of amino acids described herein: Y, G, F, M, A, S, I, L, T, V, P, K, H, Q, E, W, R, D, N, and C. Unnatural amino acids are those not listed immediately above and include, but are not limited to, modified amino acid residues, L-amino acid residues, and stereoisomers of D-amino acid residues.

[0021] A "functional variant" of a peptide is a peptide that is capable of performing essentially the same function as the peptide from which it is derived, in particular, a functional variant is capable of binding the same molecule as the peptide from which it is derived, preferably with the same affinity.

[0022] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and can refer to any nucleic acid containing the information necessary for the purpose indicated by the context. That is, a nucleic acid can be single- or double-stranded DNA or RNA, or other nucleic acid, and can include complementary sequences, such as sense and antisense strands of a nucleic acid polymer, so long as it is capable of expressing the appropriate information, e.g., in relation to an encoded peptide.

[0023] As used herein, the term "binding site" refers to a region of a molecule or molecular complex that, as a result of its shape, preferably associates with another molecule, molecular complex, chemical or compound. The polypeptides of the present disclosure described herein contain carbohydrate binding sites, i.e., binding sites capable of associating with sugars, such as monosaccharides, disaccharides and polysaccharides.

[0024] As used herein, the term "hydrogel" or "hydrophilic gel" refers to a continuous phase of hydrophilic polymers that can swell upon contact with water and other hydrophilic swelling agents. The term is used regardless of hydration state. "Hydrogel" refers to a solid or semi-solid textured material that contains water. A hydrogel is formed by a three-dimensional network of molecular structures within which a substance such as water can be held. The three-dimensional molecular network can be held together by covalent chemical bonds, or ionic bonds, or any combination thereof.

[0025] "Polysaccharide", "Carbohydrate" or "Oligosaccharide": The terms "polysaccharide", "carbohydrate" or "oligosaccharide" refer to a polymer of sugars. The terms "polysaccharide", "carbohydrate", and "oligosaccharide" may be used interchangeably. Typically, a polysaccharide or oligosaccharide contains at least three sugars. The polymer may contain natural sugars (e.g., glucose, fructose, galactose, mannose, arabinose, ribose, and xylose) and / or modified sugars (e.g., 2'-fluororibose, 2'-deoxyribose, and hexose). The term "saccharide" is used herein as a general term for a polymer of sugars and may refer to "monosaccharide", "disaccharide", "polysaccharide", "carbohydrate", or "oligosaccharide", and other synonyms.

[0026] Carbohydrate-binding polypeptides Carbohydrate-binding modules (CBMs) are small, non-catalytic proteins that can bind to specific carbohydrate ligands. CBMs are classified into families by sequence homology in the CAZy database (www.cazy.org) (Lombard et al. 2014). The mode of CBM-ligand binding is determined by the surface architecture of the CBM protein structure (Boraston et al. 2004). In most CBM families, proteins display one carbohydrate-binding site per protein.

[0027] One aspect of the present disclosure relates to a novel polypeptide belonging to a member of CBM family 92 (CBM92). In one aspect, the novel polypeptide of the present disclosure is capable of binding and cross-linking at least two polysaccharides, preferably three polysaccharides. The resulting cross-linked structure comprising the novel polypeptide of the present disclosure and two or three polysaccharides forms a hydrogel in the presence of water. Thus, in one aspect of the present disclosure, the novel polypeptide is characterized in that it comprises two or preferably three domains or regions capable of binding to a particular polysaccharide.

[0028] In one aspect, the present disclosure provides a polypeptide comprising: [ka] Repeat region 1 (R1) comprising or consisting of the amino acid sequence or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO: 8 in that the amino acid sequence of the variant comprises 1 to 6 distinct amino acid substitutions. [ka] Repeat region 2 (R2) comprising or consisting of the amino acid sequence or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO: 10 in that the amino acid sequence of the variant comprises 1 to 6 distinct amino acid substitutions. [ka] Repeat region 3 (R3) comprising or consisting of the amino acid sequence or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO: 12 in that the amino acid sequence of the variant contains 1 to 6 distinct amino acid substitutions. The present invention relates to a polypeptide comprising at least two repeat regions selected from the group consisting of:

[0029] In one aspect, the present disclosure provides a polypeptide comprising: a. SEQ ID NO:220: X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X25 X 26 X 27 X 28 X 29 X 30 X 31 X 32 X 33 X 34 WEX 37 F Repeat region 1 comprising or consisting of the amino acid sequence [In the formula, X1 is isoleucine (I) or valine (V); X2 is any amino acid, such as tryptophan (W), serine (S), threonine (T) or alanine (A); X3 is leucine (L) or isoleucine (I); X4 is lysine (K) or arginine (R) or is absent; X5 is glutamine (Q), arginine (R), alanine (A), lysine (K), or is absent; X6 is glycine (G), serine (S), tyrosine (Y), cysteine ​​(C), leucine (L) or threonine (T); X7 is any amino acid, such as phenylalanine (F) or serine (S); X8 is glycine (G) or is absent; X9 is asparagine (N), histidine (H), aspartic acid (D) or threonine (T); X 10 is asparagine (N), serine (S), threonine (T), glutamine (Q), lysine (K) or glycine (G); X 11 is lysine (K), glutamine (Q), asparagine (N) or leucine (L); X 12 is tyrosine (Y), leucine (L) or phenylalanine (F), X 13 is valine (V), leucine (L) or alanine (A), X 14is serine (S), threonine (T), cysteine ​​(C) or asparagine (N), X 15 is alanine (A), serine (S) or glycine (G), X 16 is glutamic acid (E), lysine (K), arginine (R), glutamine (Q) or aspartic acid (D), X 17 is asparagine (N), glycine (G), glutamine (Q) or proline (P), X 18 is glycine (G), asparagine (N), threonine (T) or aspartic acid (D); X 19 is leucine (L) or is absent, X 20 is glycine (G) or alanine (A) or is absent, X 21 is alanine (A) or asparagine (N) or is absent, X 22 is any amino acid, such as threonine (T), or is absent, X 23 is any amino acid, such as glycine (G), X 24 is proline (P), alanine (A), glutamine (Q) or arginine (R), X 25 is leucine (L), methionine (M), alanine (A) or isoleucine (I), X 26 is any amino acid, such as threonine (T), X 27 is alanine (A), cysteine ​​(C) or tryptophan (W), X 28 is asparagine (N), aspartic acid (D), glutamic acid (E) or arginine (R); X 29 is arginine (R), alanine (A) or serine (S), X 30 is threonine (T), aspartic acid (D), proline (P), isoleucine (I), asparagine (N) or alanine (A); X 31 is threonine (T), alanine (A), valine (V), glycine (G), lysine (K) or isoleucine (I); X 32 is alanine (A), valine (V), proline (P) or leucine (L); X 33 is glycine (G), glutamine (Q), serine (S) or aspartic acid (D), X 34 is any amino acid, such as glycine (G), X 37 is any amino acid, such as glutamine (Q) or lysine (K)]; or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO: 220 in that the amino acid sequence of the variant comprises 1 to 6 distinct amino acid substitutions. b. SEQ ID NO:221: X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 X 31 WEX 34 F Repeat region 2 comprising or consisting of the amino acid sequence [In the formula, X1 is isoleucine (I) or valine (V); X2 is alanine (A), asparagine (N), tyrosine (Y) or threonine (T); X3 is leucine (L) or isoleucine (I); X4 is glutamine (Q), arginine (R), leucine (L) or lysine (K); X5 is glycine (G), alanine (A), asparagine (N) or serine (S); X6 is any amino acid, such as alanine (A), or is absent; X7 is glycine (G) or serine (S) or is absent; X8 is asparagine (N), methionine (M), glutamine (Q), histidine (H) or serine (S); X9 is glycine (G) or alanine (A); X 10 is lysine (K), leucine (L), methionine (M), asparagine (N) or serine (S); X 11 is tyrosine (Y), X 12 is valine (V) or phenylalanine (F), X 13 is serine (S), cysteine ​​(C), lysine (K) or glutamine (Q), X 14 is alanine (A), valine (V) or serine (S), X 15 is glutamic acid (E), aspartic acid (D), glutamine (Q) or glycine (G), X 16 is asparagine (N), proline (P), glycine (G), threonine (T) or aspartic acid (D), X 17 is glycine (G), aspartic acid (D), isoleucine (I), asparagine (N), or is absent; X 18 is valine (V) or glycine (G) or is absent, X 19 is glycine (G), threonine (T), leucine (L), alanine (A), or glutamic acid (E), or is absent; X 20 is any amino acid, such as glutamine (Q), or is absent, X 21 is proline (P), alanine (A), glutamine (Q) or asparagine (N), X 22 is leucine (L), isoleucine (I), methionine (M) or valine (V); X 23 is threonine (T), asparagine (N), phenylalanine (F) or arginine (R); X 24 is alanine (A), cysteine ​​(C) or arginine (R), X 25 is asparagine (N), alanine (A), threonine (T) or glutamine (Q), X 26 is alanine (A), glycine (G) or arginine (R), X 27 is threonine (T), alanine (A), lysine (K), proline (P) or leucine (L); X 28 is any amino acid, such as alanine (A), X 29 is isoleucine (I), valine (V), proline (P), alanine (A), lysine (K), tyrosine (Y) or leucine (L); X 30 is glycine (G), alanine (A), glutamic acid (E), threonine (T), serine (S), glutamine (Q) or aspartic acid (D); X 31 is glycine (G), alanine (A), aspartic acid (D), serine (S), proline (P) or lysine (K); X 34 is any amino acid, such as lysine (K)], or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO: 221 in that the amino acid sequence of the variant comprises 1 to 6 distinct amino acid substitutions. c. SEQ ID NO:222: X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 X 31 X 32 X 33 X 34 X 35 WEX 38 F Repeat region 3 comprising or consisting of the amino acid sequence [In the formula, X1 is valine (V), isoleucine (I), tyrosine (Y) or phenylalanine (F); X2 is alanine (A), serine (S), cysteine ​​(C), threonine (T) or glycine (G); X3 is leucine (L), phenylalanine (F) or isoleucine (I); X4 is arginine (R), lysine (K) or glutamine (Q); X5 is glycine (G), alanine (A) or serine (S); X6 is arginine (R) or valine (V) or is absent; X7 is any amino acid, such as asparagine (N); X8 is asparagine (N), histidine (H), alanine (A), lysine (K) or absent; X9 is glycine (G), tryptophan (W) or asparagine (N); X 10 is lysine (K), leucine (L), methionine (M), serine (S), alanine (A) or glutamine (Q), X 11 is tyrosine (Y), tryptophan (W) or phenylalanine (F), X 12 is valine (V), leucine (L), methionine (M) or isoleucine (I), X 13 is serine (S) or glutamine (Q), X 14 is alanine (A), serine (S), histidine (H) or glycine (G), X 15 is glutamic acid (E), aspartic acid (D), glutamine (Q), serine (S) or asparagine (N); X 16 is asparagine (N), leucine (L), tryptophan (W), methionine (M) or aspartic acid (D); X 17 is glycine (G), serine (S) or aspartic acid (D), X 18 is any amino acid, such as serine (S) or threonine (T), X 19 is asparagine (N), valine (V), or aspartic acid (D), or is absent; X 20 is alanine (A) or is absent, X 21 is serine (S) or is absent, X 22 is any amino acid, such as alanine (A), or is absent, X 23is proline (P), alanine (A), arginine (R), threonine (T) or glycine (G), X 24 is leucine (L), methionine (M), valine (V) or isoleucine (I), X 25 is threonine (T), tyrosine (Y), asparagine (N), leucine (L) or glutamine (Q), X 26 is alanine (A), cysteine ​​(C), phenylalanine (F) or tryptophan (W), X 27 is asparagine (N), alanine (A), threonine (T) or serine (S), X 28 is arginine (R) or lysine (K), X 29 is aspartic acid (D) or is absent, X 30 is threonine (T), alanine (A), proline (P), serine (S) or glycine (G); X 31 is any amino acid, such as alanine (A), X 32 is isoleucine (I), glutamic acid (E), proline (P), alanine (A) or valine (V), X 33 is glycine (G), lysine (K), or aspartic acid (D), or is absent; X 34 is glycine (G), serine (S), lysine (K), aspartic acid (D) or glutamine (Q), X 35 is glycine (G), alanine (A), aspartic acid (D) or cysteine ​​(C), X 38 is any amino acid, such as lysine (K)], or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO: 222 in that the amino acid sequence of the variant contains 1 to 6 distinct amino acid substitutions. The present invention relates to a polypeptide comprising at least two repeat regions selected from the group consisting of:

[0030] In one embodiment of the disclosure, each repeat region of the polypeptide contains a key tryptophan (W) residue, preferably at position 35 in repeat region 1, position 32 in repeat region 2, and position 36 in repeat region 3, based on the amino acid numbering provided herein. The tryptophan (W) residue plays a key role in binding between the repeat region containing it and the polysaccharide or disaccharide, as evidenced by knockout binding site mutants containing tryptophan (W) to alanine (A) substitutions. The tryptophan side chain ring has a major interaction with the carbon ring of the disaccharide or polysaccharide to which the polypeptide is bound.

[0031] Thus, in one embodiment of the disclosure, the polypeptide described herein comprises a repeat region 1 comprising the amino acid sequence of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67 or SEQ ID NO:220, or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67 or SEQ ID NO:220 in that the amino acid sequence of the variant comprises 1 to 6 individual amino acid substitutions; provided, however, that the residue at position 35 of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67 or SEQ ID NO:220 or a variant thereof is tryptophan (W).

[0032] In another embodiment of the disclosure, the polypeptide comprises a repeat region 2 comprising the amino acid sequence of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68, or SEQ ID NO:221, or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68, or SEQ ID NO:221 in that the amino acid sequence of the variant comprises 1 to 6 individual amino acid substitutions; provided, however, that the residue at position 32 of the variant of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68, or SEQ ID NO:221 is tryptophan (W).

[0033] In another embodiment of the disclosure, the polypeptide comprises a repeat region 3 comprising the amino acid sequence of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66 or SEQ ID NO:69, SEQ ID NO:222, or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66 or SEQ ID NO:69, SEQ ID NO:222 in that the amino acid sequence of the variant comprises 1 to 6 individual amino acid substitutions; provided, however, that the residue at position 36 of the variants of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66 or SEQ ID NO:69, SEQ ID NO:222 is tryptophan (W).

[0034] In one embodiment of the present disclosure, each repeat region of the polypeptide further comprises, in addition to a tryptophan (W) residue at position 35 of R1, position 32 of R2 or position 36 of R3, a glutamic acid at position 36 of R1, position 33 of R2 or position 37 of R3, and / or a phenylalanine at position 38 of R1, position 35 of R2 or position 39 of R3, based on the amino acid numbering provided herein, and preferably comprises both a glutamic acid at position 36 of R1, position 33 of R2 or position 37 of R3 and a phenylalanine at position 38 of R1, position 35 of R2 or position 39 of R3. The glutamic acid and phenylalanine residues are conserved and play an important role in the binding between the repeat region containing them and polysaccharides or disaccharides. This can also be seen in FIG. 3B. Tryptophan, glutamic acid and / or phenylalanine may be present in one, two or all three repeat regions at the positions specified above.

[0035] Thus, in a further embodiment of the disclosure, the polypeptide comprises a repeat region 1 comprising the amino acid sequence of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67 or SEQ ID NO:220, or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67 or SEQ ID NO:220 in that the amino acid sequence of the variant comprises 1 to 6 individual amino acid substitutions, however, the residue at position 35 of said variant of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67 or SEQ ID NO:220 is tryptophan (W); and the residue at position 36 of said variant of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67 or SEQ ID NO:220 is glutamic acid (E); and / or provided that the residue at position 38 of said variant of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67 or SEQ ID NO:220 is phenylalanine (F).

[0036] In another embodiment of the disclosure, the polypeptide comprises a repeat region 2 comprising the amino acid sequence of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68, or SEQ ID NO:221, or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68, or SEQ ID NO:221 in that the amino acid sequence of the variant comprises 1 to 6 individual amino acid substitutions; however, the residue at position 32 of said variant of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68 or SEQ ID NO:221 is tryptophan (W); the residue at position 33 of said variant of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68 or SEQ ID NO:221 is glutamic acid (E); and / or provided that the residue at position 35 of said variant of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68 or SEQ ID NO:221 is phenylalanine (F).

[0037] In another embodiment of the disclosure, the polypeptide comprises a repeat region 3 comprising the amino acid sequence of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, or SEQ ID NO:222, or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, or SEQ ID NO:222 in that the amino acid sequence of the variant comprises 1 to 6 individual amino acid substitutions; however, the residue at position 36 of said variant of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69 or SEQ ID NO:222 is tryptophan (W); the residue at position 37 of said variant of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69 or SEQ ID NO:222 is glutamic acid (E); and / or provided that the residue at position 39 of said variant of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69 or SEQ ID NO:222 is phenylalanine (F).

[0038] In one embodiment of the present disclosure, each repeat region of the polypeptide comprises a tyrosine at position 12 of R1 or position 11 of R2 and R3, and / or a valine at position 13 of R1 or position 12 of R2 and R3, based on the amino acid numbering provided herein. These tyrosine and / or valine residues are located near the binding site in the 3D structure of the polypeptide when it binds to a disaccharide or polysaccharide, and therefore may contribute to binding of the repeat region to the disaccharide or polysaccharide.

[0039] In a further embodiment of the disclosure, the polypeptide comprises a repeat region 1 comprising the amino acid sequence of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67 or SEQ ID NO:220, or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67 or SEQ ID NO:220 in that the amino acid sequence of the variant comprises 1 to 6 individual amino acid substitutions; however, the residue at position 12 of said variant of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67 or SEQ ID NO:220 is a tyrosine (Y); and / or provided that the residue at position 13 of said variant of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67 or SEQ ID NO:220 is a valine (V).

[0040] In another embodiment of the disclosure, the polypeptide comprises a repeat region 2 comprising the amino acid sequence of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68, or SEQ ID NO:221, or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68, or SEQ ID NO:221 in that the amino acid sequence of the variant comprises 1 to 6 individual amino acid substitutions; however, the residue at position 11 of said variant of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68 or SEQ ID NO:221 is a tyrosine (Y); and / or provided that the residue at position 12 of said variant of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68 or SEQ ID NO:221 is a valine (V).

[0041] In another embodiment of the disclosure, the polypeptide comprises a repeat region 2 comprising the amino acid sequence of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:222, or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:222 in that the amino acid sequence of the variant comprises 1 to 6 individual amino acid substitutions; however, the residue at position 11 of said variants of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:222 is a tyrosine (Y); and / or Provided that the residue at position 12 of the variants of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:222 is a valine (V).

[0042] In one embodiment of the present disclosure, repeat region 1 of the polypeptide disclosed herein comprises or consists of the amino acid sequence of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67, SEQ ID NO:220, or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67, SEQ ID NO:220 in that the amino acid sequence of the variant comprises 1 to 6 individual amino acid substitutions.

[0043] In one embodiment of the present disclosure, repeat region 2 comprises or consists of the amino acid sequence of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68, SEQ ID NO:221, or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68, SEQ ID NO:221 in that the amino acid sequence of the variant comprises 1 to 6 individual amino acid substitutions.

[0044] In one embodiment of the present disclosure, repeat region 3 comprises or consists of the amino acid sequence of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:222, or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:222 in that the amino acid sequence of the variant comprises 1 to 6 individual amino acid substitutions.

[0045] In one embodiment of the disclosure, each of repeat region 1, repeat region 2 and repeat region 3 is flanked at its N-terminus by at least three amino acid residues and at its C-terminus by at least three amino acid residues.

[0046] For example, in a polypeptide of the disclosure, repeat region 1, repeat region 2 and repeat region 3 are each flanked at their N-terminus by at least three amino acid residues and at their C-terminus by at least three amino acid residues, and thus Y N -R1-Y C Z N -R2-Z C J N -R3-J C and Where: Y N , Y C , Z N , Z C , J N , and J C can each individually be an amino acid sequence comprising at least three amino acids.

[0047] This structure is just one example. N , Y C , ZN , Z C , J N , and J C may each individually be an amino acid sequence comprising more than 3 amino acids, such as more than 5 amino acids, for example more than 10 amino acids, such as more than 15 amino acids, for example more than 20 amino acids. N , Y C , Z N , Z C , J N , and J C The number of amino acids in each of the may be the same or different.

[0048] In one embodiment of the disclosure, the functional variant SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67 or SEQ ID NO:220 differs from SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67 or SEQ ID NO:220 in that the amino acid sequence of the variant comprises 5 individual amino acid substitutions, such as 4 individual amino acid substitutions, such as 3 individual amino acid substitutions, such as 2 individual amino acid substitutions, such as 1 individual amino acid substitution.

[0049] In one embodiment of the disclosure, a functional variant of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67 or SEQ ID NO:220 is selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67 or SEQ ID NO:220, It differs from SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67 or SEQ ID NO:220 in that it comprises 6 individual amino acid substitutions, such as 5 individual amino acid substitutions, such as 4 individual amino acid substitutions, such as 3 individual amino acid substitutions, such as 2 individual amino acid substitutions, such as 1 individual amino acid substitution in any one of residues 1 to 34 or 36 to 38 of SEQ ID NO:220.

[0050] In one embodiment of the disclosure, a functional variant of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67 or SEQ ID NO:220 is selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67 or SEQ ID NO:220, differs from SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67 or SEQ ID NO:220 in that it includes 6 distinct amino acid substitutions, such as 5 distinct amino acid substitutions, such as 4 distinct amino acid substitutions, such as 3 distinct amino acid substitutions, such as 2 distinct amino acid substitutions, such as 1 distinct amino acid substitution, at any one of residues 1 to 34 or 37 of SEQ ID NO:220.

[0051] In one embodiment of the disclosure, a functional variant of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68 or SEQ ID NO:221 differs from SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68 or SEQ ID NO:221 in that the amino acid sequence of the variant comprises 5 individual amino acid substitutions, such as 4 individual amino acid substitutions, such as 3 individual amino acid substitutions, such as 2 individual amino acid substitutions, such as 1 individual amino acid substitution.

[0052] In one embodiment of the disclosure, a functional variant of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68 or SEQ ID NO:221 is selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68 or SEQ ID NO:221, differs from SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68 or SEQ ID NO:221 in that it comprises 6 individual amino acid substitutions, such as 5 individual amino acid substitutions, such as 4 individual amino acid substitutions, such as 3 individual amino acid substitutions, such as 2 individual amino acid substitutions, such as 1 individual amino acid substitution in any one of residues 1 to 31 or 33 to 35 of SEQ ID NO:221.

[0053] In one embodiment of the disclosure, a functional variant of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68 or SEQ ID NO:221 is selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68 or SEQ ID NO:221, or SEQ ID NO:221 in that it comprises 6 individual amino acid substitutions, such as 5 individual amino acid substitutions, such as 4 individual amino acid substitutions, such as 3 individual amino acid substitutions, such as 2 individual amino acid substitutions, such as 1 individual amino acid substitution at any one of residues 1 to 31 or 34 of SEQ ID NO:221.

[0054] In one embodiment of the disclosure, a functional variant of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69 or SEQ ID NO:222 differs from SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69 or SEQ ID NO:222 in that the amino acid sequence of the variant comprises 5 individual amino acid substitutions, such as 4 individual amino acid substitutions, such as 3 individual amino acid substitutions, such as 2 individual amino acid substitutions, such as 1 individual amino acid substitution.

[0055] In one embodiment of the disclosure, a functional variant of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69 or SEQ ID NO:222 is selected from the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69 or SEQ ID NO:222, differs from SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69 or SEQ ID NO:222 in that it comprises 6 individual amino acid substitutions, such as 5 individual amino acid substitutions, such as 4 individual amino acid substitutions, such as 3 individual amino acid substitutions, such as 2 individual amino acid substitutions, such as 1 individual amino acid substitution, in any one of residues 1 to 35 or 37 to 39 of SEQ ID NO:222.

[0056] In one embodiment of the disclosure, a functional variant of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69 or SEQ ID NO:222 is selected from the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69 or SEQ ID NO:222, or SEQ ID NO:222, in that it comprises six individual amino acid substitutions, such as five individual amino acid substitutions, such as four individual amino acid substitutions, such as three individual amino acid substitutions, such as two individual amino acid substitutions, such as one individual amino acid substitution at any one of residues 1 to 35 or 38 of SEQ ID NO:222.

[0057] In one embodiment of the disclosure, the individual amino acid substitutions are conservative amino acid substitutions.

[0058] In one embodiment of the present disclosure, repeat region 1 of the polypeptide described herein comprises or consists of the amino acid sequence of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67.

[0059] In one embodiment of the present disclosure, repeat region 2 of the polypeptide described herein comprises or consists of the amino acid sequence of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68.

[0060] In one embodiment of the present disclosure, repeat region 3 of the polypeptide described herein comprises or consists of the amino acid sequence of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69.

[0061] In one embodiment of the disclosure, the polypeptide comprises repeat region 1 and repeat region 2, or a functional variant thereof; repeat region 1 and repeat region 3, or a functional variant thereof; repeat region 2 and repeat region 3, or a functional variant thereof; repeat region 1, repeat region 2 and repeat region 3, or a functional variant thereof.

[0062] In a further aspect, the present disclosure relates to a polypeptide comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:20 to SEQ ID NO:33, or SEQ ID NO:71 to SEQ ID NO:219, wherein the polypeptide is capable of attaching one or more saccharide units.

[0063] One embodiment of the present disclosure relates to a polypeptide comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 20 to SEQ ID NO: 33, and capable of binding one or more saccharide units.

[0064] In a further embodiment of the present disclosure, the polypeptide comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:20 to SEQ ID NO:33, such as at least 85% sequence identity with SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:20 to SEQ ID NO:33, such as at least 90% sequence identity with SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:20 to SEQ ID NO:33, such as at least 95% sequence identity with SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:20 to SEQ ID NO:33, such as at least 96% sequence identity with SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:20 to SEQ ID NO:33, such as at least 97% sequence identity with SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:20 to SEQ ID NO:33, such as at least 98% sequence identity with SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:20 to SEQ ID NO:33, such as at least 99% sequence identity with SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:20 to SEQ ID NO:33.

[0065] In another embodiment of the present disclosure, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:20-SEQ ID NO:33.

[0066] In one embodiment of the disclosure, the polypeptide comprises: a. Repeat region 1 comprising or consisting of the amino acid sequence of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64 or SEQ ID NO:67, or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64 or SEQ ID NO:67 in that the amino acid sequence of the variant comprises 1 to 6 individual amino acid substitutions, b. Repeat region 2 comprising or consisting of the amino acid sequence of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65 or SEQ ID NO:68, or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65 or SEQ ID NO:68 in that the amino acid sequence of the variant comprises 1 to 6 individual amino acid substitutions, and c. Repeat region 3 comprising or consisting of the amino acid sequence of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66 or SEQ ID NO:69, or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69 in that the amino acid sequence of the variant comprises 1 to 6 individual amino acid substitutions. The repeat region comprises at least two repeat regions selected from the group consisting of:

[0067] In one embodiment of the disclosure, the polypeptide comprises three repeat regions, each of which has a. Repeat region 1 comprising or consisting of the amino acid sequence of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64 or SEQ ID NO:67, or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64 or SEQ ID NO:67 in that the amino acid sequence of the variant comprises 1 to 6 individual amino acid substitutions, b. Repeat region 2 comprising or consisting of the amino acid sequence of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65 or SEQ ID NO:68, or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65 or SEQ ID NO:68 in that the amino acid sequence of the variant comprises 1 to 6 individual amino acid substitutions, and c. Repeat region 3 comprising or consisting of the amino acid sequence of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66 or SEQ ID NO:69, or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69 in that the amino acid sequence of the variant comprises 1 to 6 individual amino acid substitutions. is selected from the group consisting of:

[0068] In one embodiment of the present disclosure, the polypeptide has a β-trefoil fold structure, preferably consisting of six β-hairpins, each formed by two β-strands, exhibiting approximately three-fold symmetry.

[0069] One function of the polypeptides of the present disclosure is their ability to bind and even cross-link certain di- and polysaccharides, which is at least in part related to the presence in the polypeptides of the present disclosure of two or preferably three repeat regions, such as repeat region 1, repeat region 2, and / or repeat region 3, as defined herein, that are capable of recognizing and binding to di- and polysaccharides.

[0070] Thus, in one embodiment of the present disclosure, repeat region 1, repeat region 2, and repeat region 3 are each capable of binding at least one disaccharide.

[0071] In one embodiment of the present disclosure, repeat region 1, repeat region 2, and repeat region 3 are each capable of binding at least one polysaccharide.

[0072] The repeat regions of the polypeptides of the present disclosure are capable of binding di- and polysaccharides that are characterized by containing the same types of linkages between the monosaccharide units, even though they may have different amino acid sequences.

[0073] The polypeptides of the present disclosure can bind any disaccharide or longer oligosaccharide or polysaccharide that contains the specific Glc-β-1,6 linkage, and several of the polysaccharides described herein contain this linkage.

[0074] The polypeptides of the present disclosure also exhibit binding to extremely rare Glc-β-1,2-Glc structures found in aberrant bacterial secretions.

[0075] In one embodiment of the present disclosure, repeat region 1, repeat region 2 and / or repeat region 3 are each capable of binding a di- or polysaccharide comprising at least one Glc-β-1,6-Glc unit.

[0076] In another embodiment of the present disclosure, repeat region 1, repeat region 2 and / or repeat region 3 are each capable of binding a di- or polysaccharide comprising at least one Glc-β-1,2-Glc unit.

[0077] In one embodiment of the present disclosure, repeat region 1, repeat region 2 and / or repeat region 3 are each capable of binding a di- or polysaccharide comprising a glycan comprising at least one Glc-β-1,6-Glc unit.

[0078] In one embodiment of the present disclosure, repeat region 1, repeat region 2 and / or repeat region 3 are each capable of binding di- or polysaccharides having a β-1,6-glucan backbone.

[0079] In one embodiment of the present disclosure, repeat region 1, repeat region 2 and / or repeat region 3 are each capable of binding di- or polysaccharides having one or more Glc-β-1,6-Glc modifications.

[0080] In one embodiment of the present disclosure, repeat region 1, repeat region 2 and / or repeat region 3 are each capable of binding a di- or polysaccharide having one or more Glc-β-1,6-Glc modification(s) on the β-1,3-glucan backbone.

[0081] In one embodiment of the present disclosure, repeat region 1, repeat region 2 and / or repeat region 3 are each capable of binding the polysaccharide scleroglucan.

[0082] In one embodiment of the present disclosure, repeat region 1, repeat region 2 and / or repeat region 3 are each capable of binding the polysaccharide pustulan.

[0083] In one embodiment of the present disclosure, repeat region 1, repeat region 2 and / or repeat region 3 are each capable of binding the polysaccharide laminarin.

[0084] In one embodiment of the present disclosure, repeat region 1, repeat region 2 and / or repeat region 3 are each capable of binding the polysaccharide gentiobiose.

[0085] In one embodiment of the present disclosure, the saccharide is at least a disaccharide.

[0086] In one embodiment of the present disclosure, the saccharide is an oligosaccharide or polysaccharide.

[0087] Polynucleotides encoding carbohydrate-binding polypeptides In one aspect, the disclosure relates to polynucleotides encoding the polypeptides described herein.

[0088] In one embodiment of the disclosure, the polynucleotide is a polypeptide, a. SEQ ID NO:220: X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 X 31 X 32 X 33 X 34 WEX 37 F Repeat region 1 comprising or consisting of the amino acid sequence [In the formula, X1 is isoleucine (I) or valine (V); X2 is any amino acid, such as tryptophan (W), serine (S), threonine (T) or alanine (A); X3 is leucine (L) or isoleucine (I); X4 is lysine (K) or arginine (R) or is absent; X5 is glutamine (Q), arginine (R), alanine (A), lysine (K), or is absent; X6 is glycine (G), serine (S), tyrosine (Y), cysteine ​​(C), leucine (L) or threonine (T); X7 is any amino acid, such as phenylalanine (F) or serine (S); X8 is glycine (G) or is absent; X9 is asparagine (N), histidine (H), aspartic acid (D) or threonine (T); X 10 is asparagine (N), serine (S), threonine (T), glutamine (Q), lysine (K) or glycine (G); X 11 is lysine (K), glutamine (Q), asparagine (N) or leucine (L); X 12 is tyrosine (Y), leucine (L) or phenylalanine (F), X 13 is valine (V), leucine (L) or alanine (A), X 14 is serine (S), threonine (T), cysteine ​​(C) or asparagine (N), X 15 is alanine (A), serine (S) or glycine (G), X 16 is glutamic acid (E), lysine (K), arginine (R), glutamine (Q) or aspartic acid (D), X 17is asparagine (N), glycine (G), glutamine (Q) or proline (P), X 18 is glycine (G), asparagine (N), threonine (T) or aspartic acid (D); X 19 is leucine (L) or is absent, X 20 is glycine (G) or alanine (A) or is absent, X 21 is alanine (A) or asparagine (N) or is absent, X 22 is any amino acid, such as threonine (T), or is absent, X 23 is any amino acid, such as glycine (G), X 24 is proline (P), alanine (A), glutamine (Q) or arginine (R), X 25 is leucine (L), methionine (M), alanine (A) or isoleucine (I), X 26 is any amino acid, such as threonine (T), X 27 is alanine (A), cysteine ​​(C) or tryptophan (W), X 28 is asparagine (N), aspartic acid (D), glutamic acid (E) or arginine (R); X 29 is arginine (R), alanine (A) or serine (S), X 30 is threonine (T), aspartic acid (D), proline (P), isoleucine (I), asparagine (N) or alanine (A); X 31 is threonine (T), alanine (A), valine (V), glycine (G), lysine (K) or isoleucine (I); X 32is alanine (A), valine (V), proline (P) or leucine (L); X 33 is glycine (G), glutamine (Q), serine (S) or aspartic acid (D), X 34 is any amino acid, such as glycine (G), X 37 is any amino acid, such as glutamine (Q) or lysine (K)]; or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO: 220 in that the amino acid sequence of the variant comprises 1 to 6 distinct amino acid substitutions. b. SEQ ID NO:221: X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 X 31 WEX 34 F Repeat region 2 comprising or consisting of the amino acid sequence [In the formula, X1 is isoleucine (I) or valine (V); X2 is alanine (A), asparagine (N), tyrosine (Y) or threonine (T); X3 is leucine (L) or isoleucine (I); X4 is glutamine (Q), arginine (R), leucine (L) or lysine (K); X5 is glycine (G), alanine (A), asparagine (N) or serine (S); X6 is any amino acid, such as alanine (A), or is absent; X7 is glycine (G) or serine (S) or is absent; X8 is asparagine (N), methionine (M), glutamine (Q), histidine (H) or serine (S); X9 is glycine (G) or alanine (A); X 10 is lysine (K), leucine (L), methionine (M), asparagine (N) or serine (S); X 11 is tyrosine (Y), X 12 is valine (V) or phenylalanine (F), X 13 is serine (S), cysteine ​​(C), lysine (K) or glutamine (Q), X 14 is alanine (A), valine (V) or serine (S), X 15 is glutamic acid (E), aspartic acid (D), glutamine (Q) or glycine (G), X 16 is asparagine (N), proline (P), glycine (G), threonine (T) or aspartic acid (D), X 17 is glycine (G), aspartic acid (D), isoleucine (I), asparagine (N), or is absent; X 18 is valine (V) or glycine (G) or is absent, X 19 is glycine (G), threonine (T), leucine (L), alanine (A), or glutamic acid (E), or is absent; X 20 is any amino acid, such as glutamine (Q), or is absent, X21 is proline (P), alanine (A), glutamine (Q) or asparagine (N), X 22 is leucine (L), isoleucine (I), methionine (M) or valine (V); X 23 is threonine (T), asparagine (N), phenylalanine (F) or arginine (R); X 24 is alanine (A), cysteine ​​(C) or arginine (R), X 25 is asparagine (N), alanine (A), threonine (T) or glutamine (Q), X 26 is alanine (A), glycine (G) or arginine (R), X 27 is threonine (T), alanine (A), lysine (K), proline (P) or leucine (L); X 28 is any amino acid, such as alanine (A), X 29 is isoleucine (I), valine (V), proline (P), alanine (A), lysine (K), tyrosine (Y) or leucine (L); X 30 is glycine (G), alanine (A), glutamic acid (E), threonine (T), serine (S), glutamine (Q) or aspartic acid (D); X 31 is glycine (G), alanine (A), aspartic acid (D), serine (S), proline (P) or lysine (K); X 34 is any amino acid, such as lysine (K)], or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO: 221 in that the amino acid sequence of the variant comprises 1 to 6 distinct amino acid substitutions. c. SEQ ID NO:222: X1X2X3X4X5X6X7X8X9X 10X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 X 31 X 32 X 33 X 34 X 35 WEX 38 F Repeat region 3 comprising or consisting of the amino acid sequence [In the formula, X1 is valine (V), isoleucine (I), tyrosine (Y) or phenylalanine (F); X2 is alanine (A), serine (S), cysteine ​​(C), threonine (T) or glycine (G); X3 is leucine (L), phenylalanine (F) or isoleucine (I); X4 is arginine (R), lysine (K) or glutamine (Q); X5 is glycine (G), alanine (A) or serine (S); X6 is arginine (R) or valine (V) or is absent; X7 is any amino acid, such as asparagine (N); X8 is asparagine (N), histidine (H), alanine (A), lysine (K) or absent; X9 is glycine (G), tryptophan (W) or asparagine (N); X 10 is lysine (K), leucine (L), methionine (M), serine (S), alanine (A) or glutamine (Q), X11 is tyrosine (Y), tryptophan (W) or phenylalanine (F), X 12 is valine (V), leucine (L), methionine (M) or isoleucine (I), X 13 is serine (S) or glutamine (Q), X 14 is alanine (A), serine (S), histidine (H) or glycine (G), X 15 is glutamic acid (E), aspartic acid (D), glutamine (Q), serine (S) or asparagine (N); X 16 is asparagine (N), leucine (L), tryptophan (W), methionine (M) or aspartic acid (D); X 17 is glycine (G), serine (S) or aspartic acid (D), X 18 is any amino acid, such as serine (S) or threonine (T), X 19 is asparagine (N), valine (V), or aspartic acid (D), or is absent; X 20 is alanine (A) or is absent, X 21 is serine (S) or is absent, X 22 is any amino acid, such as alanine (A), or is absent, X 23 is proline (P), alanine (A), arginine (R), threonine (T) or glycine (G), X 24 is leucine (L), methionine (M), valine (V) or isoleucine (I), X 25 is threonine (T), tyrosine (Y), asparagine (N), leucine (L) or glutamine (Q); X 26is alanine (A), cysteine ​​(C), phenylalanine (F) or tryptophan (W), X 27 is asparagine (N), alanine (A), threonine (T) or serine (S), X 28 is arginine (R) or lysine (K), X 29 is aspartic acid (D) or is absent, X 30 is threonine (T), alanine (A), proline (P), serine (S) or glycine (G); X 31 is any amino acid, such as alanine (A), X 32 is isoleucine (I), glutamic acid (E), proline (P), alanine (A) or valine (V), X 33 is glycine (G), lysine (K), or aspartic acid (D), or is absent; X 34 is glycine (G), serine (S), lysine (K), aspartic acid (D) or glutamine (Q), X 35 is glycine (G), alanine (A), aspartic acid (D) or cysteine ​​(C), X 38 is any amino acid, such as lysine (K)], or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO: 222 in that the amino acid sequence of the variant contains 1 to 6 distinct amino acid substitutions. The polypeptide encodes a polypeptide comprising at least two repeat regions selected from the group consisting of:

[0089] In one embodiment of the disclosure, the polynucleotide is codon optimized for expression in a host cell.

[0090] In another aspect, the present disclosure relates to a recombinant vector comprising a nucleic acid sequence encoding a polynucleotide described herein or a carbohydrate binding polypeptide according to any of the embodiments described herein. Examples of recombinant vectors include, but are not limited to, plasmids, viral vectors, cosmids, and artificial chromosomes.

[0091] In one embodiment of the present disclosure, the vector is a plasmid vector.

[0092] In a further aspect, the present disclosure relates to host cells (eg, standard expression strains of E. coli) that express the recombinant vectors described herein.

[0093] Hydrogel As provided herein, the polypeptides of the present disclosure can bind polysaccharides. Each of the polypeptides of the present disclosure can bind up to three polysaccharide chains. Furthermore, when multiple polypeptides of the present disclosure are present with a polysaccharide, the polypeptides of the present disclosure may not only bind the polysaccharides but also cross-link them. Thus, the polypeptides of the present disclosure can bind and cross-link specific polysaccharides. Specific polysaccharides are described herein.

[0094] In one aspect, the present disclosure relates to the use of a polypeptide as described in any of the embodiments herein to cross-link two or more polysaccharides.

[0095] Another aspect of the present disclosure relates to a composition comprising at least one polypeptide according to any of the embodiments described herein and two or more polysaccharides, where the polysaccharides are cross-linked by the polypeptide, e.g., the polypeptide is a cross-linker that links the polysaccharides together.

[0096] In another aspect, the present disclosure relates to a hydrogel comprising at least one polypeptide according to any of the embodiments described herein and two or more polysaccharides, the hydrogel having a cross-linked structure.

[0097] In another aspect, the present disclosure relates to a hydrogel comprising at least two polypeptides according to any of the embodiments described herein and three or more polysaccharides, the hydrogel having a cross-linked structure.

[0098] Preferably, the present disclosure also relates to a hydrogel comprising a plurality of polypeptides and a plurality of polysaccharides according to any of the embodiments described herein, said hydrogel having a cross-linked structure.

[0099] Indeed, the polypeptides according to any of the embodiments described herein function as crosslinkers in the hydrogels of the present disclosure.

[0100] In one embodiment of the present disclosure, the hydrogel is crosslinked with a bifunctional or polyfunctional linker, e.g., a linker capable of binding to two or more polysaccharide chains, that is a polypeptide as described herein.

[0101] In one embodiment of the disclosure, the hydrogel comprises one or more polysaccharides, each of which has a size of at least 20.000 Da, such as at least 50.000 Da, for example at least 70.000 Da, such as at least 100.000 Da, for example at least 150.000 Da, such as at least 200.000 Da, for example at least 300.000 Da, such as at least 500.000 Da, for example at least 700.000 Da, such as at least 800.000 Da, for example at least 900.000 Da, for example at least 1.000.000 Da, for example at least 2.000.000 Da. Preferably, the polysaccharide is at least 100.000 Da, such as at least 500.000 Da, for example at least 1.000.000 Da, for example at least 2.000.000 Da.

[0102] In one embodiment of the present disclosure, the hydrogel comprises a polysaccharide that is water dispersible. In a further embodiment, the polysaccharide has a water content of 70 μg water / mg saccharide or more, preferably 74 μg water / mg saccharide or more, for example 77 μg water / mg saccharide.

[0103] In one embodiment of the present disclosure, the hydrogel comprises one or more polysaccharides that are biopolymers, such as scleroglucan or schizophyllan, each of which contains one or more β-1,3-glucan units having β-1,6 linked glucose modifications to the backbone.

[0104] In one embodiment of the present disclosure, the hydrogel comprises 0.1 g / L to 10 g / L of a polypeptide of the present disclosure and 2 g / L to 10 g / L of a polysaccharide as defined herein. Preferably, the hydrogel comprises 0.15 g / L to 5 g / L of a polypeptide of the present disclosure and 3 g / L to 5 g / L of a polysaccharide as defined herein.

[0105] In one embodiment of the disclosure, the hydrogel comprises at least 0.1 g / L of a polypeptide of the disclosure, such as at least 0.2 g / L of a polypeptide, such as at least 0.3 g / L of a polypeptide, such as at least 0.5 g / L of a polypeptide, such as at least 0.8 g / L of a polypeptide, such as at least 1 g / L of a polypeptide, such as at least 1.3 g / L of a polypeptide, such as at least 1.7 g / L of a polypeptide, such as at least 2 g / L of a polypeptide, such as at least 2.5 g / L of a polypeptide, such as at least 3 g / L of a polypeptide, such as at least 3.5 g / L of a polypeptide, For example, at least 4 g / L of a polypeptide, such as at least 4.5 g / L of a polypeptide, for example at least 5 g / L of a polypeptide, such as at least 5.5 g / L of a polypeptide, for example at least 6 g / L of a polypeptide, such as at least 6.5 g / L of a polypeptide, for example at least 7 g / L of a polypeptide, such as at least 7.5 g / L of a polypeptide, for example at least 8 g / L of a polypeptide, such as at least 8.5 g / L of a polypeptide, for example at least 9 g / L of a polypeptide, such as at least 9.5 g / L of a polypeptide, for example at least 10 g / L of a polypeptide of the present disclosure.

[0106] In one embodiment of the present disclosure, the hydrogel comprises up to 10 g / L of a polypeptide of the present disclosure.

[0107] In one embodiment of the disclosure, the hydrogel comprises at least 2 g / L of polysaccharide, such as at least 3 g / L of polysaccharide, and at most 5 g / L of polysaccharide, such as at most 6 g / L of polysaccharide, such as at most 7 g / L of polysaccharide, such as at most 8 g / L of polysaccharide, such as at most 9 g / L of polysaccharide, 10 g / L of polysaccharide. Preferably, in one embodiment of the disclosure, the hydrogel comprises at least 3 g / L of polysaccharide, and at most 5 g / L of polysaccharide, the polysaccharide comprising at least one Glc-β-1,6-Glc unit.

[0108] In one embodiment of the disclosure, the hydrogel comprises at least 2.5 g / L polysaccharide, such as at least 3 g / L polysaccharide, such as at least 3.5 g / L polysaccharide, such as at least 4 g / L polysaccharide, such as at least 4.5 g / L polysaccharide, such as at least 5 g / L polysaccharide, such as at least 5.5 g / L polysaccharide, such as at least 6 g / L polysaccharide, such as at least 6.5 g / L polysaccharide, such as at least 7 g / L polysaccharide, such as at least 7.5 g / L polysaccharide, such as at least 8 g / L polysaccharide, such as at least 8.5 g / L polysaccharide, such as at least 9 g / L polysaccharide, such as at least 9.5 g / L polysaccharide, such as up to 10 g / L polysaccharide, wherein the polysaccharide comprises at least one Glc-β-1,6-Glc unit.

[0109] In one embodiment of the disclosure, the hydrogel comprises at least 2 g / L scleroglucan, such as at least 3 g / L scleroglucan, and at most 10 g / L scleroglucan, such as at most 6 g / L scleroglucan, such as at most 7 g / L scleroglucan, such as at most 8 g / L scleroglucan, such as at most 9 g / L scleroglucan. Preferably, in one embodiment of the disclosure, the hydrogel comprises at least 3 g / L scleroglucan, and at most 5 g / L scleroglucan.

[0110] In one embodiment of the disclosure, the hydrogel comprises at least 2.5 g / L of scleroglucan, such as at least 3 g / L of scleroglucan, such as at least 3.5 g / L of scleroglucan, such as at least 4 g / L of scleroglucan, such as at least 4.5 g / L of scleroglucan, such as at least 5 g / L of scleroglucan, such as at least 5.5 g / L of scleroglucan, such as at least 6 g / L of scleroglucan, For example, at least 6.5 g / L scleroglucan, such as at least 7 g / L scleroglucan, for example at least 7.5 g / L scleroglucan, such as at least 8 g / L scleroglucan, for example at least 8.5 g / L scleroglucan, such as at least 9 g / L polysaccharide, for example at least 9.5 g / L polysaccharide, for example up to 10 g / L polysaccharide, wherein the polysaccharide comprises at least one Glc-β-1,6-Glc unit.

[0111] For example, as shown in FIG. 5 and Example 5 of the present disclosure, the hydrogel can include 3-5 g / L of the scleroglucan polysaccharide, such as about 4 g / L of the scleroglucan polysaccharide.

[0112] In an embodiment of the present disclosure, the hydrogel comprises at least 90% water by weight, such as at least 91% water by weight, such as at least 92% water by weight, for example at least 93% water by weight, such as at least 94% water by weight, for example at least 95% water by weight, such as at least 96% water by weight, for example at least 97% water by weight, such as at least 98% water by weight, for example at least 99% water by weight, such as at least 99.5% water by weight.

[0113] The hydrogel according to one embodiment of the present disclosure can be dried out in air at room temperature and then rehydrated upon contact with water. In one embodiment, the rehydrated gel has a lower water content than the original hydrogel, e.g., the rehydrated gel can contain 70% to 90% water by weight. For example, the rehydrated hydrogel can be in the form of a film.

[0114] In one embodiment, the rehydrated gel has a water content similar to the original hydrogel, for example, the rehydrated gel may contain at least 70% water by weight, such as at least 80% water by weight, for example at least 90% water by weight.

[0115] In one embodiment of the present disclosure, the hydrogel can be dehydrated and then rehydrated or reswelled while retaining the crosslinked structure, the processes of dehydration and rehydration referring to the change in physical form of the hydrogel due to loss of water and regain of water content, such as reswelling, respectively.

[0116] Dehydration and rehydration of the hydrogels of the present disclosure can be accomplished by any method known in the art.

[0117] In one embodiment of the present disclosure, the hydrogel is a rehydrated hydrogel, which comprises at least 70% water by weight, such as at least 73% water by weight, for example at least 75% water by weight, such as at least 78% water by weight, for example at least 80% water by weight, such as at least 83% water by weight, for example at least 85% water by weight, such as at least 88% water by weight, for example at least 90% water by weight.

[0118] In one embodiment of the disclosure, the hydrogel comprises a polypeptide of the disclosure and a polysaccharide in a ratio of 1:20 to 1:2 polypeptide:polysaccharide, such as a ratio of 1:20 to 1:3, such as a ratio of 1:20 to 1:5, such as a ratio of 1:20 to 1:7, such as a ratio of 1:20 to 1:10, such as a ratio of 1:20 to 1:12, such as a ratio of 1:20 to 1:15, such as a ratio of 1:20 to 1:18, such as a ratio of 1:18 to 1:2, such as a ratio of 1:15 to 1:2, such as a ratio of 1:13 to 1:2, such as a ratio of 1:10 to 1:2, such as a ratio of 1:8 to 1:2, such as a ratio of 1:6 to 1:2, such as a ratio of 1:4 to 1:2. For example, the polysaccharide can be scleroglucan. These ratios are volume:volume ratios based on a 5 g / L stock solution of polysaccharide in water at room temperature and a 4 g / L stock solution of a polypeptide of the disclosure in water at room temperature.

[0119] In one embodiment of the present disclosure, the hydrogel comprises: A polypeptide comprising: a. SEQ ID NO:220: X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 X 31 X 32 X 33 X 34 WEX 37 F Repeat region 1 (R1) comprising or consisting of the amino acid sequence [In the formula, X1 is isoleucine (I) or valine (V); X2 is any amino acid, such as tryptophan (W), serine (S), threonine (T) or alanine (A); X3 is leucine (L) or isoleucine (I); X4 is lysine (K) or arginine (R) or is absent; X5 is glutamine (Q), arginine (R), alanine (A), lysine (K), or is absent; X6 is glycine (G), serine (S), tyrosine (Y), cysteine ​​(C), leucine (L) or threonine (T); X7 is any amino acid, such as phenylalanine (F) or serine (S); X8 is glycine (G) or is absent; X9 is asparagine (N), histidine (H), aspartic acid (D) or threonine (T); X 10 is asparagine (N), serine (S), threonine (T), glutamine (Q), lysine (K) or glycine (G); X 11 is lysine (K), glutamine (Q), asparagine (N) or leucine (L); X 12 is tyrosine (Y), leucine (L) or phenylalanine (F), X 13 is valine (V), leucine (L) or alanine (A), X 14 is serine (S), threonine (T), cysteine ​​(C) or asparagine (N), X 15 is alanine (A), serine (S) or glycine (G), X 16 is glutamic acid (E), lysine (K), arginine (R), glutamine (Q) or aspartic acid (D), X 17 is asparagine (N), glycine (G), glutamine (Q) or proline (P), X 18 is glycine (G), asparagine (N), threonine (T) or aspartic acid (D); X 19 is leucine (L) or is absent, X 20 is glycine (G) or alanine (A) or is absent, X 21 is alanine (A) or asparagine (N) or is absent, X 22 is any amino acid, such as threonine (T), or is absent, X 23 is any amino acid, such as glycine (G), X 24is proline (P), alanine (A), glutamine (Q) or arginine (R), X 25 is leucine (L), methionine (M), alanine (A) or isoleucine (I), X 26 is any amino acid, such as threonine (T), X 27 is alanine (A), cysteine ​​(C) or tryptophan (W), X 28 is asparagine (N), aspartic acid (D), glutamic acid (E) or arginine (R); X 29 is arginine (R), alanine (A) or serine (S), X 30 is threonine (T), aspartic acid (D), proline (P), isoleucine (I), asparagine (N) or alanine (A); X 31 is threonine (T), alanine (A), valine (V), glycine (G), lysine (K) or isoleucine (I), X 32 is alanine (A), valine (V), proline (P) or leucine (L); X 33 is glycine (G), glutamine (Q), serine (S) or aspartic acid (D), X 34 is any amino acid, such as glycine (G), X 37 is any amino acid, such as glutamine (Q) or lysine (K)]; or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO: 220 in that the amino acid sequence of the variant comprises 1 to 6 distinct amino acid substitutions. b. SEQ ID NO:221: X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 X 31 WEX 34 F Repeat region 2 (R2) comprising or consisting of the amino acid sequence [In the formula, X1 is isoleucine (I) or valine (V); X2 is alanine (A), asparagine (N), tyrosine (Y) or threonine (T); X3 is leucine (L) or isoleucine (I); X4 is glutamine (Q), arginine (R), leucine (L) or lysine (K); X5 is glycine (G), alanine (A), asparagine (N) or serine (S); X6 is any amino acid, such as alanine (A), or is absent; X7 is glycine (G) or serine (S) or is absent; X8 is asparagine (N), methionine (M), glutamine (Q), histidine (H) or serine (S); X9 is glycine (G) or alanine (A); X 10 is lysine (K), leucine (L), methionine (M), asparagine (N) or serine (S); X 11 is tyrosine (Y), X 12 is valine (V) or phenylalanine (F), X 13is serine (S), cysteine ​​(C), lysine (K) or glutamine (Q), X 14 is alanine (A), valine (V) or serine (S), X 15 is glutamic acid (E), aspartic acid (D), glutamine (Q) or glycine (G), X 16 is asparagine (N), proline (P), glycine (G), threonine (T) or aspartic acid (D), X 17 is glycine (G), aspartic acid (D), isoleucine (I), asparagine (N), or is absent; X 18 is valine (V) or glycine (G) or is absent, X 19 is glycine (G), threonine (T), leucine (L), alanine (A), or glutamic acid (E), or is absent; X 20 is any amino acid, such as glutamine (Q), or is absent, X 21 is proline (P), alanine (A), glutamine (Q) or asparagine (N), X 22 is leucine (L), isoleucine (I), methionine (M) or valine (V); X 23 is threonine (T), asparagine (N), phenylalanine (F) or arginine (R); X 24 is alanine (A), cysteine ​​(C) or arginine (R), X 25 is asparagine (N), alanine (A), threonine (T) or glutamine (Q), X 26 is alanine (A), glycine (G) or arginine (R), X 27is threonine (T), alanine (A), lysine (K), proline (P) or leucine (L); X 28 is any amino acid, such as alanine (A), X 29 is isoleucine (I), valine (V), proline (P), alanine (A), lysine (K), tyrosine (Y) or leucine (L); X 30 is glycine (G), alanine (A), glutamic acid (E), threonine (T), serine (S), glutamine (Q) or aspartic acid (D); X 31 is glycine (G), alanine (A), aspartic acid (D), serine (S), proline (P) or lysine (K); X 34 is any amino acid, such as lysine (K)], or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO: 221 in that the amino acid sequence of the variant comprises 1 to 6 distinct amino acid substitutions. c. SEQ ID NO:222: X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 X 31 X 32 X 33 X 34 X 35 WEX 38 F Repeat region 3 (R3) comprising or consisting of the amino acid sequence [In the formula, X1 is valine (V), isoleucine (I), tyrosine (Y) or phenylalanine (F); X2 is alanine (A), serine (S), cysteine ​​(C), threonine (T) or glycine (G); X3 is leucine (L), phenylalanine (F) or isoleucine (I); X4 is arginine (R), lysine (K) or glutamine (Q); X5 is glycine (G), alanine (A) or serine (S); X6 is arginine (R) or valine (V) or is absent; X7 is any amino acid, such as asparagine (N); X8 is asparagine (N), histidine (H), alanine (A), lysine (K) or absent; X9 is glycine (G), tryptophan (W) or asparagine (N); X 10 is lysine (K), leucine (L), methionine (M), serine (S), alanine (A) or glutamine (Q), X 11 is tyrosine (Y), tryptophan (W) or phenylalanine (F), X 12 is valine (V), leucine (L), methionine (M) or isoleucine (I), X 13 is serine (S) or glutamine (Q), X 14 is alanine (A), serine (S), histidine (H) or glycine (G), X 15 is glutamic acid (E), aspartic acid (D), glutamine (Q), serine (S) or asparagine (N); X 16is asparagine (N), leucine (L), tryptophan (W), methionine (M) or aspartic acid (D); X 17 is glycine (G), serine (S) or aspartic acid (D), X 18 is any amino acid, such as serine (S) or threonine (T), X 19 is asparagine (N), valine (V), or aspartic acid (D), or is absent; X 20 is alanine (A) or is absent, X 21 is serine (S) or is absent, X 22 is any amino acid, such as alanine (A), or is absent, X 23 is proline (P), alanine (A), arginine (R), threonine (T) or glycine (G), X 24 is leucine (L), methionine (M), valine (V) or isoleucine (I), X 25 is threonine (T), tyrosine (Y), asparagine (N), leucine (L) or glutamine (Q); X 26 is alanine (A), cysteine ​​(C), phenylalanine (F) or tryptophan (W), X 27 is asparagine (N), alanine (A), threonine (T) or serine (S), X 28 is arginine (R) or lysine (K), X 29 is aspartic acid (D) or is absent, X 30 is threonine (T), alanine (A), proline (P), serine (S) or glycine (G); X 31is any amino acid, such as alanine (A), X 32 is isoleucine (I), glutamic acid (E), proline (P), alanine (A) or valine (V), X 33 is glycine (G), lysine (K), or aspartic acid (D), or is absent; X 34 is glycine (G), serine (S), lysine (K), aspartic acid (D) or glutamine (Q), X 35 is glycine (G), alanine (A), aspartic acid (D) or cysteine ​​(C), X 38 is any amino acid, such as lysine (K)], or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO: 222 in that the amino acid sequence of the variant contains 1 to 6 distinct amino acid substitutions. and a polypeptide comprising at least two repeat regions selected from the group consisting of: and polysaccharides comprising one or more β-1,3-glucan units having β-1,6 linked glucose modifications.

[0120] In one embodiment of the present disclosure, the hydrogel comprises: A polypeptide comprising: [ka] Repeat region 1 (R1) comprising or consisting of the amino acid sequence or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO: 8 in that the amino acid sequence of the variant comprises 1 to 6 distinct amino acid substitutions. [ka] Repeat region 2 (R2) comprising or consisting of the amino acid sequence or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO: 10 in that the amino acid sequence of the variant comprises 1 to 6 distinct amino acid substitutions. [ka] Repeat region 3 (R3) comprising or consisting of the amino acid sequence or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO: 12 in that the amino acid sequence of the variant contains 1 to 6 distinct amino acid substitutions. and a polypeptide comprising at least two repeat regions selected from the group consisting of: and polysaccharides comprising one or more β-1,3-glucan units having β-1,6 linked glucose modifications.

[0121] In one embodiment of the present disclosure, the hydrogel comprises: A polypeptide comprising: a. SEQ ID NO:220: X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 X 31 X 32 X 33 X 34 WEX 37 F Repeat region 1 (R1) comprising or consisting of the amino acid sequence [In the formula, X1 is isoleucine (I) or valine (V); X2 is any amino acid, such as tryptophan (W), serine (S), threonine (T) or alanine (A); X3 is leucine (L) or isoleucine (I); X4 is lysine (K) or arginine (R) or is absent; X5 is glutamine (Q), arginine (R), alanine (A), lysine (K), or is absent; X6 is glycine (G), serine (S), tyrosine (Y), cysteine ​​(C), leucine (L) or threonine (T); X7 is any amino acid, such as phenylalanine (F) or serine (S); X8 is glycine (G) or is absent; X9 is asparagine (N), histidine (H), aspartic acid (D) or threonine (T); X 10 is asparagine (N), serine (S), threonine (T), glutamine (Q), lysine (K) or glycine (G); X 11 is lysine (K), glutamine (Q), asparagine (N) or leucine (L); X 12 is tyrosine (Y), leucine (L) or phenylalanine (F), X 13 is valine (V), leucine (L) or alanine (A), X 14 is serine (S), threonine (T), cysteine ​​(C) or asparagine (N), X 15 is alanine (A), serine (S) or glycine (G), X 16 is glutamic acid (E), lysine (K), arginine (R), glutamine (Q) or aspartic acid (D), X 17is asparagine (N), glycine (G), glutamine (Q) or proline (P), X 18 is glycine (G), asparagine (N), threonine (T) or aspartic acid (D); X 19 is leucine (L) or is absent, X 20 is glycine (G) or alanine (A) or is absent, X 21 is alanine (A) or asparagine (N) or is absent, X 22 is any amino acid, such as threonine (T), or is absent, X 23 is any amino acid, such as glycine (G), X 24 is proline (P), alanine (A), glutamine (Q) or arginine (R), X 25 is leucine (L), methionine (M), alanine (A) or isoleucine (I), X 26 is any amino acid, such as threonine (T), X 27 is alanine (A), cysteine ​​(C) or tryptophan (W), X 28 is asparagine (N), aspartic acid (D), glutamic acid (E) or arginine (R); X 29 is arginine (R), alanine (A) or serine (S), X 30 is threonine (T), aspartic acid (D), proline (P), isoleucine (I), asparagine (N) or alanine (A); X 31 is threonine (T), alanine (A), valine (V), glycine (G), lysine (K) or isoleucine (I), X 32is alanine (A), valine (V), proline (P) or leucine (L); X 33 is glycine (G), glutamine (Q), serine (S) or aspartic acid (D), X 34 is any amino acid, such as glycine (G), X 37 is any amino acid, such as glutamine (Q) or lysine (K)]; or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO: 220 in that the amino acid sequence of the variant comprises 1 to 6 distinct amino acid substitutions. b. SEQ ID NO:221: X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 X 31 WEX 34 F Repeat region 2 (R2) comprising or consisting of the amino acid sequence [In the formula, X1 is isoleucine (I) or valine (V); X2 is alanine (A), asparagine (N), tyrosine (Y) or threonine (T); X3 is leucine (L) or isoleucine (I); X4 is glutamine (Q), arginine (R), leucine (L) or lysine (K); X5 is glycine (G), alanine (A), asparagine (N) or serine (S); X6 is any amino acid, such as alanine (A), or is absent; X7 is glycine (G) or serine (S) or is absent; X8 is asparagine (N), methionine (M), glutamine (Q), histidine (H) or serine (S); X9 is glycine (G) or alanine (A); X 10 is lysine (K), leucine (L), methionine (M), asparagine (N) or serine (S); X 11 is tyrosine (Y), X 12 is valine (V) or phenylalanine (F), X 13 is serine (S), cysteine ​​(C), lysine (K) or glutamine (Q), X 14 is alanine (A), valine (V) or serine (S), X 15 is glutamic acid (E), aspartic acid (D), glutamine (Q) or glycine (G), X 16 is asparagine (N), proline (P), glycine (G), threonine (T) or aspartic acid (D), X 17 is glycine (G), aspartic acid (D), isoleucine (I), asparagine (N), or is absent; X 18 is valine (V) or glycine (G) or is absent; X 19 is glycine (G), threonine (T), leucine (L), alanine (A), or glutamic acid (E), or is absent; X 20 is any amino acid, such as glutamine (Q), or is absent, X21 is proline (P), alanine (A), glutamine (Q) or asparagine (N), X 22 is leucine (L), isoleucine (I), methionine (M) or valine (V); X 23 is threonine (T), asparagine (N), phenylalanine (F) or arginine (R); X 24 is alanine (A), cysteine ​​(C) or arginine (R), X 25 is asparagine (N), alanine (A), threonine (T) or glutamine (Q), X 26 is alanine (A), glycine (G) or arginine (R), X 27 is threonine (T), alanine (A), lysine (K), proline (P) or leucine (L); X 28 is any amino acid, such as alanine (A), X 29 is isoleucine (I), valine (V), proline (P), alanine (A), lysine (K), tyrosine (Y) or leucine (L); X 30 is glycine (G), alanine (A), glutamic acid (E), threonine (T), serine (S), glutamine (Q) or aspartic acid (D); X 31 is glycine (G), alanine (A), aspartic acid (D), serine (S), proline (P) or lysine (K); X 34 is any amino acid, such as lysine (K)], or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO: 221 in that the amino acid sequence of the variant comprises 1 to 6 distinct amino acid substitutions. c. SEQ ID NO:222: X1X2X3X4X5X6X7X8X9X 10X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 X 31 X 32 X 33 X 34 X 35 WEX 38 F Repeat region 3 (R3) comprising or consisting of the amino acid sequence [In the formula, X1 is valine (V), isoleucine (I), tyrosine (Y) or phenylalanine (F); X2 is alanine (A), serine (S), cysteine ​​(C), threonine (T) or glycine (G); X3 is leucine (L), phenylalanine (F) or isoleucine (I); X4 is arginine (R), lysine (K) or glutamine (Q); X5 is glycine (G), alanine (A) or serine (S); X6 is arginine (R) or valine (V) or is absent; X7 is any amino acid, such as asparagine (N); X8 is asparagine (N), histidine (H), alanine (A), lysine (K) or absent; X9 is glycine (G), tryptophan (W) or asparagine (N); X 10 is lysine (K), leucine (L), methionine (M), serine (S), alanine (A) or glutamine (Q), X11 is tyrosine (Y), tryptophan (W) or phenylalanine (F), X 12 is valine (V), leucine (L), methionine (M) or isoleucine (I), X 13 is serine (S) or glutamine (Q), X 14 is alanine (A), serine (S), histidine (H) or glycine (G), X 15 is glutamic acid (E), aspartic acid (D), glutamine (Q), serine (S) or asparagine (N); X 16 is asparagine (N), leucine (L), tryptophan (W), methionine (M) or aspartic acid (D); X 17 is glycine (G), serine (S) or aspartic acid (D), X 18 is any amino acid, such as serine (S) or threonine (T), X 19 is asparagine (N), valine (V), or aspartic acid (D), or is absent; X 20 is alanine (A) or is absent, X 21 is serine (S) or is absent, X 22 is any amino acid, such as alanine (A), or is absent, X 23 is proline (P), alanine (A), arginine (R), threonine (T) or glycine (G), X 24 is leucine (L), methionine (M), valine (V) or isoleucine (I), X 25 is threonine (T), tyrosine (Y), asparagine (N), leucine (L) or glutamine (Q); X 26is alanine (A), cysteine ​​(C), phenylalanine (F) or tryptophan (W), X 27 is asparagine (N), alanine (A), threonine (T) or serine (S), X 28 is arginine (R) or lysine (K), X 29 is aspartic acid (D) or is absent, X 30 is threonine (T), alanine (A), proline (P), serine (S) or glycine (G); X 31 is any amino acid, such as alanine (A), X 32 is isoleucine (I), glutamic acid (E), proline (P), alanine (A) or valine (V), X 33 is glycine (G), lysine (K), or aspartic acid (D), or is absent; X 34 is glycine (G), serine (S), lysine (K), aspartic acid (D) or glutamine (Q), X 35 is glycine (G), alanine (A), aspartic acid (D) or cysteine ​​(C), X 38 is any amino acid, such as lysine (K)], or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO: 222 in that the amino acid sequence of the variant contains 1 to 6 distinct amino acid substitutions. and a polypeptide comprising at least two repeat regions selected from the group consisting of: and the polysaccharide scleroglucan.

[0122] In one embodiment of the present disclosure, the hydrogel comprises: A polypeptide comprising: [ka] Repeat region 1 (R1) comprising or consisting of the amino acid sequence or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO: 8 in that the amino acid sequence of the variant comprises 1 to 6 distinct amino acid substitutions. [ka] Repeat region 2 (R2) comprising or consisting of the amino acid sequence or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO: 10 in that the amino acid sequence of the variant comprises 1 to 6 distinct amino acid substitutions. [ka] Repeat region 3 (R3) comprising or consisting of the amino acid sequence or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO: 12 in that the amino acid sequence of the variant contains 1 to 6 distinct amino acid substitutions. and a polypeptide comprising at least two repeat regions selected from the group consisting of: and the polysaccharide scleroglucan.

[0123] In one embodiment of the present disclosure, the hydrogel comprises: A polypeptide comprising an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 3 or SEQ ID NO: 4, SEQ ID NO: 20 to SEQ ID NO: 33, or SEQ ID NO: 71 to SEQ ID NO: 219, and capable of binding one or more saccharide units; and polysaccharides comprising one or more β-1,3-glucan units having β-1,6 linked glucose modifications.

[0124] In one embodiment of the present disclosure, the hydrogel comprises: A polypeptide comprising an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 3 or SEQ ID NO: 4, SEQ ID NO: 20 to SEQ ID NO: 33, or SEQ ID NO: 71 to SEQ ID NO: 219, and capable of binding one or more saccharide units; and the polysaccharide scleroglucan.

[0125] In one embodiment of the present disclosure, the hydrogel comprises: A polypeptide comprising an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 3 or SEQ ID NO: 4, or SEQ ID NO: 20 to SEQ ID NO: 33, and capable of binding one or more saccharide units; and polysaccharides comprising one or more β-1,3-glucan units having β-1,6 linked glucose modifications.

[0126] In one embodiment of the present disclosure, the hydrogel comprises: A polypeptide comprising an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 3 or SEQ ID NO: 4, or SEQ ID NO: 20 to SEQ ID NO: 33, and capable of binding one or more saccharide units; and the polysaccharide scleroglucan.

[0127] In another aspect, the present disclosure relates to a crosslinker for crosslinking polysaccharides to form hydrogels, the crosslinker being a polypeptide as described herein.

[0128] In yet another aspect, the present disclosure provides a method of making a hydrogel, comprising: a. preparing a solution containing a polysaccharide; b. providing a solution comprising a polypeptide described herein; c. mixing the solution at room temperature with continuous stirring; The present invention relates to a method for obtaining a hydrogel through cross-linking of the polysaccharide.

[0129] In one embodiment of the present disclosure, the hydrogel comprises a polysaccharide as described herein. EXAMPLES

[0130] Example 1: Protein domains identified as part of the CBM family. A database search for CBM domain homologues was performed using BLASTP with the C. pinensis Cp-F1 protein as a query against a nonredundant protein sequence dataset from the Genbank database at the National Center for Biotechnology Information (NCBI) (http: / / www.ncbi.nlm.nih.gov). Sequences containing homologues of Cp-F1 were selected to generate a subset of CBM-containing proteins for further analysis. This subset was assessed using the NCBI taxonomy browser (http: / / www.ncbi.nlm.nih.gov / Taxonomy). Incomplete and redundant entries were removed. Furthermore, only one representative species was selected from each genus, resulting in a final dataset containing 164 sequences.

[0131] All identified carbohydrate binding protein modules are summarized in Table 1. The table includes, for all identified carbohydrate binding protein modules, the gene accession number, the species name, a short identifier, and a sequence identifier with reference to the attached ST.26 sequence listing. The identifier includes the abbreviation of the species name and the number of amino acids in the full-length multi-module protein that contains one of the carbohydrate binding domains. In the natural form, all these carbohydrate binding domains are part of a multi-module protein with carbohydrate degradation function. TIFF2024543518000013.tif242165TIFF2024543518000014.tif247165TIFF2024543518 000015.tif243165TIFF2024543518000016.tif249165TIFF2024543518000017.tif57165

[0132] Example 2: Recombinant production of CBM92 domain proteins of the present disclosure. Certain genes investigated in this study were synthesized in proprietary vectors by ThermoFisher GeneArt. These were then subcloned into the expression vector pET21a (ThermoFisher) with a C-terminal His6 tag and conferring ampicillin resistance (indicated as "commercially synthesized" in Table 2). Other genes were cloned in-house from C. pinensis genomic DNA (DSMZ, Germany) (indicated as "cloned from gDNA into pLATE31" in Table 2).

[0133] Recombinant proteins were generated following the cloning strategy described in Table 2. CBM domains were cloned from large multi-module genes. Thus, the nucleotide ranges specified refer to fragments cloned from the full-length gene. Sizes (kDa) refer to the resulting recombinant CBM proteins. gDNA = genomic DNA. TIFF2024543518000018.tif158165TIFF2024543518000019.tif53165

[0134] Results: Protein production and purification were analyzed by SDS-PAGE at each time. Protein yield was measured at each time using Bradford assay and / or Nanodrop. Successful production and purification of all constructs was confirmed by SDS-PAGE analysis (data not shown). Typical protein yield of Cp-F1 is 150 mg pure protein from 1 L culture of overexpressing E. coli cells. Other proteins were produced and purified with yields of 25-75 mg pure protein and up to 150 mg from 1 L culture.

[0135] Example 3: CBM92 domain proteins of the present disclosure can bind the polysaccharide scleroglucan. Twelve proteins identified as part of the CBM92 family (see Table 3) and five F1 variants in which tryptophan (W / Trp) in the binding site "WExF" motif was converted to alanine (A / Ala) by site-directed mutagenesis in one (α, β, or γ), two (β / γ), or three (α / β / γ) binding domains were tested for binding to water-insoluble polysaccharides by pull-down assays (Figure 7). Proteins and excess polysaccharide were incubated together in water at room temperature in a slowly rotating incubator. After 2-3 hours of incubation, samples were briefly centrifuged to pellet the insoluble polysaccharides. All proteins bound to the polysaccharides were pulled down in this pellet, whereas unbound proteins remained in the supernatant. Samples were taken from the supernatant and analyzed by SDS-PAGE. A different assay, affinity gel electrophoresis, was used to test binding to laminarin, a polysaccharide with a low molecular weight and therefore high solubility (see also Tomme et al. 2020). TIFF2024543518000020.tif120165

[0136] Results: The absence of protein bands on SDS-PAGE indicated that the proteins were successfully bound to polysaccharides (Table 4). Consistent binding affinity was shown to polysaccharides containing Glc-β-1,6-Glc linkages, namely pustulan (a linear β-1,6-glucan), as well as scleroglucan and yeast β-glucan, both of which consist of β-1,3-glucan chains containing a single substitution of β-1,6-linked glucosyl residues. Some of the CBM proteins tested also showed some binding to curdlan, a linear β-1,3-glucan, and in some cases, to lichenan, which contains β-1,3- and β-1,4-linked glucosyl residues. There was also weak binding to birch xylan in some cases, suggesting the possibility of relatively loose specificity in certain CBM proteins. Notably, Dm-F1, which naturally lacks all of the Trp residues at the binding site, did not bind to any of the polysaccharides tested (Figure 7). For all proteins tested, no binding was observed to cellulose, other types of xylans, various types of β-mannans, chitin, starch, chitosan, or mixed linkage β-1,3:1,4-glucans of plant origin.

[0137] It was also confirmed that all wild-type proteins that showed binding to scleroglucan were gel-forming. TIFF2024543518000021.tif174165

[0138] Example 4: A CMB92 domain protein of the present disclosure can bind three scleroglucan molecules. Sequence analysis of Cp-F1 (SEQ ID NO:3, SEQ ID NO:20) and Cp-F2 (SEQ ID NO:4, SEQ ID NO:21) and their homologs revealed three repeat regions (SEQ ID NO:8, 10 and 12 for Cp-F1, SEQ ID NO:9, 11 and 13 for Cp-F2), each of which contained one conserved tryptophan binding site. The same repeat regions were identified in other proteins of the CBM92 family (proteins corresponding to SEQ ID NO:22 to SEQ ID NO:33 in Table 1), which each had three repeat regions corresponding to the amino acid sequences of SEQ ID NO:34 to SEQ ID NO:69. Structural analysis of the protein by X-ray crystallography showed three obvious binding pockets, two of which could be crystallized with the glucose monosaccharide ligand. All three putative binding sites were explored and confirmed by site-directed mutagenesis. Rheological analysis of mutant variants of the Cp-F1 protein showed that all three binding sites must be functional for stable hydrogel formation to occur. For each mutation, tryptophan (W / Trp) in the binding site “WExF” motif was converted to alanine (A / Ala) by site-directed mutagenesis, and the experiment was performed at a final concentration of 4 g / L scleroglucan in the gel and a protein loading of 50% (i.e., final protein concentration of 2 g / L).

[0139] result: Figure 2 shows the superimposed crystal structures of Cp-F1 and Cp-F2. Glucose monosaccharide ligands are visible in two of the binding sites. Binding and gel formation were reduced in the single and double mutants and abolished in the triple mutant. This confirmed that three sites contribute to binding and thus three polysaccharide chains can be bound. See also Figure 3 (A and B) which shows a magnified image of the binding site in repeat region 1 of Cp-F2 showing some of the amino acids involved. Rheological data of the mutant variants of Cp-F1 revealed that CP-F1 variants variant no. 1 with Ala at the first binding site in the protein sequence (binding site α, SEQ ID NO: 5) and variant no. 3 with Ala at the third binding site in the protein sequence (binding site γ, SEQ ID NO: 7) showed only slight increase in polysaccharide viscosity, whereas variant no. 2 with Ala at the second binding site in the protein sequence (binding site β, SEQ ID NO: 6) and the triple mutant (SEQ ID NO: 19) were similar to the negative control without protein.

[0140] Example 5: CBM92 domain proteins of the present disclosure can crosslink scleroglucan to form hydrogels. Protein Cp-F1, at a starting stock concentration of 4 mg / ml in water (higher starting stock concentrations could also be used), was mixed with scleroglucan at a starting stock concentration of 5 g / L in water at room temperature. The two solutions were quickly mixed by adding the protein solution to the polysaccharide solution, which was continuously vortexed. A hydrogel formed spontaneously within a few seconds, reaching maximum stiffness after about 1 h. To vary the stiffness of the hydrogel, consistent stock concentrations of both scleroglucan (e.g., 5 mg / ml as stock solution) and protein (4–30 mg / ml as stock solutions) were used and these were mixed in different ratios. The following final protein concentrations were tested: 0.08 g / L, 0.2 g / L, 0.4 g / L, 0.8 g / L, 1.2 g / L, 1.6 g / L, and 2 g / L. This corresponds to a protein content of 2%, 5%, 10%, 20%, 30%, 40%, and 50% relative to the polysaccharide content, respectively (see FIG. 6A). Scleroglucan was at a final concentration of 4 g / L in all cases.

[0141] Alternatively, one would use a consistent scleroglucan concentration (e.g., 4 mg / ml in the final mixture and 5 mg / ml in the starting stock solution) and vary the protein concentration by pre-preparing protein stock solutions of different concentrations.

[0142] Example: 0.8 ml of scleroglucan (5 mg / ml solution in water) was mixed with 0.2 ml of F1 protein (10 mg / ml solution in water) to produce 1 ml of hydrogel with final concentrations of 4 mg / ml scleroglucan and 2 mg / ml F1 protein.

[0143] The rheological properties of the hydrogels were measured at 25° C. using a rheometer in cone-plate configuration. For each gel, multiple concentrations and ratios of protein and polysaccharide were measured and compared in each case to the rheological measurements of "polysaccharide without protein." For each rheological experiment, both the shear viscosity and the dynamic shear properties, given by the storage modulus G' and loss modulus G", were determined.

[0144] Results: When Cp-F1 or Cp-F2 was mixed with scleroglucan, hydrogels formed spontaneously within minutes and reached near maximum stiffness in about 1 h (Figure 4). The storage modulus increased linearly with increasing protein amount, supporting the protein function being involved in gel stabilization (Figure 6A). The storage modulus of the hydrogels correlated linearly with increasing protein concentration (Figure 6B). The analysis was repeated with other CMB92 domain proteins (Table 2), and all wild-type (native) proteins that showed binding to scleroglucan also showed gel formation with scleroglucan. Notably, all other CMB92 domain proteins listed in Table 2 that have tryptophan (W) at each of the three binding sites in the three repeat regions formed reliable and stable hydrogels. Proteins that are missing one or more tryptophans (W) from one or more of the three binding sites in the three repeat regions may form unstable hydrogels. None of the Cp-F1 variants formed gels.

[0145] Increasing the protein:saccharide ratio increased the solidity of the gel and decreased its transparency (Figure 5), and also increased the strength of the gel, as shown in Figure 6A.

[0146] Overview of Arrays [SEQ ID NO: 1] The complete nucleic acid sequence encoding Cp-F1 [SEQ ID NO:2] The complete nucleic acid sequence encoding Cp-F2 [SEQ ID NO:3] Full sequence of Cp-F1: MASMTGGQQMGRGSVGKTIWLQGFNNKYVNSKNGQGAMWCDSDAPQAWELFTVVDAGNGKIALRGNNGMYVSSENGEQAITCNRPAIQGWEAFDWLETADGKVSLRGSNGLFISSENGAAAMTCTRPTASGWEAFGYSVVGNALEHHHHH [SEQ ID NO: 4] Full sequence of Cp-F2: MPIGKTIWLQGFNSKYVNSRNGQGAMWCDSDTPQAWELFTVIDAGNGKIALRGNNGLYVSSENGEQAMTCNRPAIDGWEVFDWISNSDGSVSLRGSNGMYVSSENGEQAITCNRPAIDGWERFNWAAATALTGHHHHHHG [SEQ ID NO: 5] Full sequence of Cp-F1 with the binding site of repeat region 1 knocked out: MASMTGGQQMGRGSVGKTIWLQGFNNKYVNSKNGQGAMWCDSDAPQAAELFTVVDAGNGKIALRGNNGMYVSSENGEQAITCNRPAIQGWEAFDWLETADGKVSLRGSNGLFISSENGAAAMTCTRPTASGWEAFGYSVVGNALEHHHHHH [SEQ ID NO: 6] Full sequence of Cp-F1 with the binding site of repeat region 2 knocked out: MASMTGGQQMGRGSVGKTIWLQGFNNKYVNSKNGQGAMWCDSDAPQAWELFTVVDAGNGKIALRGNNGMYVSSENGEQAITCNRPAIQGAEAFDWLETADGKVSLRGSNGLFISSENGAAAMTCTRPTASGWEAFGYSVVGNALEHHHHHH [SEQ ID NO: 7] The full sequence of Cp-F1 with the binding site of repeat region 3 knocked out: MASMTGGQQMGRGSVGKTIWLQGFNNKYVNSKNGQGAMWCDSDAPQAWELFTVVDAGNGKIALRGNNGMYVSSENGEQAITCNRPAIQGWEAFDWLETADGKVSLRGSNGLFISSENGAAAMTCTRPTASGAEAFGYSVVGNALEHHHHHH [ka] [SEQ ID NO: 14] Repeat region 1-consensus X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 WEX 32 F [SEQ ID NO: 15] Repeat region 2-consensus X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 WEX 32 F [SEQ ID NO: 16] Repeat region 3-consensus X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 WEX 32 F [SEQ ID NO: 17] Double mutant Cp-F1-W90AW132A in which the second and third key Trp are Ala MASMTGGQQMGRGSVGKTIWLQGFNNKYVNSKNGQGAMWCDSDAPQAWELFTVVDAGNGKIALRGNNGMYVSSENGEQAITCNRPAIQGAEAFDWLETADGKVSLRGSNGLFISSENGAAAMTCTRPTASGAEAFGYSVVGNALEHHHHHH [SEQ ID NO: 18] Double mutant Cp-F1-W48AW132A in which the first and third key Trp are changed to Ala MASMTGGQQMGRGSVGKTIWLQGFNNKYVNSKNGQGAMWCDSDAPQAAELFTVVDAGNGKIALRGNNGMYVSSENGEQAITCNRPAIQGWEAFDWLETADGKVSLRGSNGLFISSENGAAAMTCTRPTASGAEAFGYSVVGNALEHHHHHH [SEQ ID NO: 19] Triple mutant, Cp-F1-W48AW90AW132A MASMTGGQQMGRGSVGKTIWLQGFNNKYVNSKNGQGAMWCDSDAPQAAELFTVVDAGNGKIALRGNNGMYVSSENGEQAITCNRPAIQGAEAFDWLETADGKVSLRGSNGLFISSENGAAAMTCTRPTASGAEAFGYSVVGNALEHHHHHH [SEQ ID NO: 20] Full sequence of Cp-F1 (without N-terminal and C-terminal tags) VGKTIWLQGFNNKYVNSKNGQGAMWCDSDAPQAWELFTVVDAGNGKIALRGNNGMYVSSENGEQAITCNRPAIQGWEAFDWLETADGKVSLRGSNGLFISSENGAAAMTCTRPTASGWEAFGYSVVGNA [SEQ ID NO: 21] Full sequence of Cp-F2 without the C-terminal tag PIGKTIWLQGFNSKYVNSRNGQGAMWCDSDTPQAWELFTVIDAGNGKIALRGNNGLYVSSENGEQAMTCNRPAIDGWEVFDWISNSDGSVSLRGSNGMYVSSENGEQAITCNRPAIDGWERFNWAAATALT [SEQ ID NO: 22] The entire sequence of Aa-F1 IANRENISIRALSNNLYVSSENGLRPITCNRTKVDIWERFSIESVGGGRVAIKGSNGSYFSSEDGLKPMRCNRKKAEAWEEFTLEPLGGDLYAIKGNNGAYVSHNNGSVEGLTCNKSIVGDQEKFIIKDAASK [SEQ ID NO: 23] The entire sequence of Aa-F2 TTDAPIGKTISLRKTNGDKKYVTAEQTANNKQLIARAIAVQGWEKFSIETHPDGGIALKALSNANYVQVQGINVNAAAAKKDKLTQFIWKNKGNGKVALKSVSANKWIQASWSSDNAVLFAKGIEDKGWETFDWKIENTQK [SEQ ID NO: 24] The entire sequence of Cp-F3 TGPIGQSVTLKGFNNQYVSSENGTQAMNCNRPTASGWETFSVVDAGGGKIALLSQGKYVSSENGTQAMTCNRLTIGDWEKFDWVVNADGKISLRGSNGQYVSSENGTQPMNCNRATISGWEAFGVNQ [SEQ ID NO: 25] The entire sequence of Cp-F4 PVGQIITLRGSNNLYVSGENGTKAMTCERTAPQTWEQFSVWNAGPGKVNLRSMGKYVSSENGLQAITCNRTTAASYEAFDWISNADGTVSLRGNNGLYVSSENGAAAMTCTRPTIDGWEKFNFTIVGPA [SEQ ID NO: 26] The entire sequence of Cp-F5 GGTAPIGSTITIKGSNGLFASGENGAQAMTCNRPTAQAWEQFTVVDAGGGKVALRSQGMYVSSENGAQAMTCSRPTIQDWEKFNWIDNGDGTFSLRGNNGSYVSSENGTQAMTCNRPTIQGWEKFTR [SEQ ID NO: 27] The entire sequence of Cp-F6 IGQTVTIKGFNNQYVCSEGNTQPMICNRAVAQSWEQFTVVDAGGGRVALRNQGNYVCSENGTQAVNCNRASVGPWEQFEWISNSDGTISFRGNNGAYLSAEDGMARMTCTKTTIGAAEKFKINQ [SEQ ID NO: 28] The entire sequence of Cp-F7 VGSIIYLYHDTLLVCSENGTQAMNCNRTGLGPWEKFEVVDAGNNTVALKGNNGLYVKAGNPVFCTGTALDSSTCFNWISLSSNTVALQSGKGLYMSSENGTQAMNWNRTAIGGWETFRWGTTTAA [SEQ ID NO: 29] The entire sequence of Ea-F1 PSIDIDEGKIALKSVHGKYLSAQPDGRAEWNRNIASEWEYFHLEKRQGDKITLKGAHGMYVSAQPDGEVQINRQAAPPTGWEEFTVEDRGNNVICLKSIHWKYLSAQMDGTVQWNRDSAPKGGWEEFQIVRPGGPG [SEQ ID NO: 30] Full sequence of Mr-F1 SGAPIGSTITLKGFNNQYVSSENGTQAMNCNRPTASAWEQFLVVDAGAGKIALQSMGKYVSSENGTQAITCNRTTYGDWEKFDWVPTTDGKVTLRGNNGKFISSENGTQAMTCTRATASGWEAFGVNQ [SEQ ID NO: 31] Full sequence of Mr-F2 PAPPIGTVISLKGFNGKYVSGENGTQAMTCNRTVAGDWEHFTVLDAGNGKIYLRSMGKYVSSENGTQAITCNRTTPSDWEKFDWIVTTDGKITLRGNNGKFISSENGTQAMTCNRTTASGWEAFGLNQ [SEQ ID NO: 32] Full sequence of Mr-F3 TPPIGQTITLKGSNNNYVSSENGTQPMNCNRPTAGGWEQFTIVNAGSGKVALLNSGKYVSSENGTQAINCNRTSVGPWEQFDWVGNADGTVSFRGSNGKYISGENGTQAMTCNRATIGGWESFRVNQ [SEQ ID NO: 33] Complete sequence of Pc-F1 AVAAPVGQTIWLKACSTQQFVSADQNLGATAPLVANRATVQGWEQFQVVDAGGGTIALRATGSGLYVSADTNVGGQLTANRPTIQDWERFEWVELGNGSIGLKARSNGLYVSADLGRNASAPLYASRASIGGCWEAFTWGSVGG [ka] JPEG2024543518000024.jpg228159JPEG2024543518000025.jpg228159[SEQ ID NO:70] Complete sequence of Dm-F1 non-binding, non-gel-forming protein SYIGIGDNVWFEAYNGSFISSENGASPMTCNTFTVGETEVFTIVDAGDGKIALLGNNGKYVSSNNGTTSMTCTKDEIGETEKFYWINLSNGQMALLGKGGFVSMEGGSSPINANRNAIDGWEIYSWGKQDIQT [SEQ ID NO: 220] Repeat region 1-consensus X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 X 31 X 32 X 33 X 34 WEX 37 F [SEQ ID NO: 221] Repeat region 2-consensus X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 X 31 WEX 34 F [SEQ ID NO: 222] Repeat region 3-consensus X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 X 31 X 32 X 33 X 34 X 35 WEX 38 F

[0147] References Boraston AB, Bolam DN, Gilbert HJ, Davies GJ. Carbohydrate-binding modules: fine-tuning polysaccharide recognition. Biochem J. 2004;382(Pt 3):769-81. Coviello T, Grassi M, Rambone G, Santucci E, Carafa M, Murtas E, et al. Novel hydrogel system from scleroglucan: synthesis and characterization. Journal of Controlled Release. 1999;60(2):367-78 Coviello T, Grassi M, Rambone G, Alhaique F. A crosslinked system from Scleroglucan derivative: preparation and characterization. Biomaterials. 2001;22(13):1899-909 Coviello T, Grassi M, Palleschi A, Bocchinfuso G, Coluzzi G, Banishoeib F, et al. A new scleroglucan / borax hydrogel: swelling and drug release studies. International Journal of Pharmaceutics. 2005;289(1):97-107 Crescenzi V, Gamini A, Paradossi G, Torri G. Solution properties of a new polyelectrolyte derived from the polysaccharide scleroglucan. Carbohydr Pol. 1983;3(4):273-86 Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Henrissat B. The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res. 2014;42(Database issue):D490-5 Maeda H, Rambone G, Coviello T, Yuguchi Y, Urakawa H, Alhaique F, et al. Low-degree oxidized scleroglucan and its hydrogel. International Journal of Biological Macromolecules. 2001;28(5):351-8 Palleschi A, Bocchinfuso G, Coviello T, Alhaique F. Molecular dynamics investigations of the polysaccharide scleroglucan: first study on the triple helix structure. Carbohydr Res. 2005;340(13):2154-62 Palleschi A, Coviello T, Bocchinfuso G, Alhaique F. Investigation on a new scleroglucan / borax hydrogel: Structure and drug release. International Journal of Pharmaceutics. 2006;322(1):13-21 Schmid J, Meyer V, Sieber V. Scleroglucan: biosynthesis, production and application of a versatile hydrocolloid. Appl Microbiol Biotechnol. 2011;91(4):937-47 Tomme P., Boraston A., Kormos JM et al. Affinity electrophoresis for the identification and characterization of soluble sugar binding by carbohydrate-binding modules. Enzyme and Microbial Technology 2000:27(7):453-458.

[0148] Terms 1. A polypeptide comprising: [ka] Repeat region 1 (R1) comprising or consisting of the amino acid sequence or a functional variant thereof, wherein the amino acid sequence of said variant differs from SEQ ID NO:X in that the amino acid sequence of said variant comprises 1 to 6 distinct amino acid substitutions. [ka] Repeat region 2 (R2) comprising or consisting of the amino acid sequence or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO: 10 in that the amino acid sequence of the variant comprises 1 to 6 distinct amino acid substitutions. [ka] Repeat region 3 (R3) comprising or consisting of the amino acid sequence or a functional variant thereof, wherein the amino acid sequence of said variant differs from SEQ ID NO:XXX in that the amino acid sequence of said variant contains 1 to 6 distinct amino acid substitutions. The polypeptide comprising at least two repeat regions selected from the group consisting of:

[0149] 2. A polypeptide comprising: a. SEQ ID NO:14: X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 WEX 32 F Repeat region 1 (R1) comprising or consisting of the amino acid sequence [In the formula, X1 is isoleucine (I) or valine (V); X2 is any amino acid, such as tryptophan (W), serine (S), threonine (T) or alanine (A); X3 is leucine (L), isoleucine (I) or phenylalanine (F); X4 is glutamine (Q), arginine (R) or lysine (K); X5 is glycine (G), alanine (A), serine (S), asparagine (N) or lysine (K); X6 is any amino acid, such as alanine (A); X7 is asparagine (N), histidine (H), serine (S) or threonine (T); X8 is asparagine (N) or glycine (G); X9 is lysine (K), glutamine (Q) or leucine (L); X 10 is tyrosine (Y) or phenylalanine (F), X 11 is valine (V), leucine (L), isoleucine (I) or methionine (M), X 12 is serine (S), threonine (T), cysteine ​​(C) or asparagine (N), X 13 is alanine (A), serine (S) or glycine (G), X 14 is glutamic acid (E) or aspartic acid (D), X 15 is asparagine (N) or aspartic acid (D), X 16 is glycine (G), proline (P), asparagine (N) or valine (V), X 17 is any amino acid, such as threonine (T), X 18 is any amino acid, such as glycine (G), X 19 is proline (P), alanine (A) or arginine (R), X 20 is leucine (L), methionine (M), alanine (A), valine (V) or isoleucine (I), X 21 is any amino acid, such as threonine (T), X 22 is alanine (A), cysteine ​​(C) or tryptophan (W), X 23 is asparagine (N), aspartic acid (D), threonine (T) or serine (S); X 24 is arginine (R), alanine (A) or serine (S), X 25 is threonine (T), aspartic acid (D) or alanine (A), X 26 is threonine (T), alanine (A) or serine (S), X 27 is alanine (A), valine (V) or isoleucine (I), X 28 is glycine (G), glutamine (Q), serine (S) or aspartic acid (D), X 29 is any amino acid, such as glycine (G), X 32 is any amino acid, such as glutamine (Q) or lysine (K)]; or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO: 14 in that the amino acid sequence of the variant comprises 1 to 6 distinct amino acid substitutions. b. SEQ ID NO:15: X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 WEX 32 F Repeat region 2 (R2) comprising or consisting of the amino acid sequence [In the formula, X1 is isoleucine (I), valine (V) or phenylalanine (F); X2 is alanine (A), serine (S) or threonine (T); X3 is leucine (L) or phenylalanine (F); X4 is glutamine (Q), arginine (R) or lysine (K); X5 is glycine (G), alanine (A) or serine (S); X6 is any amino acid, such as alanine (A); X7 is asparagine (N), methionine (M), histidine (H) or threonine (T); X8 is glycine (G), asparagine (N) or aspartic acid (D); X9 is lysine (K), leucine (L), methionine (M) or glutamine (Q); X 10 is tyrosine (Y) or phenylalanine (F), X 11 is valine (V), leucine (L) or isoleucine (I), X 12 is serine (S), cysteine ​​(C) or threonine (T), X 13 is alanine (A) or serine (S), X 14 is glutamic acid (E), aspartic acid (D), glutamine (Q) or asparagine (N), X 15 is asparagine (N), proline (P), glycine (G), threonine (T), glutamine (Q) or aspartic acid (D); X 16 is glycine (G), aspartic acid (D), alanine (A), asparagine (N), threonine (T) or arginine (R); X 17is glycine (G), threonine (T), leucine (L), alanine (A), valine (V) or glutamine (Q), X 18 is any amino acid, such as serine (S), X 19 is proline (P), alanine (A), glutamine (Q) or lysine (K), X 20 is leucine (L), isoleucine (I), methionine (M) or valine (V); X 21 is threonine (T), isoleucine (I), asparagine (N), valine (V) or alanine (A); X 22 is alanine (A), cysteine ​​(C) or tryptophan (W), X 23 is asparagine (N), aspartic acid (D), threonine (T) or serine (S); X 24 is alanine (A), cysteine ​​(C) or arginine (R), X 25 is threonine (T), alanine (A), asparagine (N), proline (P) or aspartic acid (D), X 26 is any amino acid, such as alanine (A), X 27 is isoleucine (I), valine (V), proline (P), alanine (A), or leucine (L); X 28 is glycine (G), serine (S), glutamine (Q), or aspartic acid (D), X 29 is glycine (G), alanine (A), aspartic acid (D), glutamic acid (E), proline (P) or arginine (R); X 32 is any amino acid, such as lysine (K)], or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO: 15 in that the amino acid sequence of the variant comprises 1 to 6 distinct amino acid substitutions. c. SEQ ID NO:16: X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 WEX 32 F Repeat region 3 (R3) comprising or consisting of the amino acid sequence [In the formula, X1 is valine (V), isoleucine (I), tyrosine (Y), phenylalanine (F) or glutamic acid (E); X2 is alanine (A), serine (S), cysteine ​​(C) or glycine (G); X3 is leucine (L), phenylalanine (F) or isoleucine (I); X4 is arginine (R), lysine (K), glutamine (Q), leucine (L) or methionine (M); X5 is glycine (G), alanine (A) or serine (S); X6 is any amino acid, such as asparagine (N); X7 is asparagine (N), histidine (H) or threonine (T); X8 is glycine (G) or asparagine (N); X9 is lysine (K), leucine (L), arginine (R), glutamine (Q) or asparagine (N); X10 is tyrosine (Y) or phenylalanine (F), X 11 is valine (V), leucine (L) or isoleucine (I), X 12 is serine (S), threonine (T), cysteine ​​(C) or glutamine (Q), X 13 is alanine (A), serine (S), histidine (H), threonine (T), methionine (M) or glutamine (Q), X 14 is glutamic acid (E), aspartic acid (D), glutamine (Q) or asparagine (N), X 15 is asparagine (N), leucine (L) or aspartic acid (D), X 16 is glycine (G), asparagine (N), alanine (A), or aspartic acid (D); X 17 is any amino acid, such as glycine (G), X 18 is any amino acid, such as alanine (A), X 19 is proline (P), alanine (A), glutamine (Q) or glycine (G), X 20 is leucine (L), methionine (M), glutamine (Q), valine (V) or isoleucine (I), X 21 is threonine (T), isoleucine (I), asparagine (N), leucine (L), or methionine (M); X 22 is alanine (A), cysteine ​​(C) or arginine (R), X 23 is asparagine (N), aspartic acid (D), threonine (T) or serine (S); X 24 is arginine (R), histidine (H), alanine (A), serine (S) or lysine (K); X 25 is threonine (T), alanine (A), proline (P), serine (S) or aspartic acid (D); X 26 is any amino acid, such as alanine (A), X 27 is isoleucine (I), alanine (A) or valine (V), X 28 is glycine (G), serine (S) or glutamine (Q), X 29 is glycine (G), alanine (A), proline (P) or cysteine ​​(C), X 32 is any amino acid, such as lysine (K)], or a functional variant thereof, wherein the amino acid sequence of the variant differs from SEQ ID NO: 16 in that the amino acid sequence of the variant comprises 1 to 6 distinct amino acid substitutions. The polypeptide comprising at least two repeat regions selected from the group consisting of:

Claims

【Request Item 1】 【Chemistry 1】 or a functional variant thereof, wherein the amino acid sequence of said variant differs from SEQ ID NO: 8 in that it comprises 1 to 6 individual amino acid substitutions; 【Chemistry 2】 or a functional variant thereof, wherein the amino acid sequence of said variant differs from SEQ ID NO: 10 in that it comprises 1 to 6 individual amino acid substitutions; 【Transformation 3】 or a functional variant thereof, wherein the amino acid sequence of said variant differs from SEQ ID NO: 12 in that it comprises 1 to 6 individual amino acid substitutions. A hydrogel comprising at least one polypeptide comprising at least two repeat regions selected from the group consisting of: Each of the repeat region 1, the repeat region 2, and the repeat region 3 can bind at least one polysaccharide, and the hydrogel has a cross-linked structure. Hydrogel.

2. a. SEQ ID NO: 220: X １ X ２ X ３ X ４ X ５ X ６ X ７ X ８ X ９ X １０ X １１ X １２ X １３ X １４ X １５ X １６ X １７ X １８ X １９ X ２０ X ２１ X ２２ X ２３ X ２４ X ２５ X ２６ X ２７ X ２８ X ２９ X ３０ X ３１ X ３２ X ３３ X ３４ WEX ３７ F The amino acid sequence of [In the formula, X １ is isoleucine (I) or valine (V), X ２ is any amino acid, X ３ is leucine (L) or isoleucine (I), X ４ is lysine (K) or arginine (R) or is absent, X ５ is glutamine (Q), arginine (R), alanine (A), or lysine (K), or is absent; X ６ is glycine (G), serine (S), tyrosine (Y), cysteine ​​(C), leucine (L) or threonine (T), X ７ is any amino acid, X ８ is glycine (G) or is absent, X ９ is asparagine (N), histidine (H), aspartic acid (D) or threonine (T), X １０ is asparagine (N), serine (S), threonine (T), glutamine (Q), lysine (K) or glycine (G), X １１ is lysine (K), glutamine (Q), asparagine (N) or leucine (L), X １２ is tyrosine (Y), leucine (L) or phenylalanine (F), X １３ is valine (V), leucine (L) or alanine (A), X １４ is serine (S), threonine (T), cysteine ​​(C) or asparagine (N), X １５ is alanine (A), serine (S) or glycine (G), X １６ is glutamic acid (E), lysine (K), arginine (R), glutamine (Q) or aspartic acid (D), X １７ is asparagine (N), glycine (G), glutamine (Q) or proline (P), X １８ is glycine (G), asparagine (N), threonine (T) or aspartic acid (D), X １９ is leucine (L) or is absent, X ２０ is glycine (G) or alanine (A) or is absent, X ２１ is alanine (A) or asparagine (N) or is absent, X ２２ is any amino acid or is absent, X ２３ is any amino acid, X ２４ is proline (P), alanine (A), glutamine (Q) or arginine (R), X ２５ is leucine (L), methionine (M), alanine (A) or isoleucine (I), X ２６ is any amino acid, X ２７ is alanine (A), cysteine ​​(C) or tryptophan (W), X ２８ is asparagine (N), aspartic acid (D), glutamic acid (E) or arginine (R), X ２９ is arginine (R), alanine (A) or serine (S), X ３０ is threonine (T), aspartic acid (D), proline (P), isoleucine (I), asparagine (N) or alanine (A), X ３１ is threonine (T), alanine (A), valine (V), glycine (G), lysine (K) or isoleucine (I), X ３２ is alanine (A), valine (V), proline (P) or leucine (L), X ３３ is glycine (G), glutamine (Q), serine (S) or aspartic acid (D), X ３４ is any amino acid, X ３７ is any amino acid], or a functional variant thereof, wherein the amino acid sequence of said variant differs from SEQ ID NO: 220 in that it comprises 1 to 6 individual amino acid substitutions; b. SEQ ID NO: 221: X １ X ２ X ３ X ４ X ５ X ６ X ７ X ８ X ９ X １０ X １１ X １２ X １３ X １４ X １５ X １６ X １７ X １８ X １９ X ２０ X ２１ X ２２ X ２３ X ２４ X ２５ X ２６ X ２７ X ２８ X ２９ X ３０ X ３１ WEX ３４ F The amino acid sequence of [In the formula, X １ is isoleucine (I) or valine (V), X ２ is alanine (A), asparagine (N), tyrosine (Y) or threonine (T), X ３ is leucine (L) or isoleucine (I), X ４ is glutamine (Q), arginine (R), leucine (L) or lysine (K), X ５ is glycine (G), alanine (A), asparagine (N) or serine (S), X ６ is any amino acid or is absent, X ７ is glycine (G) or serine (S) or is absent, X ８ is asparagine (N), methionine (M), glutamine (Q), histidine (H) or serine (S), X ９ is glycine (G) or alanine (A), X １０ is lysine (K), leucine (L), methionine (M), asparagine (N) or serine (S), X １１ is tyrosine (Y), X １２ is valine (V) or phenylalanine (F), X １３ is serine (S), cysteine ​​(C), lysine (K) or glutamine (Q), X １４ is alanine (A), valine (V) or serine (S), X １５ is glutamic acid (E), aspartic acid (D), glutamine (Q) or glycine (G), X １６ is asparagine (N), proline (P), glycine (G), threonine (T) or aspartic acid (D), X １７ is glycine (G), aspartic acid (D), isoleucine (I), or asparagine (N), or is absent; X １８ is valine (V) or glycine (G) or is absent, X １９ is glycine (G), threonine (T), leucine (L), alanine (A), or glutamic acid (E), or is absent; X ２０ is any amino acid or is absent, X ２１ is proline (P), alanine (A), glutamine (Q) or asparagine (N), X ２２ is leucine (L), isoleucine (I), methionine (M) or valine (V), X ２３ is threonine (T), asparagine (N), phenylalanine (F) or arginine (R), X ２４ is alanine (A), cysteine ​​(C) or arginine (R), X ２５ is asparagine (N), alanine (A), threonine (T) or glutamine (Q), X ２６ is alanine (A), glycine (G) or arginine (R), X ２７ is threonine (T), alanine (A), lysine (K), proline (P) or leucine (L), X ２８ is any amino acid, X ２９ is isoleucine (I), valine (V), proline (P), alanine (A), lysine (K), tyrosine (Y) or leucine (L), X ３０ is glycine (G), alanine (A), glutamic acid (E), threonine (T), serine (S), glutamine (Q) or aspartic acid (D), X ３１ is glycine (G), alanine (A), aspartic acid (D), serine (S), proline (P) or lysine (K), X ３４ is any amino acid], or a functional variant thereof, wherein the amino acid sequence of said variant differs from SEQ ID NO: 221 in that it comprises 1 to 6 individual amino acid substitutions; c. SEQ ID NO: 222: X １ X ２ X ３ X ４ X ５ X ６ X ７ X ８ X ９ X １０ X １１ X １２ X １３ X １４ X １５ X １６ X １７ X １８ X １９ X ２０ X ２１ X ２２ X ２３ X ２４ X ２５ X ２６ X ２７ X ２８ X ２９ X ３０ X ３１ X ３２ X ３３ X ３４ X ３５ WEX ３８ F The amino acid sequence of [In the formula, X １ is valine (V), isoleucine (I), tyrosine (Y) or phenylalanine (F), X ２ is alanine (A), serine (S), cysteine ​​(C), threonine (T) or glycine (G), X ３ is leucine (L), phenylalanine (F) or isoleucine (I), X ４ is arginine (R), lysine (K) or glutamine (Q), X ５ is glycine (G), alanine (A) or serine (S), X ６ is arginine (R) or valine (V) or is absent; X ７ is any amino acid, X ８ is asparagine (N), histidine (H), alanine (A), lysine (K), or is absent; X ９ is glycine (G), tryptophan (W) or asparagine (N), X １０ is lysine (K), leucine (L), methionine (M), serine (S), alanine (A), or glutamine (Q), X １１ is tyrosine (Y), tryptophan (W) or phenylalanine (F), X １２ is valine (V), leucine (L), methionine (M) or isoleucine (I), X １３ is serine (S) or glutamine (Q), X １４ is alanine (A), serine (S), histidine (H) or glycine (G), X １５ is glutamic acid (E), aspartic acid (D), glutamine (Q), serine (S) or asparagine (N), X １６ is asparagine (N), leucine (L), tryptophan (W), methionine (M) or aspartic acid (D), X １７ is glycine (G), serine (S) or aspartic acid (D), X １８ is any amino acid, X １９ is asparagine (N), valine (V), or aspartic acid (D), or is absent; X ２０ is alanine (A) or is absent, X ２１ is serine (S) or is absent, X ２２ is any amino acid or is absent, X ２３ is proline (P), alanine (A), arginine (R), threonine (T) or glycine (G), X ２４ is leucine (L), methionine (M), valine (V) or isoleucine (I), X ２５ is threonine (T), tyrosine (Y), asparagine (N), leucine (L) or glutamine (Q), X ２６ is alanine (A), cysteine ​​(C), phenylalanine (F) or tryptophan (W), X ２７ is asparagine (N), alanine (A), threonine (T) or serine (S), X ２８ is arginine (R) or lysine (K), X ２９ is aspartic acid (D) or is absent, X ３０ is threonine (T), alanine (A), proline (P), serine (S) or glycine (G), X ３１ is any amino acid, X ３２ is isoleucine (I), glutamic acid (E), proline (P), alanine (A) or valine (V), X ３３ is glycine (G), lysine (K), or aspartic acid (D), or is absent; X ３４ is glycine (G), serine (S), lysine (K), aspartic acid (D) or glutamine (Q), X ３５ is glycine (G), alanine (A), aspartic acid (D) or cysteine ​​(C), X ３８ is any amino acid], or a functional variant thereof, wherein the amino acid sequence of said variant differs from SEQ ID NO: 222 in that it comprises 1 to 6 individual amino acid substitutions. A hydrogel comprising at least one polypeptide comprising at least two repeat regions selected from the group consisting of: Each of the repeat region 1, the repeat region 2, and the repeat region 3 can bind at least one polysaccharide, and the hydrogel has a cross-linked structure. Hydrogel.

3. a. SEQ ID NO: 220: X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 X13 X14 X15 X16 X17 X18 X19 X20 X21 X22 X23 X24 X25 X 26 X 27 X 28 X 29 X 30 X 31 X 32 X 33 X 34 WEX 37 F The amino acid sequence of [In the formula, X 1 is isoleucine (I) or valine (V); X2 is tryptophan (W), serine (S), threonine (T) or alanine (A); X 3 is leucine (L) or isoleucine (I); X 4 is lysine (K) or arginine (R) or is absent; X 5 is glutamine (Q), arginine (R), alanine (A), or lysine (K), or is absent; X 6 is glycine (G), serine (S), tyrosine (Y), cysteine ​​(C), leucine (L) or threonine (T); X 7 is phenylalanine (F) or serine (S); X 8 is glycine (G) or is absent; X 9 is asparagine (N), histidine (H), aspartic acid (D) or threonine (T); X 10 is asparagine (N), serine (S), threonine (T), glutamine (Q), lysine (K) or glycine (G); X 11 is lysine (K), glutamine (Q), asparagine (N) or leucine (L); X 12 is tyrosine (Y), leucine (L) or phenylalanine (F); X 13 is valine (V), leucine (L) or alanine (A); X 14 is serine (S), threonine (T), cysteine ​​(C) or asparagine (N); X 15 is alanine (A), serine (S) or glycine (G); X 16 is glutamic acid (E), lysine (K), arginine (R), glutamine (Q) or aspartic acid (D); X 17 is asparagine (N), glycine (G), glutamine (Q) or proline (P); X 18 is glycine (G), asparagine (N), threonine (T) or aspartic acid (D); X 19 is leucine (L) or is absent; X20 is glycine (G) or alanine (A) or is absent; X 21 is alanine (A) or asparagine (N), or is absent; X22 is threonine (T) or is absent; X 23 is glycine (G); X24 is proline (P), alanine (A), glutamine (Q) or arginine (R); X 25 is leucine (L), methionine (M), alanine (A) or isoleucine (I); X 26 is threonine (T); X 27 is alanine (A), cysteine ​​(C) or tryptophan (W); X 28 is asparagine (N), aspartic acid (D), glutamic acid (E) or arginine (R); X 29 is arginine (R), alanine (A) or serine (S); X 30 is threonine (T), aspartic acid (D), proline (P), isoleucine (I), asparagine (N) or alanine (A); X 31 is threonine (T), alanine (A), valine (V), glycine (G), lysine (K) or isoleucine (I); X 32 is alanine (A), valine (V), proline (P) or leucine (L); X 33 is glycine (G), glutamine (Q), serine (S) or aspartic acid (D); X 34 is glycine (G); X 37 is glutamine (Q) or lysine (K)], or a functional variant thereof, wherein the amino acid sequence of said variant differs from SEQ ID NO: 220 in that it comprises 1 to 6 individual amino acid substitutions; b. SEQ ID NO: 221: X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 X13 X14 X15 X16 X17 X18 X19 X20 X21 X22 X23 X24 X25 X 26 X 27 X 28 X 29 X 30 X 31 WEX 34 F The amino acid sequence of [In the formula, X 1 is isoleucine (I) or valine (V); X2 is alanine (A), asparagine (N), tyrosine (Y) or threonine (T); X 3 is leucine (L) or isoleucine (I); X 4 is glutamine (Q), arginine (R), leucine (L) or lysine (K); X5 is glycine (G), alanine (A), asparagine (N) or serine (S); X 6 is alanine (A) or is absent; X 7 is glycine (G) or serine (S) or is absent; X 8 is asparagine (N), methionine (M), glutamine (Q), histidine (H) or serine (S); X 9 is glycine (G) or alanine (A); X 10 is lysine (K), leucine (L), methionine (M), asparagine (N) or serine (S); X 11 is tyrosine (Y); X 12 is valine (V) or phenylalanine (F); X 13 is serine (S), cysteine ​​(C), lysine (K) or glutamine (Q); X 14 is alanine (A), valine (V) or serine (S); X 15 is glutamic acid (E), aspartic acid (D), glutamine (Q) or glycine (G); X 16 is asparagine (N), proline (P), glycine (G), threonine (T) or aspartic acid (D); X 17 is glycine (G), aspartic acid (D), isoleucine (I), or asparagine (N), or is absent; X 18 is valine (V) or glycine (G) or is absent; X 19 is glycine (G), threonine (T), leucine (L), alanine (A), or glutamic acid (E), or is absent; X 20 is glutamine (Q) or is absent; X 21 is proline (P), alanine (A), glutamine (Q) or asparagine (N); X 22 is leucine (L), isoleucine (I), methionine (M) or valine (V); X 23 is threonine (T), asparagine (N), phenylalanine (F) or arginine (R); X24 is alanine (A), cysteine ​​(C) or arginine (R); X 25 is asparagine (N), alanine (A), threonine (T) or glutamine (Q); X 26 is alanine (A), glycine (G) or arginine (R); X 27 is threonine (T), alanine (A), lysine (K), proline (P) or leucine (L); X 28 is alanine (A); X 29 is isoleucine (I), valine (V), proline (P), alanine (A), lysine (K), tyrosine (Y) or leucine (L); X 30 is glycine (G), alanine (A), glutamic acid (E), threonine (T), serine (S), glutamine (Q) or aspartic acid (D); X 31 is glycine (G), alanine (A), aspartic acid (D), serine (S), proline (P) or lysine (K); X 34 is lysine (K)], or a functional variant thereof, wherein the amino acid sequence of said variant differs from SEQ ID NO: 221 in that it comprises 1 to 6 individual amino acid substitutions; c. SEQ ID NO: 222: X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 X13 X14 X15 X16 X17 X18 X19 X20 X21 X22 X23 X24 X25 X 26 X 27 X 28 X 29 X 30 X 31 X 32 X 33 X 34 X 35 WEX 38 F The amino acid sequence of [In the formula, X 1 is valine (V), isoleucine (I), tyrosine (Y) or phenylalanine (F); X2 is alanine (A), serine (S), cysteine ​​(C), threonine (T) or glycine (G); X 3 is leucine (L), phenylalanine (F) or isoleucine (I); X 4 is arginine (R), lysine (K) or glutamine (Q); X5 is glycine (G), alanine (A) or serine (S); X 6 is arginine (R) or valine (V) or is absent; X 7 is asparagine (N); X 8 is asparagine (N), histidine (H), alanine (A), lysine (K), or is absent; X 9 is glycine (G), tryptophan (W) or asparagine (N); X 10 is lysine (K), leucine (L), methionine (M), serine (S), alanine (A) or glutamine (Q); X 11 is tyrosine (Y), tryptophan (W) or phenylalanine (F); X 12 is valine (V), leucine (L), methionine (M) or isoleucine (I); X 13 is serine (S) or glutamine (Q); X 14 is alanine (A), serine (S), histidine (H) or glycine (G); X 15 is glutamic acid (E), aspartic acid (D), glutamine (Q), serine (S) or asparagine (N); X 16 is asparagine (N), leucine (L), tryptophan (W), methionine (M) or aspartic acid (D); X 17 is glycine (G), serine (S) or aspartic acid (D); X 18 is serine (S) or threonine (T); X 19 is asparagine (N), valine (V), or aspartic acid (D), or is absent; X20 is alanine (A) or is absent; X 21 is serine (S) or is absent; X22 is alanine (A) or is absent; X 23 is proline (P), alanine (A), arginine (R), threonine (T) or glycine (G); X 24 is leucine (L), methionine (M), valine (V) or isoleucine (I); X 25 is threonine (T), tyrosine (Y), asparagine (N), leucine (L) or glutamine (Q); X 26 is alanine (A), cysteine ​​(C), phenylalanine (F) or tryptophan (W); X 27 is asparagine (N), alanine (A), threonine (T) or serine (S); X 28 is arginine (R) or lysine (K); X 29 is aspartic acid (D) or is absent; X 30 is threonine (T), alanine (A), proline (P), serine (S) or glycine (G); X 31 is alanine (A); X 32 is isoleucine (I), glutamic acid (E), proline (P), alanine (A) or valine (V); X 33 is glycine (G), lysine (K), or aspartic acid (D), or is absent; X 34 is glycine (G), serine (S), lysine (K), aspartic acid (D) or glutamine (Q); X 35 is glycine (G), alanine (A), aspartic acid (D) or cysteine ​​(C); X 38 is lysine (K)], or a functional variant thereof, wherein the amino acid sequence of said variant differs from SEQ ID NO: 222 in that it comprises 1 to 6 individual amino acid substitutions. A hydrogel comprising at least one polypeptide comprising at least two repeat regions selected from the group consisting of: Each of the repeat region 1, the repeat region 2, and the repeat region 3 can bind at least one polysaccharide, and the hydrogel has a cross-linked structure. Hydrogel.

4. A hydrogel described in claim 1 or 2, wherein the polypeptide is a bifunctional or polyfunctional linker.

5. A hydrogel described in claim 1 or 2, wherein the polysaccharide contains at least one Glc-β-1,6-Glc unit and / or at least one Glc-β-1,2-Glc unit.

6. The hydrogel described in claim 4, wherein the polysaccharide contains one or more β-1,3-glucan units having β-1,6-linked glucose modifications.

7. A hydrogel described in claim 1 or 2, wherein the polysaccharide is scleroglucan.

8. A hydrogel described in claim 1 or 2, wherein the hydrogel contains at least 0.1 g / L of polypeptide.

9. A hydrogel as described in claim 1 or 2, wherein the hydrogel contains at least 2 g / L and at most 10 g / L of polysaccharides.

10. The hydrogel of claim 1 or 2, wherein the hydrogel contains at least 90% water by weight.

11. A hydrogel described in claim 1 or 2, wherein the ratio of polypeptide to polysaccharide is 1:20 to 1:

2.

12. The polypeptide of claim 1, a.

1. Repeat region 1 comprising the amino acid sequence of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67 or SEQ ID NO:220 or a functional variant thereof, wherein the amino acid sequence of said variant differs from SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67 or SEQ ID NO:220 in that the amino acid sequence of said variant comprises 1 to 6 individual amino acid substitutions, with the proviso that the residue at position 35 of said functional variant of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67, SEQ ID NO:220 is tryptophan (W). b. Repeat region 2 comprising the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO: 59, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 68 or SEQ ID NO: 221 or a functional variant thereof, wherein the amino acid sequence of the variant is the same as SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44 in that the amino acid sequence of the variant comprises 1 to 6 individual amino acid substitutions. , SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68 or SEQ ID NO:221, with the proviso that the residue at position 32 of said functional variant of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68, SEQ ID NO:221 is tryptophan (W); and / or c. Repeat region 3 comprising the amino acid sequence of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69 or SEQ ID NO:222 or a functional variant thereof, wherein the amino acid sequence of said variant is the same as SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69 or SEQ ID NO:222 in that the amino acid sequence of said variant comprises 1 to 6 individual amino acid substitutions. 45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69 or SEQ ID NO:222, with the proviso that the residue at position 36 of said functional variant of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66 or SEQ ID NO:69, SEQ ID NO:222 is tryptophan (W). The hydrogel of claim 1 or 2, comprising:

13. The polypeptide of claim 12, a. Repeat region 1 comprising the amino acid sequence of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67 or SEQ ID NO:220 or a functional variant thereof, wherein the amino acid sequence of said variant differs from SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67 or SEQ ID NO:220 in that the amino acid sequence of said variant comprises 1 to 6 individual amino acid substitutions; provided that the residue at position 35 of the functional variants of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67, and SEQ ID NO:220 is tryptophan (W); the residue at position 36 of said functional variants of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:220, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67 is glutamic acid (E); and / or Repeat region 1, provided that the residue at position 38 of said functional variants of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67, SEQ ID NO:220 is phenylalanine (F); b. Repeat region 2 comprising the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO: 59, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 68 or SEQ ID NO: 221 or a functional variant thereof, wherein the amino acid sequence of said variant differs from SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO: 59, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 68 or SEQ ID NO: 221 in that the amino acid sequence of said variant comprises 1 to 6 individual amino acid substitutions; provided that the residue at position 32 of the functional variants of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO: 59, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 68, and SEQ ID NO: 221 is tryptophan (W); the residue at position 33 of said functional variants of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68, SEQ ID NO:221 is glutamic acid (E); and / or Repeat region 2, provided that the residue at position 35 of said functional variants of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO: 59, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 68, SEQ ID NO: 221 is phenylalanine (F); and / or c. Repeat region 3 comprising the amino acid sequence of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69 or SEQ ID NO:222 or a functional variant thereof, wherein the amino acid sequence of said variant differs from SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69 or SEQ ID NO:222 in that the amino acid sequence of said variant comprises 1 to 6 individual amino acid substitutions; provided that the residue at position 36 of the functional variants of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 45, SEQ ID NO: 48, SEQ ID NO: 51, SEQ ID NO: 54, SEQ ID NO: 57, SEQ ID NO: 60, SEQ ID NO: 63, SEQ ID NO: 66, SEQ ID NO: 69, and SEQ ID NO: 222 is tryptophan (W); the residue at position 37 of said functional variants of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:222 is glutamic acid (E); and / or Repeat region 3 of the functional variants of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 45, SEQ ID NO: 48, SEQ ID NO: 51, SEQ ID NO: 54, SEQ ID NO: 57, SEQ ID NO: 60, SEQ ID NO: 63, SEQ ID NO: 66, SEQ ID NO: 69, SEQ ID NO: 222, provided that the residue at position 39 is phenylalanine (F). The hydrogel of claim 1 or 2, comprising:

14. The polypeptide of claim 1, a. Repeat region 1 comprising the amino acid sequence of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67 or SEQ ID NO:220 or a functional variant thereof, wherein the amino acid sequence of said variant differs from SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67 or SEQ ID NO:220 in that the amino acid sequence of said variant comprises 1 to 6 individual amino acid substitutions; with the proviso that the residue at position 12 of said functional variants of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67, SEQ ID NO:220 is tyrosine (Y); and / or Repeat region 1, provided that the residue at position 13 of said functional variants of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67, SEQ ID NO:220 is valine (V); b. Repeat region 2 comprising the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO: 59, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 68 or SEQ ID NO: 221 or a functional variant thereof, wherein the amino acid sequence of said variant differs from SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO: 59, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 68 or SEQ ID NO: 221 in that the amino acid sequence of said variant comprises 1 to 6 individual amino acid substitutions; with the proviso that the residue at position 11 of said functional variants of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO: 59, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 68, SEQ ID NO: 221 is tyrosine (Y); and / or Repeat region 2, provided that the residue at position 12 of said functional variants of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO: 59, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 68, SEQ ID NO: 221 is valine (V); and / or c. Repeat region 3 comprising the amino acid sequence of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:222 or a functional variant thereof, wherein the amino acid sequence of said variant differs from SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:222 in that the amino acid sequence of said variant comprises 1 to 6 individual amino acid substitutions; with the proviso that the residue at position 11 of said functional variants of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 45, SEQ ID NO: 48, SEQ ID NO: 51, SEQ ID NO: 54, SEQ ID NO: 57, SEQ ID NO: 60, SEQ ID NO: 63, SEQ ID NO: 66, SEQ ID NO: 69, SEQ ID NO: 222 is tyrosine (Y); and / or Repeat region 3 of the functional variants of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 45, SEQ ID NO: 48, SEQ ID NO: 51, SEQ ID NO: 54, SEQ ID NO: 57, SEQ ID NO: 60, SEQ ID NO: 63, SEQ ID NO: 66, SEQ ID NO: 69, SEQ ID NO: 222, provided that the residue at position 12 is valine (V). The hydrogel of claim 1 or 2, comprising:

15. The hydrogel of claim 1 or 2, wherein the polypeptide comprises repeat region 1, repeat region 2 and repeat region 3.

16. Each of repeat region 1, repeat region 2 and repeat region 3 is flanked at its N-terminus by at least three amino acid residues and at its C-terminus by at least three amino acid residues, and therefore Y Ｎ -R1-Y Ｃ Z Ｎ -R2-Z Ｃ J Ｎ -R3-J Ｃ where Y Ｎ , Y Ｃ , Z Ｎ , Z Ｃ , J Ｎ , and J Ｃ 16. The hydrogel of claim 15, wherein each of the amino acid sequences individually comprises at least three amino acids.

17. 3. Use of at least one polypeptide for cross-linking two or more polysaccharides to produce a hydrogel according to claim 1 or 2, wherein said polypeptide is as defined in claim 1 or 2.

18. A method for producing the hydrogel of claim 1 or 2, comprising: a. providing a solution comprising a polysaccharide; b. Providing a solution comprising a polypeptide as defined in claim 1 or 2; c. Mixing the solution at room temperature with continuous stirring. Including, Thereby, the hydrogel is obtained.

Images