INCORPORATION BY REFERENCE

[0001] A PCT Request Form is filed concurrently with this specification as part of the present application. Each application that the present application claims benefit of or priority to as identified in the concurrently filed PCT Request Form is incorporated by reference herein in their entireties and for all purposes.

FIELD OF THE DISCLOSURE

[0002] The present disclosure relates to bio-based compositions of biopolymers connected by carbohydrate binding domains. In particular embodiments, the binding domains are linking units, which in turn interact with the biopolymers within the composition.

BACKGROUND

[0003] Biological polymers (or biopolymers), such as cellulose and chitin, are abundant starting materials for use in material science. The polysaccharide cellulose is the most common biopolymer on earth. Although it is mostly found in plant biomass, it is also produced by animals, fungi and bacteria. Cellulose is a crystalline assembly of cellobiose subunits which are made from glucose. Due to its crystalline structure, cellulose has high tensile strength and elasticity approaching that of synthetic carbon fibers, and it has a very favorable strength/weight ratio compared to, for example, steel. In plant cell walls, cellulose is found as a composite with other polysaccharides such as hemicellulose, pectin, lignin, enzymes and structural proteins. These molecules link the cellulose microfibrils improving the mechanism of load transfer when the cell is subjected to mechanical stress. Tuning biopolymers by cross-linking them to improve their physical properties as biofibers would be desirable. Such cross-linked biopolymers could be beneficial components of consumer products as strengthened, glues, thickeners, absorbents or controlled release agents.

[0004] The background description provided herein is for the purposes of generally presenting the context of the disclosure. Work of the presently named inventors, to the extent it is described in this background section, as well as aspects of the description that may not otherwise qualify as prior art at the time of filing, are neither expressly nor impliedly admitted as prior art against the present disclosure. SUMMARY

[0005] The present disclosure relates to bio-based compositions of biopolymers joined together using binding domains which are connected to each other by polypeptide tethers. The bio-based compositions may include a plurality of biopolymers which are bound together by way of linkers composed of carbohydrate binding domains. The physical properties of the biopolymers so modified by joining them together may be advantageously enhanced.

[0006] Accordingly, in a first aspect, the present invention encompasses a composition. In some embodiments, the composition includes a plurality of biopolymers having at least one first biopolymer and at least one second biopolymer; and a linker having a first binding domain attached to a portion of the at least one first biopolymer, a second binding domain attached to a portion of the at least one second biopolymer, and a tether disposed between the first and second binding domains, wherein each of the first and second binding domains includes, independently, a carbohydrate binding domain and wherein the tether comprises a polypeptide.

[0007] In some embodiments, the at least one first biopolymer includes chitin, chitosan, cellulose, amorphous cellulose, crystalline cellulose, starch, dextran, glucan, alginate, hyaluronic acid or a mixture thereof.

[0008] In some embodiments, the at least one second biopolymer is chitin, chitosan, cellulose, amorphous cellulose, crystalline cellulose, starch, dextran, glucan, alginate, hyaluronic acid or a mixture thereof.

[0009] In some embodiments, the at least one first biopolymer and the at least one second biopolymer are the same biopolymer or different biopolymers and the first and second binding domains are the same binding domain or different binding domains.

[0010] In some embodiments, the first and second binding domains are the same binding domain.

[0011] In some embodiments, the binding domain is a cellulose binding domain or a chitin binding domain.

[0012] In some embodiments, the binding domain is a polypeptide sequence having at least 90% sequence identity to any one of the following SEQ ID NOs: 1-19, 30-43, 49- 60, and 70-104.

[0013] In some embodiments, the binding domain is a polypeptide sequence having at least 90% sequence identity to any one of the following SEQ ID NOs: SEQ ID NQ:20, in which X at each position of SEQ ID NO:20 is an amino acid present at a position in one of SEQ ID NOs:l-19 when optimally aligned with SEQ ID NO:20; or SEQ ID NO:21, in which X at each position of SEQ ID NO:21 is an amino acid present at a position in one of SEQ ID NOs:l-19 or one of SEQ ID NOs:l-3 and 6-11 when optimally aligned with SEQ ID NO:21; or SEQ ID NO:44, in which X at each position of SEQ ID NO:44 is an amino acid present at a position in one of SEQ ID NOs:30-43 when optimally aligned with SEQ ID NO:44; or SEQ ID NO:45, in which X at each position of SEQ ID NO:45 is an amino acid present at a position in one of SEQ ID NOs:30-43 or one of SEQ ID NOs:30-34 when optimally aligned with SEQ ID NO:45; or SEQ ID NO:61, in which X at each position of SEQ ID NO:61 is an amino acid present at a position in one of SEQ ID N0s:50-60 when optimally aligned with SEQ ID NO:61; or SEQ ID NO:62, in which X at each position of SEQ ID NO:62 is an amino acid present at a position in one of SEQ ID N0s:50-60 or one of SEQ ID NOs:50-52, 54, and 56 when optimally aligned with SEQ ID NO:62; or SEQ ID NO: 105, in which X at each position of SEQ ID NO: 105 is an amino acid present at a position in one of SEQ ID N0s:70-104 or one of SEQ ID NOs:70-77, 82, 84, 86, 88, 89-91, and 104 when optimally aligned with SEQ ID NO: 105; or SEQ ID NO: 106, in which X at each position of SEQ ID NO: 106 is an amino acid present at a position in one of SEQ ID N0s:70-104 or one of SEQ ID NOs:70-72, 82, 86, 88, and 104 when optimally aligned with SEQ ID NO:106.

[0014] In some embodiments, the tether is a polypeptide sequence having at least 90% sequence identity to any one of the following SEQ ID NOs:120-126.

[0015] In some embodiments, the first binding domain is attached to a portion of the first biopolymer by hydrogen bonding, and wherein the second binding domain is attached to a portion of the second biopolymer by hydrogen bonding.

[0016] In some embodiments, the composition is a textile, a formulation, a food product, a cosmetic product, a consumer product, a packaging material, an absorbent article, a film, a barrier material, an adhesive, a biodegradable product, a gel, a capsule, a pharmaceutical composition, a nanocomposite, a matrix, a laminate, or a surgical product.

[0017] In a second aspect, the present disclosure encompasses a biopolymer linker. In some embodiments, a structure of formula wherein BD ¹ is a first binding domain; BD ² is a second binding domain; BD ^m is a third binding domain; T is a first tether; T ^m is a second tether; and n is 0, 1, 2, 3, 4, 5, or more.

[0018] In some embodiments, the first tether and the second tether each are independently a polypeptide.

[0019] In some embodiments, the first tether has a polypeptide sequence having at least 90% sequence identity to any one of the following SEQ ID NOs: 120-126.

[0020] In some embodiments, the first, second and third binding domains each independently have a polypeptide sequence having at least 90% sequence identity to any one of the following SEQ ID NOs: 1-19, 30-43, 49-60, and 70-104.

[0021] In some embodiments, the first, second and third binding domains each independently have a polypeptide sequence having at least 90% sequence identity to any one of the following SEQ ID NOs: SEQ ID NO:20, in which X at each position of SEQ ID NO:20 is an amino acid present at a position in one of SEQ ID NOs: 1-19 when optimally aligned with SEQ ID NO:20; or

SEQ ID NO:21, in which X at each position of SEQ ID NO:21 is an amino acid present at a position in one of SEQ ID NOs:l-19 or one of SEQ ID NOs:l-3 and 6-11 when optimally aligned with SEQ ID NO:21; or SEQ ID NO:44, in which X at each position of SEQ ID NO:44 is an amino acid present at a position in one of SEQ ID NOs:30-43 when optimally aligned with SEQ ID NO:44; or SEQ ID NO:45, in which X at each position of SEQ ID NO:45 is an amino acid present at a position in one of SEQ ID NOs:30-43 or one of SEQ ID NOs:30-34 when optimally aligned with SEQ ID NO:45; or SEQ ID NO:61, in which X at each position of SEQ ID NO:61 is an amino acid present at a position in one of SEQ ID N0s:50-60 when optimally aligned with SEQ ID NO:61; or SEQ ID NO:62, in which X at each position of SEQ ID NO:62 is an amino acid present at a position in one of SEQ ID NQs:50-60 or one of SEQ ID NQs:50-52, 54, and 56 when optimally aligned with SEQ ID NO:62; or SEQ ID NO: 105, in which X at each position of SEQ ID NO: 105 is an amino acid present at a position in one of SEQ ID N0s:70-104 or one of SEQ ID NOs:70-77, 82, 84, 86, 88, 89-91, and 104 when optimally aligned with SEQ ID NO: 105; or SEQ ID NO: 106, in which X at each position of SEQ ID NO: 106 is an amino acid present at a position in one of SEQ ID N0s:70-104 or one of SEQ ID NOs:70-72, 82, 86, 88, and 104 when optimally aligned with SEQ ID NO: 106.

[0022] In some embodiments, the biopolymer linker has a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 300 or SEQ ID NO: 301.

[0023] In some embodiments, the biopolymer linker includes an adhesive, a thickener, an additive, an absorbent, a coating, or a filler.

[0024] In some embodiments, the biopolymer linker is configured to be biodegradable or bio-releasable.

[0025] In some embodiments, the biopolymer linker is configured to be degraded or to be released in the presence of an enzyme.

[0026] In a third aspect, the present invention encompasses a method of making a bio-based composition. In some embodiments, the method includes providing a linker in the presence of a plurality of biopolymers, the plurality of biopolymers including at least one first biopolymer and at least one second biopolymer, to form a bio-based composition; and optionally exposing the composition to an enzyme, thereby degrading the linker and/or the biopolymers; wherein the linker includes a first binding domain, a second binding domain and a tether disposed between the first and second binding domains.

[0027] In some embodiments, the plurality of biopolymers is an amorphous nanocellulose or a crystalline nanocellulose.

[0028] In some embodiments, providing is mixing the linker with the plurality of biopolymers or applying the linker to a surface comprising the plurality of biopolymers. [0029] In some embodiments, the method also includes casting, spinning, laminating, molding, or weaving the composition to form an article.

[0030] In some embodiments, the article is a textile, a formulation, a food product, a cosmetic product, a consumer product, a packaging material, an absorbent article, a film, a barrier material, an adhesive, a biodegradable product, a gel, a capsule, a pharmaceutical composition, a nanocomposite, a foam, or a surgical product.

[0031] In some embodiments, each of the first and second binding domains is, independently, a carbohydrate binding domain and the tether is a polypeptide.

[0032] In some embodiments, the linker is a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 300 or SEQ ID NO: 301. [0033] In some embodiments, the at least one first biopolymer is chitin, chitosan, cellulose, amorphous cellulose, crystalline cellulose, starch, dextran, glucan, alginate, hyaluronic acid or a mixture thereof.

[0034] In some embodiments, the at least one second biopolymer is chitin, chitosan, cellulose, amorphous cellulose, crystalline cellulose, starch, dextran, glucan, alginate, hyaluronic acid or a mixture thereof.

[0035] In some embodiments, the at least one first biopolymer and the at least one second biopolymer are the same biopolymer or different biopolymers and the first and second binding domains are the same binding domain or different binding domains. [0036] In some embodiments, the first and second binding domains are the same binding domain.

[0037] In some embodiments, the binding domain includes a cellulose binding domain or a chitin binding domain.

[0038] In some embodiments, the binding domain has a polypeptide sequence having at least 90% sequence identity to any one of the following SEQ ID NOs: 1-19, 30-43, 49- 60, and 70-104.

[0039] In some embodiments, the binding domain includes a polypeptide sequence having at least 90% sequence identity to any one of the following SEQ ID NOs: SEQ ID NO:20, in which X at each position of SEQ ID NO:20 is an amino acid present at a position in one of SEQ ID NOs: 1-19 when optimally aligned with SEQ ID NO:20; or SEQ ID NO:21, in which X at each position of SEQ ID NO:21 is an amino acid present at a position in one of SEQ ID NOs:l-19 or one of SEQ ID NOs:l-3 and 6-11 when optimally aligned with SEQ ID NO:21; or SEQ ID NO:44, in which X at each position of SEQ ID NO:44 is an amino acid present at a position in one of SEQ ID NOs:3Q-43 when optimally aligned with SEQ ID NO:44; or SEQ ID NO:45, in which X at each position of SEQ ID NO:45 is an amino acid present at a position in one of SEQ ID NOs:30-43 or one of SEQ ID NOs:30-34 when optimally aligned with SEQ ID NO:45; or SEQ ID NO:61, in which X at each position of SEQ ID NO:61 is an amino acid present at a position in one of SEQ ID NQs:50-60 when optimally aligned with SEQ ID NO:61; or SEQ ID NO:62, in which X at each position of SEQ ID NO:62 is an amino acid present at a position in one of SEQ ID N0s:50-60 or one of SEQ ID NOs:50-52, 54, and 56 when optimally aligned with SEQ ID NO:62; or SEQ ID NO: 105, in which X at each position of SEQ ID NO: 105 is an amino acid present at a position in one of SEQ ID N0s:70-104 or one of SEQ ID NOs:70-77, 82, 84, 86, 88, 89-91, and 104 when optimally aligned with SEQ ID NO: 105; or SEQ ID NO: 106, in which X at each position of SEQ ID NO: 106 is an amino acid present at a position in one of SEQ ID N0s:70-104 or one of SEQ ID NOs:70-72, 82, 86, 88, and 104 when optimally aligned with SEQ ID NO: 106.

[0040] In some embodiments, the tether has a polypeptide sequence having at least 90% sequence identity to any one of the following SEQ ID NOs:120-126.

[0041] In some embodiments, the first binding domain attaches to a portion of the first biopolymer by hydrogen bonding, and wherein the second binding domain attaches to a portion of the second biopolymer by hydrogen bonding.

[0042] These and other aspects are described further below with reference to the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0043] FIG. 1 shows an illustrative schematic of a non-limiting bio-based composition in accordance with certain disclosed embodiments.

[0044] FIGS. 2A-2D show amino acid sequences for binding domains in accordance with certain disclosed embodiments.

[0045] FIGS. 3A-3D show amino acid sequences for binding domains in accordance with certain disclosed embodiments.

[0046] FIGS. 4A-4C show amino acid sequences binding domains in accordance with certain disclosed embodiments.

[0047] FIGS. 5A-5E shows amino acid sequences for binding domains in accordance with certain disclosed embodiments.

[0048] FIG. 6A shows the results of S DS -PAGE separation of binding domain proteins based on size in accordance with certain disclosed embodiments.

[0049] FIGS. 6B-6E are graphical illustrations of fluorescence data for binding domain proteins in accordance with certain disclosed embodiments.

[0050] FIG. 7 is a graphical illustration of rheology data in accordance with certain disclosed embodiments.

[0051] FIG. 8 shows water contact angle measurements for films formed on a glass substrate from various mixtures including the linker proteins in accordance with certain disclosed embodiments.

[0052] FIG. 9A shows the amino acid sequence SEQ ID No. 300 representing purified binding domain protein Sl-v2. [0053] FIG. 9B shows the amino acid sequence SEQ ID No. 301 representing purified binding domain protein Sl-vl.

DETAILED DESCRIPTION

[0054] In the following description, numerous specific details are set forth to provide a thorough understanding of the presented embodiments. The disclosed embodiments may be practiced without some or all of these specific details. In other instances, well- known process operations have not been described in detail to not unnecessarily obscure the disclosed embodiments. While the disclosed embodiments will be described in conjunction with the specific embodiments, it will be understood that it is not intended to limit the disclosed embodiments.

Definitions

[0055] By ‘ ‘protein,” “peptide,” or “polypeptide,” as used interchangeably, is meant any chain of more than two amino acids, regardless of post-translational modification (e.g., glycosylation or phosphorylation), constituting all or part of a naturally occurring polypeptide or peptide, or constituting a non-naturally occurring polypeptide or peptide, which can include coded amino acids, non-coded amino acids, modified amino acids (e.g., chemically and/or biologically modified amino acids), and/or modified backbones. Non-limiting amino acids include glycine (Gly, G), alanine (Ala, A), valine (Vai, V), isoleucine (He, I), leucine (Leu, L), cysteine (Cys, C), methionine (Met, M), aspartic acid (Asp, D), glutamic acid (Glu, E), arginine (Arg, R), histidine (His, H), lysine (Lys, K), asparagine (Asn, N), glutamine (Gin, Q), serine (Ser, S), threonine (Thr, T), proline (Pro, P), phenylalanine (Phe, F), tyrosine (Tyr, Y), tryptophan (Trp, W), selenocysteine (Sec, U), and pyrrolysine (Pyl, O).

[0056] The term “modified,” as used in reference to amino acids, means an amino acid including one or more modifications, such as a post-translation modification (e.g., acetylation, methylation, phosphorylation, ubiquitination, SUMOylation, ribosylation, glycosylation, acylation, or isomerization), or including a non-natural amino acid.

[0057] The term “modified,” as used in reference to a protein, means a polypeptide sequence including one or more amino acid substitution, as compared to the reference sequence for the protein.

[0058] The term “fragment” is meant a portion of a nucleic acid or a polypeptide that is at least one nucleotide or one amino acid shorter than the reference sequence. This portion contains, preferably, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 1800 or more nucleotides; or 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 640 amino acids or more. In another example, any polypeptide fragment can include a stretch of at least about 5 (e.g., about 10, about 20, about 30, about 40, about 50, or about 100) amino acids that are at least about 40% (e.g., about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 87%, about 98%, about 99%, or about 100%) identical to any of the sequences described herein can be utilized in accordance with the invention. In certain embodiments, a polypeptide to be utilized in accordance with the invention includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations (e.g., one or more conservative amino acid substitutions, as described herein). In yet another example, any nucleic acid fragment can include a stretch of at least about 5 (e.g., about 7, about 8, about 10, about 12, about 14, about 18, about 20, about 24, about 28, about 30, or more) nucleotides that are at least about 40% (about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 87%, about 98%, about 99%, or about 100%) identical to any of the sequences described herein can be utilized in accordance with the invention.

[0059] The term “conservative amino acid substitution” refers to the interchangeability in proteins of amino acid residues having similar side chains (e.g., of similar size, charge, and/or polarity). For example, a group of amino acids having aliphatic side chains consists of glycine (Gly, G), alanine (Ala, A), valine (Vai, V), leucine (Leu, L), and isoleucine (He, I); a group of amino acids having aliphatic -hydroxyl side chains consists of serine (Ser, S) and threonine (Thr, T); a group of amino acids having amide containing side chains consisting of asparagine (Asn, N) and glutamine (Gin, Q); a group of amino acids having aromatic side chains consists of phenylalanine (Phe, F), tyrosine (Tyr, Y), and tryptophan (Trp, W); a group of amino acids having basic side chains consists of lysine (Lys, K), arginine (Arg, R), and histidine (His, H); a group of amino acids having acidic side chains consists of glutamic acid (Glu, E) and aspartic acid (Asp, D); a group of amino acids having sulfur containing side chains consists of cysteine (Cys, C) and methionine (Met, M); and a group having hydroxyl, sulfur, or selenium containing side chains consists of serine (Ser, S), threonine (Thr, T), cysteine (Cys, C), methionine (Met, M), and selenocysteine (Sec, U). Exemplary conservative amino acid substitution groups are valine-leucine-isoleucine (VLI), phenylalanine-tyrosine (FY), lysine-arginine (KR), alanine-valine (AV), glycine- serine (GS), glutamate-aspartate (ED), and asparagine-glutamine (NQ). The present disclosure encompasses any sequence having a conservative amino acid sequence of any polypeptide sequence described herein.

[0060] In some embodiments, the conservative amino acid substitution is a conservation between groups of strongly similar properties (e.g., roughly equivalent to scoring > 0.5 in the Gonnet PAM 250 matrix). In particular embodiments, such conservation groups are serine-threonine-alanine (STA), asparagine-glutamate- glutamine-lysine (NEQK), asparagine-histidine-glutamine-lysine (NHQK), asparagine- aspartate-glutamate-glutamine (NDEQ), glutamine -histidine-arginine-lysine (QHRK), methionine-isoleucine-leucine- valine (MILV) , methionine-isoleucine-leucine- phenylalanine (MILF), histidine-tryptophan (HY), and phenylalanine-tyrosine- tryptophan (FYW).

[0061] In some embodiments, the conservative amino acid substitution is a conservation between groups of weakly similar properties (e.g., roughly equivalent to scoring =< 0.5 and > 0 in the Gonnet PAM 250 matrix). In particular embodiments, such conservation groups are cysteine-serine-alanine (CSA), alanine-threonine-valine (ATV), serine-alanine-glycine (SAG), serine-threonine-asparagine-lysine (STNK), serine- threonine-proline- alanine (STPA), serine-glycine-asparagine-aspartate (SGND), serine- asparagine-aspartate-glutamate-glutamine-lysine (SNDEQK), asparagine-aspartate- glutamate-glutamine-histidine-lysine (NDEQHK), asparagine-glutamate-glutamine- histidine-arginine-lysine (NEQHRK), phenylalanine-v aline- leucine-isoleucine- methionine (FVLIM), and histidine-phenylalanine-tyrosine (HFY).

[0062] As used herein, when a polypeptide or nucleic acid sequence is referred to as having “at least X % sequence identity” to a reference sequence, it is meant that at least X percent of the amino acids or nucleotides in the polypeptide or nucleic acid are identical to those of the reference sequence when the sequences are optimally aligned. An optimal alignment of sequences can be determined in various ways that are within the skill in the art, for instance, the Smith Waterman alignment algorithm (see, e.g., Smith T F et al., J. Mol. Biol. 1981; 147:195-7) and BLAST (Basic Local Alignment Search Tool; see, e.g., Altschul S F et al., J. Mol. Biol. 1990; 215:403-10). These and other alignment algorithms are accessible using publicly available computer software such as “Best Fit” (see, e.g., Smith T F et al., Adv. Appl. Math. 1981; 2(4):482-9) as incorporated into GeneMatcher Plus™ (see, e.g., Schwarz and Dayhof, “Atlas of Protein Sequence and Structure,” ed. Dayhoff, M. O., pp. 353-358, 1979), BLAST, BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, T-COFFEE, MUSCLE, MAFFT, or Megalign (DNASTAR). In addition, those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve optimal alignment over the length of the sequences being compared. In general, for polypeptides, the length of comparison sequences can be at least five amino acids, preferably 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, or more amino acids, up to the entire length of the polypeptide. For nucleic acids, the length of comparison sequences can generally be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, or more nucleotides, up to the entire length of the nucleic acid molecule. It is understood that for the purposes of determining sequence identity when comparing a DNA sequence to an RNA sequence, a thymine nucleotide is equivalent to a uracil nucleotide.

[0063] By “substantial identity” or “substantially identical” is meant a polypeptide or nucleic acid sequence that has the same polypeptide or nucleic acid sequence, respectively, as a reference sequence, or has a specified percentage of amino acid residues or nucleotides, respectively, that are the same at the corresponding location within a reference sequence when the two sequences are optimally aligned. For example, an amino acid sequence that is “substantially identical” to a reference sequence has at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reference amino acid sequence. For polypeptides, the length of comparison sequences will generally be at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 90, 100, 150, 200, 250, 300, or 350 contiguous amino acids (e.g., a full-length sequence). For nucleic acids, the length of comparison sequences will generally be at least 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous nucleotides (e.g., the full-length nucleotide sequence).

Sequence identity may be measured using sequence analysis software on the default setting (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis., 53705). Such software may match similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications.

[0064] By “attaching,” “attachment,” or related word forms is meant any covalent or non-covalent bonding interaction between two components. Non-covalent bonding interactions include, without limitation, hydrogen bonding, ionic interactions, halogen bonding, electrostatic interactions, T bond interactions, hydrophobic interactions, inclusion complexes, clathration, van der Waals interactions, and combinations thereof. [0065] As used herein, the term “about” is understood to account for minor increases and/or decreases beyond a recited value, which changes do not significantly impact the desired function of the parameter beyond the recited value. In some cases, about encompasses +/- 10% of any recited value. As used herein, this term modifies any recited value, range of values, or endpoints of one or more ranges.

[0066] As used herein, the terms “top,” “bottom,” “upper,” “lower,” “above,” and “below” are used to provide a relative relationship between structures. The use of these terms does not indicate or require that a particular structure must be located at a particular location in the apparatus.

[0067] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having” or any other variation thereof, are intended to cover a nonexclusive inclusion. For example, a process, method, article, or apparatus that comprises a list of features is not necessarily limited only to those features but may include other features not expressly listed or inherent to such process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive -or and not to an exclusive-or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0068] It should be noted that ratios, concentrations, amounts, and other numerical data may be expressed herein in a range format. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a concentration range of “about 0.1% to about 5%” should be interpreted to include not only the explicitly recited concentration of about 0.1 wt. % to about 5 wt. %, but also include individual concentrations (e.g., 1%, 2%, 3%, and 4%) and the subranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within the indicated range. In an embodiment, the term “about” can include traditional rounding according to significant figures of the numerical value. In addition, the phrase “about ‘x’ to ‘y’” includes “about ‘x’ to about ‘y’”. [0069] This written description uses examples to disclose the embodiments, including the best mode, and also to enable those of ordinary skill in the art to make and use the invention. The patentable scope is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.

[0070] Note that not all of the activities described above in the general description or the examples are required, that a portion of a specific activity may not be required, and that one or more further activities may be performed in addition to those described. The order in which activities are listed is not necessarily the order in which they are performed.

[0071] In this specification, the concepts have been described with reference to specific embodiments. However, one of ordinary skill in the art appreciates that various modifications and changes can be made without departing from the scope of the invention as set forth in the claims below. Accordingly, the specification and figures are to be regarded in an illustrative rather than a restrictive sense, and all such modifications are intended to be included within the scope of invention.

[0072] Benefits, other advantages, and solutions to problems have been described herein with regard to specific embodiments. However, the benefits, advantages, solutions to problems, and any feature(s) that may cause any benefit, advantage, or solution to occur or become more pronounced are not to be construed as a critical, required, or essential feature of any or all the claims.

[0073] After reading the specification, skilled artisans will appreciate that certain features are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any sub-combination. Further, references to values stated in ranges include each and every value within that range.

[0074] The present disclosure relates to biobased compositions including biopolymers and linkers, as well as methods for making and using such compositions. In some instances, linkers are attached to the biopolymer, thereby forming a composite material. Using such linkers, crosslinks form between biopolymers, and properties of the compositions can be controlled. Crosslinking can include any useful attachment, including hydrogen bonding between a binding domain and a biopolymer.

[0075] To provide effective binding, the linker can include binding domains (e.g., carbohydrate binding domains). Such domains can be selected to attach to particular regions or types of biopolymer. For instance, the binding domain can bind to certain isoforms of a biopolymer (e.g., crystalline cellulose, as compared to amorphous cellulose) or certain types of biopolymer (e.g., cellulose, as compared to chitin). To form a composition including two different types of biopolymers (e.g., cellulose and chitin), the linker can include a first binding domain configured to attach to a portion of cellulose and a second binding domain configured to attach to a portion of chitin. As described herein, the linker can include two or more binding domains, in which the domains can be same or different.

[0076] FIG. 1 provides a non-limiting schematic of a biobased composition including a plurality of biopolymers, which includes at least one first biopolymer 121 and at least one second biopolymer 122. The first and second biopolymers can be the same or different. The composition can also include a linker 110, which in turn includes a first binding domain 111, a second binding domain 112, and a tether 113 disposed therein.

[0077] The first binding domain 111 binds the first biopolymer 121 and the second binding domain 122 binds the second biopolymer 122. In some embodiments, the binding domains bind to the biopolymers by hydrogen bonding.

Biopolymers

[0078] Biopolymers are naturally-occurring polymers such as proteins and carbohydrates. Examples of suitable biopolymers include, but are not limited to, collagens (including Types I, II, III, IV and V, human or non-human), recombinant collagens, denatured collagens (or gelatins), fibrin-fibrinogen, elastin, glycoproteins, alginate, chitosan, hyaluronic acid, chondroitin sulfates and glycosaminoglycans, as well as cell-interactive glycoproteins such as laminin, fibronectin or tenascin.

[0079] Non-limiting biopolymers can include cellulose (e.g., amorphous cellulose, crystalline cellulose, cellulose nanocrystals, nanocellulose, etc.), chitin (e.g., amorphous chitin, crystalline chitin, chitin nanocrystals, chitin flakes, etc.), and others. The biopolymer can have any useful form, such as an amorphous structure, crystalline structure, flakes, particles, etc. Furthermore, different allomorphs may be present, or a particular allomorph may be dominant (e.g., cellulose I).

[0080] Within the composition, a single type of biopolymer can be present. In another embodiment, two forms of a single type of biopolymer can be present (e.g., different isoforms or crystalline forms). In yet another embodiment, two different types of biopolymer can be present.

[0081] In some embodiments, the biopolymer can include chitin, chitosan, cellulose, amorphous cellulose, crystalline cellulose, starch, dextran, glucan, alginate, hyaluronic acid or a mixture thereof. In other embodiments, the biopolymer can include monosaccharides, disaccharides, glucose, xylose, xylulose, arabinose, maltose, mannose, galactose, soluble oligosaccharides, xylan, mannan, beta-glucans, mutan, cyclodextrins and mixtures thereof.

Linkers

[0082] Linkers are moieties which join, crosslink, glue or conjugate other substances together. In particular embodiment, the linker can include a plurality of binding domains (e.g., any described herein) with a tether disposed therebetween. In one embodiment, the linker includes a structure of formula (I): wherein:

BD ¹ is a first binding domain;

BD ² is a second binding domain;

BD ^m is a third binding domain; and n is 0, 1, 2, 3, 4, 5, or more.

In some embodiments, n=0 for the structure of formula (I).

In another embodiments, the linker includes a structure of formula (II):

BD ¹ - T - BD ² [ - T ^m - BD ^m ] _n (II), wherein:

BD ¹ is the first binding domain;

T is a tether;

BD ² is the second binding domain;

T ^m is a second tether;

BD ^m is a third binding domain; and n is 0, 1, 2, 3, 4, 5, or more. [0083] In formula (I), T constitutes a tether, which can be a bivalent, bivalent, tetravalent, or another n- valent (multi- valent) tether having an n number of attachment sites to bind to a binding domain. Each of BD ¹ , BD ² , and BD ^m can be the same or different. In some embodiments, the tether joins binding domains together. In some embodiments, the tether is selected based upon the amount of cross-linking desired. In some embodiments, the tether is selected based upon how tightly bound the biopolymers are in proximity to one another.

[0084] Non-limiting tethers include a polypeptide. Such polypeptide tethers can be flexible. Flexible sequences can include, e.g., (GGGGS) _n (SEQ ID NO: 120, in which n can be 1, 2, 3, 4, 5, or greater); (GGGS) _n (SEQ ID NO:121, in which n can be 1, 2, 3, 4, 5, or greater); G _n , in which n can be 4, 5, 6, 7, 8, 9, 10, or greater, etc.

[0085] In other instances, the polypeptide tether includes an alpha helix. Such polypeptide tethers can include, e.g., (EAAAK) _n (SEQ ID NO: 122, in which n can be 1, 2, 3, 4, 5, or greater); (EAAAAK) _n (SEQ ID NO:123, in which n can be 1, 2, 3, 4, 5, or greater), etc.

[0086] Yet other tethers can include any of the following: GPYGPGASAAAAAAGGYGPGSGQQGPGQQGPGQQGPGQQGPGQQ (SEQ ID NO:124, adf-3 (Araneus diadematus)' , UniProtKB Q16987, aa 341-384);

GVGVPGVGVPGVGVPGVGVP (SEQ ID NO: 125, elastin); and PGVGVAPGVGVAPGVGVAPGVG (SEQ ID NO: 126, eln (Homo sapiens), UniProtKB P15502, aa 504-535).

[0087] In yet other instances, the polypeptide tether can include a cleavage sequence, such as a proteolytic peptide sequence. Such cleavage sequence can include those sensitive to cleavage by an enzyme, such as a protease, an amylase, a chitinase, a chitosanase, a cellulase, a galactosidase, a glucanase, a glucosidase, a hydrolase, a lipase, a lyase, an oxidoreductase, and/or a pectinase. Non-limiting enzymes include bromelain, cathepsin B, collagenase, chymotrypsin, elastase, Factor FVIIa, Factor Xia, Factor Xa, fiscin, furin, keratinase, matrix metalloproteinase (e.g., MMP-1), papain, pepsin, rennin, thrombin, trypsin, and others.

Carbohydrate binding domains

[0088] The binding domains can include cellulose binding domains, chitin binding domains, or polysaccharide binding domains. Examples of binding domains include those from carbohydrate binding module family 1 (CBM1), carbohydrate binding module family 2 (CBM2), carbohydrate binding module family 3 (CBM3), carbohydrate binding module family 20 (CBM20), carbohydrate binding module family 44 (CBM44), as well as subfamilies thereof. In particular embodiments, the binding domain is from CBM1, CBM2a (e.g., which can have particular affinity for crystalline cellulose), CBM3b, CBM44 (e.g., which can have particular affinity for amorphous cellulose), and others. The binding domain may bind reversibly or irreversibly.

[0089] FIGS. 2A-2D, 3A-3D, 4A-4C, and 5A-5E provide non-limiting amino acid sequences for binding domains. Each of the SEQ ID NO: recited herein includes the recited sequence, as well as that sequence with conservative amino acid substitutions. For each of FIGS. 2C-2D, 3C-3D, 4B-4C, and 5D-5E, the asterisk (*) indicates positions that have a single, fully conserved residue; the colon (:) indicates conservation between groups of strongly similar properties (e.g., as described herein); and the period (.) indicates conservation between groups of weakly similar properties (e.g., as described herein).

[0090] Amino acid sequences are shown in FIGS. 2A-2D. The sequences are for (A,B) domains from carbohydrate binding domain family 2 (SEQ ID NOs: l-19); (C) a first consensus sequence (Consl, SEQ ID NO:20), in which each X at each position of SEQ ID NO:20 is an amino acid (or a conservative amino acid substitution thereof) present at a position in one of SEQ ID NOs: 1-19 when optimally aligned with SEQ ID NO:20; and (D) a second consensus sequence (Cons2, SEQ ID NO:21), in which each X at each position of SEQ ID NO:21 is an amino acid (or a conservative amino acid substitution thereof) present at a position in one of SEQ ID NOs:l-19 or one of SEQ ID NOs:l-3 and 6-11 when optimally aligned with SEQ ID NO:21.

[0091] In one embodiment, the binding domain (e.g., the first, second, and/or further binding domain) includes a polypeptide sequence having at least 90% (e.g., at least 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of the following SEQ ID NOs:l- 19 or a fragment thereof. In some embodiments, the binding domain includes a polypeptide sequence having at least 90% sequence identity to any one of the following SEQ ID NOs:l-19 or any one of the following SEQ ID NOs:l-19 having one or more conservative amino acid substitutions, as described herein.

[0092] In another embodiment, the binding domain includes a polypeptide sequence having at least 90% sequence identity to SEQ ID NOs:20-21, in which X at each position of SEQ ID NOs:20-21 is an amino acid present at a position in one of SEQ ID NOs:l-19 when optimally aligned with SEQ ID NOs:20-21, respectively. In yet another embodiment, each X at each position of SEQ ID NOs:20-21 includes a conservative amino acid substitution of an amino acid present at a position in one of SEQ ID NOs: 1- 19 when optimally aligned with SEQ ID NOs:20-21, respectively.

[0093] Amino acid sequences are shown in FIGS. 3A-3D. The sequences are amino acid sequences for (A,B) domains from carbohydrate binding domain family 3 (SEQ ID NOs:30-43); (C) a third consensus sequence (Cons3, SEQ ID NO:44), in which each X at each position of SEQ ID NO:44 is an amino acid (or a conservative amino acid substitution thereof) present at a position in one of SEQ ID NOs:30-43 when optimally aligned with SEQ ID NO:44; and (D) a fourth consensus sequence (Cons4, SEQ ID NO:45), in which each X at each position of SEQ ID NO:45 is an amino acid (or a conservative amino acid substitution thereof) present at a position in one of SEQ ID NOs:30-43 or one of SEQ ID NOs:30-34 when optimally aligned with SEQ ID NO:45. [0094] In one embodiment, the binding domain (e.g., the first, second, and/or further binding domain) includes a polypeptide sequence having at least 90% (e.g., at least 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of the following SEQ ID NOs:30- 43 or a fragment thereof. In some embodiments, the binding domain includes a polypeptide sequence having at least 90% sequence identity to any one of the following SEQ ID NOs:30-43 or any one of the following SEQ ID NOs:30-43 having one or more conservative amino acid substitutions, as described herein.

[0095] In another embodiment, the binding domain includes a polypeptide sequence having at least 90% sequence identity to SEQ ID NOs:44-45, in which X at each position of SEQ ID NOs:44-45 is an amino acid present at a position in one of SEQ ID NOs:30- 43 when optimally aligned with SEQ ID NOs:44-45, respectively. In yet another embodiment, each X at each position of SEQ ID NOs:44-45 includes a conservative amino acid substitution of an amino acid present at a position in one of SEQ ID NOs:30- 45 when optimally aligned with SEQ ID NOs:44-45, respectively.

[0096] Amino acid sequences are shown in FIGS. 4A-4C. The sequences are amino acid sequences for (A) domains from carbohydrate binding domain family 20 (SEQ ID NOs:49-60); (B) a fifth consensus sequence (Cons5, SEQ ID NO:61), in which each X at each position of SEQ ID NO:61 is an amino acid (or a conservative amino acid substitution thereof) present at a position in one of SEQ ID N0s:50-60 when optimally aligned with SEQ ID NO:61; and (C) a sixth consensus sequence (Cons6, SEQ ID NO:62), in which each X at each position of SEQ ID NO:62 is an amino acid (or a conservative amino acid substitution thereof) present at a position in one of SEQ ID N0s:50-60 or one of SEQ ID NOs:50-52, 54, and 56 when optimally aligned with SEQ ID NO:62.

[0097] In one embodiment, the binding domain (e.g., the first, second, and/or further binding domain) includes a polypeptide sequence having at least 90% (e.g., at least 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of the following SEQ ID NOs:49- 60 or a fragment thereof. In some embodiments, the binding domain includes a polypeptide sequence having at least 90% sequence identity to any one of the following SEQ ID NOs:49-60 or any one of the following SEQ ID NOs:49-60 having one or more conservative amino acid substitutions, as described herein.

[0098] In another embodiment, the binding domain includes a polypeptide sequence having at least 90% sequence identity to SEQ ID NOs:61-62, in which X at each position of SEQ ID NOs:61-62 is an amino acid present at a position in one of SEQ ID NOs:49- 60 when optimally aligned with SEQ ID NOs:61-62, respectively. In yet another embodiment, each X at each position of SEQ ID NOs:61-62 includes a conservative amino acid substitution of an amino acid present at a position in one of SEQ ID NOs:49- 60 when optimally aligned with SEQ ID NOs:61-62, respectively.

[0099] Amino acid sequences are shown in FIGS. 5A-5E. The sequences are amino acid sequences for (A-C) domains from carbohydrate binding domain family 1 (SEQ ID N0s:70-104); (D) a seventh consensus sequence (Cons7, SEQ ID NO:105), in which each X at each position of SEQ ID NO: 105 is an amino acid (or a conservative amino acid substitution thereof) present at a position in one of SEQ ID N0s:70-104 or one of SEQ ID NOs:70-77, 82, 84, 86, 88, 89-91, and 104 when optimally aligned with SEQ ID NO: 105; and (E) an eighth consensus sequence (Cons8, SEQ ID NO: 106), in which each X at each position of SEQ ID NO: 106 is an amino acid (or a conservative amino acid substitution thereof) present at a position in one of SEQ ID N0s:70-104 or one of SEQ ID NOs:70-72, 82, 86, 88, and 104 when optimally aligned with SEQ ID NO:62.

[0100] In one embodiment, the binding domain (e.g., the first, second, and/or further binding domain) includes a polypeptide sequence having at least 90% (e.g., at least 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of the following SEQ ID NQs:70- 104 or a fragment thereof. In some embodiments, the binding domain includes a polypeptide sequence having at least 90% sequence identity to any one of the following SEQ ID N0s:70-104 or any one of the following SEQ ID N0s:70-104 having one or more conservative amino acid substitutions, as described herein. [0101] In another embodiment, the binding domain includes a polypeptide sequence having at least 90% sequence identity to SEQ ID NOs: 105-106, in which X at each position of SEQ ID NOs: 105-106 is an amino acid present at a position in one of SEQ ID N0s:70-104 when optimally aligned with SEQ ID NOs: 105-106, respectively. In yet another embodiment, each X at each position of SEQ ID NOs: 105- 106 includes a conservative amino acid substitution of an amino acid present at a position in one of SEQ ID N0s:70-104 when optimally aligned with SEQ ID N0s:105-106, respectively.

[0102] In some embodiments, the binding domain has a relative molecular weight of about 4-40 kDa. In other embodiments, the binding domain has high affinity for crystalline cellulose and/or chitin, e.g., having a Kd ranging from about 0.5-1.5 pM or about 0.8-1.4pM.

[0103] Yet other binding domains includes a chitin binding domain (see, e.g., UniProtKB ID:Q838S1, Q47QG3, or Q9RJY2), a maltose binding domain, and others. In some embodiments, the binding domain is one that occurs in a cellulase, which binds preferentially to cellulose and/or to poly- or oligosaccharide fragments thereof. The binding domain can be a single domain polypeptide or as a dimer, a trimer, or a polymer; or as a part of a protein hybrid.

[0104] FIG. 6A shows the results of a size-based separation of purified binding domain proteins which can function as linkers or cross-linking agents between biopolymers. Two of the proteins studied had two CBMs and a tether (as illustrated in FIG. 1). They are labelled as Sl-vl (SEQ ID NO: 301) and Sl-v2 (SEQ ID No:300). A monomeric CBM labelled mono (SEQ ID No:49) was also tested. Proteins were expressed from a plasmid in an E. coli host and included a C-terminal affinity tag to facilitate purification using standard techniques.

[0105] Intrinsic tryptophan fluorescence FIGS. 6B - 6E show that linking the CBM domains does not disrupt binding relative to the monomeric CBM. FIG. 6B shows the fluorescent spectral shift of intrinsic tryptophan residues from purified Sl-v2 linked binding protein in response to non-binding (pullulans - middle 4 spectra in the 320 nm region) and binding (CMC upper 10 spectra in the 320 nm region) polysaccharides. Carboxymethylcellulose (CMC) and phosphate buffered saline (PBS) are negative controls (lowest 2 spectra in the 320 nm region). FIG. 6D shows fluorescent spectral shift of intrinsic tryptophan residues of the monomeric protein in response to CMC. For FIGS. 6B & 6E, the isobestic point near 360 nm in both figures is consistent with the observation of the same binding event occurring in the CBMs regardless of linkage. FIGS. 6C & 6E binding curves derived from tryptophan fluorescence show similar binding characteristics to carboxymethylcellulose with the half maximal signal (Kdapp) observed at 4.5 uM (linked) or 12 uM (monomeric). For both binding curves the Hill coefficient was near 1 (n = 1.3 linked and 1.2 monomer) consistent with independent binding of the linked CBM domains. Data was fit to a simple ligand binding model, the Hill equation sigma = [ligandjn / ([ligandjn + Kd) where sigma = ratio of fluorescence at 329 nm (FL329 uM CMC /FL329 0 uM CMC) n = fitted ‘hill coefficient’

Kd = fitted apparent dissociation constant

[0106] To obtain the data shown in FIG. 7, rheology experiments showing change in shear stress observed in a solution of 0.5% CMC with various amounts of linked CBM (Sl-v2 protein - PLP in the figure legend, SEQ ID No:300) from 0 - 10 mg I mL were performed. With 5 mg/ml of the linker in an 0.5% solution of CMC, the most favorable performance was obtained.

[0107] In FIG. 8, illustrates water contact angle measurements of linkers in accordance with certain embodiments. The linker alone, CBM protein Sl-v2, (CBM in the figure legend, SEQ ID No:300) was tested and compared to carboxymethyl cellulose (CMC, a binder), hydroxyethyl cellulose (HEC, a non-binder), and bovine serum albumin (BSA, no specific binding to carbohydrates CMC and HEC) and combinations thereof. The combination of linked CBM and binding carbohydrate gives films with increased hydrophobicity (greater water contact angle) relative to films formed from: non-binding carbohydrate (HEC); binding carbohydrate alone (CMC); nonspecific binding protein and either carbohydrate; CBM alone. The improved hydrophobicity would be advantageous in some embodiments for cosmetic film formation.

[0108] An amino acid sequence for a non- limiting embodiment of a linker protein is shown in FIG. 9A. In this linker, two binding domains of SEQ ID No: 49 are joined together by a flexible tether of the sequence AGGGGSGGGGSEAAAKGGGGSGGGGS (a combination of SEQ ID No: 120 and SEQ ID No: 121) and an affinity purification tag of the sequence AENLYFQSSSGHHHHHH (SEQ ID No:300).

[0109] FIG. 9B shows the amino acid sequence SEQ ID No. 301 representing purified binding domain protein Sl-vl. Enzymes

[0110] In some instances, the biobased compositions herein can be treated with one or more enzymes. Such treatment can degrade or release portions of the composition. For instance, if the linker is biodegradable or bio-releasable, then treatment with an enzyme can degrade or cleave the linker. In another instance, the biopolymer can be biodegradable, such that treatment with an enzyme can degrade or cleave the biopolymer. Treatment can include a single type of enzyme, or a combination of two or more different enzymes.

[0111] Non-limiting enzymes include a protease, an amylase, a chitinase, a chitosanase, a cellulase, a galactosidase, a glucanase, a glucosidase, a hydrolase, a lipase, a lyase, an oxidoreductase, and/or a pectinase. Yet further enzymes can an endo-P-(l,4)- D-glucanase, exo-P-(l,4)-D-glucanase, and/or P-D-glucosidase, as well as combinations thereof. Examples of cellulases include microbial cellulases, particularly bacterial or fungal cellulases. Classes of cellulases include endoglucanases, notably endo-l,4-P- glucanases (EC 3.2.1.4), particularly mono-component (recombinant) endo-l,4-P- glucanases.

Characteristics of compositions

[0112] The compositions herein can have one or more useful characteristics or properties, which can be tuned by the selection of and the amount of each component (biopolymers, binding domains and tethers). Depending upon the linker selected, the composition’s level of cross-linking can be adjusted to obtain a specific physical characteristic. In particular embodiments, the composition includes at least one property, at least two properties, or at least three properties selected from the following:

(i) a tensile strength of at least about 100 MPa or from about 100- 5000 MPa, 100-1000 MPa, or 300-600 MPa (e.g., as determined by ASTM D638-14, D3379, or D882-18);

(ii) a tensile modulus of greater than about 0.1 MPa, 0.2 MPa, 0.5 MPa, 1 MPa, 2 MPa, 5 MPa, 10 MPa, 20 MPa, 50 MPa, 60 MPa, 80 MPa, 100 MPa, 110 MPa, 120 MPa, 130 MPa, 140 MPa, 150 MPa, 200 MPa, 250 MPa, or from about 500 MPa - 5 GPa (e.g., as determined by ASTM D638-14, D3379, or D882-18);

(iii) a strain of about 10-60% or 20-60% (e.g., as determined by ASTM D638-14, D3379, or D882-18); (iv) an elongation at break of about 10-700%, 10-300%, or 10-50% (e.g., as determined by ASTM D638-14, D3379, or D882-18);

(v) an adhesion having a 90 degree peel resistance of at least about 38 N/100 mm or from about 40-500 N/100 mm (e.g., as determined by ASTM D6862-11);

(vi) an adhesion having a 180 degree peel resistance of at least about 38 N/100 mm or from about 40-500 N/100 mm (e.g., as determined by ASTM D3330);

(vii) an adhesion having a T-peel resistance of at least about 60 N/100 mm or from about 50-600 N/100 mm (e.g., as determined by ASTM D1876);

(viii) a lap shear resistance of at least about 0.1 N/mm ² (e.g., as determined by ASTM D1002, D3163, D3165, or D5868);

(ix) a lap shear strength of greater than about 50 psi, 80 psi, 100 psi, 200 psi, 500 psi, or 750 psi (e.g., as determined by ASTM D1002);

(x) a crosshatch adhesion to metal, glass, ceramic, and/or plastic of greater than 4B (e.g., as determined by ASTM D3359-17);

(xi) a peel strength of at least about 0.1 N or from about 0.1-60 N (e.g., as determined by ASTM F2256);

(xii) a lamination strength between the composition and a first test body (e.g., an outer substrate, a material layer, etc.) of at least about 1 N, 2 N, 3 N, or 4 N per 25.4 mm of sample width (e.g., as determined by ASTM F904-98);

(xiii) a measure of tack of about 0.1 -0.6 MPa (e.g., as determined by ASTM D2979-16, D6195, or D3121-17);

(xiv) a work of debonding of about 1-20 J/m ² ;

(xv) a kinetic coefficient of friction of less than about 0.5, 0.4, or 0.2 or from about 0.1-0.5, 0.2-0.5, or 0.1-0.4 (e.g., as determined by ASTM D1894-14);

(xvi) a maximum load of at least about 40 N, 50 N, or 60 N (e.g., as determined by ASTM D882-12, in which maximum load can be measured in the cross direction or the machine direction);

(xvii) a compressibility having a degree of penetration of about 20-500 tenths of a mm (e.g., as determined by penetrometry according to ASTM D2137);

(xviii) a swelling degree of about 10-130%, 30-80%, or 40-70% (e.g., as determined by a gravimetric method after submerging in distilled water for 60 minutes at room temperature); (xix) a pencil hardness that is between 3B and 7H, between 2B and 6H, or between IB and 5H (e.g., as determined by ASTM D3363-05);

(xx) a yellowness index that is less than 1.9, 1.8, 1.7. 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, or 0.8 (e.g., as determined by ASTM E313-15);

(xxi) a heat seal strength of at least about 35 N, 45 N, 55 N, 65 N, 75 N, 85 N, or 95 N per 25.4 mm width or 15 mm width using a heat sealing temperature of about 60°-120°C (e.g., as determined by ASTM F88/F88M-09);

(xxii) a solvent resistance greater than 10 rubs, 20 rubs, 30 rubs, 40 rubs, 50 rubs, 60 rubs, 80 rubs, 100 rubs, 120 rubs, 140 rubs, 160 rubs, 180 rubs, or 200 rubs (e.g., as determined by ASTM D5402-15, Method B);

(xxiii) a moisture vapor transmission rate (MVTR) of less than about 10 grams per square meter per day (g/m ² /day), 5 g/m ² /day, 2 g/m ² /day, 1 g/m ² /day, 0.6 g/m ² /day, 0.4 g/m ² /day, or 0.2 g/m ² /day (e.g., as determined by ASTM F1249-13 at about 37° C and about 90% relative humidity);

(xxiv) a work recovery of at least about 5%, 10%, 25%, 50%, 100%, 250%, or 500% (e.g., relative to the plurality of biopolymers lacking a linker or as determined by ASTM D 1774-94);

(xxv) a glass transition temperature of greater than about -40°C, -30°C, - 20°C, -10°C, 0°C, 10°C, 20°C, or 30°C (e.g., as determined by differential scanning calorimetry or ASTM D7426-08); and

(xxvi) a bio-based carbon content of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater (e.g., as determined by ASTM D6866).

[0113] In some embodiments, the composition forms an adhesion between a surface and an object, in which the peel strength is determined at room temperature, unless otherwise specified. Non-limiting methods of determining adhesion includes forming an adhesion between a surface and an object having a 90 degree peel resistance as determined according to ASTM D6862-11 of at least 38 N/100 mm. ASTM D6862-11 entitled “Standard Test Method for 90 Degree Peel Resistance of Adhesives” (11th revision) determines the peel strength of to separate the object from the surface.

[0114] In one embodiment, the 90-degree peel resistance is at least 40 N/100 mm, at least 42 N/100 mm, at least 44 N/100 mm, at least 46 N/100 mm, at least 48 N/100 mm, at least 50 N/100 mm, at least 52 N/100 mm, at least 54 N/100 mm, at least 56 N/100 mm, at least 58 N/100 mm, at least 60 N/100 mm, at least 65 N/100 mm, or at least 70 N/100 mm. In one further embodiment, the 90-degree peel resistance can be in a range between 38 N/100 mm and 1 kN/100 mm, such as between 50 N/lOOmm and 900 N/lOOmm, between 60 N/100 mm and 800 N/100 mm, or 70 N/100 mm and 700 N/100 mm.

[0115] In other embodiments, adhesion is determined by applying a formulation (e.g., including a composition herein) to a surface to form a coating, and the coating has a tape test rating as determined by ASTM D3359-17 (“Standard Test Methods for Rating Adhesion by Tape Test”) of at least 2B. In yet another embodiment, the coating has a tape test rating of at least 3B in accordance with ASTM D3359-17, at least 4B in accordance with ASTM D3359-17, or 5B in accordance with ASTM D3359-17.

[0116] In one embodiment, the composition forms an adhesion between a surface and an object having a lap shear resistance as determined by ASTM D1002-10, D3163, D3165, or D5868 of at least 0.1 N/mm ² . ASTM standards D1002-10 (“Standard Test Method for Apparent Shear Strength of Single-Lap-Joint Adhesively Bonded Metal Specimens by Tension Loading (Metal-to-Metal)”), D3163-01 (2014, “Standard Test Method for Determining Strength of Adhesively Bonded Rigid Plastic Lap-Shear Joints in Shear by Tension Loading”), D3165-07 (2014, “Standard Test Method for Strength Properties of Adhesives in Shear by Tension Loading of Single-Lap-Joint Laminated Assemblies”), and D5868-01 (2014, “Standard Test Method for Lap Shear Adhesion for Fiber Reinforced Plastic (FRP) Bonding”) are test methods for determining bonding characteristics of adhesives joining various materials. The test results are all reported in force per area, e.g., in psi or N/mm ² .

[0117] In one embodiment, the composition forms an adhesive having a lap shear resistance in accordance with ASTM D1002, D3163, D3165, or D5868 of at least 0.2 N/mm ² , at least 0.4 N/mm ² , at least 0.6 N/mm ² , at least 0.8 N/mm ² , at least 1 N/mm ² , at least 1.5 N/mm ² , at least 2 N/mm ² , at least 2.5 N/mm ² , at least 3 N/mm ² , at least

4 N/mm ² , at least 5 N/mm ² , at least 10 N/mm ² , at least 15 N/mm ² , at least 20 N/mm ² , at least 25 N/mm ² , at least 30 N/mm ² , at least 35 N/mm ² , at least 40 N/mm ² , or at least

45 N/mm ² .

[0118] In one embodiment, the composition can form an adhesive having a lap shear resistance in accordance with ASTM D1002, D3163, D3165, or D5868 of not greater than 150 N/mm ² , not greater than 100 N/mm ² , not greater than 80 N/mm ² , not greater than 60 N/mm ² , not greater than 50 N/mm ² , not greater than 40 N/mm ² , not greater than 30 N/mm ² , not greater than 20 N/mm ² , not greater than 10 N/mm ² , not greater than

5 N/mm ² , or not greater than 3 N/mm ² . In one embodiment, the composition forms an adhesive having a lap shear resistance in accordance with ASTM D1002, D3163, D3165, or D5868 in the range from 0.1 N/mm ² to 80 N/mm ² , from 0.2 N/mm ² to 50 N/mm ² , or from 0.3 N/mm ² to 30 N/mm ² .

[0119] In yet one further embodiment, the composition forms an adhesion between a surface and an object having a T-peel resistance as determined by ASTM D1876-08 (2015, “Standard Test Method for Peel Resistance of Adhesives (T-Peel Test)) of at least 60 N/100 mm. In yet another embodiment, the adhesives formed by the composition have a T-peel resistance in accordance with ASTM D1876-08 of at least 70 N/100 mm, at least 70 N/100 mm, at least 70 N/100 mm, at least 65 N/100 mm, at least 70 N/100 mm, at least 75 N/100 mm, at least 80 N/100 mm, at least 90 N/100 mm, at least 100 N/100 mm, at least 120 N/100 mm, or at least 150 N/100 mm. In one further embodiment, the adhesives formed by the composition have a T-peel resistance in accordance with ASTM D1876-08 of not greater than 300 N/100 mm, not greater than 250 N/100 mm, not greater than 200 N/100 mm, not greater than 180 N/100 mm, not greater than 160 N/100 mm, not greater than 140 N/100 mm, not greater than 120 N/100 mm, not greater than 100 N/100 mm, not greater than 95 N/100 mm, not greater than 85 N/100 mm, or not greater than 75 N/100 mm. In yet one further embodiment, the adhesives formed by the composition have a T-peel resistance in accordance with ASTM D1876-08 in the range of 60 N/100 mm and 300 N/100 mm, in the range of 65 N/100 mm and 200 N/100 mm, in the range of 70 N/100 mm and 150 N/100 mm, or in the range of 75 N/100 mm and 100 N/100 mm.

[0120] In yet one further embodiment, the composition forms an adhesion between a surface and an object having a 180 degree peel resistance as determined by ASTM D3330-04 (2010, “Standard Test Method for Peel Adhesion of Pressure-Sensitive Tape,” Test Method A) of at least 38 N/100 mm, at least 40 N/100 mm, at least 42 N/100 mm, at least 44 N/100 mm, at least 46 N/100 mm, at least 48 N/100 mm, at least 50 N/100 mm, at least 55 N/100 mm, at least 60 N/100 mm, at least 65 N/100 mm, at least 70 N/100 mm, or at least 80 N/100 mm. In one embodiment, the adhesives formed by the composition have a 180 degree peel resistance in accordance with ASTM D3330-04 of not greater than 250 N/100 mm, not greater than 230 N/100 mm, not greater than 210 N/100 mm, not greater than 190 N/100 mm, not greater than 170 N/100 mm, not greater than 150 N/100 mm, not greater than 130 N/100 mm, or not greater than 110 N/100 mm. In one embodiment, the adhesives formed by the composition have a 180 degree peel resistance in accordance with ASTM D3330-04 in the range from 38 N/100 mm to 250 N/100 mm, from 45 N/100 mm to 200 N/100 mm, from 50 N/100 mm to 100 N/100 mm.

[0121] The compositions herein can be configured to be biodegradable or bioreleasable. For instance, the linker can include one or more cleavage sites, which can be selective or specific for a particular enzyme (e.g., any herein, such as a protease). In another instance, the composition can be provided as an interlayer between a top layer and a bottom layer, in which the composition can degrade to release the top and bottom layers. Degradation can include use of enzymes to cleave the linkers and/or the biopolymers. For instance, a protease can be used to bind to and enzymatically cleave linker, and a cellulase or chitinase to degrade the biopolymer portion of the composition. [0122] In particular embodiments, the compositions herein can undergo bio-triggered degradation for debonding of adhesives, coatings, and composites. Degradation can be triggered, e.g., by employing one or more proteases, hydrolases, peptidases, and the like. Such degradation can be promoted by providing a microbe or a supernatant derived from such microbes. In use, the final enzymatic breakdown product can be the amino acids of the biopolymer and/or the linker, such that compositions herein can exhibit biodegradability. In one non-limiting instance, a compositions herein can be employed as a film, a coating, or an adhesive, which can be de-bonded on demand with an engineered microbe and be reverted back to benign starting materials (amino acids of the biopolymer and/or the linker).

[0123] In further embodiments, the compositions herein can form a hydrogel, which in turn can have at least one property selected from the group consisting of:

(i) the hydrogel is an element of a pharmaceutical formulation adapted to release an active ingredient;

(ii) the hydrogel is an element of an engineered tissue for biological tissue replacement;

(iii) the hydrogel is self-healing;

(iv) the hydrogel has a peel strength according to ASTM F2256 (2015) of at least 0.1 N, at least 0.2 N, at least 0.4 N, at least 0.6 N, at least 0.8 N, at least 1.0 N, at least 1.5 N, at least 2 N, at least 4 N, at least 6 N, at least 8 N, or at least 10 N; and

(v) any combination of (i), (ii), (iii), and (iv). [0124] In addressing the aforementioned peel strength, in some embodiments, the compositions herein may permit formation of an adhesive material that is adherent for a sufficient period to permit the tissue to repair themselves but is not necessarily designed to act as a permanent adhesive. For example, compositions herein can be selected to provide a bioadhesive that serves as a temporary interface while the body's natural healing mechanisms repair the tissue damage and reestablish the bonding of two separated tissue planes. In some instances, the adhesive may provide adherence between the tissues in situ for at least 7 days, more particularly, at least 10 days or at least 14 days. In certain embodiments, the adhesive strength of the adhesive materials may be at least 50 N as tested by ASTM F2255 dated 2003, more particularly at least 60 N as tested by ASTM F2255 dated 2003 or at least 70 N as tested by ASTM F2255 dated 2003. In certain instances, the T-peel strength as tested by ASTM F2256 dated 2005 may be at least 0.20 N, for example, at least 0.50 N as tested by ASTM F2256 dated 2005, or at least 0.70 N as tested by ASTM F2256 dated 2005. During the tissue repair process, the bioadhesive may be resorbed, degraded, etc. or may be used as a makeshift framework to permit cell ingrowth and/or stabilization during the tissue repair process. [0125] In any embodiment herein, the substrate (e.g., for an adhesion measurement) can be selected from metal, glass, ceramic, or plastic. In yet another embodiment, the metal can be selected from cold rolled steel, stainless steel, aluminum, anodized aluminum, nickel, alloys, electroless plated metal, or electroplated metal. In one further embodiment, the glass can be selected from borosilicate glass or quartz. In one more embodiment, the plastic can be selected from polycarbonate, polyethylene terephthalate, polyamide, filled polyamide, polyester, poly(methyl methacrylate), poly acrylates, polystyrene, polyvinyl, chlorinated polyvinyl, or laminates thereof.

[0126] In one further embodiment, the compositions herein can have a water vapor permeability according to ASTM D1653-13 of not more than 100 g (m ² 24h) ^-1 , not more than 80 g (m ² 24h) ^-1 , not more than 60 g (m ² 24h) ^-1 , not more than 40 g (m ² 24h) ^-1 , not more than 20 g (m ² 24h) ^-1 , not more than 15 g (m ² 24h) ^-1 , not more than 10 g (m ² 24h) ^_ \ not more than 8 g (m ² 24h) ^-1 , not more than 5 g (m ² 24h) ^-1 , not more than 3 g (m ² 24h) ^-1 , not more than 2 g (m ² 24h) ^-1 , not more than 1 g (m ² 24h) ^-1 , not more than 0.8 g (m ² 24h) ^-1 , not more than 0.5 g (m ² 24h) ^-1 , not more than 0.2 g (m ² 24h) ^-1 , not more than 0.1 g (m ² 24h) ^-1 , or not more than 0.05 g (m ² 24h) ^-1 . In one embodiment, the water permeability is at least 0.0001 mg (m ² 24h) ^-1 . [0127] In a further embodiment, the compositions herein have a bio-based carbon content of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, as determined by ASTM D6866. Bio-based carbon content as defined herein is the percentage of carbons from renewable or biogenic sources, such as plants or animals over the total number of carbons in the compound.

Applications

[0128] The compositions herein can be employed as ingredients and/or components in any useful composition for any useful application. Exemplary, non- limiting applications include adhesives, coatings, films, plastics, thickeners, additives, absorbents, coatings, fillers, textiles, formulations, food products, cosmetic products, consumer products, packaging material, absorbent articles, barrier materials, biodegradable products, gels, capsules, pharmaceutical compositions, nanocomposites, matrixes, laminates, or surgical products. Such applications can include materials for use in constructing consumer films, biodegradable products, electronics, industrial adhesives, packages, and the like.

[0129] The compositions may include from about 0.001 to about 10 mg of linker per ml of biopolymer in some embodiments.

[0130] Yet other applications include use of the composition as a polymer curative, a resin (e.g., an ion free resin), a monomer for a polymer or a copolymer, and the like. The composition can be provided in any useful form, such as a film, a composite structure, a bulk structure, a fiber, or a particle.

[0131] In some embodiments, the present disclosure encompasses methods for manufacturing any use herein (e.g., an adhesive, a coating, a film, a plastic, a composite, and the like) by applying a composition herein (e.g., any foregoing compound) in the assembly of the adhesive, the coating, the film, the plastic, or the composite. The composition can be applied to a substrate (e.g., paper, backing, plastic, etc.). In some embodiments, the composition is provided as a solution, which can be gelled or hardened. In other embodiments, the composition is provided as a layer. In yet other embodiments, the composition is provided as a polymer curative.

[0132] Methods can include providing a biopolymer and a linker, thereby forming the composition. In one embodiment, the biopolymer and linker can form a solution upon addition, thereby forming a casting solution that can be further processed by way of casting, gelling, spinning, curing, laminating, molding, weaving, and the like. Yet other processing can include solution cast lines, ink jetting, dip coating, spraying, spin coating, blow molding, extrusion, pultrusion, injection molding, melt-processing, and/or electrospinning. In some embodiments, the casting solution can be further mixed and filtered. In another embodiment, a solution including the linker is applied to a surface including the biopolymer(s). In yet another embodiment, the biopolymer and linker can form a gel upon addition, which can then cast as a film or other polymeric article. This article, in turn, can be hydrated or dehydrated in use.

[0133] Methods can include exposing the composition to an enzyme, thereby degrading the linker and/or the biopolymer. In some embodiments, the linker can be a polypeptide, which can be degraded by use of a protease. The biopolymer can be degraded by use of enzymes that can degrade that biopolymer, such as cellulases to degrade cellulose, chitinase to degrade chitin, glucanase to degrade glucan, and so forth. Biopolymers and linkers can be degraded by providing a particular enzyme, a cocktail or combination of enzymes, or upon exposure to microorganisms capable of expressing such enzyme(s).

Other embodiments

[0134] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.

[0135] Although the foregoing embodiments have been described in some detail for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims. It should be noted that there are many alternative ways of implementing the processes, systems and apparatus of the present embodiments. Accordingly, the present embodiments are considered as illustrative and not restrictive; and the embodiments are not to be limited to the details given herein.