Enzyme construct useful for surface coatings and methods of use

Abstract

Provided are constructs comprising an enzyme, a linker, a polymer or immobilization material, and optionally a peptide tag as well as compositions comprising said constructs, which construct is useful for anti-fouling, among other uses. Methods of use of the constructions or compositions, such as for coatings that reduce biological fouling and / or the growth of mold and / or mildew, are also provided.

Description

Attorney Docket No. 5218.262.WOENZYME CONSTRUCT USEFUL FOR SURFACE COATINGS AND METHODS OF USERELATED APPLICATION INFORMATION

[0001] This application cliams the benefit, under 35 U.S.C. §119(e), of U.S. Provisional Application No. 63 / 762,224, filed February 24, 2025, and U.S. Provisional Application No.63 / 867,194, filed August 20, 2025, the entire contents of each of which is incorporated by reference herein in its entirety.STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

[0002] A Sequence Listing in XML format, submitted under 37 C.F.R. § 1.821, entitled 5218-262WO_ST26.xml, 34,126 bytes in size, generated on February 11, 2026, and filed herewith, is hereby incorporated by reference into the specification for its disclosures.BACKGROUND

[0003] Antifouling and bilge coatings are applied in the recreational and commercial boating industry to minimize or eliminate the deposition of marine organisms and algae (Durr & Thomason, 2009). Without antifouling coatings, marine organisms can accumulate on a ship’s hull or other areas leading to increased hull resistance, higher fuel consumption, and elevated underwater noise. Current antifouling coatings can have a major impact on the environment through the release of copper, zinc, and other biocides, and typically have a 12-month service life (Nehring, 2001; Schiff et al., 2004). Further, countries such as the European Union and others are developing new environmental risk assessment tools that set a high standard for new product entering the market (Ytreberg et al., 2021). Therefore, there is a need for new antifouling agents that are environmentally friendly but can also meet the needs of boaters.SUMMARY

[0004] One aspect of the invention relates to a construct of Formula I:Formula IAttorney Docket No. 5218.262.WOwherein R1 is an enzyme; Pl is present or absent, and when present is a peptide tag; LI is a linker; and P2 is a polymer.

[0005] Another aspect of the invention relates to a composition comprising a construct as described herein.

[0006] Another aspect of the invention relates to a method of reducing biological fouling on a surface, the method comprising applying a composition as described herein to said surface, thereby reducing biological fouling as compared to a surface not comprising the composition.

[0007] Another aspect of the invention relates to a method of reducing mold and / or mildew growth on a surface, the method comprising applying a composition as described herein to said surface, thereby reducing mold and / or mildew growth as compared to a surface not comprising the composition.

[0008] Another aspect of the invention relates to a method of making construct as described, the method comprising providing an enzyme, optionally wherein the enzyme is conjugated to a peptide tag; conjugating the enzyme or the peptide tag to a linker; and conjugating the linker to a polymer by click chemistry.

[0009] Another aspect of the invention relates to a method of decolorizing a dye, the method comprising adding a construct or composition as described herein, and copper (II) sulfate, to a composition comprising the dye (e.g., comprising an aqueous buffer), thereby decolorizing the dye.

[0010] Another aspect of the invention relates to a method of polymerizing phenolic compounds, the method comprising adding a construct or composition as described herein, to a composition comprising said phenolic compounds, whereby the phenolic compounds are oxidatively polymerized.

[0011] Another aspect of the invention relates to a method of clarifying a cloudy aqueous solution, the method comprising adding a construct or composition as described herein, to the aqueous solution, whereby the solution is clarified.

[0012] Another aspect of the invention relates to a method of reducing the potential for discoloration, residue accumulation, or staining (permanent or temporary) associated with environmental factors, including, but not limited to microbes, fungi, organic material, mineral deposits, moisture, oils, dyes, pigments, food and beverage substances, chemicals cleaning agents, rust atmospheric pollutants, and other contaminants. Surfaces should be protected through appropriate cleaning, sealing, or treatment methods, and spills or deposits should be promptly removed to reduce the potential for discoloration, residue accumulation, or permanent staining.Attorney Docket No. 5218.262.WO

[0013] Another aspect of the invention relates to a composition comprising a xerogel (e.g., a silica xerogel) and an enzyme, optionally wherein the xerogel integrates (e.g., encapsulates) the enzyme.

[0014] Another aspect of the invention relates to a composition comprising a hydrogel and an enzyme, optionally wherein the hydrogel integrates (e.g., encapsulates) the enzyme as described herein.

[0015] Another aspect of the invention relates to a construct of Formula II:Formula IIwherein: Ri is an enzyme; Pi is present or absent, and when present is a peptide tag; Li is a first linker; and Ii is an immobilization material, wherein the " — " indicates covalent bonds.

[0016] Another aspect of the invention relates to a method of making an antifouling coating, the method comprising combining an enzyme and a composition that, when cured, will produce a xerogel (e.g., a silicate xerogel), wherein the composition can be applied to a surface, thereby providing an antifouling coating.

[0017] Another aspect of the invention relates to a method of making an antifouling coating, the method comprising providing an enzyme and encapsulating the enzyme in a hydrogel (e.g., an alginate hydrogel), wherein the hydrogel encapsulating the enzyme can be applied to a surface, thereby providing an antifouling coating.

[0018] Another aspect of the invention relates to a method of producing a purified recombinant protein, the method comprising: (a) preparing a vector comprising a polynucleotide encoding fusion protein comprising the recombinant protein, a peptide tag (e.g., a SUMO tag), and a purification tag; (b) transfecting a cell (e.g., a bacterial cell) with the vector, wherein the cell thereby produces the fusion protein; (c) isolating the fusion protein using the purification tag; (d) combining the isolated fusion protein with a construct of Formula II, wherein the enzyme of the construct is a protease and said protease cleaves the fusion protein to provide the recombinant protein; and (e) purifying the recombinant protein, thereby providing the purified recombinant protein.

[0019] These and other aspects of the invention are set forth in more detail in the description below.Attorney Docket No. 5218.262.WOBRIEF DESCRIPTION OF THE DRAWINGS

[0020] Fig. 1 is an image of a Coomassie stained SDS-PAGE gel showing the expression of Halo-tagged laccase and Halo-tagged trypsin.

[0021] Fig. 2 is an image of a Coomassie stained SDS-PAGE gel showing the expression of Halo-tagged APOL3 and Halo-tagged lysozyme.

[0022] Fig. 3 is an image of a Coomassie stained SDS-PAGE gel showing the expression of Halo-tagged xylanase.

[0023] Fig. 4 is a graph showing the activity of Halo-tagged xylanase that was captured within a polymer matrix in 96-well plates using reactive diformylbenzoate Linker 1 (OP A) or reactive chloropropylbenzoate Linker 2 (HALO) and assayed repeatedly using the XylX6 colorimetric assay system (Megazyme Inc.). Good retention of Xylanase activity was observed with both polymer matrices, while the nonspecific coating of the 96-well plate led to a significant loss of Xylanase activity over time.

[0024] Fig. 5A is a schematic of the laccase - HaloTag® fusion assembled for this study, which has a molecular weight of 87,170.5 Daltons. Laccase = laccase from Thermus thermophilus HALO = HaloTag® protein; 6XHis = polyhistidine tag, which allows for affinity purification using cobalt resin.

[0025] Fig. 5B is an SDS-PAGE gel of affinity purified Halo-Laccase fusion produced in practicable quantities (10 - 50 mg / L) in 1 L E. coli expression cultures (LB medium, induced with 0.4 mM IPTG, shaken overnight at 17 °C). A major band elutes from the cobalt column with 250 mM imidazole in the expected molecular weight region.

[0026] Fig. 5C is an image showing that the eluted laccase fraction has concentration and pH dependent activity towards DMP. Rows labeled “A” are sodium acetate at pH 5.4 and rows labeled “T” are Tris buffered saline (TBS) at pH 7.6.

[0027] Fig. 5D is a graph showing that the laccase has an absorbance at 600 nm, consistent with a copper-bound enzyme.

[0028] Fig. 5E is a graph showing the quantification of enzyme velocity plots derived from experiment shown in Fig. 5C.

[0029] Fig. 6 is a graph showing that the samples were incubated at the indicated temperatures (Fisher shaker, 600 rpm) for 8 min and the absorbance of DMP recorded at 470 nm (open circles, with enzyme; closed black circles, no enzyme). We observed maximum activity at 95 °C, consistent with the initial literature value of max activity was 92 °C. The addition of a HaloTag® does not appear to inhibit laccase activity for phenolic substrates.Attorney Docket No. 5218.262.WO

[0030] Fig. 7A is a graph showing the decolorization of indigo carmine dye with free laccase. Samples were incubated at 65 °C for 90 min and the absorbance of indigo carmine read at 600 nm. Efficient dye decolorization required the addition of copper sulfate. The addition of 50 pM copper sulfate gave robust decolorization (i.e., clear solution, lower absorbance) within 90 min.

[0031] Fig. 7B is a graph showing the decolorization of indigo carmine dye with immobilized laccase. Laccase was immobilized on magnetic beads and, with the addition of 50 pM copper sulfate, also gave robust decolorization (i.e., lower absorbance) of indigo carmine over multiple rounds of reuse (4X).

[0032] Fig. 8A presents data on the polymerization of a simple polyphenol, catechol, with free ("CATECHOL-LACCASE") and immobilized laccase ("CATECHOL-LACCASEBEADS"). Solutions of catechol were incubated with and without laccase in free and immobilized form. After the 19h incubation period (65 °C), UV / Vis spectra were acquired. Catechol exhibited increase in absorbance characteristic of polymer formation

[0013] , Assays were conducted in 0.1 MNaOAc buffer, pH 5 at 50 mM substrate concentration. Following polymerization, reactions were cooled to 4 °C for one week and centrifuged to collect the precipitated products.

[0033] Fig. 8B presents data on the polymerization of a simple polyphenol, resorcinol, with free ("RESORCINOL-LACCASE") and immobilized laccase ("RESORCINOL-LACCASEBEADS"). Solutions of resorcinol were incubated with and without laccase in free and immobilized form. After the 19h incubation period (65 °C), UV / Vis spectra were acquired. Resorcinol reacted in the presence of free laccase but not in the presence of immobilized laccase. Assays were conducted in 0.1 M NaOAc buffer, pH 5 at 50 mM substrate concentration. Following polymerization, reactions were cooled to 4 °C for one week and centrifuged to collect the precipitated products.

[0034] Fig. 8C presents data on the polymerization of a simple polyphenol, hydroquinone, with free ("HYDROQUINONE-LACCASE") and immobilized laccase ("HYDROQUINONE-LACCASEBEADS"). Solutions of hydroquinone were incubated with and without laccase in free and immobilized form. After the 19h incubation period (65 °C), UV / Vis spectra were acquired. Like catechol shown in Fig. 8A, hydroquinone exhibited increase in absorbance characteristic of polymer formation

[0013] , Assays were conducted in 0.1 M NaOAc buffer, pH 5 at 50 mM substrate concentration. Following polymerization, reactions were cooled to 4 °C for one week and centrifuged to collect the precipitated products.

[0035] Fig. 8D shows HPLC analysis of hydroquinone monomer (unreacted).

[0036] Fig. 8E shows HPLC analysis of free laccase, indicating the formation of polymeric species.Attorney Docket No. 5218.262.WO

[0037] Fig. 8F shows HPLC analysis of immobilized laccase, indicating the formation of polymeric species.

[0038] FIG. 8G shows a control HPLC analysis with no catalyst.

[0039] Fig. 9A reports on the clarification of fruit juice with free laccase. No changes in juice color were detected after 1 h of laccase treatment of unfiltered apple juice at the indicated temperatures, indicating that this particular laccase may not be ideal for use in beverage-related applications without additional optimization.

[0040] Fig. 9B reports on the clarification of fruit juice with free laccase. No changes in turbidity were detected after 1 h of laccase treatment of unfiltered apple juice at the indicated temperatures, indicating, consistent with FIG. 9A, that this particular laccase may not be ideal for use in beverage-related applications without additional optimization.

[0041] Fig. 10 is a series of cartoon schematics illustrating enzyme fusions of the present invention under variable environmental conditions using the approaches described herein.

[0042] Fig. 11 is a series of chemical structures showing the enzyme covalent capture methods used in lysine capture and HaloTag® capture approaches.

[0043] Fig. 12A is a gel image showing that HaloTag® fusions of xylanase from T. lanuginosus are produced in practicable quantities (10 - 50 mg / L) in IL E. coli expression cultures (LB medium, induced with 0.4 mM IPTG, shaken overnight at 17 °C). Protein fusions have the general form of HaloTag®-enzyme-6XHis and are readily purified using cobalt resin.

[0044] Fig. 12B is a graph showing the coupled assay (Megazyme XylX6 reagent) of HaloTag®-xylanase in both free and immobilized (Promega MagHalo beads) forms, and demonstrates activity of the enzyme and functionality of the HaloTag®.

[0045] Fig. 12C is a gel image showing that HaloTag® fusions of TEV protease are produced in practicable quantities (10- 50 mg / L) in ILL. coli expression cultures (LB medium, induced with 0.4 mM IPTG, shaken overnight at 17 °C). Protein fusions have the general form of HaloTag®-enzyme-6XHis and are readily purified using cobalt resin.

[0046] Fig. 12D is a gel image showing the assay of immobilized TEV protease (Promega MagHalo beads) in the presence of an MBP-TEVsite-GFP fusion protein. The immobilized TEV preparation retains activity after one month (Day 34) in cold storage (4 °C).

[0047] Fig. 13 is a graph showing that HaloTag®-Xylanase fusion protein was immobilized in a polymer matrix in a 96 well plate after preincubation with either HaloTag® capture linker (HALO), lysine capture linker (OP A), or no linker capture molecule (nonspecific). After 24h of polymer curing, samples were assayed with the XylX6 coupled assay 1-, 2-, 5-, and 9-days post-cure with (washed) or without (unwashed) vigorous PBS washing (3 washes x 5 min withAttorney Docket No. 5218.262.WOshaking at 300 RPM). When not being assayed at room temperature, samples were kept at 4 °C. The positive control assay was conducted using non-immobilized xylanase. Error bars represent the standard deviations of three replicate measurements per condition.

[0048] Fig. 14A is image showing fiberglass hull samples coated with xerogel formulations of Mix 1 (tetraethyl orthosilicate, triethoxyoctylsilane, ethanol, and hydrochloric acid) and Mix 2 (tetraethyl orthosilicate, (3 -chi oropropyl)tri ethoxy silane, ethanol, and hydrochloric acid), and cured for at least 24 hours.

[0049] Fig. 14B is an image showing the hull samples from Fig. 14A that have been incubated in brackish water by suspension from a floating dock.

[0050] Fig. 15 is a series of images showing the fouling assay of silica xerogels with and without entrapped enzymes on fiberglass hull samples over the indicated time period. Mix 1 (tetraethyl orthosilicate, triethoxyoctylsilane, ethanol, and hydrochloric acid) and Mix 2 (tetraethyl orthosilicate, (3-chloropropyl)triethoxysilane, ethanol, and hydrochloric acid). Day 0 image is as shown in FIG. 14B.

[0051] Fig. 16 is a series of images showing the fouling assay of silica xerogels with and without entrapped enzymes on fiberglass hull samples over the indicated time period. Mix 1 (tetraethyl orthosilicate, triethoxy octylsilane, ethanol, and hydrochloric acid), Mix 2 (tetraethyl orthosilicate, (3 -chi oropropyl)tri ethoxy silane, ethanol, and hydrochloric acid), and Mix 3 (tetramethylorthosilicate, (3-chloropropyl)triethoxysilane, ethanol, and hydrochloric acid).

[0052] Fig. 17 is a graph showing the xylanase activity measured with the XylX6 colorimetric assay system (Megazyme Inc.) on two different hull sample types (D = Daedalus yacht hull sample and R = Regulator marine hull sample). The painted enzyme-polymer matrices (immobilized xylanase) outperformed non-specific enzyme capture.

[0053] Fig. 18 is a series of images showing algae-coated fiberglass hull samples pre- and posttreatment with recombinant Halo-Xylanase and Halo-Laccase proteins.

[0054] Fig. 19 is a graph showing the activity of recombinant Halo-Xylanase and Halo-Laccase proteins after incubation in brackish water for 8 days (Pamlico Sound, Belhaven, NC) measured by XylX6 colorimetric assay system (Megazyme Inc.). The enzymes were captured within a polymer matrix and painted onto fiberglass hull sample (Daedalus yacht hull). Enzyme-linker combinations tested include Halo-Laccase = LHD; OPA-Laccase = LOD; Halo-Xylanase = XHD; Halo-Xylanase-Laccase = XLHD; Halo-OPA-Xylanase = XHOD; OPA-Xylanase = XOD; and were compared to non-specific enzyme attachment (laccase = LD; xylanase = XD; and xylanase / laccase combination = XLD) as well as a BLANK = control.Attorney Docket No. 5218.262.WO

[0055] Fig. 20 is a flowchart showing the design strategy for using immobilized enzymes according to some embodiments of the present invention to produce purified, recombinant target protein.

[0056] Fig. 21A is a cartoon schematic showing the structure of enzyme constructs according to some embodiments of the present invention, wherein the enzymes (e.g., SENP1 and / or TEV protease) are fused to a HALO tag, a linker (e.g., a chemical linker), and then to a magnetic bead that is used for immobilization.

[0057] Fig. 21B is a gel image showing the cleavage of the SUMO protein target from a SUMO-GFP fusion using a SENP1 or TEV protease immobilized with a magnetic bead.

[0058] Fig.22 is a gel image showing the cleavage of the SUMO protein target from a SUMO-GFP fusion using a SENP1 protease immobilized with a magnetic bead. The cleavage experiment was conducted sequentially four times, indicating a retention of SENP1 activity.

[0059] Fig. 23A is a cartoon schematic showing the structure of a tagged target protein (e.g., SUMO-tagged Histatin) that is to be cleaved according to some embodiments of the present invention using a protease (e.g., SENP1) immobilized with a magnetic bead.

[0060] Fig.23B is a gel image showing the cleavage of a protein target (e.g., the Pl 13 histatin peptide) from a SUMO fusion using a SENP1 protease immobilized with a magnetic bead.

[0061] Fig.23C is a graph showing a time course in hours of the cleavage a protein target (e.g., the Pl 13 histatin peptide) from a SUMO fusion using a SENP1 protease immobilized with a magnetic bead (ON = overnight).

[0062] Fig.23D is a gel image showing the cleavage of a protein target (e.g., the Pl 13 histatin peptide) from a SUMO fusion using a SENP1 protease immobilized with a magnetic bead. The cleavage experiment was conducted sequentially ten times, indicating a retention of SENP1 activity.DETAILED DESCRIPTION OF THE INVENTION

[0063] The present invention is explained in greater detail below. This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure which do not depart from the instant invention. Hence, the following specificationAttorney Docket No. 5218.262.WOis intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations and variations thereof.

[0064] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0065] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of a conflict in terminology, the present specification is controlling.

[0066] All publications, patent applications, patents, nucleotide sequences, amino acid sequences and other references mentioned herein are incorporated by reference to the extent consistent with the present disclosure.

[0067] As used in the description of the invention and the appended claims, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0068] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0069] The term “inhibit” or “reduce” or grammatical variations thereof as used herein refers to a decrease or diminishment in the specified level or activity of at least about 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more.

[0070] Nucleotide sequences are presented herein by single strand only, in the 5’ to 3’ direction, from left to right, unless specifically indicated otherwise. Nucleotides and amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the one-letter code, or the three-letter code, both in accordance with 37 C.F.R. §1.822 and established usage.Attorney Docket No. 5218.262.WO

[0071] Except as otherwise indicated, standard methods known to those skilled in the art may be used for production of recombinant and synthetic polypeptides, antibodies or antigenbinding fragments thereof, manipulation of nucleic acid sequences, production of transformed cells, the construction of rAAV constructs, modified capsid proteins, packaging vectors expressing the AAV rep and / or cap sequences, and transiently and stably transfected packaging cells. Such techniques are known to those skilled in the art. See, e.g., SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL 4th Ed. (Cold Spring Harbor, NY, 2012); F. M. AUSUBEL et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

[0072] As used herein, the terms “protein” and “polypeptide” are used interchangeably and encompass both peptides and proteins, unless indicated otherwise.

[0073] The term “fragment,” as applied to a polypeptide, will be understood to mean an amino acid sequence of reduced length relative to a reference polypeptide or amino acid sequence and having an amino acid sequence identical or almost identical (e.g., 90%, 92%, 95%, 98%, 99% identical) to the reference polypeptide or amino acid sequence. Such a polypeptide fragment according to the invention may be, where appropriate, included in a larger polypeptide of which it is a constituent. In some embodiments, such fragments can comprise, consist essentially of, and / or consist of peptides having a length of at least about 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, or more consecutive amino acids of a polypeptide or amino acid sequence according to the invention.

[0074] As used herein, the term “modified,” as applied to a polypeptide sequence, refers to a sequence that differs from a wildtype sequence due to one or more deletions, additions, substitutions, or any combination thereof.

[0075] As used herein “sequence identity” refers to the extent to which two optimally aligned polynucleotide or polypeptide sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids. “Identity” can be readily calculated by known methods including, but not limited to, those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W ., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991).Attorney Docket No. 5218.262.WO

[0076] “H” refers to a hydrogen atom. “C” refers to a carbon atom. “N” refers to a nitrogen atom. “O” refers to an oxygen atom. “Halo” refers to F, Cl, Br or I. The term “hydroxy,” as used herein, refers to an -OH moiety. “Cl” refers to a chlorine atom.

[0077] An “acyl” is intended to mean a group -C(O)-R, where R is a suitable substituent, such as alkyl, cycloalkyl, heterocycle, aryl, or heteroaryl. Examples of acyl include, but are not limited to, an acetyl group, a propionyl group, a butyroyl group, a benzoyl group, etc.

[0078] “Alkyl,” as used herein, refers to a straight or branched chain hydrocarbon containing from 1 or 2 to 10 or 20 or more carbon atoms (e.g., C2, C3, C4, C5, C6, C7, C8, C9, CIO, Cl 1, C12, C13, C14, C15, etc.). In some embodiments the alkyl can be a lower alkyl. "Lower alkyl" refers to a straight or branched chain alkyl having from 1 to 3, or from 1 to 5, or from 1 to 8 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3 -methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and the like. In some embodiments, alkyl groups as described herein are optionally substituted (e.g., from 1 to 3 or 4 times) with independently selected H, halo, hydroxy, acyl, alkyl, etc. Representative examples of halo substituted alkyls (e.g., haloalkyl, with a halo appended to the molecule through the alkyl group) include, but are not limited to, chlorine substituted alkyl.

[0079] The term “optionally substituted” indicates that the specified group is either unsubstituted, or substituted by one or more suitable substituents. A “substituent” that is “substituted” is an atom or group which takes the place of a hydrogen atom on the parent chain or cycle of an organic molecule, for example, H, halo, hydroxy, acyl, alkyl, etc.Constructs and Compositions

[0080] One aspect of the invention relates to a construct of Formula I:Formula Iwherein: Ri is an enzyme; Pi is present or absent, and when present is a peptide tag; Li is a first linker; and P2 is a polymer, wherein the " — " indicates covalent bonds.

[0081] In some embodiments, the enzyme is an anti-fouling enzyme. In some embodiments, the enzyme is an alcalase, a subtilisin, a tannase, an apolipoprotein, a lysozyme, a multicopper oxidase, axylanase, a protease, a cellulase, a laccase, and / or a chitinase. In some embodiments,Attorney Docket No. 5218.262.WOthe enzyme is apolipoprotein L3 (AP0L3). In some embodiments, the protease is thrombin, HRV14 3C, Tobacco Etch Virus (TEV) protease, factor Xa, SENP1, and / or enterokinase cleavage enzyme (EKT). In some embodiments, the protease is SENP1. In some embodiments, the enzyme is conjugated to a peptide tag (e.g., a GST tag and / or a His-tag).

[0082] In some embodiments, the enzyme comprises any one of SEQ ID NOs: 1-4 or 13. In some embodiments, the enzyme comprises a His-tag, is conjugated to a peptide tag with a linker, and comprises any one of SEQ ID NOs: 5-8 or 14. Functional fragments of these sequences may also be used as the enzyme and / or peptide tag. As used herein with respect to polypeptides, the term "functional fragment" or "active fragment" refers to polypeptide fragment that retains at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more of at least one biological activity (e.g., protease activity, oxidase activity, hydrolase activity, cellulase activity, chitinase activity, xylanase activity, and / or subtilase activity) of the full-length polypeptide. In some embodiments, the functional fragment has a higher level of at least one biological activity of the full-length polypeptide.

[0083] In some embodiments, the enzyme comprises one or more surface modifications (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 surface modifications or more). In some embodiments, the surface modification is a polyethylene glycol (PEG), a methyl, an ethyl, an isopropyl, an alkyl, and the like. In some embodiments, the PEG has a molecular weight between about 350 Daltons to about 20,000 Daltons (e.g., about 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, or 20000 Daltons).

[0084] In some embodiments, the enzyme has been optimized to increase its net surface charge ("supercharging"). As used herein, the term “supercharge” refers to any modification of a protein that results in the increase or decrease of the overall net surface charge of the protein. Modifications include, but are not limited to, alterations in amino acid sequence or addition of charged moieties (e.g., carboxylic acid groups, phosphate groups, sulfate groups, amino groups). In some embodiments, supercharging an enzyme comprises modifying the enzyme to mutate select residues (e.g., Asp, Glu, Arg, Lys, Asn, and / or Gin) of the enzyme into amino acid residues that are positively charged at physiological pH (e.g., Lys and / or Arg).SEQ ID NO: 1 APOL3 GLGQGWGWEASCFACLIRSCCQVVTFTFPFGFQGISQSLENVSGYYADARLEVGSTQ LRTAGSCSHSFKRSFLEKKRFTEEATKYFRERVSPVHLQILLTNNEAWKRFVTAAELPAttorney Docket No. 5218.262.WORDEADALYEALKKLRTYAAIEDEYVQQKDEQFREWFLKEFPQVKRKIQESIEKLRAL ANGIEEVHRGCTISNVVSSSTGAASGIMSLAGLVLAPFTAGTSLALTAAGVGLGAAS AVTGITTSIVEHSYTSSAEAEASRLTATSIDRLKVFKEVMRDITPNLLSLLNNYYEATQ TIGSEIRAIRQARARARLPVTTWRISAGSGGQAERTIAGTTRAVSRGARILSATTSGIFL ALDVVNLVYESKHLHEGAKSASAEELRRQAQELEENLMELTQIYQRLNPCHTHGGSSEQ ID NO: 2 Thermomyces lanuginosus Xylanase VGFTPVALAALAATGALAFPAGNATELEKRQTTPNSEGWHDGYYYSWWSDGGAQ ATYTNLEGGTYEISWGDGGNLVGGKGWNPGLNARAIHFEGVYQPNGNSYLAVYGW TRNPLVEYYIVENFGTYDPSSGATDLGTVECDGSIYRLGKTTRVNAPSIDGTQTFDQY WSVRQDKRTSGTVQTGCHFDAWARAGLNVNGDHYYQIVATEGYFSSGYARITVAD VGGGSSEQ ID NO: 3 Aspergillus clavatus Xylanase FAERAGTPSSTGWNNGYYYSFWTDNGGTVNYQNGNGGSYSVQWKDTGNFVGGKG WNPGSARTINYSGSFNPSGNAYLTVYGWTTNPLVEYYIVENYGTYNPGNGGTYRGS VYSDGANYNIYTATRYNAPSIEGDKTFTQYWSVRQSKRTGGTVTTANHFNAWAQL GMSLGTHNYQIVATEGYQSSGSSSITVYLEKLAAALESEQ ID NO: 4 Laccase LARRSFLQAAAGSLVLGLARAQGPSFPEPKVVRSQGGLLSLKLSATPTPLALAGQRA TLLTYGGSFPGPTLRVRPRDTVRLTLENRLPEPTNLHWHGLPISPKVDDPFLEIPPGES WTYEFTVPKELAGTFWYHPHLHGRVAPQLFAGLLGALVVESSLDAIPELREAEEHLL VLKDLALQGGRPAPHTPMDWMNGKEGDLVLVNGALRPTLVAQKATLRLRLLNASN ARYYRLALQDHPLYLIAADGGFLEEPLEVSELLLAPGERAEVLVRLRKEGRFLLQAL PYDRGAMGMMDMGGMAHAMPQGPSRPETLLYLIAPKNPKPLPLPKALSPFPTLPAP VVTRRLVLTEDMMAARFFINGQVFDHRRVDLKGQAQTVEVWEVENQGDMDHPFH LHVHPFQVLSVGGRPFPYRAWKDVVNLKAGEVARLLVPLREKGRTVFHCHIVEHED RGMMGVLEVGKLAAALESEQ ID NO: 5 Halo-tagged APOL3 MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAP THRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFH WAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGTAttorney Docket No. 5218.262.WOLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDW LHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEI ARWLSTLEISGGGSDGGSDGGSDMGLGQGWGWEASCFACLIRSCCQVVTFTFPFGF QGISQSLENVSGYYADARLEVGSTQLRTAGSCSHSFKRSFLEKKRFTEEATKYFRERV SPVHLQILLTNNEAWKRFVTAAELPRDEADALYEALKKLRTYAAIEDEYVQQKDEQ FREWFLKEFPQVKRKIQESIEKLRAL ANGIEEVHRGCTISNVVS S STGAASGIMSL AGL VLAPFTAGTSLALTAAGVGLGAASAVTGITTSIVEHSYTSSAEAEASRLTATSIDRLK VFKEVMRDITPNLLSLLNNYYEATQTIGSEIRAIRQARARARLPVTTWRISAGSGGQA ERTIAGTTRAVSRGARILSATTSGIFLALDVVNLVYESKHLHEGAKSASAEELRRQAQ ELEENLMELTQIYQRLNPCHTHGGSHHHHHH—SEQ ID NO: 6 Halo-tagged Thermomyces lanuginosus Xylanase MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAP THRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFH WAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGT LPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDW LHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEI ARWLSTLEISGGGSDGGSDGGSDMVGFTPVALAALAATGALAFPAGNATELEKRQT TPNSEGWHDGYYYSWWSDGGAQATYTNLEGGTYEISWGDGGNLVGGKGWNPGLN ARAIHFEGVYQPNGNSYLAVYGWTRNPLVEYYIVENFGTYDPSSGATDLGTVECDG SIYRLGKTTRVNAPSIDGTQTFDQYWSVRQDKRTSGTVQTGCHFDAWARAGLNVN GDHYYQIVATEGYF S SGYARIT VAD VGGGSHHHHHHSEQ ID NO: 7 Halo-tagged Aspergillus clavatus Xylanase MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAP THRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFH WAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGT LPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDW LHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEI ARWLSTLEISGGGSDGGSDGGSDFAERAGTPSSTGWNNGYYYSFWTDNGGTVNYQ NGNGGSYSVQWKDTGNFVGGKGWNPGSARTINYSGSFNPSGNAYLTVYGWTTNPL VEYYIVENYGTYNPGNGGTYRGSVYSDGANYNIYTATRYNAPSIEGDKTFTQYWSV RQSKRTGGTVTTANHFNAWAQLGMSLGTHNYQIVATEGYQSSGSSSITVYLEKLAA ALEHHHHHHAttorney Docket No. 5218.262.WOSEQ ID NO: 8 Halo-tagged Laccase MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAP THRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFH WAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGT LPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDW LHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEI ARWLSTLEISGGGSDGGSDGGSDMLARRSFLQAAAGSLVLGLARAQGPSFPEPKVV RSQGGLLSLKLSATPTPLALAGQRATLLTYGGSFPGPTLRVRPRDTVRLTLENRLPEP TNLHWHGLPISPKVDDPFLEIPPGESWTYEFTVPKELAGTFWYHPHLHGRVAPQLFA GLLGALVVESSLDAIPELREAEEHLLVLKDLALQGGRPAPHTPMDWMNGKEGDLVL VNGALRPTLVAQKATLRLRLLNASNARYYRLALQDHPLYLIAADGGFLEEPLEVSEL LLAPGERAEVLVRLRKEGRFLLQALPYDRGAMGMMDMGGMAHAMPQGPSRPETL LYLIAPKNPKPLPLPKALSPFPTLPAPVVTRRLVLTEDMMAARFFINGQVFDHRRVDL KGQAQTVEVWEVENQGDMDHPFHLHVHPFQVLSVGGRPFPYRAWKDVVNLKAGE VARLLVPLREKGRTVFHCHIVEHEDRGMMGVLEVGKLAAALEHHHHHH

[0085] In some embodiments, the peptide tag is useful for purifying polypeptides (e.g., antifouling peptides or other enzymes) and / or increasing the activity and / or stability of the polypeptides when they are conjugated to a linker. In some embodiments, the peptide tag is a small ubiquitin modifying protein (SUMO) tag, a SpyTag / SpyCatcher peptide or component thereof (e.g., a SpyTag peptide (i.e., a peptide that binds to SpyCatcher) or a SpyCatcher peptide (i.e., a modified domain from S. pyogenes surface protein)), a SNAP -tag® (i.e., a modified O6-alkylguanine-DNA alkyltransferase (AGT) enzyme), a HaloTag® (i.e., a modified haloalkane dehalogenase), and / or a CLIP -tag™ (i.e., a modified AGT enzyme). See, e.g., Tan et al. "Kinetic controlled Tag-catcher interactions for directed covalent protein assembly," PLoS One ll(10):e0165074 (2016); Los et al. "HaloTag: a novel protein labeling technology for cell imaging and protein analysis," ACS Chemical Biology. 3(6): 373-82 (2008); Zakeri et al., "Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesion," PNAS USA 109(12):E690-E697 (2012); Juillerat et al. "Directed evolution of O6-alkylguanine-DNA alkyltransferase for efficient labeling of fusion proteins with small molecules in vivo," Chemistry & Biology 10(4):313-317 (2003); Gautier et al. "An engineered protein tag for multiprotein labeling in living cells," Chemistry & BiologyAttorney Docket No. 5218.262.WO15(2): 128-136 (2008); Butt et al. "SUMO fusion technology for difficult-to-express proteins," Protein Expr Purif 43(1): 1-9 (2005).

[0086] In some embodiments, the peptide tag is useful for linking the enzyme and the polymer. For example, in some embodiments, the peptide tag is a SpyTag / SpyCatcher peptide or component thereof (e.g., a SpyTag peptide (i.e., a peptide that binds to SpyCatcher) or a SpyCatcher peptide (i.e., a modified domain from S. pyogenes surface protein)) and the SpyTag is covalently bonded to the enzyme and the SpyCatcher is covalently bonded to the polymer.

[0087] In some embodiments, the HaloTag® comprises SEQ ID NO: 9. In some embodiments, the peptide tag is conjugated to the enzyme with a peptide linker (e.g., a serine-glycine linker). In some embodiments, a serine-glycine linker comprises SEQ ID NO: 10 or 11.SEQ ID NO: 9 Halo tag MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAP THRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFH WAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGT LPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDW LHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEI ARWLSTLEISEQ ID NO: 10 Serine-glycine linker 1SGGGSDGGSDGGSDMSEQ ID NO: 11 Serine-glycine linker 2(GGSD)i-sSEQ ID NO: 12 Leucine linker(L)4-20SEQ ID NO: 13 SENP1 MEFPEITEEMEKEIKNVFRNGNQDEVLSEAFRLTITRKDIQTLNHLNWLNDEIINFYM NMLMERSKEKGLPSVHAFNTFFFTKLKTAGYQAVKRWTKKVDVFSVDILLVPIHLG VHWCLAVVDFRKKNITYYDSMGGINNEACRILLQYLKQESIDKKRKEFDTNGWQLF SKKSQEIPQQMNGSDCGMFACKYADCITKDRPINFTQQHMPYFRKRMVWEILHRKL LAttorney Docket No. 5218.262.WOSEQ ID NO: 14 Halo-SENPl MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAP THRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFH WAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGT LPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDW LHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEI ARWLSTLEISGGGSDGGSDGGSDMEFPEITEEMEKEIKNVFRNGNQDEVLSEAFRLTI TRKDIQTLNHLNWLNDEIINFYMNMLMERSKEKGLPSVHAFNTFFFTKLKTAGYQA VKRWTKKVDVFSVDILLVPIHLGVHWCLAVVDFRKKNITYYDSMGGINNEACRILL QYLKQESIDKKRKEFDTNGWQLFSKKSQEIPQQMNGSDCGMFACKYADCITKDRPI NFTQQHMPYFRKRMVWEILHRKLLGGSHHHHHHSEQ ID NO: 15 SUMO GSGDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIHFKVKMTTHLKKLKESYCQRQG VPMNSLRFLFEGQRIADNHTPKELGMEEEDVIEVYQEQTGG SEQ ID NO: 16 SUMO-P113 MHHHHHHGSGDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIHFKVKMTTHLKKLK ESYCQRQGVPMNSLRFLFEGQRIADNHTPKELGMEEEDVIEVYQEQTGGAKRHHGY KRKFHSEQ ID NO: 17 SpyTagAHIVMVDAYKPTK

[0088] Another aspect of the present invention is an expression cassette or a vector optionally codon optimized for expression in an organism, the expression cassette or vector comprising a polynucleotide encoding an enzyme as described herein or an enzyme as described herein conjugated to a peptide tag as described herein, which may be codon-optimized for expression in the organism.

[0089] In some embodiments, the linker is formed from a benzoate linker. In some embodiments, the linker is formed from a benzoate linker of Formula A:Attorney Docket No. 5218.262.WOwherein: n is an integer of from 1 to 10; and R2 and R3 are each independently a formyl, hydroxy, hydroxyalkyl, halo, or haloalkyl.

[0090] In some embodiments, the benzoate linker is prop-2-yn-l-yl 3,4-diformylbenzoate or prop-2 -yn- 1 -yl 3 -chloropropylbenzoate:prop-2-yn-1 -yl 3-chloropropylbenzoate

[0091] In some embodiments, the benzoate linker is prop-2-yn-l-yl 3-chloropropylbenzoate and the enzyme or peptide tag, when present, is conjugated to the linker by alkyl halide displacement.

[0092] In some embodiments, the polymer is a polymer formed by a click chemistry reaction. In some embodiments, the polymer is a [3+2] azide-alkyne cycloaddition polymer (e.g., an alkyne-azide polymer), a Diels- Alder based polymer, a thiol-ene based polymer (e.g., a thiolalkene radical addition based polymer), and / or a strain-promoted azide-alkyne cycloaddition (SPAAC) based polymer. In some embodiments, the polymer is formed by combining a first monomer comprising at least two azide groups and a second monomer comprising at least two activated alkyne groups. In some embodiments, the first monomer comprises a compound of Formula B:Attorney Docket No. 5218.262.WOFormula Bwherein Xi and X2 are each independently C, N, or O; and n is an integer of from 1 to 10. In some embodiments, the second monomer comprises a compound of Formula C:Formula Cwherein one of Ri or R2 is N3 and the other is hydroxy; one of R3 or R4 is N3 and the other is hydroxy, and n is an integer from 0 to 10. In some embodiments, the second monomer comprises a compound of Formula D:Formula Dwherein one of Ri or R2 is N3 and the other is hydroxy; one of R3 or R4 is N3 and the other is hydroxy, and n is an integer from 0 to 10. In some embodiments, the first monomer comprises ethane- 1,2-diyl dipropiolate:ethane-1,2-diyl dipropiolateand the second monomer comprises 2,2-bis(azidomethyl)propane-l,3-diol:2,2-bis(azidomethyl)propane-1 ,3-diol jn someembodiments, the second monomer comprises:Attorney Docket No. 5218.262.WO

[0093] In some embodiments, the linker is conjugated to the polymer by click chemistry (e.g., azide-alkyne Huisgen cycloaddition). In some embodiments, the construct has a formula:

[0094] In some embodiments, the polymer is a protein polymer. In some embodiments, the polymer is formed from cross-linked protein (e.g., oxidative cross-linked protein). In some embodiments, the protein comprises one or more post-translational modifications. In some embodiments, the post-translational modification comprises modification of one or more tyrosine residues to 3,4-dihydroxy-L-phenylalanine (L-DOPA). In some embodiments, the protein is mussel adhesive protein (MAP). In some embodiments, the MAP is mussel foot protein 3 (mfp-3 or fp-3; SEQ ID NO: 25), mussel foot protein 151 (mfp-151 or fp-151; SEQ ID NO: 26), mussel foot protein 131 (mfp-131 or fp-131; SEQ ID NO: 27), Mytilus galloprovincialis foot protein type-5 (Mgfp-5; SEQ ID NO: 28), Perna viridis foot protein 5p (PVFP-5P; SEQ ID NO: 29), or any combination thereof.

[0095] One aspect of the invention relates to a composition comprising a xerogel (e.g., a silica xerogel) and an enzyme, optionally wherein the xerogel integrates (e.g., encapsulates) the enzyme. In some embodiments, the xerogel is a tetramethyl orthosilicate (TMOS) gel or a tetraethyl orthosilicate (TEOS) gel. In some embodiments, the xerogel comprises a composition comprising an orthosilicate (e.g., tetraethyl orthosilicate (TEOS) or tetramethylorthosilicate (TMOS)), a silane (e.g., triethoxyoctylsilane or (3-chloropropyl)triethoxysilane), ethanol, and a catalytic acid (e.g., hydrochloric acid, acetic acid, or nitric acid) or catalytic base (e.g., sodium hydroxide or aqueous ammonia). In some embodiments, the orthosilicate and the silane are present in the xerogel in a molar ratio of about 3:1 to about 1:3 (orthosilicate : silane) (e.g., about 3:1, 2.5:1, 2:1, 1.5:1, 1:1, 1:1.5: 1:2, 1:2.5, or 1:3). In some embodiments, the xerogel further comprises a second orthosilicate and / or a second silane. In some embodiments, the orthosilicate, the second orthosilicate, and the silane are present in the xerogel in an amount of about 3:1:1, 1:3:1, 1:1:3, 3:3:1, 3:1:3, 1:3:3, 2:1:1, 1:2:1, 1:1:2, 2:2:1, 2:1:2, 1:2:2, 1:1:1, or any variation thereof (first orthosilicate : second orthosilicate : silane). In some embodiments, the orthosilicate, the first silane, and the second silane are present in the xerogel in an amount of about 3:1:1, 1:3:1, 1:1:3, 3:3:1, 3:1:3, 1:3:3,Attorney Docket No. 5218.262.WO2:1:1, 1:2:1, 1:1:2, 2:2:1, 2:1:2, 1:2:2, 1:1:1, or any variation thereof (orthosilicate : first silane : second silane). In some embodiments, the xerogel comprises a composition comprising TEOS, triethoxyoctylsilane, ethanol, and hydrochloric acid; TEOS, (3-chloropropyl)triethoxysilane, ethanol, and hydrochloric acid; or TMOS, (3-chloropropyl)triethoxysilane, ethanol, and hydrochloric acid.

[0096] One aspect of the invention relates to a composition comprising a hydrogel and an enzyme, optionally wherein the hydrogel integrates (e.g., encapsulates) the enzyme as described herein. In some embodiments, the hydrogel is an alginate hydrogel, optionally wherein the alginate hydrogel is a cross-linked alginate hydrogel. In some embodiments, the crosslinking is ionic crosslinking with a calcium salt (e.g., calcium chloride) or other bivalent metal cation (e.g., Mn(II), Fe(II), Co(II), Ni(II), Cu(II), Zn(II), Pd(II), or Mg(II)). In some embodiments, the crosslinking is covalent crosslinking (e.g., with glutaraldehyde, genipin, 1-Ethyl-3 -(3 -dimethylaminopropyl) carbodiimide and N-hydroxy succinimide, procyanidin, nordihydroguaiaretic acid, and the like).

[0097] One aspect of the invention relates to a construct of Formula II:Formula IIwherein: Ri is an enzyme; Pi is present or absent, and when present is a peptide tag; Li is a first linker; and Ii is an immobilization material, wherein the " — " indicates covalent bonds.

[0098] As used herein, the term "immobilization material" is intended to mean a material useful for interacting with (e.g., binding to) an immobilization surface. In some embodiments, the immobilization material is a polymer (e.g., a polymer described herein with respect to Formula I). In some embodiments, the immobilization material is a bead (e.g., a magnetic bead and / or an agarose bead (e.g., a Sepharose™ bead)), a nanoparticle (e.g., a superparamagnetic iron oxide nanoparticle (SPION)), polyvinyl chloride (PVC) surface, a fiberglass surface, a steel surface, and / or a metal alloy surface. For example, a magnetic bead may be an immobilization material that can be immobilized on a magnetic surface. In some embodiments, magnetic beads have a diameter of from about 0.1 to about 5 micrometers. In some embodiments, the beads may have a polymeric or silica coating, which coating may be functionalized with active groups for coupling to Li.

[0099] In some embodiments, the linker comprises a compound of Formula D:Attorney Docket No. 5218.262.WOFormula Dwherein n is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0100] In some embodiments, the construct of Formula II has the formula:enzyme |_|peptide tag^^ —immobilization material

[0101] Also provided herein is a composition comprising a construct as described herein. In some embodiments, the composition comprises two or more constructs as described herein, wherein the two or more constructs each independently comprise an enzyme that is an alcalase, a subtilisin, a tannase, an apolipoprotein, a lysozyme, a multicopper oxidase, a xylanase, a protease, a cellulase, a laccase, and / or a chitinase (i.e., the composition comprises an alcalase, a subtilisin, a tannase, an apolipoprotein, a lysozyme, a multicopper oxidase, a xylanase, a protease, a cellulase, a laccase, and / or a chitinase).

[0102] In some embodiments, the composition comprises the construct in an amount of about 0.5% to about 99% (e.g., about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or about 99%) by weight of the composition.

[0103] In some embodiments, the composition further comprises water, an organic solvent, a pigment, an antifoaming agent, a thickener, a surfactant, a resin, a leveling agent, a curing agent, or any combination thereof.

[0104] In some embodiments, the composition is stable at about -20 °C to about 50 °C (e.g., about -20, -15, -10, -5, 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 °C) for about 1 month to about 24 months (e.g., about 1, 2, 3, 4, 5, 6, 7 ,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months).

[0105] In some embodiments, the composition is stable in non-salinized water (e.g., fresh water) such as a river, lake, pond, lagoon, swamp, and the like. In some embodiments, the composition is stable in salinized water (e.g., salt water, ocean water, sea water, etc.) such as an ocean, a salt-water lake, a salt-water lagoon, and the like. In some embodiments, the composition may be used as a paint to coat a surface.The terms "stable" and "stability" when used herein in relation to an enzyme and / or composition of the present invention are meant to indicate a retention of the desired activity (e.g., anti -fouling activity, protease activity, decolorizing activity, polymerizing activity,Attorney Docket No. 5218.262.WOclarifying activity, and the like) of the enzyme and / or composition after a desired period of time, e.g., about 1 month to about 12 months (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months) or about 1 year to about 10 years (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years). In some embodiments, a stable enzyme and / or composition of the present invention retains about 5% to about 50% (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%) of the desired activity after a desired period of timeMethods of Use

[0106] One aspect of the invention relates to a method of reducing biological fouling on a surface, the method comprising applying a composition as described herein to said surface, thereby reducing biological fouling as compared to a surface not comprising the composition. In some embodiments, the biological fouling on the surface comprising the composition is reduced by about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to the surface not comprising the composition.

[0107] Another aspect of the invention is a method of reducing mold and / or mildew growth on a surface, the method comprising applying a composition as described herein to said surface, thereby reducing mold and / or mildew growth as compared to a surface not comprising the composition. In some embodiments, the mold and / or mildew growth on the surface comprising the composition is reduced by about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to the surface not comprising the composition.

[0108] Another aspect of the invention is a method of reducing marine growth on a surface, the method comprising applying a composition as described herein to said surface, thereby reducing marine growth as compared to a surface not comprising the composition. In some embodiments, the marine growth on the surface comprising the composition is reduced by about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to the surface not comprising the composition. In some embodiments, the marine growth comprises barnacles, mussels, tube worms, algae, grass, weeds, and the like.

[0109] In some embodiments, the method comprises applying the composition to the surface from every 1 week to about every 52 weeks (e.g., about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, or every 52 weeks).

[0110] In some embodiments, the surface is a surface on a marine vessel (e.g., a boat hull, submarine hull, deck, bridge, keel, hatch, cushion, seat, propeller, rudder, mast, and / or bilge). In some embodiments, the surface on the marine vessel is below the waterline (e.g., wherein the composition reduces biological fouling). In some embodiments, the surface on the marine vessel is above the waterline (e.g., wherein the composition reduces mold, mildew, and / orAttorney Docket No. 5218.262.WOmarine growth). In some embodiments, the surface is a water inlet, a pipe (e.g., a water pipe and / or an oil pipe), a nozzle (e.g., a sprinkler nozzle), a cable (e.g., an underwater cable), a manufacturing apparatus (e.g., a paper manufacturing apparatus), a food processing apparatus, construction equipment (e.g., underwater construction equipment), a desalination apparatus, a bioreactor (e.g., a membrane bioreactor), a water cooling system (e.g., a power station water cooling system), a drug delivery device (e.g., a microelectrochemical drug delivery device), a well, a ventilation component (e.g., an air duct and / or an air conditioning component), a humidifier, a dehumidifier, a heat exchanger, and / or a heatsink. One of ordinary skill in the art would understand that any surface that is susceptible to biological fouling, mold, mildew, and / or marine growth could benefit from a composition as described herein; such surfaces include, but are not limited to, any surface that is submerged in water, routinely exposed to water, and / or in a humid (e.g., an environment with a humidity level about 60%).[OHl] Another aspect of the invention is a method of making an antifouling coating, the method comprising providing an enzyme, optionally wherein the enzyme is conjugated to a peptide tag; conjugating enzyme or the peptide tag to a linker; and conjugating the linker to a polymer by click chemistry. In some embodiments, the conjugating the enzyme or the peptide tag to a linker is carried out by alkyl halide displacement. In some embodiments, the conjugating the linker to a polymer by click chemistry is carried out by azide-alkyne Huisgen cycloaddition.

[0112] Another aspect of the invention relates to a method of making an antifouling coating, the method comprising combining an enzyme and a composition that, when cured, will produce a xerogel (e.g., a silicate xerogel), wherein the composition can be applied to a surface, thereby providing an antifouling coating.

[0113] Another aspect of the invention relates to a method of making an antifouling coating, the method comprising providing an enzyme and encapsulating the enzyme in a hydrogel (e.g., an alginate hydrogel), wherein the hydrogel encapsulating the enzyme can be applied to a surface, thereby providing an antifouling coating.

[0114] Another aspect of the invention relates to a method of decolorizing a dye, the method comprising adding a construct or composition as described herein, and copper (II) sulfate, to a composition comprising the dye (e.g., comprising an aqueous buffer), thereby decolorizing the dye. In some embodiments, the dye comprises an indigo dye (e.g., indigo carmine). In some embodiments, the construct is recovered from the composition comprising the dye, and is reused in a second decolorizing method. In some embodiments, the construct is provided immobilized and reused with new compositions comprising dye, such as in a flow reactor. SuchAttorney Docket No. 5218.262.WOapplications may be useful for decolorizing textile effluents. In some embodiments, the construct may be reused two, three, four, or five times, or more.

[0115] Another aspect of the invention relates to a method of polymerizing phenolic compounds, the method comprising adding a construct or composition as described herein, to a composition comprising said phenolic compounds, whereby the phenolic compounds are oxidatively polymerized. In some embodiments, the phenolic compounds comprise catechol and / or hydroquinone.

[0116] Another aspect of the invention relates to a method of clarifying a cloudy aqueous solution, the method comprising adding a construct or composition as described herein, to the aqueous solution, whereby the solution is clarified. In some embodiments, the cloudy aqueous solution is a fruit juice (e.g., unfiltered apple juice).

[0117] Another aspect of the invention relates to a method of producing a purified recombinant protein, the method comprising: (a) preparing a vector comprising a polynucleotide encoding fusion protein comprising the recombinant protein, a peptide tag (e.g., a SUMO tag), and a purification tag; (b) transfecting a cell (e.g., a bacterial cell) with the vector, wherein the cell thereby produces the fusion protein; (c) isolating the fusion protein using the purification tag; (d) combining the isolated fusion protein with a construct of Formula II, wherein the enzyme of the construct is a protease and said protease cleaves the fusion protein to provide the recombinant protein; and (e) purifying the recombinant protein, thereby providing the purified recombinant protein.

[0118] In some embodiments, the fusion protein has the structure P-S-R, wherein "P" is the purification tag, "S" is the peptide tag, "R" is the recombinant protein, and indicates a covalent bond and / or a linker. In some embodiments, the construct of Formula II cleaves a covalent bond between the recombinant protein and the peptide tag, thereby producing the recombinant protein and a second fusion protein comprising the purification tag and the peptide tag. In some embodiments, the protease in the construct of Formula II produces cleaves the fusion protein such that the recombinant protein is produced with its native sequence (i.e., contains no extra amino acid residues). In some embodiments, the purification tag is a His-tag (e.g., a 6X - 10X His-tag; SEQ ID NO: 19), a FLAG-tag (SEQ ID NO: 20), an HA-tag (SEQ ID NO: 21), Strep-tag (SEQ ID NO: 22), a GST-tag (SEQ ID NO: 23), and / or a V5-tag (SEQ ID NO: 24)

[0119] Having described the present invention, the same will be explained in greater detail in the following examples, which are included herein for illustration purposes only, and which are not intended to be limiting to the invention.Attorney Docket No. 5218.262.WOEXAMPLESExample 1

[0120] Anti-fouling coatings hold enormous benefits for both commercial and recreational boaters but are currently under intense scrutiny for their lack of environmental compatibility. This has created demand for a new generation of anti-fouling coatings that deliver the same benefits as existing coatings without the release of toxic levels of heavy metals. Enzymatic anti-fouling coatings would present a solution to this problem, as they would not rely on high levels of metals and their degradation products are non-toxic. However, the application of enzymatic solutions to fouling has been hampered by the lack of effective chemical tools to incorporate them into new anti-fouling coatings.

[0121] Recently published studies have demonstrated that enzyme ‘paints’ (enzyme-polymer hybrid materials) can tether enzymes to polymer matrices via click chemistry (Bartlett et al., 2021). These materials can retain enzymatic activity when applied as a surface coating and retain activity in a simulated seawater environment (Baker-Branstetter et al., 2023). However, other approaches may show additional benefits.

[0122] This work investigates the ability of a chloropropylbenzoate linker to capture HaloTag® labeled enzymes. Without wishing to be bound by any particular theory, it is anticipated that this strategy will enhance retention of enzymatic activity in immobilized form.

[0123] Laccase, trypsin, APOL3, lysozyme, and xylanase were conjugated with a Halo tag and the expression of this construct was analyzed by SDS-PAGE (Figs. 1-3).

[0124] We will selectively modify enzymes, both tagged or not, such as lysozymes, xylanases, and proteases with a diformylbenzoate linker (e.g., prop-2-yn-l-yl 3,4-diformylbenzoate) and couple these to a polymer matrix by click chemistry. We will also clone, express, and purify HaloTag® fusions of lysozymes, xylanases, and proteases and couple these to a polymer matrix via alkyl halide displacement from chloropropylbenzoate linker (e.g., prop-2-yn-l-yl 3-chloropropylbenzoate). We will then assay the resulting immobilized preparations for retention of enzymatic activity.

[0125] The activity of an immobilized, Halo-tagged xylanase is shown in Fig. 4. Briefly, recombinant Halo-xylanase protein was captured within a polymer matrix in 96-well plates using a reactive prop-2-yn-l-yl 3,4-diformylbenzoate linker or a reactive prop-2-yn-l-yl 3-chloropropylbenzoate linker in a 96-well plate and assayed repeatedly using the XylX6 colorimetric assay system (Megazyme Inc.), a schematic of this assay is shown below. GoodAttorney Docket No. 5218.262.WOretention of xylanase activity was observed with both polymer matrices, while nonspecific coating of the 96-well plate led to a significant loss of xylanase activity over time.XylX6 colorimetric assay mechanism

[0126] We will assay immobilized enzyme activity in artificial seawater under different time conditions (e.g., 5, 15, 30, 60 or more days) and over multiple storage conditions (e.g., 4, 20, and 37 °C). These assays will demonstrate longevity and robustness of the active enzyme preparations and be performed in microwell plates coated with enzyme-polymer composite.

[0127] Preliminary tests have been conducted to examine xylanase activity in organic polymer painted on boat hull samples from two different boat manufacturers (Fig. 17). Briefly, a recombinant Halo-Xylanase protein was captured within a polymer matrix and painted onto fiberglass hull samples and assayed repeatedly using the XylX6 colorimetric assay system (Megazyme Inc.). Good retention of Xylanase activity was observed with the painted enzyme-polymer matrices on two different hull sample types. It was seen that enzyme activity wasAttorney Docket No. 5218.262.WOmaintained on fiberglass hull samples, and that covalent attachment superior to non-specific attachment of enzymes.

[0128] We will conduct anti-biofouling assays using brackish water obtained from local marine habitats. Laboratory plasticware coated with enzyme-polymer matrix will be incubated with brackish water and biofouling of treated surfaces assayed by crystal violet staining (Kamimura et al., 2022).

[0129] Preliminary tests have been conducted to examine Anti-algae activity of enzymes on algae-coated hull samples (Fig. 18). Briefly, recombinant Halo-Xylanase and Halo-Laccase proteins were applied directly onto algae coated fiberglass hull samples post-incubation in brackish water for one month (untreated water from the Pamlico Sound, Belhaven, NC). Enzymes facilitated water-debridement assisted algae removal versus buffer only controls. The combination of xylanase and laccase enzymes gave superior algae release. It was seen that topical application of enzymes facilitated algae release and demonstrated its use as a spray coating. Further, enzyme activity was retained after incubation in brackish water (Fig. 19).Briefly, recombinant Halo-Xylanase and Halo-Laccase proteins were captured within a polymer matrix and painted onto fiberglass hull samples and incubated in brackish water for 8 days (Pamlico Sound, Belhaven, NC). Enzyme activity was retained with the painted enzyme-polymer matrices on two different hull sample types and outperformed non-specific enzyme attachment.ReferencesBaker-Branstetter, R. W., Bartlett, M. E., Shuler, S. A., & Messersmith, R. E. (2023). Covalent immobilization of xylanase and lysing complex into polymer scaffolds with longterm activity retention. Journal of Coatings Technology and Research, 20(3), 973-978.Bartlett, M. E., Shuler, S. A., Rose, D. J., Gilbert, L. M., Hegab, R. A., Lawton, T. J., & Messersmith, R. E. (2021). Paintable proteins: biofunctional coatings via covalent incorporation of proteins into a polymer network. New Journal of Chemistry, 45(47), 22084-22092.Deane, D. T., Cope, T. A., Schulz, A. M., Bennett, E. T., & Hughes, R. M. (2023). Design, Heterologous Expression, and Application of an Immobilized Protein Kinase. Bioconjugate Chemistry, 37(1), 204-211.Durr, S., & Thomason, J. C. (2009). Biofouling. In Biofouling (pp. i-xix).Attorney Docket No. 5218.262.WOKamimura, R., Kanematsu, H., Ogawa, A., Kogo, T., Miura, H., Kawai, R., Hirai, N., Kato, T., Yoshitake, M., & Barry, D. M. (2022). Quantitative Analyses of Biofilm by Using Crystal Violet Staining and Optical Reflection. Materials, 75(19).Nehring, S. (2001). After the TBT era: Alternative anti-fouling paints and their ecological risks. Senckenbergiana Maritima, 37(2), 341-351.Norris, J. L., Patel, T., Dasari, A. K. R., Cope, T. A., Lim, K. H., & Hughes, R. M. (2020). Covalent and non-covalent strategies for the immobilization of Tobacco Etch Virus protease (TEVp) on superparamagnetic nanoparticles. Journal of Biotechnology, 322, 1-9.Schiff, K., Diehl, D., & Valkirs, A. (2004). Copper emissions from antifouling paint on recreational vessels. Marine Pollution Bulletin, 48(3), 371-377.Ytreberg, E., Lagerstrbm, M., Nou, S., & Wiklund, A.-K. E. (2021). Environmental risk assessment of using antifouling paints on pleasure crafts in European Union waters. Journal of Environmental Management, 281, 111846.Example 2

[0130] Laccases are copper-binding metalloenzymes with a wide range of possible applications in applied biotechnology. These applications cover a wide variety of industries, including textiles, food and beverage, and pulp and paper. However, widespread application of laccases to longstanding problems in applied biotechnology has been hampered by the relative scarcity of robust laccase formulations. One possible solution to these problems is through the development of immobilized laccases that can be recycled for multiple rounds of application.

[0131] An immobilizable laccase formulation was created by fusing a laccase from Thermus thermophilus with a HaloTag® protein. This protein fusion was expressed in E. coli and its enzymatic activity was confirmed in assays with dimethoxyphenol (DMP) and other phenolic substrates.

[0132] Here, the applicability of this laccase was assessed in both immobilized and nonimmobilized form with regard to three problems that have been proposed as potential industrial applications of laccases: decolorization of dyes in textile wastewater, the synthesis of short polymers from natural phenolic substrates, and clarification of raw beverages. Temperature and pH dependence of laccase activity were also explored.

[0133] Overall, the studies confirmed that laccase immobilization is a promising route to enhancing the application of laccases to long standing problems in industry and biotechnology.

[0134] Fusion proteins have long been used by researchers for a variety of functions including improvement of protein expression, better stability, and condensation of function to a singleAttorney Docket No. 5218.262.WOpolypeptide chain [1,2], These types of proteins serve to connect other proteins to solid supportcan be used in food manufacturing, drugs, and other biotechniques [3], This technique effectively immobilizes a particular protein of interest (POI), permitting the use of unique catalytic systems with a great range of uses. However, there is no set method for designing an immobilized fusion protein that yields consistent, robust results [4,5],

[0135] We are particularly interested in the creation of immobilized enzymes using a fusion protein approach. Laccases are copper binding proteins with oxidoreductase activity with numerous applications in biotechnology but relatively few examples of immobilized thermophilic laccases have been previously reported (6). In this work, we use a laccase from Thermus thermophilus HB2 originally characterized by Myazaki (7). This laccase had an optimal reaction temperature of 92 °C and a half-life at 80 °C of over 14h (7), making it an ideal candidate for demanding industrial applications. To immobilize this laccase, we utilize the HaloTag® protein as a fusion partner. HaloTag enables formation of a covalent bond to a haloalkane containing substrate, such as HaloLink resin (Promega, Inc.) [8,9], In this work, we investigated the activity of our laccase - HaloTag® fusion in both free and immobilized forms in three applications of industrial relevance: the decolorization of textile dyes, the clarification of beverages, and the synthesis of short polymers from phenolic substrates. This work builds upon our previous efforts in the area of enzyme immobilization [10, 11],

[0136] Materials and Methods

[0137] Halo-Laccase Expression: Heterologous expression performed in E. coli (BL21-CodonPlus(DE3)-RP (Agilent)). Starter cultures used to inoculate 50 mL Lysogeny Broth (LB) containing Kanamycin antibody (LB-Kan) and 0.2% dextrose, incubated at 30 °C overnight. Overnight cultures used to inoculate 500 mL LB-Kan cultures, grown at 37 °C (shaking at 280 rpm) until reaching an OD600 ~ 0.600. Cultures were induced with 0.4 mM IPTG and incubated at 17 °C overnight (shaking at 280 rpm). 1 mM Copper (I) chloride was included in expression cultures. Samples were lysed with B-PER Complete (ThermoFisher) supplemented with 10 mM Copper chloride and purified with cobalt resin followed by size exclusion chromatography (Akta FPLC).

[0138] Immobilization Conditions: Immobilization performed with purified Halo-Laccase (10 mL of 0.500 mg / mL protein) and 1 mL of resuspended magnetic HaloTag® beads (Promega, Inc.) via incubation at room temperature for 2 h on a sample rocker. Beads washed 3 x 5 mL with PBS after washing and resuspended in 2 mL of glycerol storage buffer.

[0139] Dimethoxy phenol (DMP) assays: DMP (Sigmal Aldrich) was dissolved in buffer (pH 5 NaOAc or pH 7.6 Tris buffered saline (TBS)) at a final concentration of 2 mM and usedAttorney Docket No. 5218.262.WOimmediately. Absorbances were measured with an M2e plate reader (Molecular Devices) at 470 nm. Readings were taken every 30 seconds for 30 minutes. Initial velocities determined by the linear region of the curve.

[0140] Indigo carmine assays: Indigo carmine (Sigma- Aldrich) was dissolved in TBS, pH 7.6 at a concentration of 0.2 mg / mL. Copper (II) sulfate was added at a final concentration of 50 uM from a 1 mM stock solution in distilled water. Reactions were initiated by addition of laccase (100 pL of 0.898 mg / mL laccase solution or 100 pL of immobilized bead suspension) and incubated for 90 min in a 65 °C heat block in 1.5 mL centrifuge tubes.

[0141] Polyphenol assays: Polyphenols (catechol, resorcinol, hydroquinone) were purchased from Sigma-Aldrich and used without further purification. Solutions were made at 50 mM concentration in a NaOAc buffer (0.1 M, pH 5.0). Reactions were initiated by addition of laccase (100 pL of 0.898 mg / mL laccase solution or 100 pL of immobilized bead suspension) and incubated overnight in a heated shaker (600 rpm) overnight.

[0142] Beverage assays: Unfiltered apple juice (Martinelli’s) was purchased locally and used without further purification. Reactions were initiated by addition of laccase (50 pL of 0.343 mg / mL laccase solution) and incubated for 1 h at the indicated temperatures.

[0143] Results

[0144] Figs. 5A-E. Expression and characterization of laccase fusion protein. Fig. 5A.Schematic of the laccase - HaloTag fusion assembled for this study. Laccase = laccase from Thermus thermophilus; HALO = HaloTag® protein; 6XHis = polyhistidine tag. Fig. 5B. SDS-P AGE gel of affinity purified Halo-Laccase fusion. A maj or band elutes from the cobalt column in 250 mM imidazole in the expected molecular weight region. Fig. 5C. The eluted laccase fraction has concentration and pH dependent activity towards DMP. Fig. 5D. The laccase has an absorbance at 600 nm, consistent with a copper-bound enzyme. Fig. 5E. Quantification of enzyme velocity plots derived from experiment shown in Fig. 5C.

[0145] Fig. 6. Temperature study of Halo-Laccase fusion with 2,6-dimethoxyphenol (DMP). A. Samples were incubated at the indicated temperatures (Fisher shaker, 600 rpm) for 8 min and the absorbance of DMP recorded at 470 nm (blue circles, with enzyme; black circles, no enzyme). We observed maximum activity at 95 °C, consistent with the initial literature value of max activity was 92 °C. The addition of a HaloTag® does not appear to inhibit laccase activity for phenolic substrates. A mechanism for laccase catalyzed DMP conjugation is shown below and can be found in further detail in reference

[0012] ,DMP conjugation assayAttorney Docket No. 5218.262.WO

[0146] Figs. 7A-B. Decolorization of indigo carmine dye with free and immobilized laccase.Fig. 7A. Samples were incubated at 65 °C for 90 min and the absorbance of indigo carmine read at 600 nm. Efficient dye decolorization required the addition of copper sulfate. The addition of 50 pM copper sulfate gave robust decolorization (i.e., clear solution) within 90 min.Fig. 7B Immobilized laccase on magnetic beads with 50 pM copper sulfate also gave robust decolorization of indigo carmine over multiple rounds of reuse (4X).

[0147] Figs. 8A-G. Polymerization of simple polyphenols with free and immobilized laccase. Solutions of catechol (Fig. 8A), resorcinol (Fig. 8B), and hydroquinone (Fig. 8C) were incubated with and without laccase in free and immobilized form. After the 19h incubation period (65 °C), UV / Vis spectra were acquired. Both catechol (Fig.8A) and hydroquinone (Fig.8C) exhibited increase in absorbance characteristic of polymer formation

[0013] ; resorcinol (Fig.8B) reacted in the presence of free laccase but not in the presence of immobilized laccase. Assays were conducted in 0.1 M NaOAc buffer, pH 5 at 50 mM substrate concentration. Following polymerization, reactions were cooled to 4 °C for one week and centrifuged to collect the precipitated products. HPLC analysis of hydroquinone products (Figs. 8D-G) indicates the formation of polymeric species by both free and immobilized laccases.

[0148] Figs. 9A-B Clarification of fruit juice with free laccase. No changes in turbidity or juice color were detected after 1 h of laccase treatment of unfiltered apple juice at the indicated temperatures, indicating that this particular laccase may not be ideal for use in beverage-related applications without additional optimization.

[0149] Discussion

[0150] We have created a Halo-Laccase fusion protein and investigated its activity in three industrial applications: dye decolorization, short polymer synthesis, and beverage clarification. This laccase retained the high thermal stability and activity profile originally reported for the wild type laccase and showed promise for 2 of the 3 applications in both free and immobilizedAttorney Docket No. 5218.262.WOforms. Interestingly, the immobilized laccase exhibited good retention of activity over multiple rounds of reuse in the dye decolorization assay. While further optimization may be required to create a version of this laccase that is robust enough for industrial settings, these initial results provide a good starting point for such investigations.Example 3

[0151] This HALO-Laccase fusion protein may be used in applications in synthetic organic chemistry

[0014] , See also Bassanini et al., Review: Biocatalysis with Laccases: An Updated Overview, Catalysts 11(26): pages 1-30, 2021.

[0152] In addition, a flow reactor containing immobilized laccase could serve as an environmentally friendly method for decolorizing textile effluents.References[1] Snapp E. Design and use of fluorescent fusion proteins in cell biology. Curr Protoc cell Biol. 2005 Jul;Chapter 21:21.4.1-21.4.13.[2] Motejadded H, Kranz B, Berensmeier S, Franzreb M, Altenbuchner J. Expression, One-Step Purification, and Immobilization of HaloTagTM Fusion Proteins on Chloroalkane-Functionalized Magnetic Beads. Appl Biochem Biotechnol [Internet], 2010 Nov 15;162(7):2098-110.[3] Weidle UH, Schneider B, Georges G, Brinkmann U. Genetically engineered fusion proteins for treatment of cancer. Cancer Genomics Proteomics. 2012 Nov;9(6):357-72.[4] Spahn C, Minteer DS. Enzyme Immobilization in Biotechnology. Vol. 2, Recent Patents on Engineering. 2008. p.195-200.[5] Homaei AA, Sariri R, Vianello F, Stevanato R. Enzyme immobilization: an update. J Chem Biol. 2013 Aug;6(4): 185-205.[6] Hengsong Sun, Fangming Jiang, Xiaorong Yang. Enhanced stability of Thermus thermophiles SG0.5JP17-16 laccase immobilized by CuF-MOF and its application in the Congo red decolorization, Biochemical Engineering Journal, Volume 209, 2024,109397, ISSN 1369-703X.[7] Miyazaki K. A hyperthermophilic laccase from Thermus thermophilus HB27. Extremophiles. 2005 Dec;9(6):415-25. doi:10.1007 / s00792-005-0458-z. Epub 2005 Jul 6. PMID: 15999224.[8] Dobber J, Pohl M. HaloTag: Evaluation of a covalent one-step immobilization for biocatalysis. J Biotechnol. 2017Jan;241: 170-4.Attorney Docket No. 5218.262.WO[9] Urh, M. ‘HaloTag, a Platform Technology for Protein Analysis’. CCG2013, 6 (1), 72-78.

[0010] Norris J, Hughes R. protaTETHER - a method for the incorporation of variable linkers in protein fusions reveals impacts of linker flexibility in a PKAc-GFP fusion protein.

[0011] Dalton T. Deane, Thomas A. Cope, Anna M. Schulz, Edward T. Bennett, and Robert M. Hughes Design, Heterologous Expression, and Application of an Immobilized Protein Kinase. Bioconjugate Chemistry 2023 34 (1), 204-211. D01:10.1021 / acs.bioconjchem.2c00485

[0012] Oluyemisi E. Adelakun, Tukayi Kudanga, Ivan R. Green, Marilize le Roes-Hill, Stephanie G. Burton. Enzymatic modification of 2,6-dimethoxyphenol for the synthesis of dimers with high antioxidant capacity. Process Biochemistry, Volume 47, Issue 12, 2012, Pages 1926-1932, ISSN 1359-5113.

[0013] Sun, X., Bai, R., Zhang, Y. et al. Laccase-Catalyzed Oxidative Polymerization of Phenolic Compounds. Appl Biochem Biotechnol 171, 1673-1680 (2013).

[0014] Zerva, A.; Simic, S.; Topakas, E.; Nikodinovic-Runic, J. Applications of Microbial Laccases: Patent Review of the Past Decade (2009-2019). Catalysts 2019, 9, 1023.Example 4

[0153] Immobilized enzymes are vital for numerous technologies, including biosensors, antifouling coatings, and bioremediation materials. Despite progress in creating enzyme-polymer hybrids, the factors underlying their stability and activity under operational conditions remains poorly understood. Without wishing to be bound by any particular theory, we hypothesize that both the molecular design of enzymes and enzyme fusions and their interaction with polymer networks determine functional activity and longevity. Using multiple immobilization methods, including HaloTag® fusion technology, we will systematically investigate orientation-specific immobilization, linker engineering, surface modification, and computationally guided structural supercharging of three model enzymes (xylanase, laccase, protease), which will inform the rational design of enzyme-polymer assemblies optimized for stability across a broad range of temperatures and humidity conditions.

[0154] Enzymes embedded within polymeric materials comprise a vibrant sector of materials science and biotechnology research, bridging the gap between living systems and synthetic environments [1-6], These hybrid systems combine the high specificity and catalytic efficiency of enzymes with the durable and tunable physical properties of polymers [7, 8], The resulting materials can perform complex chemical transformations [9], respond to environmental stimuli

[0010] , and self-degrade[l] or self-regenerate

[0011] ,Attorney Docket No. 5218.262.WO

[0155] Despite the numerous examples of enzyme-polymer hybrids in the scientific literature, the molecular and physical principles that govern enzyme stability and function in these nonnative environments remain poorly understood [12-17], Enzymes have evolved to operate in aqueous cellular environments with tightly regulated temperature, pH, and ionic conditions

[0018] , However, when transferred to abiotic matrices, they encounter microenvironments that restrict conformational flexibility, limit substrate access, and can alter charge distribution and hydration shells [12, 15, 16], As a result, many immobilized enzymes experience a rapid decline in activity due to denaturation or unfavorable polymer-protein interactions [19, 20], This loss of activity over time, particularly under extremes of temperature and humidity, constitutes a critical barrier to the development of robust enzyme-based functional materials

[0019] .

[0156] Current strategies for enzyme immobilization include non-covalent adsorption, covalent attachment via surface lysines, encapsulation within sol-gels, and covalent incorporation through click chemistry or other orthogonal coupling systems [21-25], While each strategy has demonstrated success in specific applications, there is not a generally accepted set of unifying design rules that allow prediction of which enzymes or immobilization strategies will yield stable, long-lived catalysts. Empirical optimization remains the norm, with outcomes highly dependent on enzyme type, polymer chemistry, and immobilization method

[0026] , As a result, even small changes in polymer composition or enzyme surface topology can dramatically alter long-term stability and catalytic performance

[0012] ,

[0157] We have developed several platforms for the systematic investigation of enzyme immobilization methodology [27, 28], These include a suite of HaloTag®-fused enzymes, including xylanases, laccases, and proteases, representing diverse catalytic mechanisms and structural classes. The HaloTag® system allows orientation-specific attachment of enzymes to polymers via engineered linker molecules, enabling precise control over the protein-polymer interface [29, 30], Complementing these biological tools, we have also previously investigated a clickable organic polymer-based matrix, using chloropropyl- or diformylbenzoate-based linkers, capable of forming durable enzyme “paints” that retain enzymatic activity

[0031] , This system can provide a tunable testbed for elucidating the physicochemical principles underlying enzyme-polymer compatibility. By systematically varying enzyme fusion design, linker architecture, and surface / sequence modification (Fig. 10), we will identify molecular determinants that correlate with activity retention and improved robustness under challenging environmental conditions. For example, to optimize the enzymatic activity and stability, we will investigate hybrid variants of our HaloTag-specific and Lysine-specific covalent captureAttorney Docket No. 5218.262.WOof enzymes within the clickable polymer network using varying attachment sites (Fig. 10, Aim 1); we will apply our protaTETHER method to generate libraries of protein fusions with variable linker lengths and differing degrees of flexibility (Fig. 10, Aim 2); we will investigate different surface modifications of enzyme fusions, e.g., through PEGylation (Fig. 10, Aim 3) within a clickable polymer network; and we will test computationally-guided enzyme supercharging (e.g., via the Rosetta Online Server that Includes Everyone (ROSIE)) (Fig. 10, Aim 4). In addition, the integration of computational approaches into this work, including Rosetta-guided ‘supercharging’

[0032] , will allow us to probe the structural features and residuelevel interactions that may enhance or diminish enzyme function, ultimately translating into more robust enzyme activities within polymer matrices.

[0158] Enzyme-polymer hybrid materials provide new generations of anti-fouling and anticorrosive coatings, self-decontaminating or chem-bio neutralizing surfaces [3-6, 11, 33-35], and embedded sensors in field equipment and uniforms [36, 37], Understanding the molecular mechanisms of enzyme stabilization within these systems will provide the scientific foundation necessary to engineer resilient hybrid materials capable of withstanding stressors such as temperature extremes, high humidity, and repeated use [5, 19],

[0159] Results

[0160] Synthesis and characterization of a clickable polymer matrix.

[0161] Recently published studies have demonstrated that enzyme ‘paints’ (enzyme-polymer hybrid materials) can tether enzymes to polymer matrices formed via click chemistry (Bartlett et al., 2021) by the incorporation of lysine-specific capture molecules. These materials were demonstrated to retain enzymatic activity (xylanase) when applied as a surface coating to plastic substrates (Baker-Branstetter et al., 2023). Our group has expanded this technology further by creating enzyme paints that incorporate additional enzyme types (xylanase, laccase, protease) and by creating reactive linkers that enable additional enzyme immobilization strategies. The copper-free clickable matrix consists of monomer backbones (e.g., the monomers 1 and 2, shown below,) and protein capture linkers (Lysine capture and Halo capture), shown below, that attach the polymer network to the enzymes of interest. Whereas the previously reported enzyme paint strategy used a diformylbenzoate linker (Lysine Capture linker, below) shown for non-specific capture of surface lysines, we have also synthesized a chloropropylbenzoate linker (Halo Capture linker, below) to capture HaloTag® labeled enzymes. Both linkers enable incorporation of proteins into clickable polymer matrices as shown in Fig. 11 This polymer forms under mild room temperature conditions, does not require the addition of copper to catalyze the azide-alkyne click reaction, and is compatibleAttorney Docket No. 5218.262.WOwith the addition of co-solvents (DMSO, water, etc.), making it amenable to the introduction of solvated biomolecules. We have successfully synthesized both monomers and linkers in yields consistent with literature reports

[0031] in quantities that are sufficient for hundreds of small-scale (100 - 200 pL polymerization reactions) experimental trials.Lysine capture linkerHalo capture linker

[0162] Expression and characterization of immobilized enzymes.

[0163] We will use enzymes that can be immobilized by either surface lysine capture or via highly specific HaloTag® capture. In prior studies, we have expressed and confirmed the activity of the enzyme fusions that will be used in this work (Figs. 12A-D). These include a xylanase (HaloTag-xylanase (T. lanuginosus ), a laccase (HaloTag-laccase (T. thermophilus), shown above and in Figs. 5A-E), and a protease (HaloTag-TEV protease (Tobacco Etch Virus)). We can produce ample quantities of these enzymes from 1 L shake cultures in LB medium (10 - 50 mg of purified enzyme per liter of culture).

[0164] Assay of enzymes immobilized in clickable polymer matrix.

[0165] In preliminary studies, we have used a recombinant HaloTag®-xylanase fusion to demonstrate incorporation of our enzyme into the clickable polymer matrix and retention of activity (Fig. 13). In these studies, we confirmed that the enzyme retained activity with bothAttorney Docket No. 5218.262.WOlysine capture and HaloTag® capture methods. We also demonstrated that non-specific capture of enzyme activity resulted in a steady decrease in enzyme activity over time versus covalent capture, which promotes longer lived enzyme activity.

[0166] Technical Approach

[0167] We will investigate the impacts of orientation specific (HaloTag®) vs. non-orientation specific (surface lysine capture) vs. non-specific (no covalent bond) encapsulation of a recombinant xylanase-HaloTag® fusion in a copper-free clickable organic polymer. We will use several methods for immobilizing the Xylanase-HaloTag® fusion in an organic polymer and investigate baseline activity, longevity within the matrix, and resistance to environmental conditions. This will define the relationship between enzyme immobilization method (HaloTag® capture, lysine capture, simple encapsulation) and activity within a polymer and quantify how that activity changes under various environmental stressors.

[0168] Specifically, recombinant HaloTag®-Xylanase fusion protein, purified to homogeneity, will be incubated with an equimolar amount of either the Lysine Capture linker, Halo Capture linker, or no linker (vehicle only) prior to addition to a mixture of monomers 1 and 2 and allowed to polymerize overnight in 96 well plates using 100 pL volumes (example polymer formulations include, but are not limited to, 2,2-bis(azidomethyl)propane-l,3-diol (monomer 2, 252 mg, 1.35 mmol), deionized water (1000 pL), ethane- 1,2-diyl dipropiolate (monomer 1, 283 mg, 1.70 mmol), and dimethylsulfoxide (1000 uL) are added to a vial and vortexed for 30s and used immediately). Enzyme loading amounts will be varied to give final concentrations ranging from 100 nM to 2 pM, performed in triplicate to give 15 unique preparations / plate (45 including replicates) plus enzyme free controls. This arrangement will be replicated as necessary as required for the environmental studies described below. After initial polymerization, polymers will be washed with phosphate buffered saline (pH 7.2) and an initial enzyme assay will be performed with a XylX6 coupled assay (Megazyme) that uses a nitrobenzyl functionalized xylan coupled with a P-xylosidase that releases a p-nitrobenzyl chromophore after cleavage by a xylanase. After establishing initial activity levels, plates will be subjected to various environmental conditions and assayed regularly over different time intervals, as follows: we will investigate enzyme activity under a wide range of temperatures (0 - 95 °C) and humidity conditions (20% - 90%); activity retention will be compared over various time intervals (from one week up to 6 months); polymer-enzyme hybrid reusability will also be evaluated over 10 successive trials (10 activity assays with 24h between each assay). After the conclusion of the xylanase trials, identical experiments will be conducted for polymeric preparations with embedded HaloTag®-laccase and HaloTag®-proteaseAttorney Docket No. 5218.262.WOpreparations. Assays for HaloTag®-laccase will be conducted with 2,6-dimethoxyphenol (DMP) and assays for HaloTag®-TEV protease will be conducted with a TEVsite (ENLYFQG, SEQ ID NO: 30) attached to a maltose binding protein (MBP) and GFP (MBP-TEVsite-GFP substrate) as shown in Fig. 12D.

[0169] Polymer characterization. In addition to the conditions described above, we will also characterize the physical properties of the polymer preparations as follows: Glass transition temperatures (Tg) will be measured for polymer preparations with and without immobilized enzymes using a DSC Q2000 (TA Instruments), as it is anticipated that Tg values will vary between polymer samples with embedded enzyme vs. those without enzyme. Polymer swelling will be measured using simple submersion

[0038] or spectrophotometric submersion tests

[0039] in a range of various solvents: water, phosphate buffered saline, isopropanol, and water / isopropanol mixtures, as we anticipate that different polymer preparations will have different swelling properties.

[0170] We will also test the integration of multiple enzyme types (xylanase, laccase, protease) into a single polymer matrix to investigate the preservation of multiple enzymatic functions within the same sample. We will test enzyme incorporation into cross-linked alginate hydrogels [40-42] or the incorporation of enzymes into TEOS / TMOS silicate sol-gels (xerogels)

[0043] , These methods will also comprise a valuable side-by-side comparison with the clickable polymer matrix.

[0171] Preliminary tests of silica xerogels with and without entrapped enzymes are shown in Figs. 14-16. Briefly, fiberglass hull samples were coated with silica xerogel with a paint brush, cured for at least 24 hours, and incubated in brackish, marina water by suspension from a floating dock to assess anti-fouling properties. These samples were compared to a copper bottom paint currently used for anti-fouling marine purposes. The formulations tested were labeled as Mix 1 (tetraethyl orthosilicate, triethoxyoctylsilane, ethanol, and hydrochloric acid), Mix 2 (tetraethyl orthosilicate, (3 -chi oropropyl)tri ethoxy silane, ethanol, and hydrochloric acid), and Mix 3 (tetramethylorthosilicate, (3-chloropropyl)triethoxysilane, ethanol, and hydrochloric acid). Xylanase, laccase, and alcalase enzymes were tested. Generally, Mix 1 in combination with xylanase, laccase, alcalase, or a combination of all three performed well in the onsite wipe test (gentle pressure applied with a damp cloth to remove fouling). Further, Mix 1 and Mix 2 performed best overall (least amount of fouling pre-wipe test). A fouling assay of Mix 1, Mix 2, and Mix 3 in colder water temperatures (Nov - Jan) showed that Mix 1 and Mix 2 had best performance overall, but all three mixtures performed well in the on-site wipe test.Attorney Docket No. 5218.262.WO

[0172] We will investigate the impacts of linker length and flexibility on the activity of a recombinant xylanase-HaloTag® fusion in a copper-free clickable organic polymer. We will use the protaTETHER method for creating a library of xylanase-HaloTag® fusion proteins with varying linker lengths and flexibilities. These enzymes will subsequently be investigated for their baseline activities, longevities within the matrix, and resistance to various environmental conditions. This will define how modulating the inherent flexibility within a protein fusion using a linker modification approach impacts enzyme activity within a polymer matrix.

[0173] Using our previously established method (‘protaTETHER’ [44, 45]), we will incorporate libraries for flexible and rigid linkers into the HaloTag®-xylanase protein. protaTETHER enables cloning of linker libraries at any location in the protein-encoding gene. Briefly, primers for vector amplification are designed to amplify region immediately adjacent to a variable linker region (VLR) of the vector. After amplification, the linear vector sequence is digested with Dpnl and then gel extracted. Oligomer sequences are then designed to generate multiple amplicons via reiterative sequence design. The short forward primer contains 17 bp of overlap with the target vector in addition to IX or 2X linker repeat codons, while the longer reverse primer contains 17 bp of overlap with the target vector in addition to 4 - 8X complimentary linker repeat codons. Both vector (gel purified) and linker library (crude) amplicons are combined, briefly incubated at room temperature, and transformed into ultracompetent E. coli. The resulting plasmids are screened to identify constructs containing variable linker inserts. In this case, we will design complementary DNA oligomers that contain flexible GGSD (Gly-Gly-Ser-Asp; SEQ ID NO: 11) or rigid poly L (Leu; SEQ ID NO: 12) repeat sequences and overlap with the end of the HaloTag® sequence and the beginning of the Xyl sequence, subject them to PCR overamplification, and incorporate them into the HaloTag®-Xylanase sequence using the FastCloning procedure

[0046] , Clones will be characterized by Sanger sequencing and correct clones will be carried forward to recombinant protein expression. Flexible and rigid linker-containing variants of the HaloTag®-xylanase sequences will be expressed, purified, and incorporated into clickable polymer matrices as described above. Approximately five flexible linker repeats ranging from (GGSD)i - (GGSD)s (SEQ ID NO: 11) and five rigid linker repeat sequences ranging from L)4 - (L)20 (SEQ ID NO: 12) are anticipated for each HaloTag®-enzyme fusion tested. We will then subject these enzyme-polymer hybrids to environmental condition testing as described above, investigating whether a relationship exists between protein fusion flexibility and performance within an enzyme-polymer hybrid.Attorney Docket No. 5218.262.WO

[0174] We will then test the incorporation of multiple flexibility-optimized fusions (e.g., xylanase, laccase, protease) into a single polymer matrix to investigate whether optimal functions are retained when multiple enzyme types are introduced to the same sample. We will investigate the effects of increasing the flexibility within the polymer matrix itself by synthesizing variants of monomers 1 and 2 with additional methylene groups (e.g., 2, 4, or 6) as shown below. These variants will be tested to see if they enhance the activity and stability of enzyme-polymer hybrids by introducing additional flexibility into the polymer network (e.g., to enhance tunability of the polymers and make them more favorable for the retention of enzyme activity). This approach (coupling tunable flexibility within enzyme fusions to tunable flexibility within the polymer matrix) presents a novel and potentially powerful route to optimizing immobilized enzyme function and longevity.Example monomer variants tested to introduce flexibility into the polymer matrix

[0175] We will investigate the impacts of xylanase-HaloTag® fusion modification through surface PEGylation and its activity profile in a copper-free clickable organic polymer. We will investigate the impacts of surface PEGylation of xylanase-HaloTag® fusions on their activities within the clickable polymer matrix. Protein surface PEGylation has been shown to be generally effective at protein stabilization

[0047] and has been used to stabilize enzymes

[0048] , We will investigate whether approaches such as PEGylation improve the function properties of enzymes within a polymer matrix. This will define the relationship between enzyme activity and surface alkylation and investigate how this relationship impacts retention of enzyme activity within a polymer matrix.Attorney Docket No. 5218.262.WO

[0176] Specifically, Halo-Xylanase fusions will be PEGylated using NHS ester chemistry, following literature methods

[0049] , Briefly, in a solution of phosphate buffered saline (pH 7.4), mPEG-Succinimidyl Propionate (mPEG-SPA) derivatives (Biopharma PEG) will be incubated with sub-milligram quantities of the protein fusions for 24 hours with mild agitation. Proteins will be captured by cobalt resin, washed, eluted, then dialyzed against phosphate buffered saline using Snakeskin dialysis tubing (10,000 MW cutoff). By varying the ratio of mPEG-SPA to protein, the degree of protein PEGylation can be varied

[0049] , Furthermore, varying lengths of PEG chains can be used, ranging from low MW (350 Daltons) to high MW (20,000 Daltons). In our initial experiments, we will test a range of molecular weights and ratios to generate a series of PEGylated biocatalysts. Degree of PEGylation will be determined by SDS-PAGE and MALDI-TOF mass spectrometry. After PEGylation, enzyme activities will be measured against non-PEGylated enzymes; if activity is still present, the PEGylated variants will then be incorporated into the polymer matrix and investigated for the activity and stability under the environmental conditions specified above. Even if the activities of the PEGylated enzymes are compromised relative to that of the unmodified enzymes, they will still be tested in the polymer matrix, as PEGylation could imbue properties that promote longevity within the matrix vs. that of the unPEGylated enzyme.

[0177] We will apply the optimal PEGylation methods identified for the xylanase test case to the laccase and protease enzymes. If the optimal strategy identified for xylanase does not extend to these other enzyme types, then the iterative investigation of PEG MW and PEGylation ratios would be repeated for that enzyme. Analogous to the PEGylation strategy, simple reductive alkylation of proteins may also increase the stability of proteins. If the PEGylation strategy does not yield promising results, then we will pursue simple alkylation (e.g., methylation, ethylation, isopropylation) of the protein surfaces using reductive alkylation methodology previously applied to rescue difficult protein targets

[0050] ,

[0178] We will investigate the impacts of ‘supercharging’ of the xylanase component of a xylanase-HaloTag® fusion on its activity profile in a copper-free clickable organic polymer. We will investigate the impacts of computationally guided ‘supercharging’ of surface residues within the xylanase component of the xylanase-HaloTag® fusion on its activity within the clickable polymer matrix with respect to longevity and resistance to various environmental conditions. This will define the relationship between enzyme activity and supercharging and investigate this relationship impacts retention of enzyme activity within a polymer matrix. Supercharging (strategically increasing the net surface charge of a protein) is a method for enhancing protein stability and activity that is broadly applicable to stabilizing many types ofAttorney Docket No. 5218.262.WOproteins [51, 52], Supercharging approaches are therefore anticipated to enhance at least a subset of the enzymes studied in the proposal

[0053] , It remains an open question whether supercharged variants with improved stabilities / activities in solution will translate into improved immobilized stabilities / activities. This approach provides a unique opportunity to address this question. Computational resources for choosing which residues to mutate may be used

[0054] ,

[0179] Specifically, using the open web interface Supercharge program

[0054] available via ROSIE (Fig. 10)

[0055] , we will generate supercharged variants of our xylanase enzyme. We will focus exclusively on supercharging the xylanase portion of our enzyme fusion to minimize interference with HaloTag® function. Two supercharging modes are available through this interface: the AvNAPSA mode that mutates only the most exposed polar residues to minimize structural changes (only Asp / Glu / Arg / Lys / Asn / Gln residues are mutated) and the Rosetta mode that mutates residue positions that preserve and / or add favorable surface interactions, including hydrophobic and small polar surface residues. We will use both supercharging approaches, selecting ten variants from each, generate cloned versions of the computationally generated sequences, and test their enzyme activities and thermal stabilities side-by-side. The variants exhibiting the most desirable properties in terms of activity retention / enhancement and thermal stability / enhancement will be tested within the clickable polymer matrix for increased robustness to variable environmental conditions.

[0180] We will apply the computational strategies to improve enzyme performance. We will investigate modulation of the polymer / enzyme interface via specific ion effects

[0056] , By incorporating a variety of ionic species into the polymer formulation, we will determine if Hofmeister effects can play a role in improving the performance of enzyme-polymer hybrids under variable environmental conditions.

[0181] The above research addresses core interests in advanced materials and biotechnology. Understanding enzyme activity loss and retention mechanisms directly impacts the design of long-lasting biosensors, corrosion-resistant coatings, and self-decontaminating materials. The knowledge gained by these investigations will ultimately translate to applied programs focused on chem-bio defense and durable field materials. For example, without wishing to be bound by any particular theory, we propose the following potential outcomes from this work, polymers with anti-fouling and anti-corrosion properties; polymers with chem-bio threat reduction or neutralization capabilities; and / or polymers that could act as or aid in a deployable sensor.

[0182] ReferencesAttorney Docket No. 5218.262.WO1. Guicherd M, Khaled M Ben, Gueroult M, Nomme J, Dalibey M, Grimaud F, Alvarez P, Kamionka E, Gavalda S, Noel M, Vuillemin M, Amillastre E, Labourdette D, Cioci G, Tournier V, Kitpreechavanich V, Dubois P, Andre I, Duquesne S, Marty A (2024) An engineered enzyme embedded into PLA to make self-biodegradable plastic. Nature, 631(8022):884-890.2. Rodriguez-Abetxuko A, Sanchez-deAlcazar D, Munumer P, Beloqui A (2020) Tunable Polymeric Scaffolds for Enzyme Immobilization. Frontiers in Bioengineering and Biotechnology, Volume 8-20203. Hu J, Zhang G, Liu S (2012) Enzyme-responsive polymeric assemblies, nanoparticles and hydrogels. Chemical Society Reviews, 41(18):5933-5949.4. Li M, Blum NT, Wu J, Lin J, Huang P (2021) Weaving Enzymes with Polymeric Shells for Biomedical Applications. Advanced Materials, 33(34):2008438.5. Heredero M, Beloqui A (2023) Enzyme-Polymer Conjugates for Tuning, Enhancing, and Expanding Biocatalytic Activity. ChemBioChem, 24(4):e20220061 l-e202200611.6. Eixenberger D, Kumar A, Klinger S, Scharnagl N, Dawood AWH, Liese A (2023) Polymer-Grafted 3D-Printed Material for Enzyme Immobilization — Designing a Smart Enzyme Carrier. Catalysts, 13(7)7. Gomez-Romero P, Pokhriyal A, Rueda-Garcia D, Bengoa LN, Gonzalez-Gil RM (2024) Hybrid Materials: A Metareview. Chemistry of Materials, 36(1):8— 27.8. Ouyang J, Li J, Wu C (2025) Protein-Polymer Conjugates: Advancing Enzyme Catalysis in Synthetic Chemistry. ChemCatChem, 17(2):e202401180.9. Basso A, Serban S (2019) Industrial applications of immobilized enzymes — A review. Molecular Catalysis, 479: 110607.10. Chen C, Ng DYW, Weil T (2020) Polymer bioconjugates: Modern design concepts toward precision hybrid materials. Progress in Polymer Science, 105:101241.11. Li P, Zhong Y, Wang X, Hao J (2020) Enzyme-Regulated Healable Polymeric Hydrogels. ACS Central Science, 6(9): 1507-1522.12. Cummings C, Murata H, Koepsel R, Russell AJ (2013) Tailoring enzyme activity and stability using polymer-based protein engineering. Biomaterials, 34(30):7437-7443.13. Murata H, Cummings CS, Koepsel RR, Russell AJ (2013) Polymer-Based Protein Engineering Can Rationally Tune Enzyme Activity, pH-Dependence, and Stability. Biomacromolecules, 14(6) : 1919- 1926.14. Chapman R, Stenzel MH (2019) All Wrapped up: Stabilization of Enzymes within Single Enzyme Nanoparticles. Journal of the American Chemical Society, 141(7):2754-2769.Attorney Docket No. 5218.262.WO15. Silva C, Martins M, Jing S, Fu J, Cavaco-Paulo A (2018) Practical insights on enzyme stabilization. Critical Reviews in Biotechnology, 38(3):335-350.16. Asaduzzaman F, Salmon S (2022) Enzyme immobilization: polymer-solvent-enzyme compatibility. Molecular Systems Design & Engineering, 7(11): 1385-1414.17. Wright TA, Lucius Dougherty M, Schmitz B, Burridge KM, Makaroff K, Stewart JM, Fischesser HD, Shepherd JT, Berberich JA, Konkolewicz D, Page RC (2017) Polymer Conjugation to Enhance Cellulase Activity and Preserve Thermal and Functional Stability. Bioconjugate Chemistry, 28(10):2638-2645.18. Gao J, Le S, Thayumanavan S (2021) Enzyme Catalysis in Non-Native Environment with Unnatural Selectivity Using Polymeric Nanoreactors. Angewandte Chemie International Edition, 60(52):27189-27194.19. Li T, Zhang S, Fan Y, Chen C, Niu Y, Zha B, Huo F, Zhang W (2025) Mechanically Driven Enzyme Engineering: The New Frontier Beyond Chemistry. ACS Applied Materials & Interfaces.20. Liese A, Hilterhaus L (2013) Evaluation of immobilized enzymes for industrial applications. Chemical Society Reviews, 42(15):6236-6249.Example 5

[0183] Purification of recombinant protein can also be achieved using immobilized proteases as described herein. For example, proteases immobilized on magnetic beads (e.g., magnetic Sepharose™ beads conjugated to the HaloTag via a linker as described herein) could be used repeatedly to purify recombinant proteins from a known protein tag (i.e., the protease could be recovered and washed after use to cleave a target protein using the magnetic beads, thereby being free to use again in future experiments). A workflow of this process is shown in Fig.20.Briefly, a polynucleotide encoding a peptide of interest is covalently linked to a polynucleotide encoding a peptide tag (e.g., SUMO) and inserted into an expression vector. These vectors can then be expressed in vitro (e.g., in E. colt). The resulting overexpressed protein construct is incubated with the immobilized protein, where the peptide tag is cleaved off of the protein of interest. Further purification (e.g., liquid chromatography (e.g., Reversed-Phase High-Performance Liquid Chromatography), centrifugation, dialysis, and the like) can be used if required.

[0184] We tested TEV and SENP1 proteases. SENP1, in particular, was an enticing protease as it is relatively small (its catalytic domain is 27 kD), easy to express in E. coli, has robustAttorney Docket No. 5218.262.WOactivity, and leaves native sequence post-cleavage site. In addition, a SENP1 target, SUMO, is a well-studied protein tag that can easily be attached to most peptides of interest. The immobilized protease constructs are shown in Fig. 21 A and a test of the TEV and SENP proteases on a SUMO-tagged green fluorescent protein (GFP) target is shown in Fig. 21B and confirms activity of both proteases. Importantly, we repeated the experiment with the same immobilized SENP1 over several trials and saw repeated enzymatic activity, demonstrating that the immobilized SENP1 construct was able to retain activity despite the addition of the tag, linker, and magnetic bead (Fig. 22).

[0185] We tested the above workflow on a histatin target fragment, Pl 13. Histatins are 12 histidine-rich cationic peptides present in human saliva. They are anti-microbial, with activity against Candida and antimycotic resistant strains of bacteria (e.g., S. mutans, P. gingivalis, A. actinomycetemcomitans, P. aeruginosa, E. coli, and St. aureus). Pl 13 (SEQ ID NO: 18, AKRHHGYKRKFH) is a fragment from Histatin 5, that is currently used in some consumer products to protect against plaque and gingivitis. Current purification methods for Pl 13 include cleavage of the fragment from a fusion partner using the toxic cyanogen bromide (CNBr). Our approach will entail: 1) expression of Pl 13 with SUMO tag in E. Coir, 2) immobilized metal ion affinity chromatography (IMAC) purification and dialysis; 3) cleavage with immobilized protease to produce native sequence; 4) lyophilization, resuspension, filtration of the Pl 13 fragment; and 5) RP-HPLC purification of the Pl 13 fragment. The benefits for this process include elimination of protease contamination of samples, control over the extent of proteolysis, and facilitation of screening / high throughput applications.

[0186] The SUMO-tagged Pl 13 construct and cleavage process is shown in Fig. 23A. A gel image of the successful cleavage of Pl 13 from SUMO is shown in Fig.23B. The kinematic of the cleavage is shown in Fig. 23C. As above, we repeated the experiment with the same immobilized SENP1 over ten trials and saw repeated cleavage of the Pl 13 target, indicating a retention of SENP1 activity (Fig. 23D).

[0187] The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

Attorney Docket No. 5218.262.WOWHAT IS CLAIMED IS:

1. A construct of Formula I:Formula Iwherein:Ri is an enzyme;Pi is present or absent, and when present is a peptide tag;Li is a linker; andP2 is a polymer.

2. The construct of claim 1, wherein the enzyme is a tannase, an apolipoprotein (e.g., apolipoprotein L3), a lysozyme, a multicopper oxidase, a xylanase, a protease, a cellulase, a laccase, and / or a chitinase.

3. The construct of claim 1 or 2, wherein the peptide tag is present and is a SpyTag / SpyCatcher, a SNAP tag, a Halo tag, and / or a CLIP tag.

4. The construct of any preceding claim, wherein the linker is formed from a benzoate linker.

5. The construct of claim 4, wherein the linker is formed from a benzoate linker of Formula A:wherein:n is an integer of from 1 to 10;R2 is formyl, hydroxy, hydroxyalkyl, halo, or haloalkyl; andR3 is hydrogen, formyl, hydroxy, hydroxyalkyl, halo, or haloalkyl.Attorney Docket No. 5218.262.WO6. The construct of claim 5, wherein the benzoate linker is prop-2-yn-l-yl 3,4-diformylbenzoate or prop-2-yn-l-yl 3 -chloropropylbenzoate:prop-2-yn-1 -yl 3-chloropropylbenzoate7. The construct of claim 5 or 6, wherein the benzoate linker is prop-2-yn-l-yl 3-chloropropylbenzoate and the enzyme or peptide tag, when present, is conjugated to the linker by alkyl halide displacement.

8. The construct of any preceding claim, wherein the polymer is a [3+2] azide-alkyne cycloaddition polymer (e.g., an alkyne-azide polymer), a Diels-Alder based polymer, a thiolene polymer (e.g., a thiol-alkene radical addition based polymer), and / or a strain-promoted azide-alkyne cycloaddition (SPAAC) based polymer.

9. The construct of any preceding claim, wherein the linker is conjugated to the polymer by click chemistry (e.g., azide-alkyne Huisgen cycloaddition).

10. The construct of any preceding claim, wherein the construct has a formula:Attorney Docket No. 5218.262.WO11. A composition comprising the construct of any one of claims 1-10.

12. The composition of claim 11, wherein the composition comprises the construct in an amount of about 0.5% to about 99% (e.g., about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or about 99%) by weight of the composition.

13. The composition of claim 11 or 12, wherein the composition further comprises water, an organic solvent, a pigment, an antifoaming agent, a thickener, a surfactant, a resin, a leveling agent, a curing agent, or any combination thereof.

14. The composition of any one of claims 11-13, wherein the composition is stable at about -20 °C to about 50 °C (e.g., about -20, -15, -10, -5, 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 °C) for about 1 month to about 24 months (e.g., about 1, 2, 3, 4, 5, 6, 7 ,8, 9, 10, 11, 12, 13, 14. 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months).

15. A method of reducing biological fouling on a surface, the method comprising applying a composition of any one of claims 11-14 to said surface, thereby reducing biological fouling as compared to a surface not comprising the composition.

16. The method of claim 15, wherein the method comprises applying the composition to the surface from every 1 week to about every 52 weeks (e.g., about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, or every 52 weeks).

17. The method of claim 15 or 16, wherein the surface is a surface on a marine vessel (e.g., a boat hull, submarine hull, deck, bridge, keel, hatch, cushion, seat, propeller, rudder, mast, and / or bilge), a water inlet, a pipe (e.g., a water pipe and / or an oil pipe), a nozzle (e.g., a sprinkler nozzle), a cable (e.g., an underwater cable), a manufacturing apparatus (e.g., a paper manufacturing apparatus), a food processing apparatus, construction equipment (e.g.,Attorney Docket No. 5218.262.WOunderwater construction equipment), a desalination apparatus, a bioreactor (e.g., a membrane bioreactor), a water cooling system (e.g., a power station water cooling system), a drug delivery device (e.g., a microelectrochemical drug delivery device), a well, a ventilation component (e.g., an air duct and / or an air conditioning component), a humidifier, a dehumidifier, a heat exchanger, and / or a heatsink.

18. A method of making an antifouling coating, the method comprising providing an enzyme, optionally wherein the enzyme is conjugated to a peptide tag; conjugating the enzyme or the peptide tag to a linker; and conjugating the linker to a polymer by click chemistry.

19. The method of claim 18, wherein the conjugating the enzyme or the peptide tag to a linker is carried out by alkyl halide displacement.

20. The method of claim 18 or 19, wherein the conjugating the linker to a polymer by click chemistry is carried out by azide-alkyne Huisgen cycloaddition.

21. A method of reducing mold and / or mildew growth on a surface, the method comprising applying a composition of any one of claims 11-14 to said surface, thereby reducing mold and / or mildew growth as compared to a surface not comprising the composition.

22. The method of claim 21, wherein the method comprises applying the composition to the surface from every 1 week to about every 52 weeks (e.g., about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, or every 52 weeks).

23. The method of claim 21 or 22, wherein the surface is a surface on a marine vessel (e.g., a boat hull and / or a submarine hull), a water inlet, a pipe (e.g., a water pipe and / or an oil pipe), a nozzle (e.g., a sprinkler nozzle), a cable (e.g., an underwater cable), a manufacturing apparatus (e.g., a paper manufacturing apparatus), a food processing apparatus, construction equipment (e.g., underwater construction equipment), a desalination apparatus, a bioreactor (e.g., a membrane bioreactor), a water cooling system (e.g., a power station water cooling system), a drug delivery device (e.g., a microelectrochemical drug delivery device), a well, a ventilation component (e.g., an air duct and / or an air conditioning component), a humidifier, a dehumidifier, a heat exchanger, and / or a heatsink.Attorney Docket No. 5218.262.WO24. A method of decolorizing a dye, the method comprising adding the construct of any one of claims 1-10 or the composition of any one of claims 11-14, and copper (II) sulfate, to a composition comprising said dye (e.g., comprising an aqueous buffer), thereby decolorizing the dye.

25. The method of claim 24, wherein the dye comprises an indigo dye (e.g., indigo carmine).

26. The method of claim 24 or claim 25, wherein the construct is recovered from the composition comprising the dye, and is reused in a second decolorizing method.

27. The method of any one of claims 24-26, wherein the construct is provided immobilized in a flow reactor useful for decolorizing textile effluents.

28. A method of polymerizing phenolic compounds, the method comprising adding the construct of any one of claims 1-10 or the composition of any one of claims 11-14, to a composition comprising said phenolic compounds, whereby the phenolic compounds are oxidatively polymerized.

29. The method of claim 28, wherein the phenolic compounds comprise catechol and / or hydroquinone.

30. A method of clarifying a cloudy aqueous solution, the method comprising adding the construct of any one of claims 1-10 or the composition of any one of claims 11-14, to the aqueous solution, whereby the solution is clarified.

31. The method of claim 30, wherein the cloudy aqueous solution is a fruit juice (e.g., unfiltered apple juice).

32. The construct of any one of claims 1-10, wherein the enzyme is an alcalase or a subtilisin.Attorney Docket No. 5218.262.WO33. The construct of any one of claims 2-10, wherein the protease is thrombin, HRV143C, Tobacco Etch Virus (TEV) protease, factor Xa, SENP1, and / or enterokinase cleavage enzyme (EKT).

34. The construct of any one of claims 1-10, 32, or 33, wherein the enzyme comprises a surface modification.

35. The construct of claim 34, wherein the surface modification is a polyethylene glycol (PEG), a methyl, an ethyl, an isopropyl, and / or an alkyl.

36. The construct of any one of claims 1-10 or 32-35, wherein the enzyme has been optimized to increase its net surface charge.

37. A composition comprising a xerogel (e.g., a silica xerogel) and an enzyme, optionally wherein the xerogel integrates the enzyme.

38. A composition comprising a hydrogel and an enzyme, optionally wherein the hydrogel integrates the enzyme.

39. A construct of Formula IEFormula IIwherein: Ri is an enzyme; Pi is present or absent, and when present is a peptide tag; Li is a first linker; and Ii is an immobilization material.

40. The construct of claim 39, wherein the immobilization material is a magnetic bead and / or wherein the enzyme is a protease.

41. The construct of claim 39 or 40, wherein the linker comprises a compound of Formula D:Attorney Docket No. 5218.262.WOFormula Dwherein n is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

42. The construct of claim 41, wherein the construct has a formula:enzyme |_|peptide tag^^ —immobilization material43. A method of making an antifouling coating, the method comprising providing an enzyme and encapsulating the enzyme in a xerogel (e.g., a silicate xerogel).

44. A method of making an antifouling coating, the method comprising providing an enzyme and encapsulating the enzyme in a hydrogel (e.g., an alginate hydrogel).

45. A method of producing a purified recombinant protein, the method comprising:(a) preparing a vector comprising a polynucleotide encoding fusion protein comprising the recombinant protein, a peptide tag (e.g., a SUMO tag), and a purification tag;(b) transfecting a cell (e.g., a bacterial cell) with the vector, wherein the cell thereby produces the fusion protein;(c) isolating the fusion protein using the purification tag;(d) contacting the isolated fusion protein with the construct of any one of claims 39-42, wherein the construct comprises a protease and the protease cleaves the fusion protein to provide the recombinant protein; and(e) purifying the recombinant protein, thereby providing the purified recombinant protein.

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