Carbonic anhydrase fusion proteins, compositions, and methods of use

Abstract

The present disclosure is directed to carbonic anhydrase (CA) fusion proteins, polynucleotides encoding fusion proteins, concrete and enzymatic construction material (ECM) compositions, and methods for making concrete and ECM compositions comprising CA fusion proteins, and methods for repairing a fracture in a cementitious or ECM surface, improving corrosion resistance and reducing permeability and porosity in cementitious and ECM compositions.

Description

CARBONIC ANHYDRASE FUSION PROTEINS, COMPOSITIONS, AND METHODS OF USE

[0001] This application claims the priority benefit of U.S. Provisional Patent Application Serial No.63 / 510,835, filed June 28, 2023, which is hereby incorporated by reference in its entirety.

[0002] The Sequence Listing is being submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on June 27, 2024, is named 263813000101.xml and is 121,767 bytes in Size. No new matter is being introduced. FIELD

[0003] The present disclosure relates to carbonic anhydrase fusion proteins, compositions, and methods of use for concrete corrosion inhibition, repair, and concrete alternatives. BACKGROUND

[0004] Concrete is the most widely used construction material in the world. Concrete is a general term applied to a mixture of Portland cement (~ 7-15%) and water (~14-21%) mixed with aggregate (~60-75%), typically including sand, crushed stone, and / or gravel of various sizes, and air (~8%). A gel is formed during the reaction of the Portland cement with water to bind the aggregate followed by curing to form a solid mass. Concrete is easily transported as a dry material having a powdery, gravel texture, and forms a fluidic, granular slurry type of material immediately after mixing with water. Prior to curing, the fluidic mixture may be cast or poured into any suitable shape that can then cure into a molded shape with substantial compressive strength and mass.

[0005] In the United States, annual concrete production is over 500 million tons. Although concrete has many advantages and has been widely used, cracking of this material is almost inevitable due to its low tensile strength and high brittleness. Cracks can result from various causes, such as external loads, differential settlement of structures, volume changes (expansion or contraction) due to temperature and moisture gradients (e.g., wet-dry and freeze- thaw cycling), and chemical reactions (sulfate attack, reinforcement corrosion, etc.). Cracks, even microcracks, play a critical role in the material transport properties. They permit air, water, and chemicals to penetrate into concrete, thus inducing various physical and chemical reactions, expediting the concrete deterioration, and shortening the structure service life. 172604987

[0006] In transportation infrastructure, corrosion deterioration of reinforcing steel in bridge decks is often related to the penetration of deicing chemicals through microcracks into concrete. In buildings, cracks are primarily responsible for energy loss due to air infiltration. Detecting cracks, especially microcracks, within in-service structures is very challenging as cracks are often randomly distributed and can be invisible. Without timely treatment, microcracks tend to grow, permitting more fluids to get into concrete and causing more severe corrosion and deterioration, thus requiring costly repair. It is estimated that the annual cost of concrete structure maintenance and repair is $18-21 billion in U.S. alone, highlighting the urgent need to provide corrosion and crack resistance to concrete.

[0007] Typical crack repair uses epoxy, cement grout, chemical sealants, or surface treatment agents. Not only are such crack repair techniques expensive and harmful to the environment, such a repair also requires prompt detection of cracks and estimation of the degree of damage in concrete. There is an urgent need for stopping concrete cracking using smart, eco- friendly, cost-effective, crack-controlling materials.

[0008] A major environmental impact of concrete use is also the production of 400 lbs of CO2 per cubic yard of concrete, which accounts for 8% of CO2 produced worldwide. Reducing carbon dioxide emissions due to concrete is an important task. However, methods to reduce concrete consumption with replacements such as fly ash and slag in concrete consumption are not environmentally friendly. Therefore, there is a need to develop novel, environmentally friendly methods and compositions for concrete replacement and concrete repair that reduce CO2 emissions.

[0009] This disclosure is directed to overcoming these and other deficiencies in the art. SUMMARY

[0010] One aspect of the present disclosure relates to a fusion protein comprising a carbonic anhydrase domain and one or more of a calcium carbonate binding domain, a calcium binding domain, an iron binding domain, a silicatein mimetic domain, and a silica binding domain.

[0011] Another aspect of the present disclosure relates to a concrete composition comprising a fusion protein comprising a carbonic anhydrase domain and one or more of a calcium carbonate binding domain, a calcium binding domain, an iron binding domain, a silicatein mimetic domain, and a silica binding domain; and cement.

[0012] A further aspect of the present disclosure relates to an enzymatic construction material (“ECM”) composition comprising a fusion protein comprising a carbonic anhydrase 172604987domain and one or more of a calcium carbonate binding domain, a calcium binding domain, an iron binding domain, a silicatein mimetic domain, and a silica binding domain; and a particulate substrate.

[0013] Yet another aspect of the present disclosure relates to a method of repairing a fracture in a cementitious or ECM surface. This method involves contacting a fracture in a cementitious or ECM surface with a fusion protein composition as described herein under conditions effective to repair the fracture in the cementitious surface or ECM surface.

[0014] Another aspect of the present disclosure relates to a method of making a cement mixture. This method involves combining the composition of a fusion protein composition as described herein with a cement to form a cement mixture. In some embodiments, the method further involves curing the cement mixture comprising the composition to make a durable corrosion resistant concrete.

[0015] A further aspect of the present disclosure relates to a method for making an enzymatic construction material (ECM) composition. This method involves combining a fusion protein composition as described herein with a particulate substrate to form an ECM mixture. In some embodiments, the method further involves curing the ECM mixture comprising the composition to make a durable corrosion resistant ECM.

[0016] Yet another aspect of the present disclosure relates to a polynucleotide molecule for expressing a carbonic anhydrase fusion protein comprising a polynucleotide encoding a carbonic anhydrase domain and one or more of a calcium carbonate binding domain, a calcium binding domain, an iron binding domain, a silicatein mimetic domain, and a silica binding domain and a promoter operably linked to the polynucleotide.

[0017] Other aspects of the present disclosure relate to vectors comprising the polynucleotide molecules described herein and host cells comprising the polynucleotides or vectors described herein.

[0018] The present disclosure relates to novel carbonic anhydrase (“CA”) fusion proteins that catalyze the covalent condensation of carbon dioxide and water or the fixing of carbon dioxide from the atmosphere, to promote the precipitation of calcium ions as calcium carbonate crystals, and the use of these fusion proteins in cementitious and ECM (concrete alternative) materials to promote self-healing and repair properties, corrosion resistance, and carbon sequestration. These carbonic anhydrase fusion proteins provide increased stability, solubility, and scalability compared to other CA proteins as well as new functions such as iron binding, calcium carbonate binding, and silica binding for increased structural support in materials comprising the CA fusions. For example, bovine carbonic anhydrase is difficult to purify (see 172604987Bering et al., “Purification of Bovine Carbonic Anhydrase by Affinity Chromatography: An Undergraduate Biochemistry Laboratory Experiment,” Journal of Chemical Education 75.8:1021 (1998), which is hereby incorporated by reference in its entirety) and is less stable at high temperatures.

[0019] The novel carbonic anhydrase (CA) fusion proteins of the present disclosure provide many advantages over the use of other carbonic anhydrases such as those disclosed for use in enzymatic construction material for repair and corrosion resistance and durability (e.g., in U.S. Patent Application Publication Nos. US2023 / 0192548 and US2023 / 0117517, each of which is hereby incorporated by reference in its entirety). In comparison to bovine CA, the CA fusion proteins described herein retain activity at high temperature having thermostability (e.g., see FIGs.1-2). Compositions comprising the CA fusion proteins described herein can be used to prevent the formation of cracks and provide self-repair capabilities and improve longevity of the concrete. In addition to the benefits of self-healing properties, CA fusion compositions can provide corrosion resistance and replace the need to use corrosion inhibitors such as calcium nitrate to reduce permeability and porosity, which has numerous downsides such as having negative effects on the mechanical strength of concrete. For corrosion inhibition, the CA fusion enzyme uses water as part of the hydration of carbon dioxide. This dehydrates the area leading to less exposure to water by the rebar (since water can rust rebar) and the calcium carbonate crystals generated from the CA enzyme plug the porous concrete leading to less oxygen being able to enter, thus protecting the rebar from further rusting. The CA fusion proteins disclosed herein can create microbarriers in the porous voids of concrete to prevent rebar corrosion, thus improving the strength and longevity of the concrete. For repair of cementitious surfaces, epoxy systems and acrylic resins are currently commonly used, but these chemicals are all environmentally unfriendly, and often cause delamination or cracking between the original concrete matrix and the repair material. By contrast, the CA fusion proteins and compositions of the present disclosure can eliminate the need to use epoxy to repair concrete and provide a stronger product. The CA fusion proteins can also be used in new enzymatic construction material (ECM) that do not include cement. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG.1 is a graph of the carbonic anhydrase activity and thermostability of bovine carbonic anhydrase, the Masa CA fusion protein C-variant 1 (SEQ ID NO:53), and the Td CA fusion protein C-variant 1 (SEQ ID NO:55) after incubation for 20 minutes at 65oC (see Example 1). 172604987

[0021] FIG.2 is a graph of the carbonic anhydrase activity of bovine carbonic anhydrase, the Masa CA fusion protein C-variant 1 (SEQ ID NO:53), and the Td CA fusion protein C- variant 1 (SEQ ID NO:55) after incubation for 45 days at 65oC in solution prior to testing (see Example 1).

[0022] FIG.3 is a photograph of a protein gel showing expression levels and solubility of various carbonic anhydrase fusion proteins including: a bdSUMO Masa fusion protein (lane 1; SEQ ID NO:56), a Cytoplasmic MBP Masa CA fusion protein (lane 2; SEQ ID NO:57), an sfGFP Masa CA fusion protein (lane 3; SEQ ID NO:58), and an sfGFP Td fusion protein (lane 4; SEQ ID NO:59).

[0023] FIG.4 is a graph of Accelerated Corrosion Testing (“ACT”) of concrete with and without bovine carbonic anhydrase and a fusion protein CA as described herein. Crack starts in samples after 14 days are shown. The identity of the samples from left to right are as follows: control (standard Portland cement concrete), FA (fly ash concrete), CNI (calcium nitrate / nitrite), CA (bovine carbonic anhydrase at 20 mg / cubic yard of concrete), CA 5x (bovine carbonic anhydrase at 100 mg / cubic yard of concrete), CA 10X (bovine carbonic anhydrase at 200 mg / cubic yard of concrete), Isaac 1X (sfGFP Masa CA fusion protein (SEQ ID NO:58)) at 20 mg / cubic yard of concrete), Isaac 5X (sfGFP Masa CA fusion protein at 100 mg / cubic yard of concrete), and Isaac 10X (sfGFP Masa CA fusion protein at 200 mg / cubic yard of concrete).

[0024] FIG.5 is a graph showing Accelerated Corrosion Testing (ACT) showing crack starts in samples after 28 days. The identity of the samples from left to right are as follows: control (standard Portland cement concrete), FA (fly ash concrete), CNI (calcium nitrate / nitrite), CA 1x (bovine carbonic anhydrase at 20 mg / cubic yard of concrete), CA 5x (bovine carbonic anhydrase at 100 mg / cubic yard of concrete), CA 10X (bovine carbonic anhydrase at 200 mg / cubic yard of concrete), and Isaac 1X (sfGFP Masa CA fusion protein (SEQ ID NO:58)) at 20 mg / cubic yard of concrete).

[0025] FIGs.6A-B show the results of an Accelerated Corrosion Test (ACT) on different 48-day samples. FIG.6A is a photograph of the Accelerated Corrosion Test, which uses concrete samples that are cast into 4x8 in. cylinders with a ½ in. wire-brushed steel rebar embedded in the center, a plastic tank containing five wt.% NaCl solution, two stainless steel plates (the cathodes), and a 30 V DC power supply to accelerate the corrosion where the reinforcing bar is the anode in the setup. FIG.6B is a graph showing crack starts in various samples including a control (standard Portland cement concrete), FA (fly ash concrete), CNI (calcium nitrate / nitrite), and CA (bovine carbonic anhydrase) at various amounts. The samples with enzyme (CA-1X represents: 49 mg enzyme / 1 cubic yard cement) initiated a crack later than 172604987the control samples with Fly ash and CNI additives, respectively. Before the sample cracks, the brown-colored liquid was bled from the interface between the top of the cylinder and rebar. The crack starts from the axial edge of concrete, indicated by the deflection point where the voltage began to increase.

[0026] FIG.7 is a graph showing the compressive strength of 1 day cement mortar samples. Samples are (from left to right) control (regular cement), cement with 3.5% LUDOX®colloidal silica, cement with sfGFP Masa CA fusion protein (SEQ ID NO:58) at 245 mg / cubic yard (or 1.35 mg / kg), cement with Masa CA and 3.5% LUDOX®, and cement with 3.5% LUDOX®. LUDOX®is a colloidal silica. This experiment aimed to investigate whether carbonic anhydrase could replace LUDOX®or enhance its benefits in concrete, potentially further improving compressive strength.

[0027] FIGs.8A-C show compressive strength results of self-healing enzymatic concrete for control, notched, and cement paste samples containing bovine CA (CA-MOD-5x; bovine carbonic anhydrase at 245 mg / cubic yard of concrete). FIG.8A is a graph showing a comparison of compressive strengths of square-shaped samples. The mean for each data set is shown above each sample. The CA-Mod-5x samples (ESH, right side) with a 3 × 1 mm vertically oriented elliptical flaw obtained 97.5% of the original strength of the control samples after 24 hours, whereas the strength of notched samples (NH, middle) was only 75% of the intact control specimens (control, left side). FIG.8B shows photographic images of the control, notched flaw, and CA-MOD-5x samples (from left to right, respectively). FIG.8C shows scanning electron micrograph images showing calcium carbonate crystal growth on the crack tip region.

[0028] FIG.9 shows dye penetration testing in 28-day cement mortar (M) and cement paste (CP) specimens prepared with bovine CA. FIG.9A is a graph showing diffusion coefficients of dye in various samples in cement paste and cement mortar to investigate the depth that ions penetrate. The penetration depth of 10 mL water-soluble dye was measured from the top surface and indicated by black arrow bar. Images of the CP and M compositions with and without CA are shown in the insets of FIG.9.

[0029] FIG.10 shows typical m-CT scans of (left) control vs. (right) bovine CA- Modified Sample. μ-CT images of the cubic enzymatic cement paste samples (control and bovine CA-5X) are displayed in the z-direction with the scale bar positioned on the left side. The control sample exhibit a porosity of 0.35, while CA-modified sample demonstrates a reduced porosity of 0.24, indicating the CA’s impact on porosity reduction.

[0030] FIG.11 is a graphic illustration of the various amino acid domains comprising the Masa CA fusion protein C-variant 1 (SEQ ID NO:53). 172604987

[0031] FIG.12 is a graphic illustration of the various amino acid domains comprising the sfGFP Masa CA fusion protein (SEQ ID NO:58). DETAILED DESCRIPTION

[0032] This disclosure relates to carbonic anhydrase fusion proteins, concrete and ECM compositions comprising these fusion proteins, and methods for the use of these fusion proteins for concrete corrosion inhibition, concrete repair and longevity, concrete alternatives, and carbon sequestration.

[0033] Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present disclosure herein described for which they are suitable as would be understood by a person skilled in the art.

[0034] Singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and / or steps of the type described herein and / or which will become apparent to those persons skilled in the art upon reading this disclosure. In another example, reference to “a compound” includes both a single compound and a plurality of different compounds.

[0035] The term “about” includes being within a statistically meaningful range of a value. Such a range can be within an order of magnitude, such as within 10% or within 5% of a given value or range.

[0036] The term “and / or” as used herein means that the listed items are present, or used, individually or in combination. In effect, this term means that “at least one of” or “one or more” of the listed items is used or present.

[0037] In understanding the scope of the present disclosure, the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “involving”, “having”, and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but exclude the presence of other unstated features, elements, components, groups, integers and / or steps.

[0038] Certain terms employed in the specification, examples, and claims are collected herein. Unless defined otherwise, all technical and scientific terms used in this disclosure have 172604987the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0039] A “protein” or “polypeptide” is a naturally occurring or synthetic peptide, oligopeptide, polypeptide, gene product, expression product, fusion protein, or protein comprising an amino acid sequence, where the amino acids are joined to each other by peptide bonds or modified peptide bonds.

[0040] The term “fragment” when referring to a polypeptide or protein will be understood to mean a polypeptide or protein sequence of reduced length relative to the reference polypeptide or protein sequence (e.g., a wild type or full-length fusion protein) and comprising, over the common portion, polypeptide or protein sequence identical to the reference sequence. Such a fragment according to the present disclosure may be, where appropriate, included in a larger polypeptide or protein of which it is a constituent. Such fragments comprise, or alternatively consist of, polypeptides or proteins ranging in length from at least 8, 10, 12, 15, 18, 20 to 25, 30, 40, 50, 70, 80, 100, 200, 500, 1000, 1500, or any number or range therein, consecutive amino acids of a polypeptide or protein described herein.

[0041] A “reference sequence” means a nucleic acid or amino acid sequence used as a comparator for another nucleic acid or amino acid sequence, respectively, when determining sequence identity. A reference sequence can be a wild type sequence or a sequence that has not been modified as described herein.

[0042] “Sequence identity,” “percent identity,” or “% identical” refers to the exactness of a match between a reference sequence and a sequence being compared to it when optimally aligned. For example, sequence alignments and percent identity calculations may be determined using a variety of comparison methods designed to detect homologous sequences including, but not limited to, the Multalin program (Corpet, “Multiple Sequence Alignment with Hierarchical Clustering,” Nucleic Acids Res.16:10881-90 (1988), which is hereby incorporated by reference in its entirety). Sequences may also be aligned using algorithms known in the art including, but not limited to, CLUSTAL V algorithm or the BLASTN or BLAST 2 sequence programs. Preferences and options for a given aspect, feature, embodiment, or parameter of the disclosure should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences and options for all other aspects, features, embodiments, and parameters of the disclosure. Carbonic Anhydrase Fusion Proteins

[0043] One aspect of the present disclosure relates to a fusion protein comprising a carbonic anhydrase domain and one or more of a calcium carbonate binding domain, a calcium 172604987binding domain, an iron binding domain, a silicatein mimetic domain, and a silica binding domain.

[0044] This aspect of the present disclosure can be carried out with any of the embodiments disclosed herein.

[0045] A “fusion protein” as used herein is a protein or polypeptide sequence comprising one or more amino acid domains. A fusion protein also optionally comprises protein or polypeptide sequences that are proteolytically processed. A “domain” as used herein refers to a portion of a fusion protein (i.e., an amino acid sequence shorter than the entire amino acid sequence of the entire fusion protein) that has a specific function or functions or activity. As non-limiting examples, a domain can have an enzymatic function, a chemical or ion binding function, a linker function to link one or more domains, a purification or affinity tag function, a detection function, a protease cleavage site function, and / or a secretory signal peptide function.

[0046] As used herein, a “carbonic anhydrase domain” or “CA domain” within a fusion protein encompasses both full-length enzymatic carbonic anhydrase protein sequences as well as partial protein sequences or fragments of the full-length protein sequence compared to a wild- type carbonic anhydrase protein sequence that have carbonic anhydrase enzymatic activity. Carbonic anhydrase (EC 4.2.1.1) plays a role in a carbon dioxide (CO2) transport, CO2metabolism, and pH homeostasis (see Supuran and Capasso, “An Overview of the Bacterial Carbonic Anhydrases,” Metabolites 7:56 (2017), which is hereby incorporated by reference in its entirety). Carbonic anhydrase catalyzes the following reversible reaction: H2O + CO2⇄ H++ HCO3-

[0047] distinct classes named alpha, beta, gamma, and delta. These classes evolved from independent origin (Bacteria, Archaea, Eukarya) and have no significant sequence or structural identity except for the presence of a single zinc atom at the catalytic site (see Tripp et al., “Carbonic Anhydrase: New Insights for an Ancient Enzyme - 210,” J. Biol. Chem, 276(52):48615-48618 (2001), which is hereby incorporated by reference in its entirety). Alpha class CA are present in mammals where more than 11 isozymes have been identified, beta classes are present in algae and plants, and gamma classes are present in prokaryotes and Archaea. Many prokaryotes contain carbonic anhydrase genes from more than one class or more than one gene of a certain class.

[0048] The conversion of CO2 by carbonic anhydrase to bicarbonate and / or carbonate ions, allows the precipitation as carbonate salts for CO2capture and utilization.

[0049] Non-limiting examples of carbonic anhydrases and carbonic anhydrase domains are provided as follows (e.g., SEQ ID NOs:1-3, 5, 7-13, 16, and 17). 172604987

[0050] The full length alpha carbonic anhydrase sequence from Aliivibrio salmonicida (GenBank Accession No. WP_012549813, which is hereby incorporated by reference in its entirety) is as follows (SEQ ID NO:1): MKKIITTLAA LTVLSTSYTV SASEWSYSGE TAPTNWHGTC QSGVNQSPID ITHAIESTLD PIIFNYSKAG KNVVNNGHTV QVNFDAKQSI QIEGKTFTLL QLHFHAPSEN LIEGHSFPLE MHLVHADKDG NLAVIGVMLK EGKPNSELTK IWAKMPQSGQ VDLENQLDLT KLLPRDQAYY RFNGSLTTPP CSEGVTWLVM KNPIEISSEQ LAQFTALYNG NNCPTQAINA RPILK

[0051] The full length alpha carbonic anhydrase sequence from Thermosulfurimonas dismutans (GenBank Accession No. WP_068668235, which is hereby incorporated by reference in its entirety) is as follows (SEQ ID NO:2): MKKFIGGLVG SLMIGGVALA GGGHVVKWGY VGKIGPAHWG DLAHEYFMCK VGKNQSPVDI NSSVTIEAQL EPINFHYRDQ ISGEIVNNGH TIMVVPKEDN YIVVDGKKFH LKQFHFHSPS EHTVEGKYYL LELHFVHQAD DGQLAVIGVV FDRGAEHPEI AKLWKEAPEH EGKKELKSLV NMQALLPENL DYYRYSGSLT TPPCSEGVIW LFLKNPLQIS EAQAEKFKKI MGFENNRPVQ PVNARKILK

[0052] The “Masa CA”, an alpha carbonic anhydrase domain sequence derived from Aliivibrio salmonicida, is as follows (SEQ ID NO:3): ASEWSYSGET APTNWHGTCQ SGVNQSPIDI THAIESTLDP IIFNYSKAGK NVVNNGHTVQ VNFDAKQSIQ IEGKTFTLLQ LHFHAPSENL IEGHSFPLEM HLVHADKDGN LAVIGVMLKE GKPNSELTKI WAKMPQSGQV DLENQLDLTK LLPRDQAYYR FNGSLTTPPC SEGVTWLVMK NPIEISSEQL AQFTALYNGN NCPTQAINAR PILK

[0053] The Masa CA domain does not include the signal peptide at the N-terminus of the Aliivibrio salmonicida CA protein, MKKIITTLAA LTVLSTSYTV S (SEQ ID NO:4). In some embodiments, the carbonic anhydrase domain does not include a signal peptide. In some embodiments, the carbonic anhydrase domain comprises the Masa CA domain of SEQ ID NO:3. In some embodiments, the Td CA domain does not comprise SEQ ID NO:4.

[0054] “Td CA”, an alpha carbonic anhydrase domain sequence derived from Thermosulfurimonas dismutans, is as follows (SEQ ID NO:5): GGGHVVKWGY VGKIGPAHWG DLAHEYFMCK VGKNQSPVDI NSSVTIEAQL EPINFHYRDQ ISGEIVNNGH TIMVVPKEDN YIVVDGKKFH LKQFHFHSPS EHTVEGKYYL LELHFVHQAD DGQLAVIGVV FDRGAEHPEI AKLWKEAPEH EGKKELKSLV NMQALLPENL DYYRYSGSLT TPPCSEGVIW LFLKNPLQIS EAQAEKFKKI MGFENNRPVQ PVNARKILK

[0055] The Td CA domain does not include the signal peptide at the N-terminus of the Thermosulfurimonas dismutans CA protein, MKKFIGGLVG SLMIGGVALA (SEQ ID NO:6). In some embodiments, the carbonic anhydrase domain comprises the Td CA domain of SEQ ID NO:5. In some embodiments, the Td CA domain does not comprise SEQ ID NO:6. 172604987

[0056] In some embodiments, the carbonic anhydrase domain comprises amino acids 22- 235 of an Aliivibrio salmonicida carbonic anhydrase of SEQ ID NO:1 or amino acids 21-249 of of a Thermosulfurimonas dismutans carbonic anhydrase of SEQ ID NO:2, or a carbonic anhydrase domain having at least 95% sequence identity to amino acids 22-235 of SEQ ID NO:1 or amino acids 21-249 of SEQ ID NO:2.

[0057] In some embodiments, the carbonic anhydrase domain comprises an asparagine to cysteine mutation, a threonine to cysteine mutation, or a glutamine to cysteine mutation compared to a wild type carbonic anhydrase domain, where the fusion protein is capable of forming dimer-inducing disulfide bonds. In some embodiments, a mutation to introduce a cysteine may be made in a carbonic anhydrase domain sequence at one or more locations.

[0058] Such an alteration allows formation of disulfide bonds between carbonic anhydrase domains to promote covalent dimerization for enhanced lattice formation and thermostability of the fusion proteins. Lattice formation in proteins plays an important role in enhancing the stability and overall structural integrity of CA fusion proteins. Proteins are composed of long chains of amino acids that fold into specific three-dimensional structures. The lattice refers to the intricate network of noncovalent interactions that stabilize these folded structures. This lattice is primarily formed by hydrogen bonds, electrostatic interactions, van der Waals forces, and hydrophobic interactions.

[0059] The lattice provides stability to proteins in several ways. First, it helps to maintain the correct conformation of the protein, ensuring that it retains its functional shape. The lattice interactions hold the amino acid residues in their proper positions, preventing unwanted conformational changes and maintaining the overall structural stability of the protein. Additionally, the lattice contributes to the resistance of proteins against denaturation and unfolding under various environmental conditions. The intricate network of interactions in the lattice provides a strong framework that can withstand external stresses, such as changes in temperature, pH, and / or exposure to denaturing agents. By reinforcing the folded structure, the lattice helps proteins maintain their biological activity and prevents them from losing their functional properties.

[0060] The lattice formation also aids in the proper folding of newly synthesized proteins. During protein synthesis, nascent polypeptide chains need to navigate through a crowded cellular environment to adopt their native conformations. The lattice interactions guide the folding process, ensuring that the protein attains its correct structure by minimizing misfolding and aggregation events. 172604987

[0061] Thus, lattice formation in proteins aids in their stability. It confers structural integrity, resists denaturation, and facilitates proper folding. The intricate network of noncovalent interactions within the lattice plays a fundamental role in maintaining the functional and three-dimensional characteristics of proteins, enabling them to carry out their biological roles effectively.

[0062] Exemplary CA domain sequences with cysteine mutations (called “C-variants”) that promote dimerization are now described.

[0063] “Masa CA C-variant 1” is an alpha carbonic anhydrase domain sequence derived from Aliivibrio salmonicida, which has an asparagine to cysteine mutation at amino acid position 24 (N24C) relative to SEQ ID NO:3. The Masa CA C-variant 1 amino acid sequence is as follows with the N24C mutation indicated in bold text (SEQ ID NO:7): ASEWSYSGET APTNWHGTCQ SGVCQSPIDI THAIESTLDP IIFNYSKAGK NVVNNGHTVQ VNFDAKQSIQ IEGKTFTLLQ LHFHAPSENL IEGHSFPLEM HLVHADKDGN LAVIGVMLKE GKPNSELTKI WAKMPQSGQV DLENQLDLTK LLPRDQAYYR FNGSLTTPPC SEGVTWLVMK NPIEISSEQL AQFTALYNGN NCPTQAINAR PILK

[0064] “Masa CA C-variant 2” is an alpha carbonic anhydrase domain sequence derived from Aliivibrio salmonicida, which has a threonine to cysteine mutation at amino acid position 37 (T37C) relative to SEQ ID NO:3. The Masa CA C-variant 2 amino acid sequence is as follows with the T37C mutation indicated in bold text (SEQ ID NO:8): ASEWSYSGET APTNWHGTCQ SGVNQSPIDI THAIESCLDP IIFNYSKAGK NVVNNGHTVQ VNFDAKQSIQ IEGKTFTLLQ LHFHAPSENL IEGHSFPLEM HLVHADKDGN LAVIGVMLKE GKPNSELTKI WAKMPQSGQV DLENQLDLTK LLPRDQAYYR FNGSLTTPPC SEGVTWLVMK NPIEISSEQL AQFTALYNGN NCPTQAINAR PILK

[0065] The Masa CA C-variant 2 mutation (T37C) promotes the tetrameric formation of the enzyme through disulfide bond formation.

[0066] “Td CA C-variant 1”, an alpha carbonic anhydrase domain sequence derived from Thermosulfurimonas dismutans, has an asparagine to cysteine mutation at amino acid position 34 (N34C) relative to SEQ ID NO:5. The Td CA C-variant 1 amino acid sequence is as follows with the N34C mutation indicated in bold text (SEQ ID NO:9): GGGHVVKWGY VGKIGPAHWG DLAHEYFMCK VGKCQSPVDI NSSVTIEAQL EPINFHYRDQ ISGEIVNNGH TIMVVPKEDN YIVVDGKKFH LKQFHFHSPS EHTVEGKYYL LELHFVHQAD DGQLAVIGVV FDRGAEHPEI AKLWKEAPEH EGKKELKSLV NMQALLPENL DYYRYSGSLT TPPCSEGVIW LFLKNPLQIS EAQAEKFKKI MGFENNRPVQ PVNARKILK

[0067] “Td CA C-variant 2”, an alpha carbonic anhydrase domain sequence derived from Thermosulfurimonas dismutans, has a glutamine to cysteine mutation at amino acid position 49 172604987(Q49C) relative to SEQ ID NO:5. The Td CA C-variant 2 amino acid sequence is as follows with the Q49C mutation indicated in bold text (SEQ ID NO:10): GGGHVVKWGY VGKIGPAHWG DLAHEYFMCK VGKNQSPVDI NSSVTIEACL EPINFHYRDQ ISGEIVNNGH TIMVVPKEDN YIVVDGKKFH LKQFHFHSPS EHTVEGKYYL LELHFVHQAD DGQLAVIGVV FDRGAEHPEI AKLWKEAPEH EGKKELKSLV NMQALLPENL DYYRYSGSLT TPPCSEGVIW LFLKNPLQIS EAQAEKFKKI MGFENNRPVQ PVNARKILK

[0068] In some embodiments, the expressed CA fusion protein comprises one or more disulfide bridges.

[0069] The “Masa CA G-variant” is an alpha carbonic anhydrase domain sequence derived from Aliivibrio salmonicida, which has an asparagine to glycine mutation at amino acid position 110 (N110G) relative to SEQ ID NO:3. The Masa CA G-variant N110G mutation is provided to enhance thermostability of the carbonic anhydrase domain and fusion proteins comprising the N110G CA domain. The Masa CA G-variant amino acid sequence is as follows with the N110G mutation indicated in bold text (SEQ ID NO:11): ASEWSYSGET APTNWHGTCQ SGVNQSPIDI THAIESTLDP IIFNYSKAGK NVVNNGHTVQ VNFDAKQSIQ IEGKTFTLLQ LHFHAPSENL IEGHSFPLEM HLVHADKDGG LAVIGVMLKE GKPNSELTKI WAKMPQSGQV DLENQLDLTK LLPRDQAYYR FNGSLTTPPC SEGVTWLVMK NPIEISSEQL AQFTALYNGN NCPTQAINAR PILK

[0070] “Masa CA P-variant” is an alpha carbonic anhydrase domain sequence derived from Aliivibrio salmonicida that has a glutamate to proline mutation at amino acid position 143 (E143P) relative to SEQ ID NO:3. The Masa CA P-variant E143P mutation is provided to enhance esterase activity of the carbonic anhydrase domain and fusion proteins comprising the E143P CA domain. The Masa CA P-variant amino acid sequence is as follows with the E143P mutation indicated in bold text (SEQ ID NO:12): ASEWSYSGET APTNWHGTCQ SGVNQSPIDI THAIESTLDP IIFNYSKAGK NVVNNGHTVQ VNFDAKQSIQ IEGKTFTLLQ LHFHAPSENL IEGHSFPLEM HLVHADKDGN LAVIGVMLKE GKPNSELTKI WAKMPQSGQV DLPNQLDLTK LLPRDQAYYR FNGSLTTPPC SEGVTWLVMK NPIEISSEQL AQFTALYNGN NCPTQAINAR PILK

[0071] “Masa CA H-variant” is an alpha carbonic anhydrase domain sequence derived from Aliivibrio salmonicida that has a glutamate to histidine mutation at amino acid position 3 (E3H) relative to SEQ ID NO:3. The Masa CA H-variant was made to further stabilize the Masa CA enzyme domain. The Masa CA H-variant amino acid sequence is as follows with the E3H mutation indicated in bold text (SEQ ID NO:13): ASHWSYSGET APTNWHGTCQ SGVNQSPIDI THAIESTLDP IIFNYSKAGK NVVNNGHTVQ VNFDAKQSIQ IEGKTFTLLQ LHFHAPSENL IEGHSFPLEM HLVHADKDGN LAVIGVMLKE 172604987GKPNSELTKI WAKMPQSGQV DLENQLDLTK LLPRDQAYYR FNGSLTTPPC SEGVTWLVMK NPIEISSEQL AQFTALYNGN NCPTQAINAR PILK

[0072] In some embodiments, the carbonic anhydrase domain comprises one or more of: an asparagine to cysteine mutation corresponding to amino acid position 24 (N24C) of SEQ ID NO:3, a threonine to cysteine mutation corresponding to amino acid position 37 (T37C) of SEQ ID NO:3, an asparagine to cysteine mutation corresponding to amino acid position 34 (N34C) of SEQ ID NO:5, a glutamine to cysteine mutation corresponding to amino acid position 49 (Q49C) of SEQ ID NO:5, an asparagine to glycine mutation corresponding to amino acid position 110 (N110G) of SEQ ID NO:3, a glutamate to proline mutation corresponding to amino acid position 143 (E143P) of SEQ ID NO:3, and a glutamate to histidine mutation corresponding to amino acid position 3 (E3H) of SEQ ID NO:3.

[0073] In some embodiments, the carbonic anhydrase domain comprises all or part of SEQ ID NOs:1 or 2, or a carbonic anhydrase having at least 95% sequence identity to any one of SEQ ID NOs: 1 or 2. In some embodiments, the carbonic anhydrase domain comprises any one of SEQ ID NOs:3, 5, or 7-13 or a carbonic anhydrase domain having at least 95% sequence identity to any one of SEQ ID NOs: 3, 5, or 7-13. Due to genetic variation that can occur, the carbonic anhydrase domain may comprise at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the carbonic anhydrase domains described herein.

[0074] Chimeric combinations of the various CA domains disclosed herein are also contemplated. For example, the N-terminal loop of the Td CA sequence can be inserted into the N-terminal portion of the Masa CA sequence as shown below in SEQ ID NOs:16 and 17. In some embodiments, the N-terminal portion of SEQ ID NOs:3, 7, 8, or 11-13 comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 160 amino acids, or any number or range therein of SEQ ID NO:5, 9, or 10.

[0075] In some embodiments, the carbonic anhydrase domain comprises a “loop insert”. The loop insert can aid in structural stability of the active site of carbonic anhydrase and may promote enzymatic function at even higher temperatures and more extreme conditions. Exemplary loop insert sequences include: loop 1 GDLAHE (SEQ ID NO:14) and loop 2 GDLKDE (SEQ ID NO:15). In some embodiments, the carbonic anhydrase domain comprises SEQ ID NO:14 or SEQ ID NO:15. Exemplary CA domain sequences with an added loop insert sequences are now described.

[0076] “Masa CA loop 1”, an alpha carbonic anhydrase domain sequence derived from Aliivibrio salmonicida, is as follows with the loop sequence indicated in bold text (SEQ ID NO:16): 172604987ASEWSYSGET APTNWGDLAHEHGTCQSGVN QSPIDITHAI ESTLDPIIFN YSKAGKNVVN NGHTVQVNFD AKQSIQIEGK TFTLLQLHFH APSENLIEGH SFPLEMHLVH ADKDGNLAVI GVMLKEGKPN SELTKIWAKM PQSGQVDLEN QLDLTKLLPR DQAYYRFNGS LTTPPCSEGV TWLVMKNPIE ISSEQLAQFT ALYNGNNCPT QAINARPILK

[0077] “Masa CA loop 2”, an alpha carbonic anhydrase domain sequence derived from Aliivibrio salmonicida (GenBank Accession No. WP_012549813, which is hereby incorporated by reference in its entirety) is as follows (SEQ ID NO:17) with the loop sequence indicated in bold text: ASEWSYSGET APTNWGDLKDE HGTCQSGVN QSPIDITHAI ESTLDPIIFN YSKAGKNVVN NGHTVQVNFD AKQSIQIEGK TFTLLQLHFH APSENLIEGH SFPLEMHLVH ADKDGNLAVI GVMLKEGKPN SELTKIWAKM PQSGQVDLEN QLDLTKLLPR DQAYYRFNGS LTTPPCSEGV TWLVMKNPIE ISSEQLAQFT ALYNGNNCPT QAINARPILK

[0078] In some embodiments, the carbonic anhydrase domain comprises SEQ ID NO:16 or SEQ ID NO:17, or a carbonic anhydrase domain having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:16 or SEQ ID NO:17.

[0079] In some embodiments, the carbonic anhydrase domain comprises any one of SEQ ID NOs:3, 5, 7-13, 16, or 17, or a carbonic anhydrase domain having at least 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs:3, 5, 7-13, 16, or 17.

[0080] In some embodiments, the fusion protein further comprises one or more of a secretory domain, a protease cleavage site domain, a detection and / or purification tag domain, and a linker domain.

[0081] In some embodiments, the fusion protein further comprises a secretory domain sequence. The fusion proteins described herein may also comprise a “secretory domain sequence” with a secretory peptide or secretory signal sequence at the N-terminus to promote secretion of the fusion protein from the host cell in which it is being expressed. Secretory domain sequences may be present in the fusion protein and / or may be removed after the protein is secreted from the host cell during fusion protein production. In some embodiments, in addition to providing a secretory function, exemplary secretory domains disclosed herein may also improve solubility of the fusion proteins. In some embodiments, a secretory domain may be used for affinity purification. See, e.g., Kimple et al., “Overview of Affinity Tags for Protein Purification,” Curr. Protoc. Protein Sci.73:9.9.1-9.9.23 (2013), doi: 10.1002 / 0471140864.ps0909s73, which is hereby incorporated by reference in its entirety. In some embodiments, the fusion protein comprises a secretory domain sequence. Exemplary secretory domain sequences are now described. 172604987

[0082] An exemplary secretory domain sequence useful for embodiments of the present disclosure is the osmotically-inducible protein Y (osmY) protein, or fragment thereof, from Escherichia coli (UniProt Accession No. P0AFH8, which is hereby incorporated by reference in its entirety). The sequence for wild type osmY is as follows (SEQ ID NO:18): MTMTRLKISK TLLAVMLTSA VATGSAYAEN NAQTTNESAG QKVDSSMNKV GNFMDDSAIT AKVKAALVDH DNIKSTDISV KTDQKVVTLS GFVESQAQAE EAVKVAKGVE GVTSVSDKLH VRDAKEGSVK GYAGDTATTS EIKAKLLADD IVPSRHVKVE TTDGVVQLSG TVDSQAQSDR AESIAKAVDG VKSVKNDLKT

[0083] A mutant osmY protein sequence can also be used in the fusion proteins of the present disclosure. The sequence for mutant osmY is as follows (SEQ ID NO:19): MRGSQQQQQH GSMTRPKISK TLLAVMLTSA VATGSAYAEN NAQTTNESAG QKADSSMNKV GNFMDDSAIT AKVKAALVDH DNIKSTDISV KTDQKVVTLS GFVESQAQAE EAVKVAKGVE GVTSVSDKLH VRDAKEGSVK GYAGDTATTS EIKAKLLADD IVPRRHVKVE TTDGVVQLSG TVDSQAQSDR AESIAKAVDG EKSVKNDLKT K

[0084] Another secretory domain sequence useful for protein fusions described herein is from Thermococcus kodakarensis (see Muhammad et al., “Complete Signal Peptide of Tk1884, an α-amylase from Thermococcus kodakarensis, is Not Necessary for Extracellular Secretion of the Enzyme by Escherichia coli,” Amylase 1(1):75-81 (2017), which is hereby incorporated by reference in its entirety) whose sequence is MKKFVALLIT MFFVVSMAAV AQPASAA (SEQ ID NO:20).

[0085] A further secretory domain sequence is a cleavage signal peptide or a pelb signal peptide as described in Singh et al., “Effect of Signal Peptide on Stability and Folding of Escherichia coli Thioredoxin,” PloS One 8.5: e63442 (2013), which is hereby incorporated by reference in its entirety).

[0086] Another secretory domain sequence useful for bacterial periplasm localization and lattice formation of the protein fusions of the present disclosure comprises the superfolder green fluorescent protein sequence (sfGFP) (in bold text) derived from Aequorea victoria (see fpbase.org / protein / superfolder-gfp / and Dinh and Bernhardt “Using Superfolder Green Fluorescent Protein for Periplasmic Protein Localization Studies,” J. Bacteriol.193(18):4984-7 (2011), each of which is hereby incorporated by reference in its entirety) and having a D117C mutation as follows (SEQ ID NO:21): MRGSQQQQQQ GSMSKGEELF TGVVPILVEL DGDVNGHKFS VRGEGEGDAT NGKLTLKFIC TTGKLPVPWP TLVTTLTYGV QCFSRYPDHM KRHDFFKSAM PEGYVQERTI SFKDDGTYKT RAEVKFEGCT LVNRIELKGI DFKEDGNILG HKLEYNFNSH NVYITADKQK NGIKANFKIR HNVEDGSVQL ADHYQQNTPI GDGPVLLPDN HYLSTQSVLS KDPNEKRDHM VLLEFVTAAG ITHGMDELYK 172604987

[0087] In some embodiments, a polylinker MRGSQQQQQQGS (SEQ ID NO:62) is added at the amino terminus of the fusion protein to lower the activation energy required for transcription.

[0088] A further exemplary secretory domain sequence that also enhances solubility is the maltose binding protein (“MBP”) (see Pennati et al., “Maltose-Binding Protein Fusion Allows for High Level Bacterial Expression and Purification of Bioactive Mammalian Cytokine Derivatives,” PLoS One 9.9: e106724 (2014), which is hereby incorporated by reference in its entirety). In some embodiments, the MBP is cytoplasmic. The sequence for the secretory domain sequence of cytoplasmic MBP is as follows (SEQ ID NO:22): MKIEEGKLVI WINGDKGYNG LAEVGKKFEK DTGIKVTVEH PDKLEEKFPQ VAATGDGPDI IFWAHDRFGG YAQSGLLAEI TPDKAFQDKL YPFTWDAVRY NGKLIAYPIA VEALSLIYNK DLLPNPPKTW EEIPALDKEL KAKGKSALMF NLQEPYFTWP LIAADGGYAF KYENGKYDIK DVGVDNAGAK AGLTFLVDLI KNKHMNADTD YSIAEAAFNK GETAMTINGP WAWSNIDTSK VNYGVTVLPT FKGQPSKPFV GVLSAGINAA SPNKELAKEF LENYLLTDEG LEAVNKDKPL GAVALKSYEE ELAKDPRIAA TMENAQKGEI MPNIPQMSAF WYAVRTAVIN AASGRQTVDE ALKDAQTRIT K

[0089] In some embodiments, the MBP is periplasmic. The sequence for the secretory domain sequence of periplasmic MBP is as follows (SEQ ID NO:23): MKIKTGARIL ALSALTTMMF SASALAKIEE GKLVIWINGD KGYNGLAEVG KKFEKDTGIK VTVEHPDKLE EKFPQVAATG DGPDIIFWAH DRFGGYAQSG LLAEITPDKA FQDKLYPFTW DAVRYNGKLI AYPIAVEALS LIYNKDLLPN PPKTWEEIPA LDKELKAKGK SALMFNLQEP YFTWPLIAAD GGYAFKYENG KYDIKDVGVD NAGAKAGLTF LVDLIKNKHM NADTDYSIAE AAFNKGETAM TINGPWAWSN IDTSKVNYGV TVLPTFKGQP SKPFVGVLSA GINAASPNKE LAKEFLENYL LTDEGLEAVN KDKPLGAVAL KSYEEELAKD PRIAATMENA QKGEIMPNIP QMSAFWYAVR TAVINAASGR QTVDEALKDA QTRITK

[0090] Another exemplary secretory domain sequence is trigger factor (see De Geyter et al., “Trigger Factor is a Bona Fide Secretory Pathway Chaperone that Interacts with SecB and the Translocase,” EMBO Rep.21(6):e49054 (2020), which is hereby incorporated by reference in its entirety).

[0091] In some embodiments, the secretory domain sequence comprises any one of SEQ ID NOs:18-23, or a secretory domain sequence having at least 95% sequence identity to any one of SEQ ID NOs:18-23.

[0092] In some embodiments, the fusion protein further comprises one or more protease cleavage site domains. A “protease cleavage site domain” may also be optionally present in the fusion proteins described herein. A protease cleavage domain may be used to cleave off, for example, the secretory domain sequence and / or other domain sequences to produce a processed fusion protein (i.e., a fragment of the fusion protein). In some embodiments, the protease 172604987cleavage domain is located between the secretory domain sequence and the CA domain sequence. Suitable protease cleavages sites are well known to those of skill in the art. In some embodiments, the one or more protease cleavage site domains comprises any one or more of a rhinovirus protease site, a potyvirus NIa protease site, a potyvirus HC protease site, a potyvirus P1 protease site, a byovirus NIa protease site, a byovirus RNA-2-encoded protease site, an aphthovirus L protease site, an enterovirus 2A protease site, a rhinovirus 2A protease site, a picorna 3C protease site, a comovirus 24K protease site, a nepovirus 24K protease site, a RTSV (rice tungro spherical virus) 3C-like protease site, a PYVF (parsnip yellow fleck virus) 3C-like protease site, a thrombin, factor Xa protease site, a trypsin site, a Genenase protease site, a WELQut protease site, a TEV sequence, a SUMO protease site, and an enterokinase site. In order to use one of these cleavage sites, it may be necessary to remove unwanted cleavage sites from within the desired domain sequences by mutation. In some embodiments, the protease cleavage site domain comprises a rhinovirus protease cleavage site: LEVLFQGP (SEQ ID NO:24).

[0093] In some embodiments, the expressed CA fusion protein is contacted with a highly specific protease (e.g., rhinovirus protease, SUMO protease, enterokinase, TEV sequence, or WELQut protease) under conditions effective to enable cleavage at the protease cleavage site of the CA fusion protein. By this means, the CA fusion protein is not cleaved by proteases endogenous to the host cell. In some embodiments, the CA fusion protein is a proteolytic fragment.

[0094] In some embodiments, the CA fusion protein or proteolytic fragment thereof, comprises one or more of a calcium carbonate binding domain, a calcium binding domain, an iron binding domain, a silicatein mimetic domain, and a silica binding domain. Non-limiting examples of combinations of these domains with CA domains and secretory domains are provided herein (e.g., see SEQ ID NOs:51-61). In some embodiments, one or more of each of the calcium carbonate binding domain, the calcium binding domain, the iron binding domain, the silicatein mimetic domain, and / or the silica binding domain can be located before the CA domain sequence or after the CA domain sequence or a combination of both. In some embodiments, the CA fusion protein or proteolytic fragment thereof does not comprise one or more of a calcium carbonate binding domain, a calcium binding domain, an iron binding domain, a silicatein mimetic domain, and a silica binding domain.

[0095] In some embodiments, the fusion protein or fragment thereof comprises one or more “detection and / or purification tag domain”. The detection and / or purification tag domain assists in detection and / or purification of the fusion protein or fragment thereof. Exemplary and non-limiting detection and / or purification tag domains include the His-tag (hexahistidine) tag 172604987HHHHHH (SEQ ID NO:25) or His-tag pentahistidine tag HHHHH (SEQ ID NO:26), the bd SUMO tag HINLKVKGQD GNEVFFRIKR STQLKKLMNA YCDRQSVDMT AIAFLFDGR RLRAEQTPDE LEMEDGDEID AMLHQTGG (SEQ ID NO:27), FLAG tag DYKDDDDK (SEQ ID NO:28), Strep II tag WSHPQFEK (SEQ ID NO:29), streptavidin-binding peptide (SBP) tag MDEKTTGWRG GHVVEGLAGE LEQLRARLEH HPQGQREP (SEQ ID NO:30), calmodulin-binding peptide (CBP) GVMPREETDS KTASPWKSAR LMVHTVATFN SIKELNERWR SLQQLA (SEQ ID NO:31), glutathione S-transferase (GST) tag MSPILGYWKI KGLVQPTRLL LEYLEEKYEE HLYERDEGDK WRNKKFELGL EFPNLPYYID GDVKLTQSMA IIRYIADKHN MLGGCPKERA EISMLEGAVL DIRYGVSRIA YSKDFETLKV DFLSKLPEML KMFEDRLCHK TYLNGDHVTH PDFMLYDALD VVLYMDPMCL DAFPKLVCFK KRIEAIPQID KYLKSSKYIA WPLQGWQATF GGGDHPPKSD (SEQ ID NO:32), maltose-binding protein (MBP) tag YNGLAEVGKK FEKDTGIKVT VEH (SEQ ID NO:33), S-tag KETAAAKFER QHMDS (SEQ ID NO:34), HA tag YPYDVPDYA (SEQ ID NO:35), calmodulin binding peptide tag GVMPREETDS KTASPWKSAR LMVHTVATFN SIKELNERWR SLQQLA (SEQ ID NO:36), AviTag GLNDIFEAQK IEWHE (SEQ ID NO:37), and c-myc tag EQKLISEEDL (SEQ ID NO:38).

[0096] In some embodiments, the one or more detection and / or purification tag domains comprises any one or more of a His-tag, a bd SUMO tag, a FLAG tag, a Strep II tag, a streptavidin-binding peptide tag, a calmodulin-binding peptide tag, a glutathione S-transferase tag, a maltose-binding protein tag, a calmodulin binding peptide tag, and a c-myc tag.

[0097] In some embodiments, one or more “linker domains” are used in the fusion or fragment thereof. In some embodiments, the fusion protein comprises a linker domain between at least one of the secretory domain, the CA domain, the calcium carbonate binding domain, the calcium binding domain, the iron binding domain, the silicatein mimetic domain, and / or the silica binding domain, if present. In some embodiments, the fusion protein comprises a linker domain between each of the secretory domain, the CA domain, the calcium carbonate binding domain, the calcium binding domain, the iron binding domain, the silicatein mimetic domain, and / or the silica binding domain, if present.

[0098] A peptide linker can range from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, to 40 or more amino acids or any value or range therein. In some embodiments, a linker domain can range from 1 amino acid to 60 or more amino acids, and in certain embodiments a peptide linker ranges from 2 amino acids to 50 amino acids, from 3 to 30 amino acids, from 4 to 25 amino acids, from 5 to 25 amino acids, 10 amino acids to 60 amino acids, from 12 amino acids to 20 amino acids, from 20 amino acids to 50 amino acids, or from 25 amino acids to 35 amino acids in length. Non-limiting exemplary peptide linker domains useful in the fusion proteins described 172604987herein may comprise an amino acid sequence selected from one or more of GGGGSG (SEQ ID NO:39), GGGSG (SEQ ID NO:40), GGGGS (SEQ ID NO:41), GGGG (SEQ ID NO:42), GGG, GG, GST, S, AA, AAA, AAAAA (SEQ ID NO:43), and AAAA (SEQ ID NO:44). In some embodiments, the fusion protein comprises one or more linker domains.

[0099] In some embodiments, the fusion protein comprises one or more “calcium carbonate binding domains”. The calcium carbonate binding domain binds CaCO3. The calcium carbonate binding domain assists in binding of the fusion protein or fragment to compositions of the disclosure through binding affinity interactions to keep the carbonic anhydrase domains and calcium bicarbonate bound in concrete and limestone, and to promote structural support. In some embodiments, the calcium carbonate binding domain comprises GHTQNMRMYEPWF (SEQ ID NO:45).

[0100] In some embodiments, the fusion protein comprises one or more “calcium binding domains”. The calcium binding domain brings ionic calcium within range of the CA domain for enhanced CaCO3 formation. In some embodiments, the calcium binding domain comprises GDEEENDQVK (SEQ ID NO:46).

[0101] In some embodiments, the fusion protein or fragment thereof comprises one or more “iron binding domains”. The iron binding domain allows the fusion protein or fragment to adhere in concrete and / or rebar to iron surfaces such as iron rebar. In some embodiments, the iron binding domain comprises VNVPLY (SEQ ID NO:47).

[0102] In some embodiments, the fusion protein or fragment thereof comprises one or more “silica binding domains”. The silica binding domain binds silica dioxide for adhesion of the fusion protein or fragment in concrete. In some embodiments, the silica binding domain comprises GRARAQRQSSRGRKSLSRADHIHHHHH (SEQ ID NO:48) or SGRQSSRGRGGGC (SEQ ID NO:49).

[0103] In some embodiments, the fusion protein or fragment thereof comprises one or more “silicatein mimetic domains”. (See Natalio et al., “Silicatein-Mediated Incorporation of Titanium into Spicules from the Demosponge Suberites domuncula." Cell and Tissue Research 339.2: 429-436 (2010), which is hereby incorporated by reference in its entirety). The silicatein mimetic domain provides for covalent polymerization of free silica and / or free metal ions to make silica spicules (silica spicule polymerization). The process contributes to structural support of the concrete or ECM compositions comprising the fusion protein or fragment. In concert with one or more of an iron binding domain and silica binding domain, the silicatein mimetic domain allows the fusion protein or fragment thereof to polymerize silica sheaths and / or silica-metal 172604987hybrid sheaths around the rebar to reduce rusting from environmental conditions. In some embodiments, the silicatein mimetic domain comprises LSLHLN (SEQ ID NO:50).

[0104] An exemplary fusion protein of the present disclosure includes the “Masa CA fusion protein” as follows (SEQ ID NO:51): MRGSQQQQQH GSMTRPKISK TLLAVMLTSA VATGSAYAEN NAQTTNESAG QKADSSMNKV GNFMDDSAIT AKVKAALVDH DNIKSTDISV KTDQKVVTLS GFVESQAQAE EAVKVAKGVE GVTSVSDKLH VRDAKEGSVK GYAGDTATTS EIKAKLLADD IVPRRHVKVE TTDGVVQLSG TVDSQAQSDR AESIAKAVDG EKSVKNDLKT KGGGGSGLEV LFQGPGGGSG ASEWSYSGET APTNWHGTCQ SGVNQSPIDI THAIESTLDP IIFNYSKAGK NVVNNGHTVQ VNFDAKQSIQ IEGKTFTLLQ LHFHAPSENL IEGHSFPLEM HLVHADKDGN LAVIGVMLKE GKPNSELTKI WAKMPQSGQV DLENQLDLTK LLPRDQAYYR FNGSLTTPPC SEGVTWLVMK NPIEISSEQL AQFTALYNGN NCPTQAINAR PILKAAAAAH HHHHHGGGSG GRARAQRQSS RGRKSLSRAD HIHHHHHGGG GSGHTQNMRM YEPWFAAAAL SLHLNGGGGS VNVPLY

[0105] An exemplary “Masa CA fusion proteolytic fragment” after cleavage at the rhinovirus protease site is as follows (SEQ ID NO:52): GPGGGSG ASEWSYSGET APTNWHGTCQ SGVNQSPIDI THAIESTLDP IIFNYSKAGK NVVNNGHTVQ VNFDAKQSIQ IEGKTFTLLQ LHFHAPSENL IEGHSFPLEM HLVHADKDGN LAVIGVMLKE GKPNSELTKI WAKMPQSGQV DLENQLDLTK LLPRDQAYYR FNGSLTTPPC SEGVTWLVMK NPIEISSEQL AQFTALYNGN NCPTQAINAR PILKAAAAAH HHHHHGGGSG GRARAQRQSS RGRKSLSRAD HIHHHHHGGG GSGHTQNMRM YEPWFAAAAL SLHLNGGGGS VNVPLY

[0106] An exemplary fusion protein of the present disclosure includes the “Masa CA fusion protein C-variant 1” as follows (SEQ ID NO:53): MRGSQQQQQH GSMTRPKISK TLLAVMLTSA VATGSAYAEN NAQTTNESAG QKADSSMNKV GNFMDDSAIT AKVKAALVDH DNIKSTDISV KTDQKVVTLS GFVESQAQAE EAVKVAKGVE GVTSVSDKLH VRDAKEGSVK GYAGDTATTS EIKAKLLADD IVPRRHVKVE TTDGVVQLSG TVDSQAQSDR AESIAKAVDG EKSVKNDLKT KGGGGSGLEV LFQGPGGGSG ASEWSYSGET APTNWHGTCQ SGVCQSPIDI THAIESTLDP IIFNYSKAGK NVVNNGHTVQ VNFDAKQSIQ IEGKTFTLLQ LHFHAPSENL IEGHSFPLEM HLVHADKDGN LAVIGVMLKE GKPNSELTKI WAKMPQSGQV DLENQLDLTK LLPRDQAYYR FNGSLTTPPC SEGVTWLVMK NPIEISSEQL AQFTALYNGN NCPTQAINAR PILKAAAAAH HHHHHGGGSG GRARAQRQSS RGRKSLSRAD HIHHHHHGGG GSGHTQNMRM YEPWFAAAAL SLHLNGGGGS VNVPLY

[0107] The Masa CA fusion protein C-variant 1 of SEQ ID NO:53 comprises a mutant osmY secretory domain sequence (SEQ ID NO:19), a Masa CA C-variant 1 carbonic anhydrase domain (SEQ IQ NO:7), multiple linker domain sequences (SEQ ID NOs:39-44), a silica binding domain (SEQ ID NO:48), a HIS tag detection and / or purification tag (SEQ ID NO:25), a rhinovirus protease cleavage site (SEQ ID NO:24), a silicatein mimetic domain (SEQ ID 172604987NO:50), a calcium carbonate binding domain (SEQ ID NO:46), and an iron binding domain (SEQ ID NO:47). A graphic illustration of this fusion protein is provided in FIG.11.

[0108] Another exemplary fusion protein of the present disclosure includes the “Td CA fusion protein” as follows (SEQ ID NO:54): MRGSQQQQQQ GSMSKGEELF TGVVPILVEL DGDVNGHKFS VRGEGEGDAT NGKLTLKFIC TTGKLPVPWP TLVTTLTYGV QCFSRYPDHM KRHDFFKSAM PEGYVQERTI SFKDDGTYKT RAEVKFEGCT LVNRIELKGI DFKEDGNILG HKLEYNFNSH NVYITADKQK NGIKANFKIR HNVEDGSVQL ADHYQQNTPI GDGPVLLPDN HYLSTQSVLS KDPNEKRDHM VLLEFVTAAG ITHGMDELYK GGGGSGDEEE NDQVKGGGGS GSGDEEENDQ VKGGSTGGGG HVVKWGYVGK IGPAHWGDLA HEYFMCKVGK NQSPVDINSS VTIEAQLEPI NFHYRDQISG EIVNNGHTIM VVPKEDNYIV VDGKKFHLKQ FHFHSPSEHT VEGKYYLLEL HFVHQADDGQ LAVIGVVFDR GAEHPEIAKL WKEAPEHEGK KELKSLVNMQ ALLPENLDYY RYSGSLTTPP CSEGVIWLFL KNPLQISESQ AEKFKKIMGF ENNRPVQPVN ARKILKAAAA HHHHHHGGGG SGRQSSRGRG GGCGGGSAQK GTYDDYVEGL GGGSGLSLHL NGGGGSVNVP LY

[0109] A further exemplary fusion protein of the present disclosure includes the “Td CA fusion protein C-variant 1” as follows (SEQ ID NO:55): MRGSQQQQQQ GSMSKGEELF TGVVPILVEL DGDVNGHKFS VRGEGEGDAT NGKLTLKFIC TTGKLPVPWP TLVTTLTYGV QCFSRYPDHM KRHDFFKSAM PEGYVQERTI SFKDDGTYKT RAEVKFEGCT LVNRIELKGI DFKEDGNILG HKLEYNFNSH NVYITADKQK NGIKANFKIR HNVEDGSVQL ADHYQQNTPI GDGPVLLPDN HYLSTQSVLS KDPNEKRDHM VLLEFVTAAG ITHGMDELYK GGGGSGDEEE NDQVKGGGGS GSGDEEENDQ VKGGSTGGGG HVVKWGYVGK IGPAHWGDLA HEYFMCKVGK CQSPVDINSS VTIEAQLEPI NFHYRDQISG EIVNNGHTIM VVPKEDNYIV VDGKKFHLKQ FHFHSPSEHT VEGKYYLLEL HFVHQADDGQ LAVIGVVFDR GAEHPEIAKL WKEAPEHEGK KELKSLVNMQ ALLPENLDYY RYSGSLTTPP CSEGVIWLFL KNPLQISESQ AEKFKKIMGF ENNRPVQPVN ARKILKAAAA HHHHHHGGGG SGRQSSRGRG GGCGGGSAQK GTYDDYVEGL GGGSGLSLHL NGGGGSVNVP LY

[0110] Another exemplary fusion protein of the present disclosure includes the “bdSUMO Masa” fusion protein of FIG.3, Lane 1, which is as follows (SEQ ID NO:56): mrsqqqqqqH INLKVKGQDG NEVFFRIKRS TQLKKLMNAY CDRQSVDMTA IAFLFDGRRL RAEQTPDELE MEDGDEIDAM LHQTGGGDEE ENDQVKSGDE EENDQVKASE WSYSGETAPT NWHGTCQSGV NQSPIDITHA IESTLDPIIF NYSKAGKNVV NNGHTVQVNF DAKQSIQIEG KTFTLLQLHF HAPSENLIEG HSFPLEMHLV HADKDGNLAV IGVMLKEGKP NSELTKIWAK MPQSGQVDLE NQLDLTKLLP RDQAYYRFNG SLTTPPCSEG VTWLVMKNPI EISSEQLAQF TALYNGNNCP TQAINARPIL KAAGRARAQR QSSRGRKSLS RADHIHHHHH GHTQNMRMYE PWFGSVNVPL Y 172604987

[0111] Yet another exemplary fusion protein of the present disclosure includes the “Cytoplasmic MBP Masa” fusion protein of FIG.3, Lane 2, which is as follows (SEQ ID NO:57): MKIEEGKLVI WINGDKGYNG LAEVGKKFEK DTGIKVTVEH PDKLEEKFPQ VAATGDGPDI IFWAHDRFGG YAQSGLLAEI TPDKAFQDKL YPFTWDAVRY NGKLIAYPIA VEALSLIYNK DLLPNPPKTW EEIPALDKEL KAKGKSALMF NLQEPYFTWP LIAADGGYAF KYENGKYDIK DVGVDNAGAK AGLTFLVDLI KNKHMNADTD YSIAEAAFNK GETAMTINGP WAWSNIDTSK VNYGVTVLPT FKGQPSKPFV GVLSAGINAA SPNKELAKEF LENYLLTDEG LEAVNKDKPL GAVALKSYEE ELAKDPRIAA TMENAQKGEI MPNIPQMSAF WYAVRTAVIN AASGRQTVDE ALKDAQTRIT KGDEEENDQV KSGDEEENDQ VKASEWSYSG ETAPTNWHGT CQSGVNQSPI DITHAIESTL DPIIFNYSKA GKNVVNNGHT VQVNFDAKQS IQIEGKTFTL LQLHFHAPSE NLIEGHSFPL EMHLVHADKD GNLAVIGVML KEGKPNSELT KIWAKMPQSG QVDLENQLDL TKLLPRDQAY YRFNGSLTTP PCSEGVTWLV MKNPIEISSE QLAQFTALYN GNNCPTQAIN ARPILKAAGR ARAQRQSSRG RKSLSRADHI HHHHHGHTQN MRMYEPWFGS VNVPLY

[0112] Another exemplary fusion protein of the present disclosure includes the “sfGFP Masa” fusion protein of FIG.3, Lane 3 and FIG.4, which is as follows (SEQ ID NO:58): MRGSQQQQQQ GSMSKGEELF TGVVPILVEL DGDVNGHKFS VRGEGEGDAT NGKLTLKFIC TTGKLPVPWP TLVTTLTYGV QCFSRYPDHM KRHDFFKSAM PEGYVQERTI SFKDDGTYKT RAEVKFEGDT LVNRIELKGI DFKEDGNILG HKLEYNFNSH NVYITADKQK NGIKANFKIR HNVEDGSVQL ADHYQQNTPI GDGPVLLPDN HYLSTQSVLS KDPNEKRDHM VLLEFVTAAG ITHGMDELYK GDEEENDQVK SGDEEENDQV KASEWSYSGE TAPTNWHGTC QSGVNQSPID ITHAIESTLD PIIFNYSKAG KNVVNNGHTV QVNFDAKQSI QIEGKTFTLL QLHFHAPSEN LIEGHSFPLE MHLVHADKDG NLAVIGVMLK EGKPNSELTK IWAKMPQSGQ VDLENQLDLT KLLPRDQAYY RFNGSLTTPP CSEGVTWLVM KNPIEISSEQ LAQFTALYNG NNCPTQAINA RPILKAAGRA RAQRQSSRGR KSLSRADHIH HHHHGHTQNM RMYEPWFGSV NVPLY

[0113] Another exemplary fusion protein of the present disclosure includes the “sfGFP TdCA” fusion protein of FIG.3, Lane 4, which is as follows (SEQ ID NO:59): MRGSQQQQQQ GSMSKGEELF TGVVPILVEL DGDVNGHKFS VRGEGEGDAT NGKLTLKFIC TTGKLPVPWP TLVTTLTYGV QCFSRYPDHM KRHDFFKSAM PEGYVQERTI SFKDDGTYKT RAEVKFEGDT LVNRIELKGI DFKEDGNILG HKLEYNFNSH NVYITADKQK NGIKANFKIR HNVEDGSVQL ADHYQQNTPI GDGPVLLPDN HYLSTQSVLS KDPNEKRDHM VLLEFVTAAG ITHGMDELYK GDEEENDQVK SGDEEENDQV KGGGHVVKWG YVGKIGPAHW GDLAHEYFMC KVGKNQSPVD INSSVTIEAQ LEPINFHYRD QISGEIVNNG HTIMVVPKED NYIVVDGKKF HLKQFHFHSP SEHTVEGKYY LLELHFVHQA DDGQLAVIGV VFDRGAEHPE IAKLWKEAPE HEGKKELKSL VNMQALLPEN LDYYRYSGSL TTPPCSEGVI WLFLKNPLQI SESQAEKFKK IMGFENNRPV QPVNARKILK AAGRARAQRQ SSRGRKSLSR ADHIHHHHHG HTQNMRMYEP WFGSVNVPLY 172604987

[0114] Yet another exemplary fusion protein of the present disclosure includes the “Masa Com 1” fusion protein as follows (SEQ ID NO:60): MRGSQQQQQQ GSMSKGEELF TGVVPILVEL DGDVNGHKFS VRGEGEGDAT NGKLTLKFIC TTGKLPVPWP TLVTTLTYGV QCFSRYPDHM KRHDFFKSAM PEGYVQERTI SFKDDGTYKT RAEVKFEGDT LVNRIELKGI DFKEDGNILG HKLEYNFNSH NVYITADKQK NGIKANFKIR HNVEDGSVQL ADHYQQNTPI GDGPVLLPDN HYLSTQSVLS KDPNEKRDHM VLLEFVTAAG ITHGGDEEEN DQVKSGDEEE NDQVKASEWS YSGETAPTNW HGTCQSGVNQ SPIDITHAIE STLDPIIFNY SKAGKNVVNN GHTVQVNFDA KQSIQIEGKT FTLLQLHFHA PSENLIEGHS FPLEMHLVHA DKDGNLAVIG VMLKEGKPNS ELTKIWAKMP QSGQVDLENQ LDLTKLLPRD QAYYRFNGSL TTPPCSEGVT WLVMKNPIEI SSEQLAQFTA LYNGNNCPTQ AINARPILKA AGRARAQRQS SRGRKSLSRA DHIHHHHHGH TQNMRMYEPW FGSVNVPLY

[0115] A further exemplary fusion protein of the present disclosure includes the “Masa Com 2” fusion protein as follows (SEQ ID NO:61): MRGSQQQQQQ GSMSKGEELF TGVVPILVEL DGDVNGHKFS VRGEGEGDAT NGKLTLKFIC TTGKLPVPWP TLVTTLTYGV QCFSRYPDHM KRHDFFKSAM PEGYVQERTI SFKDDGTYKT RAEVKFEGDT LVNRIELKGI DFKEDGNILG HKLEYNFNSH NVYITADKQK NGIKANFKIR HNVEDGSVQL ADHYQQNTPI GDGPVLLPDN HYLSTQSVLS KDPNEKRDHM VLLEFVTAAG ITHGASEWSY SGETAPTNWH GTCQSGVNQS PIDITHAIES TLDPIIFNYS KAGKNVVNNG HTVQVNFDAK QSIQIEGKTF TLLQLHFHAP SENLIEGHSF PLEMHLVHAD KDGNLAVIGV MLKEGKPNSE LTKIWAKMPQ SGQVDLENQL DLTKLLPRDQ AYYRFNGSLT TPPCSEGVTW LVMKNPIEIS SEQLAQFTAL YNGNNCPTQA INARPILKAA GRARAQRQSS RGRKSLSRAD HIHHHHHGHT QNMRMYEPWF GSVNVPLYgg gGDEEENDQV KSGDEEENDQ VK

[0116] In some embodiments, the fusion protein comprises any one of SEQ ID NOs:51- 61 or a proteolytic fragment thereof, or a fusion protein having at least 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs:51-61 or a proteolytic fragment thereof.

[0117] Carbonic anhydrase enzymatic activity can be determined using commercial kits such as those described in Example 1. In some embodiments, the fusion protein or proteolytic fragment thereof has at least a 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater than 100% increase in enzymatic activity compared to wild type bovine carbonic anhydrase. In some embodiments, the fusion protein or proteolytic fragment thereof has at least a 20% increase in enzymatic activity compared to wild type bovine carbonic anhydrase. In some embodiments, the fusion protein or proteolytic fragment thereof is enzymatically active at 65oC.

[0118] In some embodiments, the fusion protein or proteolytic fragment thereof retains at least 30%, 40%, 50%, 60%, 70%, 80% or 90%, 100%, or any percentage or range therein, or more than 100% of the carbonic anhydrase enzymatic activity at 25oC after incubation at 65oC for 20 minutes. In some embodiments, the fusion protein or proteolytic fragment thereof retains 172604987at least 30%, 40%, 50%, 60%, 70%, 80% or 90%, 100%, or any percentage or range therein, or more than 100% of the carbonic anhydrase enzymatic activity at 25oC after incubation at 65oC for 45 days. In some embodiment of the present disclosure, the fusion proteins of the present disclosure retain at least 30%, 40%, 50%, 60%, 70%, 80% or 90%, 100%, or any percentage or range therein, or more than 100% of the carbonic anhydrase activity they show at 25°C, after incubation at 65°C for 1, 2, 5, 10, 15, 20, 30, 40, 45, 55, 65, 75, 95 or more than 95 days, or any number or range therein. Polynucleotides, Vectors, and Hosts & Fusion Protein Production

[0119] Another aspect of the present disclosure relates to a polynucleotide molecule for expressing a carbonic anhydrase fusion protein comprising a polynucleotide encoding a carbonic anhydrase domain and one or more of a calcium carbonate binding domain, a calcium binding domain, an iron binding domain, a silicatein mimetic domain, and a silica binding domain and a promoter operably linked to the polynucleotide.

[0120] This aspect of the present disclosure can be carried out with any of the embodiments disclosed herein.

[0121] Provided herein are polynucleotide molecules encoding the various domains and entire sequences of the CA fusion proteins and fragments described herein. In some embodiments, a polynucleotide molecule of the present disclosure comprises a nucleotide sequence that encodes the fusion protein amino acid sequence selected from any one of SEQ ID NOs:1-62, or a substantially similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs:1-62.

[0122] Also provided are vectors comprising the polynucleotide molecules encoding the various domains and entire sequence of the CA fusion proteins and fragments described herein. Exemplary polynucleotides encoding fusion proteins, carbonic anhydrase domains, secretory domains, a calcium carbonate binding domain, a calcium binding domain, an iron binding domain, a silicatein mimetic domain, a silica binding domain, a protease cleavage site domain, a detection and / or purification tag domain, and linker domains are provided herein in Table 1 as SEQ ID NOs:63-93. In some embodiments, the polynucleotide comprises any one of SEQ ID NOs:63-93, or a polynucleotide molecule having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or any number or range therein, sequence identity to any one of SEQ ID NOs:63-93. In some embodiments, the polynucleotide molecule encodes the amino acid sequence of any one of SEQ ID NOs:3, 5, 7-13, 16, 17, and 51-61 or a polynucleotide molecule having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs:3, 5, 7-13, 16, 17, and 51-61. In 172604987some embodiments, the polynucleotide further comprises a polynucleotide encoding one or more of a secretory domain, a protease cleavage site domain, a detection and / or purification tag domain, and a linker domain. In some embodiments, the polynucleotide is isolated.

[0123] A “vector” as used herein is a vehicle suitable for carrying genetic material into a host cell. A vector can include a nucleic acid vector, such as a plasmid or mRNA, or nucleic acids embedded into a bigger structure, such as a liposome or viral vector.

[0124] Once a suitable expression vector is selected, the desired polynucleotide sequences are cloned into the vector using standard cloning procedures in the art, as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1989), or U.S. Pat. No.4,237,224 to Cohen and Boyer, which are hereby incorporated by reference in their entirety. For example, the fusion protein-encoding polynucleotide, a promoter molecule of choice, optionally a suitable 3′ regulatory region, and if desired, a reporter gene, are incorporated into a vector-expression system of choice.

[0125] A vector can include one or more of the following elements: an origin of replication, one or more regulatory sequences (e.g., promoters, enhancers, terminators) that regulate the expression of a polypeptide of interest, and / or one or more selectable marker genes (such as, for example, antibiotic resistance genes and genes that can be used in colorimetric assays, for example, β-galactosidase). For DNA-based vectors, this usually includes the presence of elements for transcription (e.g., a promoter and a polyA signal) and translation (e.g., Kozak sequence). In some embodiments, the vector is an expression vector, i.e., a vector suitable for expressing an encoded polypeptide or construct under suitable conditions in a host cell. In some embodiments, the vector is then introduced to a suitable host. In some embodiments, the vector comprises the polynucleotides of the disclosure. In some embodiments, the vector is a bacterial vector.

[0126] Promoters vary in their “strength” (i.e., their ability to promote transcription). For the purposes of expressing a cloned gene, it is desirable to use strong promoters to obtain a high level of transcription and, hence, expression of the gene. Depending upon the host cell system utilized, any one of a number of suitable promoters may be used. For instance, when cloning in E. coli, its bacteriophages, or plasmids, promoters such as the PH promoter, T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the PRand PLpromoters of coliphage lambda and others, including but not limited, to lacUV5, ompF, bla, lpp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUV5 (tac) promoter or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene. In 172604987some embodiments, the promoter is heterologous to the carbonic anhydrase domain. In some embodiments, the promoter is heterologous to one or more of the calcium carbonate binding domain, the calcium binding domain, the iron binding domain, the silicatein mimetic domain, the silica binding domain, the secretory domain, the protease cleavage site domain, the detection and / or purification tag domain, and the linker domain.

[0127] In some embodiments, the present disclosure also provides host cells or host organisms that comprise the polynucleotides or vectors encoding the fusion proteins or fragments thereof described herein. Suitable host cells or host organisms can be any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation.

[0128] A variety of host-vector systems may be utilized to express the fusion protein- encoding sequence in a cell. Primarily, the vector system must be compatible with the host cell used. Host-vector systems include, but are not limited to, the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); and plant cells infected by bacteria. The expression elements of these vectors vary in their strength and specificities. Depending upon the host-vector system utilized, any one of a number of suitable transcription and translation elements can be used.

[0129] Exemplary eukaryotic cells include mammalian cells, such as primate or non- primate animal cells; fungal cells, such as yeast (e.g., Saccharomyces cerevisiae or Pichia pastoris); plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, PER.C6® cells (Crucell), COS cells, SP2 / 0 cells, and 293 and CHO cells, and their derivatives, such as 293-6E, CHO-DG44, CHO-K1, CHO-S, and CHO-DS cells. Exemplary prokaryotic cells include bacterial cells such as Escherichia coli.

[0130] Transcription of DNA is dependent upon the presence of a promoter which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis. The DNA sequences of eukaryotic promoters differ from those of prokaryotic promoters. Furthermore, eukaryotic promoters and accompanying genetic signals may not be recognized in or may not function in a prokaryotic system and, further, prokaryotic promoters are not recognized and do not function in eukaryotic cells. 172604987

[0131] Similarly, translation of mRNA in prokaryotes depends upon the presence of the proper prokaryotic signals which differ from those of eukaryotes. Efficient translation of mRNA in prokaryotes requires a ribosome binding site called the Shine-Dalgarno (“SD”) sequence on the mRNA. This sequence is a short nucleotide sequence of mRNA that is located before the start codon, usually AUG, which encodes the amino-terminal methionine of the protein. The SD sequences are complementary to the 3′-end of the 16S rRNA (ribosomal RNA) and probably promote binding of mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome. For a review on maximizing gene expression see Roberts and Lauer, Methods in Enzymology 68:473 (1979), which is hereby incorporated by reference in its entirety. In some embodiments, the cell comprises the polynucleotides or the vectors of the present disclosure. In some embodiments, the cell is a bacterial cell.

[0132] The introduction of the vector into the host cell can be carried out by any commonly known technique e.g., using competent cells, by means of electroporation, conjugation and other transformation techniques commonly known by those skilled in the art.

[0133] In certain embodiments, the fusion proteins or proteolytic fragments of the present disclosure may be purified or “isolated”. As used herein, the term “purified” or “isolated” is intended to refer to a composition that is separable from other components, wherein the fusion protein or proteolytic fragment thereof is purified to any degree relative to its naturally available state. Thus, a purified also refers to a fusion protein proteolytic fragment thereof that is removed from its naturally occurring environment, i.e., when recombinantly produced, then removed from the host cell in which it was produced. When the term “substantially purified” is used, this means a composition in which the fusion protein or proteolytic fragment thereof forms the major component of the composition, such that the fusion protein or proteolytic fragment thereof constitutes about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the composition.

[0134] Purified fusion proteins or proteolytic fragment thereof may be obtained by several methods. The fusion protein proteolytic fragment thereof may be produced in purified form (at least about 80% or 85% pure, or at least about 90% or 95% pure) by conventional techniques. Depending on whether the recombinant host cell is made to secrete the fusion protein into growth medium, for example by fermentation using Escherichia coli and secreted into the fermentation medium, the fusion protein can be isolated and purified by centrifugation (to separate cellular components from supernatant containing the secreted peptide) followed by sequential ammonium sulfate precipitation of the supernatant. The fraction containing the fusion protein is subjected to gel filtration in an appropriately sized dextran or polyacrylamide column 172604987to separate the fusion protein from other proteins. The fusion protein may be secreted into the media.

[0135] Alternatively, if the fusion protein is not secreted, it can be isolated from the recombinant cells using standard isolation and purification schemes. This includes disrupting the cells (e.g., by sonication, freezing, French press, etc.) and then recovering the fusion protein from the cellular debris. Purification can be achieved using the centrifugation, precipitation, and purification procedures described above. The use of purification and detection tag domains, described above, can simplify this process. Analytical methods particularly suitable for the preparation of pure peptides are ion exchange chromatography, exclusion chromatography; performing polyacrylamide gel electrophoresis; isoelectric focusing. Other methods of protein purification include ammonium sulfate precipitation, PEG, antibodies, etc. or by heat denaturation followed by centrifugation; gel filtration, reverse phase, hydroxyapatite and affinity chromatography; and combinations of these and other techniques.

[0136] In certain embodiments, purification is not required. Where purification is not performed, cell-free lysates can be recovered following centrifugation for removal of cellular debris. The resulting cell-free lysate can be treated with heat for a sufficient amount of time to deactivate any native proteases in the recovered fraction, e.g., 10 min at 100°C. If desired, one or more of biocidal agents, protease inhibitors, and non-ionic surfactants can be introduced to such a cell-free preparation (see U.S. Application Publ. No.2010 / 0043095, which is hereby incorporated by reference in its entirety).

[0137] In general, the ingredients of the compositions of the present disclosure may be supplied separately or mixed together in unit dosage form, e.g., in the form of a dry lyophilized powder or an anhydrous concentrate that indicates the amount of active fusion protein or proteolytic fragment thereof.

[0138] The compositions of the present disclosure may be formulated in a neutral form or in a salt form. Pharmaceutically acceptable salts include salts with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, and the like; and salts formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

[0139] Once a fusion protein is recovered, and optionally processed with a protease, it can be included in various compositions and used in various methods as described herein. Carbonic Anhydrase Compositions and Methods of Use

[0140] Another aspect of the present disclosure relates to a concrete composition comprising a fusion protein comprising a carbonic anhydrase domain and one or more of a 172604987calcium carbonate binding domain, a calcium domain, an iron binding domain, a silicatein mimetic domain, and a silica binding domain; and cement.

[0141] This aspect of the present disclosure can be carried out with any of the embodiments disclosed herein.

[0142] In some embodiments, the compositions of the present disclosure comprise the fusion protein at a concentration in a range of 100 nM to 500 nM, 500 nM to 1µM, 1 µM to 5 µM, and 5 µM to 10 µM. In some embodiments, the compositions of the present disclosure comprise the fusion protein at a concentration of about 1 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50, mg, 60, mg, 70 mg, 80 mg, 90 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250mg, 275mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1500 mg, 2000 mg, 3000 mg, 4000 mg, 5000 mg, 10000 mg, or any number or range therein, per cubic yard. In some embodiments, the fusion protein comprises about 100 mg-250 mg per cubic yard of concrete or ECM. In some embodiments, the fusion protein comprises 50 mg-350 mg per cubic yard of concrete or ECM.

[0143] As used herein the term “cement” refers to an initially dry substance that develops compressive strength or sets in the presence of water. Suitable cements are known in the art and include, but are not limited to, Portland cement, blended Portland cement, masonry cement, expansive cement, rapid setting and hardening cement, calcium aluminate cement, calcium sulfoaluminate cement, pozzolan-lime cement, slag-lime cement, supersulfated cement, natural cement, geopolymer cement, magnesium-based cement, magnesium silicate cement, magnesium phosphate cement, calcium silicate cement, aluminum silicate cement, potassium silicate cement, sodium silicate cement, lithium silicate cement, and any mixtures and combinations thereof.

[0144] In some embodiments, the composition further comprises one or more of a calcium source, buffer, and aggregate. As used herein the term “concrete” refers to a material made by mixing a cementing material, an aggregate, and sufficient water to set and bind the mixture. Exemplary aggregates include, but are not limited to, sand, gravel, and crushed stone. In some embodiments, the concrete comprises a mixture of Portland cement (about 7-15%), water (about 4-21%), aggregate (about 60-85%) (for example, sand, crushed stone, and / or gravel of various sizes), and a fusion protein of the present disclosure. In some embodiments, the concrete comprises about 12% Portland cement. In some embodiments, the concrete comprises about 6% water. In some embodiments, the concrete comprises about 82% aggregate. In some embodiments, the concrete comprises about 245 mg of a CA fusion protein or proteolytic fragment thereof per cubic yard. In some embodiments, the concrete comprises about 1.35 mg of a CA fusion protein or proteolytic fragment thereof per kg. 172604987

[0145] In some embodiments, a calcium source is added to the composition. Non- limiting examples of calcium include calcium provided as calcium chloride, calcium acetate, calcium bromide, calcium lactate, calcium citrate, calcium nitrate, calcium nitrite, calcium gluconate, or any combination thereof. In some embodiments, the calcium source comprises 1, 5, 10, 20, 50, 100, 200, 300, 400, 500, or 1000 kg / cubic yard of concrete, or any number or range therein. In some embodiments, the calcium source comprises 192 kg calcium formate / cubic yard of concrete.

[0146] In some embodiments, the concrete composition comprises a buffer. Any buffer suitable for a pH range of 6.0-8.5 can be used. In some embodiments, the buffer is a Tris buffer such as Tris base (Trizma®base). In some embodiments, the Tris base comprises 1, 2, 5, 10, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or 100 kg / cubic yard of concrete, or any number or range therein. In some embodiments, the Tris base comprises 17 kg / cubic yard of concrete.

[0147] In some embodiments, the concrete composition comprises a pH of about 6.0 to 8.5 or any pH or range therein. In some embodiments, the pH is about 7.0. In some embodiments, the concrete composition is formed at a temperature of about 20o-65oC or any temperature or range therein. In some embodiments, the concrete composition comprises CO2.

[0148] In some embodiments, the concrete composition comprises 192 kg calcium formate / cubic yard, 245 mg CA fusion protein or proteolytic fragment thereof / cubic yard, 181.5 kg cement / cubic yard, 1542 kg aggregate / cubic yard, and 17kg Tris base / cubic yard.

[0149] A further aspect of the present disclosure relates to an enzymatic construction material (“ECM”) composition comprising a fusion protein comprising a carbonic anhydrase domain and one or more of a calcium carbonate binding domain, a calcium binding domain, an iron binding domain, a silicatein mimetic domain, and a silica binding domain; and a particulate substrate.

[0150] This aspect of the present disclosure can be carried out with any of the embodiments disclosed herein.

[0151] In some embodiments, the particulate substrate includes, but is not limited to, one or more of sand, gravel, crushed stone, aggregate, soil, fibers, silicone bead, silicon dioxide, a silica gel, disiloxane, silicic acid, silanol, an organic silicon compound, gelatin, grit, cellulose, and a cellulose derivative. In some embodiments, calcium can be added to the composition, as described herein. In some embodiments, the ECM composition comprises aggregate.

[0152] In some embodiments, the ECM composition comprises a calcium source. In some embodiments, the calcium source comprises 1, 5, 10, 20, 50, 100, 200, 300, 400, 500, or 1000 kg / cubic yard of the ECM composition, or any number or range therein. In some 172604987embodiments, calcium comprises 192kg calcium formate / cubic yard of ECM. In some embodiments, the ECM composition comprises a buffer. Any buffer suitable for pH range of 6.0-8.5 can be used. In some embodiments, the buffer is a Tris buffer such as Trizma®base. In some embodiments, the Tris base comprises 1, 2, 5, 10, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or 100 kg / cubic yard of concrete, or any number or range therein. In some embodiments, the Tris base comprises 17 kg / cubic yard of ECM.

[0153] In some embodiments, the ECM composition comprises a pH of about 6.0 to 8.5 or any pH or range therein. In some embodiments, the pH is about 7.0. In some embodiments, the ECM composition is formed at a temperature of about 20o-65oC or any temperature or range therein. In some embodiments, the ECM composition comprises CO2. Exemplary components of ECM and methods of making and evaluating ECM can be found in Wang et al., “A Self- Healing Enzymatic Construction Material,” Matter 5(3):957-974 (2022), which is hereby incorporated by reference in its entirety). In some embodiments, the fusion protein comprises about 50 mg-250 mg per cubic yard of ECM. In some embodiments, the fusion protein comprises 107 mg / cubic yard of ECM.

[0154] In some embodiments, the ECM comprises 192 kg calcium formate / cubic yard, 107 mg CA fusion protein or proteolytic fragment thereof / cubic yard, 1696 kg aggregate / cubic yard, and 17kg Tris base / cubic yard.

[0155] In some embodiments, the carbonic anhydrase fusion protein is dissolved in an aqueous solvent and embedded in a matrix such as a hydrogel, agar, or semi-solid gelatin. Therefore, the fusion protein composition when used to apply to a cementitious surface or ECM surface, rather than flow diffusively through the concrete, remains mixed into the cement thereby allowing the repair reaction to continue for the lifetime of the enzyme activity.

[0156] In some embodiments, the concrete and / or ECM composition further comprises water.

[0157] In some embodiments, the concrete and / or ECM compositions of the present disclosure have improved self-healing, repair and / or durability properties compared to a concrete or ECM composition without the fusion protein. The compositions disclosed herein have reduced permeability to deleterious environmental corrosive agents. The reduced porosity results from calciferous crystallization due to the action of the carbonic anhydrase. An important consequence of reducing porosity of the resulting cementitious surfaces is decreased permeation by deleterious salt ions including chloride salts. Corrosion by chloride ions of iron rebars in reinforced concrete used in construction is thereby prevented or reduced. Also contributing to reduced porosity is the action of the silicatein mimetic domain and the silica binding domain, 172604987and the localization to the rebar due to the iron binding domain. The silicatein can aid in silica sheath and / or silica-metal hybrid sheath formation on the rebar to further protect it from the environmental rusting. Furthermore, the silicatein spicules formed can aid in concrete and / or ECM strength.

[0158] In some embodiments, the concrete and / or ECM compositions of the present disclosure have improved mechanical strength properties compared to a concrete or ECM composition without the fusion protein. In some embodiments, the concrete or ECM compositions have increased compressive strength compared to a concrete or ECM composition without the fusion protein. In some embodiments, the compressive strength is increased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200%, 300%, 500%, or 1000%, or any number or range therein, compared to a concrete or ECM composition without the fusion protein. In some embodiments, the concrete or ECM composition has a compressive strength of at least 20-55 MPa, or any number or range therein.

[0159] In some embodiments, the concrete and / or ECM compositions of the present disclosure have reduced crack starts compared to a concrete or ECM composition without the fusion protein. In some embodiments, the crack starts are reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200%, 300%, 500%, or 1000%, or any number or range therein, compared to a concrete or ECM composition without the fusion protein.

[0160] In some embodiments, the concrete and / or ECM compositions of the present disclosure have increased carbon sequestration compared to a concrete or ECM composition without the fusion protein. In some embodiments, the carbon sequestration is increased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200%, 300%, 500%, or 1000%, or any number or range therein, compared to a concrete or ECM composition without the fusion protein. In some embodiments, the fusion protein uses carbon dioxide from the atmosphere and calcium to form calcium carbonate crystals thereby sequestering carbon and obtaining a carbon sequestering concrete or ECM composition.

[0161] In some embodiments, the concrete or ECM compositions of the present disclosure have improved durability and corrosion resistance due to reduced porosity and reduced permeability. In some embodiments, the porosity of the concrete or ECM compositions are reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200%, 300%, 500%, or 1000%, or any number or range therein, compared to a concrete or ECM composition without the fusion protein. In some embodiments, the permeability of the concrete or ECM compositions are reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 172604987125%, 150%, 200%, 300%, 500%, or 1000%, or any number or range therein, compared to a concrete or ECM composition without the fusion protein.

[0162] The compositions described herein may optionally contain one or more additives, which include, but are not limited to anti-foam agents, water-proofing agents, dispersing agents, set-accelerators, set-retarders, plasticizing agents, water reducers, bonding agents, freezing point decreasing agents, adhesiveness-improving agents, and colorants. The additives are typically present at less than one percent by weight with respect to total weight of the composition but can be present at from 0.1 to 3 weight percent.

[0163] Suitable dispersing agents or plasticizers include, but are not limited to, hexametaphosphate, tripolyphosphate, polynaphthalene sulphonate, sulphonated polyamine, and combinations thereof.

[0164] Suitable plasticizing agents that can be used in embodiments of the present disclosure include, but are not limited to, polyhydroxycarboxylic acids or salts thereof, polycarboxylates or salts thereof; lignosulfonates, polyethylene glycols, and combinations thereof.

[0165] Suitable freezing point decreasing agents that can be used in embodiments of the present disclosure include, but are not limited to, ethyl alcohol, calcium chloride, potassium chloride, and combinations thereof.

[0166] Suitable adhesiveness-improving agents that can be used in embodiments of the present disclosure include, but are not limited to, polyvinyl acetate, styrene-butadiene, homopolymers and copolymers of (meth)acrylate esters, and combinations thereof.

[0167] Suitable water-repellent or water-proofing agents that can be used in embodiments of the present disclosure include, but are not limited to fatty acids (such as, stearic acid or oleic acid), lower alkyl fatty acid esters (such as, butyl stearate), fatty acid salts (such as, calcium or aluminum stearate), silicones, wax emulsions, hydrocarbon resins, bitumen, fats and oils, silicones, paraffins, asphalt, waxes, and combinations thereof.

[0168] Examples of suitable bonding agents include materials that can be either inorganic or organic and are soft and workable when fresh but set to form a hard, infusible solid on curing, either by hydraulic action or by chemical crosslinking. Non-limiting examples of such materials can include organic materials, such as, rubber, polyvinyl chloride, polyvinyl acetate, acrylics, styrene butadiene copolymers, glutaraldehyde, and various powdered polymers.

[0169] In some embodiments, the composition further comprises one or more of a calcium source, buffer, and aggregate. 172604987

[0170] In some embodiments, the composition further comprises a scaffolding material having a crosslinking agent.

[0171] Yet another aspect of the present disclosure relates to a method of repairing a fracture in a cementitious or ECM surface. This method involves contacting a fracture in a cementitious or ECM surface with a fusion protein composition as described herein under conditions effective to repair the fracture in the cementitious surface or ECM surface.

[0172] This aspect of the present disclosure can be carried out with any of the embodiments disclosed herein.

[0173] In some embodiments, the cementitious or ECM surface comprising the fracture is a cementitious or ECM surface that does not comprise a CA fusion protein. The subsequent addition of the CA fusion protein composition allows the healing of the fracture as described herein. A surface includes deep fractures as well as superficial fractures.

[0174] In some embodiments, said contacting occurs at ambient conditions of atmospheric carbon dioxide and temperature. In some embodiments, said contacting occurs at temperatures above 40oC.

[0175] In some embodiments, the fusion protein composition comprises a buffer solution, a calcium source, and a fusion protein or a proteolytic fragment thereof, where the fusion protein or a proteolytic fragment thereof comprises about 245 mg / cubic yard of concrete or 107 mg / cubic yard of ECM. For example, a 1x3mm crack will require 10ml calcium-buffer solution or 10ml calcium-enzyme-buffer solution (with approximately 0.1mg enzyme). In some embodiments, the calcium-buffer solution comprises 26g calcium formate and 2.422 g Tris base (Trizma®).

[0176] In some embodiments, the fracture is repaired in 5 minutes, 10 minutes, 30 minutes, 1 hr, 2, hr, 3 hr, 4 hr, 5, hr, 10 hr, 15 hr, 20 hr, 24 hr, 30 hr, 40 hr, 50 hr, 60 hr, 70 hr, 80 hr, 90 hr, or 100 hr, or any value or range therein. In some embodiments, the fracture is repaired in 24 hours. In some embodiments, the fracture is repaired in 1 day, 2 days, 3 days, 4 days, 5 days, 10 days, 20 days, 50 days, 100 days, 500 days or any value or range therein.

[0177] In some embodiments, the fracture is repaired at 15oC, 20oC, 25oC, 30oC, 35oC, 40oC, 45oC, 50oC, 55oC, 60oC, 65oC, 70oC, or 80oC, or any number or range therein.

[0178] Exemplary methods of testing self-healing properties using CA compositions are disclosed in U.S. Patent Application Publication Nos. US 2023 / 0192548 and US 2023 / 0117517, each of which is hereby incorporated by reference in its entirety.

[0179] Another aspect of the present disclosure relates to making a cement mixture. This method involves adding a fusion protein composition as described herein to a cement mixture. 172604987The method further comprises curing the cement mixture comprising the composition to make a durable corrosion resistant concrete.

[0180] This aspect of the present disclosure can be carried out with any of the embodiments disclosed herein.

[0181] A further aspect of the present disclosure relates to a method for making an enzymatic construction material (ECM) composition. This method involves combining a fusion protein composition as described herein with a particulate substrate to form an ECM mixture. The method further involves curing the ECM mixture comprising the composition to make a durable corrosion resistant ECM.

[0182] This aspect of the present disclosure can be carried out with any of the embodiments disclosed herein.

[0183] Also disclosed herein is a method of making a concrete or ECM form. This involves forming the concrete or ECM product into a desired shape and curing the formed concrete or ECM product.

[0184] Forming the concrete or ECM product mixture may involve providing a mold or frame and transferring the concrete or ECM product mixture to the mold or frame using a pump, by pouring, spraying, or by any other means known in the art.

[0185] The formed concrete or ECM product mixture may be compacted to remove large air voids developed during the transferring step using vibration, roller compacting, or through the use of chemical admixtures.

[0186] As used herein the term “curing” refers to the maintenance of a satisfactory moisture content and temperature in concrete or ECM for a period of time immediately following the forming the concrete or ECM product into a desired shape. Proper curing of concrete influences the properties of the hardened concrete, including durability, strength, watertightness, abrasion resistance, volume stability, and resistance to freeze-thawing.

[0187] Methods of curing are well known in the art and include, but are not limited to, ponding or immersion, spraying or fogging, covering concrete with moisture-saturated fabrics, impervious paper, plastic sheeting materials, treatments with liquid membrane-forming compounds, internal moist curing, steam curing, use of insulating blankets or covers, electrical curing, hot oil curing, or microwave curing.

[0188] In some embodiments, carbonic anhydrase (CA) fusion proteins are employed to catalyze the covalent condensation of carbon dioxide and water or the fixing of carbon dioxide from the atmosphere, to promote the precipitation of calcium ions in an aqueous solution as calcium carbonate crystals to sequester carbon. 172604987

[0189] In some embodiments, the lifespan of a concrete and / or ECM compositions of the present disclosure is increased by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 years, or any number or range therein, or more than 100 years compared to a concrete or ECM composition without the fusion protein.

[0190] Also contemplated are kits for use with the methods described above. Such kits would include CA fusion proteins for use in making concrete and / or ECM compositions. The kits will also include suitable instructions for carrying out the methods. EXAMPLES Example 1 – Enzymatic Activity of CA Fusion Proteins

[0191] Nucleotide sequences encoding Masa CA derived from Aliivibrio salmonicida carbonic anhydrase and Td CA derived from Thermosulfurimonas dismutans carbonic anhydrase without the N-terminal signal peptide sequence (SEQ ID NOs:3 and 5) were synthesized and codon optimized for E. coli. A pUC57-based vector was designed to include a LacI repressor sequence and a T7 promoter. PCR amplified gene constructs were then inserted into the custom puC57 vector. The genes were cloned upstream of the T7 promoter to ensure proper transcription. In the custom vector, the LacI repressor sequence was positioned downstream of the T7 promoter. This setup ensured that the LacI repressor regulated the activity of the T7 promoter, allowing controlled expression of the cloned genes. The successful integration of the constructs into the vector was confirmed through sequencing and restriction digestion analysis, ensuring that the genes were correctly positioned and the vector was intact.

[0192] Sequences encoding the carbonic anhydrase fusion proteins described above were cloned into a bacterial expression vector and fusion proteins were expressed using standard bacterial media and conditions. See for example, Chen “Bacterial Expression Systems for Recombinant Protein Production: E. coli and Beyond,” Biotechnology Advances 30.5: 1102-1107 (2012), which is hereby incorporated by reference in its entirety.

[0193] Bovine carbonic anhydrase from bovine erythrocytes was purchased from Millipore Sigma or VWR and used for comparisons to the Masa and Td CA fusion proteins described herein.

[0194] Thermostability data and activity longevity data for the Masa CA C-variant 1 fusion protein (SEQ ID NO:51) and the Td CA C-variant 1 fusion protein (SEQ ID NO:53) are shown in FIGs.1 and 2.

[0195] In brief, enzymes were tested in free saline solution for esterase activity. Uda et al., “Esterase Activity of Carbonic Anhydrases Serves as Surrogate for Selecting Antibodies 172604987Blocking Hydratase Activity,” J. Enzyme Inhib. Med. Chem.30(6):955-60 (2015), which is hereby incorporated by reference in its entirety.

[0196] Esterase activity, which is an indicator of CA activity was tested at 25oC (room temp) and 65oC (higher temperature). The assay was performed using the Carbonic Anhydrase (CA) Activity Assay Kit (Colorimetric) (ab284550) from Abcam ( / products / assay-kits / carbonic- anhydrase-ca-activity-assay-kit-colorimetric-ab284550), which is hereby incorporated by reference in its entirety.

[0197] As shown in FIG.1 and FIG.2, initial testing of fusion proteins was done in comparison to bovine CA. FIG.1 shows CA activity after 20 minutes at 65oC compared to the activity of the enzyme at 25oC. FIG.2 shows another test after CA fusion proteins and the bovine CA control were kept at 65oC for 45 days in solution. These results show that the CA fusion protein have much higher activity than bovine CA after 25 minutes at 65oC and also show high stability even after 45 days in solution after extended heat treatment. Example 2 – Solubility and Production of CA Fusion Proteins

[0198] Various CA fusion proteins with different solubility tags were expressed in BL21 E. coli bacteria, grown in 1 L cultures in laboratory shaker flasks. Fusion protein synthesis was induced at OD600with 0.2mM IPTG for 16 hours at 28oC and the fusion proteins were purified on Ni-NTA agarose. Various CA fusion proteins were electrophoresed on a protein gel to evaluate them for yield and solubility (see FIG.3). Three different secretory domain affinity tags were evaluated. Examples of fusion affinity tags are described in Kimple et al., “Overview of Affinity Tags for Protein Purification,” Curr. Protoc. Protein Sci.73:9.9.1-9.9.23 (2013), doi: 10.1002 / 0471140864.ps0909s73, which is hereby incorporated by reference in its entirety.

[0199] A bdSUMO Masa (FIG.3, lane 1; SEQ ID NO:56), a Cytoplasmic MBP Masa CA (FIG.3, lane 2; SEQ ID NO:57), an sfGFP Masa CA (FIG.3,lane 3; SEQ ID NO:58), and an sfGFP Td (FIG.3,lane 4; SEQ ID NO:59) were tested. Bioreactor production instead of shaker flask production is expected to substantially increase yields even more.

[0200] Recombinant 5X histidine-tagged sfGFP Masa CA protein (SEQ ID NO:58) was expressed in Escherichia coli BL21(DE3) cells transformed with a plasmid encoding the target protein with an 5X histidine tag. An overnight culture of transformed E. coli was used to inoculate 1 L of LB medium supplemented with 50 µg / mL kanamycin. The culture was grown at 37°C with shaking at 250 rpm until the optical density at 600 nm (OD600) reached 0.6-0.8. Protein expression was induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM, and the culture was incubated for an additional 4 hours at 37°C. 172604987

[0201] Cells were harvested by centrifugation at 6,000 x g for 15 minutes at 4°C. The cell pellet was resuspended in 30 mL of lysis buffer (50 mM sodium phosphate, pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM PMSF). Cells were lysed by sonication on ice with 10-second pulses and 30-second rest intervals for a total of 10 minutes. The lysate was clarified by centrifugation at 12,000 x g for 30 minutes at 4°C. The supernatant containing the soluble protein fraction was applied to a Ni-NTA agarose column pre-equilibrated with equilibration buffer (50 mM sodium phosphate, pH 8.0, 300 mM NaCl, 10 mM imidazole). The column was washed with 20 column volumes of wash buffer (50 mM sodium phosphate, pH 8.0, 300 mM NaCl, 20 mM imidazole) to remove non-specifically bound proteins. The recombinant protein was eluted with elution buffer (50 mM sodium phosphate, pH 8.0, 300 mM NaCl, 250 mM imidazole).

[0202] The eluted fractions containing the target protein were pooled and dialyzed overnight against 2 L of dialysis buffer (50 mM sodium phosphate, pH 8.0, 300 mM NaCl) at 4°C to remove imidazole. The dialyzed protein solution was concentrated using a 10 kDa molecular weight cutoff (MWCO) centrifugal filter unit. Protein purity was assessed by SDS- PAGE on a 12% polyacrylamide gel. Samples were prepared by mixing protein with SDS loading buffer and boiling for 5 minutes. Electrophoresis was performed at 120 V for 1 hour, and the gel was stained with Coomassie Brilliant Blue R-250. For Western blot analysis, proteins were transferred to a PVDF membrane, which was then blocked with 5% (w / v) non-fat dry milk in TBST (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween-20) for 1 hour at room temperature. The membrane was incubated with anti-His-tag primary antibody (1:5000 dilution in TBST) overnight at 4°C, followed by incubation with HRP-conjugated secondary antibody (1:10000 dilution in TBST) for 1 hour at room temperature. The signal was detected using an ECL detection system. Purified protein was aliquoted and stored at -80°C for future use. This protocol yields highly pure recombinant 5X histidine-tagged protein suitable for downstream applications such as structural analysis, enzymatic assays, or interaction studies. Example 3 – Concrete Corrosion Testing Using CA Fusion Proteins

[0203] Corrosion inhibition was evaluated using the Accelerated Corrosion Test (“ACT”), which is one of the most reliable tests to measure the corrosion properties of reinforced concrete. The ACT involves a plastic tank containing five wt.% NaCl solution, two stainless steel plates (the cathodes), and a 30 V DC power supply to accelerate the corrosion where the reinforcing bar is the anode in the setup. Concrete samples are cast into 4x8 in. cylinders with a ½ in. wire-brushed steel rebar embedded in the center (e.g., see FIGs.6A-B). To avoid crevice 172604987corrosion, the top of the cylinder where the rebar exits the samples is coated with epoxy. After the specified curing periods, the specimens are placed into the ACT setup, and a data acquisition system records the voltage. Since the transfer of ions is expedited by the current, the cracks will appear faster in the concrete with this setup. Hence, the initiation and crack growth can be deduced from the recorded voltage curve and continuous images of the samples.

[0204] The CA fusion enzymes showed comparable corrosion inhibition activity when compared to bovine CA and showed higher corrosion inhibition than the concrete industry standard, calcium nitrate. Crack starts in samples after 14 days are shown in FIG.4. The identity of the samples from left to right are as follows: control (standard Portland cement concrete), FA (fly ash concrete), CNI (calcium nitrate / nitrite), CA (bovine carbonic anhydrase at 20 mg / cubic yard of concrete), CA 5x (bovine carbonic anhydrase at 100 mg / cubic yard of concrete), CA 10X (bovine carbonic anhydrase at 200 mg / cubic yard of concrete), Isaac 1X (sfGFP Masa CA fusion protein (SEQ ID NO:58)) at 20 mg / cubic yard of concrete), Isaac 5X (sfGFP Masa CA fusion protein (SEQ ID NO:58)) at 100 mg / cubic yard of concrete), and Isaac 10X (sfGFP Masa CA fusion protein (SEQ ID NO:58)) at 200 mg / cubic yard of concrete.

[0205] FIGs.4 and 5 illustrate the crack initiation times observed in various concrete specimens across 14 and 28 days curing periods. Each data point represents the average crack initiation time of three specimens, with error bars indicating the variability within the samples. In 14 days, control specimens consistently exhibit lower crack initiation times compared to Fly ash (FA), CNI, CA and MASA modified mixes. The observation indicates that CA-10X and MASA-10X mixes demonstrate comparable crack initiation times, indicating significant improvement compared to both control, Fly ash, CNI, CA-1X, MASA-1X and MASA-5X mixes. This trend underscores the efficacy of CA-based corrosion inhibition (CA and MASA) in enhancing the durability and resistance to corrosion-induced damage in reinforced concrete structures.

[0206] An evaluation of crack starts in samples at 28 days is shown in FIG.5. The identity of the samples from left to right are as follows: control (standard Portland cement concrete), FA (fly ash concrete), CNI (calcium nitrate / nitrite), CA 1x (bovine carbonic anhydrase at 20 mg / cubic yard of concrete), CA 5x (bovine carbonic anhydrase at 100 mg / cubic yard of concrete), CA 10X (bovine carbonic anhydrase at 200 mg / cubic yard of concrete), and Isaac 1X (MASA-1X ; sfGFP Masa CA fusion protein (SEQ ID NO:58) fusion protein at 20 mg / cubic yard of concrete).

[0207] In FIG.5, The 28-day samples demonstrate that CA-5X has the longest onset of crack formation, indicating that it exhibits the highest durability compared to the control, CA- 1726049871X, CA-10X, and MASA-1X samples. The MASA-1X sample had improved durability compared to the control and while bovine CA had a higher onset of crack start value verses MASA CA, the MASA CA has improved thermostability and ease of production compared to bovine CA making the MASA CA an improved high performing additive.

[0208] An evaluation of crack starts in samples at 48 days is shown in FIGs.6A-B. It can be seen that the enzymatic approach using bovine CA and Masa CA fusion significantly enhances the corrosion resistance of concrete and concrete with fly ash and CNI additives. This result demonstrates the the CA fusion proteins are concrete corrosion inhibitors, protecting the rebar from corrosion rin long term samples.

[0209] While Calcium Nitrite (CNI) and fly ash offer significant benefits to concrete durability and longevity, they also present some disadvantages. Currently, CNI negatively affects the plasticity and curing time of fresh concrete. In addition, CNI can only be applied once and fail to re-protect when the protective layer experiences secondary cracking or spalling. On the other hand, fly ash, a supplementary cementitious material, can vary significantly in quality based on its source, leading to variability in concrete performance. The reaction of fly ash with cement is slower than traditional Portland cement, which might delay the strength development, especially in cold weather conditions. This delay can pose scheduling issues in construction timelines. Furthermore, the availability of fly ash is directly tied to coal power production, which is declining as renewable energy sources become more prevalent, potentially leading to supply issues in the future. Fly ash is prohibited in many states, such as New York.

[0210] Concrete modified with CA and MASA CA fusion proteins not only demonstrates higher corrosion durability but can also be reapplied to concrete surfaces to penetrate deeply into the material, thereby sustaining high levels of corrosion resistance. This effect is primarily achieved by reducing the porosity of the concrete. Additionally, MASA CA fusion proteins production is less costly than both CNI and fly ash. Example 4 – Compression Testing of Damaged Self-Healing Enzymatic Concrete Using CA Fusion Proteins

[0211] Various concrete samples were tested for compressive strength as shown in FIG.7, which is a graph showing the compressive strength of 1 day cement mortar samples. Samples were made according to the ASTM C109 Standard Test Method for Compressive Strength of Hydraulic Cement Mortars (using 2-in. or [50-mm] Cube Specimens). Samples were made with sfGFP Masa CA fusion protein (SEQ ID NO:58) at 245 mg / cubic yard (or 1.35 mg / kg). Some samples included 3.5% LUDOX®as indicated. This experiment aimed to investigate whether 172604987carbonic anhydrase could replace LUDOX®or enhance its benefits in concrete, potentially further improving compressive strength. The results showed that the MASA CA, LUDOX®, and their combinations can increase the early strength of cement mortar.

[0212] Square concrete samples of about 7.8 mm diameter were notched in the center using a blade to create an approximately 3 mm x 1 mm elliptical flaw. An enzymatic cement paste was made by mixing cement and water with a ratio of 0.45 and bovine CA (CA-MOD-5x; bovine carbonic anhydrase at 245 mg / cubic yard of concrete). A 1 x 3mm crack requires about 10 ml calcium-buffer solution if applied to a composition already comprising a CA enzyme, or 10 ml calcium-CA enzyme-buffer solution (0.1 mg enzyme) if applied to a composition (such as standard concrete) without a CA enzyme. The concrete samples were vertically oriented as shown in FIG.8B and tested for compressive strength (Mpa) using an Instron machine.

[0213] FIGs.8A shows that the concrete sample with bovine CA-Mod-5x fully regained 97.5% compressive strength compared with control samples, clearly showing the regaining of the strength in enzyme-modified cement paste samples with a millimeter-scale notch in 24 h. Under the catalysis of carbonic anhydrase, a high concentration of bicarbonate ion was able to react with calcium rapidly on the sample surfaces (FIG.8B). As it can be observed from the SEM images (FIG.8C), the self-activated healing process is accomplished through the fast growth of a dense formation of calcium carbonate crystals on the surface of the crack resulting in completely covering the notch and healing the sample in 24 h.

[0214] This result is distinct from the current bacterial self-activated healing mechanism in cementitious matrices are not capable of recovering even micron-scale flaw sizes in a similar timeframe (Jonkers et al., “Application of Bacteria as Self-Healing Agent for the Development of Sustainable Concrete,” Ecol. Eng.36:230-235 (2010), which is hereby incorporated by reference in its entirety). Here, it is clearly shown the full-strength recovery of a self-activated healing concrete sample in 24 h. Example 6 – Permeability Testing of Concrete with CA Proteins

[0215] Cement mortar (M) and cement paste (CP) was prepared with bovine CA. The penetration depth of 10 mL water-soluble dye after 28 days was measured from the top surface and indicated by black arrow bar (FIG.9 inset images). Images of the CP and M compositions with and without CA are shown in the insets of FIG.9. A 48% reduction in dye penetrability in the CP-CA-1X and 93% reduction in CA-M-5X compared to samples that did not contain bovine CA. This result indicated that penetrability was significantly reduced by CA (see Rosewitz et 172604987al., “An Enzymatic Self-Healing Cementitious Material,” Applied Materials Today, 23:101035 (2021), which is hereby incorporated by reference in its entirety). Prophetic Example 6 –AVDT Test for Permeability Evaluation of Concrete with CA Fusion Proteins

[0216] The Accelerated Vacuum Dye Test (AVDT) will be used to support the permeability test shown in FIG.9 for studies on the fusion CA proteins of the present disclosure. Cement mortar and paste samples will be cast in 50 mm long and 100 mm in diameter cylinder molds. Then, a curved watch glass (beaker cover) will be pressed into the top of the samples, convex side down. This process produces a concave top on each cylinder sample, creating a depression to retain water. Then, 10 mL of a 10,000-ppm negatively charged fluorescent red dye to mimic chloride ions are pipetted into the convex top, and the samples are placed in a glass vacuum chamber and pumped for 12 hours. The chamber is sealed, and the vacuum is maintained for 24 hours. When observing that the dye solution has fully penetrated the control samples, proceed to remove any remaining solution from the remaining samples.

[0217] In a previously conducted series of studies, it was found that 10 mL of dye solution completely adsorbs into the control cylinders. After drying the cylinders for three days, we split the cylinders following the standard ASTM C496 (Standard Test Method for Splitting Tensile Strength of Cylindrical Concrete Specimens, which is hereby incorporated by reference in its entirety) and measured the penetration of the dye into the concrete matrix by ultraviolet light. The inclusion of bovine CA significantly reduces the dye penetration in cement paste and cement mortar samples (see FIG.9). Prophetic Example 7 – Porosity Testing of Concrete with CA Fusion Proteins

[0218] For a more comprehensive analysis of porosity, pore size distribution, homogeneity, number of pores, and pore morphology within fusion CA-modified concrete, CT scanning will be employed as a non-destructive means of examination. Preliminary results on bovine CA showed CT scans on cement mortar samples with w / c of 0.45 using type I / II Portland cement and 5X of CA. For each mixture, samples were cut into 1 in3cubes cast in sealed containers and cured at 23 °C for 28-day hydration. Initial experiments focused on concrete mortar with CA (FIG.10). Further experiments are planned on cement paste and concrete samples with varying curing times. Based on these preliminary results, incorporating CA reduces the porosity, total number of pores, and pore volume in concrete. Most importantly, the 172604987inclusion of CA will plug in pores smaller than 1 mm, resulting in a significant reduction in the diffusivity of harmful ions.

[0219] The inset of FIG.10 shows samples treated with CA at 5X concentrations exhibit a reduced pore quantity compared to the control, particularly notable in pores larger than 4 mm³ (pink color in FIG.10), where a significant reduction is observed in CA -5X sample. This reduction is attributed to the volume of precipitated calcium carbonate crystals, which have an average diameter ranging from 5 to 10 μm, filling in the pores to reduce the volume. The ion transfer rate in concrete is governed by two primary factors: the size of the pores and the spacing between them. A larger distance between pores typically leads to increased resistance to ion transfer, as ions predominantly move through the liquid within the pores. Movement becomes considerably more difficult through the solid matrix, where liquid flow is restricted. FIG.10, inset, illustrates the schematic of the gaps between pores in the concrete. The center and right images clearly demonstrate that the average gap in the control sample is 150 μm, which is less than the gap size observed in the CA-5X sample. This demonstrated that carbonic anhydrase catalyzes crystal growth within the structure, filling some micro scale pores. This process randomly reduces the short-distance gaps created by these pores, thereby extending the overall gap between pores. Additionally, the gap between pores in CA sample almost triples that of control samples, measuring an average of 420 μm compared to 150 μm in control samples. The significant differences highlighted above indicate that enzyme-catalyzed crystal precipitation leads to notable alternations in pore structure and morphology, as well as increased pore spacing. Example 8 – Polynucleotides Encoding Fusion Proteins and Various Fusion Protein Domains

[0220] Polynucleotides encoding the various fusion proteins and fusion protein domains of the present disclosure are provided in Table 1 below. Table 1. Polynucleotide Sequences ID & SEQ ID NO: Polynucleotide Sequences (5’ to 3’)172604987GTAGATCTGG AGAACCAACT CGATCTGACG AAGTTACTCC CTCGTGACCA AGCCTACTAC CGTTTCAACG GCAGTCTGAC AACGCCTCCT TGTTCGGAAG GTGTGACATG GCTGGTAATG172604987GAAGCACAAG CAGAAAAGTT CAAAAAAATT ATGGGTTTCG AAAATAATCG GCCTGTGCAA CCGGTTAATG CTCGGAAGAT TTTGAAA172604987polynucleotide for “Td CA C- TATGTGCAAG GTCGGGAAAA ATCAATCCCC TGTCGATATC AATTCTAGCG TAACCATTGA AGCCTGCCTG GAACCTATCA variant 2” alpha carbonic ACTTCCACTA TCGGGATCAA ATTTCCGGTG AAATCGTAAA172604987mutation (encoding SEQ ID ATTCGTTTCC TCTTGAAATG CACCTTGTGC ACGCCGACAA AGATGGTAAC TTGGCCGTAA TCGGCGTAAT GCTTAAAGAA NO:13) GGGAAACCAA ATTCAGAGCT GACAAAAATC TGGGCTAAAA172604987GGGTCGTGCA ACTTAGCGGC ACAGTGGACT CCCAAGCGCA GAGTGATCGT GCGGAAAGCA TCGCAAAGGC TGTCGATGGC GAGAAGTCCG TTAAGAACGA CTTAAAGACC AAGGGTGGCG172604987AAAAAGTCGT AACCCTGTCG GGGTTTGTAG AGTCCCAAGC CCAGGCTGAA GAAGCTGTTA AAGTCGCGAA GGGTGTTGAG GGTGTCACCT CGGTATCTGA CAAGCTCCAT GTACGCGATG172604987AATCAATCCC CAGTAGACAT CAATTCATCT GTAACTATTG AAGCGCAGCT GGAACCAATC AATTTTCACT ATCGTGATCA GATTTCGGGT GAAATTGTAA ATAATGGCCA CACTATTATG172604987TTAAAAAGAT TATGGGGTTC GAGAACAATC GTCCGGTTCA ACCAGTCAAC GCCCGGAAAA TTTTAAAAGC AGCCGCTGCT CATCATCATC ATCATCATGG TGGCGGTGGT TCAGGGCGGC172604987TTAACAAAGA CAAGCCGTTG GGTGCAGTTG CATTAAAATC CTATGAGGAG GAACTCGCAA AGGATCCTCG TATTGCGGCG ACAATGGAGA ACGCACAGAA AGGTGAAATT ATGCCGAACA172604987GATGCTGAAA GAAGGTAAGC CAAATAGCGA ATTAACTAAG ATTTGGGCCA AGATGCCACA ATCCGGCCAA GTCGACCTCG AGAATCAGTT GGATCTGACC AAACTTCTCC CTCGTGATCA172604987polynucleotide for “Masa Com GTACGGGGCG AGGGCGAGGG CGATGCCACC AACGGCAAAC TGACCTTAAA GTTCATCTGT ACTACCGGGA AGCTGCCTGT 1” fusion protein (encoding GCCGTGGCCA ACTTTGGTTA CTACCTTAAC GTATGGCGTT172604987GAACCAGTCG CCGATCGACA TTACTCACGC TATCGAATCC ACACTGGATC CTATTATCTT TAATTATTCC AAGGCCGGCA AAAATGTTGT CAACAACGGC CACACCGTTC AGGTCAACTT172604987sfGFP having a D117C CAATGTTTCT CCCGTTACCC GGACCATATG AAGCGCCATG ACTTCTTCAA ATCGGCTATG CCAGAAGGCT ATGTCCAAGA mutation encoding SEQ ID ACGTACTATT TCTTTCAAAG ATGACGGCAC GTACAAGACC172604987CGAAAGCAGG CCTCACCTTT CTCGTAGACC TTATTAAGAA CAAACACATG AATGCTGATA CTGACTACTC TATTGCAGAA GCTGCGTTCA ATAAAGGCGA AACGGCGATG ACCATTAACG

[0000] t oug pre erred embodments ave been depcted and descr bed n deta herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow. 172604987

Claims

WHAT IS CLAIMED IS:

1. A fusion protein comprising: a carbonic anhydrase domain and one or more of a calcium carbonate binding domain, a calcium binding domain, an iron binding domain, a silicatein mimetic domain, and a silica binding domain.

2. The fusion protein of claim 1, wherein the carbonic anhydrase domain comprises amino acids 22-235 of an Aliivibrio salmonicida carbonic anhydrase of SEQ ID NO:1 or amino acids 21-249 of a Thermosulfurimonas dismutans carbonic anhydrase of SEQ ID NO:2, or a carbonic anhydrase domain having at least 95% sequence identity to amino acids 22-235 of SEQ ID NO:1 or amino acids 21-249 of SEQ ID NO:

2.

3. The fusion protein of claim 1 or claim 2, wherein the carbonic anhydrase domain comprises any one of SEQ ID NOs:3, 5, 7-13, 16, or 17, or a carbonic anhydrase domain having at least 95% sequence identity to any one of SEQ ID NOs:3, 5, 7-13, 16, or 17.

4. The fusion protein of any one of claims 1-3, wherein the carbonic anhydrase domain comprises one or more of: an asparagine to cysteine mutation corresponding to amino acid position 24 (N24C) of SEQ ID NO:3, a threonine to cysteine mutation corresponding to amino acid position 37 (T37C) of SEQ ID NO:3, an asparagine to cysteine mutation corresponding to amino acid position 34 (N34C) of SEQ ID NO:5, a glutamine to cysteine mutation corresponding to amino acid position 49 (Q49C) of SEQ ID NO:5, an asparagine to glycine mutation corresponding to amino acid position 110 (N110G) of SEQ ID NO:3, a glutamate to proline mutation corresponding to amino acid position 143 (E143P) of SEQ ID NO:3, and a glutamate to histidine mutation corresponding to amino acid position 3 (E3H) of SEQ ID NO:

3.

5. The fusion protein of any one of claims 1-4, wherein the carbonic anhydrase domain comprises: an asparagine to cysteine mutation at amino acid position 24 (N24C) corresponding to SEQ ID NO:3 and / or a threonine to cysteine mutation corresponding to amino acid position 37 (T37C) of SEQ ID NO:3; or 172604987an asparagine to cysteine mutation corresponding to amino acid position 34 (N34C) of SEQ ID NO:5 and / or a glutamine to cysteine mutation corresponding to amino acid position 49 (Q49C) of SEQ ID NO:5; wherein the carbonic anhydrase domain is capable of forming dimer-inducing disulfide bonds.

6. The fusion protein of any one of claims 1-5, wherein the carbonic anhydrase domain comprises a loop insert.

7. The fusion protein of claim 6, wherein the loop insert comprises SEQ ID NO:14 or SEQ ID NO:

15.

8. The fusion protein of any one of claims 1-7, wherein the calcium carbonate binding domain comprises SEQ ID NO:

45.

9. The fusion protein of any one of claims 1-8, wherein the calcium binding domain comprises SEQ ID NO:

46.

10. The fusion protein of any one of claims 1-9, wherein the iron binding domain comprises SEQ ID NO:

47.

11. The fusion protein of any one of claims 1-10, wherein the silica binding domain comprises SEQ ID NO:48 or SEQ ID NO:

49.

12. The fusion protein of any one of claims 1-11, wherein the silicatein mimetic domain comprises SEQ ID NO:

50.

13. The fusion protein of any one of claims 1-12 further comprising: a secretory domain sequence.

14. The fusion protein of claim 13, wherein the secretory domain sequence comprises any one of SEQ ID NOs:18-23, or a secretory domain sequence having at least 95% sequence identity to any one of SEQ ID NOs:18-23. 17260498715. The fusion protein of any one of claims 1-14 further comprising: one or more protease cleavage site domains.

16. The fusion protein of claim 15, wherein the one or more protease cleavage site domains comprises any one or more of a rhinovirus protease site, a potyvirus NIa protease site, a potyvirus HC protease site, a potyvirus P1 protease site, a byovirus NIa protease site, a byovirus RNA-2-encoded protease site, an aphthovirus L protease site, an enterovirus 2A protease site, a rhinovirus 2A protease site, a picorna 3C protease site, a comovirus 24K protease site, a nepovirus 24K protease site, a RTSV (rice tungro spherical virus) 3C-like protease site, a PYVF (parsnip yellow fleck virus) 3C-like protease site, a thrombin, factor Xa protease site, a trypsin site, a Genenase protease site, a WELQut protease site, a TEV sequence, a SUMO protease site, and an enterokinase site.

17. The fusion protein of any one of claims 1-16 further comprising: one or more detection and / or purification tag domains.

18. The fusion proteins of claim 17, wherein the one or more detection and / or purification tag domains comprises any one or more of a His-tag, a bd SUMO tag, a FLAG tag, a Strep II tag, a streptavidin-binding peptide tag, a calmodulin-binding peptide tag, a glutathione S-transferase tag, a maltose-binding protein tag, a calmodulin binding peptide tag, and a c-myc tag.

19. The fusion protein of any one of claims 1-18 further comprising: a linker domain between at least one of the secretory domain, the CA domain, the calcium carbonate binding domain, the calcium binding domain, the iron binding domain, the silicatein mimetic domain, and / or the silica binding domain, if present.

20. The fusion protein of any one of claims 1-19, wherein the fusion protein comprises any one of SEQ ID NOs:51-61 or a proteolytic fragment thereof, or a fusion protein having at least 95% sequence identity to any one of SEQ ID NOs:51-61 or a proteolytic fragment thereof. 17260498721. The fusion protein of any one of claims 1-20, wherein the fusion protein or proteolytic fragment thereof, if present, has at least a 20% increase in enzymatic activity compared to wild type bovine carbonic anhydrase.

22. The fusion protein of any one of claims 1-21, wherein the fusion protein or proteolytic fragment thereof, if present, is active at 65oC.

23. The fusion protein of any one of claims 1-22, wherein the fusion protein or proteolytic fragment thereof, if present, retains at least 70% of its carbonic anhydrase enzymatic activity after incubation at 65oC for 45 days in solution.

24. A concrete composition comprising a fusion protein comprising: a carbonic anhydrase domain and one or more of a calcium carbonate binding domain, a calcium binding domain, an iron binding domain, a silicatein mimetic domain, and a silica binding domain; and cement.

25. The concrete composition of claim 24 further comprising: one or more of a calcium source, buffer, and aggregate.

26. The concrete composition of claim 25, wherein the calcium source comprises calcium formate.

27. The concrete composition of any one of claims 24-26, wherein the carbonic anhydrase domain comprises any one of SEQ ID NOs:3, 5, 7-13, 16, or 17, or a carbonic anhydrase domain having at least 95% sequence identity to any one of SEQ ID NOs:3, 5, 7-13, 16, or 17.

28. The concrete composition of any one of claims 24-27, wherein the carbonic anhydrase domain comprises one or more of: an asparagine to cysteine mutation corresponding to amino acid position 24 (N24C) of SEQ ID NO:3, a threonine to cysteine mutation corresponding to amino acid position 37 (T37C) of SEQ ID NO:3, an asparagine to cysteine mutation corresponding to amino acid position 34 (N34C) of SEQ ID NO:5, a glutamine 172604987to cysteine mutation corresponding to amino acid position 49 (Q49C) of SEQ ID NO:5, an asparagine to glycine mutation corresponding to amino acid position 110 (N110G) of SEQ ID NO:3, a glutamate to proline mutation corresponding to amino acid position 143 (E143P) of SEQ ID NO:3, and a glutamate to histidine mutation corresponding to amino acid position 3 (E3H) of SEQ ID NO:

3.

29. The concrete composition of any one of claims 24-28, wherein the fusion protein further comprises one or more of a secretory domain, a protease cleavage site domain, a detection and / or purification tag domain, and a linker domain.

30. The concrete composition of any one of claims 24-29, wherein the concrete composition comprises the fusion protein at about 100-250 mg / cubic yard.

31. The concrete composition of any one of claims 24-30, wherein the fusion protein comprises any one of SEQ ID NOs:51-61 or a proteolytic fragment thereof, or a fusion protein having at least 95% sequence identity to any one of SEQ ID NOs:51-61 or a proteolytic fragment thereof.

32. The concrete composition of any one of claims 24-31, wherein the concrete composition has improved self-healing, repair and / or durability properties compared to a concrete composition without the fusion protein.

33. An enzymatic construction material (ECM) composition comprising: a fusion protein comprising: a carbonic anhydrase domain and one or more of a calcium carbonate binding domain, a calcium binding domain, an iron binding domain, a silicatein mimetic domain, and a silica binding domain; and a particulate substrate that is not cement.

34. The ECM composition of claim 33, wherein the particulate substrate comprises one or more of sand, gravel, crushed stone, aggregate, soil, fibers, silicone bead, silicon dioxide, a silica gel, disiloxane, silicic acid, silanol, an organic silicon compound, gelatin, grit, cellulose, and a cellulose derivative. 17260498735. The ECM composition of claim 33 or claim 34 further comprising: one or more of a calcium source, buffer, and aggregate.

36. The ECM composition of any one of claims 33-35, wherein the carbonic anhydrase domain comprises any one of SEQ ID NOs:3, 5, 7-13, 16, or 17, or a carbonic anhydrase domain having at least 95% sequence identity to any one of SEQ ID NOs:3, 5, 7-13, 16, or 17.

37. The ECM composition of any one of claims 32-36, wherein the carbonic anhydrase domain comprises one or more of: an asparagine to cysteine mutation corresponding to amino acid position 24 (N24C) of SEQ ID NO:3, a threonine to cysteine mutation corresponding to amino acid position 37 (T37C) of SEQ ID NO:3, an asparagine to cysteine mutation corresponding to amino acid position 34 (N34C) of SEQ ID NO:5, a glutamine to cysteine mutation corresponding to amino acid position 49 (Q49C) of SEQ ID NO:5, an asparagine to glycine mutation corresponding to amino acid position 110 (N110G) of SEQ ID NO:3, a glutamate to proline mutation corresponding to amino acid position 143 (E143P) of SEQ ID NO:3, and a glutamate to histidine mutation corresponding to amino acid position 3 (E3H) of SEQ ID NO:

3.

38. The ECM composition of any one of claims 32-37, wherein the fusion protein further comprises one or more of a secretory domain, a protease cleavage site domain, a detection and / or purification tag domain, and a linker domain.

39. The ECM composition of any one of claims 32-38, wherein the ECM composition comprises the fusion protein at about 100-250 mg / cubic yard.

40. The ECM composition of any one of claims 32-39, wherein the fusion protein comprises any one of SEQ ID NOs:51-61 or a proteolytic fragment thereof, or a fusion protein having at least 95% sequence identity to any one of SEQ ID NOs:51-61 or a proteolytic fragment thereof.

41. The ECM composition of any one of claims 32-40, wherein the ECM composition has improved self-healing, repair and / or durability properties compared to an ECM composition without the fusion protein. 17260498742. A method of repairing a fracture in a cementitious or ECM surface, said method comprising: contacting a fracture in a cementitious or ECM surface with the composition of any one of claims 24-41 under conditions effective to repair the fracture in the cementitious or ECM surface.

43. The method of claim 42, wherein said contacting occurs at ambient conditions of atmospheric carbon dioxide and temperature.

44. The method of claim 42 or claim 43, wherein the composition is cured to form a solid mass.

45. A method for making a cement mixture, said method comprising: combining the composition of any one of claims 1-22 with a cement to form a cement mixture.

46. The method of claim 45 further comprising: curing the cement mixture under conditions to make a durable corrosion resistant concrete.

47. A method for making an enzymatic construction material (ECM) mixture, said method comprising: combining the composition of any one of claims 1-22 a with a particulate substrate to form an ECM mixture.

48. The method of claim 47 further comprising: curing the ECM mixture comprising the composition to make a durable corrosion resistant ECM.

49. A polynucleotide molecule for expressing a carbonic anhydrase fusion protein comprising: 172604987a polynucleotide encoding a carbonic anhydrase domain and one or more of a calcium carbonate binding domain, a calcium binding domain, an iron binding domain, a silicatein mimetic domain, and a silica binding domain and a promoter operably linked to the polynucleotide.

50. The polynucleotide molecule of claim 49, wherein the polynucleotide comprises any one of SEQ ID NOs:63-93, or a polynucleotide molecule having at least 95% sequence identity to any one of SEQ ID NOs:63-93.

51. The polynucleotide molecule of claim 49 or claim 50, wherein the polynucleotide molecule encodes the amino acid sequence of any one of SEQ ID NOs:3, 5, 7-13, 16, 17, and 51-61 or a polynucleotide molecule having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs:3, 5, 7-13, 16, 17, and 51-61.

52. The polynucleotide molecule of any one of claims 49-51, wherein the polynucleotide further comprises a polynucleotide encoding one or more of a secretory domain, a protease cleavage site domain, a detection and / or purification tag domain, and a linker domain.

53. The polynucleotide molecule of any one of claims 49-52, wherein the promoter is heterologous to the carbonic anhydrase domain.

54. The polynucleotide molecule of any one of claims 49-53, wherein the promoter is heterologous to one or more of the secretory domain, the protease cleavage site domain, the detection and / or purification tag domain, and the linker domain.

55. A vector comprising the polynucleotide molecule of any one of claims 49- 54.

56. The vector according to claim 55, wherein the vector is a bacterial vector.

57. A host cell comprising the polynucleotide of any one of claims 49-54 or the vector of claim 55 or claim 56.

58. The cell according to claim 57, wherein the cell is a bacterial cell. 172604987

Images