Our biomaterial platform can uniquely incorporate active large, complex proteins via protein fusion, eliminating the need for crosslinkers. Biocatalysis requires both an enzyme, and a mechanism to physically separate the enzyme from product, usually a solid support. In our approach, both of these factors are produced in a single molecule. This rapid, single-pot, single-component approach dramatically reduces the cost of materials synthesis while simultaneously increasing the process' reliability and scalability. This same process can be used for a wide variety of enzymes, eliminating the time-consuming and difficult optimization process required by all other stabilization / immobilization techniques. For systems that chemically cross-link a protein to a surface, one concern is that the protein could leach from the materials if the cross-linked bond is degradable. In contrast, our approach connects enzymes to materials through a stable peptide bond without damaging the enzyme. Additionally, many proteins lose activity when stored dry or at room temperature; however, Bondwell materials can be stored dry at room temperature for nearly 10 years and remain active. Proteins fused to our materials are a million-fold more active than the same protein trapped in hydrogel, and have 1,000-times the binding capacity of protein cross linked to resin beads.

We have successfully demonstrated that enzymes remain active when fused to our materials. In this proposal we will demonstrate the unique ability of this technology to manufacturing drugs under storage/use conditions suitable for deep space exploration missions. The proposed plan will produce materials with the ability to produce amoxicillin, cephalosporin, and melatonin. In addition, we will test enzyme efficacy in our system and performance after storage. Our long-term goal is to combine the efficiency, specificity, and broad applicability of biocatalysis with telescoping in a sealed reactor flow chemistry system. Most natural therapies can be catalyzed by enzymes, and enzymologists are using directed evolution and artificial intelligence to rapidly create enzymes that catalyze novel reactions. We envision using each enzyme-material fusion to create a mesh of fibers shaped like a disc ~ 1 cm diameter. The drug is manufactured by placing the correct discs (enzymes) with reagents in the telescoping system and allowing the reaction to occur. Each disk can be rinsed, dried, and re-used as needed to manufacture one or more drugs. This unique system has the potential to produce chemicals, manufacture drugs, prepare food, or even generate biofuels from a small number of precursors.

To continue development of this technology into a final product suitable for space flight, additional research is required. Specifically:

1. ACV Synthetase (ACVS) is an extremely large gene and toxic to bacteria. We hope to keep trying other cloning options such as Bacillus megatarium. This is important for this pathway but also for building a complete library of drugs.

2. We will continue characterizing the enzymes that are pending. Due to time constraints, the loss of commercially available substrates (COVID related), supply chain issues, temporary closures or employee loss due to COVID, and the troubleshooting process of developing assays for our materials, the characterization process has taken longer than originally anticipated.

3. We will do more aging experiments with different enzyme-Ubx materials in different storage conditions to optimize the activity retention during simulated aging. We will also test different storage conditions to optimize reuse of the enzyme-Ubx materials.

Our biomaterial platform can incorporate active large, complex proteins via protein fusion, eliminating the need for crosslinkers. Biocatalysis requires both an enzyme, and a mechanism to physically separate the enzyme from product, usually a solid support. In our approach, both of these factors are produced in a single molecule. This rapid, single-pot, single-component approach dramatically reduces the cost of materials synthesis, while simultaneously increasing the process’ reliability and scalability. This same process can be used for a wide variety of enzymes, eliminating the time-consuming and difficult optimization process required by other stabilization / immobilization techniques. For systems that chemically cross-link a protein to a surface, one concern is that the protein could leach from the materials if the cross-linked bond is degradable. In contrast, our approach connects enzymes to materials through a stable peptide bond without damaging the enzyme. Additionally, many proteins lose activity when stored dry or at room temperature; however, Bondwell materials can be stored dry at room temperature for nearly 10 years and remain active. Proteins fused to our materials are a million-fold more active than the same protein trapped in hydrogel, and have 1,000-times the binding capacity of protein cross linked to resin beads.

Our long-term goal is to create membranes, each modified with one or more enzymes and sealed in a plastic reaction chamber. Therapeutics would be synthesized by connecting the appropriate series of reaction chambers and adding substrate and any needed cofactors to the first reaction chamber. Product will flow from the last chamber. We envision using each enzyme-material fusion to create a mesh of fibers shaped like a disc ~ 1cm diameter. The drug is manufactured by placing the correct discs (enzymes) with reagents in the telescoping system and allowing the reaction to occur. Each disk can be rinsed, dried, and re-used as needed to manufacture one or more drugs. This unique system has the potential to produce chemicals, manufacture drugs, prepare food, or even generate biofuels from a small number of precursors.

With the unique ability of our materials, we demonstrate here that our functionalized enzymatic material is able to catalyze reactions to produce therapeutic drugs under suitable conditions for deep space exploration missions. Our aim was to produce NASA-relevant therapeutics, including penicillin, cephalosporin C, amoxicillin, and melatonin, by incorporating the enzymes from the natural biosynthesis pathways into our biomaterials.

By the end of year 2, we have established these following main findings:

1. We have successfully inserted the genes of the enzymes into our biomaterial expression cassette. There are 12 total enzymes in the two natural biosynthesis pathways of Penicillin G, cephalosporin C, Amoxicillin, and melatonin. We have successfully expressed, purified, and formed materials for 11 out of the 12 enzymes.

2. We have successfully tested the activity of six out of the 12 enzymatic biomaterials. We have demonstrated that these enzymes are fully functional in Ubx materials. We were able to determine kinetic parameters for five of the enzymes while the activity of the sixth was confirmed by mass spectrometry. The kinetic parameters are all consistent with published data in the literature.

3. We have additionally tested Penicillin G Acylase (PGA) functionality with extremely harsh storage conditions. We demonstrated that PGA in our material maintains its catalytic efficiency after being stored in conditions that mimic the extremes expected in long-duration spaceflight.

4. We also demonstrated the activity and reusability of Isopenicillin N Synthase (IPNS) as well as Hydroxytryptophan Decarboxylase (HTDC). IPNS in our material is reusable and active after being left in the open air, at room temperature for 48 hours. HTDC remained reusable and remarkably active even after two weeks at room temperature in open air. We also saw that the materials stored in phosphate buffered saline (PBS) had a higher retention of activity after 48 hours.

5. We showed that a well-characterized enzyme, luciferase, was still remarkably active in our materials after three years of simulated aging, and seven months of actual aging.

6. We demonstrate telescoping synthesis of Melatonin (4 enzymes) is achievable in this system in 2 simple reactions.

In summary, this work demonstrates feasibility of this system as a biocatalysis platform for drug manufacturing in space. The system meets the requirements of spaceflight conditions and duration stated in the solicitation. Further work is required to move from a feasibility standpoint to full product development.

Our biomaterial platform can uniquely incorporate active large, complex proteins via protein fusion, eliminating the need for crosslinkers. Biocatalysis requires both an enzyme, and a mechanism to physically separate the enzyme from product, usually a solid support. In our approach, both of these factors are produced in a single molecule. This rapid, single-pot, single-component approach dramatically reduces the cost of materials synthesis while simultaneously increasing the process' reliability and scalability. This same process can be used for a wide variety of enzymes, eliminating the time-consuming and difficult optimization process required by all other stabilization / immobilization techniques. For systems that chemically cross-link a protein to a surface, one concern is that the protein could leach from the materials if the cross-linked bond is degradable. In contrast, our approach connects enzymes to materials through a stable peptide bond without damaging the enzyme. Additionally, many proteins lose activity when stored dry or at room temperature; however, Bondwell materials can be stored dry at room temperature for nearly 10 years and remain active. Proteins fused to our materials are a million-fold more active than the same protein trapped in hydrogel, and have 1,000-times the binding capacity of protein cross linked to resin beads.

We have successfully demonstrated that enzymes remain active when fused to our materials. In this proposal we will demonstrate the unique ability of this technology to manufacturing drugs under storage/use conditions suitable for deep space exploration missions. The proposed plan will produce materials with the ability to produce amoxicillin, cephalosporin, and melatonin. In addition, we will test enzyme efficacy in our system and performance after storage. Our long-term goal is to combine the efficiency, specificity, and broad applicability of biocatalysis with telescoping in a sealed reactor flow chemistry system. Most natural therapies can be catalyzed by enzymes, and enzymologists are using directed evolution and artificial intelligence to rapidly create enzymes that catalyze novel reactions. We envision using each enzyme-material fusion to create a mesh of fibers shaped like a disc ~ 1 cm diameter. The drug is manufactured by placing the correct discs (enzymes) with reagents in the telescoping system and allowing the reaction to occur. Each disk can be rinsed, dried, and re-used as needed to manufacture one or more drugs. This unique system has the potential to produce chemicals, manufacture drugs, prepare food, or even generate biofuels from a small number of precursors.

Enzymes have been used in therapeutics syntheses because they offer higher product yields, require fewer steps, create fewer undesirable side products, produce final products with higher purity, and enable easier product recovery than traditional chemical syntheses. However, enzymes have limitations, including easy degradation, loss of activity over time, and the requirement of careful storage and handling, which limit their use in the manufacturing of therapeutics. Bondwell Technologies has developed functionalized enzyme-based materials that address these limitations. These materials can immobilize and safely concentrate enzymes, by 1,000 - 10,000-fold compared to other immobilization techniques. Our platform technology is based on our success in producing fused proteins that can self-assemble into biomaterials in vitro. Proteins with a wide range of physical properties incorporated into our materials are significantly stabilized, while still remaining functional after boiling or autoclaving. Materials functionalized with proteins can be stored dry at room temperature for many years, and survive multiple drying/rehydration cycles, enabling their reuse. Additionally, enzyme-containing fusion proteins have retained their catalytic capabilities. Thus, our materials can be used to improve stability and handling of a variety of enzymes that are used in therapeutics manufacturing.

Our long-term goal is to create membranes, each modified with one or more enzymes and sealed in a plastic reaction chamber. Therapeutics would be synthesized by connecting the appropriate series of reaction chambers and adding substrate and any needed cofactors to the first reaction chamber. By the end of year 1, we have established these following main findings:

1. We have successfully inserted the genes of the enzymes into our biomaterial expression cassette. There are 12 total enzymes in the two natural biosynthesis pathways of Penicillin G, cephalosporin C, Amoxicillin, and melatonin. The status of these 12 enzymes are at various states, ranging from at gene synthesis and cloning step to expression, purification, and kinetic characterization step.

2. We have successfully expressed, purified, and formed materials for 4 out of the 12 enzymes of the two natural biosynthesis pathways of penicillin G and cephalosporin C. Those enzyme-materials include Penicillin G Acylase, Isopenicillin N Synthase, Isopenicillin N Acyltransferase, and Deacetoxycephalosporin C synthase.

3. We have tested the activity of Penicillin G Acylase in our material. Penicillin G Acylase (PGA) is a heterodimeric protein, which carries out two different reactions, i.e., Penicillin G hydrolysis and Amoxicillin synthesis. We demonstrated that the enzyme is fully functional in this material. We have fully characterized PGA in our material and determined kinetic parameters of both Penicillin G hydrolysis and Amoxicillin synthesis reactions. The kinetic parameters are similar to published data in the literature.

4. We have tested PGA functionality with extremely harsh storage conditions. We demonstrated that PGA in our material maintains its catalytic efficiency after being stored with extreme temperature conditions. We also demonstrated the activity and reusability of Isopenicillin N Synthase (IPNS). IPNS is reusable and remarkably active after being left in the open air, at room temperature for 48 hours.

Our biomaterial platform can uniquely incorporate active large, complex proteins via protein fusion, eliminating the need for crosslinkers. Biocatalysis requires both an enzyme, and a mechanism to physically separate the enzyme from product, usually a solid support. In our approach, both of these factors are produced in a single molecule. This rapid, single-pot, single-component approach dramatically reduces the cost of materials synthesis while simultaneously increasing the process’ reliability and scalability. This same process can be used for a wide variety of enzymes, eliminating the time-consuming and difficult optimization process required by all other stabilization / immobilization techniques. For systems that chemically cross-link a protein to a surface, one concern is that the protein could leach from the materials if the cross-linked bond is degradable. In contrast, our approach connects enzymes to materials through a stable peptide bond without damaging the enzyme. Additionally, many proteins lose activity when stored dry or at room temperature; however, Bondwell materials can be stored dry at room temperature for nearly 10 years and remain active. Proteins fused to our materials are a million-fold more active than the same protein trapped in hydrogel, and have 1,000-times the binding capacity of protein cross linked to resin beads.

We have successfully demonstrated that enzymes remain active when fused to our materials. In this proposal we will demonstrate the unique ability of this technology to manufacturing drugs under storage/use conditions suitable for deep space exploration missions. The proposed plan will produce materials with the ability to produce amoxicillin, cephalosporin, and melatonin. In addition, we will test enzyme efficacy in our system and performance after storage. Our long-term goal is to combine the efficiency, specificity, and broad applicability of biocatalysis with telescoping in a sealed reactor flow chemistry system. Most natural therapies can be catalyzed by enzymes, and enzymologists are using directed evolution and artificial intelligence to rapidly create enzymes that catalyze novel reactions. We envision using each enzyme-material fusion to create a mesh of fibers shaped like a disc ~1 cm diameter. The drug is manufactured by placing the correct discs (enzymes) with reagents in the telescoping system and allowing the reaction to occur. Each disk can be rinsed, dried, and re-used as needed to manufacture one or more drugs. This unique system has the potential to produce chemicals, manufacture drugs, prepare food, or even generate biofuels from a small number of precursors.