The significant resources, facilities, and toxic waste produced from traditional chemical synthesis makes production of medicines during long-term space exploration impossible. To increase efficiency and environmental safety, the multi-billion dollar biocatalysis industry leverages enzymes to produce fine chemicals, pharmaceuticals, and other industrially relevant compounds. Enzymes are efficient and highly selective reusable biocatalysts that can significantly accelerate the rate of chemical reactions. Biocatalysis offers higher yields, fewer side reactions, elimination of protection and de-protection steps, purer products, easier recovery and separation, and reduced waste. The drawbacks to using enzymes as biocatalysts are that enzymes are costly to produce, easily degraded or inactivated, and difficult to store. Despite the great potential of enzymes in pharmaceutical manufacturing, current approaches to solve the enzyme stability problem are insufficient. Bondwell Technologies has developed a low-cost platform approach to immobilize and stabilize a wide array of enzymes without a time-consuming optimization process.
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.