The overall objective of this proposal is to design and investigate novel active flexible and semi flexible polymeric nano-carrier platforms that will enable nanoscale spatial co-localization of multiple active enzymes. Several multi-enzyme complexes found in Nature are designed to ensure rapid transport of each intermediate in the reaction to the next neighboring active site, since the intermediates are often unstable. Thus, it is critical to molecularly co-localize these enzymes in nano-carriers so that the reactive intermediates can find the next active site for the desired products to be formed. While there have been numerous studies dealing with enzyme immobilization, there are no studies of using nanomaterials to co-localize multi-enzyme complexes, especially with reactive intermediates.
Thus, the focus of this proposal is to create active nanostructured environments that can modify the direction of complex conversions by confinement of the active catalytic functionality within both spatial and temporal scales. We will investigate the biosynthesis of flavan-3-ol, whose production is mediated by two enzymes with a highly reactive intermediate. Flavan-3-ols, such as (-) epicatechin, are flavonoid natural products with powerful antioxidant properties and are the major contributors to the cardioprotective and anticancer activity of various foods such as green tea and dark chocolate. The specific goals of the project are to: 1) Design and characterize novel nano carrier platforms based on self-assembling ionic and degradable copolymers to co-localize and stabilize multiple enzymes with reactive intermediates; and 2) Investigate enzymatic activity and flux in nano-carriers for flavon-3-ol biosynthesis. A diverse and interdisciplinary team of researchers has been assembled to address this problem.
Intellectual Merit: New bioinspired robust active nano carrier platforms with tailored chemistries will be designed to enable nanoscale spatio-temporal control of multiple enzymes. These materials form various stable nano-compartments/nanostructures. These platforms have been chosen to investigate how the flexibility of the nanostructure affects the co-localization and catalytic activity of the enzymes. This confinement is also expected to increase the stability of relatively delicate enzymatic biocatalysts. The structure, dynamics, transport properties, and thermodynamic interactions of the nano-compartments with enzymes will be investigated using experimental nanoscale tools and computational methods to obtain insights into the mechanisms of activity. This approach will facilitate rational design of the active nano-carrier platforms. These insights will be used to investigate the effects of nano-encapsulation on the enzymatic activity of, and product flux through, a multi enzyme complex in flavan-3-ol biosynthesis.
Broader Impact: The nano carrier platforms can be extended to effectively mediate many other important multi enzyme reactions with reactive intermediates (e.g., the tricarboxylic acid cycle) and channel reactions towards desired products that might not otherwise be possible. The dual functions of enzyme stabilization and improved flux provided by the environmentally responsive nano-carrier platforms will provide a general strategy for industrial use of enzymatic biocatalysts in cascade reactions, including their efficient (re)use under nominally harsh conditions. The nanoscale probes developed to elucidate fundamental nanostructure function relationships can be readily applied to other material/biomolecule systems. This proposal integrates research and educational initiatives to provide students multifaceted and interdisciplinary learning experiences. A diverse group of students will be proactively recruited. This work will have a nation-wide impact through the dissemination of problem-solving rubrics and bioethics case studies. This proposal will impact K-12 students through research experiences and several outreach mechanisms. Results from the project will be disseminated broadly through sessions on enzyme nanocatalysis at professional meetings, workshops, and archival publications.
This work has resulted in the development of new strategies to co-localize multiple enzymes on nanocarriers to mimic the functionalities of multi-enzyme complexes (MECs). By bringing the enzymatic catalytic active sites together in MECs, reaction intermediates can be transported rapidly among the active sites, which can reduce diffusion losses typically observed in free enzyme catalytic processes. MECs also enable the maintenance of high local concentrations of intermediates, which is especially critical for unstable intermediates. Different polymeric nano-carriers, both flexible and rigid, were investigated as platforms for co-localization, along with distinct enzyme attachment techniques to control the spatial arrangement of the multiple enzymes on the nano-carriers. When two different enzymes were co-localized on the nanoparticles, the overall product conversion rate was enhanced two-fold compared to the equivalent amount of free enzymes in solution. When DNA hybridization techniques were developed and utilized to precisely contol the spatial location of the multiple enzymes on the nanocarriers, the enhancement in the overall product conversion rate was even greater. Co-localizing the enzymes on nanoparticles as opposed to planar surfaces resulted in greater enhancement in the overall product conversion rate due to steric effects. These studies demonstrated that the concept of mimicking structure and function of MECs by co-localizing multiple enzymes on nanocarriers have clear kinetics benefits. Initial studies on the storage shelf life of co-localized enzymes also show promise in terms of their long-range storage potential for specific applications. The design of sustainable and re-usable multi-enzyme biocatalysts based on this work could lead to economically viable designs for next generation biocatalysts and biosensors.
