Engineering Enzyme Vesicles with Stimuli Responsive Partitioning Behavior for High Reactivity and Simultaneous Product Separation

DMR – 2429163: Engineering Enzyme Vesicles with Stimuli Responsive Partitioning Behavior for High Reactivity and Simultaneous Product Separation Non-technical Summary

Enzymes are used to make important products in the pharmaceutical, chemical, food, and detergent industries. Chemical reactions that use enzymes can be performed in water at moderate temperatures, leading to more environmentally friendly processes compared to traditional reactions that use solvents and high temperatures. One difficult challenge is when the starting molecules and final products do not dissolve in water, since enzymes function in water.

This project addresses that challenge by using microscopic protein containers, called vesicles. The vesicles can hold the enzymes on their outer surface in the water environment, and simultaneously hold the starting molecules and products at the inner surface surrounded by a liquid-like protein that separates from water. This approach enables both the enzyme and the starting materials to be in their own ideal environment very close to each other so the chemical reaction happens faster than if the vesicles were not used.

The vesicles also capture the products after the reaction. Experiments in this project vary the vesicle properties to understand why they improve enzyme reactions and optimize them for maximum reaction. The enzyme chosen for this work is a dehalogenase that can make useful chemicals but can also be used to degrade harmful “forever chemicals” that pollute water in some American communities.

Therefore, the results of this research can specifically lead to better processes to degrade forever chemicals or produce valuable chemicals, and the knowledge gained can be applied to a wide variety of enzymes and chemical reactions to make production of important molecules more sustainable in the future. In addition, middle school girls will visit the lab to do hands-on experiments, and local high school students will participate in the vesicle research and professional development and mentoring activities.

The proposed plan will provide technical training and critical personal mentoring of graduate and undergraduate students. Results from this work will be shared in the chemical engineering and protein engineering courses and at K-12 school visits and field trips, to inform and inspire students to pursue and persist in STEM fields.

Technical Summary

This research will create switchable, local hydrophobic environments in an aqueous system for enzymes with hydrophobic substrates to improve reactivity and enable simultaneous reaction and separation. This will be accomplished via creation of enzyme vesicles that are self-assembled from engineered proteins whose hydrophobicity can be turned “on” and “off”, eliminating the need for traditional two-phase solvent-aqueous processes and improving the sustainability of industrial biocatalysis.

The vesicle design incorporates enzymes directly on the outer surface in the bulk aqueous phase immediately adjacent to the substrate enriched, hydrophobic inner surface, which increases enzyme conversion of substrate. Dehalogenase enzyme, DmmA, will be used here for proof of concept as it can be applied for both organic synthesis and decontamination of environmental pollutants such as fluorinated compounds that disproportionally impact minority communities, contributing to the broad impact.

To achieve enzyme vesicles with controllable substrate partitioning, experimental work will (i) develop and characterize the activity of crosslinked, pH sensitive DmmA vesicles, (ii) assess partitioning of DmmA substrates with different hydrophobicity and molecular weight into vesicles made from protein sequences with varying hydrophobicity, and (iii) demonstrate cyclic DmmA reaction and separation of products by pH switching of vesicles between hydrophobic and hydrophilic states. As intellectual merit, this work will create the design space for enzyme vesicles so that for a given enzyme/substrate/product combination, the best vesicle properties can be selected for optimal aqueous reaction and product separation. In addition, middle school girls will visit the

lab to do hands-on experiments, and local high school students will participate in the vesicle research and professional development and mentoring activities. The proposed plan will provide technical training and critical personal mentoring of graduate and undergraduate students. Results from this work will be shared in the chemical engineering and protein engineering courses and at K-12 school visits and field trips, to inform and inspire students to pursue and persist in STEM fields.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.