Progress 01/15/23 to 01/14/25

Outputs
Target Audience:1. Food Science and Technology Researchers We engaged faculty and students in VT's Food Science and Technology department by presenting our results at the Spring Graduate Research Symposium. To broaden impact, the PI delivered an invited seminar at the University of Tennessee, Knoxville, followed by one-on-one discussions with faculty and graduate students, sharing detailed protocols and preliminary data. 2. Polymer Chemists At VT's Polymer Chemistry & Engineering Conference in April 2025, the PI addressed ~30 materials scientists to demonstrate how polymer backbone and surface architecture influence enzyme loading and activity at low pH. Complementing this talk, our graduate student presented a poster at both this meeting and the 2024 ACS Fall Conference in Denver, highlighting structure-function correlations in our immobilization platforms. 3. Biotech Company We met with a commercial enzyme-production firm, presenting our stability and performance data to R&D teams who supply enzymes across food and pharmaceutical sectors. These discussions covered scale-up considerations and potential integration of our immobilization methods into existing manufacturing workflows. Across these audiences, knowledge transfer occurred through targeted symposium talks, invited seminars, poster presentations, and in-depth discussions, equipping researchers and industry partners with actionable insights for enzyme deployment in acidic environments. Changes/Problems:Progress on ObjectiveIII--optimizing lactase performance in real acid whey streams--was delayed by the PI's transition to an independent research group and the need to train a new graduate student in carrying out the first two objectives. We also modified our original plan by adding new tasks under both ObjectiveI & II, specifically, testing the effect of directed immobilization chemistry on enzyme activities, which was not part of the initial approach but proved essential for achieving enzyme activity levels comparable to free lactase. Carrying out these additional tasks casued delays, which affected progress toward completing Obj. III.While these changes deviated from the original timeline, we were able to complete >80% of our original project goals over the entrire project period. What opportunities for training and professional development has the project provided?The grant fostered professional growth: the PI established an independent research group and trained one master's student and one undergraduate in enzyme technology and materials engineering, strengthening the scientific workforce in food-processing innovation. How have the results been disseminated to communities of interest?We engaged Virginia Tech's Food Science and Technology faculty and students by presenting our findings at the Spring Graduate Research Symposium, then broadened our reach with an invited seminar at the University of Tennessee, Knoxville, where the PI followed up with one-on-one protocol discussions. At VT's Polymer Chemistry & Engineering Conference in April 2025, the PI explained to roughly 30 materials scientists how polymer backbone and surface architecture govern enzyme loading and low-pH activity, while our graduate student showcased structure-function correlations in posters at that meeting and the 2024 ACS Fall Conference in Denver. Finally, we met with a commercial enzyme-production firm's R&D team, presenting stability and performance data and exploring scale-up and integration of our immobilization methods into their manufacturing workflows. A full draft of the manuscript focused on engineering microenvironments to enhance immobilized enzyme activity has been completed and will be submitted soon. In addition, two research articles on modifying nanoporous membrane microenvironments using vapor deposition have been published. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? During this reporting period, we have significantly advanced our understanding of how to sustain and enhance lactase performance under acidic conditions through tailored immobilization strategies. These insights extend beyond lactase, offering a blueprint for other enzyme systems operating in challenging environments. Our work lays the groundwork for valorizing acid whey streams--a major dairy by?product--and equips biotechnology companies with new, high-throughput methods to optimize their immobilized-enzyme platforms. Objective I. Investigate the effect of nano-confinement imposed by nanopores on the performance of immobilized lactase We evaluated lactase activity when confined within anodic aluminum oxide supports featuring pore diameters of 40, 80, 120, and 160 nm at both 37 °C and 65 °C (pH 7). In parallel, we compared two bioconjugation schemes--random immobilization (RI) and directed immobilization (DI) via SpyTag/SpyCatcher chemistry. Data collected: Quantitative activity assays and kinetic parameters across all pore sizes, temperatures, and conjugation methods. Results & discussion: Enzyme loading rose from 0.38 mg/g on 40 nm supports to 0.78 mg/g on 160 nm supports, indicating greater accessible surface area in larger pores. Directed immobilization yielded up to an 8-fold increase in activity versus random attachment, with DI-lactase matching the catalytic rates of its free-enzyme counterpart. Outcomes: We established a robust DI protocol for lactase and demonstrated that spatial control within nanopores dramatically enhances catalytic performance. These findings constitute new, publishable insights into effects of DI bioconjugation on biocatalysis and provide a promising technical prospect for practitioners to adopt DI approaches in enzyme-supported systems and a strong argument for follow-up techno-economic analyses. Objective II. Understand how the physicochemical properties of polymeric nanolayers affect the performance of immobilized lactase Using initiated chemical vapor deposition (iCVD), we synthesized enzyme supports varying in crosslinker chemistry (aromatic vs. acrylic), cationic functionality, and coating thickness (50-400 nm). We then measured lactase activity across pH 4-8 to simulate acid-whey conditions. Data collected: Activity profiles for lactase immobilized on each polymer variant at multiple pH values; comparative measurements of activity retention and pH dependence. Results & discussion: While hydrophobicity and stiffness alone did not predict activity trends, incorporation of cationic monomers was found to enable detectable enzyme activity at pH 4; as comparison, neither free enzymes nor enzymes immobilized on polymers without cationic monomers showed detectable activity. Enzyme performance improved with increasing layer thickness, plateauing at ~250 nm, suggesting a maximum diffusion depth for proton (or hydronium) and guiding future coating thickness selections. Outcomes: We identified key polymer design parameters--cationic charge density and nanolayer thickness--that enable robust lactase function in acidic media. This knowledge equips polymer chemists and process engineers with actionable guidelines for crafting next-generation immobilization materials. Objective III. Optimize the performance of lactase via nanoengineered spatial and chemical microenvironments for efficient biotransformation of lactose in acid whey We have initiated integrated testing of spatial (pore size, layer thickness) and chemical (functional-group) variables on lactase activity in simulated buffer systems. Filtration units have been procured and validated to support upcoming trials in real acid-whey matrices. Data collected: Preliminary device performance metrics and bench-scale flow rates; baseline enzyme stability in buffer over repeated use. Results & discussion: We observed encouraging retention of enzyme activity after 6 uses, up to 50-60%, in our preliminary tests. Future studies will delve into how polymer chemistry (stiffness, crosslinking degree and type of crosslinkers, cationic environment) potentially affect reusability of the immobilized enzymes. Outcomes: A rapid-testing platform and essential equipment are now operational, setting the stage for practical acid-whey processing trials.

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: H. Chen, F. Fianu, C. I. Moraru, R. Yang, Y. Cheng, Orthogonal Nano-Engineering (ONE): Modulating Nanotopography and Surface Chemistry of Aluminum Oxide for Superior Antibiofouling and Enhanced Chemical Stability. Adv. Mater. Interfaces 2024, 12, 2400287. https://doi.org/10.1002/admi.202400287

Progress 01/15/23 to 01/14/24

Outputs
Target Audience: Researchers in academia, government, and industry working on or are interested in enzyme-based technologies Food technologists, engineersand scientists Dairy processors Changes/Problems:Proposed budget reallocation: A reallocation of funds is proposed by the Principal Investigator, moving an estimated $8,000 from the equipment allocation (~$50,000) to cover a graduate student's summer stipend and associated fringe benefits for three months in 2024. This adjustment represents a minor change, involving less than 20% of the original equipment budget, and is within the allowable scope for budget reallocations. What opportunities for training and professional development has the project provided?This project has provided training for a historically underserved minority graduate student from Ghana on a variety of areas, including but not limited to: 1. Essential research skills: notekeeping, critical thinking, troubleshooting, scientific communication in both writing and speaking. The student was able to present two posters based on work supported by this grant. 2. Learning how to use initiated chemical vapor deposition reactor for synthesizing polymer thin films, how to perform enzyme kinetics characterization, how to use advanced instruments such as ATR-FTIR, SEM, and XPS to characterize the synthesized materials. How have the results been disseminated to communities of interest?The results have been disseminated to communities of interest via poster presentations performed by the graduate student. On one occasion the students presented at the annual food science and technology student poster competition; and on the other occasion the student participated in a material science and chemical engineering focused poster competition and the warm the best graduate student poster award. In both cases the presentations were given within the Virginia Tech community. What do you plan to do during the next reporting period to accomplish the goals?? For the upcoming reporting period, the team intends to focus on achieving the following objectives: Research Objectives: Investigate the effects of random versus specific orientation on the kinetics of lactase immobilized on two distinct polymer coatings. Develop a chemical environment that shields enzymes from surrounding protons, thereby widening the pH range within which lactase can effectively operate, particularly in acidic conditions. Immobilize lactase enzymes on a filter membrane utilizing the polymer chemistries mentioned above and compare the enzyme kinetics on membranes with those on 96-well plates. Training and Mentoring: A graduate student will receive mentorship from the Principal Investigator (PI) to achieve the aforementioned research goals. This student will present the project's findings at the ACS annual meeting, for which an abstract has already been submitted. Additionally, the graduate student is expected to defend her master's thesis, which focuses on enhancing lactase performance through immobilization, by the end of the year.

Impacts
What was accomplished under these goals? After moving to Virignia Tech, we had to set up the new lab, troubleshoot the instruments/machinary in the new space, and train graduate students to perform the material synthesis and enzyme immobilization. These activities, though essential, took considerable time to accomplish and consequently slowed down our progress on accomplishing the major objectives listed above. We are currently focusing on Obj. II. The major accomplishment are the followings: 1. We were able to complete the installation and troubleshooting of the initiated chemical vapor deposition reactor. 2. We accquired a Agilentt BioTek Synergy H1 plate reader for performing high throughput enzyme kinetics measurement in surface engineered 96-well plates and we developed a protocol for doing so. 3. We synthesized two types of polymer coatings, poly(glycidyl methacrylate) and poly(propargyl acrylate), for enzyme immobilization. The former coating results in randomly oriented enzymes whereas the latter specificly oriented enzymes. The contorl over orientation is achieved by introducing a non-natural p-azido-phenylalanine into lactase at site distal to the active site of the enzyme. This functional group "clicks" with the alkyne group in the propargyl acrylate to form a covalent linkage that allows precise control over orientation. We teamed up with Prof. Wei Sun in the department of biochemistry at Virginia Tech and havedeveloped the protocol for bioengineering of the lactase enzyme. Currently, we are optimizing the enzyme immobilization protocols for each polymer coating chemistry.

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: Alexandra Khlyustova, Yifan Cheng, and Rong Yang Macromolecules 2023 56 (16), 6492-6500 DOI: 10.1021/acs.macromol.3c01078
• Type: Journal Articles Status: Accepted Year Published: 2023 Citation: Mechano-Bactericidal Surfaces: Mechanisms, Nanofabrication, and Prospects for Food Applications https://doi.org/10.1146/annurev-food-060721-022330
• Type: Journal Articles Status: Published Year Published: 2023 Citation: All-dry free radical polymerization inside nanopores: Ion-milling-enabled coating thickness profiling revealed necking phenomena https://doi.org/10.1116/6.0001718
• Type: Journal Articles Status: Submitted Year Published: 2024 Citation: Orthogonal Nano-Engineering (ONE): Modulating Nanotopography and Surface Chemistry of Aluminum Oxide for Superior Antifouling and Enhanced Chemical Stability