Source: VIRGINIA POLYTECHNIC INSTITUTE submitted to
IMPROVING STABILITY AND ACTIVITY OF COVALENTLY IMMOBILIZED ENZYMES VIA NANOENGINEERED MICROENVIRONMENTS FOR FOOD WASTE VALORIZATION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1030481
Grant No.
2021-67034-39810
Cumulative Award Amt.
$77,003.78
Proposal No.
2022-11939
Multistate No.
(N/A)
Project Start Date
Jan 15, 2023
Project End Date
Jan 14, 2025
Grant Year
2023
Program Code
[A7201]- AFRI Post Doctoral Fellowships
Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061
Performing Department
(N/A)
Non Technical Summary
The rapid growth of the Greek-style yogurt market, from 1-2% of the US yogurt market in 2004 to nearly 40% in 2015, hascreated tremendous volumes of lactose-rich acid whey waste streams annually - a huge untapped goldmine for wastevalorization. For example, the lactose molecules in these waste streams can be converted to value-added products such asgalacto-oligosaccharides (GOS; prebiotic) and tagatose (non-nutritive sweeteners with a high relative sweetness value), viaenzymatic reactions. Nevertheless, three roadblocks keep such enzyme-based waste valorization approaches from becomingeconomically viable for commercial use: (i) highly acidic environment ofGreek-style yogurt acid whey (pH ~ 4) impairs enzymeactivity and stability, (ii) difficulty in reclaiming and reusing the enzymes, and (iii) amismatch between the desired temperature for processing and enzyme performance.Previous research indicated that covalent immobilization of enzymes onto solid supports enhances enzyme performance by creating more favorable local microenvironments and enabling repeated use of the same batch of enzymes. Here, we propose to develop a scalable nanoengineering platform for orthogonally controlling the chemical and physical aspects of the enzyme microenvironments with nanometric precision. We will use this platform to (i) generate fundamental insights on how spatial nano-confinement and chemical microenvironment individually and collectively influence the performance of immobilized enzymes, and (ii) design and fabricate highly efficient and sustainable enzymatic membrane reactors for acid whey valorization and beyond. Broadly speaking, this project hopes to provide scientific discoveries and technical know-hows for strengthening the applicability of enzymatic "upcycling" of waste streams - especially those with harsh conditions. The outcome of this project may improve the sustainability of Greek-style yogurt production and help reduce the environmental burden of dairy foodwastestreams, but also demonstrate a novel approach to lowering the cost of various enzyme-based solutions.
Animal Health Component
40%
Research Effort Categories
Basic
40%
Applied
40%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
4033470202040%
5013470200060%
Knowledge Area
403 - Waste Disposal, Recycling, and Reuse; 501 - New and Improved Food Processing Technologies;

Subject Of Investigation
3470 - Other dairy cattle products;

Field Of Science
2020 - Engineering; 2000 - Chemistry;
Goals / Objectives
Obj. I. Investigate the effect of nano-confinement imposed by nanopores on the performance of immobilized lactase.Obj. II. Understand how the physicochemical properties of polymeric nanolayers affect the performance of immobilized lactase.Obj. III. Optimize the performance of lactase via nanoengineered spatial and chemical microenvironments for efficient and sustainable biotransformation of lactose in acid whey.
Project Methods
In the proposed project, we plan to develop enzymatic membrane reactors (EMRs) that can efficiently and sustainably valorizechallenging food processing waste streams that are outside of the "comfort zones" of the enzymes, such as pH, temperature,polarity of the liquid. As a first step, this project will target acid whey from Greek yogurt due to its immense volume, highpotential for lactose valorization, and challenging acidity for conventional lactase-based biocatalysis.The solution to this challenge lies in a deep understanding of how interplay between spatial nano-confinement and chemicalmicroenvironments individually and collectively affect the activity and stability of covalently immobilized enzymes, with a focuson the pH and temperature tolerance of the enzymes. Recent progress in nanoengineering has offered unprecedentedopportunities to orthogonally control both aspects of the interfacial microenvironment with nanometric precision: nanoporousanodic aluminum oxide (AAO) for tailoring the spatial confinement surrounding the enzymes without altering surface chemistry,and initiated chemical vapor deposition (iCVD) for introducing desired chemical microenvironments and enzyme immobilizationsites in the form of robust polymeric nanocoatings without altering surface topography. This orthogonal nanoengineeringplatform will shed light on the decoupled role of spatial nano-confinement and chemical microenvironment inmaintaining/promoting activity and stability of immobilized enzymes, especially in the challenging conditions of food processingwaste streams.The outputs of this proposed project will be evaluated in terms of (i) fundamental scientific knowledges generated on the effectof enzyme microenvironment on the enzymatic stability and activity, (ii) design rules derived from these knowledges for enzymebiointerfaces, and (iii) performance of the EMR engineered based on the design rules.

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