Ranjita Thapa Acharya
Ryan Stewart
Dominik Konkolewicz
Polymer-protein conjugation is a strong way to enhance enzyme stability and functionality, but preserving native structure and activity remains a challenge. In this study, lysozyme was conjugated with polymers of various chain lengths and chemistries. First, monomers were synthesized and conjugated to lysozyme. The success of conjugation was assessed, followed by an activity assay to test the effectiveness of the conjugate. Finally, the structural integrity of the secondary form of lysozyme was assessed following modification. Analysis demonstrated that the strength and stability of the enzymes depended on both the polymer and its chain length. Overall, this study demonstrates that both structure and chain length of polymers will influence preservation and enzymatic function.
Attachment of which polymers, at varying chain lengths, will result in the most preservation and function of enzymes?
Reversible Addition-Fragmentation Chain Transfer polymerization (RAFT) was used to synthesize various polymers at varying chain lengths prior to conjugation. Conjugation was performed using EDC/NHS coupling. After conjugation, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was used to assess the the degree of conjugation. Activity was assessed using a Micrococcus Lysodeikticus assay (Wright et al., 2022). Circular dichroism spectroscopy (CD) was used to assess the secondary structure of the enzyme after conjugation (Micsonai et al., 2022).
·DMAm: Neutral and highly hydrophilic; forms a soft polymer shell that minimally blocks the lysozyme active site, resulting in the highest activity retention.
·NIPAm: Increased hydrophobic interactions and partial chain collapse lead to local crowding around the enzyme, causing moderate activity loss that increases with DP.
·DMAPA: Cationic charge promotes strong electrostatic interactions with the enzyme and bacterial surface, which likely hinders proper substrate access and lowers activity.
·AMPSA: Strongly anionic and highly hydrated; forms a dense steric and electrostatic barrier that severely limits substrate access, giving the lowest activity retention
We found that both polymer chain length and composition affected both structure and function. The future direction of this bioconjugation project will focus on expanding beyond chain-length variation to develop sequence-controlled, multifunctional, and stimuli-responsive polymer–enzyme conjugates. Future work will investigate advanced conjugation strategies, multi-enzyme assemblies, and cross-linked network systems to further enhance structural stability, catalytic efficiency, and environmental resilience.
I would like to thank Dr. Rick Page for access to his laboratories, Dr. Annne Carroll for her help in spectroscopy, Carolyn Craig for her constant support in joining a research lab, and the LEADS Institute for providing countless opportunities for its students.
Through my research experience, I have developed skills in career and self development, communication, critical thinking, teamwork, and technology.
1. Melissa Lucius Daughtery et al. Bioconjugate Chemistry. 2017, 28(10): 2638–2645
2.Thaiesha A. Wright et al. Biomacromolecules. 2022, 23(10): 4097-4109
3.Andras Micsonai et al. Nucleic Acids Res. 2022, 50(W1):W90-W98