Document Type

Article

Publication Date

2024

Publication Title

Langmuir

Abstract

The specificity and efficiency of enzyme-mediated reactions have the potential to positively impact many biotechnologies; however, many enzymes are easily degraded. Immobilization on a solid support has recently been explored to improve enzyme stability. This study aims to gain insights and facilitate enzyme adsorption onto gold nanoparticles (AuNPs) to form a stable bioconjugate through the installation of thiol functional groups that alter the protein chemistry. In specific, the model enzyme, horseradish peroxidase (HRP), is thiolated via Traut’s reagent to increase the robustness and enzymatic activity of the bioconjugate. This study compares HRP and its thiolated analog (THRP) to deduce the impact of thiolation and AuNP-immobilization on the enzyme activity and stability. HRP, THRP, and their corresponding bioconjugates, HRP-AuNP and THRP-AuNP, were analyzed via UV–vis spectrophotometry, circular dichroism, zeta potential, and enzyme–substrate kinetics assays. Our data show a 5-fold greater adsorption for THRP on the AuNP, in comparison to HRP, that translated to a 5-fold increase in the THRP-AuNP bioconjugate activity. The thiolated and immobilized HRP exhibited a substantial improvement in stability at elevated temperatures (50 °C) and storage times (1 month) relative to the native enzyme in solution. Moreover, HRP, THRP, and their bioconjugates were incubated with trypsin to assess the susceptibility to proteolytic digestion. Our results demonstrate that THRP-AuNP bioconjugates maintain full enzymatic activity after 18 h of incubation with trypsin, whereas free HRP, free THRP, and HRP-AuNP conjugates are rendered inactive by trypsin treatment. These results highlight the potential for protein modification and immobilization to substantially extend enzyme shelf life, resist protease digestion, and enhance biological function to realize enzyme-enabled biotechnologies.

Funding Source

This work was funded by the National Science Foundation through the Macromolecular, Supramolecular and Nanochemistry Program, Award # CHE-2203740. S.O. acknowledges support from the National Science Foundation under Award # DGE-1735252. The authors are grateful to Jonathan V. Sweedler for providing access to the MALDI-TOF MS instrument that was partially funded by P30DA018310 from the National Institute on Drug Abuse. Partial support was also provided by Illinois State University. This article was published Open Access thanks to a transformative agreement between Milner Library and ACS.

DOI

10.1021/acs.langmuir.4c01180

Comments

First published in Langmuir (2024). https://doi.org/10.1021/acs.langmuir.4c01180.

This open access article is published under a CC-BY 4.0 license.

Additional Files
Driskell_supporting_information.pdf (333 kB)
supporting information
Download

Included in

Chemistry Commons

Share

COinS