Department of Chemistry, Illinois State University, Normal, Illinois 61790, United States.
Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States.
Langmuir. 2024 Jul 9;40(27):13957-13967. doi: 10.1021/acs.langmuir.4c01180. Epub 2024 Jun 26.
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.
酶介导反应的特异性和效率具有积极影响许多生物技术的潜力；然而，许多酶很容易降解。最近已经探索了将酶固定在固体载体上以提高酶稳定性。本研究旨在深入了解并促进酶吸附到金纳米粒子（AuNP）上，通过安装改变蛋白质化学性质的硫醇官能团形成稳定的生物缀合物。具体来说，模型酶辣根过氧化物酶（HRP）通过 Traut 试剂进行硫醇化，以提高生物缀合物的稳健性和酶活性。本研究比较了 HRP 和其硫醇化类似物（THRP），以推断硫醇化和 AuNP 固定化对酶活性和稳定性的影响。通过紫外可见分光光度法、圆二色性、Zeta 电位和酶-底物动力学测定分析 HRP、THRP 及其相应的生物缀合物 HRP-AuNP 和 THRP-AuNP。我们的数据表明，THRP 在 AuNP 上的吸附量比 HRP 高 5 倍，这转化为 THRP-AuNP 生物缀合物活性增加了 5 倍。与溶液中的天然酶相比，硫醇化和固定化的 HRP 在较高温度（50°C）和储存时间（1 个月）下表现出显著的稳定性提高。此外，HRP、THRP 及其生物缀合物与胰蛋白酶孵育以评估对蛋白水解消化的敏感性。我们的结果表明，THRP-AuNP 生物缀合物在与胰蛋白酶孵育 18 小时后保持完全的酶活性，而游离 HRP、游离 THRP 和 HRP-AuNP 缀合物在胰蛋白酶处理后失活。这些结果突出了蛋白质修饰和固定化的潜力，可以大大延长酶的保质期，抵抗蛋白酶的消化，并增强生物功能，以实现酶促生物技术。
