School of Chemistry, Cardiff University Main Building, Park Place, Cardiff, CF10 3AT, UK.
Cardiff Catalysis Institute, Cardiff University Main Building, Park Place, Cardiff, CF10 3AT, UK.
Sci Rep. 2023 Jan 23;13(1):1267. doi: 10.1038/s41598-023-27780-4.
Agitation is a commonly encountered stress for enzymes during all stages of production and application, but investigations that aim to improve their tolerance using topological engineering have yet to be reported. Here, the plastic-degrading enzyme IsPETase was cyclized in a range of topologies including a cyclic monomer, cyclic dimer and catenane using SpyTag/SpyCatcher technologies, and their tolerance towards different stresses including mechanical agitation was investigated. The cyclic dimer and catenane topologies were less susceptible to agitation-induced inactivation resulting in enhancement of polyethylene terephthalate (PET) degradation. While contrary to conventional belief, cyclic topologies did not improve tolerance of IsPETase towards heat or proteolytic treatment, the close proximity of active sites in the dimeric and catenane variants was found to enhance PET conversion into small soluble products. Together, these findings illustrate that it is worthwhile to explore the topology engineering of enzymes used in heterogeneous catalysis as it improves factors that are often overlooked in homogeneous catalysis studies.
在生产和应用的各个阶段，酶都会经常遇到应激，即搅动，但目前还没有利用拓扑工程来提高其耐受性的研究。在这里，使用 SpyTag/SpyCatcher 技术，将可降解塑料的酶 IsPETase 环化，形成多种拓扑结构，包括环状单体、环状二聚体和索烃，并研究它们对包括机械搅拌在内的不同应激的耐受性。环状二聚体和索烃结构不易受到搅动引起的失活影响，从而增强了对聚对苯二甲酸乙二醇酯（PET）的降解。与传统观点相反，环状拓扑结构并没有提高 IsPETase 对热或蛋白水解处理的耐受性，但发现二聚体和索烃变体中活性位点的紧密接近会增强 PET 转化为小的可溶性产物。总之，这些发现表明，探索用于多相催化的酶的拓扑工程是值得的，因为它可以改善在均相催化研究中经常被忽视的因素。
