Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada.
Org Biomol Chem. 2020 Mar 18;18(11):2076-2084. doi: 10.1039/c9ob02757b.
An Auxiliary Activity Family 5 (AA5) copper-radical alcohol oxidase (AlcOx) with promiscuous activity towards simple alkyl and aromatic alcohols was evaluated using real-time reaction progress monitoring. Reaction kinetics using variable time normalization analysis (VTNA) were determined from reaction progress curves. By this approach, a detailed view of the entire reaction time course under various conditions was obtained and used to identify parameters that will inform further process optimization development. Optimal activity was found impacted by several factors, including reaction pH, oxygen saturation, and the source of a co-oxidant, either HRP or a chemical alternative, potassium ferricyanide. Analysis of reaction progress curves demonstrated that reaction stalling occurred as a result of oxygen depletion and from a loss of enzyme activity over time. The cooperativity between AlcOx, horseradish peroxidase (HRP), and catalase that result in enhanced reactivity was explored, with reaction pH being identified as a key factor for optimal activity. The results show that a process with HRP is more robust than with potassium ferricyanide, but that both oxidants likely activate AlcOx by a similar mechanism. The phenomenon of product inhibition was investigated for representative reactants, revealing that reaction inhibition was more significant for butyraldehyde than for benzaldehyde. Our analysis suggests that this is linked to the greater proportion in which butyraldehyde exists in the hydrated form.
一种具有杂化活性的辅助活性家族 5（AA5）铜自由基醇氧化酶（AlcOx），可对简单的烷基和芳族醇进行催化，我们使用实时反应监测对其进行了评估。通过可变时间归一化分析（VTNA）从反应进度曲线确定了反应动力学。通过这种方法，我们可以获得各种条件下整个反应时间过程的详细视图，并用于确定将为进一步的过程优化开发提供信息的参数。最佳活性受到多种因素的影响，包括反应 pH 值、氧气饱和度以及共氧化剂（HRP 或化学替代物铁氰化钾）的来源。反应进度曲线的分析表明，由于氧气耗尽和酶活性随时间的丧失，导致反应停滞。我们探索了醇氧化酶、辣根过氧化物酶（HRP）和过氧化氢酶之间的协同作用，这导致了增强的反应性，并且反应 pH 值被确定为最佳活性的关键因素。结果表明，具有 HRP 的过程比具有铁氰化钾的过程更稳健，但这两种氧化剂可能通过类似的机制激活 AlcOx。我们还研究了代表性反应物的产物抑制现象，结果表明，对于丁醛而言，反应抑制更为明显，而对于苯甲醛则不然。我们的分析表明，这与丁醛以水合形式存在的比例更大有关。
