Centro de Investigaciones Biológicas "Margarita Salas" (CIB), CSIC, Ramiro de Maeztu 9, 28040, Madrid, Spain.
Microb Cell Fact. 2021 Sep 9;20(1):180. doi: 10.1186/s12934-021-01669-1.
2,5-Furandicarboxylic acid (FDCA) is a precursor for green plastics due to its structural similarity to terephthalic acid, a common precursor of oil-derived polymers, and its potential production from sugars obtained from plant biomass. Hydroxymethylfurfural oxidase (HMFO) has been reported as a promising biocatalyst for FDCA production since it can convert bio-based 5-hydroxymethylfurfural (HMF) into FDCA building block. This three-step oxidation reaction occurs through the diformylfuran and 2,5-formylfurancarboxylic acid (FFCA) intermediates. Several efforts have been made for the development of HMFO variants that increase FDCA yields by improving their activities over the reaction intermediates. However, there is still limited insight into how operational conditions can influence these enzymatic reactions. The setup of optimal reaction conditions would enable to understand potential problems hampering the effective industrial production of this bioplastic precursor using HMFO as biocatalyst.
In this work, several parameters affecting the performance of Methylovorus sp HMFO oxidizing HMF have been analyzed for the wild-type enzyme, and its V367R and W466F single variants, V367R/W466F double variant, and I73V/H74Y/G356H/V367R/T414K/A419Y/A435E/W466F (8BxHMFO) octuple variant. Our results show how the oxidation of HMF by HMFO enzymes is highly influenced by pH, with different optimal pH values for the different improved variants. Moreover, the enzymes are not stable at high hydrogen peroxide concentrations and their activity is inhibited by the FFCA intermediate in a pH-dependent way. These limitations can be efficiently overcome with the addition of catalase to the reaction medium, which removes the hydrogen peroxide formed during the oxidations, and the controlled dosage of the substrate to limit the amount of FFCA accumulated in the reaction. The different behavior of wild-type HMFO and its variants against pH, hydrogen peroxide and FFCA highlights the importance of considering each variant as an individual enzyme with its own operational conditions for an eventual industrial FDCA production.
This work provides information of those parameters that condition a high production of FDCA by HMFO. Unraveling these factors allowed to increase the FDCA yields by using the most stable enzymes at their optimal pH for HMF oxidation, removing the peroxide with catalase, and avoiding FFCA accumulation by controlling substrate and/or enzyme concentration. These above findings will be useful when planning a future scale-up of these conversions and will provide new viewpoints for the design of HMFO variants that render a more effective performance during HMF conversion into FDCA.
2,5-呋喃二甲酸（FDCA）是一种绿色塑料的前体，因为它的结构与对苯二甲酸相似，对苯二甲酸是石油衍生聚合物的常见前体，而且它有可能从植物生物质获得的糖中生产。羟甲基糠醛氧化酶（HMFO）已被报道为生产 FDCA 的一种有前途的生物催化剂，因为它可以将生物基 5-羟甲基糠醛（HMF）转化为 FDCA 构建块。这个三步氧化反应通过二糠醛和 2,5-糠醛羧酸（FFCA）中间体发生。已经做出了一些努力来开发 HMFO 变体，通过提高它们对反应中间体的活性来提高 FDCA 的产量。然而，对于操作条件如何影响这些酶反应，我们仍然知之甚少。优化反应条件的设置将使我们能够了解使用 HMFO 作为生物催化剂有效工业化生产这种生物塑料前体时可能存在的问题。
在这项工作中，分析了影响甲基氧化单胞菌 HMFO 氧化 HMF 性能的几个参数，包括野生型酶及其 V367R 和 W466F 单变体、V367R/W466F 双变体、I73V/H74Y/G356H/V367R/T414K/A419Y/A435E/W466F（8BxHMFO）八重变体。我们的结果表明，HMFO 酶对 HMF 的氧化受 pH 值的影响很大，不同的改良变体有不同的最佳 pH 值。此外，这些酶在高过氧化氢浓度下不稳定，并且它们的活性以 pH 依赖的方式被 FFCA 中间体抑制。通过向反应介质中添加过氧化氢酶，可以有效地克服这些限制，过氧化氢酶可以去除氧化过程中形成的过氧化氢，并通过控制底物的用量来限制反应中积累的 FFCA 量。野生型 HMFO 及其变体对 pH 值、过氧化氢和 FFCA 的不同反应突出了考虑每个变体作为具有其自身操作条件的单个酶的重要性，以便最终进行工业 FDCA 生产。
这项工作提供了有关 HMFO 高产量生产 FDCA 的那些参数的信息。揭示这些因素可以通过在 HMFO 氧化的最佳 pH 值下使用最稳定的酶、用过氧化氢酶去除过氧化物以及通过控制底物和/或酶浓度避免 FFCA 积累来提高 FDCA 的产率。当计划对这些转化进行未来的放大时，这些发现将是有用的，并且将为 HMFO 变体的设计提供新的观点，从而在 HMF 转化为 FDCA 过程中实现更有效的性能。
