Biomass valorization has been suggested as a sustainable alternative to petroleum-based energy and commodities. In this context, the copper radical oxidases (CROs) from Auxiliary Activity Family 5/Subfamily 2 (AA5_2) are attractive biocatalysts for the selective oxidation of primary alcohols to aldehydes. Originally defined by the archetypal galactose 6-oxidase from Fusarium graminearum, fungal AA5_2 members have recently been shown to comprise a wide range of specificities for aromatic, aliphatic and furan-based alcohols. This suggests a broader substrate scope of native CROs for applications. However, only 10% of the annotated AA5_2 members have been characterized to date. Here, we define two homologues from the filamentous fungi Fusarium graminearum and F. oxysporum as predominant aryl alcohol oxidases (AAOs) through recombinant production in Pichia pastoris, detailed kinetic characterization, and enzyme product analysis. Despite possessing generally similar active-site architectures to the archetypal FgrGalOx, FgrAAO and FoxAAO have weak activity on carbohydrates, but instead efficiently oxidize specific aryl alcohols. Notably, both FgrAAO and FoxAAO oxidize hydroxymethyl furfural (HMF) directly to 5-formyl-2-furoic acid (FFCA), and desymmetrize the bioproduct glycerol to the uncommon L-isomer of glyceraldehyde. This work expands understanding of the catalytic diversity of CRO from AA5_2 to include unique representatives from Fusarium species that depart from the well-known galactose 6-oxidase activity of this family. Detailed enzymological analysis highlights the potential biotechnological applications of these orthologs in the production of renewable plastic polymer precursors and other chemicals.

中文翻译：

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两种对芳醇具有高活性的镰刀菌铜自由基氧化酶

生物质增值已被建议作为石油基能源和商品的可持续替代品。在这种情况下，来自辅助活性家族 5/亚家族 2 (AA5_2) 的铜自由基氧化酶 (CRO) 是用于将伯醇选择性氧化为醛的有吸引力的生物催化剂。真菌 AA5_2 成员最初由禾谷镰刀菌的原型半乳糖 6-氧化酶定义，最近已被证明对芳香族、脂肪族和呋喃基醇具有广泛的特异性。这表明用于应用的天然 CRO 的底物范围更广。然而，迄今为止，只有 10% 的带注释的 AA5_2 成员已被表征。在这里，我们从丝状真菌 Fusarium graminearum 和 F. 通过在毕赤酵母中重组生产、详细的动力学表征和酶产物分析，将尖孢菌作为主要的芳醇氧化酶 (AAO)。尽管具有与原型 FgrGalOx 大致相似的活性位点结构，但 FgrAAO 和 FoxAAO 对碳水化合物的活性较弱，但可以有效氧化特定的芳醇。值得注意的是，FgrAAO 和 FoxAAO 都将羟甲基糠醛 (HMF) 直接氧化为 5-甲酰基-2-糠酸 (FFCA)，并将生物产品甘油去对称化为不常见的甘油醛 L-异构体。这项工作扩展了对来自 AA5_2 的 CRO 催化多样性的理解，以包括与该家族众所周知的半乳糖 6-氧化酶活性不同的镰刀菌属物种的独特代表。