Institute of Biochemistry, Heinrich-Heine University Düsseldorf, Universitätsstraße 1, 40225, Düsseldorf, Germany.
Appl Microbiol Biotechnol. 2021 Nov;105(21-22):8313-8327. doi: 10.1007/s00253-021-11557-8. Epub 2021 Oct 13.
The development of enzymatic processes for the environmentally friendly production of 2,5-furandicarboxylic acid (FDCA), a renewable precursor for bioplastics, from 5-hydroxymethylfurfural (HMF) has gained increasing attention over the last years. Aryl-alcohol oxidases (AAOs) catalyze the oxidation of HMF to 5-formyl-2-furancarboxylic acid (FFCA) through 2,5-diformylfuran (DFF) and have thus been applied in enzymatic reaction cascades for the production of FDCA. AAOs are flavoproteins that oxidize a broad range of benzylic and aliphatic allylic primary alcohols to the corresponding aldehydes, and in some cases further to acids, while reducing molecular oxygen to hydrogen peroxide. These promising biocatalysts can also be used for the synthesis of flavors, fragrances, and chemical building blocks, but their industrial applicability suffers from low production yield in natural and heterologous hosts. Here we report on heterologous expression of a new aryl-alcohol oxidase, MaAAO, from Moesziomyces antarcticus at high yields in the methylotrophic yeast Pichia pastoris (recently reclassified as Komagataella phaffii). Fed-batch fermentation of recombinant P. pastoris yielded around 750 mg of active enzyme per liter of culture. Purified MaAAO was highly stable at pH 2-9 and exhibited high thermal stability with almost 95% residual activity after 48 h at 57.5 °C. MaAAO accepts a broad range of benzylic primary alcohols, aliphatic allylic alcohols, and furan derivatives like HMF as substrates and some oxidation products thereof like piperonal or perillaldehyde serve as building blocks for pharmaceuticals or show health-promoting effects. Besides this, MaAAO oxidized 5-hydroxymethyl-2-furancarboxylic acid (HMFCA) to FFCA, which has not been shown for any other AAO so far. Combining MaAAO with an unspecific peroxygenase oxidizing HMFCA to FFCA in one pot resulted in complete conversion of HMF to FDCA within 144 h. MaAAO is thus a promising biocatalyst for the production of precursors for bioplastics and bioactive compounds. KEY POINTS: • MaAAO from M. antarcticus was expressed in P. pastoris at 750 mg/l. • MaAAO oxidized 5-hydroxymethyl-2-furancarboxylic acid (HMFCA). • Complete conversion of HMF to 2,5-furandicarboxylic acid by combining MaAAO and UPO.
近年来，人们越来越关注利用酶法工艺从 5-羟甲基糠醛（HMF）环保生产 2,5-呋喃二甲酸（FDCA），这是一种可再生的生物塑料前体。芳醇氧化酶（AAO）通过 2,5-二糠醛（DFF）催化 HMF 氧化生成 5-糠醛-2-羧酸（FFCA），因此已应用于 FDCA 的酶促反应级联中。AAO 是黄素蛋白，可氧化广泛的苄基和脂肪族烯丙醇，生成相应的醛，在某些情况下进一步氧化成酸，同时将分子氧还原为过氧化氢。这些有前途的生物催化剂还可用于合成香料、香精和化学结构单元，但由于在天然和异源宿主中的产量低，其工业适用性受到限制。在这里，我们报告了来自南极 Moesziomyces antarcticus 的新型芳醇氧化酶 MaAAO 的异源表达，在甲醇营养酵母巴斯德毕赤酵母（最近重新分类为 Komagataella phaffii）中以高产率表达。重组巴斯德毕赤酵母的分批发酵每升培养物可产生约 750mg 活性酶。纯化的 MaAAO 在 pH 2-9 下高度稳定，在 57.5°C 下孵育 48 小时后，其热稳定性几乎保持 95%，残余活性接近 95%。MaAAO 可接受广泛的苄基伯醇、脂肪族烯丙醇和糠醛衍生物（如 HMF）作为底物，其一些氧化产物（如胡椒醛或香草醛）可用作药物的构建模块或具有促进健康的作用。此外，MaAAO 将 5-羟甲基-2-糠酸（HMFCA）氧化为 FFCA，这是迄今为止任何其他 AAO 都没有显示的。将 MaAAO 与一种非特异性过氧化物酶结合，将 HMFCA 氧化为 FFCA，一锅法反应可在 144 小时内将 HMF 完全转化为 FDCA。因此，MaAAO 是生产生物塑料前体和生物活性化合物的有前途的生物催化剂。关键点： • MaAAO 来自 M.antarcticus，在 P. pastoris 中表达量为 750mg/l。 • MaAAO 氧化 5-羟甲基-2-糠酸（HMFCA）。 • 通过结合 MaAAO 和 UPO 将 HMF 完全转化为 2,5-呋喃二甲酸。
