Heterogeneous Biocatalytic Reduction of 5-(Hydroxy)methyl Furfural Using two Co-immobilised Alcohol Dehydrogenases

Biocatalyst heterogenisation may enable robust processes that can be applied in biorefineries to selectively valorise highly functionalised platform chemicals. In this work, we co-immobilise two dehydrogenases and successfully apply them in the selective reduction of 5-hydroxymethylfurfural (HMF) to 2,5-bis(hydroxymethyl) furan (BHMF) with efficient in situ cofactor regeneration. First, we select the best enzyme candidates (an alcohol dehydrogenase from Escherichia coli together with a thermostable glucose dehydrogenase from Bacillus subtilis) and then screen a variety of carriers and chemistries to find the optimal individual immobilisation protocols for each dehydrogenase. As a result, methacrylate carriers (Purolite™) functionalised with either aldehydes or with epoxy and cobalt-chelate groups co-immobilise both enzymes in high yields with a sufficient activity recovery (>20%). These optimal heterogeneous biocatalysts enable the quantitative bio-reduction of HMF to BHMF with >99% selectivity in only fifteen minutes, exhibiting an outstanding reusability of >15 batch cycles with a total volumetric productivity of ∼5 g L−1 h−1 of BHMF. Preliminary experiments on a semipreparative scale with HMF loadings of 40 mM also reach high product conversions (86%). Overall, the judicious selection of enzymes, carriers and reaction conditions enables the design of robust biocatalysts that may contribute to paving the way to the valorisation of highly functionalised chemicals in biorefineries.

生物催化剂异相化可构建稳健工艺，应用于生物炼制厂中，实现高官能化平台化学品的选择性增值。本研究通过共固定两种脱氢酶，结合高效的原位辅酶因子再生策略，成功将5-羟甲基糠醛（5-hydroxymethylfurfural, HMF）选择性还原为2,5-双(羟甲基)呋喃（2,5-bis(hydroxymethyl) furan, BHMF）。首先，我们筛选得到最优酶种：大肠杆菌（Escherichia coli）来源的醇脱氢酶，以及枯草芽孢杆菌（Bacillus subtilis）来源的耐热葡萄糖脱氢酶；随后筛选多种载体与化学偶联策略，确定了两种脱氢酶各自的最优固定化方案。结果表明，经醛基、环氧基与钴螯合基团功能化的甲基丙烯酸酯载体（Purolite™）可高效共固定两种酶，且酶活回收率充足（>20%）。该最优异相生物催化剂可在15分钟内实现HMF向BHMF的定量生物还原，选择性>99%；同时展现出优异的重复使用性能，可完成超15个批次反应，BHMF的总体积产率约为5 g·L⁻¹·h⁻¹。在以40 mM HMF为投料量的半制备规模初步实验中，产物转化率同样可达86%。综上，通过审慎筛选酶种、载体与反应条件，可设计出性能稳健的生物催化剂，为生物炼制厂中高官能化化学品的增值利用铺平道路。