Various 4-alkylphenols can be easily obtained through reductive catalytic fractionation of lignocellulosic biomass. Selective dehydrogenation of 4-n-propylguaiacol results in the formation of isoeugenol, a valuable flavor and fragrance molecule and versatile precursor compound. Here we present the engineering of a bacterial eugenol oxidase to catalyze this reaction. Five mutations, identified from computational predictions, are first introduced to render the enzyme more thermostable. Other mutations are then added and analyzed to enhance chemoselectivity and activity. Structural insight demonstrates that the slow catalytic activity of an otherwise promising enzyme variant is due the formation of a slowly-decaying covalent substrate-flavin cofactor adduct that can be remedied by targeted residue changes. The final engineered variant comprises eight mutations, is thermostable, displays good activity and acts as a highly chemoselective 4-n-propylguaiacol oxidase. We lastly use our engineered biocatalyst in an illustrative preparative reaction at gram-scale. Our findings show that a natural enzyme can be redesigned into a tailored biocatalyst capable of valorizing lignin-based monophenols.

通过木质纤维素生物质的还原催化分馏，可以很容易地得到各种4-烷基酚。4 -n选择性脱氢-丙基愈创木酚导致异丁香酚的形成，异丁香酚是一种有价值的香精分子和多功能前体化合物。在这里，我们介绍了细菌丁香酚氧化酶的工程以催化该反应。首先引入从计算预测中确定的五个突变以使酶更耐热。然后添加并分析其他突变以增强化学选择性和活性。结构洞察表明，原本有前途的酶变体的缓慢催化活性是由于形成了缓慢衰减的共价底物-黄素辅助因子加合物，可以通过靶向残基变化来补救。最终的工程变体包含八个突变，具有热稳定性，显示出良好的活性并作为高度化学选择性的 4 -n-丙基愈创木酚氧化酶。最后，我们在克级的说明性制备反应中使用了我们的工程生物催化剂。我们的研究结果表明，一种天然酶可以重新设计成一种定制的生物催化剂，能够稳定基于木质素的单酚。