Institute of Technical Chemistry, Leibniz University Hannover, Callinstr. 5, 30167, Hannover, Germany.
Biocatalysis and Bioprocessing Group, Department of Biological and Chemical Engineering, Aarhus University, Gustav Wieds Vej 10, 8000, Aarhus, Denmark.
Chembiochem. 2023 Apr 17;24(8):e202200794. doi: 10.1002/cbic.202200794. Epub 2023 Mar 22.
Baeyer-Villiger monooxygenases (BVMOs) are attractive for selectively oxidizing various ketones using oxygen into valuable esters and lactones. However, the application of BVMOs is restrained by cofactor dependency and enzyme instability combined with water-related downsides such as low substrate loading, low oxygen capacity, and water-induced side reactions. Herein, we described a redox-neutral linear cascade with in-situ cofactor regeneration catalyzed by fused alcohol dehydrogenase and cyclohexanone monooxygenase in aqueous and microaqueous organic media. The cascade conditions have been optimized regarding substrate concentrations as well as the amounts of enzymes and cofactors with the Design of Experiments (DoE). The carrier-free immobilization technique, crosslinked enzyme aggregates (CLEAs), was applied to fusion enzymes. The resultant fusion CLEAs were proven to function in microaqueous organic systems, in which the enzyme ratios, water contents (0.5-5 vol. %), and stability have been systematically studied. The fusion CLEAs showed promising operational (up to 5 cycles) and storage stability.
Baeyer-Villiger 单加氧酶（BVMOs）是一类具有吸引力的酶，可以利用氧气将各种酮选择性氧化为有价值的酯和内酯。然而，BVMOs 的应用受到辅因子依赖性和酶不稳定性的限制，同时还存在与水相关的缺点，如低底物负载、低氧气容量以及水诱导的副反应。在此，我们描述了一种在水相和微水有机介质中，由融合的醇脱氢酶和环己酮单加氧酶催化的氧化还原中性线性级联反应，同时实现了原位辅因子再生。通过实验设计（DoE）对底物浓度以及酶和辅因子的用量进行了级联条件的优化。无载体固定化技术——交联酶聚集体（CLEAs）被应用于融合酶。结果表明，融合 CLEAs 可以在微水有机体系中发挥作用，并且系统地研究了酶比、含水量（0.5-5 体积%）和稳定性等因素。融合 CLEAs 表现出良好的操作稳定性（可进行 5 次循环）和储存稳定性。
