Luo Jianquan, Meyer Anne S, Mateiu R V, Pinelo Manuel
Department of Chemical and Biochemical Engineering, Center for BioProcess Engineering, Building 229, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark.
Department of Chemical and Biochemical Engineering, Center for BioProcess Engineering, Building 229, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark.
N Biotechnol. 2015 May 25;32(3):319-27. doi: 10.1016/j.nbt.2015.02.006. Epub 2015 Feb 16.
Facile co-immobilization of enzymes is highly desirable for bioconversion methods involving multi-enzymatic cascade reactions. Here we show for the first time that three enzymes can be immobilized in flat-sheet polymeric membranes simultaneously or separately by simple pressure-driven filtration (i.e. by directing membrane fouling formation), without any addition of organic solvent. Such co-immobilization and sequential immobilization systems were examined for the production of methanol from CO2 with formate dehydrogenase (FDH), formaldehyde dehydrogenase (FaldDH) and alcohol dehydrogenase (ADH). Enzyme activity was fully retained by this non-covalent immobilization strategy. The two immobilization systems had similar catalytic efficiencies because the second reaction (formic acid→formaldehyde) catalyzed by FaldDH was found to be the cascade bottleneck (a threshold substrate concentration was required). Moreover, the trade-off between the mitigation of product inhibition and low substrate concentration for the adjacent enzymes probably made the co-immobilization meaningless. Thus, sequential immobilization could be used for multi-enzymatic cascade reactions, as it allowed the operational conditions for each single step to be optimized, not only during the enzyme immobilization but also during the reaction process, and the pressure-driven mass transfer (flow-through mode) could overcome the diffusion resistance between enzymes. This study not only offers a green and facile immobilization method for multi-enzymatic cascade systems, but also reveals the reaction bottleneck and provides possible solutions for the bioconversion of CO2 to methanol.
对于涉及多酶级联反应的生物转化方法而言，简便的酶共固定化是非常可取的。在此，我们首次展示了通过简单的压力驱动过滤（即引导膜污染形成），可在不添加任何有机溶剂的情况下，将三种酶同时或分别固定在平板聚合物膜中。我们考察了这种共固定化和顺序固定化系统利用甲酸脱氢酶（FDH）、甲醛脱氢酶（FaldDH）和乙醇脱氢酶（ADH）从二氧化碳生产甲醇的情况。通过这种非共价固定化策略，酶活性得以完全保留。这两种固定化系统具有相似的催化效率，因为发现由FaldDH催化的第二步反应（甲酸→甲醛）是级联反应的瓶颈（需要阈值底物浓度）。此外，相邻酶在减轻产物抑制和低底物浓度之间的权衡可能使得共固定化变得没有意义。因此，顺序固定化可用于多酶级联反应，因为它不仅允许在酶固定化过程中，而且在反应过程中对每个单一步骤的操作条件进行优化，并且压力驱动的传质（流通模式）可以克服酶之间的扩散阻力。本研究不仅为多酶级联系统提供了一种绿色且简便的固定化方法，还揭示了反应瓶颈，并为二氧化碳生物转化为甲醇提供了可能的解决方案。
