Self-Immobilizing Fusion Enzymes for Compartmentalized Biocatalysis
Self-Immobilizing Fusion Enzymes for Compartmentalized Biocatalysis
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DOI：
10.1021/acscatal.7b02230
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发表时间：
2017-11-01
期刊：
影响因子：
12.9
通讯作者：
Niemeyer, Christof M.
中科院分区：
文献类型：
--
作者：
Peschke, Theo;Skoupi, Marc;Niemeyer, Christof M.
微流控酶级联的建立是研究和开发的热门领域，目前由于缺乏温和有效固定分离酶的方法而受到阻碍。我们在这里描述了使用自固定化融合酶的微流控填充床反应器的模块化配置。具体地，将三种不同的酶（R）-选择性醇脱氢酶LbADH、（S）-选择性甲基乙二醛还原酶Gre 2 p和NADP（H）再生酶葡萄糖1-脱氢酶GDH）与基于链霉亲和素结合肽、Spy和Halo的标签进行遗传融合，以使它们能够特异性和定向固定在涂覆有互补受体的磁性微珠上。将酶修饰的珠加载到四通道微流控芯片中以产生具有前手性C-s-对称底物S-硝基壬烷-2，8-二酮（NDK）的（R）-或（S）-选择性还原能力的隔室。通过手性HPLC对异构羟基酮和二醇产物的分析用于定量表征配置有不同量的酶的反应器的性能。长达14天的长操作时间表明稳定的酶固定化和反应器的一般鲁棒性。甚至更重要的是，通过微调隔室大小和负载，可以控制总体产物分布以选择性地产生具有几乎定量转化率（>95%）和优异的立体选择性（d.r. > 99：1）在连续流动过程中。我们相信，我们的概念将可扩展到各种其他生物催化或化学-酶级联反应。
The establishment of microfluidic enzyme cascades is a topical field of research and development, which is currently hampered by the lack of methodologies for mild and efficient immobilization of isolated enzymes. We here describe the use of self immobilizing fusion enzymes for the modular configuration of microfluidic packed-bed reactors. Specifically, three different enzymes, the (R)-selective alcohol dehydrogenase LbADH, the (S)-selective methylglyoxal reductase Gre2p and the NADP(H) regeneration enzyme glucose 1-dehydrogenase GDH, were genetically fused with streptavidin binding peptide, Spy and Halo-based tags, to enable their specific and directional immobilization on magnetic microbeads coated with complementary receptors. The enzyme-modified beads were loaded in four-channel microfluidic chips to create compartments that have the capability for either (R)- or (S) -selective reduction of the prochiral C-s-symmetrical substrate S-nitrononane-2,8-dione (NDK). Analysis of the isomeric hydroxyketone and diol products by chiral HPLC was used to quantitatively characterize the performance of reactors configured with different amounts of the enzymes. Long operating times of up to 14 days indicated stable enzyme immobilization and the general robustness of the reactor. Even more important, by fine-tuning of compartment size and loading, the overall product distribution could be controlled to selectively produce a single meso diol with nearly quantitative conversion (>95%) and excellent stereoselectivity (d.r. > 99:1) in a continuous flow process. We believe that our concept will be expandable to a variety of other biocatalytic or chemo-enzymatic cascade reactions.