<br>结合蛋白质相分离和生物正交连接以共固定酶以进行级联生物催化,Small

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结合蛋白质相分离和生物正交连接以共固定酶以进行级联生物催化
Small ( IF 13.0 ) Pub Date : 2024-08-12 , DOI: 10.1002/smll.202404018
Shujiao Wu ₁ , Lingling Luo ₁ , Houtian Luo ₁ , Li Qiao ₂ , Haomin Chen ₂ , Mijun Li ₁ , Xiaolin Pei ₂ , Tian Xie ₁ , Anming Wang ₂ , Roger A Sheldon _{3,

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Affiliation

用于载体生物催化的多种酶的设计和有序共固定化具有挑战性。在这里，蛋白质相分离和生物正交连接的组合用于生成含有共固定化酶的沸石咪唑框架（ZIF-8）。Zn²⁺ 离子诱导最小蛋白质模块的聚集，例如 6-His 标签、富含脯氨酸的基序（PRM）和 SRC 同源 3 （SH3）结构域，并允许偶联的醛酮还原酶（AKR）和醇脱氢酶（ADH）在低浓度下进行相分离。这是通过将 SpyCatcher 和 PRM-SH3-6His 肽片段分别融合到 AKR 的 C 端和 N 末端，以及将 SpyTag 融合到 ADH 来实现的。添加 2-甲基咪唑导致液滴形成，并能够原位空间包埋重组 AKR 和 ADH，以产生封装在 ZIF-8 （AAE@ZIF）中的级联生物解析系统。在合成（S）-1-（2-氯苯基）乙醇时，经过 6 次循环，收率仍可达 91%，每个循环的对映体过量（ee）值为 99.99%。然而，当使用传统封装的 AKR 和 ADH 在 ZIF-8 中时，产率只能达到 72.9%。因此，这项工作表明，蛋白质相分离和生物正交连接的结合能够在 ZIF-8 中原位创建稳定且空间组织良好的双酶系统，并增强通道效应。

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Combining Protein Phase Separation and Bio-orthogonal Linking to Coimmobilize Enzymes for Cascade Biocatalysis

The designed and ordered co-immobilization of multiple enzymes for vectorial biocatalysis is challenging. Here, a combination of protein phase separation and bioorthogonal linking is used to generate a zeolitic imidazole framework (ZIF-8) containing co-immobilized enzymes. Zn²⁺ ions induce the clustering of minimal protein modules, such as 6-His tag, proline-rich motif (PRM) and SRC homology 3 (SH3) domains, and allow for phase separation of the coupled aldoketoreductase (AKR) and alcohol dehydrogenase (ADH) at low concentrations. This is achieved by fusing SpyCatcher and PRM-SH3-6His peptide fragments to the C and N termini of AKR, respectively, and the SpyTag to ADH. Addition of 2-methylimidazole results in droplet formation and enables in situ spatial embedding the recombinant AKR and ADH to generate the cascade biocalysis system encapsulated in ZIF-8 (AAE@ZIF). In synthesizing (S)-1-(2-chlorophenyl) ethanol, ater 6 cycles, the yield can still reach 91%, with 99.99% enantiomeric excess (ee) value for each cycle. However, the yield could only reach 72.9% when traditionally encapsulated AKR and ADH in ZIF-8 are used. Thus, this work demonstrates that a combination of protein phase separation and bio-orthogonal linking enables the in situ creation of a stable and spatially organized bi-enzyme system with enhanced channeling effects in ZIF-8.

更新日期：2024-08-12

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