Abstract The assembly of multiple enzymes has received increasing attention due to the promising application in improving biocatalytic process. In this study, supramolecular nanoreactors were engineered by assembly of polyphosphate kinase (PPK) and bifunctional glutathione synthetase (GshF) for adenosine triphosphate (ATP) regeneration in vitro. Tetrameric PPK and dimeric GshF were first self-assembled via peptide-peptide interaction of fused leucine zipper domains to form multienzyme complexes (MECs) with plate-like morphology. Further, under the driving force of polymerization of PPK driven by substrate polyphosphate (polyP), MECs were assembled into multienzyme nanoreactors (MENRs) with branch-like morphology. MENRs were applied to a two-enzyme cascade reaction in vitro. The ATP regeneration efficiency of MENRs system was 57 % higher than that of unassembled enzymes mixture, and the thermal stability was improved by 46 %. When reused for ATP regeneration, the assemblies retained more than 70 % of the initial activity even after seven cycles. This is the first report in which an artificial complex was constructed by substrate-driven assembly of multiple enzymes for cascade reaction. The strategy demonstrated potential application of multienzyme assembly in synthetic biotechnology and nanotechnology.

中文翻译：

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通过组装多种酶构建超分子纳米反应器用于体外 ATP 再生

摘要 由于在改进生物催化过程中的应用前景，多种酶的组装受到越来越多的关注。在这项研究中，超分子纳米反应器是通过组装多磷酸激酶 (PPK) 和双功能谷胱甘肽合成酶 (GshF) 来设计的，用于体外三磷酸腺苷 (ATP) 再生。四聚体 PPK 和二聚体 GshF 首先通过融合亮氨酸拉链结构域的肽-肽相互作用自组装，形成具有板状形态的多酶复合物 (MEC)。此外，在底物多磷酸盐（polyP）驱动的PPK聚合的驱动力下，MECs组装成具有分支状形态的多酶纳米反应器（MENRs）。MENRs 被应用于体外的双酶级联反应。MENRs系统的ATP再生效率比未组装的酶混合物高57%，热稳定性提高46%。当重新用于 ATP 再生时，即使经过七个循环，该组件仍保留了超过 70% 的初始活性。这是第一个通过底物驱动的多酶组装来构建人工复合物的报告，用于级联反应。该策略展示了多酶组装在合成生物技术和纳米技术中的潜在应用。这是第一个通过底物驱动的多酶组装来构建人工复合物的报告，用于级联反应。该策略展示了多酶组装在合成生物技术和纳米技术中的潜在应用。这是第一个通过底物驱动的多酶组装来构建人工复合物的报告，用于级联反应。该策略展示了多酶组装在合成生物技术和纳米技术中的潜在应用。