Access to efficient enzymatic channeling is desired for improving all manner of designer biocatalysis. We demonstrate that enzymes constituting a multistep cascade can self-assemble with nanoparticle scaffolds into nanoclusters that access substrate channeling and improve catalytic flux by orders of magnitude. Utilizing saccharification and glycolytic enzymes with quantum dots (QDs) as a model system, nanoclustered-cascades incorporating from 4 to 10 enzymatic steps are prototyped. Along with confirming channeling using classical experiments, its efficiency is enhanced several fold more by optimizing enzymatic stoichiometry with numerical simulations, switching from spherical QDs to 2-D planar nanoplatelets, and by ordering the enzyme assembly. Detailed analyses characterize assembly formation and clarify structure-function properties. For extended cascades with unfavorable kinetics, channeled activity is maintained by splitting at a critical step, purifying end-product from the upstream sub-cascade, and feeding it as a concentrated substrate to the downstream sub-cascade. Generalized applicability is verified by extending to assemblies incorporating other hard and soft nanoparticles. Such self-assembled biocatalytic nanoclusters offer many benefits towards enabling minimalist cell-free synthetic biology.

需要获得有效的酶促通道以改进所有方式的设计者生物催化。我们证明构成多步级联的酶可以与纳米颗粒支架自组装成纳米团簇，这些纳米团簇可以进入底物通道并按数量级提高催化通量。利用具有量子点 (QD) 的糖化酶和糖酵解酶作为模型系统，对包含 4 到 10 个酶促步骤的纳米簇级联进行了原型设计。除了使用经典实验确认沟道效应外，通过使用数值模拟优化酶化学计量、从球形 QD 切换到二维平面纳米片以及对酶组装进行排序，其效率提高了数倍。详细的分析表征了装配体的形成并阐明了结构-功能特性。对于具有不利动力学的扩展级联，通过在关键步骤分裂、从上游子级联纯化最终产物并将其作为浓缩底物供给下游子级联来维持通道活性。通过扩展到包含其他硬和软纳米粒子的组件来验证普遍适用性。这种自组装生物催化纳米团簇为实现极简无细胞合成生物学提供了许多好处。