Hierarchically Engineered Mesoporous Metal-Organic Frameworks toward Cell-free Immobilized Enzyme Systems

Highly efficient cell-free enzymatic systems are typically difficult to achieve in traditional immobilized enzyme systems because of the lack of optimal spatial control of enzyme localization, substrate and product diffusion, and enzyme and coenzyme accessibility. Here, we report a strategy for expanding the pore apertures (from 3.3 to 6.7 nm) of a series of Zr-based metallic-organic frameworks (MOFs) (termed NU-100x, x = 3, 4, 5, 6, 7) with interconnected hierarchical pores by maintaining precise control of torsional angles associated with the linkers. As a proof of concept, we use the expanded NU-100x MOF structures to encapsulate lactate dehydrogenase (LDH) and demonstrate the use of the captured protein in a cell-free biosynthetic catalytic system. Remarkably, LDH immobilized in the large pores of the MOF is accessible to nicotinamide adenine dinucleotide coenzymes (NAD and NADH), allowing for in situ coenzyme regeneration leading to higher activity than that of the free enzyme. Cell-free synthetic biology is an emerging field of biotechnology that is expanding the capabilities of natural biological systems, as demonstrated by the rapid development of various systems for biofuel and biochemical production. To achieve large-scale industrial production of cell-free enzymatic systems, efficient immobilization strategies are needed to overcome challenges such as high production costs of enzymes and coenzymes. Here, we create a series of ideal enzyme carriers based on metal-organic frameworks (MOFs) with interconnected hierarchical mesoporous channels. These MOF enzyme carriers show water stability, high enzyme-loading capacity, and coenzyme accessibility and demonstrate the use of the captured protein in a cell-free biosynthetic catalytic system. This work provides a strategy for constructing novel immobilized cell-free enzymatic systems for industrially relevant applications. Farha and colleagues have developed a strategy for expanding the pore apertures of csq-net Zr-based MOFs to obtain an isoreticular series of MOF structures with pore apertures ranging from 3.3 to 6.7 nm. Enzymes immobilized in the MOF are accessible to coenzymes and show higher activity than that of the free enzymes.