The chloroplast of plants is a natural microfluidic reactor for natural photosynthesis, in which the multi-enzymatic Calvin cycle is the key. In the chloroplast, the Calvin cycle enzymes are reportedly attached to the thylakoid membrane by physical interactions. To mimic this process, we physically immobilized the first enzyme of the Calvin cycle, d-ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), into microfluidic reactors by injecting 2 μg μL−1 RuBisCO for 4 h and demonstrated the successful production of the glucose precursor, 3-phosphoglycerate, at 0.145 ± 0.008 nmol min−1. Hydrophobic interactions play an important role in the physical immobilization, making the process simple and fast. The physical immobilization presents a 5.7-fold thermal stability as compared to the free RuBisCO, and shows performances inferior but close to that of chemical immobilization in enzyme kinetics, production rate and stability. Although the reactors can retain only 40% of the initial activity after 10 cycles of reusing, the physical immobilization has an interesting special feature that the enzyme can be desorbed to refresh the reactor for new immobilization. Experiments show that >95% activity can be restored after 5 cycles of refreshing. With the merits of reusing and refreshing, up to 5 mL of 3-PGA can be produced by continuously injecting the reactant mixture with great sustainability and cost-effectiveness. The reactors are also scaled out to two and six parallel reactors as a proof of concept of large-scale synthesis. Physical immobilization in microfluidic reactors is highly suitable for the multi-enzymatic, cascaded reactions in the Calvin cycle and facilitates the future study of artificial synthesis of glucose.
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