In Vitro Biosynthesis of Isobutyraldehyde Through the Establishment of a One-Step Self-Assembly-Based Immobilization Strategy.

The in vitro biosynthesis of high-value compounds has become popular and attractive. The convenient and simple strategy of enzyme immobilization has been significant for continuous and efficient in vitro biosynthesis. On the basis of that, this work established a one-step self-assembly-based immobilization strategy to efficiently biosynthesize isobutyraldehyde in vitro. Isobutyraldehyde is a crucial precursor for the synthesis of foods and spices. The established CipA scaffold-based strategy can express and immobilize enzymes at the same time, and purification requires only one centrifugation step. Structural simulations indicated that this scaffold-dependent self-assembly did not influence the structure or catalytic mechanisms of the isobutyraldehyde production-related enzymes leucine dehydrogenase (LeuDH) and ketoisovalerate decarboxylase (Kivd). Immobilized LeuDH and Kivd displayed a higher conversion capacity and thermal stability than the free enzymes. Batch conversion experiments demonstrated that the recovered immobilized LeuDH and Kivd have similar conversion capacities to the enzymes used in the first round of reaction. The continuous production of isobutyraldehyde was achieved by filling the immobilized enzymes into the column of a constructed device. This study not only expands the application range of self-assembly systems but also provides guidance for the in vitro production of value-added compounds.