Cell-free system for one-pot production of protocatechuate via a two-enzyme cascade with coenzyme regeneration

Protocatechuate, an aromatic compound with significant industrial application, has garnered increasing interest in its production through various metabolic engineering methods. However, these methods still face limitations that hinder their feasibility for large-scale industrial application. Therefore cell-free systems could serve as a better alternative in this field. Here, we describe a two-enzyme cascade system utilizing benzaldehyde dehydrogenase (AspBADH) and p-hydroxybenzoate hydroxylase (AspPHBH) for protocatechuate production from 4-hydroxybenzaldehyde (4-HBAL), with coenzyme regeneration enabled within the system. We first characterized the enzymatic activity of AspBADH and then integrated AspPHBH into an in-vitro system for protocatechuate production. Optimal conditions were identified as pH 7.4 and a temperature range of 20-40 °C, achieving approximately 50 % substrate conversion with a 1:1 enzyme ratio at a concentration of 15 mg/ml. Notably, increasing the ratio of AspBADH to AspPHBH further enhanced conversion efficiency. To improve industrial applicability, we immobilized the enzymes in glutaraldehyde-crosslinked chitosan beads, which enhanced reusability and stability. The immobilized enzyme cascade exhibited over 90 % substrate conversion in a single cycle and a 60 % total yield after purification. Although the specific activity decreased compared to the free enzymes, the immobilized system demonstrated reusability, maintaining activity through 8 cycles.