One-pot synthesis of β-alanine from 1,3-diaminopropane using two-enzyme cascade in cell-free biotransformation

A novel two-enzyme cascade one-pot synthesis of β-alanine from 1,3-diaminopropane (DAP) has been developed. In the first step, DAP was oxidized to 3-aminopropionaldehyde (3-APAL) by diamine oxidase (DAO). In the second step, 3-APAL was oxidized to β-alanine by 3-APAL dehydrogenase (APALDH). Catalase and NADH oxidase were employed to degrade the by-product, H₂O₂, and to regenerate the cofactor, NAD⁺. DAO specific to DAP has not been reported in any prokaryote. Therefore, initial proof of concept was established using commercial eukaryotic DAO_pk (from porcine kidney) and catalase (from bovine liver), along with recombinant APALDH and NOX enzymes sourced from Arthrobacter crystallopoietes and Lactococcus lactis, respectively. β-Alanine was successfully produced via this pathway; 12.2 mM (1.1 g/L) was formed in 41 h with 12 % conversion. To increase the reaction rate, DAO with higher specific activity was identified from Arthrobacter pascens (DAO_Ap). The optimum pH and temperature of DAO_Ap were determined to be 9.0 and 37 °C, respectively. Batch enzymatic biotransformation achieved 6 % conversion, yielding 0.33 mM (29 mg/L) β-alanine in 4 h. The low titre in batch conversion was attributed to substrate inhibition affecting DAO_Ap, NOX, and catalase. Fed-batch enzymatic biotransformation was conducted to overcome substrate inhibition, yielding 47 % conversion, with 2.34 mM (63 mg/L) β-alanine formation in 4 h. Approximately a 7.5-fold increase in conversion was achieved using fed-batch enzymatic biotransformation. This study accomplished a novel two-enzyme cascade biotransformation strategy for one-pot β-alanine synthesis from DAP.