An Engineered β‐Aminopeptidase With Better Product Selectivity for L‐Carnosine Production

Efficient enzymaticsynthesis of L‐carnosine (L‐Car) is often limited by competing hydrolysis and side reactions that reduce substrate utilization and product yield. In this study, a β‐aminopeptidase ( Cb AP) from Caulobacter sp. was systematically engineered to enhance catalytic selectivity toward L‐Car using β‐alaninamide and L‐histidine as substrates. Analysis of β‐alaninamide‐derived byproducts revealed two major competing pathways, namely β‐alaninamide hydrolysis and self‐ligation into short oligopeptides. To evaluate the balance between L‐Car synthesis and competing reactions, a composite apparent selectivity index ( R S ) was introduced. Structure‐guided mutagenesis targeting residues within 4–6 Å of the active site identified several improved variants. Iterative combination of beneficial mutations yielded the optimal mutant Cb AP Q138FF96Y ( Cb AP M2 ), exhibiting an approximately 2.3‐fold increase in R S relative to Q138F and an approximately 3.1‐fold increase relative to the wild‐type enzyme, with substantial suppression of undesired side reactions. Molecular dynamics simulations indicated that these improvements arise from reduced substrate binding stability and disrupted hydrogen‐bonding interactions with β‐alaninamide and L‐Car. Under optimized 10 mL‐scale conditions, Cb AP M2 produced 37.3 mM L‐Car within 1 h, corresponding to a 74.6% yield based on β‐alanine methyl ester, with an STY of 8.44 g L − 1 h −1 .