Chembiochem. 2026 Sep 14;27(17):e70530. doi: 10.1002/cbic.70530.

ABSTRACT

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 (CbAP) 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 CbAP_Q138FF96Y (CbAP_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, CbAP_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.

PMID:42734018 | DOI:10.1002/cbic.70530