Development of a platform for the production of rare α-hydroxyacids

Details

Supervisors

Desguin, Benoît

Faculty

Faculté des sciences

Degree label

Master [120] en biochimie et biologie moléculaire et cellulaire, à finalité spécialisée: biotechnologie

Abstract

Alpha-hydroxy acids (AHAs) are essential compounds with significant applications in pharmaceuticals, cosmetics, and food industries. However, producing rare AHAs in an efficient and sustainable manner remains a major challenge due to the limitations of traditional chemical synthesis methods, including low yields, high costs, and environmental concerns. This study explores the potential of LarAH enzymes, specifically LarAH26 and LarAH31, expressed in Lactococcus lactis, to catalyze the racemization and epimerization of common AHAs into rarer, high-value ones. LarAH enzymes rely on the Nickel-Pincer Nucleotide (NPN) cofactor as part of their holoprotein structure to facilitate a proton-coupled hydride transfer mechanism, allowing continuous reactions without the need for external cofactor replenishment. The activity of L. lactis cell suspensions expressing LarAH31 was characterized, demonstrating optimal activity at pH 7.5 and 30°C. Despite optimized conditions, the racemic equilibrium for lactate was not reached. Additionally, both the purified enzyme and the bacterial expression system exhibited instability beyond 120 hours. In contrast, L. lactis expressing LarAH26 initially showed activity on D-mannonate, but later became inactive during the study. Subsequent analysis revealed that the commercially sourced substrate, originally believed to be D-mannonate, was actually its L-enantiomer. Future work can focus on improving enzyme stability and addressing challenges in substrate import and product export through the engineering of transporters. Directed evolution could enhance enzyme stability, activity, and cofactor utilization, as well as broaden substrate tolerance, making LarAH enzymes more effective and reliable for industrial-scale applications. Additionally, integrating LarAH enzymes into dynamic kinetic resolution workflows could significantly enhance their industrial relevance. This study highlights the promise of LarAH enzymes for sustainable AHA production while underlining the importance of careful reagent validation. These insights provide a foundation for developing robust biocatalytic platforms for the scalable production of rare AHAs