Glutaraldehyde cross-linked Candida antarctica lipase B on NH₂-UiO-66: A high-efficiency biocatalyst for diacylglycerol synthesis

CALB@GA-NH 2 -UiO-66 exhibited 97.31% oleic acid conversion and 77.60% DAG content. • CALB@GA-NH 2 -UiO-66 retained full initial activity after 11 reuse cycles. • The immobilized CALB exhibited a single conformational basin. • Covalent enzyme–carrier conjugates were formed via Schiff base linkages. • The immobilization moderately restrained the flexibility of α-helix 5 of CALB. This study aimed to develop a robust biocatalyst for diacylglycerol (DAG) synthesis. Candida antarctica lipase B (CALB) was immobilized on NH₂-UiO-66 via glutaraldehyde (GA) cross-linking. The cross-linked CALB exhibited significantly better performance than the adsorbed counterpart. Oleic acid conversion reached 97.31% with DAG content up to 77.60%. Moreover, the immobilized CALB retained its full initial activity after 11 reuse cycles. The temperature dependence of oleic acid conversion followed the equation ln V₀  = 8.86–4.67 × 10 3 / T , with an activation energy of 38.83 kJ·Mol −1 . Molecular dynamics simulations revealed that the flexibility of α-helix 5 in CALB@GA-NH₂-UiO-66 is moderately restrained, and the immobilized CALB exhibits a single conformational basin. Additionally, immobilization induces slight contraction of the CALB active site pocket, and reduced length and increased bottleneck radius of the tunnel. This study provides molecular insights into the structure–stability relationship of immobilized enzymes, guiding the rational design of robust biocomposites We develop a robust biocatalyst via glutaraldehyde cross-linking of CALB onto NH₂-UiO-66 for DAG production by esterification. Download: Download high-res image (210KB) Download: Download full-size image