Preparation and application of lacto-N-biose synthases immobilized onto amino-modified SiO₂ nanoparticles

Lacto-N-biose (LNB) is the core structural unit of human milk oligosaccharides and plays an indispensable role in the growth and development of infants. In this study, amino-modified SiO₂ nanoparticles (SNPs-NH₂) were used as carriers to immobilize sucrose phosphorylase (SPase), UDP-galactose-4'-epimerase (GalE), galactose-1-phosphate uridylyltransferase (GalT), and lacto-N-biose phosphorylase (LNBP), and LNB was synthesized in a one-pot method with high efficiency. The results showed that after activation of SNPs for 16 h at 25 °C with 2 mol/L hydrochloric acid, and modification with 3-Aminopropyltrimethoxysilane (APTES)/SNPs (2:1, v/m) at 65 °C for 9 h, the interface -NH₂ grafting density reached to 12.04 groups/nm². Under the optimal immobilization conditions, the SPase, GalE, GalT, and LNBP were all well-loaded onto the carriers. At the same time, the temperature and pH stability of the enzymes were significantly improved, and the optimal reaction range was significantly expanded. The enzyme activity retention rate after 7 cycles of reaction was 74% to 85%, with the LNB synthesis yield rate remaining above 90%. This study effectively simplified the separation of enzymes from the reaction system during LNB synthesis and improved its stability, laying a foundation for the industrial production of LNB.