Improvement of the Transglycosylation Efficiency of a Lacto-N-Biosidase from Bifidobacterium bifidum by Protein Engineering

The lacto-N-biosidase LnbB from Bifidobacterium bifidum JCM 1254 was engineered to improve its negligible transglycosylation efficiency with the purpose of enzymatically synthesizing lacto-N-tetraose (LNT; Gal-?1,3-GlcNAc-?1,3-Gal-?1,4-Glc) in one enzymatic step. LNT is a prebiotic human milk oligosaccharide itself and constitutes structural core range more complex oligosaccharides as well. Thirteen different variants were expressed screened for activity by monitoring product formation using lacto-N-biose 1,2-oxazoline donor substrate lactose acceptor substrate. major reaction product, yet careful analysis revealed three additional isomers, which we identified have ?1,2-linkage, ?1,6-linkage, 1,1-linkage, respectively, between (Gal-?1,3-GlcNAc) lactose. Considering both maximal yield regioselectivity well minimal hydrolysis, best variant W394H, closely followed W465H Y419N. A high also obtained W394F, substitution W394 W465 subsite ?1 hydrophobic platform enzyme His dramatically impaired undesirable hydrolysis compared Phe; effect most pronounced W465. Using p-nitrophenyl-?-lacto-N-bioside manifested an important target position. optimization concentrations confirmed that initial concentration acceptor-to-donor ratio favor transglycosylation.