Human milk is considered the gold standard for infant nutrition; it is a complex mix-ture that besides providing complete nutrition to the infant, it delivers essential biomolecules. Human milk oligosaccharides (HMO) are a group of glycans that provide essential biological functions such as immune modulators, prebiotics, and nutrients for neonatal brain development .
Bifidobacterium bifidum present in the gut’s infant produces lacto-N-biosidase (LnbB), which is involved in the catabolism of HMOs. LnbB belongs to the GH20 family N-acetylhexosaminidases that catalyzes the hydrolysis of GlcNAc residues located at the non-reducing end of oligosaccharides and glycoconjugates via a retaining substrate-assisted catalytic mechanism [2,3]. LnbB catalyzes the hydrolysis of the tetrasacchride lacto-N-tetraose (LNT) to lacto-N-biose (LNB) and lactose . Our structural-functional analysis has revealed the multi-domain organization of the enzyme and the important residues for its hydrolytic activity .
The aim of this work is the production of LNT (core 1 HMO). We here report the engineering of LnbB following a semi-rational approach. To obtain a transglycosylating enzyme we are targeting the conserved residues located in the negative subsites of the binding site. Selected mutants are characterized, the hydrolytic activity of the enzymes is tested on LNB-pNP (lacto-N-biose pNP) and the transglycosylation activity is studied using lactose as acceptor and LNB-pNP as donor. Whereas the wt enzyme has no detectable transglycosylating activity, it is observed that the transglycosylation/hydrolysis ratio largely increases when the residual hydrolase activity is decreased to a window of 0.2 to 1% relative to the wt enzyme, achieving an enzyme variant with 32% yield in LNT production for further optimization as biocatalyst.
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