Transglycosylation Activity of Engineered Bifidobacterium Lacto-N-Biosidase Mutants at Donor Subsites for Lacto-N-Tetraose Synthesis

Abstract

The health benefits of human milk oligosaccharides (HMOs) make them attractive targets as supplements for infant formula milks. However, HMO synthesis is still challenging and only two HMOs have been marketed. Engineering glycoside hydrolases into transglycosylases may provide biocatalytic routes to the synthesis of complex oligosaccharides. Lacto-N-biosidase from Bifidobacterium bifidum (LnbB) is a GH20 enzyme present in the gut microbiota of breast-fed infants that hydrolyzes lacto-N-tetraose (LNT), the core structure of the most abundant type I HMOs. Here we report a mutational study in the donor subsites of the substrate binding cleft with the aim of reducing hydrolytic activity and conferring transglycosylation activity for the synthesis of LNT from p-nitrophenyl β-lacto-N-bioside and lactose. As compared with the wt enzyme with negligible transglycosylation activity, mutants with residual hydrolase activity within 0.05% to 1.6% of the wild-type enzyme result in transglycosylating enzymes with LNT yields in the range of 10-30%. Mutations of Trp394, located in subsite -1 next to the catalytic residues, have a large impact on the transglycosylation/hydrolysis ratio, with W394F being the best mutant as a biocatalyst producing LNT at 32% yield. It is the first reported transglycosylating LnbB enzyme variant, amenable to further engineering for practical enzymatic synthesis of LNT.

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

“…We also compare LNB-oxazoline and pNP-LNB as donor substrates for the best LnbB variants. Finally, we question the previous statements [9,15] that LnbB-catalyzed transglycosylation is regioselective. [25].…”

“…Transglycosylation reactions with LnbB WT and five selected mutants (W394F, W394H, Y419N, W465H and W465F) were performed using 2.5 mM pNP-LNB as donor and 200 mM lactose acceptor at pH 4.5 and 30 • C (optimal pH for pNP-LNB [3] and the same conditions as used by Castejón-Vilatersana et al [9]). For optimization of the reaction conditions with pNP-LNB donor and lactose acceptor for LnbB W394H, transglycosylation reactions were performed using varying concentrations of pNP-LNB as donor (from 2.5 to 10 mM) and varying concentrations of lactose acceptor (200 or 400 mM).…”

“…In the CAZy database of carbohydrate-active enzymes (CAZy) [4], LNBases are found in glycoside hydrolase (GH) families 20 (GH20) and 136 (GH136). The LNBases of GH136 employ a classical double-displacement mechanism possibly including water-mediated proton transfer [5], whereas the GH20 LNBases employ a substrate-assisted mechanism, where the 2-acetamido group of the substrate (typically of a GlcNAc residue in the substrate molecules) acts as a nucleophile [6][7][8][9]. This mechanism is usually (but not always) employed in enzymes active on GlcNAc-or GalNAc-containing substrates and operates through an oxazoline or oxazolinium ion intermediate, which-unlike the case for the retaining GHs that work via a classical double-displacement mechanism-is not covalently linked to the enzyme [6][7][8][9][10][11].…”

“…GlcNAc-or GalNAc-containing substrates and operates through an oxazoline or oxazolinium ion intermediate, which-unlike the case for the retaining GHs that work via a classical double-displacement mechanism-is not covalently linked to the enzyme [6][7][8][9][10][11].…”

“…Castejón-Vilatersana et al [9] recently presented a detailed study of transglycosylation by 23 different LnbB variants targeting engineering of substrate-interacting residues. In this work, we present nine additional variants and show further improvement by alternative amino acid substitutions.…”