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.
Highlights
Human milk oligosaccharides (HMOs) furnish breast-fed infants with a number of health benefits
Bifidobacterium bifidum Lacto-N-biosidase from Bifidobacterium bifidum (LnbB) [19,22] is a member of family GH20 in the Carbohydrate Active Enzymes (CAZy) classification [23] that operates by substrate-assisted catalysis
Protein engineering approaches to modulate the T/H ratio towards improved transglycosylation can be grouped in three complementary strategies: (i) decrease hydrolysis by mutations at the donor subsites of the active site that reduce transition state stabilization, (ii) enhance acceptor binding to compete with water by mutations that increase affinity to the acceptor subsites, and (iii) modify the hydrophilic/hydrophobic balance at the active site to reduce the accessibility or reactivity of the nucleophilic water
Summary
Human milk oligosaccharides (HMOs) furnish breast-fed infants with a number of health benefits. Their unique composition, differing from other mammal’s milk, drives active research to synthesize and produce the main HMO structures as supplements for infant formula milks (for recent reviews, see [1,2,3,4,5]). While there are over 150 HMO structures identified, 2’-fucosyllactose (2’-FL) and LNnT are the only commercially available HMOs for supplementing infant formula, produced by fermentation of metabolically engineered E. coli strains [2,8]. Since LNT is more abundant in human milk than LNnT (which is mainly found in other mammal’s milks), it is a current target for designing efficient production strategies. Enzymatic synthesis (biocatalysis) and fermentation (cell factory) approaches are current focus of research
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