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Author
Gómez del Campo Tirado, Fernando
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Abstract
Human breast milk is a mixture composed of complex proteins, lipids, carbohydrates and other biologically active components. Human Milk Oligosaccharides (HMOs) are complex sugar molecules that are exclusively found in high concentrations in human breast milk. They are exclusively synthesized in the mammary glands during lactation and they exert a prebiotic effect for the infant once ingested, being mainly processed by bacteria of the Bifidobacterium genus. HMOs are involved in the infant’s gut maturation, development, blocking of pathogen adhesion in the gastrointestinal tract, and aiding in immune system modulation, among other functions. This makes HMOs a highly valuable asset to be added to infant formula, and there is a considerable interest in developing cost-effective ways to produce them.
More than 200 HMOs have been characterized, and the two central core structures are the tetrasaccharides: lacto-N-tetraose (LNT), characterized by lacto-N-biose (LNB; Galβ1,3Glc- NAc) and a lactose unit, and lacto-N-neo-tetraose (LNnT), formed by N-acetyllactosamine (Galβ1,4GlcNAc) and lactose. In humans, LNT predominates over LNnT as the core structure for the HMOs present, which makes LNT interesting for applications in the food industry.
However, producing LNT through chemical synthesis poses a problem, as numerous steps are required, and low yields are achieved. However, enzymatic synthesis of LNT is an interesting alternative, as naturally occurring glycohydrolytic enzymes can be reversed-engineered to yield transglycosylation activity. Lacto-N-biosidase from Bifidobacterium bifidum (LnbB) is an exo-retaining enzyme from the GH20 family, naturally present in the human gut microbiome, that hydrolyses LNT into LNB and lactose. It possesses transglycosylation activity, albeit at very low catalytic efficiency, and the ratio of transglycosylation/hydrolysis (T/H) activity can be modified using protein engineering techniques for rational design, by mutating key amino acids on the enzyme’s active site.
With this purpose in mind, the Laboratory of Biochemistry at IQS is working on enzyme engineering of LnbB aimed at developing a biocatalyst for LNT synthesis. Based on an in-silico tool developed in the group (BINDSCAN) that predicts positions within an enzyme that are sensible to the binding of a chosen substrate, a number of target residues were identified whose mutation may increase the binding affinity for lactose as acceptor substrate, which would theoretically increase the T/H ratio by making the substrate more available in the catalytic site for transglycosylation.
Two mutants were proposed (I324W, V426W) that may increase the binding affinity of lactose in the positive subsites of LnbB (and potentially increase the T/H ratio of LNT). Here, the mutant proteins were produced and tested for transglycosylation activity in vitro and combined with a previously identified transglycosylation active mutant (W394F) and the WT in all possible combinations. Two mutants (W394F/V426W and W394F/I324W/V426W) were found to have transglycosylation activity. The former produces LNT almost immediately, and both mutants show promise in terms of LNT yield and are candidates for further enzyme engineering programs.
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