Publication Date:
2016-02-03
Description:
Human milk oligosaccharides (HMOs) are recognized as benefiting breast-fed infants in multiple ways. As a result, there is growing interest in the synthesis of HMOs mimicking their natural diversity. Most HMOs are fucosylated oligosaccharides. α- l -Fucosidases catalyze the hydrolysis of α- l -fucose from the non-reducing end of a glucan. They fall into the glycoside hydrolase GH29 and GH95 families. The GH29 family fucosidases display a classic retaining mechanism and are good candidates for transfucosidase activity. We recently demonstrated that the α- l -fucosidase from Thermotoga maritima ( Tm αFuc) from the GH29 family can be evolved into an efficient transfucosidase by directed evolution ( Osanjo et al. 2007 ). In this work, we developed semi-rational approaches to design an α- l -transfucosidase starting with the α- l -fucosidase from commensal bacteria Bifidobacterium longum subsp. infantis ( Bi AfcB, Blon_2336). Efficient fucosylation was obtained with enzyme mutants (L321P- Bi AfcB and F34I/L321P- Bi AfcB) enabling in vitro synthesis of lactodifucotetraose, lacto- N -fucopentaose II, lacto- N -fucopentaose III and lacto- N -difucohexaose I. The enzymes also generated more complex HMOs like fucosylated para -lacto- N -neohexaose (F- p -LNnH) and mono- or difucosylated lacto- N -neohexaose (F-LNnH-I, F-LNnH-II and DF-LNnH). It is worth noting that mutation at these two positions did not result in a strong decrease in the overall activity of the enzyme, which makes these variants interesting candidates for large-scale transfucosylation reactions. For the first time, this work provides an efficient enzymatic method to synthesize the majority of fucosylated HMOs.
Print ISSN:
0959-6658
Electronic ISSN:
1460-2423
Topics:
Biology
,
Medicine
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