Improved Enzymatic Production of the Fucosylated Human Milk Oligosaccharide LNFP II with GH29B α-1,3/4-ι-Fucosidases

Yaya Yang, Albert Thor Thorhallsson, Carme Rovira, Jesper Holck, Anne S. Meyer, Huan Yang, Birgitte Zeuner*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review


Five GH29B α-1,3/4-l-fucosidases (EC were investigated for their ability to catalyze the formation of the human milk oligosaccharide lacto-N-fucopentaose II (LNFP II) from lacto-N-tetraose (LNT) and 3-fucosyllactose (3FL) via transglycosylation. We studied the effect of pH on transfucosylation and hydrolysis and explored the impact of specific mutations using molecular dynamics simulations. LNFP II yields of 91 and 65% were obtained for the wild-type SpGH29C and CpAfc2 enzymes, respectively, being the highest LNFP II transglycosylation yields reported to date. BbAfcB and BiAfcB are highly hydrolytic enzymes. The results indicate that the effects of pH and buffer systems are enzyme-dependent yet relevant to consider when designing transglycosylation reactions. Replacing Thr284 in BiAfcB with Val resulted in increased transglycosylation yields, while the opposite replacement of Val258 in SpGH29C and Val289 CpAfc2 with Thr decreased the transfucosylation, confirming a role of Thr and Val in controlling the flexibility of the acid/base loop in the enzymes, which in turn affects transglycosylation. The substitution of an Ala residue with His almost abolished secondary hydrolysis in CpAfc2 and BbAfcB. The results are directly applicable in the enhancement of transglycosylation and may have significant implications for manufacturing of LNFP II as a new infant formula ingredient.
Original languageEnglish
JournalJournal of Agricultural and Food Chemistry
Issue number19
Pages (from-to)11013-11028
Publication statusPublished - 2024


  • Transglycosylation
  • Molecular dynamics
  • Protein engineering
  • Acid/base residue flexibility


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