title: Thermostable β-galactosidases for the synthesis of human milk oligosaccharides.
abstract: Human milk oligosaccharides (HMOs) designate a unique family of bioactive lactose-based molecules present in human breast milk. Using lactose as a cheap donor, some β-galactosidases (EC 3.2.1.23) can catalyze transgalactosylation to form the human milk oligosaccharide lacto-N-neotetraose (LNnT; Gal-β(1,4)-GlcNAc-β(1,3)-Gal-β(1,4)-Glc). In order to reduce reaction times and be able to work at temperatures, which are less welcoming to microbial growth, the current study investigates the possibility of using thermostable β-galactosidases for synthesis of LNnT and N-acetyllactosamine (LacNAc; Gal-β(1,4)-GlcNAc), the latter being a core structure in HMOs. Two hyperthermostable GH 1 β-galactosidases, Ttβ-gly from Thermus thermophilus HB27 and CelB from Pyrococcus furiosus, were codon-optimized for expression in Escherichia coli along with BgaD-D, a truncated version of the GH 42 β-galactosidase from Bacillus circulans showing high transgalactosylation activity at low substrate concentrations. The three β-galactosidases were compared in the current study in terms of their transgalactosylation activity in the formation of LacNAc and LNnT. In all cases, BgaD-D was the most potent transgalactosidase, but both thermostable GH 1 β-galactosidases could catalyze formation of LNnT and LacNAc, with Ttβ-gly giving higher yields than CelB. The thermal stability of the three β-galactosidases was elucidated and the results were used to optimize the reaction efficiency in the formation of LacNAc, resulting in 5-6 times higher reaction yields and significantly shorter reaction times.
authors.affiliation: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, DK-2800 Kgs., Lyngby, Denmark.
authors.forename: Birgitte
authors.initials: B
authors.lastname: Zeuner
authors.affiliation: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, DK-2800 Kgs., Lyngby, Denmark.
authors.forename: Christian
authors.initials: C
authors.lastname: Nyffenegger
authors.affiliation: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, DK-2800 Kgs., Lyngby, Denmark.
authors.forename: Jørn Dalgaard
authors.initials: JD
authors.lastname: Mikkelsen
authors.affiliation: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, DK-2800 Kgs., Lyngby, Denmark. Electronic address: am@kt.dtu.dk.
authors.forename: Anne S
authors.initials: AS
authors.lastname: Meyer
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism; D004795:Enzyme Stability; D006031:Glycosylation; D006801:Humans; D008895:Milk, Human / Q000201:enzymology*; D009844:Oligosaccharides / Q000096:biosynthesis*; D013696:Temperature; D014312:Trisaccharides / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism*
chemical_list: D009844:Oligosaccharides; D014312:Trisaccharides; C098555:lacto-N-triose II; D001616:beta-Galactosidase; D000117:Acetylglucosamine