Thermostable β-galactosidases for the synthesis of human milk oligosaccharides

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 beta-galactosidases (EC 3.2.1.23) can catalyze transgalactosylation to form the human milk oligosaccharide lacto-N-neotetraose (LNnT; Gal-13(1,4)-G1cNAc-beta(1,3)-Gal-beta(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 beta-galactosidases for synthesis of LNnT and N-acetyllactosamine (LacNAc; Gal-beta(1,4)-G1cNAc), the latter being a core structure in HMOs. Two hyperthermostable GH 1 beta-galactosidases, Tt beta-gly from Thermus thermophilus HB27 and Ce1B from Pyrococcus furiosus, were codon-optimized for expression in Escherichia coli along with BgaD-D, a truncated version of the GH 42 beta-galactosidase from Bacillus circulans showing high transgalactosylation activity at low substrate concentrations. The three beta-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 beta-galactosidases could catalyze formation of LNnT and LacNAc, with Tt beta-gly giving higher yields than Ce1B. The thermal stability of the three beta-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.