Enzymatic lacto-N-biose elongation of human milk oligosaccharides with the GH136 lacto-N-biosidase LnbX engineered for improved transglycosylation

The lacto-N-biosidase (EC 3.2.1.140) LnbX from Bifidobacterium longum subsp. longum JCM 1217, a member of the glycoside hydrolase family 136 (GH136), was used for single-step lacto-N-biose (LNB) elongation of human milk oligosaccharides (HMOs) via disaccharide transglycosylation to expand the portfolio of HMOs available from enzymatic synthesis. We used the commercially available HMOs lacto-N-tetraose (LNT) as donor and lacto-N-neotetraose (LNnT) as acceptor for the synthesis of para-lacto-N-hexaose (pLNH). To improve the transglycosylation performance of LnbX (18 % molar yield), we designed 14 single mutants using two different strategies: 1) conservative substitution of conserved residues in the active site, and 2) shielding of the active site by large, hydrophobic residues. Protein engineering improved pLNH yield 1.5-fold as compared to the wild type (to 27 %). More than a 3-fold increase was obtained when optimizing the reaction conditions using the best variant, LnbX D416N, by an experimental design including reaction temperature, pH, donor substrate concentration, acceptor-to-donor (A/D) ratio, and enzyme concentration. Higher LNT concentrations and A/D ratios led to increased pLNH yields, and a high A/D ratio also increased the proportion of pLNH among the reaction products. The maximum molar yield of 57 % was obtained after 3 h of reaction at 100 mM LNT, 500 mM LNnT, 1 μM enzyme, 35 °C and pH 6.5. The LnbX D416N enzyme, which can use lacto-N-fucopentaose III (LNFP-III) as an alternative acceptor substrate, is active and stable at industrially relevant ranges of pH and temperature. The enzyme may thus be useful for diversification of the industrially available HMO portfolio.