Enzymatic synthesis of analogous human milk oligosaccharide and its prebiotic attributes

Abstract

Human milk oligosaccharides (HMOs) are the third most abundant component of human milk and deliver a myriad of benefits to infants including inhibiting the growth of pathogens and promoting the population of Bifidobacterium. In this study, glycoside hydrolase family 94 (GH94) member cellodextrin phosphorylase (CDP) has been used to synthesize a trisaccharide (Gal-β-1,4-Gal-β-1,4-Glc) in milligrams by transferring a galactose unit on cellobiose that mimics the primary building block of HMOs. Previous study of saturation transfer difference nuclear magnetic resonance spectroscopy indicated that the phosphate group of glucose-1-phosphate (Glc-1-P) makes hydrogen bonds with the side chain of H817, R486 and S889, and the backbone of G890, as well as a salt bridge with R486. We employed site-directed mutagenesis to determine the roles of these residues in synthesizing analogue HMO using galactose-1-phosphate (Gal-1-P). We did alanine scanning of these residues and found that mutation of R486A and G890A residues led to enzyme inactivation. Whilst mutation in H817A and S889A residues led to decreased enzymatic activity. These results further suggested that Gal-1-P bound at the donor site similar to a cognate donor (Glc-1-P). Using the native CDP, 40 mg of analogue HMO trisaccharide was produced with a 66 % yield. The product was well purified and analytically characterized. This trisaccharide is found to support the growth of probiotic bacteria such as Bifidobacterium longum, Roseburia faecis, and Blautia producta.