Mutational Analysis of the Role of the Glucansucrase Gtf180-ΔN Active Site Residues in Product and Linkage Specificity with Lactose as Acceptor Substrate

[Image: see text] Glucansucrase Gtf180-ΔN from Lactobacillus reuteri uses lactose as acceptor substrate to synthesize five glucosylated lactose molecules (F1–F5) with a degree of polymerization (DP) of 3–4 (GL34) and with (α1→2)/(α1→3)/(α1→4) glycosidic linkages. Q1140/W1065/N1029 mutations significantly changed the GL34 product ratios. Q1140 mutations clearly decreased F3 3′-glc-lac with an (α1→3) linkage and increased F4 4′,2-glc-lac with (α1→4)/(α1→2) linkages. Formation of F2 2-glc-lac with an (α1→2) linkage and F4 was negatively affected in most W1065 and N1029 mutants, respectively. Mutant N1029G synthesized four new products with additional (α1→3)-linked glucosyl moieties (2xDP4 and 2xDP5). Sucrose/lactose strongly reduced Gtf180-ΔN hydrolytic activity and increased transferase activity of Gtf180-ΔN and mutant N1029G, in comparison to activity with sucrose alone. N1029/W1065/Q1140 thus are key determinants of Gtf180-ΔN linkage and product specificity in the acceptor reaction with lactose. Mutagenesis of key residues in Gtf180-ΔN may allow synthesis of tailor-made mixtures of novel lactose-derived oligosaccharides with potential applications as prebiotic compounds in food/feed and in pharmacy/medicine.