Rational designed mutagenesis of levansucrase from Bacillus licheniformis 8-37-0-1 for product specificity study

Levansucrases, which belong to the glycoside hydrolase family 68 (GH68), synthesize β (2-6)-linked fructan levan with sucrose as substrate. We described the use of a levansucrase (Bl_SacB) from Bacillus licheniformis 8-37-0-1 for catalysis of fructosyl transfer to obtain high levan yield previously. In the present study, six variants (Y246A, N251A, K372A, R369A, R369S, and R369K) were constructed through sequence alignment and structural analysis to explore the synthesis mechanism of Bl_SacB. The selected residues were predicted to localize to the substrate-entering channel of the active cavity and close to or remote from the catalytic triad. The products of these variants ranged from homopolymers levan to fructo-oligosaccharides (FOSs). The primary FOSs were identified through MS and NMR analyses as neolevan-type neokestose [β-D-Fru-(2-6)-α-D-Glc-(1-2)-β-D-Fru], levan-type 6-kestose [β-D-Fru-(2-6)-β-D-Fru-(2-1)-α-D-Glc], and inulin-type 1-kestose [β-D-Fru-(2-1)-β-D-Fru-(2-1)-α-D-Glc]. The mutation at Tyr²⁴⁶ located remote from the catalytic triad led to the production of short-chain oligosaccharides with degree of polymerization (DP) of up to 25. The replaced Arg³⁶⁹ located close to the catalytic triad resulted in either elimination of polysaccharide synthesis or complete change in the dominant linkage of the products. The Michaelis constants (K_m) of Y246A, N251A, K372A, and R369K were found to be similar to that of the wild type (WT). However, the turnover number (k_cat) and the value of transfructosylation versus hydrolysis activity of the six variants decreased compared with those of the WT. Hence, the residues located on the surface of the substrate-entering channel of Bl_SacB can be critical in product linkage type and/or elongation mechanism.