Successful Manipulation of the Product Spectrum of the Erwinia amylovora Levansucrase by Modifying the Residues around loop1, Loop 3, and Loop 4.

Levansucrase (LS, EC 2.4.1.10) catalyzes the synthesis of levan by successively transferring the fructosyl moiety from sucrose to an elongated fructan chain. Although the product distribution of LS from Erwinia amylovora ( Ea -LS) was studied under different sucrose concentrations, the effect of residues on the product formation is yet unknown. The first levanhexaose-complexed structure of LS from Bacillus subtilis ( Bs -SacB) provided information on the oligosaccharide binding sites (OB sites), from +1 to +4 subsites. Since Ea -LS would efficiently produce fructooligosaccharides, a substitution mutation of OB sites in Bs -SacB and the corresponding residues of Ea -LS were conducted to investigate how these mutants would influence the product distribution. As a result, a series of mutants with different product spectrum were obtained. Notably, the mutants of G98E, V151F, and N200T around loop 1, loop 3, and loop 4 all showed a significant increase in both the molecular mass and the yield of high-molecular-mass levan, suggesting that the product profile of Ea -LS was significantly modified.