Levansucrases (LS) are fructosyltransferases (FTFs) belonging to family 68 of glycoside hydrolases (GH68) using sucrose as substrate to synthesize levan, a fructose polymer. From a multiple sequence analysis of GH68 family proteins, nine residues were selected and their role in acceptor and product specificity, as well as in biochemical Bacillus subtilis LS properties, was investigated. A product specificity modification was obtained with mutants Y429N and R433A that no longer produce levan but exclusively oligosaccharides. An effect of the mutation S164A was observed on enzyme stability and kinetic behavior; this mutation also induces a levan activation effect that enhances the reaction rate. We report the crystallographic structure of this mutant and found that S164 is an important residue to maintain the nucleophile position in the active site. We also found evidence of the important role of Y429 in acceptor specificity: this is a key residue coordinating the sucrose position in the catalytic domain-binding pocket. Some of these mutations resulted in LS with a broad range of specificities and new biochemical properties.

A flexible tyrosine-attached chemical lid prevents premature disengagement of growing oligosaccharides and triggers the synthesis of a high molecular weight polymer.

No abstract

Background: Increasing attention has been focused on inulin and levan-type oligosaccharides, including fructosyl-xylosides and other fructosides due to their nutraceutical properties. Bacillus subtilis levansucrase (LS) catalyzes the synthesis of levan from sucrose, but it may also transfer the fructosyl moiety from sucrose to acceptor molecules included in the reaction medium. To study transfructosylation reactions with highly active and robust derivatives, cross-linked enzyme aggregates (CLEAs) were prepared from wild LS and two mutants. CLEAs combine the catalytic features of pure protein preparations in terms of specific activity with the mechanical behavior of industrial biocatalysts.