Novel product specificity toward erlose and panose exhibited by multisite engineered mutants of amylosucrase

Abstract

A computer-aided engineering approach recently enabled to deeply reshape the active site of N. polysaccharea amylosucrase for recognition of non-natural acceptor substrates. Libraries of variants were constructed and screened on sucrose allowing the identification of 17 mutants able to synthesize molecules from sole sucrose, which are not synthesized by the parental wild-type enzyme. Three of the isolated mutants as well as the new products synthesized were characterized in details. Mutants contain between 7 and 11 mutations in the active site and the new molecules were identified as being a sucrose derivative, named erlose (α-d-glucopyranosyl-(1→4)-α-d-glucopyranosyl-(1→2)-β-d-Fructose), and a new malto-oligosaccharide named panose (α-d-glucopyranosyl-(1→6)-α-d-glucopyranosyl-(1→4)-α-d-Glucose). These product specificities were never reported for none of the amylosucrases characterized to date, nor their engineered variants. Optimization of the production of these trisaccharides of potential interest as sweeteners or prebiotic molecules was carried out. Molecular modelling studies were also performed to shed some light on the molecular factors involved in the novel product specificities of these amylosucrase variants.