Generation of Amylosucrase Variants That Terminate Catalysis of Acceptor Elongation at the Di- or Trisaccharide Stage

Abstract

An amylosucrase gene was subjected to high-rate segmental random mutagenesis, which was directed toward a segment encoding amino acids that influence the interaction with substrate molecules in subsites ؊1 to ؉3. A screen was used to identify enzyme variants with compromised glucan chain elongation. With an average mutation rate of about one mutation per targeted codon, a considerable fraction (82%) of the clones that retained catalytic activity were deficient in this trait. A detailed characterization of selected variants revealed that elongation terminated when chains reached lengths of only two or three glucose moieties. Sequencing showed that the amylosucrase derivatives had an average of no more than two amino acid substitutions and suggested that predominantly exchanges of Asp394 or Gly396 were crucial for the novel properties. Structural models of the variants indicated that steric interference between the amino acids introduced at these sites and the growing oligosaccharide chain are mainly responsible for the limitation of glucosyl transfers. The variants generated may serve as biocatalysts for limited addition of glucose moieties to acceptor molecules, using sucrose as a readily available donor substrate.

Flavonoid glucosylation by non-Leloir glycosyltransferases: formation of multiple derivatives of 3,5,7,3′,4′-pentahydroxyflavane stereoisomers

“…This and the TLC mobility suggested that they contain increasing numbers of Glc units. This was further supported by the results obtained when the reaction was catalyzed by one of the previously generated Ams variants (Ams-V16) that are compromised in chain elongation (Schneider et al 2009). In this case, essentially, only Cat-A1, the acceptor product with the highest TLC mobility, was formed, even after 120 h of incubation (not shown).…”

Synthesis of novel α-glucans with potential health benefits through controlled glucose release in the human gastrointestinal tract

“…In addition to the 3 domains A, B and C common to all GH13 family members (Figure 4), AS have 2 extra domains named N and B' that are unique to these enzymes. These 3D structures have allowed the identification of key residues involved in the polymerization process, and have guided the construction of AS mutants which produce in a controlled way either short maltooligosaccharides or insoluble amylose from sucrose (Albenne et al 2004;Cambon et al 2014;Schneider et al 2009).…”