Site Directed Mutagenesis of Dextransucrase DsrM from Weissella cibaria: Transformation to a Reuteransucrase

摘要

Glucansucrases produce alpha-glucans and gluco-oligosaccharides; the linkage type and molecular weight of glucans impacts their functionality. This study compared the catalytic specificities of dextransucrase DsrM from Weissella cibaria 10M and derivatives of this enzymes with GtfA from Lactobacillus reuteri TMW1.656. The N-variable region, which is dispensable for GtfA. activity, was essential for DsrM activity. Parallel amino acid substitutions in DsrM-Delta S and GtfA-Delta N indicated that the acceptor binding site residues determining the linkage type differ in these enzymes. DsrM-V583P:V586I had comparable enzyme activity as the respective GtfA derivative but did not increase the proportion of alpha-(1 -> 4) linkages. DsrM-S622N had low enzyme activity and an unaltered proportion of alpha-(1 -> 4) linkages while the analogous GtfA-51062N maintained enzyme activity but increased the proportion of alpha-(1 -> 4) linkages. This study of dextransucrase from Weissella spp. thus elucidated differences between glucansucrases and will facilitate study of the structure-function relationships of dextran and isomalto-oligosaccharides.