Impact of G449 Single-Point Mutation on Glucansucrase URE 13-300 Enzyme Properties and Polysaccharide Structure

Abstract

High-molecular-weight glucansucrase (GS) URE 13-300 with two catalytic domains (CDs) synthesizes insoluble branched α-glucan. In the present work, we explore the role of the amino acid glycine 449 (G449) located in domain B of CD1 on the enzyme properties and polysaccharide structure. Glycine was substituted with lysine via site-directed mutagenesis and the mutant DNA was expressed in recombinant Escherichia coli BL21 (DE3). The obtained mutant glucansucrase U13M1 had a shifted optimum pH, from 5.3 to 6.5, and a decreased optimal temperature, from 30 to 20 °C. The modified glucan, synthesized using U13M1, retained the water-insoluble nature of the URE 13-300 glucan and also has altered linkage composition, with about 30% fewer α-(1 → 3) linked glucose residues in the main chain. This is the first mutagenesis study on glucansucrase with two catalytic domains in a non-truncated form.