<p>Glucansucrases catalyze the production of α-glucans using sucrose as substrate with distinct physicochemical characteristics originates from mainly the type of linkages, degree of branching and molecular mass. New forms of α-glucans can be produced by applying specific mutations to glucansucrases. In this study, a single and double mutation at Leu⁹³³ and Leu⁹³⁵ sites of glucansucrase E81 was applied and L933I and L933I-L935F sites modified glucansucrase was obtained. Structural characterisation of bioengineered α-glucans revealed that ratio of (1 → 3):(1 → 6) linked α-Glc units altered at L933I whereas no (1 → 3)-linked α-Glc units were presented for L933I-L935F modified glucansucrase detected by NMR analysis. Both bioengineered α-glucans had lower molecular weights in comparison to glucan E81 detected by GPC analysis. TGA and DSC analysis were applied to detect the thermal profiles of bioengineered α-glucans whereas FTIR and XRD analysis were applied to determine the alterations in the structural and physical nature of the bioengineered α-glucans, respectively. Finally, SEM and AFM analysis were applied to explore the morphological modifications of the bioengineered α-glucans. This study provides a better understanding for the potential role of final glucan structures on their physicochemical characteristics. Keywords: glucansucrase; carbohydrate engineering; mutation. </p>