Met349 Mutations Enhance the Activity of 1,4-α-Glucan Branching Enzyme from Geobacillus thermoglucosidans STB02.

Abstract

1,4-α-Glucan branching enzyme (GBE, EC 2.4.1.18) is used to increase the number of α-1,6 branch points in starch and glycogen. On the basis of a multiple sequence alignment of the GBEs from a variety of bacteria, residue 349 (Geobacillus thermoglucosidans STB02 numbering) in region III is generally methionine in bacteria with higher identity, while it is threonine or serine in bacteria with lower identity. Four mutants (M349T, M349S, M349H, and M349Y) were constructed by site-directed mutagenesis and characterized. M349T and M349S showed 24.5% and 21.1% increases in specific activity compared with that of wild-type GBE, respectively. In addition, M349T and M349S displayed 24.2% and 17.6% enhancements in the α-1,6-glycosidic linkage ratio of potato starch samples, respectively. However, M349Y displayed a significant reduction in activity. Moreover, the mutations at M349 have a negligible effect on substrate specificity. Thus, M349T and M349S are more suitable for industrial applications than wild-type GBE.