Improved polymerization activity of Deinococcus geothermalis amylosucrase by semi-rational design: Effect of loop flexibility on the polymerization reaction.

In this study, we improved the polymerization activity of Deinococcus geothermalis amylosucrase (DGAS) via semi-rational design to support increased potential industrial applications. Structural analysis selected three amino acid residues (Pro219, Phe225, and Ala226) in loop 3 of the B domain that were considered to be related to polymerization activity. Various DGAS variants were constructed through site-saturated mutagenesis, from which two mutants (DGAS-P219Y and DGAS-A226N) with increased polymerization activity were selected by fluorescence screening. DGAS-A226N synthesized longer amylose-like polymers from sucrose than those from wild-type (WT) DGAS. A kinetic study of sucrose as a substrate showed that the ratio of polymerization to hydrolysis activity of DGAS-A226N was significantly higher than that of WT DGAS. DGAS-A226N exhibited its highest enzymatic activity at 50 °C; its half-life was 6.8 min at 60 °C, and its melting temperature was 61.16 ± 0.09 °C. Computational simulation analysis of DGAS-A226N indicated that its conformation was more compact than that of WT DGAS, possibly owing to decreased flexibility of loops 2, 3, 4, 7, and 8. Thus, conformational changes in DGAS-A226N allowed the production of longer oligosaccharides, which may support a broader range of industrial application by affording novel or improved product characteristics.