Molecular Cloning of the Amylosucrase Gene from a Moderate Thermophilic Bacterium Deinococcus Geothermalis and Analysis of its Dual Enzyme Activity

Amylosucrase (AS; EC 2.4.1.4) is an interesting enzyme to form an insoluble amylosetype polymer from sucrose. A putative AS gene was cloned from Deinococcus geothermalis (dgas) and efficiently expressed in E. coli using the glutathione Stransferase fusion system. The optimal reaction temperature and pH for the sucrosehydrolysis activity of DGAS were determined to be 45°C and pH 8, respectively. When compared with other ASs, DGAS has exceptionally thermostable characteristics, as demonstrated by a half-life of 6.8. h at 55°C, indicating this enzyme is the most active and thermostable AS known to date. DGAS also showed transglucosylation activity using sucrose as a sole substrate. However, the optimal temperature for transglucosylation was observed to be lower (25°C) than that for hydrolysis. These results suggest that two different catalytic activities in one enzyme demonstrate an opposite trend in its optimal reaction conditions. The transglucosylation reaction of DGAS with sucrose and maltooligosaccharides (G1-G7) as donor and acceptor molecules reveals that maltooligosaccharides need to be longer than maltotriose in order to be efficient acceptors for the glucan polymerization by DGAS. It was also found that some monosaccharides other than sucrose (galactose, xylose, methylα-D-gluco-pyranoside, and methylβ-D-glucopyranoside) can be used as acceptors for the DGAS transglucosylation reaction.