Amylosucrase (ASase; EC 2.4.1.4) synthesizes alpha-1,4-glucans using sucrose as a sole substrate. The aim of this study was to compare the enzymatic properties of four recombinant ASase genes to determine the underlying mechanisms thereof. Following cloning and expression in Escherichia coli, we determined that the ASase enzyme from Deinococcus geothermalis (DGAS) had the highest thermostability whereas ASase from Neisseria polysaccharea (NPAS) showed the greatest polymerization activity. Chimeric ASases were constructed using dgas and npas genes by overlap extension polymerase chain reaction. Two of the six chimeric ASases generated, NPAS-B' and DGAS-B, showed ASase activity using sucrose as the sole substrate. However, DGAS-B was not able to produce longer alpha-1,4-glucans; the highest degree of polymerization was <12. In the kinetic study, not only the substrate binding affinity but also the production rate of DGAS-B was greater than those of DGAS. Molecular dynamic computational simulation suggested that DGAS-B could not synthesize longer glucan chains because of the change in flexibilities of loops 4, 7, and 8 as compared to those of DGAS. (C) 2016 Elsevier Inc. All rights reserved.