An unusual chimeric amylosucrase generated by domain-swapping mutagenesis

作者：Seo Dong Ho; Jung Jong Hyun; Jung Dong Hyun; Park Sunyoung; Yoo Sang Ho; Kim Young Rok; Park Cheon Seok^*

来源：Enzyme and Microbial Technology, 2016, 86: 7-16.

DOI：10.1016/j.enzmictec.2016.01.004

• Amylosucrase
• Deinococcus geothermalis
• Neisseria polysaccharea
• Domain swapping
• Chimeric enzyme

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.

• 出版日期2016-5