title: An unusual chimeric amylosucrase generated by domain-swapping mutagenesis.
abstract: Amylosucrase (ASase; EC 2.4.1.4) synthesizes α-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 α-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 8as compared to those of DGAS.
authors.affiliation: Graduate School of Biotechnology and Institute of Life Science and Resources, Kyung Hee University, Yongin 446-701, Republic of Korea; Korea Food Research Institute, Seongnam, Gyeonggi 463-746, Republic of Korea.
authors.forename: Dong-Ho
authors.initials: DH
authors.lastname: Seo
authors.affiliation: Graduate School of Biotechnology and Institute of Life Science and Resources, Kyung Hee University, Yongin 446-701, Republic of Korea; Korea Atomic Energy Research Institute, Jeongeup, Jeonbuk 580-185, Republic of Korea.
authors.forename: Jong-Hyun
authors.initials: JH
authors.lastname: Jung
authors.affiliation: Graduate School of Biotechnology and Institute of Life Science and Resources, Kyung Hee University, Yongin 446-701, Republic of Korea.
authors.forename: Dong-Hyun
authors.initials: DH
authors.lastname: Jung
authors.affiliation: Graduate School of Biotechnology and Institute of Life Science and Resources, Kyung Hee University, Yongin 446-701, Republic of Korea.
authors.forename: Sunyoung
authors.initials: S
authors.lastname: Park
authors.affiliation: Department of Food Science and Technology, Sejong University, Seoul 143-747, Republic of Korea.
authors.forename: Sang-Ho
authors.initials: SH
authors.lastname: Yoo
authors.affiliation: Graduate School of Biotechnology and Institute of Life Science and Resources, Kyung Hee University, Yongin 446-701, Republic of Korea.
authors.forename: Young-Rok
authors.initials: YR
authors.lastname: Kim
authors.affiliation: Graduate School of Biotechnology and Institute of Life Science and Resources, Kyung Hee University, Yongin 446-701, Republic of Korea. Electronic address: cspark@khu.ac.kr.
authors.forename: Cheon-Seok
authors.initials: CS
authors.lastname: Park
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D034301:Deinococcus / Q000201:enzymology / Q000235:genetics; D004795:Enzyme Stability; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D016296:Mutagenesis; D009343:Neisseria / Q000201:enzymology / Q000235:genetics; D015202:Protein Engineering; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D005964:Glucosyltransferases; C019700:amylosucrase
keywords: Amylosucrase; Chimeric enzyme; Deinococcus geothermalis; Domain swapping; Neisseria polysaccharea