title: Development and characterization of cyclodextrin glucanotransferase as a maltoheptaose-producing enzyme using site-directed mutagenesis.
abstract: Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) mainly produces cyclodextrins (CDs) using linear maltooligosaccharides. We performed site-directed saturation mutagenesis on the +1 substrate-binding residue, H233, of CGTase from alkalophilic Bacillus sp. I-5 to prepare specific-length oligosaccharides. The obtained mutant CGTase, H233Y, primarily produced maltoheptaose (G7) using β-CD via a hydrolysis reaction. A kinetic study of H233Y showed that the kcat/Km value of β-CD was 7-fold greater than that of G7, which accounts for the accumulation of G7 during the H233Y enzyme reaction. A structure comparison of CGTases with H233Y modeling suggests that the substitution of H233Y may alter the position of the +1 subsite and slow the further hydrolysis of G7 after the ring-opening reaction.
authors.affiliation: Department of Food Science and Nutrition, Hallym University, Chuncheon, Republic of Korea.
authors.forename: Ye-Seul
authors.initials: YS
authors.lastname: Koo
authors.affiliation: Department of Food Science and Nutrition, Hallym University, Chuncheon, Republic of Korea.
authors.forename: Hye-Won
authors.initials: HW
authors.lastname: Lee
authors.affiliation: Department of Food Science and Nutrition, Hallym University, Chuncheon, Republic of Korea.
authors.forename: Hye-Yeon
authors.initials: HY
authors.lastname: Jeon
authors.affiliation: Department of Food Science and Nutrition, Hallym University, Chuncheon, Republic of Korea.
authors.forename: Hye-Jeong
authors.initials: HJ
authors.lastname: Choi
authors.affiliation: Department of Food Science and Nutrition, Hallym University, Chuncheon, Republic of Korea.
authors.forename: Woo-Jae
authors.initials: WJ
authors.lastname: Choung
authors.affiliation: Department of Food Science and Nutrition, Hallym University, Chuncheon, Republic of Korea Department of Chemistry, University of British Columbia, Vancouver, BC, Canada jhshim@hallym.ac.kr.
authors.forename: Jae-Hoon
authors.initials: JH
authors.lastname: Shim
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005936:Glucans / Q000378:metabolism*; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D005936:Glucans; D011994:Recombinant Proteins; C031146:maltoheptaose; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase
keywords: cyclodextrin; cyclodextrin glucanotransferase; maltoheptaose