title: Development and Application of Cyclodextrin Hydrolyzing Mutant Enzyme Which Hydrolyzes β- and γ-CD Selectively.
abstract: Cyclodextrins (CDs) are produced from starch by cyclodextrin glucanotransferase (CGTase), which has cyclization activity. Specifically, α-CD is an important biomolecule, as it is a molecular carrier and soluble dietary fiber used in the food industry. Upon inspection of the conserved regions of the glycoside hydrolase (GH) 13 family amylases, the amino acids K232 and H233 of CGTase were identified as playing an important role in enzyme reaction specificity. A novel CD hydrolyzing enzyme, cyclodextrin glycosyl transferase (CGTase)-alpha, was developed using site-directed mutagenesis at these positions. Action pattern analysis using various substrates revealed that CGTase-alpha was able to hydrolyze β- and γ-CD, but not α-CD. This selective CD hydrolyzing property was employed to purify α-CD from a CD mixture solution. The α-CD that remained after treatment with CGTase-alpha and exotype glucoamylase was purified using hydrophobic interaction chromatography with 99% purity.
authors.affiliation: Department of Food Science and Nutrition and Center for Aging and Health Care, Hallym University , Hallymdaehak-gil 1, Chuncheon, Gangwon-do 24252, Korea.
authors.forename: Ye-Seul
authors.initials: YS
authors.lastname: Koo
authors.affiliation: Department of Food Science and Nutrition and Center for Aging and Health Care, Hallym University , Hallymdaehak-gil 1, Chuncheon, Gangwon-do 24252, Korea.
authors.forename: Dam-Seul
authors.initials: DS
authors.lastname: Ko
authors.affiliation: Department of Food Science and Nutrition and Center for Aging and Health Care, Hallym University , Hallymdaehak-gil 1, Chuncheon, Gangwon-do 24252, Korea.
authors.forename: Da-Woon
authors.initials: DW
authors.lastname: Jeong
authors.affiliation: Department of Food Science and Nutrition and Center for Aging and Health Care, Hallym University , Hallymdaehak-gil 1, Chuncheon, Gangwon-do 24252, Korea.
authors.forename: Jae-Hoon
authors.identifier: http://orcid.org/0000-0002-0230-1769
authors.initials: JH
authors.lastname: Shim
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016415:Sequence Alignment; D013379:Substrate Specificity; D047391:alpha-Cyclodextrins / Q000737:chemistry* / Q000378:metabolism; D047408:gamma-Cyclodextrins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D047391:alpha-Cyclodextrins; D047408:gamma-Cyclodextrins; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase
keywords: CGTase; amylase; bioconversion; cyclodextrins; site-directed mutagenesis; α-cyclodextrin