title: Mutation of tyrosine167histidine at remote substrate binding subsite -6 in α-cyclodextrin glycosyltransferase enhancing α-cyclodextrin specificity by directed evolution.
abstract: α-Cyclodextrin glycosyltransferase (α-CGTase) can convert starch into α-cyclodextrin with various proportions of β-cyclodextrin and/or γ-cyclodextrin in the products. To improve the α-cyclodextrin-forming specificity, directed evolution on the wild-type α-CGTase was performed by constructing mutant library with error-prone PCR method. The positive mutant strains were selected in combination of starch plate screening with HPLC detection of the products. An α-CGTase from the mutant strain (assigned No. 95) was found to be able to increase the α:β ratio in product mixture from 3.4 to 7.8 in comparison with the wild-type α-CGTase. Sequence alignment indicated that two mutations occurred in the No. 95 mutant α-CGTase, which were Y167H and A536V. Reverse mutation revealed that Y167H was responsible for this change. A series of 167 site-substituted mutants could improve the α:β ratio to different extents as indicated by saturated mutagenesis, with Y167H as the best substitution. In conclusion, Y167 was confirmed to be one of the main subsites in the -6 domain of α-CGTase that is responsible for the α:β ratio in the product mixture. Y167H is most preferable among all types of mutant enzymes tested at this site. The reconstructed Y167H (i.e., No. 95) α-CGTase showed better potential for α-cyclodextrin production on industrial scale.
authors.affiliation: State Key Laboratories of Transducer Technology, National Engineering Lab for Industrial Enzymes, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China, 2005sbh@163.com.
authors.forename: Binghong
authors.initials: B
authors.lastname: Song
authors.forename: Yang
authors.initials: Y
authors.lastname: Yue
authors.forename: Ting
authors.initials: T
authors.lastname: Xie
authors.forename: Shijun
authors.initials: S
authors.lastname: Qian
authors.forename: Yapeng
authors.initials: Y
authors.lastname: Chao
mesh_terms: D019943:Amino Acid Substitution; D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D019020:Directed Molecular Evolution; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism; D008958:Models, Molecular; D008968:Molecular Conformation; D016296:Mutagenesis; D010802:Phylogeny; D017354:Point Mutation; D017433:Protein Structure, Secondary; D013213:Starch / Q000378:metabolism*; D013379:Substrate Specificity; D014443:Tyrosine / Q000378:metabolism; D047391:alpha-Cyclodextrins / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D047391:alpha-Cyclodextrins; D014443:Tyrosine; D006639:Histidine; D013213:Starch; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; C032613:alpha-cyclodextrin
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