title: Mutations of Lysine 47 in cyclodextrin glycosyltransferase from Paenibacillus macerans enhance beta-cyclodextrin specificity.
abstract: The nature of amino acid residue 47 shows a clear discrimination between the different groups of cyclodextrin glycosyltransferase (CGTase). The effects of amino acid side chain at position 47 on cyclodextrin product specificity were investigated by replacing Lys47 in the CGTase from Paenibacillus macerans strain JFB05-01 with arginine, histidine, threonine, serine, or leucine. All of the mutations reduced alpha-cyclodextrin-forming activity, whereas significant increases in beta-cyclodextrin-forming activity were achieved. Especially, the mutations of Lys47 into threonine, serine, or leucine converted P. macerans CGTase from alpha-type to beta/alpha-type. As a result, all of the mutants displayed a shift in product specificity toward the production of beta-cyclodextrin. Thus, they were more suitable for the industrial production of beta-cyclodextrin than the wild-type enzyme. The enhancement of beta-cyclodextrin specificity might be due to weakening or removal of hydrogen-bonding interactions between the side chain of residue 47 and the bent intermediate specific for alpha-cyclodextrin formation.
authors.affiliation: State Key Laboratory of Food Science and Technology, People's Republic of China.
authors.forename: Zhao-Feng
authors.initials: ZF
authors.lastname: Li
authors.forename: Jia-Yu
authors.initials: JY
authors.lastname: Zhang
authors.forename: Qi
authors.initials: Q
authors.lastname: Sun
authors.forename: Miao
authors.initials: M
authors.lastname: Wang
authors.forename: Zheng-Biao
authors.initials: ZB
authors.lastname: Gu
authors.forename: Guo-Cheng
authors.initials: GC
authors.lastname: Du
authors.forename: Jing
authors.initials: J
authors.lastname: Wu
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
mesh_terms: D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics; D007930:Leucine / Q000737:chemistry; D008239:Lysine / Q000737:chemistry / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D056507:Paenibacillus / Q000201:enzymology*; D012694:Serine / Q000737:chemistry; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013912:Threonine / Q000737:chemistry; D047391:alpha-Cyclodextrins / Q000378:metabolism; D047392:beta-Cyclodextrins / Q000378:metabolism*
chemical_list: D047391:alpha-Cyclodextrins; D047392:beta-Cyclodextrins; D013912:Threonine; D012694:Serine; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; D007930:Leucine; C031215:betadex; D008239:Lysine; C032613:alpha-cyclodextrin