title: Altered large-ring cyclodextrin product profile due to a mutation at Tyr-172 in the amylomaltase of Corynebacterium glutamicum.
abstract: Corynebacterium glutamicum amylomaltase (CgAM) catalyzes the formation of large-ring cyclodextrins (LR-CDs) with a degree of polymerization of 19 and higher. The cloned CgAM gene was ligated into the pET-17b vector and used to transform Escherichia coli BL21(DE3). Site-directed mutagenesis of Tyr-172 in CgAM to alanine (Y172A) was performed to determine its role in the control of LR-CD production. Both the recombinant wild-type (WT) and Y172A enzymes were purified to apparent homogeneity and characterized. The Y172A enzyme exhibited lower disproportionation, cyclization, and hydrolysis activities than the WT. The k(cat)/K(m) of the disproportionation reaction of the Y172A enzyme was 2.8-fold lower than that of the WT enzyme. The LR-CD product profile from enzyme catalysis depended on the incubation time and the enzyme concentration. Interestingly, the Y172A enzyme showed a product pattern different from that of the WT CgAM at a long incubation time. The principal LR-CD products of the Y172A mutated enzyme were a cycloamylose mixture with a degree of polymerization of 28 or 29 (CD28 or CD29), while the principal LR-CD product of the WT enzyme was CD25 at 0.05 U of amylomaltase. These results suggest that Tyr-172 plays an important role in determining the LR-CD product profile of this novel CgAM.
authors.affiliation: Starch and Cyclodextrin Research Unit, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand.
authors.forename: Wiraya
authors.initials: W
authors.lastname: Srisimarat
authors.forename: Jarunee
authors.initials: J
authors.lastname: Kaulpiboon
authors.forename: Kuakarun
authors.initials: K
authors.lastname: Krusong
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Zimmermann
authors.forename: Piamsook
authors.initials: P
authors.lastname: Pongsawasdi
mesh_terms: D003001:Cloning, Molecular; D048230:Corynebacterium glutamicum / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D003505:Cyclodextrins / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006004:Glycogen Debranching Enzyme System / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D050505:Mutant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020125:Mutation, Missense; D010957:Plasmids
chemical_list: D003505:Cyclodextrins; D006004:Glycogen Debranching Enzyme System; D050505:Mutant Proteins; C020569:4 alpha-glucanotransferase