title: Mutagenesis for improvement of activity and thermostability of amylomaltase from Corynebacterium glutamicum.
abstract: This work aims to improve thermostability of amylomaltase from a mesophilic Corynebacterium glutamicum (CgAM) by random and site-directed mutagenesis. From error prone PCR, a mutated CgAM with higher thermostability at 50 °C compared to the wild-type was selected and sequenced. The result showed that the mutant contains a single mutation of A406V. Site-directed mutagenesis was then performed to construct A406V and A406L. Both mutated CgAMs showed higher intermolecular transglucosylation activity with an upward shift in the optimum temperature and a slight increase in the optimum pH for disproportionation and cyclization reactions. Thermostability of both mutated CgAMs at 35-40 °C was significantly increased with a higher peak temperature from DSC spectra when compared to the wild-type. A406V had a greater effect on activity and thermostability than A406L. The catalytic efficiency values kcat/Km of A406V- and A406L-CgAMs were 2.9 and 1.4 times higher than that of the wild-type, respectively, mainly due to a significant increase in kcat. LR-CD product analysis demonstrated that A406V gave higher product yield, especially at longer incubation time and higher temperature, in comparison to the wild-type enzyme.
authors.affiliation: Program in Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand; Starch and Cyclodextrin Research Unit, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
authors.forename: Pitchanan
authors.initials: P
authors.lastname: Nimpiboon
authors.affiliation: Department of Pre-Clinical Science, Biochemistry, Faculty of Medicine, Thammasat University, Pathumthani 12120, Thailand.
authors.forename: Jarunee
authors.initials: J
authors.lastname: Kaulpiboon
authors.affiliation: Starch and Cyclodextrin Research Unit, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
authors.forename: Kuakarun
authors.initials: K
authors.lastname: Krusong
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan.
authors.forename: Shigeyoshi
authors.initials: S
authors.lastname: Nakamura
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan.
authors.forename: Shun-ichi
authors.initials: S
authors.lastname: Kidokoro
authors.affiliation: Starch and Cyclodextrin Research Unit, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand. Electronic address: piamsook.p@chula.ac.th.
authors.forename: Piamsook
authors.initials: P
authors.lastname: Pongsawasdi
mesh_terms: D048230:Corynebacterium glutamicum / Q000201:enzymology*; D003505:Cyclodextrins / Q000378:metabolism; D004795:Enzyme Stability; D015971:Gene Expression Regulation, Enzymologic; D006004:Glycogen Debranching Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D003505:Cyclodextrins; D006004:Glycogen Debranching Enzyme System; C020569:4 alpha-glucanotransferase
keywords: Amylomaltase; Corynebacterium glutamicum; Large-ring cyclodextrin; Random mutagenesis; Site-directed mutagenesis; Thermostability