title: Effects of N-/C-Terminal Extra Tags on the Optimal Reaction Conditions, Activity, and Quaternary Structure ofBacillus thuringiensisGlucose 1-Dehydrogenase.
abstract: Glucose dehydrogenase (GDH) is an oxidoreductase enzyme and is used as a biocatalyst to regenerate NAD(P)H in reductase-mediated chiral synthesis reactions. In this study, the glucose 1-dehydrogenase B gene (gdhB) was cloned fromBacillus thuringiensissubsp.kurstaki, and wild-type (GDH-BTWT) and His-tagged (GDH-BTN-His, GDH-BTC-His) enzymes were produced inEscherichia coliBL21 (DE3). All enzymes were produced in the soluble forms fromE. coli. GDH-BTWTand GDH-BTN-Hisshowed high specific enzymatic activities of 6.6 U/mg and 5.5 U/mg, respectively, whereas GDH-BTC-Hisshowed a very low specific enzymatic activity of 0.020 U/mg. These results suggest that the intact C-terminal carboxyl group is important for GDH-BT activity. GDH-BTWTwas stable up to 65°C, whereas GDH-BTN-Hisand GDH-BTC-Hiswere stable up to 45°C. Gel permeation chromatography showed that GDH-BTWTis a dimer, whereas GDH-BTN-Hisand GDH-BTC-Hisare monomeric. These results suggest that the intact N- and C-termini are required for GDH-BT to maintain thermostability and to form its dimer structure. The homology model of the GDH-BTWTsingle subunit was constructed based on the crystal structure ofBacillus megateriumGDH (PDB ID 3AY6), showing that GDH-BTWThas a Rossmann fold structure with its N- and C-termini located on the subunit surface, which suggests that His-tagging affected the native dimer structure. GDH-BTWTand GDH-BTN-Hisregenerated NADPH in a yeast reductase-mediated chiral synthesis reaction, suggesting that these enzymes can be used as catalysts in fine-chemical and pharmaceutical industries.
authors.affiliation: Division of Biotechnology, The Catholic University of Korea, Bucheon 14662, Republic of Korea.
authors.forename: Jeongwoo
authors.initials: J
authors.lastname: Hyun
authors.affiliation: Division of Biotechnology, The Catholic University of Korea, Bucheon 14662, Republic of Korea.|Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia, Jakarta 12930, Indonesia.
authors.forename: Maria
authors.initials: M
authors.lastname: Abigail
authors.affiliation: Division of Biotechnology, The Catholic University of Korea, Bucheon 14662, Republic of Korea.
authors.forename: Jin Woo
authors.initials: JW
authors.lastname: Choo
authors.affiliation: Division of Biotechnology, The Catholic University of Korea, Bucheon 14662, Republic of Korea.
authors.forename: Jin
authors.initials: J
authors.lastname: Ryu
authors.affiliation: Division of Biotechnology, The Catholic University of Korea, Bucheon 14662, Republic of Korea.
authors.forename: Hyung Kwoun
authors.initials: HK
authors.lastname: Kim
mesh_terms: D001413:Bacillus thuringiensis / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D042843:Glucose 1-Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D009249:NADP / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D009249:NADP; D042843:Glucose 1-Dehydrogenase
keywords: Bacillus thuringiensis; Glucose dehydrogenase; His-tag; NADPH regeneration; homology model