Effects of N-/C-Terminal Extra Tags on the Optimal Reaction Conditions, Activity, and Quaternary Structure of Glucose 1-Dehydrogenase.

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 () was cloned from subsp. , and wild-type (GDH-BT) and His-tagged (GDH-BT, GDH-BT) enzymes were produced in BL21 (DE3). All enzymes were produced in the soluble forms from . GDH-BT and GDH-BT showed high specific enzymatic activities of 6.6 U/mg and 5.5 U/mg, respectively, whereas GDH-BT showed 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-BT was stable up to 65°C, whereas GDH-BT and GDH-BT were stable up to 45°C. Gel permeation chromatography showed that GDH-BT is a dimer, whereas GDH-BT and GDH-BT are 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-BT single subunit was constructed based on the crystal structure of GDH (PDB ID 3AY6), showing that GDH-BT has 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-BT and GDH-BT regenerated NADPH in a yeast reductase-mediated chiral synthesis reaction, suggesting that these enzymes can be used as catalysts in fine-chemical and pharmaceutical industries.