Hyun Jeongwoo, Abigail Maria, Choo Jin Woo, Ryu Jin, Kim Hyung Kwoun
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.
J Microbiol Biotechnol. 2016 Oct 28;26(10):1708-1716. doi: 10.4014/jmb.1603.03021.
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.
葡萄糖脱氢酶（GDH）是一种氧化还原酶，在还原酶介导的手性合成反应中用作生物催化剂以再生NAD(P)H。在本研究中，从 亚种中克隆了葡萄糖1 -脱氢酶B基因（），并在BL21（DE3）中表达了野生型（GDH - BT）和His标签型（GDH - BT、GDH - BT）酶。所有酶均以可溶形式从 中产生。GDH - BT和GDH - BT分别显示出6.6 U/mg和5.5 U/mg的高比酶活性，而GDH - BT显示出非常低的比酶活性，为0.020 U/mg。这些结果表明完整的C末端羧基对GDH - BT活性很重要。GDH - BT在高达65°C时稳定，而GDH - BT和GDH - BT在高达45°C时稳定。凝胶渗透色谱表明GDH - BT是二聚体，而GDH - BT和GDH - BT是单体。这些结果表明完整的N末端和C末端是GDH - BT维持热稳定性和形成其二聚体结构所必需的。基于 GDH的晶体结构（PDB ID 3AY6）构建了GDH - BT单亚基的同源模型，表明GDH - BT具有Rossmann折叠结构，其N末端和C末端位于亚基表面，这表明His标签影响了天然二聚体结构。GDH - BT和GDH - BT在酵母还原酶介导的手性合成反应中再生NADPH，表明这些酶可作为精细化工和制药行业的催化剂。
