title: Cloning, expression, and characterization of a thermostable glucose-6-phosphate dehydrogenase from Thermoanaerobacter tengcongensis.
abstract: Glucose-6-phosphate dehydrogenases (G6PDs) are important enzymes widely used in bioassay and biocatalysis. In this study, we reported the cloning, expression, and enzymatic characterization of G6PDs from the thermophilic bacterium Thermoanaerobacter tengcongensis MB4 (TtG6PD). SDS-PAGE showed that purified recombinant enzyme had an apparent subunit molecular weight of 60 kDa. Kinetics assay indicated that TtG6PD preferred NADP(+) (k cat/K m = 2618 mM(-1) s(-1), k cat = 249 s(-1), K m = 0.10 ± 0.01 mM) as cofactor, although NAD(+) (k cat/K m = 138 mM(-1) s(-1), k cat = 604 s(-1), K m = 4.37 ± 0.56 mM) could also be accepted. The K m values of glucose-6-phosphate were 0.27 ± 0.07 mM and 5.08 ± 0.68 mM with NADP(+) and NAD(+) as cofactors, respectively. The enzyme displayed its optimum activity at pH 6.8-9.0 for NADP(+) and at pH 7.0-8.6 for NAD(+) while the optimal temperature was 80 °C for NADP(+) and 70 °C for NAD(+). This was the first observation that the NADP(+)-linked optimal temperature of a dual coenzyme-specific G6PD was higher than the NAD(+)-linked and growth (75 °C) optimal temperature, which suggested G6PD might contribute to the thermal resistance of a bacterium. The potential of TtG6PD to measure the activity of another thermophilic enzyme was demonstrated by the coupled assays for a thermophilic glucokinase.
authors: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, People's Republic of China. lizilong03@163.com.|State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People's Republic of China. lizilong03@163.com.
authors: Zilong
authors: Z
authors: Li
authors: Department of Industrial Microbiology and Biotechnology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People's Republic of China.
authors: Ning
authors: N
authors: Jiang
authors: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People's Republic of China. yangkq@im.ac.cn.
authors: Keqian
authors: K
authors: Yang
authors: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, People's Republic of China. jtzheng@ipe.ac.cn.
authors: Jianting
authors: J
authors: Zheng
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D013379:Substrate Specificity; D046968:Thermoanaerobacter / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D005954:Glucosephosphate Dehydrogenase
keywords: Coupling enzyme; Dual-coenzyme specific; Glucose-6-phosphate dehydrogenase; Kinetics; Thermoanaerobacter tengcongensis; Thermostable