Heterologous production of extreme alkaline thermostable NAD+-dependent formate dehydrogenase with wide-range pH activity from Myceliophthora thermophila |
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| Institution: | 1. Yildiz Technical University, Faculty of Science and Letters, Department of Chemistry, 34210, Istanbul, Turkey;2. Gebze Technical University, Department of Chemistry, 41400 Gebze, Kocaeli, Turkey;3. Gebze Technical University, Department of Molecular Biology and Genetics, 41400 Gebze, Kocaeli, Turkey;4. Laboratory of Enzyme Technology, Department of Biotechnology, School of Food, Biotechnology and Development, Agricultural University of Athens,75 Iera Odos Street, GR-11855 Athens, Greece;5. Gebze Technical University, Department of Bioengineering, 41400 Gebze, Kocaeli, Turkey;1. Faculty of Agriculture, Shinshu University, 8304 Minamiminowa, Nagano 399-4511, Japan;2. JST, PRESTO, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan;3. Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan;1. Istanbul Technical University, Faculty of Science and Letters, Department of Molecular Biology and Genetics, 34469 Istanbul, Turkey;2. Istanbul Technical University, Molecular Biology-Biotechnology & Genetics Research Center (MOBGAM), 34469 Istanbul Turkey;3. Department of Biotechnology and Chemical Technology, School of Chemical Technology, Aalto University, FI-00076 Aalto, Finland;4. School of Chemistry, University of Manchester, Manchester Institute of Biotechnology, 131 Princess Street, M1 7DN Manchester, UK;5. Istanbul AREL University, Molecular Biology and Genetics, Tepekent, Büyükçekmece, 34537 Istanbul, Turkey;1. Clean Energy Research Centre, Korea Institute of Science and Technology, P.O. Box 131, Cheongryyang, Seoul 136 791, Republic of Korea;2. Department of Microbial Engineering, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, Republic of Korea;3. Department of Bio & Nano Chemistry, College of Natural Science, Kookmin University, 861-1, Jeongneung-dong, Sungbuk-gu, Seoul 137-702, Republic of Korea |
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| Abstract: | NAD+-dependent formate dehydrogenase(s) (EC 1.2.1.2, FDH) catalyzes the interconversion of formate anion to carbon dioxide coupled with the conversion of NAD+ or NADH. FDHs attract significant attention in biotechnology due to their potential applications in NAD(H)-dependent industrial biocatalysis as well as in the production of renewable fuels and chemicals from carbon dioxide. In the present work, a new FDH from thermophilic fungus Myceliophthora thermophile (MtFDH) was characterized. The gene of the enzyme was synthesised, cloned, expressed in E. coli, as 6His-tagged protein, and purified to homogeneity by metal chelate affinity chromatography. Kinetic analysis suggested that MtFDH exhibits higher catalytic efficiency on NaHCO3 compared to formate. Notable, recombinant MtFDH displays a pH optimum for the conversion of formate anion to carbon dioxide at extreme alkaline pH (pH 10.5). Thermal stability analysis showed that the enzyme displays good thermostability with Tm 48 °C. Homology modelling and phylogenetic analysis suggested that the enzyme belongs to the D-specific 2-hydroxy acid dehydrogenases family. The active-site residues are well conserved compared to other homologous FDHs. The results of the present work provide new knowledge on the structure, function and diversity of FDHs and indicate that MtFDH possess a huge potential for CO2 reduction or NADH generation and under extreme alkaline conditions. |
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| Keywords: | Heterologous production Extreme alkaline Computational modeling NADH regeneration CO2 reduction |
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