Identification and characterization of thermostable glucose dehydrogenases from thermophilic filamentous fungi

• Published in: Applied microbiology and biotechnology 2017-01, Vol.101 (1), p.173-183
• Main Authors: Ozawa, Kazumichi, Iwasa, Hisanori, Sasaki, Noriko, Kinoshita, Nao, Hiratsuka, Atsunori, Yokoyama, Kenji
• Format: Article
• Language: English
• Subjects: Analysis; Biomedical and Life Sciences; Biosensors; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Catalysis; Cloning; Cloning, Molecular; Coenzymes - analysis; Dehydrogenase; Dehydrogenases; E coli; Enzyme Stability; Enzymes; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Flavin-Adenine Dinucleotide - analysis; Fungi; Fungi - enzymology; Fungi - genetics; Gene Expression; Genetic recombination; Glucose; Glucose Dehydrogenases - chemistry; Glucose Dehydrogenases - genetics; Glucose Dehydrogenases - isolation & purification; Glucose Dehydrogenases - metabolism; Glucose metabolism; Hot Temperature; Life Sciences; Microbial Genetics and Genomics; Microbiology; Molds (Fungi); Observations; Oxidation; Oxidoreductases; Physiological aspects; Pichia - genetics; Pichia - metabolism; Pichia pastoris; Polymerase chain reaction; Protein synthesis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Sensors; Spectrum Analysis; Studies; Talaromyces; Talaromyces - enzymology; Talaromyces - genetics; Thermoascus - enzymology; Thermoascus - genetics
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-016-7754-7

FAD-dependent glucose dehydrogenase (FAD-GDH), which contains FAD as a cofactor, catalyzes the oxidation of d -glucose to d -glucono-1,5-lactone, and plays an important role in biosensors measuring blood glucose levels. In order to obtain a novel FAD-GDH gene homolog, we performed degenerate PCR screening of genomic DNAs from 17 species of thermophilic filamentous fungi. Two FAD-GDH gene homologs were identified and cloned from Talaromyces emersonii NBRC 31232 and Thermoascus crustaceus NBRC 9129. We then prepared the recombinant enzymes produced by Escherichia coli and Pichia pastoris . Absorption spectra and enzymatic assays revealed that the resulting enzymes contained oxidized FAD as a cofactor and exhibited glucose dehydrogenase activity. The transition midpoint temperatures ( T m ) were 66.4 and 62.5 °C for glycosylated FAD-GDHs of T. emersonii and T. crustaceus prepared by using P. pastoris as a host, respectively. Therefore, both FAD-GDHs exhibited high thermostability. In conclusion, we propose that these thermostable FAD-GDHs could be ideal enzymes for use as thermotolerant glucose sensors with high accuracy.