Identification and characterization of thermostable glucose dehydrogenases from thermophilic filamentous fungi
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- 作者：Kazumichi Ozawa ; Hisanori Iwasa ; Noriko Sasaki…
- 关键词：FAD ; dependent glucose dehydrogenase ; Gene cloning ; Recombinant protein production ; Thermostability
- 刊名：Applied Microbiology and Biotechnology
- 出版年：2017
- 出版时间：January 2017
- 年：2017
- 卷：101
- 期：1
- 页码：173-183
- 全文大小：
- 刊物类别：Chemistry and Materials Science
- 刊物主题：Microbiology; Microbial Genetics and Genomics; Biotechnology;
- 出版者：Springer Berlin Heidelberg
- ISSN：1432-0614
- 卷排序：101
文摘
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 (Tm) 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.