Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Abramoff MD, Magalhaes PJ, Ram SJ (2004) Image processing with ImageJ. Biophotonics Int 11:36–42
American Diabetes Association (2016) Standards of medical care in diabetes—2016. Diabetes Care 39:S1–S112
American Diabetes Association (2004) Hospital admission guidelines for diabetes. Diabetes Care 27:S103. doi:10.2337/diacare.27.2007.S103
Bak TG (1967a) Studies on glucose dehydrogenase of Aspergillus oryzae: II. purification and physical and chemical properties. Biochim Biophys Acta 139:277–293. doi:10.1016/0005-2744(67)90032-0
Bak TG (1967b) Studies on glucose dehydrogenase of Aspergillus oryzae: III. General enzymatic properties. Biochim Biophys Acta 146:317–327. doi:10.1016/0005-2744(67)90218-5
Berka RM, Grigoriev IV, Otillar R, Salamov A, Grimwood J, Reid I, Ishmael N, John T, Darmond C, Moisan MC, Henrissat B, Coutinho PM, Lombard V, Natvig DO, Lindquist E, Schmutz J, Lucas S, Harris P, Powlowski J, Bellemare A, Taylor D, Butler G, de Vries RP, Allijn IE, van den Brink J, Ushinsky S, Storms R, Powell AJ, Paulsen IT, Elbourne LD, Baker SE, Magnuson J, Laboissiere S, Clutterbuck AJ, Martinez D, Wogulis M, de Leon AL, Rey MW, Tsang A (2011) Comparative genomic analysis of the thermophilic biomass-degrading fungi Myceliophthora thermophila and Thielavia terrestris. Nat Biotechnol 29:922–927. doi:10.1038/nbt.1976
Breathnach R, Benoist C, O’Hare K, Gannon F, Chambon P (1978) Ovalbumin gene: evidence for a leader sequence in mRNA and DNA sequences at the exon-intron boundaries. Proc Natl Acad Sci U S A 75:4853–4857
Bretthauer RK, Castellino FJ (1999) Glycosylation of Pichia pastoris-derived proteins. Biotechnol Appl Biochem 30:193–200. doi:10.1111/j.1470-8744.1999.tb00770.x
Catterall JF, O’Malley BW, Robertson MA, Staden R, Tanaka Y, Brownlee GG (1978) Nucleotide sequence homology at 12 intron-exon junctions in the chick ovalbumin gene. Nature 275:510–513. doi:10.1038/275510a0
Crognale S, Pulci V, Brozzoli V, Petruccioli M, Federici F (2006) Expression of Penicillium variabile P16 glucose oxidase gene in Pichia pastoris and characterization of the recombinant enzyme. Enzyme Microb Technol 39:1230–1235. doi:10.1016/j.enzmictec.2006.03.005
Daly R, Hearn MTW (2005) Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J Mol Recognit 18:119–138. doi:10.1002/jmr.687
Dym O, Eisenberg D (2001) Sequence-structure analysis of FAD-containing proteins. Protein Sci 10:1712–1728. doi:10.1110/ps.12801
Ferri S, Kojima K, Sode K (2011) Review of glucose oxidases and glucose dehydrogenases: a bird’s eye view of glucose sensing enzymes. J Diabetes Sci Technol 5:1068–1076. doi:10.1177/193229681100500507
Guo Y, Lu F, Zhao H, Tang Y, Lu Z (2010) Cloning and heterologous expression of glucose oxidase gene from Aspergillus niger Z-25 in Pichia pastoris. Appl Biochem Biotechnol 162:498–509. doi:10.1007/s12010-009-8778-6
Hamamatsu N, Suzumura A, Nomiya Y, Sato M, Aita T, Nakajima M, Husimi Y, Shibanaka Y (2006) Modified substrate specificity of pyrroloquinoline quinone glucose dehydrogenase by biased mutation assembling with optimized amino acid substitution. Appl Microbiol Biotechnol 73:607–617. doi:10.1007/s00253-006-0521-4
Heller A, Feldman B (2008) Electrochemical glucose sensors and their applications in diabetes management. Chem Rev 108:2482–2505. doi:10.1021/cr068069y
Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77:61–68. doi:10.1016/0378-1119(89)90359-4
Igarashi S, Hirokawa T, Sode K (2004) Engineering PQQ glucose dehydrogenase with improved substrate specificity. Site-directed mutagenesis studies on the active center of PQQ glucose dehydrogenase. Biomol Eng 21:81–89. doi:10.1016/j.bioeng.2003.12.001
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. doi:10.1038/227680a0
Macheroux P (1999) UV-visible spectroscopy as a tool to study flavoproteins. In: Chapman SK, Reid GA (eds) Flavoprotein protocols. Humana Press, New York, pp 1–7
Maheshwari R, Bharadwaj G, Bhat MK (2000) Thermophilic fungi: their physiology and enzymes. Microbiol Mol Biol Rev 64:461–488. doi:10.1128/MMBR.64.3.461-488.2000
Maras M, van Die I, Contreras R, van den Hondel CA (1999) Filamentous fungi as production organisms for glycoproteins of bio-medical interest. Glycoconj J 16:99–107. doi:10.1023/A:1026436424881
Mori K, Nakajima M, Kojima K, Murakami K, Ferri S, Sode K (2011) Screening of Aspergillus-derived FAD-glucose dehydrogenases from fungal genome database. Biotechnol Lett 33:2255–2263. doi:10.1007/s10529-011-0694-5
Ogura Y (1951) Studies on the glucose dehydrogenase of Aspergillus oryzae. J Biochem 38:75–84
Omura H, Sanada H, Yada T, Morita T, Kuyama M, Ikeda T, Kano K, Tsujimura S (2010) Coenzyme-binding glucose dehydrogenase. EP Patent 1584675:B1
Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8:785–786. doi:10.1038/nmeth.1701
Piumi F, Levasseur A, Navarro D, Zhou S, Mathieu Y, Ropartz D, Ludwig R, Faulds CB, Record E (2014) A novel glucose dehydrogenase from the white-rot fungus Pycnoporus cinnabarinus: production in Aspergillus niger and physicochemical characterization of the recombinant enzyme. Appl Microbiol Biotechnol 98:10105–10118. doi:10.1007/s00253-014-5891-4
Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York
Satake R, Ichiyanagi A, Ichikawa K, Hirokawa K, Araki Y, Yoshimura T, Gomi K (2015) Novel glucose dehydrogenase from Mucor prainii: purification, characterization, molecular cloning and gene expression in Aspergillus sojae. J Biosci Bioeng 120:498–503. doi:10.1016/j.jbiosc.2015.03.012
Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85. doi:10.1016/0003-2697(85)90442-7
Sode K, Igarashi S, Morimoto A, Yoshida H (2002) Construction of engineered water-soluble PQQ glucose dehydrogenase with improved substrate specificity. Biocatal Biotransformation 20:405–412. doi:10.1080/1024242021000058694
Swoboda BE, Massey V (1965) Purification and properties of the glucose oxidase from Aspergillus niger. J Biol Chem 240:2209–2215
Sygmund C, Klausberger M, Felice AK, Ludwig R (2011a) Reduction of quinones and phenoxy radicals by extracellular glucose dehydrogenase from Glomerella cingulata suggests a role in plant pathogenicity. Microbiology 157:3203–3212. doi:10.1099/mic.0.051904-0
Sygmund C, Staudigl P, Klausberger M, Pinotsis N, Djinović-Carugo K, Gorton L, Haltrich D, Ludwig R (2011b) Heterologous overexpression of Glomerella cingulata FAD-dependent glucose dehydrogenase in Escherichia coli and Pichia pastoris. Microb Cell Fact 10:106. doi:10.1186/1475-2859-10-106
Sygmund C, Gutmann A, Krondorfer I, Kujawa M, Glieder A, Pscheidt B, Haltrich D, Peterbauer C, Kittl R (2012) Simple and efficient expression of Agaricus meleagris pyranose dehydrogenase in Pichia pastoris. Appl Microbiol Biotechnol 94:695–704. doi:10.1007/s00253-011-3667-7
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680. doi:10.1093/nar/22.22.4673
Tschopp JF, Sverlow G, Kosson R, Craig W, Grinna L (1987) High-level secretion of glycosylated invertase in the methylotrophic yeast, Pichia pastoris. Nat Biotechnol 5:1305–1308. doi:10.1038/nbt1287-1305
Tsujimura S, Kojima S, Kano K, Ikeda T, Sato M, Sanada H, Omura H (2006) Novel FAD-dependent glucose dehydrogenase for a dioxygen-insensitive glucose biosensor. Biosci Biotechnol Biochem 70:654–659. doi:10.1271/bbb.70.654
U.S. Food and Drug Administration (2009) FDA public health notification: potentially fatal errors with GDH-PQQ glucose monitoring technology. http://www.fda.gov/MedicalDevices/Safety/AlertsandNotices/PublicHealthNotifications/ucm176992.htm. Accessed 5 November 2015
Werner WE, Demorest DM, Stevens J, Wiktorowicz JE (1993) Size-dependent separation of proteins denatured in SDS by capillary electrophoresis using a replaceable sieving matrix. Anal Biochem 212:253–258. doi:10.1006/abio.1993.1319
Yang Y, Huang L, Wang J, Wang X, Xu Z (2014) Efficient expression, purification, and characterization of a novel FAD-dependent glucose dehydrogenase from Aspergillus terreus in Pichia pastoris. J Microbiol Biotechnol 24:1516–1524
Yang Y, Huang L, Wang J, Xu Z (2015) Expression, characterization and mutagenesis of an FAD-dependent glucose dehydrogenase from Aspergillus terreus. Enzyme Microb Technol 68:43–49. doi:10.1016/j.enzmictec.2014.10.00
Yoo EH, Lee SY (2010) Glucose biosensors: an overview of use in clinical practice. Sensors 10:4558–4576. doi:10.3390/s100504558
Yoshida H, Sakai G, Mori K, Kojima K, Kamitori S, Sode K (2015) Structural analysis of fungus-derived FAD glucose dehydrogenase. Sci Rep 5:13498. doi:10.1038/srep13498
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interests
The authors declare that they have no competing interests.
Ethical statement
This articles does not contain any studies with human participants or animals performed by any of the authors.
Additional information
Kazumichi Ozawa and Hisanori Iwasa contributed equally to this work.
Electronic supplementary material
Below is the link to the electronic supplementary material.
ESM 1
(PDF 846 kb)
Rights and permissions
About this article
Cite this article
Ozawa, K., Iwasa, H., Sasaki, N. et al. Identification and characterization of thermostable glucose dehydrogenases from thermophilic filamentous fungi. Appl Microbiol Biotechnol 101, 173–183 (2017). https://doi.org/10.1007/s00253-016-7754-7
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-016-7754-7