Publications

• Reduction of quinones and phenoxy radicals by extracellular glucose dehydrogenase from Glomerella cingulata suggests a role in plant pathogenicity.

  Sygmund C., Klausberger M., Felice A.K., Ludwig R.

  The plant-pathogenic fungus Glomerella cingulata (anamorph Colletotrichum gloeosporoides) secretes high levels of an FAD-dependent glucose dehydrogenase (GDH) when grown on tomato juice-supplemented media. To elucidate its molecular and catalytic properties, GDH was produced in submerged culture. ... >> More

  The plant-pathogenic fungus Glomerella cingulata (anamorph Colletotrichum gloeosporoides) secretes high levels of an FAD-dependent glucose dehydrogenase (GDH) when grown on tomato juice-supplemented media. To elucidate its molecular and catalytic properties, GDH was produced in submerged culture. The highest volumetric activity was obtained in shaking flasks after 6 days of cultivation (3400 U l⁻¹, 4.2 % of total extracellular protein). GDH is a monomeric protein with an isoelectric point of 5.6. The molecular masses of the glycoforms ranged from 95 to 135 kDa, but after deglycosylation, a single 68 kDa band was obtained. The absorption spectrum is typical for an FAD-containing enzyme with maxima at 370 and 458 nm and the cofactor is non-covalently bound. The preferred substrates are glucose and xylose. Suitable electron acceptors are quinones, phenoxy radicals, 2,6-dichloroindophenol, ferricyanide and ferrocenium hexafluorophosphate. In contrast, oxygen turnover is very low. The GDH-encoding gene was cloned and phylogenetic analysis of the translated protein reveals its affiliation to the GMC family of oxidoreductases. The proposed function of this quinone and phenoxy radical reducing enzyme is to neutralize the action of plant laccase, phenoloxidase or peroxidase activities, which are increased in infected plants to evade fungal attack. << Less

  Microbiology (Reading) 157:3203-3212(2011) [PubMed] [EuropePMC]

• Cloning and expression of the gene encoding catalytic subunit of thermostable glucose dehydrogenase from Burkholderia cepacia in Escherichia coli.

  Inose K., Fujikawa M., Yamazaki T., Kojima K., Sode K.

  We have cloned a 1620-nucleotide gene encoding the catalytic subunit (alpha subunit) of a thermostable glucose dehydrogenase (GDH) from Burkholderia cepacia. The FAD binding motif was found in the N-terminal region of the alpha subunit. The deduced primary structure of the alpha subunit showed abo ... >> More

  We have cloned a 1620-nucleotide gene encoding the catalytic subunit (alpha subunit) of a thermostable glucose dehydrogenase (GDH) from Burkholderia cepacia. The FAD binding motif was found in the N-terminal region of the alpha subunit. The deduced primary structure of the alpha subunit showed about 48% identity to the catalytic subunits of sorbitol dehydrogenase (SDH) from Gluconobacter oxydans and 2-keto-D-gluconate dehydrogenases (2KGDH) from Erwinia herbicola and Pantoea citrea. The alpha subunit of B. cepacia was expressed in Escherichia coli in its active water-soluble form, showing maximum dye-mediated GDH activity at 70 degrees C, retaining high thermal stability. A putative open reading frame (ORF) of 507 nucleotides was also found upstream of the alpha subunit encoding an 18-kDa peptide, designated as gamma subunit. The deduced primary structure of gamma subunit showed about 30% identity to the small subunits of the SDH from G. oxydans and 2KGDHs from E. herbicola and P. citrea. << Less

  Biochim Biophys Acta 1645:133-138(2003) [PubMed] [EuropePMC]

• Biphasic expression and function of glucose dehydrogenase in Drosophila melanogaster.

  Cavener D.R., MacIntyre R.J.

  Glucose dehydrogenase (GO) was found to be expressed during the pupal stage in both sexes of Drosophila melanogaster, but is limited to the male ejaculatory duct at the adult stage. During copulation GO is transferred from males to females. Mutational analysis of the Go locus indicates that a sing ... >> More

  Glucose dehydrogenase (GO) was found to be expressed during the pupal stage in both sexes of Drosophila melanogaster, but is limited to the male ejaculatory duct at the adult stage. During copulation GO is transferred from males to females. Mutational analysis of the Go locus indicates that a single structural gene encodes the pupal and ejaculatory duct GO. Thus an example of an enzyme structural gene switching from non-sex-limited to sex-limited expression has been found. Go mutants are recessive lethals exhibiting a late pupal effective lethal phase. These mutants can be rescued by excising the anterior end of the pupal case 0-2 days prior to the normal adult emergence time. It appears that the function of GO in pupae is to aid in the degradation of the puparium cuticle in preparation for the eclosion of the adult. << Less

  Proc Natl Acad Sci U S A 80:6286-6288(1983) [PubMed] [EuropePMC]

• Heterologous overexpression of Glomerella cingulata FAD-dependent glucose dehydrogenase in Escherichia coli and Pichia pastoris.

  Sygmund C., Staudigl P., Klausberger M., Pinotsis N., Djinovic-Carugo K., Gorton L., Haltrich D., Ludwig R.

  <h4>Background</h4>FAD dependent glucose dehydrogenase (GDH) currently raises enormous interest in the field of glucose biosensors. Due to its superior properties such as high turnover rate, substrate specificity and oxygen independence, GDH makes its way into glucose biosensing. The recently disc ... >> More

  <h4>Background</h4>FAD dependent glucose dehydrogenase (GDH) currently raises enormous interest in the field of glucose biosensors. Due to its superior properties such as high turnover rate, substrate specificity and oxygen independence, GDH makes its way into glucose biosensing. The recently discovered GDH from the ascomycete Glomerella cingulata is a novel candidate for such an electrochemical application, but also of interest to study the plant-pathogen interaction of a family of wide-spread, crop destroying fungi. Heterologous expression is a necessity to facilitate the production of GDH for biotechnological applications and to study its physiological role in the outbreak of anthracnose caused by Glomerella (anamorph Colletotrichum) spp.<h4>Results</h4>Heterologous expression of active G. cingulata GDH has been achieved in both Escherichia coli and Pichia pastoris, however, the expressed volumetric activity was about 4800-fold higher in P. pastoris. Expression in E. coli resulted mainly in the formation of inclusion bodies and only after co-expression with molecular chaperones enzymatic activity was detected. The fed-batch cultivation of a P. pastoris transformant resulted in an expression of 48,000 U L⁻¹ of GDH activity (57 mg L⁻¹). Recombinant GDH was purified by a two-step purification procedure with a yield of 71%. Comparative characterization of molecular and catalytic properties shows identical features for the GDH expressed in P. pastoris and the wild-type enzyme from its natural fungal source.<h4>Conclusions</h4>The heterologous expression of active GDH was greatly favoured in the eukaryotic host. The efficient expression in P. pastoris facilitates the production of genetically engineered GDH variants for electrochemical-, physiological- and structural studies. << Less

  Microb Cell Fact 10:106-106(2011) [PubMed] [EuropePMC]

• Alteration in FAD-glucose dehydrogenase activity and hemocyte behavior contribute to initial disruption of Manduca sexta immune response to Cotesia congregata parasitoids.

  Lovallo N., Cox-Foster D.L.

  Cotesia congregata and Manduca sexta were used as a model system to study the mechanism and effect of a polydnavirus (PDV). We hypothesized that (1) FAD-glucose dehydrogenase (GLD) (EC 1.1.99.10) hemolymph titer would increase in response to parasitism, (2) hemocyte targeting behavior would be alt ... >> More

  Cotesia congregata and Manduca sexta were used as a model system to study the mechanism and effect of a polydnavirus (PDV). We hypothesized that (1) FAD-glucose dehydrogenase (GLD) (EC 1.1.99.10) hemolymph titer would increase in response to parasitism, (2) hemocyte targeting behavior would be altered by parasitism, and (3) changes observed in GLD activity and hemocyte behavior immediately post-parasitization would be due to the presence of PDV. GLD specific activity was measured at four time points early during parasitism using a spectrophotometric enzyme assay. Hemocyte behavior was measured using direct observations of hemocyte response to a foreign target in vitro. Results demonstrate that GLD increases immediately post-oviposition and post-injection of purified PDV, indicating that virions elicit nonself recognition. This increase relative to unparasitized controls also occurs in response to trioxsalen-UV inactivated virus, indicating that the initial disruption of the host immune response is not dependent upon viral transcription. Further, we demonstrate that plasmatocytes are actively spreading and aggregating but are not targeting nonself material in both parasitized and polydnavirus treatments. These results indicate that purified PDV is recognized as nonself and is triggering an immediate cellular immune response prior to viral transcription. << Less

  J Insect Physiol 45:1037-1048(1999) [PubMed] [EuropePMC]

• Studies on glucose dehydrogenase of Aspergillus oryzae. II. Purification and physical and chemical properties.

  Bak T.G.

  Biochim Biophys Acta 139:277-293(1967) [PubMed] [EuropePMC]