title: pH and heat-dependent behaviour of glucose oxidase down to single molecule level by combined fluorescence spectroscopy and molecular modelling.
abstract: BACKGROUND
  In the food industry, glucose oxidase (GOX) is used to improve the shelf life of food materials. The pH- and heat-induced conformational changes of glucose oxidase from Aspergillus niger were quantified by means of fluorescence spectroscopy and molecular dynamics simulations.
  
  
  RESULTS
  The phase diagram showed an all-or-none transition process, indicating that pH and temperature largely influence the conformational state of GOX. Shifts in maximum wavelength of Trp, Tyr were registered as the protein encounters a lower pH (pH 4.0), suggesting significant changes of the polarity around the chromophore molecule. Quenching experiments using KI showed higher quenching constants of Trp and flavin adenine dinucleotide upon heating or by changing pH value, and were mainly correlated with the conformational changes upon protein matrix. Finally, valuable insights into the thermal behaviour of GOX were obtained from molecular modelling results.
  
  
  CONCLUSIONS
  The conformation and structure of GOX protein is dependent upon the pH and heat treatment applied. Molecular dynamics simulation indicated significant changes in the substrate binding region at temperatures over 60 °C that might affect enzyme activity. Moreover, an important alteration of the small pocket hosting the positively charged His(516) residue responsible for oxygen activation appears evident at high temperatures.
authors.affiliation: Dunarea de Jos University of Galati, Faculty of Food Science and Engineering, Domneascã Street 111, 800201, Galati, Romania.
authors.forename: Loredana
authors.initials: L
authors.lastname: Dumitraşcu
authors.affiliation: Dunarea de Jos University of Galati, Faculty of Food Science and Engineering, Domneascã Street 111, 800201, Galati, Romania.
authors.forename: Nicoleta
authors.initials: N
authors.lastname: Stănciuc
authors.affiliation: Dunarea de Jos University of Galati, Faculty of Food Science and Engineering, Domneascã Street 111, 800201, Galati, Romania.
authors.forename: Gabriela Elena
authors.initials: GE
authors.lastname: Bahrim
authors.affiliation: Dunarea de Jos University of Galati, Faculty of Food Science and Engineering, Domneascã Street 111, 800201, Galati, Romania.
authors.forename: Alexandrina
authors.initials: A
authors.lastname: Ciumac
authors.affiliation: Dunarea de Jos University of Galati, Faculty of Food Science and Engineering, Domneascã Street 111, 800201, Galati, Romania.
authors.forename: Iuliana
authors.initials: I
authors.lastname: Aprodu
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology; D003198:Computer Simulation; D005949:Glucose Oxidase / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D005949:Glucose Oxidase
keywords: fluorescence measurement; glucose oxidase; molecular modelling; structural changes