Structural features of a hyperthermostable endo-beta-1,3-glucanase in solution and adsorbed on "invisible" particles.
Conformational characteristics and the adsorption behavior of endo-beta-1,3-glucanase from the hyperthermophilic microorganism Pyrococcus furiosus were studied by circular dichroism, steady-state and time-resolved fluorescence spectroscopy, and calorimetry in solution and in the adsorbed state. The adsorption isotherms were determined on two types of surfaces: hydrophobic Teflon and hydrophilic silica particles were specially designed so that they do not interact with light and therefore do not interfere with spectroscopic measurements. We present the most straightforward method to study structural features of adsorbed macromolecules in situ using common spectroscopic techniques. The enzyme was irreversibly adsorbed and immobilized in the adsorbed state even at high temperatures. Adsorption offered further stabilization to the heat-stable enzyme and in the case of adsorption on Teflon its denaturation temperature was measured at 133 degrees C, i.e., the highest experimentally determined for a protein. The maintenance of the active conformation and biological function particularly at high temperatures is important for applications in biocatalysis and biotechnology. With this study we also suggest that nature may employ adsorption as a complementary mode to maintain structural integrity of essential biomolecules at extreme conditions of temperature.
Koutsopoulos
Sotirios
S
Laboratory of Physical Chemistry and Colloid Science, Wageningen University, Wageningen, The Netherlands. sotirios.koutsopoulos@wur.nl
van der Oost
John
J
Norde
Willem
W
D000327:Adsorption; D016880:Anisotropy; D002151:Calorimetry; D002152:Calorimetry, Differential Scanning; D002384:Catalysis; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D043326:Glucan 1,3-beta-Glucosidase / Q000737:chemistry*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008027:Light; D008958:Models, Molecular; D010710:Phosphates / Q000737:chemistry; D011138:Polytetrafluoroethylene / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D020027:Pyrococcus furiosus / Q000201:enzymology; D012822:Silicon Dioxide / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013696:Temperature; D013997:Time Factors; D014466:Ultraviolet Rays
D010710:Phosphates; D012822:Silicon Dioxide; D011138:Polytetrafluoroethylene; D043326:Glucan 1,3-beta-Glucosidase; C018279:sodium phosphate