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Structural Features of a Hyperthermostable Endo-ß-1,3-glucanase in Solution and Adsorbed on "Invisible" Particles

Sotirios Koutsopoulos ^*, John van der Oost  and Willem Norde ^* 

^* Laboratory of Physical Chemistry and Colloid Science, and Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands; and Department of Biomedical Engineering, University of Groningen, Groningen, The Netherlands

Correspondence: Address reprint requests to Sotirios Koutsopoulos, Laboratory of Physical Chemistry & Colloid Science, Wageningen University, PO Box 8038, 6700 EK Wageningen, The Netherlands. Tel.: +31-317-482178; Fax: +31-317-483777; E-mail: sotiris.koutsopoulos{at}wur.nl.

Conformational characteristics and the adsorption behavior of endo-ß-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°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.