In Situ Structure and Activity Studies of an Enzyme Adsorbed on Spectroscopically Undetectable Particles

Abstract

The structural characteristics and the activity of a hyperthermophilic endoglucanase were investigated upon adsorption. Silica (hydrophilic) and Teflon (hydrophobic) surfaces were selected for the study. The materials were specially designed so that the interaction of the particles with light was negligible, and the enzyme conformation in the adsorbed state was monitored in situ. The adsorption isotherms were determined, and the adsorbed endoglucanase was studied using a number of spectroscopic techniques, enzymatic activity tests, and dynamic light scattering. Experiments were performed at pH values below, at, and above the isoelectric point of the enzyme. It was shown that the enzyme adsorbed on the hydrophobic surface of Teflon with higher affinity as compared to the hydrophilic silica nanoparticles. In all cases, adsorption was followed by (slight) changes in the secondary structure resulting in decreased beta-structural content. The changes were more profound upon adsorption on Teflon. The adsorbed enzyme remained active in the adsorbed state in spite of the structural changes induced when interacting with the surfaces.

Optical Spectroscopic Methods for Probing the Conformational Stability of Immobilised Enzymes

“…Previous literature on CD measurements of particulate systems was limited to proteins bound to nanoparticles and encapsulated into biomembrane fragments. [11][12][13] In order to extend the technique for the first time to typical immobilised biocatalyst particles of tens to hundreds of microns in diameter, it was necessary to tackle artefacts associated with particulate sedimentation, differential light scattering and absorption flattening. [10] Particles were kept uniformly dispersed in suspension in the light path using a specially constructed rotating sample cell holder.…”

Understanding protein adsorption phenomena at solid surfaces

“…Further, surface adsorption can stabilize the structure of proteins and hence improve their resistance to denaturation as compared to dissolved proteins [120][121][122][123].…”

Immobilization of urease enzyme on nanoceria modifies secondary and tertiary protein structures

“…The pattern of protein stability is necessary to assess the tendency of proteins to be adsorbed on surfaces. The optimal adsorption isotherm equation for describing the interaction between nanoceria and urease is the Sips equation ( Figure 2); this equation is an empirical model for heterogeneous adsorption [Koutsopoulos et al, 2005] where Q e is the adsorption amount normalized by the mass of nanoparticles, Q m is the maximum adsorption capacity, C e is the concentration of the adsorbent in the supernatant at adsorption equilibrium, K S is a constant related to the adsorption energy, and t is a heterogeneity coefficient. The magnitude of t is connected to the factors affecting heterogeneous adsorption.…”

“…4. The CD results were analyzed using the K2D2 web server to determine the change in the urease secondary structure from the CD spectra according to the published software (Koutsopoulos et al, 2005). The results showed that the secondary structure of the free urease consists of 84.27 % a-helix and 1.24 % b-sheet, whereas the remainder is random coil.…”