Simultaneous Observation of the Orientation and Activity of Surface-Immobilized Enzymes

Abstract

Surface immobilized enzymes have been widely used in many applications such as biosensors, biochips, biofuel production, and biofuel cell construction. Many factors dictate how enzymes' structure, activity, and stability may change when immobilized, including surface functionalization, immobilization chemistry, nature of the solid support, and enzyme surface density. To better understand how immobilization affects enzyme structure and activity, we have developed a method to measure both surface-sensitive protein vibrational spectra and enzymatic activity simultaneously. To accomplish this, an optical/fluorescence microscope was incorporated into a sum frequency generation (SFG) spectrometer. Using β-glucosidase (β-Glu) as a model system, enzymes were covalently tethered to a self-assembled monolayer surface using cysteine-maleimide chemistry. Their orientations were determined by SFG spectroscopy, with a single native cysteine residue oriented toward the functionalized surface, and activity measured simultaneously using a fluorogenic substrate resorufin β-d-glucopyranoside, with a loss of activity of 53% as compared to comparable solution measurements. Measuring β-Glu activity and orientation simultaneously provides more accurate information for designing and further improving enzymatic activity of surface-bound enzymes.

Proteolytic Rafts for Improving Intraparenchymal Migration of Minimally Invasively Administered Hydrogel-Embedded Stem Cells

“…This discrepancy can be explained by the fact that enzymes immobilized on HA scaffold have limited possibility to be up-taken by the cells. Also, as shown earlier, immobilized enzymes could have decreased activity in comparison to their activity in solution [27,28]. The beneficial effect of proteolytic rafts on cells survival, however small, is very promising and shows a need for further studies in this field, including new composition of enzymes and their concentrations.…”

Encapsulin Nanocontainers as Versatile Scaffolds for the Development of Artificial Metabolons

“…We speculate that the decreased binding affinities possibly arise from steric crowding of the surface-bound DHFR* enzymes, localized environmental perturbations generated in the immediate proximity of the nanocontainers (e.g., solvent viscosity, pH, etc. ), − or due to dynamic interactions between the DHFR* enzymes and the encapsulin surface (resulting from the flexible nature of the linker sequences connecting the TmE and SpyCatcher domains) leading to some percentage of the enzyme population adopting orientations that occlude access to the active site. However, given that the observed K m increases for DHF are relatively minor, we did not investigate this effect further.…”

Metal–Organic Frameworks for Enzyme Immobilization: Beyond Host Matrix Materials

“… 54 The methodology developed in those studies has been proven to have general applicability on different matrix supports and model proteins. 54 − 59 They implemented an optical/fluorescence microscope into a sum frequency generation (SFG) spectrometer. It is intrinsically surface-sensitive and powerful for studying the secondary structures of the protein/orientation of interfacial proteins.…”