Albuminated Glycoenzymes: Enzyme Stabilization through Orthogonal Attachment of a Single-Layered Protein Shell around a Central Glycoenzyme Core.

Here we demonstrate an approach to stabilize enzymes through the orthogonal covalent attachment of albumin on the single-enzyme level. Albuminated glycoenzymes (AGs) based upon glucose oxidase and catalase from Aspergillus niger were prepared in this manner. Gel filtration chromatography and dynamic light scattering support modification, with an increase in hydrodynamic radius of ca. 60% upon albumination. Both AGs demonstrate a marked resistance to aggregation during heating to 90 °C, but this effect is more profound in albuminated catalase. The functional characteristics of albuminated glucose oxidase vary considerably with exposure type. The AG's thermal inactivation is reduced more than 25 times compared to native glucose oxidase, and moderate stabilization is observed with one month storage at 37 °C. However, albumination has no effect on operational stability of glucose oxidase.

A Design Full of Holes: Functional Nanofilm-Coated Microdomains in Alginate Hydrogels.

This study demonstrates the successful manufacture and functional characterization of alginate hydrogels containing a variety of encapsulates within polyelectrolyte multilayer-coated micropores. These microporous alginate (MPA) hydrogels are prepared via one-step internal ionotropic gelation of the alginate using polyelectrolyte multilayer-coated CaCO3 microspheres along with the weak acid glucono-δ-lactone. Here, successful encapsulation of a model macromolecule and fluorescent nanoparticles within microcapsules-distributed throughout the larger alginate hydrogel-is confirmed with confocal microscopy, while the porous morphology of the MPA hydrogels is examined with scanning electron microscopy. Hydrogels constructed with uncoated CaCO3 microspheres release their contents into the surrounding environment, while those constructed with polyelectrolyte multilayer-coated CaCO3 microspheres retain the materials within the pores. MPA hydrogels containing the model enzyme glucose oxidase retained activity and are capable of reacting with small molecules from the external environment. The ability to encapsulate an assortment of functional materials within a moldable, biocompatible alginate matrix gives this approach great flexibility and potential in a wide variety of biomedical applications.

Glycosylation site-targeted PEGylation of glucose oxidase retains native enzymatic activity.

Targeted PEGylation of glucose oxidase at its glycosylation sites was investigated to determine the effect on enzymatic activity, as well as the bioconjugate's potential in an optical biosensing assay. Methoxy-poly(ethylene glycol)-hydrazide (4.5kDa) was covalently coupled to periodate-oxidized glycosylation sites of glucose oxidase from Aspergillus niger. The bioconjugate was characterized using gel electrophoresis, liquid chromatography, mass spectrometry, and dynamic light scattering. Gel electrophoresis data showed that the PEGylation protocol resulted in a drastic increase (ca. 100kDa) in the apparent molecular mass of the protein subunit, with complete conversion to the bioconjugate; liquid chromatography data corroborated this large increase in molecular size. Mass spectrometry data proved that the extent of PEGylation was six poly(ethylene glycol) chains per glucose oxidase dimer. Dynamic light scattering data indicated the absence of higher-order oligomers in the PEGylated GOx sample. To assess stability, enzymatic activity assays were performed in triplicate at multiple time points over the course of 29 days in the absence of glucose, as well as before and after exposure to 5% w/v glucose for 24h. At a confidence level of 95%, the bioconjugate's performance was statistically equivalent to native glucose oxidase in terms of activity retention over the 29 day time period, as well as following the 24h glucose exposure. Finally, the bioconjugate was entrapped within a poly(2-hydroxyethyl methacrylate) hydrogel containing an oxygen-sensitive phosphor, and the construct was shown to respond approximately linearly with a 220±73% signal change (n=4, 95% confidence interval) over the physiologically-relevant glucose range (i.e., 0-400mg/dL); to our knowledge, this represents the first demonstration of PEGylated glucose oxidase incorporated into an optical biosensing assay.