Development and Characterization of Stable Glycoenzyme Conjugates

Optical glucose biosensors are being developed for long-term monitoring in diabetic individuals. These sensors rely upon the enzyme glucose oxidase, and loss of enzymatic activity leads to a need for frequent recalibration and eventually sensor replacement. Current enzyme stabilization strategies are effective, but generally result in a large increase in size and exclusion from the solution-phase. This sacrifice of native properties precludes the stabilized enzyme from incorporation into the aforementioned sensing platform, which requires that the enzyme be homogenously distributed and entrapped within a hydrogel. It is this incompatibility which provides the motivation for the development of new non-traditional enzyme stabilization strategies.

Toward that end, this work focuses on the development and characterization of three enzyme modification strategies, all of which are intended to stabilize enzyme activity while permitting incorporation into an optical biosensing hydrogel. The first approach involves glycosylation site-targeted covalent attachment of poly(ethylene glycol) to glucose oxidase, which improves storage stability by 60%. The second approach builds upon the first, but subsequent modification of the poly(ethylene glycol)-modified glucose oxidase is performed to further stabilize the enzyme. This approach improves long-term storage stability by an order of magnitude. The final approach involves encasement of the glycoenzyme within a shell of albumin, wherein the inert protein is attached at the glycosylation sites in an orthogonal manner. This technique result in highly thermostable enzyme, retaining greater than 25 times more activity than native glucose oxidase following exposure to buffer at 60 ?C.

In summary, enzyme deactivation is expected to be a major barrier in the realization of long-term glucose sensing with fully implantable optical glucose biosensors, and this work represents a step towards overcoming that hurdle. Each enzyme modification strategy yields a stabilized enzyme under certain conditions, whether it be long-term storage, elevated temperature, or exposure to various solvents/additives. This work enables the stabilized enzymes to be incorporated into hydrogels for evaluation under simulated in vivo conditions, followed by in vivo evaluation. Finally, it is expected that these enzyme stabilization approaches will be advantageous in other applications as well, including in vitro diagnostics, tissue engineering, and therapeutic biologicals.