(375c) Enzymatic Hydrogels from Proteinaceous Bifunctional Building Blocks

Hydrogels find use in a myriad of applications including biocatalysis, tissue engineering, and drug delivery. There are many examples of protein-engineering efforts that address some of the more technologically challenging aspects of these applications, including stimuli-responsiveness, bioactivity and catalytic activity. Here we present a general method of enzymatic hydrogel design through the development of bifunctional protein building blocks. We present three examples of chimeric proteins, differing in tertiary and quaternary structures, that self-assemble into enzymatic supramolecular hydrogels. Self-assembly functionality is achieved by appending previously designed α-helical leucine zipper domains to the termini of the desired enzyme. Compatible helices allow for the co-assembly of enzymatic and inert building blocks in mixed supramolecular hydrogels. The physical properties and enzymatic activity of the hydrogels are dependent on the identity, amount and ratio of each building block thus allowing for the independent tuning of these characteristics.

An oxidoreductase, SLAC, from Streptomyces coelicolor is the basis of a hydrogel that catalyzes the reduction of dioxygen to water; an alcohol dehydrogenase, AdhD, from Pyrococcus furiosus is the basis of a thermostable hydrogel that catalyzes the oxidation of secondary alcohols; a phosphotriesterase, OPH, from Pseudomonas diminuta is the basis of an hydrogel that catalyzes the hydrolysis of acetylcholinesterase inhibitors. The structure of the appended leucine zipper domains in all building blocks is confirmed by circular dichroism spectroscopy. Physical cross-linking functionality of the helices is confirmed by small amplitude oscillatory shear experiments. The impact of the N- and C-terminal modifications on enzymatic activity is determined by evaluating the kinetic parameters through dilute solution activity assays. We also present data on the rate of hydrogel erosion in open buffer solution and demonstrate the independent tuning of hydrogel strength (in term of the storage modulus) and catalytic activity.

Preliminary data is presented on the use of the enzymatic hydrogels as electrode surface modifications; modified SLAC as a cathode for a biofuel cell, AdhD for the anodic oxidation of alcohols and OPH as a biosensor for nerve agents. These examples, in conjunction with previously work on fluorescent protein building blocks, demonstrate the broad utility of our protein engineering approach to advanced hydrogel design. The bifunctional protein building blocks exhibit dual roles, hydrogel formation and enzymatic activity, a scheme that will prove useful in not only biocatalysis and biosensing but also tissue engineering and drug delivery applications.