Zhixia Liu

Strengthening the stability of a tunnel-shaped homotetramer protein with nanogels.

Urate oxidase (UOX, EC 1.7.3.3) is effective for the treatment of gout and hyperuricaemia associated with tumor lysis syndrome. The inherent poor stability of UOX to temperature, proteolysis, and acidic environments is known to limit its efficacy. Herein, we encapsulated UOX into spherical and porous nanogels with diameters of 20-40 nm via a two-step in situ polymerization in the presence of oxonic acid potassium salt, an inhibitor of UOX. The UOX nanogel retained 70% of the initial activity but showed an expanded pH spectrum from pH 6-10 to 3-10 and an extended half-life at 37 °C from 5 min to 3 h. The enhanced pH stability, thermal stability, and enzyme resistance of the UOX nanogels were also confirmed by using fluorescence spectroscopy and enzymatic digestion. A molecular dynamics simulation was performed as a way to probe the mechanism underlying the formation of UOX nanogels as well as the strengthened stability against harsh conditions. It was shown that the encapsulation into the polyacrylamide network reinforced the intersubunit hydrogen bonding, shielded the hydrolytic reaction site, and thus protected the tertiary and quaternary structure of UOX. The UOX nanogel with enhanced stability provided a stable enzyme model that enables the exploration of UOX in the diagnosis and therapy of disorders associated with altered purine metabolism.

Preparation and Characterization of Single-Enzyme Nanogels

Enzymes have been incorporated in nanostructures in order to provide robust catalysts for valuable reactions, particularly those performed under harsh and denaturing conditions. This chapter describes the encapsulation of enzymes in polyacrylamide nanogels by a two-step in situ polymerization process for preparing robust biocatalysts. The first step in this process is the generation of vinyl groups on the enzyme surface, while the second step involves in situ polymerization using acrylamide as the monomer. Encapsulation of the enzyme in the hydrophilic, porous, and flexible polyacrylamide gel of several nanometers thick would help to both give a significantly enhanced thermostability and prevent the removal of essential water by polar solvents. The hydrophilic flexible polymer shell also allows the protein structure to undergo necessary conformational transitions during the catalytic reaction and, at the same time, impose marginal mass transfer restrictions for the substrates entering across the polymer shell. The effectiveness of this method is demonstrated with horseradish peroxidase (HRP), carbonic anhydrase, and lipase. The impacts of such an encapsulation on the activity and stability of enzymes are also discussed.