Temperature-Invariant Aqueous Microgels as Hosts for
Biomacromolecules
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Abstract
Immobilization of enzymes on solid
supports has been widely used
to improve enzyme recycling, enzyme stability, and performance. We
are interested in using aqueous microgels (colloidal hydrogels) as
carriers for enzymes used in high-temperature reactions. These microgels
should maintain their volume and colloidal stability in aqueous media
up to 100 °C to serve as thermo-stable supports for enzymes.
For this purpose, we prepared poly(<i>N</i>-hydroxyethyl
acrylamide) (PHEAA) microgels via a two-step synthesis. First, we
used precipitation polymerization in water to synthesize colloidal
poly(diethylene glycol-ethyl ether acrylate) (PDEGAC) particles as
a precursor. PDEGAC forms solvent swollen microgels in organic solvents
such as methanol and dioxane and in water at temperatures below 15
°C. In the second step, these PDEGAC particles were transformed
to PHEAA microgels through aminolysis in dioxane with ethanolamine
and a small amount of ethylenediamine. Dynamic laser scattering studies
confirmed that the colloidal stability of microgels was maintained
during the aminolysis in dioxane and subsequent transfer to water.
Characterization of the PHEAA microgels indicated about 9 mol % of
primary amino groups. These provide functionality for bioconjugation.
As proof-of-concept experiments, we attached the enzyme horseradish
peroxidase (HRP) to these aqueous microgels through (i) N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide
hydrochloride (EDC) coupling to the carboxylated microgels or (ii)
bis-aryl hydrazone (BAH) coupling to microgels functionalized with
6-hydrazinonicotinate acetone (PHEAA-HyNic). Our results showed that
HRP maintained its catalytic activity after covalent attachment (87%
for EDC coupling, 96% for BAH coupling). The microgel enhanced the
stability of the enzyme to thermal denaturation. For example, the
residual activity of the microgel-supported enzyme was 76% after 330
min of annealing at 50 °C, compared to only 20% for the free
enzyme under these conditions. PHEAA microgels in water show great
promise as hosts for enzymatic reaction, especially at elevated temperatures