Temperature-Invariant Aqueous Microgels as Hosts for Biomacromolecules.
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(N-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.
Department of Chemical Engineering and Applied Chemistry, University of Toronto , 200 College Street, Toronto ON M5S 3E5, Canada.|Department of Chemistry, University of Toronto , 80 St. George Street, Toronto ON M5S 3H6, Canada.
Sepehr
S
Mastour Tehrani
Department of Chemistry, University of Toronto , 80 St. George Street, Toronto ON M5S 3H6, Canada.
Yijie
Y
Lu
Department of Chemistry, University of Toronto , 80 St. George Street, Toronto ON M5S 3H6, Canada.
Gerald
G
Guerin
Department of Chemical Engineering and Applied Chemistry, University of Toronto , 200 College Street, Toronto ON M5S 3E5, Canada.|Department of Chemistry, University of Toronto , 80 St. George Street, Toronto ON M5S 3H6, Canada.
Mohsen
M
Soleimani
Department of Chemistry, University of Toronto , 80 St. George Street, Toronto ON M5S 3H6, Canada.
Dmitry
D
Pichugin
Department of Chemical Engineering and Applied Chemistry, University of Toronto , 200 College Street, Toronto ON M5S 3E5, Canada.|Department of Chemistry, University of Toronto , 80 St. George Street, Toronto ON M5S 3H6, Canada.
Mitchell A
MA
Winnik
D003102:Colloids; D004795:Enzyme Stability; D006735:Horseradish Peroxidase / Q000737:chemistry; D020100:Hydrogels; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D046529:Microscopy, Electron, Transmission; D011108:Polymers / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D014867:Water / Q000737:chemistry
D003102:Colloids; D020100:Hydrogels; D011108:Polymers; D014867:Water; D006735:Horseradish Peroxidase