Stability and activity of enzymes by immobilization
A polymer brush composed of statistical copolymers of hydrophilic and hydrophobic monomers for enzyme immobilization. The heterogeneous polymer brushes stabilized four different lipases against high temperatures. Notably, the statistical copolymers stabilized the four lipases to a greater extent than a homopolymer brush. Additionally, in the case of Rhizomucor miehei lipase, statistical copolymers stabilized the enzyme to a greater extent than homopolymers of either hydrophilic or hydrophobic monomers. The resulting increase in high-temperature stability enabled significant improvements in catalytic rates by operating reactions at elevated temperatures, which is desirable for enzyme catalysis and sensing applications. Additionally, stabilization against elevated temperatures implies stabilization against non-aqueous solvents, which is of critical importance to numerous applications of biocatalysts.
1 . A method of preparing an immobilized enzyme comprising the steps of
providing a monomer-containing polymerization precursor mixture, wherein the monomer mixture comprises a hydrophilic monomer and a hydrophobic monomer in a molar ratio between 100:1 and 1:100 of hydrophilic monomer: hydrophobic monomer, wherein the hydrophilic monomer is selected from the group consisting of [3-(methacryloylamino)propyl]trimethylammonium chloride, [2-(methacryloyloxy)ethyl]trimethylammonium chloride, 3-sulfopropyl methacrylate, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, methacrylic acid, 2-methacryloyloxyethyl phosphorylcholine, and corresponding acrylates and acrylamides thereof and wherein the hydrophobic monomer is a monomer selected from the group consisting of poly(ethylene glycol)methacrylate benzyl methacrylate, cyclohexyl methacrylate, 2-(diethylamino)ethyl methacrylate, 2-(diisopropylamino)ethyl methacrylate, n-isopropylacrylamide, 2-N-Morpholinoethyl methacrylate, and corresponding acrylates and acrylamides thereof;
performing a polymerization reaction with the precursor mixture in the presence of a substrate surface to yield a polymerized substrate comprising a copolymer brush; and
contacting the polymerized substrate with an enzyme under conditions effective to allow for attachment of the enzyme to the copolymer brush of the polymerized substrate.
2 . The method of preparing an immobilized enzyme according to claim 1 wherein the molar ratio of hydrophilic monomer: hydrophobic monomer is selected based upon the protein surface hydrophilicity, wherein a greater protein surface hydophilicity mandates a higher ratio of hydrophilic monomer to hydrophobic monomer in the copolymer.
3 . The method of preparing an immobilized enzyme according to claim 1 wherein the monomer mixture further commprises a reactive monomer.
4 . The method of preparing an immobilized enzyme according to claim 3 wherein the reactive monomer is a monomer selected from the group consisting of glycidyl methacrylate and corresponding acrylates and acrylamides, nitrilotriacetic acid and iminodiacetic acid functionalized monomers.
5 . The method of preparing an immobilized enzyme according to claim 1 wherein the precursor mixture includes an amine reactive cross-linking agent.
6 . The method of preparing an immobilized enzyme according to claim 5 wherein the amine reactive cross-linking agent is methacrylic acid N-hydroxysuccinimide ester (NHS-MA).
7 . The method of preparing an immobilized enzyme according to claim 1 wherein the copolymer brush is synthesized using a “grafted from” approach.
8 . The method of preparing an immobilized enzyme according to claim 1 wherein the molar ratio hydrophilic monomer: hydrophobic monomer is a ratio selected from the group consisting of about 20:1, about 15:1, about 12:1, about 10:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:10, about 1:12, about 1:15, and about 1:20.
9 . The method of preparing an immobilized enzyme according to claim 1 wherein the substrate surface is a silica surface.
10 . The method of preparing an immobilized enzyme according to claim 1 wherein the substrate is a microsphere.