title: Poly(2-hydroxyethyl methacrylate) for enzyme immobilization: impact on activity and stability of horseradish peroxidase.
abstract: On the basis of their versatile structure and chemistry as well as tunable mechanical properties, polymer brushes are well-suited as supports for enzyme immobilization. However, a robust surface design is hindered by an inadequate understanding of the impact on activity from the coupling motif and enzyme distribution within the brush. Herein, horseradish peroxidase C (HRP C, 44 kDa), chosen as a model enzyme, was immobilized covalently through its lysine residues on a N-hydroxysuccinimidyl carbonate-activated poly(2-hydroxyethyl methacrylate) (PHEMA) brush grafted chemically onto a flat impenetrable surface. Up to a monolayer coverage of HRP C is achieved, where most of the HRP C resides at or near the brush-air interface. Molecular modeling shows that lysines 232 and 241 are the most probable binding sites, leading to an orientation of the immobilized HRP C that does not block the active pocket of the enzyme. Michaelis-Menten kinetics of the immobilized HRP C indicated little change in the K(m) (Michaelis constant) but a large decrease in the V(max) (maximum substrate conversion rate) and a correspondingly large decrease in the k(cat) (overall catalytic rate). This indicates a loss in the percentage of active enzymes. Given the relatively ideal geometry of the HRPC-PHEMA brush, the loss of activity is most likely due to structural changes in the enzyme arising from either secondary constraints imposed by the connectivity of the N-hydroxysuccinimidyl carbonate linking moiety or nonspecific interactions between HRP C and DSC-PHEMA. Therefore, a general enzyme-brush coupling motif must optimize reactive group density to balance binding with neutrality of surroundings.
authors: Air Force Research Laboratory, Materials and Manufactoring Directorate, Wright-Patterson AFB, Ohio 45433-7750, United States.
authors: Sarah M
authors: SM
authors: Lane
authors: Zhifeng
authors: Z
authors: Kuang
authors: Jeannie
authors: J
authors: Yom
authors: Shafi
authors: S
authors: Arifuzzaman
authors: Jan
authors: J
authors: Genzer
authors: Barry
authors: B
authors: Farmer
authors: Rajesh
authors: R
authors: Naik
authors: Richard A
authors: RA
authors: Vaia
mesh_terms: D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000378:metabolism*; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D011102:Polyhydroxyethyl Methacrylate / Q000737:chemistry*
chemical_list: D004800:Enzymes, Immobilized; D011102:Polyhydroxyethyl Methacrylate; D006735:Horseradish Peroxidase