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.

Thicker is Better? Synthesis and Evaluation of Well‐Defined Polymer Brushes with Controllable Catalytic Loadings

“…The [Si-Initiator] samples were subsequently used for the surface-initiated atom-transfer radical polymerization (SI-ATRP) [8] of HEMA monomers. The pendant hydroxy groups of [Si-PHEMA] were then activated with N,N'-disuccinimidyl carbonate (DSC) [12] by using a standard protocol and the resulting samples directly reacted with dpa derivative 3 in DMF in the presence of Et 3 N to afford [Si-PHEMA-dpa]. Finally, the substrates were immersed in a solution of [PdA C H T U N G T R E N N U N G (OAc) 2 ] in CH 2 Cl 2 to give the catalytic surfaces [Si-PHEMA-dpa-Pd] after extensive washing in a Soxhlet apparatus.…”

Flow‐through immobilized enzyme reactors based on monoliths: II. Kinetics study and application

“…As for a turnover number of above‐mentioned monolithic biocatalysts, the values of this parameter ( k 3 ) for free and immobilized enzymes were practically always decreased . However, if for RNase and DNase, the k 3 values for free forms were, respectively, 2.5 and 5 times lower than for bound biocatalysts, in the case of lactate dehydrogenase, the drastic reduction of this characteristic (about 50 times lower for immobilized form comparing to free enzyme, e.g.…”

Polymer Brushes with Precise Architectures for Molecular Biorecognition

“…After the binding to these scaffolds, the tethered proteins must preserve their biological functions [76,[96][97][98]. The versatile structure, chemistry as well as tunable mechanical properties [99] of polymer brushes make these scaffolds very attractive candidates to bind and immobilize proteins.…”