共 42 条
Poly(2-hydroxyethyl methacrylate) for Enzyme Immobilization: Impact on Activity and Stability of Horseradish Peroxidase
被引:49
作者:
Lane, Sarah M.
[1
]
Kuang, Zhifeng
[1
]
Yom, Jeannie
[1
]
Arifuzzaman, Shafi
[2
]
Genzer, Jan
[2
]
Farmer, Barry
[1
]
Naik, Rajesh
[1
]
Vaia, Richard A.
[1
]
机构:
[1] USAF, Res Lab, Mat & Mfg Directorate, Wright Patterson AFB, OH 45433 USA
[2] N Carolina State Univ, Dept Chem & Biomol Engn, Raleigh, NC 27695 USA
关键词:
SURFACE-TETHERED DIBLOCK;
POLYMER BRUSHES;
MULTIVARIANT ASSEMBLIES;
GLUCOSE-OXIDASE;
CELL-ADHESION;
BINDING;
STABILIZATION;
IMPROVEMENT;
COPOLYMERS;
TEMPLATES;
D O I:
10.1021/bm200173y
中图分类号:
Q5 [生物化学];
Q7 [分子生物学];
学科分类号:
071010 ;
081704 ;
摘要:
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 FIRP 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 FIRP 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.
引用
收藏
页码:1822 / 1830
页数:9
相关论文