Novel method for immobilization of enzymes to magnetic nanoparticles

• Published in: Journal of nanoparticle research : an interdisciplinary forum for nanoscale science and technology Vol. 10; no. 6; pp. 1009 - 1025
• Main Authors: Johnson, Andrew K., Zawadzka, Anna M., Deobald, Lee A., Crawford, Ronald L., Paszczynski, Andrzej J.
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer Netherlands 01-08-2008; Springer Nature B.V
• Subjects: Adsorption; Characterization and Evaluation of Materials; Chemistry and Materials Science; E coli; Enzymatic activity; Enzymes; Inorganic Chemistry; Iron oxides; Lasers; Materials Science; Nanoparticles; Nanotechnology; Optical Devices; Optics; Peptides; Photonics; Physical Chemistry; Proteins; Research Paper; Silica; Surface chemistry; Enzymes; Affinity peptide; Nanomedicine; Magnetic nanoparticles; Nanobiotechnology
• ISSN: 1388-0764; 1572-896X
• DOI: 10.1007/s11051-007-9332-5

The value of coupling biological molecules such as enzymes to solid materials has long been recognized. To date, protein immobilization onto such surfaces often involves covalent coupling, encapsulation, or non-specific adsorption techniques. Here we demonstrate the feasibility of specifically attaching a haloalkane dehalogenase enzyme to silica-coated or uncoated iron oxide superparamagnetic nanoparticles using affinity peptides. The enzyme was cloned from
Xanthobacter autotrophicus
strain GJ10 into
Escherichia coli
to produce fusion proteins containing dehalogenase sequences with C-terminal polypeptide repeats that have specific affinity for either silica or iron oxide. The fusion proteins serve dual functions, allowing for specific inorganic surface binding and for enzymatic activity. The degree of fusion protein adsorption to nanoparticle surfaces was found to exceed that of enzymes that had not been activated with affinity sequences, particularly for iron-oxide nanoparticles. The ability to specifically adsorb cloned affinity-tagged dehalogenase was further demonstrated by selectively adsorbing dehalogenase fusion proteins containing an iron-oxide affinity tripeptide directly from cell lysate. The retention of enzymatic activity was found to be dependent upon the surface chemistry of the nanoparticles. An increase in activity was observed after adsorption of fusion proteins onto the surface of nanoparticles modified by treatment with hydrophilic polyethylene glycol or 3-glycidoxypropyltrimethoxysilane molecules. As a result of this work, it is possible to tag an active enzyme with specific peptides that bind to inorganic nanoparticle surfaces. Because the conjugates self assemble, the novel surface-specific conjugate formation procedure is highly efficient and easily scalable for use in large-scale applications.