Abstract
Enzyme immobilization is widely applied in biocatalysis to improve stability and facilitate recovery and reuse of enzymes. However, high cost of supporting materials and laborious immobilization procedures has limited its industrial application and commercialization. In this study, we report a novel self-assembly immobilization system using bacteriophage T4 capsid as a nanocarrier. The system utilizes the binding sites of the small outer capsid protein, Soc, on the T4 capsid. Enzymes as Soc fusions constructed with regular molecular cloning technology expressed at the appropriate time during phage assembly and self-assembled onto the capsids. The proof of principle experiment was carried out by immobilizing β-galactosidase, and the system was successfully applied to the immobilization of an important glycomics enzyme, Peptide-N-Glycosidase F. Production of Peptide-N-Glycosidase F and simultaneous immobilization was finished within seven hours. Characterizations of the immobilized Peptide-N-Glycosidase F indicated high retention of activity and well reserved deglycosylation capacity. The immobilized Peptide-N-Glycosidase F was easily recycled by centrifugation and exhibited good stability that sustained five repeated uses. This novel system uses the self-amplified T4 capsid as the nanoparticle-type of supporting material, and operates with a self-assembly procedure, making it a simple and low-cost enzyme immobilization technology with promising application potentials.
Highlights
Enzymes are capable of accelerating biochemical reactions in a highly efficient and specific manner
Researchers are making arduous efforts to develop simple and stable enzyme immobilization methods that could bring down the cost of immobilized enzymes
Bacteriophage T4 has served as an excellent model and a primary tool in molecular biology research
Summary
Enzymes are capable of accelerating biochemical reactions in a highly efficient and specific manner. Affinity immobilization utilizes highly specific interactions, gives good control of the orientation of immobilized enzymes, and allows minimal conformational changes Nanoparticles drew increasing attention because their inherently large surface area enhanced enzyme effectiveness, while immobilizing enzymes onto planar surfaces usually reduced the activity[10,11,12,13] Their application is hampered by high cost. Researchers attached affinity purification tags (e.g. glutathione S-transferase) onto the T4 capsids through Soc- or Hoc- fusions, and successfully used the tags to purify the phage on standard affinity resins[30,31] This is another demonstration of stable attaching of proteins or domains onto T4 capsid in the active conformation. The T4 capsid should be a well suited nanoparticle for affinity immobilization of enzymes
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