Fabrication and kinetic studies of a novel silver nanoparticles-glucose oxidase bioconjugate.
In this study, a new effective, pH and thermally stable glucose oxidase (GOX)-silver nanoparticles (AgNPs) bioconjugate was designed. AgNPs were synthesized based on the reduction of silver nitrate (AgNO(3)) by sodium borohydride (NaBH(4)) using two simple procedures. Periodic acid was used for oxidation of the GOX and emission of Lucifer yellow (LyCH) was monitored by spectrofluorometer for evaluation of the oxidation properties of the GOX. The oxidized GOX (Ox-GOX) was immobilized on AgNPs by its sugar moieties via 6-aminohexanoic acid (6AHA) as linker. A sample of the synthesized bioconjugate was loaded on 7.5% non-denaturing polyacrylamide gel electrophoresis (PAGE) to confirm its structural and physical stability. The results from enzymatic activity assay showed that the bioconjugate, GOX and Ox-GOX were similar in stability and activity in acidic and basic pH (optimum pH=7.0-8.0). Based on the results from thermal stability assay, it was found that the activity of the bioconjugate was found to be higher at lower temperatures. The V(max) of the bioconjugate, GOX, and Ox-GOX was estimated as 28.6, 6.2, and 6 IU microg(-1) enzyme and the K(m) was calculated as 2.7, 9, and 9.5 mM, respectively. It was found that the immobilization method improves the activity and stability of the GOX in different pH and temperatures. As a conclusion, the proposed method opens up the way to the development of a new bioconjugate with potential use in sensing, and many find potential applications in clinical diagnostics, medicine, and industries.
Hashemifard
N
N
Dept. of Biochemistry, Sciences & Research Branch of Islamic Azad University, Tehran, Iran.
Mohsenifar
A
A
Ranjbar
B
B
Allameh
A
A
Lotfi
A S
AS
Etemadikia
B
B
D001234:Aspergillus niger / Q000201:enzymology*; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D005949:Glucose Oxidase / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D053758:Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D012834:Silver / Q000737:chemistry*; D013696:Temperature
D004800:Enzymes, Immobilized; D012834:Silver; D005949:Glucose Oxidase