Immobilization ofPycnoporus sanguineuslaccase on copper tetra-aminophthalocyanine–Fe3O4 nanoparticle composite

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A magnetic nanoparticle composite of CuTAPc (copper tetra-aminophthalocyanine)–Fe3O4 was prepared byin situcomplexation and was characterized by FTIR (Fourier-transform IR) spectroscopy, X-ray diffraction, X-ray photoelectron spectroscopy, FESEM (field emission scanning electron microscopy) micrograph and hysteresis loop. The results showed that CuTAPc formed the covering layer on the surface of the composite. Using glutaraldehyde, laccase was covalently attached to the surface of the composite. When white-rot-fungus (Pycnoporus sanguineus) laccase was immobilized on the CuTAPc–Fe3O4 nanoparticle composite in PBS, the optimal reaction pH was 5.0 and the optimal temperature was 0 °C. When 2.0 mg/ml laccase solution was used, the immobilization yield was 20%. The activity,Km,kcat/Km andVmax values of the immobilized laccase were 1.43 units/mg, 2.38×10−5 mol/l, 2.1×103 mol−1·s−1·l and 1.19×10−6 mol·l−1·min−1 respectively. The system exhibited maximum enzyme activity at pH 3.0 and at 45 °C. After storage at 4 °C for 1 month, the residual activity of the immobilized laccase was 85% of its initial activity, while that of free laccase was only 30%. During 240 min incubation at 55 °C, the activity of immobilized laccase quickly increased to a maximum after 150 min and then decreased to 95% in the next 90 min, while the activity of the free enzyme decreased monotonically to 28%. After five consecutive operations, the immobilized laccase still retained 80% of its initial activity.

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