Purification, characterisation, and thermal denaturation of polyphenoloxidase from prawns (Penaeus vannamei)

Abstract

Polyphenoloxidase (PPO) from prawns (Penaeus vannamei) was purified by ammonium sulphate precipitation, ion-exchange column, and gel filtration chromatography. The specific activity of PPO increased by 9.51 fold to 575.03 U/mg, and the molecular weight of PPO was 66 kDa. The optimal temperature and pH were around 45°C and 6.8, determined by L-β-(3,4-dihydroxylphenyl) alanine (L-DOPA) as substrate. Km and Vmax values were found to be 2.5 mM and 0.062ΔA/min. The effect of thermal treatment on the activity, conformation, and microstructure of the PPO was investigated. Thermal stability of PPO was measured at 40°C, 50°C, 60°C, 70°C, and 80°C, and the half-life values of PPO were 105, 39.6, 13.4, 8.9, and 3.1 min, respectively. PPO activity slightly decreased at 60°C, while the relative enzyme activity remained 9.4% after 80°C for 10 min. The result of differential scanning calorimetry showed that the denatured temperature of PPO was 65°C. Thermal treatment resulted in the changes in the tertiary structure of PPO, and the λmax of fluorescence spectra was red-shifted with decreasing intensity. Curve-fitting analysis showed that α-helix and β-sheet decreased, but β-turns and random coil increased with increased temperature. Atomic force microscopy revealed that PPO molecules aggregated remarkably at 60°C. Moreover, the formation of large globular aggregates occurred as the temperature increased.