关键词: 金银花, 过氧化物酶, 三相分离法, 酶学性质

Abstract: Three phase partitioning was used to extract and purify peroxidase (POD) from Lonicera japonica Thunb. The optimal conditions were obtained as follows: pH 5.60, ammonium sulfate concentration 39.49 g/100 mL, and ratio of crude extract to t-butanol (V/V), 1:1.38. Under these conditions, the activity recovery was 87.64% with a purification factor of 5.849, and the specific activity of purified peroxidase was 1 021.6 U/mg. By using three phase partitioning, 92% pigment was removed. Enzymatic properties revealed that the optimum temperature for this enzyme was 30 ℃ and the optimum pH was 5. The enzyme was stable in the temperature range of 10–40 ℃ and in the pH range of 4–7. In the presence of both guaiacol and H2O2, Michaelis constants (Km) and maximum reaction rates (vmax) of the enzyme were 8.12 mmol/L and 1.71 mmol/(min·L) for guaiacol, and 0.822 mmol/L and and 1.38 mmol/(min·L) for H2O2 at a fixed concentration of the other substrate, respectively. The affinity of the enzyme to various substrates was in the decreasing order: pyrogallol > catechol > bimethoxy aniline > guaiacol. The peroxidase activity was activated by Ca2+, Cu2+ and Zn2+ but inhibited by Mg2+, Mn2+, citric acid, ascorbic acid, L-cysteine, sodium sulphite and sodium metabisulphite.

Key words: Lonicera japonica Thunb., peroxidase, three phase partitioning, enzymatic properties