苹果多酚氧化酶的纯化与表征

摘要

A novel copper-containing enzyme named PPOⅡ was isolated from acetone-emerged apple powder and purified by using extraction with phosphate buffer, centrifugation at low temperature, precipitation with ammonium sulfate, and separation by column chromatography with DEAE-Sephadex(A-50), Sephadex(G-75), and DEAE-celluse(DE-52). Methods like PAGE, SDS-PAGE, and MALDI-TOF were used to detect the enzyme-purified purity, and determine its molecular mass. A single band on both PAGE and SDS-PAGE showed that pure PPOⅡ was obtained, which was further confirmed by MALDI-TOF. The molecular mass of PPOⅡ is 38204 Da on the basis of the results of both SDS-PAGE and MALDI-TOF. The studies on the effect of pH on the enzymatic activity displayed that the optimum pH is about 6.6. The enzymatic stability to pH increased from pH 4.0 to 7.0, and then decreased from pH 7.0 to 11.0 in terms of activity. The optimal temperature of PPOⅡ is about 30℃.%运用丙酮浸漬干燥、磷酸盐缓冲液提取、低温离心、硫酸铵沉淀、DEAE-Sephadex(A-50)、Sephadex(G-75) 和DEAE-celluse(DE-52)层析等方法从苹果中分离获得一种新的含铜酶蛋白,该酶被命名为多酚氧化酶Ⅱ(polyphenol oxidase Ⅱ, PPOⅡ),纯化倍数是215,纯化收率是23%.PAGE、SDS-PAGE和MALDI-TOF 等技术用于测定所获的酶的纯度和分子量.在PAGE和SDS-PAGE 均显示一条带,表明PPOⅡ只由一个亚基组成,且已达到单一组分(MALDI-TOF的结果更证实了这一点).SDS-PAGE 和 MALDI-TOF 的结果都表明PPO的分子量为 38204 Da.pH值对酶活性和稳定性研究的结果显示,从pH值4.0～7.0随着pH值的增加,酶活性也不断增加;从pH值 7.0～11.0, 酶活性不断降低.PPOⅡ的最适pH值为6.6最适温度为30℃.