源于澳洲青苹果的多酚氧化酶（PPO）
特性及阻氧抑制技术研究
摘 要
澳洲青苹是一种优质高酸苹果，高酸度苹果汁生产的最佳原料。但在苹果汁生产中，鲜果经过破碎、压榨后，很容易发生酶促褐变，对产品的感官品质、风味和营养价值造成极大的影响，这也是阻碍我国果汁产业发展的一个重要原因。酶促褐变是在有氧条件下,多酚氧化酶氧化组织中内源性酚类物质形成邻醌，邻醌再相互聚合或与蛋白质、氨基酸等作用生成高分子络合物而使组织发生褐变的复杂生理过程。本试验成功的从澳洲青苹中分离纯化出多酚氧化酶，并对源于澳洲青苹的多酚氧化酶酶学特性进行了系统试验，建立了源于澳洲青苹的酶促反应动力学模型曲线，运用N2、CO2填充阻隔氧气来抑制多酚氧化酶的催化反应，从而有效地控制了酶促褐变的发生。
主要研究结果如下：
（1）采用丙酮粉沉淀抽滤法从澳洲青苹中提取多酚氧化酶。提取的粗酶液经30%～90%饱和度硫酸铵分级沉淀、透析除盐、交联葡聚糖G-75 凝胶色谱层析、后经PEG 6000浓缩，得到纯化的多酚氧化酶，纯化倍数为7.49。
（2）提取纯化所得的酶液经十二烷基磺酸钠-聚丙烯酰胺凝胶电泳,采用考马斯亮蓝R-250染色显示一条电泳带。其分子量介于31.0kD和43.0kD之间。
（3）通过对澳洲青苹多酚氧化酶与邻苯二酚反应产物的重复波谱扫描，方便快速的确定了量化反应产物含量的特定吸收波长λ＝410nm。
（4）澳洲青苹PPO酶学特性研究表明，PPO的最适pH值约为5.0，最适反应温度为16℃，热处理遵循一级动力；PPO对底物专一性高低的趋势为:邻苯二酚为最高，其次为绿原酸，焦性没食子酸，DL-多巴，对L-酪氨酸的活性为零，说明澳洲青苹PPO没有单元酚氧化酶活性。抑制剂对PPO活性有一定效果，其中，亚硫酸氢钠对PPO活性具有强烈的抑制作用，EDTA、抗坏血酸对PPO的抑制相对较弱。
（5）三氯乙酸是澳洲青苹PPO酶促反应有效的终止剂。降低氧气浓度可以抑制多酚氧化酶的活性，其中，4％的氧气浓度可完全抑制多酚氧化酶的活性；N2、CO2都能通过降低反应体系中氧气浓度而达到抑制多酚氧化酶活性的目的。
（6）澳洲青苹的可溶性固形物为(13.65±0.128)%，可滴定酸为(0.655±0.006)%，Vc含量为(87.67±0.220)mg/kg,具有很好的加工品质。气调破碎，通过用N2、CO2置换气体降低破碎环境中的氧气浓度能很好的减少苹果汁褐变程度。
关键词：澳洲青苹；多酚氧化酶；分离纯化；阻氧抑制
STUDIES ON THE CHARACTERISTECS AND THE INHIBITION BY BLOCKING OXYGEN OF PPO FROM GRANNY SMITH
ABSTRACT
Granny Smith is a kind of high quality apple with high acidity, which is suitable to produce the popular juice on international market. In the process of apple juice production, the fresh fruit is easy to occur enzymatic browning, which not only greatly influences the sensory quality, the flavour and nutritional value of the product but also is an important reason for blocking the development of our fruit juice industry. This browning is usually due to the catalytic action of the enzyme polyphenoloxidase(PPO).o-Diphenols, present in the tissue of fruits and vegetables, are oxidized by polyphenoloxidase in the presence of oxygen. This oxidation reaction results in the production of o-quinones, which subsequently polymerize with other o-quinones, phenolic substances, proteins, amino acids, etc. to produce brown pigments. These pigments are sometimes referred to as melanins. In this experiment, PPO was Purified from Granny Smith ,and its common enzyme properties were studied. The activity of PPO was inhibited by reducing oxygen concentration. The study shows that blocking oxygen is the effective method to control enzymatic browning. The results are summarized as follows:
(1) PPO was extracted from Granny Smith with acetone. The crude enzyme extract was fractionated with solid ammonium sulfate of 30%-90% saturation. After dialysis, the enzyme solution was purified by sephadex G-75 column chromatography respectively. The enzyme was concentrated by PEG 6000.A7.49 fold purification of PPO was obtained.
(2) The result from polyacrylamide gel electrophoresis of purified enzyme indicated that only one band was found using coomassie brilliant blue R-250 stain, and their molecular weight were 31.0kD～43.0kD.
(3) The largest absorbion of the product of the reaction between PPO and catechol was at λ=410nm.
(4) pH optimum was 5.0 with catechol, and temperature optimum was 16℃. The enzyme had different substrate specifities for dirrerent phenolic compounds, a maxium activity was showen with catechol, followed by chlorogenic acid, pyrogallol, DL-DOPA. The activity with tyrosine was not detected. Sodium bisulfite is the best inhibitor.
(5) Trichloroacetic acid is a kind of short-stopper which can stop enzymatic reaction. when the oxygen concentration was decreased to 4%,the activity of PPO was inhibited clearly.
(6) Granny Smith is one of the most suitable breeds for making concentrated apple juice. The results showed that the content of soluble is (13.65±0.128)%, the organic acid is(0.655±0.006)%, and Vc is (87.67±0.220)mg/kg. Atmosphere crushing can control the enzymatic browning of apple juice.
Key words: Granny Smith, polyphenol oxidase, purification，inhibiting by blocking oxygen
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