在优化茶树叶绿素酶提取及活性分析条件的基础上,对茶树叶绿素酶的分离纯化及其性质进行了研究,结果表明：加入与鲜叶等量PVP,用pH7.5的磷酸二氢钠-磷酸氢二钠缓冲液提取的叶绿素酶活性最高,叶绿素酶的活性分析最佳条件为在45℃条件下反应15 min;经过分离纯化后,叶绿素酶的纯度提高了55.96倍,酶的得率为9.45%,SDS-PAGE结果显示有一条蛋白质的条带,酶的亚基分子量为37.2 kD;对纯化后的叶绿素酶的性质研究表明,该酶最适pH为7.5,45℃时候反应活性最高,在4℃保存条件下,随着存放时间的延长,酶活性逐渐下降,保存一个月后叶绿素酶活性剩余45%,用2 mmol/L和7 mmol/L的金属离子（Ca2+、Mg2+、Zn2+、Fe3+）、EDTA处理,都对叶绿素酶活性有不同程度的促进作用。
Under the optimized conditions for the enzyme extraction and activity analysis, chlorophyllase was purified and its characterization was studied. The results indicated that the amount of polivinpolypyrrolidone (PVP) had great impact on chlase activity, chlase activity increased to max as pH value rose to 7.5. It was found that the enzyme reaction was stable at 45℃ in 15 min. Using the methods of ammonium sulfate precipitation and column chromatography on Sephadex G-200, Sepharose CL-6B, chlase in the tea leaves was purified. After three steps of Isolation and purification, It was purified 55.96 fold and a yield of 9.45%. there are one chlase stripe by SDS-PAGE. The subunit molecular mass was 37.2 kD. Research on the characterization of the purified chlase proved that the enzyme showed optimal activity at pH 7.5 and 45℃. The survival rate of enzyme activity remained 45% when stored 4℃ for a month. Adding metal ions Ca2+、Mg2+、Zn2+、Fe3+ and EDTA into chlase solution could show the promoting role on the activity of chlorophyllase in certain degree.
[1] Debora JW, Doron H, Elezer EC, et al. Chlorophyll breakdown by chlorophyllase: isolation and functional expression of Chlase gene from ethylene-treated Citrus fruit and its regulation during development[J]. Plant Journal, 1999, 6: 653~661.
[2] Fernandez-Lopez J A, Almela L, Sole-dad-Almansa M, et al.Partial purification and properties of chorophyllase from chlorotic Citrus limon leaves[J]. Phytochemistry, 1992, 31: 447~449.
[3] Rudiger W.Chlorophyll metabolism:from outer space down to the molecular level[J]. Phytochem, 1997, 7: 1151~1167.
[4] Garcia AL, Galind OL.Chlorophyllase in citrus leaves: localization and partial purification of the enzyme[J]. Photosynthetica, 1991, 25: 105~111.
[5] Tsuchiya T, Ohta H, Msauda T, et al. Purification and characterization of weo isozymes of chlorophyllase from mature leaves of Chenpodium album[J]. Plant Cell Physiol, 1997, 9: 1036~1031.
[6] Mingufz-Mosquera MI, Ganduljas B, Gsallardo-Guerrero L.Measurement of chlorophyllase activity in olive fruit (Olea europaea)[J]. Biochem, 1994, 116: 263~268.
[7] Nagao Ogura.Studies on chlorophyllase in tea leaves Ⅲ. Properties of soluble and insoluble chlorophyllases[J]. Plant and cell physiology, 1972, 13: 971~979.
[8] Mioko Kuroki, Yuzo Shioi and Tsutomu SASA. Purification and Properties of soluble chlorophyllase from tea leaf sprouts[J]. Plant and cell physiology, 1981, 4: 717~725.
[9] Katsunori Kohata, Kouichi Hanada, Yuji Yamauchi, et al. Pheophorbide a content and chlorophyllase activity in green tea. Biosci. Biotechnol. Biochem., 1998, 9: 1660~1663.
[10] Roger F, Mcfeeters. Substrate specificity of chlorophyllase[J]. Plant Physiology, 1975, 55: 377~381.
[11] AmirShapira D, Goldschmidt EE,AItman A, Chloropyhll catabolism in senescing plant tissue:In vivo breakdown intermediates suggest different degradative pathways for Citrus fruit and parsley leaves[J]. Proc Natl.Accad Sci USA, 1987, 84: 1901~1905.
[12] 唐蕾. 银杏叶生长过程中的叶绿素酶活性变化[J]. 植物生理学通讯, 2002, 38(6): 561~563.
浙公网安备 33019902000101号 
