Purification and properties of chlorophyllase from greened rye seedlings.
Purification and properties of chlorophyllase from greened rye seedlings.
复制标题
绿黑麦幼苗叶绿素酶的纯化及其性质。
DOI：
10.1093/oxfordjournals.jbchem.a134106
复制
发表时间：
1982
期刊：
影响因子：
--
通讯作者：
T. Horio
中科院分区：
文献类型：
--
作者：
K. Tanaka;T. Kakuno;J. Yamashita;T. Horio
1. 叶绿素酶[EC 3.1.1.14]以1%的胆酸盐从绿黑麦幼苗叶绿体的丙酮干粉中提取，纯化870倍，产率约为30%。纯化过程由丙酮和硫酸铵分馏和苯基- sepharose CL-4B柱疏水层析组成。2. 经分子筛色谱和等电电泳分析，纯化酶纯度较高。它的等电点是4.5，分子量是39000。3. 纯化后的酶在pH 6-9和4℃时稳定，在pH 7.5时，30%丙酮的存在和不存在都稳定。然而，在30℃时，超过10%的丙酮浓度就不稳定了。4. 纯化后的酶能将菠菜中的叶绿素a和b分别水解为叶绿素内酯a和叶绿素b和植物醇；和细菌叶绿素a从红红螺旋藻转化为细菌叶绿素内酯a和叶绿醇的衍生物，可能是全反式香叶酰香叶醇。在30%丙酮的存在下，水解率达到最大值；叶绿素a的最大刺激约为50%，叶绿素a的最大刺激约为400%。在pH 7.5和30℃，30%丙酮存在下，Km值和比活性分别为12微米和480 nmol。最低为1。叶绿素a为4 μ m和170 nmol。最低为1。mg-1分别用于红小檗细菌叶绿素a。
1. Chlorophyllase [EC 3.1.1.14] was extracted from the acetone-dried powder of the chloroplasts of greened rye seedlings with 1% cholate, and purified 870-fold with a yield of about 30%. The purification procedure was composed of fractionations with acetone and ammonium sulfate, and hydrophobic chromatography on a phenyl-Sepharose CL-4B column. 2. The purified enzyme was pure as analyzed by molecular-sieve chromatography and isoelectric electrophoresis. It had an isoelectric point of 4.5 and a molecular weight of 39,000. 3. The purified enzyme was stable at pH 6-9 and 4 degrees C. At pH 7.5, it was stable in the presence and absence of 30% acetone. However, at 30 degrees C, it was not stable above a 10% concentration of acetone. 4. The purified enzyme hydrolyzed chlorophylls a and b from spinach into chlorophyllides a and b and phytols, respectively; and bacteriochlorophyll a from Rhodospirillum rubrum into bacteriochlorophyllide a and a derivative of phytol, possibly all-trans-geranylgeraniol. The hydrolysis rates were stimulated to their maxima in the presence of 30% acetone; maximum stimulation was about 50% with bacteriochlorophyll a and about 400% with chlorophyll a. 5. At pH 7.5 and 30 degrees C in the presence of 30% acetone, the Km values and specific activities were 12 microM and 480 nmol . min-1 . mg-1 for chlorophylls a, and 4 microM and 170 nmol . min-1 . mg-1 for R. rubrum bacteriochlorophyll a, respectively.