绿化黑麦幼苗中叶绿素酶的纯化及性质

Purification and properties of chlorophyllase from greened rye seedlings.

Tanaka K, Kakuno T, Yamashita J, Horio T

J Biochem. 1982 Dec;92(6):1763-73. doi: 10.1093/oxfordjournals.jbchem.a134106.

Abstract

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.

摘要

叶绿素酶[EC 3.1.1.14]是从绿化黑麦幼苗叶绿体的丙酮干粉中用1%胆酸盐提取的，并通过苯基琼脂糖凝胶CL - 4B柱上的疏水色谱法纯化了870倍，产率约为30%。纯化过程包括用丙酮和硫酸铵分级分离以及疏水色谱法。
通过分子筛色谱法和等电聚焦电泳分析，纯化后的酶是纯的。其等电点为4.5，分子量为39,000。
纯化后的酶在pH 6 - 9和4℃下稳定。在pH 7.5时，无论有无30%丙酮存在都稳定。然而，在30℃时，丙酮浓度高于10%时它就不稳定了。
纯化后的酶分别将菠菜中的叶绿素a和b水解为叶绿素酸a和b以及植醇；将红螺菌中的细菌叶绿素a水解为细菌叶绿素酸a和一种植醇衍生物，可能是全反式香叶基香叶醇。在30%丙酮存在下，水解速率被刺激到最大值；细菌叶绿素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、30℃和30%丙酮存在下，叶绿素a的Km值和比活性分别为12μM和480 nmol·min⁻¹·mg⁻¹，红螺菌细菌叶绿素a的Km值和比活性分别为4μM和170 nmol·min⁻¹·mg⁻¹。