Large-scale isolation, fractionation, and purification of soluble starch-synthesizing enzymes: starch synthase and branching enzyme from potato tubers
Large-scale isolation, fractionation, and purification of soluble starch-synthesizing enzymes: starch synthase and branching enzyme from potato tubers
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DOI：
10.1016/j.carres.2010.04.022
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发表时间：
2010-07-19
影响因子：
3.1
通讯作者：
Robyt, John F.
中科院分区：
文献类型：
--
作者：
Mukerjea, Rupendra;Falconer, Daniel J.;Robyt, John F.
从白马铃薯块茎中大规模分离、分离和纯化了可溶性淀粉合成酶淀粉合成酶（SSS）和淀粉分支酶（SBE）。采用5个步骤：从2公斤去皮马铃薯中提取马铃薯块茎提取物，两次丙酮沉淀，两次在大型超滤聚砜中空纤维100 kDa滤筒上分馏。得到三种馏分：(1)SSS和SBE的混合物；(2) SSS，不含SBE；(3) SBE，不含SSS。污染酶（淀粉酶，磷酸化酶）。通过Molisch试验和淀粉三碘化试验判断，第二丙酮沉淀物和柱状馏分中不存在歧化酶)和碳水化合物。常规提取的SSS馏分活度为122%（300,000-400,000单位），SBE馏分活度为187%（40,000-50,000单位）。在甘氨酸缓冲液（pH 8.4）中加入0.04% （w/v）的聚乙烯醇50 K和1 mM的二硫苏糖醇，由于活化了无活性酶，SSS和SBE的长期稳定性和较高的产量。两个馏分的SSS和SBE部分具有很高的比活性，表明纯化程度高。对选取的SSS和SBE组分进行聚丙烯酰胺凝胶电泳，得到2 ~ 5个SSS和/或SBE活性条带，对应于第2个丙酮沉淀中出现的1 ~ 5个蛋白质条带。(C) 2010 Elsevier Ltd.版权所有。
Soluble starch-synthesizing enzymes, starch synthase (SSS) and starch-branching enzyme (SBE), were isolated, fractionated, and purified from white potato tubers (Solanum tuberosum) on a large scale. Five steps were used: potato tuber extract from 2 kg of peeled potatoes, two acetone precipitations, and two fractionations on a large ultrafiltration polysulfone hollow fiber 100 kDa cartridge. Three kinds of fractions were obtained: (1) mixtures of SSS and SBE; (2) SSS, free of SBE; and (3) SBE, free of SSS. Contaminating enzymes (amylase, phosphorylase. and disproportionating enzyme) and carbohydrates were absent from the 2nd acetone precipitate and from the column fractions, as judged by the Molisch test and starch triiodide test. Activity yields of 122% (300,000-400,000 units) of SSS fractions and 187% (40,000-50,000 units) of SBE fractions were routinely obtained from the cartridge. Addition of 0.04% (w/v) polyvinyl alcohol 50 K and 1 mM dithiothreitol to the glycine buffer (pH 8.4) gave long-term stability and higher yields of SSS and SBE, due to activation of inactive enzymes. Several SSS and SBE fractions from the two fractionations had very high specific activities, indicating high degrees of purification. Polyacrylamide gel electrophoresis of selected SSS and SBE fractions gave two to five SSS and/or SBE activity bands, corresponding to the one to five protein bands present in the 2nd acetone precipitate. (C) 2010 Elsevier Ltd. All rights reserved.