Purification of thermamylase in multicompartment electrolyzers with isoelectric membranes: The problem of protein solubility
Purification of thermamylase in multicompartment electrolyzers with isoelectric membranes: The problem of protein solubility
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DOI：
10.1002/elps.1150170712
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发表时间：
1996-07-01
期刊：
影响因子：
2.9
通讯作者：
Righetti, PG
中科院分区：
文献类型：
--
作者：
EsteveRomero, JS;Bossi, A;Righetti, PG
来自地衣芽孢杆菌的主要α-淀粉酶（由于其耐高温（90 ° C）而被称为热淀粉酶）已经在具有等电膜的多室电解槽中通过等电聚焦进行纯化。该酶倾向于沉淀，在接近其pI值（7.18）时产生严重的涂片。溶解度不能通过任何通常在等电聚焦中采用的并且与酶活性相容的已知方法来改善，例如添加中性和两性离子表面活性剂（例如，Nonidet，3-[（3-胆酰胺丙基）二甲基铵基]-1-丙烷-磺酸盐，最多2%），混合的水-有机溶剂（甘油，乙二醇，丙二醇）和添加不能形成胶束的两性离子，如牛磺酸。然而，添加糖，特别是蔗糖，山梨糖醇，并在较小程度上，山梨糖，大大提高了蛋白质的溶解度在pI附近。如果这些糖与0.2 M牛磺酸一起使用，则改善是显著的;此外，溶解度的增加（当达到40%的不同糖的水平时发生）伴随着大的pI偏移，通常使pI值降低多达0.4个pH单位（例如，从不存在添加剂时的pI 7.18到存在40%蔗糖和0.2M牛磺酸的混合物时的pI 6.80，牛磺酸是所有研究系列中最好的增溶剂）。这种明显的pI偏移不是由于添加剂的存在引起的pH梯度变化，因为在不存在和存在添加剂的情况下的pH测量给出了相同的结果。这些结果可以用Timasheff和Arakawa关于溶剂稳定蛋白质结构的理论来解释：糖（在约100 ℃）。1 M浓度）和两性离子如牛磺酸属于I类稳定剂，其特征在于与蛋白质的负结合和增加水的表面张力。因此，蛋白质处于"过度水合"状态，这可能会阻止与凝胶基质中的Immobilines结合，并可能改变蛋白质表面上的一些pK。在40%蔗糖和0.2M牛磺酸的溶液中，热淀粉酶可以在多室电解槽中被成功地纯化到单一的等电和等离子带。
The main alpha-amylase from Bacillus licheniformis (called thermamylase because of its resistance to high temperatures, 90 degrees C) has been subjected to purification by isoelectric focusing in multicompartment electrolyzers with isoelectric membranes. The enzyme tended to precipitate, producing severe smears in proximity of its pI value (7.18). Solubility could not be ameliorated by any of the known means typically adopted in isoelectric focusing and compatible with enzyme activity, such as addition of neutral and zwitterionic surfactants (e.g., Nonidet, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate, up to 2%), mixed hydro-organic solvents (glycerol, ethylene glycol, propylene glycol) and addition of zwitterions unable to form micelles, such as taurine. However, addition of sugars, notably saccharose, sorbitol, and, to a lesser extent, sorbose, greatly improved protein solubility in the pI proximity. The improvement was dramatic if these sugars were admired with 0.2 M taurine; Additionally, the increment of solubility (which occurred when reaching a level of 40% of the different sugars) was accompanied by a large pI shift, typically reducing the pI value by as much as 0.4 pH units (e.g., from a pI of 7.18 in the absence of additives to a pI of 6.80 in presence of a mixture of 40% sucrose and 0.2 M taurine, the best solubilizer in all the series investigated). This apparent pI shift was not due to a change of pH gradient caused by the presence of additives, since pH measurements in the absence as well as presence of additives gave identical results. The results are explained by the theory of Timasheff and Arakawa on stabilization of protein structure by solvents: sugars (at ca. 1 M concentration) and zwitterions such as taurine belong to class I stabilizers, characterized by negative binding to proteins and by increasing the surface tension of water. As a result, the protein is in a state of ''superhydration'' which might prevent binding to Immobilines in the gel matrix and might alter some pKs on the protein surface. In solutions of 40% saccharose and 0.2 M taurine, thermamylase could be successfully purified to a single isoelectric and isoionic band in the multicompartment electrolyzer.