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首页> 外文期刊>Separation and Purification Technology >Purification of tomato (Lycopersicon esculentum) α-galactosidase by three-phase partitioning and its characterization
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Purification of tomato (Lycopersicon esculentum) α-galactosidase by three-phase partitioning and its characterization

机译：三相分配法纯化番茄α-半乳糖苷酶及其表征

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Three-phase partitioning (TPP) was used to purify α-galactosidase from tomato (Lycopersicon esculentum). The technique is a novel separation process used for the extraction and purification of biomolecules. It involves the addition of salt (generally ammonium sulfate) to the crude extract followed by the addition of an organic solvent (generally butanol). The protein appears as an interfacial precipitate between upper organic solvent and lower aqueous phases. The various conditions required for attaining efficient purification of the α-galactosidase fractions were optimized. Under optimized conditions, it was seen that, 50% of ammonium sulfate saturation with 1:1 ratio of crude extract to t-butanol at pH 4.5 gave 4.3-fold purification with 80% activity yield of α-galactosidase. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed substantial purification and the molecular weight of α-galactosidase was nearly found to be as 34 kDa. The purified enzyme was characterized with respect to its activity and stability at various pH and temperature ranges. Optimum pH and temperature were determined at pH 4 and 37 °C, respectively. The enzyme was stable in the range of pH 3-5 and more than 60% of its initial activity was recovered. The α-galactosidase completely retained nearly about 70% of its initial activity at 40 °C. The kinetic constants; K_m and V_(max) using p-nitrophenyl-α-D-galactopyranoside (PNPG) as substrate were 1.07 mM and 0.01 U/mg, respectively. TPP is an attractive process for the purification of α-galactosidase.
机译：三相分配（TPP）用于从番茄（Lycopersicon esculentum）中纯化α-半乳糖苷酶。该技术是用于生物分子的提取和纯化的新型分离方法。它涉及将盐（通常为硫酸铵）添加至粗提物中，然后添加有机溶剂（通常为丁醇）。该蛋白质显示为上层有机溶剂和下层水相之间的界面沉淀。优化了有效纯化α-半乳糖苷酶馏分所需的各种条件。在优化的条件下，可以看出，在pH 4.5的条件下，粗提取物与叔丁醇的比例为1：1的50％硫酸铵饱和溶液可提纯4.3倍，α-半乳糖苷酶的活性收率为80％。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析显示有大量纯化，α-半乳糖苷酶的分子量几乎为34 kDa。关于纯化的酶在各种pH和温度范围内的活性和稳定性进行了表征。分别在pH 4和37°C下确定最佳pH和温度。该酶在pH值3-5范围内稳定，并且回收了超过60％的初始活性。在40°C时，α-半乳糖苷酶完全保留了其初始活性的近70％。动力学常数;使用对硝基苯基-α-D-吡喃半乳糖苷（PNPG）作为底物的K_m和V_（max）分别为1.07 mM和0.01 U / mg。 TPP是纯化α-半乳糖苷酶的一种有吸引力的方法。

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