Porfirif María C, Milatich Esteban J, Farruggia Beatriz M, Romanini Diana
Departamento de Tecnología, Facultad de Ciencias Bioquímicas y Farmacéuticas. Universidad Nacional de Rosario, IPROBYQ-CONICET, Suipacha 531 (S2002RLK), Rosario, Argentina.
Departamento de Tecnología, Facultad de Ciencias Bioquímicas y Farmacéuticas. Universidad Nacional de Rosario, IPROBYQ-CONICET, Suipacha 531 (S2002RLK), Rosario, Argentina.
J Chromatogr B Analyt Technol Biomed Life Sci. 2016 Jun 1;1022:87-92. doi: 10.1016/j.jchromb.2016.04.015. Epub 2016 Apr 8.
A one-step method as a strategy of alpha-amylase concentration and purification was developed in this work. This methodology requires the use of a very low concentration of biodegradable polyelectrolyte (Eudragit(®) E-PO) and represents a low cost, fast, easy to scale up and non-polluting technology. Besides, this methodology allows recycling the polymer after precipitation. The formation of reversible soluble/insoluble complexes between alpha-amylase and the polymer Eudragit(®) E-PO was studied, and their precipitation in selected conditions was applied with bioseparation purposes. Turbidimetric assays allowed to determine the pH range where the complexes are insoluble (4.50-7.00); pH 5.50 yielded the highest turbidity of the system. The presence of NaCl (0.05M) in the medium totally dissociates the protein-polymer complexes. When the adequate concentration of polymer was added under these conditions to a liquid culture of Aspergillus oryzae, purification factors of alpha-amylase up to 7.43 and recoveries of 88% were obtained in a simple step without previous clarification. These results demonstrate that this methodology is suitable for the concentration and production of alpha-amylase from this source and could be applied at the beginning of downstream processing.
本研究开发了一种一步法作为α-淀粉酶浓缩和纯化的策略。该方法需要使用极低浓度的可生物降解聚电解质（Eudragit® E-PO），是一种低成本、快速、易于扩大规模且无污染的技术。此外，该方法允许在沉淀后回收聚合物。研究了α-淀粉酶与聚合物Eudragit® E-PO之间可逆的可溶/不溶复合物的形成，并将其在选定条件下的沉淀应用于生物分离目的。比浊法可确定复合物不溶的pH范围（4.50-7.00）；pH 5.50时系统浊度最高。培养基中NaCl（0.05M）的存在会使蛋白质-聚合物复合物完全解离。当在这些条件下向米曲霉液体培养物中加入适当浓度的聚合物时，无需预先澄清，只需一步即可获得高达7.43的α-淀粉酶纯化因子和88%的回收率。这些结果表明，该方法适用于从该来源浓缩和生产α-淀粉酶，可应用于下游加工的起始阶段。
