A closed concept for purification of the membrane-bound cholesterol oxidase from Nocardia rhodochrous by surfactant-based cloud-point extraction, organic-solvent extraction and anion-exchange chromatography
A closed concept for purification of the membrane-bound cholesterol oxidase from Nocardia rhodochrous by surfactant-based cloud-point extraction, organic-solvent extraction and anion-exchange chromatography
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发表时间：
1996
影响因子：
2.8
通讯作者：
T. Minuth;J. Thömmes;M. Kula
中科院分区：
文献类型：
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作者：
T. Minuth;J. Thömmes;M. Kula
从玫瑰色诺卡氏菌中分离胆固醇氧化酶，并在实验室规模上纯化以产生分析级酶制剂。使用混合的非离子型聚氧乙烯表面活性剂溶液溶解的完整的膜蛋白，并选择性地提取温度诱导的相分离后，从未澄清的玫瑰色诺卡氏菌肉汤。我们提出了一个简单的两步法回收用于蛋白质提取的洗涤剂，并证明了成功的耦合的常规技术，如阴离子交换色谱的初始表面活性剂为基础的蛋白质提取步骤的最终产品纯化。2-甲基丙-1-醇用于萃取共沸物（表面活性剂富集）相以除去和再循环表面活性剂。经阴离子交换层析后，获得了适合于分析应用的160倍纯化的酶（sp.活性为16单位/mg），总产率为80%。
Cholesterol oxidase was isolated from Nocardia rhodochrous and purified on a bench scale to produce an analytical-grade enzyme preparation. The integral membrane protein was solubilized using a mixed non-ionic polyoxyethylene surfactant solution and selectively extracted after temperature-induced phase separation starting from unclarified broth of Nocardia rhodochrous. We present a simple two-step method for recycling the detergent used for protein extraction and demonstrate the successful coupling of conventional techniques such as anion-exchange chromatography to an initial surfactant-based protein-extraction step for final product purification. 2-Methylpropan-1-ol was employed for extraction of the coazervate (surfactant-enriched) phase to remove and recycle the surfactant. After anion-exchange chromatography, a 160-fold purified enzyme (sp. activity of 16 units/mg) suitable for analytical applications was obtained with an overall yield of 80%.