A fusion protein designed for noncovalent immobilization: Stability, enzymatic activity, and use in an enzyme reactor
A fusion protein designed for noncovalent immobilization: Stability, enzymatic activity, and use in an enzyme reactor
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DOI：
10.1038/nbt0496-481
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发表时间：
1996-04-01
影响因子：
46.9
通讯作者：
Rudolph, R
中科院分区：
文献类型：
--
作者：
Stempfer, G;HollNeugebauer, B;Rudolph, R
我们设计了一种新的固定化酶的方法，利用在其C-末端含有聚阳离子六精氨酸融合肽的酵母cli-葡萄糖苷酶融合蛋白。这种融合蛋白可以直接吸附从粗细胞提取物上的聚阴离子矩阵在一个特定的，定向的方式。在通过聚离子相互作用非共价固定后，融合蛋白的稳定性不受pH-、尿素-或热变性的影响。此外，酶性质（在增加酶浓度下的比活性、米氏常数或酶反应的活化能）不受这种非共价偶联的影响。然而，与可溶形式相比，偶联酶在连续底物转化条件下的操作稳定性显著增加。含有聚离子肽序列的融合蛋白被提出作为生产固定化酶催化剂的通用工具。
We have designed a new method for enzyme immobilization using a fusion protein of yeast cli-glucosidase containing at its C-terminus a polycationic hexa-arginine fusion peptide. This fusion protein can be directly adsorbed from crude cell extracts on polyanionic matrices in a specific, oriented fashion. Upon noncovalent immobilization by polyionic interactions, the stability of the fusion protein is not affected by pH-, urea-, or thermal-denaturation. Furthermore, the enzymatic properties (specific activity at increasing enzyme concentration, Michaelis constant, or activation energy of the enzymatic reaction) are not influenced by this noncovalent coupling. The operational stability of the coupled enzyme under conditions of continuous substrate conversion is, however, increased significantly compared to the soluble form. Fusion proteins containing polyionic peptide sequences are proposed as versatile tools for the production of immobilized enzyme catalysts.