Stempfer G, Höll-Neugebauer B, Kopetzki E, Rudolph R
Boehringer Mannheim Therapeutics, Pennzberg, Germany.
Nat Biotechnol. 1996 Apr;14(4):481-4. doi: 10.1038/nbt0496-481.
We have designed a new method for enzyme immobilization using a fusion protein of yeast alpha-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.
我们设计了一种新的酶固定化方法，该方法使用酵母α-葡萄糖苷酶的融合蛋白，其C末端含有一个聚阳离子六聚精氨酸融合肽。这种融合蛋白可以以特定的、定向的方式从粗细胞提取物中直接吸附到聚阴离子基质上。通过聚离子相互作用进行非共价固定后，融合蛋白的稳定性不受pH、尿素或热变性的影响。此外，酶学性质（酶浓度增加时的比活性、米氏常数或酶促反应的活化能）不受这种非共价偶联的影响。然而，与可溶性形式相比，在连续底物转化条件下偶联酶的操作稳定性显著提高。含有聚离子肽序列的融合蛋白被认为是生产固定化酶催化剂的通用工具。
