Kamiya N, Inoue M, Goto M, Nakamura N, Naruta Y
Department of Chemical Systems & Engineering, Graduate School of Engineering, Kyushu University, Hakozaki, Fukuoka 812-8581, Japan.
Biotechnol Prog. 2000 Jan-Feb;16(1):52-8. doi: 10.1021/bp990125b.
A surfactant-horseradish peroxidase (HRP) complex that is catalytically active in organic media has been successfully prepared by a method utilizing water-in-oil (W/O) emulsions. To optimize conditions for preparation of the HRP complex, the effects of some key parameters in the aqueous phase of W/O emulsions were investigated. The surfactant-HRP complex prepared with a nonionic surfactant exhibited a high catalytic activity compared to those with a cationic or anionic surfactant in anhydrous benzene. At the preparation step, the pH of the aqueous solution had a prominent effect on the enzymatic activity of the HRP complex in organic media. Several kinds of salts present in the HRP complex could be employed to enhance the catalytic performance in organic media. However, anionic ions present in the preparation process appeared to lower the catalytic activity owing to the complexation with heme iron. UV-visible absorption spectra of the HRP complex in benzene, which were prepared from a KCN solution (pH 7.0) or an alkaline solution (pH 12), were comparable with those of native HRP in aqueous solution under the same conditions. Resonance Raman spectroscopic studies also revealed that no significant change in the coordination state of the heme iron occurred even after coating the enzyme with surfactant molecules, lyophilization, and solubilization in nonaqueous media.
一种在有机介质中具有催化活性的表面活性剂 - 辣根过氧化物酶（HRP）复合物已通过利用油包水（W/O）乳液的方法成功制备。为了优化HRP复合物的制备条件，研究了W/O乳液水相中一些关键参数的影响。与在无水苯中用阳离子或阴离子表面活性剂制备的复合物相比，用非离子表面活性剂制备的表面活性剂 - HRP复合物表现出高催化活性。在制备步骤中，水溶液的pH对HRP复合物在有机介质中的酶活性有显著影响。HRP复合物中存在的几种盐可用于增强在有机介质中的催化性能。然而，制备过程中存在的阴离子由于与血红素铁络合而似乎降低了催化活性。由KCN溶液（pH 7.0）或碱性溶液（pH 12）制备的苯中HRP复合物的紫外 - 可见吸收光谱与相同条件下水溶液中天然HRP的光谱相当。共振拉曼光谱研究还表明，即使在用表面活性剂分子包被酶、冻干并溶解于非水介质后，血红素铁的配位状态也没有发生显著变化。
