In this study, ene reductase (ER) was entrapped in polyvinyl alcohol hydrogel, adsorbed on montmorillonite and immobilized covalently on glutaraldehyde activated 3-aminopropyl-functionalized silica gel. Although protein recovery yields were at least 85% for adsorption and covalent immobilization, only the encapsulated ER showed activity. The activity of free and entrapped ER preparations was measured by following NADPH-dependent reduction of 2-cyclohexen-1-one. The both protein recovery and activity recovery yields were calculated as 100% when 1 mg protein was used for immobilization. The both free and entrapped ER preparations showed the same optimum pH and temperature as 7.0 and 30 °C, respectively. The entrapped ER showed 34.4-fold more thermal stability than that of the free ER at 30 °C. Michaelis-Menten constant and maximum velocity values were 0.25 mM and 1.2 U/mg protein, respectively for the free ER towards 2-cyclohexen-1-one. The corresponding values were 1.5 mM and 0.9 U/mg protein for the entrapped ER. The results of time-course reduction of 2-cyclohexen-1-one showed that the entrapped ER catalyzed the reaction as effectively as the free ER. The entrapped ER remained 85% of its initial activity after 10 reused cycles.

在这项研究中，烯还原酶 (ER) 被包裹在聚乙烯醇水凝胶中，吸附在蒙脱石上并共价固定在戊二醛活化的 3-氨基丙基功能化硅胶上。尽管吸附和共价固定的蛋白质回收率至少为 85%，但只有封装的 ER 显示出活性。游离和包埋的 ER 制剂的活性是通过 2-环己烯-1-one 的 NADPH 依赖性还原来测量的。当使用 1 mg 蛋白质进行固定时，蛋白质回收率和活性回收率均计算为 100%。游离和包埋的 ER 制剂显示出相同的最佳 pH 值和温度，分别为 7.0 和 30°C。在 30 °C 下，包埋的 ER 比游离 ER 的热稳定性高 34.4 倍。Michaelis-Menten 常数和最大速度值分别为 0.25 mM 和 1.2 U/mg 蛋白质，对于 2-cyclohexen-1-one 的游离 ER。包埋 ER 的相应值为 1.5 mM 和 0.9 U/mg 蛋白质。2-cyclohexen-1-one 的时间过程还原结果表明，包埋的 ER 与游离 ER 一样有效地催化反应。在 10 次重复使用后，被困的 ER 仍保持其初始活性的 85%。