Key Laboratory of Subtropical Agriculture and Environment, Ministry of Agriculture, College of Resources and Environment, Huazhong Agricultural University, Wuhan 430070, China.
Colloids Surf B Biointerfaces. 2010 Jun 1;77(2):166-73. doi: 10.1016/j.colsurfb.2010.01.020. Epub 2010 Feb 4.
Acid phosphatase was immobilized on layered double hydroxides of uncalcined- and calcined-Mg/Al-CO(3) (Unc-LDH-CO(3), C-LDH-CO(3)) by the means of direct adsorption. Optimal pH and temperature for the activity of free and immobilized enzyme were exhibited at pH 5.5 and 37 degrees C. The Michaelis constant (K(m)) for free enzyme was 1.09 mmol mL(-1) while that for immobilized enzyme on Unc-LDH-CO(3) and C-LDH-CO(3) was increased to 1.22 and 1.19 mmol mL(-1), respectively, indicating the decreased affinity of substrate for immobilized enzymes. The residual activity of immobilized enzyme on Unc-LDH-CO(3) and C-LDH-CO(3) at optimal pH and temperature was 80% and 88%, respectively, suggesting that only little activity was lost during immobilization. The deactivation energy (E(d)) for free and immobilized enzyme on Unc-LDH-CO(3) and C-LDH-CO(3) was 65.44, 35.24 and 40.66 kJ mol(-1), respectively, indicating the improving of thermal stability of acid phosphatase after the immobilization on LDH-CO(3) especially the uncalcined form. Both chemical assays and isothermal titration calorimetry (ITC) observations implied that hydrolytic stability of acid phosphatase was promoted significantly after the immobilization on LDH-CO(3) especially the calcined form. Reusability investigation showed that more than 60% of the initial activity was remained after six reuses of immobilized enzyme on Unc-LDH-CO(3) and C-LDH-CO(3). A half-life (t(1/2)) of 10 days was calculated for free enzyme, 55 and 79 days for the immobilized enzyme on Unc-LDH-CO(3) and C-LDH-CO(3) when stored at 4 degrees C. Therefore, immobilization of acid phosphatase on Unc-LDH-CO(3) and C-LDH-CO(3) by direct adsorption is an effective means and would have promising potential for the practical application in agricultural production and environmental remediation.
酸磷酸酶通过直接吸附固定在未煅烧和煅烧的 Mg/Al-CO（3）层状双氢氧化物（Unc-LDH-CO（3），C-LDH-CO（3））上。游离酶和固定化酶的最佳 pH 和温度分别为 pH5.5 和 37°C。游离酶的米氏常数（K（m））为 1.09mmol mL（-1），而固定在 Unc-LDH-CO（3）和 C-LDH-CO（3）上的固定化酶的 K（m）增加到 1.22 和 1.19mmol mL（-1），表明固定化酶对底物的亲和力降低。固定在 Unc-LDH-CO（3）和 C-LDH-CO（3）上的固定化酶在最佳 pH 和温度下的剩余活性分别为 80%和 88%，表明固定化过程中仅损失了少量活性。游离酶和固定在 Unc-LDH-CO（3）和 C-LDH-CO（3）上的固定化酶的失活动力学能（E（d））分别为 65.44、35.24 和 40.66kJ mol（-1），表明固定在 LDH-CO（3）上，特别是未煅烧的形式后，酸磷酸酶的热稳定性得到提高。化学测定和等温滴定量热法（ITC）观察均表明，固定在 LDH-CO（3）上特别是煅烧形式后，酸磷酸酶的水解稳定性得到显著提高。重复使用性研究表明，固定在 Unc-LDH-CO（3）和 C-LDH-CO（3）上的固定化酶经过六次重复使用后，仍保留初始活性的 60%以上。游离酶的半衰期（t（1/2））为 10 天，固定在 Unc-LDH-CO（3）和 C-LDH-CO（3）上的固定化酶的半衰期分别为 55 天和 79 天，当储存在 4°C 时。因此，通过直接吸附将酸磷酸酶固定在 Unc-LDH-CO（3）和 C-LDH-CO（3）上是一种有效的方法，在农业生产和环境修复的实际应用中具有广阔的应用前景。
