Shi Qian, Chen Junhui, Wang Yanlong, Li Zhaoyong, Li Xianguo, Sun Chengjun, Zheng Li
College of Chemistry and Chemical Engineering, Ocean University of China, Qingdao 266100, China.
Research Center for Marine Ecology, The First Institute of Oceanography, State Oceanic Administration, Qingdao 266061, China.
Molecules. 2015 Nov 5;20(11):19971-83. doi: 10.3390/molecules201119670.
In this study, immobilized COX-2 was successfully constructed through glutaraldehyde-mediated covalent coupling on functional silica gel microspheres. The optimum conditions, properties, and morphological characteristics of the immobilized COX-2 were investigated. The optimal immobilization process was as follows: about 0.02 g of aminated silica gel microspheres was activated by 0.25% GA solution for 6 h and mixed with 5 U of free recombinant COX-2 solution. Then, the mixture was shaken for 8 h at 20 °C. Results showed that the immobilized COX-2 produced by this method exhibited excellent biocatalytic activity, equivalent to that of free COX-2 under the test conditions employed. The best biocatalytic activity of immobilized COX-2 appeared at pH 8.0 and still maintained at about 84% (RSD < 7.39%, n = 3) at pH 10.0. For temperature tolerance, immobilized COX-2 exhibited its maximum biocatalytic activity at 40 °C and about 68% (RSD < 6.99%, n = 3) of the activity was maintained at 60 °C. The immobilized COX-2 retained over 85% (RSD < 7.26%, n = 3) of its initial biocatalytic activity after five cycles, and after 10 days storage, the catalytic activity of immobilized COX-2 still maintained at about 95% (RSD < 3.08%, n = 3). These characteristics ensured the convenient use of the immobilized COX-2 and reduced its production cost.
在本研究中，通过戊二醛介导的共价偶联在功能性硅胶微球上成功构建了固定化COX-2。研究了固定化COX-2的最佳条件、性质和形态特征。最佳固定化过程如下：约0.02 g胺化硅胶微球用0.25%的GA溶液活化6 h，并与5 U游离重组COX-2溶液混合。然后，混合物在20℃下振荡8 h。结果表明，该方法制备的固定化COX-2表现出优异的生物催化活性，在所采用的测试条件下与游离COX-2相当。固定化COX-2的最佳生物催化活性出现在pH 8.0时，在pH 10.0时仍保持在约84%（相对标准偏差<7.39%，n = 3）。对于温度耐受性，固定化COX-2在40℃时表现出最大生物催化活性，在60℃时保持约68%（相对标准偏差<6.99%，n = 3）的活性。固定化COX-2在五个循环后保留了超过85%（相对标准偏差<7.26%，n = 3）的初始生物催化活性，在储存10天后，固定化COX-2的催化活性仍保持在约95%（相对标准偏差<3.08%，n = 3）。这些特性确保了固定化COX-2的使用便利性并降低了其生产成本。
