Beijing National Laboratory for Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, China.
Anal Chim Acta. 2011 Apr 29;692(1-2):131-7. doi: 10.1016/j.aca.2011.03.001. Epub 2011 Mar 5.
A new kind of immobilized trypsin reactor based on sub-micron skeletal polymer monolith has been developed. Covalent immobilization of trypsin on this support was performed using the epoxide functional groups in either a one- or a multi-step reaction. The proteolytic activity of the immobilized trypsin was measured by monitoring the formation of N-α-benzoyl-L-arginine (BA) which is the digestion product of a substrate N-α-benzoyl-L-arginine ethyl ester (BAEE). Results showed that the digestion speed was about 300 times faster than that performed in free solution. The performance of such an enzyme reactor was further demonstrated by digesting protein myoglobin. It has been found that the protein digestion could be achieved in 88 s at 30°C, which is comparable to 24 h digestion in solution at 37°C. Furthermore, the immobilized trypsin exhibits increased stability even after continuous use compared to that in free solution. The present monolithic enzyme-reactor provides a promising platform for the proteomic research.
一种基于亚微米骨架聚合物整体的新型固定化胰蛋白酶反应器已经被开发出来。使用环氧官能团，通过一步或多步反应，将胰蛋白酶共价固定在该载体上。通过监测 N-α-苯甲酰-L-精氨酸（BA）的形成来测量固定化胰蛋白酶的蛋白水解活性，BA 是一种 N-α-苯甲酰-L-精氨酸乙酯（BAEE）的水解产物。结果表明，固定化胰蛋白酶的消化速度比在游离溶液中快约 300 倍。通过消化蛋白质肌红蛋白进一步证明了这种酶反应器的性能。已经发现，在 30°C 下可以在 88 秒内完成蛋白质消化，这与在 37°C 下在溶液中 24 小时消化相当。此外，与游离溶液相比，固定化胰蛋白酶即使在连续使用后也表现出更高的稳定性。本整体式酶反应器为蛋白质组学研究提供了一个有前途的平台。
