Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
Molecules. 2018 Jan 19;23(1):192. doi: 10.3390/molecules23010192.
Chitosan macro-particles prepared by the neutralization method were applied to Sortase A (SrtA) immobilization using glutaraldehyde as a crosslinking agent. The particles were characterized by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Response surface methodology (RSM) was employed to optimize the immobilization process. An average specific activity of 3142 U (mg protein) was obtained under optimized immobilization conditions (chitosan concentration 3%, SrtA concentration 0.5 mg·mL, glutaraldehyde concentration 0.5%, crosslinking and immobilization at 20 °C, crosslinking for 3 h, and an immobilization time of 8 h). The transpeptidase activity of immobilized SrtA was proved by a peptide-to-peptide ligation with a conversion yield approximately at 80%, and the immobilized catalyst was successfully reused for five cycles without obvious activity loss. Moreover, the scale-up capability of using immobilized SrtA to catalyze a head-to-tail peptide cyclization was investigated in a batch reaction and the conversion yield was more than 95% when using 20 mg of peptide as a substrate.
采用中性化法制备的壳聚糖大颗粒，并用戊二醛作为交联剂固定化 Sortase A（SrtA）。通过傅里叶变换红外光谱（FTIR）和扫描电子显微镜（SEM）对颗粒进行了表征。采用响应面法（RSM）对固定化过程进行了优化。在优化的固定化条件下（壳聚糖浓度为 3%，SrtA 浓度为 0.5mg·mL，戊二醛浓度为 0.5%，在 20°C 下交联和固定化，交联 3 小时，固定化时间为 8 小时），获得了平均比酶活 3142U（mg 蛋白）。通过肽对肽的连接证明了固定化 SrtA 的转肽酶活性，转化率约为 80%，固定化催化剂成功重复使用了五次，没有明显的活性损失。此外，还研究了在间歇反应中使用固定化 SrtA 催化从头至尾肽环化的放大能力，当使用 20mg 肽作为底物时，转化率超过 95%。
