快速膜乳化法制备温敏微球及其固定胰蛋白酶

Preparation of Thermo-sensitive Microspheres by Premix Membrane Emulsification for Immobilization of Trypsin

采用快速膜乳化法并结合低温聚合法制备了尺寸均一、重复性较好的聚(异丙基丙烯酰胺丙烯酸)[P(NIPAM-co-AAc)]微球.结果表明,所制微球平均粒径为5.2m,多分散性指数为0.0323.对微球温敏响应性质的研究表明,加入亲水性单体会降低微球的低临界共溶解温度(LCST),且加入量越多LCST降低程度越大,交联剂加入量增大,微球的LCST升高.加入亲水性共聚单体,微球的响应时间增加.P(NIPAM-co-AAc)微球用于胰蛋白酶固定化,在1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐浓度0.9mg/mL、胰蛋白酶浓度1.8mg/mL及磷酸盐缓冲液浓度70mmol/L时,可得到固载量为276mg/g、活性回收率达75.07%的最优值,固定化胰蛋白酶的最适pH值为8、最适温度为37℃.

Thermo-sensitive poly（N-isopropyl acrylamide-co-acrylic acid） [P（NIPAM-co-AAc）] microspheres with narrow size distribution were successfully prepared by premix membrane emulsification at room temperature. The results show that the mean diameter of microspheres was 5.2 μm, their polydispersity index as low as 0.0323. The temperature-responsive behaviors of prepared microspheres were characterized. It was confirmed that the lower critical solution temperature （LCST） of P（NIPAM-co-AAc） microspheres decreased with addition of hydrophilic monmer. The higher the monmer amount, the lower the LCST of P（NIPAM-co-AAc） microspheres. The LCST of P（NIPAM-co-AAc） microspheres increased with more addition of N,N＇-methylenebisacrylamide. The respond time increased with addition of hydrophilic monmer. Trypsin was covalently immobilized onto the microspheres. The result showed that when the concentrations of 1-ethyl-3-（3-dimethylaminopropyl）-carbodiimide hydrochloride, trypsin and PBS were 0.9 mg/mL, 1,8 mg/mL and 70 mmol/L, the optimal value of trypsin loading and activity yield, 276.10 mg/g and 75.07% respectively, was reached. The highest activity of immobilized trypsin was obtained at pH 8 and 37 ℃.