温度响应型HRP纳米胶囊用于酶的高效固定和回收

以辣根过氧化物酶(horseradish peroxidase,HRP)为目标蛋白,N-异丙基丙烯酰胺(NIPAM)为单体,采用原位沉淀聚合法,在37℃,NIPAM和HRP质量比为6∶1的条件下,制备了粒径大小为13.7 nm,zeta电位是(-3.7±0.3)mV的温度响应型辣根过氧化物酶纳米胶囊(nHRP).采用基质辅助激光解吸电离飞行时间质谱仪(MALDI-TOF MS)、透射电镜(TEM)、动态光散射仪(DLS)和紫外-可见光分光光度计(UV-Vis)等对HRP的修饰度,nHRP的微观结构、粒径大小、催化活性、温度响应性能和热稳定性能进行了表征.研究表明,制备的辣根过氧化物酶纳米胶囊单分散性较好;在环境温度高于33℃时,nHRP出现响应变化,其粒径大小变化显著,且呈可逆性;nHRP相比HRP原酶热稳定性显著提高;50℃时,将nHRP高速离心,可使酶多次有效地分离和回收.

THERMOSENSITIVE NANOCAPSULES FOR EFFECTIVE IMMOBILIZATION AND RECYCLING OF HORSERADISH PEROXIDASE

Thermoresponsive horseradish peroxidase nanocapsules(nHRP) were prepared via in situ precipitation polymerization using N-isopropyl acrylamide(NIPAM) as the monomer.As an example,nHRP was prepared by a NIPAM/HRP mass ratio of 6,under the reaction temperature of 37 ℃,it showed a monodispersed size distribution centered at 13.7 nm and a negative zeta potential centered at(-3.7±0.3) mV.The modification degrees of HRP were characterized by MALDI-TOF mass spectrometer(MALDI-TOF MS),while the enzymatic activity of the free native HRP,modified HRP and encapsulated nHRP were determined by conducting a standard enzyme activity assay and followed by monitoring absorbance change through UV-Vis spectrometer.The particle size and morphology of nHRP were characterized using dynamic light scattering(DLS) and transmission electron microscopy(TEM).The results showed a monodispersed nHRP was prepared successfully.Compared with the native HRP,nHRP exhibits significantly improved thermal stability,according to the HRP activity assay.Thermoresponsive behavior of nHRP was examined from 25 ℃ to 45 ℃ by DLS studies,where nHRP showed a responsive temperature at 33 ℃ and a reversible response property.Accordingly,high-speed centrifugation was applied to separate and recycle nHRP at 50 ℃ with a well-retained activity.This work provides a new delicate way to immobilize enzyme based on thermoresponsive enzyme nanocapsules,which is of great interest for effective enzyme separation and recycling and other applications.