介孔碳的快速水相合成及对辣根过氧化物酶的固定

Rapid Aqueous Synthesis of Mesoporous Carbon and Their HRP Immobilization Performance

以间苯三酚-甲醛为碳源前驱体,嵌段聚合物F127(PEO106PPO70PEO106)为模板剂,在水相中快速合成了介孔碳材料.将其应用于辣根过氧化物酶的固定化,并初步研究了固定化对酶稳定性的影响.利用TEM和SEM观察该材料的微观结构,通过N2吸附-脱附技术对介孔碳材料的孔结构和孔容等进行了表征.结果表明,介孔碳材料具有蠕虫状介孔结构,孔径可在5.6～7.6 nm之间调变,相应的比表面积介于691.1～787.8 m2/g,且随着反应体系中盐酸浓度的增加,所得材料的孔径、比表面积和孔容等均呈现减小的趋势.固定在介孔碳中的辣根过氧化物酶,保持了其蛋白质二级结构,且与游离酶相比,固定化酶的热稳定性、pH稳定性和操作稳定性都有明显的提高.经多次循环操作,固定化酶依然保持较高的活性,说明其具有良好的可重复利用价值.

Mesoporous carbons have been rapidly synthesized by an aqueous way using polymerization of phloroglucinol and formaldehyde in the presence of triblock copolymer F127(PEO106PPO70PEO106) under different conditions of hydrochloric acid.The micromorphology,pore structure of the obtained materials were characterized in detail by TEM,SEM and N2 adsorption-desorption.The obtained mesoporous carbons were utilized to adsorb the horseradish peroxidase(HRP) and the thermal stability,pH stability and storage stability of the immobilized HRP activity were also investigated.The results reveal the as-synthesized mesoporous carbon has a wormhole-like mesopore structure.The concentration of hydrochloric acid is a key factor influencing the polymerization rate of phenolic resins.With the concentrations of hydrochloric acid increasing from 0.5 mol/L to 2.5 mol/L,the pore size of mesoporous carbons decreased from 7.6 nm to 5.6 nm,the BET surface areas decreased from 787.8 m2/g to 691.1 m2/g,and pore volumes decreased from 0.64 cm3/g to 0.49 cm3/g.During the process of polymerization,the phase separation appeared in 20 min,while in the reported work,the polymerization of resorcinol and phenol with formaldehyde took much longer time.After incubation at 70 ℃ for 30 min,the activity of immobilized HRP decreased slowly than that of the free HRP.The free and immobilized HRP remained 12.33% and 56.22% of their initial activity,respectively.Compare to free HRP,the immobilized HRP exhibited higher retention activity with a wider pH range between 4.0 and 10.0.The activity of immobilized HRP was also tested for both operation and storage stability.The enzyme activity of immobilized HRP decreased with increasing amount of recycle times.After recycled for 10 d,the activity of immobilized HRP retains 29% of its initial activity.The activity of immobilized enzyme could retain as high as 62% of its initial activity after 50 d of storage at 4 ℃,while the free HRP only retains 31%.The results have shown that the thermal stability,pH stability and storage stability of immobilized enzyme were significantly improved in comparison with free HRP.