磁性交联核酸酶P1聚集体的制备及性质研究

Preparation and Property of Magnetic Cross-Linked Nuclease P1 Aggregations

采用磁性纳米颗粒与酶蛋白共沉淀后经戊二醛交联的方法,制备了磁性交联核酸酶P1聚集体,并且对比分析了游离酶和固定化酶的部分酶学性质.优化的最佳制备条件为:硫酸铵质量浓度为0.8 g/mL,沉淀时间为0.5 h,戊二醛体积分数为0.6%,交联时间为2 h,所制得的固定化酶活性回收率为32.4%.酶学性质研究表明,固定化酶的Km值(30.7 mmol/L)明显高于游离酶的(7.27 mmol/L),二者最适反应温度分别为90℃和75℃,最适pH值均为5.2,固定化核酸酶P1对热和酸碱的耐受性明显增强,连续反应6次后酶活力仍保留70%,良好的操作稳定性和磁响应性有利于核酸酶P1的工业化应用.

The magnetic cross-linked enzyme aggregations （CLEAs） were prepared by depositing of nuclease P1 with magnetic nanoparticles and cross-linked with glutaraldehyde, and some enzymetic properties were compared between nuclease P1 and CLEAs. The optimal conditions of preparing magnetic CLEAs are sought out： （NH4）2SO4 80% saturation, depositing time 0.5 h, glutaraldehyde 0.6%, and cross-linking time 2 h. The activity recovery reaches to 32.4% under the above conditions. Investigation of enzymetic properties suggests that Km of magnetic CLEAs （30.7 mmol/L） gets higher than soluble enzyme （7.27 mmol/L）, and the optional reacting temperature of immobilized nuclease P1 is appropriate at 90 ℃, while optional reacting pH approaches to 5.2. Once immobilized, the resistance to heat, acid and alkali of nuclease P1 is remarkably improved, and the magnetic CLEAs maintained 70% activity after six consecutive reactions. Good operational stability and magnetic response are beneficial for industrial utilization of nuclease P1.