磁响应交联糖化酶聚集体的制备及催化特性

提出了一种采用羧基磁性纳米粒子制备杂化磁响应交联酶聚集体（M-CLEAs）的方法。表面羧基修饰的约10 nm的磁性纳米粒子与酶分子表面的氨基位点通过静电相互作用，形成复合物，在磁场作用下可将磁性纳米粒子-酶复合物从溶液中分离，经戊二醛交联即形成M-CLEAs。传统的表面氨基修饰的磁性纳米粒子与酶需在沉淀剂作用下，从溶液中分离，而后采用戊二醛共交联，而本方法无须沉淀剂，过程更为简化。以糖化酶为对象，对该过程的影响因素（交联时间、pH、酶浓度、戊二醛浓度等条件）进行了探索，并对制得的M-CLEAs的酶学性质进行了较为详细考察。结果表明，最优制备条件为：酶浓度1 mg·ml^-1，磁流体浓度10 mg·ml^-1，戊二醛浓度0.25%（质量体积比），在pH 6.0下交联反应6 h，最终载酶量可达80 mg·g^-1、比活为50 U·mg^-1。制得的固定化酶pH稳定性、热稳定性和储存稳定性均显著改善，可实现糖化酶重复使用10次，仍保留接近60%的酶活。

Preparation and characterization of magnetic cross-linked glucoamylase aggregates

The paper presents a method for preparing hybrid magnetic cross-linked enzyme aggregates (M-CLEAs) using carboxyl magnetic nanoparticles (MNP). The magnetic nanoparticle-enzyme complexes induced by electrostatic interaction between carboxyl group of magnetic particle and amino group of enzyme were separated from the solution under magnetic field, and then were cross-linked by glutaraldehyde. The traditional method of M-CLEAs preparation from amino-modified magnetic nanoparticles and the enzyme need the precipitant to prompt the separation of enzyme and magnetic nanoparticle from the solution. The method here proposed does not need the precipitant, thereof the process is greatly simplified. In this paper, the factors of cross-linking time, pH, enzyme concentration and concentration of glutaraldehyde in M-CLEAs preparation process were studied, and then the enzymatic properties of M-CLEAs were also investigated in detail. The results showed that the optimum conditions were as follows: enzyme concentration of 1 mg·ml^-1, magnetic fluid concentration of 10 mg·ml^-1, glutaraldehyde concentration of 0.25% and crosslinking reaction at pH 6.0 for 6 h. The final enzyme loading reached 80 mg·g^-1 and the activity of M-CLEAs was 50 U·mg^-1. The pH stability, thermostability and storage stability of the immobilized enzyme improved significantly, and M-CLEAs still remained nearly 60% of activity after 10 cycles.