CIESC Journal ›› 2017, Vol. 68 ›› Issue (7): 2763-2770.DOI: 10.11949/j.issn.0438-1157.20161565

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Preparation and characterization of magnetic cross-linked glucoamylase aggregates

ZHANG Shuangzheng1, CHEN Guo1,2,3, SU Pengfei1   

  1. 1 Department of Bioengineering and Biotechnology, College of Chemical Engineering, Huaqiao University, Xiamen 361021, Fujian, China;
    2 Fujian Provincial Key Laboratory of Biochemical Technology (Huaqiao University), Xiamen 361021, Fujian, China;
    3 Instrumental Analysis Center, Huaqiao University, Xiamen 361021, Fujian, China
  • Received:2016-11-07 Revised:2017-03-31 Online:2017-07-05 Published:2017-07-05
  • Contact: 10.11949/j.issn.0438-1157.20161565
  • Supported by:

    supported by the National Natural Science Foundation of China (21576108), Program for New Century Excellent Talents in Fujian Province University, and Research Innovation Ability Cultivation Program for Graduate Student of Huaqiao University.

磁响应交联糖化酶聚集体的制备及催化特性

张双正1, 陈国1,2,3, 苏鹏飞1   

  1. 1 华侨大学化工学院生物工程与技术系, 福建 厦门 361021;
    2 福建省生物化工技术重点实验室(华侨大学), 福建 厦门 361021;
    3 华侨大学分析测试中心, 福建 厦门 361021
  • 通讯作者: 陈国
  • 基金资助:

    国家自然科学基金面上基金项目(21576108);福建省新世纪优秀人才项目;华侨大学研究生科研创新能力培育计划资助项目。

Abstract:

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.

Key words: biocatalysis, enzyme, immobilization, magnetic cross-linked enzyme aggregates, preparation method, glucoamylase, characterization of enzyme

摘要:

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。

关键词: 生物催化, 酶, 固定化, 磁交联酶聚集体, 制备方法, 糖化酶, 酶学特性

CLC Number: