磁响应交联糖化酶聚集体的制备及催化特性

doi:10.11949/j.issn.0438-1157.20161565

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

• 催化、动力学与反应器 • 上一篇下一篇

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 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

收稿日期:2016-11-07 修回日期:2017-03-31 出版日期:2017-07-05 发布日期:2017-07-05
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。

Preparation and characterization of magnetic cross-linked glucoamylase aggregates

ZHANG Shuangzheng¹, CHEN Guo^1,2,3, SU Pengfei¹

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.

摘要/Abstract

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

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

中图分类号:

Q814

张双正, 陈国, 苏鹏飞. 磁响应交联糖化酶聚集体的制备及催化特性[J]. 化工学报, 2017, 68(7): 2763-2770.

ZHANG Shuangzheng, CHEN Guo, SU Pengfei. Preparation and characterization of magnetic cross-linked glucoamylase aggregates[J]. CIESC Journal, 2017, 68(7): 2763-2770.

参考文献 30

[1]	孙建华, 戴荣继, 邓玉林. 酶固定化技术研究进展[J]. 化工进展, 2010, 29(4): 715-721.SUN J H, DAI R J, DENG Y L. Progress in enzyme immobilization technique[J]. Chemical Industry and Engineering Progress, 2010, 29(4): 715-721.
[2]	SCHOEVAART R, WOLBERS M W, GOLUBOVIC M, et al. Preparation, optimization, and structures of cross-linked enzyme aggregates (CLEAs)[J]. Biotechnology and Bioengineering, 2004, 87(6): 754-762.
[3]	SHELDON R A. Cross-linked enzyme aggregates (CLEA(R)s): stable and recyclable biocatalysts[J]. Biochemical Society Transactions, 2007, 35: 1583-1587.
[4]	CUI J D, JIA S R. Optimization protocols and improved strategies of cross-linked enzyme aggregates technology: current development and future challenges[J]. Critical Reviews in Biotechnology, 2015, 35(1): 15-28.
[5]	TALEKAR S, JOSHI A, JOSHI G, et al. Parameters in preparation and characterization of cross linked enzyme aggregates (CLEAs)[J]. RSC Advances, 2013, 3(31): 12485-12511.
[6]	SHAARANI S M, JAHIM J M, RAHMAN R A, et al. Silanized maghemite for cross-linked enzyme aggregates of recombinant xylanase from Trichoderma reesei[J]. Journal of Molecular Catalysis B-Enzymatic, 2016, 133: 65-76.
[7]	GUPTA K, JANA A K, KUMAR S, et al. Solid state fermentation with recovery of Amyloglucosidase from extract by direct immobilization in cross linked enzyme aggregate for starch hydrolysis[J]. Biocatalysis and Agricultural Biotechnology, 2015, 4(4): 486-492.
[8]	ZHOU L Y, TANG W, JIANG Y J, et al. Magnetic combined cross-linked enzyme aggregates of horseradish peroxidase and glucose oxidase: an efficient biocatalyst for dye decolourization[J]. RSC Advances, 2016, 6(93): 90061-90068.
[9]	吴星涛, 薛原楷, 石贤爱. 磁性交联β-葡萄糖苷酶聚集体的制备及性质[J]. 生物物理学报, 2013, 29(10): 769-778.WU X T, XUE Y K, SHI X A. The preparation and properties of magnetic cross-linked β-glucosidase aggregates[J]. Acta Biophysica Sinica, 2013, 29(10):769-778.
[10]	SOJITRA U V, NADAR S S, RATHOD V K. A magnetic tri-enzyme nanobiocatalyst for fruit juice clarification[J]. Food Chemistry, 2016, 213: 296-305.
[11]	LIU L L, CEN Y, LIU F, et al. Analysis of alpha-amylase inhibitor from corni fructus by coupling magnetic cross-linked enzyme aggregates of alpha-amylase with HPLC-MS[J]. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, 2015, 995: 64-69.
[12]	KUMAR V V, SIVANESAN S, CABANA H. Magnetic cross-linked laccase aggregates - bioremediation tool for decolorization of distinct classes of recalcitrant dyes[J]. Science of the Total Environment, 2014, 487: 830-839.
[13]	CUI J D, CUI L L, JIA S R, et al. Hybrid cross-linked lipase aggregates with magnetic nanoparticles: a robust and recyclable biocatalysis for the epoxidation of oleic acid[J]. Journal of Agricultural and Food Chemistry, 2016, 64(38): 7179-7187.
[14]	KOPP W, DA COSTA T P, PEREIRA S C, et al. Easily handling penicillin G acylase magnetic cross-linked enzymes aggregates: catalytic and morphological studies[J]. Process Biochemistry, 2014, 49(1): 38-46.
[15]	BHATTACHARYA A, PLETSCHKE B I. Magnetic cross-linked enzyme aggregates (CLEAs): a novel concept towards carrier free immobilization of lignocellulolytic enzymes[J]. Enzyme and Microbial Technology, 2014, 61/62: 17-27.
[16]	NADAR S S, RATHOD V K. Magnetic macromolecular cross linked enzyme aggregates (CLEAs) of glucoamylase[J]. Enzyme and Microbial Technology, 2016, 83: 78-87.
[17]	KUMAR P, SATYANARAYANA T. Microbial glucoamylases: characteristics and applications[J]. Critical Reviews in Biotechnology, 2009, 29(3): 225-255.
[18]	SU P F, CHEN G, ZHAO J. Convenient preparation and characterization of surface carboxyl-functioned Fe3O4 magnetic nanoparticles[J]. Chemical Journal of Chinese Universities, 2011, 32(7): 1472-1477.
[19]	CHEN G, MA Y H, SU P F, et al. Direct binding glucoamylase onto carboxyl-functioned magnetic nanoparticles[J]. Biochemical Engineering Journal, 2012, 67: 120-125.
[20]	TALEKAR S, GHODAKE V, GHOTAGE T, et al. Novel magnetic cross-linked enzyme aggregates (magnetic CLEAs) of alpha amylase[J]. Bioresource Technology, 2012, 123: 542-547.
[21]	PALDI T, LEVY I, SHOSEYOV O. Glucoamylase starch-binding domain of Aspergillus niger B1: molecular cloning and functional characterization[J]. Biochemical Journal, 2003, 372: 905-910.
[22]	CHRISTENSEN U. pH-dependence of the fast step of maltose hydrolysis catalysed by glucoamylase G1 from Aspergillus niger[J]. Biochemical Journal, 2000, 349: 623-628.
[23]	YU S, CHOW G M. Carboxyl group (-CO2H) functionalized ferrimagnetic iron oxide nanoparticles for potential bio-applications [J]. Journal of Materials Chemistry, 2004, 14(18): 2781-2786.
[24]	KRALJ S, DROFENIK M, MAKOVEC D. Controlled surface functionalization of silica-coated magnetic nanoparticles with terminal amino and carboxyl groups[J]. Journal of Nanoparticle Research, 2011, 13(7): 2829-2841.
[25]	CUI J D, CUI L L, ZHANG S P, et al. Hybrid magnetic cross-linked enzyme aggregates of phenylalanine ammonia lyase from Rhodotorula glutinis[J]. Plos One, 2014, 9(5): e97221.
[26]	HU T G, CHENG J H, ZHANG B B, et al. Immobilization of alkaline protease on amino-functionalized magnetic nanoparticles and its efficient use for preparation of oat polypeptides[J]. Industrial & Engineering Chemistry Research, 2015, 54(17): 4689-4698.
[27]	KIM M, PARK J M, UM H J, et al. Immobilization of cross-linked lipase aggregates onto magnetic beads for enzymatic degradation of polycaprolactone[J]. Journal of Basic Microbiology, 2010, 50(3): 218-226.
[28]	PARK J M, KIM M, PARK J Y, et al. Immobilization of the cross-linked para-nitrobenzyl esterase of Bacillus subtilis aggregates onto magnetic beads[J]. Process Biochemistry, 2010, 45(2): 259-263.
[29]	CRUZ-IZQUIERDO A, PICO E A, LOPEZ C, et al. Magnetic cross-linked enzyme aggregates (mCLEAs) of Candida antarctica lipase: an efficient and stable biocatalyst for biodiesel synthesis[J]. Plos One, 2014, 9(12): e115202.
[30]	ARCA-RAMOS A, KUMAR V V, EIBES G, et al. Recyclable cross-linked laccase aggregates coupled to magnetic silica microbeads for elimination of pharmaceuticals from municipal wastewater[J]. Environmental Science and Pollution Research, 2016, 23(9): 8929-8939.

磁响应交联糖化酶聚集体的制备及催化特性

Preparation and characterization of magnetic cross-linked glucoamylase aggregates

PDF (PC)

可视化

被引次数

摘要/Abstract

引用本文

使用本文

参考文献 30

相关文章 15

编辑推荐

Metrics

本文评价

[1]	程业品, 胡达清, 徐奕莎, 刘华彦, 卢晗锋, 崔国凯. 离子液体基低共熔溶剂在转化CO₂中的应用[J]. 化工学报, 2023, 74(9): 3640-3653.
[2]	孟令玎, 崇汝青, 孙菲雪, 孟子晖, 刘文芳. 改性聚乙烯膜和氧化硅固定化碳酸酐酶[J]. 化工学报, 2023, 74(8): 3472-3484.
[3]	陈雅鑫, 袁航, 刘冠章, 毛磊, 杨纯, 张瑞芳, 张光亚. 蛋白质纳米笼介导的酶自固定化研究进展[J]. 化工学报, 2023, 74(7): 2773-2782.
[4]	汤晓玲, 王嘉瑞, 朱玄烨, 郑仁朝. 基于Pickering乳液的卤醇脱卤酶催化合成手性环氧氯丙烷[J]. 化工学报, 2023, 74(7): 2926-2934.
[5]	毛磊, 刘冠章, 袁航, 张光亚. 可捕集CO₂的纳米碳酸酐酶粒子的高效制备及性能研究[J]. 化工学报, 2023, 74(6): 2589-2598.
[6]	张兰河, 赖青燚, 王铁铮, 关潇卓, 张明爽, 程欣, 徐小惠, 贾艳萍. H₂O₂对SBR脱氮效率和污泥性能的影响[J]. 化工学报, 2023, 74(5): 2186-2196.
[7]	贾露凡, 王艺颖, 董钰漫, 李沁园, 谢鑫, 苑昊, 孟涛. 微流控双水相贴壁液滴流动强化酶促反应研究[J]. 化工学报, 2023, 74(3): 1239-1246.
[8]	刘瑞琪, 周栖桐, 张悦, 贺莹, 高静, 马丽. 基于金纳米颗粒修饰二氧化硅纳米花的生物传感器构建及应用[J]. 化工学报, 2023, 74(3): 1247-1259.
[9]	苏伟怡, 丁佳慧, 李春利, 王洪海, 姜艳军. 酶促反应结晶研究进展[J]. 化工学报, 2023, 74(2): 617-629.
[10]	刘昕, 戈钧, 李春. 光驱动微生物杂合系统提高生物制造水平[J]. 化工学报, 2023, 74(1): 330-341.
[11]	胡阳, 孙彦. 酶分子的自驱动及其介导的微纳马达[J]. 化工学报, 2023, 74(1): 116-132.
[12]	谭卓涛, 齐思雨, 许梦蛟, 戴杰, 朱晨杰, 应汉杰. 辅酶自循环的氧化还原级联体系在生物催化过程中的应用：机遇与挑战[J]. 化工学报, 2023, 74(1): 45-59.
[13]	安绍杰, 许洪峰, 李思, 许远航, 李佳锡. 利用分子机器的组装与分解构建pH敏感性谷胱甘肽过氧化物人工酶[J]. 化工学报, 2022, 73(8): 3669-3678.
[14]	张劢, 田瑶, 郭之旗, 王叶, 窦广进, 宋浩. 光催化-生物杂合系统设计优化用于燃料和化学品绿色合成[J]. 化工学报, 2022, 73(7): 2774-2789.
[15]	孙甲琛, 孙文涛, 孙慧, 吕波, 李春. 甘草黄酮合酶Ⅱ催化甘草素特异性合成7，4′-二羟基黄酮[J]. 化工学报, 2022, 73(7): 3202-3211.