氨基树脂表面活化提高固定化核酸酶P1连续催化性能

doi:10.11949/j.issn.0438-1157.20160280

化工学报 ›› 2016, Vol. 67 ›› Issue (9): 3850-3860.DOI: 10.11949/j.issn.0438-1157.20160280

氨基树脂表面活化提高固定化核酸酶P₁连续催化性能

何林姣^1,2, 刘晓静^1,2, 黄金莎^1,2, 庄伟^1,2,3, 应汉杰^1,2,3

1 南京工业大学材料化学工程国家重点实验室, 江苏南京 210009;
2 南京工业大学制药与生物工程学院国家生化工程技术研究中心, 江苏南京 211816;
3 南京工业大学江苏先进生物与化学制造协同创新中心, 江苏南京 211816

收稿日期:2016-03-09 修回日期:2016-04-28 出版日期:2016-09-05 发布日期:2016-09-05
通讯作者: 应汉杰
基金资助:
国家重点基础研究发展计划项目（2013CB733501）；国家自然科学基金项目（21506090）；江苏省自然科学基金项目（BK20130929）；江苏高校优势学科建设工程项目。

Fortified continous catalytic properties of immobilized nuclease P₁ with surface activated HA amino resin

HE Linjiao^1,2, LIU Xiaojing^1,2, HUANG Jinsha^1,2, ZHUANG Wei^1,2,3, YING Hanjie^1,2,3

1 Sate Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, Nanjing 210009, Jiangsu, China;
2 National Engineering Research Center for Biotechnology, School of Biological and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, Jiangsu, China;
3 The Synergetic Innovation Center for Advanced Materials, Nanjing Tech University, Nanjing 211816, Jiangsu, China

Received:2016-03-09 Revised:2016-04-28 Online:2016-09-05 Published:2016-09-05
Supported by:
supported by the National Basic Research Program of China (2013CB733501), the National Natural Science Foundation of China (21506090), the Natural Science Foundation of Jiangsu Province (BK20130929) and Priority Academic Program Development of Jiangsu Higher Education Institutions.

摘要/Abstract

摘要：

考察了固定化酶常用载体-氨基树脂几何结构及表面活化过程对固定化核酸酶P₁性能的影响，并进行了动力学和连续催化稳定性研究。FESEM、BET及FTIR等表征发现，氨基树脂具有大量酶可利用的孔，在固定化过程中核酸酶P₁主要利用的孔径范围为4～30 nm。相对游离的核酸酶P₁而言，所得固定酶耐酸、耐热性增强；米氏动力学研究表明各组固定酶对底物的亲和力下降，最大反应速率下降；后交联组的重复利用性相对物理吸附、化学交联组明显增强。优化的固定化条件为：酶和载体比例为3：20（质量比），酶浓度为0.8 g·L^-1，酶液pH为6.0，固定时间为10 h，在优化条件下所得固定酶的单位载体酶活为10013 U·g^-1。同时设计及优化柱连续反应器操作条件，确定反应温度为65℃，进料流量为0.75 ml·min^-1，使得产品的核苷酸浓度维持30 g·L^-1（水解率为60%）以上的累计时长达120 h，有利于核酸酶P₁连续生产核苷酸的工业化应用。

关键词: 氨基树脂, 核酸酶P₁, 交联, 表面活化, 柱连续反应器

Abstract:

The effect of HA amino resin (immobilized enzyme widely-used carrier) geometry and surface activated process on the immobilized nuclease P₁'s properties was studied. In addition, the dynamic analysis and continuous catalytic properties were also investigated. FESEM, BET and FTIR characterization were utilized to verify that HA amino resin had a great deal of enzymes available hole and the scope of the main hole was 4-30 nm in the immobilized process. Compared with free nuclease P₁, the acid resistance and heat resistance of the immobilized enzymes were improved. The study on Michael's Mention kinetics indicated that the substrate affinity and maximum reaction rate of immobilized enzymes decreased; the reusability of after crosslinking group was significantly enhanced compared to physical adsorption and chemical crosslinking groups. The optimized conditions of immobilization were as follows:the enzyme to carrier ratio of 3:20 (mass ratio), enzyme concentration of 0.8 g·L^-1 and 10 h immobilized time at pH 6.0. Under these conditions, the immobilized enzyme activity was about 10013 U·g^-1. Furthermore, the operating conditions of column flow reactor were designed and optimized. The continuous running time of the reactor was up to 120 h at 30 g·L^-1 products nucleotide concentration (hydrolysis rate of 60%), when the reaction temperature was 65℃ with substrate flow rate of 0.75 ml·min^-1. This work would be beneficial to the application of nuclease P₁ in nucleotide continuous industrial production.

Key words: amino resin, nuclease P₁, crosslinking, surface activation, column flow reactor

中图分类号:

Q814

何林姣, 刘晓静, 黄金莎, 庄伟, 应汉杰. 氨基树脂表面活化提高固定化核酸酶P₁连续催化性能[J]. 化工学报, 2016, 67(9): 3850-3860.

HE Linjiao, LIU Xiaojing, HUANG Jinsha, ZHUANG Wei, YING Hanjie. Fortified continous catalytic properties of immobilized nuclease P₁ with surface activated HA amino resin[J]. CIESC Journal, 2016, 67(9): 3850-3860.

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氨基树脂表面活化提高固定化核酸酶P₁连续催化性能

Fortified continous catalytic properties of immobilized nuclease P₁ with surface activated HA amino resin

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