重组葡萄糖异构酶的固定化研究

doi:10.7506/spkx1002-6630-201309034

食品科学 ›› 2013, Vol. 34 ›› Issue (9): 164-169.doi: 10.7506/spkx1002-6630-201309034

重组葡萄糖异构酶的固定化研究

邓辉1,2，陈晟1,2，陈坚1,2，吴敬1,2,*

1.江南大学食品科学与技术国家重点实验室，江苏无锡 214122；
2.江南大学生物工程学院，工业生物技术教育部重点实验室，江苏无锡 214122

收稿日期:2012-09-06 修回日期:2013-04-11 出版日期:2013-05-15 发布日期:2013-05-07
通讯作者: 吴敬 E-mail:jingwu80@hotmail.com
基金资助:
国家“863”计划项目(2012AA021500)；中央高校基本科研业务费专项(JUDCF10070)

Immobilization of Cells Producing Glucose Isomerases

DENG Hui1,2，CHEN Sheng1,2，CHEN Jian1,2，WU Jing1,2,*

1. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China；2. Key Laboratory of
Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China

Received:2012-09-06 Revised:2013-04-11 Online:2013-05-15 Published:2013-05-07
Contact: WU Jing E-mail:jingwu80@hotmail.com

摘要/Abstract

摘要：

采用壳聚糖絮凝和戊二醛交联的方法，对表达生产Thermobifida fusca葡萄糖异构酶(GIase)的重组大肠杆菌细胞进行固定化工艺研究，以期获得高性能和低成本的固定化产酶细胞方法。考察影响细胞絮凝和交联的各种因素，并对固定化酶制剂的理化性质、动力学常数进行测定。结果表明：最佳的固定化条件为在高密度发酵液中添加0.6%硅藻土和5mmol/L Mg2+，经60℃热处理30min后，在壳聚糖终质量分数0.1‰、pH5.5、充分搅拌条件下絮凝，酶活回收率达98%；絮凝产物在戊二醛体积分数0.25%、pH6.5、轻微搅拌条件下交联3h，以未交联的样品作参照(100%)，交联样品的酶活保留率达80%。相对于游离GIase，此条件下制备的固定化GIase的最适温度仍为80℃、最适pH值从10降低至9；在工业生产应用条件下，固定化GIase的初始酶活力达到356U/g，半衰期为61d，能够满足高果糖浆工业的生产要求。

关键词: 葡萄糖异构酶, 重组大肠杆菌细胞, 固定化, 壳聚糖絮凝, 戊二醛交联

Abstract:

In the present study, immobilization technology of recombinant Escherichia coli cells producing
Thermobifida fusca glucose isomerase was studied using chitosan flocculation and glutaraldehyde cross-linking
methods. In order to obtain the immobilization method for GIase with high performance and low cost, various factors
for affecting flocculation and crosslinking were investigated, and the physical and chemical properties, dynamic
constants and operation stability of immobilized enzyme preparation were determined. The results showed that the
optimal immobilization conditions were diatomaceous earth addition of 0.6% and 5 mmol/L Mg2+ in high-density
fermentation broth. After heat treatment at 60 ℃ for 30 min, cells and debris were flocculated by 0.1‰ chitosan
solution at pH 5.5 under fully rabbling, and recovery rate of GIase reached up to 98%. Flocculation was cross-linked
for 3 h in 0.25% glutaraldehyde solution at pH 6.5 under slight rabbling, and the enzyme activity reached up to 80%
of flocculation. Compared to free GIase, the optimal temperature was 80 ℃, and the optimal pH reduced from 10 to 9.
The initial enzyme activity 356 U/g, and its half-life reached up to 61 d.

Key words: glucose isomerase, recombinant Escherichia coli cells, immobilization, chitosan flocculation, glutaraldehyde cross-linking

中图分类号:

Q814.2

邓辉1,2，陈晟1,2，陈坚1,2，吴敬1,2,*. 重组葡萄糖异构酶的固定化研究[J]. 食品科学, 2013, 34(9): 164-169.

DENG Hui1,2，CHEN Sheng1,2，CHEN Jian1,2，WU Jing1,2,*. Immobilization of Cells Producing Glucose Isomerases[J]. FOOD SCIENCE, 2013, 34(9): 164-169.

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重组葡萄糖异构酶的固定化研究

Immobilization of Cells Producing Glucose Isomerases

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