水凝胶包埋酶(单凝胶和双网络凝胶)及磁性粒子固定化酶的制备及评价

doi:10.5246/jcps.2011.03.028

水凝胶包埋酶(单凝胶和双网络凝胶)及磁性粒子固定化酶的制备及评价

闵俊哲^*, 秋本真友子, 李翠苓, 加藤大, 豊岡利正^*

1. 静冈县立大学药学院, 日本静冈 4228526
2. 杭州海王生物工程有限公司, 中国浙江杭州 311101
3. 东京大学药学院, 日本东京 1130033

收稿日期:2010-11-29 修回日期:2011-03-20 出版日期:2011-05-06 发布日期:2011-05-06
通讯作者: 闵俊哲*, 豊岡利正*

Preparation and evaluation of enzyme encapsulated hydrogels (single gels and double network gels) and enzyme immobilized magnetic beads

Jun Zhe Min^*, Mayuko Akimoto, Cui-Ling Li, Masaru Kato, Toshimasa Toyo'oka^*

1. Laboratory of Analytical and Bio-Analytical Chemistry, School of Pharmaceutical Sciences, and Global COE Program, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan
2. Hangzhou Neptunus Bioengineering Co., Ltd, Zhejiang 311101, China
3. Graduate School of Pharmaceutical Sciences and Global COE Program, the University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo 113-0033, Japan

Received:2010-11-29 Revised:2011-03-20 Online:2011-05-06 Published:2011-05-06
Contact: Jun Zhe Min*, Toshimasa Toyo'oka*

摘要/Abstract

摘要： 酶固定化过程中, 固定化酶的方法及其载体的选择是酶固定化过程的关键因素, 适宜的固定化法和良好的载体微环境对酶活保持率提高和稳定性增强尤其重要。在本研究中, 利用96孔微分析板评价了用于高通量筛选的水凝胶包埋酶(单凝胶和双网络凝胶)及磁性粒子固定化酶的制备方法及其对酶活性(保存时间, 精度和重现性)的影响。胰蛋白酶(trypsin)成功地包埋在单凝胶和双网络凝胶中并且固定在磁性粒子上, 然而, 包埋在单凝胶和双网络凝胶, 或固定在磁性粒子上的胃蛋白酶无法与底物反应。在对酶的适应性方面, 与双网络凝胶比较, 单凝胶和磁性粒子固定化法更加优越, 适用于多种酶 (如: 胰蛋白酶,葡糖苷酸酶, CYP1A1)的固定。然而, 我们也发现浸置后, 以单凝胶包埋的固定酶有较多的损失。双网络凝胶包埋法只限于包埋胰蛋白酶, 无法用于包埋其它酶, 例如葡糖苷酸酶、CYP1A1和胃蛋白酶, 因为双网络凝胶包埋法会由于丙烯酰胺和过硫酸胺的存在而使酶失去活性。在三种酶固定方法中, 磁性粒子固定酶的方法能够最好地保留酶活性。另外, 磁性粒子固定酶的稳定性比其他两种方法好, 存放一周后胰蛋白酶和葡糖苷酸酶的活性没有任何下降。其次, 磁性粒子固定法的重复利用重现性也良好。此外, 尽管双网络凝胶法包埋酶的种类有限, 但是我们认为通过改变丙烯酰胺等载体的选择和设计, 改进载体微环境, 可以使包埋酶的效率得到提高。

关键词: 固定化酶, 包埋酶, 单凝胶, 双网络凝胶, 磁性粒子, 胰蛋白酶, 葡糖苷酸酶, CYP1A1

Abstract:

In the present research, enzyme encapsulated hydrogels (single gels and double network gels) and enzyme immobilized magnetic beads, which allow high-throughput screening, were fabricated and evaluated in terms of the preservation, precision, and repeatability of enzyme activity. The fabricated gels and magnetic beads were analyzed in a 96-well microassay plate. Trypsin was successfully encapsulated in both types of gels and immobilized to the magnetic beads. However, pepsin, either encapsulated in the gels or immobilized to the magnetic beads, could not react with its substrates. The adaptability to various enzymes (e.g., trypsin, β-glucuronidase, and CYP1A1) in the single gels and magnetic beads was superior to that in double network gels. However, the soak out of the enzymes was observed in the single gels. The double network gels could encapsulate trypsin, whereas the fabrication of the other enzymes (e.g. β-glucuronidase, CYP1A1, and pepsin) failed because of the inactivation of the enzymes by acryl amide and ammonium peroxodisulfate, which are the components of the gel formulation. The enzyme reaction in the magnetic beads exhibited the highest efficiency among the three fabrication methods. Furthermore, the stability of the enzymes immobilized to the magnetic beads was better than that fabricated by the other methods, and the activities of trypsin and β-glucuronidase did not decline for up to one week. In addition, in the magnetic beads, the activities of trypsin and β-glucuronidase can be well repeated. Hence, although the adaptability of the double network gels to various enzymes is currently limited, the efficiency of the enzyme encapsulation can be improved by optimizing the formulation of acryl amide gels.

Key words: Immobilized enzyme, Encapsulated enzyme, Single gel, Double network gel, Magnetic bead, Trypsin, β-Glucuronidase, CYP1A1

中图分类号:

R915.12

Supporting: Foundation item: The Global COE Program from the Ministry of Education, Science, Sports, and Culture of Japan.
^*Corresponding author. Tel./fax: 81-54-264-5655 (Jun Zhe Min); Tel.: 81-54-264-5656; fax: 81-54-264-5593 (Toshimasa Toyo'oka)

闵俊哲^*, 秋本真友子, 李翠苓, 加藤大, 豊岡利正^*. 水凝胶包埋酶(单凝胶和双网络凝胶)及磁性粒子固定化酶的制备及评价[J]. , DOI: 10.5246/jcps.2011.03.028.

Jun Zhe Min^*, Mayuko Akimoto, Cui-Ling Li, Masaru Kato, Toshimasa Toyo'oka^* . Preparation and evaluation of enzyme encapsulated hydrogels (single gels and double network gels) and enzyme immobilized magnetic beads [J]. , DOI: 10.5246/jcps.2011.03.028.

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