A new application of aptamer: One-step purification and immobilization of enzyme from cell lysates for biocatalysis
A new application of aptamer: One-step purification and immobilization of enzyme from cell lysates for biocatalysis
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适体的新应用：细胞裂解液中酶的一步纯化和固定化用于生物催化
DOI：
10.1016/j.jbiotec.2015.03.014
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发表时间：
2015-06-10
影响因子：
4.1
通讯作者：
Li, Chun
中科院分区：
文献类型：
--
作者：
Qiao, Lifeng;Lv, Bo;Li, Chun
适体是基于核酸的高亲和力配体，其能够通过分子识别捕获其相应的靶标。本研究利用改进的SELEX方法获得了几种对β-葡萄糖醛酸苷酶（PGUS-E）具有高亲和力和特异性的DNA适体。其中，选择Apt 5和Apt 9作为代表，并分别与磁珠共价连接。对核酸适体修饰的磁性微球进行了表征，并成功地应用于从复杂细胞裂解液中一步纯化和固定PGUS-E。通过方便地调节pH和离子强度，PGUS-E纯度对于Apt 5修饰的珠粒达到84%，对于Apt 9修饰的珠粒达到88%。此外，发现Apt 5和Apt 9修饰的磁珠的最大PGUS-E捕获能力分别为31.75 μ g/mg和32.95 μ g/mg。结果表明，磁性微球固定化PGUS-E具有良好的重复使用性，7次循环后，PGUS-E的转化率仍保持在70%以上。此外，适配子修饰的珠粒载体可以容易地再生，并且在再生的第7个循环之后，白藜芦醇（GL）的转化率仍为62%。该研究可推广到其他酶体系，为开发基于适体的酶固定化技术开辟了一条通用途径。(C)2015 Elsevier B. V.版权所有。
Aptamers are nucleic acid-based high affinity ligands that are able to capture their corresponding target through molecular recognition. In this study, several DNA aptamers with high affinity and specificity for beta-glucuronidases (PGUS-E) were obtained by our modified SELEX method. Among them, Apt5 and Apt9 were selected as representatives and covalently linked to magnetic beads, respectively. The aptamer-modified magnetic beads were characterized and successfully applied to one-step purification and immobilization of PGUS-E from the complex cell lysates. By conveniently adjusting the pH and ion strength, the PGUS-E purities reached 84% for Apt5-modified beads and 88% for Apt9-modified beads. Moreover, the maximum PGUS-E capturing capacity of the Apt5 and Apt9 modified magnetic beads were found to be 31.75 mu g/mg and 32.95 mu g/mg, respectively. The immobilized PGUS-E on aptamer-based magnetic beads showed good reusability, and the conversion of glycyrrhizin still remained more than 70% after 7 cycles. In addition, the aptamer-modified beads support can be easily regenerated, and the conversion rate of glycyrrhizin (GL) was still 62% after the 7th cycle of regeneration. This investigation can be easily extended to other enzyme systems and may help open a generic route to develop a novel enzyme immobilization technology for biocatalysis based on aptamer. (C) 2015 Elsevier B.V. All rights reserved.