Immobilization of lambda exonuclease onto polymer micropillar arrays for the solid-phase digestion of dsDNAs.
Immobilization of lambda exonuclease onto polymer micropillar arrays for the solid-phase digestion of dsDNAs.
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DOI：
10.1021/ac5002965
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发表时间：
2014-05-06
影响因子：
7.4
通讯作者：
Soper, Steven A.
中科院分区：
文献类型：
--
作者：
Oliver-Calixte, Nyote J.;Uba, Franklin I.;Battle, Katrina N.;Weerakoon-Ratnayake, Kumuditha M.;Soper, Steven A.
将酶固定在固体载体上进行生物反应的过程与基于溶液的过程相比具有一些引人注目的优势，包括酶与产物的容易分离，消除酶的自消化，以及增加酶的稳定性和活性。我们报道了将λ-外切酶固定在聚甲基丙烯酸甲酯（PMMA）微柱上的微流控装置，用于芯片上双链DNA的消化。利用3-（3-二甲氨基丙基）碳二亚胺/ n -羟基琥珀酰亚胺（EDC/NHS）偶联羧酸功能化PMMA微柱成功实现了酶固定化。我们的研究结果表明，与自由溶液酶切相比，λ-外切酶在固体载体上催化dsDNA酶切的效率更高，包括其加工效率和反应速率。λ-核酸外切酶酶切λ-DNA （48.5 kbp）的剪接率为1.0 × 103个核苷酸s-1。固相反应器的动力学行为可以用分形Michaelis-Menten模型来描述，催化效率比均相液相反应提高了近17%。这项工作的结果将对采用游离单核苷酸鉴定的新单分子DNA测序策略产生重要影响。
The process of immobilizing enzymes onto solid supports for bioreactions has some compelling advantages compared to their solution-based counterpart including the facile separation of enzyme from products, elimination of enzyme autodigestion, and increased enzyme stability and activity. We report the immobilization of λ-exonuclease onto poly(methylmethacrylate) (PMMA) micropillars populated within a microfluidic device for the on-chip digestion of double-stranded DNA. Enzyme immobilization was successfully accomplished using 3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) coupling to carboxylic acid functionalized PMMA micropillars. Our results suggest that the efficiency for the catalysis of dsDNA digestion using λ-exonuclease, including its processivity and reaction rate, were higher when the enzyme was attached to a solid support compared to the free solution digestion. We obtained a clipping rate of 1.0 × 103 nucleotides s–1 for the digestion of λ-DNA (48.5 kbp) by λ-exonuclease. The kinetic behavior of the solid-phase reactor could be described by a fractal Michaelis–Menten model with a catalytic efficiency nearly 17% better than the homogeneous solution-phase reaction. The results from this work will have important ramifications in new single-molecule DNA sequencing strategies that employ free mononucleotide identification.
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影响因子：
3.4
作者：
Aissaoui, Nesrine;Landoulsi, Jessem;Lambert, Jean-Francois
通讯作者：
Lambert, Jean-Francois
DOI：
10.1016/j.jasms.2008.03.015
发表时间：
2008-07-01
影响因子：
3.2
作者：
Lee, Jeonghoon;Musyimi, Harrison K.;Murray, Kermit K.
通讯作者：
Murray, Kermit K.
影响因子：
46.2
作者：
Chantiwas R;Park S;Soper SA;Kim BC;Takayama S;Sunkara V;Hwang H;Cho YK
通讯作者：
Cho YK
DOI：
10.1073/pnas.68.7.1639
发表时间：
1971-01-01
影响因子：
11.1
作者：
CASSUTO, E;LASH, T;RADDING, CM
通讯作者：
RADDING, CM
影响因子：
4.1
作者：
HORNBY, WE;LILLY, MD;CROOK, EM
通讯作者：
CROOK, EM