Functionalized Anodic Aluminum Oxide Membrane鈥揈lectrode System for Enzyme Immobilization

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• 作者：Zhiqiang Chen ; Jianjun Zhang ; Shanteri Singh ; Pauline Peltier-Pain ; Jon S. Thorson ; Bruce J. Hinds
• 刊名：ACS Nano
• 出版年：2014
• 出版时间：August 26, 2014
• 年：2014
• 卷：8
• 期：8
• 页码：8104-8112
• 全文大小：499K
• ISSN：1936-086X

文摘

A nanoporous membrane system with directed flow carrying reagents to sequentially attached enzymes to mimic nature鈥檚 enzyme complex system was demonstrated. Genetically modified glycosylation enzyme, OleD Loki variant, was immobilized onto nanometer-scale electrodes at the pore entrances/exits of anodic aluminum oxide membranes through His₆-tag affinity binding. The enzyme activity was assessed in two reactions鈥攁 one-step 鈥渞everse鈥?sugar nucleotide formation reaction (UDP-Glc) and a two-step sequential sugar nucleotide formation and sugar nucleotide-based glycosylation reaction. For the one-step reaction, enzyme specific activity of 6鈥?0 min^鈥? on membrane supports was seen to be comparable to solution enzyme specific activity of 10 min^鈥?. UDP-Glc production efficiencies as high as 98% were observed at a flow rate of 0.5 mL/min, at which the substrate residence time over the electrode length down pore entrances was matched to the enzyme activity rate. This flow geometry also prevented an unwanted secondary product hydrolysis reaction, as observed in the test homogeneous solution. Enzyme utilization increased by a factor of 280 compared to test homogeneous conditions due to the continuous flow of fresh substrate over the enzyme. To mimic enzyme complex systems, a two-step sequential reaction using OleD Loki enzyme was performed at membrane pore entrances then exits. After UDP-Glc formation at the entrance electrode, aglycon 4-methylumbelliferone was supplied at the exit face of the reactor, affording overall 80% glycosylation efficiency. The membrane platform showed the ability to be regenerated with purified enzyme as well as directly from expression crude, thus demonstrating a single-step immobilization and purification process.

Keywords:

biomimetic enzyme immobilization; membrane鈭抏lectrode; glycosyltransferase; glycosylation; enzyme purification; sequential reactions