Polymer monoliths as efficient solid phases for enzymatic polynucleotide degradation followed by fast HPLC analysis

Two ribonuclease A bioreactors based on lab‐made macroporous monolithic columns and intended for polynucleotide degradation were prepared using in situ free‐radical polymerization. Different methods of enzyme immobilization were applied. In the first case, the biocatalyst molecule was attached to th...

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Bibliographic Details
Published in:Journal of separation science 2013-09, Vol.36 (17), p.2793-2805
Main Authors: Volokitina, M. V., Vlakh, E. G., Platonova, G. A., Vinokhodov, D. O., Tennikova, T. B.
Format: Article
Language:English
Subjects:
Anion-exchange HPLC
Biodegradation
Bioreactors
Chromatography, High Pressure Liquid - instrumentation
CIM-DEAE disks
Computing time
Degradation
Degradation of polynucleotides
Enzymes
Enzymes, Immobilized - chemistry
Flow-through immobilized enzyme reactors
Hydrolysis
Kinetics
Mathematical analysis
Polymer monoliths
Polymerization
Polymers
Polynucleotides
Polynucleotides - chemistry
Ribonuclease A
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Summary:Two ribonuclease A bioreactors based on lab‐made macroporous monolithic columns and intended for polynucleotide degradation were prepared using in situ free‐radical polymerization. Different methods of enzyme immobilization were applied. In the first case, the biocatalyst molecule was attached to the solid surface via direct covalent binding, while in the second bioreactor the flexible‐chain synthetic polymer was used as an intermediate spacer. The effect of temperature, substrate flow rate, and loaded sample volume on the biocatalytic efficiency of the immobilized enzyme was examined. The kinetic parameters of the enzymatic degradation of synthetic polycytidylic acid were calculated and compared to those found for hydrolysis with soluble ribonuclease A. The monitoring of substrate splitting was carried out by means of fast anion‐exchange HPLC on an ultra‐short monolithic column (disk) using off‐ and on‐line analytical approaches.
ISSN:1615-9306
1615-9314
DOI:10.1002/jssc.201300406