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Glycosylation site-targeted PEGylation of glucose oxidase retains native enzymatic activity

Paper ID Volume ID Publish Year Pages File Format Full-Text
17373 42663 2013 7 PDF Available
Title
Glycosylation site-targeted PEGylation of glucose oxidase retains native enzymatic activity
Abstract

Targeted PEGylation of glucose oxidase at its glycosylation sites was investigated to determine the effect on enzymatic activity, as well as the bioconjugate's potential in an optical biosensing assay. Methoxy-poly(ethylene glycol)-hydrazide (4.5 kDa) was covalently coupled to periodate-oxidized glycosylation sites of glucose oxidase from Aspergillus niger. The bioconjugate was characterized using gel electrophoresis, liquid chromatography, mass spectrometry, and dynamic light scattering. Gel electrophoresis data showed that the PEGylation protocol resulted in a drastic increase (ca. 100 kDa) in the apparent molecular mass of the protein subunit, with complete conversion to the bioconjugate; liquid chromatography data corroborated this large increase in molecular size. Mass spectrometry data proved that the extent of PEGylation was six poly(ethylene glycol) chains per glucose oxidase dimer. Dynamic light scattering data indicated the absence of higher-order oligomers in the PEGylated GOx sample. To assess stability, enzymatic activity assays were performed in triplicate at multiple time points over the course of 29 days in the absence of glucose, as well as before and after exposure to 5% w/v glucose for 24 h. At a confidence level of 95%, the bioconjugate's performance was statistically equivalent to native glucose oxidase in terms of activity retention over the 29 day time period, as well as following the 24 h glucose exposure. Finally, the bioconjugate was entrapped within a poly(2-hydroxyethyl methacrylate) hydrogel containing an oxygen-sensitive phosphor, and the construct was shown to respond approximately linearly with a 220 ± 73% signal change (n = 4, 95% confidence interval) over the physiologically-relevant glucose range (i.e., 0–400 mg/dL); to our knowledge, this represents the first demonstration of PEGylated glucose oxidase incorporated into an optical biosensing assay.

Graphical abstractFigure optionsDownload full-size imageDownload as PowerPoint slideHighlights► Glucose oxidase from Aspergillus niger was PEGylated at its glycosylation sites. ► Mass spectrometry proved six polyethylene glycol chains per glucose oxidase dimer. ► Enzymatic activity assayed in the absence (29 days) and presence (24 h) of glucose. ► Bioconjugate's performance was statistically equivalent to native glucose oxidase. ► Bionconjugate incorporated into optical biosensor demonstrated linear response.

Keywords
PEG, poly(ethylene glycol); PEG-Hz, poly(ethylene glycol)-hydrazide; GOx, glucose oxidase; pHEMA, poly(2-hydroxyethyl methacrylate)Glucose oxidase; PEGylation; PEG-hydrazide; Operational stability; Glucose sensing
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Glycosylation site-targeted PEGylation of glucose oxidase retains native enzymatic activity
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 52, Issues 4–5, 10 April 2013, Pages 279–285
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us