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Stabilization of the hexameric glutamate dehydrogenase from Escherichia coli by cations and polyethyleneimine

Paper ID Volume ID Publish Year Pages File Format Full-Text
17363 42663 2013 7 PDF Available
Title
Stabilization of the hexameric glutamate dehydrogenase from Escherichia coli by cations and polyethyleneimine
Abstract

•GDH activity does not depend on the presence of cations, but its stability improves in the presence of Li+.•GDH is a hexameric enzyme, inactivation rate depends on enzyme concentration.•GDH is readily inactivated by aldehyde reagents and sodium borohydride.•PEI stabilizes GDH and minimizes the effects of enzyme dilution on GDH stability.•PEI also greatly increases GDH stability in stirred systems.

The enzyme glutamate dehydrogenase (GDH) from Escherichia coli is a hexameric protein. The stability of this enzyme was increased in the presence of Li+ in concentrations ranging from 1 to 10 mM, 1 M of sodium phosphate, or 1 M ammonium sulfate. A very significant dependence of the enzyme stability on protein concentration was found, suggesting that subunit dissociation could be the first step of GDH inactivation. This effect of enzyme concentration on its stability was not significantly decreased by the presence of 10 mM Li+. Subunit crosslinking could not be performed using neither dextran nor glutaraldehyde because both reagents readily inactivated GDH. Thus, they were discarded as crosslinking reagents and GDH was incubated in the presence of polyethyleneimine (PEI) with the aim of physically crosslinking the enzyme subunits. This incubation does not have a significant effect on enzyme activity. However, after optimization, the PEI-GDH was found to almost maintain the full initial activity after 2 h under conditions where the untreated enzyme retained only 20% of the initial activity, and the effect of the enzyme concentration on enzyme stability almost disappeared. This stabilization was maintained in the pH range 5–9, but it was lost at high ionic strength. This PEI-GDH composite was also much more stable than the unmodified enzyme in stirred systems. The results suggested that a real adsorption of the PEI on the GDH surface was required to obtain this stabilizing effect. A positive effect of Li+ on enzyme stability was maintained after enzyme surface coating with PEI, suggesting that the effects of both stabilizing agents could not be exactly based on the same mechanism. Thus, the coating of GDH surface with PEI seems to be a good alternative to have a stabilized and soluble composite of the enzyme.

Keywords
Enzyme stabilization; Multimeric enzymes; Stabilization by cations; Polymer coating; Polyethyleneimine; Chemical inactivation
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Stabilization of the hexameric glutamate dehydrogenase from Escherichia coli by cations and polyethyleneimine
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 52, Issues 4–5, 10 April 2013, Pages 211–217
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