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

• Published in: Enzyme and microbial technology 2013-04, Vol.52 (4-5), p.211-217
• Main Authors: Garcia-Galan, Cristina, Barbosa, Oveimar, Fernandez-Lafuente, Roberto
• Format: Article
• Language: English
• Subjects: Adsorption; ammonium sulfate; cations; Cations, Monovalent - pharmacology; Chemical inactivation; coatings; cross-linking reagents; Cross-Linking Reagents - pharmacology; crosslinking; dextran; dissociation; enzyme activity; enzyme stability; Enzyme Stability - drug effects; Enzyme stabilization; Escherichia coli; Escherichia coli - enzymology; Escherichia coli Proteins - antagonists & inhibitors; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - metabolism; glutamate dehydrogenase; Glutamate Dehydrogenase (NADP+) - antagonists & inhibitors; Glutamate Dehydrogenase (NADP+) - chemistry; Glutamate Dehydrogenase (NADP+) - metabolism; glutaraldehyde; Hydrogen-Ion Concentration; ionic strength; lithium; Lithium - pharmacology; Models, Molecular; Multimeric enzymes; Polyethyleneimine; Polyethyleneimine - pharmacology; Polymer coating; Protein Structure, Quaternary; protein subunits; sodium phosphate; Stabilization by cations
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/j.enzmictec.2013.02.014

•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 10mM, 1M of sodium phosphate, or 1M 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 10mM 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 2h 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.