Homodimeric glucose
oxidase (GOX) from Aspergillus
niger is a prominent enzyme used for a number of applications
in biotechnology and clinical diagnostics. For robust and long-term
functional applications of GOX, the stability of the protein is of
utmost importance. In vitro, GOX is irreversibly inactivated over
time by a mechanism that is poorly understood, and hence, it presents
a significant drawback for the development of strategies to stabilize
the enzyme. We show that the nonequilibrium stability of GOX is fully
described by a one-step conformational unfolding kinetics. To explore
the strategies for improving GOX nonequilibrium stability, the effect
of salts of the Hofmeister series is examined using microcalorimetry.
We obtain activation energies E
a and inactivation
temperatures T
k (at which the irreversible
step is 1.0 min–1) as a function of the salt types
and concentrations. Based on the analysis by the extended Langmuir
model, we find that at high salt concentrations (>1 M) the Hofmeister
effect on inactivation temperature is determined by the universal
ion-specific effect on the protein/water interface, which apparently
does not depend significantly on a particular amino-acid sequence
and 3D protein structure. Our findings identify protein/water interfacial
tension as a critical physicochemical attribute of excipients that
is crucial for increasing enzyme kinetic stability.
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