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Thermal Inactivation of Glucose Oxidase: Mechanism and Stabilization Using additives

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JBC-13-03
 
Title Thermal Inactivation of Glucose Oxidase: Mechanism and Stabilization Using additives
 
Creator Mudeppa Devaraja, Gouda
Sridevi Annapurna, Singh
Appu Rao, A. G.
Karanth, N. G.
Thakur, M. S.
 
Subject 04 Fermentation Technology
 
Description Thermal inactivation of glucose oxidase (GOD; _-Dglucose:
oxygen oxidoreductase), from Aspergillus niger,
followed first order kinetics both in the absence and
presence of additives. Additives such as lysozyme, NaCl,
and K2SO4 increased the half-life of the enzyme by 3.5-,
33.4-, and 23.7-fold respectively, from its initial value at 60°C. The activation energy increased from 60.3 kcal
mol_1 to 72.9, 76.1, and 88.3 kcal mol_1, whereas the
entropy of activation increased from 104 to 141, 147, and
184 cal_mol_1_deg_1 in the presence of 7.1 _ 10_5 M lysozyme, 1 M NaCl, and 0.2 M K2SO4, respectively. The
thermal unfolding of GOD in the temperature range of
25–90°C was studied using circular dichroism measurements
at 222, 274, and 375 nm. Size exclusion chromatography
was employed to follow the state of association
of enzyme and dissociation of FAD from GOD. The midpoint
for thermal inactivation of residual activity and
the dissociation of FAD was 59°C, whereas the corresponding midpoint for loss of secondary and tertiary
structure was 62°C. Dissociation of FAD from the holoenzyme was responsible for the thermal inactivation
of GOD. The irreversible nature of inactivation was
caused by a change in the state of association of apoenzyme. The dissociation of FAD resulted in the loss of
secondary and tertiary structure, leading to the unfolding
and nonspecific aggregation of the enzyme molecule
because of hydrophobic interactions of side chains. This
confirmed the critical role of FAD in structure and activity. Cysteine oxidation did not contribute to the nonspecific aggregation. The stabilization of enzyme by
NaCl and lysozyme was primarily the result of charge
neutralization. K2SO4 enhanced the thermal stability by
primarily strengthening the hydrophobic interactions
and made the holoenzyme a more compact dimeric
structure.
 
Date 2003-07
 
Type Article
PeerReviewed
 
Format application/pdf
 
Language en
 
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Identifier http://ir.cftri.com/1625/1/JBC_278_24324_-_24333_2003.pdf
Mudeppa Devaraja, Gouda and Sridevi Annapurna, Singh and Appu Rao, A. G. and Karanth, N. G. and Thakur, M. S. (2003) Thermal Inactivation of Glucose Oxidase: Mechanism and Stabilization Using additives. Journal of Biological Chemistry, 278 (27). pp. 24324-24333.