Thermal Inactivation of Glucose Oxidase

Abstract

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 K 2 SO 4 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 cal⅐mol ؊1 ⅐deg ؊1 in the presence of 7.1 ؋ 10 ؊5 M lysozyme, 1 M NaCl, and 0.2 M K 2 SO 4 , 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. K 2 SO 4 enhanced the thermal stability by primarily strengthening the hydrophobic interactions and made the holoenzyme a more compact dimeric structure.

Irreversible Thermal Denaturation of Glucose Oxidase from Aspergillus niger Is the Transition to the Denatured State with Residual Structure

“…This finding is contrary to the suggestion that dissociation of FAD induces GOX monomerization (15,50). Recently presented results indicate that after the dissociation of FAD from the holoenzyme, the apoenzyme is not in a monomeric state but tends to form aggregates (21). An alternative explanation of the reported gel filtration experiments would be the formation of an extended molten globule-like structure.…”

“…GOX contains one reduced cysteine residue in the structure that is not exposed to solvent after thermal denaturation (21). In fact, heating of the protein in the presence of iodoacetamide had no significant effect on the reversibility of the transition or on the presence of the residual structure (data not shown).…”

Thermostability and thermodynamic characterization of sprouted pearl millet ?-amylases for its biotechnological applications

“…A similar trend was observed in peroxidase, lipoxygenase and polyphenol oxidase from white yam (Eze et al, 2010) and xylanase from Aspergillus niger (Pal and Khanum, 2011) but the magnitude was higher in enzymes from white yam, showing that they undergo more aggregation and compactation during inactivation. The differences observed here could be as a result of change in the conformation of the enzymes studied (Gouda et al, 2003).…”

A Bioelectrocatalysis Method for the Kinetic Measurement of Thermal Inactivation of a Redox Enzyme, Bilirubin Oxidase

“…For example, the half life at 60˚C was calculated to be 7.5 min, while the half life at 25˚C is 39 days. We calculated the activation enthalpy (ΔH 23 and EA = 195 and 145 kJ mol −1 , ΔS ≠ = 94 and 237 J mol −1 K −1 for peroxidase isozymes (Eupatorium odoratum). 24 BOD exists in solution as a monomer of molecular weight 59950, composed of 534 amino acid residues containing one cysteine.…”