Thermal inactivation of glucose oxidase. Mechanism and stabilization using additives.
Thermal inactivation of glucose oxidase (GOD; beta-d-glucose: 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 degrees 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 x mol-1 x deg-1 in the presence of 7.1 x 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 degrees 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 degrees C, whereas the corresponding midpoint for loss of secondary and tertiary structure was 62 degrees 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.
Gouda
Mudeppa Devaraja
MD
Department of Fermentation Technology and Bioengineering, Central Food Technological Research Institute, Mysore 570013, India.
Singh
Sridevi Annapurna
SA
Rao
A G Appu
AG
Thakur
Munna Singh
MS
Karanth
Naikankatte Ganesh
NG
D001234:Aspergillus niger / Q000201:enzymology; D004789:Enzyme Activation; D004795:Enzyme Stability / Q000187:drug effects; D005949:Glucose Oxidase / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D009113:Muramidase / Q000378:metabolism / Q000494:pharmacology; D011487:Protein Conformation; D012965:Sodium Chloride / Q000378:metabolism / Q000494:pharmacology; D013696:Temperature
D012965:Sodium Chloride; D005949:Glucose Oxidase; D009113:Muramidase