Thermal stability of glucose oxidase and its admixtures with synthetic polymers

O’Malley, James J.; Ulmer, R.

doi:10.1002/bit.260150509

Cited by 58 publications

(32 citation statements)

References 9 publications

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“…In particular, half-lives of GOX I and GOX II at 45°C were 12 and 49 h, respectively. The results are comparable to those reported for an A. niger cell-bound GOX studied by O'Malley and Ulmer [44]. Enzymes from different A. niger strains studied by Kalisz et al [29], and Underkofler [42] exhibited higher thermal stabilities.…”

Section: Determination Of Optimum Ph-temperaturesupporting

confidence: 92%

Cell Bound and Extracellular Glucose Oxidases from Aspergillus niger BTL: Evidence for a Secondary Glycosylation Mechanism

Hatzinikolaou¹,

Mamma

Christakopoulos

et al. 2007

Appl Biochem Biotechnol

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Two glucose oxidase (GOX) isoforms where purified to electrophoretic homogeneity from the mycelium extract (GOXI) and the extracellular medium (GOXII) of Aspergillus niger BTL cultures. Both enzymes were found to be homodimers with nonreduced molecular masses of 148 and 159 kDa and pI values of 3.7 and 3.6 for GOXI and GOXII, respectively. The substrate specificity and the kinetic characteristics of the two GOX forms, as expressed through their apparent K m values on glucose, as well as pH and T activity optima, were almost identical. The only structural difference between the two enzymes was in their degrees of glycosylation, which were determined equal to 14.1 and 20.8% (w/w) of their molecular masses for GOXI and GOXII, respectively. The above difference in the carbohydrate content between the two enzymes seems to influence their pH and thermal stabilities. GOXII proved to be more stable than GOXI at pH values 2.5, 3.0, 8.0, and 9.0. Half-lives of GOXI at pH 3.0 and 8.0 were 8.9 and 17.5 h, respectively, whereas the corresponding values for GOXII were 13.5 and 28.1 h. As far as the thermal stability is concerned, GOXII was also more thermostable than GOXI as judged by the deactivation constants determined at various temperatures. More specifically, the half-lives of GOXI and GOXII, at 45 degrees C, were 12 and 49 h, respectively. These results suggest A. niger BTL probably possesses a secondary glycosylation mechanism that increases the stability of the excreted GOX.

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Section: Determination Of Optimum Ph-temperaturesupporting

confidence: 92%

Cell Bound and Extracellular Glucose Oxidases from Aspergillus niger BTL: Evidence for a Secondary Glycosylation Mechanism

Hatzinikolaou¹,

Mamma

Christakopoulos

et al. 2007

Appl Biochem Biotechnol

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“…Dialysis of the thermally denatured protein resulted in loss of the flavin molecule as revealed by the absence of the flavin fluorescence and absorbance (data not shown). Thus, the dissociation constant for the flavin molecule was strongly perturbed upon thermal denaturation, and the flavin cofactor dissociated from the active site as was previously suggested (3,35). This is in contrast with some other reports (36,37).…”

Section: Resultsmentioning

confidence: 62%

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

Žoldák

Zubrik

Musatov

et al. 2004

Journal of Biological Chemistry

127

105

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Glucose oxidase (GOX; ␤-D-glucose:oxygen oxidoreductase) from Aspergillus niger is a dimeric flavoprotein with a molecular mass of 80 kDa/monomer. Thermal denaturation of glucose oxidase has been studied by absorbance, circular dichroism spectroscopy, viscosimetry, and differential scanning calorimetry. Thermal transition of this homodimeric enzyme is irreversible and, surprisingly, independent of GOX concentration (0.2-5.1 mg/ml). It has an apparent transition temperature of 55.8 ؎ 1.2°C and an activation energy of ϳ280 kJ/mol, calculated from the Lumry-Eyring model. The thermally denatured state of GOX after recooling has the following characteristics. (i) It retains ϳ70% of the native secondary structure ellipticity; (ii) it has a relatively low intrinsic viscosity, 7.5 ml/g; (iii) it binds ANS; (iv) it has a low Stern-Volmer constant of tryptophan quenching; and (v) it forms defined oligomeric (dimers, trimers, tetramers) structures. It is significantly different from chemically denatured (6.67 M GdmHCl) GOX. Both the thermal and the chemical denaturation of GOX cause dissociation of the flavin cofactor; however, only the chemical denaturation is accompanied by dissociation of the homodimeric GOX into monomers. The transition temperature is independent of the protein concentration, and the properties of the thermally denatured protein indicate that thermally denatured GOX is a compact structure, a form of molten globulelike apoenzyme. GOX is thus an exceptional example of a relatively unstable mesophilic dimeric enzyme with residual structure in its thermally denatured state.Glucose oxidase (GOX 1 ; ␤-D-glucose:oxygen oxidoreductase, EC 1.1.3.4) is a flavoenzyme that catalyzes oxidation of ␤-Dglucose by molecular oxygen to ␦-gluconolactone, which subsequently hydrolyzes spontaneously to gluconic acid and hydrogen peroxide. The enzyme contains one tightly, noncovalently bound FAD cofactor per monomer and is a homodimer with a molecular mass of 160 kDa, depending on the extent of glycosylation (1). Glucose oxidase from Aspergillus niger is glycosylated by neutral sugars (mostly mannose-like sugars) and by amino sugars (2). Several reports find that the sugar content may vary from 11 up to 30% (3-5).GOX is of considerable commercial importance. The enzyme has applications in the food and fermentation industry, in the textile industry, and as a molecular diagnostic and analytical tool in medical and environmental monitoring applications (6 -10).The study of GOX and its applications is limited by its conformational instability. A significant effort has been made to increase the stability of GOX. It is known that both the thermal stability and the dynamic properties of the enzyme depend on its redox state (11, 12). Protein glycosylation affects the conformational dynamics of the active site and thus the activity of the enzyme (13). However, the way in which glycosylation affects the stability of GOX is not known. On the other hand, modification of the glucose oxidase surface by artificial long polyethylene-glycol ...

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“…Size exclusion chromatography and CD measurements clearly established the change in the state of association of apoenzyme following the dissociation of FAD (because of thermal unfolding). However, there are conflicting reports on the dissociation of FAD from GOD because of thermal unfolding (11,23). The dissociation of FAD from GOD leads to considerable loss of secondary and tertiary structure (there was a loss of ␣-helix, 36% of the total helical content).…”

Section: Discussionmentioning

confidence: 99%

Thermal Inactivation of Glucose Oxidase

Gouda

Singh

Rao

et al. 2003

Journal of Biological Chemistry

172

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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.

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Thermal stability of glucose oxidase and its admixtures with synthetic polymers

Cited by 58 publications

References 9 publications

Cell Bound and Extracellular Glucose Oxidases from Aspergillus niger BTL: Evidence for a Secondary Glycosylation Mechanism

Cell Bound and Extracellular Glucose Oxidases from Aspergillus niger BTL: Evidence for a Secondary Glycosylation Mechanism

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

Thermal Inactivation of Glucose Oxidase

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