The molten-globule residual structure is critical for reflavination of glucose oxidase

Garajová, Katarína; Zimmermann, Martina; Petrenčáková, Martina; Dzurová, Lenka; Nemergut, Michal; Škultéty, Ľudovít; Sedlák, Erik

doi:10.1016/j.bpc.2017.08.009

Cited by 9 publications

(7 citation statements)

References 35 publications

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“…The obtained rate constants, k , were evaluated as a function of urea concentration [urea]: k = k 0 (H 2 O)exp( m u [urea]), where k 0 (H 2 O) is the microscopic unfolding rate constant in the absence of urea. DSC experiments were performed as described elsewhere , using a Microcal VP-capillary DSC (Malvern).…”

Section: Methodsmentioning

confidence: 99%

“…As published previously, 11 lyophilized glucose oxidase (E.C. 1.1.3.4) from A. niger was obtained from Sigma-Aldrich (GOX2) with GenBank accession number X16061 or J05242, and UniProt number P13006.…”

Section: ■ Methodsmentioning

confidence: 99%

“…In our previous thermal stability study, we found that GOX undergoes irreversible denaturation into a molten-globule-like state (Figure b), which does not refold into the native protein even in the presence of an excess of flavin cofactor. , The molecular mechanism behind the irreversible transformation remained unclear, which provided a roadblock for the development of rational strategies to optimize long-term enzyme stability. Such an improvement would support biotechnology applications by extending the range of working conditions.…”

Section: Introductionmentioning

confidence: 99%

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Ion-Specific Protein/Water Interface Determines the Hofmeister Effect on the Kinetic Stability of Glucose Oxidase

et al. 2019

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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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Section: Methodsmentioning

confidence: 99%

Section: ■ Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Ion-Specific Protein/Water Interface Determines the Hofmeister Effect on the Kinetic Stability of Glucose Oxidase

et al. 2019

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“…[65,66] In addition, GOx exhibits absorption bands at l = 375 and 450 nm that are characteristico ft he flavin adenine dinucleotide cofactor bound to the protein structure. [67] The spectrum of the supernatant showed am uch smallera bsorption in the UV and visible regionst han that of as olution containing only GOx and Ins at the same final concentration of the colloidal sample (0.39 mg mL À1 ). This result indicates that the protein cargo is efficiently encapsulated inside the GI-PSA structure and stabilized by supramolecular interactions and that undesired protein release does not occurs ( Figure S2 in the SupportingI nformation).…”

Section: Gi-psa Characterization and Ph-triggereddisassemblymentioning

confidence: 95%

Insulin Delivery from Glucose‐Responsive, Self‐Assembled, Polyamine Nanoparticles: Smart “Sense‐and‐Treat” Nanocarriers Made Easy

Agazzi

Herrera

Cortez

et al. 2020

Chemistry A European J

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Polyamine–salt aggregates (PSA) are biomimetic soft materials that have attracted great attention due to their straightforward fabrication methods, high drug‐loading efficiencies, and attractive properties for pH‐triggered release. Herein, a simple and fast multicomponent self‐assembly process was used to construct cross‐linked poly(allylamine hydrochloride)/phosphate PSAs (hydrodynamic diameter of 360 nm) containing glucose oxidase enzyme, as a glucose‐responsive element, and human recombinant insulin, as a therapeutic agent for the treatment of diabetes mellitus (GI‐PSA). The addition of increasing glucose concentrations promotes the release of insulin due to the disassembly of the GI‐PSAs triggered by the catalytic in situ formation of gluconic acid. Under normoglycemia, the GI‐PSA integrity remained intact for at least 24 h, whereas hyperglycemic conditions resulted in 100 % cargo release after 4 h of glucose addition. This entirely supramolecular strategy presents great potential for the construction of smart glucose‐responsive delivery nanocarriers.

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“…It is a homodimer of ∼80 kDa monomers, each of which contains two structural domains, one containing the substrate binding site, and the other housing tightly, but non-covalently, bound flavin adenine dinucleotide (FAD) as a coenzyme. Previous results have suggested that the dissociation of FAD resulted in the destabilized tertiary structures of GOD and it has a critical role in the structure and activity of GOD [ 18 , 19 ]. Therefore, the binding strength of FAD with its surrounding amino acids may also affect the thermostability of GOD.…”

Section: Introductionmentioning

confidence: 99%

Enhanced Thermostability of Glucose Oxidase through Computer-Aided Molecular Design

Ning

Yuan

et al. 2018

IJMS

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Glucose oxidase (GOD, EC.1.1.3.4) specifically catalyzes the reaction of β-d-glucose to gluconic acid and hydrogen peroxide in the presence of oxygen, which has become widely used in the food industry, gluconic acid production and the feed industry. However, the poor thermostability of the current commercial GOD is a key limiting factor preventing its widespread application. In the present study, amino acids closely related to the thermostability of glucose oxidase from Penicillium notatum were predicted with a computer-aided molecular simulation analysis, and mutant libraries were established following a saturation mutagenesis strategy. Two mutants with significantly improved thermostabilities, S100A and D408W, were subsequently obtained. Their protein denaturing temperatures were enhanced by about 4.4 °C and 1.2 °C, respectively, compared with the wild-type enzyme. Treated at 55 °C for 3 h, the residual activities of the mutants were greater than 72%, while that of the wild-type enzyme was only 20%. The half-lives of S100A and D408W were 5.13- and 4.41-fold greater, respectively, than that of the wild-type enzyme at the same temperature. This work provides novel and efficient approaches for enhancing the thermostability of GOD by reducing the protein free unfolding energy or increasing the interaction of amino acids with the coenzyme.

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The molten-globule residual structure is critical for reflavination of glucose oxidase

Cited by 9 publications

References 35 publications

Ion-Specific Protein/Water Interface Determines the Hofmeister Effect on the Kinetic Stability of Glucose Oxidase

Ion-Specific Protein/Water Interface Determines the Hofmeister Effect on the Kinetic Stability of Glucose Oxidase

Insulin Delivery from Glucose‐Responsive, Self‐Assembled, Polyamine Nanoparticles: Smart “Sense‐and‐Treat” Nanocarriers Made Easy

Enhanced Thermostability of Glucose Oxidase through Computer-Aided Molecular Design

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