Thermostabilization of erythrocyte carbonic anhydrase by polyhydric additives

Cioci, Federico

doi:10.1016/0141-0229(94)00079-7

Cited by 10 publications

(9 citation statements)

References 21 publications

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“…Because of the complexity of the system and the use of simpli®ed classic models, the present work should be considered as an attempt to explore this biocatalyst from the kinetic point of view rather than an absolute kinetic study and the results should be relative. Nevertheless, it is one of the few attempts made to separate the eects of reversible unfolding and irreversible denaturation of a biocatalyst on its activity (Baptista et al, 2000;Cioci, 1995).…”

Section: Resultsmentioning

confidence: 99%

Reactivity and stability of mycelium‐bound carboxylesterase from Aspergillus oryzae

Converti

Borghi

Gandolfi

et al. 2001

Biotech & Bioengineering

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The reactivity and thermostability of a novel mycelium-bound carboxylesterase from lyophilized cells of Aspergillus oryzae are explored in organic solvent. Ethanol acetylation was selected as reference esterification reaction. High carboxylesterase activity cells were used as biocatalyst in batch esterification tests at 12.5 < S(o) < 125 mmol L(-1), 5.0 < X(o) < 30 g L(-1), 0.49 < log P < 4.5 and 30 < T < 80 degrees C, as well as in residual activity tests after incubation at 40 < T < 90 degrees C. The starting rates of product formation were used to estimate with the Arrhenius model the apparent activation enthalpies of the enzymatic reaction (29-33 kJ mol(-1)), the reversible unfolding (56-63 kJ mol(-1)), and the irreversible denaturation (22 kJ mol(-1)) of the biocatalyst.

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Section: Resultsmentioning

confidence: 99%

Reactivity and stability of mycelium‐bound carboxylesterase from Aspergillus oryzae

Converti

Borghi

Gandolfi

et al. 2001

Biotech & Bioengineering

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“…This fact is probably related to the low degrees of reversibility achieved after the thermal unfolding and the non-first-order kinetics for irreversible inactivation pathways. Favorable cases which allow the study of both processes, such as a study of the erythrocyte carbonic anhydrase, have reported similar results, but the contribution of thermal unfolding to irreversible inactivation was not quantified (Cioci, 1995).…”

Section: Discussionmentioning

confidence: 99%

Trehalose delays the reversible but not the irreversible thermal denaturation of cutinase

Baptista

Cabral

Melo

2000

Biotechnol. Bioeng.

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The effect of trehalose (0.5 M) on the thermal stability of cutinase in the alkaline pH range was studied. The thermal unfolding induced by increasing temperature was analyzed in the absence and in the presence of trehalose according to a two‐state model (which assumes that only the folded and unfolded states of cutinase were present). Trehalose delays the reversible unfolding. The midpoint temperature of the unfolding transition (Tm) increases by 4.0°C and 2.6°C at pH 9.2 and 10.5, respectively, in the presence of trehalose. At pH 9.2 the thermal unfolding occurs at higher temperatures (Tm is 52.6°C compared to 42.0°C at pH 10.5) and a refolding yield of around 80% was obtained upon cooling. This pH value was chosen to study the irreversible inactivation (long‐term stability) of cutinase. Temperatures in the transition range from folded to unfolded state were selected and the rate constants of irreversible inactivation determined. Inactivation followed first‐order kinetics and trehalose reduced the observed rate constants of inactivation, pointing to a stabilizing effect on the irreversible inactivation step of thermal denaturation. However, if the contribution of reversible unfolding on the irreversible inactivation of cutinase was taken into account, i.e., considering the fraction of cutinase molecules in the reversible unfolded conformation, the intrinsic rate constants can be calculated. Based on the intrinsic rate constants it was concluded that trehalose does not delay the irreversible inactivation. This conclusion was further supported by comparing the activation energy of the irreversible inactivation in the absence and in the presence of trehalose. The apparent activation energy in the absence and in the presence of trehalose were 67 and 99 Kcal/mol, respectively. The activation energy calculated from intrinsic rate constants was higher in the absence (30 Kcal/mol) than in the presence of trehalose (16 Kcal/mol), showing that kinetics of the irreversible inactivation step increased in the presence of trehalose. In fact, trehalose stabilized only the reversible step of thermal denaturation of cutinase. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 70: 699–703, 2000.

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“…Finally, Figure illustrates the stability behavior of bovine erythrocyte carbonic anhydrase in pure buffer and in media containing ethanol, 1-propanol, ethanediol, 1,2-propanediol, xylitol, glucose, fructose, maltose, and sucrose. The experimental melting temperatures were determined by Cioci and by Cioci et al. using UV difference spectroscopy.…”

Section: Protein Unfolding In Aqueous Systems Containing Hydroxylic A...mentioning

confidence: 99%

Effect of Surface Tension on the Stability of Heat-Stressed Proteins: A Molecular Thermodynamic Interpretation

Cioci

1996

J. Phys. Chem.

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The stability behavior of four globular proteins (glucose oxidase, ribonuclease, lysozyme, and carbonic anhydrase) in pure buffer and in the presence of water-miscible hydroxylic additives (alcohols, polyols, and sugars) was analyzed. Attention was focused on the influence of these compounds on the melting temperature of the proteins. For all of the proteins examined, this latter quantity was found to be linearly related to the bulk surface tension of the mixed solvent. To provide a quantitative interpretation to the above observation, a molecular thermodynamic model, based on the additive-induced perturbation of the equilibrium between the folded and the unfolded protein forms, was developed. It is shown that, under some limiting conditions, the Gibbs equilibrium criterion applied to the two-state unfolding process yields a linear dependence of the melting temperature on the bulk surface tension, as observed for the proteins considered. The results obtained appear to indicate that the conformational stability of heat-stressed proteins in water-hydroxylic cosolvent mixtures does not rely on any special property of these substances but rather on their ability to affect the interfacial free energy between the protein and the solvent through perturbations of the surface tension of water. The model proposed can be used for interpretation and correlation of thermal unfolding data and, as a diagnostic tool, to assess whether the surface tension mechanism provides the overwhelming contribution to protein unfolding. IntroductionThe elaborate molecular mechanisms causing the transformation of the random-coil polypeptide chain into the compact, highly ordered, structure of proteins are mainly mediated by hydrophobic interactions 1 and by nonlocal entropic effects due to steric constraints in the folded state. 2 Their contributions to the free energy of stabilization are nearly equal in magnitude, 3 but whereas hydrophobic interactions favor the native conformation, nonlocal entropic effects destabilize this state. The interplay of these two major and opposing driving forces manifests itself in the small free energies of stabilization commonly observed: 4-6 typical values for globular proteins are in the range 5-15 kcal/mol. Native proteins, therefore, are only marginally stable, and this intrinsic lability makes them easily susceptible to denaturation.Thermal inactivation is one of the most important forms of protein denaturation. 7,8 Although there is still considerable uncertainty about the mechanisms by which the polypeptide chain loses its compactness and biological activity, it now seems to be ascertained that the first and, perhaps, the only universal step in thermal inactivation is represented by partial unfolding. [9][10][11] Valuable information about the physical bases of this process can be gained by perturbing the protein environment, for instance by addition of a water-miscible component, and analyzing the resulting changes in stability.The stability behavior of several proteins in the presence of stabilizers ...

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Thermostabilization of erythrocyte carbonic anhydrase by polyhydric additives

Cited by 10 publications

References 21 publications

Reactivity and stability of mycelium‐bound carboxylesterase from Aspergillus oryzae

Reactivity and stability of mycelium‐bound carboxylesterase from Aspergillus oryzae

Trehalose delays the reversible but not the irreversible thermal denaturation of cutinase

Effect of Surface Tension on the Stability of Heat-Stressed Proteins: A Molecular Thermodynamic Interpretation

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