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ABSTRACT: The conformational transitions of thermophilic β-glycosidase fromSulfolobus solfataricus and the mechanism of its thermal and chemical activation were studied by electron paramagnetic resonance (EPR) of nitroxide
spin labels immobilized on the protein matrix. For this purpose, β-glycosidase was covalently modified by maleimide nitroxide
spin label (MAR⋅) and iodoacetamide nitroxide spin label (IAR⋅), both specific for -SH groups. The degree of modification was found to be independent of the temperature as well as of the
presence of two enzyme activators, sodium dodecyl sulphate (SDS) and butanol. In addition, a dansyl-piperidine nitroxide radical
probe (DR⋅), which has an affinity to the hydrophobic surfaces of proteins, was used in this study. The noncovalent binding of DR⋅ results in immediate formation of a probe-enzyme complex. At room temperature, the rotation frequency of the immobilized
labels decreases in order of IAR⋅ > MAR⋅ ≥ DR⋅. The temperature measurements of rotation correlation frequencies (v
c
) display values ranging from 6·107 to 2·108 s−1 and indicate a discontinuity with the inflection point at temperatureT
in in a range from 312 to 313 K. The observed enthalpies (ΔH
≠) and entropies (ΔS
≠) of the activation of spin label rotation were derived from the Arrhenius plots. The activation parameters were found to
be typical for rigid model systems. The addition of SDS and butanol produced a slight shift of the inflection point and changes
of spin-label mobility. A correlation between conformational transitions and enzyme thermal activation was discussed.
Applied Magnetic Resonance 04/2012; 18(4):515-526. · 0.75 Impact Factor
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ABSTRACT: Enzymatic synthesis of different β-D-glycosides was obtained using as biocatalyst immobilized cells, crude homogenate, and homogeneous native and recombinat β-glycosidase activity of the thermophilic archaeon Sulfolobus solfataricus. In particular our investigation was concerned with the selectivity in the glycosylation of hydroxybenzyl alcohols, salicin, 1,2-propanediol, and more complex polyols as well as the use of immobilized cells for the synthesis of hexyl-β-D-glucoside. The aromatic glucosides obtained by enzyme-catalyzed transglucosylation were used for kinetic studies of purified Sulfolobus solfataricus enzyme in the hydrolysis reaction.
07/2009; 10(1-4):195-210.
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ABSTRACT: The main reaction products obtainable by the hydrolysis of commercially available oleuropein by hyperthermophilic beta-glycosidase were purified and structurally characterized by UV and 1H and 13C NMR analyses. Their antioxidant activity, in particular their capacity to inhibit the fatty acid peroxidation rate, was studied. The molecular structures assigned revealed the presence of two elenolic acid forms presenting different antioxidant abilities closely correlated to their molecular structures, as well as an unstable elenolate which is a rearrangement product of the oleuropein aglycon. This molecule, under the reaction conditions (pH 7.0, 60 degrees C) required for beta-glycosidase activity, rapidly gives rise to 3,4-dihydroxy-phenylethanol (hydroxytyrosol).
Journal of Agricultural and Food Chemistry 08/2001; 49(7):3198-203. · 2.82 Impact Factor
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ABSTRACT: A general survey is carried out on the theoretical grounds for methods of spin, luminescence and Mössbauer labels, as well as their application in the study of protein intramolecular dynamics. When combined, these methods allow the protein dynamics to be investigated within a wide range of correlation times (tau c = 10(2) - 10(-10) s) and amplitudes. The purposeful application of the methods to various proteins at different temperatures (30-330 K), water content, substrate addition, etc., revealed a number of dynamical processes and conformational transitions in proteins. The experiments indicated correlations between the local segmental mobility of protein globules in a nanosecond temporal scale and biochemical reactions, such as long-distance electron transfer, hydrolysis and photoreactions. The biophysical labelling methods results were analysed together with the data on dynamics obtained using complementary physico-chemical methods and theoretical calculations. Special emphasis is given to recent results on proteins from thermophylic micro-organisms. The mechanisms of protein intramolecular dynamics and their role in the stability and functions of proteins and enzymes are discussed.
Spectrochimica Acta Part A Molecular and Biomolecular Spectroscopy 10/2000; 56A(10):2011-31. · 2.10 Impact Factor
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R Briante,
F La Cara,
F Febbraio,
R Barone,
G Piccialli,
R Carolla,
P Mainolfi,
L De Napoli,
M Patumi,
G Fontanazza, R Nucci
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ABSTRACT: The recombinant beta-glycosidase (EcS beta gly) from Sulfolobus solfataricus was immobilised on chitosan to perform the enzymatic hydrolysis of commercial oleuropein (heterosidic ester of elenolic acid and 3,4-dihydroxy-phenylethanol (hydroxytyrosol)) at two temperatures (60 and 70 degrees C). Interestingly, on the basis of the reasonable assumption that the enzyme hydrolyses only the sugar linkage, the biotransformation produces unstable aglycone species formed by oleuropein hydrolysis that, differently from some commercially available beta-glucosidases tested, give rise to the formation of hydroxytyrosol, at the operative temperatures of the bioreactor. The results of the biotransformation at 70 degrees C showed that the main products are hydroxytyrosol, and glucose, being the oleuropein aglycone present in low amount at the end of reaction. Both in single step approach or in recycle approach the amounts of glucose and oleuropein aglycone were lightly dependent from flow rate. The amount of hydroxytyrosol, increased on decreasing the flow rate of bioreactor in recycle approach, following a non-linear trend and obtaining the highest value at a flow rate of 15 ml h-1 while in the single step approach the 3,4-dihydroxy-phenylethanol was at its maximum at higher flow rate (16 ml h-1). For the hydrolysis of the oleuropein by bioreactor at 60 degrees C we used lower molar ratio oleuropein/enzyme only by the single step approach. In these conditions it is possible to obtain high amounts of only two products (glucose and hydroxytyrosol) in short time (2 h). The stability of the bioreactor at the operative temperatures showed a t1/2 of 30 days at 70 degrees C and a t1/2 of 56 days at 60 degrees C.
Journal of Biotechnology 03/2000; 77(2-3):275-86. · 3.05 Impact Factor
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ABSTRACT: Enzymes from thermophilic organisms are stable and active at temperatures which rapidly denature mesophilic proteins. However, there is not yet a complete understanding of the structural basis of their thermostability and thermoactivity since for each protein there seems to exist special networks of interactions that make it stable under the desired conditions. Here we have investigated the activity and conformational dynamics above 100 degrees C of the beta-glycosidase isolated from the hyperthermophilic archaeon Sulfolobus solfataricus. This has been made possible using a special stainless steel optical pressure cell which allowed us to perform enzyme assays and fluorescence measurements up to 160 degrees C without boiling the sample. The beta-glycosidase from S. solfataricus showed maximal activity at 125 degrees C. The time-resolved fluorescence studies showed that the intrinsic tryptophanyl fluorescence emission of the protein was represented by a bimodal distribution with Lorential shape and that temperature strongly affected the protein conformational dynamics. Remarkably, the tryptophan emission reveals that the indolic residues remain shielded from the solvent even at 125 degrees C, as shown by shielding from quenching and restricted tryptophan solubility. The relationship between enzyme activity and protein structural dynamics is discussed.
Biophysical Chemistry 10/1999; 81(1):23-31. · 2.20 Impact Factor
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ABSTRACT: beta-Glycosidase from the extreme thermophilic archaeon Sulfolobus solfataricus is a tetrameric protein with a molecular mass of 240 kDa, stable in the presence of detergents, and with a maximal activity at temperatures above 95 degrees C. Understanding the structure-activity relationships of the enzyme under different conditions is of fundamental importance for both theoretical and applicative purposes. In this paper we report the effect of methanol, ethanol, 1-propanol, and 1-butanol on the activity of S. solfataricus beta-glycosidase expressed in Escherichia coli. The alcohols stimulated the enzyme activity, with 1-butanol producing its maximum effect at a lower concentration than the other alcohols. The structure of the enzyme was studied in the presence of 1-butanol by circular dichroism, and Fourier-transform infrared and fluorescence spectroscopies. Circular dichroism and steady-state fluorescence measurements revealed that at low temperatures the presence of the alcohol produced no significant changes in the tertiary structure of the enzyme. However, time-resolved fluorescence data showed that the alcohol modifies the protein microenvironment, leading to a more flexible enzyme structure, which is probably responsible for the enhanced enzymatic activity.
Journal of Biochemistry 10/1999; 126(3):545-52. · 2.37 Impact Factor
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ABSTRACT: Purification of a lipoxygenase enzyme from the cultivar Tresor of durum wheat semolina (Triticum turgidum var. durum Desf) was reinvestigated furnishing a new procedure. The 895-fold purified homogeneous enzyme showed a monomeric structure with a molecular mass of 95 +/- 5 kDa. Among the substrates tested, linoleic acid showed the highest k(cat)/K(m) value; a beta-carotene bleaching activity was also detected. The enzyme optimal activity was at pH 6. 8 on linoleic acid as substrate and at pH 5.2 for the bleaching activity on beta-carotene, both assayed at 25 degrees C. The dependence of lipoxygenase activity on temperature showed a maximum at 40 degrees C for linoleic acid and at 60 degrees C for bleaching activity on beta-carotene. The amino acid composition showed the presence of only one tryptophan residue per monomer. Far-UV circular dichroism studies carried out at 25 degrees C in acidic, neutral, and basic regions revealed that the protein possesses a secondary structure content with a high percentage of alpha- and beta-structures. Near-UV circular dichroism, at 25 degrees C and at the same pH values, pointed out a strong perturbation of the tertiary structure in the acidic and basic regions compared to the neutral pH condition. Moreover, far-UV CD spectra studying the effects of the temperature on alpha-helix content revealed that the melting point of the alpha-helix is at 60 degrees C at pH 5.0, whereas it was at 50 degrees C at pH 6.8 and 9.0. The NH(2)-terminal sequence allowed a homology comparison with other lipoxygenase sequences from mammalian and vegetable sources.
Journal of Agricultural and Food Chemistry 06/1999; 47(5):1924-31. · 2.82 Impact Factor
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ABSTRACT: The tryptophanyl emission decay of beta-glycosidase from the extremophilic archaeon Sulfolobus solfataricus (Sbetagly) has been investigated by frequency domain fluorometry. The data were analyzed in terms of sum of discrete lifetimes as well as in terms of quasi- continuous lifetime distributions of different shape. At neutral pH the emission decay is characterized by two components: a long-lived component, centered at 7.4 ns, and a short one at 2.7 ns, irrespective of the decay scheme used for the interpretation of the experimental results. The effects of an irreversible inhibitor, that is, cyclophellitol, and that of a powerful denaturant such as guanidinium hydrochloride on the dynamics of Sbetagly has been investigated by observing the changes induced in the two components of the tryptophanyl emission decay. The addition of cyclophellitol to native Sbetagly reduces the contribution of the short-lived component but does not affect the long-lived one. Increasing concentrations of guanidinium hydrochloride differently affect the contributions of the two emission components. Higher concentrations were required to unfold the molecular regions containing the long-lived indolic fluorophores. These results indicate that the long-lived contribution arises from tryptophanyl residues deeply clustered in the interior of the protein matrix, whereas the short-lived one includes residues located in less rigid and more solvent accessible regions, some of which might be located in functionally important parts of protein. The knowledge of the crystallographic structure of Sbetagly allowed us to evaluate some average parameters for each tryptophanyl microenvironment in the Sbetagly such as hydrophobicity, structural flexibility, and ability of side chains to act as fluorescence quenchers. These results permitted to divide the tryptophanyl fluorescence of Sbetagly in the contribution of two emitting groups: one consisting of eight closely clustered tryptophans, that is, Trp 33, 36, 60, 84, 151 174, 425, and 433, responsible for the long-lived emission component and the other one, composed of nine tryptophans nearer to the subunit surface, that is, Trp 12, 156, 192, 287, 288, 316, 361, 376, 455, associable to the short-lived emission component. Finally, the examination of the tryptophanyl emission decay of the mesophilic beta-galactosidase from Escherichia coli (Cbetagal) and the Arrhenius analysis of its dependence on temperature indicated that the tryptophanyl environments of the mesophilic enzyme are rather homogeneous in consequence of a larger protein dynamics.
Proteins Structure Function and Bioinformatics 06/1999; 35(2):163-72. · 3.39 Impact Factor
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ABSTRACT: beta-Glycosidase from the extreme thermophilic archaeon Sulfolobus solfataricus is a thermostable tetrameric protein with a molecular mass of 240 kDa which is stable in the presence of detergents and has a maximal activity above 95 degrees C. An understanding of the structure-function relationship of the enzyme under different chemical-physical conditions is of fundamental importance for both theoretical and application purposes. In this paper we report the effect of basic pH values on the structural stability of this enzyme. The structure of the enzyme was studied at pH 10 and in the temperature range 25-97.5 degrees C using circular dichroism, Fourier-transform infrared and fluorescence spectroscopy. The spectroscopic data indicated that the enzyme stability was strongly affected by pH 10 suggesting that the destabilization of the protein structure is correlated with the perturbation of ionic interactions present in the native protein at neutral pHs. These experiments give support to the observation derived from the 3D-structure, that large ion pair networks on the surface stabilize Sulfolobus solfataricus beta-glycosidase.
Biochimie 12/1998; 80(11):949-57. · 3.02 Impact Factor
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ABSTRACT: Guanidine-induced denaturation of Sulfolobus solfataricus beta-glycosidase expressed in Escherichia coli, Sbetagly, was investigated at pH 6.5 and 25 degreesC by means of circular dichroism and fluorescence measurements. The process proved reversible when the protein concentration was lower than 0.01 mg mL-1. Moreover, the transition curves determined by fluorescence did not coincide with those determined by circular dichroism, and the GuHCl concentration corresponding at half-completion of the transition increased on raising the protein concentration in the range 0.001-0.1 mg mL-1. Gel filtration chromatography experiments showed that, in the range 2-4 M GuHCl, there was an equilibrium among tetrameric, dimeric, and monomeric species. These findings, unequivocally, indicated that the guanidine-induced denaturation of Sbetagly was not a two-state transition with concomitant unfolding and dissociation of the four subunits. A mechanism involving a dimeric intermediate species was proposed and was able to fit the experimental fluorescence intensity transition profiles, allowing the estimation of the total denaturation Gibbs energy change at 25 degreesC and pH 6.5. This figure, when normalized for the number of residues, showed that, at room temperature, Sbetagly has a stability similar to that of mesophilic proteins.
Biochemistry 11/1998; 37(41):14484-90. · 3.42 Impact Factor
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ABSTRACT: Catalytic membranes, obtained by immobilizing thermophilic beta-glycosidase onto nylon supports, were used in a nonisothermal bioreactor to study the effect of temperature gradients on the rate of enzyme reaction. Two experimental approaches were carried out to explain the molecular mechanisms by which the temperature gradients affect enzyme activity. The results showed that the thermophilic enzyme behaved as the mesophilic beta-galactosidase, exhibiting an activity increase which was linearly proportional to the transmembrane temperature difference. The efficiency of the system proposed was determined by calculating two constants, alpha and beta, which represent respectively the percentage increase of enzyme activity when a temperature difference of 1 degrees C or a temperature gradient of 1 degrees C cm-1 were applied across the catalytic membrane. The increase of enzyme activity in nonisothermal bioreactors entailed a proportional reduction of production times. The advantages in using thermophilic enzymes immobilized in nonisothermal bioreactors are also discussed.
Biotechnology and Bioengineering 08/1998; 59(1):108-15. · 3.95 Impact Factor
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ABSTRACT: Enzymes from hyperthermophilic organisms must operate at temperatures which rapidly denature proteins from mesophiles. The structural basis of this thermostability is still poorly understood. Towards a further understanding of hyperthermostability, we have determined the crystal structure of the beta-glycosidase (clan GH-1A, family 1) from the hyperthermophilic archaeon Sulfolobus solfataricus at 2.6 A resolution. The enzyme is a tetramer with subunit molecular mass at 60 kDa, and crystallises with half of the tetramer in the asymmetric unit. The structure is a (betaalpha)8 barrel, but with substantial elaborations between the beta-strands and alpha-helices in each repeat. The active site occurs at the centre of the top face of the barrel and is connected to the surface by a radial channel which becomes a blind-ended tunnel in the tetramer, and probably acts as the binding site for extended oligosaccharide substrates. Analysis of the structure reveals two features which differ significantly from mesophile proteins; (1) an unusually large proportion of surface ion-pairs involved in networks that cross-link sequentially separate structures on the protein surface, and (2) an unusually large number of solvent molecules buried in hydrophilic cavities between sequentially separate structures in the protein core. These factors suggest a model for hyperthermostability via resilience rather than rigidity.
Journal of Molecular Biology 10/1997; 271(5):789-802. · 4.00 Impact Factor
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ABSTRACT: The effects of temperature and SDS on the three-dimensional organization and secondary structure of beta-glycosidase from the thermophilic archaeon Sulfolobus solfataricus were investigated by CD, IR spectroscopy and differential scanning calorimetry. CD spectra in the near UV region showed that the detergent caused a remarkable change in the protein tertiary structure, and far-UV CD analysis revealed only a slight effect on secondary structure. Infrared spectroscopy showed that low concentrations of the detergent (up to 0.02%) induced slight changes in the enzyme secondary structure, whereas high concentrations caused the alpha-helix content to increase at high temperatures and prevented protein aggregation.
Biochemical Journal 06/1997; 323 ( Pt 3):833-40. · 4.90 Impact Factor
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ABSTRACT: Sulfolobus solfataricus beta-glycosidase expressed in Escherichia coli was fully inactivated at 65 degrees C, according to pseudo-first-order kinetics, by [3H]conduritol B epoxide (DL-1,2 anhydro-myo-inositol) synthesized as the active site directed inhibitor by a slight modification of Legler's procedure [Legler, G. (1977) Methods Enzymol. 46, 368-381]. The determination of kinetic constants for the inactivation showed that the process took place through the formation of a stabilized inhibitor-enzyme intermediate. Inactivation and reactivation studies suggested that the inhibitor-enzyme intermediate complex was formed more rapidly and hydrolyzed at a lower rate than it was for other glycosidases. Moreover, the stoichiometry of the binding, determined by electrospray mass spectrometric analysis, revealed that one molecule of the inhibitor was covalently bound to each enzyme subunit. The binding site for [3H]conduritol B epoxide was identified by the isolation and partial sequence analysis of the radioactive peptide obtained by cyanogen bromide and pepsin digests. Electrospray tandem mass analysis of the labeled peptide showed that the inhibitor was covalently bound to E387. This result, in agreement with data obtained from sequence alignments of S. solfataricus beta-glycosidase with other gluco- and galactosidases of the glycosyl hydrolase family 1 [Henrissat, B. (1991) Biochem. J. 280, 309-316], indicates that the conserved E387 is the nucleophilic amino acid residue in the active site of the enzyme.
Biochemistry 04/1997; 36(11):3068-75. · 3.42 Impact Factor
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ABSTRACT: Sulfolobus solfataricus β-glycosidase expressed in Escherichia coli was fully inactivated at 65 °C, according to pseudo-first-order kinetics, by [3H]conduritol B epoxide (dl-1,2 anhydro-myo-inositol) synthesized as the active site directed inhibitor by a slight modification of Legler's procedure [Legler, G. (1977) Methods Enzymol. 46, 368−381]. The determination of kinetic constants for the inactivation showed that the process took place through the formation of a stabilized inhibitor−enzyme intermediate. Inactivation and reactivation studies suggested that the inhibitor−enzyme intermediate complex was formed more rapidly and hydrolyzed at a lower rate than it was for other glycosidases. Moreover, the stoichiometry of the binding, determined by electrospray mass spectrometric analysis, revealed that one molecule of the inhibitor was covalently bound to each enzyme subunit. The binding site for [3H]conduritol B epoxide was identified by the isolation and partial sequence analysis of the radioactive peptide obtained by cyanogen bromide and pepsin digests. Electrospray tandem mass analysis of the labeled peptide showed that the inhibitor was covalently bound to E387. This result, in agreement with data obtained from sequence alignments of S. solfataricus β-glycosidase with other gluco- and galactosidases of the glycosyl hydrolase family 1 [Henrissat, B. (1991) Biochem. J. 280, 309−316], indicates that the conserved E387 is the nucleophilic amino acid residue in the active site of the enzyme.
03/1997;
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ABSTRACT: The emission decay of intrinsic fluorescence of the extremely thermophilic beta-glycosidase from Sulfolobus solfataricus has been investigated as functions of temperature and of iodide-quencher concentration by frequency-domain fluorometry. This protein contains 68 tryptophans and provides a matrix for correlation of the average spectroscopic behaviour with solvent exposure and local dynamics. At each temperature, the emission is very heterogeneous and interpretable in terms of quasicontinuous bimodal distribution of fluorescence lifetimes. We associate the component of the bimodal distribution to two distinct classes of tryptophanyl residues that differ in microenvironmental characteristics. Temperature and quenching experiments show that the long-lived component includes tryptophanyl residues located in buried regions with high rigidity; the short distributional component corresponds to tryptophans embedded in more flexible and exposed regions. This proposal has been confirmed by examination of the crystallographic structure. The data suggest that, at least for this protein, there is a good correlation between residue exposure and lifetime distributional components. The conformational dynamics of the two classes of tryptophanyl residues is affected differently by temperature, suggesting that the protein regions in which they are located give different contributions to enzyme properties, such as flexibility, stability and function.
European Journal of Biochemistry 03/1997; 244(1):53-8. · 3.58 Impact Factor
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ABSTRACT: The conformational dynamics of beta-glycosidase from Sulfolobus solfataricus was investigated by following the emission decay arising from the large number of tryptophanyl residues that are homogeneously dispersed in the primary structure. The fluorescence emission is characterized by a bimodal lifetime distribution, suggesting that the enzyme structure contains rigid and flexible regions, properly located in the macromolecule. The enzyme activity and thermostability appear to be related to the dynamic properties of these regions as evidenced by perturbation studies of the enzyme structure at alkaline pH and by addition of detergents such as SDS. The pH increase affects the protein dynamics with a remarkable loss of thermal stability and activity; these changes occur without any significant variation in the secondary structure as revealed by far-UV dichroic measurements. In the presence of 0.02% (w/v) SDS at alkaline pH, the enzymatic activity and thermostability are recovered. Under these conditions, the conformational dynamics appear to be similar to that evidenced at neutral pH. Further increases in SDS concentration, at alkaline pH, render the activity and thermostability of beta-glycosidase similar to those observed in the absence of detergent.
Proteins Structure Function and Bioinformatics 02/1997; 27(1):71-9. · 3.39 Impact Factor
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ABSTRACT: The beta-glycosidase isolated from the extreme thermophilic archaeon Sulfolobus solfataricus, grown at 87 degrees C, is a tetrameric protein with a molecular mass of 240 kDa. This enzyme is barely active at 30 degrees C and has optimal activity, over 95 degrees C, at pH 6.5. Its thermal stability was investigated at pH 10.1 and 10.6 by means of functional studies, circular dichroism and differential scanning calorimetry. There was no evidence of thermal activation of the enzyme and the temperature-induced denaturation was irreversible and not well represented by the two-state transition model. A more complex process occurred, involving the dissociation and unfolding of subunits, and subsequent nonspecific association and/or aggregation. Denaturation temperature was around 85 degrees C, depending on protein concentration. The denaturation enthalpy change was between 7,500 and 9,800 kJ.mol-1, depending on the pH. The collapse of the native structure around 85 degrees C was confirmed by circular dichroism measurements and time-dependent activity studies. Finally, preliminary investigations were performed on the recombinant enzyme expressed in Escherichia coli.
Journal of Biochemistry 09/1996; 120(2):292-300. · 2.37 Impact Factor
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ABSTRACT: A protein with beta-glycosidase activity from Sulfolobus solfataricus (S beta gly) was expressed in the yeast Saccharomyces cerevisiae. The purification procedure was made fast and easy by employing a single chromatographic step. After 5.8-fold purification, the cell extract gave a homogeneous enzyme at 166 U/mg. The recombinant enzyme was functionally and structurally similar to the wild-type enzyme. Kinetic experiments showed the same wide substrate specificity; in fact, the expressed enzyme hydrolyzed beta-D-gluco-, fuco-, and galactosides and a large number of glucoside dimers and oligomers, linked beta 1 -> 4. Moreover, the molecular mass of the enzyme was estimated to be 60 kDa by SDS-PAGE and 240 kDa by gel filtration, glycerol gradient, and ultracentrifugation analyses, indicating that the enzyme has a tetrameric structure. The N-terminal amino acid sequence, the temperature dependent activity, and content of secondary structure were similar to those of the wild-type enzyme. CD spectral and kinetic analyses showed that the only differences from the wild-type enzyme consist of the absence of lysine methylation, the presence of some glycosylated amino acid residues, and lower thermostability. Furthermore, calorimetric analyses on the expressed protein indicated values of delta dH = 5072 kJ/ mol and delta (d)C(p)= 100 kJ/mol, appreciably lower than those of the wild-type protein.
Protein Expression and Purification 06/1996; 7(3):299-308. · 1.59 Impact Factor
