Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Dimerization of Proline Dehydrogenase from Thermus thermophilus Is Crucial for Its Thermostability
Huijbers, Mieke M.E. ; Wu, Jenny W. ; Westphal, Adrie H. ; Berkel, Willem J.H. van - \ 2019
Biotechnology Journal 14 (2019)5. - ISSN 1860-6768
flavoprotein - protein oligomerization - thermostability - Thermus thermophilus - triosephosphate isomerase barrel

Thermus thermophilus proline dehydrogenase (TtProDH) catalyzes the first step in proline catabolism. The thermostable flavoenzyme consists of a distorted triosephosphate isomerase (TIM) barrel and three N-terminal helices: αA, αB, and αC. Using maltose-binding protein (MBP) fused constructs, it has been recently demonstrated that helix αC is crucial for TtProDH catalysis and for tetramerization through positioning of helix α8. Here, the structural features that determine the thermostability of TtProDH are reported. Selective disruption of two ion pairs in the dimerization interface of several MBP-TtProDH variants result in the formation of monomers. The newly created monomers have improved catalytic properties but their melting temperatures are decreased by more than 20 °C. Sequence comparison suggests that one of the ion-pairs involved in dimerization is unique for ProDHs from Thermus species. In summary, intermolecular ion-pairs improve the thermostability of TtProDH and a trade-off is made between thermostability and catalytic activity.

Molecular characterization of the glucose isomerase from the thermophilic bacterium Fervidobacterium gondwanense
Kluskens, L.D. ; Zeilstra, J.B. ; Geerling, A.C.M. ; Vos, W.M. de; Oost, J. van der - \ 2010
Environmental Technology 31 (2010)10. - ISSN 0959-3330 - p. 1083 - 1090.
d-xylose isomerase - biochemical-characterization - thermotoga-neapolitana - thermus-thermophilus - escherichia-coli - thermostability - cloning - purification - expression - fructose
The gene coding for xylose isomerase from the thermophilic bacterium Fervidobacterium gondwanense was cloned and overexpressed in Escherichia coli. The produced xylose isomerase (XylA), which closely resembles counterparts from Thermotoga maritima and T. neapolitana, was purified and characterized. It is optimally active at 70 degrees C, pH 7.3, with a specific activity of 15.0 U/mg for the interconversion of glucose to fructose. When compared with T. maritima XylA at 85 degrees C, a higher catalytic efficiency was observed. Divalent metal ions Co2+ and Mg2+ were found to enhance the thermostability
Increased susceptibility of ß-glucosidase from the hyperthermophile Pyrococcus furiosus to thermal inactivation at higher pressures
Bruins, M.E. ; Meersman, F. ; Janssen, A.E.M. ; Heremans, K. ; Boom, R.M. - \ 2009
FEBS Journal 276 (2009). - ISSN 1742-464X - p. 109 - 117.
enzyme inactivation - escherichia-coli - proteins - stabilization - temperature - myoglobin - thermostability - denaturation - spectroscopy - aggregation
The stability of ß-glucosidase from the hyperthermophile Pyrococcus furiosus was studied as a function of pressure, temperature and pH. The conformational stability was monitored using FTIR spectroscopy, and the functional enzyme stability was monitored by inactivation studies. The enzyme proved to be highly piezostable and thermostable, with an unfolding pressure of 800 MPa at 85 °C. The tentative pressure¿temperature stability diagram indicates that this enzyme is stabilized against thermal unfolding at low pressures. The activity measurements showed a two-step inactivation mechanism due to pressure that was most pronounced at lower temperatures. The first part of this inactivation took place at pressures below 300 MPa and was not visible as a conformational transition. The second transition in activity was concomitant with the conformational transition. An increase in pH from 5.5 to 6.5 was found to have a stabilizing effect
A stochastic model for predicting dextrose equivalent and saccharide composition during hydrolysis of starch by alpha-amylase
Besselink, T. ; Baks, T. ; Janssen, A.E.M. ; Boom, R.M. - \ 2008
Biotechnology and Bioengineering 100 (2008)4. - ISSN 0006-3592 - p. 684 - 697.
monte-carlo-simulation - bacillus-licheniformis - enzymatic-hydrolysis - soluble starch - kinetic-model - potato starch - amylopectin - amylolysis - enzymes - thermostability
A stochastic model was developed that was used to describe the formation and breakdown of all saccharides involved during -amylolytic starch hydrolysis in time. This model is based on the subsite maps found in literature for Bacillus amyloliquefaciens -amylase (BAA) and Bacillus licheniformis -amylase (BLA). Carbohydrate substrates were modeled in a relatively simple two-dimensional matrix. The predicted weight fractions of carbohydrates ranging from glucose to heptasaccharides and the predicted dextrose equivalent showed the same trend and order of magnitude as the corresponding experimental values. However, the absolute values were not the same. In case a well-defined substrate such as maltohexaose was used, comparable differences between the experimental and simulated data were observed indicating that the substrate model for starch does not cause these deviations. After changing the subsite map of BLA and the ratio between the time required for a productive and a non-productive attack for BAA, a better agreement between the model data and the experimental data was observed. Although the model input should be improved for more accurate predictions, the model can already be used to gain knowledge about the concentrations of all carbohydrates during hydrolysis with an -amylase. In addition, this model also seems to be applicable to other depolymerase-based systems
Glucosylation of beta-lactoglobulin lowers the heat capacity change of unfolding; a unique way to affect protein thermodynamics
Teeffelen, A.M.M. Van; Broersen, K. ; Jongh, H.H.J. de - \ 2005
Protein Science 14 (2005)8. - ISSN 0961-8368 - p. 2187 - 2194.
guanidine-hydrochloride - amino-acids - m-values - stability - thermostability - glycosylation - denaturation - spectroscopy - surface - identification
Chemical glycosylation of proteins occurs in vivo spontaneously, especially under stress conditions, and has been linked in a number of cases to diseases related to protein denaturation and aggregation. It is the aim of this work to study the origin of the change in thermodynamic properties due to glucosylation of the folded -lactoglobulin A. Under mild conditions Maillard products can be formed by reaction of -amino groups of lysines with the reducing group of, in this case, glucose. The formed conjugates described here have an average degree of glycosylation of 82%. No impact of the glucosylation on the protein structure is detected, except that the Stokes radius was increased by ~3%. Although at ambient temperatures the change in Gibbs energy of unfolding is reduced by 20%, the denaturation temperature is increased by 5°C. Using a combination of circular dichroism, fluorescence, and calorimetric approaches, it is shown that the change in heat capacity upon denaturation is reduced by 60% due to the glucosylation. Since in the denatured state the Stokes radius of the protein is not significantly smaller for the glucosylated protein, it is suggested that the nonpolar residues associate to the covalently linked sugar moiety in the unfolded state, thereby preventing their solvent exposure. In this way coupling of small reducing sugar moieties to solvent exposed groups of proteins offers an efficient and unique tool to deal with protein stability issues, relevant not only in nature but also for technological applications
Temperature-dependent structural and functional features of a hyperthermostable enzyme using elastic neutron scattering
Koutsopoulos, S. ; Oost, J. van der; Norde, W. - \ 2005
Proteins : Structure, Function, and Bioinformatics 61 (2005)2. - ISSN 0887-3585 - p. 377 - 384.
archaeon pyrococcus-furiosus - crystal-structure - protein dynamics - 2.5-angstrom resolution - angstrom resolution - citrate synthase - thermostability - dehydrogenase - stability - fluctuations
The dynamic behavior of an endoglucanase from the hyperthermophilic microorganism Pyrococcus furiosus was investigated using elastic neutron scattering. The temperature dependence of the atomic motions was correlated with conformational and functional characteristics of the enzyme. The onset of biological function at temperatures higher than approximately 25°C (the hyperthermostable enzyme is essentially inactive at room temperature) was associated with a dynamical transition in the anharmonic motions domain. This transition from the nonactive to the enzymatically active conformation involved structurally similar conformational substates in the energy landscape. From the mean-square displacement of the protein atoms, the molecular flexibility and the effective force constants were calculated at different temperature zones. The results showed that the activity increases at higher temperatures where the intramolecular bonds are weakened and the overall rigidity of the protein is decreased. Further temperature increase resulted in significantly increased atomic fluctuations featuring heat denaturation of the protein
Modification of beta-lactoglobulin by oligofructose: Impact on protein adsorption at the air-water interface
Trofimova, D. ; Jongh, H.H.J. de - \ 2004
Langmuir 20 (2004)13. - ISSN 0743-7463 - p. 5544 - 5552.
air/water interface - functional-properties - maillard reaction - stability - ovalbumin - thermostability - glycation - spectra
Maillard products of -lactoglobulin (Lg) and fructose oligosaccharide (FOS) were obtained in different degrees of modification depending on incubation time and pH. By use of a variety of biochemical and spectroscopic tools, it was demonstrated that the modification at limited degrees does not significantly affect the secondary, tertiary, and quaternary structure of Lg. The consequence of the modification on the thermodynamics of the protein was studied using differential scanning calorimetry, circular dichroism, and by monitoring the fluorescence intensity of protein samples with different concentrations of guanidine-HCl. The modification leads to lowering of the denaturation temperature by 5 C and a reduction of the free energy of stabilization of about 30%. Ellipsometry and drop tensiometry demonstrated that upon adsorption to air-water interfaces in equilibrium modified Lg exerts a lower surface pressure than native Lg (16 versus 22 mN/m). Moreover, the surface elastic modulus increased with increasing surface pressure but reached significantly smaller values in the case of FOS-Lg. Compared to native Lg, modification of the protein with oligofructose moieties results in higher surface loads and thicker surface layers. The consequences of these altered surface rheological properties are discussed in view of the functional behavior in technological applications.
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