- M. Fulde (1)
- R. Goethe (1)
- A. Greeff de (1)
- H. Gruppen (1)
- O. Gutierrez (1)
- A.E.M. Janssen (2)
- S. Koutsopoulos (1)
- J.F.T. Lieshout van (1)
- J. Oost van der (1)
- A. Planas (1)
- H.E. Smith (1)
- P. Valentin-Weigand (1)
- W.M. Vos de (1)
- W. Vroom (1)
- J. Willenborg (1)
Thermal Stabilization of an Endoglucanase by Cyclization
Lieshout, J.F.T. van; Gutierrez, O. ; Vroom, W. ; Planas, A. ; Vos, W.M. de; Oost, J. van der; Koutsopoulos, S. - \ 2012
Applied Biochemistry and Biotechnology 167 (2012)7. - ISSN 0273-2289 - p. 2039 - 2053.
site-directed mutagenesis - beta-glucosidase celb - bacillus-licheniformis - circular proteins - in-vivo - pyrococcus-furiosus - crystal-structure - pi-pfui - stability - intein
An intein-driven protein splicing approach allowed for the covalent linkage between the N- and C-termini of a polypeptide chain to create circular variants of the endo-ß-1,3-1,4-glucanase, LicA, from Bacillus licheniformis. Two circular variants, LicA-C1 and LicA-C2, which have connecting loops of 20 and 14 amino acids, respectively, showed catalytic activities that are approximately two and three times higher, respectively, compared to that of the linear LicA (LicA-L1). The thermal stability of the circular variants was significantly increased compared to the linear form. Whereas the linear glucanase lost half of its activity after 3 min at 65 °C, the two circular variants have 6-fold (LicA-C1) and 16-fold (LicA-C2) increased half-life time of inactivation. In agreement with this, fluorescence spectroscopy and differential scanning calorimetry studies revealed that circular enzymes undergo structural changes at higher temperatures compared to that of the linear form. The effect of calcium on the conformational stability and function of the circular LicAs was also investigated, and we observed that the presence of calcium ions results in increased thermal stability. The impact of the length of the designed loops on thermal stability of the circular proteins is discussed, and it is suggested that cyclization may be an efficient strategy for the increased stability of proteins
ArgR is an essential local transcriptional regulator of the arcABC-operon in Streptococcus suis and crucial for biological fitness in acidic environment
Fulde, M. ; Willenborg, J. ; Greeff, A. de; Benga, L. ; Smith, H.E. ; Valentin-Weigand, P. ; Goethe, R. - \ 2011
Microbiology 157 (2011)2. - ISSN 1350-0872 - p. 572 - 582.
arginine deiminase system - escherichia-coli - pseudomonas-aeruginosa - bacillus-licheniformis - lactococcus-lactis - lactobacillus-sakei - virulence factor - catabolism - expression - repressor
Streptococcus suis is one of the most important pathogens in pigs and can also cause severe infections in humans. Despite its clinical relevance very little is known about the factors contributing to its virulence. Recently, we identified a new putative virulence factor in Streptococcus suis, the arginine deiminase system (ADS), an arginine catabolic enzyme system encoded by the arcABC-operon, which enables Streptococcus suis to survive in acidic environment. In this study, we focused on ArgR, an ADS associated regulator belonging to the ArgR/AhrC arginine repressor family. Using an argR knock-out strain we could show that ArgR is essential for arcABC-operon expression and necessary for the biological fitness of Streptococcus suis. By cDNA expression microarray analyses and quantitative real time RT-PCR we found that the arcABC-operon is the only gene cluster regulated by ArgR, which is in contrast to many other bacteria. Reporter gene analysis with gfp under the control of the arcABC promoter demonstrated that ArgR is able to activate the arcABC promoter. Electrophoretic mobility shift assays with fragments of the arcABC promoter and recombinant ArgR, and chromatin immunoprecipitation with antibodies directed against ArgR revealed that ArgR interacts with the arcABC promoter in vitro and in vivo by binding to a region from -147 to 72 bp upstream of the transcriptional start point. Overall our results show that in Streptococcus suis ArgR is an essential, system specific transcriptional regulator of the ADS directly interacting with the arcABC promoter in vivo.
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
Enzyme-induced aggregation and gelation of proteins
Creusot, N.P. ; Gruppen, H. - \ 2007
Biotechnology Advances 25 (2007)6. - ISSN 0734-9750 - p. 597 - 601.
protease-induced aggregation - bacillus-licheniformis - beta-lactoglobulin - alpha-lactalbumin - whey proteins - identification - hydrolysis - mechanism - glu
This paper provides a brief overview of the effects of protein hydrolysis on aggregation and gel forming properties of protein systems. Among the food globular proteins, whey proteins and soy proteins are the most extensively studied for their ability to form different textures upon proteolysis. Recent studies were focused on identifying aggregating peptides and on mechanisms of aggregation and gelation.
The effect of carbohydrates on alpha-amylase activity measurements
Baks, T. ; Janssen, A.E.M. ; Boom, R.M. - \ 2006
Enzyme and Microbial Technology 39 (2006)1. - ISSN 0141-0229 - p. 114 - 119.
bacillus-licheniformis - starch - inactivation - hydrolysis - assay
The Ceralpha method can be used for ¿-amylase activity measurements during the hydrolysis of starch at high substrate concentrations (>40 wt.%). However, the results are affected by the carbohydrates present in the samples. The effect of carbohydrates on the Ceralpha ¿-amylase activity measurements was measured over a broad concentration range. It was found that starch has the largest influence and glucose has the lowest influence on the Ceralpha assay procedure. These results were explained by considering substrate inhibition and substrate competition. A simple kinetic model was used to describe the observed phenomena quantitatively. This model was also used to estimate the Michaelis¿Menten constant for a large number of substrates and it requires only a single experiment for each Km determination.