Zoekresultaten

Saturation mutagenesis reveals the importance of residues alphaR145 and alphaF146 of penicillin acylase in the synthesis of beta-lactam antibiotics

Penicillin acylase (PA) from Escherichia coli can catalyze the coupling of an acyl group to penicillin- and cephalosporin-derived beta-lactam nuclei, a conversion that can be used for the industrial synthesis of beta-lactam antibiotics. The modest synthetic properties of the wild-type enzyme make it desirable to engineer improved mutants. Analysis of the crystal structure of PA has shown that residues alphaR145 and alphaF146 undergo extensive repositioning upon binding of large ligands to the active site, suggesting that these residues may be good targets for mutagenesis aimed at improving the catalytic performance of PA. Therefore, site-saturation mutagenesis was performed on both positions and a complete set of all 38 variants was subjected to rapid HPLC screening for improved ampicillin synthesis. Not less than 33 mutants showed improved synthesis, indicating the importance of the mutated residues in PA-catalyzed acyl transfer kinetics. In several mutants at low substrate concentrations, the maximum level of ampicillin production was increased up to 1.5-fold, and the ratio of the synthetic rate over the hydrolytic rate was increased 5-15-fold. Moreover, due to increased tendency of the acyl-enzyme intermediate to react with beta-lactam nucleophile instead of water, mutants alphaR145G, alphaR145S and alphaR145L demonstrated an enhanced synthetic yield over wild-type PA at high substrate concentrations. This was accompanied by an increased conversion of 6-APA to ampicillin as well as a decreased undesirable hydrolysis of the acyl donor. Therefore, these mutants are interesting candidates for the enzymatic production of semi-synthetic beta-lactam antibiotics.

A prediction model for the demand of charging stations for electric vehicles in the municipality of Amsterdam

Connecting past, present and future

Using the past to orientate on the present and the future can be seen as one of history’s main contributions to educating future citizens of democratic societies. This thesis defines and explores aims and methods that may support teachers and students in making meaningful connections between the past, the present and the future in history class. Measurements with the Relevance of History Measurement Scale (RHMS), which was specifically developed for the purpose of this thesis, revealed that this type of history teaching positively affects students’ views on the relevance of history. This is an important outcome, because young students in particular have difficulty seeing the benefits of studying the past. Enabling them to see the relevance of history may be an important means to stimulate their motivation and engagement, because students’ appreciation of the value of school subjects is key to their commitment in school work.

Expanding the environmental scope

BackgroundIdentifying modifiable factors associated with well-being is of increased interest for public policy guidance. Developments in record linkage make it possible to identify what contributes to well-being from a myriad of factors. To this end, we link two large-scale data resources; the Geoscience and Health Cohort Consortium, a collection of geo-data, and the Netherlands Twin Register, which holds population-based well-being data.ObjectiveWe perform an Environment-Wide Association Study (EnWAS), where we examine 139 neighbourhood-level environmental exposures in relation to well-being.MethodsFirst, we performed a generalized estimation equation regression (N = 11,975) to test for the effects of environmental exposures on well-being. Second, to account for multicollinearity amongst exposures, we performed principal component regression. Finally, using a genetically informative design, we examined whether environmental exposure is driven by genetic predisposition for well-being.ResultsWe identified 21 environmental factors that were associated with well-being in the domains: housing stock, income, core neighbourhood characteristics, livability, and socioeconomic status. Of these associations, socioeconomic status and safety are indicated as the most important factors to explain differences in well-being. No evidence of gene-environment correlation was found.SignificanceThese observed associations, especially neighbourhood safety, could be informative for policy makers and provide public policy guidance to improve well-being. Our results show that linking databases is a fruitful exercise to identify determinants of mental health that would remain unknown by a more unilateral approach.

Enhancing a simulation-based Allocator Software with machine learning and genetic algorithms for improving the gate assignment problem

Assigning gates to flights considering physical, operational, and temporal constraints is known as the Gate Assignment Problem. This article proposes the novelty of coupling a commercial stand and gate allocation software with an off-the-grid optimization algorithm. The software provides the assignment costs, verifies constraints and restrictions of an airport, and provides an initial allocation solution. The gate assignment problem was solved using a genetic algorithm. To improve the robustness of the allocation results, delays and early arrivals are predicted using a random forest regressor, a machine learning technique and in turn they are considered by the optimization algorithm. Weather data and schedules were obtained from Zurich International Airport. Results showed that the combination of the techniques result in more efficient and robust solutions with higher degree of applicability than the one possible with the sole use of them independently.