Redirected evolution of a proline-based tautomerase:New tools for carboligation reactions

Enzymes are highly specialized catalysts, used by all living organisms to catalyze chemical reactions necessary to sustain life. Although enzymes in the course of evolution became highly specialized catalysts towards one specific chemical reaction, many enzymes are capable of catalyzing one or more unnatural reactions. These promiscuous reactions can be highly interesting, especially when the product of a promiscuous reaction forms a facile building block for the synthesis of particular pharmaceuticals. The focus of this thesis is the enzyme 4-oxalocrotonate tautomerase, which is capable of catalyzing several highly interesting carbon-carbon bond-forming reactions. The products of these reactions can be used to synthesize gamma-aminobutyric-acid analogues (GABA analogues), a highly important class of pharmaceuticals. Via extensive mutagenesis and by means of a novel color-based screenings method, enzyme variants were identified that were able to catalyze these reactions much quicker and moreover would almost exclusively synthesize the desired enantiomer. These improved enzyme variants were combined with other enzymes and a chemical catalyst in one pot, showing that all steps in this novel synthesis route could be performed without intermediate purification. Although this synthesis route needs to be further improved before it can be applied industrially, it highlights the exciting opportunities available to search for new enzyme variants and develop new sustainable catalysts that could applied in the synthesis of pharmaceuticals

Is de vrijloopstal haalbaar in Nederland?

Vrijloopstallen zijn stallen, waarin de koe vrij kan rondlopen. Ze hebben geen ligboxen. De koeien lopen en liggen op een zachte bodem. De stal biedt kansen voor het dierenwelzijn en de diergezondheid. In het buitenland zijn ervaringen met bodems van compost, zand en gedroogde mest. Voor het Productschap Zuivel en het ministerie van LNV is een haalbaarheidsstudie gedaan naar de mogelijkheden in Nederlan

In Situ Acetaldehyde Synthesis for Carboligation Reactions

The enzyme 4-oxalocrotonate tautomerase (4-OT) can promis-cuously catalyze various carboligation reactions using acetalde-hyde as a nucleophile. However, the highly reactive nature ofacetaldehyde requires intricate handling, which can impede itsusage in practical synthesis. Therefore, we investigated threeenzymatic routes to synthesize acetaldehyde in situ in one-potcascade reactions with 4-OT. Two routes afforded practicalacetaldehyde concentrations, using an environmental pollu-tant,trans-3-chloroacrylic acid, or a bio-renewable, ethanol, asstarting substrate. These routes can be combined with 4-OTcatalyzed Michael-type additions and aldol condensations inone pot. This modular systems biocatalysis methodology pro-vides a stepping stone towards the development of larger arti-ficial metabolic networks for the practical synthesis of impor-tant chemical synthons

Prediction of outcome in autoimmune hepatitis and variant syndromes

In general the prognosis of treated autoimmune hepatitis is comparable to the general population. The knowledge on risk factors predicting long term survival is still limited. Better knowledge on risk factors can lead to tailored treatment of patients in the future, preventing side effects in patients currently overtreated and preventing disease progression in patients currently undertreated.At diagnosis age, ethnicity, cirrhosis and ALT level were the main predictors for long-term survival. During treatment ALT level but not IgG level was associated with long-term survival. In patients presenting with acute or acute severe AIH improvement of liver function within 2 weeks after start of treatment was the main predictor for survival. In patients with AIH-PBC variant syndrome more patients than the guidelines indicate are treated with a combination of immunosupression and ursodeoxycholic acid.New treatment options are urgently needed in AIH which requires a better understanding of changes in the immune system. To our suprise complement did not play a role in autoimmune hepatitis. Using B-cell related cytokines, patients could be divided into immunological subgroups with each a different presentation, treatment response and outcome.LUMC / Geneeskund

Corporate social responsibility of African and Middle East mobile operators towards climate change and the potential impact of its carbon footprint

Research report, presented to the SBL Unisa, Midrand.The current and future anticipated changes in the earth’s climate are a concern that has captured business’s and governments’ global attention. Climate change and its potential impacts cannot be ignored as there is ample evidence that global warming is indeed the result of anthropogenic greenhouse gas emissions. The mobile operator in Africa and the Middle East (ME) operates on continents and in parts of the world, predicted by scientists as the most vulnerable to the effects of climate change. The mobile operator in Africa and the Middle East is moreover an emitter of significant amounts of CO2 and this exacerbates the serious environmental climate change problem that humankind faces. This research paper addresses the Corporate Social Responsibility of African and Middle East (ME) mobile operators, and its Carbon Footprint. The main objectives of the research are to identify strategic risks and opportunities and the implications for the mobile operator and to determine its Greenhouse Gas emissions. The performance against targets and plans to reduce GHG emissions are also reviewed. The research is based on the questionnaire of the Carbon Disclosure Project (CDP) initiative. A shortened and modified version of the CDP was designed and emailed to two major mobile telecom operators both operating in Africa and the Middle East. It is postulated that the telecommunications industry is at an inflection point where significant changes must take place in the way energy requirements are managed. This in turn could have a positive effect on reducing its carbon footprint, benefit corporate reputation and at the same time earn “green miles” in the subscriber’s minds. The research reached the main conclusion that the mobile operators’ investigated do not yet have strategies, systems and reporting in place to be counted as “good corporate citizens” concerning their environmental responsibility. The research further concluded that a proactive strategic intent is a necessity to achieve this goal. In short: The Corporate Social Responsibility of African and Middle East mobile operators indeed has a positive effect on its Carbon Footprint.Graduate School of Business LeadershipM.B.L

Moderne huisvesting melkvee

Brochure met praktische adviezen om de melkveehouder op een overzichtelijke en prikkelende manier te informeren over een goede huisvesting van melkvee waarmee het welzijn van de koeien verbeterd kan worden. Er wordt nader ingegaan op regelgeving en subsidies, erfinrichting, de uitvoering van de ligboxenstal, het melken en stalsysteme

High-techbedrijf naar vrij koeverkeer

Koeien op het high-techbedrijf blijven het hele jaar binnen, het dierenwelzijn in de stal krijgt daarom extra aandacht. Op het bedrijf zijn er de afgelopen jaren verschillende vormen van koeverkeer toegepast, waaronder zowel vrij koeverkeer als gestuurd koeverkeer. De laatste jaren is gewerkt met gestuurd koeverkeer en een selectie-unit. Begin april 2003 is op het high-techbedrijf een nieuwe proef gestart met vrij koeverkeer. De eerste resultaten zijn goed te noemen het aantal melkingen blijft net als de productie op peil

Smaakstof verbetert bezoekgedrag niet

De koeien op het High-techbedrijf blijven het hele jaar binnen. In de afgelopen jaren was er zowel vrij als gestuurd koeverkeer. In april 2003 startten we hier een proef met vrij koeverkeer. Daarin keken we ook naar het effect van toedienen van smaakstof in het automatisch melksysteem op het bezoek. We kunnen echter geen effect aantonen

Selective Colorimetric “Turn-On” Probe for Efficient Engineering of Iminium Biocatalysis

The efficient engineering of iminium biocatalysis has drawn considerable attention, with many applications in pharmaceutical synthesis. Here, we report a tailor-made iminium-activated colorimetric "turn-on" probe, specifically designed as a prescreening tool to facilitate engineering of iminium biocatalysis. Upon complexation of the probe with the catalytic Pro-1 residue of the model enzyme 4-oxalocrotonate tautomerase (4-OT), a brightly colored merocyanine-dye-type structure is formed. 4-OT mutants that formed this brightly colored species upon incubation with the probe proved to have a substantial activity for the iminium-based Michael-type addition of nitromethane to cinnamaldehyde, whereas mutants that showed no staining by the probe exhibited no or very low-level "Michaelase" activity. This system was exploited in a solid-phase prescreening assay termed as activated iminium colony staining (AICS) to enrich libraries for active mutants. AICS prescreening reduced the screening effort up to 20-fold. After two rounds of directed evolution, two artificial Michaelases were identified with up to 39-fold improvement in the activity for the addition of nitromethane to cinnamaldehyde, yielding the target γ-nitroaldehyde product with excellent isolated yield (up to 95%) and enantiopurity (up to >99% ee). The colorimetric activation of the turn-on probe could be extended to the class I aldolase 2-deoxy-d-ribose 5-phosphate aldolase, implicating a broader application of AICS in engineering iminium biocatalysis