Heterologe Produktion basidiomycetischer Peptidasen zur Reduktion von Speisesalz in Lebensmitteln

In der Vergangenheit rückte das Thema eines überhöhten Salzkonsums und damit einhergehend verschiedener chronischer Erkrankungen in den Fokus der Wissenschaft. Enzyme wie Hydrolasen und speziell Peptidasen werden bereits industriell eingesetzt, da sie keine Cofaktoren benötigen, oft bei hohen pH-Werten stabil sind und eine breite Spaltspezifität haben. Eine neue Anwendung von Peptidasen könnte die Generierung von L-Arginyl-dipeptiden sein. Diese Dipeptide haben keinen Eigengeschmack, sind aber in der Lage, den vorhandenen Salzgeschmack zu intensivieren. Um diese Dipeptide zu generieren, sollten in der vorliegenden Arbeit arginylspezifische Peptidasen aus Basidiomyceten isoliert und identifiziert werden. Zunächst wurden verschiedene Peptidasegene aus Trametes versicolor, Phanerochaete chrysosporium und Schizophyllum commune isoliert und in Escherichia coli bzw. Komagataella phaffii exprimiert. Eine Serinpeptidase (ABB73029) aus P. chrysosporium sowie eine Aspartatpeptidase aus T. versicolor (EIW62808) wurden erfolgreich gereinigt. Aktivitätsassays zeigten, dass es sich bei der Aspartatpeptidase nicht um eine arginylspezifische Peptidase handelt, während die Schnittstellenanalyse für die Serinpeptidase aus P. chrysosporium auf eine Exopeptidase hindeutet. In einem zweiten Ansatz wurden 29 Basidiomyceten in Minimalmedium mit 1 % Gluten über einen Zeitraum von 24 Tagen kultiviert und hinsichtlich ihrer Endo- und Exopeptidaseaktivität untersucht. Von den 29 Pilzen wurden insgesamt fünf interessante Spezies mit arginylspezifischer Peptidaseaktivität ausgewählt, darunter Agrocybe aegerita, Fomitopsis pinicola, Flammulina velutipes, Hypholoma sublateritium und Pleurotus eryngii. Nach Reinigung des Kulturüberstandes mittels Größenausschlusschromatographie, wurden aktive Fraktionen mit dem Substrat Casein inkubiert und die Spaltprodukte analysiert. Nur für Peptidasen aus A. aegerita, F. velutipes und P. eryngii wurden Schnittstellen detektiert. Da neben arginylspezifischen Schnittstellen auch ein hoher Anteil unerwünschter Exo-peptidaseschnittstellen ermittelt wurde, handelt es sich bei den Enzymen vermutlich um Exopeptidasen anstatt arginylspezifische Peptidasen. Die Generierung salzgeschmackverstärkender Dipeptide sollte final mit Hilfe einer Dipeptidylpeptidase V (DPPV) aus Pleurotus floridanus erfolgen. PflDPPV wurde heterolog in E. coli produziert und zeigte Optima bei pH 8,5 und 60 °C gegenüber dem Substrat H-L-Ala-L-Ala-para-Nitroanilin. Zudem wurde die Aktivität durch keinen Peptidaseinhibitor vollständig inhibiert

Evaluation of the develoPPP.de programme

In the past two decades, development cooperation actors have launched wide-reaching approaches to strengthen cooperation with the private sector as an active partner in financing and implementing development projects. Development partnerships with the private sector are intended to pool public and private resources, making it possible to use business know-how and capital for economic and social development in partner countries. DEval has evaluated the develoPPP.de programme, the largest programme set up by the German Federal Ministry for Economic Cooperation and Development (BMZ) to promote such partnerships. The evaluation comprised document and literature analyses, a portfolio review of all develoPPP.de projects since 2009, expert interviews and company surveys as well as 12 comprehensive case studies in four countries. The data provide key findings with regard to the way in which the programme was steered and implemented, and its results and sustainability. The findings will be used to further develop the programme. They will also be used at policy and implementation level, and enable BMZ to comply with its accountability obligations

Country Portfolio Reviews: A tool for strategic portfolio analysis in German development cooperation

Background and objectives The 2030 Agenda is making new demands on international cooperation International development cooperation is frequently criticised for being of limited effectiveness, and therefore doing little to actually promote sustainable development. It is undisputed that shortcomings in policy and institutional frameworks in partner countries, and inefficient structures and processes in international cooperation, prevent development cooperation from being more effective. In particular, partner country institutions face the challenges created by the growing fragmentation of development cooperation. More and more stakeholders are getting involved in development cooperation and playing an active role in more and more projects. In some partner countries many donors operate in the same sectors instead of complementing each other, which makes the task of coordination more complex for partner governments. In many cases, donors' working structures also act as a constraint on more effective development cooperation. Existing projects and programmes are continued for political reasons or due to the self-interest of implementing organisations, rather than in response to changing contextual factors. This means that the decisions taken are not always evidence-based. These challenges often prevent development cooperation from responding coherently and effectively to current development needs in a partner country, and prevent governments in partner countries from assuming ownership of joint development projects. Since 2015 the 2030 Agenda has provided a guiding framework for action by international cooperation. Through it the international community has agreed on a new understanding of development as well as 17 Sustainable Development Goals (SDGs). It emphasises the alignment of international cooperation with partner country priorities and needs. The Agenda aims to achieve a holistic perspective on development challenges, and to take greater account of the interactions between the social, economic and environmental dimensions of development than has so far been the case. Against this background, ensuring the relevance and ultimately also the effectiveness of bilateral development cooperation will require strategic steps to be taken on various levels. This will involve focusing bilateral development cooperation as a whole (macro level), ensuring strategic alignment and coherence at country level (meso-level), and enhancing planning and management at programme and project level (micro level). The BMZ has responded to these new demands on international development cooperation With this very much in mind, over the last few years the German Federal Ministry for Economic Cooperation and Development (BMZ) has launched processes of structural change for the strategic planning and management of its bilateral development cooperation. Alongside efforts made by the BMZ at the macro level to focus bilateral development cooperation, an increasing number of changes are being implemented at the meso-level. As also emphasised by the strategy paper Entwicklungspolitik 2030 ['Development Policy in 2030' – currently only available in German] published by the BMZ in 2018, rather than pursuing a compartmentalised focus on individual programmes and projects, it is envisaged that portfolio management will now focus on integrated and holistic country-level approaches. These changes at the meso-level reflect the understanding of development inherent in the 2030 Agenda. Be it the coherent design of country portfolios, focusing on macro-level development needs and trends in the partner country, responding to current reform momentum and government priorities, or including interactions between the social, environmental and economic dimensions of development – needed management decisions cannot be taken at the level of individual projects. They can only be made at the portfolio level. Accordingly, the BMZ has been seeking to strengthen the coherence of country portfolios through country strategies ever since 2012

Entwicklungshelferinnen und Entwicklungshelfer: ein Personalinstrument der deutschen Entwicklungszusammenarbeit

Seit mehr als 50 Jahren werden Entwicklungshelferinnen und Entwicklungshelfer von staatlichen und nichtstaatlichen deutschen Trägern in alle Welt entsandt. Hat das Modell nach so vielen Jahren ausgedient, gerade angesichts grundlegender Veränderungen im globalen Süden? Diese Frage bildet den Ausgangspunkt der ersten institutionenübergreifenden Evaluierung der entwicklungspolitischen Wirksamkeit von EHs als Personalvermittlungsinstrument der deutschen Entwicklungszusammenarbeit

Cumulative mutagenesis of the basic residues in the 201-218 region of insulin-like growth factor (IGF)-binding protein-5 results in progressive loss of both IGF-I binding and inhibition of IGF-I biological action

We have reported previously that mutation of two conserved nonbasic amino acids (G203 and Q209) within the highly basic 201–218 region in the C-terminal domain of IGF-binding protein-5 (IGFBP-5) decreases binding to IGFs. This study reveals that cumulative mutagenesis of the 10 basic residues in this region, to create the C-Term series of mutants, ultimately results in a 15-fold decrease in the affinity for IGF-I and a major loss in heparin binding. We examined the ability of mutants to inhibit IGF-mediated survival of MCF-7 cells and were able to demonstrate that this depended not only upon the affinity for IGF-I, but also the kinetics of this interaction, because IGFBP-5 mutants with similar affinity constants (KD) values, but with different association (Ka) and dissociation (Kd) rate values, had markedly different inhibitory properties. In contrast, the affinity for IGF-I provided no predictive value in terms of the ability of these mutants to enhance IGF action when bound to the substratum. Instead, these C-Term mutants appeared to enhance the actions of IGF-I by a combination of increased dissociation of IGF-IGFBP complexes from the substratum, together with dissociation of IGF-I from IGFBP-5 bound to the substratum. These effects of the IGFBPs were dependent upon binding to IGF-I, because a non-IGF binding mutant (N-Term) was unable to inhibit or enhance the actions of IGF-I. These results emphasize the importance of the kinetics of association/dissociation in determining the enhancing or inhibiting effects of IGFBP-5 and demonstrate the ability to generate an IGFBP-5 mutant with exclusively IGF-enhancing activity

Specific amino acid substitutions determine the differential contribution of the N- and C-terminal domains of insulin-like growth factor (IGF)-binding protein-5 in binding IGF-I

We have previously reported that two highly conserved amino acids in the C-terminal domain of rat insulin-like growth factor-binding protein (IGFBP)-5, Gly203 and Gln209, are involved in binding to insulin-like growth factor (IGF)-1. Here we report that mutagenesis of both amino acids simultaneously (C-Term mutant) results in a cumulative effect and an even greater reduction in IGF-I binding: 30-fold measured by solution phase IGF binding assay and 10-fold by biosensor analysis. We compared these reductions in ligand binding to the effects of specific mutations of five amino acids in the N-terminal domain (N-Term mutant), which had previously been shown by others to cause a very large reduction in IGF-I binding (1). Our results confirm this as the major IGF-binding site. To prove that the mutations in either N- or C-Term were specific for IGF-I binding, we carried out CD spectroscopy and showed that these alterations did not lead to gross conformational changes in protein structure for either mutant. Combining these mutations in both domains (N+C-Term mutant) has a cumulative effect and leads to a 126-fold reduction in IGF-I binding as measured by biosensor. Furthermore, the equivalent mutations in the C terminus of rat IGFBP-2 (C-Term 2) also results in a significant reduction in IGF-I binding, suggesting that the highly conserved Gly and Gln residues have a conserved IGF-I binding function in all six IGFBPs. Finally, although these residues lie within a major heparin-binding site in IGFBP-5 and -3, we also show that the mutations in C-Term have no effect on heparin binding