Identification of Mechanism-Based Inactivation in P450-Catalyzed Cyclopropanation Facilitates Engineering of Improved Enzymes

Following the recent discovery that heme proteins can catalyze the cyclopropanation of styrenyl olefins with high efficiency and selectivity, interest in developing new enzymes for a variety of non-natural carbene transfer reactions has burgeoned. The fact that diazo compounds and other carbene precursors are known mechanism-based inhibitors of P450s, however, led us to investigate if they also interfere with this new enzyme function. We present evidence for two inactivation pathways that are operative during cytochrome P450-catalyzed cyclopropanation. Using a combination of UV–vis, mass spectrometry, and proteomic analyses, we show that the heme cofactor and several nucleophilic side chains undergo covalent modification by ethyl diazoacetate (EDA). Substitution of two of the affected residues with less-nucleophilic amino acids led to a more than twofold improvement in cyclopropanation performance (total TTN). Elucidating the inactivation pathways of heme protein-based carbene transfer catalysts should aid in the optimization of this new biocatalytic function

Embracing open innovation to acquire external ideas and technologies and to transfer internal ideas and technologies outside

The objective of this dissertation is to increase understanding of how organizations can embrace open innovation in order to acquire external ideas and technologies from outside the organization, and to transfer internal ideas and technologies to outside the organization. The objective encompasses six sub-objectives, each addressed in one or more substudies. Altogether, the dissertation consists of nine substudies and a compendium summarizing the substudies. An extensive literature review was conducted on open innovation and crowdsourcing literature (substudies 1–4). In the subsequent empirical substudies, both qualitative research methods (substudies 5–7) and quantitative research methods (substudies 8–9) were applied. The four literature review substudies provided insights on the body of knowledge on open innovation and crowdsourcing. These substudies unveiled most of the influential articles, authors, and journals of open innovation and crowdsourcing disciplines. Moreover, they identified research gaps in the current literature. The empirical substudies offer several insightful findings. Substudy 5 shows how non-core ideas and technologies of a large firm can become valuable, especially for small firms. Intermediary platforms can find solutions to many pressing problems of large organizations by engaging renowned scientists from all over world (substudy 6). Intermediary platforms can also bring breakthrough innovations with novel mechanisms (substudy 7). Large firms are not only able to garner ideas by engaging their customers through crowdsourcing but they can also build long-lasting relations with their customers (substudies 8 and 9). Embracing open innovation brings challenges for firms too. Firms need to change their organizational structures in order to be able to fully benefit from open innovation. When crowdsourcing is successful, it produces a very large number of new ideas. This has the consequence that firms need to allocate a significant amount of resources in order to identify the most promising ideas. In an idea contest, customarily, only one or a few best ideas are rewarded (substudy 7). Sometimes, no reward is provided for the selected idea (substudies 8 and 9). Most of the ideas that are received are not implemented in practice

Properties, production, and applications of camelid single-domain antibody fragments

Camelids produce functional antibodies devoid of light chains of which the single N-terminal domain is fully capable of antigen binding. These single-domain antibody fragments (VHHs or Nanobodies®) have several advantages for biotechnological applications. They are well expressed in microorganisms and have a high stability and solubility. Furthermore, they are well suited for construction of larger molecules and selection systems such as phage, yeast, or ribosome display. This minireview offers an overview of (1) their properties as compared to conventional antibodies, (2) their production in microorganisms, with a focus on yeasts, and (3) their therapeutic applications

Exomorfología y anatomía de órganos vegetativos aéreos en especies de Flourensia DC. (Asteraceae) con importancia fitoquímica

El género Flourensia DC. es americano y comprende 32 especies. Son arbustos resinosos utilizados en medicina popular; poseen un potencial valor económico, por lo que, en algunas especies representantes, se han caracterizado sus compuestos químicos y determinado algunos posibles usos. En el presente trabajo, se estudió la exomorfología y anatomía de órganos vegetativos de F. hirta S. F. Blake, F. leptopoda S. F. Blake, F. niederleinii S. F. Blake y F. tortuosa Griseb., especies endémicas del centro de Argentina. Se observó que todas son similares en cuanto a la anatomía de hoja y tallo. En cuanto al estudio de la epidermis foliar, se concluye que es posible diferenciar las especies teniendo en cuenta la frecuencia estomática y la presencia de tricomas. Finalmente, las diferencias más notables se encontraron en la exomorfología foliar ya que se observó una gran variación en cuanto a la forma de las hojas y tamaño de las láminas y pecíolos. Con respecto a las estructuras secretoras, se encontraron tricomas y conductos secretores esquizógenos en las hojas y los tallos de todas las especies, las cuales serían responsables de la producción y secreción de las resinas.The genus Flourensia DC. is American and comprises 32 species. They are resiniferous shrubs used in folk medicine. As they have potential economic value, its chemical compounds have been characterized and some possible uses identified. In this work, the exomorphology and anatomy of vegetative organs of F. hirta S. F. Blake, F. leptopoda S. F. Blake, F. niederleinii S. F. Blake and F. tortuosa Griseb., endemic species from central Argentina, have been studied. All species are similar in leaf and stem anatomy. Regarding the epidermis, it is possible to differentiate the species according to the stomatal frequency and the presence of trichomes. Finally, the most remarkable differences were found in leaf exomorphology, since there was a large variation in shape and size of blades and petioles. Concerning the secretory structures, trichomes and secretory ducts were found in the leaves and stems of all species, which would be responsible for the production and secretion of the resins