Chemo-enzymatic epoxidation-process options for improving biocatalytic productivity

The reactor choice is crucial when designing a process where inactivation of the biocatalyst is a problem. The main bottleneck for the chemo-enzymatic epoxidation has been found to be enzyme inactivation by the hydrogen peroxide, H(2) O(2) , substrate. In the work reported here, the effect of reaction parameters on the reaction performance have been investigated and used to establish suitable operating strategies to minimize the inactivation of the enzyme, using rapeseed methyl ester (RME) as a substrate in a solvent-free system. The use of a controlled fed-batch reactor for maintaining H(2) O(2) concentration at 1.5 M resulted in increased productivity, up to 76 grams of product per gram of biocatalyst with higher retention of enzyme activity. Further investigation included a multistage design that separated the enzymatic reaction and the saturation of the RME substrate with H(2) O(2) into different vessels. This setup showed that the reaction rate as well as enzyme inactivation is strongly dependent on the H(2) O(2) concentration. A 20-fold improvement in enzymatic efficiency is required for reaching an economically feasible process. This will require a combination of enzyme modification and careful process design.     

COEVOLUTIONARY FEEDBACKS BETWEEN FAMILY INTERACTIONS AND LIFE HISTORY

Families with parental care show a parent–offspring conflict over the amount of parental investment. To date, the resolution of this conflict was modeled as being driven by either purely within‐brood or between‐brood competition. In reality the partitioning of parental resources within‐ versus between‐broods is an evolving life history trait, which can be affected by parent–offspring interactions. This coevolutionary feedback between life history and family interactions may influence the evolutionary process and outcome of parent–offspring coadaptation. We used a genetic framework for a simulation model where we allowed parental parity to coevolve with traits that determine parental investment. The model included unlinked loci for clutch size, parental sensitivity, baseline provisioning, and offspring begging. The simulation showed that tight coadaptation of parent and offspring traits only occurred in iteroparous outcomes whereas semelparous outcomes were characterized by weak coadaptation. When genetic variation in clutch size was unrestricted in the ancestral population, semelparity and maximal begging with poor coadaptation evolved throughout. Conversely, when genetic variation was limited to iteroparous conditions, and/or when parental sensitivity was treated as an evolutionarily fixed sensory bias, coadapted outcomes were more likely. Our findings show the influence of a feedback between parity, coadaptation, and conflict on the evolution of parent–offspring interactions.     

Model of Tryptophan Metabolism,Readily Scalable Using Tissue-specific Gene Expression Data

Tryptophan is utilized in various metabolic routes including protein synthesis, serotonin, and melatonin synthesis and the kynurenine pathway. Perturbations in these pathways have been associated with neurodegenerative diseases and cancer. Here we present a comprehensive kinetic model of the complex network of human tryptophan metabolism based upon existing kinetic data for all enzymatic conversions and transporters. By integrating tissue-specific expression data, modeling tryptophan metabolism in liver and brain returned intermediate metabolite concentrations in the physiological range. Sensitivity and metabolic control analyses identified expected key enzymes to govern fluxes in the branches of the network. Combining tissue-specific models revealed a considerable impact of the kynurenine pathway in liver on the concentrations of neuroactive derivatives in the brain. Moreover, using expression data from a cancer study predicted metabolite changes that resembled the experimental observations. We conclude that the combination of the kinetic model with expression data represents a powerful diagnostic tool to predict alterations in tryptophan metabolism. The model is readily scalable to include more tissues, thereby enabling assessment of organismal tryptophan metabolism in health and disease.     

The Structure of the Cytochrome P450cam–Putidaredoxin Complex Determined by Paramagnetic NMR Spectroscopy and Crystallography

Cytochrome P450cam catalyzes the hydroxylation of camphor in a complex process involving two electron transfers (ETs) from the iron-sulfur protein putidaredoxin. The enzymatic control of the successive steps of catalysis is critical for a highly efficient reaction. The injection of the successive electrons is part of the control system. To understand the molecular interactions between putidaredoxin and cytochrome P450cam, we determined the structure of the complex both in solution and in the crystal state. Paramagnetic NMR spectroscopy using lanthanide tags yielded 446 structural restraints that were used to determine the solution structure. An ensemble of 10 structures with an RMSD of 1.3 Å was obtained. The crystal structure of the complex was solved, showing a position of putidaredoxin that is identical with the one in the solution structure. The NMR data further demonstrate the presence of a minor state or set of states of the complex in solution, which is attributed to the presence of an encounter complex. The structure of the major state shows a small binding interface and a metal-to-metal distance of 16 Å, with two pathways that provide strong electronic coupling of the redox centers. The interpretation of these results is discussed in the context of ET. The structure indicates that the ET rate can be much faster than the reported value, suggesting that the process may be gated.     

Proteomic analysis of the Cyanophora paradoxa muroplast provides clues on early events in plastid endosymbiosis

Glaucophytes represent the first lineage of photosynthetic eukaryotes of primary endosymbiotic origin that diverged after plastid establishment. The muroplast of Cyanophora paradoxa represents a primitive plastid that resembles its cyanobacterial ancestor in pigment composition and the presence of a peptidoglycan wall. To attain insights into the evolutionary history of cyanobiont integration and plastid development, it would thus be highly desirable to obtain knowledge on the composition of the glaucophyte plastid proteome. Here, we provide the first proteomic analysis of the muroplast of C. paradoxa. Mass spectrometric analysis of the muroplast proteome identified 510 proteins with high confidence. The protein repertoire of the muroplast revealed novel paths for reduced carbon flow and export to the cytosol through a sugar phosphate transporter of chlamydial origin. We propose that C. paradoxa possesses a primordial plastid mirroring the situation in the early protoalga.     

Staphylococcal Phage 2638A endolysin is lytic for Staphylococcus aureus and harbors an inter-lytic-domain secondary translational start site

Staphylococcus aureus is a notorious pathogen highly successful at developing resistance to virtually all antibiotics to which it is exposed. Staphylococcal phage 2638A endolysin is a peptidoglycan hydrolase that is lytic for S. aureus when exposed externally, making it a new candidate antimicrobial. It shares a common protein organization with more than 40 other reported staphylococcal peptidoglycan hydrolases. There is an N-terminal M23 peptidase domain, a mid-protein amidase 2 domain (N-acetylmuramoyl-L-alanine amidase), and a C-terminal SH3b cell wall-binding domain. It is the first phage endolysin reported with a secondary translational start site in the inter-lytic-domain region between the peptidase and amidase domains. Deletion analysis indicates that the amidase domain confers most of the lytic activity and requires the full SH3b domain for maximal activity. Although it is common for one domain to demonstrate a dominant activity over the other, the 2638A endolysin is the first in this class of proteins to have a high-activity amidase domain (dominant over the N-terminal peptidase domain). The high activity amidase domain is an important finding in the quest for high-activity staphylolytic domains targeting novel peptidoglycan bonds.     

Viral lysis of Micromonas pusilla: impacts on dissolved organic matter production and composition

The viral mediated transformation of phytoplankton organic carbon to dissolved forms (“viral shunt”) has been suggested as a major source of dissolved organic carbon (DOC) in marine systems. Despite the potential implications of viral activity on the global carbon fluxes, studies investigating changes in the DOC composition from viral lysis is still lacking. Micromonas pusilla is an ecologically relevant picoeukaryotic phytoplankter, widely distributed in both coastal and oceanic marine waters. Viruses have been found to play a key role in regulating the population dynamics of this species. In this study we used axenic cultures of exponentially growing M. pusilla to determine the impact of viral lysis on the DOC concentration and composition, as estimated from lysate-derived production of transparent exopolymer particles (TEP) and two fractions of fluorescent dissolved organic matter (DOM): aromatic amino acids (excitation/emission; 280/320 nm; F(280/320)) and marine humic-like fluorescent DOM (320/410 nm; F(320/410)). DOC concentration increased 4.5 times faster and reached 2.6 times higher end concentration in the viral infected compared with the non-infected cultures. The production of F(280/320) and F(320/410) were 4.1 and 2.8 times higher in the infected cultures, and the elevated ratio between F(280/320) and F(320/410) in lysates suggested a higher contribution of labile (protein) components in viral produced DOM than in algal exudates. The TEP production was 1.8 times faster and reached a 1.5 times higher level in the viral infected M. pusilla culture compared with the non- infected cultures. The measured increase in both DOC and TEP concentrations suggests that viral lysis has multiple and opposite implications for the production and export processes in the pelagic ocean: (1) by releasing host biomass as DOC it decreases the organic matter sedimentation and promotes respiration and nutrient retention in the photic zone, whereas (2) the observed enhanced TEP production could stimulate particle aggregation and thus carbon export out of the photic zone.     

On-line identification of P-glycoprotein substrates by monitoring of extracellular acidification and respiration rates in living cells

The influence of P-glycoprotein (ABCB1) in drug resistance as well as drug absorption and disposition is an important factor to be considered during the development of new drugs. Thus, the early identification and exclusion of compounds showing a high affinity towards P-glycoprotein can help to select drug candidates. The aim of our study was to implement a label-free assay for the identification of P-glycoprotein substrates in living cells. For this approach, a multiparametric, chip-based sensor system was used to determine extracellular acidification, cell respiration and adhesion upon stimulation with P-glycoprotein substrates. Using L-MDR1 cells, a human P-glycoprotein overexpressing cell line, the influence of P-glycoprotein activity was determined for seven different compounds, demonstrating the applicability of the system for P-glycoprotein substrate identification. Effects were concentration dependent, as shown for the P-glycoprotein substrate verapamil, and were associated with cellular acidification and respiration. P-glycoprotein ATPase activation by verapamil could be described by a Michaelis-Menten type kinetic profile showing saturation at high substrate concentrations. The Michaelis-Menten constants K(M) were determined to be 0.92μM (calculated based on extracellular acidification) and 4.9μM (calculated based on cellular respiration). Control experiments using 100nM of the P-glycoprotein inhibitor elacridar indicated that the observed effects were related to P-glycoprotein ATPase activity. In contrast, wild-type LLC-PK1 cells not expressing P-glycoprotein were not responsive towards stimulation with different P-glycoprotein substrates. Summarizing these findings, the used microsensor system is a generic system suitable for the identification of P-glycoprotein substrates. In contrast to biochemical P-glycoprotein assays, activation of the drug efflux pump can be monitored on-line in living cells to identify P-glycoprotein substrates and to study the molecular mechanisms of adenosintriphosphate-dependent active transport.     

Skin Filling and Firming Activity of a Hyaluronic Acid Inducing Synthetic Tripeptide

International Journal of Peptide Research and Therapeutics - Aging of skin manifests in loss of volume and firming due to degradation of extracellular matrix components such as collagen and...