Asymmetric synthesis of d-glyceric acid by an alditol oxidase and directed evolution for enhanced oxidative activity towards glycerol

Glycerol as a by-product of biodiesel production is an attractive precursor for producing d-glyceric acid. Here, we demonstrate the successful production of d-glyceric acid based on glycerol via glyceraldehyde in a two-step enzyme reaction with the FAD-dependent alditol oxidase from Streptomyces coelicolor A3(2). The hydrogen peroxide generated in the reaction can be used in detergent, food, and paper industry. In order to apply the alditol oxidase in industry, the enzyme was subjected to protein engineering. Different strategies were used to enhance the substrate specificity towards glycerol. Initial attempts based on rational protein design in the active site region were found unsuccessful to increase activity. However, through directed evolution, an alditol oxidase double mutant (V125M/A244T) with 1.5-fold improved activity for glycerol was found by screening 8,000 clones. Further improvement of activity was achieved by combinatorial experiments, which led to a quadruple mutant (V125M/A244T/V133M/G399R) with 2.4-fold higher specific activity towards glycerol compared to the wild-type enzyme. Through studying the effects of mutations created, we were able to understand the importance of certain amino acids in the structure of alditol oxidase, not only for conferring enzymatic structural stability but also with respect to their influence on oxidative activity.     

Beyond diversity: functional microbiomics of the human colon

Molecular tools have revealed wide microbial diversity in the human alimentary tract. Most intestinal microorganisms have not been cultured and the in situ functions of distinct groups of the intestinal microbiota are largely unknown but pivotal to understanding the role of these microorganisms in health and disease. Promising strategies to gain more insight into the functionality of the complex microbial communities in the human alimentary tract, including fermentation processes in the colon, are discussed. These research approaches could provide a basis for the definition of a healthy gut based on key properties of microbial functionality. This will also enable the development of direct nutritional strategies for intestinal disease prevention and health promotion.     

Listeriolysin O as cytotoxic component of an immunotoxin

Monoclonal antibodies (mAbs) have been developed over the past years as promising anticancer therapeutics. The conjugation of tumor specific mAbs with cytotoxic molecules has been shown to improve their efficacy dramatically. These bifunctional immunotoxins, consisting of covalently linked antibodies and protein toxins, possess considerable potential in cancer therapy. Many of them are under investigation in clinical trials. As a result of general interest in new toxic components, we describe here the suitability of the bacterial protein Listeriolysin O (LLO) as cytotoxic component of an immunotoxin. Unique characteristics of LLO, such as its acidic pH optimum and the possibility to regulate the cytolytic activity by cysteine‐oxidation, make LLO an interesting toxophore. Oxidized LLO shows a substantially decreased cytolytic activity when compared with the reduced protein as analyzed by hemolysis. Both oxidized and reduced LLO exhibit a cell‐type‐unspecific toxicity in cell culture with a significantly higher toxicity of reduced LLO. For cell‐type‐specific targeting of LLO to tumor cells, LLO was coupled to the dsFv fragment of the monoclonal antibody B3, which recognizes the tumor‐antigen Lewis Y. The coupling of LLO to dsFv‐B3 was performed via cysteine‐containing polyionic fusion peptides that act as a specific heterodimerization motif. The novel immunotoxin B3‐LLO could be shown to specifically eliminate antigen positive MCF7 cells with an EC₅₀ value of 2.3 nM, whereas antigen negative cell lines were 80‐ to 250‐fold less sensitive towards B3‐LLO.     

Long-term performance and microbial community analysis of a full-scale synthesis gas fed reactor treating sulfate- and zinc-rich wastewater

The performance of a full-scale (500 m³) sulfidogenic synthesis gas fed gas-lift reactor treating metal- and sulfate-rich wastewater was investigated over a period of 128 weeks. After startup, the reactor had a high methanogenic activity of 46 Nm³·h⁻¹. Lowering the carbon dioxide feed rate during the first 6 weeks gradually lowered the methane production rate. Between weeks 8 and 93, less than 1% of the hydrogen supplied was used for methanogenesis. Denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified 16S rRNA gene fragments showed that the archaeal community decreased in diversity but did not disappear completely. After the carbon dioxide feed rate increased in week 88, the methane production rate also increased, confirming that methane production was carbon dioxide limited. Even though lowering the carbon dioxide feed appeared to affect part of the sulfate-reducing community, it did not prevent achieving the desired rates of sulfate reduction. The average sulfate conversion rate was 181 kg∙h⁻¹ for the first 92 weeks. After 92 weeks, the sulfate input rate was increased and from week 94 to 128, the average weekly sulfate conversion rate was 295 kg·h⁻¹ (SD ± 87). Even higher sulfate conversion rates of up to 400 kg·h⁻¹ could be sustained for weeks 120–128. The long-term performance and stability together with the ability to control methanogenesis demonstrates that synthesis gas fed reactor can be used successfully at full scale to treat metal and sulfate-rich wastewater.     

Myelin Proteomics: Molecular Anatomy of an Insulating Sheath

Fast-transmitting vertebrate axons are electrically insulated with multiple layers of nonconductive plasma membrane of glial cell origin, termed myelin. The myelin membrane is dominated by lipids, and its protein composition has historically been viewed to be of very low complexity. In this review, we discuss an updated reference compendium of 342 proteins associated with central nervous system myelin that represents a valuable resource for analyzing myelin biogenesis and white matter homeostasis. Cataloging the myelin proteome has been made possible by technical advances in the separation and mass spectrometric detection of proteins, also referred to as proteomics. This led to the identification of a large number of novel myelin-associated proteins, many of which represent low abundant components involved in catalytic activities, the cytoskeleton, vesicular trafficking, or cell adhesion. By mass spectrometry-based quantification, proteolipid protein and myelin basic protein constitute 17% and 8% of total myelin protein, respectively, suggesting that their abundance was previously overestimated. As the biochemical profile of myelin-associated proteins is highly reproducible, differential proteome analyses can be applied to material isolated from patients or animal models of myelin-related diseases such as multiple sclerosis and leukodystrophies.     

Photonic crystal nanostructures for optical biosensing applications

We present the design, fabrication and optical investigation of photonic crystal (PhC) nanocavity drop filters for use as optical biosensors. The resonant cavity mode wavelength and Q-factor are studied as a function of the ambient refractive index and as a function of adsorbed proteins (bovine serum albumin) on the sensor surface. Experiments were performed by evanescent excitation of the cavity mode via a PhC waveguide. This in turn is coupled to a ridge waveguide that allows the introduction of a fluid flow cell on a chip. A response of ∂λ/∂c=(4.54±0.66)×10⁵ nm/M is measured leading to a measured detection limit as good as  fg or  pg/mm²in the sensitive area.     

Host-specific symbiotic requirement of BdeAB, a RegR-controlled RND-type efflux system in Bradyrhizobium japonicum

Multidrug efflux systems not only cause resistance against antibiotics and toxic compounds but also mediate successful host colonization by certain plant-associated bacteria. The genome of the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum encodes 24 members of the family of resistance/nodulation/cell division (RND) multidrug efflux systems, of which BdeAB is genetically controlled by the RegSR two-component regulatory system. Phylogenetic analysis of the membrane components of these 24 RND-type transporters revealed that BdeB is more closely related to functionally characterized orthologs in other bacteria, including those associated with plants, than to any of the other 23 paralogs in B. japonicum. A mutant with a deletion of the bdeAB genes was more susceptible to inhibition by the aminoglycosides kanamycin and gentamicin than the wild type, and had a strongly decreased symbiotic nitrogen-fixation activity on soybean, but not on the alternative host plants mungbean and cowpea, and only very marginally on siratro. The host-specific role of a multidrug efflux pump is a novel feature in the rhizobia-legume symbioses. Consistent with the RegSR dependency of bdeAB, a B. japonicum regR mutant was found to have a greater sensitivity against the two tested antibiotics and a symbiotic defect that is most pronounced for soybean.     

Effect of collagen I and fibronectin on the adhesion, elasticity and cytoskeletal organization of prostate cancer cells

The adipokine adiponectin circulates in high concentration, and activates the classical pathway of complement by binding C1q, leading to the activation of C3 and formation of the membrane attack complex. Such behaviour is potentially pathophysiological. However, we showed adiponectin captured the complement inhibitor Factor H both as a pure protein and from human serum. Both heparin and a homologue of C3b, substrates binding to the C-terminus of Factor H, were inhibitory of the interaction, as was EDTA. Factor H bound equivalently to high and low molecular weight serum adiponectin, and to an N-terminal 16 kDa cyanogen bromide cleavage product of adiponectin. The binding of Factor H inhibited both the C3 and C5 convertases generated from complement activation by adiponectin, so reducing potentially pathophysiological consequences such as the deposition of C5b-9, while allowing opsonisation of target molecules with C3b.     

Isolation of RNA from bacterial samples of the human gastrointestinal tract

The human gastrointestinal (GI) tract contains a complex microbial community that consists of numerous uncultured microbes. Therefore, nucleic-acid-based approaches have been introduced to study microbial diversity and activity, and these depend on the proper isolation of DNA, rRNA and mRNA. Here, we present an RNA isolation protocol that is suitable for a wide variety of GI tract samples. The procedure for isolating DNA from GI tract samples is described in another Nature Protocols article. One of the benefits of our RNA isolation protocol is that sampling can be performed outside the laboratory, which offers possibilities for implementation in large intervention studies. The RNA isolation is based on mechanical disruption, followed by isolation of nucleic acids using phenol:chloroform:isoamylalcohol extraction and removal of DNA. In our laboratory, this protocol has resulted in the isolation of rRNA and mRNA of sufficient quality and quantity for microbial diversity and activity studies. Depending on the number of samples, the sample type and the quenching procedure chosen, the whole procedure can be performed within 2.5-4 h.