Autodisplay for the co-expression of lipase and foldase on the surface of E. coli: washing with designer bugs

Background: Lipases including the lipase from Burkholderia cepacia are in a main focus in biotechnology research since many years because of their manifold possibilities for application in industrial processes. The application of Burkholderia cepacia lipase for these processes appears complicated because of the need for support by a chaperone, the lipase specific foldase. Purification and reconstitution protocols therefore interfere with an economic implementation of such enzymes in industry. Autodisplay is a convenient method to express a variety of passenger proteins on the surface of E. coli. This method makes subsequent purification steps to obtain the protein of interest unnecessary. If enzymes are used as passengers, the corresponding cells can simply be applied as whole cell biocatalysts. Furthermore, enzymes surface displayed in this manner often acquire stabilization by anchoring within the outer membrane of E. coli. Results: The lipase and its chaperone foldase from B. cepacia were co-expressed on the surface of E. coli via autodisplay. The whole cell biocatalyst obtained thereby exhibited an enzymatic activity of 2.73 mU mL-1 towards the substrate p-nitrophenyl palmitate when applied in an OD578 =1. Outer membrane fractions prepared from the same culture volume showed a lipase activity of 4.01 mU mL-1. The lipase-whole cell biocatalyst as well as outer membrane preparations thereof were used in a standardized laundry test, usually adopted to determine the power of washing agents. In this test, the lipase whole cell biocatalyst and the membrane preparation derived thereof exhibited the same lipolytic activity as the purified lipase from B. cepacia and a lipase preparation which is already applied in commercial washing agents. Conclusions: Co-expression of both the lipase and its chaperone foldase on the surface of E. coli yields a lipid degrading whole cell biocatalyst. Therefore the chaperone supported folding process, absolutely required for the lipolytic activity appears not to be hindered by surface display. Furthermore, the cells and the membrane preparations appeared to be stable enough to endure a European standard laundry test and show efficient fat removal properties herein.<br

Proof of concept for the simplified breakdown of cellulose by combining Pseudomonas putida strains with surface displayed thermophilic endocellulase, exocellulase and β-glucosidase

BACKGROUND: The production and employment of cellulases still represents an economic bottleneck in the conversion of lignocellulosic biomass to biofuels and other biocommodities. This process could be simplified by displaying the necessary enzymes on a microbial cell surface. Such an approach, however, requires an appropriate host organism which on the one hand can withstand the rough environment coming along with lignocellulose hydrolysis, and on the other hand does not consume the generated glucose so that it remains available for subsequent fermentation steps. RESULTS: The robust soil bacterium Pseudomonas putida showed a strongly reduced uptake of glucose above a temperature of 50 °C, while remaining structurally intact hence recyclable, which makes it suitable for cellulose hydrolysis at elevated temperatures. Consequently, three complementary, thermophilic cellulases from Ruminiclostridium thermocellum were displayed on the surface of the bacterium. All three enzymes retained their activity on the cell surface. A mixture of three strains displaying each one of these enzymes was able to synergistically hydrolyze filter paper at 55 °C, producing 20 μg glucose per mL cell suspension in 24 h. CONCLUSION: We could establish Pseudomonas putida as host for the surface display of cellulases, and provided proof-of-concept for a fast and simple cellulose breakdown process at elevated temperatures. This study opens up new perspectives for the application of P. putida in the production of biofuels and other biotechnological products.<br

Functional display of heterotetrameric human protein kinase CK2 on Escherichia coli: a novel tool for drug discovery

Background: Human protein kinase CK2 represents a novel therapeutic target for neoplastic diseases. Inhibitors are in need to explore the druggability and the therapeutic options of this enzyme. A bottleneck in the search for new inhibitors is the availability of the target for testing. Therefore an assay was developed to provide easy access to CK2 for discovery of novel inhibitors. Results: Autodisplay was used to present human CK2 on the surface of Escherichia coli. Heterotetrameric CK2 consists of two subunits, α and β, which were displayed individually on the surface. Co-display of CK2α and CK2β on the cell surface led to the formation of functional holoenzyme, as demonstrated by NaCl dependency of enzymatic activity, which differs from that of the catalytic subunit CK2α without β. In addition interaction of CK2α and CK2β at the cell surface was confirmed by co-immunoprecipitation assays. Surface displayed CK2 holoenzyme enabled an easy IC50 value determination. The IC50 values for the known CK2 inhibitors TBB and Silmitasertib were determined to be 50 and 3.3 nM, respectively. Conclusion: Surface-displayed CK2α and CK2β assembled on the cell surface of E. coli to an active tetrameric holoenzyme. The whole-cell CK2 autodisplay assay as developed is suitable for inhibition studies. Furthermore, it can be used to determine quantitative CK2 inhibition data such as IC50 values. In summary, this is the first report on the functional surface display of a heterotetrameric enzyme on E. coli.<br

Autoantibodies to aS1-Casein Are Induced by Breast-Feeding

Background: The generation of antibodies is impaired in newborns due to an immature immune system and reduced exposure to pathogens due to maternally derived antibodies and placental functions. During nursing, the immune system of newborns is challenged with multiple milk-derived proteins. Amongst them, caseins are the main constituent. In particular, human aS1-casein (CSN1S1) was recently shown to possess immunomodulatory properties. We were thus interested to determine if auto-antibodies to CSN1S1 are induced by breast-feeding and may be sustained into adulthood. Methods: 62 sera of healthy adult individuals who were (n = 37) or were not (n = 25) breast-fed against human CSN1S1 were investigated by a new SD (surface display)-ELISA. For cross-checking, these sera were tested for anti Epstein-Barr virus (EBV) antibodies by a commercial ELISA. Results: IgG-antibodies were predominantly detected in individuals who had been nursed. At a cut-off value of 0.4, the SDELISA identified individuals with a history of having been breast-fed with a sensitivity of 80% and a specificity of 92%. Under these conditions, 35 out of 37 sera from healthy donors, who where breast-fed, reacted positively but only 5 sera of the 25 donors who were not breast-fed. The duration of breast-feeding was of no consequence to the antibody reaction as some healthy donors were only short term breast-fed (5 days minimum until 6 weeks maximum), but exhibited significant serum reaction against human CSN1S1 nonetheless. Conclusion: We postulate that human CSN1S1 is an autoantigen. The antigenicity is orally determined, caused by breastfeeding, and sustained into adulthood

Benchmarking High-Field Few-Electron Correlation and QED Contributions in Hg⁷⁵⁺ to Hg⁷⁸⁺ Ions. II. Theory

Theoretical resonance energies for KLL dielectronic recombination into He-, Li-, Be-, and B-like Hg ions are calculated by various means and discussed in detail. We apply the multiconfiguration Dirac-Fock and the configuration interaction Dirac-Fock-Sturmian methods, and quantum electrodynamic many-body theory. The different contributions such as relativistic electron interaction, quantum electrodynamic contributions, and finite nuclear size and mass corrections are calculated and their respective theoretical uncertainties are estimated. Our final results are compared to experimental data from the preceding paper. The comparison of theoretical values with the experimental energies shows a good overall agreement for most transitions and illustrates the significance of relativistic electron interaction contributions including correlation, magnetic, and retardation effects and quantum electrodynamic corrections. A few discrepancies found in specific recombination resonances for initially Li- and Be-like Hg ions are pointed out, suggesting the need for further theoretical and experimental studies along these isoelectronic sequences

Autoantibodies to αS1-Casein Are Induced by Breast-Feeding

BACKGROUND: The generation of antibodies is impaired in newborns due to an immature immune system and reduced exposure to pathogens due to maternally derived antibodies and placental functions. During nursing, the immune system of newborns is challenged with multiple milk-derived proteins. Amongst them, caseins are the main constituent. In particular, human αS1-casein (CSN1S1) was recently shown to possess immunomodulatory properties. We were thus interested to determine if auto-antibodies to CSN1S1 are induced by breast-feeding and may be sustained into adulthood. METHODS: 62 sera of healthy adult individuals who were (n = 37) or were not (n = 25) breast-fed against human CSN1S1 were investigated by a new SD (surface display)-ELISA. For cross-checking, these sera were tested for anti Epstein-Barr virus (EBV) antibodies by a commercial ELISA. RESULTS: IgG-antibodies were predominantly detected in individuals who had been nursed. At a cut-off value of 0.4, the SD-ELISA identified individuals with a history of having been breast-fed with a sensitivity of 80% and a specificity of 92%. Under these conditions, 35 out of 37 sera from healthy donors, who where breast-fed, reacted positively but only 5 sera of the 25 donors who were not breast-fed. The duration of breast-feeding was of no consequence to the antibody reaction as some healthy donors were only short term breast-fed (5 days minimum until 6 weeks maximum), but exhibited significant serum reaction against human CSN1S1 nonetheless. CONCLUSION: We postulate that human CSN1S1 is an autoantigen. The antigenicity is orally determined, caused by breast-feeding, and sustained into adulthood

Non-Sequential Double Ionization of Ne in Intense Laser Pulses: A Coincidence Experiment

The dynamics of Neon double ionization by 25 fs, 1.0 PW/cm2 laser pulses at 795 nm has been studied in a many particle coincidence experiment. The momentum vectors of all ejected atomic fragments (electrons and ions) have been measured using combined electron and recoil-ion momentum spectroscopy. Electron emission spectra for double and single ionization will be discussed. In both processes the mean electron energies differ considerably and high energetic electrons with energies of more than 120 eV have been observed for double ionization. The experimental results are in qualitative agreement with the rescattering model

Separation of Recollision Mechanisms in Nonsequential Strong Field Double Ionization of Ar: The Role of Excitation Tunneling

Vector momentum distributions of two electrons created in double ionization of Ar by 25 fs, 0.25PW/cm2 laser pulses at 795 nm have been measured using a “reaction microscope.” At this intensity, where nonsequential ionization dominates, distinct correlation patterns are observed in the two-electron momentum distributions. A kinematical analysis of these spectra within the classical “recollision model” revealed an (e,2e)-like process and excitation with subsequent tunneling of the second electron as two different ionization mechanisms. This allows a qualitative separation of the two mechanisms demonstrating that excitation-tunneling is the dominant contribution to the total double ionization yield

Matching the photocurrent of perovskite/organic tandem solar modules by varying the cell width

Photocurrent matching in conventional monolithic tandem solar cells is achieved by choosing semiconductors with complementary absorption spectra and by carefully adjusting the optical properties of the complete top and bottom stacks. However, for thin film photovoltaic technologies at the module level, another design variable significantly alleviates the task of photocurrent matching, namely the cell width, whose modification can be readily realized by the adjustment of the module layout. Herein we demonstrate this concept at the experimental level for the first time for a 2T-mechanically stacked perovskite (FAPbBr3)/organic (PM6:Y6:PCBM) tandem mini-module, an unprecedented approach for these emergent photovoltaic technologies fabricated in an independent manner. An excellent Isc matching is achieved by tuning the cell widths of the perovskite and organic modules to 7.22 mm (PCEPVKT-mod= 6.69%) and 3.19 mm (PCEOPV-mod= 12.46%), respectively, leading to a champion efficiency of 14.94% for the tandem module interconnected in series with an aperture area of 20.25 cm2. Rather than demonstrating high efficiencies at the level of small lab cells, our successful experimental proof-of-concept at the module level proves to be particularly useful to couple devices with non-complementary semiconductors, either in series or in parallel electrical connection, hence overcoming the limitations imposed by the monolithic structure

Progress at the Heidelberg EBIT

Two years after the relocation of the Heidelberg EBIT, several experiments are already in operation. Spectroscopic measurements in the optical region have delivered the most precise reported wavelengths for highly charged ions, in the case of the forbidden transitions of Ar XIV and Ar XV. The lifetimes of the metastable levels involved in those transitions has been determined with an error of less than 0.2%. A new, fully automatized x-ray crystal spectrometer allows systematic measurements with very high precision and reproducibility. Absolute measurements of the Lyman series of H-like ions are currently underway. Dielectronic recombination studies have yielded information on rare processes, as two-electron-one photon transitions in Ar16+, or the interference effects between dielectronic and radiative recombination in Hg77+. The apparatus can now operate at electron beam currents of more than 500 mA, and energies up to 100 keV. A further beam energy increase is planned in the near future. Ions can be extracted from the trap and transported to external experiments. Up to 4 x 107 Ar16+ ions per second can be delivered to a 1 cm diameter target at 10 m distance. Charge-exchange experiments with U64+ colliding with a cold He atomic beam have been carried out, as well as experiments aiming at the optimization of the charge state distribution of the extracted via dielectronic recombination. Two new EBITs, currently in advanced state of construction in Heidelberg, will be used for experiments at the VUV free electron laser at TESLA (Hamburg) and for the charge breeding of short-lived radioactive isotopes at the TRIUMF ISAC facility