Engineering and assaying of cytochrome P450 biocatalysts

Cytochrome P450s constitute a highly fascinating superfamily of enzymes which catalyze a broad range of reactions. They are essential for drug metabolism and promise industrial applications in biotechnology and biosensing. The constant search for cytochrome P450 enzymes with enhanced catalytic performances has generated a large body of research. This review will concentrate on two key aspects related to the identification and improvement of cytochrome P450 biocatalysts, namely the engineering and assaying of these enzymes. To this end, recent advances in cytochrome P450 development are reported and commonly used screening methods are surveyed.     

Adsorption mechanism of arsenate by zirconyl-functionalized activated carbon

Arsenate [As(V)] and arsenite [As(III)] sorption at the solid-water interface of activated carbon impregnated with zirconyl nitrate (Zr-AC) was investigated using X-ray absorption spectroscopy (XAS) and surface complexation modeling. The XAS data at the Zr K-edge suggest that the structure of the zirconyl nitrate coating is built from chains of edge-sharing ZrO8 trigonal dodecahedra bound to each other through two double hydroxyl bridges. The 8-fold coordination of each Zr atom is completed by four O atoms, which share a bit less than the two theoretically possible bidentate nitrate groups. On impregnation, two of the O atoms may lose their nitrate group and be transformed to hydroxyl groups ready for binding to carboxylic or phenolic ligands at the AC surface. As K-edge XANES results showed the presence of only As(V) on adsorption regardless of the initial As oxidation state. Oxidation to As(V) is probably mediated by available carbon species on the AC surface as found by batch titration. Zr K-edge EXAFS data indicate that arsenate tetrahedra form monodentate mononuclear surface complexes with free hydroxyl groups of zirconyl dodecahedra, whereby each bidentate nitrate group is exchanged by up to two arsenate groups. The inner-sphere arsenate binding to the Zr-AC surface sites constrained with the spectroscopic results was used in the formulation of a surface complexation model to successfully describe the adsorption behavior of arsenate in the pH range between 4 and 12. The results suggest therefore that Zr-AC is an effective adsorbent for arsenic removal due to its high surface area and the presence of high affinity surface hydroxyl groups.     

Solid-Phase Synthesis and Purification of Protein–DNA Origami Nanostructures

We present a facile method for the combined synthesis and purification of protein-decorated DNA origami nanostructures (DONs). DONs bearing reductively cleavable biotin groups in addition to ligands for ligation of recombinant proteins are bound to magnetic beads. Protein immobilization is conducted with a large protein excess to achieve high ligation yields. Subsequent to cleavage from the solid support, pure sample solutions are obtained which are suitable for direct AFM analysis of occupation patterns. We demonstrate the method's utility using three different orthogonal ligation methods, the “halo-based oligonucleotide binder” (HOB), a variant of Halo-tag, the “SpyTag/SpyCatcher” (ST/SC) system, and the enzymatic “ybbR tag” coupling. We find surprisingly low efficiency for ST/SC ligation, presumably due to electrostatic repulsion and steric hindrance, whereas the ybbR method, despite its ternary nature, shows good ligation yields. Our method is particularly useful for the development of novel ligation methods and the synthesis of mechanically fragile DONs that present protein patterns for surface-based cell assays.     

On the Plasma Chemistry of an RF Discharge Containing Aluminium Tri‐Isopropoxide Studied by FTIR Spectroscopy

In Ar and Ar/N₂ radio frequency (RF) discharges with admixtures of aluminium tri‐isopropoxide (ATI) the fragmentation of this metal‐organic precursor was studied by means of Fourier Transform Infrared (FTIR) spectroscopy using an optical long path cell providing an optical length of l = 17.2 m. The experiments were performed in an asymmetric capacitively coupled process plasma at a frequency of f = 13.56 MHz and at pres‐sure values in the range of p = 1–10.5 Pa. The discharge power was chosen between P = 10–100 W. Using FTIR spectroscopy the evolution of the concentrations of ATI and of six stable molecules, CH₄, C₂H₂, C₂H₄, C₂H₆, CO and HCN, was monitored under flowing conditions at gas flows of Φ_total = 0.5–14.5 sccm in the discharge. The concentrations of the reaction products were measured tobe between 2 x 10¹² molecules cm^–3 as e.g. found for C₂H₄and C₂H₆, and 5 x 10¹³ molecules cm^–3, as e.g. in the case of CO. In the plasma a complete dissociation of the precursor ATI was found at a power value of about P = 80 W independent on the admixture of Ar or N₂. The fragmentation efficiency (F_E) of the reaction products which originate from the ATI molecules ranges between 0.2 and 4 x 10¹⁶ molecules J^–1 while the fragmentation rate (F_R) reached up to 2.5 x 10¹⁸ molecules s^–1. The multi component detection ability of the spectrometer served to analyse the carbon balance of the by‐product formation. For all experiments, the carbon balance never exceeded 25%. Therefore, in the plasmas the majority of the provided carbon is most likely deposited at the reactor walls or forms dust particles or higher molecular C_xH_y. The conversion efficiencies (C_E) of the produced molecular species ranges between 0.1 x 10¹⁵ molecules J^–1 for C₂H₄ and 5 x 10¹⁵ molecules J^–1 for C₂H₆ depending on the discharge conditions of the RF plasma. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Evaluation of the Performance of Small Diode Pumped UV Solid State (DPSS) Nd:YAG Lasers as New Radiation Sources for Atmospheric Pressure Laser Ionization Mass Spectrometry (APLI-MS)

The performance of a KrF^* bench top excimer laser and a compact diode pumped UV solid state (DPSS) Nd:YAG laser as photo-ionizing source in LC-APLI MS is compared. The commonly applied bench-top excimer laser, operating at 248 nm, provides power densities of the order of low MW/cm² on an illuminated area of 0.5 cm² (8 mJ/pulse, 5 ns pulse duration, beam waist area 0.5 cm², 3 MW/cm²). The DPSS laser, operating at 266 nm, provides higher power densities, however, on a two orders of magnitude smaller illuminated area (60 μJ/pulse, 1 ns pulse duration, beam waist area 2 × 10^–3 cm², 30 MW/cm²). In a common LC-APLI MS setup with direct infusion of a 10 nM pyrene solution, the DPSS laser yields a significantly smaller ion signal (0.9%) and signal to noise ratio (1.4%) compared with the excimer laser. With respect to the determined low detection limits (LODs) for PAHs of 0.1 fmol using an excimer laser, LODs in DPSS laser LC-APLI MS in the low pmol regime are expected. The advantages of the DPSS laser with respect to applicability (size, cost, simplicity) may render this light source the preferred one for APLI applications not focusing on ultimately high sensitivities. Furthermore, the impact of adjustable ion source parameters on the performance of both laser systems is discussed in terms of the spatial sensitivity distribution described by the distribution of ion acceptance (DIA) measurements. Perspectives concerning the impact on future APLI-MS applications are given.