Separation of enzymes from Candida boidinii crude extract by continuous flow zone electrophoresis.

Separation of the enzymes formate dehydrogenase, formaldehyde dehydrogenase and methanol oxidase from Candida boidinii crude extract has been explored using continuous flow zone electrophoresis in the VaP-22 and the scaled-up VaP-220 electrophoresis apparatus. Yields up to 95% and purification factors between 3 and 7 were obtained, together with separation of cell debris from the enzymes. Multiple injections of sample were used to obtain a protein throughput of 46.2 mg/h in the VaP-22. A tenfold higher throughput was achieved using the VaP-220. Correlation of the electrophoretic mobility in continuous flow zone electrophoresis with the elution behavior in ion-exchange chromatography confirmed the primary role of net surface charge in the separation of biological molecules. Proteins and enzymes with differences greater than 0.05 M elution molarities in ion-exchange chromatography can be separated. This corresponds to a preparative scale (mg/h or g/h) separation of proteins and enzymes whose difference in apparent electrophoretic mobility is greater than 0.70 x 10(-5) cm2/(V.s).     

Correlation of migration behavior in free-flow zone electrophoresis and electrophoretic titration curve

The correlation of electrophoretic migration behavior in free-flow zone electrophoresis (FFZE) and electrophoretic titration curve (ETC) has been explored. It is shown that the ETC of a protein or a mixture of proteins can be used to predict the fraction numbers at which those proteins elute in a preparative scale FFZE experiment. The ETC is a quick and effective way to choose optimal buffer conditions in FFZE. FFZE is employed to determine the isoelectric points (pI) of proteins whose pIs lie beyond the range of IEF 3-9 gels. It is found that separations in FFZE are governed by the net surface charge of the proteins.     

Long Distance Measurements up to 160 Å in the GroEL Tetradecamer Using Q‐Band DEER EPR Spectroscopy

Current distance measurements between spin‐labels on multimeric protonated proteins using double electron–electron resonance (DEER) EPR spectroscopy are generally limited to the 15–60 Å range. Here we show how DEER experiments can be extended to dipolar evolution times of ca. 80 μs, permitting distances up to 170 Å to be accessed in multimeric proteins. The method relies on sparse spin‐labeling, supplemented by deuteration of protein and solvent, to minimize the deleterious impact of multispin effects and substantially increase the apparent spin‐label phase memory relaxation time, complemented by high sensitivity afforded by measurements at Q‐band. We demonstrate the approach using the tetradecameric molecular machine GroEL as an example. Two engineered surface‐exposed mutants, R268C and E315C, are used to measure pairwise distance distributions with mean values ranging from 20 to 100 Å and from 30 to 160 Å, respectively, both within and between the two heptameric rings of GroEL. The measured distance distributions are consistent with the known crystal structure of apo GroEL. The methodology presented here should significantly expand the use of DEER for the structural characterization of conformational changes in higher order oligomers.     

Use ofK L a as a criterion for scaling up the inulinase fermentation process

Applied Biochemistry and Biotechnology - The scale-up of inulinase production in aerated cultures ofCandida kefyr DSM 70106 was studied, taking into account the criterion of maintainingK L a...     

Direct Observation of Dynamic Mechanical Regulation of DNA Condensation by Environmental Stimuli

Gene delivery is a promising way to treat hereditary diseases and cancer; however, there is little understanding of DNA:carrier complex mechanical properties, which may be critical for the protection and release of nucleic acids. We applied optical tweezers to directly measure single‐molecule mechanical properties of DNA condensed using 19‐mer poly‐L ‐lysine (PLL) or branched histidine–lysine (HK) peptides. Force–extension profiles indicate that both carriers condense DNA actively, showing force plateaus during stretching and relaxation cycles. As the environment such as carrier concentration, pH, and the presence of zinc ions changes, DNA:HK complexes showed dynamically regulated mechanical properties at multiple force levels. The fundamental knowledge from this study can be applied to design a mechanically tailored complex which may enhance transfection efficiency by controlling the stability of the complex temporally and spatially.     

Quantitative In Situ Studies of Dynamic Fracture in Brittle Solids Using Dynamic X-ray Phase Contrast Imaging

We demonstrate the use of X-ray phase contrast imaging with sub-microsecond temporal resolution to obtain quantitative visualization of dynamic fracture processes in brittle solids. We examine an amorphous solid (fused silica), a ceramic single crystal (single-crystal quartz), and a polycrystalline ceramic (boron carbide), in the form of single-edge notched specimens loaded using a three-point apparatus at nominal strain rates up to \(\sim \)800 s^?1. We observe that the crack tip speed for boron carbide is relatively independent of mode I stress intensity factor rate (\(\dot {K}_{\mathrm {I}}\)) for these rates of loading, while that of fused silica and single-crystal quartz increases with \(\dot {K}_{\mathrm {I}}\). Further, for the amorphous and single crystal cases, we observe the development of a crack tip instability in the form of crack branching as the crack tip speed approaches 45% of the Rayleigh wave speed. This suggests that strain-rate-dependent mechanisms contribute to crack branching. Such mechanisms may, in turn, affect the macroscopic fracture properties of these materials.     

Formulation of Zeeman modulation as a signal filter

The Bloch equation containing a Zeeman modulation field is solved analytically by treating the Zeeman modulation frequency as a perturbation. The absorption and dispersion signals at both 0 degrees and 90 degrees modulation phase are obtained. The solutions are valid to first order in the modulation frequency, but are otherwise valid for any value of modulation amplitude or microwave amplitude. A first order treatment of modulation frequency is shown to be a valid approximation over a wide range of typical experimental EPR conditions. The solutions derived from the Bloch equation suggest that the effect of over-modulation on first and second harmonic EPR spectra can be formulated as a mathematical filter that smoothes and broadens the under-modulated signal. The only adjustable filter parameter is a width that is equivalent to the applied peak-to-peak modulation amplitude. The true spin-spin and spin-lattice relaxation rates are completely determined from the under-modulated spectrum. The filters derived from the analytic solutions of the Bloch equation in the linear limit of modulation frequency are tested against numerical solutions of the Bloch equation that are valid for any modulation frequency to show their applicability. The filters are further tested using experimental EPR spectra. Experimental under-modulated spectra are mathematically filtered and compared with the experimental over-modulated spectra. The application of modulation filters to STEPR spectra is explored and limitations are discussed.