RESUMEN
Aim: The main purpose of this work was to develop new protocols for high yield purification of secretory phospholipase A2 (PLA2) and to investigate its biophysical properties.Materials and methods: We have used a Pichia pastoris expression system for PLA2 expression and two-stage chromatography for its purification. The biophysical properties of PLA2 were investigated by circular dichroism.Results: A scalable method for high yield purification of recombinant Streptomyces violaceruber PLA2 was developed. The PLA2 from S. violaceruber was expressed in the methylotrophic yeast P. pastoris. Functional active phospholipase A2 with specific activity 73 U/mg was purified with a concentration of at least 3 mg/mL. The role of different divalent ions in PLA2 thermostability were evaluated. Ca2+ and Ba2+ ions significantly increased thermostability of the enzyme.
Asunto(s)
Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Fosfolipasas A2/aislamiento & purificación , Fosfolipasas A2/metabolismo , Pichia/genética , Pichia/metabolismo , Streptomyces/enzimología , Streptomyces/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Bario/química , Calcio/química , Cationes Bivalentes/química , Cromatografía/métodos , Dicroismo Circular/métodos , Genes Bacterianos , Concentración de Iones de Hidrógeno , Fosfolipasas A2/química , Fosfolipasas A2/genética , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , TemperaturaRESUMEN
We describe the results of our investigations of the generation of a ribbon electron beam (10 × 220 mm2) by a two-stage discharge system based on a hollow-cathode glow discharge plasma. The source design enables operation in the pressure range 2 × 10-2 to 10 Pa. At a beam accelerating voltage of 8 kV, the beam current is 450 mA at a pressure of 2 × 10-2 Pa and 150 mA at a pressure of 10 Pa. To achieve a uniform current density distribution of the beam over its cross-sectional area, a special design of emission electrode was employed. This enabled us to reduce non-uniformities of the beam current density distribution to a level of 10%.
RESUMEN
We describe a plasma-cathode electron beam source based on a hollow cathode glow discharge and operating in the forevacuum pressure range that produces a steady-state ribbon beam. The electron beam is generated in the pressure range of 10-30 Pa. A multi-aperture electron extraction and beam formation system is used to provide beam stability and enhanced uniformity of beam current density, allowing the use of this kind of device for beam-plasma surface modification over relatively large areas.
RESUMEN
We describe a plasma-cathode electron beam source based on a hollow-cathode discharge that is capable of generating a 9 kW dc electron beam at an accelerating voltage of 20 kV, with helium as a working gas at a pressure of 30 Pa. A test run of â¼50 operational hours did not indicate any significant degradation of the electron source extraction system or other structural components, and we estimate the operational lifetime of the source at about 100-120 h.
RESUMEN
We present the results of our investigations of magnetic focusing of the electron beam generated by a plasma-cathode electron source in the forevacuum pressure range (10-30 Pa). We show that a magnetic double-focusing system employing two separate field coils with the main magnetic coil located close to the beam collector at the focal plane provides effective and efficient focusing of the electron beam. With our e-beam source, this focusing system produces a power density of more than 1 MW/cm2 at the electron beam focus with an accelerating voltage of 30 kV and a beam current up to 60 mA. For comparison, the maximum beam power density provided by plasma-cathode electron sources at pressures of less than 0.1 Pa is at the level of 10 MW/cm2.