Cloning, overexpression, and characterization of a thermoactive nitrilase from the hyperthermophilic archaeon Pyrococcus abyssi.

Four open reading frames encoding putative nitrilases were identified in the genomes of the hyperthermophilic archaea Pyrococcus abyssi, Pyrococcus horikoshii, Pyrococcus furiosus, and Aeropyrum pernix (growth temperature 90-100 degrees C). The nitrilase encoding genes were cloned and overexpressed in Escherichia coli. Enzymatic activity could only be detected in the case of Py.

pHluorin-based in vivo assay for hydrolase screening.

pHluorin, a pH-sensitive mutant of green fluorescent protein (GFP), acts as a sensor for intracellular pH shifts, triggered by hydrolytic enzymes. This principle was used to develop a pHluorin-based in vivo assay for hydrolase screening. The presented assay was evaluated for Escherichia coli (E.

Oxygen transfer phenomena in 48-well microtiter plates: determination by optical monitoring of sulfite oxidation and verification by real-time measurement during microbial growth.

Oxygen limitation is one of the most frequent problems associated with the application of shaking bioreactors. The gas-liquid oxygen transfer properties of shaken 48-well microtiter plates (MTPs) were analyzed at different filling volumes, shaking diameters, and shaking frequencies. On the one hand, an optical method based on sulfite oxidation was used as a chemical model system to determine the maximum oxygen transfer capacity (OTR(max)).

The first aminoacylase-catalyzed enantioselective synthesis of aromatic beta-amino acids.

The first aminoacylase-catalyzed enantioselective synthesis of aromatic beta-amino acids is reported. The presence of an N-chloroacetyl group as acyl group in the substrate as well as the use of porcine kidney acylase I as a suitable enzyme component are prerequisites for this resolution process whereby optically active beta-amino acids are formed with high enantioselectivities of >98% ee.

Purification and properties of an enantioselective and thermoactive amidase from the thermophilic actinomycete Pseudonocardia thermophila.

A constitutively expressed thermoactive amidase from the thermophilic actinomycete Pseudonocardia thermophila was purified to homogeneity by applying hydrophobic interaction, anion exchange and gel filtration chromatography, giving a yield of 26% and a specific activity of 19.5 units mg(-1). The purified enzyme has an estimated molecular mass of 108 kDa and an isoelectric point of 4.

Rapid evaluation of oxygen and water permeation through microplate sealing tapes.

Eight commercially available microplate sealing tapes and 10 other suitable materials (transparent wound dressings) are compared qualitatively in terms of their ability to minimize water evaporation from a multiwell plate while maintaining the oxygen supply as high as possible, which is necessary for applications like aerobic growth. The transparency and sterility of the products are considered as well. All evaluated commercially available sealing tapes fall into one of the following two classes: (1) O(2) transfer is comparable to that of an unsealed plate, but water vapor retention is relatively low, or (2) O(2) transfer via the sealing is slower, but the water retention capability is comparably high.

Base-Catalyzed Synthesis of N-(2-Arylethyl)anilines and Base-Promoted Domino Synthesis of 2,3-Dihydroindoles.

The base is the key to the new one-pot reaction, which leads to pharmacologically interesting 2,3-dihydroindoles. The use of potassium tert-butylalcoholate enables the selective addition of primary anilines to substituted styrenes and a new domino reaction, consisting of a hydroamination and an aryne cyclization (see scheme).