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The P450 cytochrome monooxygenase CYP102A1 from Bacillus megaterium, better known as P450BM3, is a heme‐thiolate enzyme that catalyzes the hydroxylation of numerous substrates. Many of the resulting products are of commercial interest to the pharmaceutical and fine chemical industries. Unlike most other P450 cytochromes, P450BM3 is both soluble and fused to its natural redox partner, which supplies the necessary electrons from NADPH to drive its reaction forward. However, the industrial use of this enzyme is limited by its poor stability and its expensive cofactor. In this work, we explore the effects of buffer formulation and temperature on the stability of wildtype and P450BM3 mutant R966D/W1046S as well as on the stability of nicotinamide cofactors NADPH, NADH, and the biomimetic cofactor N‐benzyl‐1,4‐dihydronicotinamide. We demonstrate that cofactor stability is more important to increase product yield than that of the enzyme. We also demonstrate that low temperatures enhance oxido‐reduction reactions coupling, thus resulting in an increase in the molar ratio of p‐nitrophenolate produced from 10‐pNCA per oxidized cofactor. Overall, the optimized reaction conditions lead to a 2 to 2.6‐fold increase in total product output when wildtype P450BM3 or R966D/W1046S mutant is used with either of these three aforementioned cofactors. [ABSTRACT FROM AUTHOR]

The interaction between the 20 natural amino acids and the surfaces of sphalerite, pyrite, and chalcopyrite was examined in the light of density functional theory (DFT) simulations. The electronic properties of the active functional groups and the equilibrium geometries of the sorbed amino acid species were characterized as a function the 65 species resulting from their (de)protonations. Covalent bonds are predicted between the surface metallic atoms and the carboxyl groups and, though to a lesser extent, with the side and main amino groups. Chemisorption as a determining factor in the uptake of amino acid species was assessed in terms of sorption affinities for the mineral surfaces. The largest affinities result from the carboxyl groups of the deprotonated species at intermediate pH, whereas the fully protonated amino acids in acidic media predict the lowest affinities. Affinity of amino acids towards the mineral surfaces followed the trend: pyrite >> chalcopyrite > sphalerite with speciation‐dependent decreasing sequence: ∼2.5 < pH < ∼9.5; pH > ∼9.5; pH < ∼2.5. By targeting the most suitable amino acid building blocks in bio‐sourced collectors, either high collecting powers or superior selectivities can be aimed at depending on pH for improved separation of sulphidic minerals in flotation processes. [ABSTRACT FROM AUTHOR]

Abstract Background Despite the powerful impact in recent years of gene expression markers like the green fluorescent protein (GFP) to link the expression of recombinant protein for selection of high producers, there is a strong incentive to develop rapid and efficient methods for isolating mammalian cell clones secreting high levels of marker-free recombinant proteins. Recently, a method combining cell colony growth in methylcellulose-based medium with detection by a fluorescently labeled secondary antibody or antigen has shown promise for the selection of Chinese Hamster Ovary (CHO) cell lines secreting recombinant antibodies. Here we report an extension of this method referred to as fluorescent labeling in semi-solid medium (FLSSM) to detect recombinant proteins significantly smaller than antibodies, such as IGF-E5, a 25 kDa insulin-like growth factor derivative. Results CHO cell clones, expressing 300 μg/ml IGF-E5 in batch culture, were isolated more easily and quickly compared to the classic limiting dilution method. The intensity of the detected fluorescent signal was found to be proportional to the amount of IGF-E5 secreted, thus allowing the highest producers in the population to be identified and picked. CHO clones producing up to 9.5 μg/ml of Tissue-Plasminogen Activator (tPA, 67 kDa) were also generated using FLSSM. In addition, IGF-E5 high-producers were isolated from 293SF transfectants, showing that cell selection in semi-solid medium is not limited to CHO and lymphoid cells. The best positive clones were collected with a micromanipulator as well as with an automated colony picker, thus demonstrating the method's high throughput potential. Conclusion FLSSM allows rapid visualization of the high secretors from transfected pools prior to picking, thus eliminating the tedious task of screening a high number of cell isolates. Because of its rapidity and its simplicity, FLSSM is a versatile method for the screening of high producers for research and industry.