Application of surface response analysis to the optimization of penicillin acylase purification in aqueous two-phase systems

Penicillin acylase purification from an Escherichia coli crude extract using PEG 3350 – sodium citrate aqueous two phase systems was optimized. An experimental design was used to evaluate the influence of PEG, sodium citrate and sodium chloride on the purification parameters. A central composite design was defined centred on the previously found conditions for highest purification from an osmotic shock extract. Mathematical models for the partition coefficient of protein and enzyme, balance of protein and enzyme, yield and purification were calculated and statistically validated. Analysis of the contours of constant response as a function of PEG and sodium citrate concentrations for three different concentrations of NaCl revealed different effects of the three factors on the studied parameters. A maximum purification factor of 6.5 was predicted for PEG 3350, Sodium Citrate and NaCl concentrations of 15.1%, 11.0% and 8.52% respectively. However under these conditions the predicted yield was 61%. A better compromise between these two parameters can be found by superimposing the contour plots of the purification factor and yield for 10.3% NaCl. A region in the experimental space can be defined where the purification factor is always higher than 5.5 with yields exceeding 80%

Partial purification of penicillin acylase from Escherichia Coli in poly(ethylene glycol)-sodium citrate aqueous two-phase systems

Studies on the partition and purification of penicillin acylase from osmotic shock extract Escherichia coli were performed in poly (ethylene glycol)- citrate systems. Both partition behavior of the enzyme and total protein are similar to those described in other reports increasing ,with pH and tie-line length and decreasing with PEG molecular weight . However, some selectivity could be attained with PEG 1000 systems and long tie-line at pH 6.9. In these conditions 2.6 fold purification with 83% yield were achieved. Influence of pH on partition shows that is the composition of the system and not the net charge of the enzyme that determine the behaviour in these conditions. Addition of NaCl to PEG 3350 systems significantly increases the partition of the enzyme. Althought protein partition also increased, purification conditions were possible with 1.5 M NaCl where 5.7 fold purification and 85% yield was obtained. This was possible due to the higher hydrophobicity of the enzyme compared to that of most of contaminants proteins

Production of 6-aminopenicillanic acid in aqueous two-phase systems by recombinant Escherichia coli with intracellular penicillin acylase

Bioconversion of Penicillin G in PEG 20000-Dextran T 70 aqueous two-phase systems was achieved using the recombinant Escherichia coli A56 (ppA22) with intracellular penicillin acylase as catalyst. The best conversion conditions were attained for: 7%(w/v) substrate (penicillin G), enzyme activity in bottom phase 52 U/ml, pH 7.8, temperature 37°C, reaction time 40 min. Five repeated batches could be performed in these conditions. Conversions ratios between 0.902-0.985mol of 6-aminopenicillanic acid (6-APA) per mol of penicillin G, were obtained and specific productivity was 3.6-4.6 μmol/min•ml. In addition the product 6-APA could directly be crystallized from the top phase with a purity of 96.2%.Science & Technological Commission of Shanghai Municipal People’s Government

Synthesis and H+, Cu2+, and Zn2+ Coordination Behavior of a Bis(fluorophoric) Bibrachial Lariat Aza-Crown

The synthesis, protonation behavior, and Cu2+ and Zn2+ coordination chemistry of the novel bibrachial aza lariat ether (naphthalen-1-ylmethyl)[2-(20-{2-[(naphthalen-1-ylmethyl)amino]ethyl}-3,6,9,17,20,23,29,30-octaazatricyclo[23.3.1.1*11,15*]triaconta-1(29),11(30),12,14,25,27-hexaen-6-yl)ethyl]amine (L) are discussed. The macrocycle, which has two aminoethyl naphthyl moieties symmetrically appended to a 2:2 azapyridinophane structure, displays, in the pH range 2−11, six protonation steps that correspond to the protonation of the secondary amino groups. Steady-state fluorescence measurements show emissions due to the monomer and to the excimer formed between the two naphthalene fragments of the macrocycle. The time-resolved fluorescence data, obtained by the time-correlated single photon counting technique, show that a significant percentage of excimer is preformed as ground-state dimers. The ligand L forms with the metal ions Cu2+ and Zn2+ mono- and dinuclear complexes in aqueous solution. The influence of metal coordination in the fluorescence emission of L is analyzed. The acid−base, coordination capabilities, and emissive behavior of L are compared with those presented by its synthetic precursor L1, which has a tripodal tris(2-aminoethyl)amine structure functionalized at one of its terminal amino groups with a naphthyl moiet

Long Range Electron Transfer Quenching in Polyamine Chains Bearing a Terminal Naphthalene Unit

The fluorescence emission of a naphthalene unit attached to a polyamine chain is quenched by intramolecular electron transfer from the deprotonated amines to the excited fluorophore. Measurements of the respective quenching rate constants as a function of the distance, reveal an exponential dependence with β = 0.45 Å-1. Identical measurements carried out in deuterated water have shown a similar dependence with the distance β = 0.49 Å-1 but an average reduction of the absolute values of the rate constants of ca. 1.2. The polyamine chains seem to constitute a bridge through which the electron can find a route to its movement, more efficiently than through space

Ulocuplumab (BMS-936564 / MDX1338): a fully human anti-CXCR4 antibody induces cell death in chronic lymphocytic leukemia mediated through a reactive oxygen species-dependent pathway.

The CXCR4 receptor (Chemokine C-X-C motif receptor 4) is highly expressed in different hematological malignancies including chronic lymphocytic leukemia (CLL). The CXCR4 ligand (CXCL12) stimulates CXCR4 promoting cell survival and proliferation, and may contribute to the tropism of leukemia cells towards lymphoid tissues. Therefore, strategies targeting CXCR4 may constitute an effective therapeutic approach for CLL. To address that question, we studied the effect of Ulocuplumab (BMS-936564), a fully human IgG4 anti-CXCR4 antibody, using a stroma--CLL cells co-culture model. We found that Ulocuplumab (BMS-936564) inhibited CXCL12 mediated CXCR4 activation-migration of CLL cells at nanomolar concentrations. This effect was comparable to AMD3100 (Plerixafor--Mozobil), a small molecule CXCR4 inhibitor. However, Ulocuplumab (BMS-936564) but not AMD3100 induced apoptosis in CLL at nanomolar concentrations in the presence or absence of stromal cell support. This pro-apoptotic effect was independent of CLL high-risk prognostic markers, was associated with production of reactive oxygen species and did not require caspase activation. Overall, these findings are evidence that Ulocuplumab (BMS-936564) has biological activity in CLL, highlight the relevance of the CXCR4-CXCL12 pathway as a therapeutic target in CLL, and provide biological rationale for ongoing clinical trials in CLL and other hematological malignancies

In situ AFM observations of mineral replacement reactions on sulphate and carbonate surfaces

Depto. de Mineralogía y PetrologíaFac. de Ciencias GeológicasTRUEpu

Optical and photovoltaic properties of thieno[3,2-b]thiophene-based push–pull organic dyes with different anchoring groups for dye-sensitized solar cells

The effect of anchoring groups on the optical and electrochemical properties of triphenylamine-thienothiophenes, and on the photovoltaic performance of DSSCs photosensitized with the prepared dyes, was studied using newly synthesized compounds with cyanoacetic acid or rhodanine-3-acetic acid groups. Precursor aldehydes were synthesized through Suzuki cross-coupling, whereas Knoevenagel condensation of these with 2-cyanoacetic acid or rhodanine-3-acetic acid afforded the final push–pull dyes. A comprehensive photophysical study was performed in solution and in the solid state. The femtosecond time-resolved transient absorption spectra for the synthesized dyes were obtained following photoexcitation in solution and for the dyes adsorbed to TiO2 mesoporous films. Information on conformation, electronic structure, and electron distribution was obtained by density functional theory (DFT) and time-dependent DFT calculations. Triphenylamine–thienothiophene functionalized with a cyanoacetic acid anchoring group displayed the highest conversion efficiency (3.68%) as the dye sensitizer in nanocrystalline TiO2 solar cells. Coadsorption studies were performed for this dye with the ruthenium-based N719 dye, and they showed dye power conversion efficiencies enhanced by 20–64%. The best cell performance obtained with the coadsorbed N719 and cyanoacetic dye showed an efficiency of 6.05%.The authors thank Fundacao para a Ciencia e Tecnologia (FCT) for PhD grants to S.S.M.F. (SFRH/BD/87786/2012) and M.C.R.C. (SFRH/BD/78037/2011) and postdoctoral grants to L.L.G.J. (SFRH/BPD/97026/2013) and J.P. (SFRH/BPD/108469/2015). The authors also thank FEDER-COMPETE for the financial support through the CQ/UM PEst-C/QUI/UI0686/2013 (FCOMP-01-0124-FEDER-037302). The NMR spectrometer Bruker AVANCE III 400 is part of the National NMR Network and was purchased within the framework of the National Program for Scientific Re-equipment, contract REDE/1517/RMN/2005 with funds from POCI 2010 (FEDER) and FCT. The group in Coimbra are grateful for funding from "Centro de Quimica de Coimbra," which is supported by the Fundacao para a Ciencia e a Tecnologia (FCT), Portuguese Agency for Scientific Research, through the programs UID/QUI/UI0313/2013 and COMPETE. The authors also thank the Laboratory for Advanced Computing at the University of Coimbra for providing computing resources that have contributed to the research results reported in this paper (URL http://www.lca.uc.pt). This work was performed under the project "SunStorage. harvesting and storage of solar energy,"with reference POCI-01-0145-FEDER-016387, funded by the European Regional Development Fund (ERDF), through COMPETE 2020. Operational Programme for Competitiveness and Internationalization (OPCI), and by national funds through FCT. The research leading to these results has received funding from Laserlab-Europe (grant agreement no. 284464, EC's Seventh Framework Programme). A.I.P. would like to acknowledge the European Commission through the Seventh Framework Programme, the Specific Programme "Ideas" of the European Research Council for research and technological development as part of an Advanced Grant under grant agreement no. 321315, which also partially funded this work. The authors would also like to acknowledge L. Andrade and I. Mesquita for their valuable contribution for the article.info:eu-repo/semantics/publishedVersio

Prediction of Children\u27s Early Academic Adjustment from Their Temperament: The Moderating Role of Peer Temperament

The goal of the study was to examine whether target children’s temperamental negative emotional expressivity (NEE) and effortful control in the fall of kindergarten predicted academic adjustment in the spring and whether a classmate’s NEE and effortful control moderated these relations. Target children’s NEE and effortful control were measured in the fall via multiple methods, academic adjustment was measured via reading and math standardized tests in the spring, and observations of engagement in the classroom were conducted throughout the year. In the fall, teachers nominated a peer with whom each target child spent the most time and rated that peer’s temperament. Target children with high effortful control had high reading and math achievement (ps = .04 and \u3c .001, respectively), and children with low NEE increased in engagement during the year (p \u3c .001). Peers’ temperament did not have a direct relation to target children’s academic adjustment. Peers’ NEE, however, moderated the relation between target children’s effortful control, as well as NEE, and changes in engagement (ps = .03 and .05, respectively). Further, peers’ effortful control moderated the relations between target children’s NEE and reading and changes in engagement (ps = .02 and .04, respectively). In each case, target children’s temperament predicted the outcome in expected directions more strongly when peers had low NEE or high effortful control. Results are discussed in terms of how children’s temperamental qualities relate to academic adjustment, and how the relation between NEE and changes in engagement, in particular, depends on peers’ temperament