Optimization of critical medium components for the maximal production of gentamicin by Micromonospora echinospora ATCC 15838 using response surface methodology

Optimization of the fermentation medium components for maximum gentamicin production by Micromonospora echinospora ATCC 15838 was carried out. Response surface methodology was applied to optimize the medium constituents. A 2⁴full-factorial central composite design was chosen to explain the combined effects of the four medium constituents, viz. starch, soyabean meal, K₂HPO₄, and CoCl₂ and to design a minimum number of experiments. A second order model was developed and fitted using least square method. The R ² value of the model was 0.9723, which shows that model is best fit for the present studies. The results of analysis of variance and regression of a second order model showed that the linear effects of starch (p<0.001697) and CoCl₂(p<7.99E-13), and cross product effects of starch and soyabean meal (p<0.029876) and soyabean meal and CoCl₂ (p<0.008909) were more significant, suggesting that these were critical variables having the greatest effect on the production of gentamicin in the production medium. The optimized medium consisting of 9 g/L starch, 3 g/L soyabean meal, 0.9 g/L K₂HPO₄, and 0.01 g/L CoCL₂ predicted 850 mg/L of gentamicin which was almost 110% higher than that of the unoptimized medium. The amounts of starch, soyabean meal, and K₂HPO₄ required were also reduced with RSM.     

Optimized chromatographic and bioluminescent methods for inorganic pyrophosphate based on its conversion to ATP by firefly luciferase

Two new methods for inorganic pyrophosphate (PPi) quantification are described. They are based on the enzymatic conversion of PPi into ATP by firefly luciferase (Luc, E.C. 1.13.12.7) in the presence of dehydroluciferyl-adenylate (L-AMP) followed by the determination of ATP by one of two different procedures, either UV-monitored (260 nm) ion-pair-HPLC (IP-HPLC) (method A) or luciferase-dependent bioluminescence in the presence of its substrate, firefly luciferin (d-LH₂) (method B). These methods were subjected to optimization using experimental design methodologies to obtain optimum values for the selected factors: method A—incubation time (t_inc = 15 min), inactivation time of the enzyme (t_inac = 2 min), pH of the reaction mixture (pH 7.50) and the concentrations of L-AMP ([L-AMP] = 40 μM) and luciferase ([Luc] = 0.1 μM); method B—concentrations of L-AMP ([L-AMP] = 2 μM), luciferase ([Luc] = 50 nM) and luciferin ([LH₂] = 30 μM). Method A has a linear response over the range of 0.1-20 μM of PPi, with a limit of detection (LOD) of 0.5 μM and a limit of quantitation (LOQ) of 1.8 μM. Precision, expressed as relative standard deviation (R.S.D.), is 7.4% at 1 μM PPi and 5.9% at 8 μM PPi. Method B has a linear response over the range of 0.75-6.0 μM of PPi, with LOD and LOQ of 0.624 and 2.23 μM, respectively, and a R.S.D. of 5.1% at 2.5 μM PPi and 4.9% at 5 μM PPi. Under optimized conditions sensitive and robust methods can be obtained for the analysis of PPi impurities in commercial nucleotides and tripolyphosphate (P₃).     

Taguchi OA16 orthogonal array design for the optimization of cloud point extraction for selenium determination in environmental and biological samples by tungsten-modified tube electrothermal atomic absorption spectrometry

Orthogonal array design (OAD) was applied for the first time to optimize cloud point extraction (CPE) conditions for Se(IV) determination by electrothermal atomic absorption spectrometry (ETAAS) in environmental and biological samples. Selenium was reacted with o-phenylenediamine to form piazselenol in an acidic medium (pH 2). Using Triton X-114, as surfactant, piazselenol was quantitatively extracted into small volume (about 30 μL) of the surfactant-rich phase after centrifugation. Five relevant factors, i.e. surfactant concentration, pH, ionic strength, equilibrium time and temperature were selected and the effects of each factor were studied at four levels on the extraction efficiency of Se(IV) and optimized. The statistical analysis revealed that the most important factors contributing to the extraction efficiency are ionic strength, pH and percent of surfactant. Based on the results obtained from the analysis of variance (ANOVA), the optimum conditions for extraction were established as: pH 6; vial temperature = 50 °C; extraction time = 7 min and 0.3% (w/v) of Triton X-114. The method was permitted to obtain a detection limit of 0.09 ng mL⁻¹ and two linear calibration ranges from 0.6 to 1.0 and 1.0 to 80.0 ng mL⁻¹ Se. The precision (%RSD) of the extraction and determination for the six replicates of Se at 20 ng mL⁻¹ was better than 3.6% and the enrichment factor of 63.5 was achieved. The studied analyte was successfully extracted and determined with high efficiency using cloud point extraction method in water and biological matrices.     

Silver(I) Complexes with a Bulky Anthracene‐Based Dicarboxylic Ligand: Syntheses,Crystal Structures,and Luminescent Properties

To explore the influence of anthracene skeleton with a larger conjugated π‐system on the structures and properties of its complexes, two Ag^I–carboxylate complexes based on anthracene‐9,10‐dicarboxylate (L) were synthesized and characterized: [{[Ag(L)][Ag(dmpy)₂]}_∞] ( 1 ) and [{[Ag₂(L)(bipy)₂]}_∞] ( 2 ) (dmpy = 2,6‐dimethylpyridine and bipy = 4,4′‐bipyridine). Complex 1 has an interesting framework consisting of anionic chains {[Ag(L)]^–}_∞ and the mononuclear cationic units [Ag(dmpy)₂]⁺, which is further assembled to form networks along the different crystallographic directions by the intermolecular C–H ··· Ag hydrogen‐bonding interactions. Complex 2 takes a ladder‐like chain structure by incorporating 4,4′‐bipyridine (bipy) as a bridging co‐ligand, which is further interlinked to generate a planar network through interchain Ag–Ag bonding contacts. The steric bulk of anthracene ring in L may play an important role in the formation of 1 and 2 . Moreover, the luminescent properties of the 1 and 2 were investigated in detail.     

Binding Mechanisms in Supramolecular Complexes

Forces to reckon with : Supramolecular complexes, such as the one shown, are normally based on a combination of different interactions such as ion pairing, hydrogen bonds, and stacking interactions. The not always simple characterization of the nature and strength of intermolecular forces provides assistance to the understanding of biomimetic systems, as well as for the design of synthetic receptors, drugs, and intelligent materials.

     

Designed Topology and Site‐Selective Metal Composition in Tetranuclear [MM′⋅⋅⋅M′M] Linear Complexes

The ligand 1,3‐bis[3‐oxo‐3‐(2‐hydroxyphenyl)propionyl]benzene (H₄L), designed to align transition metals into tetranuclear linear molecules, reacts with M^II salts (M=Ni, Co, Cu) to yield complexes with the expected [MM???MM] topology. The novel complexes [Co₄L₂(py)₆] ( 2 ; py=pyridine) and [Na(py)₂][Cu₄L₂(py)₄](ClO₄) ( 3 ) have been crystallographically characterised. The metal sites in complexes 2 and 3 , together with previously characterised [Ni₄L₂(py)₆] ( 1 ), favour different coordination geometries. These have been exploited for the deliberate synthesis of the heterometallic complex [Cu₂Ni₂L₂(py)₆] ( 4 ). Complexes 1 , 2 , 3 and 4 exhibit antiferromagnetic interactions between pairs of metals within each cluster, leading to S=0 spin ground states, except for the latter cluster, which features two quasi‐independent S=1/2 moieties within the molecule. Complex 4 gathers the structural and physical conditions, thus allowing it to be considered as prototype of a two‐qbit quantum gate.     

Determination of selected polychlorinated biphenyls in water samples by ultrasound-assisted emulsification-microextraction and gas chromatography-mass-selective detection

Ultrasound-assisted emulsification-microextraction (USAEME) procedure was developed for the determination of selected polychlorinated biphenyls (PCBs) in 10 mL of water samples by gas chromatography-mass-selective detection. After determination of the most suitable solvent and extraction time, several other parameters including solvent volume, centrifugation time and ionic strength of the sample were optimized using a 2³ factorial experimental design. The optimized USAEME procedure used 200 μL of chloroform as extraction solvent, 10 min of extraction with no ionic strength adjustment at 25 °C and 5 min of centrifugation at 4000 rpm. The limits of detection ranged from 14 ng L⁻¹ (for PCB153) to 30 ng L⁻¹ (for PCB101). Recoveries of PCBs from fortified distilled water are over 80% for three different fortification levels between 0.1 and 5 μg L⁻¹ and relative standard deviations of the recoveries are below 10%. The performance of the proposed method was compared with those involving traditional liquid-liquid extraction (LLE) and solid phase extraction (SPE) on the real water samples (i.e., tap and well water as well as domestic and industrial wastewaters, etc.) and comparable efficiencies were obtained. The proposed USAEME procedure has been demonstrated to be viable, simple, rapid and easy to use for residue analysis of PCBs in water samples.     

Allosteric Switches: Remote Controls for Proteins

Long‐distance service : Finding a selective small‐molecule modulator for every domain of every human protein is a Herculean task. Wouldn't it be much easier to control the activity of any protein of choice by simply fusing it with a generally adaptable switch protein and controlling protein activity indirectly?

     

Development and validation of an ultra-high-performance liquid chromatography coupled to time-of-flight mass spectrometry method to quantify benzoic acid and long-chain monocarboxylic acids (C12–C28) in atmospheric aerosols

An ultra-high-performance liquid chromatography/time-of-flight mass spectrometry (with negative ion electrospray ionization) methodology was developed for the simultaneous quantification of benzoic acid and 15 long-chain monocarboxylic acids (MCAs) in ambient aerosols. A fast and quantitative pressurized fluid extraction procedure was optimized using experimental design and the extracts were analyzed without any further clean-up step. Chromatographic separation was achieved on a BEH-C18 column with a mobile phase consisting of 5 mM formic acid in water/acetonitrile (90:10, v/v) and methanol. Excellent precision and accuracies in the MS mass measurements were observed. The method was validated using actual samples spiked with a solution containing either standards or ¹³C-surrogates. Matrix effects were observed for the C₁₄ MCA only. It was then applied to air particulate reference materials and atmospheric samples collected by a low-volume sampler.     

Statistical mixture design development of digestion methods for Oyster tissue using inductively coupled plasma optical emission spectrometry for the determination of metallic ions

The quantitative determination of chemical elements in organic or biological samples is an important analytical problem. Normally the elements to be determined in the organic matrix must be transformed into a simple inorganic form. A digestion method by heating on a block digestor has been developed for the determination of Al, As, Ba, Ca, Cd, Co, Cu, Fe, Mg, Mn, V and Zn in Oyster tissue by ICP OES. A simplex centroid statistical mixture design has been used to study the effects of changing HNO₃, HCl and H₂O₂ reagent proportions on the digestion of these samples. Response surface and principal component analyses show that the species Ca, Cd, Cu, Fe, Mg, Mn and Zn have very similar analytical tendencies under this experiment. By means of mixture modeling maximum recoveries for these ions were predicted using 19%, 18% and 63% of the HCl, HNO₃ and H₂O₂ pseudocomponent mixtures, respectively. This corresponds to 21.4%, 30.8% and 47.8% of the HCl, HNO₃ and H₂O₂ commercial solutions. Furthermore the As, Co and V ions present large recoveries for these mixtures as well. The Al and Ba ion recoveries are seen to be independent of the mixture proportions. The analysis of Oyster tissue reference material (SRM 1566b - NIST) under optimized conditions at the selected wavelengths resulted in ion recoveries between 90% and 100%.