Isolation,Purification and Characterisation of an Organic Solvent-Tolerant Ca2+-Dependent Protease from Bacillus megaterium AU02

A new organic solvent-tolerant strain Bacillus megaterium AU02 which secretes an organic solvent-tolerant protease was isolated from milk industry waste. Statistical methods were employed to achieve optimum protease production of 43.6 U/ml in shake flask cultures. The productivity of the protease was increased to 53 U/ml when cultivated under controlled conditions in a 7-L fermentor. The protease was purified to homogeneity by a three-step process with 24 % yield and specific activity of 5,375 U/mg. The molecular mass of the protease was found to be 59 kDa. The enzyme was active over a wide range of pH (6.0–9.0), with an optimum activity at pH 7.0 and temperature from 40 to 70 °C having an optimum activity at 50 °C. The thermal stability of the enzyme increased significantly in the presence of CaCl₂, and it retained 90 % activity at 50 °C for 3 h. The K _m and V _max values were determined as 0.722 mg/ml and 0.018 U/mg respectively. The metalloprotease exhibited significant stability in the presence of organic solvents with log P values more than 2.5, nonionic detergents and oxidising agent. An attempt was made to test the synthesis of aspartame precursor (Cbz-Asp-Phe-NH₂) which was catalysed by AU02 protease in the presence of 50 % DMSO. These properties of AU02 protease make it an ideal choice for enzymatic peptide synthesis in organic media.     

Structural,optical, and charge transport properties of cyclopentadithiophene derivatives: a theoretical study

Density functional theory calculations were carried out to investigate the structural and opto-electronic properties of cyclopenta[2,1-b:3,4-b′]dithiophene (CPDT) derivatives. The ground state, cationic and anionic geometries of cyclopentadithiophene derivatives were optimized at B3LYP/6-311G(d,p) level of theory. Based on these geometries, ionic state properties such as ionization potentials, electron affinities, hole extraction potential, and electron extraction potential of cyclopentadithiophene derivatives have been calculated. The charge transfer integral, spatial overlap integral, and site energy were calculated from the matrix elements of Kohn–Sham Hamiltonian. Computed results show that the mobility of charge carriers in CPDT derivatives is strongly affected by the substitution of electron withdrawing group at bridge-head and α-positions. The excited state geometry of CPDT derivatives were optimized using configuration interaction singles method. On the basis of ground and excited states geometry, absorption and emission spectra of cyclopentadithiophene derivatives were calculated using the time-dependent density functional theory method. It has been observed that the substitution of EWG in cyclopentadithiophene core alters the absorption and emission spectra. The nonlinear optical property of CPDT derivatives have been studied through computed static polarizability and first hyperpolarizability.     

Nickel(II) Complexes of Pentadentate N5 Ligands as Catalysts for Alkane Hydroxylation by Using m‐CPBA as Oxidant: A Combined Experimental and Computational Study

A new family of nickel(II) complexes of the type [Ni(L)(CH₃CN)](BPh₄)₂, where L=N‐methyl‐N,N′,N′‐tris(pyrid‐2‐ylmethyl)‐ethylenediamine (L1, 1 ), N‐benzyl‐N,N′,N′‐tris(pyrid‐2‐yl‐methyl)‐ethylenediamine (L2, 2 ), N‐methyl‐N,N′‐bis(pyrid‐2‐ylmethyl)‐N′‐(6‐methyl‐pyrid‐2‐yl‐methyl)‐ethylenediamine (L3, 3 ), N‐methyl‐N,N′‐bis(pyrid‐2‐ylmethyl)‐N′‐(quinolin‐2‐ylmethyl)‐ethylenediamine (L4, 4 ), and N‐methyl‐N,N′‐bis(pyrid‐2‐ylmethyl)‐N′‐imidazole‐2‐ylmethyl)‐ethylenediamine (L5, 5 ), has been isolated and characterized by means of elemental analysis, mass spectrometry, UV/Vis spectroscopy, and electrochemistry. The single‐crystal X‐ray structure of [Ni(L3)(CH₃CN)](BPh₄)₂ reveals that the nickel(II) center is located in a distorted octahedral coordination geometry constituted by all the five nitrogen atoms of the pentadentate ligand and an acetonitrile molecule. In a dichloromethane/acetonitrile solvent mixture, all the complexes show ligand field bands in the visible region characteristic of an octahedral coordination geometry. They exhibit a one‐electron oxidation corresponding to the Ni^II/Ni^III redox couple the potential of which depends upon the ligand donor functionalities. The new complexes catalyze the oxidation of cyclohexane in the presence of m‐CPBA as oxidant up to a turnover number of 530 with good alcohol selectivity (A/K, 7.1–10.6, A=alcohol, K=ketone). Upon replacing the pyridylmethyl arm in [Ni(L1)(CH₃CN)](BPh₄)₂ by the strongly σ‐bonding but weakly π‐bonding imidazolylmethyl arm as in [Ni(L5)(CH₃CN)](BPh₄)₂ or the sterically demanding 6‐methylpyridylmethyl ([Ni(L3)(CH₃CN)](BPh₄)₂ and the quinolylmethyl arms ([Ni(L4)(CH₃CN)](BPh₄)₂, both the catalytic activity and the selectivity decrease. DFT studies performed on cyclohexane oxidation by complexes 1 and 5 demonstrate the two spin‐state reactivity for the high‐spin [(N5)Ni^II?O^.] intermediate (ts1_hs, ts2_doublet), which has a low‐spin state located closely in energy to the high‐spin state. The lower catalytic activity of complex 5 is mainly due to the formation of thermodynamically less accessible m‐CPBA‐coordinated precursor of [Ni^II(L5)(OOCOC₆H₄Cl)]⁺ ( 5 a ). Adamantane is oxidized to 1‐adamantanol, 2‐adamantanol, and 2‐adamantanone (3°/2°, 10.6–11.5), and cumene is selectively oxidized to 2‐phenyl‐2‐propanol. The incorporation of sterically hindering pyridylmethyl and quinolylmethyl donor ligands around the Ni^II leads to a high 3°/2° bond selectivity for adamantane oxidation, which is in contrast to the lower cyclohexane oxidation activities of the complexes.     

Development and validation of a HPLC method for simultaneous quantitation of gatifloxacin, sparfloxacin and moxifloxacin using levofloxacin as internal standard in human plasma: application to a clinical pharmacokinetic study

A highly selective, sensitive and accurate HPLC method has been developed and validated for the estimation of three fluoroquinolones (FQs) viz., gatifloxacin (GFC), sparfloxacin (SFC) and moxifloxacin (MFC) with 500 microL human plasma using levofloxacin (LFC) as an internal standard (IS). The sample preparation involved simple liquid-liquid extraction of GFC, SFC, MFC and IS from human plasma with ethyl acetate. The resolution of peaks was achieved with phosphate buffer (pH 2.5)-acetonitrile (80:20, v/v) at a flow rate of 1 mL/min on a Kromasil C(18) column. The total chromatographic run time was 18.0 min and the simultaneous elution of GFC, SFC, MFC and IS occurred at approximately 10.8, 12.8, 17.0 and 6.0 min, respectively. The method proved to be accurate and precise at linearity range of 100-10,000 ng/mL with a correlation coefficient (r) of > or =0.999. The limit of quantitation for each of the FQs studied was 100 ng/mL. The intra- and inter-day precision and accuracy values found to be within the assay variability limits as per the FDA guidelines. The developed assay method was applied to a pharmacokinetic study in human volunteers following oral administration of 400 mg GFC tablet.     

Rapid resolution of carbohydrate isomers by electrospray ionization ambient pressure ion mobility spectrometry-time-of-flight mass spectrometry (ESI-APIMS-TOFMS)

Carbohydrates are an extremely complex group of isomeric molecules that have been difficult to analyze in the gas phase by mass spectrometry because (1) precursor ions and product ions to successive stages of MS(n) are frequently mixtures of isomers, and (2) detailed information about the anomeric configuration and location of specific stereochemical variants of monosaccharides within larger molecules has not been possible to obtain in a general way. Herein, it is demonstrated that gas-phase analyses by direct combination of electrospray ionization, ambient pressure ion mobility spectrometry, and time-of-flight mass spectrometry (ESI-APIMS-TOFMS) provides sufficient resolution to separate different anomeric methyl glycosides and to separate different stereoisomeric methyl glycosides having the same anomeric configuration. Reducing sugars were typically resolved into more than one peak, which might represent separation of cyclic species having different anomeric configurations and/or ring forms. The extent of separation, both with methyl glycosides and reducing sugars, was significantly affected by the nature of the drift gas and by the nature of an adducting metal ion or ion complex. The study demonstrated that ESI-APIMS-TOFMS is a rapid and effective analytical technique for the separation of isomeric methyl glycosides and simple sugars, and can be used to differentiate glycosides having different anomeric configurations.     

Synthesis, spectroscopic characterization and DNA nuclease activity of Cu(II) complexes derived from pyrazolone based NSO-donor Schiff base ligands

Two neutral mononuclear Cu(II) complexes have been prepared in EtOH using Schiff bases derived from 4-toluoyl pyrazolone and thiosemicarbazide. Both the ligands have been characterized on the basis of elemental analysis, IR, (1)H NMR, (13)C NMR and mass spectral data. The molecular geometry of one of these ligands has been determined by single crystal X-ray study. It reveals that these ligands exist in amine-one tautomeric form in the solid state. Microanalytical data, Cu-estimation, molar conductivity, magnetic measurements, IR, UV-Visible, FAB-Mass, TG-DTA data and ESR spectral studies were used to confirm the structures of the complexes. Electronic absorption and IR spectra of the complexes suggest a square-planar geometry around the central metal ion. The interaction of complexes with pET30a plasmid DNA was investigated by spectroscopic measurements. Results suggest that the copper complexes bind to DNA via an intercalative mode and can quench the fluorescence intensity of EB bound to DNA. The interaction between the complexes and DNA has also been investigated by agarose gel electrophoresis, interestingly, we found that the copper(II) complexes can cleave circular plasmid DNA to nicked and linear forms.     

Charge transport and optical properties of discotic liquid crystalline molecules THDDP and substituted THDP

The theoretical investigations have been carried out on the discotic liquid crystalline molecules, 2,3,6,7‐tetrakis‐hexyloxy‐9,16‐diaza‐dibenzo[a,c]phenazine (THDDP) and different substituted 2,3,6,7‐tetrakis‐hexyloxy‐dibenzo[a,c]phenazine (THDP) to study their charge transport and optical properties. The key parameters of charge transport such as charge transfer integrals and site energies have been calculated from the matrix elements of Kohn‐Sham Hamiltonian. The reorganization energy for the presence of excess charge and the rate of charge transfer calculated from Marcus theory have been used to find the mobility of the charge carrier in the studied molecules. The results show that the substitution and stacking angle change strongly affect the charge carrier mobility in π‐stacked THDDP and substituted THDP molecules. Molecular dynamics simulations have been performed to find the most favorable conformation. The time‐dependent density functional theory (TDDFT) calculations reveal that for these molecules the different substitutions does not alter the main features of optical properties and the molecules may be used as blue light emitters. © 2011 Wiley Periodicals, Inc. Int J Quantum Chem, 2011     

Mechanism and kinetics of the atmospheric degradation of 2-formylcinnamaldehyde with O3 and hydroxyl OH radicals – a theoretical study

In the present investigation, the reaction mechanism and kinetics of 2-formylcinnamaldehyde (2-FC) with O₃ and hydroxyl OH radicals were studied. The reaction of 2-FC with O₃ radical are initiated by the formation of primary ozonide, whereas the reaction of 2-FC with the hydroxyl OH radical are initiated by two different ways: (1). H-atom abstraction by hydroxyl OH radical from the –CHO and –CH = CHCHO group of 2-FC (2). Hydroxyl OH addition to the –CH = CHCHO group to the ring-opened 2-FC. These reactions lead to the formation of an alkyl radical. The reaction pathways corresponding to the reactions between 2-FC with O₃ and hydroxyl OH radicals have been analysed using density functionals of B3LYP and M06-2X level of methods with the 6-31+G(d,p) basis set. Single-point energy calculations for the most favourable reactive species are determined by B3LYP/6-311++G(d,p) and CCSD(T)/6-31+G(d,p) levels of theory. From the obtained results, the hydroxyl OH addition at C8 position of 2-FC are most favourable than the C9 position of 2-FC. The subsequent reactions of the alkyl radicals, formed from the hydroxyl OH addition at C8 position, are analysed in detail. The individual and overall rate constant for the most favourable reactions are calculated by canonical variational transition theory with small-curvature tunnelling corrections over the temperature range of 278–350 K. The calculated theoretical rate constants are in good agreement with the available experimental data. The Arrhenius plot of the rate constants with the temperature are fitted and the atmospheric lifetimes of the 2-FC with hydroxyl OH radical reaction in the troposphere calculate for the first time, which can be applied to the study on the atmospheric implications. The condensed Fukui function has been verified for the most favourable reaction sites. This study can be regarded as an attempt to investigate the O₃-initiated and hydroxyl OH-initiated reaction mechanisms of 2-FC in the atmosphere.     

Ferroelectric polymer nanocomposite alignment layer in twisted nematic liquid crystal devices for reducing switching voltage

Twisted nematic liquid crystal device (TNLCD) was fabricated using a ferroelectric zinc oxide (ZnO)-doped polyimide alignment layer. The ferroelectric nanoparticle can produce a local electric field, which can trigger the orientation of liquid crystal molecule and reduces the switching voltage. The uniform dispersion of ferroelectric ZnO nanoparticles in the alignment layer was studied using field emission scanning electron microscopy and atomic force microscopy. The ferroelectric property of ZnO-doped polyimide was investigated using dynamic contact electrostatic force microscopy. An increased local electric field due to the presence of nano ZnO was confirmed with the help of scanning tunnelling microscopy. An augmentation of capacitance was observed with an increase in concentration, which substantiates the reduction of switching voltage of TNLCD with the modification of ferroelectric nanoparticle-doped alignment layer.