Engineering cytochrome P-450s

Cytochrome P-450 isozymes represent a critical component of nature’s spectrum of detoxification catalysts that could be exploited for bioremediation. The ethanol-inducible human cytochrome P-450 2E1 serves as a model eukaryotic P-450 that complements the bacterial P-450 cam in dehalogenation and detoxification of environmental pollutants. We explored the construction of novel chimeric P-450s using cytochrome P-450 camC and 2E1 genes. For construction of chimera 1 (478 amino acids, 55.14 kDa), 145 amino acids from the N-terminus of P-450 2E1 protein (493 amino acids, 56.84 kDa) were replaced with 130 amino acids from the N-terminus of P-450 camC protein (415 amino acids, 46.66 kDa). In chimera 2 (525 amino acids, 60.24 kDa) the strategy involves replacement of 28 amino acids in the C-terminus of chimera 1 with 75 amino acids from the C-terminus of P-450 camC gene. Homology models of both the chimeric proteins were developed using SWISS-MODEL based on the known crystal structure of cytochrome P-450 camC, BM-3, 1DT6A, and 2C17A. The models indicated that the proposed heme-binding site was intact, which is inevitable for catalytic activity of cytochrome P-450s. The expression of chimera 1 and 2 genes in Escherichia coli DH5α was evident from light-pink cell pellets, protein band in sodium dodecyl sulfate polyacrylamide gel electrophoresis, and diagnostic carbon monoxide-difference spectra. Our studies show that strategies can be developed to exploit the natural diversity of the P-450 superfamily to generate chimeric biocatalysts that would provide new templates amenable to directed evolution.     

Acidity and Catalytic Activity of Sulfate Modified Mixed Oxides of Tin and Praseodymium

A comparitive study of the acid-base properties and physico-chemical characteristics of pure SnO₂, sulfated SnO₂ and sulfated binary oxides of Sn and Pr was done. The data were correlated with the catalytic activity of the oxides towards benzoylation of toluene.     

Synthesis, spectral characterization and crystal structure of copper(II) complexes of 2-benzoylpyridine-N(4)-phenylsemicarbazone

An interesting series of nine new copper(II) complexes [Cu₂L₂(OAc)₂]·H₂O (1), [CuLNCS]·½H₂O (2), [CuLNO₃]·½H₂O (3), [Cu(HL)Cl₂]·H₂O (4), [Cu₂(HL)₂(SO₄)₂]·4H₂O (5), [CuLClO₄]·½H₂O (6), [CuLBr]·2H₂O (7), [CuL₂]·H₂O (8) and [CuLN₃]·CH₃OH (9) of 2-benzoylpyridine-N(4)-phenyl semicarbazone (HL) have been synthesized and physico-chemically characterized. The tridentate character of the semicarbazone is inferred from IR spectra. Based on the EPR studies, spin Hamiltonian and bonding parameters have been calculated. The g values, calculated for all the complexes in frozen DMF, indicate the presence of the unpaired electron in the d_x²-y² orbital. The structure of the compound, [Cu₂L₂(OAc)₂] (1a) has been resolved using single crystal X-ray diffraction studies. The crystal structure revealed monoclinic space group P2₁/n. The coordination geometry about the copper(II) in 1a is distorted square pyramidal with one pyridine nitrogen atom, the imino nitrogen, enolate oxygen and acetate oxygen in the basal plane, an acetate oxygen form adjacent moiety occupies the apical position, serving as a bridge to form a centrosymmetric dimeric structure.     

p-Nitrophenol Biodegradation by Aerobic Microbial Granules

Mixed microbial consortia in the form of aerobic microbial granules (AMG) capable of xenobiotic degradation can be developed from activated sludge or by adaptation of microbial granules pre-grown on labile carbon sources. Both of these approaches were investigated for the cultivation of AMG capable of p-nitrophenol (PNP) biodegradation. Attempts to cultivate AMG from activated sludge using PNP as the sole carbon source were not successful due to poor microbial growth and washout of the inoculated activated sludge. As part of the second approach, parallel sequencing batch reactors (SBRs) were inoculated with pre-grown AMG and operated by feeding both acetate and PNP together (RA), PNP alone (RB) or acetate alone (RC). Acetate/PNP mineralization and nitrification were monitored in the three SBRs. PNP biodegradation was quickly established in both RA and RB. PNP removal rates were found to be 47 and 55?mg g VSS^?1 h^?1 in RA and RB, respectively. PNP biodegradation during the SBR cycle consisted of distinct lag, exponential and deceleration phases. However, with higher concentrations of PNP (>50?mg l^?1), disintegration of granules was observed in RA and RB. When PNP was the sole carbon source, it inhibited the development of aerobic granules from activated sludge and caused disintegration of pre-cultivated aerobic granules. When PNP was the co-substrate along with acetate, the structural and functional integrity (including nitrification) of the granular sludge was maintained. This report highlights the importance of a labile co-substrate for maintaining the physical and functional integrity of granular sludge, when used for toxic waste degradation.     

A convenient protocol for C-H oxidation mediated by an azido radical culminating in Ritter-type amidation

Cerium(IV) ammonium nitrate in combination with sodium azide reacts with unactivated hydrocarbons in acetonitrile to furnish acetamides in one pot. The strategy can be used to introduce nitrogen functionality into a variety of compounds; a carboxylic ester directly afforded the corresponding α-amino acid.     

A High-Performance Catalytic and Recyclability of Phyto-Synthesized Silver Nanoparticles Embedded in Natural Polymer

The present work deals with phytogenic synthesis of Ag NPs in the natural polymer alginate as support material using Aglaia elaeagnoidea leaf extract as a reducing, capping, and stabilizing agent. Ag nanoparticles embedded in alginate were characterized using UV–Vis absorption spectroscopy, Fourier transform infrared spectroscopy, field emission scanning electron microscopy, energy dispersive X-ray spectroscopy, X-ray diffraction, transmission electron microscopy techniques and selected area electron diffraction techniques. The formation of AgNPs embedded in the polymer was in spherical shape with an average size of 12 nm range has been noticed. The prepared embedded nanoparticles in polymer were evaluated as a solid heterogeneous catalyst for the reduction of 4-nitrophenol (4-NP) to 4-aminophenol (4-AP) and methylene blue to leuco methylene blue in the liquid phase using sodium borohydride (NaBH₄) as reducing agent. The silver nanoparticles embedded polymer exhibited extraordinary catalytic efficacy in reduction of 4-NP to 4-AP and the rate constant is 0.5054 min^?1 at ambient conditions. The catalyst was recycled and reused up to 10 cycles without significant loss of catalytic activity. The preparation of Ag–CA composite was facile, stable, efficient, eco-friendly, easy to recycle, non-toxic, and cost effective for commercial application.     

Density functional theory studies on vibrational and electronic spectra of 2-chloro-6-methoxypyridine

The Fourier transform infrared (FTIR) and FT-Raman spectra of 2-chloro-6-methoxypyridine have been recorded in the range 3700-400 and 3700-100 cm(-1), respectively. The complete vibrational assignment and analysis of the fundamental modes of the compound was carried out using the observed FTIR and FT-Raman data. The vibrational frequencies determined experimentally were compared with the theoretical frequencies computed by DFT gradient calculations (B3LYP method) employing the 6-31G(d,p), cc-pVTZ and/6-311++G(d,p) basis sets for the optimised geometry of the compound. The geometry and normal modes of vibration obtained from the DFT methods are in good agreement with the experimental data. The normal co-ordinate analysis was also carried out using DFT force fields utilising Wilson's FG matrix method. The influence of the substituents bulky chlorine atom and the methoxy group on the spectral characteristics of the compound has been discussed. The electronic spectrum determined by TD-DFT method is compared with the observed electronic spectrum.     

Drug Design,Synthesis and Biological Evaluation of Heterocyclic Molecules as Anti-Inflammatory Agents

Non-steroidal anti-inflammatory drugs (NSAIDs) are generally utilized for numerous inflammatory ailments. The long-term utilization of NSAIDs prompts adverse reactions such as gastrointestinal ulceration, renal dysfunction and hepatotoxicity; however, selective COX-2 inhibitors prevent these adverse events. Various scientific approaches have been employed to identify safer COX-2 inhibitors, as in any case, a large portion of particular COX-2 inhibitors have been retracted from the market because of severe cardiovascular events. This study aimed to develop and synthesize a novel series of indomethacin analogues with potential anti-inflammatory properties and fewer side effects, wherein carboxylic acid moiety was substituted using DCC/DMAP coupling. This study incorporates the docking of various indomethacin analogues to detect the binding interactions with COX-2 protein (PDB ID: 3NT1). MD simulation was performed to measure the stability and flexibility of ligand–protein interactions at the atomic level, for which the top-scoring ligand–protein complex was selected. These compounds were evaluated in vitro for COX enzymes inhibition. Likewise, selected compounds were screened in vivo for anti-inflammatory potential using the carrageenan-induced rat paw oedema method and their ulcerogenic potential. The acute toxicity of compounds was also predicted using in silico tools. Most of the compounds exhibited the potent inhibition of both COX enzymes; however, 3e and 3c showed the most potent COX-2 inhibition having IC50 0.34 µM and 1.39 µM, respectively. These compounds also demonstrated potent anti-inflammatory potential without ulcerogenic liability. The biological evaluation revealed that the compound substituted with 4-nitrophenyl was most active.     

Liquid phase benzylation of toluene using benzyl alcohol over iron promoted sulfated zirconia

Concentrated (0.2 M) aqueous solutions of HP-acids, such as H_3+x+mPV^IV _mV^V _x-mMo_12-xO₄₀ and their analogues with an excess VO²⁺ cation, are oxidized by dioxygen at 343 K and atmospheric pressure through intermediate active complexes (IAC) [H_x+m-1PV^IV _mV^V _x-mMo_12-xO₄₀ ^{4 -}] · [VO²⁺]_y · O₂, where m + y ≥ 3. The electron transfer to the coordinated O₂ molecule inside AC is the limiting stage at high m. At low m, the formation of IAC becomes the limiting stage that results in a sharp decrease in the oxidation rate. This revised version was published online in August 2006 with corrections to the Cover Date.