Electrochemical characterization of biodeterioration of paint films containing cadmium yellow pigment

The voltammetry of microparticles (VMP) methodology was used to characterize the biological attack of different bacteria and fungi to reconstructed egg tempera and egg–linseed oil emulsion paint films containing cadmium yellow (CdS), which mimic historical painting techniques. When these paint films are in contact with aqueous acetate buffer, different cathodic signals are observed. As a result of the crossing of VMP data with attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), scanning electrochemical microscopy (SECM), field emission scanning electron microscopy (FESEM), and atomic force microscopy (AFM), these voltammetric signals can be associated with the reduction of CdS and different complexes associated to the proteinaceous and fatty acid fractions of the binders. After biological attack with different fungi (Acremonium chrysogenum, Aspergillus niger, Mucor rouxii, Penicillium chrysogenum, and Trichoderma pseudokoningii) and bacteria (Arthrobacter oxydans, Bacillus amyloliquefaciens, and Streptomyces cellulofans), the observed electrochemical signals experience specific modifications depending on the binder and the biological agent, allowing for an electrochemical monitoring of biological attack.     

Quantum-Chemical Study on Positional Selectivity in the Trimethylsilylation and Sulfonation of Pyrrole and <Emphasis Type="Italic">N</Emphasis>-Alkylpyrroles

Positional selectivity (α:β ratio) of electrophilic substitution in pyrrole, N-methylpyrrole, and N-tert-butylpyrrole was analyzed by ab initio [RHF/6-31G(d), MP2/6-31G(d)//RHF/6-31G(d)] and DFT [B3LYP/6-31G(d)] calculations of some σ-complexes derived from the substrates. The results of calculations with the use as model electrophilic species of trimethylsilyl cation [MP2/6-31G(d)//RHF/6-31G(d) and B3LYP/6-31G(d)] and SO₃ molecule [B3LYP/6-31G(d)] instead of proton are fairly consistent with the experimental data, according to which trimethylsilylation of pyrrole and its N-substituted derivatives with trimethylsilyl trifluoromethanesulfonate, as well as sulfonation with pyridine-sulfur trioxide complex, gives the corresponding β-substituted products.     

Comparative Study on Different Expression Hosts for Alkaline Phytase Engineered in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The application of alkaline phytase as a feed additive is restricted by the poor specific activity. Escherichia coli is a frequently used host for directed evolution of proteins including alkaline phytase towards improved activity. However, it is not suitable for production of food-grade products due to potential pathogenicity. To combine the advantages of different expression systems, mutants of the alkaline phytase originated from Bacillus subtilis 168 (phy168) were first generated via directed evolution in E. coli and then transformed to food-grade hosts B. subtilis and Pichia pastoris for secretory expression. In order to investigate the suitability of different expression systems, the phy168 mutants expressed in different hosts were characterized and compared in terms of specific activity, pH profile, pH stability, temperature profile, and thermostability. The specific activity of B. subtilis-expressed D24G/K70R/K111E/N121S mutant at pH 7.0 and 60 °C was 30.4 U/mg, obviously higher than those in P. pastoris (22.7 U/mg) and E. coli (19.7 U/mg). Moreover, after 10 min incubation at 80 °C, the B. subtilis-expressed D24G/K70R/K111E/N121S retained about 70 % of the activity at pH 7.0 and 37 °C, whereas the values were only about 25 and 50 % when expressed in P. pastoris and E. coli, respectively. These results suggested B. subtilis as an appropriate host for expression of phy168 mutants and that the strategy of creating mutants in one host and expressing them in another might be a new solution to industrial production of proteins with desired properties.     

CO<Subscript>2</Subscript> Biofixation of <Emphasis Type="Italic">Actinobacillus succinogenes</Emphasis> Through Novel Amine-Functionalized Polystyrene Microsphere Materials

CO₂-derived succinate production was enhanced by Actinobacillus succinogenes through polystyrene (PSt) microsphere materials for CO₂ adsorption in bioreactor, and the adhesion forces between A. succinogenes bacteria and PSt materials were characterized. Synthesized uniformly sized and highly cross-linked PSt microspheres had high specific surface areas. After modification with amine functional groups, the novel amine-functionalized PSt microspheres exhibited a high adsorption capacity of 25.3 mg CO₂/g materials. After addition with the functionalized microspheres into the culture broth, CO₂ supply to the cells increased. Succinate production by A. succinogenes can be enhanced from 29.6 to 48.1 g L^?1. Moreover, the characterization of interaction forces between A. succinogenes cells and the microspheres indicated that the maximal adhesive force was about 250 pN. The amine-functionalized PSt microspheres can adsorb a large amount of CO₂ and be employed for A. succinogenes anaerobic cultivation in bioreactor for high-efficiency production of CO₂-derived succinate.     

Effect of Plant Growth Promoting Bacteria Associated with Halophytic Weed (<Emphasis Type="Italic">Psoralea corylifolia</Emphasis> L) on Germination and Seedling Growth of Wheat Under Saline Conditions

Halotolerant bacteria associated with Psoralea corylifolia L., a luxuriantly growing annual weed in salinity-affected semi-arid regions of western Maharashtra, India were evaluated for their plant growth-promoting activity in wheat. A total of 79 bacteria associated with different parts viz., root, shoot and nodule endophytes, rhizosphere, rhizoplane, and leaf epiphytes, were isolated and grouped based on their habitat. Twelve bacteria isolated for their potential in plant growth promotion were further selected for in vitro studies. Molecular identification showed the presence of the genera Bacillus, Pantoea, Marinobacterium, Acinetobacter, Enterobacter, Pseudomonas, Rhizobium, and Sinorhizobium (LC027447-53; LC027455; LC027457, LC027459, and LC128410). The phylogenetic studies along with carbon source utilization profiles using the Biolog® indicated the presence of novel species and the in planta studies revealed promising results under salinity stress. Whereas the nodule endophytes had minute plant growth-promoting (PGP) activity, the cell free culture filtrates of these strains enhanced seed germination of wheat (Triticum aestivum L). The maximum vigor index was monitored in isolate Y7 (Enterobacter sp strain NIASMVII). Indole acetic acid (IAA) production by the isolates ranged between 0.22 and 25.58 μg mL^?1. This signifies the need of exploration of their individual metabolites for developing next-generation bio-inoculants through co-inoculation with other compatible microbes. This study has potential in utilization of the weed-associated microbiome in terms of alleviation of salinity stress in crop plants.     

Viscosities and Surface Tensions of Phenetole with <Emphasis Type="Italic">N</Emphasis>-Methyl-2-pyrrolidone, <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-Dimethylformamide and Tetrahydrofuran Binary Systems at Three Temperatures

Viscosities, η, and surface tensions, σ, of binary systems of phenetole (ethoxy benzene or ethyl phenyl ether) with N-methyl-2-pyrrolidone, N,N-dimethylformamide or with tetrahydrofuran were measured over the entire mole fraction range and at (298, 303 and 308) K. The experimental data was used to compute the deviations in viscosity, Δη, and surface tension, Δσ. Values of the excess Gibbs energy of activation G*^E, surface entropy S _σ and surface enthalpy H _σ were calculated. Viscosity data of the binary systems were calculated using the Grunberg and Nissan and the three-body and four-body McAllister correlations. The Redlich–Kister method was used for evaluation of coefficients and standard deviations for Δη, Δσ and G*^E. The results were interpreted in terms of the probable effect of molecular interactions between components as well as polarity.     

Ab Initio Theoretical Studies on the Kinetics of Hydrogen Abstraction Type Reactions of Hydroxyl Radicals with CH<Subscript>3</Subscript>CCl<Subscript>2</Subscript>F and CH<Subscript>3</Subscript>CClF<Subscript>2</Subscript>

The hydrogen abstraction reactions from CH₃Cl₂F (R-141b) and CH₃CClF₂ (R-142b) by OH radicals are studied theoretically by semi-classical transition state theory. The stationary points for the reactions are located by using KMLYP density functional method along with 6-311++G(2d,2p) basis set and MP2 method along with 6-311+G(d,p) basis set. Single-point energy calculations are performed by the CBS-Q and G4 combination methods on the geometries optimized at the KMLYP/6-311++G(2d,2p) level of theory. Vibrational anharmonicity coefficients, x _ij , which are needed for semi-classical transition state theory calculations, are computed at the KMLYP/6-311++G(2d,2p) and MP2/6-311+G(d,p) levels of theory. The computed barrier heights are slightly sensitive to the quantum-chemical method. Thermal rate coefficients are computed over the temperature range from 200 to 2000 K and they are shown to be in accordance with available experimental data. On the basis of the computed rate coefficients, the tropospheric lifetime of the CH₃CCl₂F and CH₃CClF₂ are estimated to be about 6.5 and 12.0 years, respectively.     

Effect of Extracellular Tyrosinase on the Expression Level of P450, Fpr,and Fdx and Ortho-hydroxylation of Daidzein in <Emphasis Type="Italic">Streptomyces avermitilis</Emphasis>

We have reported that the expression of CYP105D7 in Streptomyces avermitilis produces 112.5 mg L^?1 of 7,3′,4′-trihydroxyisoflavone (3'ODI) in 15 h of the reaction time, when 7,4′-dihydroxyisoflavone (daidzein) is used as a substrate. Although production is significant, rapid degradation of 3'ODI after 15 h was observed in a whole-cell biotransformation system, suggesting the further modification of 3'ODI by endogenous enzymes. In this present study, the effect of deletion of extracellular tyrosinase (melC2) in S. avermitilis for 3'ODI production as well as the expressions of CYP105D7, ferredoxin (Fdx), and ferredoxin reductase (Fpr) were investigated. The result revealed that daidzein hydroxylation activity in the ?melC2 mutant decreased by 40% compared with wild-type S. avermitilis. Further, melC2 deletion significantly affects the messenger RNA (mRNA) expression profile of CYP105D7 and its electron transfer counterparts. Real-time PCR analysis of 9 Fdx, 6 Fpr, and CYP105D7 revealed a significant decrease in mRNA expression level compared to wild-type S. avermitilis. The result clearly shows that the decrease in daidzein hydroxylation activity is due to the lower expression level of CYP105D7 and its electron transfer counterpart in the ?melC2 mutant. Furthermore, melC2 deletion prevents the degradation of 3'ODI.     

Specific detection and effective inhibition of a single bacterial species <Emphasis Type="Italic">in situ</Emphasis> using peptide mineralized Au cluster probes

Increasingly serious microbial infections call for the development of new simpler methods for the precise diagnosis and specific inhibition of such pathogens. In this work, a peptide mineralized Au cluster probe was applied as a new simplified strategy to both recognize and inhibit a single bacteria species of Staphylococcus aureus (S. aureus) simultaneously. The probes are composed of peptides and Au clusters. Moreover, the peptides specifically target S. aureus cells and the Au clusters provide fluorescent imaging and have an antibacterial effect. These new probes enable the simultaneous specific detection and effective destruction S. aureus cells in situ.     

Efficient Biotransformation of Phytosterols to Dehydroepiandrosterone by <Emphasis Type="Italic">Mycobacterium</Emphasis> sp.

In this study, a method for the efficient production of dehydroepiandrosterone (DHEA) from phytosterols in a vegetable oil/aqueous two-phase system by Mycobacterium sp. was developed. After the 3-hydroxyl group of phytosterols was protected, they could be converted into DHEA with high yield and productivity by Mycobacterium sp. NRRL B-3683. In a shake flask biotransformation, 15.05 g l^?1 of DHEA and a DHEA yield of 85.39% (mol mol^?1) were attained after 7 days with an initial substrate concentration of 25 g l^?1. When biotransformation was carried out in a 30-l stirred bioreactor with 25 g l^?1 substrate, the DHEA concentration and yield was 16.33 g l^?1 and 92.65% (mol mol^?1) after 7 days, respectively. The results of this study suggest that inexpensive phytosterols could be utilized for the efficient production of DHEA.     

Production of <Emphasis Type="Italic">R</Emphasis>-Mandelic Acid Using Nitrilase from Recombinant <Emphasis Type="Italic">E. coli</Emphasis> Cells Immobilized with Tris(Hydroxymethyl)Phosphine

Recombinant Escherichia coli cells harboring nitrilase from Alcaligenes faecalis were immobilized using tris(hydroxymethyl)phosphine (THP) as the coupling agent. The optimal pH and temperature of the THP-immobilized cells were determined at pH 8.0 and 55 °C. The half-lives of THP-immobilized cells measured at 35, 40, and 50 °C were 1800, 965, and 163 h, respectively. The concentration of R-mandelic acid (R-MA) reached 358 mM after merely 1-h conversion by the immobilized cells with 500 mM R,S-mandelonitrile (R,S-MN), affording the highest productivity of 1307 g L^?1 day^?1 and the space-time productivity of 143.2 mmol L^?1 h^?1 g^?1. The immobilized cells with granular shape were successfully recycled for 60 batches using 100 mM R,S-MN as substrate at 40 °C with 64% of relative activity, suggesting that the immobilized E. coli cells obtained in this study are promising for the production of R-MA.     

Experimental and Theoretical Vibrational Spectra of Sideridiol Isolated from <Emphasis Type="Italic">Sideritis</Emphasis> Species

Sideridiol (ent-7α,18β-dihydroxykaur-15-ene) one of the ent-kaurene diterpenoid, is isolated from the genus Sideritis L. belongs to the family of Lamiaceae. The vibrational frequencies of sideridiol in the ground state have been calculated using the Density Functional Theory (DFT) method with the 6-31G(d) and 6 31+G(d,p) basis sets. The calculated vibrational frequencies have been compared with that of obtained experimental IR spectrum.     

EPR study of the nature of the impurity centers responsible for the scintillation properties of the Li<Subscript>2</Subscript>Zn<Subscript>2</Subscript>(MoO<Subscript>4</Subscript>)<Subscript>3</Subscript> crystal

The binary molybdate Li₂Zn₂(MoO₄)₃ of a new crystal type was characterized by EPR, optical spectroscopy, and X-ray diffraction methods. The crystals have the Pnma symmetry group and the lattice parameters a = 5.1139(5) Å, b = 10.4926(13) Å, c = 17.6445(22) Å; Z = 4. The crystals possess scintillation properties; emission is caused by the presence of impurity levels in the forbidden band. The EPR studies of the nature of the impurity centers responsible for the scintillation characteristics of the crystal showed that the centers were Cu²⁺ ions substituted for zinc ions in the oxygen octahedra. The directions of the main values of the g and tensors (g _zz , A _zz ) correspond to the direction of O-Cu-O of the oxygen octahedron distorted along the Z axis. The EPR spectra of the copper ions are described by the spin Hamiltonian with the parameters g _‖ = 2.38, g _⊥ = 2.06; A _‖ = 116 G, A _⊥ = 0 G.     

Structure,bioactivity and theoretical study of 1-cyano-<Emphasis Type="Italic">N</Emphasis>-p-tolylcyclo-propanecarboxamide

A novel cyclopropane derivative, 1-cyano-N-p-tolylcyclopropanecarboxamide (C₁₂H₁₂N₂O, Mr = 200.24) was synthesized and its structure was studied by X-ray diffraction, FTIR, ¹H and ¹³C NMR spectrum and MS. The crystals are monoclinic, space group P2_1/c with a = 7.109 (4), b = 13.758 (7), c = 11.505 (6) Å, α = 90.00, β = 102.731 (8), γ = 90.00 °, V = 1097.6 (9) ų, Z = 4, F(000) = 312, D _c  = 1.212 g/cm³, μ = 0.0800 mm^?1, the final R = 0.0490 and wR = 0.1480 for 1,375 observed reflections with I > 2σ(I). A total of 6,109 reflections were collected, of which 2,290 were independent (R _int = 0.0290). Theoretical calculation of the title compound was carried out with HF/6-31G (d,p), B3LYP/6-31G (d,p), MP2/6-31G (d,p). The full geometry optimization was carried out using 6-31G(d,p) basis set, and the frontier orbital energy. Atomic net charges were discussed, and the structure-activity relationship was also studied. The preliminary biological test showed that the synthesized compound is bioactive against the KARI of Escherichia coli.     

In Vitro Biotransformation,Safety, and Chemopreventive Action of Novel 8-Methoxy-Purine-2,6-Dione Derivatives

Metabolic stability, mutagenicity, antimutagenicity, and the ability to scavenge free radicals of four novel 8-methoxy-purine-2,6-dione derivatives (compounds 1–4) demonstrating analgesic and anti-inflammatory properties were determined. Metabolic stability was evaluated in Cunninghamella and microsomal models, mutagenic and antimutagenic properties were assessed using the Ames and the Vibrio harveyi tests, and free radical scavenging activity was evaluated with 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay. In the Cunninghamella model, compound 2 did not undergo any biotransformation; whereas 3 and 4 showed less metabolic stability: 1–9 and 53–88% of the parental compound, respectively, underwent biotransformation reactions in different Cunninghamella strains. The metabolites detected after the biotransformation of 3 and 4 were aromatic hydroxylation and N-dealkylation products. On the other hand, the N-dealkylation product was the only metabolite formed in microsome assay. Additionally, these derivatives do not possess mutagenic potential in microbiological models (Vibrio harveyi and Salmonella typhimurium) considered. Moreover, all compounds showed a strong chemopreventive activity in the modified Vibrio harveyi strains BB7X and BB7M. However, radical scavenging activity was not the mechanism which explained the observed chemopreventive activity.     

Development of nitrilase-mediated process for phenylacetic acid production from phenylacetonitrile

This study aimed at developing an efficient biotransformation process for phenylacetic acid production from phenylacetonitrile by using recombinant Escherichia coli M15 harboring a double mutant MG nitrilase (I113M/Y199G) from Burkholderia cenocepacia J2315. A yield of 2310 U/mL nitrilase was obtained by fermentation after the optimization of cultivation conditions, with a specific activity of 64 U/mg dcw. The MG nitrilase showed high substrate tolerance and completely hydrolyzed 100 mM phenylacetonitrile in 30 min under optimal conditions. To alleviate substrate inhibition, periodic or continuous batch-feeding of substrate was used during the biotransformation. Up to 164 g/L substrate was completely hydrolyzed in 9 h with continuous batch-feeding using resting cells, corresponding to 400 U/mL of nitrilase activity, and leading to production of 163.4 g/L phenylacetic acid. The hydrolysis process has potential application for phenylacetic acid production on a large scale.     

Properties and <Emphasis Type="Italic">N</Emphasis>-glycosylation of recombinant endoglucanase II from <Emphasis Type="Italic">Penicillium verruculosum</Emphasis>

Cellulases are the main components of enzyme complexes used in biotransformation processes of plant raw materials into valuable commercial products. Endoglucanase II (EG II) from the Penicillium verruculosum fungus was cloned into Penicillium canescens. The homogeneous recombinant EGII form is isolated and its properties are studied in comparison with the native enzyme. The N-glycosylation sites and the structure of the N-linked glycans are been determined using mass spectrometry. The biochemical and catalytic properties, as well as the N-glycosylation type of the obtained recombinant EGII form, appear to be close to the native enzyme. At the two potential N-glycosylation sites (N42 and N194) of both forms of the enzyme, N-linked high mannose glycans (or their enzymatic “trimming” products) according to the general formula (Man)_1–9(GlcNAc)₂ are detected. No glycosylation is found at the third potential site (N19).     

Synthesis and in vitro antibacterial evaluation of 6-substituted 4-amino-pyrazolo[3,4-<Emphasis Type="Italic">d</Emphasis>]pyrimidines

Pyrazolo[3,4-d]pyrimidines are one of the most important classes of fused heterocyclic compounds which exhibit a broad range of biological and medicinal properties. They are known as anticancer, antifungal, antibacterial, antiviral and anti-inflammatory agents. In this study, some new 6-substituted 4-amino-pyrazolo[3,4-d]pyrimidine derivatives were prepared via reaction of 5-amino-3-methyl-1-phenyl-1H-pyrazole-4-carbonitrile with various nitriles in the presence of sodium ethoxide as catalyst. The inhibitory properties of synthesized compounds were studied according to CLSI guidelines against some pathogenic bacteria including four gram-positive strains (Streptococcus pyogenes, Staphylococcus aureus, Bacillus cereus and Bacillus subtilis subsp. spizizenii) and three gram-negative strains (Pseudomonas aeruginosa, Shigella flexneri and Salmonella enterica subsp. enterica). The antibacterial effects of all derivatives were compared with those of antibiotics belonging to different classes. The values were reported as inhibition zone diameter (IZD), minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The effect of substituents on the biological activity of derivatives was discussed as well. The inhibitory effect of compound 6a, was shown to be the most, with MIC values in the range of 32–4096 μg/mL. Since most of the synthesized compounds were effective against Streptococcus pyogenes and Pseudomonas aeruginosa, they can be considered as inhibitors of these two bacteria.     

Characterization of Truncated <Emphasis Type="Italic">dsz</Emphasis> Operon Responsible for Dibenzothiophene Biodesulfurization in <Emphasis Type="Italic">Rhodococcus</Emphasis> sp. FUM94

Numerous desulfurizing bacteria from the Rhodococcus genus harbor conserved dsz genes responsible for the degradation of sulfur compounds through 4S pathway. This study describes a newly identified desulfurizing bacterium, Rhodococcus sp. FUM94, which unlike previously identified strains encodes a truncated dsz operon. DNA sequencing revealed a frameshift mutation in the dszA gene, which led to an alteration of 66 amino acids and deletion of other C-terminal 66 amino acids. The resulting DszA polypeptide was shorter than DszA in Rhodococcus sp. IGTS8 reference strain. Despite the truncation, desulfurizing activity of the operon was observed and attributed to the removal of an overlap of dszA and dszB genes, and lack of active site in the altered region. Desulfurization experiments resulted in specific production rate of 6.3 mmol 2-hydroxy biphenyl (kgDCW)^?1 h^?1 at 2 g l^?1 biocatalyst concentration and 68.8% biodesulfurization yield at 20 g l^?1 biocatalyst concentration, both at 271 μM dibenzothiophene concentration which is comparable to similar wild-type biocatalysts.     

Robust Parameter Identification to Perform the Modeling of <Emphasis Type="Italic">pta</Emphasis> and <Emphasis Type="Italic">poxB</Emphasis> Genes Deletion Effect on <Emphasis Type="Italic">Escherichia Coli</Emphasis>

The aim of this study was to design a robust parameter identification algorithm to characterize the effect of gene deletion on Escherichia coli (E. coli) MG1655. Two genes (pta and poxB) in the competitive pathways were deleted from this microorganism to inhibit pyruvate consumption. This condition deviated the E. coli metabolism toward the Krebs cycle. As a consequence, the biomass, substrate (glucose), lactic, and acetate acids as well as ethanol concentrations were modified. A hybrid model was proposed to consider the effect of gene deletion on the metabolism of E. coli. The model parameters were estimated by the application of a least mean square method based on the instrument variable technique. To evaluate the parametric identifier method, a set of robust exact differentiators, based on the super-twisting algorithm, was implemented. The hybrid model was successfully characterized by the parameters obtained from experimental information of E. coli MG1655. The significant difference between parameters obtained with wild-type strain and the modified (with deleted genes) justifies the application of the parametric identification algorithm. This characterization can be used to optimize the production of different byproducts of commercial interest.