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The exposed and transformed groups of fibrous brucite, wollastonite, chrysotile asbestos, sepiolite, palygorskite, clinoptilolite, crocidolite and diatomaceous earth mineral materials are analyzed by IR spectra after acid and alikali etching, strong mechanical and polarity molecular interaction. The results show the active sites concentrate on the ends in stick mineral materials and on the defect or hole edge in pipe mineral materials. The inside active site of mineral materials plays a main role in small molecular substance. The shape of minerals influence their distribution and density of active site. The strong mechanical impulsion and weak chemical force change the active site feature of minerals, the powder process enables minerals exposed more surface group and more combined types. The surface processing with the small polarity molecular or the brand of middle molecular may produce ionation and new coordinate bond, and change the active properties and level of original mineral materials.

Hydroxylation of DHEA to 7α,15α-diOH-DHEA was catalyzed by NADPH-dependent cytochrome P450 monooxygenase from Colletotrichum lini. By adding coenzyme precursor nicotinic acid, the NADPH/NADP ratio was significantly increased, and the 7α,15α-diOH-DHEA molar conversion was enhanced from 37.37% to 50.85%. To improve the availability of intracellular NADPH, a dual cosubstrate-coupled system consisting of nicotinic acid and glucose was investigated in C. lini. Using 20mM nicotinic acid and 15g/L glucose as cosubstrate for NADPH regeneration, the 7α,15α-diOH-DHEA molar conversion was dramatically increased by 74.58%. The conversion course was simultaneously shortened by 30h. Moreover, a fed-batch transformation model was established to diminish DHEA toxicity to C. lini and further increase DHEA concentration. The maximum concentration of DHEA was elevated to 15g/L using a three-batch transformation in a coenzyme regeneration system, and 7α,15α-diOH-DHEA production of 11.21g/L could be achieved after 60h of biotransformation. These results demonstrated that this strategy was promising for steroids hydroxylation.

A comprehensive study on formation and characteristics of soluble microbial products (SMP) during drinking water biofiltration was made in four parallel pilot-scale ceramic biofilters with acetate as the substrate. Excellent treatment performance was achieved while microbial biomass and acetate carbon both declined with the depth of filter. The SMP concentration was determined by calculating the difference between the concentration of dissolved organic carbon (DOC), biodegradable dissolved organic carbon (BDOC) and acetate carbon. The results revealed that SMP showed an obvious increase from 0 to 100 cm depth of the filter. A rising specific ultraviolet absorbance (SUVA) was also found, indicating that benzene or carbonyl might exist in these compounds. SMP produced during this drinking water biological process were proved to have weak mutagenicity and were not precursors of by-products of chlorination disinfection. The volatile parts of SMP were half-quantity analyzed and most of them were dicarboxyl acids, others were hydrocarbons or benzene with 16-17 carbon atoms.

Pseudomonas putida CGMCC3830 harboring nitrilase was used in isonicotinic acid production from 4-cyanopyridine. This nitrilase showed optimum activities towards 4-cyanopyridine at pH 7.5 and 45°C. The half-life of P. putida nitrilase was 93.3 h, 33.9 h, and 9.5 h at 30°C, 38°C, and 45°C, respectively. 4-Cyanopyridine (100 mM) was fully converted into isonicotinic acid within 20 min. The bench-scale production of isonicotinic acid was carried out using 3 mg of resting cells per mL in a 1 L system at 30°C and finally, 123 g L−1 of isonicotinic acid were obtained within 200 min without any by-products. The conversion reaction suffered from the product inhibition effect after the tenth feeding. The volumetric productivity was 36.9 g L−1 h−1. P. putida shows significant potential in nitrile hydrolysis for isonicotinic acid production. This paper is the first report on isonicotinic acid biosynthesis using Pseudomonas putida and it represents the highest isonicotinic acid production reported so far.

An investigation of the ignition delay time for oil–water emulsion with different additional more volatile liquid fuels as single droplets is conducted. The measurement is made by the suspended droplets. In the experiments, the ratio of n-dodecane, water and n-heptane (more volatile fuel) is as follows: 6:4:0, 5:4:1, 4:4:2, 3:4:3. The experimental and calculated results show that the ignition delay time decreases with the amount of the more volatile component increasing, and the trend will be smooth for high amount of more volatile component. This result is a very useful method to solve the normal ignition for emulsified oil mixed with a large amount of water in oil in diesel engines.

Soluble turnip cytochrome f has been purified from the periplasmic fraction of Escherichia coli expressing a truncated petA gene encoding the precursor protein lacking the C-terminal 33 amino-acid residues. The protein is identical [as judged by 1H-NMR spectroscopy, midpoint redox potential (+ 365 mV) and electron transfer reactions with plastocyanin] to cytochrome f purified from turnip leaves. Several residues in the hydrophobic patch surrounding the haem group have been changed by site-directed mutagenesis, and the proteins purified from E. coli. The Y1F and Q7N mutants showed only minor changes in the plastocyanin-binding constant Ka and the second-order rate constant for electron transfer to plastocyanin, whereas the Y160S mutant showed a 30% decrease in the overall rate of electron transfer caused in part by a 60% decrease in binding constant and partially compensated by an increased driving force due to a 27-mV decrease in redox potential. In contrast, the F4Y mutant showed increased rates of electron transfer which may be ascribed to an increased binding constant and a 14-mV decrease in midpoint redox potential. This indicates that subtle changes in the hydrophobic patch can influence rates of electron transfer to plastocyanin by changing the binding constants and altering the midpoint redox potential of the cytochrome haem group.

Dissolved organic matters (DOM) are critical in the formation of the mutagenic disinfection byproducts (DBPs). In this study, five DOM fractions were isolated and investigated from a contaminated river and a clean reservoir source waters using resin adsorption. The DOM fractions were characterized with excitation-emission matrix, and several typical DBPs formation potentials and the mutagenicity of each DOM fraction were measured. Among these fractions, hydrophobic neutrals (HON), hydrophilic acids (HIA) and hydrophilic bases (HIB) generated the highest carbon-containing DBPs in the river source water, as did HIB and hydrophobic bases (HOB) in reservoir water. Hydrophobic acids (HOA), HON, and HIA were the three most important fractions forming nitrogen-containing DBPs. Following chlorination, the mutagenicity of HON, HIA, HOA and HIB was 1503, 626, 422 and 116 ng 4-NQO/mg DOC in river water, respectively. Only HIA and HOA were mutagenic with 85 and 10 ng 4-NQO/mg DOC in reservoir water, respectively. The soluble microbial products like substances and aromatic proteins contributed significantly to the mutagenicity of river water; whereas the humic acid-like and fulvic acid-like substances were the primary contributors to the mutagenicity of reservoir water.

It has been proved that sevoflurane postconditioning (SpostC) could protect the heart against myocardial ischemia/reperfusion injury, however, there has been few research focused on the electrophysiological effects of SpostC. The objective of the study was to investigate the effects of SpostC on action potential duration (APD) and L-type calcium current (I(Ca, L)) in isolated cardiomyocytes.Langendorff perfused SD rat hearts were randomly assigned to one of the time control (TC), ischemia/reperfusion (I/R, 25 minutes of ischemia followed by 30 minutes of reperfusion), and SpostC (postconditioned with 3% sevoflurane) groups. At the end of reperfusion, epicardial myocytes were dissociated enzymatically for patch clamp studies.Sevoflurane directly prolonged APD and decreased peak I(Ca, L) densities in epicardial myocytes of the TC group (P0.05). I/R injury shortened APD and decreased peak I(Ca, L) densities in epicardial myocytes of the I/R group (P0.05). SpostC prolonged APD and increased peak I(Ca, L) densities in epicardial myocytes exposed to I/R injury (P0.05). SpostC decreased intracellular reactive oxygen species (ROS) levels, reduced the incidence of ventricular tachycardia and ventricular fibrillation, and decreased reperfusion arrhythmia scores compared with the I/R group (all P0.05).SpostC attenuates APD shortening and I(Ca, L) suppression induced by I/R injury. The regulation of APD and I(Ca, L) by SpostC might be related with intracellular ROS modulation, which contributes to the alleviation of reperfusion ventricular arrhythmia.

Over the past decades, nitrilases have drawn considerable attention because of their application in nitrile degradation as prominent biocatalysts. Nitrilases are derived from bacteria, filamentous fungi, yeasts, and plants. In-depth investigations on their natural sources function mechanisms, enzyme structure, screening pathways, and biocatalytic properties have been conducted. Moreover, the immobilization, purification, gene cloning and modifications of nitrilase have been dwelt upon. Some nitrilases are used commercially as biofactories for carboxylic acids production, waste treatment, and surface modification. This critical review summarizes the current status of nitrilase research, and discusses a number of challenges and significant attempts in its further development. Nitrilase is a significant and promising biocatalyst for catalytic applications.

Nitrilase-mediated biocatalysis has attracted substantial attention for its application in carboxylic acid production in recent years. In the present study, the fungus CA3-1 was isolated and identified as Gibberella intermedia based on its morphology, its 18S ribosomal DNA (rDNA), and internal transcribed spacer (ITS) sequences. The enzymatic properties of G. intermedia resting cells were determined, and the optimum activity was achieved at 40 °C with pH 7.6. The half-lives of the nitrilase at 30, 40, and 50 °C were 231.1, 72.9, and 6.4 h, respectively. This Gibberella nitrilase showed a wide substrate spectrum with high specificity for heterocyclic and aliphatic nitriles. It remained extremely active in 5% propanol. The presence of Ag(+), Hg(2+), and excess substrate inhibited the nitrilase activity, whereas Fe(2+), Mn(2+), and Li(+) improved enzyme activity. 3-Cyanopyridine (50 mM) was hydrolyzed into nicotinic acid within 30 min, whereas only5% of nicotinamide was detected. The results show that this fungal nitrilase is a promising candidate for commercial application in nicotinic acid production.

BackgroundNitrilase is an important member of the nitrilase superfamiliy. It has attracted substantial interest from academia and industry for its function of converting nitriles directly into the corresponding carboxylic acids in recent years. Thus nitrilase has played a crucial role in production of commercial carboxylic acids in chemical industry and detoxification of nitrile-contaminated wastes. However, conventional studies mainly focused on the bacterial nitrilase and the potential of fungal nitrilase has been far from being fully explored. Research on fungal nitrilase gene expression will advance our understanding for its biological function of fungal nitrilase in nitrile hydrolysis.Methodology/principal findingsA fungal nitrilase gene from Gibberella intermedia was cloned through reverse transcription-PCR. The open reading frame consisted of 963 bp and potentially encoded a protein of 320 amino acid residues with a theoretical molecular mass of 35.94 kDa. Furthermore, the catalytic triad (Glu-45, Lys-127, and Cys-162) was proposed and confirmed by site-directed mutagenesis. The encoding gene was expressed in Escherichia coli Rosetta-gami (DE3) and the recombinant protein with His(6)-tag was purified to electrophoretic homogeneity. The purified enzyme exhibited optimal activity at 45°C and pH 7.8. This nitrilase was specific towards aliphatic and aromatic nitriles. The kinetic parameters V(max) and K(m) for 3-cyanopyridine were determined to be 0.81 µmol/min·mg and 12.11 mM through Hanes-Woolf plot, respectively. 3-Cyanopyridine (100 mM) could be thoroughly hydrolyzed into nicotinic acid within 10 min using the recombinant strain with the release of about 3% nicotinamide and no substrate was detected.Conclusions/significanceIn the present study, a fungal nitrilase was cloned from the cDNA sequence of G. intermedia and successfully expressed in E. coli Rosetta-gami (DE3). The recombinant strain displayed good 3-cyanopyridine degradation efficiency and wide substrate spectrum. This fungal nitrilase might be a potential candidate for industrial applications in carboxylic acids production.

To prepare monoclonal antibodies against HBsAg and estimate its binding capacity to both wild-type and varies of immune escape mutant-type HBsAg.Using self-made the anti-HBs G6 mAb and HRP-labeled goat anti-HBs antibody, A sandwich ELISA for detection both wild-type and varies of immune escape mutant-type HBsAg has been developed. Applying this assay, to detect 17 species of whole gene wild-type and recombination expressed HBsAg which varied in "a" determinant. to evaluate its clinical application characteristic of this assay, we used the other commercial reagents to Comparing with it.One strain hybridoma cell lin secreted anti-HBs mAb (defined G6), was obtained. It grew stably and the titers of the culture supernatant and hydroperitoneum were 2 048 and 4 096x10(3). The sensitivity to the wild-type HBsAg of this assay was less than 0.125 microg/L. This anti-HBs mAb can bind with 12 in 15 species mutant HBsAg (P/Nor =2.5), and 2 of them were low absorbent value at 450 nm(A(450)) group which was 7.55 percent of the wild-type, 1 of them was middle A(450) value group which was 59.40 percent of the wild-type, 9 of them were high A(450) value group which was 92.1-109.4 percent of the wild-type. The low A(450) value group and the negative group were diversified in 120-124 basyl in I loop of the HBV "a" determinant. We also used other commercial reagents, which was widely used in clinic, to detect partial species mutant HBsAg, the average A(450) value was less than foregoing assay.The G6 anti-HBs mAb was able to bind most of the immune escape mutant HBsAg in the test. And there would be some special regulations between the mutant site and the recombination expressed HBsAg binding capability.

To search for and verify some common B cell epitopes in the core proteins of woodchuck hepatitis virus and human hepatitis B virus.Monoclonal antibodies against both core proteins of woodchuck hepatitis virus (WHV) and human hepatitis B virus (HBV) were prepared by inoculating Balb/c mice with denatured recombination WHV and HBV core proteins. ELISA and immunoblotting assays for WHcAg and HBcAg were carried out by using these antibodies. Immunohistochemistry was carried out with liver tissue sections of both WHV-infected woodchucks and chronic HBV-infected patients. The epitopes were mapped with the mouse mAbs (6D1 and 1H4) by using a panel of 24 16mer overlapping peptides covering the entire WHcAg. The amino acid sequences of WHcAg and HBcAg were compared.Cross-reactions were observed between mAbs (6D1 and 1H4) and WHcAg and between Mabs and HBcAg/HBcAg in ELISA and immunoblotting assay. Liver tissue sections of both WHV-infected woodchucks and chronic HBV-infected patients could be stained specifically by mAbs. The epitopes were mapped at aa1-8 (6D1) and aa125-140 (1H4) of the core proteins of both WHV and HBV by using ELISA assay. WHcAg and HBcAg share similar amino acids sequences at aa1-8 and aa125-140 respectively.The core proteins of woodchuck hepatitis virus and human hepatitis B virus share common linear B cell epitopes which span aa1-8 and aa125-140 respectively.

The cytochrome b(6)f complex is an obligatory electron transfer and proton-translocating enzyme in all oxygenic photosynthesis. Its operation has been described by the "Q-cycle." This model proposes that electrons are transferred from plastoquinol to plastocyanin (the reductant of P700 in Photosystem I) through, obligatorily in series, the iron-sulfur and the cytochrome f redox centers in the cytochrome b(6)f complex. However, here we demonstrate that (a) the iron-sulfur center-dependent reductions of plastocyanin and P700 are much faster than cytochrome f reduction, both in Chlamydomonas reinhardtii cytochrome f mutants and in the wild type, and (b) the steady-state photosynthetic electron transport does not correlate with strongly inhibited cytochrome f reduction kinetics in the mutants. Thus, cytochrome f is not an obligatory intermediate for electrons flowing through the cytochrome b(6)f complex. The oxidation equivalents from Photosystem I are delivered to the high potential chain of the cytochrome b(6)f complex both at the cytochrome f level and, independently, at another site connected to the quinol-oxidizing site, possibly the iron-sulfur center.

The lumen segment of cytochrome f consists of a small and a large domain. The role of the small domain in the biogenesis and stability of the cytochrome b(6)f complex and electron transfer through the cytochrome b(6)f complex was studied with a small domain deletion mutant in Chlamydomonas reinhardtii. The mutant is able to grow photoautotrophically but with a slower rate than the wild type strain. The heme group is covalently attached to the polypeptide, and the visible absorption spectrum of the mutant protein is identical to that of the native protein. The kinetics of electron transfer in the mutant were measured by flash kinetic spectroscopy. Our results show that the rate for the oxidation of cytochrome f was unchanged (t(12) = approximately 100 micros), but the half-time for the reduction of cytochrome f is increased (t(12) = 32 ms; for wild type, t(12) = 2.1 ms). Cytochrome b(6) reduction was slower than that of the wild type by a factor of approximately 2 (t(12) = 8.6 ms; for wild type, t(12) = 4.7 ms); the slow phase of the electrochromic band shift also displayed a slower kinetics (t(12) = 5.5 ms; for wild type, t(12) = 2.7 ms). The stability of the cytochrome b(6)f complex in the mutant was examined by following the kinetics of the degradation of the individual subunits after inhibiting protein synthesis in the chloroplast. The results indicate that the cytochrome b(6)f complex in the small domain deletion mutant is less stable than in the wild type. We conclude that the small domain is not essential for the biogenesis of cytochrome f and the cytochrome b(6)f complex. However, it does have a role in electron transfer through the cytochrome b(6)f complex and contributes to the stability of the complex.

The reactive microglias induced by 25 micromol of beta-amyloid peptides (Abeta25-35) and/or IFN-gamma can initiate the microglial respiratory burst and release NO, respectively. Oxidative stress and inflammatory function have been implicated in Alzheimer's disease (AD). We showed that 10 micromol 17-beta-estradiol (E2) and 1-10 micromol ginsenoside Rg1 (Rg1) could prevent the toxicity of Abeta25-35 and/or IFN-gamma to microglias, inhibit the microglial respiratory burst activity and decrease the accumulation of NO. These results demonstrated the protectional effect of E2 or Rg1 on neuron from damaging by reactive microglias in AD.