Purification and renaturation of recombinant human lymphotoxin (tumour necrosis factor beta) expressed in Escherichia coli as inclusion bodies

High level expression of recombinant human tumour necrosis factor β (rh TNF-β) in Escherichia coli results in the formation of two portions of protein, namely soluble active protein and insoluble protein which is inactive and aggregates in the form of inclusion bodies (IBs). In this study, a procedure for purification and renaturation of rh TNF-β from inclusion bodies has been designed and verified experimentally with a product purity of more than 90% and a recovery of about 30%. The procedure includes washing of IBs with specific wash buffer (Triton X-100/EDTA/lysozyme/PMSF), their solubilization with 8 mol dm^?3 alkaline urea, purification with ion-exchange columns, refolding with renaturation buffer and finally concentration and desalination with an ultrafiltration membrane. The characteristics of the renatured protein were identical with those of purified protein from the soluble fraction as demonstrated by (1) SDS-PAGE, (2) cytotoxic activity on mouse L929 cells, (3) N-terminal amino acid sequence, and (4) gel filtration chromatography.     

Liquid–liquid extraction of a recombinant protein with reverse micelles

Recombinant cytochrome bb>5b> was extracted into the reversed micelle phase of an anionic surfactant (AOT) in octane and back-extracted to a final aqueous phase. The extraction of the protein was controlled by an electrostatic mechanism, since it was dependent on the global charge of the protein. This was directly demonstrated by experiments with native and mutant cytochromes obtained by site directed mutagenesis. The back-extraction of cytochrome bb>5b> to a fresh aqueous phase was decreased by factors that reduced the size of the water pool of the organic phase, such as high salt concentrations (1–2 mol dm^?3 NaCl) and low temperatures (4°C), probably because of an increase in a favourable interaction of this protein with the surfactant at closer distances.     

Direct atomic scale analysis of the distribution of Cu valence states in Cu/γ-Al2O3 catalysts

In this paper, the microstructure of a 1 wt.% Cu/b3;-Alb>2b>Ob>3b> catalyst that was reduced in a 4% hydrogen/argon atmosphere at temperatures of 523, 773 and 1073 K, is studied by Z-contrast imaging and electron energy-loss spectroscopy (EELS) in a scanning transmission electron microscope (STEM). Results show that the copper species are well dispersed when the catalyst is reduced below 523 K. At 773 K, separated Cu(I) and Cu(0) species are found existing as ring-like and bulk-like particles. This appears to indicate that the copper has not been reduced to its metallic form due to the interaction between the copper oxide and the support. Large spherical particles having core-shell structures with Cu(I) in the shells and Cu(0) in the cores are generated when the catalyst is reduced at 1073 K. The formation of partially oxidized copper species upon reduction at 1073 K is attributed to the metallic copper interaction with the alumina support. This study also demonstrates that high-spatial resolution Z-contrast imaging and EELS performed simultaneously can provide unique information on the morphology and chemistry of metal species in supported metal catalysts.     

An improved cryofixation method: cryoquenching of small tissue blocks during microwave irradiation

The metal contact method of rapid freezing is greatly improved by irradiating the specimen with microwaves at 2.45 GHz for a short period of time (50 ms), while pushing the specimen onto the surface of the copper block cooled by liquid Nb>2b>. The microwave irradiation, together with two technical improvements (a light-mass plunger and a recently developed β-gel shock absorber) for preventing bounce, produces a good freezing zone for squid retina, with high reproducibility for each experimental trial, extending from the contact surface to a depth of about 15 μm, which is comparable to the depth obtained by the metal contact method using liquid He in the absence of microwave irradiation. A good freezing zone was also experimentally demonstrated in specimens of rat liver and heart muscle. Microwave irradiation does not have appreciable effects on the ultrastructure of squid retina. The mechanism underlying the improvement in the rapid freezing under the microwave irradiation is discussed.     

Effects of irradiation in combination with waxing on the essential oils in orange peel

The study evaluated the effects of waxing, irradiation dose and post‐irradiation storage time on the essential oils in orange peel. Mature oranges (Maroc late) waxed or unwaxed were treated with 1 or 2 kGy radiation and stored for 9 weeks at 20 °C and 40–50% RH. Essential oils from the peel were extracted and analysed by GC. Results showed that D ‐limonene was significantly lower in waxed oranges; levels in samples treated with 2 kGy were higher than those in non‐irradiated or treated with 1 kGy. Linalool, methyl anthranilate and 3,7‐dimethyl‐2,6‐octadienal decreased as the dose increased. Levels of linalool increased with storage time but D ‐limonene was unaffected. These results may aid in guaranteeing the quality of orange essential oils during storage time. Copyright © 2004 Society of Chemical Industry     

Photocatalytic degradation of 2,2‐bis(4‐hydroxy‐3‐methylphenyl) propane (BPP) based on molecular recognition interaction

BACKGROUND: Endocrine disruptors in the aquatic environment and their potential adverse effects are currently issues of concern. One of these endocrine disruptors is 2,2‐bis(4‐hydroxy‐3‐methylphenyl)propane (BPP). In this work the molecular recognition interaction of BPP with β‐cyclodextrin (β‐CD) was studied using IR spectroscopy and steady state fluorescence spectroscopy, and the photocatalytic degradation behaviour of BPP based on molecular recognition interaction was investigated in a TiOb>2b>/UV–visible (λb>maxb> = 365 nm) system. This might provide a new method for the treatment of some organic pollutants in wastewater. RESULTS: β‐CD reacts with BPP to form a 1:1 inclusion complex, the formation constant of which is 4.94 × 10³ L mol^?1. The photodegradation rate constant of BPP after molecular recognition by β‐CD showed a 1.42‐fold increase in the TiOb>2b>/UV–visible (λb>maxb> = 365 nm) system. The photodegradation of BPP depended on the concentration of β‐CD, the pH value, the gaseous medium and the initial concentration of BPP. The photodegradation efficiency of BPP with molecular recognition was higher than that without molecular recognition. After 100 min of irradiation the mineralisation efficiency of BPP after molecular recognition by β‐CD reached 94.8%, whereas the mineralisation efficiency of BPP before molecular recognition by β‐CD was only 40.6%. CONCLUSION: The photocatalytic degradation of BPP after molecular recognition by β‐CD can be enhanced in the TiOb>2b>/UV‐visible (λb>maxb> = 365 nm) system. This enhancement is dependent on the enhancement of the adsorption of BPP, the moderate inclusion depth of BPP in the β‐CD cavity and the increase in the frontier electron density of BPP after molecular recognition. Copyright © 2008 Society of Chemical Industry     

One‐step production of 2,3‐butanediol from starch by secretory over‐expression of amylase in Klebsiella pneumoniae

BACKGROUND: 2,3‐Butanediol (2,3‐BD) is a valuable chemical that can be biosynthesized from many kinds of substrates. For commercial biological production of 2,3‐BD, it is desirable to use cheap substrate without pretreatment, such as starch. However, there have been few reports on the production of 2,3‐BD directly from starch. RESULTS: In this work, gene malS coding for α‐amylase (EC 3.2.1.1) precursor was inserted into plasmid pUC18K, and secretory over‐expression of α‐amylase was achieved by engineered Klebsiella pneumoniae. The extracellular recombinant amylase accelerated the hydrolyzation of starch, and one‐step production of 2,3‐BD from starch was carried out by engineered K. pneumoniae. A 2,3‐BD concentration of 3.8 g L^?1 and yield of 0.19 g 2,3‐BD g^?1 starch were obtained after 24 h fermentation. CONCLUSION: The one‐step production of 2,3‐BD from starch was achieved by secretory over‐expression of amylase in K. pneumoniae. This would simplify the process and reduce the production cost considerably by enabling use of starch with minimal pretreatment. Copyright © 2008 Society of Chemical Industry     

Kinetics study of Ti[O(CH2)4OCHCH2]4 initiated ring‐opening polymerization of ε‐caprolactone by differential scanning calorimetry

Chemical shrinkage was used for the in situ measurement of the progressing chemical stabilization reactions and the influence of ozone during the stabilization of polyacrylonitrile. A method for evaluating the activation energy through the sensitivity temperature is presented. The calculated results show that the activation energies were 161.57 kJ/mol in air and 181.23 kJ/mol in ozone-enriched air. Therefore, the chemical reactions were postponed during stabilization in ozone-enriched air. Ozone seemed to act in three ways: first, ozone promoted the formation of the serious skin–core structure. Second, ozone accelerated the chemical reactions and shortened the stabilization time at lower heating rates. Third, ozone postponed the chemical reactions. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci 2008     

Maghnite‐H+, a solid catalyst for the cationic polymerization of α‐methylstyrene

The polymerization of α‐methylstyrene (AMS) catalyzed by Maghnite‐H⁺ (Mag‐H) was investigated. Mag‐H is a montmorillonite sheet silicate clay, exchanged with protons. It was found that the cationic polymerization of AMS is initiated by Mag‐H at ambient temperature in bulk and in solution. The effect of the amount of Mag‐H, the temperature, and the solvent was studied. The polymerization rate increased with increase in the temperature and the proportion of catalyst, and it was larger in nonpolar solvents. These results indicated the cationic nature of the polymerization. It may be suggested that the polymerization is initiated by proton addition to monomer from Mag‐H. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

基于互信息域的L2S接口方法研究

分析了基于互信息域的有效信噪比映射的L2S(link level to system level)接口方法,研究了信道相关带宽对接口复杂度的影响,在不明显降低预测准确度的前提下,验证了利用信道相关带宽来简化接口计算复杂度的可能性。