A two-phase partitioning bioreactor system for treating benzene-contaminated soil

Benzene-contaminated topsoil, with an organic content of 42%, was treated by an air volatilization process, followed by a two-phase partitioning bioreactor to allow benzene mineralization. The effects of moisture content and temperature on the adsorption and desorption of benzene on to soil were investigated, and 95% of the benzene (at a concentration equivalent to 3.7 kg benzene m^–3 soil^–1) was removed at 50°C by air volatilization. When 30 g soil was contaminated with 1000 mg benzene (a concentration 3 times higher), 93% of the benzene was removed by the air volatilization technique, of which 91% was consumed in a two-phase partitioning bioreactor within 2 h.     

Effect of short interspersed element sequences on the integration and expression of a reporter gene in the preimplantation-stage mouse embryos

Based on the assumption that foreign DNA sequences may have increased chance of integration into the host genome if they are flanked by high copy-numbered genomic sequences such as SINEs (short interspersed elements), we investigated the integration frequency of Lac Z reporter gene flanked by a fused B1/B2 in an in vivo system using pronuclear microinjection technique in the mouse. The SINE-flanked DNA showed a 4-fold increased integration frequency of the reporter gene than the control DNA (63% vs. 16%). Moreover, the level of beta-galactosidase expression, estimated from the X-Gal staining intensity in transgenic embryos, was greatly higher in SINE-carrying DNA. These results suggest that the SINE sequences can serve a very useful tool in improving the efficiency of current transgenic animal technology.     

Recycling wasted biomaterial,crab shells,as an adsorbent for the removal of high concentration of phosphate

In this study, crab shells were recycled as an adsorbent for the removal of phosphate. The effects of shell particle size, temperature, pH and phosphate concentration on phosphate removal were investigated. Shell particles less than 1000 μm in diameter removed more than 85% of 500 mg/L phosphate in 24 h while particles 3350 μm in diameter exhibited only 50% removal efficiency. Temperature showed negligible effect on phosphate removal in the range of 15–45 °C. Although removal efficiency was highest at pH 2.0, the efficiency remained 50–60% at pH of 4.0–10.0. The maximum removal capacity was calculated as 108.9 mg/g through Langmuir isotherm plotting, which was 17.0 and 4.7 times higher than those of coal fly ash and scallop shells, respectively. Although calcium carbonate played an active role in the removal of phosphate, both proteins composing 12.5% of crab shells and cellulose-like backbone of the crab shells also played an important role in phosphate removal.     

NDRG3-mediated lactate signaling in hypoxia

Hypoxia is associated with many pathological conditions as well as the normal physiology of metazoans. We identified a lactate-dependent signaling pathway in hypoxia, mediated by the oxygen- and lactate-regulated protein NDRG family member 3 (NDRG3). Oxygen negatively regulates NDRG3 expression at the protein level via the PHD2/VHL system, whereas lactate, produced in excess under prolonged hypoxia, blocks its proteasomal degradation by binding to NDRG3. We also found that the stabilized NDRG3 protein promotes angiogenesis and cell growth under hypoxia by activating the Raf-ERK pathway. Inhibiting cellular lactate production abolishes NDRG3-mediated hypoxia responses. The NDRG3-Raf-ERK axis therefore provides the genetic basis for lactate-induced hypoxia signaling, which can be exploited for the development of therapies targeting hypoxia-induced diseases in addition to advancing our understanding of the normal physiology of hypoxia responses. [BMB Reports 2015; 48(6): 301-302]     

E.?coli Outer Membrane and Interactions with OmpLA

The outer membrane of Gram-negative bacteria is a unique asymmetric lipid bilayer composed of phospholipids (PLs) in the inner leaflet and lipopolysaccharides (LPSs) in the outer leaflet. Its function as a selective barrier is crucial for the survival of bacteria in many distinct environments, and it also renders Gram-negative bacteria more resistant to antibiotics than their Gram-positive counterparts. Here, we report the structural properties of a model of the Escherichia coli outer membrane and its interaction with outer membrane phospholipase A (OmpLA) utilizing molecular dynamics simulations. Our results reveal that given the lipid composition used here, the hydrophobic thickness of the outer membrane is ∼3 Å thinner than the corresponding PL bilayer, mainly because of the thinner LPS leaflet. Further thinning in the vicinity of OmpLA is observed due to hydrophobic matching. The particular shape of the OmpLA barrel induces various interactions between LPS and PL leaflets, resulting in asymmetric thinning around the protein. The interaction between OmpLA extracellular loops and LPS (headgroups and core oligosaccharides) stabilizes the loop conformation with reduced dynamics, which leads to secondary structure variation and loop displacement compared to that in a DLPC bilayer. In addition, we demonstrate that the LPS/PL ratios in asymmetric bilayers can be reliably estimated by the per-lipid surface area of each lipid type, and there is no statistical difference in the overall membrane structure for the outer membranes with one more or less LPS in the outer leaflet, although individual lipid properties vary slightly.     

Substrate specificity of ribose-5-phosphate isomerases from <Emphasis Type="Italic">Clostridium difficile</Emphasis> and <Emphasis Type="Italic">Thermotoga maritima</Emphasis>

The activity of ribose-5-phosphate isomerases (RpiB) from Clostridium difficile for d-ribose isomerization was optimal at pH 7.5 and 40°C, while that from Thermotoga maritima for l-talose isomerization was optimal at pH 8.0 and 70°C. C. difficile RpiB exhibited activity only with aldose substrates possessing hydroxyl groups oriented in the right-handed configuration (Fischer projections) at the C2 and C3 positions, such as d-ribose, d-allose, l-talose, l-lyxose, d-gulose, and l-mannose. In contrast, T. maritima RpiB displayed activity only with aldose substrates possessing hydroxyl groups configured the same direction at the C2, C3, and C4 positions, such as the d- and l-forms of ribose, talose, and allose.     

Mannose production from fructose by free and immobilized <Emphasis Type="SmallCaps">d</Emphasis>-lyxose isomerases from <Emphasis Type="Italic">Providencia stuartii</Emphasis>

A recombinant d-lyxose isomerase from Providencia stuartii was immobilized on Duolite A568 beads which gave the highest conversion of d-fructose to d-mannose among the various immobilization beads evaluated. Maximum activities of both the free and immobilized enzymes for fructose isomerization were at pH 7.5 and 45°C in the presence of 1 mM Mn²⁺. Enzyme half-lives were 14 and 30 h at 35°C and 3.4 and 5.1 h at 45°C, respectively. The immobilized enzyme in 300 g fructose/l (replaced hourly), produced 75 g mannose/l at 35°C = 25% (w/w) yield with a productivity of 75 g mannose l⁻¹ h⁻¹ after 23 cycles.     

Substrate specificity of a recombinant d‐lyxose isomerase from Serratia proteamaculans that produces d‐lyxose and d‐mannose

Aims: Characterization of substrate specificity of a d ‐lyxose isomerase from Serratia proteamaculans and application of the enzyme in the production of d ‐lyxose and d ‐mannose. Methods and Results: The concentrations of monosaccharides were determined using a Bio‐LC system. The activity of the recombinant protein from Ser. proteamaculans was the highest for d ‐lyxose among aldoses, indicating that it is a d‐ lyxose isomerase. The native recombinant enzyme existed as a 54‐kDa dimer, and the maximal activity for d‐ lyxose isomerization was observed at pH 7·5 and 40°C in the presence of 1 mmol l^?1 Mn²⁺. The K_m values for d ‐lyxose, d ‐mannose, d ‐xylulose, and d ‐fructose were 13·3, 32·2, 3·83, and 19·4 mmol l^?1, respectively. In 2 ml of reaction volume at pH 7·5 and 35°C, d ‐lyxose was produced at 35% (w/v) from 50% (w/v) d ‐xylulose by the d‐ lyxose isomerase in 3 h, while d ‐mannose were produced at 10% (w/v) from 50% (w/v) d ‐fructose in 5 h. Conclusions: We identified the putative sugar isomerase from Ser. proteamaculans as a d ‐lyxose isomerase. The enzyme exhibited isomerization activity for aldose substrates with the C2 and C3 hydroxyl groups in the left‐hand configuration. High production rates of d‐ lyxose and d ‐mannose by the enzyme were obtained. Significance and Impact of the Study: A new d‐ lyxose isomerase was found, and this enzyme had higher activity for d ‐lyxose and d ‐mannose than previously reported enzymes. Thus, the enzyme can be applied in industrial production of d ‐lyxose and d ‐mannose.     

Enantioselective production of 2,2-dimethylcyclopropane carboxylic acid from 2,2-dimethylcyclopropane carbonitrile using the nitrile hydratase and amidase of Rhodococcus erythropolis ATCC 25544

The enantioselective production of (S)-2,2-dimethylcyclopropane carboxylic acid was investigated in 53 Rhodococcus and Pseudomonas related strains. Rhodococcus erythropolis ATCC 25544 was selected as it showed the highest enantioselectivity. The enantioselectivity was due to the amidase activity in a two-step reaction involving nitrile hydratase. The enantiomeric excess of the amidase was highest at pH 7.0 and decreased significantly above 20 °C. For the enantioselective production of (S)-2,2-dimethylcyclopropane carboxylic acid, the optimum reaction conditions of the cells were determined to be pH 7.0, 20 °C, and 10% (v/v) methanol and were the same as the optimum pH and temperature for the enantioselective conversion by the amidase. Under these conditions, the R. erythropolis ATCC 25544 cells, which harbored nitrile hydratase and amidase enzymes, produced 45 mM (S)-2,2-dimethylcyclopropane carboxylic acid from racemic 100 mM 2,2-dimethylcyclopropane carbonitrile with an 81.8% enantiomeric excess after 64 h.