Preparation of Black Copper Electrolyte by Acid Oxidation Leaching of Refining Slag

This is an article in the field of metallurgical engineering. The preparation of black copper electrolyte by removing copper from secondary refining slag of high copper and high tin anode furnace by hydrogen peroxide oxidation and acid leaching was studied. The process conditions related to the removal efficiency of the copper and arsenic were researched including the hydrogen peroxide dosage, reaction temperature, sulfuric acid concentration and reaction time. Results show that, under the following optimal conditions, including refining slag of 20.0 g, 2.5 mol/L sulfuric acid of 200 mL, solid-liquid ratio of 1/10, temperature of 65 ℃, dosage of hydrogen peroxide of 10.0 mL, stirring speed of 400 r/min and leaching time of 60 min, the leaching ratios of Cu and As can reach 88.98% and 87.33%, respectively, and the leaching ratios of Ni and Sn were only 7.72% and 1.34%, respectively. The concentration of copper ion in the leaching solution was 48.48 g/L, which can be used as the supplement of black copper electrolyte

Biochemical characterization of a haloalkane dehalogenase DadB from Alcanivorax dieselolei B-5.

Recently, we found that Alcanivorax bacteria from various marine environments were capable of degrading halogenated alkanes. Genome sequencing of A. dieselolei B-5 revealed two putative haloalkane dehalogenase (HLD) genes, which were supposed to be involved in degradation of halogenated compounds. In this report, we confirm for the first time that the Alcanivorax bacterium encodes a truly functional HLD named DadB. An activity assay with 46 halogenated substrates indicated that DadB possesses broad substrate range and has the highest overall activity among the identified HLDs. DadB prefers brominated substrates; chlorinated alkenes; and the C2-C3 substrates, including the persistent pollutants of 1,2-dichloroethane, 1,2-dichloropropane and 1,2,3-trichloropropane. As DadB displays no detectable activity toward long-chain haloalkanes such as 1-chlorohexadecane and 1-chlorooctadecane, the degradation of them in A. dieselolei B-5 might be attributed to other enzymes. Kinetic constants were determined with 6 substrates. DadB has highest affinity and largest k cat/K m value toward 1,3-dibromopropane (K(m) = 0.82 mM, k(cat)/K(m) = 16.43 mM(-1) · s(-1)). DadB aggregates fast in the buffers with pH ≤ 7.0, while keeps stable in monomer form when pH ≥ 7.5. According to homology modeling, DadB has an open active cavity with a large access tunnel, which is supposed important for larger molecules as opposed to C2-C3 substrates. Combined with the results for other HLDs, we deduce that residue I247 plays an important role in substrate selection. These results suggest that DadB and its host, A. dieselolei B-5, are of potential use for biocatalysis and bioremediation applications

Biochemical Characterization of a Haloalkane Dehalogenase DadB from Alcanivorax dieselolei B-5

National Science Foundation of China [41176154]; Public Welfare Project of SOA [201005032]; COMRA project [DY125-15-R-01]; International Sci & Tech Cooperation Program of China [2010DFB23320]Recently, we found that Alcanivorax bacteria from various marine environments were capable of degrading halogenated alkanes. Genome sequencing of A. dieselolei B-5 revealed two putative haloalkane dehalogenase (HLD) genes, which were supposed to be involved in degradation of halogenated compounds. In this report, we confirm for the first time that the Alcanivorax bacterium encodes a truly functional HLD named DadB. An activity assay with 46 halogenated substrates indicated that DadB possesses broad substrate range and has the highest overall activity among the identified HLDs. DadB prefers brominated substrates; chlorinated alkenes; and the C-2-C-3 substrates, including the persistent pollutants of 1, 2-dichloroethane, 1,2-dichloropropane and 1,2,3-trichloropropane. As DadB displays no detectable activity toward long-chain haloalkanes such as 1-chlorohexadecane and 1-chlorooctadecane, the degradation of them in A. dieselolei B-5 might be attributed to other enzymes. Kinetic constants were determined with 6 substrates. DadB has highest affinity and largest k(cat)/K-m value toward 1,3-dibromopropane (K-m = 0.82 mM, k(cat)/K-m = 16.43 mM(-1).s(-1)). DadB aggregates fast in the buffers with pH = 7.5. According to homology modeling, DadB has an open active cavity with a large access tunnel, which is supposed important for larger molecules as opposed to C-2-C-3 substrates. Combined with the results for other HLDs, we deduce that residue I247 plays an important role in substrate selection. These results suggest that DadB and its host, A. dieselolei B-5, are of potential use for biocatalysis and bioremediation applications

SDS-PAGE analysis of the expression and purification of DadB.

<p>M, marker; lane 1, supernatant of <i>E. coli</i> BL21(DE3) without any vectors; lane 2, supernatant before induction; lane 3: supernatant after induction; lane 4, resuspended precipitate; lane 5, flow-through after binding; and lane 6, purified DadB.</p

Active sites and access tunnels of LinB, DadB and DhaA.

<p>Yellow, cyan, green and red indicate the halide-binding residues, the basic residues, the acidic residues, and the nucleophile residues, respectively. Magenta indicates the two residues at the adjacent position of the main tunnel and the slot tunnel. The pictures were produced with Swiss-PdbViewer 4.04.</p

Kinetic parameters of DadB and other HLDs.

a<p>Chovancova, 2011(Doctoral dissertation).</p>b<p>Jesenska et al., 2005.</p>c<p>Sato et al., 2005.</p>d<p>Nagata et al., 1999.</p>e<p>Chovancova, 2003.</p

Specific activity (nmol·s⁻¹·mg⁻¹) of DadB and comparison with other HLDs.

<p>Group A, chlorinated or brominated alkanes longer than C₃; Group B, chlorinated or brominated C₂ and C₃ alkanes; Group C, iodinated alkanes and halogenated cycloalkanes, alkenes, ethers, nitriles; Group D, other substrates used in activity assay of DadB. ND, no detectable activity. NA, no available activity. 2-Iodobutane is unstable and its spontaneous dehalogenation interferes the detection of iodine ion concentration seriously, so accurate activity to 2-iodobutane was not obtained.</p>a<p>The activity data of these five HLDs were collected from the paper of Koudelakova et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089144#pone.0089144-Koudelakova1" target="_blank">[18]</a>.</p

Transcriptome Analysis Reveals a Promotion of Carotenoid Production by Copper Ions in Recombinant <i>Saccharomyces cerevisiae</i>

We previously constructed a Saccharomyces cerevisiae carotenoid producer BL03-D-4 which produced much more carotenoid in YPM (modified YPD) media than YPD media. In this study, the impacts of nutritional components on carotenoid accumulation of BL03-D-4 were investigated. When using YPM media, the carotenoid yield was increased 10-fold compared to using the YPD media. To elucidate the hidden mechanism, a transcriptome analysis was performed and showed that 464 genes changed significantly in YPM media. Furthermore, inspired by the differential gene expression analysis which indicated that ADY2, HES1, and CUP1 showed the most remarkable changes, we found that the improvement of carotenoid accumulation in YPM media was mainly due to the copper ions, since supplementation of 0.08 mM CuSO4 in YPD media could increase carotenoid yield 9.2-fold. Reverse engineering of target genes was performed and carotenoid yield could be increased 6.4-fold in YPD media through overexpression of ACE1. The present study revealed for the first time the prominent promotion of carotenoid yield by copper ions in engineered S. cerevisiae and provided a new target ACE1 for genetic engineering of S. cerevisiae for the bioproduction of carotenoids

Identification of a novel metabolic engineering target for carotenoid production in Saccharomyces cerevisiae via ethanol-induced adaptive laboratory evolution

Abstract Carotenoids are a large family of health-beneficial compounds that have been widely used in the food and nutraceutical industries. There have been extensive studies to engineer Saccharomyces cerevisiae for the production of carotenoids, which already gained high level. However, it was difficult to discover new targets that were relevant to the accumulation of carotenoids. Herein, a new, ethanol-induced adaptive laboratory evolution was applied to boost carotenoid accumulation in a carotenoid producer BL03-D-4, subsequently, an evolved strain M3 was obtained with a 5.1-fold increase in carotenoid yield. Through whole-genome resequencing and reverse engineering, loss-of-function mutation of phosphofructokinase 1 (PFK1) was revealed as the major cause of increased carotenoid yield. Transcriptome analysis was conducted to reveal the potential mechanisms for improved yield, and strengthening of gluconeogenesis and downregulation of cell wall-related genes were observed in M3. This study provided a classic case where the appropriate selective pressure could be employed to improve carotenoid yield using adaptive evolution and elucidated the causal mutation of evolved strain

Long-Term Effect against Methicillin-Resistant Staphylococcus aureus of Emodin Released from Coaxial Electrospinning Nanofiber Membranes with a Biphasic Profile

Methicillin-resistant Staphylococcus aureus (MRSA) is a serious and rapidly growing threat to human beings. Emodin has a potent activity against MRSA; however, its usage is limited due to high hydrophobicity and low oral bioavailability. Thus, the coaxial electrospinning nanofibers encapsulating emodin in the core of hydrophilic poly (vinylpyrrolidone), with a hygroscopic cellulose acetate sheath, have been fabricated to provide long-term effect against MRSA. Scanning electron microscopy and transmission electron microscopy confirmed the nanofibers had a linear morphology with nanometer in diameter, smooth surface, and core-shell structure. Attenuated total reflection-Fourier transform infrared spectra, X-ray diffraction patterns, and differential scanning calorimetric analyses verified emodin existed in amorphous form in the nanofibers. The nanofibers have 99.38 &plusmn; 1.00% entrapment efficiency of emodin and 167.8 &plusmn; 0.20% swelling ratio. Emodin released from nanofibers showed a biphasic drug release profile with an initial rapid release followed by a slower sustained release. CCK-8 assays confirmed the nontoxic nature of the emodin-loaded nanofibers to HaCaT cells. The anti-MRSA activity of the nanofibers can persist up to 9 days in AATCC147 and soft-agar overlay assays. These findings suggest that the emodin-loaded electrospun nanofibers with core-shell structure could be used as topical drug delivery system for wound infected by MRSA