直链醇对反胶束体系中木素过氧化物酶催化活性的影响

根据研究发现, 在有醇作助表面活性剂的CTAB反胶束中木素过氧化物酶(LiP)不能表现活力, 而在水介质中CTAB对LiP的催化活性影响又不是很大. 为了揭示其中醇的影响, 本工作就不同碳链长度的醇对LiP酶催化性能的影响进行了研究. 由于CTAB反胶束体系中醇浓度较高, 且碳原子数大于4的直链醇在水中的溶解度又很小, 为此采用了LiP可在其中显示催化活性的CTAB正胶束、AOT反胶束和Brij30反胶束作介质, 通过研究这些介质中不同链长的醇对LiP催化活力的影响, 来探讨CTAB反胶束中木素过氧化物酶(LiP)不能表现活力的原因. 结果表明, 不管表面活性剂聚集体的结构、电性质及反胶束大小如何, 只要醇的浓度超过500 mmol•L^－1 (丁醇≥1200 mmol•L^－1), LiP在上述原本可显示活力的介质中均无催化活性. 据此推测CTAB反胶束中木素过氧化物酶(LiP)不能表现活力的原因主要是由助表面活性剂醇造成的.     

Lignin peroxidase and protease production by Streptomyces viridosporus T7A in the presence of calcium carbonate

Streptomyces are good producers of enzymes of industrial interest, such as lignin peroxidase (LiP) and proteases. To optimize production of these enzymes by Streptomyces viridosporus T7A, two parameters were evaluated: carbon sources and calcium carbonate. Shake-flask fermentations were performed using culture media, with and without CaCO₃, contained yeast extract, mineral salts and either glucose, lactose, galactose, or corn oil. In the absence of calcium carbonate, the maximum values for LiP and protease activities occurred during the idiophase with LiP activity being favored by glucose, corn oil, and galactose, and protease activity being favored only by corn oil. Calcium carbonate affected the cell morphology by reducing the size of the pellets. Moreover, in the presence of the salt, LiP production was growth-associated in all media but the glucose medium. Higher enzyme levels were observed when galactose and glucose were used as carbon sources. Protease activity was repressed by both glucose and galactose, whereas corn oil was the best carbon source for the enzyme production. Calcium carbonate increased LiP production by up to 2.6-fold. Such improvement was not observed for protease production, suggesting a selective effect of CaCO₃ on LiP activity.     

Purification and characterization of organic solvent stable serine alkaline protease from newly isolated Bacillus circulans M34

A protease from newly isolated Bacillus circulans M34 was purified by Q‐Sepharose anion exchange chromatography and Sepharose–bacitracin affinity chromatography followed by (NH₄)₂SO₄ precipitation. The molecular mass of the purified enzyme was determined using SDS–PAGE. The optimum pH and temperature for protease activity were 11 and 50°C, respectively. The effect of various metal ions on protease activity was investigated. Alkaline protease from Bacillus circulans M34 wase activated by Zn²⁺, Cu²⁺ and Co²⁺ up to 31%. The purified protease was found to be stable in the organic solvents, surfactants and oxidizing agent. The substrate specificity of purified protease was investigated towards different substrates. The protease was almost completely inhibited by the serine protease inhibitor phenylmethanesulfonyl fluoride. The kinetic parameters of the purified protease, maximum rate (V_max) and Michaelis constant (K_m), were determined using a Lineweaver–Burk plot. Copyright © 2015 John Wiley & Sons, Ltd.     

Use of steam explosion liquor from sugar cane bagasse for lignin peroxidase production by Phanerochaete chrysosporium

The possibility of using two by-products of the sugar cane industry, molasses and bagasse steam explosion liquor (SEL), for lignin peroxidase (LiP) production by Phanerochaete chrysosporium was investigated. For comparison, the fungus was initially cultivated in synthetic media containing either glucose, sucrose, xylose, or xylan as sole carbon sources. The effect of veratryl alcohol (VA) was also investigated in relation to the enzyme activity levels. Results showed that sucrose was not metabolized by this fungus, which precluded the use of molasses as a carbon source. Glucose, xylose, and xylan promoted equivalent cell growth. Enzyme levels in the absence of VA were lower than 28 UI/L and in the presence of VA reached 109 IU/L with glucose and 85 IU/L with xylose or xylan. SEL was adequate for P. chrysosporium LiP production as LiP activity reached 90 IU/L. When VA was added to this medium, enzyme concentration increased to 155 IU/L.     

Purification and Characterization of Thermostable α-Galactosidase from <Emphasis Type="Italic">Aspergillus terreus</Emphasis><Subscript>GR</Subscript>

An extracellular thermostable α-galactosidase producing Aspergillus terreus _GR strain was isolated from soil sample using guar gum as sole source of carbon. It was purified to apparent homogeneity by acetone precipitation, gel filtration followed by DEAE-Sephacel chromatographic step. The purified enzyme showed a single band after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the purified enzyme after SDS-PAGE was 108 kDa. The enzyme showed optimum pH and temperature of 5.0 and 65 °C, respectively, for artificial substrate pNPαGal. α-Galactosidase from A. terreus _GR is found to be thermostable, as it was not inactivated after heating at 65 °C for 40 min. The K _m for pNPαGal, oNPαGal, raffinose, and stachyose are 0.1, 0.28, 0.42, and 0.33 mM, respectively. Inhibitors such as 1,10-phenanthroline, phenylmethylsulfonyl fluoride, ethylenediaminetetraacetic acid, mercaptoethanol, and urea have no effect, whereas N-bromosuccinamide inhibited enzyme activity by 100%. Among metal ions tested, Mg²⁺, Ni²⁺, Ca²⁺, Co²⁺, and Mn²⁺ had no effect on enzyme activity, but Ag⁺, Hg²⁺, and Cu²⁺ have inhibited complete activity.     

Chemically bonded multiwalled carbon nanotubes as efficient material for solid phase extraction of some metal ions in food samples

Multiwalled carbon nanotubes chemically functionalized with 2-((3-silylpropylimino) methyl) phenol (SPIMP-MWCNT) and successfully applied for the solid phase extraction (SPE) of some metal ions in food samples. The influences of the analytical parameters including pH, amounts of solid phase, eluent conditions (type, volume and concentrations), sample volume and interference of some metal ions on the recoveries of ions Cu²⁺, Pb²⁺, Fe²⁺, Ni²⁺ and Zn²⁺ ion were investigated. The metal ions retained on SPIMP-MWCNT was eluted using 6?mL of 4?mol?L^?1 HNO₃ solution and their content was determined by flame atomic absorption spectrometry (FAAS) with recoveries more than 95% and relative standard deviations (n?=?5) between 2.4–3.4% for both reproducibility and repeatability. The detection limit of this metal ions was between 1.0–2.6?ng?mL^?1 (3S _b , n?=?10) and their preconcentration factor was 100, while their loading capacity was above 32.9?mg?g^?1 of SPIMP-MWCNT. The proposed method was successfully applied for the preconcentration and determination of analytes in different samples.     

金属离子对氯过氧化物酶在25℃到55℃激活作用以及机理的研究

基于氯过氧化物酶（CPO）的卤化活性分析,发现某些碱土金属（Ca2+, Mg2+）和过渡金属（Co2+, Ni2+）对CPO具有明显的激活及稳定化作用。例如25 ºC时与CPO在纯缓冲溶液中相比,在75 μmol·L-1 Ca2+,90 μmol·L-1 Mg2+,90 μmol·L-1 Ni2+及105 μmol·L-1 Co2+存在时CPO可分别获得1.33,1.37,1.34 及1.27倍的最大相对活性。而在55 ºC,没有金属离子存在时,CPO 30分钟后仅能保留40%的活性,但在Ca2+,Mg2+离子的介质中,CPO的活性可分别保留81% 和 75%。推测这是由于金属离子结合在CPO活性中心周围的酸-碱催化位点Glu183, His105 and Asp106上,通过底物浓集和诱导有利构象来激活CPO. 同时动力学研究表明金属离子对CPO的激活归因于催化效率（kcat）的提高,以及CPO对底物亲和性及选择性的改善。     

Production and Physico-chemical Characterization of a Biosurfactant Produced by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> OBP1 Isolated from Petroleum Sludge

Pseudomonas aeruginosa strain OBP1, isolated from petroleum sludge, was used to produce biosurfactant from a modified mineral salt medium with 2% n-hexadecane as sole source of carbon. The crude biosurfactant was fractionated using TLC and HPLC. Using FTIR spectroscopy, ¹H NMR, and LC-MS analyses, the chemical structure of the purified fraction of crude biosurfactant was identified as rhamnolipid species. The LC-MS spectra show that monorhamnolipid (l-rhamnopyranosyl-β-hydroxydecanoyl-β- hydroxydecanoate, Rha-C₁₀-C₁₀) was produced in abundance with the predominant congener [M–H]⁻ ions for l-rhamnopyranosyl-l-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoate (Rha-Rha-C₁₀-C₁₀). Seven different carbon substrates and five nitrogen sources were examined for their effect on rhamnolipid production. Using n-hexadecane (20 g/l) as carbon substrate and urea along with (NH₄)₂SO₄ (2 g/l each) as nitrogen source was found to be the best, with a maximum yield of 4.8 g/l. The biosurfactant reduced the surface tension of water to 31.1 mNm⁻¹ with a critical micelle concentration of 45 mg/l. The biosurfactant showed a better emulsifying activity against a variety of hydrocarbon and achieved a maximum emulsion index of 82% for diesel. The purified biosurfactant showed a significant antibacterial activity against Staphylococcus aureus at a minimum inhibitory concentration of 8 μg/ml.     

CuAg nanoparticles formed in situ on electrochemically pre‐anodized screen‐printed carbon electrodes for the detection of nitrate and nitrite anions

CuAg nanoparticles (CuAgNPs) were electrochemically formed in situ on pre‐anodized, screen‐printed carbon electrodes (SPCEs) that possessed many oxygen‐containing functional groups capable of adsorbing metal ions, namely Cu²⁺ and Ag⁺. Pre‐anodization was achieved using continuous cyclic voltammetry in the range of potential 0.3–2.0 V under a scan rate of 50 mV/s. Cu²⁺ and Ag⁺ ions were adsorbed on the pre‐anodized SPCE by immersing the electrode in solutions containing both metal ions, and then CuAgNPs were formed in situ via electrochemical reduction in a deaerated, neat NaClO₄ solution after the electrode was ultrasonicated to remove physically adsorbed metal ions. Although CuNPs showed higher activity than AgNPs toward both nitrate (NO₃⁻) and nitrite (NO₂⁻) ions, the instability of CuNPs hindered the application, so CuAgNPs were employed to achieve a compromise between sensitivity and stability. The SPCE/anodized/CuAgNP electrodes showed activity toward the electrochemical reduction of NO₃⁻ and NO₂⁻, respectively, with the limit of detection (LOD) of 15.6 μM (0.97 ppm) and 11.1 μM (0.51 ppm), which is sufficient to fit the allowed values (50 and 3 ppm, respectively) in drinking water as suggested by the World Health Organization (WHO).     

Xylanase production by the thermophilic mold Humicola lanuginosa in solid-state fermentation

Among the lignocellulosic substrates tested, wheat bran supported a high xylanase (EC 3.2.1.8) secretion by Humicola lanuginosa in solid-state fermentation (SSF). Enzyme production reached a peak in 72 h followed by a decline thereafter. Enzyme production was very high (7832 U/g of dry moldy bran) when wheat bran was moistened with tap water at a substrate-to-moistening agent ratio of 1:2.5 (w/v) and an inoculum level of 3 × 10⁶ spores/10 g of wheat bran at a water activity (a _w ) of 0.95. Cultivation of the mold in large enamel trays yielded a xylanase titer comparable with that in flasks. Parametric optimization resulted in a 31% increase in enzyme production in SSF. Xylanase production was approx 23-fold higher in SSF than in submerged fermentation (SmF). A threshold constitutive level of xylanase was secreted by H. lanuginosa in a medium containing glucose as the sole carbon source. The enzyme was induced by xylose and xylan. Enzyme synthesis was repressed beyond 1.0% (w/v) xylose in SmF, whereas it was unaffected up to 3.0% (w/w) in SSF, suggesting a minimization of catabolite repression in SSF.     

Effect of intercalation of metal ions on the colloidal and solid properties of vanadium pentaoxide hydrate,V2O5 nH2O

The colloidal stability of V₂O₅ nH₂O was studied on the basis of the measurements of critical flocculation concentration (CFC) by metal ions, amount of ions exchanged (or intercalated), and -potential. In total, the CFC values obeyed the Schulze Hardy law and strong Hofmeister's series was found in the systems including alkaline ions. The sequence of colloidal stability of V₂O₅ nH₂O in the electrolyte solutions was related to the intercalation of metal ions in the interlayer spaces of the solid. The largest CFC value for Li⁺ (87 mmol dm^–3) was explained by smaller affinity of Li⁺ to be intercalated in V₂O₅ nH₂O as well as smaller Hamaker constant of the intercalated solid compared to the other systems.Effect of intercalation of metal ions on the crystalline properties of the materials was measured by use of XRD and electron microscope. Under highly dehydrated condition the ions whose radii are smaller than 0.1 nm are captured in the structure of V₂O₅ nH₂O without changing interlayer distances, while those larger than 0.1 nm increase the interlayer distance. In a saturated H₂O vapor interlayer distances increased with increasing charge of intercalated ions. However, when intercalated with ions carrying the same valency the interlayer distances of the sample decreased with decrease in the hydration property of ions. Hydrolyzable Cr³⁺ gave exceptionally larger interlayer distances, both in a vacuum and in H₂O vapor.     

Synthesis,spectral characterization,redox and antimicrobial activity of Schiff base transition metal(II) complexes derived from 4-aminoantipyrine and 3-salicylideneacetylacetone

A novel tetradentate N₂O₂ type Schiff base, synthesized from 1-phenyl-2,3-dimethyl-4-aminopyrazol-5-one(4-aminoantipyrine) and 3-salicylidene-acetylacetone, forms stable complexes with transition metal ions such as Cu ^II , Ni ^II , Co ^II and Zn ^II in ethanol. Microanalytical data, magnetic susceptibility, IR, UV-Vis.,¹H-NMR, ESR and Mass spectral techniques were used to confirm the structures. Electronic absorption spectra of the complexes suggest a square-planar geometry around the central metal ion. These complexes show higher conductance values, supporting their electrolytic nature. The monomeric nature of the complexes was confirmed from their magnetic susceptibility values. Cyclic voltammogram of the copper(II) and nickel(II) complexes in DMSO solution at 300 K were recorded and the results are discussed. The X-band ESR spectra of the copper complex were recorded and the molecular orbital coefficient values were calculated from the spectra. The in vitro antimicrobial activities of the investigated compounds were tested against bacteria such as Klebsiella pneumoniae, Staphylococcus aureus, Bacillus subtilis and Escherichia coli and fungi like Aspergillus niger and Rhizoctonia bataicola. Most of the metal chelates show higher antimicrobial activity for the above microorganisms than that of the free ligand.     

Lithium,rubidium and cesium ion removal using potassium iron(III) hexacyanoferrate(II) supported on polymethylmethacrylate

Potassium iron(III) hexacyanoferrate(II) supported on poly methyl methacrylate, has been developed and investigated for the removal of lithium, rubidium and cesium ions. The material is capable of sorbing maximum quantities of these ions from 5.0, 2.5 and 4.5 M HNO₃ solutions respectively. Sorption studies, conducted individually for each metal ion, under optimized conditions, demonstrated that it was predominantly physisorption in the case of lithium ion while shifting to chemisorption with increasing ionic size. Distribution coefficient (K _d) values followed the order Cs⁺ > Rb⁺ > Li⁺ at low concentrations of metal ions. Following these findings Cs⁺ can preferably be removed from 1.5 to 5 M HNO₃ nuclear waste solutions.     

Application of capillary electrophoresis coupling with electrochemiluminescence detection to estimate activity of leucine aminopeptidas

A new method to estimate the leucine aminopeptidase (LAP, EC 3.4.11.1) activity using capillary electrophoresis coupled with electrochemiluminescence (ECL) is described. The liberated proline produced by LAP catalyzing the hydrolysis reaction of leucin–proline was used as an ECL coreagent to enhance Ru(bpy)₃²⁺ ECL signals efficiently. The detection limit for proline was 2.88 × 10^?6 m (signal‐to‐noise ratio 3), which was equal to 9.60 × 10^?8 units of LAP being used to catalyze leucin–proline for 1 min. The Michaelis constant K_m (2.07 × 10^?2 mol/L) and the maximum reaction velocity V_max (1.06 × 10^?5 mol/L/min) of LAP for leucin–proline are reported. The reaction conditions including the concentration of metal ions, incubation temperature and pH were optimized. This method was successfully applied to detect LAP activity in plasma and the results were in good agreement with that obtained by the clinical method. Copyright © 2013 John Wiley & Sons, Ltd.     

CaII Binding Regulates and Dominates the Reactivity of a Transition‐Metal‐Ion‐Dependent Diesterase from Mycobacterium tuberculosis

The diesterase Rv0805 from Mycobacterium tuberculosis is a dinuclear metallohydrolase that plays an important role in signal transduction by controlling the intracellular levels of cyclic nucleotides. As Rv0805 is essential for mycobacterial growth it is a promising new target for the development of chemotherapeutics to treat tuberculosis. The in vivo metal‐ion composition of Rv0805 is subject to debate. Here, we demonstrate that the active site accommodates two divalent transition metal ions with binding affinities ranging from approximately 50 nm for Mn^II to about 600 nm for Zn^II. In contrast, the enzyme GpdQ from Enterobacter aerogenes, despite having a coordination sphere identical to that of Rv0805, binds only one metal ion in the absence of substrate, thus demonstrating the significance of the outer sphere to modulate metal‐ion binding and enzymatic reactivity. Ca^II also binds tightly to Rv0805 (K_d≈40 nm ), but kinetic, calorimetric, and spectroscopic data indicate that two Ca^II ions bind at a site different from the dinuclear transition‐metal‐ion binding site. Ca^II acts as an activator of the enzymatic activity but is able to promote the hydrolysis of substrates even in the absence of transition‐metal ions, thus providing an effective strategy for the regulation of the enzymatic activity.     

Recombination System Based on Cre α Complementation and Leucine Zipper Fusions

A gene encoding β-1,3-1,4-glucanase was cloned by polymerase chain reaction (PCR) from Bacillus subtilis MA139. Sequencing result showed 97% homology to the corresponding gene from Bacillus licheniformis. The open reading frame (ORF) of the gene contained 690 bp coding for a 226 amino-acid matured protein with the estimated molecular weight of 24.44 kDa. The β-1,3-1,4-glucanase gene was subcloned into an expression vector of pET28a and expressed in Escherichia coli BL21 and then purified by metal affinity chromatography using a nickel–nitrilotriacetic acid (Ni–NTA) column. The purified β-1,3-1,4-glucanase demonstrated 24.05 and 12.52 U ml^-1 activities for the substrates of barley β-glucan and lichenan, respectively, and the specific activities were 728.79 and 379.1 U mg^-1 for them, respectively. The optimal temperature and pH of the purified enzyme were 40°C and 6.4, respectively. When barley β-glucan was used as the substrate, K _m was 5.34 mg ml^-1, and K _cat showed 7,206.71 S^-1, thus the ratio of K _cat and K _m was 1,349.67 ml s^-1 mg^-1. The activity of β-1,3-1,4-glucanase was affected by a range of metal ions or ethylenediaminetetraacetic acid (EDTA).     

取代基对有机钼化合物中α-氢转移反应势垒和产物稳定性的影响

使用B3LYP方法研究了发生在有机钼化合物R³R⁴Mo(≡CH)(CHR¹R²)和R³R⁴Mo(＝CH₂)(＝CR¹R²)之间的α-氢转移反应, 探讨了R¹, R², R³和R⁴位置上不同取代基对α-氢转移反应势垒和产物稳定性的影响. 研究发现, 金属钼有机化合物中, 发生α-氢转移的碳原子在过渡态中采用sp²杂化. R¹和R²位置上取代基对α-氢转移反应势垒的影响取决于取代基对过渡态中碳原子的未参与sp²杂化的p_z轨道上单电子的离域作用. 当R¹, R²位置是甲基时, 由于碳原子的p_z轨道与甲基的一个C—H键轨道间存在强的超共轭效应, 从而可以较大程度地降低α-氢转移反应的势垒. 研究结果还表明, 当R³, R⁴位置为SiH₃时的反应势垒较低. 所以当R¹和R² 位置为Me, R³和R⁴位置为SiH₃时, 反应势垒最低. 第一个甲基取代R¹或R²位置的H时, 反应势垒降低很大; 第二个甲基继续取代时, 反应势垒的降低约为第一个甲基的一半. 第一个SiH₃取代R³或R⁴位置的甲基时, 反应势垒降低较大; 第二个SiH₃继续取代时, 反应势垒的降低小于第一个SiH₃的一半. 对反应物和产物的相对稳定性的研究表明, 第一个甲基和第二个甲基对产物的相对能量的降低几乎相同; 第一个SiH₃降低产物的相对能量, 但是第二个SiH₃使产物的相对能量升高, 从而抵消了第一个SiH₃对产物的稳定作用.     

Kinetics of the reaction of sodium arylthiolates with nitro-carboxybenzyl halide derivatives

The rate constants for the reactions of 4-halomethyl-3-nitrobenzoic acids, the nonnitro derivatives, and their ethyl esters with arylthiolates were measured at different temperatures. It was found that the retardation in rate constants compared to benzyl halides is due to the electrostatic repulsion between the electronegative substituents (COO⁻ and/or NO₂) in the substrates and thiolate ions. Good correlations between log k₂ values of the acids and carbon basicities of thiolates were found while log k₂ values of the esters show good straight lines with Hammett σ constants, pk_a, and carbon basicities of arylthiolates. © 1996 John Wiley & Sons Inc     

Evaluation of Native and Chemically Modified Sargassum glaucescens for Continuous Biosorption of Co(II)

In the present study, biosorption of stable cobalt was studied in an up-flow fixed-bed column using the brown alga Sargassum glaucescens treated with formaldehyde (FA) or MgCl₂. Notable increase in cobalt removal was observed for FA-treated biosorbent with 2.7 and 1.4 times higher dynamic capacity (DC) and uptake capacity (UC) than native alga, respectively. Consequently, FA-treated S. glaucescens was selected for further investigations. In particle size experiments, the DCs of 0.5–1 and 1–2 mm particles were both equal to 27.6 mg/g, and corresponding UCs were 34 and 38 mg/g, respectively. The maximum DC was obtained at residence time of 2.5 min. Studying the effect of additional ions indicated partial effect of Na⁺ and K⁺ ions on DC and UC, Mg²⁺ reduced highly the DC and slightly the UC while heavy metal ions (Ni²⁺, Cd²⁺, Cu²⁺, Zn²⁺, Pb²⁺ and Cr³⁺) caused decrease in both DC and UC about 1.5–4.7 and 1.8–3.2 times, respectively. Moreover, the column regeneration studies were carried out for four sorption–desorption cycles. The DC and the UC highly decreased in the second cycle, partially decreased or remained constant in the third and in the fourth one.