模拟微重力下影响重组Escherichia coli外源蛋白质表达途径分析

空间微重力对航天医学、材料学等领域发展起到极其重要的推动作用,然而在生物化工领域的研究尚处于起步阶段。微生物在微重力环境中培养时,其生长代谢和基因转录表达都与地球重力场中截然不同。前期研究表明表达重组β-葡萄糖醛酸苷酶的大肠杆菌在模拟微重力(simulated microgravity,SMG)环境中菌体生长和外源蛋白质表达量均比正常重力对照(normal gravity,NG)环境要高。为深入分析SMG环境效应对菌株在外源蛋白质表达过程中的影响,对该重组大肠杆菌菌株在SMG和NG培养过程中加入IPTG诱导外源蛋白质表达4 h 后的全细胞蛋白质酶解产物, 通过三重稳定同位素二甲基标记方法并结合质谱分离鉴定技术,在肽段水平上标记后进行差异定量蛋白质组学分析。结果表明:与NG对照相比,SMG条件下共有124个蛋白质表达水平发生显著变化,其中92个蛋白质上调,6个蛋白质下调。上调蛋白质集中在细胞压力胁迫、氨基酸代谢、翻译转录和碳代谢等途径,这些相关途径的蛋白质表达水平变化可以影响大肠杆菌生理代谢及外源蛋白质表达效率。     

Enzymatic Synthesis of Galactosylated Serine/Threonine Derivatives by β-Galactosidase from Escherichia coli

The transgalactosylations of serine/threonine derivatives were investigated using β-galactosidase from Escherichia coli as biocatalyst. Using ortho-nitrophenyl-β-d-galactoside as donor, the highest bioconversion yield of transgalactosylated N-carboxy benzyl l-serine benzyl ester (23.2%) was achieved in heptane:buffer medium (70:30), whereas with the lactose, the highest bioconversion yield (3.94%) was obtained in the buffer reaction system. The structures of most abundant galactosylated serine products were characterized by MS/MS. The molecular docking simulation revealed that the binding of serine/threonine derivatives to the enzyme’s active site was stronger (−4.6~−7.9 kcal/mol) than that of the natural acceptor, glucose, and mainly occurred through interactions with aromatic residues. For N-tert-butoxycarbonyl serine methyl ester (6.8%) and N-carboxybenzyl serine benzyl ester (3.4%), their binding affinities and the distances between their hydroxyl side chain and the 1′-OH group of galactose moiety were in good accordance with the quantified bioconversion yields. Despite its lower predicted bioconversion yield, the high experimental bioconversion yield obtained with N-carboxybenzyl serine methyl ester (23.2%) demonstrated the importance of the thermodynamically-driven nature of the transgalactosylation reaction.     

5α,8α-Epidioxycholest-6-en-3-β-ol from three cone snails of the Indian ocean

Three cone snail species (Conus ebraeus, C. leopardus, C. tessulatus, family Conidae) have been investigated for their sterolic fraction. Besides cholesterol, 5α,8α-epidioxycholest-6-en-3-β-ol has been isolated from each species and the structure determined using two-dimensional nuclear magnetic resonance. The occurrence of 5α,8α-epidioxycholest-6-en-3-β-ol in high amounts in the crude extract (0.5%), constitutes an interesting source of this compound.     

Occurrence of 5α-cholesta-7,24-dien-3β-ol and 23-dehydrolophenol in the bean lipids of Vanilla madagascariensis

Eleven 4-desmethylsterols, four 4,4-dimethylsterols, and twelve 4-methylsterols were identified in Vanilla madagascariensis beans. The 4-desmethylsterol fraction was characterized by a high level of 5α-cholesta-7,24(25)-dien-3β-ol (35.3%). The 4-methylsterol fraction was characterized by a high level of 31-norcycloartenol (57.5%) and the presence of 23-dehydrolophenol (9.4%). Cycloartenol (72.9%), cyclosadol (12.7%), parkeol (9.7%), and 24-dehydrotirucallol (4.7%) were identified in the 4,4-dimethylsterol fraction.     

Photocatalytic Disinfection of Escherichia coli over Titanium (IV) Oxide Supported on Hβ Zeolite

Fe loaded Al-MCM-41, (Si/Al = 25, 50, 75 and 100) catalysts were synthesized by hydrothermal method and characterized by the XRD, BET (surface area), FT-IR, and UV- vis and Mössbauer techniques. The liquid phase hydroxylation of phenol with hydrogen peroxide was studied and exclusive formation of dihydroxybenzene was observed. The phenol conversion was found to be almost same at various reaction temperatures viz 40, 60 and 80 °C, but at room temperature only about 30% conversion was recorded. The activity followed the order Fe/Al-MCM-41 (25) > Fe/Al-MCM-41(50) > Fe/Al-MCM-41 (75) > Fe/Al-MCM-41 (100) which was also the order of acidity. Effects of Fe content in Fe/Al-MCM-41 catalysts, solvent, phenol/H₂O₂ mole ratio on phenol conversion was examined. The reaction was also carried out over 10% iron loaded mordenite and the results are compared.     

Comparing the Catalytic and Structural Characteristics of a ‘Short’ Unspecific Peroxygenase (UPO) Expressed in Pichia pastoris and Escherichia coli

Unspecific peroxygenases (UPOs) have emerged as valuable tools for the oxygenation of non-activated carbon atoms, as they exhibit high turnovers, good stability and depend only on hydrogen peroxide as the external oxidant for activity. However, the isolation of UPOs from their natural fungal sources remains a barrier to wider application. We have cloned the gene encoding an ‘artificial’ peroxygenase (artUPO), close in sequence to the ‘short’ UPO from Marasmius rotula (MroUPO), and expressed it in both the yeast Pichia pastoris and E. coli to compare the catalytic and structural characteristics of the enzymes produced in each system. Catalytic efficiency for the UPO substrate 5-nitro-1,3-benzodioxole (NBD) was largely the same for both enzymes, and the structures also revealed few differences apart from the expected glycosylation of the yeast enzyme. However, the glycosylated enzyme displayed greater stability, as determined by nano differential scanning fluorimetry (nano-DSF) measurements. Interestingly, while artUPO hydroxylated ethylbenzene derivatives to give the (R)-alcohols, also given by a variant of the ‘long’ UPO from Agrocybe aegerita (AaeUPO), it gave the opposite (S)-series of sulfoxide products from a range of sulfide substrates, broadening the scope for application of the enzymes. The structures of artUPO reveal substantial differences to that of AaeUPO, and provide a platform for investigating the distinctive activity of this and related'short’ UPOs.     

Antimicrobial Effect of α- or β-Cyclodextrin Complexes with Trans-Cinnamaldehyde Against Staphylococcus aureus and Escherichia coli

Molecular inclusion or encapsulation based on cyclodextrin (CD) is the most widely used method in encapsulation technology. α- and β-CD complexes consisting of antibacterial trans-cinnamaldehyde were prepared by the molecular inclusion method. The characteristics of the complex were determined, including examination of their antimicrobial effects after the drying process. The particle sizes of the trans-cinnamaldehyde–CD complexes were observed in the range of 300 to 500 nm after production. Particle sizes of both trans-cinnamaldehyde–CD complexes were slightly increased increasing in a dose-dependent manner of trans-cinnamaldehyde. Although β-CD complexes were larger than α-CD complexes, they possessed a lower polydispersity index with a narrow size distribution. The encapsulation efficiency of trans-cinnamaldehyde–CD complexes was >90% in all formulations. Trans-cinnamaldehyde affected reduction of Staphylococcus aureus and Escherichia coli, with antibacterial activity increasing in a dose-dependent manner of trans-cinnamaldehyde concentration. In addition, β-CD complexes showed more effective antimicrobial effects compared to α-CD complexes.     

β-Apo-8′-Carotenoic acid and its esters in sunflower oil oxidation

The oxidation kinetics of sunflower oil (SO) and pure triacylglycerols of sunflower oil (TGSO) in the presence of different concentrations (0.0008–0.02%, 1.9–32.7×10⁻⁵ M) of β-apo-8′-carotenoic acid (CA), ethyl β-apo-8′-carotenoate (EC), and β-apo-8′-carotenoylglycerol (CG) were studied. The process was performed at high (kinetic regime) and low (diffusion regime) oxygen concentrations at room temperature and at 100°C and in the dark and in daylight. CA, EC, and CG were not antioxidants in TGSO systems. However, the carotenoid derivatives, especially CA, increased the stability of tocopherol-containing SO at room temperature and in daylight. The stabilization effect was more evident in a kinetic regime of oxidation. The synergism between the carotenoids and tocopherols was characterized by the increase of the stabilization factor F and activity A. F and A were highest for CA (F=1.2–5.5, A=2.4–78.6), followed by EC (F=1.2–3.5, A=1.7–14.6) and CG (F=1.1–2.1, A=1.6–5.5) in the kinetic regime for SO exposed to daylight at room temperature. Presented at the 91st AOCS Annual Meeting & Expo, April 25–28, 2000, San Diego, California.     

Production of cyclic adenosine-3′,5′-monophosphate by whole cell catalysis using recombinant Escherichia coli overexpressing adenylate cyclase

Adenylate cyclase (EC 4.6.1.1) catalyzes the formation of cyclic adenosine-3′,5′-monophosphate (cAMP) from adenosine 5′-triphosphate (ATP). Recombinant Escherichia coli overexpressing adenylate cyclase was used to synthesize cAMP by whole cell catalysis. Some key parameters were examined during the catalytic process, while pH and Mg²⁺ were found to influence cAMP production significantly. Optimum conditions were pH 8.52 and 30 °C with 77.2 mM Mg²⁺ in 100 mM Tris-HCl buffer, including 0.25% Triton-X 100 as detergent and 30 mM pyruvate sodium as enzyme activator for 6 h. 14.93 g/L of cAMP was produced with a conversion rate of 91.5%. The current work provided a potential way for the industrial production of cAMP.     

人肝细胞生长因子β链在E.coli中的表达、纯化及活性测定

目的 为获得重组人肝细胞生长因子β(rhHGF-β)的高效表达体系,分离纯化rhHGF-β,以期研究其在生物学和医学中的作用。方法 从人胎肝组织中提取总RNA,采用RT-PCR技术,对HGF-β基因加工修饰,与载体PBV220重组,构建rhHGF-β的表达载体,转化大肠杆菌,筛选rhHGF-β的高效表达菌株。对rhHGF-β进行粗纯化,透析复性后MTT法检测生物活性。结果 获得了rhHGF-β完整的cDNA序列,重组克隆的酶切结果与设计一致。目的 蛋白的表达量占全菌蛋白的30％,粗纯化后纯度达90％,复性产物能刺激大鼠肝细胞的增殖。结论 建立了rhHGF-β的表达体系,获得了具有生物活性的rhHGF-β,为进一步大量制备和研究HGF的生物学功能提供了重要的实验数据。     

Isolation of 8-hydroxygeraniol-8-O-β-d-glucoside,a probable intermediate in biosynthesis of iridoid monoterpenes,from defensive secretions ofPlagiodera versicolora andGastrophysa viridula (Coleoptera: Chrysomelidae)

8-Hydroxygeraniol and its 8-O--d-glucoside have been found as trace components in the defensive secretions ofPlagiodera versicolora andGastrophysa viridula larvae. This discovery supports the hypothesis that the evolution of the utilization of plant precursors by some chrysomelid species was favored by the plesiomorphic occurrence of a-glucosidase and an oxidase in the defensive secretion of iridoid-producing species.     

TGF-β1 Protection against Aβ1–42-Induced Neuroinflammation and Neurodegeneration in Rats

Transforming growth factor (TGF)-β1, a cytokine that can be expressed in the brain, is a key regulator of the brain’s responses to injury and inflammation. Alzheimer’s disease (AD), the most common neurodegenerative disorder, involves inflammatory processes in the brain in addition to the hallmarks, amyloid-β (Aβ) plaques and neurofibrillary tangles. Recently, we have shown that T-helper (Th) 17 cells, a subpopulation of CD4⁺ T-cells with high proinflammation, also participate in the brain inflammatory process of AD. However, it is poorly known whether TGF-β1 ameliorates the lymphocyte-mediated neuroinflammation and, thereby, alleviates neurodegeneration in AD. Herein, we administered TGF-β1 via the intracerebroventricle (ICV) in AD model rats, by Aβ_1–42 injection in both sides of the hippocampus, to show the neuroprotection of TGF-β1. The TGF-β1 administration after the Aβ_1–42 injection ameliorated cognitive deficit and neuronal loss and apoptosis, reduced amyloid precursor protein (APP) expression, elevated protein phosphatase (PP)2A expression, attenuated glial activation and alleviated the imbalance of the pro-inflammatory/anti-inflammatory responses of T-lymphocytes, compared to the Aβ_1–42 injection alone. These findings demonstrate that TGF-β1 provides protection against AD neurodegeneration and suggest that the TGF-β1 neuroprotection is implemented by the alleviation of glial and T-cell-mediated neuroinflammation.     

Elektrostatisch unterstützte Nassabscheidung in einem Windkanal: Ermittlung des Fraktionsabscheidegrads

In the range of small particle sizes, the probability of separation decreases due to lower inertia, so that electrostatic charging of a generated water spray mist is suitable to improve the emission-reducing effect. Based on an application scenario, the separation mechanisms caused by inertia, turbulent mixing, and electrostatic attraction were studied to approximate the fractional separation efficiency. The approach illustrates the size-dependent dominance of the separation mechanisms and allows a qualitative comparison with practical results.     

β-Mercaptoalkanoic carboxylic esters: versatile synthons in heterocyclic chemistry

This review presents approaches to the synthesis of heterocyclic annelated thiophenes of the last 35 years. Annelated thiophenes have been reported by the cyclocondensation reaction of β -mercaptoalkanoic carboxylic esters with 1,3-bifunctional substrates such as α -haloenones, β -haloenones, α -halogenoacrylic acid derivatives. The reaction takes place by nucleophilic addititon, followed by Dieckmann cyclization with the elimination of hydrogen halide. In case of other substrates such as β -chlorocinnamonitriles, β -haloacrylonitriles, arylsulfonylacrylonitriles, α,β -unsaturated nitriles, Dieckmann–Thorpe–Ziegler cyclization occurs resulting in aminothiophene derivatives. Functionalized annulated thiophene derivatives have potent industrial applications because of significant biological properties.

     

Chemical Defenses Against Fish Predation in Three Brazilian Octocorals: 11β,12β-Epoxypukalide as a Feeding Deterrent in Phyllogorgia dilatata

The feeding-deterrence properties of crude extracts of three Brazilian octocoral species, Neospongodes atlantica Kükenthal (Alcyonacea, Nephtheidae), Plexaurella regia Castro (Gorgonacea, Plexauridae), and Phyllogorgia dilatata Esper (Gorgonacea, Gorgoniidae), were investigated. All the extracts were incorporated into food strips at the concentrations occurring in the living organisms. Crude extract and its ethyl acetate fraction obtained from P. dilatata collected in Armação dos Búzios (Rio de Janeiro State), when incorporated into artificial diets and tested in the habitat of origin, reduced consumption of food strips by fishes, relative to controls. Crude extracts from two octocoral species collected at the National Marine Park of Abrolhos (Bahia State), N. atlantica and P. regia, had no apparent feeding-deterrence properties; in fact, they seemed to stimulate feeding. Bioassay-guided fractionation of the bioactive P. dilatata crude extract revealed that the deterrent property was restricted to a medium polarity fraction. Field palatability experiments with two pure compounds isolated from this fraction revealed that the furanocembranolide 11,12-epoxypukalide is a potent feeding deterrent produced by P. dilatata against fish. Apparently, furanocembranolides are a particular class of compounds with feeding deterrent properties, protecting some octocorals from potential fish predator species in both tropical and temperate environments.     

Triethanolamine Stabilization of Methotrexate-β-Cyclodextrin Interactions in Ternary Complexes

The interaction of methotrexate (MTX) with beta-cyclodextrin (β-CD) in the presence of triethanolamine (TEA) was investigated with the aim to elucidate the mechanism whereby self-assembly cyclodextrin systems work in association with this third component. Solubility diagram studies showed synergic increment of the MTX solubility to be about thirty-fold. Experiments using 2D ROESY and molecular modeling studies revealed the inclusion of aromatic ring III of the drug into β-CD cavity, in which TEA contributes by intensifying MTX interaction with β-CD and stabilizes MTX:β-CD:TEA ternary complex by electrostatic interaction. The maintenance of these interactions in solid phase was also studied in ternary MTX:β-CD:TEA and comparisons were made with freeze dried binary MTX:β-CD and physical mixtures. FTIR studies evidenced that MTX–β-CD interaction remained in solid ternary complexes, which was also supported by thermal (differential scanning calorimetry (DSC), thermogravimetric analysis (TG)/first derivative of TG analysis (DTG) and C,N,H elementary analysis) and structural (X-ray diffraction analysis, (XRD)) studies, mainly regarding the increment of drug stability. The efficient in vitro drug dissolution studies successfully demonstrated the contribution of ternary complexes, which highlights the importance of this possible new raw material for further applications in drug delivery systems.     

Protein Ligation-Assisted Reconstitution of Split HRP Fragments for Facile Production of HRP Fusion Proteins in E. coli

Horseradish peroxidase (HRP) is a pivotal biocatalyst for biosensor development and fine chemical synthesis. HRP proteins are mostly extracted and purified from the roots of horseradish because the solubility and productivity of recombinant HRP in bacteria are significantly low. In this study, we investigate the reconstitution system of split HRP fragments to improve its soluble expression levels in E. coli allowing the cost-effective production of bioactive HRPs. To promote the effective association between two HRP fragments (HRPn and HRPc), we exploit SpyTag-SpyCatcher chemistry, a versatile protein coupling method with high affinity and selectivity. Each HRP fragment was genetically fused with SpyTag and SpyCatcher, respectively, exhibiting soluble expression in the E. coli cytoplasm. The engineered split HRPs were effectively and irreversibly reconstituted into a biologically active and stable assembly that can catalyze intrinsic enzymatic reactions. Compared to the chaperone co-expression system, our approach shows that the production yield of soluble HRP is comparable, but the purity of the final product is relatively high. Therefore, our results can be applied to the high-yield production of recombinant HRP variants and other difficult-to-express proteins in bacteria without complex downstream processes.     

Kinetics of extraction of Ti(IV) from SO42– medium by Cyanex 301 dissolved in kerosene containing 5% heptan-1-ol

The kinetics of Ti(IV) extraction by Cyanex 301 (HA) were investigated by measuring initial flux of Ti(IV) transfer (F, kmol/m²s), using a Lewis cell, operated at 3 Hz. The empirical flux equation at 298 K is found to be as follows: F (kmol/m²s) = 10^–4.288 [Ti(IV)] (1 + 447 [H⁺])^–1 [HA]_(o) (1 + 1.18[SO₄^2–])^–1. The activation energy, E_a, has been measured to be within 37–60 kJ/mol, depending on experimental parameters and temperature region. The ΔS^± value is always highly negative. Analysis of the flux equation has been done, given various parametric conditions, to elucidate the mechanism of extraction. The rate-determining chemical reaction step, in most parametric conditions, appears to be as follows: TiO²⁺ + A^– → TiOA⁺; and this step occurs via an S_N2 mechanism as suggested by high negative ΔS^± values. However, in certain cases, the extraction process appears as intermediate controlled as supported by E_a value of less than ~48 kJ/mol.