Production of Lewis x tetrasaccharides by metabolically engineered Escherichia coli

Two tetrasaccharides carrying the trisaccharidic Lewis x motif on a GlcNAc or a Gal residue were produced on the gram-scale by high-cell-density cultures of metabolically engineered Escherichia coli strains that overexpressed the Helicobacter pylori futA gene for alpha-3 fucosyltransferase and the Neisseria meningitidis lgtB gene for beta-4 galactosyltransferase. The first compound Galbeta-4(Fucalpha-3)GlcNAcbeta-4GlcNAc was produced by glycosylation of chitinbiose, which was endogenously generated in the bacterial cytoplasm by the successive action of the rhizobial chitin-synthase NodC and the Bacillus circulans chitinase A1, whose genes were additionally expressed in the E. coli strain. The second compound, Galbeta-4(Fucalpha-3)GlcNAcbeta-3Gal, was produced from exogenously added Gal by a strain that was deficient in galactokinase activity and overexpressed the additional N. meningitidis lgtA gene for beta-3 N-acetylglucosaminyltransferase.     

Enhancement of bioconversion gangliosides to monosialotetrahexosylganglioside by in situ sialic acid removal and recovery

Monosialotetrahexosylganglioside (GM1) production via bioconversion from gangliosides is promising for industrial application because it has the advantages of a high GM1 yield and an environmentally friendly process. Sialidase hydrolyzes gangliosides to GM1 producing sialic acid as a by‐product, which inhibits the sialidase activity, while the incomplete conversion of gangliosides was indicated by thin‐layer chromatography (TLC) in the presence of sialic acid. The sialic acid showed competitive inhibition on the sialidase activity with an inhibition constant of 0.75 mmol/L. By harnessing the in situ product removal (ISPR) technique, 50 g/L of crude gangliosides was completely converted to GM1 after a 12 h conversion. The GM1 concentration increased from 0.42 to 10.88 g/L in the ISPR system, which was 59.1 % higher than that of the control (6.84 g/L GM1). In addition, sialic acid was recovered simultaneously with a yield of 74.7 %. In summary, the ISPR system improved the bioconversion from gangliosides to GM1 and recovered sialic acid within a one‐step bioprocess.     

Fe3+交联聚乙烯亚胺-单宁酸复合纳滤膜研究

利用聚乙烯亚胺(PEI)、单宁酸(TA)与Fe³⁺之间的螯合作用,通过层层自组装技术,制备了PEI-TA复合纳滤膜,对其表面形态及性能进行了表征,并研究了TA、Fe³⁺含量和组装层数对膜性能的影响。结果表明,当PEI、TA、Fe³⁺的质量浓度分别为2、3、3 g/L,组装层数为6层时,复合膜性能为优。优化膜对Na₂SO₄、MgCl₂、NaCl的截留率分别为97.34%、65.46%、73.11%,水通量达24.91 L/(m²·h)。该复合膜制备工艺简单、绿色,在脱盐、水质软化等水处理领域具有应用潜力。     

Iterative One‐Pot α‐Glycosylation Strategy: Application to Oligosaccharide Synthesis

Based on the combined use of dimethylformamide (DMF) modulation and neighboring group participation, three iterative one‐pot α‐glycosylation methods, i.e., one‐pot (α,α)‐, one‐pot (β,α)‐, and one‐pot (α,β)‐glycosylations, were developed. These methods are applicable to a range of thioglycosyl donors, confer stereocontrol in α‐/β‐glycosidic bond formation, and thus provide for rapid access to oligosaccharides with various permutations of anomeric configurations. The utility of these one‐pot glycosylation methods is demonstrated in the synthesis of eight non‐natural and natural oligosaccharide targets, including the core 1 serine conjugate, core 8 serine conjugate, the D ‐Gal‐α(1→3)‐D ‐Glc‐α(1→3)‐L ‐Rha trisaccharide unit of the cell wall component in Streptococcus pneumoniae, and the D ‐Glc‐α(1→2)‐D ‐Glc‐α(1→3)‐D ‐Glc trisaccharide terminus of the N‐linked glycan precursor. Confirmation of the anomeric configurations of these oligosaccharides is evidenced by ¹H, ¹³C, ¹³C‐non‐proton decoupling, and heteronuclear correlation 2D NMR experiments. Global deprotection of selected oligosaccharide targets is illustrated.     

双酚单丙烯酸酯类抗氧剂GM的合成

以2,2′-甲撑双(4-甲基-6-特丁基苯酚)(简称2246)、丙烯酸、氯氧化磷等为反应原料,通过酯化反应合成了GM,考察了合成GM的适宜工艺条件。在n(2246)∶n(丙烯酸)∶n(氯氧化磷)=1∶1.25∶0.90、催化剂m(三乙胺)∶m(2246)=1∶1.0X、反应温度105℃、反应时间2.5 h的合成条件下,原料2246转化率大于96.0%;GM产品选择性大于91.0%;GM产品摩尔收率大于83.0%;GM产品的液相色谱纯度大于99.0%。采用该工艺合成抗氧剂GM的特点是将酰卤化过程和脱卤化氢过程合二为一,由2246和丙烯酸直接进行酯化反应合成GM。工艺流程短,产品收率高、质量稳定,有可能便于工业化生产。     

虎杖中抗氧化成分的提取分离及其活性研究

提取了虎杖中具有抗氧化活性的总蒽醌(TA),并从总蒽醌中依次分离出强酸成分(SAP)、中酸成分(MAP)和弱酸成分(WAP)。然后采用二苯代苦味酰自由基(DPPH)法对不同质量浓度的TA、SAP、MAP和WAP进行了抗氧化活性测定。TA、SAP、MAP和WAP质量浓度分别为0.2 g/L、0.5 g/L、0.8 g/L、1.2 g/L时,对自由基的最大清除率依次分别为:29.5%、67.1%、85.7%、87.5%;34.0%、74.2%、86.5%、95.4%;9.8%、24.0%、35.2%、47.3%;6.5%、11.1%、19.6%、19.9%。TA、SAP、MAP和WAP对自由基均有程度不同的清除作用,其中SAP的效果最佳,当其质量浓度为1.2 g/L时,最大清除率可高达95.4%。     

Effect of stoichiometric imbalances on the melt condensation polymerization of poly(dodecamethylene terephthalamide) studied by intrinsic viscosity and 13C‐NMR

Poly(dodecamethylene terephthalamide) (PA‐12,T) was synthesized by melt condensation polymerization of 12,T salt with 0, 1, 3, 5, or 10% molar excess of 1,12‐diaminododecane (DA), terephthalic acid (TA), or benzoic acid (BA). Intrinsic viscosities (IV) (0.5 g/dL in 96% H₂SO₄ at 25°C) were measured to determine relative molecular weight differences. IV was highest for reactions containing 1 and 3 mol % excess DA (1.36 and 1.31 dL/g, respectively), followed by the product of pure 1 : 1 salt (1.25 dL/g). For all concentrations of excess TA and BA, IV was decreased. ¹³C‐NMR chemical shifts for DA, TA, and BA end groups were identified and their concentrations determined by comparison with the intensity of main chain polymer peaks. A log–log plot of IV versus number average molecular weight calculated from ¹³C‐NMR data shows a linear trend with Mark‐Houwink constants of K = 55.8 × 10^?5 dL/g and α = 0.81. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

Longitudinal Evolution of the Concentration of Gangliosides GM3 and GD3 in Human Milk

It has been reported that dietary gangliosides may have an important role in preventing infections and in brain development during early infancy. However, data related to the evolution of their concentration over the different stages of lactation are scarce. Liquid chromatography coupled with electrospray ionization high resolution mass spectrometer (LC/ESI‐HR‐MS) has been optimized to quantify the two major ganglioside classes, i.e., aNeu5Ac(2‐8)aNeu5Ac(2‐3)bDGalp(1‐4)bDGlcp(1‐1)Cer (GD3) and aNeu5Ac(2‐3)bDGalp(1‐4)bDGlcp(1‐1)Cer (GM3) in human milk. Gangliosides were extracted using chloroform and methanol, further purified by solid‐phase extraction and separated by reversed‐phase liquid chromatography. Repeatability, intermediate reproducibility, and recovery values were assessed to validate the method. In human milk, GD3 and GM3 could be quantified at the level of 0.1 and 0.2 μg/mL, respectively, with relative standard deviation of repeatability [CV(r)] and intermediate reproducibility [CV(iR)] values ranging from 1.9 to 15.0 % and 1.9 to 22.5 %, respectively. The described method was used to quantify GD3 and GM3 in human milk samples collected from 450 volunteers between 0 and 11 days and at 30, 60 and 120 days postpartum, providing for the first time the concentration of these minor lipids in a large cohort. The content of total gangliosides ranged from 8.1 and 10.7 μg/mL and the mean intake of gangliosides in infants 30, 60 and 120 days postpartum could be estimated at about 5.5, 7.0 and 8.6 mg of total gangliosides per day, respectively, when infants were exclusively breastfed.     

Sphingolipids: Key Regulators of Apoptosis and Pivotal Players in Cancer Drug Resistance

Drug resistance elicited by cancer cells still constitutes a huge problem that frequently impairs the efficacy of both conventional and novel molecular therapies. Chemotherapy usually acts to induce apoptosis in cancer cells; therefore, the investigation of apoptosis control and of the mechanisms used by cancer cells to evade apoptosis could be translated in an improvement of therapies. Among many tools acquired by cancer cells to this end, the de-regulated synthesis and metabolism of sphingolipids have been well documented. Sphingolipids are known to play many structural and signalling roles in cells, as they are involved in the control of growth, survival, adhesion, and motility. In particular, in order to increase survival, cancer cells: (a) counteract the accumulation of ceramide that is endowed with pro-apoptotic potential and is induced by many drugs; (b) increase the synthesis of sphingosine-1-phosphate and glucosylceramide that are pro-survivals signals; (c) modify the synthesis and the metabolism of complex glycosphingolipids, particularly increasing the levels of modified species of gangliosides such as 9-O acetylated GD3 (αNeu5Ac(2-8)αNeu5Ac(2-3)βGal(1-4)βGlc(1-1)Cer) or N-glycolyl GM3 (αNeu5Ac (2-3)βGal(1-4)βGlc(1-1)Cer) and de-N-acetyl GM3 (NeuNH(2)βGal(1-4)βGlc(1-1)Cer) endowed with anti-apoptotic roles and of globoside Gb3 related to a higher expression of the multidrug resistance gene MDR1. In light of this evidence, the employment of chemical or genetic approaches specifically targeting sphingolipid dysregulations appears a promising tool for the improvement of current chemotherapy efficacy.     

Application of a New Engineered Strain of Yarrowia lipolytica for Effective Production of Calcium Ketoglutarate Dietary Supplements

The present study aimed to develop a technology for the production of dietary supplements based on yeast biomass and α-ketoglutaric acid (KGA), produced by a new transformant of Yarrowia lipolytica with improved KGA biosynthesis ability, as well to verify the usefulness of the obtained products for food and feed purposes. Transformants of Y. lipolytica were constructed to overexpress genes encoding glycerol kinase, methylcitrate synthase and mitochondrial organic acid transporter. The strains were compared in terms of growth ability in glycerol- and oil-based media as well as their suitability for KGA biosynthesis in mixed glycerol–oil medium. The impact of different C:N:P ratios on KGA production by selected strain was also evaluated. Application of the strain that overexpressed all three genes in the culture with a C:N:P ratio of 87:5:1 allowed us to obtain 53.1 g/L of KGA with productivity of 0.35 g/Lh and yield of 0.53 g/g. Finally, the possibility of obtaining three different products with desired nutritional and health-beneficial characteristics was demonstrated: (1) calcium α-ketoglutarate (CaKGA) with purity of 89.9% obtained by precipitation of KGA with CaCO₃, (2) yeast biomass with very good nutritional properties, (3) fixed biomass-CaKGA preparation containing 87.2 μg/g of kynurenic acid, which increases the health-promoting value of the product.     

Tailored Synthesis of 162‐Residue S‐Monoglycosylated GM2‐Activator Protein (GM2AP) Analogues that Allows Facile Access to a Protein Library

A synthetic protocol for the preparation of 162‐residue S‐monoglycosylated GM2‐activator protein (GM2AP) analogues bearing various amino acid substitutions for Thr69 has been developed. The facile incorporation of the replacements into the protein was achieved by means of a one‐pot/N‐to‐C‐directed sequential ligation strategy using readily accessible middle N‐sulfanylethylanilide (SEAlide) peptides each consisting of seven amino acid residues. A kinetically controlled ligation protocol was successfully applied to the assembly of three peptide segments covering the GM2AP. The native chemical ligation (NCL) reactivities of the SEAlide peptides can be tuned by the presence or absence of phosphate salts. Furthermore, NCL of the alkyl thioester fragment [GM2AP (1–31)] with the N‐terminal cysteinyl prolyl thioester [GM2AP (32–67)] proceeded smoothly to yield the 67‐residue prolyl thioester, with the prolyl thioester moiety remaining intact. This newly developed strategy enabled the facile synthesis of GM2AP analogues. Thus, we refer to this synthetic protocol as “tailored synthesis” for the construction of a GM2AP library.     

One‐Pot Conversion of L‐Threonine into L‐Homoalanine: Biocatalytic Production of an Unnatural Amino Acid from a Natural One

A novel biocatalytic process for production of L ‐homoalanine from L ‐threonine has been developed using coupled enzyme reactions consisting of a threonine deaminase (TD) and an ω‐transaminase (ω‐TA). TD catalyzes the dehydration/deamination of L ‐threonine, leading to the generation of 2‐oxobutyrate which is asymmetrically converted to L ‐homoalanine via transamination with benzylamine executed by ω‐TA. To make up the coupled reaction system, we cloned and overexpressed a TD from Escherichia coli and an (S)‐specific ω‐TA from Paracoccus denitrificans. In the coupled reactions, L ‐threonine serves as a precursor of 2‐oxobutyrate for the ω‐TA reaction, eliminating the need for employing the expensive oxo acid as a starting reactant. In contrast to α‐transaminase reactions in which use of amino acids as an exclusive amino donor limits complete conversion, amines are exploited in the ω‐TA reaction and thus maximum conversion could reach 100%. The ω‐TA‐only reaction with 10 mM 2‐oxobutyrate and 20 mM benzylamine resulted in 94% yield of optically pure L ‐homoalanine (ee>99%). However, the ω‐TA‐only reaction did not produce any detectable amount of L ‐homoalanine from 10 mM L ‐threonine and 20 mM benzylamine, whereas the ω‐TA reaction coupled with TD led to 91% conversion of L ‐threonine to L ‐homoalanine.     

Enhanced Anti-Atherosclerotic Efficacy of pH-Responsively Releasable Ganglioside GM3 Delivered by Reconstituted High-Density Lipoprotein

Recently, the atheroprotective role of endogenous GM3 and an atherogenesis-inhibiting effect of exogenous GM3 suggested a possibility of exogenous GM3 being recruited as an anti-atherosclerotic drug. This study seeks to endow exogenous GM3 with atherosclerotic targetability via reconstituted high-density lipoprotein (rHDL), an atherosclerotic targeting drug nanocarrier. Unloaded rHDL, rHDL loaded with exogenous GM3 at a low concentration (GM3_L-rHDL), and rHDL carrying GM3 at a relatively high concentration (GM3_H-rHDL) were prepared and characterized. The inhibitory effect of GM3-rHDL on lipid deposition in macrophages was confirmed, and GM3-rHDL did not affect the survival of red blood cells. In vivo experiments using ApoE^−/− mice fed a high fat diet further confirmed the anti-atherosclerotic efficacy of exogenous GM3 and demonstrated that GM3 packed in HDL nanoparticles (GM3-rHDL) has an enhanced anti-atherosclerotic efficacy and a reduced effective dose of GM3. Then, the macrophage- and atherosclerotic plaque-targeting abilities of GM3-rHD, most likely via the interaction of ApoA-I on GM3-rHDL with its receptors (e.g., SR-B1) on cells, were certified via a microsphere-based method and an aortic fragment-based method, respectively. Moreover, we found that solution acidification enhanced GM3 release from GM3-rHDL nanoparticles, implying the pH-responsive GM3 release when GM3-rHDL enters the acidic atherosclerotic plaques from the neutral blood. The rHDL-mediated atherosclerotic targetability and pH-responsive GM3 release of GM3-rHDL enhanced the anti-atherosclerotic efficacy of exogenous GM3. The development of the GM3-rHDL nanoparticle may help with the application of exogenous GM3 as a clinical drug. Moreover, the data imply that the GM3-rHDL nanoparticle has the potential of being recruited as a drug nanocarrier with atherosclerotic targetability and enhanced anti-atherosclerotic efficacy.     

Preparation of defined molecular species of lactosylceramide by chemical deacylation and reacylation withN-succinimidyl fatty acid esters

A procedure for the preparation of specific molecular species ofd-erythro-lactosylceramide involving deacylation and reacylation of lactosylceramide prepared from bovine brain gangliosides is described. Lactosylceramide wasN-deacylated by alkaline hydrolysis and the resulting four lysolactosylceramides, which contained d18∶1, d20∶1, d18∶0 and d20∶0 long-chain bases, were simultaneously re-N-acylated with theN-succinimidyl ester of either 16∶0, 18∶0, 20∶0, 24∶0, 20∶1, 22∶1 or 24∶1 fatty acid. The resulting lactosylceramide contained four molecular species of lactosylceramides, i.e., d18∶1, d20∶1, d18∶0 and d20∶0 long-chain bases coupled with the fatty acid that was introduced. Lactosylceramides prepared in this manner were separated into four individual molecular species by high-performance liquid chromatography (HPLC). Each of the purified molecular species of lactosylceramide was quantitated by HPLC after derivatization with benzoylchloride and was characterized by mass spectrometry. The yields of reacylated lactosylceramide were 38–58% relative to the starting lactosylceramide; the purity of each of the molecular species of lactosylceramide was greater than 95%. The glycosphingolipid nomenclature is as recommended by the IUPAC-IUB Commission on Biochemical Nomenclature (1). GalCer, galactosylceramide, Gal(β1-1)Cer; GlcCer, glucosylceramide, Glc(β1-1)Cer; LacCer, lactosylceramide, Gal(β1-4)GlcCer; GbOse₃Cer, globotriaosylceramide, Gal(α1-4)Gal(β1-4)GlcCer; GbOse₄Cer, globotetraosylceramide, GalNAc(β1-3)Gal(α1-4)-Gal(β1-4)GlcCer; GgOse₃Cer, gangliotriaosylceramide, GalNAc-(β1-4)Gal(β1-4)GlcCer; GgOse₄Cer, gangliotetraosylceramide, Gal(β1-3)GalNAc(β1-4)Gal(β1-4)GlcCer; GM3, (NeuAcα2-3)-Galβ1-4GlcCer; GM1, Galβ1-3GalNAcβ1-4(NeuAcα2-3)Galβ1-4-GlcCer. The molecular species abbreviations suggested by Breimeret al. (2) are used. For example, in the notation d18∶1−18∶0, the d18∶1 represents the long-chain base sphingosine (d-erythro-1,3-dihydroxy-2-amino-trans-4-octadecene) and 18∶0 represents the fatty acid (octadecanoic acid).     

Chemical Synthesis of GM2 Glycans,Bioconjugation with Bacteriophage Qβ, and the Induction of Anticancer Antibodies

The development of carbohydrate‐based antitumor vaccines is an attractive approach towards tumor prevention and treatment. Herein, we focused on the ganglioside GM2 tumor‐associated carbohydrate antigen (TACA), which is overexpressed in a wide range of tumor cells. GM2 was synthesized chemically and conjugated with a virus‐like particle derived from bacteriophage Qβ. Although the copper‐catalyzed azide–alkyne cycloaddition reaction efficiently introduced 237 copies of GM2 per Qβ, this construct failed to induce significant amounts of anti‐GM2 antibodies compared to the Qβ control. In contrast, GM2 immobilized on Qβ through a thiourea linker elicited high titers of IgG antibodies that recognized GM2‐positive tumor cells and effectively induced cell lysis through complement‐mediated cytotoxicity. Thus, bacteriophage Qβ is a suitable platform to boost antibody responses towards GM2, a representative member of an important class of TACA: the ganglioside.     

抗氧剂GM对ABS热氧稳定性能的影响

2-叔丁基-6-(3-叔丁基-2-羟基-5-甲苯甲基)-4-甲基苯酚丙烯酸酯(GM)是一种新型双酚单丙烯酸酯类抗氧剂,其在聚合物加工过程中的热稳定性能尤为显著.本文通过测定添加不同含量的抗氧剂2246和抗氧剂GM的ABS树脂的氧化诱导参数及其经热氧老化后的力学性能以及色差值AE,分析研究了抗氧剂2246和抗氧剂GM在抗...     

酶法分离制备γ-氨基丁酸和L-天冬氨酸

以L-谷氨酸(L-G lu)和L-天冬氨酸(L-Asp)两种混合酸性氨基酸〔m(L-G lu)∶m(L-Asp)=1∶1〕为原料,利用大肠杆菌菌体内脱羧酶对L-谷氨酸的专一脱羧作用,酶法分离制备了γ-氨基丁酸和L-天冬氨酸。考察了转化体系温度、pH等影响L-谷氨酸脱羧酶活力的主要因素,实验表明,最佳工艺条件为:温度35℃,转化体系pH=5.0,ρ(菌体)=6 g/L,ρ(Tween80)=0.15 g/L,菌龄16 h,ρ(底物)=60 g/L。L-谷氨酸脱羧酶在最适转化条件下比酶活高达15 036 U。1 g湿菌体可重复使用3次共转化L-谷氨酸和L-天冬氨酸混合物30 g,其中L-谷氨酸完全转化为γ-氨基丁酸。γ-氨基丁酸及L-天冬氨酸的总收率可分别达到理论收率的88%和90%。     

Improvement of fermentative production of exopolysaccharides from Aureobasidium pullulans under various conditions

The optimization of exopolysaccharide (EPS) production was investigated in the polymorphic fungal strain of Aureobasidium pullulans (KCTC 6081) with varying pH, nutrients concentration, and mixing parameters in batch fermentation condition. The maximum production of EPS (～7.5 g/L) was observed at pH 4, while optimum nutrient concentration of carbon (sucrose), nitrogen (NaNO₃), phosphorous (K₂HPO₄), and ascorbic acid was 50 g/L, 5 g/L, 1 g/L and 2 g/L, respectively. Interestingly, EPS productivity under non pH controlled fermentation conditions was 0.12 g/L/h with 400 rpm mixing, while under a controlled pH of 4, the EPS productivity was 0.21 g/L/h with 600 rpm, respectively. The fed-batch fermentation increased the EPS productivity up to 0.345 g/L/h with changing mixing conditions from 200 to 600 rpm and reached 47 g/L with 88% pullulan. Thus, pH and mixing were the key parameters for enhancing EPS production from A. pullulans. It is expected that these optimized parameters can be well used for enhanced industrial production of pullulan.     

负载光敏剂的壳寡糖衍生物纳米胶束的制备及性能

通过酰化反应将亚油酸引入到壳寡糖(COS)中得到两亲性衍生物N-亚油酰壳寡糖(LCOS)。用IR、元素分析和凝胶渗透色谱进行了结构表征。运用芘荧光探针法研究LCOS在水溶液中的胶束化行为,并制备LCOS负载四苯基卟啉(TPP)的载药胶束(TPP-LCOS)。通过等温滴定微量热法(ITC)测定了LCOS对TPP负载过程的热力学参数。结果表明,产物取代度约为42.1%;LCOS的临界胶束质量浓度CMC为2.00×10-3g/L,可有效负载TPP成纳米球形胶束,粒径在60~100 nm。并且LCOS与TPP之间中等强度的结合(Ka=2.87×106L/mol),有望使TPP缓慢释放,增加药效时间。     

Bacillus sp.W112产生表面活性剂条件优化及其特性

从大庆油田油水混合样中分离得到一株芽孢杆菌(Bacillus sp. W112). 菌株发酵液具有较高的表面活性,而且其表面活性剂具有较好的温度、pH和盐稳定性. 对表面活性剂产生条件进行优化,确定了最佳碳源为30 g/L麦芽糖,最佳氮源为1.5 g/L的混合氮源[硫酸铵+硝酸钠(1:2, j)],培养温度为37℃,在500 mL三角瓶中的装液量为100 mL,pH值为7.0,接种量为10%(j). 通过盐酸沉淀-化学试剂萃取法提取得到浅黄色固体产物,通过红外光谱分析,初步确定为一种脂肽类表面活性剂.