阿魏侧耳多糖的分离纯化与抗肿瘤活性的研究*

从阿魏侧耳 (Pleurotusferulae)子实体中用热水浸提出粗多糖PFW ,将PFW脱蛋白后 ,经DEAE—纤维素柱层析 ,得两种多糖PF1 和PF2 。用光散射和GPC联机测定 :PF1 含有两种组分其重均分子量分别为 7 875× 1 0 5 和 3 2 4 5× 1 0 4 ;PF2 重均分子量为 3 1 87× 1 0 6。用IR和GC分析它们组成和结构 ,结果表明 :PF1 由鼠李糖、葡萄糖、半乳糖和甘露糖组成 ,含有 β 糖苷键和甘露糖苷 ;PF2 由鼠李糖、木糖、葡萄糖、半乳糖和甘露糖组成 ,含有α 糖苷键。给小鼠腹腔注射多糖 ,对移植性肿瘤S 1 80均有一定的抑制作用     

海藻糖微生物酶法合成机制的研究*

来源于嗜酸热古菌芝田硫化叶菌 (Sulfolobusshibatae)B1 2的麦芽寡糖基海藻糖合酶(MTSase)和麦芽寡糖基海藻糖海藻糖水解酶 (MTHase)基因在大肠杆菌中获得表达。将获得纯化的两个酶 ,分别以麦芽寡糖和淀粉为转化底物 ,在pH5 5 ,6 0℃条件下合成海藻糖。从反应产物分析结果可知 ,两个酶合成海藻糖时能利用的最小底物是麦芽四糖 ,海藻糖产率与麦芽寡糖链长正相关。同时还发现两个酶都具有轻微的α 1 ,4 葡萄糖苷酶活性 ,能在麦芽寡糖还原末端水解α 1 ,4糖苷键     

蛹虫草胞外多糖的研究I．半乳甘露聚糖(CM-I)的纯化和结构研究

从蛹虫草菌体培养液中提取水溶性粗多糖经分离纯化,得一种含有少量蛋白的半乳甘露聚糖CM-I,分子量2．7×10’,[a]19d=十54．7°。糖组成摩尔比,半乳糖： 甘露糖=6：5。经高碘酸氧化,Smith降解,部分酸水解,半乳糖苷酶解,1H-NMR分析,完全甲基化与GC及GC-MS分析,证明：多糖CM-I具有高度分枝结构,其以β-(1→2)连接的甘露糖为主干,枝链由较大量的β-(1→6)半乳糖和较大最的β-(1→2)呋喃半乳糖构成,分别连接在主干的0-4和0一6上。     

Ralstonia eutropha CH34酯酶基因的克隆和序列分析

采用含α 乙酸萘酯和固兰RR的表面琼脂法从RalstoniaeutrophaCH34的基因文库中筛选酯酶基因estA ,对含有estA的 1 7kbDNA片段的核甘酸序列分析表明 ,该基因全长82 5bp,编码由 2 75个氨基酸组成的EstA蛋白 ,分子量为 30 785D。经推导氨基酸序列的同源性分析 ,发现EstA与参与芳香化合物代谢中间位裂解途径的水解酶有很高的同源性。     

一株产微生物絮凝剂菌株的分离鉴定及特性*

从活性污泥中分离筛选到一株产絮凝剂的细菌 A2 5,鉴定为巨大芽孢杆菌 Bacillusmegaterium。该菌株产絮凝剂的最适碳源为麦芽糖 ,最适氮源为酵母提取物 ,最适 p H为 7.0～ 10 .0。絮凝剂的形成与菌体生长同步 ,均在 10 h达到最高值。该絮凝剂主要分布在发酵液中 ,另外还有一部分存在于菌体上。所产絮凝剂对供试的各种悬浮液和菌悬液都具有良好的絮凝效果。     

假单细菌GX4-1利用鱼粉废水产絮凝剂的研究

研究了假单胞菌 (Pseudomonassp .)GX4 1利用鱼粉废水产生絮凝剂的条件 ,结果表明 ,适宜条件为废水COD浓度为 1 0g/L左右、初始pH为 7～ 9、培养温度为 30℃、摇床转速为 1 0 0～ 2 50r/min ,此时的絮凝率可高达 99 5%。同时发现在废水培养基不灭菌的条件下 ,该菌仍能产生高效的絮凝剂。该菌利用鱼粉废水产生的絮凝剂对高岭土悬液、土壤悬液和细活性炭粉末悬液和电瓷厂污水均有较好的絮凝作用。     

花鲈(Lateolabrax japonicus)VAMP-2基因的特征研究

应用反转录PCR和锚定PCR方法克隆了全长715个碱基的花鲈(Lateolabrax japonicus)SPV2基因, 证明其编码110个氨基酸的VAMP-2蛋白. 运用生物信息学研究工具进行的一级结构分析显示: SPV2蛋白含有SNARE基序及其核心精氨酸残基, 其序列N端有一N-糖肽键的糖基化位点, C端有一跨膜结构域. 高级结构预测揭示SPV2蛋白的主要空间构象是一个组成SNARE基序的α螺旋卷曲骨架, 其主要细节都与X晶体衍射方法得到的大鼠VAMP-2结构相一致. 半定量PCR结果表明: 在mRNA水平上, VAMP-2基因在鱼体各组织中广泛表达, 其中以脑组织最为突出, 而肌肉组织则相对最少.     

利用Ac/Ds转座子系统在水稻中获得无选择标记转基因植株的方法

获得无选择标记转基因植株是进行重复转基因及消除转基因植株中标记基因潜在危害性的关键。实验采用了Ac/Ds转座子系统在水稻(Oryza sativa L.)中进行无hpt选择标记的转基因。将含有目的基因bar的Ds元件和hpt标记基因置于同一个T-DNA中,通过农杆菌(Agrobacterium tumefaciens) EHA105介导将Ac-T-DNA及Ds-T-DNA分别转入到不同的水稻植株,再将单拷贝的Ac-T-DNA植株与单拷贝的Ds-T-DNA植株杂交得到同时含有Ac和Ds元件的F₁植株,F_{1自交产生F2后代,F2植株中转座后的Ds元件与T-DNA独立分离,在总共100株F2水稻植株中筛选得到2株只含有Ds元件插入而无hpt标记基因的转基因水稻植株。结果表明,利用Ac/Ds转座子系统在水稻中获得无选择标记的转基因植株是可行的。}     

深层培养裂褶菌胞外多糖的提取及结构研究

对深层培养裂褶菌 (Schizophyllumcommune)胞外多糖的提取工艺及多糖结构进行了初步研究。将等电点法与Sevag法相结合可高效的去除多糖中的蛋白 ,其方法简单有效。纯多糖经凝胶柱层析 ,聚丙烯酰胺凝胶电泳 ,高效液相色谱分析为均一组分 ,分子量 4×1 0 4D。通过完全水解 ,纸层析 ,气相色谱分析单糖组分 ,红外光谱 ,酶解反应 ,高碘酸氧化分析结构 ,证明了裂褶菌多糖是以葡萄糖为单一组分 ,β (1 3)和β (1 6)糖苷键组成的β D葡聚糖。     

阿魏侧耳酸提水溶性多糖的研究

从热水浸提过的阿魏侧耳（Pleurotus ferulae）子实体中用3%的三氯乙酸提取出另一种水溶性粗多糖PFA,将PFA经Sevage法脱蛋白、透析、CTAB络合去沉淀后得多糖PF3。苯酚硫酸法测得PF3糖含量为94.1%;用光散射和GPC联机测得PF3重均分子量为1.965×10.6;用IR和GC分析了组成和结构,PF3由鼠李糖、木糖、葡萄糖、半乳糖和甘露糖组成,含有α-糖苷键。同时研究了PF3对核糖核酸酶的抑制作用,表明在浓度5mg/mL时其抑制率可达44.4%。     

Flocculant and Chemical Properties of a Polysaccharide from Pullularia pullulans

An extracellular polysaccharide, PP-floc, was synthesized from glucose by Pullularia pullulans (or Aureobasidium pullulans) in a pilot plant batch fermentor containing 175 liters of culture medium. At 58 h of fermentation, the concentration of PP-floc was 1.03 g/100 ml, giving a 25.8% conversion of initial glucose to polysaccharide. The flocculant activity of the culture medium increased during the fermentation process and reached its maximum at 50 h of culture age. Less PP-floc (0.33 lb/ton of slimes [approximately 149.7 g/0.907 t]) was required to give the same flocculant activity as a synthetic polymer of acrylamide, Separan NP-10 (0.5 lb/ton of slimes [approximately 226.8 g/0.907 t]), at all temperatures from 25 to 100 C. The degree of inactivation of PP-floc and Separan NP-10 at elevated temperatures was almost identical, and they were completely inactivated at about the same temperature (80 C). PP-floc also gave better compaction of slimes than Separan NP-10 at all temperatures tested. PP-floc was soluble in water and its specific optical rotation was [alpha](D) (25) + 194 degrees in water (c, 0.4). PP-floc contained 83.3% carbohydrate, 3.2% protein, and 8.1% water. Glucose was found to be the principal sugar monomer with traces (>5%) of galactose and mannose present. Structural studies on the fractions of purified polysaccharide by methylation and by periodate oxidation techniques prove that PP-floc is linear and composed of alpha-(1 --> 4) and alpha-(1 --> 6) glucopyranosyl units in the approximate ratio of 2:1. The action of pullulanase on crude PP-floc suggested the ordered arrangement of two consecutive alpha-(1 --> 4)-linked glucopyranosyl units flanked by alpha-(1 --> 6)-linked glucopyranose residues.     

Purification and Chemical Properties of a Flocculant Produced by Paecilomyces

A new flocculant for microbial cells was purified by ethanol precipitation and gel chromatography from the culture fluid of Paecilomyces sp. I-1. Isoelectric focusing of the flocculant (PF-101) showed a single band at pH 8.5, and its molecular weight was estimated to be over 300,000 daltons by the ultra-filtration method. The results of elemental analysis, the IR spectrum and investigation of the acid hydrolysate by gas and liquid chromatography and colorimetrie analysis suggested that PF-101 was a polysaccharide composed of galactosamine. About 80% of the galactosamine residues were N-unsubstituted and 8% were N-acetylated. Studies on deaminative cleavage, periodate oxidation and Smith degradation suggested that the galactosamine residues were mainly linked by α (→4)-linkaees.     

Isolation and structural study of polysaccharides from campion Silene vulgaris

Silenan SV, a pectic polysaccharide, was isolated from the aerial part of Silene vulgaris (Moench) Garke (Oberna behen (L.) Ikonn.), widespread through the European North of Russia. The polysaccharide was found to contain residues of galacturonic acid (63%), arabinose, galactose, and rhamnose as the main constituents. The results of a partial acidic hydrolysis, pectinase digestion, and NMR studies of silenan SV indicated that its molecule contains a linear alpha-1,4-D-galacturonan backbone and ramified regions. The core of the ramified regions is composed of residues of alpha-1,4-D-galacturonic acid along with 2-substituted alpha-rhamnopyranose residues. The NMR data showed that the silenan SV side chains are composed of the blocks built from the terminal alpha-1,5-linked arabinofuranose and beta-1,4-linked galactopyranose residues; these most likely are the side chains of rhamnogalacturonan, characteristic of other pectic polysaccharides. The nonreducing ends of these side chains contain alpha-arabinofuranose residues.     

Isolation of a coaggregation-inhibiting cell wall polysaccharide from Streptococcus sanguis H1.

Coaggregation between Streptococcus sanguis H1 and Capnocytophaga ochracea ATCC 33596 cells is mediated by a carbohydrate receptor on the former and an adhesin on the latter. Two methods were used to release the carbohydrate receptor from the gram-positive streptococcus, autoclaving and mutanolysin treatment. The polysaccharide released from the streptococcal cell wall by either treatment was purified by ion-exchange chromatography; this polysaccharide inhibited coaggregation when preincubated with the gram-negative capnocytophaga partner. After hydrolysis of the polysaccharide by hydrofluoric acid (HF), the major oligosaccharide of the polysaccharide was purified by high-performance liquid chromatography. By analysis of the HF hydrolysis of the polysaccharide and the purified oligosaccharide, this major oligosaccharide appeared to be the repeating unit of the polysaccharide, with minor components resulting from internal hydrolysis of the major oligosaccharide. Gas chromatography results showed that the oligomer was a hexasaccharide, consisting of rhamnose, galactose, and glucose, in the ratio of 2:3:1, respectively. By weight, the purified hexasaccharide was a fourfold-more-potent inhibitor of coaggregation than the native polysaccharide. Resistance to hydrolysis by sulfuric acid alone and susceptibility to hydrolysis by HF suggested that oligosaccharide chains of the polysaccharide are linked by phosphodiester bonds. Studies with a coaggregation-defective mutant of S. sanguis H1 revealed that the cell walls of the mutant contained neither the polysaccharide nor the hexasaccharide repeating unit. The purification of both a polysaccharide and its constituent hexasaccharide repeating unit, which both inhibited coaggregation, and the absence of this polysaccharide or hexasaccharide on a coaggregation-defective mutant strongly suggest that the hexasaccharide derived from the polysaccharide functions as the receptor for the adhesin from C. ochracea ATCC 33596.     

Isolation, characterization, and antitumor activities of the cell wall polysaccharides from Elsinoe leucospila.

A cell-wall preparation from the cells of Elsinoe leucospila, which produces elsinan extracellularly when grown on sucrose or glucose-potato extract medium, was fractionated systematically. The heteropolysaccharide that was released by treatment with Actinase E digestion, comprised D-mannose, D-galactose, and D-glucose (molar ratio, 1.5:1.0:0.1). Methylation, mild acid hydrolysis, and 13C-NMR studies suggested that the polysaccharide contains a backbone of alpha-(1----6)-linked D-mannose residues having two kinds of side chains, one attached at the O-4 with single or short beta-(1----6)-linked D-galactofuranosyl residues, and the other attached at O-2 with short side chains, most probably, of alpha-(1----3)-linked D-mannopyranosyl residues. A moderately branched D-glucan fraction, obtained from the cold alkali extract, was fractionated to give an antitumor-active purified beta-(1----3)-glucan having branches of single beta-D-glucosyl groups, one out of eight D-glucose residues being substituted at the O-6.     

Acetyl-coenzyme A:polysialic acid O-acetyltransferase from K1-positive Escherichia coli. The enzyme responsible for the O-acetyl plus phenotype and for O-acetyl form variation

The capsular polysaccharide of Escherichia coli K1 is a linear polymer of N-acetylneuraminic acid in alpha-2,8 linkage. Certain substrains of E. coli K1 (designated OAc+) modify the polysaccharide by O-acetylation of the sialic acids. We demonstrate here an acetyl-coenzyme A: polysialosyl O-acetyltransferase activity that is found only in E. coli K1 OAc+ substrains. When form variation between the O-acetyl-positive and -negative states occurred in strain D698:K1, the fluctuations were accompanied by appropriate changes in the expression of enzyme activity. Thus, expression of this enzyme can account for the OAc+ phenotype and for the form variation between OAc+ and OAc-. The enzyme was solubilized in nonionic detergent and freed of endogenous acceptor activity by DEAE-cellulose chromatography, and its general properties were determined. Analysis of the reaction product showed a highly preferential acetylation reaction that was confined to polysialosyl units of greater than 14 residues. Acetyl groups were shown to be transferred to both the 7- and the 9-positions of the sialic acid residues. The partially purified enzyme was stable even after prolonged incubation at 57 degrees C. In contrast, any further purification resulted in loss of activity, even at 4 degrees C. Treatment of the stable enzyme with a polysialic acid-specific endoneuraminidase caused a similar loss of enzyme stability. This effect of the endoneuraminidase could be protected against by the addition of exogenous polysialic acid. This indicates that the partially purified enzyme contains traces of endogenous polysialic acid substrate that are required for the stability of the enzyme. Finally, the enzyme can O-acetylate the polysialic acid chains on the eucaryotic protein neural cell adhesion molecule, suggesting that enzymatic recognition of the substrate requires only the polysialic acid sequence.     

Macromolecular properties and end-group analysis of heparin isolated from bovine liver capsule.

Glycosaminoglycans were extracted from bovine liver capsule with 4 M-guanidinium chloride, resulting in solubilization of approx. 90% of the total uronic acid-containing polysaccharide of the tissue. The extracted polysaccharide was purified and fractionated by anion-exchange chromatography on DEAE-cellulose, density-gradient ultracentrifugation in CsCl and finally gel chromatography on Sepharose 4B. By using these procedures, the two major polysaccharide components, dermatan sulphate and heparin, which constituted 55 and 30% respectively of the total glycosaminoglycan content of the tissue, were separated from each other. Analysis of the macromolecular properties of the two polysaccharides showed that heparin existed exclusively as single polysaccharide chains, whereas dermatan sulphate occurred largely as a proteoglycan (protein content, 74% dry wt.). The purified heparin preparation was subjected to sedimentation-equilibrium ultracentrifugation, indicating a molecular weight of 8800. Analysis for neutral sugars (by g.l.c.) showed 0.1 residue of xylose and 0.2 residue of galactose/polysaccharide chain; serine amounted to 0.3 residue/polysaccharide chain. Reduction of the heparin with NaB3H4 resulted in incorporation of 3H, approximately corresponding to one reducible group/polysaccharide chain. The 3H-labelled sugar residue was liberated by a combination of acid hydrolysis and deaminative cleavage of the polysaccharide with HNO2; it was subsequently identified as an aldonic acid by paper electrophoresis. Most of the heparin chains thus contained a uronic acid residue in reducing position. It is suggested that heparin isolated from bovine liver capsule is a degradation product released from larger molecules by an endo-glycuronidase.     

Structure and physiological activity of lemnan, Lemna minor L. pectin

A pectic polysaccharide, lemnan, was isolated from freshly collected duckweed Lemna minor L. Its sugar chain was shown to be mainly composed of the residues of D-galacturonic acid (64%), galactose, arabinose, xylose, and D-apiose, a branched chain sugar. The high content of D-apiose (25%) indicated that lemnan is an apiogalacturonan type pectin similar to zosteran, a pectic polysaccharide from a sea phanerogam of the Zosteraceae family. The results of partial acidic hydrolysis, pectinase digestion, and NMR studies of lemnan demonstrated that its macromolecule contains regions of the linear alpha-1,4-D-galacturonan and branched apiogalacturonan. The side chains of apiogalacturonan were found to be formed of single and 1,5-linked residues of D-apiofuranose attached to 2- and 3-positions of the D-galacturonic acid residues of the apiogalacturonan backbone. Lemnan was shown to exhibit an immunomodulatory effect by activating the system of phagocytosis.     

Structural analysis of the extracellular polysaccharide produced by Sphaerotilus natans

An extracellular polysaccharide (EPS) was recovered and purified from the culture fluid of a sheathed bacterium, Sphaerotilus natans. Glucose, rhamnose, and aldobiouronic acid were detected in the acid hydrolysate of EPS by thin-layer chromatography (TLC). The aldobiouronic acid was found to be composed of glucuronic acid and rhamnose by TLC and gas-liquid chromatography analyses of the corresponding neutral disaccharide. The structure of EPS was identified by methylation linkage analysis and nuclear magnetic resonance. Additionally, partial acid hydrolysates of EPS were prepared and put through fast atom bombardment-mass spectrometry to determine the sugar sequence of EPS. The resulting data showed that EPS produced by S. natans is a new gellan-like polysaccharide constructed from a tetrasaccharide repeating unit, as shown below. -->4)-alpha-D-Glcp-(1-->2)-beta-D-GlcA p-(1-->2)-alpha-L-Rha p-(1-->3)-beta-L-Rha p-(1-->.     

Isolation and purification of cell wall polysaccharide of Bacillus anthracis (Δ Sterne)

A polysaccharide fraction was isolated form sodium-dodecyl-sulfate (SDS) treated cell walls of Bacillus anthracis (delta Sterne) by hydrofluoric acid (HF) hydrolysis and ethanolic precipitation. The polysaccharide fraction was subsequently purified by several washings with absolute ethanol. Purity of the isolated polysaccharide was tested using the anthrone assay and amino acid analyzer. The molecular mass of the polysaccharide fraction as determined by gel filtration chromatography was about 12000 Da. Preliminary analyses of the polysaccharide was done using thin layer chromatography and amino acid analyzer, and results obtained from these analyses were further confirmed by gas liquid chromatography and 13C-NMR spectroscopy. Results showed that the polysaccharide moiety contained galactose, N-acetylglucosamine, and N-acetylmannosamine in an approximate molar ratio of 3:2:1. This moiety was devoid of muramic acid, alanine, diaminopimelic acid, glutamic acid, and lipid, thus indicating that the isolated polysaccharide was of pure quality.