氯代十六烷基吡啶在聚唾液酸分离纯化中的应用

利用氯代十六烷基吡啶对聚唾液酸特异性的络合效应,分离E．Coli发酵液中的聚唾液酸。通过单因素实验和正交实验分析确定氯代十六烷基吡啶与聚唾液酸发生络合和解离反应的最优工艺条件：络合时NaCl浓度0．05mol／L,pH6．0,加入的氯代十六烷基吡啶的质量为聚唾液酸质量的3倍;解离时NaCl浓度0．8mol／L,解离温度50℃。得到的聚唾液酸纯度大于95％,回收率大于80％。     

端基法测定聚唾液酸平均聚合度

建立了端基法测定聚唾液酸平均聚合度的方法.采用间苯二酚比色法和丙二腈荧光法分别测定聚唾液酸中唾液酸总量和还原端唾液酸残基含量,两者之比即为平均聚合度.研究表明,在pH 9.5的硼酸缓冲液中,80℃水浴下聚唾液酸还原端唾液酸残基与丙二腈反应25 min后生成荧光物质,其荧光强度与还原端唾液酸残基含量呈线性正相关关系,线性范围在1～ 20 mg/L之间,变异系数和检出限分别为3.7％和0.36 mg/L.端基法测定大肠杆菌发酵液中聚唾液酸的平均聚合度为45.76,与高效液相凝胶色谱法比较,误差为3.2％.该法可以简便快速地测定发酵液中聚唾液酸的平均聚合度,有利于聚唾液酸生产过程分析及产品性能评估.     

丙酮酸钠对E．coli CCTCCM208088发酵生产聚唾液酸的影响

研究了丙酮酸钠对E．coliCCTCCM208088发酵生产聚唾液酸的影响。首先在摇瓶水平上优化了丙酮酸钠的添加策略：发酵初始培养基中添加6g／L丙酮酸钠,聚唾液酸的产量达到最高水平3．47g／L,相比空白相比提高了73％,聚唾液酸产率（YP／（x．s））提高了125％。在30L发酵罐中,添加丙酮酸钠使聚唾液酸产量达到了6．6g／L,相对照提高了53．5％。通过有机酸分析,添加丙酮酸钠增强了菌体内三羧酸循环．可能导致胞内能荷水平的提高．从而促进了聚唾液酸的合成。     

两阶段搅拌转速控制策略发酵生产聚唾液酸

目的:优化聚唾液酸发酵过程的搅拌转速.方法:比较不同搅拌转速对大肠杆菌Escherichia coli K235分批发酵生产聚唾液酸过程的影响.结果:根据发酵前、后期菌体细胞比生长速率和聚唾液酸比合成速率达到最大值所需搅拌转速的不同,提出了两阶段搅拌转速控制策略:发酵前期(0～15h)控制搅拌转速500r/min,发酵中后期控制搅拌转速700r/min.结论:两阶段搅拌转速控制策略使聚唾液酸产量达到3 966mg/L,比恒定搅拌转速500r/min和700r/min分别提高了31.8%和49.3%.将两阶段搅拌转速控制策略与分批补料发酵技术结合,聚唾液酸产量提高到5 108mg/L,山梨醇的转化率达到0.12g/g.     

聚唾液酸,一种非GAGs、非免疫原性生物材料的应用研究进展

聚唾液酸是一种由N-乙酰神经氨酸连接、电负性的线性同聚物,存在于人体、动物细胞和少数致病菌中,主要以糖蛋白(神经细胞粘附分子)和糖脂形式存在,是一种非糖胺聚糖(GAGs)、非免疫原性、生物可降解的优良生物材料。聚唾液酸可用作组织工程和药物缓释材料,也可以与其它大分子复合形成功能材料。对聚唾液酸生物学功能、发酵生产及应用进行概述,以期为聚唾液酸的进一步应用研究提供参考。     

嗜热脂肪芽孢杆菌的β—半乳糖苷酶合成低聚兰乳糖动力学模型

根据反应机理和产物的化学结构,建立了嗜热脂肪芽孢来源的β－半乳糖苷酶催化乳糖水解和转半乳糖苷反应偶合的动力学模型,模型中包含了低聚三糖和低聚四糖的生成。通过优化计算,估算反应动力学参数,结果表明该模型能较好地与实验结果相吻合。     

聚唾液酸与唾液酸的研究进展

唾液酸是一族神经氨酸(Neuraminic acid)的衍生物。聚唾液酸(Polysialic acid)是唾液酸(Sialic acid)单体以α-2,8或α-2,9键连接的直链同聚物,是一些哺乳动物细胞中糖蛋白的组成部分和少数几种细菌的胞外多糖组分。综述了唾液酸和聚唾液酸的结构、性质、生物学功能、生物合成和生产应用。     

嗜热脂肪芽孢杆菌β-半乳糖苷酶合成低聚半乳糖动力学模型

根据反应机理和产物的化学结构,建立了嗜热脂肪芽孢来源的β-半乳糖苷酶催化乳糖水解和转半乳糖苷反应偶合的动力学模型,模型中包含了低聚三糖和低聚四糖的生成。通过优化计算,估算反应动力学参数,结果表明该模型能较好地与实验结果相吻合。     

聚唾液酸高产菌株的高通量诱变选育及发酵工艺优化

从自然水体中筛选得到1株产聚唾液酸的微生物菌株SA-1,经Biolog生化和分子生物学分析,鉴定其为大肠杆菌。以大肠杆菌SA-1为出发菌株,通过常压室温等离子体(ARTP)诱变育种,借助酸碱透明圈和高通量自动挑选仪筛选获得高产聚唾液酸的突变菌株SA-18。摇瓶发酵结果显示,大肠杆菌突变株SA-18的聚唾液酸产量可达0.95 g/L,是出发菌株产量(0.20 g/L)的4.75倍;进一步,通过调控优化K₂HPO₄浓度实现了突变大肠杆菌SA-18的高密度发酵：当K₂HPO₄质量浓度为5.0 g/L时,在10 L发酵罐中,36 h时聚唾液酸产量达到12.20 g/L,继续补料发酵,至60 h时,聚唾液酸产量最高可达16.10 g/L。     

聚谷氨酸批式生物合成的主要影响因素研究

采用Bacillus subtilisNX-2菌株,在5 L发酵罐批式操作过程中生物合成γ-聚谷氨酸,实验考察了搅拌转速(300~1 000 r/min)及底物质量浓度对生物合成γ-聚谷氨酸产率的影响。结果表明:转速为400 r/min时,γ-聚谷氨酸产率最高,可高达21.78 g/L。在转速400 r/min的条件下,考察了葡萄糖和谷氨酸浓度对聚谷氨酸生物合成的影响,根据葡萄糖初始质量浓度为40 g/L的实验数据建立了动力学方程。并在葡萄糖初始质量浓度为30,40,50,60g/L的范围内,验证所建动力学方程的稳定性和实用性。结果表明,在以上葡萄糖初始质量浓度范围内,所建模型对B.subtilisNX-2批式生物合成γ-聚谷氨酸产率的预测值与实验值能较好的吻合。     

Neural cell adhesion molecule-associated polysialic acid inhibits NR2B-containing N-methyl-D-aspartate receptors and prevents glutamate-induced cell death

The neural cell adhesion molecule (NCAM) and its associated glycan polysialic acid play important roles in the development of the nervous system and N-methyl-D-aspartate(NMDA)receptor-dependent synaptic plasticity in the adult. Here, we investigated the influence of polysialic acid on NMDA receptor activity. We found that glutamate-elicited NMDA receptor currents in cultured hippocampal neurons were reduced by approximately 30% with the application of polysialic acid or polysialylated NCAM but not by the sialic acid monomer, chondroitin sulfate, or non-polysialylated NCAM. Polysialic acid inhibited NMDA receptor currents elicited by 3 microm glutamate but not by 30 microm glutamate, suggesting that polysialic acid acts as a competitive antagonist, possibly at the glutamate binding site. The polysialic acid induced effects were mimicked and fully occluded by the NR2B subunit specific antagonist, ifenprodil. Recordings from single synaptosomal NMDA receptors reconstituted in lipid bilayers revealed that polysialic acid reduced open probability but not the conductance of NR2B-containing NMDA receptors in a polysialic acid and glutamate concentration-dependent manner. The activity of single NR2B-lacking synaptosomal NMDA receptors was not affected by polysialic acid. Application of polysialic acid to hippocampal cultures reduced excitotoxic cell death induced by low micromolar concentration of glutamate via activation of NR2B-containing NMDA receptors, whereas enzymatic removal of polysialic acid resulted in increased cell death that occluded glutamate-induced excitotoxicity. These observations indicate that the cell adhesion molecule-associated glycan polysialic acid is able to prevent excitotoxicity via inhibition of NR2B subunit-containing NMDA receptors.     

An IgG monoclonal antibody to group B meningococci cross-reacts with developmentally regulated polysialic acid units of glycoproteins in neural and extraneural tissues

The structurally similar polysialic acid capsules of group B meningococci and Escherichia coli K1 are poor immunogens, and attempts are currently being made to improve their immunogenicity by chemical modifications. An IgG monoclonal antibody to these polysialic acid capsules was used for the study of the presence of structurally similar components in tissue glycoproteins to investigate the reasons for the poor immunogenicity and to evaluate potential dangers in active or passive immunization. By immunoblotting polysialic acid was detected outside the brain in newborn rat kidney, heart, and muscle. It appeared in immunoblots as one component and with similar mobility to the neural cell adhesion molecule N-CAM. Specificity studies of the antibody and endosialidase treatment showed that the polysialic acid glycans detected were composed of chains as long as eight sialic acid residues or more. The polysialic acid was not detected in the corresponding tissues of the adult animal. These results indicate that polysialic acid units are developmentally regulated components of both neural and extraneural tissues, and are bound to components with properties similar to a known cell-adhesion molecule. This together with the presence of low amounts of polysialic acid even in the adult brain, suggests potential hazards in vaccination trials and suggested immunotherapy of meningitis caused by group B meningococci or E. coli K1, which should be carefully assessed.     

Immunohistochemical localization of polysialic acid in tissue sections: differential binding to polynucleotides and DNA of a murine IgG and a human IgM monoclonal antibody.

For immunolocalization of alpha(2-8)-linked polysialic acid, which forms part of the neural cell adhesion molecule (N-CAM), two monoclonal antibodies, MAb735 and IgMNOV, were employed. Both antibodies have previously been shown to bind the extremely low immunogenic capsular polysaccharide of group B meningococci, which also consists of alpha(2-8) polysialic acid, but not to other, even closely related forms of polysialic acid. Despite the identical polysaccharide specificity of these two MAb, we observed marked differences of the staining pattern in tissue sections. We showed that these differences in immunostaining were due to the crossreactivity of IgMNOV with polynucleotides and DNA. MAb735, however, was shown to react exclusively with alpha(2-8) polysialic acid. Moreover, the specificity of MAb735 proved to be unique among eleven other MAb directed against various bacterial polysaccharides, as it was the only one unreactive with polynucleotides. Thus, MAb735, the only IgG type mouse monoclonal antibody to polysialic acid thus far reported, can be considered a specific probe for the unambiguous detection of alpha(2-8) polysialic acid in tissue sections, and should therefore help to further elucidate the role of polysialic acid in developmental processes.     

Mutant bacteriophage with non-catalytic endosialidase binds to both bacterial and eukaryotic polysialic acid and can be used as probe for its detection

There is a molecular mimicry between the polysialic acid polysaccharide of bacterial pathogens causing sepsis and meningitis, and the carbohydrate units of the neural cell adhesion molecule NCAM. We investigated whether bacteriophage mutants with catalytically disabled endosialidase, which bind but do not cleave polysialic acid, could recognise and bind to bacterial and eukaryotic polysialic acid. In nitrocellulose dot blot assay the mutant bacteriophages, but not the wild-type phages, remained specifically bound to polysialic acid–containing bacteria including Escherichia coli K1 and K92, group B meningococci, Mannheimia (Pasteurella) haemolytica A2, and Moraxella nonliquefaciens. A minimum binding requirement was determined to be 10 sialyl residues in the polysialic acid chain. In Western blots the mutant phages specifically bound to the embryonic polysialylated form of NCAM, but not to the adult less sialylated form of the molecule. The mutant phages together with secondary anti-phage antibodies were subsequently successfully used in fluorescence microscopy of cultured cells and light microscopy of paraffin-embedded tissue sections as a probe for the eukaryotic polysialic acid. Thus, mutant bacteriophages of meningitis causing bacteria bind to and detect the molecularly mimicked polysialic acid of the neural cell adhesion molecule in host tissues.     

Biochemical engineering of surface alpha 2-8 polysialic acid for immunotargeting tumor cells

To target tumor cells for immunotherapy, we evaluated the feasibility of altering the epitopes on the surface polysialic acid of tumor cells. A precursor (N-propionylmannosamine), when incubated with leukemic cells, RBL-2H3 and RMA, resulted in substitution of the N-acetyl groups of surface alpha2-8 polysialic acid with N-propionyl groups. Expression of the altered alpha2-8 N-propionylpolysialic acid on the surface of tumor cells induced their susceptibility to cell death mediated by monoclonal antibody 13D9 (mAb 13D9), which specifically recognizes alpha2-8 N-propionylated polysialic acid. The expression of alpha2-8 N-propionylated polysialic acid and the lysis of tumor cells by antibody-dependent cytotoxicity depended on the time and dose of incorporation of N-propionylated mannosamine. In vivo, mAb 13D9 effectively controlled metastasis of leukemic cells RMA when mice were administered the precursor N-propionylated mannosamine.     

NMR study into the mechanism of recognition of the degree of polymerization by oligo/polysialic acid antibodies

Oligo/polysialic acids consisting of consecutive α(2,8)-linkages on gangliosides and glycoproteins play a role in cell adhesion and differentiation events in a manner that is dependent on the degree of polymerization (DP). Anti-oligo/polysialic acid antibodies often have DP-dependent antigenic specificity, and such unique antibodies are often used in biological studies for the detection and differentiation of oligo/polysialic acids. However, molecular mechanisms remain unclear. We here use NMR techniques to analyze the binding epitopes of the anti-oligo/polysialic acid monoclonal antibodies (mAb) A2B5 and 12E3. The mAb A2B5, which has a preference for trisialic acid, recognizes sialic acid residues at the non-reducing terminus and those in nascent units. On the other hand, mAb 12E3, which prefers oligo/polysialic acids of more than six sugar units, recognizes inner sialic acid residues. In both structural complexes, the interresidue transferred NOE correlations are significantly different from those arising from analogs of the free states, indicating that the bound and free sugar conformations are distinct. The ability of the two mAbs to distinguish the chain lengths comes from different binding epitopes and possibly from the conformational differences in the oligo/polysialic acids. Information on the recognition modes is needed for the structural design of immunoreactive antigens for the development of high-affinity anti-polysialic acid antibodies and of related vaccines against pathogenic, polysialic acid-coated bacteria.