紫皮石斛寡糖的酶法制备及单糖组成分析

从多糖提取后的紫皮石斛（Dendrobium devonianum Paxt.）渣上分离到一株能够水解石斛多糖的菌株,为丰富寡糖食品新原料种类,对该菌株的发酵产酶活性进行分析。利用酶法制备紫皮石斛寡糖,并进行小试和中试,采用硅胶柱色谱对寡糖进行初步分离,PMP衍生化后通过HPLC分析单糖组成。结果表明,水解石斛多糖的菌株为粗糙脉孢菌（Neurospora crassa）。该菌株液态发酵至第4天酶活最高,为0.1 U/μL,最佳酶解时间为24 h,粗酶液能够耐受30%的乙醇。通过粗酶液生物转化石斛多糖的小试和中试成功制备了大量的石斛寡糖,并对各组分进行了初步分离,单糖组成分析表明其主要由甘露糖组成,并含有少量的葡萄糖或果糖。通过粗糙脉孢菌酶法制备得到的紫皮石斛寡糖主要为甘露寡糖。     

HPLC检测肠灌流模型中PMP衍生化葡寡糖含量的方法学建立

目的:建立肠灌流模型中1-苯基-3-甲基-5-吡唑啉酮(PMP)衍生化葡寡糖含量的检测方法,并进行方法学考察.方法:采用HPLC法测定,色谱条件为,色谱柱:phenomenex C18(250×4.60mm,51μm);流动相:100mMNaAc/乙腈(梯度洗脱);流速:1.0mL·min,-1;紫外检测波长245nm;进样量:20μL;柱温:40℃.结果:PMP衍生化葡寡糖在此色谱条件下获得良好分离,肠灌流液、血浆样品及酚红对10种化合物的检测均无影响.结论:该方法准确可靠,重复性良好,为寡糖的吸收研究提供了技术支持.     

酶促反应制备壳寡糖条件的研究

通过膜分离法从Microbacterium sp.OU01的发酵液中分离到内切壳聚糖酶ChiN,并对其酶解制备壳寡糖条件进行了优化,获得的酶解产物数均分子量为1200。经薄层层析与HPLC分析,降解产物中不含单糖,初步说明降解产物为壳寡糖。酶解制备壳寡糖反应条件温和,操作简便,为实现壳寡糖产业化奠定了基础。     

两步法硫酸水解热凝胶生产β-1,3-葡寡糖

对硫酸水解热凝胶制备β-1,3-葡寡糖进行了研究。首先建立了葡寡糖的分析方法,采用半制备HPLC分离热凝胶水解液得到β-1,3-葡寡糖,利用ESI-MS和TLC技术对分离组分进行了成分鉴定。考察了反应温度和反应时间对热凝胶寡糖收率的影响,结果表明,两步法硫酸水解获取低聚合度β-1,3-热凝胶寡糖(DP 2~6)的较优水解条件为:1.0 mol/L硫酸于70℃水解1%热凝胶6h,回收水解残留物后于80℃继续水解3 h;对于较高聚合度寡糖(DP 7~10)两步水解时间分别为4 h和1 h较为合适。     

用于荧光辅助糖电泳寡糖标尺的构建

荧光辅助糖电泳(FACE)是一种简洁廉价的分离糖类方法。寡糖首先与8-氨基萘基-1,3,6-三磺酸(ANTS)反应标记,然后,ANTS标记的寡糖通过在32％丙烯酰胺-2．4％双丙烯酰胺组成的分离胶上电泳从而得以相互分离。结果表明,电泳图谱能准确反映寡糖的聚合度梯度,因而,一种具有连续聚合度的淀粉水解液的荧光标记电泳图谱可以作为荧光辅助糖电泳的分子量标尺。     

荧光标记电泳对透明质酸寡糖片段鉴定的方法建立

近年来,透明质酸寡糖片段（hyaluronan oligosaccharides, o-HA）的生物学活性引起国外学者的重视,因为o-HA具有一定的生物学活性,如参与免疫调节、刺激新生血管形成等.本研究建立一种经济、简便的ANTS（8-氨基奈-1,3,6-三磺酸）荧光标记电泳对透明质酸寡糖片段大小鉴定的新实验方法.实验原理为,ANTS能与糖分子发生还原反应,在反应时提供3个电子和1个荧光基团,通过高浓度PAGE分离,在特定波长下呈现颜色反应.采用酶消化法得到不同分子量大小的o-HA片段,测得不同片段大小的o-HA聚合度,分别与高效液相色谱（high-performance liquid chromatography, HPLC）和静电喷雾电离质谱（electrospray ionization mass spectrometry, ESI-MS）进行比较,结果吻合.研究提示,用荧光标记电泳法分析寡糖分子量,操作简单、设备低廉、灵敏度较高且检测速度快,是一种检测鉴定寡糖分子的较好方法.     

糖类结构研究的若干进展

近几年糖类的结构测定有一定的进展。本文介绍了高效液相层析(HPLC)在糖类结构研究中的若干新的应用,其中包括:单糖和寡糖的高效阴离子交换层析和脉冲安培检测器的应用;寡糖的二维HPLC以及HPLC和电子计算机联用测定影响HPLC的一些参数,进而利用有关参数和HPLC的行为预测寡糖的结构。     

夏枯草多糖分离纯化、相关性质及药效

研究了夏枯草多糖的提取工艺、分离纯化、部分理化性质以及夏枯草多糖的免疫学活性。夏枯草经水提醇沉,去蛋白、脱色、凝胶柱色谱等步骤分离纯化得到夏枯草精多糖（Pure polysaccahrides of Spica prunellae,PPSP）。HPGPC法测其相对分子质量,红外光谱法对其结构进行初步分析,柱前对氨基苯甲酸衍生化HPLC法测其单糖组成。免疫学实验证明夏枯草精多糖单独作用或协同ConA均可显著促进小鼠脾淋巴细胞增殖,夏枯草精多糖在200μg／mL质量浓度下对正常小鼠的廓清指数和吞噬指数有明显促进作用。     

Man8GlcNAc2糖基化的酿酒酵母菌株的构建

酿酒酵母糖蛋白的N-糖基化经过高尔基体的修饰后形成聚合度约150-200的甘露寡糖,高尔基体N-糖基化的糖基转移酶Mnn1p和Och1p在甘露寡糖的形成过程中起关键作用。通过同源重组置换敲除了酵母中的MNN1和OCH1基因阻断高尔基体N-糖基化修饰,分离纯化了mnn1 och1突变株中的N-糖蛋白,糖酰胺酶PNGaseF酶解释放的N-糖链经过2-氨基吡啶衍生后,利用HPLC和MALDITOF/MS结合的方法分析了突变株糖蛋白上的N-糖链。结果显示mnn1 och1突变株中的糖蛋白的N-糖链为结构单一的糖链,分子量为1794.66,推测为Man8GlcNAc2。     

红花菜豆凝集素的糖结合专一性

经肼解、Ｂｉｏ－Ｇｅｌ Ｐ－２柱层析、ＮａＢ＾３Ｈ４和ＮａＢＨ４还原,制备各种来源的、氚标记在还原末端的、还原末端为Ｎ－乙酰氨基葡萄糖醇的混合寡糖,经Ｂｉｏ－Ｇｅｌ Ｐ－４凝胶柱分离,以及用糖苷酶酶解,制备了各种不同类型的氚标记的寡糖。这些寡糖在固定化的ＰＣＬ－Ｓｅｐｈａｒｏｓｅ柱上亲和层析,根据各种类型寡糖在ＰＣＬ－Ｓｅｐｈａｒｏｓｅ柱上的层析行为,确定红花菜豆（矮生红花变种）凝集素（ＰＣＬ）的     

Cellulose as Extracellular Polysaccharide of Hot Spring Sulfur-turf Bacterial Mat

The carbohydrate fraction of a hot spring sulfur-turf bacterial mat was shown to contain cellulose by the examination of neutral sugar composition, methylation analysis, and the identification of free oligosacchrides obtained from an acetolyzate of the desulfurized sulfur-turf mat. This suggested that the sulfur-oxidizing bacteria composing the sulfur-turf were producers of cellulose.     

壳寡糖作用于巨噬细胞的机制初探

研究壳寡糖对免疫系统中巨噬细胞作用的具体机制.结合流式细胞仪和激光共聚焦实验检测壳寡糖与巨噬细胞相互作用,通过凝胶阻滞实验在体外验证壳寡糖的胞内定位.实验结果证明壳寡糖与巨噬细胞相互作用过程如下,先与细胞膜结合,然后进入细胞内,最后定位在细胞核的核酸(DNA/RNA)上.     

Cellulose as extracellular polysaccharide of hot spring sulfur-turf bacterial mat

The carbohydrate fraction of a hot spring sulfur-turf bacterial mat was shown to contain cellulose by the examination of neutral sugar composition, methylation analysis, and the identification of free oligosacchrides obtained from an acetolyzate of the desulfurized sulfur-turf mat. This suggested that the sulfur-oxidizing bacteria composing the sulfur-turf were producers of cellulose.     

人胚肺成纤维细胞株纤连蛋白结构域N─糖链结构研究

本文应用明胶、肝素亲和层析二步法首先纯化了人胚肺成纤维细胞培养液的纤连蛋白（Ｆｉｂｒｏｎｅｏｔｈｅ,Ｆｎ）,经ＳＤＳ－ＰＡＧＥ鉴定为一条带,然后用胰糜蛋白酶消化纯化的Ｆｎ所获得的酶解波,经分离分别得到明胶结合片段和肝素结合片段,再应用凝集素－ＨＲＰ染色的Ｗｅｓｔｅｎ转移电泳法研究糖链结构,结果证实：１．Ｆｎ中明胶结合片段（４４ｋｄ）中含有二天线和多天线复杂型糖链,并接有平分型ｇｌｃＮＡｃ糖基、核心力Ｆｕｃ。２肝素结合片段（３０ｋｄ）只含有二天线复杂型糖链,不含平分型ＧｌｃＮＡｃ糖基及核心Ｆｕｃ．     

Application of microbore HPLC in combination with tandem MS for the quantification of rosuvastatin in human plasma

The potential of microbore high-performance liquid chromatography (HPLC) in combination with tandem mass spectrometry (MS/MS) for the sensitive detection of rosuvastatin (Crestor) in human plasma was investigated. Three microbore HPLC columns with internal diameters (i.d.) of 0.5, 1.0 and 2.0 mm were evaluated for column efficiency and mass sensitivity, and compared to a conventional 4.6 mm i.d. column. The 2.0 and 1.0 mm i.d. columns performed very well while the 0.5 mm i.d. column was slightly less efficient, this is probably due to a lower packing density. Good results with respect to gains in mass sensitivity compared to the conventional analytical column were achieved with the 2.0 and 1.0 mm columns. Thus, the 2.0 mm i.d. column had an improved signal-to-noise (S/N) ratio of 16 whilst the 1.0 mm i.d. column had an improved S/N ratio of greater than 70. Experiments with the 1.0 mm i.d. HPLC column were performed to determine the robustness of the microbore method for human plasma extracts after sample preparation using solid-phase extraction (SPE). A number of problems were encountered with extracts including high backgrounds, the blocking of the column and a rapid deterioration in column performance. The blocking of the column by particulates was solved by off-line filtration of the sample extracts. Peak tailing of the analytes and high background, both of which were due to endogenous interferences in the extracts, were eliminated using gradient elution. Using these approaches over 500 injections of plasma extracts were achieved without significant deterioration in assay performance. Quantities of rosuvastatin of 0.3 pg on-column could be detected and cross-validation experiments demonstrated that the conventional and the microbore HPLC-MS/MS methods provided similar information on the concentration of rosuvastatin but with greatly reduced sample consumption using the microbore method.     

Forensic analysis of eleven cyclic antidepressants in human biological samples using a new reversed-phase chromatographic column of 2 μm porous microspherical silica gel

A high-performance liquid chromatographic method has been developed for the forensic analysis of eleven frequently used cyclic antidepressant drugs (ADSs) (amitriptyline, amoxapine, clomipramine, desipramine, dosulepine, doxepin, imipramine, maprotiline, melitracen, mianserine and nortriptyline) using a recently developed reversed-phase column with 2 μm particles for the analysis of biological samples. The separation was carried out using two different C₈ reversed-phase columns (column 1: 100 mm × 4.6 mm I.D., particle size 2 μm, TSK gel Super-Octyl; column 2: 100 mm × 4.6 mm I.D., particle size 5 μm, Hypersil MOS-C₈) for comparison. The mobile phase was composed of methanol-20 mM KH₂PO₄ (pH 7) (60:40, v/v) and the flow-rate was 0.6 ml/min for both columns. The absorbance of the eluent was monitored at 254 nm. When the eleven drugs were determined, the sensitivity with the 2 μm particles was about five times greater than with the 5 μm particles. Retention times on column 1 were shorter than those on column 2. These results show that the new ODS column packing with a particle size of 2 μm gives higher sensitivity and a shorter analysis time than the conventional ODS column packing when applied to the analysis of biological samples.     

RNA extracts with polyadenylic acid sequences transfer specific sensitivity for a low molecular weight antigen (MW 486).

RNA prepared from the lymphoid tissues of guinea pigs specifically sensitized to mono(p-azobenzene-arsonate)-N-chloroacetyl-l-tyrosine (ARSNAT) (MW = 486) was fractionated by oligo(dT)-cellulose affinity chromatography. Two fractions designated as I and II were eluted from the column. Fraction II, binding to the column and containing polyadenylic acid sequences, transferred ARSNAT or keyhole-limpet-hemocyanin (KLH) sensitivity to nonsensitized guinea pig peritoneal exudate cells (GP-PEC) in 14 experiments. Fraction I was unable to transfer KLH or ARSNAT sensitivity to GP-PEC. The amount of Fraction II needed to transfer ARSNAT sensitivity was 10 times less than previously reported. Synthetic nonlymphoid cell poly(A) tested in this system failed to transfer ARSNAT or KLH sensitivity to GP-PEC. Both Fractions I and II were inactivated by ribonuclease. The results suggest a possible messenger function for the poly (A)-containing RNA fractions.     

Isolation of a malonyl-CoA-sensitive CPT/beta-oxidation enzyme complex from heart mitochondria

The goal of this study was to establish conditions for solubilization and characterization of CPTo, the malonyl-CoA sensitive form of mitochondrial carnitine palmitoyltransferase. CPTo of heart mitochondria is soluble in 1% octyl glucoside with retention of malonyl-CoA sensitivity. The degree of malonyl-CoA sensitivity is dependent on both the concentration of octyl glucoside and the presence of salt (KCl). In mannitol-sucrose, 0.5-1% octyl glucoside solubilizes CPTo without loss of malonyl-CoA sensitivity; however, either increasing the detergent concentration or addition of KCl promotes loss of malonyl-CoA sensitivity. The immunoglobulin fraction from immune serum obtained from rabbits immunized with the malonyl-CoA-insensitive form of CPT (CPTi) purified from beef heart mitochondria was used for preparation of an affinity column. The antibody column retained both malonyl-CoA-sensitive and -insensitive CPT activity without apparent selectivity. In addition to CPT, several other major protein bands were detected when the antibody column eluates were subjected to SDS-PAGE; however, native gel electrophoresis gives a large, high molecular weight, diffuse band. After elution of the antibody-CPT column with salt, a 68,000-Da protein is retained by the column. The retained protein contains the CPT activity, but it is not inhibited by malonyl-CoA. Thus, salt elution separates catalysis from inhibition. When the salt eluate is subjected to affinity chromatography using agarose-CoA, two protein peaks are obtained; both bind malonyl-CoA. One of the two fractions contains beta-hydroxyacyl-CoA dehydrogenase, beta-ketothiolase, and crotonase activity. These data show that octyl glucoside solubilized CPTo and CPTi are associated with a complex that contains beta-oxidation enzymes.     

寡聚糖生物农药对棉花体内细菌数量和作物生长的作用

通过常规分离方法,测定寡聚糖生物农药对中棉12品种的棉茎体内细菌群落数量的影响。2批实验结果表明喷寡聚糖后棉茎体内细菌群落数量均低于对照,喷寡聚糖10d后棉茎体内细菌群落数量显著下降(8.0×10     

利用重组大肠杆菌进行寡糖合成的研究进展

随着更多寡糖生物学活性的阐明,寡糖合成研究已成为糖生物学研究的热点之一,其中,以重组大肠杆菌作为酶盒或生物反应器,利用Leloir途径合成寡糖的方法,是近年来发展起来的一类重要的寡糖生物合成技术,并取得了较多的进展。将从细菌糖基转移酶的表达和鉴定、糖核苷酸的供给和寡糖的合成途径等几个方面,关注利用细菌功能尤其是利用重组大肠杆菌合成寡糖的研究进展,并分析各技术手段的优缺点及其应用前景。