湖南尖吻蝮蛇毒两个出血毒素的纯化和理化性质

经ＳｅｐｈａｄｅｘＧ－７５凝胶过滤,ＱＡＥ－ＳｅｐｈａｄｅｘＡ－５０和ＣＭ－ＳｅｐｈａｄｅｘＣ－２５离子交换层析的步骤,从湖南产尖吻蝮（Ｄｉｅｎａｇｋｉｓｔｒｏｄｏｎａｃｕｔｕｓ）蛇毒中纯化出两个出血毒素（ＤａＨＴ－１和ＤａＨＴ－２）．ＳＤＳ－ＰＡＧＥ测得分子量均为２３．５ｋＤ,ＩＥＦ－ＰＡＧＥ测得等电点分别为５．６和５．２,两者具有相似的氨基酸组成,其中酸性氨基酸（Ａｓｘ,Ｇｌｘ）分别占２３％和２４％,ＤａＨＴ－１和ＤａＨＴ－２的最小出血剂量（ＭＨＤ）分别为０．５μｇ和０．８μｇ。都具蛋白水解酶活性,无对ＴＡＭＥ,ＢＡＥＥ的水解活性和ＰＬＡ２酶活性．两者的蛋白水解酶活力与出血活性并非正相关．ＤａＨＴ－１和ＤａＨＴ－２的最适温度分别为３５℃和４０℃,最适ｐＨ为６－９,对热均不稳定,温度高于６０℃活性完全丧失。金属离子的分析显示每摩尔毒素蛋白约含０．５ｍｏｌ的Ｚｎ,１ｍｏｌ的Ｃａ,较多的Ｎａ、Ｋ、Ｍｇ,不含Ｃｏ。     

慈菇（Sagittaria safittifolia）α－半乳糖苷酶的纯化与性质

以对硝基苯糖苷基为底物,测定了慈菇的１２种糖苷酶,其中α－甘露糖苷酶、α－和β－半乳糖苷酶活力较高;经硫酸铵分级沉淀,ＳｅｐｈａｄｅｘＧ－１５０分子筛层析,ＣｏｎＡＳｅｐｈａｒｏｓｅ４Ｂ亲和层析,ＤＥＡＥ－ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ离子交换层析,从慈菇抽提液纯化了α－半乳糖苷酶。纯化酶的比活提高１０７２倍,活力回收１５．６％,在圆盘聚丙烯酰胺凝胶电泳和ＳＤＳ－ＰＡＧＥ上均显示１条蛋白质带,在α－半乳糖苷酶浓度为１５０ｍＵ／ｍｌ的溶液中测不到其他糖苷酶的活力。慈菇α－半乳糖苷酶的分子量用ＳｅｐｈａｄｅｘＧ－１００凝胶过滤柱测定或在ＳＤＳ－ＰＡＧＥ上测定均为６０ｋＤ,酶反应的最适ｐＨ在５．８附近,最适温度为６０℃。该酶分解对硝基苯基－α－半乳糖苷的Ｋ＿ｍ值为３．７×１０￣（－４）ｍｏｌ／Ｌ,Ｖ＿ｍ值为２．１×１０￣（－４）ｍｏｌ／Ｌ。银离子、汞离子显著抑制酶活力,Ｄ－半乳糖和密二糖均竞争性地抑制该酶水解对硝基苯基α－Ｄ－半乳糖苷的活力,根据Ｄｉｘｏｎ作图求得其Ｋ＿ｉ值分别为０．９２×１０￣（－３）ｍｏｌ／Ｌ和１．９８×１０￣（－３）ｍｏｌ／Ｌ。２－脱氧－Ｄ－半乳糖和Ｌ－岩藻糖为酶活力的非竞争性抑制剂。化学修饰     

枸杞子糖蛋白的分离纯化、物化性质及糖肽键特征

从宁夏枸杞子提取得到的粗多糖,经ＤＥＡＥ－Ｃｅｌｌｕｌｏｓｅ和ＳｅｐｈａｄｅｘＧ－１００柱层析,得到均一的枸杞子糖蛋白ＬｂＧＰ。分子量由ＳＤＳ－ＰＡＧＥ测定为８８ｋｄ,糖含量为７０％,糖组成为Ａｒａ：Ｇａｌ：Ｇｌｃ＝２．５：１．０：１．０（摩尔比）,并含有其他１８种天然氨基酸。初步分析表明ＬｂＧＰ是Ｏ－连接的糖蛋白。     

湖南尖吻蝮蛇毒两个出血毒素的纯化和理化性质

经ＳｅｐｈａｄｅｘＧ－７５凝胶过滤,ＱＡＥ－ＳｅｐｈａｄｅｘＡ－５０和ＣＭ－Ｓｅｐｈａｄｅｘ Ｃ－２５离子交换层析的步骤,从湖南产尖吻蝮蛇毒中纯化出两个出血毒素。ＳＤＳ－ＰＡＧＥ测得分子量均为２３．５ｋＤ,ＩＥＦ－ＰＡＧＥ测得等电点分别为５．６和５．２,两者具有相似的氨基酸组成,其中酸性氨基酸分别占２３％和２４％。ＤａＨＴ－１和ＤａＨＴ－２的最小出血剂量（ＭＨＤ）分别为０．５μｇ和０．８μｇ。都具     

枸杞子糖蛋白的分离纯化,物化性质及糖肽键特征

从宁夏枸杞子提取得到的粗多糖,经ＤＥＡＥ－Ｃｅｌｌｕｌｏｓｅ和ＳｅｐｈａｄｅｘＧ－１００柱层析,得到均一的枸杞子糖蛋白ＬｂＧＰ。分子量由ＳＤＳ－ＰＡＧＥ测定为８８ｋｄ,糖含量为７０％,糖组成为Ａｒａ：Ｇａｌ：Ｇｌｃ＝２．５：１．０：１．０,并含有其他１８种天然氨基酸。初步分析表明ＬｂＧＰ是Ｏ－连接的糖蛋白。     

浒苔多糖的分离、纯化和分析

浒苔（Ｅｎｔｅｒｏｍｏｒｐｈａｐｒｏｌｉｆｅｒａ）经热水提取,Ｓｅｖａｇｅ法除去蛋白质,用乙醇沉淀,ＳｅｐｈａｄｅｘＧ－１００柱层析,得浒苔多糖（简称ＥＰ）精制品。经ＳｅｐｈａｄｅｘＧ－２００柱层析鉴定为单一对称性洗脱峰。红外光谱分析具有多糖特征吸收峰,紫外光谱分析未见有核酸和蛋白质的特征吸收峰。总糖含量为８８．８％,其中糖醛酸含量为３３．６％。单糖组成为Ｌ－阿拉伯糖、Ｌ－岩藻糖、Ｄ－甘露糖、Ｄ－半乳糖及Ｄ－葡萄糖,平均分子量为２５０００。     

当归水溶性多糖级分As-Ⅲa和As-Ⅲb的纯化鉴定与结构研究

当归热水抽提得到的粗多糖,经乙醇分级沉淀,ＤＥＡＥ纤维素分离和ＳｅｐｈａｄｅｘＧ１５０柱层析纯化,得到水洗脱级分Ａｓ—Ⅲａ和碱洗脱级分Ａｓ—Ⅲｂ两个多糖级分。经测定这两级分为均一组分。红外光谱呈现出典型的多糖吸收峰。气相色谱分析表明Ａｓ—Ⅲａ由葡萄糖组成,Ａｓ—Ⅲｂ由葡萄糖、甘露糖和阿拉伯糖组成。高碘酸氧化及Ｓｍｉｔｈ降解分析表明Ａｓ—Ⅲａ的单体通过α（１→３）糖苷键相连,Ａｓ—Ⅲｂ主要通过（１→４）和（１→６）糖苷键相连。     

双乙酰还原酶的分离纯化研究

比较了５种细菌、４种酵母菌和鸡肝中双乙酰还原酶含量,其中粪肠杆菌（Ｅｎｔｅｒｏ－ｃｏｃｃｕｓｆａｅｃａｌｉｓ）ＡＳ１．５９５中含量较高,为９．６ＩＵ／ｇ湿菌体。并对ＡＳ１．５９５菌酶和鸡肝酶进一步经ＳｅｐｈａｄｅｘＧ－１００和ＤＺＡＥ柱分离纯化,提纯倍数分别为４５、８９３,最后收率分别为１８．５％、５．１％。     

地衣芽孢杆菌β－甘露聚糖酶的纯化及酶学性质

地衣芽孢杆菌（Ｂａｃｉｌｌｕｓｌｉｃｈｅｎｉｆｏｒｍｉｓ）ＮＫ－２７菌株发酵产生的β－甘露聚糖酶（β－ｍａｎｎａｎａｓｅ）经硫酸铵盐析沉淀,两次ＤＥＡＥ纤维素和ＳｅｐｈａｄｅｘＧ－１００离子交换柱层析以及制备ＰＡＧＥ筹步骤,获得了凝胶电泳均一的样品。用ＳＤＳ－凝胶电泳测得纯化后的β－甘露聚糖酶分子量为２６ｋＤ,用凝胶聚焦电泳测得等电点ＰＩ为５．０。酶反应的最适ｐＨ为９．０,最后温度为６０℃,稳定ｐＨ为６．０—９．０,稳定温度为４０℃。金属离子中Ｍｇ￣（２＋）、Ｃａ￣（２＋）、Ｆｅ￣（２＋）、Ｎｉ￣（２＋）对该酶有一定的激活作用;而Ｓｎ￣（２＋）、Ｚｎ￣（２＋）、Ａｌ￣（３＋）、Ａｇ￣＋和Ｈｇ￣（２＋）对该酶有强烈的抑制作用。ＮＫ－２７菌株的β－甘露聚糖酶对魔芋葡萄甘露聚糖和角豆胶半乳甘露聚糖的Ｋｍ值分别为７．１４和５．５６ｍｇ·ｍｌ￣（－１）;Ｖ＿（ｍａｘ）分别为２００．５３和１５７．４５μｍｏｌ·ｍｇ￣（－１）·ｍｉｎ￣（－１）。     

黑曲霉ρ-葡萄糖甘酶的提纯与性质

从黑曲霉Ａｓｐｅｒｇｉｌｕｓｎｉｇｅｒ发酵液中分离提纯了β－葡萄糖苷酶。提纯步骤通过（ＮＨ４）２ＳＯ４分级沉淀,ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－５０和ＳｅｐｈａｄｅｘＧ－１００等三步纯化,得到凝胶电泳均一的β－葡萄糖苷酶。该酶的最适ｐＨ４．５,最适温度６０℃,Ｋｍ为０．４４（ＮＰＧ）,并有较好的热稳定性。用ＳＤＳ－凝胶电泳法和凝胶色谱法测得该酶的分子量为１２００００     

Structure of the capsular polysaccharide of Klebsiella serotype K40

The capsular polysaccharide from Klebsiella Serotype K40 contains D-galactose, D-mannose, L-rhamnose, and D-glucuronic acid in the ratios of 4:1:1:1. Methylation analysis of the native and carboxyl-reduced polysaccharide provided information about the glycosidic linkages in the repeating unit. Degradation of the permethylated polymer with base established the identity of the sugar unit preceding the glycosyluronic acid residue. The modes of linkages of different sugar residues were further confirmed by Smith degradation and partial hydrolysis of the K40 polysaccharide. The anomeric configurations of the different sugar residues were determined by oxidation of the peracetylated native and carboxyl-reduced polysaccharide with chromium trioxide. Based on all of these results, the heptasaccharide structure 1 was assigned to the repeating unit of the K40 polysaccharide. (Formula: see text)     

Structural features of a sulphated,fucose-containing polysaccharide from the brown seaweed dictyota dichotoma

A sulphated heteropolysaccharide (～15% of the acid-extractable material) isolated from the brown alga Dictyota dichotoma contains residues of D-glucuronic acid, D-galactose, D-mannose, D-xylose, and L-fucose¹. Partial hydrolysis of the polysaccharide with acid gave one neutral and two acidic oligosaccharides. The behaviour towards periodate of the polysaccharide before and after partial hydrolysis, alkali-treatment, and methanolysis has been studied. Evidence is thereby provided that the polysaccharide is partially sulphated and composed of (1→4)-linked residues of D-glucuronic acid, D-galactose, D-mannose, and D-xylose, and (1→2)-linked L-fucose.     

Occurrence of two extracellular acidic polysaccharides as products of Rhizobium meliloti 201

The extracellular polysaccharide of Rhizobium meliloti 201 consists of two acidic polysaccharides, APS-I and APS-II. APS-I is composed of D-glucose, D-mannose and D-glucuronic acid in a molar ratio of 3:3:2, whereas APS-II is composed of D-glucose, D-galactose, D-mannose and pyruvic acid in a molar ratio of 4:3:2:1.APS-II was separated from the extracellular polysaccharide preparation by hydrolysing APS-I to its octasaccharide repeating unit with a specific enzyme. APS-I and APS-II were also separated by treatment with cetylpyridinium chloride and by paper electrophoresis of the depyruvylated polysaccharide.     

Structural analysis of anti-tumor heteropolysaccharide GFPS1b from the cultured mycelia of Grifola frondosa GF9801

A 21-kDa heteropolysaccharide, coded as GFPS1b, was obtained from the cultured mycelia of Grifola frondosa GF9801 by hot-water extraction, ethanol precipitation, and fractioned by DEAE Sepharose Fast-flow, followed by the purification with Sephadex G-100 column chromatography using an AKTA purifier. It exhibited more potent anti-proliferative activity on MCF-7 cells than other polysaccharide fractions. GFPS1b was an acidic polysaccharide with approximately 16.60% protein and 4.3% uronic acid. Gas chromatography of absolute acid hydrolysate of GFPS1b suggested that it was composed of D-glucose, D-galactose, and L-arabinose with a molar ratio of 4:2:1. Periodate oxidation, Smith degradation, partial acid hydrolyzation, methylation analysis, FT-IR, and (1)H, (13)C NMR spectroscopy analysis revealed that GFPS1b had a backbone consisting of alpha-(1-->4)-linked D-galacopyranosyl and alpha-(1-->3)-linked D-glucopyranosyl residues substituted at O-6 with glycosyl residues composed of alpha-L-arabinose-(1-->4)-alpha-D-glucose (1--> linked residues.     

The structure of unit B-type glycopeptides from porcine thyroglobulin

The structure of Unit B-type glycopeptides (monosialo-type and disialo-type) was investigated by Smith degradation, methyllation, and mass spectral analysis. These glycopeptides contain three peripheral sugar chains. Two are composed of D-galactose residues linked at C-6 and 2-acetamido-2-deoxy-D-glucose residues linked at C-4, and the other is composed of a D-galactose residues linked at C-6, a 2-acetamido-2-deoxy-D-glucose residues linked at C-4, and a D-mannose residue linked at C-2. Most of these peripheral sugar chains are linked to two inner D-mannose residues which are substituted at C-3 and C-6, and constitute branching points. L-Fucose and N-acetyl-neuraminic acid residues are nonreducing terminal groups, and a di-N-acetylchitobiose moiety is linked to an asparagine residue in the peptide moiety. By methylation analysis of the oligosaccharide obtained by hydrazinolysis of the disialoglycopeptide, the L-fucose residues was found to be linked to C-6 of the 2-acetamido-2-deoxy-D-glucose residue linked to the asparagine residue. From these results, and from the previously reported data on the sugar sequence and the anomeric configurations of the linkages between sugar residues, structures for these glycopeptides are proposed.     

猴头多糖HEP-2化学成分研究

猴头菌(Hericium erinaceus (Bull.) Pers．)的菌丝体培养物经水溶、乙醇沉淀、除蛋白等步骤得到猴头多糖(HEP),利用柱层析从HEP中分离得到两个组分HEP-1和HEP-2。用高效液相凝胶法(HPGPC)鉴定了HEP-2的纯度及分子量,通过有机元素分析、IR、ICP等多种分析方法确定HEP-2为一种硫酸化复合多糖,首次确定了猴头多糖的基本化学组成。     

Antigenic polysaccharides of bacteria. 33. The structure of O-specific polysaccharide chain of lipopolysaccharide from Pseudomonas cepacia serotype 6

On mild acid degradation of the Pseudomonas cepacia serotype 6 lipopolysaccharide, the O-specific polysaccharide was obtained, which contains D-mannose and D-galactose residues in the ratio approximately 1:1, as well as O-acetyl groups. On the basis of 1H and 13C NMR analysis, calculation of specific optical rotation, and methylation, it was concluded that the polysaccharide possesses the following structure: (formula; see text) Regularities in glycosidation effects in 13C NMR spectra of 1,3-linked disaccharides containing furanoside residues are discussed.     

Structural analysis of an acidic, fatty acid ester-bonded extracellular polysaccharide produced by a pristane-assimilating marine bacterium, Rhodococcus erythropolis PR4

Rhodococcus erythropolis PR4 is a marine bacterium that can degrade various alkanes including pristane, a C(19) branched alkane. This strain produces a large quantity of extracellular polysaccharides, which are assumed to play an important role in the hydrocarbon tolerance of this bacterium. The strain produced two acidic extracellular polysaccharides, FR1 and FR2, and the latter showed emulsifying activity toward clove oil, whereas the former did not. FR2 was composed of D-galactose, D-glucose, D-mannose, D-glucuronic acid, and pyruvic acid at a molar ratio of 1:1:1:1:1, and contained 2.9% (w/w) stearic acid and 4.3% (w/w) palmitic acid attached via ester bonds. Therefore, we designated FR2 as a PR4 fatty acid-containing extracellular polysaccharide or FACEPS. The chemical structure of the PR4 FACEPS polysaccharide chain was determined by 1D (1)H and (13)C NMR spectroscopies as well as by 2D DQF-COSY, TOCSY, HMQC, HMBC, and NOESY experiments. The sugar chain of PR4 FACEPS was shown to consist of tetrasaccharide repeating units having the following structure: [structure: see text].     

Structural elucidation of a 3-O-methyl-D-galactose-containing neutral polysaccharide from the fruiting bodies of Phellinus igniarius

PIP60-1, a novel heteropolysaccharide isolated from fruiting bodies of the medicinal fungus, Phellinus igniarius, has a molecular weight of 1.71 x 10(4)Da and is composed of L-fucose, D-glucose, D-mannose, D-galactose and 3-O-Me-D-galactose in a ratio of 1:1:1:2:1. A structural investigation of PIP60-1 carried out using sugar and methylation analyses, combined with (1)H and (13)C NMR spectroscopy, including COSY, TOCSY, NOESY, HSQC and HMBC experiments, established the repeating unit of the polysaccharide as the following: [structure: see text]