金针菇(Flammulina Velutipes(Curt.ex Fr.)Sing.)子实体多糖PA_(5)DE的提取及性质研究

 从金针菇Flammulina velutipes(Curt.ex Fr.)Sing.子实体中提取水溶性多糖,经乙醇分级,DEAE-Sephadex A-25纯化,得PA_5DE。以聚丙烯酰胺凝胶电泳和凝胶柱Sepharose 4B层析证明是化学均一性多糖,分子量是47.1万。用GLC、I.R、~13C-N.M.R.分析表明含有D-葡萄糖、D-甘露糖、D-岩藻糖。PA_5DE分子可能具有分支结构,含β-型糖苷键,存在β(1→3)和β(1→6)型糖苷键连接,并有抑制肿瘤S-180的活性。     

白魔芋和花魔芋葡甘露聚糖研究

从白魔芋和花魔芋的块茎中分离纯化出两种魔芋葡甘露聚糖(Konjac Glucomannan,简称KGM),分别名为白魔芋葡甘露聚糖(aKGM)和花魔芋葡甘露聚糖(rKGM)。通过超离心、玻璃纤维纸电泳和凝胶过沪证明aKGM和rKGM都是均一的多糖。这两种多糖都由甘露糖(M)和葡萄糖(G)组成,其克分子比G/M:aKGM为1:1.69,rKGM为1:1.60,分子量前者为8.09×10~5,后者为7.37×10~5。酶水解实验和红外光谱分析说明aKGM和rKGM都是由甘露糖和葡萄糖以β-1,4-糖苷键连接的杂多糖;有O-乙酰基的特征吸收峰,说明在某些糖残基上可能有乙酰基团。     

一种水溶性党参多糖的分离纯化及结构分析

采用水提醇沉法得到党参粗多糖(COP),采用Sevag法除去蛋白成份,接着通过Sephacry1 S-200HR及Sephadex G-25凝胶柱色谱分离得到均一多糖COP-1。凝胶渗透色谱法(HPGPC)测定其纯度和平均分子量。高效液相色谱法(HPLC)确定其单糖的组成。红外光谱及核磁推测COP-1结构。结果表明COP-1平均分子量约为2.1×10~3 Da,且均由阶D-(2→1)呋喃果糖组成。     

安络小皮伞中水溶性多糖的研究——Ⅰ.甘露聚糖 FP_1 的纯化与结构研究

从安络小皮伞水溶性多糖中分离纯化得一甘露聚糖 FP_1。分子量约为24万。经红外光谱、~+H-NMR 谱和亲和层析指明为β-甘露聚糖。结构分析采用高碘酸氧化、Smith 降解、完全甲基化 GC、GC-MS 与~(13)C-NMR 分析,分子的主链是β-D-(1→6)连接的甘露糖,支链为β-(1→3),β(1→2)甘露糖,分别连接在主链的 O-3和 O-2上。     

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。     

人参叶水溶性多糖——杂多糖P_N的纯化与结构初步确定

 从人参叶中提取的水溶性多糖经分离纯化得杂多糖P_N。P_N的分子量约为190万,单糖组成为阿拉伯糖、鼠李糖、木糖、半乳糖醛酸、半乳糖、葡萄糖及少量未知糖,单糖的摩尔比依次为8.1:0.8:1.0:1.6:12.5:4.1(未知糖除外)。经超离心分析,琼脂糖4B柱分析,玻璃纤维纸电泳和醋酸薄膜电泳鉴定等证明P_N为均一组份。经果胶酶降解,部分酸水解,高碘酸盐氧化,Smith降解,甲基化及其产物气相色谱(GLC)、气相色谱-质谱联用(GLC-MS)等结构分析表明P_N为多分支结构,分子的主链主要是由β-(1→3)连接的半乳糖组成,并在4—0和6—0上带有分支,平均每三个半乳糖有二个分支。     

小刺猴头菌丝体多糖的结构研究

对小刺猴头过滤掉发酵液的发酵菌丝体,水提和碱提后获得的均一组分多糖HMP-w1.1和HMP-a1.1进行结构性质的研究。结果表明:HMP-w1.1是分子量为36.3 kD的α型吡喃糖,单糖组成为甘露糖(Man),葡萄糖(Glc),半乳糖(Gal),岩藻糖(Fuc);HMP-a1.1是分子量为42.8 kD的β型吡喃糖,单糖组成为甘露糖(Man),半乳糖醛酸(GalUA),葡萄糖(Glc),半乳糖(Gal),岩藻糖(Fuc)。综合高碘酸氧化和Smith降解的试验结果,推断HMP-w1.1的糖苷键构型可能为1→、1→4、1→4,6、1→6、1→2、1→2,6;HMP-a1.1的糖苷键构型可能为1→6、1→2、1→2,6。     

银耳碱提孢子多糖A-BTF的分离与结构研究

银耳孢子发酵粉(Tremella fuciformisBerk),用热水煮提后,除去水溶性多糖。其沉淀用1M NaOH提取,Sevage法除去蛋白,用乙醇沉淀得到粗多糖。粗多糖经DEAE-32-cellulose和sephadex G-200反复分离后,纯化得到分布均一的多糖A-BTF。HPGPC测定A-BTF的分子量为67000,糖组成分析显示主要由葡萄糖组成。多糖A-BTF的甲基化产物,经水解、还原、乙酰化,通过GC-MS分析表明,主要含有1,6连接的葡萄糖和1,3,6连接的甘露糖,另外还有1,4连接的葡萄糖少量的半乳糖和1-NH2-来苏糖,末端为端基连接的葡萄糖。     

长松萝多糖的研究

长松萝经三氯甲烷抽提后风干,用热水提取,乙醇沉淀,经微晶纤维素柱层析纯化,得白色粉末状多糖USL。USL经Sephadex G-150柱层析证明为一组均匀多糖,其糖的含量为89.52％。经气相色谱检定,由阿拉伯糖(Ara)、木糖(Xy1)、甘露糖(Man)、葡萄糖(Glc)聚合而成,其克分子比为0.31:0.05:1.00:18.10。经Sephadex G-200柱层析测定,平均分子量为30×10~4,经高碘酸氧化,Smith降解,有甲酸、丙三醇、赤藓醇生成。红外光谱在896cm-~1处有吸收,证明USL多糖结构主要以β(1→4)、β(1→6)甙键连接而成的杂多糖。     

白芍多糖分离纯化及性质分析

采用热水浸提.乙醇沉淀法中药白芍(Paeonia Albiflora Pall)水提取液中分离纯化得到一种多糖蛋白复合物,命名为PAⅡ(Paeonia Albiflora Pall PolysaccharideⅡ,PAⅡ).电泳、FPLC和HPLC检测其纯度;红外光谱和气相色谱法对其结构和组成进行初步分析.结果表明PAⅡ为均一多糖组分,是以D-葡萄吡喃糖为主的多糖-蛋白复合物,总糖含量为85.0%,蛋白含量为13.2%;该糖为吡喃型结构,糖肽键为非O-型;单糖组分为葡萄糖、阿拉伯糖、甘露糖、鼠李糖、木糖,摩尔比为153:2.6:1.25:1:1,平均分子量为5.25×104.     

朱桔Mn-SOD的纯化、鉴定及浓度梯度胶电泳在其中的应用

朱桔叶中存在Mn-SOD、Fe-SOD和CuZn-SOD三种类型,在10%的PAGE中有三条活性带,其中Mn-SOD的Rf值大于Fe-SOD与CuZn-SOD₁的Rf值相同;在4%~35%的梯度胶电泳中有四条活性带,而Mn-SOD的R_f值最小,表明该酶带有较高的电荷密度。Mn-SOD占总活性的20%左右,已被纯化到均一程度。该酶的比活性为1 249 U/mg,分子量和亚基分子量分别为54.0 kD和26.6 kD,在紫外区最大吸收值为280 nm,在95℃处理15 min仍保留了46%的酶活性,等电点为5.06。该酶的活性不被KCN、H₂O₂抑制,但对1% SDS和氯仿-乙醇液敏感。     

Isolation and characteristics of benzoyl-DL-arginl-p-nitroanilide-hydrolysing enzyme (BAPAase) from vetch seedlings]

The enzyme hydrolysing N-benzoyl-D,L-arginine-p-nitroanilide (BAPA). is isolated from vetch seedlings and 1600-fold purified by means of chromatography on DEAE-cellulose, hdroxyapatite and gel filtration through Sephadex G-100. The preparation is chromatographically homogenous, but disc electrophoresis in polyacrylamide gel revealed an insignificant contamination by inactive proteins. The data of disc electrophoresis in polyacrylamide gel in the presence of sodium dodecylsulphate have shown that BAPAase has a quaternary structure containing, probably, four subunits identical in their molecular weight. BAPAase has a narrow substrate specificity: it hydrolyses BAPA, benzoyl-D,L,-argininenaphtylamide, benzoyl-L-arginyglycine CBZ-L-arginylglycine histones and protamine, but does not attack L-arginyl-p-nitroanilide benzoyl-L-arginineamide, tosyl-L-arginine methyl ester and casein.     

Glycosyltransferases of Streptococcus mutans strain Ingbritt.

The glycosyltransferases of S. mutans strain Ingbritt have been resolved by SDS-polyacrylamide gel electrophoresis, followed by incubation in the presence of non-ionic detergent to restore enzyme activity. A group of high molecular weight proteins synthesizing glucans has been identified, as well as three distinct fructan-synthesizing activities. The glucan-forming enzymes have been purified by affinity chromatography on insoluble glucan, followed by gel chromatography in SDS, and antiserum to the purified enzymes has shown that they are antigenically identical within serotypes c, e and f, and cross-react strongly with serotype b.     

Purification and some physicochemical properties of staphylococcal enterotoxin D.

A method was developed for the isolation of staphylococcal enterotoxin D in highly purified form from cultures of Staphylococcus aureus strain 1151m. The method involves removal of the toxin from the culture supernatant fluid with the ion-exchange resin CG-50 followed by chromatography on carboxymethylcellulose (twice) and by gel filtration on Sephadex G-75 (twice). The purified toxin is homogeneous by polyacrylamide gel and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and double gel diffusion tests. It is a simple, colorless, antigenic protein with an isoelectric point of 7.4 as determined by isoelectric focusing. Its molecular weight was determined to be 27 300 +/- 700 by molecular sieve chromatography on Sephadex G-100 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its serological activity is stable over a wide range of pH values (1.2--10.7). The enterotoxin consists of 236 amino acid residues and contains no free sulfhydryl groups. End-group analysis showed serine to be the NH2-terminal amino acid and lysine to be the COOH-terminal amino acid.     

松杉灵芝发酵菌丝体中水溶多糖的分离纯化与结构的比较研究

松杉灵芝发酵菌丝体经热水提取,冻融分级及乙醇二次分级,分离纯化出GFb级份,电泳及凝胶柱层析示其为均一多糖,分子量为9.8万。小于子实体多糖相应级份。 GFb经红外光谱,气相色谱,气质联机,碳13核磁共振,高碘酸盐氧化,Smith降解,甲基化及部分酸水解分析,确定其基本结构中主链为1→6葡萄糖基和1→6半乳糖基构戍,二者之比为1∶1,分支点在0-3位上,分枝点率为50％,与子实体多糖GF_3相同,侧链由1→3葡萄糖基,1→4葡萄糖基,末端葡萄糖基及末端半乳糖基构成,分子中分枝率为55.6％,较子实体多糖GF_3分枝率略低,分枝链略短。     

Cryptomonad biliprotein: phycocyanin-645 from a Chroomonas species.

The properties of phycocyanin-645 from the fresh water cryptomonad Chroomonas spec. were investigated after the pigment was isolated and purified by a combination of differential ammonium sulphate fractionation, gel filtration chromatography and ammonium sulphate gradient elution. Phycocyanin-645 is characterized by absorption maxima at 645 nm, 584 nm, 369 nm, 275 nm and shoulders at 340 nm and 620 nm. The CD spectrum has a negative maximum at 645 nm and a positive maximum at 584 nm with a shoulder at 610 nm. The fluorescence emission spectrum is asymmetrical and shows a maximum at 660 nm and a shoulder at approximately 715 nm. The molecular weight of the native phycocyanin-645, estimated by gel filtration, is 45000 for all multiple pigment forms below. Phycocyanin-645 is heterogeneous as revealed by isoelectric focusing with pIs at 7.03, 6.17, 5.75, 5.25 and 4.88, respectively, the main bands lying at pI 7.03 and pI 6.17. This was confirmed by polyacrylamide gel electrophoresis; five pigment compoents differing in mobility were found. We propose the term "multiple pigment forms" for these five phycocyanin-645 modifications. Calibrated SDS gel electrophoresis shows phycocyanin-645 to consist of three subunits, two light chains (alpha1, alpha2), having molecular weights of 9200 and 10400, respectively, and one heavy chain (beta), having a molecular weight of 15 500. Suggesting a 1:1:2 ratio between the subunits, the quaternary structure of the pigment molecule is alpha1beta--alpha2beta1.     

Potato phosphorylase isoenzymes

Potato phosphorylase isoenzymes were separated by gel electrophoresis, and DEAE-Sephadex chromatography. Electrofocusing experiments showed a heterogeneity in isolelectric point. Molecular weights and Stokes-radii were estimated using Sephadex G200. The adsorption on glycogen of two low molecular weight forms, probably dimers, was investigated by means of gel electrophoresis. The dissociation constants were 5 × 10⁻⁵ and 2 × 10⁻³% glycogen.     

Purification and characterization of amphiphilic trehalase from rabbit small intestine

Rabbit intestinal trehalase (alpha,alpha-trehalose glucohydrolase, EC 3.2.1.28) was solubilized with Triton X-100 and purified in the presence of EDTA. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis in the presence of Triton X-100 or SDS. It showed amphiphilic properties on gel filtration. polyacrylamide gel electrophoresis, charge-shift electrophoresis and phenyl-Sepharose chromatography. Its molecular weight was estimated to be about 330 000 by gel filtration under nondenaturing conditions and in the presence of Triton X-100, the value being in satisfactory agreement with the sum of the weight of one Triton X-100 micelle and twice the molecular weight (105 000) of purified hydrophilic trehalase which had been deprived of the anchor segment. The two purified trehalases gave almost the same molecular weights (about 75 000) on SDS-polyacrylamide gel electrophoresis. These results suggest that intestinal trehalase consists of two subunits with a molecular weight of 75 000 and that its anchor segment is small (less than 5000). Triton X-100 extracts freshly prepared from intestinal microvilli essentially showed one form of trehalase, which behaved on phenyl-Sepharose and Con A-Sepharose chromatography in the same manner as purified amphiphilic trehalase.     

delta-Aminolevulinic acid synthetase from rat liver mitochondria. Purification and properties.

delta-Aminolevulinic acid synthetase has been purified from liver mitochondria of young, uninduced rats. After nonionic detergent solubilization of mitochondrial inner membrane-matrix fractions, the enzyme was purified to a specific activity of approximately 2,000 nmol of delta-aminolevulinic acid formed/h/mg of protein at 30 degrees C, by means of ammonium sulfate precipitation, diethylaminoethyl cellulose chromatography, Sephacryl chromatography, and preparative gel electrophoresis. The purified enzyme preparation thus obtained was apparently homogeneous as judged by its migration as a single band with a molecular weight of 58,000 +/- 6,000 upon electrophoresis in sodium dodecyl sulfate polyacrylamide gels. The native enzyme probably exists as a dimer with a molecular weight of approximately 120,000. A pH optimum of 7.5 and an isoelectric point of 4.5 were also determined. Both monovalent cations and hemin strongly inhibited the activity of the purified enzyme.     

Microbial Utilization of Benzenesulfonic Acid

An enzyme which degrades yeast glucan and yeast cells in the logarithmic phase of growth (log yeast cells) and produces protoplasts from log yeast cells has been crystallized from the culture filtrate of a strain belonging to Fungi Imperfecti.

Analyses by ultracentrifugation and disc gel electrophoresis showed the crystalline enzyme to be homogeneous. Its molecular weight was found to be 24,500. The hydrolysis of laminarin, pachyman and yeast glucan was catalysed by the enzyme to produce a mixture of laminaridextrins. The conversion of log yeast cells to protoplasts was obtained by the addition of only this enzyme, the addition of mercaptoethanol or phosphomannanase to the enzyme promoted the conversion.