海洋真菌抗病原菌生物活性物质的研究

对分离自大连海域的海洋生物共附生真菌进行了抗菌活性的初筛与复筛,发现有6株具有抗表皮葡萄球菌和铜绿假单胞菌的活性,发现培养基成分及培养方式对活性菌株抗菌物质的产生有显著影响。菌株11-N2抗菌活性最强,其发酵液抗表皮葡萄球菌的效价相当于为氨苄青霉素11.4 U/mL,抗铜绿假单胞菌的效价相当于氨苄青霉素6.0 U/mL。通过硅胶柱层析对该菌株提取物的抗菌活性成分进行了初步追踪,发现二氯甲烷∶甲醇=15∶1洗脱组分具有最强活性,薄层层析显示该组分主要成分在254 nm紫外光下具有强吸收,薄层显色反应提示其主要成分可能为含胺基的甾体或三萜类、有机胺类、吲哚衍生物类化合物。     

五步蛇毒降纤酶的快速分离与纯化

目的：建立快速分离纯化降纤酶工艺方法适合于规模化生产。方法以五步蛇毒为原料。采用ＥＤＴＡ络合－硫氰酸钾沉淀初步分离与快速离子交换和凝胶层析化相结合。分离纯化出符合新部颁标准的降纤酶成分。结果经初步分离五步蛇粗毒可使出血毒去除９２％,非类凝血酶（ＴＬＥ）成分去除６５．１７％,ＴＬＥ活性回收保持９２．６８％以上。层析组份中至少含有４种ＴＬＥ,其中１种为降酶成分。初步化学分离,。离子交换柱层析和凝胶过滤     

灵芝发酵菌丝体中灵芝酸的分离纯化及生物活性检测

灵芝深层发酵菌丝体经甲醇提取得到粗灵芝酸,以甲苯:乙酸乙酯:乙酸(12:4:0.5)为展开剂,通过硅胶薄层层析法分离得到三种灵芝酸,命名为M_1,M_2和M_3,其R_f值分别为0.56,0.49,0.17.在优化的硅胶柱层析条件下,即以CH_3OH:CHCl_3=3:97,5:95,1:9为洗脱剂,进行分段洗脱;进样量:硅胶量=1:60;在30m×500mm的层析柱上纯化得到三种灵芝酸.抗菌实验显示,三种四环三萜酸具有抑制大肠杆菌、产气杆菌、肠炎杆菌、金黄色葡萄球菌和枯草芽孢杆菌生长的活性.     

大蹼铃蟾皮肤分泌液中抗菌活性肽的分离纯化及其性质

通过ＳｅｐｈａｄｅｘＧ－７５,Ｇ－５０和Ｇ－２５洗脱等步骤,从云南产大蹼铃蟾（Ｂｏｍｂｉｎａｍａｘｉｎａ）皮肤分泌中分离得到了一种有广泛抗菌活性的多肽,被命名为ＭＳＰ－Ⅶ,用快原子轰击质谱（ｆ．ａ．ｂ．ｍ．ｓ）分析其Ｍ＋／Ｚ＝２６４８。即此活性肽的分子量为２６４７Ｄ。用自动氨基酸分析仪分析,含有２６个氨基酸,抗菌实验结果表明,此多肽对格兰氏阴性和格兰氏阳性细胞以及真菌都有较强的抗菌活性,溶血实验表     

曲氏传药中乌头碱的薄层色谱测定

在硅胶Ｇ薄层板上用氯仿：１,２．二氯乙烷：甲醇：氨水（９：１：０．９：０．０７,ｖ／ｖ）为展开剂分离中草药复方曲氏传药中的生物碱,并用改良碘化铀钾试剂显色后对其中的乌头碱进行直接扫描测定。此法简便、快速、准确、灵敏且稳定。     

滑桃树种子内生菌Streptomyces sp.WXC抗菌化合物的分离和结构鉴定

本文对分离自滑桃树种仁的内生菌WXC进行初步的分类研究,其形态特征和系统发育分析表明该菌属于链霉菌属(Streptomyces sp.)。其发酵产物具有抗菌活性,以抗菌活性为指导,采用硅胶层析、凝胶层析和反相中压柱层析对Streptomyces sp.WXC发酵代谢产物进行分离纯化,得到2个吡喃萘醌类化合物,通过核磁共振、质谱等波谱技术确定它们分别为frenolicin B(1)和deoxyfrenolicin(2)。     

昆虫谷胱甘肽S-转移酶分离纯化的新方法

谷胱甘肽Ｓ－转移酶（ｇｌｕｔａｔｈｉｏｎｅＳ－ｔｒａｎｓｆｅｒａｓｅｓ,ＧＳＴ）是一类具有多种生理功能的同功酶．从蜡螟幼虫（Ｇａｌｅｒｉａｍｅｌｏｎｅｌａ）的提取液中分离纯化谷胱甘肽Ｓ－转移酶的基本方法如下：首先将冷冻的蜡螟幼虫在磷酸缓冲液中匀桨,经１００００ｇ和１０００００ｇ分级离心;取上清液通过ＱＡＥ－ＳｅｐｈａｄｅｘＡ－２５离子交换柱层析除去部分色素和杂蛋白;然后采用谷胱甘肽－琼脂糖凝胶亲和层析（ＧＳＨ－ＱＴ４）,四溴酚酞二磺酸盐－琼脂糖凝胶亲和层析（ＢＳＰ－ＱＴ４）,铜离子－琼脂糖凝胶螯合层析（Ｃｕ２＋－ＱＴ４）及ＰＢＥ９４－Ｓｅｐｈａｒｏｓｅ（ＰＢＥ９４）聚焦层析等层析技术进一步分离纯化．将上述方法获得的色谱峰以ＣＤＮＢ和ＤＣＮＢ为底物检测生物活性．具有生物活性部分的蛋白质,通过ＳＤＳ－ＰＡＧＥ测定其分子量．实验结果表明,采用ＧＳＨ－ＱＴ４亲和层析法获得的活性峰,在ＳＤＳ－ＰＡＧＥ图谱上呈现出两条带,分子量为２４ｋＤ,２４．５ｋＤ左右;Ｃｕ２＋－ＱＴ６螯合层析法分离的活性峰,呈现出一条带,分子量为２４ｋＤ左右;ＰＢＥ９４－聚焦层析法分离获得三个活性峰：第一色谱峰,呈现出一条带,分子量为２３ｋＤ左右     

苦荞种子胰蛋白酶抑制剂的分离纯化及部分性质研究

采用凝胶层析及离子交换层析等方法,从苦荞种子中分离出一组胰蛋白酶抑制剂（ＴＢＴＩ－Ⅰ、Ⅱ）．对其性质研究表明：两个组分均对胰蛋白酶有较强的抑制作用,对胰凝乳蛋白酶抑制作用较弱,其中ＴＢＴＩ－Ⅱ的抑制作用大于ＴＢＴＩ－Ⅰ,两者对胃蛋白酶、木瓜蛋白酶及枯草杆菌蛋白酶均无抑制作用．用ＳＤＳ－聚丙烯酰胺凝胶电泳和ＳｅｐｈａｄｅｘＧ－１００凝胶层析分别对纯化产物进行分析得出ＴＢＴＩ－Ⅰ和ＴＢＴＩ－Ⅱ的近似分子量分别为１５．０ｋＤ和１８．０ｋＤ．ＴＢＴＩ－Ⅰ、Ⅱ都具有较高的热稳定性,在１００℃处理１０ｍｉｎ后可保留８６％左右的抑制活性．ＴＢＴＩ在酸性环境下较为稳定,在ｐＨ２．０条件下保温１ｈ,仍保留７５％的抑制活性．用Ｌｉｎｅｖｅａｅｒ－Ｂｕｒｋ作图法得知,该抑制剂属竞争性抑制类型,ＴＢＴＩ－Ⅱ的Ｋｉ值为３．５９×１０－７ｍｏｌ／Ｌ（以ＢＡＰＮＡ为底物）,对胰蛋白酶的摩尔抑制比为１∶１．４．     

抗水稻黄单胞菌的苏云金芽胞杆菌菌株筛选及其 活性物质

【目的】从400株苏云金芽胞杆菌菌株中筛选出拮抗水稻黄单胞菌活性最好的菌株YBT-2532,并对其抑菌活性物质进行分离。【方法】对苏云金芽胞杆菌YBT-2532产生的活性物质理化特性进行测定。【结果】该活性物质对温度、蛋白酶、pH均不敏感,70 °C处理1 h仍保留有75%的活性;活性物质在pH 2.0?12.0较稳定;该活性物质溶于甲醇、微溶于乙醇、不溶于丙酮、二氯甲烷和氯仿。利用凝胶过滤、离子交换层析、固相萃取、高效液相色谱技术,对抑菌组分进行分离,并通过HPLC-IT-MS方法确定其分子量。纯化的活性组分是一种分子量为797.8 Da的强极性水溶性小分子。【结论】该活性物质性质与已知的来源于苏云金芽胞杆菌的抗菌活性物质不同,可能为新型抗菌物质。     

氧化铝层析从云南红豆杉植物中转化提取紫杉醇

采用氧化铝层析从云南红豆杉植物汉膏中转化生成和分离提纯了紫杉醇。考察了氧化铝的类型、流动相中水和甲醇的添中、反应时间以及洗脱剂组成等层析操作条件对紫杉醇回收和分离的影响。研究表明,采用碱性氧化铝柱层析分离和回收紫杉醇效果显著,通过对紫杉醇转化和分离条件的优化,经一步氧化铝柱层析,可以使紫杉醇的含量从小于１．０％提高到大于２７％,紫杉醇的回收大于１７０％。     

寄生隐丛赤壳菌Cryphonectria parasitica致病毒素Cp-I的分离纯化和结构分析

寄生隐丛赤壳菌Cryphonectria parasitica菌株经液体培养,石油醚萃取其发酵液获得对板栗带叶嫩枝具有致萎活性的粗提物,以氯仿:石油醚:甲醇(6:2:2)作洗脱剂,粗提物经硅胶色谱分离,共得到3组纯组份,其中第1组份(Cp-I)对板栗幼苗致萎活性较高.质谱、核磁共振和红外光谱测定表明Cp-I分子量为278,化学式为C<,16>H<,22>O<,4>.     

Identification and Purification of Calcium-Independent Phospholipase A2 from Bovine Brain Cytosol

Substantial amounts of phospholipase A2 activity were detected in bovine brain cytosol. The major phospholipase A2 activity was present in the precipitate at 40% saturation with solid ammonium sulfate. After the desaltate of the precipitate was loaded onto an Ultrogel AcA 54 gel filtration column, almost all the activity eluted in the void volume when chromatographed without 1 M KCl. However, when buffer with 1 M KCl was used as the eluent, two active peaks were obtained. One peak (peak I) eluted in the void volume, and the other (peak II) eluted with an apparent molecular mass of 39 kDa as compared with standards. The former was active with diacylglycero-3-phosphoethanolamine, whereas the latter was active with both diacylglycero-3-phosphoethanolamine and 1-alk-1'-enyl-2-acylglycero-3-phosphoethanolamine (plasmenylethanolamine). The apparent molecular mass of peak I was estimated to be 110 kDa as compared with standards on an Ultrogel AcA 34 gel filtration column. Both peaks were purified further with a hydrophobic chromatography column (AffiGel 10 coupled with plasmenylethanolamine) and then by high-resolution liquid chromatography on an MA7Q column. The phospholipase A2 obtained from peak II migrated as one main band with a 40-kDa molecular mass and two minor bands with 14- and 25-kDa molecular masses. Phospholipase A2 obtained from peak I eluted as a single peak on high-resolution liquid chromatography but contained two bands with apparent molecular masses of 100 and 110 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

大孔吸附树脂结合硅胶柱层析纯化环孢菌素A

采用大孔吸附树脂层析结合硅胶柱层析,对环孢菌素A的分离纯化进行研究,确定了最佳层析条件,建立了工业化制备环孢菌素A的工艺。大孔吸附树脂层析选用D101树脂作为吸附介质,提取液丙酮含量控制在50%,最大吸附量为35 mg/g湿树脂,洗脱剂选用丙酮;硅胶柱层析选用42~64μm硅胶作为层析介质,最优层析条件为柱床高径比10∶1,流动相配比V(石油醚)∶V(丙酮)=70∶30,流速80 mL/m in,环孢菌素A上样质量浓度100 g/L,硅胶层析平均收率为84.2%,环孢菌素A纯度可达到97%以上,整个工艺总收率为65%~70%。     

山黧豆毒素的薄层分离

通过对山黧豆中常见的20几种游离氨基酸薄层层析发现,其毒素ODAP的Rf值与好几种相关氨基酸的接近,而用阳离子交换柱对氨基酸抽提样进行简单的分离纯化,第一个洗脱出来的即为ODAP,这样在排除其它氨基酸干扰的情况下再进行ODAP的薄层分析将得到准确的结果。     

寄生隐丛赤壳菌致病毒素Cp-I的分离纯化和结构分析

寄生隐丛赤壳菌Cryphonectria parasitica菌株经液体培养,石油醚萃取其发酵液获得对板栗带叶嫩枝具有致萎活性的粗提物,以氯仿:石油醚:甲醇(6:2:2)作洗脱剂,粗提物经硅胶色谱分离,共得到3组纯组份,其中第1组份(Cp-I)对板栗幼苗致萎活性较高。质谱、核磁共振和红外光谱测定表明Cp-I分子量为278,化学式为C16H22O4。     

Rapid isolation of kestose by low-pressure chromatography after enzymatic synthesis with invertase

By using a two-step protocol involving flash chromatography on silica gel-60 and gel filtration on Biogel P2 we isolated 56 g pure kestose/L after enzymatic synthesis with 115 U invertase in media containing 50% (w/w) sucrose and 90% (v/v) butyl acetate. The isolated product is homogenous after high pH anion exchange chromatography with pulsed amperometric detection and is suitable for downstream utilization without further purification.     

以聚乙二醇为层析伴侣同时制备SOD、过氧化氢酶和血红蛋白

发展了一条从红细胞裂解液中同时制备超氧化物歧化酶(SOD)、过氧化氢酶和血红蛋白的新工艺。采用0 75 %的聚乙二醇600作为层析伴侣,使血红蛋白直接流过阴离子交换层析柱,同时吸附SOD和过氧化氢酶。经过梯度洗脱获得SOD和过氧化氢酶组分,再经过疏水性相互作用层析与凝胶过滤层析相串联,使SOD和过氧化氢酶得到纯化。纯化后的SOD和过氧化氢酶的比活力分别达到15932u/mg和65918u/mg ,血红蛋白的纯度达到99.9%以上。总回收率为:SOD ,47.4% ;过氧化氢酶,29.6% ;血红蛋白,88.7%。     

The Azure Dyes their Purification and Physicochemical properties. II. Purification of Azure B

A method is described for the purification of the dye azure B in quantities sufficient for biological staining experiments on a larger scale. The method is based on the use of column chromatography. Two columns are employed. In column A with silica gel as adsorbent the azure B fraction is isolated from a suitable substrate ('technical' azure B gained by a modification of Bernthsen's synthesis of methylene blue, or plychrome methylene blue) using an acetate-formate mixture as eluent. In column B, on an Amberlite polyineric adsorbent (XAD-2) the acetate-formate anions are exchanged for chloride. Regeneration of both columns is possible: KMnO₄, Na₂S₂O₄ and water are run through column A, 5% NaOH, methanol and water through column B. Purification of azure B on economic terms is thus attained. The opinion is expressed that this method is also applicable to the purification of other cationic dyes.     

The azure dyes: their purification and physicochemical properties. II. Purification of azure B.

A method is described for the purification of the dye azure B in quantities sufficient for biological staining experiments on a larger scale. The method is based on the use of column chromatography. Two columns are employed. In column A with silica gel as adsorbent the azure B fraction is isolated from a suitable substrate ('technical' azure B gained by a modification of Bernthsen's synthesis of methylene blue, or polychrome methylene blue) using an acetate-formate mixture as eluent. In column B, on an Amberlite polymeric adsorbent (XAD-2) the acetate-formate anions are exchanged in chloride. Regeneration of both columns is possible: KMnO4, Na2S2O4 and water are run through column A; 5% NaOH, methanol and water through column B. Purification of azure B on economic terms is thus attained. The opinion is expressed that this method is also applicable to the purification of other cationic dyes.     

大豆异黄酮的分离与纯化

天然大豆异黄酮作为健康食品在防治骨质疏松和癌症方面具有一定功效。由于化合物结构相似,异黄酮甙元特别是高纯度黄豆黄素（glycitein）的获得有一定难度,文献报道大多是通过盐酸水解异黄酮甙的方法获得,而这种方法对环境污染和工厂生产设备腐蚀较大。本文报道了用醇溶剂进行固相提取以及硅胶柱色谱方对含有三种大豆异黄酮甙元的混合物产品进行分离。通过低成本和无环境污染的固相提取方法得到纯度为97％的黄豆黄素和纯度超过95％的大豆黄素（daidzein）;95％纯度的另一种大豆甙元金雀异黄素（genistein）则通过硅胶柱色谱分离得到。应用硅胶柱色谱,一次性分离了一种含有两个异黄酮甙：大豆甙（daidzin）和黄豆甙（glycitin）的产品。