Polypeptide antibiotic 26a from Bacillus subtilis. II. Isolation and purification procedures.

Both the analytical and preparative methods by which the preparations of 26a bydrochloride salt with a high antibacterial activity and 20--30% recovery have been obtained from the fermentation fluids of Bacillus subtilis are presented. On an industrial scale the antibiotic can be yielded by absorption of bioactivity on Amberlite CG-50I column and precipitation with picric acid of crude substance from active elutes as adduct which was divided on equilibrated CM--cellulose and finally purified by gel filtration on Sephadex G-25 column. The purified preparation gave a single band by SDS-polyacrylamide gel electrophoresis and one ninhydrin-positive spot by thin-layer chromatography on silica gel G plates corresponding to single zones of bioactivity on bioautograms, and moved as a single peak of almost constant antibacterial activity on Sephadex G-75, G-100 and G-200 columns. The potency of the purest preparations, lot Sephadex G-25, was 6,500--7,000 arbitrary units/mg, and were characterized as follows: purification factor, 57; purity of 98--100% by densitometer scans of SDS-polyacrylamide gels; MIC for Sarcina lutea by twofold agar dilution assay, 0.306 microgram/ml.     

季也蒙假丝酵母胞外多糖的分离纯化及抗氧化活性研究

对一株源于海参肠道的季也蒙假丝酵母产胞外多糖进行分离纯化并对其抗氧化活性进行研究.采用乙醇沉淀、Sevag除蛋白等方法得到胞外粗多糖EPS.EPS经Sepharose强阴离子交换层析分离后,分别得到3个组分EPS1、EPS2和EPS3,对抗氧化活性较高的EPS2采用Sephacryal凝胶过滤层析进行纯化,得到1个单一组分EPS2-1,采用气相色谱法分析其单糖组成,并验证其抗氧化活性.结果表明:EPS2-1是由木糖、甘露糖、葡萄糖和半乳糖组成的;它具有较强的抗氧化活性,当其浓度为0.36 mg/mL时,对羟基自由基的清除率可达100％,明显高于同浓度下Vc对羟基自由基的清除率14.42％;当EPS2-1浓度为0.60 mg/mL时,对超氧阴离子自由基的清除率可达53.22％;同时EPS2-1表现出一定的还原力.研究证明该活性多糖具有很好的应用潜力,值得进一步研究开发.     

Isolation, purification and antitumor activity of lipopolysaccharide from cow placenta

Lipopolysaccharide from cow placenta (LPS-CP-2) has been isolated and purified by hot phenol-water extraction, enzyme hydrolysis, chloroform-petroleum ether method, ion-exchange and gel-filtration chromatography. Also, LPS-PS-2 was evaluated for antitumor activity against Ehrlich ascites carcinoma (EAC)-bearing Swiss albino mice. LPS-PS-2 caused significant (P<0.05) decrease in tumor volume, and viable cell count; and it prolonged the life span of EAC-tumor-bearing mice. Hematological profile indicates that LPS-CP-2 possessed protective action on the haemopoietic system. Further, administration of LPS-CP-2 reduced the tumor volume of both DLA and EAC cell lines in a dose-dependent way. The LPS-PS was found to be devoid of pyrogenic response in the rabbits. These results indicate that LPS-PS exhibited significant antitumor activity without pyrogenic response, suggesting its potential as antitumor agent.     

皱皮木瓜多糖的分离、纯化及抗氧化活性研究

为了研究木瓜多糖的提取、分离、纯化与抗氧化活性,采用水提醇沉法提取皱皮木瓜中的多糖,得多糖Ⅰ;利用Sevag法除去多糖中的蛋白质后得多糖Ⅱ;以30%H_2O_2脱除色素后再次醇沉得到精制多糖Ⅲ;透析除去小分子后利用AB-8大孔树脂进行分离以水、30%、50%、70%和95%乙醇洗脱,其中水洗脱部分多糖为Ⅳ。用苯酚-硫酸法测定多糖含量。多糖Ⅰ得率为9.83%,多糖含量(纯度,下同)为64.45%;脱蛋白后多糖Ⅱ中多糖含量为78.23%;经脱色后多糖Ⅲ含量达88.39%;大孔树脂水洗脱部分多糖Ⅳ含量为89.74%。以DPPH(2,2-二苯基-1-苦肼基)清除率和Fe~(3+)还原力方法测定木瓜多糖的抗氧化活性,木瓜多糖均体现出一定的抗氧化作用,呈浓度依赖性增强,其中多糖Ⅰ、Ⅱ表现出更好的作用。     

中华蜂体内放线菌的分离、多样性及抗菌活性研究

【目的】探索药用昆虫中华蜂(Apis cerana cerana Fabricius)体内可培养放线菌的分离方法,研究其物种多样性及抗菌活性,挖掘更多微生物资源。【方法】选用7种分离培养基对中华蜂样品体内放线菌进行分离;通过采用16S rRNA PCR-RFLP和16S rRNA基因序列分析方法研究其多样性;选用4种致病菌对菌株进行抗菌活性初探。【结果】共分离得到180株放线菌,根据菌落的形态和细胞特征观察结果,从中选取84株作为代表菌株,其分属于3个目、4个科中的4个属,其中6株为潜在新种;最适的表面消毒方法:浓度为0.2%的Cl O2(二氧化氯)作为消毒剂,作用60 s;拮抗实验显示,31.0%、48.8%、27.4%、16.7%的代表菌株分别对大肠杆菌、枯草芽孢杆菌、玉米弯孢病菌、西瓜枯萎病菌有不同程度的抗菌活性,71.4%的代表菌株对至少一种病原菌有拮抗作用。【结论】分离方法的选择对昆虫体内放线菌的分离效果影响较大;中华蜂体内放线菌具有丰富的多样性,展现出很好的抗菌活性,表明其在发现新型生物活性物质中具有很大的潜力。     

羊肚菌多糖的分离纯化及组成结构分析

液体深层发酵所得的羊肚菌（Ｍｏｒｃｈｅｌｌａ ｅｓｓｕｌｅｎｔａ Ｌ．）营养液经超过滤法去掉小分子量组分,脱脂,脱色,脱蛋白质,减压低温浓缩,用超速离心法分离提纯,再用乙醇沉淀法反复多次将所要的多糖沉淀提纯,得到羊肚菌多糖（ＭＥＰ）。对其中分子量为１００００￣１０００００的多糖（ＭＥＰ－ＳＰ）,采用ＤＥＡＥ－纤维素离子交换柱,以不同浓度ＮａＣｌ为洗脱液,进行纯化分离,再经Ｓｅｐｈａｒｏｓｅ ＣＬ－     

Isolation and purification of calmodulin from the shrimp, Crangon crangon.

Calmodulin was isolated and purified from shrimp abdominal muscle by heat precipitation, ion exchange and hydrophobic interaction chromatography. The purified calmodulin was homogeneous when evaluated by polyacrylamide gel electrophoresis. A still remaining contaminant was eliminated by high performance liquid chromatography on a phenyl column. The biological and physicochemical properties of shrimp calmodulin such as amino acid composition, molecular weight and the ability to activate calmodulin-deficient bovine heart phosphodiesterase were compared to those of other invertebrate calmodulins.     

Isolation, purification, and immunomodulatory activity in vitro of three polysaccharides from roots of Cudrania tricuspidata

Three water-soluble polysaccharides (CTPS-1A, CTPS-2B, and CTPS-3A) were obtained from roots of Cudrania tricuspidata (Carr.) Bur. in this study. The homogeneity of polysaccharides was determined, and the average molecular weight, ultraviolet, infrared, monosaccharide composition, and methylation analyses were carried out. Immunomodulatory activity assays in vitro showed that the three polysaccharides could directly stimulate the proliferation of mouse splenocytes alone or combining with concanavalin A or lipopolysaccharide. Furthermore, their stimulating activities were higher than that of the widely clinically used lentinan at optimal concentrations. CTPS-1A and CTPS-2B also enhanced the pinocytic activity of mouse peritoneal macrophages.     

分枝杆菌脂聚糖的分离、纯化及其结构和功能分析

【目的】建立分离、纯化分枝杆菌脂聚糖的方法,初步比较分析不同菌株来源的脂阿拉伯甘露聚糖(Lipoarabinomannan,LAM)和脂甘露聚糖(Lipomannan,LM)的结构差异及研究脂聚糖刺激对巨噬细胞环氧合酶-2(Cyclooxygenase-2,COX-2)蛋白表达的影响。【方法】应用Triton X-114液相法提取脂聚糖,电洗脱法分离纯化,基质辅助激光解析电离串联飞行时间质谱(MALDI-TOF/TOF-MS)进行分子量鉴定;基于特异性识别非还原性末端α-D-甘露糖基的刀豆球蛋白(Concanavalin A,Con A)分析新诺分枝杆菌JDM601、结核分枝杆菌H37Rv标准株和耻垢分枝杆菌mc2155脂聚糖的结构差异;进一步用Western blot检测脂聚糖刺激的RAW 264.7巨噬细胞COX-2蛋白的表达。【结果】通过电洗脱法成功纯化出3种菌株脂聚糖;MALDI-TOF/TOF-MS鉴定发现,分子量从小到大依次为新诺分枝杆菌JDM601、耻垢分枝杆菌mc2155和结核分枝杆菌H37Rv来源的脂聚糖。Western blot显示,Con A能与结核分枝杆菌H37Rv标准株来源的LAM相互作用,而不能与新诺分枝杆菌JDM601和耻垢分枝杆菌来源的LAM相互作用;并且发现Con A与新诺分枝杆菌JDM601来源的LM有很强的反应,然而与其余两种来源的LM反应很弱。3种菌株来源的脂聚糖均能刺激RAW 264.7巨噬细胞COX-2蛋白的表达。【结论】首次成功对来源于中国临床分枝杆菌分离株的脂聚糖进行了分离纯化,初步探讨了不同菌株来源分枝杆菌脂聚糖的结构差异,并表明LAM和LM均能刺激巨噬细胞诱导COX-2蛋白的表达,为进一步研究其对宿主的毒力和免疫机制奠定了基础。     

Isolation, purification and chemical composition of insoluble collagen from Guerin epithelioma.

1. The insoluble collagen from Guerin epithelioma was isolated and its chemical composition was determined. The unusually high histidine content is accompanied in tumour collagen by a relatively small amount of lysine and arginine. 2. The isolated protein was strongly bound to glycoprotein, which could not be removed by EDTA treatment unless this procedure was preceded by digestion of the complex with trypsin.     

Isolation, structure, and antibacterial activity of thiazomycin A, a potent thiazolyl peptide antibiotic from Amycolatopsis fastidiosa

Thiazolyl peptides are a class of thiazole-rich macrocyclic potent antibacterial agents. Recently, we described thiazomycin, a new member of thiazolyl peptides, discovered by a thiazolyl peptide specific chemical screening. This method also allowed for the discovery of a new thiazolyl peptide, thiazomycin A, which carries modification in the oxazolidine ring of the amino sugar moiety. Thiazomycin A is a specific inhibitor of protein synthesis (IC(50) 0.7 microg/mL) and a potent Gram-positive antibacterial agent with minimum inhibitory concentration (MIC) ranging 0.002-0.25 microg/mL. The isolation and structure elucidation and biological activities of thiazomycin A are described.     

龙须藤叶粗提物的分离纯化及生物活性初探

为进一步研究和开发新植物源农药,拓宽龙须藤(Bauhinia championii)的生物活性研究,探索其不同组分潜在的杀菌和除草活性,该研究通过常温冷浸提取法提取、真空浓缩得到甲醇提取物。结果表明:用硅胶柱层析分离纯化,经TLC检识和碘缸显色后整合得到9个组分。反复重结晶7号和8号组分中析出的物质,经TLC检识和测熔点,得到1个纯化合物,编为33号,经波谱数据分析与molbase库对照,鉴定该化合物为(1R,2S,3S,4S,5S,6S)-6-甲氧基-1,2,3,4,5-环己烷五醇,是一种重要的工业原料。杀菌和除草活性试验结果显示,粗提物在1 000μg·m L~(-1)时对水稻稻瘟病菌的抑制率为(40.84±1.00)%,对稗草根的抑制率为(49.18±2.33)%;各组分在500μg·m L~(-1)时,4号组分对水稻稻瘟病菌的抑制率达到(44.19±0.76)%,2号和3号组分对稗草根的抑制率分别为(88.92±1.31)%和(90.99±1.45)%,3号和6号组分对马齿苋根的抑制率分别为(72.06±1.31)%和(89.92±1.73)%。这表明龙须藤叶提取物对水稻稻瘟病菌、稗草根和马齿苋根有良好的抑制效果,可进一步分离2号、3号、4号、6号组分,以获得高活性的单体化合物。     

Isolation, purification and properties of aortic elastase.

An elastase from pig aorta has been partially purified and characterised; it exhibits immunological cross reaction with pig pancreatic elastase. Its proteolytic (k-elastin gel and polymeric elastin substrates) and esterolytic (N-succinoyl-trialanine paranitroanilide) activities as well as its degree of inhibition by serum protease inhibitors (alpha1-antitrypsin and alpha2-macro-globulin) differ sensibly from those of pancreatic elastase [14,16].     

Isolation, purification, and reconstitution of a proline carrier protein from Mycobacterium phlei.

Membrane vesicles from Mycobacterium phlei contain carrier proteins for proline, glutamine, and glutamic acid. The transport of proline is Na+ dependent and required substrate oxidation. A proline carrier protein was solubilized from the membrane vesicles by treatment with cholate and Triton X-100. Electron microscopic observation of the detergent-treated membrane vesicles showed that they are closed structures. The detergent-extracted proteins were purified by means of sucrose density gradient centrifugation, followed by gel filtration and isoelectric focusing. A single protein with a molecular weight of 20,000 +/- 1000 was found on polyacrylamide gel electrophoresis. Reconstitution of proline transport was demonstrated when the purified protein was incubated with the detergent-extracted membrane vesicles. This reconstituted transport system was specific for proline and required substrate oxidation and Na+. The purified protein was also incorporated into liposomes, and proline uptake was demonstrated when energy was supplied as a membrane potential introduced by K+ diffusion via valinomycin. The uptake of proline was Na+ dependent and was inhibited by uncoupler or by sulfhydryl reagents.     

芦荟多糖的分离纯化及性质研究

采用水提醇沉法提取芦荟多糖,经DEAE-C32柱层析分离,Sephades G-100进一步纯化,得AⅠ、AⅡ和AⅢ三种芦荟多糖。Sephadex G-100凝胶色谱表明,AⅠ组分为均一组分,其分子量约为3.8×10~4。借助气相色谱技术,研究了芦荟粗多糖和AⅠ组分的单糖组成。另外,红外光谱鉴定芦荟多糖主要为吡喃多糖。