海南红树林淡紫拟青霉胞外多糖提取条件的优化

为了探讨影响红树林淡紫拟青霉胞外多糖提取的因素,确定最佳提取方案,设置不同的发酵液浓缩倍数、三氯乙酸用量等因素,设计单因素实验测定多糖提取最佳条件。然后设计正交试验,检测多糖在不同条件下的提取率,以获得最佳提取工艺。结果发现,发酵液浓缩3~5倍时多糖提取率最高,10%的三氯乙酸对蛋白质脱除效果最好;正交试验表明影响多糖提取的因素依次为乙醇用量、沉淀时间和温度,最优方案为3倍95%乙醇、4 ℃沉淀24 h。该条件下,多糖提取率可达57.835%±1.206%。研究结果为红树林淡紫拟青霉胞外多糖的提取研究提供了参考。     

一株胞外多糖产生菌的筛选鉴定及其多糖成分研究

以菌落周围有粘稠分泌物为初筛标准从土壤中筛选胞外多糖产生菌,苯酚-硫酸法测定初筛菌胞外多糖的产量;将胞外多糖产生菌LE-3进行形态学观察、BIOLOG分析与16S rDNA鉴定;提取菌株LE-3胞外粗多糖,该粗多糖经Sevage法除蛋白、透析和CM-琼脂糖凝胶FF、DEAE-琼脂糖凝胶FF、丙烯葡聚糖凝胶S-200HR柱层析进行纯化,傅里叶红外光谱法、薄层层析法和高效液相色谱法分析胞外多糖的单糖组成。结果表明:从土壤中筛出26株符合初筛标准的菌株,3株产糖量在5 g/L以上,其中菌株LE-3产量为5.29 g/L;根据形态学特征、BIOLOG和分子生物学分析,初步鉴定菌株LE-3为一株解淀粉芽孢杆菌亚种(Bacillus amyloliquefaciens subsp.amyloliquefaciens);该菌株胞外粗多糖经Sevage法除蛋白、透析和三次柱层析纯化后,得到单一组分的LE-3胞外多糖;傅里叶红外光谱法、薄层层析法和高效液相色谱法分析确定该多糖为果聚糖。     

斜链拟青霉多糖活性组分的分离及其清除自由基活性

对斜链拟青霉胞外多糖进行体外清除自由基活性研究。利用醇沉法从斜链拟青霉发酵液中醇沉获得斜链拟青霉胞外多糖,经DEAE-纤维素-52柱层析后得到1个主峰P3和2个小峰P1与P2,对主峰P3进行Sephadex G-100柱层析,表明P3为纯化物; 应用DPPH-酶标法和化学发光法分别对P3进行清除DPPH·、·OH和O-2·能力测试。结果表明：P3对DPPH·、·OH和O-2·均具有明显的清除能力,且与浓度呈量效关系,IC50分别为0.083、0.121和0.214 mg/mL。     

根癌农杆菌介导的增强型绿色荧光蛋白基因在淡紫拟青霉中的转化

为了实现增强型绿色荧光蛋白基因 (egfp) 在生防真菌淡紫拟青霉9410菌株中的转化,借助中间质粒pcDNA3.1(-) 构建nptⅡ-egfp融合基因的表达载体pUPNGT,然后采用根癌农杆菌介导的转化法将egfp基因转化到淡紫拟青霉9410菌株中。PCR检测和Southern blotting分析结果表明,egfp基因以单拷贝形式整合到淡紫拟青霉9410的基因组中。荧光显微镜观察结果显示,转化子在488 nm下能产生绿色荧光。这些结果说明egfp基因已成功转化至淡紫拟青霉9410菌株并获得表达。这些工作可为淡紫拟青霉在不同条件下的防效评价、环境安全评价等提供新的途径和方法。     

沙漠生物结皮芽孢杆菌产胞外多糖的纯化及其絮凝性的研究

【目的】以新疆古尔班通古特沙漠的生物结皮为样品,通过培养、筛选、分离得到一株高产胞外多糖(EPS)的菌株XJ-27,对XJ-27菌株所产的胞外多糖进行分离纯化,并对其絮凝性进行研究。【方法】利用DEAE sepharose CL-6B阴离子层析和Sephadex G100凝胶层析的方法对胞外多糖进行纯化,通过紫外分析方法和高效凝胶渗透色谱进行纯度的测定,利用高效凝胶渗透色谱法(HP-GPC)测定其分子量,以高岭土为体系对其絮凝性进行研究。【结果】利用层析分离的方法共得到2个胞外多糖的组分,对其中一个组分进一步纯化,得到组分EPS-I。结果表明,EPS-I纯度较高,分子量为575 kD。同时对胞外多糖的絮凝性进行了研究,结果表明该胞外多糖对高岭土为体系的絮凝率为80.4%。【结论】菌株XJ-27产胞外多糖,其胞外多糖具有絮凝性,对该胞外多糖进行分离纯化后,得到分子量为575 kD的多糖组分EPS-I。     

聚乙二醇修饰重组溶葡球菌酶的初步研究

目的:探讨聚乙二醇(PEG)修饰重组溶葡球菌酶(lysostaphin)的反应条件以及修饰后产物的纯化方法.方法:采用超声波细胞粉碎机进行菌体破碎,阳离子交换层析、疏水层析进行蛋白纯化;在不同条件下,将活化的单甲氧基聚乙二醇琥珀酰亚胺丙酸酯(mPEG-SPA)与纯化后的lysostaphin反应,以单个PEG-Lysostaphin的比例为指标,用SDS-PAGE、MALDI-TOF-MS方法确定其在修饰产物中的所占比例;采用Sephacryl S-200分子筛凝胶层析法对修饰产物进行分离纯化.结果:mPEG-SPA修饰lysostaphin的反应条件为pH 8.0,温度4℃,lysostaphin与mPEG-SPA的质量比为1∶5,反应时间2.0h;反应产物经一步Sephacryl S-200分子筛凝胶层析纯化后,初步实现分离.结论:初步确定了聚乙二醇修饰lysostaphin的反应条件及修饰产物的纯化方法.     

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。     

鲍姆木层孔菌多糖对HepG2细胞增殖及侵袭相关能力的抑制作用

证实了鲍姆木层孔菌多糖对肝癌细胞系HepG2细胞增殖及侵袭的抑制作用。鲍姆木层孔菌提取物经乙醇分级沉淀和DEAE-Sepharoes F.F.离子柱层析以及Sephacryl S-200凝胶柱层析纯化,获得均一多糖PLP60-B1,该多糖具有体外活性。MTT法及流式细胞仪技术证实多糖PLP60-B1通过致使HepG2细胞阻滞于S期而显著抑制HepG2细胞的增殖和细胞集落的形成,且也可显著抑制细胞的粘附及侵袭能力。     

苦瓜多糖的提取、分离纯化及组成性质

正交实验确定提取工艺后,用热水提取法得到苦瓜多糖(MCP).对MCP进行DEAE-32离子交换层析分离,得到3个多糖组分MCP1、MCP2和MCP3. 进一步采用Sephacryl S-400凝胶层析进行分离,经凝胶层析和高效液相色谱检测表明,MCP1、MCP2为均一性多糖组分.通过高效液相凝胶色谱法测定了两者的相对分子质量分别为1.16×106和7.45×105.用PMP衍生化法测定其单糖,结果表明: MCP1系由Man、Rham、GlcUA、GalUA、Glu、Gal、Xyl、Ara等单糖组成的杂多糖,摩尔比为1.03:2.93:1.00:14.95:2.16:30.70:2.85:4.50.MCP2系由Rham、GalUA、Gal、Xyl、Ara等单糖组成的杂多糖,对应的摩尔比为1.63:21.88:4.66:1.00:1.29.紫外光谱表明该多糖不含蛋白质和核酸.     

淡紫拟青霉固体发酵条件初步研究

淡紫拟青霉是一种具有防治植物线虫作用的生防菌株。为了获得淡紫拟青霉固体发酵的最佳条件,采用单因素实验和正交实验对淡紫拟青霉固体发酵的培养基组成及培养条件进行摸索。淡紫拟青霉固体发酵的最佳培养基组成为麸皮和玉米粉4:1、料水比1:0.6、氯化锰含量为0.6%、初始pH值为6.0,固体发酵最佳的培养条件为接种量6%,培养温度25℃,培养时间为9 d,在此条件下淡紫拟青霉固体发酵的分生孢子数为13.5×10~9 CFU/g。     

Components and antioxidant activity of the polysaccharide from Streptomyces virginia H03

A polysaccharide was isolated from the broth of cultured Streptomyces virginia H03 which was treated by ethanol deposition and savage method to remove the protein, and was purified using Sephadex G-150 column chromatography. The components of the polysaccharide were determined by gas chromatography. The purified polysaccharide was made up of mannose, glucose and galactose, in a 2:1:1 proportion. Its average apparent molecular weight was 3.76 x 10(4) Da which was determined by gel permeation chromatography. In addition, several antioxidant assays were adopted to investigate the antioxidant activity of the polysaccharide in vitro. The results indicated that the purified polysaccharide showed significant antioxidant activity against superoxide anion, hydrogen peroxide and 1,1-diphenyl-2-picrylhydrazyl radical, and lipid peroxidation as with standard antioxidants such as vitamin C. Furthermore, the polysaccharide had a better heat stability than vitamin C, which suggested that the polysaccharide might be a potent useful antioxidant.     

Ultrasound complex enzymes assisted extraction and biochemical activities of polysaccharides from Epimedium leaves

Ultrasound complex enzymes (UCE) were used to extract crude polysaccharides (CEP) from Epimedium leaves. The response surface methodology (RSM) based on Box–Behnken Design (BBD) was employed to further optimize the extraction conditions. The optimum conditions were: complex enzyme dose of 50, 250, 200, 100 U/g (papain, pectase, cellulase and α-amylase), extraction temperature 46.8 °C, ultrasound time 42.3 min, pH 4.28, and ultrasound power 311 W. Under these conditions, the experimental yield of CEP was 5.98%, which was well matched with the value predicted by the model. Three major polysaccharide fractions (EP-1, EP-2 and EP-3) were obtained by ultrafiltration, chromatography on DEAE-Sepharose Fast Flow and Sephadex G-100 column. Their chemical–physical characteristics were investigated using chemical methods, gas chromatography (GC) and high-performance size-exclusion chromatography (HPSEC). Biochemical characterization of the polysaccharide fractions was evaluated on the basis of free radical scavenging, ferric-reducing antioxidant power (FRAP), lymphocyte proliferation, macrophage phagocytosis and NK cell activity assay in vitro. The results showed that all fractions contained higher galacturonic acid (GalA) and galactose (Gal), possessed considerable antioxidant activity, and could promote proliferation of splenic lymphocytes, activate macrophages and enhance NK cells’ activity. UCE as a more effective and environment-friendly technique could be employed for extracting active ingredients from plant materials, extensively applied in pharmaceutical and food industries.     

Purification and characterization of a potential antifungal protein from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> E1R-J against <Emphasis Type="Italic">Valsa mali</Emphasis>

In order to identify the antagonistic substances produced by Bacillus subtilis E1R-J as candidate of biocontrol agents for controlling Apple Valsa Canker, hydrochloric acid precipitation, reverse phase chromatography, gel filtration, and ion exchange chromatography were used. The purified fraction EP-2 showed a single band in native-polyacrylamide gel electrophoresis (native-PAGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Fraction EP-2 was eluted from native-PAGE and showed a clear inhibition zone against V. mali 03-8. These results prove that EP-2 is one of the most important antifungal substances produced by B. subtilis E1R-J in fermentation broth. SDS-PAGE and Nano-LC–ESI–MS/MS analysis results demonstrated that EP-2 was likely an antifungal peptide (trA0A086WXP9), with a relative molecular mass of 12.44 kDa and isoelectric point of 9.94. The examination of antagonistic mechanism under SEM and TEM showed that EP-2 appeared to inhibit Valsa mali 03-8 by causing hyphal swelling, distortion, abnormality and protoplasts extravasation. Inhibition spectrum results showed that antifungal protein EP-2 had significantly inhibition on sixteen kinds of plant pathogenic fungi. The stability test results showed that protein EP-2 was stable with antifungal activity at temperatures as high as 100 °C for 30 min and in pH values ranging from 1.0 to 8.0, or incubated with each 5 mM Cu²⁺, Zn²⁺, Mg²⁺, or K⁺. However, the antifungal activity was negatively affected by Proteinase K treatment.     

Isolation and characterization of a succinylated polysaccharide from the cell wall of Micrococcus agilis

A polysaccharide consisting of rhamnose, galactose, glucosamine and ester-linked succinic acid was extracted from the isolated cell walls of Micrococcus agilis by the hot water-phenol and 5% trichloroacetic acid (TCA) extraction methods. The hot water-phenol extractable polysaccharide accounted for 30% of the weight of the wall, with 23% by the TCA method. Phosphorus contents were less than 0.01% of the polysaccharide. Succinyl residues released by alkali treatment (0.1 N NaOH, 30 min, 37 degrees C) were identified by gas-liquid chromatography, and accounted for 6.3% and 5.1% of the polysaccharide purified from the hot water-phenol and TCA extracts, respectively. The polysaccharide was not bound when chromatography on Concanavalin A-Sepharose 4B (Con A/Sepharose 4B) columns was performed and it could thus be separated from any residual membrane lipomannan. The purified polysaccharide behaved as a negatively-charged polymer on electrophoresis in 1% agarose (at pH 8.6). A strong cross-reaction, unaffected by removal of the succinyl groups, was observed with type XXIII pneumococcal polysaccharide antiserum indicating the presence of L-rhamnose, linked through non-reducing, lateral end groups.     

Isolation of a coaggregation-inhibiting cell wall polysaccharide from Streptococcus sanguis H1.

Coaggregation between Streptococcus sanguis H1 and Capnocytophaga ochracea ATCC 33596 cells is mediated by a carbohydrate receptor on the former and an adhesin on the latter. Two methods were used to release the carbohydrate receptor from the gram-positive streptococcus, autoclaving and mutanolysin treatment. The polysaccharide released from the streptococcal cell wall by either treatment was purified by ion-exchange chromatography; this polysaccharide inhibited coaggregation when preincubated with the gram-negative capnocytophaga partner. After hydrolysis of the polysaccharide by hydrofluoric acid (HF), the major oligosaccharide of the polysaccharide was purified by high-performance liquid chromatography. By analysis of the HF hydrolysis of the polysaccharide and the purified oligosaccharide, this major oligosaccharide appeared to be the repeating unit of the polysaccharide, with minor components resulting from internal hydrolysis of the major oligosaccharide. Gas chromatography results showed that the oligomer was a hexasaccharide, consisting of rhamnose, galactose, and glucose, in the ratio of 2:3:1, respectively. By weight, the purified hexasaccharide was a fourfold-more-potent inhibitor of coaggregation than the native polysaccharide. Resistance to hydrolysis by sulfuric acid alone and susceptibility to hydrolysis by HF suggested that oligosaccharide chains of the polysaccharide are linked by phosphodiester bonds. Studies with a coaggregation-defective mutant of S. sanguis H1 revealed that the cell walls of the mutant contained neither the polysaccharide nor the hexasaccharide repeating unit. The purification of both a polysaccharide and its constituent hexasaccharide repeating unit, which both inhibited coaggregation, and the absence of this polysaccharide or hexasaccharide on a coaggregation-defective mutant strongly suggest that the hexasaccharide derived from the polysaccharide functions as the receptor for the adhesin from C. ochracea ATCC 33596.     

野生糙皮侧耳多糖的提取、分离纯化和组分分析

以野生糙皮侧耳子实体为材料,用水浸法及乙醇沉淀法提取多糖;用过氧化氢、重蒸酚、不同含量的活性碳进行脱色;然后用氢氧化锌法、三氯醋酸法、sevag法、胰蛋白酶与sevag法相结合的方法进行脱蛋白;再通过透析和柱层析作进一步分离纯化;最后以纸层析法进行组成糖分析.结果表明:野生糙皮侧耳的水溶性多糖以1.5%活性碳脱色效果最...     

千斤拔多糖的提取纯化及其结构鉴定

利用正交试验考察千斤拔多糖的提取工艺,并比较脱蛋白方法中的Sevag法和三氯乙酸法的纯化效果,总糖含量测定采用苯酚-硫酸法,蛋白质含量测定采用考马斯亮蓝法;采用DEAE-52纤维柱法来分离多糖,并运用HPLC色谱来分析千斤拔多糖中的单糖成分。结果表明:经正交试验得出千斤拔多糖的最佳提取条件为时间2.5 h,料液比为1∶30,温度80℃,其多糖得率为8.558%。对比两种脱蛋白的方法,Sevage法萃取3次时蛋白的脱除效果最好。经DEAE-52纤维柱来分离多糖共分得7个组分。经HPLC色谱鉴定出有葡萄糖,甘露糖和阿拉伯糖,主要单糖成分为葡萄糖。     

库克Noni果汁多糖含量及分子量测定

建立测定库克Noni果汁中多糖含量及分子量的方法。通过醇沉法分离Noni果汁多糖,进一步分离纯化得到均一多糖组分;用精制Noni果汁多糖测得该多糖对葡萄糖的换算因子,对多糖含量进行定量测定;并通过SE-HPLC(Size Exclusion-High Performance Liquid Chromatography)法测定了该多糖的相对分子量。结果表明,多糖含量测定方法简便可行,供试液在4 h内显色稳定,重现性较好,平均回收率为99.3%,RSD为1.93%(n=3);测得该多糖的相对分子量为1140 KDa。     

Isolation and purification of cell wall polysaccharide of Bacillus anthracis (Δ Sterne)

A polysaccharide fraction was isolated form sodium-dodecyl-sulfate (SDS) treated cell walls of Bacillus anthracis (delta Sterne) by hydrofluoric acid (HF) hydrolysis and ethanolic precipitation. The polysaccharide fraction was subsequently purified by several washings with absolute ethanol. Purity of the isolated polysaccharide was tested using the anthrone assay and amino acid analyzer. The molecular mass of the polysaccharide fraction as determined by gel filtration chromatography was about 12000 Da. Preliminary analyses of the polysaccharide was done using thin layer chromatography and amino acid analyzer, and results obtained from these analyses were further confirmed by gas liquid chromatography and 13C-NMR spectroscopy. Results showed that the polysaccharide moiety contained galactose, N-acetylglucosamine, and N-acetylmannosamine in an approximate molar ratio of 3:2:1. This moiety was devoid of muramic acid, alanine, diaminopimelic acid, glutamic acid, and lipid, thus indicating that the isolated polysaccharide was of pure quality.     

Purification and characterization of a novel polysaccharide involved in the pellicle produced by a thermotolerant Acetobacter strain

Acetobacter strains able to produce a thick pellicle at 37 degrees C were screened among many thermotolerant strains isolated from fruits in Thailand. As a result, Acetobacter sp. SKU 1100 was selected as the producer of a relatively thick pellicle even when cultured at higher temperatures such as 37 degrees C or 40 degrees C. This strain could produce a pellicle polysaccharide in a shaking submerged culture as well as under static culture conditions. The polysaccharide was found to be attached to the bacterial cells. Although the polysaccharide production was higher at 30 degrees C than at 37 degrees C in shaking submerged culture, the productivity in static culture was not decreased even at higher temperatures. The membrane-attached polysaccharide was purified from the SKU 1100 strain by cell disruptions using either ultrasonic treatment or lysozyme treatment, followed by ultracentrifugation, enzyme treatments, dialysis against SDS, DEAE-cellulose column chromatography, alcohol precipitation, and gel filtration chromatography. The polysaccharide purified by the sonic treatment and also by the mild conditions using lysozyme treatment had the same average molecular mass of 120 kDa. The purified polysaccharide was composed of three different monosaccharides; glucose, galactose, and rhamnose, in an approximately equimolar ratio of 1:1:1.