真空冷冻干燥后藻蓝蛋白提取工艺及稳定性的研究

为拓宽极大螺旋藻藻蓝蛋白的应用范围,研究了温度、时间、pH值、固液比对其提取工艺及金属离子、食品添加剂等因素对其稳定性的影响。结果表明：藻蓝蛋白的最佳提取工艺为温度30℃、反应时间15h、Na2HPO4-柠檬酸缓冲液pH值70,固液比1∶60,在此条件下藻蓝蛋白的提取率达最大值。藻蓝蛋白在pH值50~70,温度30℃,室内可见光或暗光条件下较稳定;淀粉、蔗糖、明胶、苯甲酸钠等食品添加剂与低浓度条件下的氧化剂和还原剂对其稳定性影响不显著;金属离子Na+、K+、Mg2+和低浓度的Ca2+、Zn2+、Al3+、Fe3+、Cu2+对其稳定性无影响。因此,藻蓝蛋白在弱酸性环境、低金属离子浓度及常用食品添加剂等条件下较稳定,可将其作为天然色素广泛应用于食品工业中。     

别藻蓝蛋白的聚集态研究

为了研究鱼腥藻PCC7120（Anabaena sp．PCC7120）中别藻蓝蛋白（APC）α和β亚基（α-APC和β-APC）中藻蓝胆素（PCB）与脱辅基蛋白的生物合成,并在蓝藻体外对这两种色素蛋白PCB—ApcA和PCB-ApcB合成时聚集过程进行分析,通过多种组合的质粒在大肠杆菌体内共同表达进行重组。色素蛋白的吸收和荧光光谱以及Zn电泳表明,在大肠杆菌体内同时得到色素蛋白PCB—ApcA和PCB—ApcB,并且体内重组色素蛋白的细胞荧光光谱显示,色素蛋白以三聚体的形式存在,而破碎细胞后所得上清液所显示的光谱特征为单聚体的特征。     

螺旋藻藻蓝蛋白的稳定性及抗癌活性研究

螺旋藻藻蓝蛋白提取物在pH4.0~8.5、40℃以下和弱光环境中表现稳定,并通过MTT比色法测定其对肝癌细胞SMMC-7721和喉癌细胞HEp-2的生长抑制作用,研究了其抗癌活性。     

表达重组别藻蓝蛋白质粒在工程菌株中的遗传稳定性研究

将含有表达重组别藻蓝蛋白基因的重组质粒pMal-2X的工程菌株P1分别在含有Amp的LB固体培养基上采用划线法连续传代至100代,其生长速度（在LB培养基中）、菌落形态和抗生素抗性等方面与原始种子库无明显差异;取第10,20,50,100代提取质粒DNA经EcoRI限制性内切酶酶切检查,酶切图谱没有改变。DNA测序未见apc基因变异。原代菌株与传代第10,20,50和100代菌株经诱导培养,其rAPC表达水平、菌体蛋白的SDS—PAGE图谱及重组蛋白的免疫原性均无明显差异。以上结果表明,重组质粒pMal-2X在工程菌株P1中具有良好的遗传稳定性。     

从一种富含藻胆蛋白的螺旋藻中大量提取和纯化藻蓝蛋白的研究

采用新鲜藻丝为原料和分段梯度盐析分离纯化钝顶螺旋藻Sp(NS)-90020的藻蓝蛋白（PC）,经羟基磷灰石一次层析,能使提取的PC的纯度大于普遍认可的标准,由于该工艺流程较为简单,适合藻蓝蛋白的大量生产,藻胆蛋白经Sephadex凝胶过滤后的可达电泳纯度标准,经SDS-PAE测得PC,APC的分子量分别约为38.33kD。     

柱孢鱼腥藻的藻蓝蛋白亚基和F_(678)色素蛋白

柱孢鱼腥藻的藻蓝蛋白包含有两个亚基。β亚基具有578nm吸收峰和600nm荧光发射峰,分子量19.80±0.40KD,可表明β亚基仅有一个载色团。α亚基具有578nm和630nm吸收峰及645nm的荧光发射峰,分子量17.35±0.38,α亚基可能有两个载色团。别藻蓝蛋白有635nm和650nm吸收蜂及664nm的荧光发射峰。具藻胆体的类囊体膜从高盐浓度缓冲液移至低盐浓度缓冲液时表现678nm荧光发射峰,可能柱孢鱼腥藻存在的F_(678)色素蛋白相当于GLazer(1975)的别藻蓝蛋白B。     

别藻蓝蛋白藻蓝胆素发色团分子构象研究

主要研究了蓝绿藻污棕席藻(Phormidium luridum)别藻蓝蛋白在不同 pH值条件下的吸收光谱和共振拉曼光谱.发现低聚化的结果导致了三聚体别藻蓝蛋白 650nm 特征吸收峰的消失和一些共振拉曼带强度和位置的移动.结果表明在低 pH 值作用下的低聚化的别藻蓝蛋白中藻蓝胆素发色团分子的构象和自由胆素分子类似,比三聚体的别藻蓝蛋白的发色团分子更趋于卷曲,折叠的构象态.而三聚体的别藻蓝蛋白,主要的拉曼带 1645cm^-1是其发色团分子构象处于更线性延展的标志,其光谱行为和吸收光谱 A_vis/A_uv所表征的发色团分子构象的结果相一致.     

超声波对蔷薇藻生长及生理特性的影响

目的:用超声波处理蔷薇藻(Rhodella reticulata),研究超声波电流、处理时间和脉冲时间对蔷薇藻生长代谢的影响.方法:通过均匀设计实验结合DPS软件对均匀设计实验结果进行二次多项式模型分析和拟合,以生物量、胞外多糖和藻蓝蛋白为目标建立回归模型,优化超声波处理条件.结果:以生物量为目标:电流39%,超声时间123.27s,脉冲时间0.55s,干重的预测值为2.42g/L;以胞外多糖为目标:电流21%,超声时间245s,脉冲时间9.5s,胞外多糖的预测值为656.22mg/L;以藻蓝蛋白为目标:电流21%,超声时间5.0s,脉冲时间0.5s,藻蓝蛋白的预测值为63.53mg/L;优化后生物量、胞外多糖和藻蓝蛋白的产量比未经优化的分别提高了13.3%、11.4%和31.1%.结论:一定强度的超声波促进了蔷薇藻的生长和胞外多糖及藻蓝蛋白的积累.     

藻蓝蛋白的最新药理活性及作用机制研究

藻蓝蛋白是一种重要的光合辅助色素,包含一个开链的藻蓝素发色团,首先在螺旋藻中被发现。藻蓝蛋白由于具有抗癌、抗氧化等多种生物活性,成为近年天然海洋药物研究的热点。本文对近几年来有代表性的中英文文献进行分析、归纳,介绍关于藻蓝蛋白的最新药理活性的研究状况及可能的机制,为进一步研究其各种生物学功能提供参考。     

不同硝酸钾浓度对蔷薇藻生长及生理特性的影响

蔷薇藻(Rhodella reticulata)是属于红藻门的一种海洋单细胞微藻,其生长过程中产生藻胆蛋白、胞外多糖等生物活性物质.研究了不同硝酸钾浓度对蔷薇藻生长代谢的影响,分析测定蔷薇藻比生长速率、胞外多糖产量、藻蓝蛋白含量、色素含量、硝酸还原酶活性和SOD活性等参数.结果表明,与无氮培养基相比,蔷薇藻在以硝酸钾为氮源,浓度为0.75 g/L的条件下生长最好,硝酸还原酶活性最大,分别得到最大的藻蓝蛋白产量(24 mg/L)和类胡萝卜素含量(1.42 mg/L);当KNO3浓度为7.5 g/L时,获得最高叶绿素a含量为1.91 mg/L;高氮源有利于产糖,当KNO3浓度为30 g/L时,得到最高胞外多糖产量为1.633 g/L;SOD活性随硝酸钾浓度增加而增大.     

B-Phycoerythrin from Rhodella violacea

Two isoproteins of the native B-phycoerythrin of the red alga, Rhodella violacea, were purified from crude extracts by preparative polyacrylamide gel electrophoresis and subsequently characterized. The slower moving pigment in gel electrophoresis was designated B-PE I, the faster as B-PE II. Both were found to occur in about equal amounts.B-PE I has a molecular weight of about 280000 and an IEP at 4.39, B-PE II a molecular weight of nearly 265000 and an IEP at 4.23. B-PE I and II are characterized by absorption maxima at 568 and 542 nm and a shoulder at 500 nm in the visible part of the absorption spectra. Their absorption coefficients at 542 nm differ with values of 5.54 and 5.63, respectively. The fluorescence emission spectra show a single maximum at 575 for B-PE I and at 578 nm for B-PE II. Both spectra have a shoulder at 630 nm. The fluorescence yield of B-PE II is lower by 25%.In calibrated SDS gel electrophoresis of the purified pigments B-PE I and II show two subunits of molecular weights of 18 900 and 29 200 and 18 500 and 29 900, respectively.Quantitative amino acid analyses indicated, that the isoproteins are very similar. B-PE II, however, has a significantly higher content of acidic amino acids and a lower percentage of basic residues, which is in keeping with its lower isoelectric point.Functional aspects of the occurrence of two isoproteins of B-phycoerythrin are discussed.Abbreviations PE phycoerythrin - MG molecular weight - IEP isoelectric point - SDS sodium dodecyl sulphate - AA acrylamide - BIS N,N-methylenebisacrylamide - Tris Tris(hydroxymethyl)aminomethan     

A bacterial pathogen of Rhodella reticulata

A bacterium was isolated which caused disease in a laboratory culture of the red alga, Rhodella reticulata. The organism was an aerobic Gram-negative rod, aflagellate andpigmented. The absorption maximum of the pigments in methanol was 460 nm. The bacteriumshowed hydrolytic activity towards agar and produced extracellular agarase. The organismutilized monosaccharide constituents of the polysaccharide of R. reticulata , as well asthe whole polysaccharide of alga, as a sole carbon source. The bacterium was identified as Cytophaga sp. LR₂.     

The Discovery of Tetraploid Camellia reticulata and Its Implication in Studies on the Origin of This Species

Camellia reticulata and its two allied species, C. saluenensis and C. pitardii,all from the Jinshajiang Valley of Yunnan and Sichuan, were cytologically studied in this work. Among 34 populations of C.reticulata studied here, 21 were found to be tetraploids (2n=60), 11 hexaploids (2n = 90), and the remaining two diploids (2n = 30). Tetraploid was reported in C. reticulata for the first time and its karyomophology was studied. In meiosis of pollen mother cells of the tetraploids and the hexaploids, only bivalents were observed in most of the populations, although in several populations or individuals a few univalents or tetravalents appeared. In the hexaploids, hexavalents were not observed. Thus, the meiotic chromosome cofigurations showed that the tetraploids and hexaploids in C.reticulata allopolyploids, and the appearance of a few tetravalents in some populations might indicatehomology of chromosomes in different genomes to some extent. The characteristics of the resting nuclei and the prophase chromosomes of all the tetraploids and hexaploids were quite similar to each other. The tetraploids of C.reticulata from the localities with the altitude of over 1800 m were gross-morphologically similar to the hexaploids, but those growing between 1100 ～1800 m were different to some extent from the hexaploids. In geographical distribution, the tetraploids and the hexaploids were continuous, and overlapped with C. saluenensis and C. pitardii.     

Influence of viscous Rhodella grisea (Rhodophyceae) proteoglycan on chemically induced cough reflex

An algal extracellular biopolymer (over 8.5 × 10⁵ Da) composed of carbohydrates (52%) and protein (∼13%) has been isolated from a red alga Rhodella grisea growing in natural conditions by concentration of water medium, alcohol precipitation, dialysis and freeze-drying. This mucilagineous biopolymer contained xylose and its 3-O- and 4-O-methyl derivatives (∼63%), galactose (∼12%), glucuronic acid (11-12%), glucose (∼5%), rhamnose (∼4%), fucose (∼3-4%) and low content of others accompaning sugars. When tested on the citric acid-induced cough and reactivity of airways smooth muscle in vivo in the test system guinea pigs, this biopolymer assigned a significant cough suppressing effect. The reactivity of airways smooth muscle was not affected indicating that expectoration effect was not suppressed by biopolymer application, which is important from the pharmacological point of view.     

蚯蚓纤溶酶的纯化及稳定性研究

蚯蚓纤溶酶（ＥＰＡ）抽提液经３０％ ～７０％ 饱和度的（ＮＨ４）２ＳＯ４ 盐析、ＤＥＡＥ—纤维素柱层析、Ｓｅｐｈａｄｅｘ Ｇ－７５葡聚糖凝胶过滤等纯化步骤,得到了具有纤溶活性的洗脱峰,置凝胶电泳后,得到四个活性组分,它们经５０℃保温６ｈ,活力上升６４％ ;在２ ｍ ｏｌ／Ｌ盐酸胍存在时,活力仅保存７．２％ ,当其浓度降低时,活力可恢复至９０％ ;在１％ ＳＤＳ存在时,活力仅保存１２．１％ ,但当ＳＤＳ除去时,活力又可恢复。因此,盐酸胍、ＳＤＳ均为ＥＰＡ 可逆性抑制剂。另外,ＥＰＡ 中含有较高的糖链（占总量的４５％ ）,具有良好的抵抗自水解作用。     

Influence of physiological factors on the lysis effect of Cytophaga on the red microalga Rhodella reticulata

The influence of different factors on the lysis of the red microalga, Rhodella reticulata, by Cytophaga sp. LR2 was studied. The pathogenic bacterial strain was more resistant than the alga to the physiological parameters studied, which assured long-term survival of bacteria in algal cultures. Cytophaga sp. LR2 infected R. reticulata at temperatures between 15 and 30 degrees C, in the illuminated as well as the non-illuminated cultures, at pH values between 5.0 and 9.0, and in the presence of NaCl and CaCl2 in the culture medium. SEM showed a different morphology of the bacteria in algal cultures from those of axenic cultures of Cytophaga. Observations of specific associations between algal and bacterial cells revealed that the role of the slime extrusions on the bacterial surface was attachment of Cytophaga to algal cells, and that their clumping leads to rapid lysis.     

Biliprotein assembly in the disc-shaped phycobilisomes of Rhodella violacea electron microscopical and biochemical analyses of C-phycocyanin and allophycocyanin aggregates

C-phycocyanin and allophycocyanin from the red alga Rhodella violacea were investigated by electron microscopy and biochemical methods using samples taken from the same fractions.The molecular weights of the native biliprotein aggregates C-phycocyanin and allophycocyanin are about 139,000 (140,000) and 130,000 (145,000) as revealed by calibrated gel chromatography, gradient gel electrophoresis and morphological measurements on the basis of an average protein packing density. These molecular weights are direct evidence for a trimeric aggregation form ()₃ of these biliproteins. Independently, their monomers were determined to be about 34,400 (C-phycocyanin) and 33,900 (allophycocyanin).C-phycocyanin and allophycocyanin are ringshaped, six-membered, biliprotein aggregates with dimensions of about 10.2×3.0 nm and 10.0×3.0 nm, respectively. The aggregates are made up of six subunits, 3 and 3, which are assumed to be associated in alternating positions. They are arranged in regular hexagons in C₆ symmetry. Hexameric aggregates ()₆, so far only isolated for C-phycocyanin, originate by face to face association of two trimeric aggregates.     

Interactions between the unicellular red alga Rhodella reticulata (Rhodophyta) and contaminated bacteria

AIMS: To define the role of the bacterial strains LR1 and LR3 in the Rhodella cell destruction caused by Cytophaga sp.LR2. METHODS AND RESULTS: The bacteria were obtained from algal culture with destruction. They were isolated in pure culture and tested for biochemical activities using Polymicrotest. The ability of bacteria to degrade and utilize the algal polysaccharide was investigated. The bacteria were grown in a media containing Rhodella polysaccharide as a sole carbon source. The level of the reducing sugars in the culture media was determined. Scanning electron microscopy (SEM) was used to define the location of bacteria in extensively and intensively cultivated Rhodella reticulata previously infected by Cytophaga sp. LR2. CONCLUSIONS: The lysis of Rhodella reticulata cells is due to the joint action of the three bacterial strains with the former pathogen Cytophaga sp. LR2 playing the main role. The accumulation of the polysaccharide and the excreted metabolites of the strains LR1 and LR3 stimulated the development of Cytophaga sp. LR2. The adaptation of the strain to particular conditions of alga cultivation and the utilization of polysaccharide as a sole carbon source supported its stable growth in alga suspension and destruction of Rhodella cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The predominance of Cytophaga sp. LR2 over the two other contaminants and the lysis of Rhodella reticulata cells resulted from the ability of the bacterium to attach to the algal polysaccharide sheath. The formation of slime and extrusions facilitated the phenomenon of bacterial adhesion to the algal surface as well as the formation of colonial alga - bacterial spherules. The sedimentation of these aggregates decreased the ability of the algal strain to photosynthesize, led to the lysis of the cells and finally caused the death of Rhodella.