基于蛋白质组学对螺旋藻砷胁迫响应机制的研究

通过研究螺旋藻(Spirulina sp.)在砷离子胁迫下的蛋白质组变化,从蛋白质表达水平解释螺旋藻对砷离子胁迫的响应机理。螺旋藻经过不同浓度砷离子胁迫7 d后,提取蛋白质进行凝胶电泳,并对差异蛋白进行质谱分析。结果表明螺旋藻在2.0 ppm砷酸盐中暴露10 min光合放氧速率降低27.3%,培养24 h后细胞内的金属硫蛋白、叶绿素、类胡萝卜素及藻胆蛋白相对含量均明显降低。蛋白组学共鉴定出75个差异蛋白,其中26个显著上调,49个呈现下调。这些差异蛋白表明砷离子主要通过破坏螺旋藻光合色素蛋白,干扰电子传递过程,导致能量合成受损,使得依赖光合作用产生能量进行的跨膜运动、蛋白质合成等相关过程受到影响;同时,活性氧清除与防御相关蛋白呈现上调,螺旋藻细胞内抗氧化系统被激活。     

Purification of C-phycocyanin from Spirulina (Arthrospira) fusiformis

C-phycocyanin was purified from Spirulina (Arthrospira) fusiformis by a multi-step treatment of the crude extract with rivanol in a ratio 10:1 (v/v), followed by 40% saturation with ammonium sulfate. After removal of rivanol by gel-filtration on Sephadex G-25, the pigment solution was saturated to 70% with ammonium sulfate. After the last step of purification, C-phycocyanin had an emission and absorption maxima at 620 and 650 nm, respectively and absorbance ratio A(620)/A(280) of 4.3, which are specific for the pure biliprotein. Its homogeneity was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, yielding two bands of molecular masses 19500 and 21500 kDa, corresponding to alpha and beta subunits of the pigment, respectively. The yield of C-phycocyanin was approximately 46% from its content in the crude extract.     

低温胁迫对螺旋藻脱氢酶活性的影响

低温胁迫试验结果表明,处理的温度越低、天数越长,鄂尔多斯高原碱湖的钝顶螺旋藻S1、引进的钝顶螺旋藻S2和极大螺旋藻S3细胞内脱氢酶活性越低。在每一种处理下,脱氧酶活性高低的排列顺序均为S1〉S2〉S3。脱氢酶活性的Q10值变化范围排列为S1〈S2〈S3。S1的生理活动临界低温为5℃,S2和S3的为15℃。S1对低温有较强的适应性,为耐寒植物;S2和S3为冷敏感植物。     

环境因子与螺旋藻(节旋藻)分布及丰度的相关性分析

【目的】2015年7月至2015年9月,对海岸线较长、湿地类型比较多样化的渤海湿地及周边水域中螺旋藻(节旋藻)的分布进行研究,并对其与环境因子的相互关系进行分析。【方法】采用相关分析和灰关联分析对螺旋藻(节旋藻)数量与环境因子关系进行分析。【结果】螺旋藻(节旋藻)数量与pH、盐度、总氮(TN)、总磷(TP)、化学需氧量(COD)、氨氮(NH_3-N)呈显著正相关(P0.05,P0.01),与溶解氧(DO)呈显著负相关(P0.05,P0.01),与温度呈负相关,但相关不显著(P0.05);灰关联分析结果表明:各环境因素对螺旋藻数量的关联程度依次为:CODpHTN温度TPDO盐度NH_3-N。【结论】渤海海滨湿地及周边水域中螺旋藻(节旋藻)资源丰富,对螺旋藻(节旋藻)分布影响最大的环境因子是COD,最小是NH_3-N。     

The Temperature Dependent Proteomic Analysis of Thermotoga maritima

Thermotoga maritima (T. maritima) is a typical thermophile, and its proteome response to environmental temperature changes has yet to be explored. This study aims to uncover the temperature-dependent proteins of T. maritima using comparative proteomic approach. T. maritima was cultured under four temperatures, 60°C, 70°C, 80°C and 90°C, and the bacterial proteins were extracted and electrophoresed in two-dimensional mode. After analysis of gel images, a total of 224 spots, either cytoplasm or membrane, were defined as temperature-dependent. Of these spots, 75 unique bacterial proteins were identified using MALDI TOF/TOF MS. As is well known, the chaperone proteins such as heat shock protein 60 and elongation factor Tu, were up-regulated in abundance due to increased temperature. However, several temperature-dependent proteins of T. maritima responded very differently when compared to responses of the thermophile T. tengcongensis. Intriguingly, a number of proteins involved in central carbohydrate metabolism were significantly up-regulated at higher temperature. Their corresponding mRNA levels were elevated accordingly. The increase in abundance of several key enzymes indicates that a number of central carbohydrate metabolism pathways of T. maritima are activated at higher temperatures.     

螺旋藻乙醇酸氧化酶(GO)的研究

采用比色法对鄂尔多斯高原碱湖的钝顶螺旋藻(S1)与国外引进的钝顶螺旋藻(S2)和极大螺旋藻(S3)的乙醇酸氧化酶(GO)进行了比较研究。结果表明:在25℃、pH 8.0条件下,S1、S2和S3的GO活性分别为70.9 U/gFW、59.6 U/gFW和80.9 U/gFW;最适温度均为30℃;在0℃~35℃(30℃)范围内比较稳定;最适pH值分别为8.6、8.2和8.4;pH值稳定范围,S1为7.6~10.0、S2为8.0~9.0;S3为8.0~8.6。S1的GO对温度和pH适应范围最宽,且在低温、高温、强酸和强碱下的活性均比引进种的高。     

Analysis of the promoter region of the RuBisCO gene of Arthrospira (Spirulina) platensis

Ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO), a key enzyme involved with photosynthetic carbon assimilation, catalyzes the carboxylation and oxidization of ribulose-1,5-bisphosphate. Interestingly, the promoter region of this gene from Arthrospira platensis could drive the expression of a downstream gene in Escherichia coli. In this study, using green fluorescent protein as a reporter of gene expression, the structure and function of the promoter region of the RuBisCO gene of A. platensis was analyzed. There are three hypothetical promoter elements predicted in the 200 bp upstream of the open reading frame (ORF) of RuBisCO. Through deletion analysis of the promoter, it was demonstrated that one of these elements was the active promoter, which was located between ?94 and the ORF of RuBisCO. The ?35 box of the RuBisCO promoter (TTGACT) was very similar to that of the rpoD gene (TTGACA) of E. coli, with only the sixth nucleotide divergent. Site-directed mutational analysis showed that when the sixth nucleotide (T) was changed to A, the activity of the promoter remained unchanged. However, when the first and second Ts were mutated, the activities of the promoters decreased drastically. Determining the structure and function of promoters would help elucidate the molecular mechanisms of the gene expression and regulation.     

Physiological behaviour of Arthrospira (Spirulina) maxima during acclimation to changes in irradiance

The physiological behaviour of Arthrospira (Spirulina) maxima during acclimation to sudden changes in irradiance from high (HL) to low light (LL) and vice versa was studied by following parameters concerning growth rate, pigment, carbohydrate and protein cell contents. Applying first order kinetics, the specific acclimation rates for the parameters considered were calculated. During HL to LL shift, pigments increased to compensate for a reduction in growth irradiance in order to maintain relatively high growth rates, whereas carbohydrates decreased at the highest rate. The synthesis of phycobiliproteins proceeded at a rate similar or little higher than that of chlorophyll a, indicating their importance in the light harvesting at low irradiance. During LL to HL shift, carbohydrate biosynthesis was increased, whereas pigment and protein cell contents decreased. The kinetic analysis suggested that the pigment decrease could be accounted for both by dilution through growth and in vivo degradation. During this transition, the initially high cell pigment content gave rise to a very heavy carbohydrate synthesis, which for a short time, after the shift to HL conditions, overshot the final steady-state. In the same period the specific growth rate also increased notably, overshooting the μmax. The acclimation rates of the measured parameters were faster during LL to HL transition then during the reverse. The physiological response of A. maxima during the acclimation to sudden irradiance shifts points out the ability of this cyanobacterium to alter light harvesting and highlights again the key role of carbohydrates when the cells underwent an energy crisis during down-shift. This revised version was published online in August 2006 with corrections to the Cover Date.     

低温对螺旋藻可溶性蛋白和游离氨基酸外渗的影响

以鄂尔多斯高原碱湖特有的钝顶螺旋藻S1、引进的钝顶螺旋藻S2和极大螺旋藻S3为材料,采用Folin法和茚三酮法分别测定低温下细胞外渗液中可溶性蛋白和游离氮基酸含量。结果表明：处理温度越低,时间越长,螺旋藻细胞外渗液中可溶性蛋白和游离氨基酸的含量越高。S1膜损伤程度最轻,比引进种S2、S3有较强的抗寒能力。判断低温对螺旋藻膜损伤的程度,用可溶性蛋白较游离氨基酸更接近于实际。     

Arthrospira (Spirulina) platensis: An effective biosorbent for nutrients

Arthrospira (Spirulina) platensis was tested for biosorption properties. Preliminary experiments concerning biosorption kinetics were performed on Cr(III) ions. Equilibrium of biosorption was tested for Cr(III), Mn(II) and Mg(II) ions, since these elements are crucial for animals with metabolic disorders. In our study, Spirulina was proposed as a feed additive for animals suffering from diseases characterized by insulin dysregulation, abnormal adipose distribution and a high risk for laminitis. Maximum biosorption capacity of A. platensis, determined from Langmuir equation, was 45.2 for Cr(III), 44.3 for Mn(II) and 42.0 mg/g for Mg(II) ions. Biosorption of Mg(II) ions by microalga has never been studied so far. Finally, the raw and enriched microalgal biomass was examined by ICP-OES to determine its multielamental analysis before and after biosorption, FTIR to indicate functional groups that participated in biosorption and SEM-EDX to illustrate the binding of metal ions on the surface of algal biomass. ICP-OES showed that the content of elements significantly increased in the enriched A. platensis. FTIR spectroscopy evidenced that biosorption of metal ions was mainly due to carboxylate groups present on the microalgal cell wall. SEM analysis clearly showed that biosorption occurred. Arthrospira platensis turned out to be a good biosorbent of metal ions.     

Factors indicating culture status during cultivation of Spirulina (Arthrospira) platensis

Factors indicating culture status of two Spirulina platensis strains were monitored in a batch mode cultivation for 36 days. Changing mode in all factors showed a common turning point, indicating shift of cell or culture status. Mean biomass productivity was highly sustained until day 22, chlorophyll a concentration peaked on day 22, pH value was >12 on day 22, coil number was abruptly shortened on day 22, and floating activity was sustained at greater than 79% after day 22, indicating that day 22 is a criterion reflecting phase-transfer in cell physiology in a batch culture system. Many of these changes may have been caused by increased pH, suggesting that pH control is essential for mass production of S. platensis. Fluctuations in floating activity were likely induced by the number of cellular gas vacuoles. Consequently, coil number per trichome and floating activity of S. platensis could readily act as simple indicators for determination of culture status or harvesting time of cells.     

Spirulina (Arthrospira): potential application as an animal feed supplement

Spirulina has been used as human food supplement for over 20 years, but its use as an animal feed supplement is relatively recent. This paper reviews earlier studies on its more established effects on growth, survival and tissue quality in a whole range of animals and more recent studies on its immunomodulatory, anti-viral and anti-cancer effects. The basis for potential application as an animal feed supplement is discussed.     

Simultaneous use of urea and potassium nitrate for Arthrospira (Spirulina) platensis cultivation

Urea has been considered as a promising alternative nitrogen source for the cultivation of Arthrospira platensis if it is possible to avoid ammonia toxicity; however, this procedure can lead to periods of nitrogen shortage. This study shows that the addition of potassium nitrate, which acts as a nitrogen reservoir, to cultivations carried out with urea in a fed-batch process can increase the maximum cell concentration (X(m) ) and also cell productivity (P(X) ). Using response surface methodology, the model indicates that the estimated optimum X(m) can be achieved with 17.3 mM potassium nitrate and 8.9 mM urea. Under this condition an X(m) of 6077 ± 199 mg/L and a P(X) of 341.5 ± 19.1 mg L(-1) day(-1) were obtained.     

First description of a cyanophage infecting the cyanobacterium Arthrospira platensis (Spirulina)

Cyanobacteria constitute a versatile group of photosynthetic bacteria of immense commercial and ecological importance. Some species of this group are cultivated and sold as food because of their high nutritional value. This is typically the case for Arthrospira platensis. We describe, for the first time, a virus infecting this economically important filamentous cyanobacterium isolated from culture pools located in the South of France. This virus could be observed and discriminated easily from other particles with flow cytometry. Based on morphology and molecular investigation, it was proposed that the virus belongs to the cyanopodovirus group with a capsid and short tail of about 120 and 20 nm, respectively. Finally, the virus appeared to be highly specific (very narrow host range) to A. platensis.     

Zinc removal from model solution and wastewater by Arthrospira (Spirulina) Platensis biomass

The biosorption of zinc from model solution as well as wastewater by Arthrospira (Spirulina) platensis biomass was studied. Adsorption capacity of the biosorbent was investigated as a function of contact time between adsorbent and zinc, the initial metals and sorbent concentration, pH value, and temperature. The ability of Arthrospira biomass for zinc biosorption exhibited a maximum at the pH range 4–8. Equilibrium data fitted well with the Langmuir model as well as the Freundlich model with maximum adsorption capacity of 7.1 mg/g. The pseudo second-order model was found to correlate well with the experimental data. Different thermodynamic parameters, ΔG°, ΔH° and ΔS° were evaluated and it has been found that the sorption was feasible, spontaneous, and endothermic in nature. The process of zinc removal from industrial effluent was studied at different time of sorbat–sorbent interaction and different sorbent dosage. Maximum zinc removal (83%) was obtained at sorbent concentration 60 g/L during 1-h experiment. The results indicate that Arthrospira platensis biomass could be effectively used for zinc removal from industrial effluents.     

Photosynthetic and respiratory electron transport in the alkaliphilic cyanobacterium Arthrospira (Spirulina) platensis

Photosynthetic and respiratory electron transport and their interplay with ion transport have been studied in Arthrospira platensis, a filamentous alkaliphilic cyanobacterium living in hypersaline lakes. As typical for alkaliphiles, A. platensis apparently does not maintain an outward positive pH gradient at its plasma membrane. Accordingly, sodium extrusion occurs via an ATP-dependent primary sodium pump, in contrast to the Na⁺/H⁺ antiport in most cyanobacteria. A. platensis is strongly dependent on sodium/bicarbonate symport for the uptake of inorganic carbon. Sodium extrusion in the presence of the Photosystem II inhibitor diuron indicates that a significant amount of ATP is supplied by cyclic electron transport around Photosystem I, the content of which in A. platensis is exceptionally high. Plastoquinol is oxidized by two parallel pathways, via the cytochrome b ₆ f complex and a putative cytochrome bd complex, both of which are active in the light and in the dark. This revised version was published online in August 2006 with corrections to the Cover Date.     

Polyhydroxyalkanoates and pigments coproduction by Arthrospira (Spirulina) platensis cultivated in crude glycerol

Journal of Applied Phycology - Valorization of industrial waste as an alternative carbon source for microalgae mixotrophic cultivation and co-production of high-value compounds are strategies to...     

Characterization of the iron-containing superoxide dismutase and its response to stress in cyanobacterium Spirulina (Arthrospira) platensis

Superoxide dismutase (SOD) is considered a primary antioxidant which defends against reactive oxygen species that are induced by environmental stress. In this study, we examined changes in SOD activity and expression in the cyanobacterium Spirulina (Arthrospira) platensis under iron and salinity stress; we characterized its induction under these stress conditions and we overexpressed the enzyme in a bacterial host for preliminary characterization. Analysis of SOD isoforms concludes that S. platensis was found to regulate only the iron-containing SOD isoform (FeSOD) in response to two types of stress that were tested. The FeSOD expression (on the level of both mRNA and enzyme activity) was induced by the stress conditions of salinity and iron levels. The FeSOD from S. platensis was overexpressed in Escherichia coli BL21. The recombinant FeSOD protein (about 23 kDa) was purified for characterization. It showed high specific activity and pH stability at 6.0–9.0, and it is relatively thermostable, retaining 45 % of its activity after 30 min at 90 °C. Phylogenetic analysis reveals that S. platensis FeSOD is grouped with the FeSODs from other cyanobacterial species and separated from those of the eukaryotic Chlorophyta, suggesting that the FeSOD gene may be used as a molecular marker in physiological, phylogenetic, and taxonomic studies. This study also suggests that the increased activity and expression of SOD may play a role in algal survival under stress conditions.     

Purification and characterization of phycoferritin from the blue-green alga, Arthrospira (Spirulina) platensis

Phycoferritin from the nutritionally important blue-green alga Arthrospira platensis has been isolated, by application of conventional biochemical techniques. The molecular mass, yield, iron and total neutral carbohydrate contents of the purified protein were 470 kDa, 0.044 mg g⁻¹ of Arthrospira, 1.4 and 20%, respectively. The iron content was much lower when compared to bacterial and mammalian ferritins. The P: Fe ratio of Arthrospira phycoferritin was 1: 3.5, a value akin to bacterioferritins. Native gel-electrophoresis revealed the presence of isoforms. Subunit analysis by SDS-PAGE and Western blotting showed a protein subunit with an apparent molecular mass of 18 kDa. Oligomeric forms of the protein subunit were also present. The phycoferritin exhibited cross-reactivity with anti-pea seed ferritin suggesting phylogenetic relationship with that of higher plants. Carbohydrate analysis of phycoferritin by GC-MS revealed the presence of sugars such as galactose, glucose and mannose similar to that of mammalian ferritins. Interestingly, the analysis also revealed sugars such as rhamnose, xylose and talose, which has not been reported in the structure of ferritins. Except for very low histidine content in phycoferritin, the rest of the amino acid composition resembled to ferritins of other species. UV-visible spectral analysis of the phycoferritin revealed the presence of haem groups, a property characteristic of bacterioferritins. The fluorescence intensity of phycoferritin was higher than equine spleen ferritin. Circular dichroic spectra revealed a lower degree of helicity.