Study on Separation of Phycoerythrin by Q-Sepharose Fast Flow

[Objective] This study aimed to establish an efficient process for separation of phycoerythrin by using Q Sepharose Fast Flow resin and verity its feasibility for scale-up. [Method] Elution gradient, sample volume and flow rate were optimized to determine the optimal separation condition, under which the scale-up process was verified. [Result] The optimal condition for separation of phycoerythrin by using Q Sepharose FF resin was investigated： 30 ml of laver extract was loaded to the Q Sepharose FF column with a bed volume of 8 ml; subsequently, the column was stepwise eluted with 0-0.10-0.35-1.00 mol/L NaCI solution （pH 6.0） at a constant flow rate of 1 ml/min; the elution peak under 0.35 mol/L NaCI solution was collected, and the recovery rate and purity coefficient （A565/A280） of phycoerythrin were determined as 44.3 and 1.15, respectively. Based on the established process, 75 ml of phycoerythrin extract was loaded to the Q Sepharose FF column with a bed volume of 20 ml for separation, while no significant variation was observed in the separation result. [Conclusion] Phycoerythrin can be well separated from laver extract by using Q Sepharose FF resin and the process is feasible for scale-up.

CLONING AND ANALYSIS OF PHYCOERYTHRIN GENES IN GRACILARIA LEMANEIFORMIS (RHODOPHYCEAE)

Cloning and sequencing of the genes coding for the α and β subunit of phycoerythrin (PE) of a red alga- Gracilaria lemaneiformis (GL) are reported. Alignment of 1084 nucleotides sequenced with three known red algal PE genes, Rhodella violacea (RV), Polysiphonia boldii (PB) and Aglaothamnion neglectum (AN), showed high level of conservation, and similarities of 77.6% (between GL and RV), 77.9% (GL and AN) and 79.0% (GL and PB). The similarities of amino acids were 84.8% (between GL and RV), 85.7% (GL and PB), and 80.6% (AN and GL), higher than those among nucleotides.

Purification and Photodynamic Bioactivity of Phycoerythrin and Phycocyanin from Porphyra yezoensis Ueda

Phycoerythrin and phycocyanin were purified from Porphyra yezoensis Ueda with their bioactivity determined in this study. Continuous precipitation with ammonium sulfate at different concentrations(10%, 20%, 40% and 50%) increased the purity(A564:A280) of phycoerythrin to 1.49, 3.92 fold of the raw extract(0.38) and the purity(A615:A280) of phycocyanin to 0.70, 3.33 fold of the raw extract(0.21). Two more times of chromatography with hydroxylapatites finally made the purity of phycoerythrin and phycocyanin reach 5.50, 14.47 fold of the raw extract, and 5.10, 24.29 fold of the raw extract, respectviely. The yield of high purity phycoerythrin and phycocyanin were 0.21% and 0.09% of dried P. yezoensis blade, respectively. The photodynamic cytotoxic experiment showed that both phycoerythrin and phycocyanin inhibited the growth of liver tumor cells significantly. It was found that 250 mg L-1 purified phycoerythrin and phycocyanin inhibited the growth of hepatocellular carcinoma cells 24 h after laser-irradiation by 80% and 59%, respectively, and 100 mg L-1 purified phycoerythrin and phycocyanin induced the apoptosis of 31.54% and 32.54% of the cells, respectively, 8 h after photodynamic therapy. Oue findings demonstrated that P. yezoensis can serve as photosensitizer(phycoerythrin and phycocyanin) producer.

Effects of Different Extraction and Purification Methods on the Acquisition of Phycoerythrin from Porphyridium purpureum

Phycoerythrin, as the main light-harvesting antenna in Porphyridium purpureum, exists at the outermost end of the phycobilisome. It has advantages of good fluorescence intensity, anti-oxidation, scavenging free radicals, and high chroma, so it has been widely used in food, cosmetics, pharmaceuticals, and other industries. In this study, the effects of different extraction(ultrasonic breaking method, bead grinding method, liquid nitrogen grinding method, and freezing-thawing method) and purification methods(salting out method, ultrafiltration method, and combination of salting out and ultrafiltration method) on the acquisition of phycoerythrin from P. purpureum were studied, and the characteristics of phycoerythrin in the P. purpureum were identified. The results showed that the freezing-thawing method could extract phycoerythrin from the powder of P. purpureum to the utmost extent, and the concentration of the extracted phycoerythrin was up to 0.036 g/L. The salting out method could most effectively purify phycoerythrin, and the purity index was 2.216. The identification of phycoerythrin by ultraviolet absorption spectroscopy and fluorescence spectroscopy indicated that the phycoerythrin had the maximum absorption peak at 545 nm, and the maximum Stokes shift was up to 79 nm. Due to its high fluorescence characteristics, it can be used as a fluorescent marker in the fields of molecular biology and clinical medicine, and can also be used as a good photosensitizer in tumor therapy.

R-phycoerythrin标记抗猪瘟病毒荧光抗体的制备及其与FITC标记荧光抗体检测的比较研究

用摩尔比100∶1的化学交联剂SPDP分别将RPE、抗猪瘟病毒抗体衍生,两种衍生物再以摩尔比1∶1液相交联,HPLC纯化制备出RPE标记的抗猪瘟病毒荧光抗体。对制备的荧光抗体从荧光特性、抗体活性、稳定性及纯度等方面进行鉴定。以溴化氰活化的琼脂糖微球为固相载体,采用固相免疫荧光检测法,以制备的RPE、FITC分别标记的荧光抗体检测猪瘟病毒细胞毒。结果表明,RPE标记的荧光抗体检测阳性微球在蓝绿光激发下发射桔黄色荧光,荧光明亮,无本底光干扰,检测灵敏度高达2.67×10-6mg.mL-1,是FITC标记的荧光抗体检测灵敏度的10倍。RPE可替代FITC或与FITC等荧光染料一起用于多色荧光免疫检测。

Temporal and Spatial Variations of Abundance of Phycocyaninand Phycoerythrin-Rich Synechococcus in Pearl River Estuary and Adjacent Coastal Area

Three surveys were carried out in Pearl River Estuary and adjacent coastal area in May, August, and November, 2013, to investigate the temporal and spatial variations of abundance of phycoerythrin-rich Synechococcus(PE-rich SYN) and phycocyanin-rich Synechococcus(PC-rich SYN). The effects of environmental factors on the alternation of the different Synechococcus groups were also elucidated. PE-rich SYN was detected in three surveys, whereas PC-rich SYN was detected in May and August, but not in November. The highest abundances of PE-rich SYN and PC-rich SYN were recorded in August and May, with mean values of 74.17×103 and 189.92×103 cells m L-1, respectively. From May to November, the relative abundance of PE-rich SYN increased, whereas that of PC-rich SYN declined. PE-rich and PC-rich SYN presented similar horizontal distributions with high abundance in the southern estuary in May, and in the western estuary in August. The abundances of PE-rich and PC-rich SYN were high at 27–32℃and salinity of 10–20. PC-rich SYN was not detected at < 24℃, and PC:PE-rich SYN decreased in abundance with salinity increase. When less than 20 mg L-1, suspended particulate matter(SPM) was helpful for Synechococcus growth. PE-rich SYN decreased in abundance when the concentration of dissolved inorganic nitrogen increased in May and November, and the concentration of phosphate increased in November. However, PC-rich SYN abundance and nutrients showed no correlation. Principal component analysis and regression analysis indicated that PE-rich SYN significantly correlated with the principal components that were affected by environmental factors.

Isolation, properties and spatial site analysis of γ subunits of B-phycoerythrin and R-phycoerythrin

Polysiphonia urceolata R phycoerythrin and Porphyridium cruentum B phycoerythrin were degraded with proteinaseK, and then the nearly native γ subunits were isolated from the reaction mixture. The process of degradation of phycoerythrin with proteinaseK showed that the γ subunit is located in the central cavity of (αβ) 6 hexamer of phycoerythrin. Comparative analysis of the spectra of the native phycoerythrin, the phycoerythrin at pH 12 and the isolated γ subunit showed that the absorption peaks of phycoerythrobilins on α or β subunit are at 535 nm (or 545 nm) and 565 nm, the fluorescence emission maximum at 580 nm; the absorption peak of phycoerythrobilins on the isolated γ subunit is at 589 nm, the fluorescence emission peak at 620 nm which overlaps the absorption maximum of C phycocyanin and perhaps contributes to the energy transfer with high efficiency between phycoerythrin and phycocyanin in phycobilisome; the absorption maximum of phycourobilin on the isolated γ subunit is at 498 nm, which is the same as that in native phycoerythrin, and the fluorescence emission maximum at 575 nm.

Langmuir-Blodgett films of phycobiliproteins (I)——Monomolecular film and conformational studies of R-phycoerythrin

The surface pressure-area (π-A) isotherm of R-phycoerythrin (R-PE) at the air-water interface has been measured. The results indicate that R-PE can form the monomolecular film. Moreover, the molecule-occupied area extrapolating the linear part of the n-A isotherm is identical with that when an R-PE molecule is located at the interface with its disk plane parallel to the air-water interface. The transmission electron micrograph (TEM) and the measurement of the thickness of the protein monolayer by ellipsometry show that the orientation of R-PE disk plane on the substrate is parallel to the plane of substrate. Absorption and fluorescence spectra of R-PE LB multilayers were obtained through transferring R-PE monolayer at the air-water interface to the substrates at the proper surface pressure by Langmuir-Blodgett (LB) technique. These spectra of R-PE LB films do not show distinct differences from those in aqueous solution. Comparative studies of circular dichroism (CD) spectra of the protein between in aqueous solution and in LB film show that the changes of the secondary structure of R-PE take place, i.e. the β-sheet component of the protein increases in the LB films.

Spectra of excitons in R-phycoerythrin microcrystals

The antenna system of photosynthetic organisms serves to absorb light energy andtransfer excitation energy to the reaction centers. Energy transfer in the antenna system hasbeen found to be very efficient and rapid. The overall transfer efficiency exceeds 90％, andthe whole process of energy transfer from the phycoerythrin (PE) to the reaction center iscompleted within 50 ps in intact cells of red algae and cyanobacteria. Phycobilisome. the important photosynthetic antenna in red algae and cyanobacteria,consists of antenna polypeptide rods and core. One side of the core attaches to thethylakoid membrane and connects with the reaction center. the other side connects with

Langmuir-Blodgett films of phycobiliproteins (II) --Photoelectrochemical property of R-phycoerythrin

Through an electrochemical cell deposited with R-phycoerythrin (R-PE) monolayer on SnO2 optically transparent electrode (SnO2 OTE), charge transport phenomenon and the photoelectrochemical behavior of R-PE have been investigated. The experimental results indicate that the cell is able to generate photocurrent; moreover, the signal increases apparently in the presence of electron donor or acceptor in the electrolyte solution, showing that the photocurrent of R-PE would originate from its charge transfer. Further comparative test showed that the photocurrent came from the photo-induced charge separation property of the chromorphores attached covalently to the apoprotein of R-PE. The photocurrent spectrum of R-PE LB films verified the above viewpoint, from which the mechanism of photo-induced charge transfer of R-PE is suggested. The quantum yield for photoelectric conversion of R-PE LB films was measured to be φ520nm=3.4% and the photovoltage approached 400 mV. Moreover, the protein is stable for a long irradiation time. In addition, the effects of pH value of the electrolyte solution, the concentration of electron donor, the number of layers of R-PE LB films and the irradiation time on the photocurrent are studied.

Cultivation of Anabena variabilis,Synechococcus elongatus,Spirulina platensis for the production of C-Phycocyanin,C-Phycoerythrin and Thalassiosira,Skeletonema,Chaetoceros for fucoxanthin

The pigments are vital parts of photosynthetic machinery in algae and exhibit a myriad of applications as nutraceuticals,cosmetics,colorants,which find huge applications in many industires,including the paint industry and paper industry.The antimicrobial,antioxidative,anti-inflammatory,and anti-cancer potential of microalgal pigments contribute towards their varied industrial applications.In this study,pigments(Fucoxanthin,C-phycocyanin,and C-phycoerythrin),extracted from microalgae were investigated to check the antimicrobial efficacy using agar well diffusion and disc diffusion methods against pathogenic bacteria:Staphylococcus aureus and Escherichia coli.The fucoxanthin pigment isolated from diatom Thalassiosira sp.exhibited the highest antibacterial activity against Staphylococcus aureus(17±1.53 mm)and in contrast fucoxanthin pigment isolated from Chaetoceroes sp.exhibited the highest bactericidal activity against Escherichia coli(18±1).The pigment extract of Spirulina plantesis showed the highest antibacterial activity against E.coli(41±0.3)and S.elongatus also exhibited high antibacterial activity against E.coli(32±0.5)while Anabaena variabilis showed the highest antibacterial activity against Staphylococcus aureus(34±0.5).The potential of microalgal pigments is highly valuable and further extensive studies can elaborate the underlying mechanisms.

Isolation of Picocyanobacteria (Order Synechococcales) and Occurrence of the Cyanotoxin Anatoxin-A in a Shallow Mesotrophic Pond

We have verified the use of a serial filtration method to isolate picocyanobacteria for analysis. We used eDNA metabarcoding to confirm the picocyanobacteria as members of the Order Synechococcales, Genus Cyanobium, specifically Cyanobium 6307. Fluorometric analysis using accessory pigments phycocyanin and phycoerythrin described periods of excess biomass, where the net growth rate model confirmed these conditions. The total anatoxin-a concentrations in the picocyanobacterial sample ranged from 0.0074 - 6.41 μg·L-1 representing a 40-fold difference over the entire sampling season. Sampling frequency of every three days appeared to be an important factor in capturing these changes in anatoxin-a concentration. During a period of excess biomass, we were able to establish a linear correlation between cyanobacterial biomass and Anatoxin-a concentrations.

Isolation of Picocyanobacteria (Order Synechococcales) and Occurrence of Cyanotoxins (Anatoxin-a) in Saline Microhabitats at Martha’s Vineyard, MA

We have used serial filtration to isolate picocyanobacteria from brackish and marine microhabitats for analysis. We used 16s metabarcoding to confirm the picocyanobacteria as members of the Order Synechococcales, Genus Cyanobium 6307 (Upper Chilmark Pond) and differing abundances of Cyanobium 6307 and Synechococcus 9902 (Chilmark Pond, Edgartown Great Pond, Tisbury Great Pond and Tashmoo Pond). The proportion and composition of (pico)cyanobacteria in water samples were influenced by the salinity concentrations at various sites, as evidenced by fluorometry and 16s metabarcoding analysis. The cyanobacterial neurotoxin anatoxin-a was present in the picocyanobacterial samples from all studied sites. Additional analyses using fluorometry and 16s metabarcoding described members of the Order Nostocales, including a halotolerant population of Dolichospermum sp., Sphaerospermopsis spp. and Nodularia spp. in Upper Chilmark Pond. We were able to establish a positive linear correlation between cyanobacterial biomass (phycocyanin) and anatoxin-a concentrations using samples taken from Upper Chilmark Pond.

Comparison of Phycobiliproteins from Gracilaria lemaneiformis (Rhodophyceae) and Its Pigment Mutants in Spectral and Molecular Respects

Comparative studies of absorption spectra of phycobiliproteins of Gracilaria lemaneiformis Greville and its pigmental mutants were conducted in this study. The results showed that the absorption spectra of phycoerythrins ( PE) from different material changed significantly, while those of phycocyanins (PC) and allophycocyanins (APC) were basically similar. In order to disclose the essence of die difference, partial sequences of die subunit genes of PE of Qingdao strain of G. lemaneiformis (qd) and its pigmental mutants were determined. The amino acid sequences were deduced and used to explain spectral shifts of PE from the pigmental mutants. The amino acid sequences of PE resembled each other, and several residues changed among qd and its pigmental mutants. Residue substitutions were found in a region consisting of amino acids which determined are secondary structure and subunits interactions, thus might influence the confirmation and interaction of subunits, and further caused spectral deviation.

响应面法优化复合酶法提取紫菜藻红蛋白工艺

研究复合酶法提取紫菜藻红蛋白的最优工艺,筛选了复合酶的配比,并选取酶解温度、提取时间、加酶量和p H值为自变量,采用响应面法研究各因素及其交互作用对紫菜藻红蛋白得率和纯度的影响。结果表明,紫菜藻红蛋白复合酶法提取的最优酶配比为纤维素酶和果胶酶质量比7∶3,通过响应面优化,确定藻红蛋白最优提取工艺为:p H?6.8、酶解温度39℃、提取时间7.2?h、在5?g样品中加酶0.04?g。在此条件下藻红蛋白得率为2.257%,纯度达到1.656。经过验证,所得工艺参数准确,可用于复合酶法生产紫菜藻红蛋白。

藻红蛋白介导的光敏反应可诱导人肝癌7721细胞凋亡

目的 :探讨藻红蛋白介导的光动力效应对肝癌细胞的杀伤机制是否与诱导瘤细胞凋亡有关。方法 :对培养的人肝癌细胞进行光动力治疗处理 ,通过观察DNALadders的形成和形态学改变 ,判断凋亡的发生与否。结果 :可观察到在经过光动力治疗处理一定时间后 ,772 1细胞出现典型的凋亡形态学改变 ,同时伴有特征性的DNALadders出现。结论 :藻红蛋白介导的光敏反应杀伤肿瘤细胞与诱导人肝癌细胞凋亡相关 ,是一种很有前景的光动力药物。

紫球藻及其应用研究

紫球藻是一种有较大经济价值的海洋单细胞红藻,其生长过程中合成的藻红蛋白、高不饱和脂肪酸和紫球藻多糖等多种生物活性产物具有广阔的应用前景及较大的潜在市场。国外有关学者已经对紫球藻进行了相关的开发研究并取得了良好的进展,但国内在该领域的研究较少.本文综述了紫球藻的生物学和培养特性、经济价值和开发前景等相关问题,以期引起国内有关学者对紫球藻的研究和开发兴趣。

R-藻红蛋白光动力杀伤的形态学机制研究

在荧光显微镜下观察新型光敏剂Ｒ藻红蛋白的荧光在肿瘤细胞中的分布，探讨以藻红蛋白为光敏药物的光动力损伤可能发生的细胞水平机制，以及培养液酸度和藻红蛋白浓度对其与细胞结合、进入及光动力杀伤的关系．结果表明在ｐＨ５ 时，藻红蛋白与瘤细胞有最好的结合．其结合部位随藻红蛋白浓度的增加，逐渐以分布在胞膜、胞浆和胞核为主，表现为一个动态进入细胞的过程．光动力杀伤作用与其进入细胞的数量成正相关，从形态学上证明了Ｒ藻红蛋白介导的光动力治疗作用与藻红蛋白进入瘤细胞状态紧密相关．

龙须菜藻胆蛋白抗突变与抗肿瘤作用的研究

初步观察了龙须菜藻红蛋白(PE)粗提物对环磷酰胺(CP)诱发小鼠染色体突变的抑制作用和对荷S180实体瘤小鼠的抑瘤作用。试验结果表明,藻红蛋白粗提物可使环磷酰胺诱发的小鼠骨髓嗜多染红细胞(PCE)微核率明显降低,具有显著的抗突变作用,且呈现一定的剂量效应关系;其对小鼠S180实体瘤的抑瘤率为44%。

紫球藻藻红蛋白的分离纯化及光谱特性研究

紫球藻（Ｐｏｒｐｈｙｒｉｄｉｕｍ ｃｒｕｅｎｔｕｍ）的水溶性色素粗提物经过固体硫酸铵沉淀和羟基磷灰石（ＨＡ）柱层析后，可以分离出较纯的Ｂ－藻红蛋白（Ｂ－ＰＥ），纯度（ＯＤ５４５ｎｍ／ＯＤ２８０ｎｍ）为３．０。测定了Ｂ－ＰＥ的紫外可见光吸收光谱和荧光光谱以及紫球藻活体吸收光谱。Ｂ－ＰＥ在５４５ｎｍ和５６３ｎｍ各有一个峰，在４９８ｎｍ有一肩峰，与活体吸收光谱相比较只是最大吸收峰向短波方向移动了５ｎｍ。激发光谱在５５３～５６７ｎｍ有一峰，在５００ｎｍ和５３６ｎｍ各有一肩峰，室温荧光发射峰在５７８ｎｍ左右。