一步柱层析纯化螺旋藻藻蓝蛋白

采用硫酸铵盐析结合疏水层析技术分离纯化螺旋藻中的藻蓝蛋白.试验结果表明,在磷酸盐缓冲体系下藻蓝蛋白粗提液经1.25 mol/L硫酸铵盐析处理后离心脱气,只需采用一步Macro-Prep Methyl 疏水层析,藻蓝蛋白的纯度（A620/A280）可提高到4.017,回收率为19.38%.特征吸收峰和荧光光谱证实纯化后的产物符合藻蓝蛋白的性质,Native-PAGE电泳只出现单一染色带,表明纯化得到的藻蓝蛋白是均一的;SDS-PAGE电泳出现分子量为15.4 kDa、17.3 kDa的2条染色带,分别为藻蓝蛋白的α亚基与β亚基.     

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.     

Rapid and selective extraction of phycocyanin from Spirulina platensis with ultrasonic cell disruption

A study was conducted on the efficiency of phycocyanin extraction from Spirulina platensis (Arthrospira platensis) cells disrupted by ultrasonic irradiation. Extraction followed first-order kinetics with respect to the length of time for irradiation. The first-order rate constant increased linearly with the output of ultrasonic irradiation. In order to extract phycocyanin there was an appropriate range of ultrasonic frequency, f_u. In addition the most important finding is that the purity of phycocyanin in its crude extract depended on f_u. For example, phycocyanin was extracted with higher purity at f_u = 28 kHz than at f_u = 20 kHz. It is suggested that rapid and selective extraction of phycocyanin from S. platensis may be possible if an optimized ultrasonic application is developed for a given suspension.     

Strategy for a protein purification design using C-phycocyanin extract

A variety of techniques have been developed for the separation and recovery of proteins. The cost of purifying the product is frequently determined by the desired quality of the final product, which is evaluated by measuring the purity. In this work the design of a protein purification process for C-phycocyanin, a phycobiliprotein that can be used in the food and medical industries, was established. The study evaluated the use of ammonium sulfate precipitation, ion exchange chromatography and gel filtration to purify C-phycocyanin in a variety of sequences. The final design included the C-phycocyanin extraction step, precipitation with ammonium sulfate and ion exchange chromatography. When the elution step was studied, the kind of elution and pH were considered in order to obtain a product with a final purity of 4.0 with a purification factor of 6.35, so that, at the end of the strategy, C-phycocyanin of analytical grade would be obtained.     

Ammonium chloride: a novel effective and inexpensive salt solution for phycocyanin extraction from <Emphasis Type="BoldItalic">Arthrospira</Emphasis> (<Emphasis Type="BoldItalic">Spirulina</Emphasis>) <Emphasis Type="BoldItalic">platensis</Emphasis>

Phycocyanin, a blue pigment, is a type of phycobiliproteins. Because of its various potential properties, phycocyanin is applied to various fields, such as nutraceutical, pharmaceutical, medicine, cosmetics, and biotechnological research. The cost and application of phycocyanin are highly dependent on its purity index. In this study, ammonium chloride is presented as a novel, effective, and inexpensive salt for phycocyanin extraction. Compared with sodium phosphate, which is commonly used during phycocyanin extraction process, ammonium chloride solution efficiently extracted phycocyanin with high purity from Arthrospira platensis FACHB-314. In addition, ammonium phosphate solution is also presented as an alternative precipitation agent in phycocyanin purification that may replace the widely used ammonium sulfate. Statistical analysis shows that there is no significant difference in phycocyanin concentration between crude extracts (overall mean of 0.208 and 0.215 for extraction using sodium phosphate and ammonium chloride, respectively). However, the difference in phycocyanin purity ratio (A₆₂₀/A₂₈₀) between these two extractions is significant (overall mean of 0.742 and 1.428 for extraction using sodium phosphate and ammonium chloride, respectively). With ammonium chloride, the purity indexes of phycocyanin are 1.5 and 2.81 after the optimum extraction step, and precipitation used as the primary purification step, respectively. The present study describes a novel purification method to achieve phycocyanin with analytical grade without multiple purification steps.     

Large-scale recovery of C-phycocyanin from Spirulina platensis using expanded bed adsorption chromatography

C-phycocyanin was purified on a large scale by a combination of expanded bed adsorption, anion-exchange chromatography and hydroxyapatite chromatography from inferior Spirulina platensis that cannot be used for human consumption. First, phycobiliproteins were extracted by a simple, scaleable method and then were recovered by Phenyl-Sepharose chromatography in an expanded bed column. The purity (the A(620)/A(280) ratio) of C-phycocyanin isolated with STREAMLINE column was up to 2.87, and the yield was as high as 31 mg/g of dried S. platensis. After the first step, we used conventional anion-exchange chromatography for the purification steps, with a yield of 7.7 mg/g of dried S. platensis at a purity greater than 3.2 and with an A(620)/A(650) index higher than 5.0. The fractions from anion-exchange chromatography with a level of purity that did not conform to the above standard were subjected to hydroxyapatite chromatography, with a C-PC yield of 4.45 mg/g of dried S. platensis with a purity greater than 3.2. The protein from both purification methods showed one absolute absorption peak at 620 nm and a fluorescence maximum at 650 nm, which is consistent with the typical spectrum of C-phycocyanin. SDS-PAGE gave two bands corresponding to 21 and 18 kDa. In-gel digestion and LC-ESI-MS showed that the protein is C-phycocyanin.     

条斑紫菜藻红、藻蓝蛋白逐级放大的纯化工艺

采用“破碎-盐析-层析”的方法纯化条斑紫菜藻胆蛋白,并在提取规模上逐步放大。首先在综合比较凝胶层析去盐效率后,从Sephadex G-25、G-100、S-300和CL-6B中选择G-25作为实验流程中的去盐填料,其次将提取流程的初试原料条斑紫菜量逐步放大,选取了1g、20g和400g三个量,结果表明随着初试紫菜量逐步放大,最终所得藻胆蛋白中吸收光谱纯度>3.2的蛋白产率依次提高,其中400g冻干紫菜的藻红蛋白产率为0.323％,藻蓝蛋白产率为0.148％。由此认为该实验工艺流程具有规模放大的潜力,这为高纯度藻胆蛋白的规模生产提供了一条可行的方案。     

A novel method of single step hydrophobic interaction chromatography for the purification of phycocyanin from Phormidium fragile and its characterization for antioxidant property

Phycocyanin--a major phycobiliprotein constitutively produced by many cyanobacteria--holds several promising applications in diagnostics, biomedical research, and therapeutics. This paper discusses a novel rapid method for the purification of cyanobacterial phycocyanin (C-PC) from Phormidium fragile using hydrophobic interaction chromatography. The protein was extracted and concentrated by grinding under liquid nitrogen and ammonium sulfate fractionation. C-PC was purified by single step hydrophobic interaction chromatography. Purified phycocyanin showed absorbance maximum (lambda(max)) at 624 nm. The criterion of purity (R) achieved was 4.52. Phycocyanin to phycoerythrin and phycocyanin to allophycocyanin purity ratio were 3.85 and 7.49, respectively. The purified protein showed a pI of 5.2 and has two subunits with molecular mass of 19 and 20 kDa each, corresponding to its highly reported alpha and beta subunits. The subunits of phycocyanin were confirmed by their bilin fluorescence using zinc assisted fluorescence enhancement technique. Intact C-PC was of 125 kDa as determined by HPLC, suggested the (alphabeta)(3) subunit assembly. Results obtained by this method in terms of purity, recovery, process time, simplicity, and efficacy are much better than previous methodologies. Purified phycocyanin was further scrutinized for its antioxidant capacity and judged against five non-enzymatic antioxidants by FRAP assay.     

Potential Aqueous Two‐Phase Processes for the Primary Recovery of Colored Protein from Microbial Origin

The primary recovery of c‐phycocyanin and b‐phycoerythrin from Spirulina maxima and Porphyridium cruentum, respectively, using an established extraction strategy was selected as a practical model system to study the generic application of polyethylene glycol (PEG)‐phosphate aqueous two‐phase systems (ATPS). The generic practical implementation of ATPS extraction was evaluated for the recovery of colored proteins from microbial origin. A comparison of the influence of system parameters, such as PEG molecular mass, concentration of PEG as well as salt, system pH and volume ratio, on the partition behavior of c‐phycocyanin and b‐phycoerythrin was carried out to determine under which conditions target colored protein and contaminants concentrate to opposite phases. One‐stage processes are proposed for the primary recovery of the colored proteins. PEG1450‐phosphate ATPS extraction (volume ratio (V_R) equal to 0.3, tie‐line length (TLL) of 34 % w/w and system pH 7.0) for the recovery of c‐phycocyanin from Spirulina maxima resulted in a primary recovery process that produced a protein purity of 2.1 ± 0.2 (defined as the relationship of 620 nm to 280 nm absorbance) and a product yield of 98 % [w/w]. PEG1000‐phosphate ATPS extraction (i.e., V_R = 1.0, PEG 1000, TLL 50 % w/w and system pH 7.0) was preferred for the recovery of b‐phycoerythrin from Porphyridium cruentum, which resulted in a protein purity of 2.8 ± 0.2 (defined as the relationship of 545 nm to 280 nm absorbance) and a product yield of 82 % [w/w]. The purity of c‐phycocyanin and b‐phycoerythrin from the crude extract increased 3‐ and 4‐fold, respectively, after ATPS. The results reported herein demonstrated the benefits of the practical generic application of ATPS for the primary recovery of colored proteins from microbial origin as a first step for the development of purification processes.     

A large-scale purification of paclitaxel from cell cultures of Taxus chinensis

A large-scale purification method was developed for producing paclitaxel, to guarantee high purity and yield from plant cell cultures. The complete method for mass production was a simple and efficient procedure, for the isolation and purification of paclitaxel from the biomass of Taxus chinensis, consisting of solvent extraction, synthetic adsorbent treatment, and two steps of precipitation, followed by two steps of high performance liquid chromatography (HPLC). The organic solvent extraction of biomass obtained crude extract containing paclitaxel. The use of synthetic adsorbent treatment and precipitation in the prepurification process allows for rapid and efficient separation of paclitaxel from interfering compounds and dramatically increases the yield and purity of crude paclitaxel for HPLC purification steps compared to alternative processes. This prepurification process serves to minimise solvent usage, size, and complexity of the HPLC operations for paclitaxel purification. The paclitaxel of over 99.5% purity can be simply obtained with high yield from crude paclitaxel by HPLC using reverse-phase separation on C18 as the first step and normal-phase separation on silica as the second step.     

Purification of the Proteinaceous α-Amylase Inhibitor from Phaseolus Vulgaris by Affinity Chromatography with Immobilized Enzyme or Antibody

A protein inhibiting salivary and pancreatic a-amylase of mammalian origin is contained in dry seeds of beans (Phaseolus vulgaris). Starting from a crude bean extract, the amylase inhibitor may be purified about 30fold in one step to apparent homogeneity by chromatography on matrix-bound salivary amylase. Compared with protein obtained by a conventional purification procedure and in similar yield, the amylase inhibitor obtained by affinity chromatography had the same specific activity (4.5 (akat inhibitor units/mg protein). A one step purification from crude extracts to homogenous inhibitor with the same specific activity was achieved by immuno-affinity chromatography on immobilized rabbit antibody raised against pure amylase inhibitor. The yield was 60 % that of a conventional purification. Criteria of purity of the inhibitor protein were thin-layer electrofocussing and immuno-electrophoresis.     

Optimization of phycocyanin extraction from Spirulina platensis using factorial design

Phycocyanin extraction from cyanobacteria Spirulina platensis was optimized using factorial design and response surface techniques. The effects of temperature and biomass-solvent ratio on phycocyanin concentration and extract purity were evaluated to determine the optimum conditions for phycocyanin extraction. The optimum conditions for the extraction of phycocyanin from S. platensis were the highest biomass-solvent ratio, 0.08 gmL(-1), and 25 degrees C. Under these conditions it's possible to obtain an extract of phycocyanin with a concentration of 3.68 mgmL(-1) and purity ratio (A(615)/A(280)) of 0.46.     

Recombinant phycobiliproteins. Recombinant C-phycocyanins equipped with affinity tags, oligomerization, and biospecific recognition domains

A family of specific cloning vectors was constructed to express in the cyanobacterium Anabaena sp. PCC7120 recombinant C-phycocyanin subunits with one or more different tags, including the 6xHis tag, oligomerization domains, and the streptavidin-binding Strep2 tag. Such tagged alpha or beta subunits of Anabaena sp. PCC7120 C-phycocyanin formed stoichiometric complexes in vivo with appropriate wild-type subunits to give constructs with the appropriate oligomerization state and normal posttranslational modifications and with spectroscopic properties very similar to those of unmodified phycocyanin. All of these constructs were incorporated in vivo into the rod substructures of the light-harvesting complex, the phycobilisome. The C-terminal 114-residue portion of the Anabaena sp. PCC7120 biotin carboxyl carrier protein (BCCP114) was cloned and overexpressed and was biotinylated up to 20% in Escherichia coli and 40% in wild-type Anabaena sp. His-tagged phycocyanin beta--BCCP114 constructs expressed in Anabaena sp. were >30% biotinylated. In such recombinant phycocyanins equipped with stable trimerization domains, >75% of the fusion protein was specifically bound to streptavidin- or avidin-coated beads. Thus, the methods described here achieve in vivo production of stable oligomeric phycobiliprotein constructs equipped with affinity purification tags and biospecific recognition domains usable as fluorescent labels without further chemical manipulation.     

Interaction of C-phycocyanin with lipid monolayers under nitrogen and in the presence of air

The surface interaction of C-phycocyanin with lipids was studied using the monolayer technique. The surface activity of the protein was found to be higher at the lipid-water interface than at the nitrogen-water interface, particularly at high surface pressures of the lipid monolayer. The maximum initial surface pressures beyond which phycocyanin could not penetrate the dipalmitoylphosphatidylcholine and monogalactosyldiglycerol monolayers were 27 and 30 mN m-1, respectively. Below these values the protein demonstrated preferential interaction with the monogalactosyldiglycerol monolayer. The surface properties of the unfolded protein at pH 2.5 at the lipid-water interface were compared with those of the protein at pH 7.0. Higher affinity of the three-dimensional structure of the protein to lipid monolayers was observed, in particular by high subphase protein concentration. When the lipid films were subjected to oxidation stress by exposure to air, the surface properties of C-phycocyanin and dipalmitoylphosphatidylcholine were not greatly affected but the surface activity of monogalactosyldiacylglycerol was reduced dramatically by autoxidation. The oxidation of monogalactosyldiacylglycerol could not be prevented by the introduction of C-phycocyanin molecules at the lipid-water interface.     

The characterization of C-phycocyanin from an extremely halo-tolerant blue–green alga, Coccochloris elabens

C-Phycocyanin was isolated and purified from a uni-algal culture of an extremely halo-tolerant blue-green alga, Coccochloris elabens. This alga can be grown under laboratory conditions in 25% (w/v) NaCl. Purified halophile phycocyanin was characterized by amino acid analysis and the measurement of sedimentation velocity, fluorescence polarization and immunodiffusion as a function of protein concentration, pH and ionic strength. The results were compared with those of studies of phycocyanin isolated from Plectonema calothricoides and from several other sources. The states of aggregation previously characterized as being present in other C-phycocyanins, monomer, trimer and hexamer, were present in halophile phycocyanin and were characterized as antigenically related to all C-phycocyanins tested. The equilibrium between 3S monomer and 11S hexamer at low concentrations in halophile phycocyanin was quantitatively similar to that for other phycocyanins. The effect of pH and ionic strength on the 6S (trimer) and 11S (hexamer) aggregation of halophile phycocyanin was markedly salt-dependent and the relative amount of each aggregate in the presence of 2m-NaCl was like that of C-phycocyanin from mesophiles, in the absence of additional salt. In antigenic relationship and aggregation properties, the phycocyanin from C. elabens appeared to be most closely related to that isolated from the thermophilic blue-green alga, Synechococcus lividus. Amino acid content of the halophile phycocyanin indicated the presence of a significantly larger number of acidic residues than that found in mesophiles. Explanations of the properties of the halophile protein require consideration of a strong contribution of hydrophobic forces and utilize both charge-shielding and salting-out effects.     

Spectroscopic properties of tetrapyrroles on denatured biliproteins

Four biliproteins (phycoerythrin 545, phycocyanin 612, phycocyanin 645, and C-phycocyanin) were denatured by a high concentration of urea and then studied by absorption spectroscopy. Low pH and high protein concentrations conserved the tetrapyrroles' color, and mercaptoethanol and dithiothreitol promoted bleaching. One of these tetrapyrroles, cryptoviolin, appeared not to be hypochromic in the presence of depleting phycocyanobilin, but its absorbance did decay when phycocyanobilin is absent. The product from the treatment of phycocyanobilin with mercaptoethanol or dithiothreitol overlapped spectrally with cryptoviolin and gave the false appearance of maintaining a constant cryptoviolin concentration. Failure to note this effect could result in erroneous cryptoviolin/phycocyanibilin ratios.     

A simple method for efficient extraction and purification of C-phycocyanin from Spirulina platensis Geitler

Phycocyanin is a major light harvesting accessory pigment of red algae and cyanobacteria. In the light of its many commercial applications in food and pharmaceutical industry, purity of the pigment plays a major role. Pharmaceutical industry demands a highly pure phycocyanin with A620/280 ratio of 4 and food industry a ratio of 2. In the present study phycocyanin was extracted in sodium phosphate buffer (pH 7) after macerating in liquid nitrogen. The crude phycocyanin thus extracted was precipitated with 50% ammonium sulphate, purified by dialysis and finally by gel filtration chromatography. Pure phycocyanin was finally obtained with an A620/A280 value of 4.98.     

R-phycocyanin II, a new phycocyanin occurring in marine Synechococcus species. Identification of the terminal energy acceptor bilin in phycocyanins

A new member of the phycocyanin family of phycobiliproteins, R-phycocyanin II (R-PC II) has been discovered in several strains of marine Synechococcus sp. R-PC II has absorption maxima at 533 and 554 nm, a subsidiary maximum at 615 nm, and a fluorescence emission maximum at 646 nm. It is the first phycoerythrobilin (PEB)-containing phycocyanin of cyanobacterial origin. The purified protein is made up of alpha and beta subunits in equal amounts and is in an (alpha beta)2 aggregation state. The alpha and beta subunits of this protein are homologous to the corresponding subunits of previously described C- and R-phycocyanins as assessed by amino-terminal sequence determination and analyses of sequences about sites of bilin attachment. R-PC II carries phycocyanobilin (PCB) at beta-84 and PEB at alpha-84 and beta-155 (residue numbering is that for C-phycocyanin), whereas in C-phycocyanin PCB is present at all three positions. In R-phycocyanin, the bilin distribution is alpha-84 (PCB), beta-84 (PCB), beta-155 (PEB). In both R-phycocyanin and R-phycocyanin II excitation at 550 nm, absorbed primarily by PEB groups, leads to emission at 625 nm from PCB. These comparative data support the conclusion that the invariant beta-84 PCB serves as the terminal energy acceptor in phycocyanins.     

Physical-chemical properties of C-phycocyanin isolated from an acido-thermophilic eukaryote, Cyanidium caldarium.

C-Phycocyanin from an acido-thermophilic eukaryotic alga, Cyanidium caldarium, was characterized with respect to subunit structure, absorption spectrum and fluorescence properties and was found to be similar to C-phycocyanins from mesophilic sources. The pH-dependence of fluorescence polarization and the changes in sedimentation velocity as a function of pH, concentration and temperature indicate the presence of extremely large amounts of unusually stable 19S aggregates. It was not possible to disaggregate this phycocyanin completely to monomer under normal conditions. The amino acid composition is similar to that of phycocyanins from other thermophilic and halophilic sources. The isoelectric point of this C-phycocyanin was 5.11, an unusually high value. The properties of this C-phycocyanin suggest an increase in protein stability as its mode of adaptation to the environmental stress of high temperature.     

发菜藻蓝蛋白分离纯化的研究

以发菜为材料,比较了提取液类型和饱和硫酸铵浓度对藻蓝蛋白提取的影响,并对藻蓝蛋白的提取程序和部分特性进行了研究。结果表明:50 mmol/L KP缓冲液(pH值7.2)是合适的提取液,体积分数为40%~50%饱和硫酸铵盐析效果优于其它浓度。经过DEAE-Toyopeal 650 S离子交换层析和SuperdexTM200凝胶过滤层析后,藻蓝蛋白纯度达6.2,最大吸收峰位于615 nm,荧光发射峰位于649 nm,由α和β2个亚基组成,其分子质量分别为18 051.17和19 142.27 Da。因此,发菜藻蓝蛋白分离纯化较为理想的程序为:藻粉→50 mmol/L KP缓冲液(pH值7.2)浸泡→French pressure(1 500 kg/cm2)破碎细胞→40%~50%饱和硫酸铵盐析→DEAE-Toyopeal 650 S离子交换层析→SuperdexTM200凝胶过滤层析→较纯的藻蓝蛋白。