Large-scale isolation and purification of R-phycoerythrin from red alga Palmaria palmata using the expanded bed adsorption method

R-phycoerythrin, a light-harvesting protein in some marine algae, and can be widely used in medicine, was isolated and purified from a red alga, Palmaria palmata (Lannaeus) Kuntze, using the streamline column (expanded bed adsorption) combined with ion-exchange chromatography. Because the crude extract was applied to the column upwardly, the column would not be blocked by polysaccharides usually very abundant in the extract of marine alga, this kind of blockage could hardly lie overcome in ordinary chromatographic column. After applying the crude extract containing 0.5 mol/L (NH4)(2)SO4, (NH4)(2)SO4 solution of different concentrations (0.2 mol/L, 0.1 mol/L and 0.05 mol/L) was used to elute the column downwardly and the eluates were collected and desalted. The desalted eluates were then applied onto all ion-exchange chromatographic column loaded with Q-sepharose for further purification of the R-phycoerythrin. Through these two steps, the purity (OD565/OD280) of the R-phycoerythrin from P. palmata was up to 3.5, more than 3.2, the commonly accepted criterion for purity, and the yield of the purified R-phycoerythrin could reach 0.122 mg/g of frozen P. palmata, much higher than that of phycobiliproteins purified with the previous methods. The result indicated that the cost of R-phycoerythrin will drop down with the method reported in this article.

Expression of TNF-alpha gene in Anabaena sp PCC 7120 and purification of its product by affinity chromatography

The effects of N (NaNO3) and C (NaAc) source in medium on the expression of tumor necrosis factor-alpha (TNF-alpha) gene in transgenic Anabaena sp. PCC 7120 were compared. The data showed that N source stabilized the expression of foreign protein and C source altered the synthesis of cell walls. Comparing several methods for breaking the cells, supersonic was able to extract TNF-alpha better than others. For purification of TNF-alpha, transgenic Anabaena cells were broken, the extracts were precipitated with ammonia sulfate, and the impure TNF-alpha was eluted from DEAE ion exchange chromatography. Electrophoresis (PAGE-SDS) showed a single band at 17 kD position.

Heterologous expression and purification of recombinant allophycocyanin in marine Streptomyces sp isolate M097

Allophycocyanin is one of the most important marine active peptides. Previous studies suggested that recombinant allophycocyanin (rAPC) could remarkably inhibit the S-180 carcinoma in mice, indicating its potential pharmaceutical uses. Based on intergeneric conjugal transfer, heterologous expression of rAPC was first achieved in marine Streptomyces sp. isolate M097 through inserting the apc gene into the thiostrepton-induced vector pIJ8600. The transformation frequency for this system was approximately 10(-4) exconjugants/recipient. In the transformed Streptomyces sp. isolate M097, the yield of purified rAPC could amount to about 38 mg/l using a simple purification protocol, and HPLC analysis showed that the purity of the protein reached about 91.5%. In vitro activity tests also revealed that the purified rAPC had effective scavenging abilities on superoxide and hydroxyl radicals. This would widen the usefulness of the marine Streptomyces as a host to express the rAPC and to offer industrial strain for the production of rAPC.

Method for large-scale isolation and purification of R-phycoerythrin from red alga Polysiphonia urceolata Grev

R-phycoerythrin was isolated and purified from a red alga, Polysiphonia urceolata Grev, using Streamline column combined with ion-exchange chromatography or hydroxyapatite chromatography. The purity of R-phycoerythrin isolated by Streamline column was up to 1.66 and the yield of R-phycoerythrin could be as high as 0.68 mg/g frozen P. urceolata. All the eluates from Streamline column were divided into two equivalent parts, respectively. One part was pumped into the ion-exchange column loaded with Q-Sepharose and the other was applied to the adsorption column loaded with hydroxyapatite. The purities of R-phycoerythrin purified using these two methods were both up to 3.26, more than 3.2 the commonly accepted criterion. The yield of purified R-phycoerythrin from the ion-exchange chromatography was 0.40 mg/g frozen P. urceolata and that from the hydroxyapatite chromatography could reach 0.34 mg/g frozen P. urceolata. The purified protein had three absorption peaks at 498, 535, and 565 nm and displayed a fluorescence maximum at 580 nm, which was consistent with the typical spectrum of R-phycoerythrin. The purified R-PE was also identified with electrophoresis. Only one single protein band appeared on native-PAGE with silver staining. SDS-PAGE demonstrated the presence of one 20 kDa major subunit, and one low intensity band corresponding to 33 kDa subunit. The results indicate that using the expanded bed adsorption combined with ion-exchange chromatography or hydroxyapatite chromatography, R-phycoerythrin can be purified from frozen P. urceolata on large scale. (c) 2006 Elsevier Inc. All rights reserved.

An efficient combination of supercritical fluid extraction and high-speed counter-current chromatography to extract and purify homoisoflavonoids from Ophiopogon japonicus (Thunb.) Ker-Gawler

Supercritical fluid extraction (SFE) was used to extract homoisoflavonoids from Ophiopogon japonicus (Thunb.) Ker-Gawler. The optimization of parameters was carried out using an orthogonal test L-9 (3)(4) including pressure, temperature, dynamic extraction time and the amount of modifier. The process was then scaled up by 100 times with a preparative SFE system under the optimized conditions of 25 MPa, 55 degrees C, 4.0 h and 25% methanol as a modifier. Then crude extracts were separated and purified by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane/ethyl acetate/methanol/ACN/water (1.8:1.0:1.0:1.2:1.0 v/v). There three homoisoflavonoidal compounds including methylophiopogonanone A 6-aldehydo-isoophiopogonone A, and 6-formyl-isoophiopogonanone A, were successfully isolated and purified in one step. The collected fractions were analyzed by HPLC. In each operation, 140 mg crude extracts was separated and yielded 15.3 mg of methylophiopogonanone A (96.9% purity), 4.1 mg of 6-aldehydo-isoophiopogonone A (98.3% purity) and 13.5 mg of 6-formyl-isoophiopogonanone A (97.3% purity) respectively. The chemical structure of the three homoisoflavonoids are identified by means of ESI-MS and NMR analysis.

One step isolation and purification of liquiritigenin and isoliquiritigenin from Glycyrrhiza uralensis Risch using high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) technique in semi-preparative scale has been successfully applied to the separation of bioactive flavonoid compounds, liquiritigenin and isoliquiritigenin in one step from the crude extract of Glycyrrhiza uralensis Risch. The HSCCC was performed using a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-acetonitrile-water (2:2:1:0.6:2, v/v). Yields of liquiritigenin (98.9% purity) and isoliquiritigenin (98.3% purity) obtained were 0.52% and 0.32%. Chemical structures of the purified liquiritigenin and isoliquiritigenin were identified by electrospray ionization-MS (ESI-MS) and NMR analysis. (c) 2005 Published by Elsevier B.V.

Carbon nanotubes as adsorbent of solid-phase extraction and matrix for laser desorption/ionization mass spectrometry

A method with carbon nanotubes functioning both as the adsorbent of solid-phase extraction (SPE) and the matrix for matrix assisted laser desorption/ ionization mass spectrometry (MALDI-MS) to analyze small molecules in solution has been developed. In this method, 10 muL suspensions of carbon nanotubes in 50% (vol/vol) methanol were added to the sample solution to extract analytes onto surface of carbon nanotubes because of their dramatic hydrophobicity. Carbon nanotubes in solution are deposited onto the bottom of tube with centrifugation. After removing the supernatant fluid, carbon nanotubes are suspended again with dispersant and pipetted directly onto the sample target of the MALDI-MS to perform a mass spectrometric analysis. It was demonstrated by analysis of a variety of small molecules that the resolution of peaks and the efficiency of desorption/ ionization on the carbon nanotubes are better than those on the activated carbon. It is found that with the addition of glycerol and sucrose to the dispersant, the intensity, the ratio of signal to noise (S/N), and the resolution of peaks for analytes by mass spectrometry increased greatly. Compared with the previously reported method by depositing sample solution onto thin layer of carbon nanotubes, it is observed that the detection limit for analytes can be enhanced about 10 to 100 times due to solid-phase extraction of analytes in solution by carbon nanotubes. An acceptable result of simultaneously quantitative analysis of three analytes in solution has been achieved. The application in determining drugs spiked into urine has also been realized. (C) 2004 American Society for Mass Spectrometry.

沙冬青抗冻组份的分离纯化和afp基因克隆的尝试

本文用三种方法分离纯化沙冬青（Ammopiptanthus mongolicus）叶片抗冻蛋白质。从70%硫酸铵沉淀后的上清液中得到了一种碱性的小分子物质，UiS，具有较高的溶解度( 260 mg/ml)和较强的降低溶液冰点的能力（200 mg/ml，冰点小于-20℃）：从10%硫酸铵和26%氯化铵共沉淀物中分离到一种抗冻蛋白-B3，B3分子量为39.8 kDa，热滞活性为0.45℃(10 mg/ml)：从沙冬青叶片热稳定蛋白质中用双向电泳一电泳回收法分离到afp,其分子量为40 kDa,pl为9.0。热滞活性为0.9℃(20 mg/ml)，和其他抗冻蛋白质进行比较，没有发现相同的类型。afp和uis在叶片含量都很高，可能是沙冬青抗冻生理过程中两种主要物质，它们对于沙冬青抵御-20℃左右的冷冻温度具有重要的意义。afp N端序列为SDDL SFTF NKFV PCQT DILF，据此合成了六段5’引物和一段3’引物，用RT - PCR方法从叶片中扩增出一段200 bp左右的片段。

Synergistic extraction of rare earths using acid-base coupling extractants of calix[4]arene carboxyl derivative and primary amine N1923

Synergistic effect in the extraction of rare earth (RE) metals by the acid-base coupling (ABC) extractants of calix[4]arene carboxyl derivative Bu-t[4]CH2COOH (H(4)A) and primary amine N1923 (RNH2) has been investigated. The extraction of RE was enhanced by the addition of sodium cations into the aqueous phase not only in the extraction system of Bu-t[4]CH2COOH alone but also in the mixture of Bu-t[4]CH2COOH and N1923. The separation factors (SFs) indicating the extraction selectivity of adjacent RE elements became higher in the mixture system.

Effects of ultrahigh pressure extraction conditions on yields and antioxidant activity of ginsenoside from ginseng

Ultrahigh pressure technique was employed to extract ginsenosides from roots of ginseng (Panax ginseng C.A. Meyer). The optimal conditions for ultrahigh pressure extraction (UPE) of total ginsenosides were quantified by UV-vis spectrophotometry with the ginsenoside Re as standard, the signal ginsenosides were quantified by HPLC and ELSD with ginsenosides Re, Rg(1), Rb-1, Rc and Rb-2 as standards. Orthogonal design was applied to evaluate the effects of four independent factors (extraction pressure, extraction temperature, extraction time and ethanol concentration) on the yield and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of ginsenoside, which are based on microwave extraction (ME), ultrasound extraction (UE), soxhlet extraction (SE) and heat reflux extraction (HRE) method. The results showed that UPE method can produce ginsenoside with the highest yield and the best radical scavenging activity compared to other used ones. Scanning electron microscopic (SEM) images of the plant cells after ultrahigh pressure treatment was obtained to provide visual evidence of the disruption effect.

Synergistic extraction of rare earths by mixture of bis(2,4,4-trimethylpentyl)phosphinic acid and Sec-nonylphenoxy acetic acid

Synergistic extraction of trivalent rare earths (RE=Sc, Y, La, Gd, Yb) from hydrochloride medium using mixture of bis(2,4,4-trimethylpentyl)phosphinic acid (HL, Cyanex272) and Sec-nonylphenoxy acetic acid (HA, CA-100) in n-heptane has been studied. The synergistic enhancement coefficients were observed for La (1.30), Gd (1.97), Y (3.59), Yb (8.21) and Sc (14.41). The results indicated yttrium was extracted into n-heptane as YH(5)A(4)L(4) mixed species instead of Y(HL2)(3), Y(OH)(2)A(HA)(3) which were extracted by Cyanex272 and CA-100, respectively. A cation exchange mechanism was proposed and further clarified by IR spectra. The equilibrium constants, formation constants and thermodynamic functions such as Delta G, Delta H and Delta S were determined. The Cyanex272 + CA-100 system not only enhanced the extraction efficiency of RE but also improved the selectivities significantly. The mutual separation factors of these ions suggested the mixture system would be of practical value in extraction and separation of rare earths.