WO2018227664A1 - 一种葛仙米藻胆蛋白的提取、纯化方法及纯化的藻红蛋白 - Google Patents

一种葛仙米藻胆蛋白的提取、纯化方法及纯化的藻红蛋白 Download PDF

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WO2018227664A1
WO2018227664A1 PCT/CN2017/090808 CN2017090808W WO2018227664A1 WO 2018227664 A1 WO2018227664 A1 WO 2018227664A1 CN 2017090808 W CN2017090808 W CN 2017090808W WO 2018227664 A1 WO2018227664 A1 WO 2018227664A1
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buffer
conductivity
phycoerythrin
nacl
exchange column
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French (fr)
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余佳
张瑞华
王玉兰
占豪
陈盛
王维
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湖南炎帝生物工程有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/405—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from algae

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  • the invention relates to a method for efficiently extracting and purifying phycoerythrin and purified phycoerythrin.
  • Nostoc sphaeroids Kutzing the scientific name of the genus Saccharomyces cerevisiae, belongs to the genus Nostocaceae (Nostoc.), which belongs to the genus of the genus. It is called the celestial rice and the celestial genus. It is also known as the water fungus, the wormwood, and the earth. Novoca commune vauch is a precious and edible nitrogen-and-nitrogen algae that is traditionally exported in China.
  • Phycobiliprotein is a light-harvesting pigment protein widely found in phycobilisomes (PBS) of red algae, blue-green algae and cryptophyta, including phycocyanin (PC) and phycoerythrim (PE). Phycoerythrocyanin (PEC) and Allphycocyanin (APC). Gexian rice is rich in biliary protein and is superior to other algae. Lycoerythrin is a kind of fluorescein protein with high added value, which can be used for immunoassay, soluble antigen, antibody detection diagnostic reagent, fluorescent probe, fluorescent label and so on. At present, the price of phycoerythrin with a purity of more than 95% in the market is more than 5,000 yuan, and the higher the purity, the higher the price.
  • the purification of phycobiliproteins usually requires first purification, such as salting out, isoelectric point or crystallization, to remove the heteroproteins, followed by column chromatography, including hydroxy limestone adsorption chromatography, cellulose series ion exchange. Chromatography, affinity chromatography and size exclusion chromatography.
  • the present invention provides a method for extracting phycobiliprotein of genus, comprising the steps of: (a) adding dry powder of cemetery to water; (b) adding liquid nitrogen, continuously stirring, and volatilizing liquid nitrogen Completely; (c) centrifugal filtration to obtain a gentamicin extract; (d) freeze-drying to obtain a crude extract of genomic protein.
  • the water may be pure water, deionized water, distilled water or the like.
  • the mass ratio of the dry powder of the genus and the water is 1:40 to 1:70; preferably, 1:50, or 1:60.
  • the liquid nitrogen is added in an amount of 0.5% to 1% of the total mass of the dried starch and water.
  • the stirring time is from 4 h to 10 h; preferably, 6 h.
  • the centrifugal rotation speed is 5000 rpm-8000 rpm; the centrifugation time is 20 min-30 min; and the centrifugal filtration can be performed by a disc centrifuge.
  • the invention also provides a method for purifying phytoene red albumin, comprising the following steps: (1) using an anion exchange column to pass the column; (2) disposing PB buffer Buffer A and PB-NaCl buffer Buffer B, filtering (3) Puxian rice phycobiliprotein crude powder is dissolved in PB buffer Buffer A, centrifuged, filtered; (4) wash the pump, balance the ion exchange column with PB buffer Buffer A; (5) step (3) The prepared phycobiliprotein prepared by dissolving in PB buffer Buffer A is directly loaded, and the gradient is eluted by PB-NaCl buffer Buffer B, and the eluent is collected; (6) the ion exchange column is washed with a salt solution, and then used.
  • the alkali solution is backwashed; (7) the eluate of step (5) is subjected to ultrafiltration concentration and dialysis to obtain a purified aqueous solution of phycoerythrin; (8) the genus The aqueous solution of erythroprotein is stored at 0 to 10 °C.
  • the 20% ethanol water in the anion exchange column is replaced with deionized water before the column is passed, and the purpose is to store the anion exchange column with 20% ethanol water, which should be replaced before use.
  • the anion exchange column is an Agarosix FF-DEAE anion exchange column.
  • the medium of the anion exchange column is composed of an agarose gel having a particle diameter of 50 to 150 ⁇ m and a degree of crosslinking of 6%; preferably, a crosslinked agarose gel having a particle diameter of 90 ⁇ m and a crosslinking degree of 6%.
  • Composition the agarose gel relies on a secondary chain such as a hydrogen bond between sugar chains to maintain a network structure, and the density of the network structure depends on the concentration of agarose.
  • the structure of the agarose gel is stable and can be used under many conditions (such as water, salt solution in the range of pH 4-9); the agarose gel starts to melt above 40 °C, can not be autoclaved, available chemistry Sterilization treatment.
  • Cross-linked agarose gel is a commonly used chromatographic matrix in biological separation. It uses different functional groups to modify the hydroxyl groups on the surface to prepare commercial fillers such as hydrophobic chromatography, ion exchange chromatography and affinity chromatography.
  • the pH of the PB buffer is 5 to 6.5, and the conductivity is 5 to 10 ⁇ s/cm; preferably, the pH is 6 to 6.5, the conductivity is 6 to 8 ⁇ s/cm; further preferably, the pH is The conductivity was 5, 6, 6.3, and 6.5, and the electrical conductivity was 8.5 ⁇ s/cm, 8.0 ⁇ s/cm, 7.1 ⁇ s/cm, and 6.0 ⁇ s/cm, respectively.
  • the pH of the PB-NaCl buffer is 5 to 6.5, and the conductivity is 5 to 10 ⁇ s/cm; preferably, the pH is 6 to 6.5, and the conductivity is 6 to 8 ⁇ s/cm; further preferably The pH was 5, 6, 6.3, and 6.5, and the electrical conductivity was 8.5 ⁇ s/cm, 8.0 ⁇ s/cm, 7.1 ⁇ s/cm, and 6.0 ⁇ s/cm, respectively.
  • the conductivity is 8.5 ⁇ s/cm
  • the pH of the PB-NaCl buffer is 5, and the conductivity is 8.5 ⁇ s/cm; or, the pH of the PB buffer
  • the conductivity is 8.0 ⁇ s/cm
  • the pH of the PB-NaCl buffer is 6, and the conductivity is 8.0 ⁇ s/cm; or, when the pH of the PB buffer is 6.3, the conductivity is 7.1 ⁇ s/cm, PB.
  • PB-NaCl buffer pH 6.3 conductivity 7.1 ⁇ s / cm; or, PB buffer pH 6.5, conductivity 6.0 ⁇ s / cm
  • the PB-NaCl buffer had a pH of 6.5 and a conductivity of 6.0 ⁇ s/cm.
  • the PB-NaCl buffer is prepared by adding NaCl solids in the configured PB buffer, and measuring the conductivity while the conductivity is up to the required level.
  • the filtration means that the solid membrane is removed by filtration under reduced pressure with a 0.2 ⁇ m filter membrane; and the pressure is reduced by a vacuum pump under reduced pressure filtration.
  • the crude extract of the genus Phytophthora bilirubin may be prepared according to the above method; or the crude extract powder of the genus Phytophthora glutamate is composed of dry powder of pure sage and pure water (liquid to liquid ratio 1 : 60) Extraction for 6 h, filtration, and lyophilization.
  • the amount of the crude extract of the phycocyanin protein and the PB buffer is 5 g: 1 L to 15 g: 1 L; preferably, 10 g: 1 L.
  • the centrifugal rotation speed may be, but not limited to, 5000 to 10000 r/min, such as 8000 r/min.
  • the centrifugation time is 20 min to 40 min; preferably, 30 min.
  • the filtration is preferably filtered using a 0.2 ⁇ m filter.
  • the pump is washed with deionized water for 1 to 1.5 column volumes; preferably, 1 column volume is washed with deionized water.
  • the equilibration process uses 3 to 10 column volumes of PB buffer; preferably, 4 to 6 column volumes of PB buffer; further preferably, 5 column volumes of PB buffer Buffer A.
  • the pH of the PB buffer is 5 to 6.5, and the conductivity is 5 to 10 ⁇ s/cm; preferably, the pH is 6 to 6.5, the conductivity is 6 to 8 ⁇ s/cm; further preferably, the pH is The ratios were 6, 6.3, and 6.5, and the electrical conductivity was 8.0 ⁇ s/cm, 7.1 ⁇ s/cm, and 6.0 ⁇ s/cm, respectively.
  • the pH of the PB-NaCl buffer is 5 to 6.5, and the conductivity is 5 to 10 ⁇ s/cm; preferably, the pH is 6 to 6.5, and the conductivity is 6 to 8 ⁇ s/cm; further preferably The pH was 6, 6.3, and 6.5, and the electrical conductivity was 8.0 ⁇ s/cm, 7.1 ⁇ s/cm, and 6.0 ⁇ s/cm, respectively.
  • the pH of the PB buffer is 5, the conductivity is 8.5 ⁇ s/cm, the pH of the PB-NaCl buffer is 5, and the conductivity is 8.5 ⁇ s/cm; or, the pH of the PB buffer is 6.
  • the conductivity is 8.0 ⁇ s/cm
  • the pH of the PB-NaCl buffer is 6, and the conductivity is 8.0 ⁇ s/cm; or, the pH of the PB buffer is 6.3, and the conductivity is 7.1 ⁇ s/cm
  • the PB-NaCl buffer The pH of the solution was 6.3, the conductivity was 7.1 ⁇ s/cm; or, the pH of the PB buffer was 6.5, and the conductivity was 6.0 ⁇ s/cm, the pH of the PB-NaCl buffer was 6.5, and the conductivity was 6.0 ⁇ s/cm.
  • the loading amount is 2 to 4 column volumes; preferably, 2, 2.5, 3, 4 column volumes.
  • the collected eluate is 4 to 5 column volumes of 20 to 100% of an eluent, preferably 40 to 100% of an eluent.
  • the salt solution is preferably NaCl or KCl, and the concentration of the salt solution is 0.02 to 0.1 M; Ground, 0.05M.
  • the salt solution is used in an amount of from 1 to 2 column volumes.
  • the alkali solution is preferably NaOH or KOH, and the concentration of the alkali solution is 0.1 to 0.5 M; preferably, 0.1 M.
  • the alkali solution is used in an amount of from 3 to 4 column volumes.
  • the ultrafiltration membrane has a pore diameter of from 1000 D to 8000 D; preferably, it is 3000 D.
  • the dialysis can be carried out by any conventional dialysis method.
  • the temperature at which the aqueous solution is stored is preferably 4 °C.
  • the method for purifying phycoerythrin of the present invention comprises the steps of: dissolving crude extract of phycobiliprotein of genus Phytophthora with PB buffer Buffer A having a pH of 6 to 6.5 and an electric conductivity of 6 to 8 ⁇ s/cm, 8000 r Centrifuge at /min for 30 min, filter with 0.2 ⁇ m filter; Agarosix FF-DEAE anion exchange column was passed through the column, 6% cross-linked agarose gel medium was replaced with deionized water, and column was deionized with 1 column volume.
  • washing pump 5 column volume of PB buffer Buffer A equilibrated, loading 4 column volumes, collecting gradient elution of PB-NaCl buffer Buffer B with pH 6-6.5 and conductivity 6-8 ⁇ s/cm 4 to 5 column volumes, concentrated by ultrafiltration with 3000D ultrafiltration membrane, dialyzed, and stored in an aqueous solution at 4 °C.
  • the invention also provides a phycoerythrin purified by the above purification method; the phycoerythrin phycoerythrin has a relative molecular weight of 67KD-70KD.
  • the anion exchange column selected in the present invention such as the Agarosix FF-DEAE anion exchange column, significantly improves the purification efficiency, eliminates the need for ammonium sulfate precipitation in the existing purification method, and greatly increases the concentration and purity of phycoerythrin.
  • the method is simple, the effect is obvious, and the processing capacity is large, which provides a basis for industrial application.
  • the loading conditions selected by the invention have been proved by experiments to be large, and the number of protein samples that can be purified at one time is simple, the purification method is simple, the cost is low, the purification efficiency of phycoerythrin is greatly improved, and the basis for industrialized large production is provided.
  • the present invention can remove most of the impurities in the phycobiliprotein crude powder, so that the extraction rate of phycoerythrin is close to 100%, and the amount of loss is small.
  • the concentration and purity of the phycoerythrin obtained by the present invention are significantly improved, and the purity of the phycoerythrin can be increased to 90% or more.
  • the purified phycoerythrin of the invention has high added value and can be applied to the fields of cosmetics, food, health food and biomedicine, and has broad development prospects.
  • Figure 1 shows a gel filtration chromatogram of the purified phycoerythrin in Example 7.
  • Gel filtration chromatography peaks at different times depending on the molecular weight of the protein, and the purity of each test sample is calculated by the ratio of the peak area to the total area.
  • the relative molecular weight of phycoerythrin was estimated to be 67 KD-70 KD based on the peak position.
  • Figure 2 shows the ultraviolet spectrum (1. phycoerythrin; 2. phycoerythrin standard) of the purified phycoerythrin and phycoerythrin reference substance in Example 7.
  • the peak positions of the two samples are basically the same, and there are similar conjugate structures, which are similar samples.
  • Figure 3 shows the fluorescence spectra of the purified phycoerythrin and phycoerythrin reference substance in Example 7 (1. phycoerythrin; 2. phycoerythrin standard). By comparison, the peak positions of the two samples are the same, indicating that they are homogeneous samples.
  • Figure 4 shows a three-component SGS-PAGE analysis diagram (1: Marker (Mr is 14.3 kD, 20.1 kD, 29.0 kD, 44.3 kD, 66.4 kD, 97.2 kD from bottom to top, respectively); 2: prepared according to the above method of the present invention The phycobiliprotein crude powder; 3: the phycoerythrin purified in Example 7; 4: phycocyanin) The subunit molecular weight of the phycoerythrin was analyzed to be about 14 kD based on electrophoresis data.
  • the detection method of the phycoerythrin concentration of the present invention is Coomassie Brilliant Blue G250 staining method, and the absorbance value at a wavelength of 595 nm is measured, and a standard curve is drawn and calculated according to the method described by Li Hesheng, "Plant Physiology and Biochemistry Experiment Principles and Techniques", and the algae is purchased.
  • the purity of the phycoerythrin of the present invention is detected by an ultraviolet absorption spectrometer, and the purity is calculated as:
  • Lycopene concentration (g/mL) purity purity(%) the first time 0.96 4.87 83 the second time 0.96 4.87 83 the third time 0.96 4.87 83
  • 50.11g, 50.07g, and 49.91g of dried cemetery powder were separately added to 2500 mL of pure water, stirred uniformly, and then added with 20 mL of liquid nitrogen, and vigorously stirred while adding. After the liquid nitrogen is completely evaporated, stirring is continued, the ice is completely dissolved, centrifugally filtered, and freeze-dried to obtain 9.97 g, 10.03 g, and 10.01 g of the crude extract powder of genus phycobiliprotein, respectively, with a pH of 7.0 and conductivity.
  • Buffer A of 5.7 ⁇ s/cm PB buffer was dissolved, centrifuged at 8000 r/min for 30 min, filtered through a 0.2 ⁇ m filter; column was passed through a DEAE-Cpato anion exchange column: 1) The pump was washed with 1 column volume of deionized water; ) equilibrate with 5 column volumes of PB buffer Buffer A; 3) load 2 column volumes.
  • a PB-NaCl eluate with a pH of 7.0 and a conductivity of 5.7 ⁇ s/cm was collected from 40 to 100%. The results are shown in Table 2 below:
  • the average concentration of phycoerythrin was 1.01 g/mL and the average purity was 5.21 (83.9%). After three repeated experiments, the experiment was very reproducible. Similarly, the purity and concentration of purified phycoerythrin are low, and it can be seen that the purification of phycoerythrin by DEAE-Cpato column is not efficient and the effect is poor.
  • 49.98 g, 50.13 g, and 49.92 g of dried cemetery powder were separately added to 2500 mL of pure water, stirred uniformly, and then added with 20 mL of liquid nitrogen, and vigorously stirred while adding. After the liquid nitrogen is completely evaporated, stirring is continued, the ice is completely dissolved, centrifugally filtered, and freeze-dried to obtain 9.96 g, 9.97 g, and 10.02 g of crude extract of phycobiliprotein, respectively, with a pH of 7.0 and conductivity.
  • PB buffer Buffer A was dissolved, centrifuged at 8000 r/min for 30 min, filtered through a 0.2 ⁇ m filter; column was passed through a DEAE-Sephadex Fast Flow anion exchange column: 1) The pump was washed with 1 column volume of deionized water. 2) Balance with 5 column volumes of PB buffer Buffer A; 3) Load 1 column volume, collect 40 to 100% PB-NaCl eluent with a pH of 7.0 and a conductivity of 5.7 ⁇ s/cm. See Table 3 below:
  • Lycopene concentration (g/mL) purity purity(%) the first time 1.02 6.02 85.8 the second time 1.02 6.02 85.8 the third time 1.02 6.02 85.8
  • the average phycoerythrin concentration obtained by three purifications was 1.02 g/mL, and the average purity was 6.02 (85.8%). After three repeated experiments, the experiment was very reproducible. However, the purity and concentration of purified phycoerythrin are not high. It can be seen that although the purification of phycoerythrin by DEAE-Sephadex Fast Flow column is slightly higher than that of the above two anion exchange columns, the efficiency is still not satisfactory and the effect is poor.
  • Buffer A of 5.7 ⁇ s/cm PB buffer was dissolved, centrifuged at 8000 r/min for 30 min, and filtered through a 0.2 ⁇ m filter; the column was passed through an Agarosix FF-DEAE anion exchange column: 1) The pump was washed with 1 column volume of deionized water; 2) Balance with 5 column volumes of PB buffer Buffer A; 3) Load 2 column volumes. A 40-100% PB-NaCl eluate with a pH of 7.0 and a conductivity of 5.7 ⁇ s/cm was collected. The results are shown in Table 4 below:
  • Lycopene concentration (g/mL) purity purity(%) the first time 2.09 9.68 90.6 the second time 2.09 9.68 90.6 the third time 2.12 9.68 90.6
  • the average concentration of phycoerythrin obtained by three purifications was 2.10 g/mL, and the average purity was 9.68 (94.1%). After three repeated experiments, the experiment was very reproducible. In addition, the purity and concentration of phycoerythrin purified by the Agarosix FF-DEAE anion exchange column were significantly improved. It can be seen that the Agarosix FF-DEAE anion exchange column is suitable for the purification of phycoerythrin.
  • 60.01 g, 60.11 g, and 60.03 g of dried cemetery powder were separately added to 3000 mL of pure water, stirred uniformly, and then 25 mL of liquid nitrogen was added, and vigorously stirred while adding. After the liquid nitrogen is completely evaporated, stirring is continued, the ice is completely dissolved, centrifugally filtered, and freeze-dried to obtain 12.51 g, 12.47 g, and 12.53 g of crude phycobiliproteins, respectively, with a pH of 5.0 and conductivity.
  • Buffer A of 8.5 ⁇ s/cm PB buffer was dissolved, centrifuged at 8000 r/min for 30 min, and filtered through a 0.2 ⁇ m filter; the column was passed through an Agarosix FF-DEAE anion exchange column: 1) The pump was washed with 1 column volume of deionized water; 2) Balance with 5 column volumes of PB buffer Buffer A; 3) Load 2.5 column volumes.
  • the PB-NaCl eluent with 40-100% pH 5.0 and conductivity 8.5 ⁇ s/cm was collected. The results are shown in Table 5 below:
  • the average concentration of phycoerythrin was 2.51 g/mL and the average purity was 10.01 (90.9%). After three repeated experiments, the experiment was very reproducible. In addition, the purity and concentration of phycoerythrin purified by the Agarosix FF-DEAE anion exchange column were significantly improved. It can be seen that the Agarosix FF-DEAE anion exchange column is suitable for the purification of phycoerythrin.
  • Buffer A of 8.0 ⁇ s/cm PB buffer was dissolved, centrifuged at 8000 r/min for 30 min, and filtered through a 0.2 ⁇ m filter; the column was passed through an Agarosix FF-DEAE anion exchange column: 1) The pump was washed with 1 column volume of deionized water; 2) Balance with 5 column volumes of PB buffer Buffer A; 3) Load 3 column volumes.
  • a PB-NaCl eluate with a pH of 6.0 to 100% and a conductivity of 8.0 ⁇ s/cm was collected. The results are shown in Table 6 below:
  • Lycopene concentration (g/mL) purity purity(%) the first time 3.03 13.62 93.2 the second time 3.03 13.62 93.2 the third time 3.03 13.62 93.2
  • the average concentration of phycoerythrin obtained by three purifications was 3.03 g/mL, and the average purity was 13.62 (93.2%). After three repeated experiments, the experiment was very reproducible. In addition, the purity and concentration of phycoerythrin purified by the Agarosix FF-DEAE anion exchange column were significantly improved. It can be seen that the Agarosix FF-DEAE anion exchange column is suitable for the purification of phycoerythrin.
  • 100.01 g, 100.13 g, and 100.09 g of dried cemetery powder were separately added to 5000 mL of pure water, stirred uniformly, and then added with 40 mL of liquid nitrogen, and vigorously stirred while adding. After the liquid nitrogen is completely evaporated, stirring is continued, the ice is completely dissolved, centrifugally filtered, and lyophilized to obtain 20.02 g, 19.98 g, 20.01 g of crude extract of genomic protein of Gesmanthus chinensis, respectively, with a pH of 6.3 and conductivity.
  • Buffer A of 7.1 ⁇ s/cm PB buffer was dissolved, centrifuged at 8000 r/min for 30 min, and filtered through a 0.2 ⁇ m filter; the column was passed through an Agarosix FF-DEAE anion exchange column: 1) The pump was washed with 1 column volume of deionized water; 2) Balance with 5 column volumes of PB buffer Buffer A; 3) Load 4 column volumes. A 40-100% PB-NaCl eluate with a pH of 6.3 and a conductivity of 7.1 ⁇ s/cm was collected. The results are shown in Table 7 below:
  • the average concentration of phycoerythrin obtained by three purifications was 4.21 g/mL, and the average purity was 16.43 (94.3%). After three repeated experiments, the experiment was very reproducible. It was found that when the loading conditions were as follows: Buffer A was dissolved in PB buffer Buffer A with a pH of 6.3 and a conductivity of 7.1 ⁇ s/cm, the volume of the four columns was up to 4, and the highest concentration of phycoerythrin was 4.21.
  • the gel filtration chromatogram of the purified phycoerythrin in Example 7 is shown in FIG.
  • Gel filtration chromatography peaks at different times depending on the molecular weight of the protein, and the purity of each test sample is calculated by the ratio of the peak area to the total area.
  • the peak position is about 8 mL.
  • the molecular weight of about 9 mL is about 67 KD
  • the relative molecular weight of phycoerythrin is estimated to be 67 KD-70 KD.
  • the ultraviolet spectrum of the purified phycoerythrin and phycoerythrin reference substance in Example 7 is shown in Fig. 2.
  • the peak positions of the two samples were substantially coincident, and the absorption peaks were 542 nm and 565 nm, and there were similar conjugate structures, which were judged to be similar samples.
  • Buffer A of 6.0 ⁇ s/cm PB buffer was dissolved, centrifuged at 8000 r/min for 30 min, and filtered through a 0.2 ⁇ m filter; the column was passed through an Agarosix FF-DEAE anion exchange column: 1) The pump was washed with 1 column volume of deionized water; 2) Balance with 5 column volumes of PB buffer Buffer A; 3) Load 3 column volumes.
  • a PB-NaCl eluate with a pH of 6.5 and a conductivity of 6.0 ⁇ s/cm was collected from 40 to 100%. The results are shown in Table 8 below:
  • Lycopene concentration (g/mL) purity purity(%) the first time 2.11 3.71 78.7 the second time 2.11 3.71 78.7 the third time 2.11 3.71 78.7
  • PB buffer Buffer A 9.2 ⁇ s/cm PB buffer Buffer A was dissolved, centrifuged at 8000r/min for 30min, filtered with 0.2 ⁇ m filter membrane; wash pump with 1 column volume of deionized water, 5 column volume of PB buffer Buffer A balance, upper For 4 column volumes, collect 40 to 100% PB-NaCl eluent with a pH of 9.3 and a conductivity of 9.5 ⁇ s/cm.
  • Table 10 The results are shown in Table 10 below:
  • Lycopene concentration (g/mL) purity purity(%) the first time 2.09 2.74 73.2 the second time 2.09 2.74 73.2 the third time 2.11 2.74 73.2
  • the average concentration of phycoerythrin obtained by three purifications was 2.10 g/mL, and the average purity was 2.74 (73.2%). After three repeated experiments, the experiment was very reproducible.
  • the concentration of the obtained phycoerythrin was significantly lower than that of the other examples, indicating that the pH range of the loading and elution was inappropriate, and the suitable elution condition was pH of the PB buffer Buffer A of 5 to 6.5, conductivity.
  • the loading condition is 5 to 10 ⁇ s/cm
  • the loading condition is PB-NaCl buffer Buffer B having a pH of 5 to 6.5 and an electric conductivity of 5 to 10 ⁇ s/cm.
  • Lycopene concentration (g/mL) purity purity(%) the first time 0.60 4.3 81.1 the second time 0.59 4.3 81.1 the third time 0.60 4.3 81.1
  • the average concentration of phycoerythrin obtained by three purifications was 0.597 g/mL, and the average purity was 4.3 (81.1%), and the concentration and purity were significantly lower than those of Examples 1 to 4. After three repeated experiments verified.
  • the buffer system Tris-HCl and pH selected in Comparative Example 1 made the purification effect poor, and the purification operation steps were complicated, which increased the cost of actual operation.
  • the PB buffer system selected by the method of the invention is more suitable for the purification of phycoerythrin, and the purification process can be completed by one-time chromatography, and the purity and concentration of phycoerythrin are higher than that of the comparative example 1. Therefore, the method of the present invention has high operability, simple operation, low use cost, and high concentration and purity of the purified phycoerythrin.
  • the average concentration of phycoerythrin obtained by three purifications was 0.67 g/mL, and the average purity was 8.1 (89%), and the concentration and purity were significantly lower than those of Examples 3 to 4. After three repeated experiments verified.
  • the PB buffer system selected in Comparative Example 2 has a lower pH and the system is acidic, which makes the purification of phycoerythrin after two Purification step, but the purity of the purification is not ideal, up to 89%.
  • the purity of the phycoerythrin obtained by the method of the invention is significantly higher than that of the comparative example 2, and the purification process can be completed by one method of the method of the present invention, and the purity and concentration of the phycoerythrin obtained are higher than that of the comparative example 2. Therefore, the method of the present invention has high operability, simple operation, low use cost, high phycoerythrin concentration and high purity.
  • the filler of the Agarosix FF-DEAE anion exchange column selected for the present invention is a 6% cross-linking degree agarose gel, which exhibits high load in purification.
  • the experiment proves that the method of the invention is suitable for using the resin for purifying phycoerythrin, and the effect is obviously superior to other DEAE anion exchange columns (including DEAE-52 cellulose column and DEAE-Sephadex Fast Flow column); and the loss of phycoerythrin protein is small.
  • the large amount of sample loading and high purity may be due to the fact that the ion exchange column ligand used in the present invention binds to phycoerythrin, and does not bind to other impurity proteins, thereby increasing the adsorption amount of phycoerythrin; (2) in buffer pH.
  • the purity of the phycoerythrin obtained by the method of the present invention is significantly higher than that of the comparative example because the negatively charged portion of the phosphate ion-encapsulated phycoerythrin in the PB system is only in the appropriate pH range.
  • phycoerythrin having high purity and high yield can be isolated by one step of Agarosix FF-DEAE, which greatly simplifies the experimental procedure.
  • the phycoerythrin having a concentration of 2.51 g/mL or more and a purity of 10.01 (90.9%) or more can be obtained from the crude extract powder of genus Phytophthora, and the method can be loaded by the method of the present invention.

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Abstract

提供了一种葛仙米藻红蛋白的纯化方法，包括：(1)选用阴离子交换柱过柱；(2)配置PB缓冲液BufferA和PB-NaCl缓冲液Buffer B；(3)葛仙米藻胆蛋白粗提粉末用PB缓冲液BufferA溶解，离心，过滤；(4)洗泵，用PB缓冲液BufferA平衡离子交换柱；(5)将步骤(3)制备的溶解于PB缓冲液BufferA中的葛仙米藻胆蛋白直接上样，PB-NaCl缓冲液Buffer B梯度洗脱，收集洗脱液；(6)用盐溶液清洗离子交换柱，然后用碱溶液反向冲洗；(7)对淋洗液进行超滤浓缩，透析，得到纯化的葛仙米藻红蛋白水溶液。此外，还提供了一种葛仙米藻胆蛋白的提取方法。

Description

一种葛仙米藻胆蛋白的提取、纯化方法及纯化的藻红蛋白 技术领域
本发明涉及一种高效提取及纯化葛仙米藻红蛋白的方法和纯化的藻红蛋白。
背景技术
葛仙米(Nostoc sphaeroids Kutzing)，学名拟球状念珠藻，属蓝藻门(Nostocaceae)念珠藻属(Nostoc.)，与发菜同属，古称天仙米、天仙菜，又名水木耳、田木耳、地木耳(Nostoc commune vauch)，是我国传统出口的珍贵药食两用固氮蓝藻。它是一种多细胞的丝状植物，其细胞结构简单、个体由圆球形细胞组成不分枝的单列丝状体，丝状体呈念珠状，群体呈胶质状、球状或其他不规则形状，蓝绿色或黄褐色，肉眼可见，具有固氮能力。《本草纲目》、《药性考》、《全国中草药汇编》等称葛仙米的功效为明目益气、令人有子、解热清隔、利肠胃、痰火能疗、久食延年、消除疲劳、收敛、治夜盲症、烫火伤等。
藻胆蛋白是广泛存在于红藻、蓝绿藻和隐藻的藻胆体(Phycobilisomes，PBS)中的捕光色素蛋白，包括藻蓝蛋白(Phycocyanin，PC)、藻红蛋白(Phycoerythrim，PE)、藻红蓝蛋白(Phycoerythrocyanin，PEC)和别藻胆蛋白(Allphycocyanin，APC)四类。葛仙米藻胆蛋白含量丰富，优于其他藻类。藻红蛋白是一类附加值非常高的荧光色素蛋白，可用于免疫分析中可溶性抗原、抗体检测诊断试剂、荧光探针、荧光标记等。目前市面上1mg纯度在95％以上的藻红蛋白售价达到5000元以上，纯度越高售价剧增。
藻胆蛋白的纯化通常首先需要经过如盐析法、等电点法或结晶法等初步分离除去杂蛋白后，再通过柱层析法纯化，包括羟基石灰石吸附层析法、纤维素系列离子交换层析法、亲和层析和分子排阻层析法。
在藻胆蛋白粗提粉中，杂蛋白含量极高。要分离得到商品应用标准的藻红蛋白，用已报道的藻胆蛋白分离纯化方法，操作复杂，导致生产成本太高，无法大量制备以满足潜在的市场需求。因此，有必要开发一种简单有效的适合大量制备分离纯化藻红蛋白的方法，为实现葛仙米藻红蛋白的工业化生产提供技术基础。
发明内容
本发明的目的是提供一种高效提取及纯化葛仙米藻红蛋白的方法和纯化的藻红蛋白。
为实现上述目的，本发明提供了一种葛仙米藻胆蛋白的提取方法，包括以下步骤：(a)将葛仙米干粉加入水中；(b)加入液氮，不断搅拌，待液氮挥发完全；(c)离心过滤，得到葛仙米藻胆蛋白提取液；(d)冷冻干燥得到葛仙米藻胆蛋白粗提粉末。
步骤(a)中，所述水可以是纯水、去离子水、蒸馏水等。
步骤(a)中，所述葛仙米干粉和水的质量比为1:40～1:70；优选地，为1:50，或1:60。
步骤(b)中，所述液氮的加入量为葛仙米干粉和水总质量的0.5％～1％。
步骤(b)中，所述搅拌的时间为4h-10h；优选地，为6h。
步骤(c)中，所述离心的转速为5000rpm-8000rpm；离心的时间为20min-30min；可以采用碟式离心机离心过滤。
本发明还提供了一种葛仙米藻红蛋白的纯化方法，包括以下步骤：(1)选用阴离子交换柱过柱；(2)配置PB缓冲液Buffer A和PB-NaCl缓冲液Buffer B，过滤；(3)葛仙米藻胆蛋白粗提粉末用PB缓冲液Buffer A溶解，离心，过滤；(4)洗泵，用PB缓冲液Buffer A平衡离子交换柱；(5)将步骤(3)制备的溶解于PB缓冲液Buffer A中的葛仙米藻胆蛋白直接上样，PB-NaCl缓冲液Buffer B梯度洗脱，收集洗脱液；(6)用盐溶液清洗离子交换柱，然后用碱溶液溶液反向冲洗；(7)对步骤(5)的洗脱液进行超滤浓缩，透析，得到纯化的葛仙米藻红蛋白水溶液；(8)步骤(7)得到的葛仙米藻红蛋白水溶液0～10℃保存。
步骤(1)中，所述过柱前先用去离子水置换阴离子交换柱中的20％的乙醇水，其目的是阴离子交换柱用20％的乙醇水保存，使用前应该将其置换掉。
步骤(1)中，所述阴离子交换柱为Agarosix FF-DEAE阴离子交换柱。所述阴离子交换柱的介质为粒径为50～150μm，交联度为6％的琼脂糖凝胶组成；优选地，为粒经为90μm，交联度为6％的交联琼脂糖凝胶组成；所述琼脂糖凝胶是依靠糖链之间的次级链如氢键来维持网状结构，网状结构的疏密依靠琼脂糖的浓度。一般情况下，琼脂糖凝胶的结构是稳定的，可以在许多条件下使用(如水，pH4-9范围内的盐溶液)；琼脂糖凝胶在40℃以上开始融化，不能高压消毒，可用化学灭菌活处理。交联琼脂糖凝胶是生物分离中常用的色谱基质，利用不同的功能基对其表面的羟基进行修饰，进而制备出疏水色谱、离子交换色谱和亲和色谱等商业填料。
步骤(2)中，所述PB缓冲液的pH为5～6.5，电导率为5～10μs/cm；优选地，pH为6～6.5，电导率为6～8μs/cm；进一步优选地，pH为5、6、6.3、6.5，电导率分别为8.5μs/cm、8.0μs/cm、7.1μs/cm、6.0μs/cm。
步骤(2)中，所述PB-NaCl缓冲液的pH为5～6.5，电导率为5～10μs/cm；优选地，pH为6～6.5，电导率为6～8μs/cm；进一步优选地，pH为5、6、6.3、6.5，电导率分别为8.5μs/cm、8.0μs/cm、7.1μs/cm、6.0μs/cm。
步骤(2)中，进一步优选地，PB缓冲液pH为5时，电导率为8.5μs/cm时，PB-NaCl缓冲液pH为5，电导率为8.5μs/cm；或，PB缓冲液pH为6时，电导率为8.0μs/cm时，PB-NaCl缓冲液pH为6，电导率为8.0μs/cm；或，PB缓冲液pH为6.3时，电导率为7.1μs/cm时，PB-NaCl缓冲液pH为6.3，电导率为7.1μs/cm；或，PB缓冲液pH为6.5时，电导率为6.0μs/cm 时，PB-NaCl缓冲液pH为6.5，电导率为6.0μs/cm。
步骤(2)中，所述PB-NaCl缓冲液的配制方法为在配置好的PB缓冲液中添加NaCl固体，边加边测电导率，直至电导率达到要求。
步骤(2)中，所述过滤是指用0.2μm的过滤膜减压过滤，除去固体杂质；采用减压泵减压抽滤的方法进行减压
步骤(3)中，所述葛仙米藻胆蛋白粗提粉末可以按上述方法制备得到；或所述葛仙米藻胆蛋白粗提粉末是由葛仙米干粉和纯水(料液比1:60)提取6h，过滤，冷冻干燥得到。
步骤(3)中，所述葛仙米藻胆蛋白粗提粉末、PB缓冲液的用量比5g:1L～15g:1L；优选地，为10g:1L。
步骤(3)中，所述离心转速可以但不限于5000～10000r/min，如8000r/min。
步骤(3)中，所述离心时间为20min～40min；优选地，为30min。
步骤(3)中，所述过滤优选采用0.2μm的滤膜过滤。
步骤(4)中，所述洗泵时用去离子水洗1～1.5个柱体积；优选地，用去离子水洗1个柱体积。
步骤(4)中，所述平衡过程用3～10个柱体积的PB缓冲液；优选地，用4～6个柱体积的PB缓冲液；进一步优选地，用5个柱体积的PB缓冲液Buffer A。
步骤(5)中，所述PB缓冲液的pH为5～6.5，电导率为5～10μs/cm；优选地，pH为6～6.5，电导率为6～8μs/cm；进一步优选地，pH为6、6.3、6.5，电导率分别为8.0μs/cm、7.1μs/cm、6.0μs/cm。
步骤(5)中，所述PB-NaCl缓冲液的pH为5～6.5，电导率为5～10μs/cm；优选地，pH为6～6.5，电导率为6～8μs/cm；进一步优选地，pH为6、6.3、6.5，电导率分别为8.0μs/cm、7.1μs/cm、6.0μs/cm。
步骤(5)中，进一步优选地，PB缓冲液pH为5，电导率为8.5μs/cm时，PB-NaCl缓冲液pH为5，电导率为8.5μs/cm；或，PB缓冲液pH为6，电导率为8.0μs/cm时，PB-NaCl缓冲液pH为6，电导率为8.0μs/cm；或，PB缓冲液pH为6.3，电导率为7.1μs/cm时，PB-NaCl缓冲液pH为6.3，电导率为7.1μs/cm；或，PB缓冲液pH为6.5，电导率为6.0μs/cm时，PB-NaCl缓冲液pH为6.5，电导率为6.0μs/cm。
步骤(5)中，所述上样量为2～4个柱体积；优选地，为2、2.5、3、4个柱体积。
步骤(5)中，所述收集的洗脱液为4～5个柱体积20～100％的洗脱液，优选为40～100％的洗脱液。
步骤(6)中，所述盐溶液优选为NaCl或KCl，所述盐溶液的浓度为0.02～0.1M；优选 地，为0.05M。
步骤(6)中，所述盐溶液的用量为1～2个柱体积。
步骤(6)中，所述碱溶液优选为NaOH或KOH，所述碱溶液的浓度为0.1～0.5M；优选地，为0.1M。
步骤(6)中，所述碱溶液的用量为3～4个柱体积。
步骤(7)中，所述超滤膜孔径为1000D～8000D；优选地，为3000D。
步骤(7)中，所述透析可采用任何常规透析方法进行。
步骤(8)中，所述水溶液保存的温度优选为4℃。
具体地，本发明藻红蛋白的纯化方法，包括以下步骤：将葛仙米藻胆蛋白粗提粉末用pH为6～6.5、电导率为6～8μs/cm的PB缓冲液Buffer A溶解，8000r/min离心30min，用0.2μm的滤膜过滤；Agarosix FF-DEAE阴离子交换柱过柱，用去离子水置换的6％的交联琼脂糖凝胶介质灌柱，用1个柱体积的去离子水洗泵，5个柱体积的PB缓冲液Buffer A平衡，上样4个柱体积，收集pH为6～6.5、电导率为6～8μs/cm的PB-NaCl缓冲液Buffer B的梯度洗脱液4～5个柱体积，用3000D的超滤膜超滤浓缩，透析，水溶液4℃保存。
本发明还提供了一种由上述纯化方法纯化得到的葛仙米藻红蛋白；所述藻红蛋白的藻红蛋白的相对分子量为67KD-70KD。
与现有技术相比，本发明的有益效果在于：
i)本发明选用的阴离子交换柱如Agarosix FF-DEAE阴离子交换柱显著提高了纯化效率，省去现有纯化方法中需采用硫酸铵沉淀的步骤，并且大大提高了藻红蛋白的浓度和纯度，方法简单，效果明显，可处理量大，为工业化应用提供了基础。
ii)本发明选用的上样条件经实验证明上样量大，一次可纯化的蛋白样品多，纯化方法简单，成本低，大大提高了藻红蛋白的纯化效率，为实现工业化大生产提供基础。
iii)本发明可以去除藻胆蛋白粗提粉末中大部分的杂质，使得藻红蛋白的提取率接近100％，损失量少。
iv)本发明得到的葛仙米藻红蛋白浓度和纯度都得到显著提高，可以将藻红蛋白的纯度提高到90％以上，甚至更高。
v)本发明纯化的藻红蛋白，产品附加值高，可应用于化妆品、食品、保健食品以及生物医药等领域，有广阔的开发前景。
附图说明
图1表示实施例7中纯化的藻红蛋白的凝胶过滤色谱图。凝胶过滤层析根据蛋白质的分子量的不同在不同的时间出峰，经过峰面积与总面积的比值计算得到每次检测样品的纯度。 根据出峰位置估算藻红蛋白的相对分子量为67KD-70KD。
图2表示实施例7中纯化的藻红蛋白与藻红蛋白对照品的紫外光谱(1.藻红蛋白；2.藻红蛋白标准品)。通过对比，两个样品的出峰位置基本相同，有相近的共轭结构，是同类样品。
图3表示实施例7中纯化的藻红蛋白与藻红蛋白对照品的荧光光谱(1.藻红蛋白；2.藻红蛋白标准品)。通过对比，两个样品的出峰位置相同，说明是同类样品。
图4表示三组分SGS-PAGE分析图(1：Marker(Mr从下至上分别是14.3kD、20.1kD、29.0kD、44.3kD、66.4kD、97.2kD)；2：按本发明上述方法制备的藻胆蛋白粗提粉末；3：实施例7纯化得到的藻红蛋白；4：藻蓝蛋白)根据电泳数据分析藻红蛋白的亚基分子量约为14kD。
具体实施方式
结合以下具体实施例和附图，对本发明作进一步的详细说明。实施本发明的过程、条件、实验方法等，除以下专门提及的内容之外，均为本领域的普遍知识和公知常识，本发明没有特别限制内容。实施例中未注明的具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购买得到的常规产品。
本发明藻红蛋白浓度的检测方法为考马斯亮蓝G250染色法，测定波长为595nm处的吸光值，按李合生的《植物生理生化实验原理和技术》记载方法绘制标准曲线并计算，购买藻红蛋白标准品所测得的藻红蛋白标准曲线为OD595＝0.8096c+0.0084，R2＝0.9986；浓度的计算方法为：浓度c＝(OD595-0.0084)÷0.8096。
本发明藻红蛋白的纯度由紫外吸收光谱仪检测，纯度的计算方法为：
纯度＝A545/A280；纯度％＝A545/(A545+A280)×100％。
实施例1 采用DEAE-Cellulose 52阴离子交换柱进行实验室小试纯化藻红蛋白
将50.01g、50.03g、49.99g葛仙米干粉分别加入2500mL纯水，搅拌均匀后加入20mL液氮，边加边剧烈搅拌。待液氮挥发完全后，继续搅拌，待碎冰完全溶解，离心过滤，冷冻干燥分别得到10.02g、9.98g、10.01g葛仙米藻胆蛋白粗提粉末。分别用pH为7.0、电导率为5.7μs/cm的PB缓冲液Buffer A溶解，8000r/min离心30min，用0.2μm的滤膜过滤；采用DEAE-Cellulose 52阴离子交换柱过柱：1)用1个柱体积的去离子水洗泵；2)用5个柱体积的PB缓冲液Buffer A平衡；3)上样2个柱体积。收集40～100％pH为7.0、电导率为5.7μs/cm的PB-NaCl洗脱液，结果见下表1：
表1
  藻红蛋白浓度(g/mL) 纯度 纯度(％)
第一次 0.96 4.87 83
第二次 0.96 4.87 83
第三次 0.96 4.87 83
由上表1可见，三次纯化得到藻红蛋白浓度均值为0.96g/mL，纯度均值为4.87(83％)。经过三次重复实验验证了本实验有很好的重现性。但是纯化的藻红蛋白的纯度和浓度偏低，可见DEAE-Cellulose 52阴离子交换柱纯化藻红蛋白效率不高，效果较差。
实施例2 采用DEAE-Cpato阴离子交换柱进行实验室小试纯化藻红蛋白
将50.11g、50.07g、49.91g葛仙米干粉分别加入2500mL纯水，搅拌均匀后加入20mL液氮，边加边剧烈搅拌。待液氮挥发完全后，继续搅拌，待碎冰完全溶解，离心过滤，冷冻干燥分别得到9.97g、10.03g、10.01g葛仙米藻胆蛋白粗提粉末，分别用pH为7.0、电导率为5.7μs/cm的PB缓冲液Buffer A溶解，8000r/min离心30min，用0.2μm的滤膜过滤；采用DEAE-Cpato阴离子交换柱过柱：1)用1个柱体积的去离子水洗泵；2)用5个柱体积的PB缓冲液Buffer A平衡；3)上样2个柱体积。收集40～100％pH为7.0、电导率为5.7μs/cm的PB-NaCl洗脱液，结果见下表2：
表2
  藻红蛋白浓度(g/mL) 纯度 纯度(％)
第一次 1.01 5.21 83.9
第二次 1.01 5.21 83.9
第三次 1.01 5.21 83.9
由上表2可见，三次纯化得到藻红蛋白浓度均值为1.01g/mL，纯度均值为5.21(83.9％)。经过三次重复实验验证了本实验有很好的重现性。同样，纯化的藻红蛋白的纯度和浓度偏低，可见DEAE-Cpato柱纯化藻红蛋白效率不高，效果较差。
实施例3 采用DEAE-Sephadex Fast Flow阴离子交换柱进行实验室小试纯化藻红蛋白
将49.98g、50.13g、49.92g葛仙米干粉分别加入2500mL纯水，搅拌均匀后加入20mL液氮，边加边剧烈搅拌。待液氮挥发完全后，继续搅拌，待碎冰完全溶解，离心过滤，冷冻干燥分别得到9.96g、9.97g、10.02g葛仙米藻胆蛋白粗提粉末，分别用pH为7.0、电导率为5.7μs/cm的PB缓冲液Buffer A溶解，8000r/min离心30min，用0.2μm的滤膜过滤；采用DEAE-Sephadex Fast Flow阴离子交换柱过柱：1)用1个柱体积的去离子水洗泵；2)用5个柱体积的PB缓冲液Buffer A平衡；3)上样1个柱体积，收集40～100％pH为7.0、电导率为5.7μs/cm的PB-NaCl洗脱液，结果见下表3：
表3
  藻红蛋白浓度(g/mL) 纯度 纯度(％)
第一次 1.02 6.02 85.8
第二次 1.02 6.02 85.8
第三次 1.02 6.02 85.8
由上表3可见，三次纯化得到藻红蛋白浓度均值为1.02g/mL，纯度均值为6.02(85.8％)。经过三次重复实验验证了本实验有很好的重现性。但纯化的藻红蛋白的纯度和浓度不高，可见DEAE-Sephadex Fast Flow柱纯化藻红蛋白虽然较以上两种阴离子交换柱纯度稍有提高，但是效率仍然不理想，效果较差。
实施例4 采用Agarosix FF-DEAE阴离子交换柱进行实验室小试纯化藻红蛋白
将50.09g、50.03g、49.99g葛仙米干粉分别加入2500mL纯水，搅拌均匀后加入20mL液氮，边加边剧烈搅拌。待液氮挥发完全后，继续搅拌，待碎冰完全溶解，离心过滤，冷冻干燥分别得到9.96g、9.91g、10.01g葛仙米藻胆蛋白粗提粉末，分别用pH为7.0、电导率为5.7μs/cm的PB缓冲液Buffer A溶解，8000r/min离心30min，用0.2μm的滤膜过滤；采用Agarosix FF-DEAE阴离子交换柱过柱：1)用1个柱体积的去离子水洗泵；2)用5个柱体积的PB缓冲液Buffer A平衡；3)上样2个柱体积。收集40～100％pH为7.0、电导率为5.7μs/cm的PB-NaCl洗脱液，结果见下表4：
表4
  藻红蛋白浓度(g/mL) 纯度 纯度(％)
第一次 2.09 9.68 90.6
第二次 2.09 9.68 90.6
第三次 2.12 9.68 90.6
由上表4可见，三次纯化得到藻红蛋白浓度均值为2.10g/mL，纯度均值为9.68(94.1％)。经过三次重复实验验证了本实验有很好的重现性。另外，经Agarosix FF-DEAE阴离子交换柱纯化的藻红蛋白的纯度和浓度均有显著提高，可见Agarosix FF-DEAE阴离子交换柱适用于藻红蛋白的纯化。
实施例5 采用本发明方法进行实验室小试纯化藻红蛋白
将60.01g、60.11g、60.03g葛仙米干粉分别加入3000mL纯水，搅拌均匀后加入25mL液氮，边加边剧烈搅拌。待液氮挥发完全后，继续搅拌，待碎冰完全溶解，离心过滤，冷冻干燥分别得到12.51g、12.47g、12.53g葛仙米藻胆蛋白粗提粉末，分别用pH为5.0、电导率为8.5μs/cm的PB缓冲液Buffer A溶解，8000r/min离心30min，用0.2μm的滤膜过滤；采用Agarosix FF-DEAE阴离子交换柱过柱：1)用1个柱体积的去离子水洗泵；2)用5个柱体积的PB缓冲液Buffer A平衡；3)上样2.5个柱体积。收集40～100％pH为5.0、电导率为8.5μs/cm的PB-NaCl洗脱液，结果见下表5：
表5
  藻红蛋白浓度(g/mL) 纯度 纯度(％)
第一次 2.51 10.01 90.9
第二次 2.51 10.01 90.9
第三次 2.51 10.01 90.9
由上表5可见，三次纯化得到藻红蛋白浓度均值为2.51g/mL，纯度均值为10.01(90.9％)。经过三次重复实验验证了本实验有很好的重现性。另外，经Agarosix FF-DEAE阴离子交换柱纯化的藻红蛋白的纯度和浓度均有显著提高，可见Agarosix FF-DEAE阴离子交换柱适用于藻红蛋白的纯化。
实施例6 采用本发明方法进行实验室小试纯化藻红蛋白
将75.01g、75.03g、75.99g葛仙米干粉分别加入3750mL纯水，搅拌均匀后加入30mL液氮，边加边剧烈搅拌。待液氮挥发完全后，继续搅拌，待碎冰完全溶解，离心过滤，冷冻干燥分别得到15.00g、14.97g、15.03g葛仙米藻胆蛋白粗提粉末，分别用pH为6.0、电导率为8.0μs/cm的PB缓冲液Buffer A溶解，8000r/min离心30min，用0.2μm的滤膜过滤；采用Agarosix FF-DEAE阴离子交换柱过柱：1)用1个柱体积的去离子水洗泵；2)用5个柱体积的PB缓冲液Buffer A平衡；3)上样3个柱体积。收集40～100％pH为6.0、电导率为8.0μs/cm的PB-NaCl洗脱液，结果见下表6：
表6
  藻红蛋白浓度(g/mL) 纯度 纯度(％)
第一次 3.03 13.62 93.2
第二次 3.03 13.62 93.2
第三次 3.03 13.62 93.2
由上表6可见，三次纯化得到藻红蛋白浓度均值为3.03g/mL，纯度均值为13.62(93.2％)。经过三次重复实验验证了本实验有很好的重现性。另外，经Agarosix FF-DEAE阴离子交换柱纯化的藻红蛋白的纯度和浓度均有显著提高，可见Agarosix FF-DEAE阴离子交换柱适用于藻红蛋白的纯化。
实施例7 采用本发明方法进行实验室小试纯化藻红蛋白
将100.01g、100.13g、100.09g葛仙米干粉分别加入5000mL纯水，搅拌均匀后加入40mL液氮，边加边剧烈搅拌。待液氮挥发完全后，继续搅拌，待碎冰完全溶解，离心过滤，冷冻干燥分别得到20.02g、19.98g、20.01g葛仙米藻胆蛋白粗提粉末，分别用pH为6.3、电导率为7.1μs/cm的PB缓冲液Buffer A溶解，8000r/min离心30min，用0.2μm的滤膜过滤；采用Agarosix FF-DEAE阴离子交换柱过柱：1)用1个柱体积的去离子水洗泵；2)用5个柱体积的PB缓冲液Buffer A平衡；3)上样4个柱体积。收集40～100％pH为6.3、电导率为7.1μs/cm的PB-NaCl洗脱液，结果见下表7：
表7
  藻红蛋白浓度(g/mL) 纯度 纯度(％)
第一次 4.21 16.43 94.3
第二次 4.21 16.43 94.3
第三次 4.21 16.43 94.3
由上表7可见，三次纯化得到藻红蛋白浓度均值为4.21g/mL，纯度均值为16.43(94.3％)。经过三次重复实验验证了本实验有很好的重现性。实验发现，当上样条件为：用pH为6.3、电导率为7.1μs/cm的PB缓冲液Buffer A溶解时，可以达到上样4个柱体积，所得到的藻红蛋白浓度最高，为4.21g/mL；同时，洗脱条件为pH为6.3、电导率为7.1μs/cm的PB-NaCl缓冲液Buffer B梯度淋洗可以得到纯度为16.43的藻红蛋白，大大提高了纯化效率。
实施例7中纯化的藻红蛋白的凝胶过滤色谱图如图1所示。凝胶过滤层析根据蛋白质的分子量的不同在不同的时间出峰，经过峰面积与总面积的比值计算得到每次检测样品的纯度。出峰位置约为8mL，根据标准图谱，9mL左右分子量约为67KD，估算藻红蛋白的相对分子量为67KD-70KD。
实施例7中纯化的藻红蛋白与藻红蛋白对照品的紫外光谱如图2所示。通过对比，两个样品的出峰位置基本重合，吸收峰为542nm和565nm，有相近的共轭结构，判断为同类样品。
实施例7中纯化的藻红蛋白与藻红蛋白对照品的荧光光谱如图3所示。通过对比，两个样品的特征出峰位置相同(575nm)，说明是同类样品。
实施例8 采用本发明方法进行实验室小试纯化藻红蛋白
将75.11g、75.03g、74.99g葛仙米干粉分别加入3750mL纯水，搅拌均匀后加入30mL液氮，边加边剧烈搅拌。待液氮挥发完全后，继续搅拌，待碎冰完全溶解，离心过滤，冷冻干燥分别得到15.02g、14.92g、15.03g葛仙米藻胆蛋白粗提粉末，分别用pH为6.5、电导率为6.0μs/cm的PB缓冲液Buffer A溶解，8000r/min离心30min，用0.2μm的滤膜过滤；采用Agarosix FF-DEAE阴离子交换柱过柱：1)用1个柱体积的去离子水洗泵；2)用5个柱体积的PB缓冲液Buffer A平衡；3)上样3个柱体积。收集40～100％pH为6.5、电导率为6.0μs/cm的PB-NaCl洗脱液，结果见下表8：
表8
  藻红蛋白浓度(g/mL) 纯度 纯度(％)
第一次 3.02 14.12 93.4
第二次 3.02 14.12 93.4
第三次 3.02 14.12 93.4
由上表8可见，三次纯化得到藻红蛋白浓度均值为3.02g/mL，纯度均值为14.12(93.4％)。经过三次重复实验验证了本实验有很好的重现性。
实施例9 采用本发明方法进行实验室小试纯化藻红蛋白
将50.08g、50.30g、49.94g葛仙米干粉分别加入2500mL纯水，搅拌均匀后加入20mL液氮，边加边剧烈搅拌。待液氮挥发完全后，继续搅拌，待碎冰完全溶解，离心过滤，冷冻干燥分别得到10.02g、9.91g、10.01g葛仙米藻胆蛋白粗提粉末用pH为7.5、电导率为5.8μs/cm的PB缓冲液Buffer A溶解，8000r/min离心30min，用0.2μm的滤膜过滤；采用Agarosix FF-DEAE阴离子交换柱过柱：1)用1个柱体积的去离子水洗泵；2)用5个柱体积的PB缓冲液Buffer A平衡；3)上样2个柱体积。收集40～100％pH为7.5、电导率为5.9μs/cm的PB-NaCl洗脱液，结果见下表9：
表9
  藻红蛋白浓度(g/mL) 纯度 纯度(％)
第一次 2.11 3.71 78.7
第二次 2.11 3.71 78.7
第三次 2.11 3.71 78.7
由上表9可见，三次纯化得到藻红蛋白浓度均值为2.11g/mL，纯度均值为3.71(78.7％)。经过三次重复实验验证了本实验有很好的重现性。但是PB缓冲液Buffer A和PB-NaCl缓冲液Buffer B的pH超过6.5时，纯化效果明显下降。
实施例10 采用本发明方法进行实验室小试纯化藻红蛋白
将50.02g、49.93g、49.99g葛仙米干粉分别加入2500mL纯水，搅拌均匀后加入20mL液氮，边加边剧烈搅拌。待液氮挥发完全后，继续搅拌，待碎冰完全溶解，离心过滤，冷冻干燥分别得到9.98g、9.95g、10.03g葛仙米藻胆蛋白粗提粉末，分别用pH为9.3、电导率为9.2μs/cm的PB缓冲液Buffer A溶解，8000r/min离心30min，用0.2μm的滤膜过滤；用1个柱体积的去离子水洗泵，5个柱体积的PB缓冲液Buffer A平衡，上样4个柱体积，收集40～100％pH为9.3、电导率为9.5μs/cm的PB-NaCl洗脱液，结果见下表10：
表10
  藻红蛋白浓度(g/mL) 纯度 纯度(％)
第一次 2.09 2.74 73.2
第二次 2.09 2.74 73.2
第三次 2.11 2.74 73.2
由上表10可见，三次纯化得到藻红蛋白浓度均值为2.10g/mL，纯度均值为2.74(73.2％)。经过三次重复实验验证了本实验有很好的重现性。得到的藻红蛋白的浓度均显著低于其他实施例，说明该上样和洗脱的pH范围是不合适的，适宜的洗脱条件为PB缓冲液Buffer A的pH为5～6.5，电导率为5～10μs/cm，上样条件为PB-NaCl缓冲液Buffer B的pH为5～6.5，电导率为5～10μs/cm。
对比例1
将10.03g、9.96g、10.02g葛仙米藻胆蛋白粗提粉末按文献“李邵蓉，Rhodosorus marinus中藻红蛋白的纯化及其性质的研究”，用Tris-HCl缓冲液(pH＝8.4、0.1mol/L，下同)溶解，用1个柱体积的去离子水洗泵，5个柱体积的Tris-HCl缓冲液平衡，上样4个柱体积，用0.1mol/L、0.2mol/L、0.5mol/L的NaCl溶液分步洗脱，收集红色样品对Tris-HCl缓冲液透析，再通过一次同样的DEAE纤维柱，收集红色样品，将收集的样品对PB缓冲液(pH＝6.6，0.1mol/L)充分透析，上Bio-gel p300柱，收集两条红色带，检测结果见下表11：
表11
  藻红蛋白浓度(g/mL) 纯度 纯度(％)
第一次 0.60 4.3 81.1
第二次 0.59 4.3 81.1
第三次 0.60 4.3 81.1
由上表11可见，三次纯化得到藻红蛋白浓度均值为0.597g/mL，纯度均值为4.3(81.1％)，其浓度以及纯度均显著低于实施例1～4。经过三次重复实验验证。
对比例1中选用的缓冲体系Tris-HCl及pH使得纯化效果较差，纯化操作步骤繁杂，增加了实际操作的成本。本发明方法选用的PB缓冲体系更适合藻红蛋白的纯化，一次层析即可完成纯化过程，而且藻红蛋白纯度、浓度高于对比例1。因此，本发明的方法可操作性强，操作简便，使用成本低，纯化得到的藻红蛋白的浓度和纯度高。
对比例2
将10.01g、10.04g、10.03g葛仙米藻胆蛋白粗提粉末按文献“程凌江，条斑紫菜中R-藻红蛋白的纯化及其和亚基的分离与发色团含量的测定”，用15％的NH4SO4盐析除杂，用Bio-gel p300柱进一步纯化，洗脱液为pH＝6.8的0.1mol/L磷酸盐缓冲液(含1mmol/L叠氮钠和mmol/L β-巯基乙醇)，洗脱液流速15mL/h，收集红色带，检测结果见下表12：
表12
  藻红蛋白浓度(g/mL) 纯度 纯度(％)
第一次 0.67 8.1 89
第二次 0.67 8.1 89
第三次 0.67 8.1 89
由上表12可见，三次纯化得到的藻红蛋白的浓度均值为0.67g/mL，纯度均值为8.1(89％)，其浓度以及纯度均显著低于实施例3～4。经过三次重复实验验证。
对比例2中选用的PB缓冲体系pH较低，体系偏酸性，使得藻红蛋白的纯化虽然经过两 步纯化，但纯化的纯度不理想，最高仅为89％。本发明方法得到的藻红蛋白的纯度显著高于对比例2，而且本发明方法通过一次层析即可完成纯化过程，得到的藻红蛋白纯度和浓度均高于对比例2。因此，本发明的方法可操作性强，操作简便，使用成本低，得到的藻红蛋白浓度高、纯度高。
由实施例1～10和对比例1～2可见，(1)本发明所选用的Agarosix FF-DEAE阴离子交换柱的填料是6％的交联度琼脂糖凝胶，在纯化中表现出高载量(120mg/mL)和高稳定性(操作温度4～40℃，操作压力≦3Bar)，性能更好。实验证明本发明方法适于采用该树脂用于纯化藻红蛋白，效果明显优于其他DEAE阴离子交换柱(包括DEAE-52纤维素柱以及DEAE-Sephadex Fast Flow柱)；且藻红蛋白质损失少，上样量大，纯度高，可能是因为本发明采用的离子交换柱配基与藻红蛋白结合，而与其他杂质蛋白不结合，提高了藻红蛋白的吸附量；(2)在缓冲液pH值为5～6.5时，本发明方法得到的藻红蛋白的纯度显著高于对比例，是因为只有在该合适的pH范围内，PB体系中的磷酸盐离子包裹的藻红蛋白的带负电部分裸露，使得藻红蛋白尽可能多地吸附于Agarosix FF-DEAE柱上，而其他的杂蛋白不被吸附，在上样平衡时就会被排除在柱外；Tris-HCl体系则很难使藻红蛋白吸附。(3)本发明可以通过一步Agarosix FF-DEAE分离得到纯度高、产量高(上样量大，得到的藻红蛋白浓度高)的藻红蛋白，大大简化了实验步骤。
综上所述，采用本发明方法可以从葛仙米藻胆蛋白粗提粉末中得到浓度在2.51g/mL以上，纯度在10.01(90.9％)以上的藻红蛋白，并且本发明方法上样量大、方法简单、工艺周期短；同时本发明方法还将纯化的藻红蛋白的纯度从已有文献中报道的4.3(81.1％)提高到16.43(94.3％)，显著提高了藻红蛋白的纯度，具有很大的应用前景。
本发明的保护内容不局限于以上实施例。在不背离发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求书为保护范围。

Claims (12)

  1. 一种葛仙米藻红蛋白的纯化方法，其特征在于，包括步骤：(1)选用阴离子交换柱过柱；(2)配置PB缓冲液Buffer A和PB-NaCl缓冲液Buffer B；(3)葛仙米藻胆蛋白粗提粉末用PB缓冲液Buffer A溶解，离心，过滤；(4)洗泵，用PB缓冲液Buffer A平衡离子交换柱；(5)将步骤(3)制备的溶解于PB缓冲液Buffer A中的葛仙米藻胆蛋白直接上样，PB-NaCl缓冲液Buffer B梯度洗脱，收集洗脱液；(6)用盐溶液清洗离子交换柱，然后用碱溶液反向冲洗；(7)对步骤(5)的洗脱液进行超滤浓缩，透析，得到纯化的葛仙米藻红蛋白水溶液；其中，PB缓冲液Buffer A的pH为5～6.5；PB-NaCl缓冲液Buffer B的pH为5～6.5。
  2. 如权利要求1所述的方法，其特征在于，步骤(2)中，PB缓冲液Buffer A的电导率为5～10μs/cm；PB-NaCl缓冲液Buffer B的电导率为5～10μs/cm。
  3. 如权利要求1所述的方法，其特征在于，步骤(2)中，所述Buffer A缓冲液和Buffer B缓冲液的pH为6～6.5，电导率为6～8μs/cm。
  4. 如权利要求1所述的方法，其特征在于，步骤(2)中，所述PB缓冲液pH为5，电导率为8.5μs/cm时，PB-NaCl缓冲液pH为5，电导率为8.5μs/cm；或，PB缓冲液pH为6，电导率为8.0μs/cm时，PB-NaCl缓冲液pH为6，电导率为8.0μs/cm；或，PB缓冲液pH为6.3，电导率为7.1μs/cm时，PB-NaCl缓冲液pH为6.3，电导率为7.1μs/cm；或，PB缓冲液pH为6.5，电导率为6.0μs/cm时，PB-NaCl缓冲液pH为6.5，电导率为6.0μs/cm。
  5. 如权利要求1所述的方法，其特征在于，步骤(1)中，所述阴离子交换柱为Agarosix FF-DEAE阴离子交换柱。
  6. 如权利要求1所述的方法，其特征在于，步骤(1)中，所述阴离子交换柱的介质为粒径为50～150μm，交联度为6％的琼脂糖凝胶。
  7. 如权利要求1所述的方法，其特征在于，所述步骤(1)过柱前先用去离子水置换离子交换柱介质中的20％的乙醇水。
  8. 如权利要求1所述的方法，其特征在于，步骤(3)中，所述葛仙米藻胆蛋白粗提粉末、PB缓冲液的用量比5g:1L～15g:1L。
  9. 如权利要求1所述的方法，其特征在于，步骤(4)中，所述超滤膜孔径为1000D～8000D。
  10. 如权利要求1所述的方法，其特征在于，步骤(6)中，所述NaCl或KCl的浓度为0.02～0.1M；所述NaOH或KOH的浓度为0.1～0.5M。
  11. 一种由权利要求1～10之任一项所述方法得到的葛仙米藻红蛋白。
  12. 一种葛仙米藻胆蛋白的提取方法，其特征在于，所述方法包括以下步骤：(a)将葛仙米干粉加入水中；(b)加入液氮，搅拌，待液氮挥发完全；(c)离心过滤，得到葛仙米藻胆蛋白提取液；(d)冷冻干燥得到葛仙米藻胆蛋白粗提粉末。
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