A kind of method of extracting Polysialic acid
Technical field
A kind of method of extracting Polysialic acid belongs to technical field of bioengineering.
Background technology
Sialic acid is the derivative of a class neuraminic acid, and its chemical structure is an acid aminosugar that contains 9 carbon atoms and have the pyranose structure, and systematic naming method is a 5-amino-3,5-dideoxy-D-glycerine D-gala ketononose.Constituted different sialic acid derivatives according to different linking group on No. 5 carbon potentials, topmost two kinds of sialic acids are 5-acetylaminohydroxyphenylarsonic acid 3,5-dideoxy-D-glycerine-D-gala ketononose (NANA, Neu5Ac, the N-n acetylneuraminic acid n) and 3-deoxidation-D-glycerine-D-gala ketononose (KDN), remaining sialic acid all is to derive by these two kinds to form.Wherein, the N-n acetylneuraminic acid n is more common in the vertebrates body, as animals such as fish, batrachians, reptiles, birds.Ubiquity sialic acid in the mammalian body is maximum in cerebrospinal fluid and mucus comparatively speaking.The baby is higher at the young stage sialic acid content, and is significant to its body development.
Polysialic acid (Polysialic acid, Colominic acid) is the linear homopolymer of sialic acid with α-2,8 and/or the connection of α-2,9 ketoside key, is the exocellular polysaccharide component of a few bacterium.Nineteen fifty-seven Barry and Goebel at first find this polymkeric substance in Escherichia coli K-235 and K-1, later on again successively at Neisseria miningitidis B, Salmonella toucra 048 finds this material among the Citrobacter freundii 05.α-2, terminal two the adjacent monomers of the Polysialic acid that 8 ketoside keys connect form lactone (the hydroxyl dehydrating condensation on monomeric No. 9 carbon potentials of monomeric carboxyl of first and second forms), the stable of Polysialic acid played a role, this also is the one of the main reasons that most Polysialic acid connects with α-2,8 ketoside key.
Sialic general structure
Polysialic acid is colourless, and is soluble in water, very stable in the aqueous solution, and the storage several months does not change in the time of 4 ℃.If contain very micro-organic acid or pH in the solution greater than 10, its stability just is subjected to very big influence.The free sialic acid can suppress common cold virus and other pathogenic micro-organisms invasion red corpuscle effectively.Sialic analogue Zanamivir (N-ethanoyl-2; 3-dideoxy-4-guanidine radicals sialic acid) the anti influenza mechanism with Tamiflu (oseltamivir phosphate) is the same; the activity that can suppress sialidase on the influenza virus is by the specifics of U.S. FDA official approval for the treatment bird flu.Sialic acid derivative SialylLewis
XAs important anti-adhesive medicine, can effectively stop the excessive gathering of white corpuscle, diseases such as treatment rheumatoid arthritis, septic shock.Simultaneously, sialic acid is a kind of cough-relieving apophlegmatic agent, and is all effective in cure for center and peripheral nerve disease and demyelinating disease.Can treat senile dementia as the sialic acid cholesterol.In addition, the content of sialic acid in brain is very high, may participate in ion outflow and nervous excitation as nerve conduction matter.Monosialoganglioside has certain function for treatment cerebral ischemia, parkinsonism and neural wound.Discovered in recent years, sialic acid also have immunosuppressive action.It with the sialic acid frontier that exploration that the guide carries out biologically active substance has become new drug research.Polysialic acid can be used as the sustained release dosage of some medicines, also can be used for fixing enzyme being used for diagnosis, or some medicament production are carried out chemically modified.The side effect that Polysialic acid can avoid other cellular component such as lipopolysaccharides or protein to cause as the vaccine of treatment some diseases.
In recent years, biodegradable polymer more and more attracts attention in the drug delivery system research field, and partial polymer has been applied to various formulations.That more successfully be used for polypeptide and protein drug transfer system at present is polyoxyethylene glycol (PEG); But PEG is a synthetic, nondegradable in vivo, easily in bladder accumulation, but also may produce the antibody of anti-PEG.In seeking the degradable polymer that substitutes PEG, the Polysialic acid (PSA) that α-2,8 connects is to find the best polypeptide and the control-released agent of protein medicaments at present.The LipoXen company clinical trial of Britain Polysialic acid Interferon, rabbit, Regular Insulin, asparaginase and HRBC growth hormone etc., find its effect than PEGization effective, the transformation period is longer; Wherein mainly use molecular weight and be about the Polysialic acid that 7000Da~15000Da, α-2,8 connects.Along with the fast development at field of medicaments of genetically engineered and protein engineering, following increasing medicine will be range protein and polypeptide drug, so Polysialic acid will have huge market potential as medicine intermediate in future.And also can carry out the large-scale production of Polysialic acid in the world at present without any company, the technology of therefore developing the separation and purification of high purity Polysialic acid is significant.
Summary of the invention
The purpose of this invention is to provide a kind of method of extracting Polysialic acid,, reach the requirement that makeup and medicine are produced, make its competitive power stronger to improve the purity of Polysialic acid.
Technical scheme of the present invention: a kind of method of extracting Polysialic acid, fermented liquid with strain intestinal bacteria (Escherichiacoli) K235-JY II-74 is a raw material, remove thalline through Plate Filtration, supernatant liquor adds an amount of sodium-chlor and alcohol solvent is settled out Polysialic acid, the dissolving of Polysialic acid precipitate with deionized water, remove denatured protein with Plate Filtration again, filtrate is handled through Sumizyme MP, chloro-hexadecane yl pyridines or cetyl trimethylammonium bromide complex-precipitation and dissociate after, go out Polysialic acid with an amount of ethanol sedimentation again, the Polysialic acid precipitation is dissolved with ultrapure water, carry out grading purification by chromatography column, collect elutriant through dialysis, lyophilize gets the Polysialic acid product; Each process condition is:
(1) filter: add 80~200 purpose pearlite filtering aids of 10~20g/L in the fermented liquid, fermented liquid is removed thalline through Plate Filtration, and filter type is selected 110 order industrial filter cloths;
(2) precipitation: the clear liquid that filtration is removed behind the thalline adds sodium-chlor, make the sodium-chlor mass concentration reach 2%~5%, the ethanol that adds 95% mass concentration of 2~4 times of volumes after the dissolving again, be settled out Polysialic acid, and overnight leaving standstill, by decant or centrifugal acquisition Polysialic acid precipitation, use the washing with alcohol 2~4 times of a small amount of 75% mass concentration again;
(3) dissolving, filtration: the dissolving of Polysialic acid precipitate with deionized water, 80~200 purpose pearlite filtering aids of adding 10~20g/L filter by Plate Filtration or diatomite filter, remove partly denatured protein matter by filtering;
(4) enzyme is handled: filter the filtrate of removing behind the partly denatured protein and add Sumizyme MP and handles, its technology is: basic protein enzyme activity 2~50,000 U/mL, and pH 7~8, and dosage is the 10mL/kg Polysialic acid, 30~70 ℃ of enzymolysis 3~7 hours;
(5) complex-precipitation: the Polysialic acid solution that enzyme was handled adds excessive chloro-hexadecane yl pyridines or cetyl trimethylammonium bromide and carries out complex-precipitation, chloro-hexadecane yl pyridines or cetyl trimethylammonium bromide dosage are 1~3g/g Polysialic acid, and temperature is 10~15 ℃;
(6) dissociate: the Polysialic acid precipitation that obtains through complexing adds 2~4 times of sodium chloride solutions of measuring 0.5~0.8M, and 40~60 ℃ of water-baths were dissociated 3~8 hours;
(7) ethanol sedimentation: the solution after dissociating adds the ethanol of 95% mass concentration of 2~4 times of volumes again, is settled out Polysialic acid, and overnight leaving standstill, and by decant or centrifugal acquisition Polysialic acid precipitation, uses the washing with alcohol 2~4 times of a small amount of 75% mass concentration again;
(8) dissolving, column chromatography: the Polysialic acid that ethanol sedimentation goes out carries out chromatographic separation after dissolving with ultrapure water, its technology is: the chromatography column filler is DEAE-Mierocrystalline cellulose or dextrane gel, chromatography column carries out balance with 0.01~0.05M phosphate buffered saline buffer, elutriant is 0.02~0.08M sodium chloride solution, elution speed is 1~5mL/min, collects the Polysialic acid of molecular weight 7000~15000Da and the molecular weight Polysialic acid greater than 15000Da respectively;
(9) dialysis: the Polysialic acid of molecular weight 7000~15000Da that the chromatography collection obtains and molecular weight are that 7000Da, 12000~14000Da dialysis membrane were dialysed 48~72 hours with molecular weight cut-off respectively greater than the Polysialic acid of 15000Da;
(10) lyophilize: two kinds of dialysis liquids obtain the Polysialic acid product of two kinds of specifications respectively through lyophilize.
Beneficial effect of the present invention: the present invention proposes a kind of effective Polysialic acid separation purification method.Fermented liquid precipitates Polysialic acid and protein with excess ethyl alcohol simultaneously, makes with Plate Filtration behind the resolution of precipitate the easier removal of protein of sex change to help the subsequent extracted purifying.Sumizyme MP adds the remaining protein of can degrading in the Polysialic acid solution to, and chloro-hexadecane yl pyridines or cetyl trimethylammonium bromide can form precipitation with the complexing of Polysialic acid specificity, help Polysialic acid and some other macromole, small-molecule substance are separated.Polysialic acid can be collected the Polysialic acid of different molecular weight ranges further by chromatographic separation, utilizes dialysis can remove some small-molecule substances, and through after the lyophilize, the purity of Polysialic acid reaches more than 97%.
Description of drawings
The extraction schematic flow sheet of Fig. 1 Polysialic acid.
The biological material specimens explanation
Intestinal bacteria (Escherichia coli) K235-JY II-74 Southern Yangtze University's biotechnology institute biochemical engineering laboratory preservation and providing, this bacterial strain are open at " Wuxi Light Industry Univ.'s journal " 2002,21 (5), 456~459.According to the relevant regulations of patent examination, the applicant guarantees that this bacterial classification provided to the public in 20 years applyings date.
Embodiment
Embodiment 1. Polysialic acid preparation of fermentation liquid
Bacterial strain intestinal bacteria (Escherichia coli) K235-JY II-74.
Fermention medium (g/L) sorbyl alcohol 20, ammonium chloride 2.0, dipotassium hydrogen phosphate 1.5, MgSO
40.9, Tryptones 1.5, pH 7.8.
Utilize the 30L fermention medium, on 50L B.Braum mechanical agitator tank, adopt the pH control method to ferment, inoculum size 4%, add a small amount of polyoxyethylene polyoxypropylene glyceryl ether (bubble enemy) before the sterilization with the foam in the control fermenting process, fermentor tank rotating speed 300~500rpm, ventilation is 30L/min, gauge pressure 0.05Mpa, 37 ℃ of leavening temperatures.The ammoniacal liquor of regulating the pH value is strong aqua, concentration of hydrochloric acid 3M.The pH in thalli growth stage drops to 6.4 naturally, with ammoniacal liquor and hydrochloric acid the pH value is controlled at about 6.4 then, until fermentation ends.In the fermenting process, certain hour sampling and measuring sorbitol concentration at interval, fermentation begins to add sorbyl alcohol to 12h.According to the variation of concentration of substrate, estimate the wear rate of thalline to substrate, adjust control feed supplement liquid stream rate of acceleration, make carbon source concentration be controlled at 5g/L between the 20g/L.Following jar of fermentation culture 60 hours is measured Polysialic acid output and is reached 3.5g/L.
The extraction of embodiment 2. Polysialic acids
Get the 5L fermented liquid, remove thalline through Plate Filtration, gained is crossed cleaner liquid adding 100-250g sodium-chlor (NaCl) and is stirred and makes its dissolving, the clear liquid of sodium chloride-containing adds the ethanol of 95% mass concentration of 2~4 times of volumes again, be settled out Polysialic acid, and overnight leaving standstill, by decant or centrifugal acquisition Polysialic acid precipitation, use a small amount of 75% mass concentration washing with alcohol 2~4 times again, the Polysialic acid precipitation is dissolved in the proper amount of deionized water more then, the gained troubled liquor is removed partial protein through Plate Filtration, the filtrate that obtains is regulated pH 7~8 and is added about 50mL Sumizyme MP (enzyme 2~50,000 U/mL alive), under 30~70 ℃ of water bath with thermostatic control conditions, handled 3~7 hours, add excessive chloro-hexadecane yl pyridines or cetyl trimethylammonium bromide again and carry out complexing, after overnight the leaving standstill, decant or centrifugal collecting precipitation, the sodium chloride solution that precipitation adds 0.5~0.8M dissociates, 40~60 ℃ of water-baths were dissociated 3~8 hours, dissociate and obtain the ethanol that clear liquid adds 95% mass concentration of 2~4 times of volumes, be settled out Polysialic acid, and overnight leaving standstill, by decant or centrifugal acquisition Polysialic acid precipitation, use a small amount of 75% mass concentration washing with alcohol 2~4 times again, the Polysialic acid precipitation is dissolved in an amount of ultrapure water more then, obtain clarified liq, this solution enters the good chromatography column of balance by peristaltic pump, carry out wash-out with 0.02~0.08M sodium chloride solution, elution speed is 1~5mL/min, collect the Polysialic acid of molecular weight 7000~15000Da and molecular weight Polysialic acid respectively greater than 15000Da, be that 7000Da or 12000~14000Da dialysis membrane were dialysed 48~72 hours with molecular weight cut-off respectively, last dialysis liquid gets the Polysialic acid product through lyophilize, and purity reaches more than 97%.
Table 1 Polysialic acid extracts the yield of purifying
Step |
Filtration sterilization |
Ethanol sedimentation-filtration |
Protease treatment |
Complexing is dissociated |
Chromatography |
Dialysis |
Lyophilize |
The protein removal rate |
84.1% |
89.5% |
92.4% |
98.5% |
99% |
99.5% |
99.8% |
The Polysialic acid yield |
94.5% |
95.2% |
96.1% |
95% |
90% |
98% |
|