TW200521136A - Method of purification for a lectin of marine microalgae - Google Patents

Abstract

A method of purification for a lectin of marine microalgae that separates and purifies the agglutinins from Chlorella luteovirides with high activity. Firstly, an extract is obtained by a method of cytoclasis, which is glass bead with ultrasonication. Then, the extract is divided by a ammonium sulfate solution with 60-80% saturated concentration, and a protein is precipitated and collected. Next, the precipitable protein is dialyzed by phosphate buffer saline, moreover; a suppressor is collected by centrifugation. Subsequently, the suppressor is added to DEAE-C-52 ion exchange chromatography to separate and purify, and an active protein solution is collected. Following that, the protein solution is separated and purified again by CM-C-52 ion exchange chromatography. Finally, the purification is performed by Superdex 75 gel filtration, and the method of a lectin of marine microalgae is successively accomplished.

Description

200521136 V. Description of the invention (1) 1. [Technical field to which the invention belongs] The present invention relates to a method for purifying algae, and more particularly to a method for purifying chlorella lectin. 2. [Prior art] Lectin (agg 1 utinins) refers to an active substance that can bind to red blood cells or normal and other deformed cells to cause agglutination. Lectin is a protein, of which lectin and carbohydrate Compound binding, lectins are distributed in various organisms in nature, such as plants, bacteria, lichens, fungi, earthworms, snails, eels, catfish, shellfish, mammals, insects. Lectin in marine life includes seaweed, sea cucumber, coral and sponge, etc., their structure is very different between different organisms, and the activity of lectin will vary depending on the growth site. At present, the plant lectin has been isolated. More than 200 species are mainly distributed in seeds, but are distributed in smaller amounts in roots, leaves and other parts. Glycoproteins have the ability to combine with red blood cells and other cell surface carbohydrate structures. In various biological species such as chicken bead, Ganoderma lucidum, Flammulina velutipes, ricinus, and castor beans, proteins with immunomodulatory functions and hemolytic proteins can be separated and purified. It can agglutinate human red blood cells and has good immunosuppressive power. In the study of marine algae proteolysis, hemolytic proteins and high coagulation have also been found.

Page 5 200521136 " " '一' ~ ------------ V. Description of Invention (2) " Most lectins contain 2-50% carbohydrates, and some lectins do not contain enzymes. Lectins can react with specific sugars, similar to the binding properties of antibody specific (an 11 b 〇dyspecificity), so Generally, this property is used as an indicator for the isolation or analysis of glycoproteins. Lectins are immune and specific, and are selective for the agglutination of human red blood cells, that is, cells of one blood type, but not agglutinated cells of another blood type. Therefore, it can be used to distinguish red blood cells of different blood types . Lis and Sharon in 1986 AD and Go 1 lob in 1959 AD pointed out that some lectins belonging to glycoproteins can be used in blood group classification, determination of glycoproteins and sugars Fat, stimulation of spleen cells and lymphatic mitosis, detection of normal or malignant tumor cells, induction of cell wall production of degrading enzymes, activation of signal transduction pathways and anti-tumor functions. AD 丨 In 1998, Sparvoli and others found that green bean (iima-bean) lectin would agglutinate type A blood cells, but not type B or 0 blood cells. In addition to S, lectin has an agglutination effect on red blood cells, and agglutination can also occur on other cells, such as lymphocytes, bacteria, fungi, and sperm cells. In 1933, Nathan and others divided the current lectin types into (1) plant sources: (A) cereals. · The protein-bound sugars were mostly ^ N-acetylglucosamine And acety lneuramic acid, and the protein is composed of two units, each unit has two binding sites, contains disulfide bonds and does not require metals to communicate with

200521136 V. Description of the invention (3); (B) Leguminosae: the saccharides they bind are less specific, consisting of 2 or 4 units with a molecular weight of 25-30 kilodaltons (kDa), each A unit body has a binding site, without disulfide bonds, and requires a positive divalent I bow ion (Ca 2+) and a positive monopyridium ion (Μ η 2+) to participate in the agglutination reaction. (II) Animal source: (a ) C-type (C-type): there is no specific carbohydrates, the number of units is different, but the molecular weight of each unit is not more than 15 thousand, each unit has 1-8 binding positions, Has a disulfide bond and needs to be combined with a positive divalent feed ion; (B) S type (S -1 ype): its bound bile is D-galactose (d -galactose) 'The number of constituent units is unknown' Each unit has a binding site, no disulfide bonds and no metal ions. The research history of algae lectins can be traced back to Boyd et al. In 1966 AD. They have detected that the cell fluid (cel 1 sap) of 24 kinds of Puerto Rican algae can aggregate human red blood cells. In other words, the seaweed extract contains Contains lectin components. In addition, they also found that the degree of agglutination activity of different seaweeds varies according to blood type. For example, Spiridiafi 1 ament 〇sa only aggregates type A red blood cells, but Codium isthmoclaclum, Turbinaria t urb i nata, and Sargassum natans can aggregate A. , 0, and B type red blood cells, but the activity is too weak and heat-resistant during testing, so it has doubts about its applicability and value, and no further research on biochemical characteristics has been done. In 1970, B1 unde η and Rogers investigated British algae one after another, and found that some species of algae have high agglutination activity. At this time, many scholars were interested, and research on algae was started. The field of lectins. At that time, they used red blood cell suspensions (s a 1 丨 n e s u s p e n s i ο η) made of gen saline to make a living.

200521136 --------- V. Description of the invention (4) '" " ---- Physical detection, in which ii 1. A red bath Pt 1 lota plumosa contains anti-type B red blood cell coagulation Ϊ ^^ More special. They tested more than a hundred kinds of seaweeds, and concluded that only a few seaweeds contain lectins, and the agglutination activity is very low, and there is no significant difference in the agglutination activity detected in different parts of the same type of bath. West, = 81 years later (Hori) et al. Detected 43 species of seaweed along the coast of Japan, and their 'capsules /' have 14 kinds of red blood cell activity in spinal mice, but & 8 kinds of human red blood cells have The activity 'where U1 arusak 丨 丨 has more affinity for type A and type 0 red blood cells' has shown that the algae has a rare anti- (A + H) (anti — (A + H)) specificity. As bioassays show that many algae extracts have low or no agglutination activity on red blood cell solutions, in order to improve and increase the detection effect, many scholars have used different enzymes to treat red blood cells in addition to changing the blood source to enhance the binding of lectin to the surface of red blood cells. ability. In 1977, Rogers prepared red blood cells in addition to saline, and set up another set of albumins or red blood cells that had been pretreated with papain before detecting seaweed. As a result of the extraction, the algae agglutination activity was significantly improved after the latter two treatments. Since then, many scholars have used different enzymes, such as trypsin and neuraminidase, to test the activity. As a result, it is found that the algae extract and the red blood cell solution after enzyme treatment are compared with After any treatment, the agglutination activity of the red blood cell solution is significantly improved. After enzyme treatment, some proteins on the surface of red blood cells are destroyed by enzymes. Therefore, the glycosyl groups that can be recognized by lectin have more opportunities to be exposed on the cell surface and be recognized and bound by lectin.

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V. Description of the invention (5) The agglutination activity was significantly enhanced, and the scholars began to mistake the specificity of the affinity of different sugars and found the structure of lectin. To achieve the effect of improving detection. From this kind of treatment, we have learned about the characteristics of algae agglutinin. The algae agglutinin is a kind of glycoprotein produced by glycoproteins. Researchers and isolates agglutinate them.

^ Lectins will recognize specific glycosyl groups and bind them. This kind of biological force is b. Therefore, it can be used to explore the chemical structure of cell surface bile molecules and cell membranes and cell biological functions.咛 多 研 :: Using the special binding property of lectin, the surface of the object cells has sugar-containing substances with important biological functions. In addition, some of the phytochemicals have been used in clinical medicine as anti-cancer research. The toxic protein in ricin agglutinin has been used to treat small whites containing abdominal tumors. The number of tumors in the abdominal cavity has been reduced in 48 hours To the one in the control group, and found that the mice treated with lectin did not die or develop within 5 months. In the meantime, ^ ^ ball bath has the advantages of easy cultivation and rapid reproduction. 'In a short period of time, eggs have a lot of biomass.' Chlorella, in addition to rich carbohydrate and half of the quality and vitamins, its protein content almost accounts for Chlorella cell cell protein i (40 ~ 60% of dry weight) is a kind of highly valuable single water treatment and ball bath. It is commonly used to produce health food, organic matter and waste metals. It is also widely used in health care. , Such as improving antioxidant capacity, regulating shellfish, strengthening immune function and improving intestinal function. Chlorella except SCp). ， 处 ’is the English title " single cell protein, (referred to as

200521136

It can be used as a raw material for bio-fertilizers, biotin, algin, cosmetics or pharmaceuticals as a gas or liquid. Also small and antitumor activity. With the study of sugar biotins. In view of the new chlorella, biomedical and biopharmaceutical, a new algae purification method was developed. Or as a source of natural pigments, fatty acids and vitamins, or as a source of antibacterial function in extractable chlorella extracts. As a result of scientific development, more people have noticed that it has been used in blood grouping and biochemical medicine. It can be used for various purposes as described above. It can be applied to the above-mentioned various research and development. [Summary of the Invention] / One of the purposes of the present invention is to provide a method for purifying microalgal glycoproteins. A purified chlorella agglutinin was obtained by exchange chromatography and colloidal filtration chromatography to facilitate various biomedical, blood group identification, health food, and related research and applications.

2. Another object of the present invention is to provide a method for purifying microalgae glycoproteins. The method is to use a aqueous ammonium sulfate solution with an appropriate concentration range to precipitate the chlorella agglutinin for analysis to develop chlorella agglutination. Optimum conditions for the separation and purification of vegans. Another object of the present invention is to provide a method for purifying microalgal glycoproteins. The method is to detect the agglutination activity of a human or animal red blood cell.

Page 10 200521136 V. Description of the invention (7) Test, as a basis for judging whether it is purified or not. According to the above-mentioned object, the present invention provides a method for purifying microalgae glycoprotein, which comprises performing an extraction step of chlorella somatic cells, wherein a phosphate buffer saline is used. The desired chlorella lectin is separated and purified, and the obtained algae extract is used for ammonium sulfate aqueous solution (ammo n ium sul fate) to immerse the protein, and the precipitated ammonium sulfate aqueous solution with a concentration of 0-80% is collected. Protein for chlorin lectin isolation and purification. The obtained protein Shen Dian was placed in a phosphate buffer solution for dialysis and desalting. After centrifugation, the upper protein clarified solution was taken, and a DEAE-C-52 (diethyl amin〇ethyl_ ^ cellulose-52) column was used for an anion exchange column. Analysis and use a concentration gradient method (from Oi Moore concentration to 0.5 Moore concentration) to collect the active protein 'which is collected from the 0.2 Moore concentration of sodium vapor, The active protein was then dialyzed through Sodlum Acetate Buffer to remove salts, and then analyzed by CM-C-5 2 (carb〇xymethyhcellulose-52) f exchange column. Carved in: the second middle school uses emulsified sodium water and valley liquid to collect a solid protein by using a concentration gradient method (from 0.1 Moore concentration to 0.5 Moore concentration), and then uses dex technology …, 4 瞢 know 厣, i, 八, 秘, 八, 八, 用, 75, 75, 75, 75, 用, 75, 用, 用, 75, 八, 八 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, b, All steps go through one, two, and blood Cells to determine their hemagglutinating activity and purified or not.

200521136

Another aspect of the present invention provides a method for purifying red or white of human or animal treated with enzyme by using microalgae to separate the agglutination activity of protein for judging whether it is purified or not-basis Wherein, the enzyme may be pancreatic protein or chain protein (papa i η). ypsin) 4. [Embodiment] In the method for purifying microalgae glycoprotein of the present invention, since chlorella has a tough cell wall, this cell wall is made of cellulose, hemicellulose, etc., so it needs to be used A method of cell disruption breaks the cell wall of the chlorella alga so as to facilitate the extraction of lectin. Among them, different cell disruption methods have different agglutination activities on the chlorella extract. The cell breaking method used in the present invention includes (1) liquid nitrogen method: the chlorella somatic cells are ground into liquid powder by liquid nitrogen at a temperature of liquid nitrogen (minus 196 degrees Celsius). (2) glass bead method (g 1 assbead) ····························································································································· of-bakes · Dry Bath · 0.2g One minute, hit 20 times in total; (3) Ultrasound treatment (ul trasoni cat ion): using a cell grinder (cel 1 disrupter) in the ice bath to destroy the chlorella algal somatic cells, in which the broken cells The condition is that the ultrasound enhancement value (u 11 ras ο nicintensifierva 1 ue) is set to 50, and the frequency of breaking cells is to rest for 2 minutes every 3 minutes, breaking cells 10 times in total; (4) Tissue fragmentation method (War i ng-b 1 ender): Set the dial

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To the required speed grade, the time of each crushing is 5 seconds, leap seconds, 4 crushing 6 times. 5) The first grinding method: the chlorella steamed body two cells and alumina (A! 2%) by weight Ratio (w / w) 1: 3, uniformly mix it, and use a glass rod to grind it in an ice bath for 10 minutes; (six) the second grinding method: a small ball bathing the body cells and the sea sand in a weight ratio (w / w ) 1: 3 uniformly mixed using a glass * rod in an ice bath for 10 minutes; (7) Enzyme method: the two yeasts were mixed at a volume ratio of 1: 1 for two hours at 37 ° C, where The two enzymes are a volume percentage of Macrozyme R-10 and distilled water of 2% (w / v) and a volume percentage of giucuronidase and distilled water of 2% (v / v). In the method for purifying glycoproteins of the microbath according to the present invention, human blood of type A, B, 0, AB and animal blood, such as pigs, cattle, and sheep, are used to separate the protein collected in each separation and purification step with one The agglutination activity of erythrocyte suspension was used as a basis for judging whether the protein was purified or not. The preparation of this erythrocyte suspension was made by the following methods: · The collected erythrocytes were added to the pH value of 7. 2, 50 mmol In mM phosphate buffer (Phosphate Buffer Saline, PBS), wash three times by centrifugation. The centrifugation condition is 20000 rotation speed (20000 r pm) per minute, continuous rotation for 10 minutes. ; Take down the red blood cell concentrated solution, and then dilute with phosphate buffered saline (PBS) to a 2% (v / v) red blood cell suspension, and reserve it at 4 ° C for biological detection. The agglutination activity is determined by separating eggs obtained in a purification and separation step.

Page 13 200521136 V. Description of the invention (10) White matter, placed in the first hole of a 96-well micro-V-plates at 50 microliters (micro 1 iter, // L) In addition, 50 microliters of phosphate buffered saline (PBS) was placed in each well of a 96-well blood plate, and the protein in the first well was uniformly mixed with the phosphate buffered solution. Take 50 microliters of the mixed solution in the first hole into the second hole and make one-half continuous dilution, that is, after performing the above steps, the concentration of the second hole mixed solution is in the first hole One and a half, after that, 2% red blood cell suspension was added to each hole, and after homogeneous mixing, it was left at room temperature for 2 hours, and its agglutination reaction was observed with the naked eye. The size of agglutination activity is expressed by the agglutination valence (ti ter). The expression of agglutination 饧 is the reciprocal of the final dilution dilution when erythrocyte agglutination is observed. For example, the protein still has Activity, so the cohesive force valence at this time is 64, which is 26. It should be noted that in the method for purifying the microalgal glycophilic protein of the present invention, a erythrocyte enzyme suspension and the protein collected in each purification and separation step may be used to perform an agglutination activity test, in order to improve the detection result The use of enzyme-treated red blood cells, such as pronase, trypsin, or papin, proteinases can increase the red blood cell surface's acceptance position (receptor sit to help The establishment of agglutination activity. And boll a discusses each,… six seal sheets, f · take off a button I and the preparation of red blood cell enzyme suspension is as follows; L enzyme, such as papain, add monolithic acid In bee buffer and configured as one of 2% (v / v) by volume of enzyme solution: take a red blood cell suspension and add it to this enzyme solution, where red blood cells and enzymes

200521136 V. Description of the invention (11) The volume ratio of the solution is 1 · 1 0 ′ and the reaction is performed at 37 ° C for 1 hour to remove the sialic acid or glycoprotein on the surface of red blood cells, and placed at 4. The test of its agglutination activity stored under 〇 is to place 50 microliters of the protein obtained in a purification and separation step on the first hole of a 96-well blood cell calculation disk, and in addition, take 50 microliters The phosphate buffer solution is placed in each hole in the 96-well blood well calculation plate, and the protein in the first hole is uniformly mixed with the phosphate buffer solution. After that, the mixed solution in the first hole is taken as 50. Slightly dilute to the second hole and make one-half serial dilution, that is, after performing the above steps, the concentration of the second hole mixed solution is half of that in the first hole, and then add 2% red blood cell enzyme suspension After being uniformly mixed in each hole, the system was left at room temperature for 2 hours, and the agglutination reaction was observed with the naked eye. The purification method 10 of the bathed pro-enzyme protein of the present invention is to extract a part of the protein from the chlorella extract with a sulfuric acid aqueous solution and use it to determine the optimal conditions for the separation and purification of chlorella agglutinin. Among them, the agglutination activity of the precipitated protein in different ammonium sulfate aqueous solutions of different degrees = ^ degrees is not the same as shown in Table 1. Among them, 0-20% saturated ammonium sulfate aqueous solution precipitates proteins with the highest agglutination activity, which are π agglutination valence (tt π 'and 20-40%, 40-60% and 60_80% saturated ammonium sulfate aqueous solution ^ The precipitated protein also has partial agglutination activity, which is between 22 and 25 agglutination force, while 8 0-100% has no agglutination activity.

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200521136 V. Description of the invention (12) The ammonium sulfate content to be added is as described in Table 2. For example, if you want to configure a 0-20% saturated ammonium sulfate aqueous solution, refer to the initial saturation concentration of ammonium sulfate as 0%, When the final saturated concentration of ammonium sulfate is 20%, under this condition, the content of solid ammonium sulfate required in a 1 liter solution is 106 grams, and the other ammonium sulfate content required in the other concentration ranges can be deduced by analogy. Table I

Saturated concentration range of sulfuric acid aqueous solution (%) Protein concentration (mg / ml) Cohesion value (tite) Coarse extraction 0.168 26 0-20 0.573 29 20-40 0.4 11 25 40-60 0.244 No agglutination activity 60 -80 0.266 22 80-100 0.2 1 No agglutination activity Page 16 200521136 V. Description of the invention (13) Table final concentration of ammonium sulfate (%) 20 25 30 35 40 45 50 55 60 65 70 75 80 90 100 Add one liter Solid ammonium sulfate content in solution (g) Initial saturation concentration of sulfuric acid% 0 106 134 164 194 226 258 291 326 361 398 436 476 476 516 603 697 0 20 0 27 55 83 113 143 175 207 241 276 312 349 387 469 557 20 25 0 27 56 84 115 146 179 211 245 280 317 355 436 522 25 30 0 28 56 86 117 148 181 214 249 285 323 402 488 30 35 0 28 57 87 118 151 184 218 254 291 369 453 35 40 0 29 58 89 120 153 187 222 258 335 418 40 45 0 29 59 90 123 156 190 226 302 383 45 50 0 30 60 92 125 159 194 268 348 50 55 0 31 61 93 127 161 235 313 55 60 0 31 62 95 129 201 279 60 65 0 31 63 97 168 244 65 70 0 32 65 134 209 70 75 0 32 101 174 75 80 0 34 139 80 90 0 70 90 100 0 100 The purification method of the microalgal glycoprotein of the present invention will be described in the following specific examples Do a detailed explanation. In a specific embodiment of the method for purifying the microalgae glycoprotein of the present invention, iiii page 17 200521136 V. Description of the invention (14) A chlorella bead, such as c. Lute〇viridis luteoviridis, is used (Ch1 ore 11 a glass bead method, the key to this chlorella algae body: skin treatment plus glassy agglutination agglutination activity, so, because the cell-breaking time is a matter of minutes, take a two-minute rest with a machine shock To avoid the loss of agglutination activity, and break the cells to collect a protein, six γ ^ t, to separate and purify the small globules of the chlorella somatic cells after the break ♦ 2 I + 疋 protein shell bath L is as follows: # 先 ， ； 〇ΓΛν The step of the quality solution is to take 0.5 grams of broken Chlorella alga cells, and add 1 M to the pellet. Extraction of algae agglutinin, such as 5 ml of M salt buffer (PBS). The extraction conditions are extraction at 4t for 24 hours, and then centrifugation at a speed of 20,000 rpm for 20 minutes. Remove the residue. Next, take the supernatant of the upper layer and add one sulfur of different concentration range In the ammonium aqueous solution, for example, -20%, 20-40%, 40%, 60%, 600 ~ 80%, and 80% -1000% saturated ammonium sulfate aqueous solution to cut off the desired A protein solution from Shen Dian, this protein contains the desired chlorella lectin. The steps for determining the lectin separation and purification conditions with different concentrations of ammonium sulfate aqueous solution are as follows: 0-20% saturated concentration Ammonium sulfate was dissolved in water and added to the chlorella extract, and equilibrated in an ice bath for one hour, and the protein to be separated was precipitated. At 4 ° C, it was centrifuged at 15,000 rpm for 30 minutes. After that, the supernatant of the upper layer was taken, and a 20-40% saturated aqueous solution of sulfuric acid was added to obtain eggs precipitated in this concentration range.

Page 18 200521136 V. Description of the invention (15) White matter. Then, the above steps and conditions are repeated with a saturation concentration of 20% each. Therefore, five divided protein precipitates are obtained. After the division is completed, the protein precipitated in each division is dissolved in phosphate buffered saline (PBS) and placed in a dialysis bag. It is dialyzed for 48-72 hours. The purpose of dialysis is to remove Salts such as acid salts, in which the dialysis solution is changed every four hours. Next, after dialysis is completed, rotate at 15,000 rpm (rpm) and centrifuge for 30 minutes to remove impurities of insoluble matter, then take the upper clear solution and measure its agglutination activity, where 0-20% Saturated concentration of ^ ammonium sulfate aqueous solution has the highest agglutination activity of the protein, and 20-8 ()% ammonium sulfate aqueous solution of the protein precipitated also has some agglutination activity. Therefore, 0-80% saturated concentration of ammonium sulfate aqueous solution is taken The precipitated eggs continued to scramble. Beimujin followed by 'pass the 0-80% saturated concentration of ammonium sulfate aqueous solution of the protein precipitated into a DEAE-C-52 anion exchange chromatography column separation and purification, the purification and separation step is under 4 t get on. Among them, the size of this stage ion exchange chromatography column is 2. 6αηχ 18cm, and this $ column first uses a phosphate buffer solution (ph0sphate buffe with a pH value of 8.0, the absorption value of 28nm is zero, Then the protein to be separated is passed into the column, wherein the flow rate is 1.5 ml per minute. First, the recoverable effluent (based on the agglutination activity with red blood cells) is used. After the bran, is it used? The degree of gradient method is to transfer the concentration range of the gasified sodium hydroxide aqueous solution in the desired egg eight from 0 mol concentration to 0.5 mol concentration (M).

Page 19 200521136 V. Description of the invention (16) ^ points, and the collected protein is divided into 5 ml per minute and 3 ml per minute. The collected proteins were tested for their agglutination activity, and their absorption values were detected at a wavelength of 280 nm. Among them, the protein extracted from the aqueous sodium carbonate solution of 0.2 Moore ^ had the largest value at the same time. The absorption of ^^ and agglutination activity, while the protein effluent collected by other divisions has two absorbance and agglutination activity, as shown in the first figure. Therefore, the protein effluent, which was extracted with an aqueous solution of sodium chloride at a concentration of 0 mole, was collected. · After concentrating the protein effluent collected at this concentration, a sodium acetate buffer (Sodium Acetate Buffer) is used as a dialysis solution. The pH of the dialysis solution is 4.0 and the concentration is 50 millimolar (mM). Its volume is 1000 times the volume of the collected protein effluent, and its dialysis time is more than 12 hours. After that, it is centrifuged at 15,000 rpm to remove insoluble precipitates and impurities to achieve the purpose of purification and to remove proteins without agglutination activity, which is beneficial to the next step of purification and separation. The DEAE-C-52 anion exchange chromatography method was used to extract the obtained protein solution with an aqueous sodium chloride solution at a concentration of 0.2 Moore, and passed through a CM-C-5 2 cation exchange chromatography column. For the next step of isolation and purification. Among them, the configuration method of the cation exchange chromatography column is as follows: First, weigh out an appropriate amount of dry powder of CM-cellulose-52, and soak it with 50 times the volume of 0.5 equivalent sodium hydroxide (NaoH) aqueous solution for 30 minutes. It was washed with deionized water to neutrality (its pH value was lower than 8.0), and then immersed in 50 times the volume of 0.5 equivalent hydrogen chloride (HC1) aqueous solution for 30 minutes, and then deionized water.

Page 20 200521136 V. Description of the invention (17) Wash to neutral (its pH value is higher than 4. (0, acetic acid buffer solution with a concentration of 10 > 1 > Mohr), after several hours, slowly pour off the upper layer Remove the suspended particles and pour into the column. After precipitation to the required height, fully equilibrate with a 10j sodium acetate buffer solution with a concentration of 4.0, and use the anion exchange chromatography of DEAE-C-52. The neutral protein solution collected in the method was passed into the cation exchange chromatography column of this CM-C-52. Ϊ́ —As shown in the figure, Bin " First, it is-sodium acetate buffer solution (10 mM) Mo: =, PΗ value is 4 ·.) First put forward the absorption value of unadsorbed protein ^ at the wavelength. The protein effluent has absorption at this wavelength but has no agglutination activity when detected by red blood cells. Therefore, Friend = aqueous solution ' Concentration gradient for production: Protein is extracted. Among them, this aqueous solution of sodium chloride: produces, Moore (degrees) to 0.5 Moore concentration ⑷ 'set per ΐ milliliter ml ... 1 liter of red blood cells隹 is a knife brake, which detects the protein and white matter output of each division collected. Egg water absorbed at each concentration range; = Absorptive, but chlorinated at a concentration of ° .2 Mohr and collected at 0? Tir / has the highest agglutinating activity, so protein, The next step is the purification and separation step of the next step of purification of the gasified sodium aqueous solution, which has the activity to enter the lamp. To be explained, the protein,, capacity, and cold #field of the set II; the anion exchange chromatography method, the A sodium acetate buffer solution was used for dialysis at night. Therefore, its egg 200521136 V. Description of the invention (18) The white shell solution will have more negative electrical properties. Therefore, when passed CM-52 ce 1 1 u 1 〇se When a cation exchange column is used, its lectin will be adsorbed on the fibers of this column, and then it is extracted with different concentrations of sodium chloride aqueous solution to achieve the purpose of separation and remove proteins without agglutination activity. -C-52 cation-exchange chromatography-collected protein with active protein 'was centrifuged at rpm (rpm) for 20 minutes to remove insoluble precipitate. Then, a Superdex 75 colloid tube was used. Zhujin !! Purification and separation steps, in which the Superdex 75 colloid and the official column are first placed in a phosphate buffer solution with a concentration of 10 · Moore and a pH value of 7.4, and the buffer solution is extracted under 4 C until the wavelength The absorption value of 28〇ηιη is zero. Afterwards, the volume of the active protein effluent collected in the cation exchange chromatography column of CM-C-52 is used to concentrate the volume to 3%. Milliliter to 3 milliliter, and then passed into this Superdex 75 ^ body tube column, where 0.3 milliliter per minute, each milliliter is a minute to collect the effluent protein solution. As shown in the third figure, the absorption value and agglutination activity of each protein effluent at a wavelength of 28011111 are detected. After being separated and purified by the colloidal chromatography method, two kinds of effluents with J = mass can be obtained. Therefore, the protein effluent and red blood cells were tested for y activity, and the purification step of chlorella agglutinin was completed. One of the proteins has agglutination with red blood cells, but the other protein can be learned through the above-mentioned process of purifying chlorella lectin, and the present invention provides

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V. Description of the invention (19) The lectin purification method is simple, straightforward, and easy to operate, and chlorella and t can be used in other biochemical functions and health foods, etc. Inch is highly economical. The above description is only a specific embodiment of the present invention, and is not intended to limit the scope of the patent application for the invention. Any other equivalent changes or modifications made outside the scope of the present disclosure should be applied. Contained within the scope of the main spirit described below. T% patent

200521136_ Brief description of the diagram V. [Simplified illustration of the diagram] The first diagram is a graph of the purification and separation of chlorella lectin by C. luteoviridis using anion exchange chromatography column method of DEAE-C-52. It is a graph of chlorella C. luteoviridis using CM-C-52 cation exchange chromatography column method to purify and isolate chlorella lectin; and the third graph is of chlorella C. luteoviridis using colloidal chromatography of Superdex 75 Graph of purification and separation of chlorella lectin by filtration method.

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Claims (1)

200521136 6. Application patent scope 1. A method for purifying microalgae glycoprotein, comprising: providing a chlorella sample, the chlorella sample system comprises a plurality of chlorella somatic cells; performing the chlorella sample A cell breaking step to break the plurality of chlorella somatic cells; perform a collection step on the broken plurality of chlorella somatic cells; perform an anion exchange column chromatography step; perform a cation exchange A column chromatography step, wherein the cation exchange column chromatography step is performed after the anion exchange column chromatography step; and a colloid filtration chromatography step is performed to obtain a lectin. 2. The method for purifying microalgal glycoproteins as described in item 1 of the scope of the patent application, wherein after the lysis step described above, the method further comprises performing a plurality of tests of red blood cell agglutination activity. 3. The method for purifying microalgal glycoproteins as described in item 1 of the scope of the patent application, wherein the step of breaking cells is achieved by a liquid nitrogen method. 4. The method for purifying microalgal glycoproteins as described in item 1 of the scope of the patent application, wherein the step of breaking cells is achieved by a glass bead method. 5. Purification method of microalgal glycoprotein as described in item 1 of the scope of patent application 200521136 6. The scope of the patent application is completed, in which the above-mentioned cell-breaking step is performed by the ultrasonic treatment method 6. The microalgae sugar as described in item 1 of the scope of patent application | The steps are achieved by the / organization method. 7. The microalgae glycoprotein described in item 1 of the patent application scope, wherein the above-mentioned cytolytic step is achieved by the / mill method. Purification method 8. The microalgae glycoprotein Xi Nuo & K K < purification method as described in item 1 of the scope of the patent application, wherein the cell breaking step described above is achieved by an enzyme method. 9. The method for purifying microalgal glycoproteins as described in item 1 of the scope of the patent application, wherein the above-mentioned collection step comprises using a phosphate buffer saline as an extraction solution. 10 · The method for purifying a microalgal glycoprotein as described in item 1 of the scope of the patent application, wherein the above-mentioned collection step includes adding an ammonium monosulfate aqueous solution. 11. Purification method of microalgal glycoprotein as described in item 10 of the scope of patent application Method 'wherein the saturated concentration range of the above-mentioned ammonium sulfate aqueous solution is selected from any one of the following: · The weight percentage concentration is 0-20%, 20-40%, 40-60%, and 60-80%. 012. Purification method of microalgal glycoproteins as described in the first patent application scope Page 26 200521136 VI. Patent application method, wherein the above-mentioned anion exchange column chromatography method comprises extracting a protein substance with an aqueous solution of sodium chloride. 13. The method for purifying microalgal glycoproteins according to item 1 of the scope of the patent application, wherein the method of cation exchange column chromatography described above comprises eluting a protein substance with an aqueous solution of sodium chloride. 14. The method for purifying a slightly glycophilic protein as described in item 12 of the scope of the patent application, further comprising extracting the protein substance by a concentration gradient method. 15. The method for purifying a microalgal glycoprotein as described in item 13 of the scope of the patent application, further comprising extracting the protein substance by a concentration gradient method. 16. The method for purifying a microalgal glycoprotein according to item 12 of the patent application scope, further comprising using a sodium acetate buffer (Sodium Acetate Buffer) as a dialysis solution. 17 · The method for purifying a microalgal glycoprotein as described in item 13 of the scope of the patent application, further comprising using a dish buffer solution as a dialysis solution. 18. A method for purifying microalgae glycoproteins, comprising: providing a chlorella sample, the chlorella sample system comprising a plurality of globules without somatic cells; performing an sonication step of mixed glass beads, and Break that Page 27 200521136 VI. Application scope of a plurality of chlorella somatic cells; performing an extraction step on the broken chlorella somatic cells to obtain an extract; performing a precipitation of the extract Step to obtain a first protein solution, wherein the step of precipitating comprises adding a solution of ammonium sulfate to perform a dialysis on the first protein solution to obtain a second protein solution, wherein in the dialysis step It includes adding a sodium acetate buffer solution; performing a first column chromatography step to obtain a third protein solution, wherein a first sodium chloride having a different concentration is added in the first column chromatography step An aqueous solution; performing a second column chromatography step to obtain a fourth protein solution, wherein in the second column chromatography step, a second aqueous solution of sodium gasification having a different concentration is added; and The protein solution is subjected to a colloidal filtration chromatography step to obtain a lectin. 19. The method for purifying microalgal glycoproteins as described in item 18 of the scope of patent application, wherein the weight percentage concentration of the above ammonium sulfate aqueous solution is 0-80% 20. As described in item 18 of the scope of patent application A method for purifying microalgae glycoprotein, wherein after the cell breaking step described above, the method further comprises performing a plurality of red blood cells 200521136 6. Scope of patent application Testing of agglutination activity. 21. The method for purifying microalgal glycoproteins as described in item 18 of the scope of patent application, wherein the above-mentioned extraction step includes adding a phosphate buffer saline (Phosphate buffer saline) 〇 22. As the scope of patent application No. 1 The method for purifying a microalgal glycophilic protein according to item 8, wherein the first aqueous sodium chloride solution further comprises extracting a protein substance by a concentration gradient method. 23. The method for purifying a microalgal glycoprotein as described in claim 18, wherein the second aqueous sodium chloride solution further comprises extracting a protein substance by a concentration gradient method. Page 29