CN100591696C - Method for separation purifying hyaluronic acid - Google Patents

Method for separation purifying hyaluronic acid Download PDF

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CN100591696C
CN100591696C CN 200710017757 CN200710017757A CN100591696C CN 100591696 C CN100591696 C CN 100591696C CN 200710017757 CN200710017757 CN 200710017757 CN 200710017757 A CN200710017757 A CN 200710017757A CN 100591696 C CN100591696 C CN 100591696C
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hyaluronic acid
ha
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added
purification
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CN101045754A (en
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李东亮
江元汝
郭育涛
斌 陈
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西安建筑科技大学
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Abstract

This invention relates to a separating and purifying method of hyaluronic acid. Add hyaluronic acid extract to alkaline amortization water solution, and join tryptic enzyme, whip at ordinary temperature; add halogen benzene to above mixed liquor, whip, then centrifugalize and abstract supernatant; add sodium chloride of 0.15 mol / L to 0.25 mol / L to above supernatant, whip for dissolving, then add at least 2.5 times volume ethanol of not less than 95 percent, whip, stand ing for deposition, then centrifugalize, obtain precipitate which is hyaluronic acid.

Description

透明质酸分离纯化的方法 The method of separation and purification of hyaluronic acid

技术领域 FIELD

本发明涉及一种分离纯化透明质酸(Hyaluronic acid,简称HA) 的方法。 The present invention relates to an isolated purified hyaluronic acid (Hyaluronic acid, referred to as HA) method. 背景技术 Background technique

HA是一种由葡萄糖醛酸和N-乙酰葡萄糖胺为双糖单位聚合而成的大分子生物多糖。 HA is a glucuronic acid and N- acetylglucosamine disaccharide units of biological macromolecules obtained by polymerizing a polysaccharide. HA有生物相容性、生物降解性及光学活性等。 HA with a biocompatible, biodegradable and other optically active. HA主要用于临床治疗、化妆品、医疗用品研究等方面。 HA is mainly used in the clinical treatment, cosmetics, medical products research. 在生物分离工程领域,寻求生物物质分离新技术、新方法是长期以来关注的重点之一。 In the field of bio-separation project, looking for new technologies and new methods of biomass separation is one of the key long-standing concern. 对HA分离纯化方法的研究从1952年以来不断有相关的论文发表和专利报道。 Research on HA separation and purification methods since 1952 there have been papers published and patents related reports.

用活性炭和粉末纤维素吸附剂对微生物发酵HA提取物的分离纯化方法(Biochimica et Biophysica Acta, 1957. Vol.24: 397-400)。 Powdered cellulose and activated carbon adsorbents for separation and purification methods HA microbial fermentation extract (Biochimica et Biophysica Acta, 1957. Vol.24: 397-400). 该方法的最大缺点是:随着蛋白质被吸附剂的除去,部分HA随之带入而损失掉。 The biggest drawback of this method is that: the adsorbent is removed as the protein, followed by portion into the HA is lost. 此外,由于动物组织中的HA是以与蛋白质聚糖的形式存在,在对动物源HA分离纯化中,用此法会在除去蛋白质的过程中损失大量的HA。 Furthermore, since the HA is present in the form of protein glycans and animal tissues, separation and purification of HA in animal sources, this method will lose a lot in the process of removing HA protein. 因此该法不宜用于动物源HA的分离纯化。 This method should not be so isolated and purified HA for animal origin.

用氯仿结合膜滤法从牛眼球玻璃体中分离纯化HA的方法(US Pat. No4, 141, 973),该法的最大缺点是:分离过程中使用常规方法不易得到的DNase及RNase两类核酸酶。 Method (. US Pat No4, 141, 973) isolated from bovine vitreous HA purified with chloroform binding membrane filtration, the biggest drawback of the method is that: the separation process using two types of RNase and DNase nuclease conventional methods is not readily available .

用胰蛋白酶水解法从牛关节液中制取HA的方法(WO 86/06728)。 Method (WO 86/06728) by trypsin hydrolysis HA preparation from bovine articular fluid. 该法的最大缺点是:分离过程中多次在磷酸盐溶液中进行酶分解及其附属过程,因此,操作过程复杂。 The biggest drawback of this method is that: a plurality of times during the separation and its subsidiary enzymatic degradation process in a phosphate solution, therefore, complicated operation. 用溴化十六垸基三甲铵(CTAB)从发酵液中分离HA的方法(US No. 4, 782, 046); —种用超滤与氯化十六烷基吡啶(CPC)沉淀结合的方法从发酵液中分离HA的方法(WO 92/08799)。 The method of separating from a fermentation broth HA with sixteen embankment trimethylammonium bromide (CTAB) (US No. 4, 782, 046); - precipitants ultrafiltration and hexadecyl pyridinium chloride (CPC) binds methods (WO 92/08799) HA isolated from the fermentation broth. 该类方法最大的缺点是:在实施用CTAB或CPC分离纯化HA过程中,要经过多次CTAB或CPC沉淀HA及盐溶过程,操作过程繁杂。 The biggest drawback of such a method is: with CTAB or CPC separation and purification process HA embodiment, the CPC or CTAB to precipitate after several HA and salt dissolution process, complicated process operation. 有的得到的HA产物既含有蛋白质又含有核酸(WO 92/08799)。 Some resultant HA product contains both proteins and nucleic acids comprising (WO 92/08799).

用CPC结合DEAE-纤维素从人脐带、牛眼玻璃体、鸡冠中分离纯化HA的方法(生物化学杂志,1996年第12巻第2期)。 Purification HA from human umbilical cord, bovine vitreous, combs with CPC DEAE- cellulose binding method (Journal of Biochemistry, 1996, Volume 12, No. 2). 该方法的最大缺点是:分离纯化过程中在除去蛋白质的同时造成大量HA的损失。 The biggest drawback of this process is: the process of separation and purification of a large amount of loss at the same time removing the HA protein. 此外,操作复杂。 Moreover, complicated operation.

用氯仿、链霉蛋白酶及CPC从鸡冠中制备HA的方法(医药工业, 1986, 17.7)。 Method, and CPC pronase preparation of HA from rooster combs with chloroform (Pharmaceutical Industry, 1986, 17.7). 该法的最大缺点是:由于先在pH4.5-5.0之间,用氯仿变性除去蛋白质造成HA随部分蛋白质的除去而损失及HA分子发生降解。 The biggest drawback of this method is that: due to between pH4.5-5.0, with chloroform to remove protein denaturation resulting in removal of the protein with the HA loss and degradation of HA molecules. 此外,由于使用CPC,操作过程复杂。 Further, since the CPC, the operation complicated.

一种利用离子交换树脂层析分离纯化HA的方法(离子交换与吸附1999.15.4)及(中国医药工业杂志,.2001.32.17)。 Method (1999.15.4 adsorption and ion exchange), and (Chinese Journal of Pharmaceuticals, .2001.32.17) An isolated and purified by using ion exchange resin chromatography HA. 该类方法目前仅处于实验室研究阶段。 Such methods currently available only in the laboratory research stage. 发明内容 SUMMARY

本发明要解决的技术问题是:提供一种透明质酸分离纯化的方法, 该方法操作步骤少,操作简便;成本费用低;产品质量好。 The present invention is to solve the technical problem are: to provide a process for isolation and purification of hyaluronic acid, which fewer steps, simple operation; low cost; good quality product. 本发明解决的技术问题的技术方案是:它包括以下步骤: The present invention solves the technical problem aspect is that: it comprises the steps of:

(1) 将得到的透明质酸提取物加入到碱性缓冲水溶液中,并加入胰蛋白酶,在常温下搅拌; (1) hyaluronic acid extract obtained was added to an alkaline aqueous buffer, trypsin added, and the mixture was stirred at room temperature;

(2) 向上述混合液中加入卤苯,搅拌,然后离心抽取上清液;(3 ) 向上述清液中加入氯化钠,得到浓度为0.15mol/L~0. 25mol/L的氯化钠溶液,并搅拌溶解,然后加至少2. 5 倍体积的95%以上的乙醇并搅动,静置至沉淀发生后离心,沉淀物即为分解蛋白质的透明质酸; (2) added to the above mixture halophenyl, stirred and then centrifuged to extract the supernatant; (3) Sodium chloride was added to the supernatant to give a concentration of 0.15mol / L ~ 0 25mol / L chloride. soda solution, and stirred to dissolve, then add 95% ethanol at least 2.5 times the volume of agitated and allowed to stand until precipitation occurs after the centrifugation, the precipitate is the decomposition of hyaluronic acid protein;

(4) 加分解蛋白质的透明质酸到由NaCl、 NaH2P0jQ N&HP04组成的,或NaCl、 KH2P04和K2HP04组成的离子溶液中搅拌溶解,然后加至少2. 5倍体积的95%以上的乙醇并搅动,静置至沉淀发生后离心, 沉淀物为固性物,该固性物含至少42%的透明质酸; (4) adding to the protein by the decomposition of hyaluronic acid NaCl, NaH2P0jQ N & HP04 composition, or NaCl, KH2P04 and K2HP04 solution consisting of ionic dissolved with stirring, then add at least 95% ethanol and 2.5 volumes of agitation, after standing to precipitation centrifugation, the precipitate is a curable composition, the curable composition containing at least 42% of hyaluronic acid;

(5) 将步骤(4)的沉淀物加入由NaCl、 NaH2P0jn Na2HP04组成的,或NaCl、 KH2P04和K2HP04组成的离子溶液后搅拌溶解,然后加入活性炭,或活性炭和高岭土,搅拌、离心,上清液经滤膜抽滤,滤液即为透明质酸溶液,然后加至少2.5倍体积的95%以上的乙醇并搅动, 静置至沉淀发生后离心,沉淀物中透明质酸含量至少60%。 (5) The step (4) was added to the precipitate NaCl, NaH2P0jn Na2HP04 composition, or NaCl, KH2P04 ionic composition of the solution and the rear K2HP04 dissolved with stirring, followed by addition of activated carbon, kaolin or activated carbon and the mixture was stirred, centrifuged, the supernatant by membrane filtration, the filtrate is hyaluronic acid solution, then add 95% ethanol at least 2.5 times the volume of agitated and allowed to stand until precipitation occurred after centrifugation, the precipitate hyaluronic acid content of at least 60%.

(6) 将所述步骤(5)的经滤膜抽滤后所得的透明质酸溶液用35000Da 透析袋在去离子水中透析3~4h,然后,向透析后的透明质酸溶液中加羧甲基纤维素并搅拌,最后离心或抽滤得到滤液;羧甲基纤维素的使用量为10〜30g/l; (6) The obtained after said step (5) by membrane filtration using 35000Da acid solution 3 ~ 4h dialysis bag dialyzed in deionized water, then added to the hyaluronic acid solution carboxymethylcellulose after dialysis cellulose and stirred filtrate was finally obtained by centrifugation or filtration; the amount of carboxymethyl cellulose is 10~30g / l;

(7) 将上述滤液,冷冻,真空干燥,得无色透明片状透明质酸产物, 或向上述滤液中加氯化钠至0. 15mol/L〜0.25mol/L ,加至少2. 5倍体积的95%以上的乙醇并搅动,静置至沉淀发生后离心,沉淀物经冷冻,真空干燥,得到白色粉末状透明质酸产物。 (7) The above filtrate was freeze-dried in vacuo to give a colorless transparent sheet-like product of hyaluronic acid, or sodium chloride was added to the filtrate to 0. 15mol / L~0.25mol / L, at least 2.5-fold increase more than 95% by volume of ethanol and stirred, allowed to stand until precipitation occurs after the centrifugation, the precipitate was freeze-dried in vacuo to give a white powder of hyaluronic acid product.

所述步骤(1)的碱性缓冲水溶液化学药品是由NaHC03和Na2C03 组成,或由NaHC03和NaOH组成;NaHC03的使用量为0. 01~120g/L,碳酸钠Na2C03的用量为0〜100g/L,或NaOH为0~lg/L。 Said step (1) of the basic aqueous buffer solution of NaHC03 and Na2C03 chemicals is composed of, or consisting of NaOH and NaHC03; NaHC03 used in an amount of 0. 01 ~ 120g / L, an amount of sodium carbonate Na2C03 0~100g / L, or NaOH is 0 ~ lg / L.

所述碳酸氢钠和碳酸钠的浓度是l~10g/L。 The concentration of the sodium bicarbonate and sodium carbonate are l ~ 10g / L.

所述步骤(1)所用的胰蛋白酶为粗制胰蛋白酶或精制胰蛋白酶, 用量范围为透明质酸提取物中每毫克蛋白质用2~100个活力单位的酶量,酶的分解温度为20-42°C,分解时间为0. 5〜90h。 Said step (1) to be used as a crude trypsin or trypsin purified trypsin, hyaluronidase extract an amount in the range of the amount of enzyme per mg of protein were used in 2 to 100 units of activity, the decomposition temperature of the enzyme is 20 42 ° C, the decomposition time is 0. 5~90h.

所述酶的分解温度范围是30〜33°C,酶加量是按透明质酸提取物中每毫克蛋白质用8~25个活力单位的酶量计,时间是24h以内。 The decomposition temperature range of the enzyme is 30~33 ° C, the enzyme dosage is based on hyaluronic acid per mg of protein extract with 8-25 units of enzyme activity meter, less time is 24h.

步骤(2)加入卤苯是氯苯、溴苯、氟苯或碘苯;加入的体积比为:5〜25%。 Step (2) was added benzene halide is chlorobenzene, bromobenzene, fluorobenzene or iodobenzene; the volume ratio of added: 5~25%.

步骤(4)的离子溶液中NaCl的浓度为5.8~18g/l, NaH2P04 的浓度为0. 01~10g/l, 12朋04的浓度为0. 01g/l。 NaCl concentration of ions in the solution in step (4) is 5.8 ~ 18g / l, a concentration of NaH2P04 is 0. 01 ~ 10g / l, a concentration of 04 to 12 Four 0. 01g / l.

所述步骤(5)中活性炭用量范围为10〜120g/l,;高岭土的使用量为l~100g/l,;活性炭与高岭土的比为l: 0〜2。 Said step (5) the amount of active carbon ranges 10~120g / l ,; kaolin used in an amount of l ~ 100g / l ratio of kaolin to activated carbon ,; l: 0~2.

所述步骤(5)的滤膜的孔径为0.22/zm以下。 Membrane pore size of said step (5) was 0.22 / zm or less. . 本发明的有益效果是- Advantageous effect of the invention is -

利用碳酸盐控制水溶液的pH,用胰蛋白酶水解HA提取物中的蛋白质,然后把经乙醇回收的分解蛋白质后的HA,加到含氯化钠、磷酸二氢钠及磷酸氢二钠的溶液中形成一种离子溶液,向该离子溶液加入乙醇,大量的蛋白质成分不但不沉淀反而溶解,从而一次分离去除大量蛋白质,并为以后的HA高回收率创造了条件。 PH control using carbonate aqueous solution, hydrolyzed with trypsin HA protein extract, and then the decomposed HA protein is recovered ethanol, containing added sodium chloride, sodium dihydrogen phosphate and disodium hydrogen phosphate solution an ion is formed in solution, ethanol was added to the ionic solution, not only a large number of protein components but does not dissolve the precipitate, thereby separating and removing a large number of proteins, and to create the conditions for a high recovery rate after HA. 此后,经过活性碳混合高岭土吸附,残留的大量蛋白质成分被去除及微量核酸被全部除去。 Thereafter, the activated carbon to kaolin adsorption, residual amounts of protein and trace components have been removed nucleic acid is completely removed. 最后,经过透析及羧甲基纤维素处理,微量蛋白质成分进一步减少。 Finally, after the dialysis treatment, and carboxymethyl cellulose, trace protein component is further reduced. 结果,发酵源HA提取物经本发明方法分离纯化纯度达到99. 97%以上,动物源的达到99. 5%以上。 As a result, fermentation derived HA extract isolated by the method of the present invention The purity of more than 99.97%, or more of animal origin reached 99.5%.

与本发明相比,目前普遍使用的CPC等季胺盐类沉淀HA法,要达到99.5%以上纯度,至少需要三次CPC反复沉淀和溶解(朱宛中等, 眼用透明质酸制备工艺改进,中国生物制品学杂志,1996年第3期), 酶水解后需要2-3次氯仿处理;这些在操作步骤上及操作难度上均超过本发明。 Compared with the present invention, CPC and other quaternary ammonium salts are currently widely used HA precipitation method, to reach a purity of 99.5%, at least three times of repeated CPC precipitation and dissolution (Zhu Wan medium, hyaluronic ophthalmic preparation process improvement, China Journal of biological products Science, No. 3, 1996), the enzymatic hydrolysis process requires 2-3 times with chloroform; the present invention are more than these steps and on the difficulty of the operation. 本发明在操作步骤上及操作方便上均有优势。 The present invention is in operation and the operation step are convenient advantages.

本发明方法中使用的碳酸盐及磷酸盐均是普通化学试剂,价格低, 使用量少,及本发明在稳定条件下操作;而使用季胺盐方法中的CPC 等的价格很高,多次使用,用量较大,加之CPC类方法操作难度大。 Carbonates, and phosphates used in the method of the present invention are common chemicals and low price, a small amount, and the present invention is operated under stable conditions; and quaternary amine salts of such methods CPC price is very high, multiple uses, a larger amount, plus class method CPC operation is difficult. 这使得本发明在生产成本上占有优势。 This makes the present invention an advantage in production cost.

本发明操作均在PH>7及温和条件下进行,避免对HA长链分子造成不良影响的操作的被使用。 Operation of the present invention are carried out in the PH> 7 and mild conditions, avoiding long chain molecules of HA caused by the adverse effects of operation is used.

依据本发明方法,最终产物的掺杂物蛋白质达到瑞典Headon小于0. 5%的要求。 The method according to the present invention, the final protein product dopant reaches Sweden Headon requirements of less than 0.5%. 特别是本发明的发酵源HA纯度达到高纯度99. 9鄉以上。 Particular fermentation derived HA purity according to the present invention achieves a high purity more than 99.9 township. 本发明的产物之一,具体步骤(5)的结果,发酵源纯度可达到99. 6% 以上,动物源纯度可达90.8%以上,因此,可根据化妆品及药品对HA 纯度要求的不同制备不同纯度的产品。 One product of the invention, particularly the result of step (5), the fermentation source than the purity of 99.6% can be achieved, animal sources purity of more than 90.8%, therefore, may be different according to different cosmetic and pharmaceutical purity requirements of the prepared HA the purity of the product.

此外,本发明中使用了卤苯,不但在于卤苯对蛋白质的变性及溶解脂的能力与通常使用的氯仿相近,而且氯苯的沸点比氯仿高。 Further, the present invention uses a halogen benzene, halobenzene not only similar to that commonly used chloroform ability of denatured protein and lipid is dissolved, and a boiling point higher than chlorobenzene chloroform. 这样使用氯苯比使用氯仿自然挥发量少的多,因此既减少了试剂的用量, 又减少了对环境的污染。 Such chlorobenzene less than natural evaporation using chloroform more, thus not only reducing the amount of reagents, and reduced environmental pollution.

动物源HA及发酵源HA提取物主要分离纯化步骤后的结果,分别见表1及表2: And animal sources fermentation derived HA HA extraction result after the primary separation and purification steps were, respectively, shown in Table 1 and Table 2:

表1来自牛眼玻璃体的HA提取物主要分离纯化步骤后的结果<table>table see original document page 10</column></row> <table> Purification main results of Table HA bovine vitreous extract from step 1 after the <table> table see original document page 10 </ column> </ row> <table>

注:①*表示绝干物。 Note: ① * represents the absolute dry matter.

②纯化倍数系纯化前后单位体积或质量中HA与蛋白质之比的比的倒数。 The reciprocal ratio of volume mass ② fold purification system before and after purification unit or HA-protein ratio.

表2来自发酵HA提取物主要分离纯化步骤后的结果 Results from Table HA Purification main fermentation extract after step 2

<table>table see original document page 10</column></row> <table> <Table> table see original document page 10 </ column> </ row> <table>

具体实施方式 Detailed ways

本发明的具体步骤如下- Specific steps of the present invention are as follows -

(1) 将得到的HA提取物加入到碱性缓冲水溶液中,并加入胰蛋白酶,在一定温度下搅拌一定时间。 (1) The HA obtained extract was added to the basic aqueous buffer solution, trypsin was added and stirred for a certain time at a certain temperature.

(2) 向上述搅拌液中加入卤苯,然后强烈搅拌30min,最后, 离心并抽取上清液。 (2) was added to the stirred solution halobenzene, followed by vigorous stirring 30min, and finally, extracting the supernatant and centrifuged.

(3) 向上述清液中加氯化钠至0.2mol/L并搅拌溶解,然后加2. 5倍以上体积的95%以上的乙醇或无水乙醇并搅动,静置沉淀发生后离心。 (3) was added and dissolved with stirring to the supernatant was added sodium chloride to 0.2mol / L, then add more than 2.5 times the volume of 95% ethanol or absolute ethanol and stirred, allowed to stand after the occurrence of precipitation by centrifugation. 沉淀物既为分解蛋白质的HA。 HA is an exploded precipitate both proteins.

(4) 加分解蛋白质的HA到一种离子溶液中搅拌溶解,然后加至少2.5倍体积的95%以上的乙醇并搅动,静置至沉淀发生后离心,沉 (4) HA protein was added to decompose an ionic solution and stirred to dissolve, then add 95% ethanol at least 2.5 times the volume of agitated and allowed to stand until precipitation centrifugation, Shen

淀物既为分解蛋白质的透明质酸; Both for the decomposition of hyaluronic acid precipitate protein;

(5)将分解蛋白质的透明质酸溶入与(4)相同的离子溶液中, 然后加入活性炭及高岭土,搅拌lh后离心,上清液经滤膜抽滤。 (5) The decomposition of hyaluronic acid and dissolved protein (4) the same ions in the solution, and then adding activated carbon and kaolin, after centrifugation LH stirring, the supernatant by suction filtration membrane. 滤液既为HA溶液(加乙醇回收可得HA含量609()以上的产物)。 The filtrate was either HA solution (product above was added ethanol recovery available HA content 609 ()).

(6)将步骤(5)的HA溶液用35000Da透析袋在去离子水透析3-4h,然后,向透析后的溶液中加羧甲基纤维素并搅拌,最后离心或抽滤(加乙醇回收可得HA含量9(F。以上的产物)。 (6) The step (5) was treated with HA 35000Da dialysis bag 3-4h dialyzed in deionized water, then added to the carboxymethylcellulose solution after dialysis and stirred, and finally by centrifugation or filtration (ethanol recovery HA content available 9 (F. or more products).

(7) 将上述滤液,冷冻,真空干燥,得无色透明片状透明质酸(HA)产物。 (7) The above filtrate was freeze-dried in vacuo to give a colorless transparent sheet-hyaluronic acid (HA) product. 或向其中加氯化钠至0.15mol/L〜0.25mol/L ,加至少2.5倍体积的95%以上的乙醇并搅动,静置至沉淀发生后离心,沉淀物经冷冻,真空干燥,得到白色粉末状透明质酸产物。 Thereto was added sodium chloride, or to 0.15mol / L~0.25mol / L, plus at least 2.5 times the volume of 95% ethanol and stirred, allowed to stand until precipitation occurs after the centrifugation, the precipitate was freeze-dried in vacuo to give a white powdery hyaluronic acid product.

所述将步骤(1)碱性缓冲水溶液是由NaHC03和Na2C03或NaHC03 和NaOH, NaHC03的使用量为0. 01~120g/l,碳酸钠Na2C03的用量为0~100g/l,或NaOH为0~lg/l;所述碳酸氢钠及碳酸钠最佳浓度是l-10g/L。 Said step (1) is a basic aqueous buffer solution of NaHC03 and Na2C03 and NaHC03 or NaOH, is used in an amount NaHC03 0. 01 ~ 120g / l, the amount of sodium carbonate Na2C03 is 0 ~ 100g / l, or NaOH 0 ~ lg / l; the optimum concentration of sodium bicarbonate and sodium carbonate is l-10g / L.

本发明所用的碱性缓冲水溶液是指由碳酸氢钠和碳酸钠组成,或碳酸氢钠和氢氧化钠组成(或相应的钾盐),这里碳酸氢钠的使用量为0.01~120g/L,碳酸钠的用量为0~100g/L,氢氧化钠的用量为0~lg/L。 Basic aqueous buffer solution used in the present invention refers to a composition consisting of sodium bicarbonate and sodium carbonate, sodium hydroxide or sodium bicarbonate and the composition (or the corresponding potassium salt), an amount of sodium bicarbonate used herein is 0.01 ~ 120g / L, sodium carbonate is used in an amount of 0 ~ 100g / L, sodium hydroxide in an amount of 0 ~ lg / L.

本发明所用的胰蛋白酶为粗制胰蛋白酶(生物化学实验指导,清华大学出版社.2004年1月1版:199~202页)或精制胰蛋白酶。 The trypsin used in the present invention as a crude trypsin (Biochemical Experiment guide, Tsinghua University Press, January 2004 edition: 199 to 202) or purified trypsin. 用量为HA提取物中每毫克蛋白质加2~100活力个单位的量(pH7.5,温度为37t:条件,每分钟分解产生l/^g酪氨酸为l个单位),酶的分解温度为20~42卩,分解时间为0. 5~90h,较好的温度范围是30〜33。 Extract in an amount of HA per milligram of protein plus 2 to 100 units of activity (pH 7.5, temperature 37t: condition, decomposition of produced per minute l / ^ g l tyrosine units), the decomposition temperature of the enzyme Jie 20 to 42, the decomposition time is 0. 5 ~ 90h, the temperature range is preferably 30~33. C,较好的酶加量是HA提取物中每毫克蛋白质加8~25个活力单位的酶量,时间最好控制24h以内。 C, the amount of enzyme added is preferably HA protein extract enzyme activity an amount of 8-25 mg per unit time is preferably controlled within 24h.

为了减少试剂用量和减少环境污染,本发明向酶水解液中加入的非极性溶剂卤苯是氯苯或溴苯以代替通常的氯仿。 In order to reduce the amount of reagent and reduce environmental pollution, the present invention is added to the enzymatic hydrolysis of non-polar solvent solution halobenzene is chlorobenzene or bromobenzene in place of the usual chloroform. 这一步的离心,工业化时可用碟片式离心机半连续工作。 This centrifugation step can be used semi-continuous disc centrifuge industrial work.

本发明溶解纯化产物I的离子溶液为由氯化钠及磷酸二氢钠(或钾)和磷酸氢二钠(或钾)组成的溶液。 The present invention was dissolved ions I of purified product by sodium chloride and sodium dihydrogen phosphate (or potassium) and disodium hydrogen phosphate solution (or potassium) thereof. 氯化钠较好的浓度范围为5~18g/L,磷酸二氢钠或磷酸二氢钾较好的浓度范围0.01~0.5g/L, 磷酸氢二钠或磷酸氢二钾较好的浓度为0.1~2. 5g/L。 Preferably sodium chloride concentration ranging from 5 ~ 18g / L, potassium dihydrogen phosphate or sodium dihydrogen phosphate concentration range of preferably 0.01 ~ 0.5g L, disodium hydrogen phosphate or dipotassium hydrogen phosphate is preferably a concentration / is 0.1 ~ 2. 5g / L.

本发明向HA水溶液中加入的活性炭、高岭土及羧甲基纤维素介质均经过稀盐酸浸泡、稀碱浸泡、抽滤、用去离子水洗至中性和干燥过程处理。 The present invention is added to an aqueous solution of HA of activated carbon, kaolin, and carboxymethyl cellulose media are soaked through with dilute hydrochloric acid, dilute alkali soaking, suction filtration, washed with deionized water to neutrality and drying process. 活性炭用量较好的范围为30~150g/L;高岭土的使用量为40~80g/L 。 Activated carbon is preferably used in an amount in the range of 30 ~ 150g / L; the amount of kaolin was 40 ~ 80g / L. 羧甲基纤维素较好的用量范围为l~5g/L。 Carboxymethyl cellulose is preferably used in an amount in the range of l ~ 5g / L.

HA的定量分析法用咔唑法(Analytical Biochemistry. 1962.4:330〜334 ), 分子量用粘度法(Biochem. Biophys. 1960.42.476~486),蛋白质的测定用Folin-酚法,核酸的检査用二苯胺法、地衣酚法和紫外扫描。 Inspection, the molecular weight viscosity method, determination of protein by Folin- phenol method, nucleic acid: HA quantitative analysis method using carbazole method (330~334 Analytical Biochemistry 1962.4.) (Biochem Biophys 1960.42.476 ~ 486..) diphenylamine method, the orcinol method, and UV scanning. 结构检査用IR。 Structure inspection IR.

本发明不但可以在实验室实施,而且容易实现工业化,酶分解及加卤苯等过程只需一个搅拌釜。 The present invention can be implemented not only in the laboratory, and easy to industrialize and enzymatic degradation process just like adding a halobenzene stirred tank. 卤苯与水相的分离只需一台碟片式离心机。 Separating the aqueous phase halophenyl only a disc centrifuge. 固液分离时需要一台常用离心过滤机和一个抽滤装置,透析需要一台中空纤维透析装置。 Need a common centrifugal filter device and a solid-liquid separation by suction filtration, hollow fiber dialysis station requires a dialysis apparatus. 所有这些都表明,本发明是容易实现从实验室到工业生产过程的放大。 All this suggests that the present invention can be readily made from laboratory to industrial processes amplification.

本发明HA提取物的取得是由原材料经水溶液提取,制得HA提取液后,向提取液中加入氯化钠、乙醇,经沉淀、离心或过滤获得的。 HA obtain extract of the present invention is extracted from raw materials by an aqueous solution, the extract was prepared HA, sodium chloride, ethanol extract by sedimentation, centrifugation or filtration obtained.

从不同原材料中提取HA提取液的方法是:(1)从微生物发酵液中制取HA提取液:参见(无锡轻工大学学报.2000. Vol.19 No. 5 :437-439)。 The method of extracting HA extracts from different raw materials are: (1) from a microbial fermentation broth extract HA preparation: see (Light Industry University OF .2000 Vol.19 No. 5:. 437-439). (2)从动物眼玻璃体中制取HA提取液:剥去眼球外层皮革及除去晶状体,加入0. 37g/L的EDTA-Na,组织捣碎机捣1 ~2min,后于2000r/min离心10分钟或抽滤,得HA提取液。 (2) from the animal vitreous extract in the preparation of HA: leather and stripped to remove the outer layer of the eye lens, was added 0. 37g / L of EDTA-Na, tissue compactors tamper 1 ~ 2min, after 2000r / min centrifugal 10 minutes or filtration, to obtain HA extract. (3)从人脐带中制取HA提取液:取冷冻人脐带切片后,按重量的4~6倍加入去离子水,并按0. 37g/L的浓度加入EDTA-Na,缓慢搅拌2~3h,离心或过滤得HA提取液。 (3) from the human umbilical cord HA extract preparation: After taking frozen sections of human umbilical cord, 4 to 6 times by weight of deionized water, press 0. 37g / L concentration of added EDTA-Na, was slowly stirred for 2 ~ 3h, centrifugation or filtration to obtain HA extract. (4)从鸡冠中制取HA提取液:新鲜公鸡冠冷冻后切成片,加入6倍体积的无水乙醇或95%的乙醇,搅拌10~20min, 离心或抽滤,按重量向离心得到的固体物中加入5-8倍的去离子水, 并按0. 37g/L的浓度加入EDTA-Na,搅拌3~4h,离心或抽滤,得HA (4) Preparation of HA from rooster combs extract: Fresh sliced ​​rooster combs frozen after addition of 6 volumes of absolute ethanol or 95% ethanol, stirred for 10 ~ 20min, centrifugation or suction filtration, centrifugation by weight to give the solid was added deionized water 5-8 times, press 0. 37g / L concentration of added EDTA-Na, was stirred for 3 ~ 4h, centrifugation or suction filtration to obtain HA

本发明所用的HA提取物均为从提取液中直接分离得到的湿样固体物,所用的水都为去离子水,所有数据都是三次测定的平均值。 HA used in the present invention are isolated directly extract from the extract obtained in wet-like solids, the water used is deionized water, all the data are the average of triplicate determinations. 百分比是以质量计的。 The percentage is based on a mass basis. 最终HA产品的IR谱图与相同来源的生化试剂HA的IR谱图在波形及吸收峰上是一致的;浓度l-3g/L的HA水溶液的核酸测定的吸收值均为零,紫外区扫描均无核酸吸收峰。 The final HA product IR IR spectrum of the HA and biochemical reagents same source spectrum and absorption peak in the waveform is the same; absorbance measured the concentration of nucleic acid l-3g / L of an aqueous solution of HA are zero, the scanning UV region no nucleic acid absorption peak.

通过下面的从HA提取物中分离纯化HA的过程概要及详细实施例中,能更好地理解本发明,它们叙述了从发酵液、牛眼玻璃体、人脐带及公鸡冠HA提取物中分离纯化HA的具体过程。 By following the process of separation and purification of HA from the HA extracts summary and detailed embodiments of the present invention will be better understood, a description thereof is separated from the fermentation broth, bovine vitreous, rooster combs and human umbilical cord HA purified extract specific process of HA. 但本发明并不仅适用于从上述四种原料中分离纯化HA。 However, the present invention is applicable not only to the above four isolated and purified from the raw material HA.

从HA提取物中分离纯化HA过程概要: Extract isolated from HA HA purification process Summary:

具体步骤(1)中,在不同的碱性缓冲溶液中酶解蛋白质,其它条件相同而结果是不同的。 DETAILED step (1), the alkaline hydrolysis of proteins in different buffer solution, and the result is different from the same other conditions. 例如,使用0.01~0.1mol/L的磷酸二氢钠和磷酸氢二钠组成的缓冲溶液酶解牛盐源HA提取物中蛋白质,结果步骤(4) HA的纯度仅19~25%;步骤(5) HA纯度仅35~40%,蛋白质的去除率60~65%, HA的损失率20~33%。 For example, use 0.01 ~ 0.1mol / L sodium dihydrogen phosphate and disodium hydrogen phosphate buffer solution consisting of bovine enzyme extract yanyuan HA protein, the result of step (. 4) HA purity of only 19 to 25%; Step ( . 5) HA purity is only 35% to 40%, the removal rate of 60 to 65% protein, 20 to HA loss rate of 33%. 使用碳酸氢钠与碳酸钠,或碳酸氢钠和氢氧化钠,或单一碳酸氢钠,或碳酸氢钠和乙酸(调pH) 组成的缓冲溶液,结果步骤(4) HA的纯度为40〜68y。 Sodium bicarbonate and sodium carbonate, and sodium hydroxide or sodium bicarbonate, or a single bicarbonate, or bicarbonate and a buffer solution of acetic acid (adjusted pH) composed of the result of step (4) Purity of HA 40~68y . 之间,步骤(5) HA的纯度为60〜98%之间。 Between Step (5) purity of the HA is between 60~98%. 其中效果最好的是碳酸氢钠与碳酸钠组成的碱性缓冲溶液。 Which it is the best solution of sodium bicarbonate and sodium carbonate alkaline buffer composition. 例如,在碳酸氢钠浓度为0.3g/L,碳酸钠浓度为0.7g/L时,步骤(4)區的纯度为45%, HA的损失率12呢;步骤(5) HA的纯度为769&, HA的损失率6《。 For example, sodium bicarbonate in a concentration of 0.3g / L, sodium carbonate concentration of 0.7g / L, the step (4) was a purity of 45% area loss rate of 12 HA it; Step (5) 769 & purity of HA loss rate of 6 HA. " 又例如,碳酸氢钠浓度为30g/L , 碳酸钠浓度为70g/L时,步骤(4)HA的纯度为50%, HA的损失率12%; 步骤(5)HA的纯度为66%,HA的损失率4%。 As another example, sodium bicarbonate at a concentration of 30g / L, sodium carbonate concentration was 70g / L, the step (4) was a purity of 50% HA, HA loss rate of 12%; purity Step (. 5) 66% HA, 4% loss of HA. 效果较好的浓度是l~10g/L 的碳酸氢钠及碳酸钠浓度,实例见后面实施例。 Better concentrations are 10g / L concentration of sodium carbonate and sodium bicarbonate l ~, see examples later examples.

具体步骤(1)中,酶的加量、酶水解的温度及水解的时间是分离纯化HA要选取的条件之一,选取这些条件不同结果不同。 DETAILED step (1), the amount of enzyme added, the temperature and time of hydrolysis is enzymatic hydrolysis of the selected one of the conditions for separation and purification of HA, select a different outcome of these different conditions. 例如, 对牛眼源HA的分离纯化,酶的加量选每毫克蛋白质加2个活力单位的酶量,酶的水解温度选37°C,水解时间选60h ,步骤(4) HA的纯度为48%, HA的损失率13%;步骤(5) HA的纯度为78%, HA的损失率5%。 For example, separation and purification of bovine-derived HA, selected from an enzyme dosage per mg of protein plus 2 units of the amount of enzyme activity, the enzyme is selected from the hydrolysis temperature 37 ° C, 60h purity hydrolysis time is selected, step (. 4) of HA 48%, 13% loss of HA; purity step (. 5) 78% HA, HA loss rate of 5%. 酶的加量选每毫克蛋白质加60个活力单位的酶量,酶的水解温度选3(TC,水解时间选10h ,步骤(4) HA的纯度为58%, HA 的损失率10%;步骤(5) HA的纯度为9线HA的损失率鄉。三者协调较好的是:酶的加量选用HA提取物中每毫克蛋白质,加12个活力单位的酶量,酶的水解温度选33t:,水解时间选20 h,结果见后面详细实例。具体步骤(2)中,使用氯苯或溴苯,用量可以在0.2-0.3倍体积的水解液量范围。对发酵源HA提取物的纯化,步骤(5)纯度要求《99. 3%及纯化产物111纯度要求《99. 65%的,具体步骤(2)可以跳过。 Adding amount of the enzyme is selected from per mg of protein plus 60 activity units of the amount of enzyme, enzymatic hydrolysis temperature is selected from 3 (TC, hydrolysis time selected 10H, step (4) Purity of HA was 58% loss of HA 10%; Step (5) purity of HA HA 9 line loss rate is preferably coordinated three rural: HA selection plus the amount of enzyme per milligram of protein extract, the amount of enzyme activity added 12 units of enzyme hydrolysis temperature is selected from 33t :, hydrolysis time is selected from 20 h, the detailed results are shown in examples below. specifically in step (2), a bromobenzene or chlorobenzene, an amount of the hydrolyzate in an amount ranging from 0.2 to 0.3 times in volume. fermentation derived HA extract purification step (5) the purity requirements "and 99.3 percent purity product was purified 111" 99.65%, the specific step (2) can be skipped.

具体步骤(4)中,离子溶液中各种离子的浓度不同时纯化的结果不同。 DETAILED step (4), different concentrations of various ions in the solution of ions simultaneously without purification results. 例如,氯化钠的浓度为5.8g/L,磷酸氢二钠的浓度为0.15g/L,磷酸氢二钠的浓度为0.02g/L时,源自牛眼玻璃体HA提取物的纯化结果是:纯化I的纯度为58%及回收率为86%,纯化II的纯度为91%及回收率为90% 。 For example, the concentration of sodium chloride 5.8g / L, disodium hydrogen phosphate to a concentration of 0.15g / L, the concentration of disodium hydrogen phosphate 0.02g / L when purified from bovine vitreous HA results extract is : purification I of 58% purity and 86% recovery, purified purity of II and 91% recovery is 90%. 又例如,氯化钠的浓度为17.4g/L,磷酸氢二钠的浓度为1.5g/L,磷酸氢二钠的浓度为0.4g/L时,源自牛眼玻璃体HA提取物的纯化结果是:步骤(4)的纯度为639&及回收率为87%,步骤(5)的纯度为93%及回收率为95%。 When another example, the concentration of sodium chloride 17.4g / L, the concentration of disodium hydrogen phosphate 1.5g / L, the concentration of disodium hydrogen phosphate 0.4g / L, the results derived from purified bovine vitreous extract HA They are: purity step (4) is 639 and the recovery was 87% & purity of step (5) was 93% and the recovery was 95%. 最佳的离子溶液浓度为氯化钠的浓度为12. 4g/L,磷酸氢二钠的浓度为0. 5g/L,磷酸氢二钠的浓度为0.1g/L,结果见后面详细实例。 The optimum concentration of sodium ion concentration of the solution was 12. 4g / L, disodium hydrogen phosphate to a concentration of 0. 5g / L, disodium hydrogen phosphate to a concentration of 0.1g / L, the results see detailed example below.

具体步骤5中,活性炭起吸附核酸及残余蛋白质作用,高岭土其辅助作用,这一步采用离心过滤,高岭土可不加。 Step 5 In particular, from the activated carbon adsorption of residual protein and nucleic acid action, kaolin its secondary effect, which centrifugal filtration step, the kaolin may be added. 若前述的具体步骤(1)中酶水解不好时,这一步的HA回收率低,这是由于仍有较多的HA与蛋白质共价相连。 When the concrete steps (1) during the enzymatic hydrolysis in poor recovery of low HA this step, it is still more due to the protein covalently linked HA. 这一步中,活性炭的加量根据步骤(5)中蛋白质的含量及HA的浓度变化,0.1%~0. 3%的动物源HA加8~12%的活性炭,发酵源HA加3~5%的活性炭,活性炭超过15%不利于这一步的HA回收率。 This step, plus the amount of activated carbon according to step (5) changes in the concentration of content of the protein and the HA of 0.1% ~ 0.3% of animal-derived HA plus 8 to 12% activated carbon, fermentation derived HA plus 3-5% the activated carbon, more than 15% HA is not conducive to the recovery of this step. 较好加量见详细实施例。 See detail preferred embodiments of dosage.

具体步骤(6)中,透析时间发酵源HA采用3h,动物源HA采用4h,透析时间长HA纯度不增加,HA回收率有所降低。 DETAILED step (6), duration of dialysis employed fermentation derived HA 3h, use of animal-derived HA 4h, dialysis for a long time does not increase the purity of HA, HA recovery rate decreased. 例如透析6h, 发酵源HA的回收率为93%,动物源HA的回收率为90%。 For example dialysis 6h, recovery of fermentation derived HA was 93% and the recovery of animal origin HA was 90%. 羧甲基纤维素对HA纯度的提高有一定作用,尤其对动物源HA。 Carboxymethyl cellulose has a role to improve the purity of the HA, HA especially of animal origin. 对于要求纯度99. 95%的发酵源HA还可以不加羧甲基纤维素,这一步羧甲基纤维素的加量为l〜2g/L,恥的纯度可以提高0.02%以上。 99.95% purity requirements of fermentation derived HA may be used without further carboxymethyl cellulose, plus the amount of this step is carboxymethylcellulose l~2g / L, the purity can be increased shame 0.02% or more. 动物源HA,这一步加量为1~2 g/L的羧甲基纤维素,HA的纯度提高0. 02%以上。 Animal sources HA, this step is added in an amount of 1 ~ 2 g / L carboxymethyl cellulose, HA purity increased by 0.02%. 动物源HA,这一步加量为3〜4 g/L的羧甲基纤维素,HA的纯度提高0.18%。 Animal sources HA, this step is added in an amount of 3~4 g / L carboxymethyl cellulose, HA increase the purity of 0.18%. 较好的加量和结果见下面详细实施例。 The dosage and the preferred embodiments detailed results, see below Examples. 实施例: Example:

实施例1 :从牛眼玻璃体HA提取物中分离纯化操作条件较好时的一例 Example 1: one case when the operating condition is preferably isolated from purified bovine vitreous extract HA

步骤l、取10g (未干燥样)牛眼源HA提取物,加浓度为8g/L 的碳酸氢钠(NaHC03)水溶液450ml和浓度为2. 6g/L的碳酸钠(化20)3) 水溶液50ml,再加入0. 25 g的胰蛋白酶,在33°C、 120r/tnin下搅拌20h。 Step L, taking 10g (undried sample) bovine-derived HA extract added at a concentration of 8g / L sodium bicarbonate (or NaHCO3 in) an aqueous solution of a concentration of 450ml 2. 6g / L of sodium carbonate (of 20) 3) aqueous solution 50ml, was added 0. 25 g of trypsin, stirring at 33 ° C, 120r / tnin 20h.

步骤2、向上述溶液中加入150ml氯苯于1000r/min搅拌30min, 然后与2500r/min离心20min,抽取上清液。 Step 2, was added to the above solution 150ml chlorobenzene at 1000r / min was stirred for 30min, then with 2500r / min centrifugal 20min, the supernatant was extracted.

步骤3、然后加入5g氯化钠,搅拌溶解,再加入1500ml的无水乙醇或95%的乙醇,搅动混合,静置lh,后于2000rin/min离心10min, 得到粗品HA。 Step 3, and 5g of sodium chloride was added, stirred to dissolve, then add 1500ml anhydrous ethanol or 95% ethanol, mixed by agitation, left to stand LH, after 2000rin / min centrifugal 10min, to obtain a crude product HA.

步骤4、粗品HA加入到氯化钠浓度为10. 6g/L、磷酸氢二钠浓度为0. 2g/L、磷酸二氢钠的浓度为0. 06g/L的500ml水溶液中,搅拌溶解,再向其中加入1500ml无水乙醇,搅动混合,静置lh,后于2000rin/min离心10min,并用75%的乙醇50ml洗涤沉淀HA(即纯化产物I)。 Step 4, the crude product was added to a sodium chloride concentration of HA 10. 6g / L, disodium hydrogen phosphate at a concentration of 0. 2g / L, the concentration of sodium dihydrogen phosphate was 0. 06g / L of 500ml water and stirred to dissolve, 1500ml of anhydrous ethanol was added thereto, mixed by agitation, left to stand LH, after 2000rin / min centrifugal 10min, and the precipitate was washed with 75% ethanol 50ml HA (i.e. purified product I).

步骤5、 将上述沉淀HA溶于100ml的氯化钠磷酸盐溶液(浓度同步骤4)中,搅拌溶解,再向溶液中加入4g活性炭和8g高岭土,搅拌lh,然后经0.22^m的滤膜抽滤,收集滤液。 Step 5, the above-described HA precipitated sodium chloride was dissolved in 100ml phosphate solution (with a concentration step 4), was dissolved with stirring, 4g of activated carbon was added again and 8g kaolin, LH stirring, and then the membrane was 0.22 ^ m filtration, the filtrate collected.

步骤6、将上述滤液于35000Da的透析袋中于500ml去离子水中透析4h,然后保留中加入0.3g羧甲基纤维素,搅拌混合30min, 经0.22pm的滤膜抽滤,收集滤液。 Step 6, the above filtrate was 35000Da dialysis bags in 500ml deionized water and dialyzed 4h, then added 0.3g reservation carboxymethyl cellulose, mixed by stirring 30min, after 0.22pm membrane filtration, and the filtrate was collected.

步骤7、向滤液中加入lg氯化钠,搅拌溶解,然后再加入3倍体积的95%的乙醇,搅动混合,静置lh,抽滤,并用20ml丙酮洗涤固体HA。 Step 7, lg of sodium chloride was added to the filtrate, stirred to dissolve, then adding 95% ethanol three times the volume of the mixture agitated and allowed to stand LH, suction filtered and the solid washed with 20ml of acetone HA. 将固体HA经冷冻,真空干燥,得到白色粉末状HA产物。 The HA solid was freeze-dried in vacuo to give a white powder product HA. 这一实施例的纯化效果见表l。 Purification effect of this embodiment are shown in Table l. 最终结果如下(本发明所提纯度指透明质酸与蛋白质相加透明质酸之后比值的百分数): The final results are as follows (referred to in the present invention refers to the ratio of the purity of the protein after the addition of hyaluronic acid hyaluronic acid percentage):

HA纯度99. 63% HA purity of 99.63%

收得率83. 81% Yield 83.81%

分子量6. lx105 6. lx105 molecular weight

实施例2 :从发酵HA提取物中分离纯化操作条件较好时的一例 Example 2: Purification one case when the operating condition is preferably isolated from the fermentation extract HA

改变实施例1中:歩骤(1)的酶加量为0.15g及水解时间12h; 步骤(5)的氯化钠磷酸盐溶液的量为500ml,活性炭的量为25g和高岭土的量为40g;步骤(6)的透析用离子水为1500ml,透析时间为3h;步骤(7)的氯化钠为4g。 Changes in Example 1: ho step (1) is the amount of enzyme added and the time of hydrolysis 12h 0.15g; the amount of sodium phosphate solution in step (5) is 500ml, the amount of activated carbon in an amount of 25g and 40g of kaolin ; dialysis step (6) with deionized water to 1500ml, duration of dialysis 3H; chloride in step (7) of 4g. 这一实施例的纯化效果见表2。 Purification effect of this embodiment are shown in Table 2. 最终结果如下: The final results are as follows:

HA纯度99.96% HA purity 99.96

收得率90.6% Yield 90.6%

分子量5.8x105 实施例3 : 从人脐带HA提取物中分离纯化HA Molecular weight of 5.8x105 Example 3: extract from human umbilical cord HA HA was isolated and purified

步骤及操作同实施例l,结果如下: Steps and operations as in Example l, the following results:

HA纯度99. 51%收得率97.17 % HA purity 99.51% 97.17% yield

分子量4.5xl05 实施例4从公鸡HA提取物中分离纯化操作条件较好时的一例 Example 4.5xl05 molecular separation and purification of one case when operating conditions is preferably from rooster HA extract 4

改变实施例1中:步骤(1)碳酸氢钠的浓度为10g/L,碳酸钠的浓度为1.6g/L;步骤(4)的氯化钠浓度为11.6g/L、磷酸氢二钠浓度为0.15g/L、磷酸二氢钠的浓度为0.10g/L;步骤(5)的活性炭的量为10g和高岭土的量为20g;步骤(6)的羧甲基纤维素的量为0.5g。 Change Example 1: Step (1) the concentration of sodium bicarbonate was 10g / L, the concentration of sodium carbonate was 1.6g / L; step (4) sodium chloride at a concentration of 11.6g / L, the concentration of disodium hydrogen phosphate of 0.15g / L, the concentration of sodium dihydrogen phosphate was 0.10g / L; the amount of activated carbon in step (5) in an amount of 10g and 20g of kaolin; carboxymethylcellulose is step (6) is 0.5g . 结果如下: The results are as follows:

HA纯度99. 52% HA purity of 99.52%

收得率83. 6% Yield 83.6%

分子量4.8xl05 Molecular weight 4.8xl05

本发明碱性缓冲水溶液中盐可以为相应的钾盐,碱性缓冲水溶液还可以是乙酸钠与碳酸氢钠组成的溶液。 The present invention is an alkaline aqueous buffer salts may be the corresponding potassium salt, an alkaline aqueous solution may also be buffered sodium acetate and sodium hydrogencarbonate solution.

Claims (10)

1、一种透明质酸分离纯化的方法,其特征是:它包括以下步骤: (1)将得到的透明质酸提取物加入到碱性缓冲水溶液中,并加入胰蛋白酶,在常温下搅拌; (2)向上述混合液中加入卤苯,搅拌,然后离心抽取上清液; (3)向上述清液中加入氯化钠,得到浓度为0.15mol/L~0.25mol/L的氯化钠溶液,并搅拌溶解,然后加至少2.5倍体积的95%以上的乙醇并搅动,静置至沉淀发生后离心,沉淀物即为分解蛋白质的透明质酸; (4)加分解蛋白质的透明质酸到由NaCl、NaH2PO4和Na2HPO4组成的,或NaCl、KH2PO4和K2HPO4组成的离子溶液中搅拌溶解,然后加至少2.5倍体积的95%以上的乙醇并搅动,静置至沉淀发生后离心,沉淀物为固性物,该固性物含至少42%的透明质酸; (5)将步骤(4)的沉淀物加入由NaCl、NaH2PO4和Na2HPO4组成的,或NaCl、KH2PO4和K2HPO4组成的离子溶液后搅拌溶解,然后加入活 1. A method for separation and purification of hyaluronic acid, characterized in that: it comprises the following steps: (1) The resulting hyaluronic acid was added to extract the basic aqueous buffer solution, trypsin was added and the mixture was stirred at room temperature; (2) added to the above mixture halophenyl, stirred and then centrifuged to extract the supernatant; (3) sodium chloride was added to the supernatant to give a concentration of 0.15mol / L ~ 0.25mol / L sodium chloride solution, and stirred to dissolve, then add 95% ethanol at least 2.5 times the volume of agitated and allowed to stand until precipitation occurs after the centrifugation, the precipitate is the decomposition of hyaluronic acid protein; hyaluronic acid (4) plus protein degradation to the NaCl, NaH2PO4 and Na2HPO4 composition, or NaCl, KH2PO4 and K2HPO4 solution consisting of ions dissolved with stirring, and then 95% ethanol was added, and at least 2.5 times the volume of agitated, allowed to stand until precipitation occurs after the centrifugation, the precipitate is curable composition, the curable composition containing at least 42% of hyaluronic acid; (5) in step (4) was added to the precipitate NaCl, NaH2PO4 and Na2HPO4 composition, or NaCl, KH2PO4 and K2HPO4 stirred solution consisting of ionic was dissolved, followed by addition of live 炭,或活性炭和高岭土,搅拌、离心,上清液经滤膜抽滤,滤液即为透明质酸溶液,然后加至少2.5倍体积的95%以上的乙醇并搅动,静置至沉淀发生后离心,沉淀物中透明质酸含量至少60%。 Charcoal, or activated carbon and kaolin, stirred, centrifuged, the supernatant was the membrane filtration, the filtrate is hyaluronic acid solution, then add 95% ethanol at least 2.5 volumes and agitated, allowed to stand until precipitation occurred after centrifugation , precipitates at least 60% acid content.
2、 根据权利要求1所述的透明质酸分离纯化的方法,其特征是:它还包以下步骤:(6)将所述步骤(5)的经滤膜抽滤后所得的透明质酸溶液用35000Da透析袋在去离子水中透析3~4h,然后,向透析后的透明质酸溶液中加羧甲基纤维素并搅拌,最后离心或抽滤得到滤液;羧甲基纤维素的使用量为10~30g/l;(7)将上述滤液,冷冻,真空干燥,得无色透明片状透明质酸产物,或向上述滤液中加氯化钠至0. 15mol/L~0. 25mol/L ,加至少2. 5倍体积的95%以上的乙醇并搅动,静置至沉淀发生后离心,沉淀物经冷冻,真空干燥,得到白色粉末状透明质酸产物。 2. The method of separation and purification of hyaluronic acid according to claim 1, characterized in that: the package further steps of: (6) obtained after said step (5) the hyaluronic acid solution by membrane filtration 35000Da dialysis bag with dialysis in deionized water 3 ~ 4h, then added to the hyaluronic acid, carboxymethyl cellulose dialysis solution and stirred, and finally the filtrate obtained by centrifugation or filtration; the amount of carboxymethyl cellulose 10 ~ 30g / l;. (7) the above filtrate was freeze-dried in vacuo to give a colorless transparent sheet-like product of hyaluronic acid, or sodium chloride was added to the filtrate to 0. 15mol / L ~ 0 25mol / L , plus at least 95% ethanol and 2.5 volumes of agitated, centrifuged, the precipitate was freeze-dried in vacuo until precipitation occurred after standing, hyaluronic acid product was obtained as a white powder.
3、 根据权利要求1所述的透明质酸分离纯化的方法,其特征是-所述步骤(1)的碱性缓冲水溶液化学药品是由NaHC03和Na2C03组成,或由NaHC03和NaOH组成;NaHC03的使用量为0. 01~120g/L,碳酸钠Na2C03的用量为0〜100g/L,或NaOH为0~lg/L。 3. The method of separation and purification of hyaluronic acid according to claim 1, characterized in that - said step (1) of the basic aqueous buffer solution of NaHC03 and Na2C03 chemicals is composed of, or consisting of NaOH and NaHC03; of NaHC03 used in an amount of 0. 01 ~ 120g / L, an amount of sodium carbonate Na2C03 0~100g / L, or NaOH is 0 ~ lg / L.
4、 根据权利要求3所述的透明质酸分离纯化的方法,其特征是:所述碳酸氢钠和碳酸钠的浓度是l〜10g/L。 4. The method of separation and purification of hyaluronic acid according to claim 3, characterized in that: the concentration of the sodium bicarbonate and sodium carbonate are l~10g / L.
5、 根据权利要求1所述的透明质酸分离纯化的方法,其特征是:所述步骤(1)所用的胰蛋白酶为粗制胰蛋白酶或精制胰蛋白酶,用量范围为透明质酸提取物中每毫克蛋白质用2-100个活力单位的酶量,酶的分解温度为20〜42'C,分解时间为0.5〜90h。 5. The method of separation and purification of hyaluronic acid according to claim 1, wherein: said step (1) to be used as a crude trypsin or trypsin purified trypsin, hyaluronidase amount ranging extract the amount of enzyme used per mg of protein in activity units 2-100, the decomposition temperature of the enzyme is 20~42'C, the decomposition time is 0.5~90h.
6、 根据权利要求5所述的透明质酸分离纯化的方法,其特征是:所述酶的分解温度范围是30~33°C,酶加量是按透明质酸提取物中每毫克蛋白质用8〜25个活力单位的酶量计,时间是24h以内。 6. The method of separation and purification of hyaluronic acid according to claim 5, characterized in that: the decomposition temperature range of the enzyme is 30 ~ 33 ° C, the enzyme dosage is based on hyaluronic acid per mg of protein extract with 8~25 a meter enzyme activity unit of time is less than 24h.
7、 根据权利要求1所述的透明质酸分离纯化的方法,其特征是:所述步骤(2)加入卤苯是氯苯、溴苯、氟苯或碘苯;加入的体积比为:5〜25%。 7. The method of separation and purification of hyaluronic acid according to claim 1, wherein: said step (2) was added benzene halide is chlorobenzene, bromobenzene, fluorobenzene or iodobenzene; the volume ratio of added: 5 ~ 25%.
8、 根据权利要求1所述的透明质酸分离纯化的方法,其特征是-所述步骤(4)的离子溶液中NaCl的浓度为5.8~18g/l, NaH2P04的浓度为0. 01~10g/l,船,04的浓度为0. 01g/l。 8. The method of separation and purification of hyaluronic acid according to claim 1, characterized in that - said concentration step (4) of the ionic solution of NaCl is 5.8 ~ 18g / l, a concentration of NaH2P04 is 0. 01 ~ 10g / l, the boat, the concentration of 04 to 0. 01g / l.
9、 根据权利要求1所述的透明质酸分离纯化的方法,其特征是-所述步骤(5)中活性炭用量范围为10〜120g/l,;高岭土的使用量为l~100g/l,;活性炭与高岭土的比为l: 0~2。 9. The method of separation and purification of hyaluronic acid according to claim 1, characterized in that - said step (5) the amount of active carbon ranges 10~120g / l ,; kaolin used in an amount of l ~ 100g / l, ; ratio of activated carbon and kaolin to l: 0 ~ 2.
10、 根据权利要求1所述的透明质酸分离纯化的方法,其特征是:所述步骤(5)的滤膜的孔径为0.22/zm以下。 10. The method of separation and purification of hyaluronic acid according to claim 1, characterized in that: said step a pore size filter (5) is 0.22 / zm or less.
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