CN1138776C - Extraction method of pyrroquinolinequinone - Google Patents
Extraction method of pyrroquinolinequinone Download PDFInfo
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- CN1138776C CN1138776C CNB001146394A CN00114639A CN1138776C CN 1138776 C CN1138776 C CN 1138776C CN B001146394 A CNB001146394 A CN B001146394A CN 00114639 A CN00114639 A CN 00114639A CN 1138776 C CN1138776 C CN 1138776C
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- pqq
- pyrroloquinoline quinone
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- adsorption column
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- 238000000605 extraction Methods 0.000 title abstract 2
- 238000000034 method Methods 0.000 claims abstract description 20
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims abstract description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000007788 liquid Substances 0.000 claims abstract description 9
- 239000012043 crude product Substances 0.000 claims abstract description 8
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims abstract description 6
- MMXZSJMASHPLLR-UHFFFAOYSA-N pyrroloquinoline quinone Chemical compound C12=C(C(O)=O)C=C(C(O)=O)N=C2C(=O)C(=O)C2=C1NC(C(=O)O)=C2 MMXZSJMASHPLLR-UHFFFAOYSA-N 0.000 claims description 82
- 238000005406 washing Methods 0.000 claims description 12
- 238000010521 absorption reaction Methods 0.000 claims description 9
- 238000001179 sorption measurement Methods 0.000 claims description 9
- 238000001035 drying Methods 0.000 claims description 8
- 238000002425 crystallisation Methods 0.000 claims description 4
- 230000008025 crystallization Effects 0.000 claims description 4
- 238000009736 wetting Methods 0.000 claims description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 abstract description 12
- 239000000047 product Substances 0.000 abstract description 5
- 238000011084 recovery Methods 0.000 abstract description 5
- 150000003839 salts Chemical class 0.000 abstract description 4
- 238000001953 recrystallisation Methods 0.000 abstract description 2
- 150000001450 anions Chemical class 0.000 abstract 1
- 235000019441 ethanol Nutrition 0.000 abstract 1
- 238000000855 fermentation Methods 0.000 abstract 1
- 230000004151 fermentation Effects 0.000 abstract 1
- 238000011031 large-scale manufacturing process Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 14
- 238000005251 capillar electrophoresis Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- IKHGUXGNUITLKF-UHFFFAOYSA-N Acetaldehyde Chemical compound CC=O IKHGUXGNUITLKF-UHFFFAOYSA-N 0.000 description 2
- 102000015336 Nerve Growth Factor Human genes 0.000 description 2
- 108010025020 Nerve Growth Factor Proteins 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 229940053128 nerve growth factor Drugs 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000011027 product recovery Methods 0.000 description 2
- 239000012266 salt solution Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 241000862974 Hyphomicrobium Species 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000006189 pyrroloquinoline quinone biosynthetic process Effects 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000002594 sorbent Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Nitrogen Condensed Heterocyclic Rings (AREA)
Abstract
The present invention discloses an extraction method of PQQ. PQQ fermentation liquid adsorbed with hydrophilic anion exchanger is washed, eluted and concentrated in vacuum to obtain PQQ crude products, and then, the recrystallization is carried out for removing more than 70 % of salt in the crude products. A reverse-phase adsorbing column of a Seppak C18 column is used, and ethyl alcohol containing 5% pyridine flows through one-fold column volume and then is eluted by a 70 % methanol solution; thereby, the recovery rate is raised by about 30 %, and the cost is reduced by 30 to 40 %. The method has the characteristics of simple technological operation, low cost, high recovery rate of products, high purity, etc. and can be used for the post-treatment process of the large-scale production of PQQ.
Description
The present invention relates to a kind of extracting method of pyrroloquinoline quinone.
Pyrroloquinoline quinone (Pyrroloquinoline Quinone is called for short PQQ) is a kind of micromolecular compound.PQQ can stimulate some microorganisms and plant-growth, promote female mouse upgrowth breeding, acceleration extracellular matrix maturation, strengthen immunity of organisms, remove and regulate interior free yl, improve neurocyte and nerve growth factor (NGF) and generate, prevent alcohol, acetaldehyde [Van kleef.M.A.G﹠amp such as infringement liver; Duine JA., Factors relevant inbacterial PQQ.Appl.Environ.Microbiol.1989.55.1209-1213], so PQQ can be used as a kind of useful starting material of healthcare products, novel drugs exploitation.
PQQ can pass through chemical synthesis or biological synthesis process [Ameyama.M, Hayashi.M et al., Microbial production of PQQ.Agric.Biol.Chem.1984,48:561-565; Van.Kleef.M.A.G﹠amp; Duine.J.A., L-Tyr is the precursor of PQQ biosynthesis in Hyphomicrobium X.FEBS 1988,237:91-97; JP. clear 63-267287 C12P 17/18] obtain, because the former has complex process, isomer is difficult to drawbacks such as separation, cost height.Therefore, PQQ mainly adopts biological synthesis process to carry out at present.Its extracting method is: PQQ fermented liquid → wetting ability ion-exchanger absorption → less salt solution washing → high level salt solution wash-out → accent pH ≈ 2 → anti-phase adsorption column (0.75cm
3) → 70% methanol-eluted fractions → straight empty dry.But this method only is suitable for a small amount of fermented liquid (tens of liter) preparation PQQ, and still is in laboratory advanced development on a small scale, product price extremely expensive (39 dollars/mg of Germany, Japanese 150 Renminbi/mg), therefore greatly limited the Application and Development of PQQ.
The object of the present invention is to provide the extracting method of a kind of pyrroloquinoline quinone (PQQ), it is simple, with low cost that this method should have technological operation, and characteristics such as product recovery rate and the equal height of purity can be used for the last handling process of PQQ scale operation.
For achieving the above object, the technical solution adopted in the present invention is as follows:
A kind of extracting method of pyrroloquinoline quinone, after the absorption of pyrroloquinoline quinone fermented liquid usefulness wetting ability anionite, through washing, wash-out, with anti-phase adsorption column absorption, methanol-eluted fractions, obtain pyrroloquinoline quinone after the vacuum concentration drying again, it is characterized in that: behind washing, wash-out, carry out the vacuum concentration drying, obtain the pyrroloquinoline quinone crude product, dissolving reaches the KCl saturation ratio, adjust pH to 3.5 is in crystallization below 10 ℃; Anti-phase adsorption column is 20cm
3Post; Use the one times of column volume of the anti-phase adsorption column of washing with alcohol that contains 5% pyridine before the methanol-eluted fractions.
The present invention's technical scheme more specifically is as follows: the extracting method of a kind of pyrroloquinoline quinone (PQQ), after PQQ fermented liquid usefulness DEAE-Portugal's coagel (wetting ability anionite) absorption, with the 20mmol/LTris-HCl solution washing that contains 0.6mol/L KCl, with the 20mmol/LTris-HCl eluant solution that contains 0.6～1.2mol/LKCl, the vacuum concentration drying obtains the PQQ crude product again; This dissolving crude product is reached the KCl saturation ratio, and solution is transferred pH ≈ 3.5,＜10 ℃ of following crystallizations, and the centrifugal crystal PQQ that gets; This being dissolved in water, transferring pH ≈ 3.5, is 20cm with column volume
3Seppak C
18Post carries out anti-phase absorption, with the Tris solution washing of pH ≈ 2.5, crosses one times of column volume with the ethanol stream that contains 5% pyridine, uses 70% methanol solution wash-out again, and the vacuum concentration drying obtains product P QQ.The PQQ that extracts with aforesaid method detects through HPLC and capillary electrophoresis, and the result shows that its purity is respectively 98.3% and 95%.The nutrient solution of collecting from the laboratory, the PQQ of preparation detects its purity through capillary electrophoresis and reaches 100%.
In the extracting method of the present invention, behind the high salt concentration eluant solution, dissociated PQQ liquor capacity is big from the sorbent material, obtains containing the PQQ goods of high salt through vacuum concentration, adopts recrystallization method can remove the salt more than 70% in the thick PQQ goods again.Simultaneously, anti-phase adsorption column changes 0.75cm
3Pillar is 20cm
3Post, the former be applicable to purifying contain 10 surplus milligram PQQ fermented liquid, then a post then can contain hundreds of milligrams of PQQ fermented liquids by purifying.PQQ can be adsorbed by the reversed phase chromatography post effectively, if only use 70% methanol solution wash-out, even slow down elution speed, prolongs elution time, also is difficult to the complete wash-out of PQQ is come out.Adopt earlier among the present invention with the one times of column volume of the anti-phase adsorption column of washing with alcohol that contains 5% pyridine, use 70% methanol solution wash-out again, not only make the rate of recovery improve about 30% (bringing up to about 1.3mg/L) by the original 1mg/L rate of recovery, and this reverse post can use repeatedly, thereby reduces cost 30-40%.Therefore, it is simple, with low cost that present method has technological operation, characteristics such as product recovery rate and purity height, and can be used for the last handling process of PQQ scale operation.
Below in conjunction with specific embodiment technical scheme of the present invention is further described:
Embodiment:
With the PQQ fermented liquid collected with the coagel absorption of DEAE-Portugal after, with the 20mmol/L Tris-HCl solution washing that contains 0.6mol/L KCl, then with the 20mmol/L Tris-HCl eluant solution of 0.6～1.2mol/L KCl, the vacuum concentration drying obtains the PQQ crude product; This dissolving crude product is reached KCl saturation ratio solution (transferring pH ≈ 3.5),＜10 ℃ of following crystallizations, the centrifugal crystal PQQ that gets; With this accent pH ≈ 3.5 that is dissolved in water, use SeppakC
18The anti-phase absorption of post, pH ≈ 2.5 solution washings are crossed one times of column volume with the ethanol stream that contains 5% pyridine, use 70% methanol-eluted fractions again, and the vacuum concentration drying obtains product P QQ.Detect its purity through capillary electrophoresis and can reach 95%～100%.The rate of recovery of PQQ improves 30%.
Claims (1)
1. the extracting method of a pyrroloquinoline quinone, after the absorption of pyrroloquinoline quinone fermented liquid usefulness wetting ability anionite, through washing, wash-out, with anti-phase adsorption column absorption, methanol-eluted fractions, obtain pyrroloquinoline quinone after the vacuum concentration drying again, it is characterized in that: behind washing, wash-out, carry out the vacuum concentration drying, obtain the pyrroloquinoline quinone crude product, dissolving reaches the KCl saturation ratio, adjust pH to 3.5 is in crystallization below 10 ℃; Anti-phase adsorption column is 20cm
3Post; Use the one times of column volume of the anti-phase adsorption column of washing with alcohol that contains 5% pyridine before the methanol-eluted fractions.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB001146394A CN1138776C (en) | 2000-06-20 | 2000-06-20 | Extraction method of pyrroquinolinequinone |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB001146394A CN1138776C (en) | 2000-06-20 | 2000-06-20 | Extraction method of pyrroquinolinequinone |
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| CN1329004A CN1329004A (en) | 2002-01-02 |
| CN1138776C true CN1138776C (en) | 2004-02-18 |
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| CNB001146394A Expired - Fee Related CN1138776C (en) | 2000-06-20 | 2000-06-20 | Extraction method of pyrroquinolinequinone |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1814761B (en) * | 2005-01-31 | 2010-04-21 | 中国人民解放军军事医学科学院生物工程研究所 | Pyrroloquinoline quinone synthesis related gene and its coding protein |
| CN102960350A (en) * | 2012-11-30 | 2013-03-13 | 上海医学生命科学研究中心有限公司 | Application of pyrroloquinoline quinone (PQQ) active materials in aspect of preventing and treating bacterial wilt of plants |
| CN112194658A (en) * | 2020-10-23 | 2021-01-08 | 内蒙古拜克生物有限公司 | Separation and purification method of pyrroloquinoline quinone |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102061278B (en) * | 2010-11-17 | 2013-01-23 | 中国人民解放军军事医学科学院生物工程研究所 | Methylovorus sp. MP688 and application thereof |
| CN103239451A (en) * | 2012-02-10 | 2013-08-14 | 上海医学生命科学研究中心有限公司 | Application of pyrroloquinoline quinone in treatment and/or prevention of liver fibrosis |
| CN103224965B (en) * | 2013-03-27 | 2015-04-01 | 中国人民解放军军事医学科学院生物工程研究所 | Method for producing pyrroloquinoline quinine through microbial fermentation and fermentation medium used in same |
| CN104328155B (en) * | 2014-11-20 | 2019-06-28 | 郑州轻工业学院 | Utilize the method and application of bacillus of oxidizing glucose production pyrroloquinoline quinone |
| CN104892597B (en) * | 2015-05-14 | 2016-05-04 | 郑州轻工业学院 | PQQ in complexing abstraction separation and purification zymotic fluid |
| CN105294687B (en) * | 2015-12-02 | 2016-11-16 | 郑州轻工业学院 | The method of ion pair Bi-aqueous extraction pyrroloquinoline quinone |
| CN110698472B (en) * | 2019-09-11 | 2020-09-04 | 北大方正集团有限公司 | Purification method of pyrroloquinoline quinone |
| CN111635402B (en) * | 2020-06-24 | 2021-04-30 | 北大方正集团有限公司 | Separation and purification method of pyrroloquinoline quinone |
| CN112500407B (en) * | 2020-12-25 | 2022-03-11 | 西安蓝晓科技新材料股份有限公司 | Purification method of pyrroloquinoline quinone disodium salt |
| CN113337432B (en) * | 2021-06-04 | 2023-06-02 | 华熙生物科技股份有限公司 | Methylophilus for producing pyrroloquinoline quinone and application thereof |
-
2000
- 2000-06-20 CN CNB001146394A patent/CN1138776C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1814761B (en) * | 2005-01-31 | 2010-04-21 | 中国人民解放军军事医学科学院生物工程研究所 | Pyrroloquinoline quinone synthesis related gene and its coding protein |
| CN102960350A (en) * | 2012-11-30 | 2013-03-13 | 上海医学生命科学研究中心有限公司 | Application of pyrroloquinoline quinone (PQQ) active materials in aspect of preventing and treating bacterial wilt of plants |
| CN102960350B (en) * | 2012-11-30 | 2014-09-17 | 上海医学生命科学研究中心有限公司 | Application of pyrroloquinoline quinone (PQQ) active materials in aspect of preventing and treating bacterial wilt of plants |
| CN112194658A (en) * | 2020-10-23 | 2021-01-08 | 内蒙古拜克生物有限公司 | Separation and purification method of pyrroloquinoline quinone |
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| CN1329004A (en) | 2002-01-02 |
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