CN1329004A

CN1329004A - Extraction method of pyrroquinolinequinone

Info

Publication number: CN1329004A
Application number: CN 00114639
Authority: CN
Inventors: 赵永芳; 王银善; 吴波; 成汇; 徐宁; 闵向荣
Original assignee: Wuhan University WHU
Current assignee: Wuhan University WHU
Priority date: 2000-06-20
Filing date: 2000-06-20
Publication date: 2002-01-02
Anticipated expiration: 2020-06-20
Also published as: CN1138776C

Abstract

An extraction method of PQQ includes the following steps: using hydrophilic anion exchanger to adsorb PQQ fermentation liquor, then washing, eluting, vacuum concentrating and drying to obtain PQQ crude product, recrystallizing to remove above 70% of salt content from crude product, then using Seppak C18 column reversed phase adsorbing column and making ethyl alcohol containing 5% pyridine pass through one-fold column volume, and using 70% methyl alcohol solution to make elution to raise recovery rate by about 30% and reduce cost by 30-40%. This invention features simple operation process, low cost and high product recovery rate and high purity. It can be used for after-treatment process of PQQ large-scale production.

Description

The extracting method of pyrroloquinoline quinone

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 J A, Factors relevant inbacterial PQQ.Appl.Envirom 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.JA., L-Tyr is the precursor of PQQ biosynthesis in HyphomicrobiumX. 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:

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/L Tris-HCl solution washing that contains 0.6mol/L KCl, with the 20mmol/L Tris-HCl eluant solution that contains 0.6～1.2mol/L KCl, 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. 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.