Extraction method of pyrroquinolinequinone

The present invention relates to a kind of pyrroquinolinequinone extraction method.

Pyrroquinolinequinone (  Quinone Pyrroloquinoline, abbreviated as PQQ) is a small molecular compound. Some microorganisms and PQQ can stimulate plant growth, promoting the growth and reproduction of the female RAT, mature extracellular matrix is accelerated, enhanced immunity, removing and adjusting free radicals in vivo, the nerve cell and the nerve growth factor is generated (NGF), prevent alcohol, damage of the liver of acetaldehyde [Van   kleef.M.A.G & Duine   J   A, Factors   relevant   in bacterial   PQQ.Appl.Envirom   Microbiol. 1989.55.1209-1213], PQQ can be used as a health-care product therefore, new drug development of a useful raw material.

PQQ can be by chemical synthesis or biological synthesis [Ameyama.M, Hayashi.M   et   al. ,   Production   of   PQQ Microbial   Agric.Biol.Chem. 1984, 48 : 561-565; Van.Kleef. &Duine.JA M.A.G. ,   Is   PQQ L-Tyr   precursor   in   the   biosynthesis   of   FEBS   Hyphomicrobium X.   1988,237: 91-97 ; JP. Manifest 63-267287   C12P   17/18] to obtain, because the process is complicated, difficult to separate isomers, drawback of higher cost. Therefore, the current main PQQ to the biological synthesis. Its extraction method is as follows: the fermented solution PQQ → hydrophilic ion exchange agent adsorption → low-salt solution to wash → high salt solution elution → adjusting pH≈ 2 → reversed phase column (0.75 cm³) <AOX1AAO >70% methanol elution → direct air drying. However, the method only suitable for a small amount of the fermented solution preparation PQQ (tens of litres), and is still in the laboratory stage small-scale trial, the product price extremely expensive (German 39 US dollar/mg, Japan 150 renminbi/mg), thereby greatly limiting the development and application of PQQ.

The purpose of this invention is to provide a method for extracting (PQQ) pyrroquinolinequinone, the method should be of a simple process operation, low cost, product recovery and purity and the like, can be used for post-treating process of the mass production of PQQ.

To achieve the above object, the technical proposal adopted by the invention are as follows:

A method for extracting (PQQ) pyrroquinolinequinone, the fermentation broth with PQQ DEAE- portugal gathers gel (hydrophilic anion exchanger) after absorption, containing 0.6mol/L of   KCl 20mmol/L   Tris-HCl solution, re-containing 0.6-1.2mol/L of   KCl 20mmol/L   Tris-HCl solution elution, PQQ crude product by vacuum concentration and drying; dissolving the crude this saturation KCl, solution to adjust pH≈ 3.5, in <the 10 [...] lower crystallization, centrifugation to obtain crystal PQQ; the water and dissolved, adjusting pH≈ 3.5, to volume column for 20 cm³ the Seppak   C₁₈ column for reverse phase adsorption, pH≈ 2.5 for the washing solution Tris, containing 5% pyridine flowing through a fold column volume of ethanol, then using 70% methanol solution elution, PQQ product is obtained by vacuum concentration and drying. PQQ of extracting the above-mentioned method for the detection and capillary electrophoresis HPLC, the result shows that the purity of 98.3% and 95%. Fluid collected from the laboratory, by PQQ preparation of capillary electrophoresis detection with a purity of up to 100%.

In the extracting method of the present invention, the high-concentration salt solution for eluting after, PQQ from absorbent solution from the subclass of large volume, through the vacuum and concentrated by PQQ products containing high salt, then the re-crystallization method in the coarse PQQ products can remove 70% salt of the above. At the same time, the opposite phase to the absorption column 0.75 cm³ the columella 20 cm³ column, the former is suitable for purifying containing 10 milligrams of the fermented solution PQQ more, and then a column containing hundreds of milligrams of purified to PQQ fermentation liquid. PQQ can effectively be reversed-phase chromatographic column to adsorption, if for only 70% methanol solution elution, even if slow down the elution speed, the elution time, it is difficult to completely elute out the PQQ. In this invention the first containing 5% ethanol-washing of pyridine-phase adsorption column a times the column volume, then using 70% methanol solution elution, not only the recovery rate 30% the left and right (from the original 1 mg/L increase the recovery rate of 1.3 mg/L left and right), and the reverse column can be used repeatedly, thereby reducing the cost 30-40%. Therefore, this method has the advantages that the process operation is simple, low cost, high purity and the characteristics of the product recovery rate, but also can be used for post-treating process of the mass production of PQQ.