CN1814766A - Preparing pravastatin - Google Patents

Abstract

The present invention relates to a new microbial process for the preparation of compound formula (I) from a compound of general formula (II) wherein R stands for an alkali metal or ammonium ion, by the submerged cultivation of a mold strain able to 6 beta -hydroxylate a compound of Formula (II) in aerobic fermentation and by the separation and purification of the product of Formula (I) formed in the course of the bioconversion. The process comprises cultivating a strain of Mortierella maculata filamentous mold species that is able to 6 beta -hydroxylate a compound of general Formula (II), on a nutrient medium containing assimilable carbon and nitrogen sources and mineral salts and separating the product formed from the fermentation broth, then isolating the compound of formula (I) and purifying the same. Novel strains of Mortierella maculata are also disclosed.

Description

Culture

The application is that application number is dividing an application of the female case of CN200310123383.3. The applying date of this mother's case is on February 3rd, 2000; Denomination of invention is " culture ".

Technical field

The present invention relates to a kind of new culture, and for the preparation of the method for Pravastatin, specifically, relate to the microbial process with the industrial-scale production Pravastatin.

Background technology

Atherosclerotic, the especially the most dangerous factor of coronary vasodilator obturation are the elevated cholesterol of blood plasma. Recent two decades has carried out in depth research to the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (EC.1.1.1.34) as Biosynthesis of cholesterol speed limit key enzyme. Pravastatin, the compound of a kind of formula I,

And other related compound (ML-236B, Mevacor (mevinolin), Simvastatin) is that [1346-1348 (1976) for A.Endo etc., J.Antibiot.29 for the competitive inhibitor of HMG-CoA reductase; A.Endo etc., FEBS Lett.72,323-326 (1976); C.H.Kuo etc., J.Org.Chem.48,1991 (1983)].

Pravastatin from dog urine is separated (Arai, M. etc., Sankyo Kenkyusyo Nenpo, 40,1-38,1988) at the research ML-236B between metabilic stage by (undocumented results) such as M.Tanaka at first. At present, Pravastatin is a kind of gemfibrozil, and it has best mechanism of action in treatment. Its most important feature is tissue selectivity, namely for example its suppresses the synthetic of cholesterol at liver with in small intestine at two main positions of cholesterol generation (cholesterogenesis), and is difficult to detect the endocellular enzyme of restriction at other organ. Yet the Biosynthesis of cholesterol restriction of Mevacor and Simvastatin but is significant (T.Koga etc., Biochim.Biophys.Acta, 1045,115-120,1990) in most of organs.

Pravastatin is different from Mevacor and the Simvastatin that has more the lipophilic feature in essence aspect chemical constitution. In the situation of rear two kinds of compounds, the substituent end that is connected with the C-1 carbon atom of hexahydro-naphthalenc skeleton is arranged in hexa-atomic lactonic ring, and in the situation of Pravastatin, do not have described lactonic ring, but there is the sodium-salt form of bioactive open chain dihydroxy acid. Another important structural difference is that Mevacor and Simvastatin are methyl in the C-6-position of hexahydro naphthalene nucleus, and can see a hydroxyl in Pravastatin, and this causes further strengthening in its hydrophilic characteristic aspect.

As the result of said structure difference, Pravastatin penetrates the lipophilic film (A.T.M., Serajuddin etc., J.Pharm.Sci.80,830-834,1991) of peripheral cells with being merely able to utmost point low degree.

Can reach the suitability for industrialized production Pravastatin by two sweats. In first sweat, the ML-236B in preparation microorganism stage then in second sweat, on 6 beta-positions, by the microorganism hydroxylation, makes the sodium salt as the ML-236B acid of substrate be converted into Pravastatin.

According to disclosed patent, with belonging to the mould bacterial classification that do not belong to together and with the trichobacteria that belongs to Nocardia (Nocardia), with actinomadura (Actinomadura) and streptomyces (Streptomyces), the microorganism hydroxylation of ML-236B (No. the 895090th, belgian patent specification, Japanese patent specification the 5th can be finished in varying degrees, 810, No. 572, United States Patent (USP) the 4th, 537,859 and 4,346, No. 227 and disclosed European Patent Application No. 0605230). For example mucor hiemalis (Mucor hiemalis), Syncephalastrum nigricans, cunninghamella echinulata (Cunninghamella echinulata) carry out the bio-transformation of ML-236B substrate with 500 μ g/ml concentration with the bacterial strain that belongs to procaryotic Nocardia, actinomadura and streptomyces with 2000-4000 μ g/ml to disclose the use filamentary mould.

A FAQs that experiences in the situation with filamentary mould production Pravastatin is because the antifungic action of ML-236B, causing that microorganism can not tolerate adds in the culture even the ML-236B substrate (Serizawa etc. of low concentration, J.Antibiotics, 36,887-891,1983). When the Japanology personnel further investigate hydroxylation with Streptomyces carbophilus, also observe the cytotoxicity (M.Hosobuchi etc., Biotechnology and Bioengineering, 42,815-820,1993) of this substrate.

The Japan author has managed to improve with recombinant DNA technology the hydroxylation ability of Streptomyces carbophilus bacterial strain. The hydroxylation of ML-236B needs cytochrome P-450 monooxygenase system (Matsuoka etc., Eur.J.Biochem.184,707-713,1989). Yet, according to described author's research, in the cytochrome P-450 monooxygenase system of bacterium, not a kind of but several albumen work in the electronics transmission, this is so that the application of described dna technique is more difficult. Developing for the production of the effective microorganism hydroxylation of the cost of Pravastatin method is extremely difficult and complicated work.

The objective of the invention is to describe in detail and a kind ofly produce the new microbial process of Pravastatin for commercial scale from ML-236B, described method can more favorably produced Pravastatin under the condition than those methods of previously known. During our research work, we have attempted above all researchs, might be suitable for making the ML-236B microbial conversion as the microbial strains of the hydroxylase of the Pravastatin of high concentration to find to contain.

Summary of the invention

The present invention relates to a kind of for the substrate compounds from formula (II)

Wherein R represents a kind of alkali metal or ammonium ion,

The microbial process of preparation formula (I) compound,

Said method comprising the steps of: (a) on the nutrient medium that contains assimilable Carbon and nitrogen sources and inorganic salts, cultivation can be with the bacterial strain of the Mortierella maculata filamentary mould bacterial classification of formula (II) compound 6 β-hydroxylation; (b) substrate to be transformed is added in the formed Mortierella maculata culture; (c) allow described fermenting substrate until till the bio-transformation end; (d) compound of separate type (I) from described culture meat soup; (e) compound of the described formula of separation and purification (I).

The invention still further relates to and be preserved in the biologically pure culture that the state-run agricultural of Budapest, HUN and industrial microorganism preservation center, preserving number are the Mortierella maculata n.sp.E-97 bacterial strain of NCAIM (P) F 001266; And the biologically pure culture of the mutant strain Mortierella maculata n.sp.E-97/15/13 bacterial strain of described bacterial strain, this mutant strain is preserved in the state-run agricultural of Budapest, HUN and industrial microorganism preservation center, and preserving number is NCAIM (P) F 001267.

Description of drawings

Fig. 1 illustrates the physical features of Mortierella maculata n.sp.E-97.

The specific embodiment

In the process of our screening sequence, covered about 5500 kinds of protokaryons and eucaryon bacterial strain, selected 23 kinds of microorganisms, what described 23 kinds of microorganisms conversely can the hydroxylation ML-236B. In these bacterial strains, a kind of filamentary mould proves and is more suitable in producing Pravastatin because with from the known bacterial strain of publication relatively, it is higher to the resistance of ML-236B. According to means of taxonomic research, this bacterial strain proves a kind of new representative bacterial classification (Mortierella maculata n.sp.) that belongs to Mortierella (Mortierella). From selected mould, on the one hand, by using mutant-system of selection, on the other hand, by inducing the hydroxylase of described bacterial strain, be separated to a kind of new bacterial strain, described new bacterial strain can make ML-236B substrate hydroxylation become Pravastatin with higher concentration by discloseder bacterial strain than up to now. As mutagens, can use physics and chemistry mutagens (UV irradiation, methyl mesylate, N-methyl-N '-nitro-N-nitrosoguanidine). After the mutagenic treatment, in order to prepare haploid cell, spore suspension is coated on the agar plate that contains benomyl, then in order to induce hydroxylase, the colony inoculation that forms is contained 8-at 100 μ g/ml to be taken off-(2-methyl-butyryl)-ML-236B or contain on the agar plate of ML-236B. By the application of these methods, prepare mutant strain from can ML-236B being converted into the new bacterial strain of Pravastatin in the degree that is significantly higher than parental strain.

In the process of Optimal Experimental, the best approach that we have determined to be used for the most useful inoculum of ML-236B hydroxylation and best biotransformation medium and have repeated to add ML-236B with high concentration.

Therefore, the present invention is based on the called after E-97 of the mould of the separation of Mortierella maculata by name and the discriminating of E-97/15/13 bacterial strain, described bacterial strain is preserved in state-run agricultural and industrial microorganism preservation center (Department of Microbiology and Biotechnology. University of Horticulture and the Food Industry Budapest), its preserving number is respectively NCAIM (P) F 001266 and NCAIM (P) F 001267, they are under suitable fermentation condition, can produce Pravastatin high-levelly, and during bio-transformation, only obtain a small amount of or micro-unwanted related compound, 6 Alpha-hydroxies of sour form-ML-236B for example, 2 α-hydroxyl-ML-236B, 8-takes off-(2-methyl-butyryl)-ML-236B, 3 α, 5 beta-dihydroxies-5,6-dihydro-different ML-236B, 8a, the hydroxylation derivative on the position 2 and 3 of beta-hydroxy-ML-236B and the 2-methyl that is positioned at ML-236B-butyryl side chain. Therefore, these bacterial strains are particularly suitable for the industrial-scale production Pravastatin.

The commercial scale economical production of considering described active ingredient changes with the ML-236B concentration of substrate, therefore importantly has the bacterial strain that can tolerate high compact rhzomorph and Pravastatin concentration. Therefore, an again pith of the present invention is to recognize: the hydroxylation ability that can improve original mould separator by using mutant-selection and enzyme induction method, and by the suitable substrate adding method of exploitation, can in one step, realize a large amount of ML-236B hydroxylations are become Pravastatin. In a word, the new mutation bacterial strain of called after Mortierella maculata n.sp.E-97/15/13 is particularly suitable for producing Pravastatin.

Hereinafter summarized the most important discriminating attribute taxology feature relatively of new mould bacterial classification and the known Mortierella bacterial classification of described separation.

The taxology of holotype bacterial strain Mortierella maculata nov.spec.E-97 is described

On starch-casein-brewer's wort-agar medium, form well aerial mycelium (the thick cover layer of 10 μ m of surpassing is arranged on the substrate mycelium). During beginning, it shows as the closely white net of braiding of mycelia, afterwards, the sparse light yellow sporogenesis spot with several millimeters straight warps (newname " maculatus " refers to above-mentioned spot) occurs. This pale yellow color can occupy the larger continuous surface that gas is given birth to net sometimes. The color great majority of substrate mycelium are colourless or light yellow on Czapek agar medium, blood-Czapek agar medium, tyrosine agar medium, starch-casein agar culture medium, wort agar culture medium etc. The color of substrate mycelium net is pale red on yeast extract-glucose-protein culture medium. In fact do not produce at above listed culture medium and can spread and soluble pigment, perhaps seldom produce very shallow light yellow at these culture mediums. The bacterium colony of bacterial strain E-97, because it produces volatile oil, and similar to other bacterial classification (except the bacterial classification of Mortierella isabellina group (section Isabellina)) of many Mortierellas, can distribute a kind of very distinctive strong fragrance.

By the sporangiophore of the Ref. No. 1-7 among Fig. 1 name, usually (and less on the substrate mycelium) on the aerial hyphae with a large amount of mutually between very different local formation of distance. They are branch not, but majority is straight or crooked. Their length is generally 60-80 μ m. Starting point in most cases is a substantially living mycelia zone of netting of short but gas that strongly expand, and they are separated by cell wall. Sporangiophore self also can expand (being consumingly sometimes), shown in Ref. No. 6, but on sporangial direction, they by 5.0-9.0 μ m gradually constriction to 1.0-2.0 μ m. Important taxology feature is under described sporangiophore, and they never widen (referring to Ref. No. 8).

Sporangium is spherical; Be the spheroid that slightly flattens in some cases. They directly through being about 6.0-17.0 μ m, less than the measured value of other Mortierella bacterial classification. In the sporangium many spores can be arranged, also exist but only have in the sporangium of a spore. Spore 9 is cylindrical or not too oval. Their size is 3.0-5.0 * 1.5-2.0 μ m. In single spore, can there be 1 or 2 little dark-coloured spherical oil droplets 10. Because sporangial wall is very easy to decompose, therefore in wet environment, spore can scatter soon. After sporangium disintegrates, sometimes at the end of sporangiospore, can observe tiny tuning fork sample " capsule neck " and a kind of very (but not being typical) columella of short degeneration. Spherical or columniform gemma 15-28 can form at most of different differential mediums. Common size is 10-25 μ m. In described culture medium, also can see the chain, blastogenesis cell of spherical gemma 13, the spore 15-23 of sprouting, a mycelia and merge spline structure and giant cell etc. around mycelia association 11, the mycelia of the specific spiral growth of another mycelia. In aerial mycelium, can also observe large (directly through 50-250pm) very fine and close mycelia net 14, but not have detectable joint element to exist.

The culture of bacterial strain E-97 can make nitrate reduction become nitrite, not hydrolyzed starch, aesculin, arginine or gelatin, but hydrolysis tween polysorbates does not decompose the paraffin hydrocarbon class. The culture of bacterial strain E-97 has urease activity, shows good growth between pH7.0-9.0, tolerates maximum 2%NaCl. The effect of xanthine, hypoxanthine, lecithin, tyrosine and adenine is negative effect. From glucose, fructose, glycerine and galactolipin, detect the strong acid that culture produces, but do not produced or produced seldom strong acid from wood sugar, arabinose, gossypose, D-sorbite, inositol, inulin etc. Detect weak growth at acetonate and acetate, but can not grow when finding to contain benzoate, salicylate, citrate, lactate, succinate, tartrate and malonate. Well-grown when observing with glucose and fructose as the sole carbon source of culture medium. Wood sugar, arabinose, rhamnose, sucrose, gossypose, sweet mellow wine and inositol utilize evidence negative. Described culture is decomposition of cellulose not.

The systematics position: bacterial strain E-97 belongs to Mortierellaceae, and it is a prototypical member of Mortierella: many spores are generally arranged in the sporangium, and columella is extremely degenerated, and usually has gemma, do not detect zygosporic existence, bacterium colony distributes a kind of very distinctive strong aroma. In Mortierella, bacterial strain E-97 is the Typical Representative of " Mortierella alpina group (Section Alpina) ". The latter to be characterized as sporangiophore extremely short, branch (maximum length is 200 μ m) not, and the small (Zycha of sporangium, H.und Siepmann, R, Mucorales.Eine Beschreibung aller Gattungen und Ar dieser Pilzgruppe.D-3301 Lehre, Verl.von J.Cramer.1969). In the member of Mortierella alpina group, bacterial strain E-97 and bacterial classification Sa Ke Mortierella (M.thaxteri Bj rling 1936) and kidney spore Mortierella (M.renispora Dixon-Stewart 1932) show the highest similitude. Yet the data of Table I clearly illustrate that the difference of the identification feature aspect of bacterial strain E-97 and these two kinds. Therefore, as a novel species, we are with described bacterial strain called after Mortierella maculata nov.spec.E-97 thus.

Table 1

According to key identification feature, to the holotype of Mortierella maculata n.sp.

Bacterial strain E-97 and bacterial classification kidney spore Mortierella and Sa Ke Mortierella compare

	Kidney spore Mortierella	Mortierella bacterial strain E-97	The Sa Ke Mortierella
				The origin of sporangiophore	Common, however mycelia is more roomy than normal mycelia, and side is from enlarging the mycelia of expanding	Non-from giving birth to regional adnation from (expand or normally) aerial hyphae or substrate mycelium.	From aerial hyphae to expand from tight knot section or substrate mycelium be non-from tight knot section adnation
The shape of sporangiophore and size	To top orientation gradually by 10 μ m shrinking to 3 μ m. Length is about 200 μ m.	Majority is straight or crooked, not branch. Width to the top gradually by 5-9 μ m shrinking to 1.5-2.5 μ m. Length is about 60-80 μ m. In not overstriking of top.	The about 60-90 μ of length m. The width of starting point is about 5-7 μ m, in the top shrinking to 1.5-2 μ m. In the immediately overstriking of sporangium place.
				Sporangial shape and size	Colourless, directly through being 25 μ m.	Majority is spherical (directly through 6-17 μ m), and few for not too flat. Generally contain many spores, seldom contain a spore.	Spherical (directly through 12-20 μ m). They contain many spores, but on some culture medium the sporangium that only contains a spore are arranged also.
Sporangial wall (film)	Expanded film. Still keep capsule neck spline structure after the disintegration.	Disintegrate. Keep tuning fork sample capsule neck.	Disintegrate, keep the capsule neck of small back-flexing.
				The shape of spore	Kidney shape roughly. Transparent knot	Cylindrical. Length (3-5 μ m)	Oval transparent spore, size

And size	Structure, size are 2 * 4 μ m.	Twice that can greater than width (1.5-2 μ m).	Be 3.5-4 * 1.5-2 μ m.
				Gemma	Generate at most of different culture mediums.	Usually be present on the very different culture mediums, majority is present in the aerial mycelium. Spherical or elongated (10-25 μ m).	The oval gemma (10-14 μ m) of giving birth in the middle of the substrate mycelium.
Joint element	Usually be present on all differential mediums. Add cover mycelia directly through being about 500 μ m, be about 30 μ m when not having them.	Do not detect.	Do not observe.
				The fine and close kitchen range (foci) of mycelia net		Usually be the large fine and close mycelia kitchen range (50-250 μ m) that weaves, and without joint element.	In old culture, for the fine and close mycelia net of large (directly through 100-125 μ m) ecru, without joint element.
The color of aerial mycelium and habit	Loose and always white the mycelia net	White and light yellow spot.	Begin to be the cobweb sample, finer and close afterwards.

Preparing according to the present invention in the method for Pravastatin, preferably using the fungal strain culture of called after Mortierella maculata n.sp.E-97 or its called after E-97/15/13 mutant strain. So selected bacterial strain is because its Fast Growth is highly preferred bacterial strain. As carbon source, it utilizes glucose, glycerine, fructose or galactolipin easily. As nitrogenous source, can utilize yeast extract, peptone, casein, meat extract, soy meal, corn steep liquor, sodium nitrate or ammonium sulfate.

In the culture medium for the production of Pravastatin, except above Carbon and nitrogen sources, can also contain inorganic salts (for example potassium dihydrogen phosphate, magnesium chloride, magnesium sulfate), trace element (ferrous salt, magnesium salts), amino acid and defoamer.

According to a preferred embodiment of the present invention, will be inoculated in the seed culture medium by the spore suspension of the Mortierella maculata n.sp. agar slant culture preparation of the mutant strain [NCAIM (P) F 001267] of called after E-97 bacterial strain or its called after E-97/15/13; Then, will approximately 25-30 ℃, preferably approximately 24-28 ℃, most preferably cultivate 3 days 10% inoculum transferred speciess in biotransformation medium down at about 28 ℃. Then with its approximately 25-28 ℃, be preferably in about 28 ℃ and cultivated 4 days, then the sodium salt with glucose and ML-236B acid adds in the described culture. According to the concentration of the ML-236B substrate that adds, will cultivate and under aerobic conditions continue to carry out again 2-12 days, pH is remained between the 5.5-7.5, be preferably in 7.0. Under stirring and aeration condition, this moment, air velocity was 0.2vvm, and the rotating speed of agitator is 400/ minute, carries out bio-transformation.

During the fermentation, after the bio-transformation of ML-236B substrate, carry out again high pressure liquid chromatography (HPLC) method (HPLC). According to the method, with 2 times of described meat soup Sample Dilutions, then centrifugal with methyl alcohol, supernatant is used for HPLC analyzes under following parameter: Waters analytic type HPLC instrument; Pillar: Nucleosil C₁₈10 μ m; Detect wavelength: 238nm; Volume injected: 20 μ l, flow velocity: 1ml/ minute; Adopt gradient elution, eluent: A=0.05% phosphate aqueous solution, B=acetonitrile.

Gradient:

Time (minute)	Eluent A (%)	Eluent B (%)
			0	70	30
20	0	100
			25	0	100
25.1	70	30
			35	70	30

Approximate retention time: Pravastatin 8.6-9.0 minute; ML-236B acid 11.6-12.0 minute; Pravastatin lactone 15.0-15.5 minute, ML-236B 16.5-17.0 minute.

In order to produce Pravastatin, added the sodium-salt aqueous solution of ML-236B acid at the 96th hour that cultivates. For the method, followingly prepare described substrate with solid form. In 40 ℃, in the 0.2M sodium hydroxide solution with ML-236B lactone hydrolysis 2 hours, then with hydrochloric acid the pH of reactant mixture is transferred to 7.5, the solution of described neutralization is layered on the Diaion HP-20 adsorption column; Remove the sodium chloride that generates during the neutralization reaction by washing pillar with water, then use the sodium salt of 50% aqueous acetone solution wash-out ML-236B acid from pillar. After this, vacuum distillation washings and with the aqueous residue freeze-drying. After the neutralization reaction, also can be with the aqueous solution of the basic hydrolysis product of ML-236B directly as substrate. In this case, measure the sodium salt content of ML-236B acid in the hydrolysate by HPLC, and before using always with described solution preservation in 20 ℃.

PH to the 4th day meat soup that ferments is higher, and is more favourable to the hydroxylation of ML-236B substrate. When the pH of meat soup surpasses 6.3, allow to begin to add the ML-236B substrate. At the 4th day of fermentation, the sodium-salt aqueous solution that adds as required the ML-236B acid of aseptic filtration was 500 μ g/ml to reaching concentration. Followingly also will be added in the described culture at 25 minutes 50% glucose solutions of 121 ℃ of sterilizations: if the pH value of meat soup is higher than 6.7, then add 1% glucose corresponding to described meat soup volume, if and pH is in the scope of 6.3-6.7, the amount of the glucose that then adds is 0.5%. From meat soup, consume the sodium salt of ML-236B acid after 24 hours, by its conversion of HPLC determination and analysis. In this case, every milliliter of meat soup adds 500 μ g ML-236Bs in addition. Except the ML-236B substrate, also add as mentioned above glucose. The morphological feature of 120 hours cultures is spherula (pellet) growth (spheroid is straight through being 0.5-3.0mm). After 24 hours, consume the substrate of the second dosage from meat soup, therefore, adding generation concentration in full meat soup is the sodium salt of another part ML-236B acid of 500 μ g/ml again, and according to the parallel interpolation glucose of the pH value of meat soup. From beginning in the 4th day of fermentation, the frequency with every day repeats to add described substrate and glucose as mentioned above, until 17-18 days of fermenting.

In order from meat soup, to reclaim described product, preferably consider during bio-transformation, to generate with its sour form the fact of Pravastatin, therefore by its absorption on anion-exchange resin column, it can be separated from the filtrate of meat soup. In order to separate described product, preferably utilize strong-base anion-exchange resin, it is the polystyrene-divinylbenzene polymer with the quaternary ammonium active group. Be mixed into by the anion exchange resin with OH-form in the filtrate of meat soup, described product is directly adsorbed from described filtrate. With the acetone-water mixture of acetic acid or sodium chloride-containing, preferably with containing acetone-water (1: the 1) mixture of 1% sodium chloride, the product wash-out that can be from described pillar will be adsorbed at anion exchange resin out. Merge the flow point that contains Pravastatin, vacuum distillation goes out the acetone in the washings. With 15% sulfuric acid the pH of concentrate is transferred to the scope of 3.5-4.0, aqueous solution ethyl acetate extraction. Wash acetic acid ethyl ester extract with water, and use anhydrous sodium sulfate drying. Then prepare lactone derivatives with Pravastatin. Under continuous stirring, induce lactone to generate by the trifluoroacetic acid that exists in order to catalytic amount, in the ethyl acetate solution in drying under the room temperature, carry out the closure of lactonic ring. Check conversion process by thin layer chromatography analysis (TLC). After finishing the lactone generation, ethyl acetate solution at first with the washing of 5% sodium bicarbonate aqueous solution, then washes with water, uses anhydrous sodium sulfate drying and vacuum evaporation again. Residue after the evaporation is used charcoal treatment in acetone soln, and then evaporation, and from the aliphatic alcohol that contains 1-4 carbon atom, recrystallization from ethanol preferably. As eluent, use silica gel column chromatography, with the evaporation residue purifying of recrystallization mother liquor with the ethyl acetate that uses ethyl acetate content to increase gradually-n-hexane mixture.

By in the acetone at room temperature with the sodium hydroxide hydrolysis of equivalent, with after the recrystallization and the pravastatin lactone that obtains of chromatographic purifying prepare Pravastatin. After the generation of finishing sodium salt of pravastatin, reactant mixture dilute with water and neutralization, vacuum distillation acetone content. On the post that contains Diaion HP-20 resin, from resulting aqueous residue, adsorb Pravastatin, with deionized water washing, use again acetone-deionized water mixture wash-out from the described pillar. The flow point that then will contain Pravastatin merges, and distills out the acetone content, can obtain the high-purity Pravastatin after with the aqueous residue freeze-drying, can be with its recrystallization from the ethyl acetate-ethanol mixture.

In the process of described method, can adsorb the Pravastatin of whole amounts. At the pravastatin lactone period of contact, also may generate 3 Alpha-hydroxies-different-ML-236B and other accessory substance. Although the reaction of these back reduces the yield of described separation, those compounds can separate by above-mentioned purification process, therefore can produce by this way the Pravastatin of pharmaceutically acceptable quality.

After finishing bio-transformation, perhaps from fermentation broth or the filtrate that after separating thread fungal cell, obtains, can extract Pravastatin. Perhaps by filtering or can removing the filamentary mould cell by centrifugal; Yet, especially in commercial scale, preferably prepare full meat soup extract. Before the extraction, with inorganic acid, preferably use dilute sulfuric acid, with or the pH of the filtrate of fermentation broth or meat soup transfer to 3.5-3.7. With the ester of acetic acid and contain the aliphatic alcohol of 24 carbon atoms, preferably with ethyl acetate or isobutyl acetate, carry out described extraction. Extraction step should be carried out very soon, to avoid generating described lactone derivatives by Pravastatin under acid pH.

Can make the sour form of Pravastatin transfer to aqueous phase as sodium salt from the organic solvent extraction thing. For example, can from acetic acid ethyl ester extract, extract Pravastatin with 5% sodium acid carbonate or the alkalescent water (pH 7.5-8.0) of 1/10 and 1/20 volume ratio. Find from the above alkaline aqueous extract that obtains, can be recovered to the Pravastatin of pure form by using the column chromatography of non-ionic adsorption resin. A kind of method for optimizing is at first to remove the solvent that is soluble in the aqueous phase by vacuum distillation from alkaline aqueous extract, then aqueous extract is loaded onto Diaion HP-20 post.

The sodium salt of pravastatin that is just adsorbing at described pillar carries out wash-out by the content of acetone that increases gradually the aqueous solution and is purified, and the Main Current that then will contain Pravastatin divides and merges and Vacuum Concentration. Aqueous concentrates further by at another Diaion HP-20 column chromatography and purifying, obtains to contain the washings of pure Pravastatin, with its with activated carbon clarification and freeze-drying after, can obtain the Pravastatin of pharmaceutically acceptable quality.

This separation method comprises still less step than previous method, generates and hydrolysis because do not relate to the lactone of Pravastatin in described method. Between described separation period, make Pravastatin under acid condition, only expose finite time, because therefore stable low than in neutrality or alkaline solution of its stability, in fact do not generate artefact (artefacts) in this separation method under acid condition.

In addition, the purification by chromatography Pravastatin on the Sephadex LH-20 sephadex (hydroxypropyl derivative) is preferably used in discovery. By using the method, can produce the Pravastatin that purity surpasses 99.5% (measuring by HPLC).

In our experimentation, have realized that with described filamentary mould or filamentous bacterial strain and especially can with the organic solvent extraction thing of the meat soup of the Mortierella maculata n.sp. bacterial strain of compound 6 β of general formula (II)-hydroxylation or meat soup nitrate, preferably with ethyl acetate or isobutyl acetate extract, can Pravastatin be precipitated out as crystal salt with secondary amine. Also find the generation for salt, several secondary amine that contain alkyl substituent, naphthenic substituent, aralkyl substituting group or aryl substituent are suitable. In them, select convenient nontoxic secondary amine, for example dioctylamine, dicyclohexyl amine, dibenzylamine. Be the dibenzylamine of 1.5 a great deal oves by the Pravastatin content that adds with respect to extract, carry out for example separation of dibenzyl amine salt of described organic secondary amine salt intermediate, then make extract be concentrated into 5% of its initial volume by vacuum distillation, join in the described concentrate with the dibenzylamine of 0.2 a great deal of ratio with another amount more at last. Crystal dibenzyl amine salt is precipitated out from described concentrate. Filter crystal raw product and vacuum drying. Then with active carbon with its clarification and in acetone recrystallization.

Comprise therein in the previous described method of organic solvent extraction and reextraction under the alkaline pH that the separation method that forms based on secondary amine salt can also be used to replace the purifying of column chromatography. In this case, preferably from the isobutyl acetate extract that after alkaline aqueous extract acidifying, obtains, precipitate Pravastatin dibenzyl amine salt.

With NaOH or sodium alkoxide, caustic alcohol preferably, can make the organic secondary amine salt of Pravastatin change into Pravastatin.

Described conversion is described in detail in the situation of Pravastatin dibenzyl amine salt. In isobutyl acetate-aqueous mixtures, be 8.0-8.5 by the scope that under agitation keeps pH with the dibenzylamine salt suspension of recrystallization then, be added in the described suspension with the NaOH of the aqueous solution with equivalent. After the dissolving of described suspension, separation of phases contains the aqueous solution isobutyl acetate washed twice of Pravastatin. The aqueous solution is clarified and freeze-drying with active carbon, obtains the Pravastatin of pharmaceutically acceptable quality.

Being used for a kind of method for optimizing that Pravastatin dibenzyl amine salt is converted into Pravastatin is that dibenzylamine salt suspension with recrystallization is in ethanol, then under agitation equivalent or excessive slightly caustic alcohol are added in this suspension, then Vacuum Concentration reactant mixture, and by adding acetone, Pravastatin is precipitated out from concentrate with crystal form.

Being used for another method for optimizing that Pravastatin dibenzyl amine salt is converted into Pravastatin is that dibenzyl amine salt with recrystallization is dissolved in the ethyl acetate-ethanol mixture, then the ethanolic solution with equivalent or excessive slightly NaOH is added in the described solution, makes the Pravastatin precipitation.

Separate Pravastatin by secondary amine salt intermediate, all simpler than the separation method of any previously known. In this separation method, do not generate artifact (artifacts), and do not need to use any chromatography method, just can solve from the bio-transformation accessory substance with from by separating Pravastatin the synthetic various metabolites of hydroxylation microorganism biological.

The structure of the secondary amine salt that separates of Pravastatin, pravastatin lactone and Pravastatin by UV, IR,¹H-NMR、 ¹³C-NMR and mass spectrography confirm.

Embodiment

More fully describe and understand the present invention with reference to following examples, described embodiment provides as an illustration rather than is used for limiting the scope of the invention by any way.

Embodiment 1

Prepare spore suspension with 5ml 0.9% sodium chloride solution, this solution derives from can be with Mortierella maculata nov.spec.E-97[NCAIM (P) F 001266 of ML-236B 6 β-hydroxylation] the 7-10 brewer's wort in age of bacterial strain-yeast extract agar slant culture, with the 100ml seed culture medium PI that sterilizes in the described suspension inoculation 500ml conical flask.

The composition of culture medium PI:

Glucose 50g

Soy meal 20g

Be dissolved in the 1000ml running water.

PH with culture medium before the sterilization transfers to 7.0, then sterilizes 25 minutes for 121 ℃. In 28 ℃ culture is gone up jolting 3 days at rotary shaker (250rpm, 2.5cm amplitude), the culture transferred species that then 10ml is obtained is in 25 minutes 100-100ml biotransformation medium MU/4 of 121 sterilizations in the 500ml conical flask.

The composition of culture medium MU/4:

Glucose 40g

Soy meal 20g

Casein-peptone 1g

Asparagine 2g

Potassium dihydrogen phosphate 0.5g

Be dissolved in the 1000ml running water.

PH with culture medium before the sterilization transfers to 7.0, then sterilizes 25 minutes for 121 ℃.

In 25 ℃ with conical flask at rotary shaker (250rpm, 2.5cm amplitude) upper jolting is 4 days, then the aqueous solution form with aseptic filtration is added to 50-50mg ML-236B substrate (sodium salt of ML-236B acid) in the culture, then proceeds to cultivate. Equally, at the 5th day, the sodium salt of other 50-50mg ML-236B acid is added in this mycotic culture thing, and makes fermentation proceed again 24 hours. Measure Pravastatin content in the meat soup by HPLC. Fermentation is proceeded 168 hours. When bio-transformation finished, the Pravastatin mean concentration was 620 μ g/ml in the fermentation broth.

Embodiment 2

In the fermentation tank of 5 liters of working volumes of laboratory scale, preparation MU/S biotransformation medium adds the nutrient media components corresponding to 5 liters of volumes, but 4.5 liters of application of samples at the most only, then sterilized 45 minutes for 121 ℃, use the 500ml inoculum inoculation according to embodiment 1 preparation.

The composition of culture medium MU/8:

Glucose 20g

Glycerine 20g

Soy meal 20g

Peptone 5g

Potassium dihydrogen phosphate 0.5g

Polypropylene glycol 2000 1g

Be dissolved in the 1000ml running water.

PH with culture medium before the sterilization transfers to 7.0 values.

Be 400rpm and be under 60 l/hs from the Ventilation Rate of bottom direction in 28 ℃, stir speed (S.S.), described fermentation was carried out 4 days. After the transferred species the 2nd day, culture begins serious foaming, and this foam can reduce by adding polypropylene glycol 2000 again. At the initial stage (16-20 hour) of fermentation, pH is reduced to 5.0-5.5 from 6.5 initial value, then reaches 6.3-7.5 since increase in the 3rd day and at the 4th day. If the pH of meat soup greater than 6.3, then allows to begin to add the ML-236B substrate. At the 4th day that ferments, add 2.5g ML-236B substrate with the aseptic filtration aqueous solution. Calculate the volume of meat soup, be parallel to the substrate that adds, according to pH, with 25 minutes 50% solution form of 121 ℃ of sterilizations, 0.5-1.0% glucose is joined in the culture. After 24 hours, consume the ML-236B substrate from culture, this can detect by HPLC by the sample from take from fermentation tank. In this case, add as mentioned above 2.5g ML-236B substrate and glucose, and when described substrate is converted into Pravastatin, this bio-transformation was carried out 24 hours again.

After finishing described fermentation, 5.1 liters of meat soups that contain 630 μ g/ml Pravastatins filter with filter cloth. 2 premium on currency are joined in the mycelium of separation, then the mycelium suspension was stirred 1 hour, then filter. These two kinds of filtrates are mixed, and (pillar was directly through 3.4cm at the pillar that contains 138g (250 ml) Dowex A1 400 (OH) resin with flow velocity 500ml/ hour, resin bed height 28 cm) pass through on, then resin bed washs with the 300ml deionized water. Subsequently, with 1 liter of acetone-water (1: 1) mixture that contains 10g sodium chloride resin is carried out wash-out. The volume of flow point is each flow point 100ml. By following thin-layer chromatography (TLC) methods analyst washings: adsorbent: Kieselgel 60 F₂₅₄DC (Merck) aluminium foil; Solvent: Acetone-Benzene-acetic acid (50: 50: 3) mixture; Detect: use Sonnenschein's reagent. The R of Pravastatin_fValue is 0.5. Merge the flow point that contains described product, vacuum distillation goes out acetone. With 15% sulfuric acid the pH of 400ml concentrate is transferred to 3.5-4.0, then use the 150ml ethyl acetate extraction 3 times. Acetic acid ethyl ester extract is merged, use anhydrous sodium sulfate drying. Subsequently, under room temperature, continuous stirring, by adding trifluoroacetic acid with catalytic amount from Pravastatin acid preparation pravastatin lactone. The generation of pravastatin lactone is by the TLC control (R of the pravastatin lactone in the above-mentioned TLC system_fValue is 0.7). After lactone generated and finishes, then described ethyl acetate used the 50ml water washing with the washing of 2 * 50ml, 5% sodium bicarbonate aqueous solution, with anhydrous sodium sulfate drying and vacuum evaporation. The evaporation residue of the 3g that obtains amount is dissolved in the 100 ml acetone and with the 0.3g active carbon clarifies. Then filter and remove active carbon, vacuum evaporation acetone. The raw product that obtains crystallization from 20ml ethanol. The crystal pravastatin lactone of precipitation is filtered out, wash vacuum drying under room temperature with the 30ml n-hexane at filter. Obtain so the chromatographically pure pravastatin lactone of 1.5g. Fusing point 140-142 ℃, [α]_D=+194 ° (c=0.5, methyl alcohol). The vacuum evaporating crystalization mother liquor obtains the 1.2g evaporation residue, and this residue is at the pillar that contains 24g Kieselgel 60 adsorbents (the straight warp of pillar: 1.6cm, the height of bed: 20 cm) upper chromatography. The raw product that will be dissolved in 5ml benzene is layered on this pillar. Use ethyl acetate-n-hexane mixture to carry out wash-out, wherein increase gradually the content of ethyl acetate. With 60% ethyl acetate-40% n-hexane mixture can wash-out be out from this pillar with pravastatin lactone. Adopt the mixture of ethyl acetate-n-hexane (9: 1) as solvent, by TLC control flow point. Merge the flow point that contains pravastatin lactone, vacuum evaporation. According to the method, obtain the pure product of 0.3g, its quality is identical in quality with the pravastatin lactone that obtains by crystallization.

Two batches of pravastatin lactones are mixed, then prepare in accordance with the following methods sodium salt: the 1.8g pravastatin lactone is dissolved in the 20ml acetone, stirs the lower 4.5ml of adding 1M sodium hydrate aqueous solution, so at room temperature this solution was stirred 0.5 hour. After the generation of finishing salt, 20ml water is joined in the mixture, make the solution neutralization, then vacuum evaporation acetone. With pillar (pillar straight warp: 2.6cm, the height of bed: 30cm) upper chromatography of aqueous concentrates at usefulness 150ml Diaion HP 20 resin fillings. As eluant, eluent, use the mixture of acetone-deionized water, wherein the concentration of acetone increases with 5% substep. With the acetone-deionized water mixture that contains 15% acetone, Pravastatin is washed out from pillar. Analyze flow point by TLC. Merge the flow point that contains described product, vacuum evaporation acetone. By the freeze-drying aqueous residue, obtain the 1.3g Pravastatin. Crystallization goes out chromatographically pure product from the mixture of ethanol and ethyl acetate.

Fusing point: 170-173 ℃ (decomposition)

[α] ^D ₂₀=+156 °, (c=0.5 is in the water).

Ultra-violet absorption spectrum (20 μ g/ml are in the methyl alcohol): λ_max＝231，237，245nm

(logε-4.263；4.311；4.136)

Infrared absorption spectroscopy (KBr): υ OH 3415, υ CH 2965, υ C=O 1730, υ COO^-1575 cm ^-1。

¹H-NMR wave spectrum (D₂O，δ，ppm)：0.86，d，3H(2-CH ₃); 5.92, dd, J=10.0 and 5.4 Hz, 1H (3-H); 5.99, d, J=10.0Hz, 1H (4-H); 5.52, br 1H (5-H); 4.24, m 1H (6-H); 5.34, br, 1H (8-H); 4.06, m, and 1H (β-H), 3.65, m, 1H (δ-H); 1.05, d, 3H (2 '-CH₃)；0.82，t，3H(4’-H ₃)。

¹³C-NMR wave spectrum (D₂O，δ，ppm)：15.3，q(2-CH ₃)；139.5，d(C-3)；129.5，d， (C-4)；138.1，s(C-4a)，127.7，d(C-5)；66.6，d(C-6)；70.1，d(C-8)；182.6s (COO ^-)；72.6.d(C-β)；73.0，d(C-δ)；182.0，s(C-1’)18.8；q(2’-CH ₃)； 13.7，q(C-4’)。

Cation FAB mass spectrum (characteristic ion):

[M+Na] ^-469；[M+H] ⁺447

Anion FAB mass spectrum (characteristic ion):

[M-H] ^-445，[M-Na] ^-423, m/z 101[2-methyl-butyric acid-H]^-。

Embodiment 3

In 5 liters of working volume fermentation tanks of laboratory scale, prepare as described in example 1 above biotransformation medium MU/4, although its 4.5 liters of application of sample at the most, the composition meter of culture medium is by 5 liters of calculating. 121 ℃ of sterilizations were used the 500ml inoculum inoculation according to embodiment 1 preparation after 45 minutes. By using stir speed (S.S.) to be 300rpm, Ventilation Rate is 50 l/hs, and making ferments carried out 4 days in 25 ℃. After being added into 5g ML-236B substrate in the culture, carry out bio-transformation according to embodiment 2.

After finishing this generation conversion, 4.9 liters of meat soups that contain 660 μ g/ml Pravastatins are filtered, by in 2 * 1 liters of deionized waters, suspending the mycelium that washing separates. The pH of 5.6 liters of bouillon filtrate (BF)s that will mix with 20% sulfuric acid transfers to 3.5-3.7, and then this acid filtrate and 2750ml ethyl acetate stirred 30 minutes. Subsequently, separation of phases. Water is used 2 * 1375ml ethyl acetate extraction again. The 470ml deionized water is joined in the 4740ml acetic acid ethyl ester extract of mixing, then with 1M NaOH the pH of the ethyl acetate mixture aqueous solution is transferred to 7.5-8.0. Stir after 20 minutes, then separation of phases uses 2 * 235ml deionized water extraction acetic acid ethyl ester extract as mentioned above. Then with the aqueous solution Vacuum Concentration of the alkalescent 1080ml volume that mixes to the 280ml volume. Aqueous solution that this is concentrated is layered on the chromatographic column of filling with 280ml Diaion HP-20 (Mitsubishi Co., Japan) non-ionic resin (highly: the ratio of straight warp=6.5). Adsorb at this pillar with flow velocity 250-300ml/ hour, then with 840ml deionized water washing pillar. Subsequently, use in the following order 800ml 5%, 1000ml 10%, and 500ml 15% and 500ml 20% contains the water elution pillar of acetone. In the process of wash-out, collect the flow point of 50ml, described flow point is analyzed according to the TLC method that provides among the embodiment 2. To contain Pravastatin is the flow point merging of main component, and resulting solution for vacuum concentration is to the 260ml volume. Concentrated aqueous solution chromatography on the pillar of the 260ml volume that contains Diaion HP-20 resin. Behind the absorption Pravastatin, pillar is with the washing of 790ml deionized water, then with the part of 260-260ml with the following aqueous acetone solution wash-out that increases gradually content of acetone: 2.5%, 5.0%, 7.5%, 10.0%, 12.5%, 15.0% and 20.0%. In the process of column chromatography, collect the flow point of 25ml, analyze as mentioned above the Pravastatin content in the flow point. The handle of identifying by TLC contains Pravastatin as the flow point merging of one-component, and vacuum evaporation. Subsequently, the 0.3g active carbon is joined in the concentrated aqueous solution (about 30ml), make Pravastatin clarification 30 minutes under the room temperature. Then from solution, remove active carbon by filtration method, with the filtrate freeze-drying. Obtain like this Pravastatin of 1.62g lyophilized form.

Embodiment 4

With Mortierella maculata n.sp.E-97[NCAIM (P) F 001266 that cultivates 10-12 days] slant culture of bacterial strain, prepare spore suspension with aseptic 0.9% sodium chloride solution of 5ml, the 500ml VHIG seed culture medium that will in the 3000ml conical flask, sterilize with this suspension inoculation.

The composition of culture medium VHIG:

Glucose 30g

Meat extract 8g

Yeast extract 1g

Tween-80 (polyoxyethylene (20) sorbitan monooleate) 0.5g

Be dissolved in the 1000ml running water.

PH with culture medium before the sterilization transfers to 7.0,121 sterilizations 25 minutes. Upper with culture cultivation 3 days at rotary shaker (250rpm, 2.5cm amplitude), then with the fermentation tank that contains biotransformation medium PK in the 5 liters of working volumes of inoculum inoculation experiments chamber scale that obtain.

The composition of culture medium PK:

Glucose 40g

Peptone 5g

Soy meal 20g

K ₂HPO ₄              2g

KH ₂PO ₄              1g

NaNO ₃                2g

KCl                   0.5g

Be dissolved in the 1000ml running water.

PH with culture medium before the sterilization transfers to 7.0. After inoculating, cultivate, add substrate and bio-transformation according to embodiment 2, separate Pravastatin again from this meat soup, wherein the concentration of Pravastatin is 650 μ g/ml during fermentation ends.

Finish fermentation, under continuous stirring, with 2M NaOH 4.9 liters of pH that contain the meat soup of 650 μ g/ml Pravastatins are transferred to 9.5-10.0, stir after 1 hour, with 20% sulfuric acid pH is transferred to 3.5-3.7. Subsequently, with 2.45 liters of these acid solutions of ethyl acetate extraction. Separation of phases is with the extract of centrifugal process from the organic phase preparation clarification of this emulsification. According to said method, meat soup is used 2 * 1.22 liters of ethyl acetate extractions again. Acetic acid ethyl ester extract is merged, add 0.4 liter of deionized water, then with 1M NaOH the pH of mixture is transferred to 8.0-8.5. Separation of phases, as mentioned above, with pH 8.0-8.52 * 0.2 liter of deionized water extraction ethyl acetate phase. The pH of the weak alkaline aqueous solution that contains Pravastatin that will mix with 20% sulfuric acid under stirring transfers to 3.5-3.7. The acid solution that obtains is with 4 * 0.2 liters of ethyl acetate extractions. The acetic acid ethyl ester extract that mixes washs with 2 * 0.2 liters of deionized waters, then 150% (mole) dibenzylamine (according to the Pravastatin content by the HPLC measurement-calculating) is joined in the ethyl acetate solution. Ethyl acetate solution Vacuum Concentration to 0.2 is risen volume. 20% (mole) dibenzylamine is joined in the concentrate of acquisition, the solution of precipitation spends the night in 0-5 ℃ of placement again. Then the Pravastatin dibenzyl amine salt of filtering-depositing will be deposited on the filter first with cold ethyl acetate washing 1 time, then with n-hexane washing 2 times, at last in 40-50 ℃ of vacuum drying. Under the room temperature, the raw product (3.9g) that obtains is dissolved in 100ml methyl alcohol, then makes this solution clarification with the 0.45g active carbon. After this Vacuum Concentration methyl alcohol filtrate. Externally temperature 62-66 ℃ the time, the residue of evaporation is dissolved in the 120ml acetone, then solution is cooled to room temperature. Subsequently, proceeding recrystallization under 0-5 ℃ spends the night. The crystal of filtering-depositing washs crystal 2 times with cold acetone first on filter, then with n-hexane washing 2 times. With the Pravastatin dibenzylamine salt suspension of recrystallization in the mixture of 160ml isobutyl acetate and 80ml deionized water. Subsequently, under agitation with equivalent NaOH is joined in the suspension. After suspension disappears, separation of phases, the aqueous solution that contains Pravastatin washs with 2 * 30ml isobutyl acetate. The aqueous solution that obtains is clarified with active carbon. Then will contain filter liquor and be concentrated into about 20ml volume. The aqueous solution that obtains is loaded onto on the chromatographic column (highly: straight through=22) of filling with 0.4 liter of Sephadex LH-20 gel (supplier: Pharmacia, Sweden). In chromatography process, deionized water is collected the flow point of 20ml as eluent. Analyze by the TLC convection current, then adopt said method also those flow points that contain Pravastatin to be analyzed by HPLC. Merge the flow point and the freeze-drying that contain pure Pravastatin. Obtain like this 1.75g Pravastatin, the purity of measuring it by HPLC is higher than 99.5%.

Embodiment 5

With Mortierella maculata n.sp.E-97[NCAIM (P) F 001266 that cultivates 10-12 days] slant culture and aseptic 0.9% sodium chloride solution of 5ml of bacterial strain prepare spore suspension, then uses as described in Example 4 this suspension inoculation 500ml seed culture medium. In 5 liters of working volume fermentation tanks of laboratory scale, biotransformation medium PC/4 121 ℃ of sterilizations 45 minutes, is then inoculated with inoculum.

The composition of culture medium PC/4:

Brewer's wort 5.0%

Soy meal 1.0%

Peptone 1.0%

Corn steep liquor 1.0%

MgSO ₄×7H ₂O                 0.1％

Be dissolved in the 1000ml running water.

PH with culture medium before the sterilization transfers to 7.0. After inoculating, cultivate and add substrate according to embodiment 2, obtain 5.1 liters and contain the meat soup that concentration is the Pravastatin of 610 μ g/ml.

According to the method that provides among the embodiment 4, from this meat soup, produce 3.7g Pravastatin dibenzyl amine salt raw product, after its recrystallization, therefrom obtain 2.9g Pravastatin dibenzyl amine salt. The Pravastatin dibenzylamine salt suspension of this recrystallization in 45ml ethanol, is then under agitation added 110% (mole) NaOH by the ethanolic solution that adds 1M NaOH. Continued agitating solution 0.5 hour, and then the 0.3g active carbon was joined in the solution and also stirred again 0.5 hour. Filtering solution is concentrated into 15ml with filtrate. Then under 56-60 ℃, 60ml acetone is joined in the concentrate. The solution that obtains is cooled to room temperature, then spends the night in+5 ℃ of placements. Subsequently, with sedimentation and filtration, then use 2 * 20ml acetone, 2 * 20ml ethyl acetate and the washing of 2 * 20ml n-hexane, last vacuum drying. The rough Pravastatin of the 1.7g that obtains is dissolved in the ethanol, then with active carbon clarification, crystallization from Ethanol-Acetic Acid ethyl ester mixture. Obtain like this 1.54 g Pravastatins, it is identical with the product of embodiment 2.

Embodiment 6

As described in Example 4, with Mortierella maculata n.sp.E-97[NCAIM (P) F 001266 that cultivates 7-10 days] slant culture of bacterial strain, then the 500ml seed culture medium MI that sterilizes in the inoculation 3000ml conical flask cultivated 3 days at rotary shaker in 28 ℃.

The composition of culture medium MI:

Glucose 40g

Casein 5g

KCl                       0.5g

NaNO ₃                   3g

KH ₂PO ₄                 2g

MgSO ₄×7H ₂O            0.5g

FeSO ₄×7H ₂O            0.01g

Be dissolved in the 1000ml running water.

PH with culture medium before the sterilization transfers to 6.0,121 ℃ of sterilizations 35 minutes. The inoculum that obtains is inoculated among 5 liters of biotransformation medium P12 that sterilize in the fermentation tank.

The composition of culture medium P12:

Glucose 10g

Brewer's wort 50g

Yeast extract 5g

Corn steep liquor 5g

MgSO ₄×7H ₂O             1g

Tween-80 0.5g

Be dissolved in the 1000ml running water.

PH with culture medium before the sterilization transfers to 7.0, then sterilizes 45 minutes for 121 ℃. Ferment, add substrate and bio-transformation according to embodiment 2. After finishing bio-transformation, the concentration that following separation generates is the Pravastatin of 620 μ g/ml:

With 2M NaOH 5.15 liters of pH that contain the meat soup of 620 μ g/ml Pravastatins are transferred to 9.5 values, then stirred 1 hour under the room temperature. Then filtered broth, mycelium suspend in 1 * 0.5 water and wash first by suspending in 1 * 2 premium on currency. Merging filtrate transfers to 3.7 with 20% sulfuric acid with the pH value of the aqueous solution, then uses 1.5 liters of ethyl acetate extractions with 2.5 liters first. Acetic acid ethyl ester extract is merged, with adding the 1.95g dicyclohexyl amine after the washing of 2 * 0.5 premium on currency. Under 40 ℃ of decompressions, acetic acid ethyl ester extract is concentrated into 200ml, again the 0.195g dicyclohexyl amine is joined in the concentrate, then it was stirred 6 hours under 15 ℃. The crystalline solid of precipitation is filtered, then wash with 15ml ethyl acetate with 20ml first, lower dry at 40 ℃. Obtain like this 3.51g raw product. This raw product after the recrystallization, obtains 3.05g Pravastatin dicyclohexyl amine salt (fusing point: 162-168 ℃) in acetone-alcohol mixture, be translated into Pravastatin according to embodiment 5.

Embodiment 7

As described in Example 2, with Mortierella maculata n.sp.E-97[NCAIM (P) F 001266] bacterial strain ferments, adds substrate and bio-transformation. The following Pravastatin that obtains by bio-transformation that from meat soup, separates.

5 liters of culture broth that contain concentration and be 650 μ g/ml Pravastatins are filtered at filter cloth. In 2 liters of 0.1M sodium hydroxide solutions, the fungal hyphae body was stirred 1 hour, then filter. Mix two kinds of filtrates, with 15% sulfuric acid pH is transferred to 3.5-4.0. Subsequently, this solution is with 2 * 1.8 liters of ethyl acetate extractions. The ethyl acetate that mixes is used the 800ml water washing mutually. Then add the 400ml deionized water, with 1M NaOH the pH value of mixture is transferred to 8.0-8.5. Mixture was stirred 15 minutes, then separation of phases. With 300ml water join ethyl acetate mutually in, and pH transferred to 8.0-8.5. Stir after 15 minutes separation of phases. Again 300ml water is joined ethyl acetate mutually in, and pH transferred to 8.0-9.5. Then mixture was stirred 15 minutes. The Re-isolation two-phase. Mix all waters, and with 15% sulfuric acid pH is transferred to 3.5-4.0, then use 3 * 300ml ethyl acetate extraction. The ethyl acetate that mixes is used the 150ml water washing mutually, uses anhydrous sodium sulfate drying, and filters. Then 150% (mole) dioctylamine (according to Pravastatin content-calculating) is joined in the acetic acid ethyl ester extract. Ethyl acetate is evaporated to about 1/10 volume, adds acetone until precipitation occurs. Mixture spends the night in+5 ℃ of placements. Filtering-depositing on the G-4 filter, first with the washing of 20ml acetone, then with the washing of 20ml n-hexane, and vacuum drying under room temperature. The rough Pravastatin dioctylamine of the 3.3g that recrystallization obtains from 20ml acetone salt obtains the pure Pravastatin dioctylamine salt of 2.7g. Fusing point: 143-146 ℃. With the method that provides among the embodiment 5, Pravastatin dioctylamine salt is converted into Pravastatin.

Embodiment 8

By to separate from natural habitat, can be with the exploitation of the Mortierella maculata n.sp.E-97 bacterial strain hydroxylation ability of ML-236B 6 β-hydroxylation, in the Catastrophic selection that discusses in detail hereinafter and the enzyme induction experiment, obtain Mortierella maculata n.sp.E-97/15/13 [NCAIM (P) F 001267] mutant strain.

Mortierella maculata n.sp.E-97[NCAIM (P) F 001266 that will be separated by us] bacterial strain cultivated 7 days at the MS agar slant culture-medium in 28 ℃.

The composition of agar medium MS:

Glucose 4g

Brewer's wort 10g

Yeast extract 4g

Agar 20g

In 1000ml distilled water.

Spore is washed out from slant culture with 5ml 0.9% sodium chloride solution, then after spore suspension is transferred to sterile petri dish, with uviol lamp it was shone 1 minute. Subsequently, be that N-methyl-N '-nitro-N-nitrosoguanidine of 2000 μ g/ml joins in the spore suspension with final concentration. Then this suspension is transferred in the 100ml conical flask, and descended to use the speed of 150rpm with its jolting 20 minutes in 28 ℃. Subsequently, by with the speed of 400rpm centrifugal 10 minutes, make the spore precipitation, then be suspended in the 0.9% sodium chloride sterile solution. This suspension is coated on the agar plate MU-VB that contains 10 μ g/ml benomyl and 1% defibrinated blood.

The composition of agar medium MU-VB:

Glucose 40g

Asparagine 2g

Peptone 2.5g

Potassium dihydrogen phosphate 0.5g

Agar 20g

Be dissolved in the 990ml distilled water; After the sterilization, be this culture media supplemented 10ml ox blood and 10mg benomyl.

Agar plate in 28 ℃ cultivate 7 days after, with the bacterium colony of growth by selecting at random transferred species in the test tube that agar medium PS is housed.

The composition of agar medium PS:

Glucose 40g

Mycoprotein peptone 10g

Agar 15g

Be dissolved in the 1000ml distilled water.

PH value with culture medium before the sterilization transfers to 5.6-5.7. 121 sterilizations 20 minutes.

Slant culture was cultivated 12 days in 28 ℃, as described in Example 1, and the Pravastatin productivity of test cultures in shake flat experiment. Select Mortierella maculata n. sp.E-97/15/13 mutant strain by the method, this mutant strain produces the Pravastatin that surpasses 60% conversion ratio with the sodium salt of the ML-236B acid of the 1000 μ g/ml concentration of using.

By containing 100 μ g/ml 8-and taking off-the MU-VB agar medium of (2-methyl-butyryl) ML-236B and/or ML-236B cultivates, and induces the hydroxylase of Mortierella maculata n.sp.E-97/15/13 bacterial strain. After selecting at random the bacterium colony of growth, with their transferred speciess to the MU-VB inclined-plane that contains inducer. Detect the Pravastatin productivity of inclined-plane grown culture according to the method described in the embodiment 1, but following difference is arranged: from the 4th day of fermentation, add the ML-236B substrate with the amount of 500 μ g/ml, continuous adding 11 days, add gradually ML-236B sodium substrate, ML-236B sodium substrate was converted into Pravastatin fully in 12 days. When in 50 diastatochromogenes, carrying out the bio-transformation end with 30g ML-236B sodium substrate, 18.5 g Pravastatins have been generated by HPLC mensuration. In accordance with the following methods, from the fermentation broth that mixes, carry out the recovery of Pravastatin.

After finishing fermentation, the pH that will contain 5.5 liters of meat soups of 3360 μ g/ml concentration Pravastatins with 20% sulfuric acid solution transfers to 3.5-3.7. Subsequently, this acid solution is with 2.75 liters of ethyl acetate extractions. Separation of phases is by centrifugal organic phase preparation clarification extract from emulsification. As described previously, extract again meat soup 2 times with 1.37 liters of ethyl acetate. The acetic acid ethyl ester extract that mixes washs with 2 * 1.15 liters of deionized waters, then 150% (mole) dibenzylamine (according to the Pravastatin content by the HPLC measurement-calculating) is joined in the ethyl acetate solution. With the ethyl acetate solution Vacuum Concentration to about 0.23 liter of volume. Again 20% (mole) dibenzylamine is joined in the concentrate, precipitation solution is spent the night in 0-5 ℃ of placement. The dibenzyl amine salt of the Pravastatin acid of filtering-depositing, then with sediment by washing in the ethyl acetate that it is suspended in cooling, then be suspended in n-hexane washing 2 times, at last in 40-50 ℃ of vacuum drying. In 62-65 ℃ of temperature the raw product (22.4g) that obtains is dissolved in 0.67 liter of acetone, makes the solution clarification with the 2.2g active carbon. After the clarification, acetone filtrate Vacuum Concentration to 0.56 is risen volume. The crystal that will precipitate from this concentrate under said temperature dissolves again, then this solution is cooled to room temperature. Subsequently, continuing recrystallization at 0-5 ℃ spends the night. The crystal of filtering-depositing washs for 2 times by suspending in the acetone of cooling, then is suspended in the n-hexane and washs 2 times. Dibenzyl amine salt in the Pravastatin acid of 40-50 ℃ of vacuum drying recrystallization. Be dissolved in 740ml ethyl acetate-ethanol (9: the 1) mixture at the 40-44 ℃ of dibenzyl amine salt (14.8g) with the Pravastatin acid that obtains, then 110% (mole) NaOH joined in this solution of 1M ethanolic solution form. Then the precipitation solution that the continuation stirring obtains under the room temperature 0.5 hour precipitates by using or reaching in ice-cooled 1-1.5 hour fully. Subsequently, filtering-depositing is with ethyl acetate and the washing of 2 * 150ml hexane of 2 * 150ml cooling, at last in 40-50 ℃ of vacuum drying. The Pravastatin that obtains is dissolved in the ethanol, with the clarification of 1.0g active carbon, then from Ethanol-Acetic Acid ethyl ester mixture, crystallizes out. Obtain like this 9.4g Pravastatin, its physical constant is corresponding to the data that provide among the embodiment 2.

Although this paper has described some at present preferred embodiment of the present invention, but it will be apparent to one skilled in the art that in the situation that does not deviate from the spirit and scope of the present invention and can the embodiment that the present invention describes be made various changes and modifications. Therefore, the present invention only limits to appended claims and the required degree of the applicable rule of law.

Claims (2)

1. one kind is preserved in the state-run agricultural of Budapest, HUN and industrial microorganism preservation center, preserving number are the pure culture of biology of Mortierella maculata n.sp.E-97/15/13 bacterial strain of NCAIM (P) F 001267.

One kind can be with the Mortierella culture of formula (II) compound 6 β-hydroxylation,

Wherein R represents a kind of alkali metal or ammonium ion, described culture mainly is comprised of a kind of new bacterial strain Mortierella maculata n.sp.E-97/15/13, described bacterial strain is preserved in the state-run agricultural of Budapest, HUN and industrial microorganism preservation center, and preserving number is NCAIM (P) F 001267.