The purpose of this invention is to provide a kind of simple and therefore have more the technology of economic attractiveness, be used for: with sufficiently high (and preferably, improve) productive rate, prepare and have good quality (preferably, the potassium salt of penicillin of crystalline form improved quality), described penicillin is selected from the group of penicillin G and penicillin v.
" productive rate " is defined as in this article: for the penicillin that exists in the extract, and the % productive rate of penicillin in the sylvite of the penicillin of group crystalline form, that be selected from penicillin G and penicillin v.Enough height are defined as in this article: preferably at least 90%, more preferably at least 92%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, most preferably at least 98%.
" good quality " is defined as in this article: the sylvite of the penicillin of that finally obtain, crystalline form, as to be selected from penicillin G and penicillin v group contains considerably less impurity or free from foreign meter, and for example, foreign matter content is less than 5% (w/v), more preferably, be less than 4%, more preferably, be less than 3%, more preferably, be less than 2%, more preferably, be less than 1%, more preferably, be less than 0.5%, more preferably, be less than 0.25%, further more preferably, be less than 0.1%.Impurity can for example be 6-amino-penicillanic acid (6-APA), toluylic acid (PA), the penicillic acid (penicillic acid) to hydroxyl penicillin G, penicillin G, the penicilloic acid (penicilloicacid) of penicillin G and the penilloic acid (penilloic acid) and the corresponding impurity at penicillin v of penicillin G.
In one aspect, the invention provides a kind of technology, be used for: prepare the sylvite of the penicillin of group crystalline form, that be selected from penicillin G and penicillin v from following suspension, described suspension comprises sylvite, the organic solvent of the penicillin of the group that is selected from penicillin G and penicillin v and is selected from C
1, C
2And C
3The alcohol of the group that alcohol constitutes.Preferably, described penicillin is penicillin G.Surprisingly, we find, in technology according to the present invention, the productive rate of the sylvite of the penicillin of group crystalline form, that be selected from penicillin G and penicillin v is that preamble is defined sufficiently high, and equal or even be higher than technology known in the art, the crystalline quality of Huo Deing is good as the preamble definition simultaneously.Another advantage of technology of the present invention is, only needs a step separating step, and this has not only brought sufficiently high productive rate, has also brought sizable economical advantage.
In technology of the present invention, organic solvent can be any appropriate organic solvent well known by persons skilled in the art.The example of this type of appropriate organic solvent is pentyl acetate, n-butyl acetate, ethyl acetate, hexone, pimelinketone, isopropylcarbinol or propyl carbinol.Preferably, organic solvent is a n-butyl acetate.
Be selected from C according to the alcohol in the technology of the present invention
1, C
2And C
3The group that alcohol constitutes, it is methyl alcohol, ethanol, n-propyl alcohol or Virahol preferably.Most preferably, described alcohol is methyl alcohol.
In the technology of the sylvite of the penicillin of group crystalline form produced according to the present invention, that be selected from penicillin G and penicillin v, penicillin can obtain from any suitable production technique known in the art.Penicillin G can for example be to produce microorganism according to methods known in the art by fermentation penicillin G when having the side chain precursor toluylic acid, and for example the bacterial strain of Penicillium chrysogenum is produced.Can use any suitable technique, for example centrifugal or filtration from the fermentation culture separating biomass, produces the fermentation fluid that contains penicillin thus.Preferably, using filtration step comes from the nutrient solution separating biomass.Alternatively, residual solid is washed.
By adding at least a acid, for example sulfuric acid, spirit of salt or nitric acid or their any combination is acidified to fermentation culture or fluid the pH in 2 to 4 the scope, preferably, and the pH in 2.5 to 3 the scope.Then by being extracted in the organic solvent, with penicillin with separate through the acidifying water.Can use any appropriate organic solvent, for example, pentyl acetate, n-butyl acetate, ethyl acetate, hexone, pimelinketone, isopropylcarbinol or propyl carbinol.Preferably, organic solvent is a n-butyl acetate.The adding of suitable de-emulsifier may significantly improve extraction.
" extract " is defined as following organic solvent in this article, and the penicillin that is selected from the group of penicillin G and penicillin v has been extracted in the described organic solvent.Alternatively, can be according to methods known in the art, comprise the extract of penicillin with activated carbon treatment, to remove impurity.
In one embodiment of the invention, crossed by activated carbon treatment alternatively, can mix with suitable potassium source subsequently according to the extract of penicillin preamble definition, that comprise the group that is selected from penicillin G and penicillin v, penicillin with the group that will be selected from penicillin G and penicillin v is converted into corresponding potassium salt of penicillin, obtains to comprise the mixture of organic solvent and potassium salt of penicillin thus.
Preferably such with the consumption in the suitable potassium source of the extract blended of the penicillin that comprises the group that is selected from penicillin G and penicillin v: described consumption makes the potassium that adds 0.4 to 3 molar equivalent for penicillin.Preferably, for penicillin, add the potassium of 0.6 to 2 molar equivalent, more preferably, the potassium of 0.7 to 1.6 molar equivalent, more preferably, the potassium of 0.8 to 1.4 molar equivalent, more preferably, the potassium of 0.9 to 1.2 molar equivalent.Most preferably, the potassium that for penicillin, adds 1 molar equivalent.Suitable potassium source is to be preferably any sylvite of faintly acid.The example in the potassium source that this type of is suitable is potassium acetate or salt of wormwood.The potassium source can be used with solid form, perhaps can be water-soluble earlier, form the aqueous solution in potassium source.The suitable concn in potassium source is in the aqueous solution, for example, and between 10 to 60%w/v, for example, between 15 to 55%w/v, for example between 20 to 50%.
Subsequently, can from the mixture that comprises the organic solvent and the sylvite of the penicillin of the group that is selected from penicillin G and penicillin v, remove a part of anhydrating, perhaps comprise the part of the azeotrope (for example azeotrope of water and n-butyl acetate) of water and organic solvent.The water that is present in the mixture may be from fermentation culture or from organic solvent, or along with the aqueous solution in potassium source is introduced into.Preferably, after the part of water or azeotrope was removed, the water-content that comprises the organic solvent and the mixture of the sylvite of the penicillin of the group that is selected from penicillin G and penicillin v was<10% (v/v), more preferably,<5% (v/v), more preferably,<2.5% (v/v), more preferably,<1% (v/v), more preferably,<0.5% (v/v), most preferably,<0.2% (v/v).
According to mentioned above, at first after the part evaporation with the azeotrope of the part of water or water/organic solvent, in mixture, add and be selected from C
1, C
2And C
3The alcohol of the group that alcohol constitutes is with the suspension of sylvite, organic solvent and the alcohol of the penicillin that obtains to comprise the group that is selected from penicillin G and penicillin v.Alcohol can be methyl alcohol, ethanol, n-propyl alcohol or Virahol.Preferably, described alcohol is methyl alcohol.
Preferably, be selected from C
1, C
2And C
3The alcohol of the group that alcohol constitutes is (dry) that does." do " expression alcohol when using in this article and contain the water that is less than 10% (v/v), preferably, be less than the water of 5% (v/v), preferably, be less than 2% (v/v).Most preferably, the alcohol of Ganing contains the water of 0% (v/v).
After being in contact with one another, can carry out the stirring of certain hour to the suspension of sylvite, organic solvent and the alcohol of the penicillin that comprises the group that is selected from penicillin G and penicillin v.Preferably, to the suspension time of carrying out be stirring between 5 minutes to 24 hours, preferably, between 10 minutes to 12 hours, more preferably, between 20 minutes to 8 hours, more preferably, between 0.5 hour to 6 hours, more preferably, between 45 minutes to 3 hours.Preferably, to suspension stir the sylvite of penicillin of the group that is selected from penicillin G and penicillin v crystal formation till.More preferably, to suspension stir the sylvite of penicillin of the group that is selected from penicillin G and penicillin v crystal formation and do not lump till.Can under any suitable temperature, stir suspension, preferably, the temperature between 0 to 40 ℃, more preferably, between 5 to 30 ℃, most preferably, between 10 to 25 ℃.
In the suspension according to technology of the present invention, the volume of alcohol can be any suitable ratio with the ratio that comprises the organic solvent and the volume of the mixture of the sylvite of the penicillin of the group that is selected from penicillin G and penicillin v.Preferably, this ratio is between 1: 20 to 1: 3, preferably, and between 1: 15 to 1: 4, preferably, between 1: 10 to 1: 6.
During technology of the present invention, be selected from the partly or entirely crystallization of sylvite of penicillin of the group of penicillin G and penicillin v.For example, from comprising organic solvent and being selected from the mixture of sylvite of penicillin of group of penicillin G and penicillin v during the evaporation, the sylvite of penicillin that is selected from the group of penicillin G and penicillin v can begin crystallization after the extract that comprises penicillin adds suitable potassium source and/or in the part of the azeotrope of the part of water or water/organic solvent.When comprise organic solvent and be selected from penicillin G and penicillin v group penicillin sylvite (alternatively, partly or entirely crystalline) mixture be selected from C
1, C
2And C
3When the alcohol of the group that alcohol constitutes contacts, and/or during the suspension of sylvite, organic solvent and the alcohol of the penicillin that comprises the group that is selected from penicillin G and penicillin v stirred, be selected from the sylvite also further crystallization or the recrystallization of penicillin of the group of penicillin G and penicillin v.
In another embodiment, in the extract of the penicillin of the group that is selected from penicillin G and penicillin v that contains according to preamble described definition and acquisition, add suitable potassium source, and comprise after the azeotrope evaporation of organic solvent and water, for example by filtering the crystal of the sylvite of the penicillin of the group that is selected from penicillin G and penicillin v that collection obtains.Under the condition identical (for example), thus obtained crystal is suspended in again comprises previously described organic solvent and be selected from C at aspects such as the ratio of alcohol/organic solvent, time, temperature with aforementioned embodiments
1, C
2And C
3In the mixture of the alcohol of the group that alcohol constitutes.
In any embodiment of the present invention, can be by any suitable method known in the art, the sylvite of the penicillin of the group isolation of crystalline form, that be selected from penicillin G and penicillin v from suspension is for example undertaken by centrifugal or filtration.Can obtain the wet cake of the potassium salt of penicillin of crystalline form then.Alternatively, with organic solvent or organic solvent and be selected from C
1, C
2And C
3The mixture of the alcohol of the group that alcohol constitutes washs this wet cake.If used washing step, preferably, organic solvent is identical with the organic solvent that is used for extract, and, be selected from C
1, C
2And C
3The alcohol of the group that alcohol constitutes be used for the pure identical of suspension.Can use any suitable technique known in the art, come the wet cake of sylvite of the penicillin of the group dried crystals form, that be selected from penicillin G and penicillin v.In this embodiment of the present invention, penicillin is penicillin G preferably.
Alternatively, the crystalline wet cake of sylvite that will comprise the penicillin of the group that is selected from penicillin G and penicillin v mixes with water, obtains to be selected from the aqueous solution of sylvite of penicillin of the group of penicillin G and penicillin v.But extracting then (strip) aqueous solution is with the organic solvent of removing trace and/or be selected from C
1, C
2And C
3The alcohol of the group that alcohol constitutes.Extracting comprises the evaporation of water, organic solvent and/or alcohol.The aqueous solution of sylvite of penicillin that is selected from the group of penicillin G and penicillin v can be used for proceeding to by methods known in the art (for example, EP 0,977 883 is described) Enzymatic transformation of 6-amino-penicillanic acid (6-APA).Available suitable penioillin acylase is handled and is comprised the solution of sylvite of penicillin that dissolved is selected from the group of penicillin G and penicillin v; described enzyme for example is suitable penicillin G acylase; so that penicillin G deacylation; and suitable penicillin v acyltransferase, so that penicillin v deacylation.Being found the biology that can produce suitable penioillin acylase is; for example, the kind of Acetobacter, Aeromonas, Alcaligenes, Aphanocladium, Bacillus sp., Cephalosporium, Escherichia, Flavobacterium, Kluyvera, Mycoplana, Protaminobacter, Providentia, Pseudomonas or Xanthomonas.Confirm that especially the enzyme that obtains from Acetobacterpasteurioanum, Alcaligenes faecalis, Bacillus megaterium, Escherichia coli, Providentia rettgeri and Xanthomonas citrii can successfully be used for the method according to this invention.In the literature, penioillin acylase is also referred to as penicillin amidase.Penioillin acylase can be used as the free enzyme and uses, and also can be suitable for any suitable form that is fixed, and for example, describes among EP 0222462 and the WO 97/04086.By penioillin acylase the Enzymatic transformation that dissolved Pen G K carries out is caused forming 6-amino-penicillanic acid (6-APA) and toluylic acid.