CN108467880A - The preparation method of micafen sodium precursor - Google Patents

Abstract

The invention discloses a kind of methods that completely new FR901379 is converted into FR179642.During this method converts cross-linking enzyme aggressiveness technology to FR179642 applied to FR901379.This method is by the way that deacylase is prepared into deacylase cross-linking enzyme aggressiveness, which converts FR901379 to FR179642.In this way, the molar yield that FR901379 is converted into FR179642 can reach 92% or more, while simplifying production process, reduces cost.

Description

The preparation method of micafen sodium precursor

Technical field

The present invention relates to field of microbial fermentation, and in particular to the preparation of antimycotic fermentation semisynthetic drug precursor, more More particularly to the preparation method of micafen sodium precursor FR179642.

Background technology

In recent years, since immunocompromised patient increases year by year, fungal infection incidence is significantly raised, especially deep fungal The incidence and case fatality rate of infection dramatically increase.Echinocandin class antibiotic is that one group found the 1970s naturally produces Object has similar ring type polypeptide core and different fatty acid side chains, can inhibit fungal cell wall β -1 noncompetitively, The activity of 3- glucan synthases, to reach antimycotic purpose.Compared with conventional antifungal drug, such drug, which has, to be made With mechanism uniqueness, toxic side effect is low, and has very strong antibacterial activity to some azoles and the drug resistant fungi of amphotericin B, is The common drug of clinical treatment deep fungal infection at present.This kind of drug of FDA approveds listing includes Caspofungin (Caspofungin), mikafen (Micafungin) and anidulafungin (Anidulafungin).Mikafen pharmaceutical salts are Micafen sodium.

FR901379 is the important precursor for synthesizing mikafen class drug, is obtained after mutated by C.empetri Superior strain fermentation gained.FR901379 obtains mikafen parent nucleus FR179642 after removing side chain by deacylase, FR179642 is by being chemically modified to obtain micafen sodium.

The structure of FR901379 is as follows：

The structure of FR179642 is as follows：

Chinese patent 91104847.2 is disclosed passes through mycelium actinoplanes utahensis I by FR901379 FO-13244 obtains the sodium salt of FR179642 in 37 DEG C of deacylation bases.

Chinese patent CN102443050, which is disclosed, to be obtained using a kind of cyclic lipopeptide compound as precursor by digesting side chain FR179642, and report the process for separation and purification of this kind of precursor cyclic lipopeptide compound.In addition there are in document report by FR179642 prepares synthetic method (Ueda, the S. of mikafen；Ezaki,M.；Tanaka,M.；et al.Studies on a novellipopeptide acylase from Streptomyces spp.for production of FR179642,a key intermediate of antifungal lipopeptide drug FK463.38th Intersci Conf Antimicrob Agents Chemother(Sept 24 1998,San Diego)1998,Abst F-145)。

Chinese patent CN106755221 discloses a kind of preparation method of FR179642, and FR179642 bacterial strains are in culture medium Middle fermentation is added mikafen precursor FR901379, and by the method for multistage temperature control, is improved at twice during the fermentation The yield of FR179642.

But the fermentation of FR179642 rests on always a very low level for a long time, although people all for find one It is a cheap, the culture medium of high yield and effort, but produce little effect.This preparation difficulty for allowing for FR179642 compounds increases, at This raising, to make micafen sodium be chronically at a higher price indirectly.Therefore there is an urgent need to a kind of cheap height by people The method of effect synthesis FR179642.

Chinese patent CN106544382 discloses a kind of method that FR901379 is converted to FR179642, and this method is logical Cross control conversion fluid in FR901379 initial concentration and conversion process in temperature and pH control, significantly improve the conversion of enzyme Efficiency, conversion ratio may be up to 83%, but the method disclosed in the patent is to collect thalline to be converted, and transformation system exists a large amount of Microorganism, conversion rate is slower, and the stability of enzyme is poor, and purification procedures are complicated, of high cost.

East China University of Science's master thesis " structure of echinocandin deacylase genetic engineering bacterium and application study " is public A kind of research of ECB deacylations enzyme immobilizatio has been opened, since that there are storage times is short for free ECB deacylases, has recycled difficulty etc. Disadvantage, the Authors of Science Articles carry out glutaraldehyde cross-linking to LX-1000HA resins, use it for EBC deacylation enzyme immobilizatios.By this Method, complicated for operation although conversion ratio is higher, cost is higher, and the chemical reaction of immobilization process is easy to cause the mistake of enzyme part It is living.

Based on to expanding production, the needs of cost are reduced, in the production field of mikafen, it is still desirable to more preferably The method that FR901379 is converted to FR179642.

Cao of Delft Polytechnics of Holland in 2000 etc. proposes a kind of new on the basis of cross-linking enzyme and crosslinked enzyme crystal The enzyme immobilization technology of type, cross-linking enzyme aggressiveness (cross-linked enzyme aggregates, CLEAs).This passes through change Property keeps enzyme molecule close and forms aggregation and be precipitated out from solvent, is later crosslinked aggregation, forms cross-linking enzyme Aggressiveness.Cross-linking enzyme aggressiveness is immobilised enzymes of the enzyme as carrier itself, and unit volume enzyme concentration is high, and stable, recyclable, catalysis is lived Property it is high, production cost is low, has potential application prospect.

The enzyme that the cross-linking enzyme aggressiveness reported at present uses includes hydrolase, lyase, oxidoreducing enzyme, has no sending out at present The fermentation arts of ferment semisynthetic drug have been reported that, also have no the report that this technology is applied to deacylase.

Invention content

The purpose of the present invention is to provide a kind of methods that completely new FR901379 is converted into FR179642.This method will be handed over During connection enzyme aggressiveness technology is converted into FR179642 applied to FR901379.

This method is by the way that deacylase is prepared into deacylase cross-linking enzyme aggressiveness, which converts FR901379 For FR179642.In this way, the molar yield that FR901379 is converted into FR179642 can reach 92% or more, while letter Change production process, reduces cost.

The method of the present invention includes the following steps：

1) it is thick to obtain echinocandin B deacylase by echinocandin B deacylation expression of enzymes in extracellular genetic engineering bacterium for fermentation energy Enzyme；

2) preparation of deacylase cross-linking enzyme aggressiveness；

3) FR901379 and the reaction of deacylase cross-linking enzyme aggressiveness, are converted into FR179642.

Wherein, the thick enzyme of deacylase is made by producing strains fermentation in step 1), and expression can be used in the fermentation of thick enzyme producing strains In extracellular engineering strain, such as streptomyces albus engineering strain (Streptomyces albus), (referring to “Efficient Bioconversion of Echinocandin B to Its Nucleus by Overexpression of Deacylase Genes in Different Host Strains[J].Appl Environ Microbiol, 2013Feb；79(4):1126-33), fermentation preparation is carried out using condition well known to those skilled in the art, for example, including but not It is limited to fermentation process disclosed in CN102618606.

The deacylase filtering fermentation liquor being prepared according to the method described above or centrifugation are added ammonium sulfate to supernatant and carry out salt Analysis, is passed through air, is stood after stirring, filter or centrifuge, and collects precipitation, obtains the thick enzyme of deacylase.

Wherein, the dosage of the ammonium sulfate be fermented supernatant fluid 40~80% (w/v), preferably 50%；It is passed through air Flow per minute be equivalent to be added ammonium sulfate after feed liquid total volume 10~30% (v/v), preferably 20%；Mixing time is 20 ~60 minutes, preferably 30 minutes；Mixing speed is 50~150rpm；Static conditions be 4~15 DEG C of temperature, 8~16 hours time, Preferred static conditions are 8 DEG C and stand 12 hours.

In step 2), the specific method for preparing of deacylase cross-linking enzyme aggressiveness is：The deacylase that step 1) is prepared is thick After phosphate buffer is added in enzyme, it is passed through air, then crosslinking agent metacetaldehyde is added into above-mentioned reaction solution, stirring, to form deacylation Enzyme crosslinking enzyme aggressiveness.

Wherein, the phosphate buffer phosphoric acid 2~2.5g/L of potassium dihydrogen, disodium hydrogen phosphate 1~1.5g/L, pH salt Acid or sodium hydroxide are adjusted to 5.5~6.5；It is preferred that potassium dihydrogen phosphate 2.24g/L, disodium hydrogen phosphate 1.24g/L, pH 6.0.Slightly The addition of enzyme is 1~3% (w/v), preferably thick enzyme addition 2%.

The flow per minute for being passed through air is equivalent to 10~30% (v/v) of material liquid volume, and preferably 20%.The poly- second Aldehyde addition is 5~10mmol/L, preferably 5mmol/L；Whipping temp is 28~32 DEG C；Mixing speed is 50~100rpm, excellent It is 2 hours to select 50rpm, mixing time.

In step 3), to above-mentioned steps 2) mikafen precursor FR901379 is added in obtained reaction solution, stirring makes rice The fragrant net precursor FR901379 of card is converted into mikafen parent nucleus, monitors mikafen precursor FR901379 and meter Ka in transformation system Fragrant net parent nucleus concentration, when mikafen precursor FR901379 concentration is no longer reduced, when mikafen parent nucleus concentration is not further added by, knot Beam converts.

Wherein, the addition of the mikafen precursor FR901379 is 5~20g/L, preferably 20g/L, whipping temp It it is 28~32 DEG C, mixing speed is 50~150rpm, and preferably 100rpm, transformation time is 6~24 hours, terminates the index of conversion Mikafen precursor FR901379 concentration in reaction system is detected for HPLC no longer to reduce, mikafen parent nucleus concentration no longer increases Add.

FR901379 of the present invention can be used method well known to those skilled in the art and carry out fermentation preparation, for example, packet It includes but is not limited to preparation method disclosed in 201611019780.X.

Flow and mixing speed, time of the present invention by control air, form the suitable enzyme aggressiveness of particle size, both kept away Exempt from that cross-linking enzyme aggressiveness grain size is excessive so that it is excessive in turn avoid enzyme molecule from being contacted well with substrate for internal enzyme molecule Being exposed in organic solvent system causes to inactivate.Enzyme activity higher after deacylation enzyme crosslinking of the present invention.The present invention is of low cost, operation letter Just, conversion rate and the stability of enzyme are greatly improved, the cost isolated and purified is reduced.

Specific embodiment

It present invention will be described in detail below.However, the present invention may be embodied as many different forms, And it is not necessarily limited in embodiment described herein, and it is to make disclosure to provide the purpose in these embodiments More completely with comprehensively.Agents useful for same and raw material, except providing preparation method, remaining is commercially available.Unless otherwise defined, Otherwise the meaning and the normally understood meaning of claim theme technical field personnel that all scientific and technical terminologies have herein It is identical.

Embodiment 1 produces the fermentation of deacylase bacterial strain and prepared by thick enzyme

Strain：Streptomyces albus (Streptomyces albus), preserving number：ATCC21725；- 80 DEG C of cryovials preserve；

Seed culture medium：The fried soybean cake powder 0.5% (w/v, similarly hereinafter) of heat, yeast powder 0.5%, peptone 0.5%, glucose 1%, pH about 6.8~7.2,30 DEG C are cultivated 1~2 day；

Fermentation medium：The fried soybean cake powder 1% of heat, yeast powder 1%, peptone 1%, glucose 3%, sodium chloride 0.5%, Epsom salt 0.2%, dipotassium hydrogen phosphate 0.1%, pH about 6.8~7.2,30 DEG C are cultivated 2~3 days；

Streptomyces albus engineering strain is inoculated in seed culture medium, 30 DEG C are cultivated 1~2 day, then according to fermentation The 5% of volume is inoculated in fermentation medium, and 30 DEG C are cultivated 2~3 days；

After fermentation, filtering fermentation liquor is obtained into supernatant, ammonium sulfate is added according to the 50% of supernatant volume, is passed through Air, flow per minute are equivalent to the 20% of material liquid volume, while stirring 30 minutes, and 8 DEG C stand 12 hours, and it is heavy to be collected by filtration It forms sediment, obtains thick enzyme.

The preparation of 2 FR901379 of embodiment

Coleophoma empetri strains are cultivated in seed culture medium.The seed culture medium is glucose 10g/L, soluble starch 30g/L, cottonseed meal 20g/L, dry powder corn steep liquor 5g/L, dipotassium hydrogen phosphate 2g/L, calcium carbonate 3g/L, Antifoaming agent 1g/L.

Suitable seed culture medium is prepared, 120 DEG C of sterilizing 30min postcoolings are to 24-26 DEG C, by seed culture medium volume ratio The shake-flask seed in 0.5~1% inoculum concentration access 48h kind ages, then cultivates 48h for 24-26 DEG C.

Cultured seed is transferred to the hair of 120 DEG C of sterilizing 30min postcoolings to 24-26 DEG C of 12000L with 2% ratio In ferment culture medium, which forms, and quality (g)/volume (L) ratio is：Fructose 140, corn protein powder 10, casein 6, Yeast protein peptone 7, magnesium sulfate 2, dipotassium hydrogen phosphate 0.5, calcium carbonate 3, antifoaming agent 0.5, remaining is water.

24-26 DEG C of temperature in fermentation process, ventilatory capacity 1VVM, initial speed 50rpm, in the process regulate and control rotating speed 50~ Dissolved oxygen is set to be not less than 20% between 180rpm.

Fructose is configured to after 120 DEG C of sterilizing 30min of 500g/L aqueous solutions for use, hydrogen peroxide is diluted to 30% aqueous solution and waits for With.Fermentation 72h starts, and fructose and hydrogen peroxide are added in fermentation tank respectively, and additional way is continuously uninterruptedly to add, 144h Stopping is added, and controlling additional amount in the process makes total quality (g)/fermentation volume (L) ratio of adding be fructose 120, hydrogen peroxide 1.5. Fermentation period is 8 days, measures FR901379 and puts a concentration of 3.4g/L of tank.

3 deacylation enzyme crosslinking of embodiment conversion production mikafen parent nucleus

Phosphate buffer 1 000L, phosphoric acid potassium dihydrogen 2.24g/L and disodium hydrogen phosphate 1.24g/ are prepared in retort L, it is 6.0 to adjust pH with hydrochloric acid or sodium hydroxide.

The thick enzymes of 20kg are added into phosphate buffer, are passed through air, flow per minute is 200L, adds metacetaldehyde 5mmol/L, 30 DEG C of speed with 50rpm stir 2 hours.

20kg mikafen precursor FR901379 are added into reaction solution, 30 DEG C are stirred to react 6~24 hours, stirring Speed is 100rpm, and mikafen precursor FR901379 and mikafen parent nucleus are dense in HPLC monitorings transformation system in conversion process Degree, when mikafen precursor FR901379 concentration is no longer reduced, when mikafen parent nucleus concentration is not further added by, end converts.Instead It answers liquid to filter, filtrate HPLC is taken quantitatively to detect mikafen parent nucleus content, it is 92.45% to calculate molar yield.

Claims (9)

1. a kind of method that FR901379 is converted into FR179642, this method is that FR901379 and deacylase cross-linking enzyme aggressiveness are anti- It answers, is converted into FR179642,

Wherein, the preparation method of the cross-linking enzyme aggressiveness is to react the thick enzyme of deacylase with crosslinking agent metacetaldehyde, forms deacylase and hands over Join enzyme aggressiveness.

2. the preparation method of cross-linking enzyme aggressiveness as described in claim 1 is：

After phosphate buffer is added in the thick enzyme of deacylase, it is passed through air, then crosslinking agent metacetaldehyde is added into above-mentioned reaction solution, stirred It mixes, to form deacylase cross-linking enzyme aggressiveness.

3. method as claimed in claim 2, phosphate buffer phosphoric acid 2~2.5g/L of potassium dihydrogen, disodium hydrogen phosphate 1~ 1.5g/L, pH are 5.5~6.5, and the addition of thick enzyme is 1~3%, and the flow per minute for being passed through air is equivalent to material liquid volume 10~30%, metacetaldehyde addition is 5~10mmol/L, and whipping temp is 28~32 DEG C, and mixing speed is 50~100rpm, is stirred It is 2 hours to mix the time.

4. method as claimed in claim 3, phosphate buffer phosphoric acid potassium dihydrogen 2.24g/L, disodium hydrogen phosphate 1.24g/L, PH is 6.0.

5. the addition of method as claimed in claim 3, thick enzyme is 2%.

6. the addition of method as claimed in claim 3, metacetaldehyde is 5mmol/L.

7. method as claimed in claim 3, the mixing speed being added after metacetaldehyde is 50rpm.

8. the addition of the method as described in claim 1, FR901379 is 5~20g/L.

9. the addition of the method as described in claim 1, FR901379 is 20g/L.