CN108676831A - The preparation method of echinocandin B parent nucleus - Google Patents
The preparation method of echinocandin B parent nucleus Download PDFInfo
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- CN108676831A CN108676831A CN201810536352.7A CN201810536352A CN108676831A CN 108676831 A CN108676831 A CN 108676831A CN 201810536352 A CN201810536352 A CN 201810536352A CN 108676831 A CN108676831 A CN 108676831A
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/50—Cyclic peptides containing at least one abnormal peptide link
- C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
- C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
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Abstract
The invention discloses a kind of methods that completely new echinocandin B is converted into echinocandin B parent nucleus.During this method converts cross-linking enzyme aggressiveness technology to echinocandin B parent nucleus applied to echinocandin B.This method is by the way that deacylase is prepared into deacylase cross-linking enzyme aggressiveness, which converts echinocandin B to echinocandin B parent nucleus.In this way, molar yield can reach 85% or more, while simplifying production process, reduces cost.
Description
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 of anidulafungin precursor echinocandin B parent nucleus.
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).
The preparation process of anidulafungin includes following three step：(1) aspergillus nidulans (Aspergilus nidulans) fermentation production
Raw echinocandin B (Echinocandin B, ECB)；(2) pass through microorganism conversion --- the deacylation that actinoplanes fermentation generates
Change enzyme to be catalyzed echinocandin B, so that side chain is broken, generate precursor-echinocandin B parent nucleus of anidulafungin
(Echinocandin B Nucleus, ECBN)；(3) new side is added using the method for chemical modification on the basis of parent nucleus
Chain --- penta oxygen-triphen carboxyl.The preparation of wherein precursor parent nucleus is committed step.At present for the preparation of ECB parent nucleus, chemistry side
Method cost is higher, and microorganism conversion has the following advantages：Reaction condition is mild, product is single, the chemo-selective of height, region
Selectivity and enantio-selectivity are easy to purifying, are free from environmental pollution and can complete some chemical syntheses and be difficult to the reaction carried out.
The technique that United States Patent (USP) US7785826B2 discloses ECB Transformed Es CBN.This technique main flow includes：ECB is sent out
After ferment is completed, centrifugation obtains mycelium, and mycelium, which is resuspended in water then addition deacylase, to be converted, and ECB is de-
Acyl enzyme is merely with primary.But this method operation is relatively complicated, and technique conversion takes 20~30h, and conversion ratio is low, molar yield
Only 30%.
CN102618606 discloses a kind of using the full cell of actinomyces or zymotic fluid as the echinocandin of catalyst biology
Method for transformation.This method is added to a kind of cosolvent in transformation system, improves solubility of the substrate in transformation system, the hydrotropy
Agent is beta-cyclodextrin or derivatives thereof, and the advantages of this method is to improve conversion rate and conversion ratio, the disadvantage is that resting cell,
A large amount of thalline are carried in system, enzyme-to-substrate contacting efficiency is not high, and later separation purification step complexity cost is higher, does not solve
Enzyme is the easy in inactivation in containing organic solvent system the problem of.
CN103074403 discloses a kind of method converting ECB to ECBN, is related to after fermentation through the acid that phosphorates
Salt, ultrasound, centrifugation, concentrated by rotary evaporation obtain echinocandin B deacylase enzyme solution, and ECB substrates are then added and are converted.This method
It is disadvantageous in that thick enzyme extraction process is cumbersome, is unfavorable for industrial amplification production；Thick enzyme is extracted using the mode of revolving, due to
Temperature drift, easily causes protein denaturation, and the vigor of deacylase has negative effect.
CN103374593 discloses one kind, and addition echinocandin B substrate is converted into echinocandin B parent nucleus during the fermentation
Method, this method by substrate be added to deacylase generate fermented liquid transfer, there are a large amount of microorganisms for reaction system, subsequently
Purification procedures are complicated, of high cost.
CN103387975 discloses a kind of preparation method of immobilization ring grease peptidyl transferase, the ring grease acyltransferase polypeptide
Transferase is fixed on carrier；The ring grease peptidyl transferase is from natural or artificial mutant or mutation and by leading to
Cross what conversion after importing external cyclic annular acyl transferase gene obtained.ECBN is turned using immobilization ring grease peptidyl transferase
Turn to anidulafungin.In this way, although conversion ratio is higher, complicated for operation, cost is higher, and the chemistry of immobilization process is anti-
Enzyme partial inactivation should be easy to cause.
CN105648000 discloses a kind of echinocandin B microbial enzyme method for transformation, and in particular to echinocandin B converts
For the method for echinocandin B parent nucleus.It is included in after microbial fermentation generates echinocandin B deacylase and extracts thick enzyme, then passes through
The mode that thick enzyme and echinocandin B is added batch-wise is converted, and thick enzyme is added batch-wise in this method and substrate is converted, and does not solve
Certainly enzyme is exposed in organic solvent system the problem of being easy inactivation.
Based on to expanding production, the needs of cost are reduced, in the production field of anidulafungin, it is still desirable to more preferably ECBN
The method for being converted to anidulafungin.
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 echinocandin B is converted into echinocandin B parent nucleus.The party
During method converts cross-linking enzyme aggressiveness technology to echinocandin B parent nucleus applied to echinocandin B.
This method is by the way that deacylase is prepared into deacylase cross-linking enzyme aggressiveness, which turns echinocandin B
Turn to echinocandin B parent nucleus.In this way, can reach 85% molar yield, while simplifying production process, reduces into
This.
The method of the present invention includes the following steps：
1) fermentation energies obtain echinocandin B deacylase by echinocandin B deacylation expression of enzymes in extracellular genetic engineering bacterium
Thick enzyme；
2) the thick enzyme of deacylase is prepared as deacylase cross-linking enzyme aggressiveness by；
3) echinocandin B is reacted with deacylase cross-linking enzyme aggressiveness, is converted into echinocandin B parent nucleus.
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) echinocandin B is added in obtained reaction solution, it stirs, echinocandin is made to convert
For echinocandin B parent nucleus, the concentration of echinocandin B and echinocandin B parent nucleus in transformation system is monitored, when echinocandin B concentration
It no longer reduces, when echinocandin B parent nucleus concentration is not further added by, terminates conversion.
Wherein, the echinocandin B is dissolved in dimethyl sulfoxide, obtains the echinocandin B solution that mass concentration is 10%, makes
Echinocandin B addition be 5~20g/L, preferably 20g/L, whipping temp be 28~32 DEG C, mixing speed be 50~
150rpm, preferably 150rpm, transformation time are 12~48 hours, and the index for terminating conversion is that spine is white in HPLC detection reaction systems
Rhzomorph B concentration is no longer reduced, and echinocandin B parent nucleus concentration is not further added by.
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.
2 deacylation enzyme crosslinking of embodiment conversion production echinocandin B 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.
The 10% echinocandin B dimethyl sulfoxide solution containing 20kg echinocandin B is added into reaction solution, 30 DEG C of stirrings are anti-
It answers 12~48 hours, the speed of stirring is 150rpm, echinocandin B and the white bacterium of spine in HPLC monitoring transformation systems in conversion process
Plain B parent nucleus concentration terminates conversion when echinocandin concentration is no longer reduced, and echinocandin B parent nucleus concentration is not further added by.Reaction
Liquid filters, and filtrate HPLC is taken quantitatively to detect echinocandin B parent nucleus content, and it is 85.13% to calculate molar yield.
Claims (8)
1. a kind of method that echinocandin B is converted into echinocandin B parent nucleus, this method is echinocandin B and deacylase cross-linking enzyme
Aggressiveness reacts, and is converted into echinocandin B parent nucleus,
Wherein, the preparation method of the cross-linking enzyme aggressiveness is as follows：
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.
2. the method as described in claim 1, 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.
3. method as claimed in claim 2, phosphate buffer phosphoric acid potassium dihydrogen 2.24g/L, disodium hydrogen phosphate 1.24g/L,
PH is 6.0.
4. the addition of method as claimed in claim 2, thick enzyme is 2%.
5. the addition of method as claimed in claim 2, metacetaldehyde is 5mmol/L.
6. method as claimed in claim 2, the mixing speed being added after metacetaldehyde is 50rpm.
7. the addition of the method as described in claim 1, echinocandin B is 5~20g/L.
8. the addition of the method as described in claim 1, echinocandin B is 20g/L.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109593809A (en) * | 2018-12-07 | 2019-04-09 | 成都雅途生物技术有限公司 | A kind of method of immobilized microorganism enzymatic conversion echinocandin B |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102443560A (en) * | 2010-10-13 | 2012-05-09 | 上海医药工业研究院 | Gene engineering bacterium for high efficiency conversion of echinocandin B and preparation method thereof |
| CN102618606A (en) * | 2011-01-30 | 2012-08-01 | 浙江海正药业股份有限公司 | Echinocandin biotransformation method |
| CN103074403A (en) * | 2011-10-26 | 2013-05-01 | 上海医药工业研究院 | Method for converting echinocandin B by using microbial enzyme |
-
2018
- 2018-05-30 CN CN201810536352.7A patent/CN108676831A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102443560A (en) * | 2010-10-13 | 2012-05-09 | 上海医药工业研究院 | Gene engineering bacterium for high efficiency conversion of echinocandin B and preparation method thereof |
| CN102618606A (en) * | 2011-01-30 | 2012-08-01 | 浙江海正药业股份有限公司 | Echinocandin biotransformation method |
| CN103074403A (en) * | 2011-10-26 | 2013-05-01 | 上海医药工业研究院 | Method for converting echinocandin B by using microbial enzyme |
Non-Patent Citations (3)
| Title |
|---|
| LINQIU CAO等: "《Immobilised enzymes: carrier-bound or carrier-free?》", 《CURRENT OPINION IN BIOTECHNOLOGY》 * |
| 徐忠等编著: "《功能性变性淀粉》", 30 April 2010, 中国轻工业出版社 * |
| 王梦凡等: "《交联酶聚体技术研究进展》", 《中国工程院化工、冶金与材料工学部第七届学术会议》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109593809A (en) * | 2018-12-07 | 2019-04-09 | 成都雅途生物技术有限公司 | A kind of method of immobilized microorganism enzymatic conversion echinocandin B |
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Application publication date: 20181019 |