CN108753881A - The preparation method of FR179642 - Google Patents
The preparation method of FR179642 Download PDFInfo
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- CN108753881A CN108753881A CN201810536326.4A CN201810536326A CN108753881A CN 108753881 A CN108753881 A CN 108753881A CN 201810536326 A CN201810536326 A CN 201810536326A CN 108753881 A CN108753881 A CN 108753881A
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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
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- 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 preparation method of FR179642, this method includes：1), the preparation of the culture medium of fermentation FR901379；2) bacterial strain Coleophoma empetri seed culture fluids, are inoculated in fermentation medium to ferment；3), after 24~72h of fermented and cultured, methyl hexadecanoate and soybean lecithin mixture are added into zymotic fluid；4), when ferment 160h after, when the concentration of FR901379 no longer increases, fermentation ends；5) FR901379 obtained in the previous step is reacted with deacylase cross-linking enzyme aggressiveness, is converted into FR179642.
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 fermentation process 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：
USP5502033, EP0431350A1 are disclosed using Coleophoma empetri F-11899 fermenting and producings
The process of FR901379, fermentation medium include basic carbon nitrogen source, inorganic salts, unit 100mg/L.CN201010571797.2
Disclose a kind of culture medium using Coleophoma empetri F-11899 fermenting and producings FR901379, optimize carbon source and
Organic nitrogen source, unit are increased to 300~400mg/L.But in the technology of existing fermenting and producing FR901379, however it remains due to
The growth characteristics of der Pilz in zymotic fluid, zymotic fluid viscosity is excessively high, and oxygen transmission effect is poor, and dissolved oxygen is relatively low, to inhibit bacterium
Body cometabolism, the problem for causing FR901379 fermentation units relatively low.
201010571797.2 provide one kind gives birth to for bacterial strain Coleophoma empetri F-11899 or its variant
The fermentation medium of FR901379 is produced, the fermentation medium contains carbon source, organic nitrogen source, inorganic salts and metal ion.Fermentation
Potency is 300~400mg/L.
201611019780.X provides a kind of culture medium and its method of fermenting and producing FR901379, the group of the culture medium
As fructose, corn protein powder, casein, Yeast protein peptone, magnesium sulfate, dipotassium hydrogen phosphate, calcium carbonate, antifoaming agent, water, into one
Step includes adding fructose and hydrogen peroxide during the fermentation, and by the improvement to fermentation medium, the yield of FR901379 is higher,
Unit reaches 3.0~3.4g/L.But the nutrition of this method zymotic fluid is excessively abundant, it is big to put tank Fungal biodiversity, isolates and purifies into
This is higher.
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.
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.
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 preparation method of FR179642.
Invention content
The purpose of the present invention is to provide a kind of preparation methods of completely new FR179642.This method includes two main steps
Suddenly：Methyl hexadecanoate is added in the culture solution of fermentation FR901379 in the first step；Second step, FR901379 and deacylation enzyme crosslinking
Enzyme aggressiveness reacts, and obtains FR179642.
More specifically, the present invention includes the following steps：
(1) bacterial strain Coleophoma empetri F-11899 seed culture fluids fermentation medium is inoculated in send out
Ferment；
(2) after 24~72h of fermented and cultured, added into zymotic fluid 3~10% mass volume ratios methyl hexadecanoate and
Soybean lecithin mixture.In the methyl hexadecanoate and soybean lecithin mixture, mass volume ratio shared by soybean lecithin
It is 10~30%；
(3) after fermentation 160h, when the concentration of FR901379 no longer increases, fermentation ends；
(4) FR901379 and the reaction of deacylase cross-linking enzyme aggressiveness, are converted into FR179642.
Wherein, in step (1), fermentation medium can be used condition well known to those skilled in the art and carry out fermentation medium
Preparation, it may for example comprise but be not limited to fermentation medium disclosed in 201010571797.2.
In step (4), deacylase cross-linking enzyme aggressiveness is prepared via a method which：Fermentation energy is by echinocandin B deacylation
Expression of enzymes obtains the thick enzyme of echinocandin B deacylase in extracellular genetic engineering bacterium, and the thick enzyme of deacylase is prepared as deacylase cross-linking enzyme
Aggressiveness.
For more detailed,
The thick enzyme of deacylase is made by producing strains fermentation, and the fermentation of thick enzyme producing strains can be used expression in extracellular gene work
Journey bacterial 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) it, is carried out using condition well known to those skilled in the art public in fermentation preparation, such as including but not limited to CN102618606
The fermentation process opened.
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.
Deacylase cross-linking enzyme aggressiveness prepare specific method be：Phosphate buffer is added in the thick enzyme of the deacylase being prepared
Afterwards, bovine serum albumin(BSA) (BSA) is added as crosslinking protective agent, is passed through air, is stood after stirring.Again into above-mentioned reaction solution
Crosslinking agent glutaraldehyde, stirring, to form deacylase cross-linking enzyme aggressiveness is added.
Wherein, the phosphate buffer phosphoric acid 2~2.5g/L of potassium dihydrogen, disodium hydrogen phosphate 1~1.5g/L, pH adjusting
To 5.5~6.5；It is preferred that potassium dihydrogen phosphate 2.24g/L, disodium hydrogen phosphate 1.24g/L, pH 6.0.The addition of thick enzyme is 1~
3% (w/v), preferably thick enzyme addition 2%.
The addition of the bovine serum albumin(BSA) (BSA) is 1~3g/L, preferably 2g/L, is passed through the stream per minute of air
Amount is equivalent to 10~30% (v/v) of material liquid volume, and preferably 20%；Mixing time is 20~60 minutes, preferably 30 minutes；Stirring
Speed is 50~150rpm；Static conditions are 4~15 DEG C of temperature, 0.5~2 hour time, and preferred static conditions are 8 DEG C of standings
1 hour.
The glutaraldehyde addition is 10~30mmol/L, preferably 20mmol/L；Whipping temp is 28~32 DEG C；Stirring
Speed is 50~150rpm, preferably 100rpm, mixing time 1~3 hour, preferably 2 hours.
Mikafen precursor FR901379 is added in the reaction solution obtained to above-mentioned steps, stirring makes mikafen precursor
FR901379 is converted into mikafen parent nucleus, and it is dense to monitor mikafen precursor FR901379 and mikafen parent nucleus in transformation system
Degree, when mikafen precursor FR901379 concentration is no longer reduced, when mikafen parent nucleus concentration is not further added by, end 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.
Before methyl hexadecanoate is added as FR901379 fatty acid side chains in the method for the present invention in FR901379 fermentation process
Body substance promotes the biosynthesis of FR901379.It is introduced in zymotic fluid simultaneously and the immiscible soybean lecithin of reaction solution, palm fibre
Palmitic acid acid methyl esters together as the carrier of oxygen in fermentation system with soybean lecithin in addition to as fermentation precursor, also reducing gas-liquid and passing
Oxygen resistance solves the problems, such as that prior art fermentation process dissolved oxygen is insufficient, improves the fermentation yield of FR901379, reduce life
Produce cost.
On the other hand, the present invention, using cross-linking enzyme aggressiveness technology, is led to during FR901379 is converted into FR179642
Addition bovine serum albumin(BSA) (BSA) is crossed as crosslinking protective agent, while controlling flow and mixing speed, the time of air, is formed
The suitable enzyme aggressiveness of particle size, had both avoided that cross-linking enzyme aggressiveness grain size is excessive so that internal enzyme molecule is from well and bottom
Object contacts, and in turn avoids enzyme molecule and is excessively exposed to cause to inactivate in organic solvent system.Enzyme activity after deacylation enzyme crosslinking of the present invention
Higher, FR901379 are converted into the conversion ratio higher of FR179642.The present invention is of low cost, and easy to operate, reduction isolates and purifies
Cost.
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
Cultured seed is transferred to 120 DEG C of sterilizing 30min postcoolings of 30L to 24-26 DEG C with 5% (v/v) ratio
In fermentation medium, which forms, and quality (g)/volume (L) ratio is：Glucose 50, cottonseed meal 20, yeast powder
10, peptone 10, magnesium sulfate 2, dipotassium hydrogen phosphate 1, calcium carbonate 4, antifoaming agent 1, remaining is water.
24-26 DEG C of temperature is controlled in fermentation process, ventilatory capacity 1VVM, initial speed 150rpm regulate and control rotating speed 150 in the process
Dissolved oxygen is set to be not less than 20% between~600rpm.
Methyl hexadecanoate and soybean lecithin are configured to solution according to mass volume ratio shared by soybean lecithin for 20%
It is for use after 120 DEG C of sterilizing 30min.
After fermented and cultured 48h, methyl hexadecanoate and the soybean lecithin that 8% mass volume ratio is added into zymotic fluid are mixed
Close object.
Fermentation period is 8 days, measures FR901379 and puts a concentration of 3.51g/L of tank.
Embodiment 2
Cultured seed is transferred to 120 DEG C of sterilizing 30min postcoolings of 30L to 24-26 DEG C with 5% (v/v) ratio
In fermentation medium, which forms, and quality (g)/volume (L) ratio is：Glucose 50, cottonseed meal 20, yeast powder
10, peptone 10, magnesium sulfate 2, dipotassium hydrogen phosphate 1, calcium carbonate 4, antifoaming agent 1, remaining is water.
24-26 DEG C of temperature is controlled in fermentation process, ventilatory capacity 1VVM, initial speed 150rpm regulate and control rotating speed 150 in the process
Dissolved oxygen is set to be not less than 20% between~600rpm.
Methyl hexadecanoate and soybean lecithin are configured to solution according to mass volume ratio shared by soybean lecithin for 10%
It is for use after 120 DEG C of sterilizing 30min.
After fermented and cultured 48h, the methyl hexadecanoate and soybean lecithin of 10% mass volume ratio are added into zymotic fluid
Mixture.
Fermentation period is 8 days, measures FR901379 and puts a concentration of 3.24g/L of tank.
Embodiment 3
Cultured seed is transferred to 120 DEG C of sterilizing 30min postcoolings of 30L to 24-26 DEG C with 5% (v/v) ratio
In fermentation medium, which forms, and quality (g)/volume (L) ratio is：Glucose 50, cottonseed meal 20, yeast powder
10, peptone 10, magnesium sulfate 2, dipotassium hydrogen phosphate 1, calcium carbonate 4, antifoaming agent 1, remaining is water.
24-26 DEG C of temperature is controlled in fermentation process, ventilatory capacity 1VVM, initial speed 150rpm regulate and control rotating speed 150 in the process
Dissolved oxygen is set to be not less than 20% between~600rpm.
Methyl hexadecanoate and soybean lecithin are configured to solution according to mass volume ratio shared by soybean lecithin for 30%
It is for use after 120 DEG C of sterilizing 30min.
After fermented and cultured 48h, methyl hexadecanoate and the soybean lecithin that 3% mass volume ratio is added into zymotic fluid are mixed
Close object.
Fermentation period is 8 days, measures FR901379 and puts a concentration of 3.43g/L of tank.
Embodiment 4
Cultured seed is transferred to 120 DEG C of sterilizing 30min postcoolings of 30L to 24-26 DEG C with 5% (v/v) ratio
In fermentation medium, which forms, and quality (g)/volume (L) ratio is：Glucose 50, cottonseed meal 20, yeast powder
10, peptone 10, magnesium sulfate 2, dipotassium hydrogen phosphate 1, calcium carbonate 4, antifoaming agent 1, remaining is water.
24-26 DEG C of temperature is controlled in fermentation process, ventilatory capacity 1VVM, initial speed 150rpm regulate and control rotating speed 150 in the process
Dissolved oxygen is set to be not less than 20% between~600rpm.
Methyl hexadecanoate and soybean lecithin are configured to solution according to mass volume ratio shared by soybean lecithin for 15%
It is for use after 120 DEG C of sterilizing 30min.
After fermented and cultured 48h, methyl hexadecanoate and the soybean lecithin that 5% mass volume ratio is added into zymotic fluid are mixed
Close object.
Fermentation period is 8 days, measures FR901379 and puts a concentration of 3.30g/L of tank.
Embodiment 5
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.
Embodiment 6
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, 2kg bovine serum albumin(BSA)s (BSA) are passed through air, and flow per minute is
200L, while stirring 30 minutes, mixing speed 80rpm, 8 DEG C stand 1 hour.
Glutaraldehyde 20mmol/L is added into reaction solution, 30 DEG C are stirred 2 hours, mixing speed 100rpm.
The mikafen precursor FR901379 that 20kg embodiments 1 are prepared is added into reaction solution, 30 DEG C are stirred to react
6~24 hours, the speed of stirring was 100rpm, mikafen precursor FR901379 in HPLC monitoring transformation systems in conversion process
With mikafen parent nucleus concentration, when mikafen precursor FR901379 concentration is no longer reduced, mikafen parent nucleus concentration no longer increases
Added-time terminates conversion.Reaction solution filters, and filtrate HPLC is taken quantitatively to detect mikafen parent nucleus content, calculates molar yield and is
96.84%.
Embodiment 7
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, 2kg bovine serum albumin(BSA)s (BSA) are passed through air, and flow per minute is
100L, while stirring 60 minutes, mixing speed 150rpm, 8 DEG C stand 1 hour.
Glutaraldehyde 20mmol/L is added into reaction solution, 30 DEG C are stirred 2 hours, mixing speed 100rpm.
The mikafen precursor FR901379 that 20kg embodiments 1 are prepared is added into reaction solution, 30 DEG C are stirred to react
6~24 hours, the speed of stirring was 100rpm, mikafen precursor FR901379 in HPLC monitoring transformation systems in conversion process
With mikafen parent nucleus concentration, when mikafen precursor FR901379 concentration is no longer reduced, mikafen parent nucleus concentration no longer increases
Added-time terminates conversion.Reaction solution filters, and filtrate HPLC is taken quantitatively to detect mikafen parent nucleus content, calculates molar yield and is
94.19%.
Embodiment 8
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, 2kg bovine serum albumin(BSA)s (BSA) are passed through air, and flow per minute is
300L, while stirring 20 minutes, mixing speed 50rpm, 8 DEG C stand 1 hour.
Glutaraldehyde 20mmol/L is added into reaction solution, 30 DEG C are stirred 2 hours, mixing speed 100rpm.
The mikafen precursor FR901379 that 20kg embodiments 1 are prepared is added into reaction solution, 30 DEG C are stirred to react 6
~24 hours, the speed of stirring was 100rpm, mikafen precursor FR901379 in HPLC monitoring transformation systems in conversion process
With mikafen parent nucleus concentration, when mikafen precursor FR901379 concentration is no longer reduced, mikafen parent nucleus concentration no longer increases
Added-time terminates conversion.Reaction solution filters, and filtrate HPLC is taken quantitatively to detect mikafen parent nucleus content, calculates molar yield and is
92.78%.
Claims (11)
1. a kind of preparation method of FR179642, this method include：
1), the preparation of the culture medium of fermentation FR901379；
2) bacterial strain Coleophoma empetri seed culture fluids, are inoculated in fermentation medium to ferment；
3), after 24~72h of fermented and cultured, methyl hexadecanoate and soybean lecithin mixture are added into zymotic fluid；
4), when ferment 160h after, when the concentration of FR901379 no longer increases, fermentation ends；
5) FR901379 obtained in the previous step is reacted with deacylase cross-linking enzyme aggressiveness, is converted into FR179642.
2. the quality of methyl hexadecanoate and soybean lecithin mixture is added into zymotic fluid for the method as described in claim 1
Volume ratio is 3~10%.
3. method as claimed in claim 2, in methyl hexadecanoate and soybean lecithin mixture, quality shared by soybean lecithin
Volume ratio is 10~30%.
4. the group of the method as described in claim 1, fermentation medium becomes：Glucose, cottonseed meal, yeast powder, peptone,
Magnesium sulfate, dipotassium hydrogen phosphate, calcium carbonate, antifoaming agent, water.
5. the method for claim 1, wherein the preparation method of deacylase cross-linking enzyme aggressiveness is by the thick enzyme of deacylase and friendship
Join protective agent bovine serum albumin(BSA), the reaction of crosslinking agent glutaraldehyde, to form deacylase cross-linking enzyme aggressiveness.
6. method as claimed in claim 5, the preparation method of deacylase cross-linking enzyme aggressiveness is that phosphoric acid is added in the thick enzyme of deacylase
After buffer solution, bovine serum albumin(BSA) is added as crosslinking protective agent, air is passed through, is stood after stirring, then to above-mentioned reaction solution
Middle addition crosslinking agent glutaraldehyde, stirring, to form deacylase cross-linking enzyme aggressiveness.
7. method as claimed in claim 6,2~2.5g/L of phosphate buffer phosphoric acid 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 addition of bovine serum albumin(BSA) is 1~3g/L, is passed through sky
The flow per minute of gas is equivalent to the 10~30% of material liquid volume, and mixing time is 20~60 minutes, and mixing speed is 50~
150rpm, static conditions are 4~15 DEG C of temperature, 0.5~2 hour time, and glutaraldehyde addition is 10~30mmol/L, is added penta
Stirring condition after dialdehyde is 28~32 DEG C of temperature, 1~3 hour time.
8. the method for claim 7, phosphate buffer phosphoric acid potassium dihydrogen 2.24g/L, disodium hydrogen phosphate 1.24g/L,
PH is 6.0.
9. the method for claim 7, the addition of thick enzyme is 2%.
10. the method for claim 7, the addition of bovine serum albumin(BSA) is 2g/L.
11. the method for claim 7, glutaraldehyde addition is 20mmol/L.
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| CN116496911A (en) * | 2023-04-23 | 2023-07-28 | 浙江昊清生物科技有限公司 | Ricasfungin intermediate FR901379 high-yield strain and application thereof |
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| CN108085354A (en) * | 2016-11-21 | 2018-05-29 | 重庆乾泰生物医药有限公司 | A kind of culture medium and its method of fermenting and producing FR901379 |
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| CN102876745A (en) * | 2011-07-14 | 2013-01-16 | 北大方正集团有限公司 | Method for production of lipstatin through fermentation |
| CN106047848A (en) * | 2016-07-15 | 2016-10-26 | 电子科技大学 | Preparation method of crosslinking halohydrin dehalogenase aggregate |
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| CN106399431A (en) * | 2016-11-28 | 2017-02-15 | 无锡福祈制药有限公司 | Preparation method of micafungin precursor |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116496911A (en) * | 2023-04-23 | 2023-07-28 | 浙江昊清生物科技有限公司 | Ricasfungin intermediate FR901379 high-yield strain and application thereof |
| CN116496911B (en) * | 2023-04-23 | 2023-12-26 | 浙江昊清生物科技有限公司 | Ricasfungin intermediate FR901379 high-yield strain and application thereof |
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