CN105648000B - A kind of echinocandin B microbial enzyme method for transformation - Google Patents
A kind of echinocandin B microbial enzyme method for transformation Download PDFInfo
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Abstract
The invention discloses a kind of echinocandin B microbial enzyme method for transformation, and in particular to the method that echinocandin B is converted into echinocandin B parent nucleus.It is included in after microbial fermentation generates echinocandin B deacylase and extracts thick enzyme, is then converted by way of thick enzyme and echinocandin B is added batch-wise.The molar yield of the method for the present invention is high, and production concentration is high, and conducive to isolating and purifying, and echinocandin B substrate is conducive to recycling and reusing, reduces production cost.
Description
Technical field
The present invention relates to field of biological pharmacy, in particular it relates to a kind of echinocandin B microbial enzyme conversion side
Method.
Background technique
Echinocandin class (Echinocandins) is one group of natural products of phase late 1970s discovery, has class
As ring type polypeptide core and different fatty acid side chains structure feature, being capable of Noncompetition inhibition fungal cell wall β -1,3-
Glucan synthase activity, clinically uses as antifungal drug.
Echinocandin B (Echinocandin B, abbreviation ECB) is by aspergillus nidulans (Aspergilus nidulans) generation
It thanks to generation, is one of echinocandin class, structure is as follows:
Echinocandin B obtains cyclic peptide parent nucleus (Echinocandin B Nucleus, ECBN) through deacylation enzyme effect, and cyclic peptide is female
Antifungal drug anidulafungin (Anidulafungin) can be prepared using chemical modification in core.At present for ECB parent nucleus
Preparation, chemical synthesis process higher cost, mainly use microorganism conversion.Microorganism conversion has the advantage that reaction item
Part is mild, and product is single, and chemo-selective, regioselectivity and the enantio-selectivity of height are easy to purify, environmental pollution
It is small, and some chemical syntheses can be completed and be difficult to the reaction carried out.
Echinocandin B parent nucleus (ECBN) has the following structure:
The technique that US7785826B2 describes ECB Transformed E CBN in detail.This technique main flow includes:
After ECB fermentation is completed, centrifugation obtains mycelium, mycelium is resuspended in water, deacylase is then added
It is converted.But the molar yield of the method is lower, and transformation time is long, and conversion needs 20 ~ 30h of time-consuming, and production cost is higher.
CN103374593A discloses one kind, and addition echinocandin B (ECB) substrate is converted into the white bacterium of spine during the fermentation
The method of plain B parent nucleus；CN102618606B discloses a kind of bioconversion method of Echinocandin compound, including, by spine
After white bacteriums compound, buffer solution and cosolvent mixing, adds full cell deacylase and converted；Contemporary literature
These method for transformation of report, mainly actinoplanes utahensis (Actinoplanesutahensis) or other genetic engineerings
Bacterium fermentation generates deacylase, and then substrate is added in fermentation liquid and is converted.The shortcomings that this transformation system, is exist
A large amount of microorganisms, stability is poor, and being limited by fermentation liquid enzyme activity causes the ECB concentration of substrate of investment relatively low, the ECB parent nucleus of generation
Concentration is relatively low, and whole system volume is larger, is not easy to isolate and purify, and in system a large amount of insoluble matters such as thallus and ECB presence
Unconverted ECB is caused to be difficult to recycle, production cost is higher.
CN103074403B discloses a kind of method for converting ECBN for ECB, is related to after fermentation by adding phosphoric acid
Salt, ultrasound, centrifugation, concentrated by rotary evaporation obtain echinocandin B deacylase enzyme solution, and ECB substrate is then added and is 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.
Therefore need to develop that a kind of thick enzyme extracting mode is simple, mild condition, cost is relatively low, the negative effect that enzyme activity is subject to
It is smaller, while high conversion efficiency, it is easily isolated purifying, the technique that ECB is easily recycled reuse.
Summary of the invention
It is an object of the present invention to provide one kind efficiently by echinocandin B for deficiency existing for existing conversion process
The method for being converted into echinocandin B parent nucleus.
In order to achieve the above objectives, the present invention the following technical schemes are provided:
The method that echinocandin B is converted to echinocandin B parent nucleus, comprising the following steps:
1), fermentation energy obtains echinocandin B deacylase by echinocandin B deacylation expression of enzymes in extracellular genetic engineering bacterium；
2), after fermentation, by filtering fermentation liquor or centrifugation, supernatant is obtained, supernatant volume is added into supernatant
40% ~ 80% (w/v) weight non-heavy metal salt, stir 2 ~ 8 hours, filtering or centrifugation, collect precipitating, obtain thick enzyme；
3), echinocandin B is dissolved in methanol, ethyl alcohol or dimethyl sulfoxide, it is white to obtain the spine that mass concentration is 5% ~ 15%
Rhzomorph B solution；
4) phosphate, is added portionwise in the thick enzyme of the echinocandin B solution of step 3) preparation and step 2 preparation respectively
In buffer, stirring makes echinocandin B be converted into echinocandin B parent nucleus, monitors echinocandin B and the white bacterium of spine in transformation system
Plain B parent nucleus concentration terminates conversion when echinocandin B concentration is no longer reduced, and echinocandin B parent nucleus concentration is not further added by.
Wherein, it is preferable that non-heavy metal salt described in step 2 is ammonium sulfate；
Phosphate buffering liquid concentration described in step 4) is 0.05 ~ 0.15M；Further, phosphate-buffered described in step 4)
Solution salt acid for adjusting pH to 5 ~ 7, most preferably the salt acid for adjusting pH to 5.5 ~ 6.5 of phosphate buffer solution described in step 4).
Further, it is 3 ~ 6 times that thick enzyme number, which is added batch-wise, in step 4), and every minor tick 2 ~ 8 hours, each additive amount is to turn
Change 1% ~ 4% (w/v) of reaction solution volume.
Further, it is 2 ~ 5 times that echinocandin B substrate number, which is added batch-wise, in step 4), every minor tick 2 ~ 8 hours, every time
Additive amount calculates 0.1% ~ 0.4% (w/v) for being equivalent to conversion reaction liquor capacity with echinocandin B.
Further, it is reaction solution body that step 4), which is added to the primary quantity of the echinocandin B in phosphate buffer solution,
0.1% ~ 0.4% long-pending (w/v)；Be added to thick enzyme in phosphate buffer solution primary quantity be conversion reaction liquor capacity 1% ~
4%(w/v)。
Further, female using echinocandin B in HPLC monitoring transformation system and echinocandin B in step 4) conversion process
Nuclear concentration, wherein the condition of the HPLC detection of echinocandin B is as follows:
Chromatographic column specification: 2.1mm*100mm*1.8 μm of C18 column, mobile phase: methanol: acetonitrile: water=70:10:20 (V/V),
Column temperature: 40 DEG C, flow velocity: 0.3ml/min, wavelength: 210nm, retention time: about 5.5min；
The condition of the HPLC detection of echinocandin B parent nucleus is as follows:
4.6mm*250mm*5 μm of column of chromatographic column specification C18, wavelength: 210nm, column temperature: 40 DEG C, flow velocity: 1ml/min is adopted
With gradient elution, mobile phase A phase: 0.5% ammonium dihydrogen phosphate aqueous solution (W/V): methanol=95:5 (V/V), Mobile phase B phase:
0.5% ammonium dihydrogen phosphate aqueous solution (W/V): methanol=70:30 (V/V),
Retention time about 10.0min.
Wherein, in the above method, step 1), fermentation bacterial strain uses therefor is can be by echinocandin B deacylation expression of enzymes extracellular
Genetic engineering bacterium, 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, 2013 Feb; 79(4):
1126-33), the fermentation that condition well known to those skilled in the art carries out echinocandin B deacylase can be used, for example including but not
It is limited to fermentation process disclosed in CN102618606B.
Step 2 needs simply slightly to propose echinocandin B deacylase progress in fermentation liquid after fermentation.Because used in
Fermentation liquid is separated by solid-liquid separation first extracellular by the echinocandin B deacylase that bacterial strain generates, and obtains deacylase containing echinocandin B
Supernatant.Using the principle saltoutd, precipitates, separates thick enzyme；The principle of saltouing refers to the salt ion of high concentration in protein
Hydrone, which can be competed, with protein in solution reduces its solubility to destroy the hydration shell of protein surface, is allowed to from solution
In be precipitated out, while can will salt out come protein be again dissolved in water without influence crude protein property.It saltouts general
The non-heavy metal salt such as sodium chloride, ammonium chloride, sodium sulphate, ammonium sulfate etc. for selecting solubility high.Ammonium sulfate is because its solubility is big, temperature
Degree coefficient is small and is not easy to make protein denaturation and most widely used.Therefore add according to 40 ~ 80% (mass volume ratios) of supernatant volume
Enter ammonium sulfate, stir 2 ~ 8 hours, solution filtering or centrifugation collect precipitating, obtain thick enzyme.
Due to water-soluble very poor, the step 3) of echinocandin B, using methanol, ethyl alcohol or dimethyl sulfoxide as cosolvent, system
The echinocandin B solution that standby mass concentration is 5% ~ 15%, mixes well when helping for echinocandin B to be added in reaction system
And conversion.
Use concentration for 0.05 ~ 0.15M in transformation system, the phosphate buffer of pH about 5.5 ~ 6.5, provide centainly from
Sub- intensity and stable pH environment, are conducive to the stabilization of the dissolution of echinocandin B deacylase and deacylase in conversion process.
Thick enzyme and echinocandin B is added batch-wise to phosphate buffer in step 4), and 28 ~ 32 DEG C are stirred and converted.It is existing
Have in technology, in the conversion process due to echinocandin B, deacylase can be inactivated gradually, transformation efficiency decline, and the present invention, which uses, to be divided
The form criticized adds thick enzyme and is conducive to meet and maintain enzyme activity needed for substrate conversion in reaction system, improves transformation efficiency, excellent
Selection of land, adding thick enzyme number is 3 ~ 6 times, every minor tick 2 ~ 8 hours, and each additive amount is the 1% ~ 4% of conversion reaction liquor capacity
(mass volume ratio).
Since the water solubility of echinocandin B is very poor, even if being added to by cosolvent such as methanol, ethyl alcohol or dimethyl sulfoxide
Also be easy gradually to be precipitated in aqueous solution in reaction system, need to be added more cosolvent can be only achieved it is preferable as a result, and
Organic solvent excessively will cause protein denaturation, and deacylase is caused to inactivate, conversion capability decline；To solve the above problems, this hair
The bright substrate echinocandin B solution that adds in the form of in batches is conducive to keep enzyme activity, disperses echinocandin B in reaction system
It is more abundant to mix conversion, reaction system is enabled to convert more echinocandin B, the echinocandin B for obtaining higher concentration is female
Core, conducive to isolating and purifying.Preferably, addition echinocandin B substrate number is 2 ~ 5 times, and every minor tick 2 ~ 8 hours adds every time
Amount calculates 0.1% ~ 0.4% (w/v) for being equivalent to conversion reaction liquor capacity with echinocandin B.
Thick enzyme extracting mode of the invention is simple, mild condition, and cost is relatively low, and the negative effect that enzyme activity is subject to is smaller.Together
When the present invention by optimizing, the feeding mode of thick enzyme and echinocandin B improves the concentration of substrate of reaction system and conversion obtains
Parent nucleus concentration, improve transformation efficiency, be more conducive to isolating and purifying.And since the composition of entire reaction system is more simple
It is single, only include water-soluble phosphate-buffered salt, thick enzyme, echinocandin B parent nucleus and echinocandin B substrate not soluble in water, turns
Echinocandin B parent nucleus is present in aqueous solution after change terminates, and remaining a small amount of solid content is mainly echinocandin B substrate, benefit
In filtering.The extraction that echinocandin B parent nucleus solution carries out next step to echinocandin B parent nucleus can be obtained by way of filtering,
Simultaneously precipitating echinocandin B substrate can simple recycling and reusing, reduce production cost, be suitable for large-scale industrial production,
With commercial value well.In addition phosphate buffer in transformation system provides certain ionic strength and stable pH ring
Border is conducive to the stabilization of the dissolution of echinocandin B deacylase and deacylase in conversion process.
Specific embodiment
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
And not meaning that has any restrictions to the present invention.
Embodiment 1 produces the fermentation and the preparation of thick enzyme of echinocandin B deacylase bacterial strain
Strain: streptomyces albus (Streptomyces albus), deposit number: ATCC21725；- 80 DEG C of cryovials save；
Seed culture medium: the fried soybean cake powder 0.5% 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%, seven water
Magnesium sulfate 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 body
Long-pending 5% 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 50% (w/v) of supernatant volume,
Stirring 6 hours, precipitating is collected by filtration in solution, obtains thick enzyme.
The conversion of 2 echinocandin B substrate of embodiment
Phosphate buffer 1 000L is prepared in reactor tank, contains KH2PO42.24g/L and Na2HPO4·12H2O 1.24g/
L, adjusting pH with HCl is about 6.0.
Echinocandin B substrate is dissolved in methanol, concentration 10%.
200rpm is stirred in 28 ~ 32 DEG C of reactor tank temperature of control, adds thick enzyme 30kg into reactor tank for the first time, and slowly
The echinocandin B methanol solution that 30L 10% is added makes echinocandin B content 3kg in conversion reaction solution, (that is, echinocandin B
Content (kg) is equivalent to the 0.3% of conversion reaction solution volume (L)), start to convert.
After converting 4h, thick enzyme 10kg is added into reactor tank, and the echinocandin B methanol for being slowly added to 30L 10% is molten
Liquid；
After converting 8h, thick enzyme 10kg is added into reactor tank, and the echinocandin B methanol for being slowly added to 20L 10% is molten
Liquid；
After converting 12h, thick enzyme 20kg is added into reactor tank, and the echinocandin B methanol for being slowly added to 20L 10% is molten
Liquid；
Echinocandin B substrate and echinocandin B parent nucleus concentration are monitored with HPLC, when echinocandin B concentration of substrate no longer subtracts
Few, echinocandin B parent nucleus concentration is not further added by, and terminates conversion, and filtering fermentation liquor takes filtrate HPLC quantitative detection echinocandin B
Parent nucleus content, and the molar yield of echinocandin B is calculated, it is 85% (molar yield %=generation ECBN molal quantity/substrate ECB
Molal quantity * 100%).The precipitating of filtering is mainly unconverted echinocandin B substrate, can be recycled and uses in conversion next time.
The conversion of 3 echinocandin B substrate of embodiment
Phosphate buffer 3000L is prepared in reactor tank, contains KH2PO42.24g/L and Na2HPO4·12H2O 1.24g/
L, adjusting pH with HCl is about 6.0；
Echinocandin B substrate is dissolved in ethyl alcohol, concentration 10%；
200rpm is stirred in 28 ~ 32 DEG C of reactor tank temperature of control, adds thick enzyme 120kg into reactor tank for the first time, and slowly
The echinocandin B ethanol solution that 120L10% is added makes echinocandin B content 12kg in conversion reaction solution, starts to convert；
After converting 6h, thick enzyme 30kg is added into reactor tank, and the echinocandin B ethyl alcohol for being slowly added to 90L 10% is molten
Liquid；
After converting 12h, thick enzyme 30kg is added into reactor tank, and the echinocandin B ethyl alcohol for being slowly added to 90L 10% is molten
Liquid；
After converting 18h, thick enzyme 60kg is added into reactor tank, and the echinocandin B ethyl alcohol for being slowly added to 60L 10% is molten
Liquid；
After conversion for 24 hours, thick enzyme 30kg is added into reactor tank；
Echinocandin B substrate and echinocandin B parent nucleus concentration are monitored with HPLC, when echinocandin B concentration of substrate no longer subtracts
Few, echinocandin B parent nucleus concentration is not further added by, and terminates conversion, and filtering fermentation liquor takes filtrate HPLC quantitative detection echinocandin B
Parent nucleus content, and the molar yield for calculating echinocandin B is 80% (molar yield %=generation ECBN molal quantity/substrate ECB
Molal quantity * 100%).The precipitating of filtering is mainly unconverted echinocandin B substrate, can be recycled and uses in conversion next time.
Embodiment 4 produces the fermentation and the preparation of thick enzyme of echinocandin B deacylase bacterial strain
Strain: streptomyces albus (Streptomyces albus), deposit number: ATCC21725；- 80 DEG C of cryovials save；
Seed culture medium: the fried soybean cake powder 0.5% 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%, seven water
Magnesium sulfate 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 body
Long-pending 5% is inoculated in fermentation medium, and 30 DEG C are cultivated 2 ~ 3 days；
After fermentation, filtering fermentation liquor is obtained into supernatant, sodium chloride is added according to 40% (w/v) of supernatant volume,
Stirring 6 hours, precipitating is collected by filtration in solution, obtains thick enzyme.
The conversion of 5 echinocandin B substrate of embodiment
Phosphate buffer 1 000L is prepared in reactor tank, contains KH2PO42.24g/L and Na2HPO4·12H2O 1.24g/
L, adjusting pH with HCl is about 5.5；
Echinocandin B substrate is dissolved in dimethyl sulfoxide, concentration 5%；
28 ~ 32 DEG C of reactor tank temperature of control stirs 200rpm, adds into reactor tank obtain according to 4 method of embodiment for the first time
The thick enzyme 10kg obtained, and the dimethyl sulphoxide solution for being slowly added to the echinocandin B of 40L 5% makes echinocandin B content
2kg starts to convert；
After converting 2h, thick enzyme 40kg is added into reactor tank, and the echinocandin B dimethyl for being slowly added to 80L 5% is sub-
Sulfolane solution；
After converting 8h, thick enzyme 30kg is added into reactor tank, and the echinocandin B dimethyl for being slowly added to 60L 5% is sub-
Sulfolane solution；
After converting 10h, thick enzyme 20kg is added into reactor tank, and be slowly added to the echinocandin B dimethyl of 40L 5%
Sulfoxide solution；
After converting 12h, thick enzyme 10kg is added into reactor tank, and be slowly added to the echinocandin B dimethyl of 20L 5%
Sulfoxide solution；
After converting 16h, thick enzyme 10kg is added into reactor tank；
Echinocandin B substrate and echinocandin B parent nucleus concentration are monitored with HPLC, when echinocandin B concentration of substrate no longer subtracts
Few, echinocandin B parent nucleus concentration is not further added by, and terminates conversion, and filtering fermentation liquor takes filtrate HPLC quantitative detection echinocandin B
Parent nucleus content, the molar yield for calculating echinocandin B is 87%.The precipitating of filtering is mainly unconverted echinocandin B bottom
Object can be recycled and use in conversion next time.
Embodiment 6 produces the fermentation and the preparation of thick enzyme of echinocandin B deacylase bacterial strain
Strain: streptomyces albus (Streptomyces albus), deposit number: ATCC21725；- 80 DEG C of cryovials save；
Seed culture medium: the fried soybean cake powder 0.5% 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%, seven water
Magnesium sulfate 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 body
Long-pending 5% is inoculated in fermentation medium, and 30 DEG C are cultivated 2 ~ 3 days；
After fermentation, filtering fermentation liquor is obtained into supernatant, sodium sulphate is added according to 80% (w/v) of supernatant volume,
Stirring 6 hours, precipitating is collected by filtration in solution, obtains thick enzyme.
The conversion of 7 echinocandin B substrate of embodiment
Phosphate buffer 3000L is prepared in reactor tank, and KH is added2PO42.24g/L and Na2HPO4·12H2O
1.24g/L, adjusting pH with HCl is about 6.2；
Echinocandin B substrate is dissolved in ethyl alcohol, concentration 15%；
200rpm is stirred in 28 ~ 32 DEG C of reactor tank temperature of control, adds the preparation of 6 method of embodiment into reactor tank for the first time
Thick enzyme 120kg, and the echinocandin B ethanol solution for being slowly added to 80L 15% makes echinocandin B content in conversion reaction solution
0.4%, start to convert；
After converting 8h, thick enzyme 120kg is added into reactor tank, and the echinocandin B ethyl alcohol for being slowly added to 20L 15% is molten
Liquid；
After converting 16h, thick enzyme 90kg is added into reactor tank, and the echinocandin B ethyl alcohol for being slowly added to 20L 15% is molten
Liquid；
After conversion for 24 hours, thick enzyme 60kg is added into reactor tank；
Echinocandin B substrate and echinocandin B parent nucleus concentration are monitored with HPLC, when echinocandin B concentration of substrate no longer subtracts
Few, echinocandin B parent nucleus concentration is not further added by, and terminates conversion, and filtering fermentation liquor takes filtrate HPLC quantitative detection echinocandin B
Parent nucleus content, and the molar yield for calculating echinocandin B is 83%.The precipitating of filtering is mainly unconverted echinocandin B bottom
Object can be recycled and use in conversion next time.
Claims (7)
1. the method that echinocandin B is converted to echinocandin B parent nucleus, comprising the following steps:
1), fermentation energy obtains echinocandin B deacylase by echinocandin B deacylation expression of enzymes in extracellular genetic engineering bacterium；
2), after fermentation, by filtering fermentation liquor or centrifugation, supernatant is obtained, supernatant volume is added into supernatant
The non-heavy metal salt of 40%~80% (w/v) weight stirs 2~8 hours, filters or be collected by centrifugation precipitating, obtain thick enzyme；
3), echinocandin B is dissolved in methanol, ethyl alcohol or dimethyl sulfoxide, obtains the echinocandin B that mass concentration is 5~15%
Solution；
4) phosphate-buffered, is added portionwise in the thick enzyme of the echinocandin B solution of step 3) preparation and step 2) preparation respectively
In liquid, stirring makes echinocandin B be converted into echinocandin B parent nucleus, and it is female to monitor echinocandin B and echinocandin B in transformation system
Nuclear concentration terminates conversion when echinocandin B concentration is no longer reduced, and echinocandin B parent nucleus concentration is not further added by,
Wherein, it is the 0.1% of reaction solution volume that step 4), which is added to the primary quantity of the echinocandin B in phosphate buffer solution,
~0.4% (w/v)；The primary quantity for being added to thick enzyme in phosphate buffer solution is the 1%~4% of conversion reaction liquor capacity
(w/v)。
2. method as described in claim 1, which is characterized in that non-heavy metal salt described in step 2) is ammonium sulfate.
3. method as described in claim 1, which is characterized in that the step 4) phosphate buffering liquid concentration is 0.05~0.15M.
4. method as claimed in claim 3, which is characterized in that the step 4) phosphate buffer solution with salt acid for adjusting pH extremely
5.5~6.5.
5. method as described in claim 1, which is characterized in that it is 3~6 times that thick enzyme number, which is added batch-wise, in step 4), every minor tick 2
~8 hours, each additive amount was 1%~4% (w/v) of conversion reaction liquor capacity.
6. method as described in claim 1, which is characterized in that it is 2~5 times that echinocandin B solution number, which is added batch-wise, in step 4),
Every minor tick 2~8 hours, each additive amount with echinocandin B calculate be equivalent to conversion reaction liquor capacity 0.1%~
0.4% (w/v).
7. method as described in claim 1, which is characterized in that using spine in HPLC monitoring transformation system in step 4) conversion process
White rhzomorph B and echinocandin B parent nucleus concentration.
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| CN113174398B (en) * | 2021-04-22 | 2022-04-29 | 浙江工业大学 | Expression cassette for recombinant expression of echinocandin B deacylase and application |
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