CN105154424A - Preparation method of immobilized cyclic lipopeptide deacylase and application thereof - Google Patents
Preparation method of immobilized cyclic lipopeptide deacylase and application thereof Download PDFInfo
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- CN105154424A CN105154424A CN201510626188.5A CN201510626188A CN105154424A CN 105154424 A CN105154424 A CN 105154424A CN 201510626188 A CN201510626188 A CN 201510626188A CN 105154424 A CN105154424 A CN 105154424A
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- cyclic lipopeptide
- deacylase
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Abstract
The invention discloses a preparation method of immobilized cyclic lipopeptide deacylase. A cyclic lipopeptide deacylase solution is directly mixed with a porous hydrophily enzyme carrier without purification, one or more kinds of organic solutions and inorganic salt are added, and then the immobilized cyclic lipopeptide deacylase is obtained. The invention further discloses application of the method in industrial production of micafungin parent nucleuses and anidulafungin parent nucleuses. The preparation method is simple in technology, enzyme purification equipment is omitted, the recovery rate of enzyme activities is obviously increased in the immobilization process, and immobilized enzymes can be repeatedly used. The enzyme method for converting micafungin and anidulafungin intermediates has higher concentration of reaction substrates when compared with a fermentation conversion method, a few product impurities are generated, and the conversion efficiency is high.
Description
Technical field
The invention belongs to the technical field of enzyme immobilization in biotechnology; Particularly, the present invention relates to a kind of preparation method and application thereof of immobilization cyclic lipopeptide deacylase.
Technical background
Echinocandin class microbiotic is that the class that 20 century 70s find has the natural product of suppression β-1,3-glucan synthase activity, and have good anti-mycotic activity, its basic structure as shown in Equation 1.Due to the R that natural Echinocandin compound has
2fatty acid side chain, has certain hemolytic toxicity at human body, and the fragrant clean class antifungal drug such as the MFG therefore gone on the market and anidulafungin is all sloughed by original fatty acid side chain of natural compounds, changes different fatty acid side chains.The process difficult chemical method sloughed due to side chain completes, and general cyclic lipopeptide deacylase (Deacylase) that adopts carries out bio-transformation.
Formula 1: Echinocandin compound basic structure formula (R
1:-CH
3or-H; R
2: C
15-C
17chain; R
3:-CH
3or-H; R
4:-OH or-OSO
3h; R
5:-OSO
3h or-H; R
6:-OH or-H; R
7: CH
3or CH
2cONH
2)
The cyclic lipopeptide deacylase (Deacylase) reported all derives from actinoplanes utahensis (Actinoplanesutahensis) or ring streptomycete (Streptomycesanulatus).In the bioconversion method of bibliographical information, the general Precurosor fermentation method that adopts transforms, and namely first microorganism is carried out product enzyme and cultivate, then direct being added in fermentation system by substrate transforms.The method has a lot of shortcoming, as substrate in transformation system and production concentration is low, long reaction time, liquid treatment volume are large, impurity many (Media Components and organic solvent) in system, product postprocessing difficulty, purifying difficulty are large.The not reproducible use of this fermentation system in addition, cost is high.
Enzyme immobilization can solve the problem preferably.It is generally acknowledged; such as patent application " a kind of immobilization cyclic lipopeptide acyltransferase and its production and use; 201210148392.7 "; preferably first enzyme is carried out purifying (as ion-exchange, molecular sieve, hydrophobic chromatography and ultrafiltration dialysis etc. during enzyme immobilization; carry out alone or in combination as required), in enzyme liquid, foreign protein ratio is more little is more conducive to immobilization.This is because, there is the various impurity such as a lot of pigment, amino acid, cell debris, secondary metabolite in fermented liquid; Enzyme is on the low side relative to the concentration of various impurity, and impurity can suppress the vigor of fixation support; The active group simultaneously contained in impurity can combine the being at war with property of binding site of hydrophilic carrier, causes the immobilized enzyme rate of recovery alive low.
But enzyme purification process can cause the loss of enzyme, and need extra equipment, add cost.
In enzyme immobilization process, the atopic of carrier and gal4 amino acid residue is determined by chemical property and microenvironment (comprise medium forms, ionic strength, pH etc.).Follow this thinking, the object of the invention is to find suitable condition, fermented liquid is without the need to purifying, and the cyclic lipopeptide deacylase of fixation support just optionally in fermented liquid is combined, reduce the combination with impurity, obtain the immobilization cyclic lipopeptide deacylase having industrial value.
Summary of the invention
The invention discloses a kind of method of specific set collar lipopeptid deacylase from fermented liquid.
Method disclosed by the invention, comprises the following steps:
The cyclic lipopeptide deacylase solution dissociated is obtained after fermented liquid containing cyclic lipopeptide deacylase and mycelium being separated by filtration;
By not purified for free cyclic lipopeptide deacylase solution, directly mix with porous, hydrophilic enzyme carrier, and add one or more organic solutions and inorganic salt, being fixed cyclic lipopeptide deacylase.
Wherein said
Free cyclic lipopeptide deacylase solution and the blending ratio of carrier are that every gram of carrier needs enzyme to live as 9-60000U, and being preferably every gram of carrier needs enzyme to live as 45-360U.
When mixing containing free cyclic lipopeptide deacylase solution and carrier, system pH is 5-11.
It is the enzyme carrier of matrix bonding epoxide that porous, hydrophilic enzyme carrier is selected from polymethacrylate.Be particularly preferably any one in EupergitCM or LX1000-EP.
Organic solution is any one in ethanol, Virahol, normal hexane, DMSO, acetone, DMF, tetrahydrofuran (THF), pyridine, acetonitrile, is preferably any one in ethanol, Virahol, acetone.
Inorganic salt are any one in sodium-chlor, Repone K, magnesium chloride, zinc chloride, are preferably any one in Repone K, magnesium chloride.
Temperature containing free cyclic lipopeptide deacylase solution and carrier mixing is 5-80 DEG C, is preferably 10-40 DEG C, most preferably is 20-30 DEG C.
Fermented liquid containing cyclic lipopeptide deacylase refers to and derives from the microorganism strains that natural energy produces cyclic lipopeptide deacylase, or the artificial mutant of these microorganism strains or mutation, or the engineering strain containing cyclic lipopeptide deacylase encoding gene.
The invention also discloses and the preparation method of this immobilization cyclic lipopeptide deacylase is being prepared the application in echinocandin class medicine parent nucleus.
Wherein said echinocandin class medicine parent nucleus is MFG parent nucleus or anidulafungin parent nucleus.
In the present invention, above-mentioned enzyme unit definition alive is: when 40 DEG C, generate the enzyme amount needed for 1 micromole's product, be namely defined as 1U in 1 hour.Product determination step is: measure 17.5mL crude enzyme liquid respectively, phosphoric acid (0.2mol/L, pH5.5) the damping fluid 5mL containing MFG precursor or anidulafungin precursor, methyl alcohol 2.5mL.Under 40 DEG C of water bath condition, react 1 hour, deionized water suitably dilutes, and 0.22 μm of nylon membrane filters, and HPLC measures product MFG parent nucleus or anidulafungin parent nucleus concentration.
The present invention is by after the fermented liquid containing cyclic lipopeptide deacylase and mycelium simple separation, without the need to other enzyme purification steps, directly mix with fixation support, realize the efficient combination of cyclic lipopeptide deacylase and fixation support, obtain the immobilization cyclic lipopeptide deacylase having industrial value.Fixing condition provided by the present invention enhances fixation support epoxy group group and the selective binding of target enzyme, reduces the combination with impurity, overcomes crude enzyme liquid and be unfavorable for immobilized difficult point.Compared with previous methods, this preparation method's technique is simple, not only saves enzyme purification equipment, and in immobilization process, the enzyme rate of recovery alive significantly improves, and immobilized enzyme can repeatedly use.Enzymatic conversion method MFG and anidulafungin intermediate, compare microbe conversion method reaction substrate concentration high, impurity in products is few, and transformation efficiency is high.
Embodiment
Below in conjunction with specific embodiment, the present invention is further explained.
The acquisition of embodiment 1 cyclic lipopeptide deacylase solution
The cyclic lipopeptide deacylase that 1.1 actinoplanes utahensises (Actinoplanesutahensis) produce
Adopt actinoplanes utahensis IFO-13244 bacterial strain, according to the fermentation process described in embodiment in patent US5376634 1, carry out fermentation culture, obtain Mycelium culture liquid 150L.Then in the filtered on buchner funnel being covered with filter paper, collect the filtrate 120L containing cyclic lipopeptide deacylase, namely free cyclic lipopeptide deacylase solution, collect filtrate, detect through HPLC, enzyme work is 0.864 × 10
5u.
The cyclic lipopeptide deacylase that 1.2 streptomycetes (Streptomycessp.) produce
Adopt streptomycete No. 6907 bacterial strains, according to the fermentation process described in embodiment in patent WO97/32975 1, carry out fermentation culture, obtain Mycelium culture liquid 150L.Then in the filtered on buchner funnel being covered with filter paper, collect the filtrate 130L containing cyclic lipopeptide deacylase, namely free cyclic lipopeptide deacylase solution, collect filtrate, detect through HPLC, enzyme work is 0.768 × 10
5u.
The 1.3 cyclic lipopeptide deacylases produced containing the restructuring streptomycete of external source cyclic lipopeptide deacylase gene
From actinoplanes utahensis, clone cyclic lipopeptide deacylase gene by literature method (AppliedandEnvironmentalMicrobiology, 2013, V79.4,1126-1133), and restructuring is in the genome of streptomycete.The activation of picking recombinant conversion, and enlarged culturing is expressed, and obtains Mycelium culture liquid 150L.Then in the filtered on buchner funnel being covered with filter paper, collect the filtrate 120L containing cyclic lipopeptide deacylase, namely free cyclic lipopeptide deacylase solution, collect filtrate, detect through HPLC, enzyme work is 0.606 × 10
5u.
The selection of the dissimilar epoxide carrier of embodiment 2
Get in above-described embodiment 1 the cyclic lipopeptide deacylase solution obtaining prepared by 1.1 dissociating, add 1gEupergitCM or LX1000-EP respectively, 25 DEG C are stirred 24 hours, collecting by filtration immobilization cyclic lipopeptide deacylase, pure water three times, drying at room temperature 3 hours, finally carries out enzyme activity determination.
Table 1 dissimilar epoxide carrier is to the fixing situation of original resolvase liquid
As can be seen from Table 1, under same enzyme consumption condition the rate of recovery of carrier LX1000-EP all higher than carrier EupergitCM.
Table 2 epoxide carrier LX1000-EP is to the fixing situation of original resolvase liquid
As can be seen from Table 2, be within the scope of 9-60000U/g, all can realize free enzyme immobilizatio in the blending ratio of free cyclic lipopeptide deacylase solution and carrier, in the group of the enzyme rate of recovery alive more than 5%, every gram of carrier needs enzyme to live as 45-360U.
Consolidated statement 1 and table 2 can be found out, enzyme liquid is not purified directly adds vehicle treated, and in immobilization process, the enzyme rate of recovery alive is lower, can not meet the needs of practical application.
The optimization of aqueous solution polarity in embodiment 3 immobilization process
Obtain the cyclic lipopeptide deacylase solution 1L dissociated in Example 1 prepared by 1.1, add dissimilar organic solvent (comprising ethanol, Virahol, normal hexane, DMSO, acetone, DMF, tetrahydrofuran (THF), pyridine, acetonitrile) each 50mL, 100mL, 200mL or 300mL respectively.Continue to add 5gLX1000-EP carrier respectively, 25 DEG C are stirred 24 hours, and collecting by filtration immobilization cyclic lipopeptide deacylase, pure water three times, drying at room temperature 3 hours, finally carries out enzyme activity determination.
The cyclic lipopeptide deacylase immobilization of table 3 different organic solvents
As can be seen from Table 3, in free cyclic lipopeptide deacylase solution, add dissimilar organic solvent, comprise ethanol, Virahol, normal hexane, DMSO, acetone, DMF, tetrahydrofuran (THF), pyridine, acetonitrile, its immobilized enzyme rate of recovery alive all has a certain upgrade.Wherein the effect of ethanol, Virahol and acetone is obvious especially, and its suitableeest interpolation concentration is 10%.
The optimization of embodiment 4 immobilization process ionic strength and pH value
The cyclic lipopeptide deacylase solution 1L dissociated is obtained prepared by 1.1 in Example 1, add Virahol 100mL, add dissimilar inorganic salt (comprising sodium-chlor, calcium chloride, Repone K, magnesium chloride, ammonium chloride, zinc chloride) each 1mol simultaneously respectively, and be adjusted to different pH value.Continue to add 5gLX1000-EP carrier respectively, 25 DEG C are stirred 24 hours, and collecting by filtration immobilization cyclic lipopeptide deacylase, pure water three times, drying at room temperature 3 hours, finally carries out enzyme activity determination.
As can be seen from Table 4, free cyclic lipopeptide deacylase solution, when not adding inorganic salt, being within the scope of 5-11, all can realizing free enzyme immobilizatio in pH value, preferable ph is 8-10.After adding the inorganic salt such as sodium-chlor, Repone K, magnesium chloride, zinc chloride, the immobilization particle enzyme of acquisition is lived and is all had a certain upgrade.Wherein the effect of Repone K and magnesium chloride especially obviously, adds 1mol/L Repone K immobilization effect best when pH value is 9, and immobilization particle enzyme is lived as 100.8U/g, and the immobilized enzyme rate of recovery alive is 70.0%.
The optimization of temperature and enzyme liquid measure and carrier amount blending ratio in embodiment 5 immobilization process
Obtain the cyclic lipopeptide deacylase solution 1L dissociated in Example 1 prepared by 1.1, add Virahol 100mL and Repone K 1mol, and adjust ph is 9.Continue to add LX1000-EP carrier 4g, 5g, 6g or 7g respectively, at different temperatures, stir 24 hours, collecting by filtration immobilization cyclic lipopeptide deacylase, pure water three times, drying at room temperature 3 hours, finally carries out enzyme activity determination.
The cyclic lipopeptide deacylase immobilization of table 5 differing temps and carrier amount
As can be seen from Table 5, be within the scope of 5-80 DEG C at the mixing temperature of free cyclic lipopeptide deacylase solution and carrier, all can realize free enzyme immobilizatio, wherein the better effects if of 10-40 DEG C, the best results of 20-30 DEG C.Free cyclic lipopeptide deacylase solution and the blending ratio of carrier most preferably are in 1L enzyme liquid and add 6g carrier.
The purifying of embodiment 6 cyclic lipopeptide deacylase and immobilization
Reference literature method (JournalofIndustrialMicrobiology & Biotechnology, 2000,24,173-180) obtain the cyclic lipopeptide deacylase solution 120L that dissociates in purifying embodiment 1 prepared by 1.1, namely enzyme work is 0.864 × 10
5u, obtains the cyclic lipopeptide deacylase solution 3L of purifying, and detect through HPLC, enzyme work is 0.173 × 10
5u, purifying enzyme yield alive is 20.0%.
Get above-mentioned purified cyclic lipopeptide deacylase solution 1L, adjust ph is 7, adds 30gEupergitCM carrier or 30gLX1000-EP carrier respectively, 25 DEG C are stirred 24 hours, collecting by filtration immobilization cyclic lipopeptide deacylase, pure water three times, drying at room temperature 3 hours, finally carries out enzyme activity determination.
Table 6 epoxide carrier is to the fixing situation of purifying resolvase liquid
As can be seen from Table 6, the cyclic lipopeptide deacylase solution 120L dissociated is obtained prepared by 1.1 in embodiment 1, cyclic lipopeptide deacylase solution 3L is obtained after purifying, purifying enzyme yield alive is 20.0%, although immobilized enzyme is lived, the rate of recovery is very high, can 90.0% be reached, but due to purifying enzyme live yield low, enzyme live total yield only have 18.0%.
The large system immobilization cyclic lipopeptide deacylase of embodiment 7
Obtain the cyclic lipopeptide deacylase solution 100L dissociated in Example 1 prepared by 1.1, namely enzyme work is 0.720 × 10
5u, adds Virahol 10L and Repone K 100mol, and adjust ph is 9.Continue to add 600gLX1000-EP carrier, 25 DEG C are stirred 24 hours, collecting by filtration immobilization cyclic lipopeptide deacylase, pure water three times, drying at room temperature 3 hours.After measured, the immobilization cyclic lipopeptide deacylase granzyme of collection is lived as 98.6U/g, and the immobilized enzyme rate of recovery alive is 82.2%.
Embodiment 8 immobilization cyclic lipopeptide deacylase catalytic production MFG parent nucleus
2g MFG precursor and 10mL methyl alcohol are added phosphoric acid (0.1mol/L, pH5.5) damping fluid and be settled to 100mL, then adds the immobilization cyclic lipopeptide deacylase particle 10g prepared by embodiment 7.40 DEG C, 240rpm shaking table reacts.React after 8 hours, HPLC detects, and the molar yield of MFG parent nucleus is 98%.
Embodiment 9 immobilization cyclic lipopeptide deacylase catalytic production anidulafungin parent nucleus
2g anidulafungin precursor and 10mL methyl alcohol are added phosphoric acid (0.1mol/L, pH5.5) damping fluid and be settled to 100mL, then adds the immobilization cyclic lipopeptide deacylase particle 10g prepared by embodiment 7.40 DEG C, 240rpm shaking table reacts.React after 15 hours, HPLC detects, and the molar yield of anidulafungin parent nucleus is 95%.
Embodiment 10 immobilization cyclic lipopeptide deacylase batch catalytic production MFG parent nucleus
Immobilization cyclic lipopeptide deacylase catalytic production MFG parent nucleus is used by the method for embodiment 8.After terminating reaction, collecting by filtration immobilization cyclic lipopeptide deacylase, and with pure water three times, the immobilization cyclic lipopeptide deacylase of collection, continues the method catalytic production MFG parent nucleus pressing embodiment 8.Repeat 20 times, all record the output of MFG parent nucleus at every turn, as shown in table 7.
Table 7 immobilization cyclic lipopeptide deacylase batch catalytic production MFG parent nucleus
As can be seen from Table 7, immobilization cyclic lipopeptide deacylase catalytic production MFG parent nucleus is used by the method for embodiment 8.Immobilization cyclic lipopeptide deacylase can reuse 20 times, front can keep for 17 times more than 95% productive rate.
Prepare free cyclic lipopeptide deacylase solution by other two kinds of methods in embodiment 1 and repeat above test, the test-results of basic simlarity can be obtained.
Claims (10)
1. a preparation method for immobilization cyclic lipopeptide deacylase, comprises the following steps:
The cyclic lipopeptide deacylase solution dissociated is obtained after fermented liquid containing cyclic lipopeptide deacylase and mycelium being separated by filtration;
By not purified for free cyclic lipopeptide deacylase solution, directly mix with porous, hydrophilic enzyme carrier, and add one or more organic solutions and inorganic salt, being fixed cyclic lipopeptide deacylase;
The blending ratio of wherein said free cyclic lipopeptide deacylase solution and carrier is that every gram of carrier needs enzyme to live as 9-60000U; When mixing containing free cyclic lipopeptide deacylase solution and carrier, system pH is 5-11.
2. preparation method as claimed in claim 1, described porous, hydrophilic enzyme carrier is selected from: the enzyme carrier taking polymethacrylate as matrix bonding epoxide.
3. preparation method as claimed in claim 2, described porous, hydrophilic enzyme carrier is any one in EupergitCM or LX1000-EP.
4. preparation method as claimed in claim 1, described organic solution is any one in ethanol, Virahol, normal hexane, DMSO, acetone, DMF, tetrahydrofuran (THF), pyridine, acetonitrile, is preferably any one in ethanol, Virahol, acetone.
5. preparation method as claimed in claim 1, described inorganic salt are any one in sodium-chlor, Repone K, magnesium chloride, zinc chloride, are preferably any one in Repone K, magnesium chloride.
6. preparation method as claimed in claim 1, the described temperature containing free cyclic lipopeptide deacylase solution and carrier mixing is 5-80 DEG C, is preferably 10-40 DEG C, most preferably is 20-30 DEG C.
7. preparation method as claimed in claim 1, the blending ratio of described free cyclic lipopeptide deacylase solution and carrier, being preferably every gram of carrier needs enzyme to live as 45-360U.
8. preparation method as claimed in claim 1, the described fermented liquid containing cyclic lipopeptide deacylase refers to and derives from the microorganism strains that natural energy produces cyclic lipopeptide deacylase, or the artificial mutant of these microorganism strains or mutation, or the engineering strain containing cyclic lipopeptide deacylase encoding gene.
9. as method as described in arbitrary in claim 1 to 8 is preparing the application in echinocandin class medicine parent nucleus.
10. apply as claimed in claim 9, described echinocandin class medicine parent nucleus is MFG parent nucleus or anidulafungin parent nucleus.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108441529A (en) * | 2018-05-30 | 2018-08-24 | 博瑞生物医药（苏州）股份有限公司 | A kind of fermentation process of micafen sodium precursor FR179642 |
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| KR20240067913A (en) * | 2021-09-07 | 2024-05-17 | 바이오콘 리미티드 | Method for producing echinocandin nuclei |
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Patent Citations (4)
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| CN101525603A (en) * | 2008-03-04 | 2009-09-09 | 中国医药集团总公司四川抗菌素工业研究所 | Immobilized alpha-amino-acid ester hydrolase, preparation and application thereof |
| CN103387975A (en) * | 2012-05-11 | 2013-11-13 | 上海天伟生物制药有限公司 | Immobilized cyclic lipopeptide acyltransferase, preparation method thereof, and application thereof |
| CN104120120A (en) * | 2014-06-27 | 2014-10-29 | 浙江工业大学 | Immobilized recombinant penicillin G acylase and application thereof |
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