CN105154424B - A kind of preparation method and applications of immobilization cyclic lipopeptide deacylase - Google Patents
A kind of preparation method and applications of immobilization cyclic lipopeptide deacylase Download PDFInfo
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- CN105154424B CN105154424B CN201510626188.5A CN201510626188A CN105154424B CN 105154424 B CN105154424 B CN 105154424B CN 201510626188 A CN201510626188 A CN 201510626188A CN 105154424 B CN105154424 B CN 105154424B
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Abstract
The invention discloses a kind of preparation methods of immobilization cyclic lipopeptide deacylase.Cyclic lipopeptide deacylation enzyme solutions without further purification, are directly mixed with porous, hydrophilic zymophore, and one or more organic solutions and inorganic salts are added, and obtain immobilization cyclic lipopeptide deacylase；It also discloses this method and can be used for industrialized production mikafen parent nucleus and anidulafungin parent nucleus.Preparation method disclosed by the invention is simple for process, not only saves enzyme purification equipment, and the enzyme activity rate of recovery significantly improves in immobilization process, immobilised enzymes can repeatedly use.Enzymatic conversion method mikafen and anidulafungin intermediate, high compared to microbe conversion method reaction substrate concentration, impurity in products is few, and transformation efficiency is high.
Description
Technical field
The invention belongs to the technical field of enzyme immobilization in biotechnology；In particular it relates to a kind of immobilization ring
The preparation method and applications of lipopeptid deacylase.
Technical background
Echinocandin class antibiotic, which is the one kind found the 1970s, has inhibition beta-1,3-dextran synthetase activity
Property natural products, there is good antifungal activity, basic structure is as shown in Equation 1.Due to natural echinocandin class chemical combination
R possessed by object2Fatty acid side chain has certain hemolytic toxicity, therefore the mikafen and A Ni listed in human body
Fragrant only equal fragrant net class antifungal drug is all to slough original fatty acid side chain of native compound, changes different aliphatic acid sides
Chain.Since the process that side chain is sloughed is difficult to be completed with chemical method, biology is generally carried out using cyclic lipopeptide deacylase (Deacylase)
Conversion.
Formula 1：Echinocandin compound basic structural formula (R1：-CH3Or-H；R2：C15-C17Chain；R3：-CH3Or-H；R4：-
OH or-OSO3H；R5：-OSO3H or-H；R6：- OH or-H；R7：CH3Or CH2CONH2)
It has been reported that cyclic lipopeptide deacylase (Deacylase) derive from actinoplanes utahensis (Actinoplanes
) or ring streptomycete (Streptomyces anulatus) utahensis.In the bioconversion method of document report, generally
It is converted using Precurosor fermentation method, i.e., microorganism is subjected to producing enzyme culture first, then directly substrate is added in fermentation system
It is converted.This method has disadvantages that, as substrate is low with production concentration, the reaction time is long in transformation system, liquid handling body
Impurity more (Media Components and organic solvents), product postprocessing are difficult greatly, in system for product, it is big etc. to purify difficulty.In addition this
The not reproducible use of one fermentation system, it is of high cost.
Enzyme immobilization can preferably solve the above problems.It is generally believed that such as patent application《A kind of immobilization cyclic lipopeptide acyl
Based transferase and its preparation method and application, 201210148392.7》, preferably first enzyme is purified when enzyme immobilization (such as from
Sub exchanges, molecular sieve, hydrophobic chromatography and ultrafiltration dialysis etc., as needed progress alone or in combination), foreign protein ratio is got in enzyme solution
It is small to be more conducive to immobilization.This is because there are many pigments, amino acid, cell fragment, secondary metabolites etc. in zymotic fluid
Various impurity；Enzyme is relatively low relative to the concentration of various impurity, and impurity can inhibit the vigor of fixation support；Contain in impurity simultaneously
Active group the being at war with property of binding site of hydrophilic carrier can be combined, cause the immobilization enzyme activity rate of recovery low.
However enzyme purification process can cause the loss of enzyme, and additional equipment is needed, increase cost.
During enzyme immobilization, the atopic of carrier and gal4 amino acid residue is by chemical property and microenvironment
(including medium composition, ionic strength, pH etc.) is determined.This thinking is followed, the purpose of the present invention is finding suitable condition, is sent out
Zymotic fluid is just optionally combined with the cyclic lipopeptide deacylase in zymotic fluid without purifying, fixation support, is reduced and impurity
In conjunction with acquisition has the immobilization cyclic lipopeptide deacylase of industrial value.
Invention content
The method of the invention discloses a kind of fixation cyclic lipopeptide deacylase specific from zymotic fluid.
Method disclosed by the invention, includes the following steps：
Free cyclic lipopeptide deacylase is obtained after zymotic fluid containing cyclic lipopeptide deacylase and mycelium are separated by filtration
Solution；
Without further purification by free cyclic lipopeptide deacylation enzyme solutions, it is directly mixed with porous, hydrophilic zymophore, and is added one
Kind or a variety of organic solutions and inorganic salts, obtain immobilization cyclic lipopeptide deacylase.
It is wherein described
The mixed proportion of free cyclic lipopeptide deacylation enzyme solutions and carrier is that need enzyme activity be 9-60000U to every gram of carrier, preferably
It is 45-360U to need enzyme activity for every gram of carrier.
System pH is 5-11 when being mixed containing free cyclic lipopeptide deacylation enzyme solutions and carrier.
Porous, hydrophilic zymophore is selected from the zymophore that epoxides is bonded using polymethacrylates as matrix.It is especially excellent
It is selected as any one of Eupergit CM or LX1000-EP.
Organic solution is any in ethyl alcohol, isopropanol, n-hexane, DMSO, acetone, DMF, tetrahydrofuran, pyridine, acetonitrile
Kind, preferably any one of ethyl alcohol, isopropanol, acetone.
Inorganic salts are any one of sodium chloride, potassium chloride, magnesium chloride, zinc chloride, preferably in potassium chloride, magnesium chloride
It is any.
The temperature mixed containing free cyclic lipopeptide deacylation enzyme solutions and carrier is 5-80 DEG C, preferably 10-40 DEG C, optimal
It is selected as 20-30 DEG C.
Zymotic fluid containing cyclic lipopeptide deacylase refers to the microbial strains that cyclic lipopeptide deacylase is generated from natural energy,
The either artificial mutant of these microbial strains or mutation or the gene work containing cyclic lipopeptide deacylation enzyme coding gene
Journey bacterial strain.
The invention also discloses the preparation method of this immobilization cyclic lipopeptide deacylase is being prepared echinocandin class drug
Application in parent nucleus.
The wherein described echinocandin class drug parent nucleus is mikafen parent nucleus or anidulafungin parent nucleus.
In the present invention, above-mentioned enzyme-activity unit is defined as：At 40 DEG C, the enzyme needed for 1 micromole's product is generated in 1 hour
Amount, that is, be defined as 1U.Product determination step is：17.5mL crude enzyme liquids are measured respectively, contain mikafen precursor or anidulafungin
Phosphoric acid (0.2mol/L, pH5.5) buffer solution 5mL, methanol 2.5mL of precursor.Under 40 DEG C of water bath conditions, react 1 hour, deionization
Water suitably dilutes, 0.22 μm of nylon membrane filtration, and HPLC measures product mikafen parent nucleus or anidulafungin parent nucleus concentration.
The present invention is walked after the zymotic fluid containing cyclic lipopeptide deacylase and mycelium simple separation without other enzyme purifications
Suddenly, it is directly mixed with fixation support, realizes that the efficient combination of cyclic lipopeptide deacylase and fixation support, acquisition have industrial value
Immobilization cyclic lipopeptide deacylase.Fixing condition provided by the present invention enhances fixation support epoxide epoxy group group and target
The selective binding of enzyme, the combination of reduction and impurity, overcomes the difficult point that crude enzyme liquid is unfavorable for immobilization.With previous methods phase
Than this preparation method is simple for process, not only saves enzyme purification equipment, and the enzyme activity rate of recovery obviously carries in immobilization process
Height, immobilised enzymes can repeatedly use.Enzymatic conversion method mikafen and anidulafungin intermediate are reacted compared to microbe conversion method
Concentration of substrate is high, and impurity in products is few, and transformation efficiency is high.
Specific implementation mode
The present invention is further explained with reference to specific embodiment.
The acquisition of 1 cyclic lipopeptide deacylation enzyme solutions of embodiment
The cyclic lipopeptide deacylase that 1.1 actinoplanes utahensis (Actinoplanes utahensis) generate
Using actinoplanes utahensis IFO-13244 bacterial strains, according to fermentation described in embodiment 1 in patent US5376634
Method carries out fermented and cultured, obtains Mycelium culture liquid 150L.Then in the filtered on buchner funnel for being covered with filter paper, collection contains
The filtrate 120L of cyclic lipopeptide deacylase, i.e. free cyclic lipopeptide deacylation enzyme solutions, collect filtrate, are detected through HPLC, enzyme activity is
0.864×105U。
The cyclic lipopeptide deacylase that 1.2 streptomycetes (Streptomyces sp.) generate
It is sent out according to fermentation process described in embodiment 1 in patent WO97/32975 using No. 6907 bacterial strains of streptomycete
Ferment culture obtains Mycelium culture liquid 150L.Then it in the filtered on buchner funnel for being covered with filter paper, collects and contains cyclic lipopeptide deacylase
Filtrate 130L, i.e., free cyclic lipopeptide deacylation enzyme solutions collect filtrate, are detected through HPLC, and enzyme activity is 0.768 × 105U。
The cyclic lipopeptide deacylase that the recombination streptomycete of the 1.3 deacylation enzyme genes of cyclic lipopeptide containing external source generates
By literature method (Applied and Environmental Microbiology, 2013, V79.4,1126-
1133) cyclic lipopeptide deacylation enzyme gene is cloned from actinoplanes utahensis, and is recombinated into the genome of streptomycete.Picking recombinates
Transformant activates, and expands culture and expressed, and obtains Mycelium culture liquid 150L.Then in the Buchner funnel mistake for being covered with filter paper
Filter collects the filtrate 120L containing cyclic lipopeptide deacylase, i.e. free cyclic lipopeptide deacylation enzyme solutions, collects filtrate, examined through HPLC
It surveys, enzyme activity is 0.606 × 105U。
The selection of 2 different type epoxides carrier of embodiment
Free cyclic lipopeptide deacylation enzyme solutions are obtained prepared by taking 1.1 in above-described embodiment 1, are separately added into 1g
Eupergit CM or LX1000-EP, 25 DEG C are stirred 24 hours, and immobilization cyclic lipopeptide deacylase, pure water three is collected by filtration
Secondary, drying at room temperature 3 hours finally carries out enzyme activity determination.
Fixing situation of the 1 different type epoxides carrier of table to original free enzyme solution
As it can be seen from table 1 the rate of recovery of carrier LX1000-EP is above carrier Eupergit under the conditions of identical enzyme dosage
CM。
Fixing situations of the 2 epoxides carrier LX1000-EP of table to original free enzyme solution
From table 2 it can be seen that the mixed proportion in free cyclic lipopeptide deacylation enzyme solutions and carrier is 9-60000U/g models
In enclosing, free enzyme immobilizatio may be implemented, it is 45- that the enzyme activity rate of recovery every gram of carrier in 5% or more group, which needs enzyme activity,
360U。
Comprehensive Tables 1 and 2 can be seen that enzyme solution is not purified to be directly added into vehicle treated, and enzyme activity is returned in immobilization process
Yield is relatively low, cannot meet the needs of practical application.
The polar optimization of aqueous solution in 3 immobilization process of embodiment
Free cyclic lipopeptide deacylation enzyme solutions 1L is obtained prepared by 1.1 in Example 1, and it is organic to be separately added into different type
Each 50mL, 100mL of solvent (including ethyl alcohol, isopropanol, n-hexane, DMSO, acetone, DMF, tetrahydrofuran, pyridine, acetonitrile),
200mL or 300mL.Continue to be separately added into 5g LX1000-EP carriers, 25 DEG C are stirred 24 hours, and immobilization cyclic lipopeptide is collected by filtration
Deacylase, three times, drying at room temperature 3 hours finally carries out enzyme activity determination to pure water.
The cyclic lipopeptide deacylation enzyme immobilization of 3 different organic solvents of table
From table 3 it can be seen that in free cyclic lipopeptide deacylation enzyme solutions, different type organic solvent, including second is added
Alcohol, isopropanol, n-hexane, DMSO, acetone, DMF, tetrahydrofuran, pyridine, acetonitrile, the immobilization enzyme activity rate of recovery have centainly
Promotion.The effect of wherein ethyl alcohol, isopropanol and acetone is particularly evident, most suitable to add a concentration of 10%.
The optimization of embodiment 4 immobilization process ionic strength and pH value
Free cyclic lipopeptide deacylation enzyme solutions 1L is obtained prepared by 1.1 in Example 1, isopropanol 100mL is added, simultaneously
It is separately added into different type inorganic salts (including sodium chloride, calcium chloride, potassium chloride, magnesium chloride, ammonium chloride, zinc chloride) each 1mol,
And it is adjusted to different pH value.Continue to be separately added into 5g LX1000-EP carriers, 25 DEG C are stirred 24 hours, and immobilization is collected by filtration
Cyclic lipopeptide deacylase, three times, drying at room temperature 3 hours finally carries out enzyme activity determination to pure water.
From table 4, it can be seen that free cyclic lipopeptide deacylation enzyme solutions are 5-11 models in pH value when not adding inorganic salts
In enclosing, free enzyme immobilizatio, preferable ph 8-10 may be implemented.Add sodium chloride, potassium chloride, magnesium chloride, zinc chloride
After equal inorganic salts, the immobilization particle enzyme activity of acquisition all has a certain upgrade.The effect of wherein potassium chloride and magnesium chloride is especially bright
Aobvious, when pH value is 9, addition 1mol/L potassium chloride immobilization effects are best, and immobilization particle enzyme activity is 100.8U/g, immobilization
The enzyme activity rate of recovery is 70.0%.
The optimization of temperature and enzyme liquid amount and carrier amount mixed proportion in 5 immobilization process of embodiment
Free cyclic lipopeptide deacylation enzyme solutions 1L is obtained prepared by 1.1 in Example 1, and isopropanol 100mL and chlorine is added
Change potassium 1mol, and it is 9 to adjust pH value.Continue to be separately added into LX1000-EP carrier 4g, 5g, 6g or 7g and stir at different temperatures
It mixes 24 hours, immobilization cyclic lipopeptide deacylase is collected by filtration, three times, drying at room temperature 3 hours finally carries out enzyme activity survey to pure water
It is fixed.
The cyclic lipopeptide deacylation enzyme immobilization of 5 different temperatures of table and carrier amount
As can be seen from Table 5, it is within the scope of 5-80 DEG C in the mixing temperature of free cyclic lipopeptide deacylation enzyme solutions and carrier,
Free enzyme immobilizatio may be implemented, wherein 10-40 DEG C of effect is more preferable, 20-30 DEG C of best results.Free cyclic lipopeptide
The mixed proportion of deacylation enzyme solutions and carrier is most preferably addition 6g carriers in 1L enzyme solutions.
The purifying and immobilization of 6 cyclic lipopeptide deacylase of embodiment
Reference literature method (Journal of Industrial Microbiology＆Biotechnology, 2000,
24,173-180) free cyclic lipopeptide deacylation enzyme solutions 120L is obtained prepared by purifying 1.1 in embodiment 1, i.e. enzyme activity is 0.864
×105U, the cyclic lipopeptide deacylation enzyme solutions 3L purified, is detected through HPLC, and enzyme activity is 0.173 × 105U purifies enzyme activity yield
It is 20.0%.
Above-mentioned purified cyclic lipopeptide deacylation enzyme solutions 1L is taken, it is 7 to adjust pH value, is separately added into 30g Eupergit CM
Carrier or 30g LX1000-EP carriers, 25 DEG C are stirred 24 hours, and immobilization cyclic lipopeptide deacylase, pure water three is collected by filtration
Secondary, drying at room temperature 3 hours finally carries out enzyme activity determination.
Fixing situation of the 6 epoxides carrier of table to the free enzyme solution of purifying
As can be seen from Table 6,1.1 in embodiment 1 prepared by obtain free cyclic lipopeptide deacylation enzyme solutions 120L, after purification
Cyclic lipopeptide deacylation enzyme solutions 3L is obtained, purifying enzyme activity yield is 20.0%, although the immobilization enzyme activity rate of recovery is very high, can be reached
90.0%, but since purifying enzyme activity yield is low, enzyme activity overall recovery only has 18.0%.
7 big system immobilization cyclic lipopeptide deacylase of embodiment
Obtain free cyclic lipopeptide deacylation enzyme solutions 100L prepared by 1.1 in Example 1, i.e., enzyme activity be 0.720 ×
105Isopropanol 10L and potassium chloride 100mol is added in U, and it is 9 to adjust pH value.Continuously add 600g LX1000-EP carriers, 25
DEG C stirring 24 hours, is collected by filtration immobilization cyclic lipopeptide deacylase, pure water three times, drying at room temperature 3 hours.After measured, it receives
For the immobilization cyclic lipopeptide deacylase particle enzyme activity integrated as 98.6U/g, the immobilization enzyme activity rate of recovery is 82.2%.
8 immobilization cyclic lipopeptide deacylase catalytic production mikafen parent nucleus of embodiment
Phosphoric acid (0.1mol/L, pH5.5) buffer solution is added in 2g mikafen precursor and 10mL methanol and is settled to
100mL adds the immobilization cyclic lipopeptide deacylation enzyme granulate 10g prepared by embodiment 7.40 DEG C, the reaction of 240rpm shaking tables.Reaction
After 8 hours, the molar yield of HPLC detections, mikafen parent nucleus is 98%.
9 immobilization cyclic lipopeptide deacylase catalytic production anidulafungin parent nucleus of embodiment
Phosphoric acid (0.1mol/L, pH5.5) buffer solution is added in 2g anidulafungins precursor and 10mL methanol and is settled to
100mL adds the immobilization cyclic lipopeptide deacylation enzyme granulate 10g prepared by embodiment 7.40 DEG C, the reaction of 240rpm shaking tables.Reaction
After 15 hours, the molar yield of HPLC detections, anidulafungin parent nucleus is 95%.
10 immobilization cyclic lipopeptide deacylase batch catalytic production mikafen parent nucleus of embodiment
Immobilization cyclic lipopeptide deacylase catalytic production mikafen parent nucleus is used as described in Example 8.After reaction was completed,
Immobilization cyclic lipopeptide deacylase is collected by filtration, pure water is used in combination three times, the immobilization cyclic lipopeptide deacylase of collection continues by reality
Apply the method catalytic production mikafen parent nucleus of example 8.It is repeated 20 times, the yield of mikafen parent nucleus is recorded every time, such as 7 institute of table
Show.
7 immobilization cyclic lipopeptide deacylase batch catalytic production mikafen parent nucleus of table
As can be seen from Table 7, use immobilization cyclic lipopeptide deacylase catalytic production mikafen female as described in Example 8
Core.Immobilization cyclic lipopeptide deacylase may be reused 20 times, the first 17 times yields that can keep 95% or more.
Free cyclic lipopeptide deacylation enzyme solutions are prepared with other two methods in embodiment 1 and repeat the above experiment, it can
To obtain substantially similar test result.
Claims (11)
1. a kind of preparation method of immobilization cyclic lipopeptide deacylase, includes the following steps：
Free cyclic lipopeptide deacylation enzyme solutions are obtained after zymotic fluid containing cyclic lipopeptide deacylase and mycelium are separated by filtration；
Without further purification by free cyclic lipopeptide deacylation enzyme solutions, directly mix with porous, hydrophilic zymophore, and be added one kind or
A variety of organic solutions and inorganic salts obtain immobilization cyclic lipopeptide deacylase；
The mixed proportion of the wherein described free cyclic lipopeptide deacylation enzyme solutions and carrier is that need enzyme activity be 9-60000U to every gram of carrier；
System pH is 8-10 when being mixed containing free cyclic lipopeptide deacylation enzyme solutions and carrier；The porous, hydrophilic zymophore be with
Polymethacrylates is the zymophore that matrix is bonded epoxides；The organic solution is ethyl alcohol, isopropanol, acetone or DMF
Any one of, a concentration of 10-30% of addition of organic solvent；The inorganic salts are sodium chloride, potassium chloride, magnesium chloride, chlorination
Any one of zinc.
2. preparation method as described in claim 1, it is characterised in that：The free cyclic lipopeptide deacylation enzyme solutions and carrier
Mixed proportion is that need enzyme activity be 45-360U to every gram of carrier.
3. preparation method as described in claim 1, it is characterised in that：The porous, hydrophilic zymophore is Eupergit CM
Or any one of LX1000-EP.
4. preparation method as described in claim 1, it is characterised in that：The organic solution is in ethyl alcohol, isopropanol, acetone
Any, addition a concentration of 10% of organic solvent.
5. preparation method as described in claim 1, it is characterised in that：The inorganic salts are any in potassium chloride, magnesium chloride
Kind.
6. preparation method as described in claim 1, it is characterised in that：It is described to contain free cyclic lipopeptide deacylation enzyme solutions and load
The temperature of body mixing is 5-80 DEG C.
7. preparation method as claimed in claim 6, it is characterised in that：It is described to contain free cyclic lipopeptide deacylation enzyme solutions and load
The temperature of body mixing is 10-40 DEG C.
8. preparation method as claimed in claim 7, it is characterised in that：It is described to contain free cyclic lipopeptide deacylation enzyme solutions and load
The temperature of body mixing is 20-30 DEG C.
9. preparation method as described in claim 1, it is characterised in that：The zymotic fluid containing cyclic lipopeptide deacylase refers to coming
Artificial mutant or the change of the microbial strains or these microbial strains of cyclic lipopeptide deacylase are generated derived from natural energy
Kind, or the engineering strain containing cyclic lipopeptide deacylation enzyme coding gene.
10. such as application of any the method in preparing echinocandin class drug parent nucleus in claim 1 to 9.
11. application as claimed in claim 10, it is characterised in that：The echinocandin class drug parent nucleus is mikafen parent nucleus
Or anidulafungin parent nucleus.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2023037252A1 (en) * | 2021-09-07 | 2023-03-16 | Biocon Limited | Preparation method of echinocandin nucleus |
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| 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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| 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 |
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| 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 |
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