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
- Publication number
- 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
- Authority
- CN
- China
- Prior art keywords
- cyclic lipopeptide
- immobilization
- preparation
- deacylase
- enzyme
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108010028921 Lipopeptides Proteins 0.000 title claims abstract description 89
- 125000004122 cyclic group Chemical group 0.000 title claims abstract description 89
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 108090000790 Enzymes Proteins 0.000 claims abstract description 103
- 102000004190 Enzymes Human genes 0.000 claims abstract description 101
- 230000020176 deacylation Effects 0.000 claims abstract description 41
- 238000005947 deacylation reaction Methods 0.000 claims abstract description 41
- 230000000694 effects Effects 0.000 claims abstract description 38
- 238000000034 method Methods 0.000 claims abstract description 27
- 108010064760 Anidulafungin Proteins 0.000 claims abstract description 9
- JHVAMHSQVVQIOT-MFAJLEFUSA-N anidulafungin Chemical compound C1=CC(OCCCCC)=CC=C1C1=CC=C(C=2C=CC(=CC=2)C(=O)N[C@@H]2C(N[C@H](C(=O)N3C[C@H](O)C[C@H]3C(=O)N[C@H](C(=O)N[C@H](C(=O)N3C[C@H](C)[C@H](O)[C@H]3C(=O)N[C@H](O)[C@H](O)C2)[C@@H](C)O)[C@H](O)[C@@H](O)C=2C=CC(O)=CC=2)[C@@H](C)O)=O)C=C1 JHVAMHSQVVQIOT-MFAJLEFUSA-N 0.000 claims abstract description 9
- 229960003348 anidulafungin Drugs 0.000 claims abstract description 9
- 150000003839 salts Chemical class 0.000 claims abstract description 9
- 238000000746 purification Methods 0.000 claims abstract description 8
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 21
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 19
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 18
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 13
- 235000002639 sodium chloride Nutrition 0.000 claims description 12
- 238000001914 filtration Methods 0.000 claims description 9
- 239000012530 fluid Substances 0.000 claims description 9
- 239000001103 potassium chloride Substances 0.000 claims description 9
- 235000011164 potassium chloride Nutrition 0.000 claims description 9
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 108010049047 Echinocandins Proteins 0.000 claims description 7
- 235000019441 ethanol Nutrition 0.000 claims description 7
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 7
- 150000002118 epoxides Chemical class 0.000 claims description 6
- 239000003960 organic solvent Substances 0.000 claims description 5
- GSNUFIFRDBKVIE-UHFFFAOYSA-N DMF Natural products CC1=CC=C(C)O1 GSNUFIFRDBKVIE-UHFFFAOYSA-N 0.000 claims description 4
- 229940079593 drug Drugs 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 4
- 230000000813 microbial effect Effects 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- 239000011159 matrix material Substances 0.000 claims description 2
- 229920000193 polymethacrylate Polymers 0.000 claims description 2
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims 1
- 238000005660 chlorination reaction Methods 0.000 claims 1
- 239000011701 zinc Substances 0.000 claims 1
- 229910052725 zinc Inorganic materials 0.000 claims 1
- 238000006243 chemical reaction Methods 0.000 abstract description 11
- 238000011084 recovery Methods 0.000 abstract description 10
- 239000012535 impurity Substances 0.000 abstract description 9
- 238000004519 manufacturing process Methods 0.000 abstract description 9
- 239000000758 substrate Substances 0.000 abstract description 4
- 230000009466 transformation Effects 0.000 abstract description 3
- 230000002255 enzymatic effect Effects 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 33
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 230000003197 catalytic effect Effects 0.000 description 7
- 238000004128 high performance liquid chromatography Methods 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 6
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 6
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 6
- 238000001035 drying Methods 0.000 description 6
- 239000000706 filtrate Substances 0.000 description 6
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 6
- 241000187840 Actinoplanes utahensis Species 0.000 description 5
- 241001655322 Streptomycetales Species 0.000 description 5
- 239000000969 carrier Substances 0.000 description 5
- 238000000855 fermentation Methods 0.000 description 5
- 230000004151 fermentation Effects 0.000 description 5
- 239000002243 precursor Substances 0.000 description 4
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 235000011147 magnesium chloride Nutrition 0.000 description 3
- 238000005457 optimization Methods 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 3
- 239000011592 zinc chloride Substances 0.000 description 3
- 235000005074 zinc chloride Nutrition 0.000 description 3
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- 229910004727 OSO3H Inorganic materials 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 229940044613 1-propanol Drugs 0.000 description 1
- 241000187844 Actinoplanes Species 0.000 description 1
- 206010003645 Atopy Diseases 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 108010001515 Galectin 4 Proteins 0.000 description 1
- 102100039556 Galectin-4 Human genes 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 241000186986 Streptomyces anulatus Species 0.000 description 1
- 241000187180 Streptomyces sp. Species 0.000 description 1
- 102000004357 Transferases Human genes 0.000 description 1
- 108090000992 Transferases Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 230000000843 anti-fungal effect Effects 0.000 description 1
- 239000003429 antifungal agent Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 235000011148 calcium chloride Nutrition 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000002242 deionisation method Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical group 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000002949 hemolytic effect Effects 0.000 description 1
- 239000012052 hydrophilic carrier Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000012092 media component Substances 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- BDERNNFJNOPAEC-UHFFFAOYSA-N n-propyl alcohol Natural products CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 238000012805 post-processing Methods 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Enzymes And Modification Thereof (AREA)
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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510626188.5A CN105154424B (en) | 2015-09-28 | 2015-09-28 | A kind of preparation method and applications of immobilization cyclic lipopeptide deacylase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510626188.5A CN105154424B (en) | 2015-09-28 | 2015-09-28 | A kind of preparation method and applications of immobilization cyclic lipopeptide deacylase |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105154424A CN105154424A (en) | 2015-12-16 |
| CN105154424B true CN105154424B (en) | 2018-10-16 |
Family
ID=54795469
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510626188.5A Active CN105154424B (en) | 2015-09-28 | 2015-09-28 | A kind of preparation method and applications of immobilization cyclic lipopeptide deacylase |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105154424B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023037252A1 (en) * | 2021-09-07 | 2023-03-16 | Biocon Limited | Preparation method of echinocandin nucleus |
Families Citing this family (1)
| 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 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN104450662A (en) * | 2014-12-29 | 2015-03-25 | 临沂市宏昱生物科技有限公司 | Method for preparing immobilized fructosyltransferase through ion exchange fibers |
-
2015
- 2015-09-28 CN CN201510626188.5A patent/CN105154424B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN104450662A (en) * | 2014-12-29 | 2015-03-25 | 临沂市宏昱生物科技有限公司 | Method for preparing immobilized fructosyltransferase through ion exchange fibers |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023037252A1 (en) * | 2021-09-07 | 2023-03-16 | Biocon Limited | Preparation method of echinocandin nucleus |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105154424A (en) | 2015-12-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106191170B (en) | A kind of method that enzyme process prepares atriphos | |
| CN105647996B (en) | The method that immobilized enzyme method prepares atriphos | |
| CN105112476B (en) | Method for producing lipopeptide biosurfactant by fermentation | |
| CN106978368B (en) | Raoultella ornithinolytica and application thereof | |
| EP2210935A1 (en) | Methods for isolating bacteria | |
| CN111575310B (en) | Recombinant saccharomyces cerevisiae expressing caveolin and application thereof | |
| US11085059B2 (en) | Methylopila sp. and use thereof in selective resolution preparation of (S)-α-ethyl-2-oxo-1-pyrrolidineacetate | |
| CN109609582A (en) | A kind of method that microorganism catalysis deracemization prepares L-glufosinate-ammonium | |
| CN105154424B (en) | A kind of preparation method and applications of immobilization cyclic lipopeptide deacylase | |
| WO2010058427A2 (en) | Process for production and purification of polymyxin b sulfate | |
| CN106544382A (en) | A kind of preparation method of cyclohexyl peptide compounds FR179642 | |
| US20160237467A1 (en) | Method of semi-solid state fermentation for producing surfactin from a mutant strain of bacillus subtilis subsp | |
| CN112662658A (en) | Production of L-phenylpyruvic acid by immobilized recombinant escherichia coli using L-phenylalanine | |
| CN110357788B (en) | Polyketone compound and preparation method and application thereof | |
| CN106636054A (en) | Microbial catalytic carrier for converting and synthesizing organic acid and preparation method thereof | |
| CN110577936A (en) | Method for efficiently degrading tetracyclic and sulfonamide antibiotics by laccase | |
| CN112680394B (en) | Method for improving biosynthesis amount of natural product | |
| CN103833146A (en) | Mixed strain embedding immobilizing agent for treating wastewater | |
| CN113774004A (en) | Lactobacillus brevis and method for preparing gamma-aminobutyric acid by recycling whole cells of lactobacillus brevis | |
| CN103387975A (en) | Immobilized cyclic lipopeptide acyltransferase, preparation method thereof, and application thereof | |
| CN112980910A (en) | Method for microbial transformation of 11 alpha-hydroxy canrenone in phase transfer catalyst system | |
| CN113604515A (en) | Method for synthesizing phenyllactic acid by using semi-hydrophobic crystal gum base whole-cell catalyst in bioreactor | |
| Yu et al. | Biosynthesis of polyglutamic acid and its application on agriculture | |
| JP3067183B2 (en) | Method for producing FR900506 substance | |
| CN113396214A (en) | Method for producing nigericin from streptomyces sp.mcc-0151 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |