CN108911962A - A method of the high efficiency extraction Pantothenic acid from enzymolysis liquid - Google Patents
A method of the high efficiency extraction Pantothenic acid from enzymolysis liquid Download PDFInfo
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- CN108911962A CN108911962A CN201810619872.4A CN201810619872A CN108911962A CN 108911962 A CN108911962 A CN 108911962A CN 201810619872 A CN201810619872 A CN 201810619872A CN 108911962 A CN108911962 A CN 108911962A
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- pantothenic acid
- enzymolysis liquid
- high efficiency
- efficiency extraction
- liquid
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- GHOKWGTUZJEAQD-ZETCQYMHSA-N (D)-(+)-Pantothenic acid Chemical compound OCC(C)(C)[C@@H](O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-ZETCQYMHSA-N 0.000 title claims abstract description 210
- GHOKWGTUZJEAQD-UHFFFAOYSA-N Chick antidermatitis factor Natural products OCC(C)(C)C(O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-UHFFFAOYSA-N 0.000 title claims abstract description 106
- 235000019161 pantothenic acid Nutrition 0.000 title claims abstract description 106
- 239000011713 pantothenic acid Substances 0.000 title claims abstract description 105
- 229940055726 pantothenic acid Drugs 0.000 title claims abstract description 105
- 239000007788 liquid Substances 0.000 title claims abstract description 81
- 238000000034 method Methods 0.000 title claims abstract description 69
- 238000000605 extraction Methods 0.000 title claims abstract description 63
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 45
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 20
- -1 proline methyl ester hexafluorophosphate Chemical compound 0.000 claims description 41
- 239000007864 aqueous solution Substances 0.000 claims description 19
- 239000006228 supernatant Substances 0.000 claims description 18
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 14
- 239000002608 ionic liquid Substances 0.000 claims description 13
- 230000002209 hydrophobic effect Effects 0.000 claims description 12
- 238000000926 separation method Methods 0.000 claims description 11
- 239000003960 organic solvent Substances 0.000 claims description 9
- 238000013517 stratification Methods 0.000 claims description 8
- 150000001413 amino acids Chemical class 0.000 claims description 4
- 238000002425 crystallisation Methods 0.000 claims description 3
- 230000008025 crystallization Effects 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 2
- 238000003825 pressing Methods 0.000 claims description 2
- 150000002948 pantothenic acids Chemical class 0.000 claims 1
- 108090000790 Enzymes Proteins 0.000 abstract description 19
- 102000004190 Enzymes Human genes 0.000 abstract description 19
- 239000000126 substance Substances 0.000 abstract description 11
- 230000008569 process Effects 0.000 abstract description 9
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 21
- 239000012071 phase Substances 0.000 description 18
- 238000002156 mixing Methods 0.000 description 13
- 238000000855 fermentation Methods 0.000 description 12
- 230000004151 fermentation Effects 0.000 description 12
- 150000002148 esters Chemical class 0.000 description 10
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 239000000654 additive Substances 0.000 description 6
- 230000000996 additive effect Effects 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 6
- OTOIIPJYVQJATP-UHFFFAOYSA-N pantoic acid Chemical compound OCC(C)(C)C(O)C(O)=O OTOIIPJYVQJATP-UHFFFAOYSA-N 0.000 description 6
- 238000011084 recovery Methods 0.000 description 6
- 239000000758 substrate Substances 0.000 description 6
- 230000007062 hydrolysis Effects 0.000 description 5
- 238000006460 hydrolysis reaction Methods 0.000 description 5
- 235000003283 Pachira macrocarpa Nutrition 0.000 description 4
- YNCRBFODOPHHAO-YUELXQCFSA-N Phaseic acid Natural products CC(=CC(=O)O)C=C[C@@H]1[C@@]2(C)CO[C@@]1(C)CC(=O)C2 YNCRBFODOPHHAO-YUELXQCFSA-N 0.000 description 4
- 241001083492 Trapa Species 0.000 description 4
- 235000014364 Trapa natans Nutrition 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 125000004494 ethyl ester group Chemical group 0.000 description 4
- 239000000284 extract Substances 0.000 description 4
- IZGYIFFQBZWOLJ-UHFFFAOYSA-N neophaseic acid Natural products C1C(=O)CC2(C)OCC1(C)C2(O)C=CC(C)=CC(O)=O IZGYIFFQBZWOLJ-UHFFFAOYSA-N 0.000 description 4
- IZGYIFFQBZWOLJ-CKAACLRMSA-N phaseic acid Chemical compound C1C(=O)C[C@@]2(C)OC[C@]1(C)[C@@]2(O)C=CC(/C)=C\C(O)=O IZGYIFFQBZWOLJ-CKAACLRMSA-N 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 235000009165 saligot Nutrition 0.000 description 4
- OTOIIPJYVQJATP-BYPYZUCNSA-N (R)-pantoic acid Chemical compound OCC(C)(C)[C@@H](O)C(O)=O OTOIIPJYVQJATP-BYPYZUCNSA-N 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- VSGNNIFQASZAOI-UHFFFAOYSA-L calcium acetate Chemical compound [Ca+2].CC([O-])=O.CC([O-])=O VSGNNIFQASZAOI-UHFFFAOYSA-L 0.000 description 3
- 239000001639 calcium acetate Substances 0.000 description 3
- 229960005147 calcium acetate Drugs 0.000 description 3
- 235000011092 calcium acetate Nutrition 0.000 description 3
- 238000006555 catalytic reaction Methods 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000007071 enzymatic hydrolysis Effects 0.000 description 3
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 description 2
- 241000223218 Fusarium Species 0.000 description 2
- 241000233732 Fusarium verticillioides Species 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 229940024606 amino acid Drugs 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000005194 fractionation Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 208000034783 hypoesthesia Diseases 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 150000002596 lactones Chemical class 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000002002 slurry Substances 0.000 description 2
- 159000000000 sodium salts Chemical class 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- DBBFFYXLECNESF-WCCKRBBISA-N (2s)-2-amino-3-methylbutanoic acid;nitric acid Chemical compound O[N+]([O-])=O.CC(C)[C@H](N)C(O)=O DBBFFYXLECNESF-WCCKRBBISA-N 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 206010010774 Constipation Diseases 0.000 description 1
- 208000001840 Dandruff Diseases 0.000 description 1
- 206010057315 Daydreaming Diseases 0.000 description 1
- UNXHWFMMPAWVPI-UHFFFAOYSA-N Erythritol Natural products OCC(O)C(O)CO UNXHWFMMPAWVPI-UHFFFAOYSA-N 0.000 description 1
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000241413 Propolis Species 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 208000005392 Spasm Diseases 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 229930003571 Vitamin B5 Natural products 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 210000004100 adrenal gland Anatomy 0.000 description 1
- BIGPRXCJEDHCLP-UHFFFAOYSA-N ammonium bisulfate Chemical compound [NH4+].OS([O-])(=O)=O BIGPRXCJEDHCLP-UHFFFAOYSA-N 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- FAPWYRCQGJNNSJ-UBKPKTQASA-L calcium D-pantothenic acid Chemical compound [Ca+2].OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O.OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O FAPWYRCQGJNNSJ-UBKPKTQASA-L 0.000 description 1
- 229960002079 calcium pantothenate Drugs 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- UNXHWFMMPAWVPI-ZXZARUISSA-N erythritol Chemical compound OC[C@H](O)[C@H](O)CO UNXHWFMMPAWVPI-ZXZARUISSA-N 0.000 description 1
- 239000006052 feed supplement Substances 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 229940069949 propolis Drugs 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000006340 racemization Effects 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 235000014347 soups Nutrition 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000003519 ventilatory effect Effects 0.000 description 1
- 235000009492 vitamin B5 Nutrition 0.000 description 1
- 239000011675 vitamin B5 Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/42—Separation; Purification; Stabilisation; Use of additives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/42—Separation; Purification; Stabilisation; Use of additives
- C07C51/48—Separation; Purification; Stabilisation; Use of additives by liquid-liquid treatment
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to technical field of enzyme engineering, and in particular to a method of the high efficiency extraction Pantothenic acid from enzymolysis liquid.The method of the high efficiency extraction Pantothenic acid of the present invention from enzymolysis liquid, on the basis of existing Pantothenic acid extracting method, by being advanced optimized to the extractant, so that more thorough as extraction of the extractant to Pantothenic acid in enzymolysis liquid using existing ethyl acetate, for the present invention using Pantothenic acid for the technique of Objective extraction object, the interference for effectively eliminating other substances in water phase after extracting, so that subsequent refinement treatment process is more simple.
Description
Technical field
The invention belongs to technical field of enzyme engineering, and in particular to a kind of side of the high efficiency extraction Pantothenic acid from enzymolysis liquid
Method.
Background technique
Pantothenic acid, also known as pantothenic acid, vitamin B5, are widely present in living nature, are the main of synthesis coacetylase in animal body
Raw material, main function are the metabolism for participating in sugar, fat and protein.Human body lack pantothenic acid can cause DOMS or spasm,
Adrenal gland function deficiency and decline, absent minded, numbness in hands and feet, constipation, energy are the tired power of muscle after lacking, slightly taking exercise
Most, worried, the nervous and symptoms such as grind one's teeth in sleep.Pantothenic acid is widely used in medicine, feed and food etc. with its distinctive biochemical function
Field.And D-pantoyl lactone (D-PL) is then the synthesis most important hand-type intermediate of D-VB5 series of products.
It is typically only capable to that DL- pantoic acid lactone is made by chemically synthesized method in the prior art, is carrying out in DL- pantoic acid
The fractionation of ester.The method for splitting of DL- pantoic acid lactone is mostly to use chemical method, at high cost the disadvantage is that resolving agent is expensive,
And separation is difficult.In biological resolution method, DL- pantoic acid lactone is split using microbial enzyme method, because having environmental pollution and poison
Property it is few the features such as and become important directions of research.
A kind of mode that microbial enzyme method splits DL- pantoic acid lactone is to utilize the microorganism for producing L- pantoic acid lactone enzyme,
L- pantoic acid lactone in decomposing D L- pantoic acid lactone obtains undecomposed D-form pantoic acid lactone；The shortcomings that the method, is only
There is the lactone of L- configuration to be hydrolyzed thoroughly can just obtain very much the D-pantoyl lactone of high-optical-purity, and reaction time
It is long, and prolonged hydrolytic process will lead to a part of D-pantoyl lactone and hydrolyze, in the product Pantothenic acid caused
Ester yield is not high；Another way is to utilize the microorganism for producing D-pantoyl lactone enzyme, in selective hydrolysis DL- pantoic acid lactone
D-pantoyl lactone, and then obtain Pantothenic acid, then Pantothenic acid lactonize to generate D-pantoyl lactone；This method
Hydrolysis time is short, although percent hydrolysis is not high (30% or so), unhydrolysed L- pantoic acid lactone can recycle after racemization, product
Overall yield it is higher, therefore, become the main stream approach for preparing Pantothenic acid and D-pantoyl lactone in the prior art.
The bacterial strain that can generate D-pantoyl lactone hydrolase of report known in the state of the art includes fusarium moniliforme, a beading
Fusarium etc., and by the optimization of actication of culture and condition of enzyme production, so that the enzymolysis efficiency of Pantothenic acid has very greatly
Raising, still, the high efficiency extraction of target product is also a problem in Pantothenic acid enzymolysis process in enzymolysis liquid.Existing skill
The art of extraction in to(for) Pantothenic acid and/or D-pantoyl lactone is mostly carried out with organic solvent extractionprocess, i.e., is with ethyl acetate
Extractant extracts unreacted L- pantoic acid lactone and has lacked D-pantoyl lactone, and the Pantothenic acid digested then enters water
Phase, then the Pantothenic acid in water phase is subjected to the obtained D-pantoyl lactone that lactonizes.But, it is generally the case that ethyl acetate pair
Recovery rate of D-pantoyl lactone only has 70% or so, therefore, remains on a small amount of D-pantoyl lactone still
In water phase.Certainly, this is for using D-pantoyl lactone, for the technique of final product, there is no what influences, but for D-
For pantoic acid is the technique of desired end product, then it there is a problem that a recovery rate especially recovery rate is slightly lower, so that after
It is continuous also to need to carry out additional purification processing, and cause the wastage of material of D-pantoyl lactone.Although passing through in the prior art
The mode of addition sodium salt further improves ethyl acetate to the recovery rate of D-pantoyl lactone, still, due to the sodium salt of addition
It can be present in water phase, certain puzzlement is also resulted in the purification of subsequent Pantothenic acid.Furthermore using ethyl acetate as extractant
During extracting, it is necessary to the treatment process of heating enzyme deactivation is first passed through, to eliminate D-pantoyl lactone hydrolase to acetic acid second
The negative effect of generation is hydrolyzed in ester, but also extraction process is increasingly complex.Therefore, how from enzymolysis liquid high efficiency extraction D-
Pantoic acid also becomes urgent problem to be solved in Pantothenic acid enzymolysis process.
Summary of the invention
For this purpose, technical problem to be solved by the present invention lies in provide a kind of high efficiency extraction Pantothenic acid from enzymolysis liquid
Method.
In order to solve the above technical problems, a kind of method of high efficiency extraction Pantothenic acid from enzymolysis liquid of the present invention,
Include the following steps：
(1) it takes enzymolysis liquid containing Pantothenic acid to remove thallus through being separated by solid-liquid separation, collects supernatant, it is spare；
(2) organic solvent containing hydrophobic ionic liquid is added into gained supernatant to extract, after stratification, collects
Water phase, as aqueous solution containing Pantothenic acid.
In the step (2), the hydrophobic ionic liquid is hydrophobic amino acid ionic liquid.
In the step (2), the hydrophobic amino acid ionic liquid includes proline methyl ester hexafluorophosphate.
In the step (2), the volume ratio of the hydrophobic ionic liquid and the organic solvent is 3-5：100.
In the step (2), the organic solvent includes ethyl acetate.
In the step (2), the volume ratio of the organic solvent and the supernatant is 1-2：1.
In the step (1), the solid-liquid separation step is filters pressing or centrifugal filtration.
It further include that pretreated step is carried out to the enzymolysis liquid in the step (1).
The pre-treatment step includes the steps that diatomite is added into the enzymolysis liquid to be mixed.
In the step (2), further including will the aqueous solution containing Pantothenic acid that collected be concentrated, the step that refines of crystallization
Suddenly, Pantothenic acid sterling is obtained.
The method of the high efficiency extraction Pantothenic acid of the present invention from enzymolysis liquid, in the base of existing Pantothenic acid extracting method
On plinth, by being advanced optimized to the extractant, so that being extractant to the general solution of D- in enzymolysis liquid using existing ethyl acetate
The extraction of acid is more thorough, and the presence of hydrophobic ionic liquid, effectively improves such as D-pantoyl lactone, DL- pantoic acid
The dissolution situation of lactone and L- pantoic acid lactone in organic phase, so that a recovery rate of ethyl acetate greatly improves, for
The present invention using Pantothenic acid as the technique of Objective extraction object for, effectively eliminate the interference of other substances in water phase after extraction,
So that subsequent refinement treatment process is more simple；Moreover, the method for high efficiency extraction Pantothenic acid of the present invention, hydrophobicity from
The presence of sub- liquid effectively inhibits hydrolysis of the D-pantoyl lactone hydrolase for ethyl acetate, so that entire extracting method
Without carrying out additional destroy the enzyme treatment to enzymolysis liquid, the process of extraction is simplified.
Specific embodiment
Embodiment 1
The enzymolysis liquid that the Pantothenic acid is extracted in the present embodiment is to turn according to art methods enzymatic production and enzymatic hydrolysis
Change and obtain, i.e., producing enzyme fermentation is carried out with wild type Fusarium and obtain the crude enzyme liquid containing D-pantoyl lactone hydrolase.It is described
It is new that culture medium and condition of culture can refer to Southern Yangtze University's soup one《Microbial enzyme method fractionation prepares D-pantoyl lactone》Middle record
Enzyme producing method and enzymatic hydrolysis method for transformation.
Fermentative medium formula is：Glycerol 1%, yeast extract 1%, peptone 0.8%, corn pulp 0.4%, pH8.0, fermentation
Crude enzyme liquid is collected after producing enzyme culture.
DL- pantoic acid lactone prepared by chemical method is configured to the aqueous solution of concentration 200g/L, and is added 30mmol/L's
Ca2+, crude enzyme liquid conversion enzymatic hydrolysis 10h is carried out under the conditions of 30, DEG C pH7.0, obtains enzymolysis liquid.Through detecting, contain in gained enzymolysis liquid
The content for having Pantothenic acid is 35.06g/L.
Described in the present embodiment from enzymolysis liquid high efficiency extraction Pantothenic acid method, include the following steps：
(1) enzymolysis liquid containing Pantothenic acid obtained above is taken, the diatomite mixing for being added 2% carries out pretreatment 10min, with
By centrifugation removal thallus and diatomite, supernatant is collected, it is spare；
(2) according to 1：The acetic acid second containing proline methyl ester hexafluorophosphate is added into gained supernatant for 1 volume ratio
Ester (volume ratio 3：100) mixing is extracted, and is mixed well rear stratification, is then collected water phase, as water containing Pantothenic acid
Solution.Through detecting, in gained aqueous solution containing Pantothenic acid, content < 0.05g/L, the DL- pantoic acid lactone of D-pantoyl lactone
Content < 0.02g/L, L- pantoic acid lactone content < 0.02g/L, and the content > 35g/L of the Pantothenic acid.As it can be seen that
The method of the high efficiency extraction Pantothenic acid of the present invention from enzymolysis liquid can effectively promote the one of Pantothenic acid and other substances
Secondary property extraction and separation, and improve extraction efficiency.
Pantothenic acid sterling can be obtained through Conventional concentration, crystallization in gained aqueous solution containing Pantothenic acid.
Embodiment 2
Described in the present embodiment from enzymolysis liquid high efficiency extraction Pantothenic acid method, extract enzymolysis liquid and 1 phase of embodiment
Together.
Described in the present embodiment from enzymolysis liquid high efficiency extraction Pantothenic acid method, include the following steps：
(1) enzymolysis liquid containing Pantothenic acid obtained above is taken, the diatomite mixing for being added 2% carries out pretreatment 10min, with
By centrifugation removal thallus and diatomite, supernatant is collected, it is spare；
(2) according to 2：The acetic acid second containing proline methyl ester hexafluorophosphate is added into gained supernatant for 1 volume ratio
Ester (volume ratio 5：100) mixing is extracted, and is mixed well rear stratification, is then collected water phase, as water containing Pantothenic acid
Solution.Through detecting, in gained aqueous solution containing Pantothenic acid, content < 0.05g/L, the DL- pantoic acid lactone of D-pantoyl lactone
Content < 0.02g/L, L- pantoic acid lactone content < 0.02g/L, and the content > 35g/L of the Pantothenic acid.As it can be seen that
The method of the high efficiency extraction Pantothenic acid of the present invention from enzymolysis liquid can effectively promote the one of Pantothenic acid and other substances
Secondary property extraction and separation, and improve extraction efficiency.
Embodiment 3
Described in the present embodiment from enzymolysis liquid high efficiency extraction Pantothenic acid method, extract enzymolysis liquid and 1 phase of embodiment
Together.
Described in the present embodiment from enzymolysis liquid high efficiency extraction Pantothenic acid method, include the following steps：
(1) enzymolysis liquid containing Pantothenic acid obtained above is taken, the diatomite mixing for being added 2% carries out pretreatment 10min, with
By centrifugation removal thallus and diatomite, supernatant is collected, it is spare；
(2) according to 1.5：The acetic acid containing proline methyl ester hexafluorophosphate is added into gained supernatant for 1 volume ratio
Ethyl ester (volume ratio 4：100) mixing is extracted, and is mixed well rear stratification, is then collected water phase, and Pantothenic acid is as contained
Aqueous solution.Through detecting, in gained aqueous solution containing Pantothenic acid, in content < 0.03g/L, the DL- pantoic acid of D-pantoyl lactone
The content < 0.02g/L of content < 0.02g/L, the L- pantoic acid lactone of ester, and the content > 35g/L of the Pantothenic acid.It can
See, the method for the high efficiency extraction Pantothenic acid of the present invention from enzymolysis liquid can effectively promote Pantothenic acid and other substances
Disposable extraction and separation, and improve extraction efficiency.
Embodiment 4
The fermentation liquid of the hydrolase containing D-pantoyl lactone described in the present embodiment, is made in accordance with the following steps：
(1) oese of the Fusarium moniliforme by the LB plate culture and activation culture a ring to seed is inoculated with to train
It supports and carries out seed activation in base, control cultivation temperature is 28 DEG C, controls shaking speed 150rpm, and incubation time 10h is planted
Sub- liquid；
The seed culture medium includes：Glycerol 8g/L, water chestnut starch 25g/L, peptone 8g/L, asparagine 5g/L, sulfuric acid
Ammonium 3g/L adjusts pH7.0-8.0；The water chestnut slurry is, using the water chestnut for cleaning peeling as raw material, addition equivalent amount of water is through obtained by slurrying；
(2) seed liquor after activation is seeded to the 5L fermentor equipped with 2.5L fermentation medium according to 10% inoculum concentration
Middle carry out fermented and cultured, control cultivation temperature is 28 DEG C, mixing speed 150rpm, ventilatory capacity 1.5vvm, and incubation time is
40h obtains zymocyte liquid；
The fermentation medium includes：Glucose 25g/L, glycerol 8g/L, water chestnut slurry 35g/L, without amino nitrogen source 10g/L,
Yeast extract 10g/L, sodium citrate 5g/L, valine nitrate 6g/L, propolis 10g/L, antierythrite 12g/L, MgSO4.7H2O
0.6g/L, K2HPO41.0g/L adjusts pH7.0-8.0；
In fermentation process, start the glucose feed supplement for carrying out 150g/L after fermentation reaction 12h, control feed rate is
10L/h。
It takes chemically synthesized DL- pantoic acid lactone to be configured to 10% aqueous solution, and is added and accounts for the DL- pantoic acid lactone
The calcium acetate of additive amount 1% is 7.0 with NaOH or HCl solution tune pH, as enzymic catalytic reaction substrate.
It takes obtained fermentation liquid in the present embodiment to filter through Buchner funnel filter paper, obtains wet thallus；According to 1g wet thallus/
The wet thallus is added in the additive amount of 100mL substrate solution, and controlling pH is 7.0, in 28 DEG C, controls shaking table 160r/min, into
Row enzymatic conversion reaction 8 hours, obtain enzymolysis liquid.Through detecting, the content of Pantothenic acid is 31.83g/L in the enzymolysis liquid.
Described in the present embodiment from enzymolysis liquid high efficiency extraction Pantothenic acid method, include the following steps：
(1) enzymolysis liquid containing Pantothenic acid obtained above is taken, the diatomite mixing for being added 2% carries out pretreatment 10min, with
By centrifugation removal thallus and diatomite, supernatant is collected, it is spare；
(2) according to 1.5：The acetic acid containing proline methyl ester hexafluorophosphate is added into gained supernatant for 1 volume ratio
Ethyl ester (volume ratio 4：100) mixing is extracted, and is mixed well rear stratification, is then collected water phase, and Pantothenic acid is as contained
Aqueous solution.Through detecting, in gained aqueous solution containing Pantothenic acid, in content < 0.03g/L, the DL- pantoic acid of D-pantoyl lactone
The content < 0.02g/L of content < 0.02g/L, the L- pantoic acid lactone of ester, and the content > 31.75g/L of the Pantothenic acid.
As it can be seen that the method for the high efficiency extraction Pantothenic acid of the present invention from enzymolysis liquid can effectively promote Pantothenic acid and other substances
Disposable extraction and separation, and improve extraction efficiency.
Embodiment 5
The fermentation liquid of the hydrolase containing D-pantoyl lactone described in the present embodiment is same as Example 4.
It takes chemically synthesized DL- pantoic acid lactone to be configured to 70% aqueous solution, and is added and accounts for the DL- pantoic acid lactone
The calcium acetate of additive amount 2% is 7.0 with NaOH or HCl solution tune pH, as enzymic catalytic reaction substrate.
Fermentation liquid is made in Example 4 to filter through Buchner funnel filter paper, obtains wet thallus；According to 1g wet thallus/100mL
The wet thallus is added in the additive amount of substrate solution, and controlling pH is 7.0, in 28 DEG C, controls shaking table 160r/min, carries out enzyme and turns
Change reaction 8 hours.Through detecting, the content of Pantothenic acid is 207.72g/L in the enzymolysis liquid.
Described in the present embodiment from enzymolysis liquid high efficiency extraction Pantothenic acid method, include the following steps：
(1) enzymolysis liquid containing Pantothenic acid obtained above is taken, the diatomite mixing for being added 2% carries out pretreatment 10min, with
By centrifugation removal thallus and diatomite, supernatant is collected, it is spare；
(2) according to 1.5：The acetic acid containing proline methyl ester hexafluorophosphate is added into gained supernatant for 1 volume ratio
Ethyl ester (volume ratio 4：100) mixing is extracted, and is mixed well rear stratification, is then collected water phase, and Pantothenic acid is as contained
Aqueous solution.Through detecting, in gained aqueous solution containing Pantothenic acid, in content < 0.03g/L, the DL- pantoic acid of D-pantoyl lactone
The content < 0.02g/L of content < 0.02g/L, the L- pantoic acid lactone of ester, and the content > 207.5g/L of the Pantothenic acid.
As it can be seen that the method for the high efficiency extraction Pantothenic acid of the present invention from enzymolysis liquid can effectively promote Pantothenic acid and other substances
Disposable extraction and separation, and improve extraction efficiency.
Embodiment 6
The fermentation liquid of the hydrolase containing D-pantoyl lactone described in the present embodiment is same as Example 4.
It takes chemically synthesized DL- pantoic acid lactone to be configured to 40% aqueous solution, and is added and accounts for the DL- pantoic acid lactone
The calcium acetate of additive amount 1.5% is 7.0 with NaOH or HCl solution tune pH, as enzymic catalytic reaction substrate.
Fermentation liquid is made in Example 3 to filter through Buchner funnel filter paper, obtains wet thallus；According to 1g wet thallus/100mL
The wet thallus is added in the additive amount of substrate solution, and controlling pH is 7.0, in 28 DEG C, controls shaking table 160r/min, carries out enzyme and turns
Change reaction 8 hours.Through detecting, the content of Pantothenic acid is 128.38g/L in the enzymolysis liquid.
Described in the present embodiment from enzymolysis liquid high efficiency extraction Pantothenic acid method, include the following steps：
(1) enzymolysis liquid containing Pantothenic acid obtained above is taken, the diatomite mixing for being added 2% carries out pretreatment 10min, with
By centrifugation removal thallus and diatomite, supernatant is collected, it is spare；
(2) according to 1.5：The acetic acid containing proline methyl ester hexafluorophosphate is added into gained supernatant for 1 volume ratio
Ethyl ester (volume ratio 4：100) mixing is extracted, and is mixed well rear stratification, is then collected water phase, and Pantothenic acid is as contained
Aqueous solution.Through detecting, in gained aqueous solution containing Pantothenic acid, in content < 0.03g/L, the DL- pantoic acid of D-pantoyl lactone
The content < 0.02g/L of content < 0.02g/L, the L- pantoic acid lactone of ester, and the content > 128g/L of the Pantothenic acid.It can
See, the method for the high efficiency extraction Pantothenic acid of the present invention from enzymolysis liquid can effectively promote Pantothenic acid and other substances
Disposable extraction and separation, and improve extraction efficiency.
Comparative example 1
Enzymolysis liquid described in the present embodiment and from enzymolysis liquid high efficiency extraction Pantothenic acid method with embodiment 3, area
It is not only that, the extractant is only ethyl acetate.Through detecting, in gained water phase, the content > 5g/L of D-pantoyl lactone,
The content > 20g/L of content > 30g/L, the L- pantoic acid lactone of DL- pantoic acid lactone, and the content < of the Pantothenic acid
32g/L。
Comparative example 2
Enzymolysis liquid described in the present embodiment and from enzymolysis liquid high efficiency extraction Pantothenic acid method with embodiment 3, area
It is not only that, the extractant is the ethyl acetate that saturated sodium-chloride is added.Through detecting, in gained water phase, in Pantothenic acid
The content > 14g/L of content > 20g/L, the L- pantoic acid lactone of content > 3g/L, the DL- pantoic acid lactone of ester, and the D-
The content < 32g/L of pantoic acid.
It can be seen that the method for the high efficiency extraction Pantothenic acid of the present invention from enzymolysis liquid, by molten to the extraction
Agent advanced optimizes, so that it is more thorough as extraction of the extractant to Pantothenic acid in enzymolysis liquid using existing ethyl acetate, and dredge
The presence of aqueous ionic liquid, effectively improving such as D-pantoyl lactone, DL- pantoic acid lactone and L- pantoic acid lactone is having
Dissolution situation in machine phase a, so that recovery rate of ethyl acetate greatly improves, for the present invention using Pantothenic acid as target
For the technique of extract, the interference of other substances in water phase after extracting effectively is eliminated, so that subsequent refinement treatment process
It is more simple；Moreover, the method for high efficiency extraction Pantothenic acid of the present invention, the presence of hydrophobic ionic liquid effectively inhibit
Hydrolysis of the D-pantoyl lactone hydrolase for ethyl acetate so that entire extracting method be not necessarily to carry out enzymolysis liquid it is additional
Destroy the enzyme treatment simplifies the process of extraction.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or
It changes still within the protection scope of the invention.
Claims (10)
1. a kind of method of the high efficiency extraction Pantothenic acid from enzymolysis liquid, which is characterized in that include the following steps：
(1) it takes enzymolysis liquid containing Pantothenic acid to remove thallus through being separated by solid-liquid separation, collects supernatant, it is spare；
(2) organic solvent containing hydrophobic ionic liquid is added into gained supernatant to extract, after stratification, collects water
Phase, as aqueous solution containing Pantothenic acid.
2. the method for the high efficiency extraction Pantothenic acid according to claim 1 from enzymolysis liquid, which is characterized in that the step
(2) in, the hydrophobic ionic liquid is hydrophobic amino acid ionic liquid.
3. the method for the high efficiency extraction Pantothenic acid according to claim 2 from enzymolysis liquid, which is characterized in that the step
(2) in, the hydrophobic amino acid ionic liquid includes proline methyl ester hexafluorophosphate.
4. the method for the high efficiency extraction Pantothenic acid according to claim 1-3 from enzymolysis liquid, which is characterized in that
In the step (2), the volume ratio of the hydrophobic ionic liquid and the organic solvent is 3-5：100.
5. the method for the high efficiency extraction Pantothenic acid according to claim 1-4 from enzymolysis liquid, which is characterized in that
In the step (2), the organic solvent includes ethyl acetate.
6. the method for the high efficiency extraction Pantothenic acid according to claim 1-5 from enzymolysis liquid, which is characterized in that
In the step (2), the volume ratio of the organic solvent and the supernatant is 1-2：1.
7. the method for the high efficiency extraction Pantothenic acid according to claim 1-6 from enzymolysis liquid, which is characterized in that
In the step (1), the solid-liquid separation step is filters pressing or centrifugal filtration.
8. the method for the high efficiency extraction Pantothenic acid according to claim 1-7 from enzymolysis liquid, which is characterized in that
It further include that pretreated step is carried out to the enzymolysis liquid in the step (1).
9. the method for the high efficiency extraction Pantothenic acid according to claim 8 from enzymolysis liquid, which is characterized in that the pre- place
Reason step includes the steps that diatomite is added into the enzymolysis liquid to be mixed.
10. the method for -9 described in any item high efficiency extraction Pantothenic acids from enzymolysis liquid, feature exist according to claim 1
In, in the step (2), further include the steps that will the aqueous solution containing Pantothenic acid that collected it is concentrated, crystallization refine, obtain
Pantothenic acid sterling.
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