CN118125980A - Method for separating and purifying histidine betaine - Google Patents
Method for separating and purifying histidine betaine Download PDFInfo
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- GPPYTCRVKHULJH-QMMMGPOBSA-N N(alpha),N(alpha),N(alpha)-trimethyl-L-histidine Chemical compound C[N+](C)(C)[C@H](C([O-])=O)CC1=CNC=N1 GPPYTCRVKHULJH-QMMMGPOBSA-N 0.000 title claims abstract description 64
- 238000000034 method Methods 0.000 title claims abstract description 48
- 238000000746 purification Methods 0.000 claims abstract description 32
- 239000003957 anion exchange resin Substances 0.000 claims abstract description 24
- 238000011068 loading method Methods 0.000 claims abstract description 21
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 claims abstract description 19
- 238000000926 separation method Methods 0.000 claims abstract description 18
- 239000007788 liquid Substances 0.000 claims abstract description 11
- 239000006228 supernatant Substances 0.000 claims abstract description 11
- 239000003960 organic solvent Substances 0.000 claims abstract description 9
- 238000005119 centrifugation Methods 0.000 claims abstract description 6
- 238000011049 filling Methods 0.000 claims abstract description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 87
- 239000011347 resin Substances 0.000 claims description 28
- 229920005989 resin Polymers 0.000 claims description 28
- 238000010828 elution Methods 0.000 claims description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical group CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 20
- 239000008213 purified water Substances 0.000 claims description 19
- 230000008929 regeneration Effects 0.000 claims description 10
- 238000011069 regeneration method Methods 0.000 claims description 10
- 230000009466 transformation Effects 0.000 claims description 10
- 239000003208 petroleum Substances 0.000 claims description 9
- 230000007935 neutral effect Effects 0.000 claims description 8
- 230000008569 process Effects 0.000 claims description 7
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 6
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 4
- 239000003513 alkali Substances 0.000 claims description 2
- SSISHJJTAXXQAX-ZETCQYMHSA-N L-ergothioneine Chemical compound C[N+](C)(C)[C@H](C([O-])=O)CC1=CNC(=S)N1 SSISHJJTAXXQAX-ZETCQYMHSA-N 0.000 abstract description 22
- 229940093497 ergothioneine Drugs 0.000 abstract description 22
- 239000003480 eluent Substances 0.000 abstract description 8
- 239000004480 active ingredient Substances 0.000 abstract description 3
- 238000007670 refining Methods 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 88
- 238000004128 high performance liquid chromatography Methods 0.000 description 18
- 238000006243 chemical reaction Methods 0.000 description 17
- 108090000790 Enzymes Proteins 0.000 description 16
- 102000004190 Enzymes Human genes 0.000 description 16
- 238000001179 sorption measurement Methods 0.000 description 13
- 239000012295 chemical reaction liquid Substances 0.000 description 9
- 150000001413 amino acids Chemical class 0.000 description 7
- 238000000605 extraction Methods 0.000 description 7
- 102100027324 2-hydroxyacyl-CoA lyase 1 Human genes 0.000 description 6
- 101001009252 Homo sapiens 2-hydroxyacyl-CoA lyase 1 Proteins 0.000 description 6
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 6
- 239000000758 substrate Substances 0.000 description 6
- VUQUOGPMUUJORT-UHFFFAOYSA-N methyl 4-methylbenzenesulfonate Chemical compound COS(=O)(=O)C1=CC=C(C)C=C1 VUQUOGPMUUJORT-UHFFFAOYSA-N 0.000 description 5
- HYHCSLBZRBJJCH-UHFFFAOYSA-N sodium polysulfide Chemical compound [Na+].S HYHCSLBZRBJJCH-UHFFFAOYSA-N 0.000 description 5
- 238000006555 catalytic reaction Methods 0.000 description 4
- 238000006911 enzymatic reaction Methods 0.000 description 4
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000003963 antioxidant agent Substances 0.000 description 3
- 230000003078 antioxidant effect Effects 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 229910052979 sodium sulfide Inorganic materials 0.000 description 3
- GRVFOGOEDUUMBP-UHFFFAOYSA-N sodium sulfide (anhydrous) Chemical compound [Na+].[Na+].[S-2] GRVFOGOEDUUMBP-UHFFFAOYSA-N 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 108060004795 Methyltransferase Proteins 0.000 description 2
- 102000016397 Methyltransferase Human genes 0.000 description 2
- MEFKEPWMEQBLKI-AIRLBKTGSA-N S-adenosyl-L-methioninate Chemical compound O[C@@H]1[C@H](O)[C@@H](C[S+](CC[C@H](N)C([O-])=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 MEFKEPWMEQBLKI-AIRLBKTGSA-N 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- KWIUHFFTVRNATP-UHFFFAOYSA-N glycine betaine Chemical compound C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 description 2
- 229910017053 inorganic salt Inorganic materials 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 239000000376 reactant Substances 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000011593 sulfur Substances 0.000 description 2
- 229910052717 sulfur Inorganic materials 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- FVNKWWBXNSNIAR-BYPYZUCNSA-N (2s)-2-amino-3-(2-sulfanylidene-1,3-dihydroimidazol-4-yl)propanoic acid Chemical compound OC(=O)[C@@H](N)CC1=CNC(=S)N1 FVNKWWBXNSNIAR-BYPYZUCNSA-N 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 241001532621 Methanosalsum zhilinae Species 0.000 description 1
- 241000221961 Neurospora crassa Species 0.000 description 1
- 108700040121 Protein Methyltransferases Proteins 0.000 description 1
- 102000055027 Protein Methyltransferases Human genes 0.000 description 1
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229960003237 betaine Drugs 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
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- 239000002537 cosmetic Substances 0.000 description 1
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- 238000011033 desalting Methods 0.000 description 1
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- 229940079593 drug Drugs 0.000 description 1
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- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 229920001021 polysulfide Polymers 0.000 description 1
- 239000005077 polysulfide Substances 0.000 description 1
- 150000008117 polysulfides Polymers 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
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- 230000002000 scavenging effect Effects 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
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- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Landscapes
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention belongs to the technical field of active ingredient refining, and discloses a separation and purification method of histidine betaine. The purification method comprises the following steps: extracting histidine betaine to be purified by adopting an organic solvent, layering, taking a lower layer liquid, and then obtaining a supernatant by centrifugation to obtain an extract; filling a chromatographic column with a strong-alkaline anion exchange resin, loading the extract into the chromatographic column, and eluting with a soluble bicarbonate solution to obtain purified histidine betaine; the mass fraction of the soluble bicarbonate solution is 0.05% -5%. According to the separation and purification method provided by the invention, through optimizing the purification step, the soluble bicarbonate solution is selected as the eluent, and the concentration of the eluent is controlled, so that the yield and purity of histidine betaine can be improved, the separation and purification step is simplified, and the purification efficiency is improved; but also can separate and purify unreacted histidine betaine from the synthesized ergothioneine.
Description
Technical Field
The invention belongs to the technical field of active ingredient refining, and particularly relates to a separation and purification method of histidine betaine.
Background
Ergothioneine (Ergothioneine, EGT for short) is an important active substance in the body and is a special amino acid of thiohistidine betaine. Foreign researches show that the natural antioxidant can play a role in an antioxidant in organisms, has various physiological functions of scavenging free radicals, maintaining DNA biosynthesis, normal growth of cells, cellular immunity and the like, is a natural antioxidant and a potential nutritional food, and has great application prospect in industries such as foods, cosmetics, medicines and the like.
Histidine betaine is an important intermediate in the process of synthesizing ergothioneine, for example, histidine (His) is firstly used as a main substrate and a methyl donor is used as an auxiliary substrate in the process of synthesizing ergothioneine, histidine betaine (TMH) is firstly prepared under the catalysis of EgtD enzyme and methyltransferase, and then the ergothioneine is synthesized by utilizing the histidine betaine. In the synthesis process, the prepared histidine betaine is purified firstly, so that the catalytic efficiency of the subsequent reaction is improved; meanwhile, after the ergothioneine is synthesized, unreacted histidine betaine is separated and purified, so that the waste of resources can be avoided, and the cost is saved. However, the current method for separating and purifying histidine betaine is less, and the purification process is complicated, the efficiency is low, and the yield is low; and unreacted histidine betaine cannot be separated and purified from the synthesized ergothioneine.
Therefore, it is highly desirable to provide a method for separating and purifying histidine betaine, which can not only improve purification efficiency and histidine betaine yield, but also separate and purify unreacted histidine betaine from the synthesized ergothioneine.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the invention provides a separation and purification method of histidine betaine (TMH). The method provided by the invention not only can improve the purification efficiency and the histidine betaine yield, but also can separate and purify unreacted histidine betaine from the synthesized ergothioneine.
The invention provides a separation and purification method of histidine betaine (TMH).
Specifically, the separation and purification method of histidine betaine comprises the following steps:
Extracting histidine betaine to be purified by adopting an organic solvent, layering, taking a lower layer liquid, and then obtaining a supernatant by centrifugation to obtain an extract;
filling a chromatographic column with a strong-alkali anion exchange resin, loading the extract into the chromatographic column, and eluting with a soluble bicarbonate solution to obtain purified histidine betaine;
The mass fraction of the soluble bicarbonate solution is 0.05% -5%.
It is understood that the histidine betaine to be purified is crude histidine betaine produced by an enzymatic method or reaction liquid containing histidine betaine obtained by an enzymatic method or a chemical-enzymatic coupling method. The method comprises the steps of (1) preparing histidine betaine (TMH) reaction liquid by using histidine (His) as a main substrate and a methyl donor as an auxiliary substrate under the catalysis of EgtD enzyme and methyltransferase; the reaction liquid containing histidine betaine is prepared by the enzyme method, and the reaction liquid containing histidine betaine is prepared by the enzyme method.
If histidine (His) is used as a main substrate and methyl p-toluenesulfonate (MeOTs) is used as a side substrate, histidine betaine (TMH) is prepared under the catalysis of EgtD enzyme and methyltransferase (HMT enzyme); then polysulfide is added, and Ergothioneine (EGT) is generated under the catalysis of St enzyme (sulfur transfer enzyme), wherein the amino acid sequence of EgtD enzyme is shown as SEQ ID NO. 1, the amino acid sequence of methyl transfer enzyme is shown as SEQ ID NO. 2, and the amino acid sequence of St enzyme (sulfur transfer enzyme) is shown as SEQ ID NO. 3. The reaction liquid of the prepared histidine betaine and the reaction liquid of the generated ergothioneine can be purified by adopting the method provided by the invention.
SEQ ID NO：1：NADIIDIRRVAVEINLKTEITSMFRPKDGPRQLPTLLLYN ERGLQLFERITYLEEYYLTNDEIKILTKHATEMASFIPSGAMIIELGSGNLRKVNLLLEALDNAGKAIDYYALDLSREELERTLAQVPSYKHVKCHGLLGTYDDGRDWLKAPENINKQKCILHLGSSIGNFNRSDAATFLKGFTDVLGPNDKMLIGVDACNDPARVYHAYNDKVGITHEFILNGLRNANEIIGETAFIEGDWRVIGEYVYDEEGGRHQAFYAPTRDTMVMGELIRSHDRIQIEQSLKYSKEESERLWSTAGLEQVSEWTYGNEYGLHLLAKSR( Source Neurospora crassa OR a);
SEQ ID NO：2：STPSLIPSGVHEVLAKYKDGNYVDGWAELWDKSKGDR LPWDRGFPNPALEDTLIQKRAIIGGPLGQDAQGKTYRKKALVPGCGRGVDVLLLASFGYDAYGLEYSATAVDVCQEEQAKNGDQYPVRDAEIGQGKITFVQGDFFEDTWLEKLNLTRNCFDVIYDYTFFCALNPSMRPQWALRHTQLLADSPRGHLICLEFPRHKDPSVQGPPWGSASEAYRAHLSHPGEEIPYDASRQCQFDSSKAPSAQGLERVAYWQPERTHEVGKNEKGEVQDRVSIWQRPPQSSL( Source Aspergillu s clavatus NRRL 1);
SEQ ID NO：3：MKSISTDELLENLHRYKVIDIRSVDAYNGWKENGENR GGHIRSAKSLPYKWVNYIDWIEIVRSKDILPQDKLVIYGYDSEKAEEVARMFEKAGYTDLNIYPSFFEWVERNLPMDRLERYRHLVSPDWLNQLITTDNAPEYDNDKYVICHCHYRNPVDYEKGHIPGSIPLDTNSLESEDTWNRRSPEELKDALENAGISSETTVIVYGRFSYPKNDDPFPGSSAGHLGAMRCAFIMLYAGVKDVRILNGGLQSWLDAGYNVTTEPAKISKVSFGANIPLNPKIAVDLEEAKEILSDPGKKLVSVRSWREYIGEVSGYNYIEKKGRIPGSVFGDCGTDAYHMENYRNLDHTMREYHEIEDKWKELGITPEKRNAFYCGTGWRGSEAFLNAWLMGWDNAAVFDGGWFEWSNNDLPFETGVPEK( Source Methanosalsum zhilinae).
Preferably, the soluble bicarbonate solution has a mass fraction of 0.1% -3%; further preferably, the soluble bicarbonate solution has a mass fraction of 0.1% -2%.
Preferably, the soluble bicarbonate comprises at least one of NaHCO 3 solution, KHCO 3 solution, NH 4HCO3 solution; further preferably, the soluble bicarbonate is NaHCO 3 solution.
Preferably, the organic solvent is at least one selected from petroleum ether, diethyl ether, ethyl acetate and chloroform; further preferably, the organic solvent is petroleum ether.
Preferably, the centrifugation is performed by: centrifuging at 0-5deg.C at 5000-9000rpm for 10-30min; further preferably, the centrifugation is performed by: centrifuging at 7000-9000rpm and 0-5deg.C for 15-30min. The invention destroys the protein in the reaction liquid through the extraction process, so that the protein is denatured and emulsified, and then the protein in the reaction liquid can be effectively removed through low-temperature high-speed centrifugation, and the loss of active ingredients can be avoided.
Preferably, the strongly basic anion exchange resin is a D201 strongly basic anion exchange resin or a D301 strongly basic anion exchange resin.
Preferably, the strongly basic anion exchange resin is subjected to a resin transformation treatment prior to loading. Specifically, the resin transformation process comprises the following steps: the column was loaded with a strongly basic anion exchange resin and eluted sequentially with NaOH solution and purified water until the effluent conductivity was <5 uS/cm.
Preferably, the mass fraction of the NaOH solution is 1% -8%; further preferably, the mass fraction of the NaOH solution is 2% -5%.
Preferably, when the elution is carried out by using a soluble bicarbonate solution, the flow rate of the elution is 5-20mL/min; further preferably, the flow rate of the elution is 8-15mL/min.
Preferably, after the elution is completed, the strongly basic anion exchange resin may be subjected to a resin regeneration treatment. The resin regeneration process comprises the following steps: the elution is carried out sequentially with sodium hydroxide solution and purified water until the effluent is neutral. The regenerated resin after treatment can be reused without affecting the eluting effect.
Preferably, the mass fraction of the NaOH solution is 1% -8%; further preferably, the mass fraction of the NaOH solution is 2% -5%.
In order to further remove the inorganic salt impurities in the histidine betaine, a desalting treatment can be performed subsequently, and the treatment is a conventional treatment as long as the inorganic salt impurities can be further removed.
More specifically, the separation and purification method of histidine betaine (TMH) comprises the following steps:
(1) Extracting histidine betaine to be purified by adopting an organic solvent, layering, taking a lower layer liquid, centrifuging the lower layer liquid at the rotating speed of 5000-9000 and the temperature of 0-5 ℃ for 10-30min, and collecting supernatant to obtain an extract;
(2) Loading the strong-alkaline anion exchange resin into a chromatographic column, eluting by using NaOH solution and purified water in sequence until the conductivity of effluent liquid is less than 5 uS/cm; and (2) loading the extract prepared in the step (1) into the chromatographic column, and eluting with a soluble bicarbonate solution with the mass fraction of 0.05% -5% at a flow rate of 5-20mL/min to obtain the separated and purified histidine betaine.
The structure of the histidine betaine (TMH) has amphoteric groups, under the condition of alkalescence, TMH is negatively charged and can be adsorbed by strong-alkalinity anion exchange resin, and then soluble bicarbonate (which can provide HCO 3 -) is adopted for elution, HCO 3 - interacts with the resin, so that the histidine betaine is separated from impurities, and the aim of purification is achieved.
Compared with the prior art, the invention has the beneficial effects that:
the separation and purification method provided by the invention comprises the steps of extracting histidine betaine to be purified by using an organic solvent, filling a chromatographic column by using a strong-alkaline anion exchange resin, and eluting by using a soluble bicarbonate solution. The step of optimizing separation and purification is adopted, the soluble bicarbonate solution is selected as the eluent, and the concentration of the eluent is controlled, so that the yield and purity of histidine betaine can be improved, the separation and purification steps are simplified, and the purification efficiency is improved; but also can separate and purify unreacted histidine betaine from the synthesized ergothioneine.
Detailed Description
In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples will be presented. It should be noted that the following examples do not limit the scope of the invention.
The starting materials, reagents or apparatus used in the following examples are all available from conventional commercial sources or may be obtained by methods known in the art unless otherwise specified.
The TMH reaction solution to be purified used in the following examples and comparative examples may be crude histidine betaine produced by an enzymatic method or a reaction solution containing histidine betaine produced by an enzymatic method. The method comprises the following steps:
S1, preparing a reaction solution (the total reaction volume is 1L): 15.51g of histidine (His, concentration 100 mM), 587mg of adenosylmethionine p-toluenesulfonate (SAM, concentration 0.75 mM) and 3.75g of glycine (Gly, concentration 50 mM) were weighed, 800mL of pure water was taken and dissolved, and 120mL of methanol was added; then, pH was adjusted to 9.0 with 5M NaOH, and after constant volume to 1000mL, 11.17g MeOTs (60 mM concentration) was added, and finally, the mixture was preheated at 30℃for further use.
S2, adding 24.0g EgtD enzyme (the amino acid sequence is shown as SEQ ID NO:1, the concentration is 1.5g/L, BZ002-DRN-1 is marked, the enzyme load is 53.52 mg/g) and 50.6gHMT enzyme (the amino acid sequence is shown as SEQ ID NO:2, the concentration is 3g/L, BZ018-DRN-1 is marked, the enzyme load is 59.22 mg/g) into the reaction liquid prepared in the step S1, and adjusting the pH once for 0.5h to maintain the pH value to be 9.0; after 2h and 4h reactions 60mM MeOTs was added to give a final MeOTs concentration of 180mM. Sampling in 2h, 8h and 24h, diluting with 50% acetonitrile water for 100 times, and measuring His and TMH content to obtain histidine betaine (TMH reaction solution), and storing at 4deg.C for use.
S3, preparing sodium polysulfide: under the monitoring of an infrared thermal imager, preheating an electric furnace to 110 ℃, adding 5.5g of sublimed sulfur powder into a crucible, stirring until the sublimed sulfur powder is completely melted to light yellow liquid, adding 40g of sodium sulfide crystals into the crucible containing the melted sublimed sulfur powder, stirring until the sodium sulfide is completely melted, keeping stirring for 20min until the solid sodium sulfide in the reactor disappears, completely converting the light yellow melted sublimed sulfur into red brown to black liquid, and keeping the temperature (110 ℃) for 20min until the reactants in the crucible are fully combined, closing the reactor, cooling the reactor to room temperature to crystallize the reactants into solid, thus obtaining the sodium polysulfide.
Preparing 200g/L sodium polysulfide, regulating the pH to 9.0 by using HCl, and filtering by using a 0.22 mu m filter head to obtain a clear solution for later use; taking 2mL of sodium polysulfide solution with the concentration of 200g/L and 84mL of TMH reaction solution (with the concentration of 51 mM) prepared in the step S2, regulating the initial pH to 9.12 to 9 by using 5M HCl, and preheating at 30 ℃ to obtain a mixed solution;
S4, adding 13.15g St enzyme (the concentration is 4g/L, the amino acid sequence is shown as SEQ ID NO:3 and is marked as BZ 006-DRN-1) into the mixed solution obtained in the step S3, and adjusting the pH value every 0.5h to keep the pH value at 9.0; 2mL of 200g/L sodium polysulfide solution was added every 3 hours of reaction, 7 times in total. After the reaction, the ergothioneine solution is obtained.
Example 1
A method for purifying histidine betaine (TMH), comprising the steps of:
(1) Extraction: extracting TMH reaction solution to be purified (ergothioneine solution prepared in step S4 and containing unreacted histidine betaine) with petroleum ether of equal volume, standing for layering, taking out the lower layer, and centrifuging at 8000rpm and 4deg.C for 20min; centrifuging to obtain supernatant, and preparing into extractive solution;
(2) Resin transformation: d201 strongly basic anion exchange resin was loaded into a chromatographic column with a column volume of 60mL. 200mL (3 BV, three column volumes) was eluted at 10mL/min using 4% NaOH solution. After the elution is finished, continuing to use purified water to elute until the electric conductivity of the effluent is less than 5 uS/cm;
(3) Loading: loading 6mL of extract (1/10 BV) into a chromatographic column;
(4) Eluting: the purified histidine betaine was obtained by eluting with 0.2% NaHCO 3 solution at a flow rate of 10mL/min, eluting 300mL (5 BV), using 10mL (1/6 BV) as a receiving unit, and performing HPCL analysis to detect TMH content. And combining a plurality of groups of receiving units with larger peak areas and single peak according to the HPLC result, then carrying out HPLC analysis, detecting and determining that TMH is single peak, and calculating the yield and purity. The column was eluted with 4% naoh solution under the conditions as above while detecting the TMH content of the eluate by HPLC until no TMH signal peak was detected in the effluent and the dead adsorption rate of TMH was calculated.
(5) Resin regeneration: 200mL (3 BV) of the solution was eluted at a rate of 10mL/min using 4% NaOH solution, and the elution was continued using purified water until the effluent was neutral. The regenerated resin can be reused.
Example 2
A method for purifying histidine betaine (TMH), comprising the steps of:
(1) Extraction: extracting TMH reaction solution (ergothioneine solution prepared in step S4) to be purified with petroleum ether of equal volume, standing for layering, taking the lower layer, and centrifuging at 8000rpm and 4deg.C for 20min; centrifuging to obtain supernatant, and preparing into extractive solution;
(2) Resin transformation: d201 strongly basic anion exchange resin was loaded into a chromatographic column with a column volume of 60mL. 200mL (3 BV, three column volumes) was eluted at 10mL/min using 4% NaOH solution. After the elution is finished, continuing to use purified water to elute until the electric conductivity of the effluent is less than 5 uS/cm;
(3) Loading: loading 6mL of extract (1/10 BV) into a chromatographic column;
(4) Eluting: the purified histidine betaine was obtained by eluting with 0.1% NaHCO 3 solution at a flow rate of 10mL/min, eluting 300mL (5 BV), using 10mL (1/6 BV) as a receiving unit, and performing HPCL analysis to detect TMH content. And combining a plurality of groups of receiving units with larger peak areas and single peak according to the HPLC result, then carrying out HPLC analysis, detecting and determining that TMH is single peak, and calculating the yield and purity. The column was eluted with 4% naoh solution under the conditions as above while detecting the TMH content of the eluate by HPLC until no TMH signal peak was detected in the effluent and the dead adsorption rate of TMH was calculated.
(5) Resin regeneration: 200mL (3 BV) of the solution was eluted at a rate of 10mL/min using 4% NaOH solution, and the elution was continued using purified water until the effluent was neutral. The regenerated resin can be reused.
Example 3
A method for purifying histidine betaine (TMH), comprising the steps of:
(1) Extraction: extracting TMH reaction solution (ergothioneine solution prepared in step S4) to be purified with petroleum ether of equal volume, standing for layering, taking the lower layer, and centrifuging at 8000rpm and 4deg.C for 20min; centrifuging to obtain supernatant, and preparing into extractive solution;
(2) Resin transformation: d201 strongly basic anion exchange resin was loaded into a chromatographic column with a column volume of 60mL. 200mL (3 BV, three column volumes) was eluted at 10mL/min using 4% NaOH solution. After the elution is finished, continuing to use purified water to elute until the electric conductivity of the effluent is less than 5 uS/cm;
(3) Loading: loading 6mL of extract (1/10 BV) into a chromatographic column;
(4) Eluting: the purified histidine betaine was obtained by eluting with 0.5% NaHCO 3 solution at a flow rate of 10mL/min, eluting 300mL (5 BV), using 10mL (1/6 BV) as a receiving unit, and performing HPCL analysis to detect TMH content. And combining a plurality of groups of receiving units with larger peak areas and single peak according to the HPLC result, then carrying out HPLC analysis, detecting and determining that TMH is single peak, and calculating the yield and purity. The column was eluted with 4% naoh solution under the conditions as above while detecting the TMH content of the eluate by HPLC until no TMH signal peak was detected in the effluent and the dead adsorption rate of TMH was calculated.
(5) Resin regeneration: 200mL (3 BV) of the solution was eluted at a rate of 10mL/min using 4% NaOH solution, and the elution was continued using purified water until the effluent was neutral. The regenerated resin can be reused.
Example 4
A method for purifying histidine betaine (TMH), comprising the steps of:
(1) Extraction: extracting TMH reaction solution (ergothioneine solution prepared in step S4) to be purified with petroleum ether of equal volume, standing for layering, taking the lower layer, and centrifuging at 8000rpm and 4deg.C for 20min; centrifuging to obtain supernatant, and preparing into extractive solution;
(2) Resin transformation: d201 strongly basic anion exchange resin was loaded into a chromatographic column with a column volume of 60mL. 200mL (3 BV, three column volumes) was eluted at 10mL/min using 4% NaOH solution. After the elution is finished, continuing to use purified water to elute until the electric conductivity of the effluent is less than 5 uS/cm;
(3) Loading: loading 6mL of extract (1/10 BV) into a chromatographic column;
(4) Eluting: the purified histidine betaine was obtained by eluting with 1.0% NaHCO 3 solution at a flow rate of 10mL/min, eluting 300mL (5 BV), using 10mL (1/6 BV) as a receiving unit, and performing HPCL analysis to detect TMH content. And combining a plurality of groups of receiving units with larger peak areas and single peak according to the HPLC result, then carrying out HPLC analysis, detecting and determining that TMH is single peak, and calculating the yield and purity. The column was eluted with 4% naoh solution under the conditions as above while detecting the TMH content of the eluate by HPLC until no TMH signal peak was detected in the effluent and the dead adsorption rate of TMH was calculated.
(5) Resin regeneration: 200mL (3 BV) of the solution was eluted at a rate of 10mL/min using 4% NaOH solution, and the elution was continued using purified water until the effluent was neutral. The regenerated resin can be reused.
Example 5
A method for purifying histidine betaine (TMH), comprising the steps of:
(1) Extraction: extracting TMH reaction solution (ergothioneine solution prepared in step S4) to be purified with petroleum ether of equal volume, standing for layering, taking the lower layer, and centrifuging at 8000rpm and 4deg.C for 20min; centrifuging to obtain supernatant, and preparing into extractive solution;
(2) Resin transformation: d201 strongly basic anion exchange resin was loaded into a chromatographic column with a column volume of 60mL. 200mL (3 BV, three column volumes) was eluted at 10mL/min using 4% NaOH solution. After the elution is finished, continuing to use purified water to elute until the electric conductivity of the effluent is less than 5 uS/cm;
(3) Loading: loading 6mL of extract (1/10 BV) into a chromatographic column;
(4) Eluting: the purified histidine betaine was obtained by eluting with 1.5% NaHCO 3 solution at a flow rate of 10mL/min, eluting 300mL (5 BV), using 10mL (1/6 BV) as a receiving unit, and performing HPCL analysis to detect TMH content. And combining a plurality of groups of receiving units with larger peak areas and single peak according to the HPLC result, then carrying out HPLC analysis, detecting and determining that TMH is single peak, and calculating the yield and purity. The column was eluted with 4% naoh solution under the conditions as above while detecting the TMH content of the eluate by HPLC until no TMH signal peak was detected in the effluent and the dead adsorption rate of TMH was calculated.
(5) Resin regeneration: 200mL (3 BV) of the solution was eluted at a rate of 10mL/min using 4% NaOH solution, and the elution was continued using purified water until the effluent was neutral. The regenerated resin can be reused.
Example 6
A method for purifying histidine betaine (TMH), comprising the steps of:
(1) Extraction: extracting TMH reaction solution (ergothioneine solution prepared in step S4) to be purified with petroleum ether of equal volume, standing for layering, taking the lower layer, and centrifuging at 8000rpm and 4deg.C for 20min; centrifuging to obtain supernatant, and preparing into extractive solution;
(2) Resin transformation: d201 strongly basic anion exchange resin was loaded into a chromatographic column with a column volume of 60mL. 200mL (3 BV, three column volumes) was eluted at 10mL/min using 4% NaOH solution. After the elution is finished, continuing to use purified water to elute until the electric conductivity of the effluent is less than 5 uS/cm;
(3) Loading: loading 6mL of extract (1/10 BV) into a chromatographic column;
(4) Eluting: the purified histidine betaine was obtained by eluting with 2.0% NaHCO 3 solution at a flow rate of 10mL/min, eluting 300mL (5 BV), using 10mL (1/6 BV) as a receiving unit, and performing HPCL analysis to detect TMH content. And combining a plurality of groups of receiving units with larger peak areas and single peak according to the HPLC result, then carrying out HPLC analysis, detecting and determining that TMH is single peak, and calculating the yield and purity. The column was eluted with 4% naoh solution under the conditions as above while detecting the TMH content of the eluate by HPLC until no TMH signal peak was detected in the effluent and the dead adsorption rate of TMH was calculated.
(5) Resin regeneration: 200mL (3 BV) of the solution was eluted at a rate of 10mL/min using 4% NaOH solution, and the elution was continued using purified water until the effluent was neutral. The regenerated resin can be reused.
Example 7
Example 7 differs from example 1 in that the TMH reaction solution to be purified used is histidine betaine prepared in step S2, and the rest of the steps are the same as in example 1. The purity after purification was tested separately and the TMH yield and TMH dead adsorption rate were calculated.
Comparative example 1
On the basis of example 1, the eluent 0.2% NaHCO 3 solution was replaced by 0.1%, 0.2%, 1.0% and 4% NaOH solution respectively, and the rest of the procedure was the same as in example 1. The purity after purification was tested separately and the TMH yield and TMH dead adsorption rate were calculated.
Comparative example 2
On the basis of example 1, the eluent 0.2% nahco 3 solution was replaced by 0.2%, 1.0%, 2.0% and 3.0% NH 4 OH solution, respectively, and the rest of the procedure was the same as in example 1. The purity after purification was tested separately and the TMH yield and TMH dead adsorption rate were calculated.
Comparative example 3
The remainder of the procedure was as in example 1, except that the eluent 0.2% NaHCO 3 solution was replaced by purified water on the basis of example 1. The purity after purification was tested separately and the TMH yield and TMH dead adsorption rate were calculated.
Comparative example 4
On the basis of example 1, the eluent 0.2% NaHCO 3 solution was replaced by 8% NaHCO 3 solution, the rest of the procedure being as in example 1. The purity after purification was tested separately and the TMH yield and TMH dead adsorption rate were calculated.
Product effect test
The results of the TMH purity, yield and TMH dead adsorption rate in examples 1-6 and comparative examples 1-4 are shown in tables 1 and 2, after testing and calculation.
TABLE 1
| Group of | TMH yield/% | TMH dead adsorption rate/% | TMH purity/% |
| Example 1 | 94.94 | 4.38 | 98.79 |
| Example 2 | 90.56 | 8.11 | 98.06 |
| Example 3 | 91.20 | 7.21 | 98.56 |
| Example 4 | 93.49 | 5.18 | 97.69 |
| Example 5 | 92.03 | 6.84 | 97.11 |
| Example 6 | 84.17 | 12.82 | 90.32 |
| Example 7 | 94.51 | 3.89 | 98.12 |
TABLE 2
As shown in Table 1, the purification method provided by the invention can effectively improve the yield of TMH, and the yield is more than 84.0%, up to 94.94%, and the purity is more than 90%. When the NaHCO 3 solution is replaced by NaOH solution, NH 4 OH solution or water, the yield is obviously reduced, and the TMH loss is larger.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. The separation and purification method of histidine betaine is characterized by comprising the following steps:
Extracting histidine betaine to be purified by adopting an organic solvent, layering, taking a lower layer liquid, and then obtaining a supernatant by centrifugation to obtain an extract;
filling a chromatographic column with a strong-alkali anion exchange resin, loading the extract into the chromatographic column, and eluting with a soluble bicarbonate solution to obtain purified histidine betaine;
The mass fraction of the soluble bicarbonate solution is 0.05% -5%.
2. The method according to claim 1, wherein the mass fraction of the soluble bicarbonate solution is 0.1% -3%.
3. The separation and purification method according to claim 1 or 2, wherein the organic solvent is at least one selected from petroleum ether, diethyl ether, ethyl acetate, chloroform.
4. The separation and purification method according to claim 1, wherein the centrifugation is performed by: centrifuging at 0-5deg.C at 5000-9000rpm for 10-30min.
5. The separation and purification method according to claim 1 or 2, wherein the strongly basic anion exchange resin is D201 strongly basic anion exchange resin.
6. The method according to claim 1, wherein the strongly basic anion exchange resin is subjected to a resin transformation treatment prior to loading; the resin transformation process comprises the following steps: the column was loaded with a strongly basic anion exchange resin and eluted sequentially with NaOH solution and purified water until the effluent conductivity was <5 uS/cm.
7. The separation and purification method according to claim 6, wherein the mass fraction of the NaOH solution is 1% -8%; further preferably, the mass fraction of the NaOH solution is 2% -5%.
8. The method according to claim 1 or 2, wherein the flow rate of elution is 5-20mL/min when elution is performed with a soluble bicarbonate solution.
9. The separation and purification method according to any one of claims 1, 2 and 6, wherein the strongly basic anion exchange resin is subjected to a resin regeneration treatment after the elution is completed; the resin regeneration process comprises the following steps: the elution is carried out sequentially with sodium hydroxide solution and purified water until the effluent is neutral.
10. The separation and purification method according to any one of claims 1,2, 4, 6, 7, comprising the steps of:
(1) Extracting histidine betaine to be purified by adopting an organic solvent, layering, taking a lower layer liquid, centrifuging the lower layer liquid at the rotating speed of 5000-9000rpm and the temperature of 0-5 ℃ for 10-30min, and collecting supernatant to obtain an extract;
(2) Loading the strong-alkaline anion exchange resin into a chromatographic column, eluting by using NaOH solution and purified water in sequence until the conductivity of effluent liquid is less than 5 uS/cm; and (2) loading the extract prepared in the step (1) into the chromatographic column, and eluting with a soluble bicarbonate solution with the mass fraction of 0.05% -5% at a flow rate of 5-20mL/min to obtain purified histidine betaine.
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