CN111549015B - Process for separating and removing citrinin in nuclease liquid by utilizing chromatographic technique - Google Patents
Process for separating and removing citrinin in nuclease liquid by utilizing chromatographic technique Download PDFInfo
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- CN111549015B CN111549015B CN202010459562.8A CN202010459562A CN111549015B CN 111549015 B CN111549015 B CN 111549015B CN 202010459562 A CN202010459562 A CN 202010459562A CN 111549015 B CN111549015 B CN 111549015B
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- CQIUKKVOEOPUDV-IYSWYEEDSA-N antimycin Chemical compound OC1=C(C(O)=O)C(=O)C(C)=C2[C@H](C)[C@@H](C)OC=C21 CQIUKKVOEOPUDV-IYSWYEEDSA-N 0.000 title claims abstract description 81
- CQIUKKVOEOPUDV-UHFFFAOYSA-N citrinine Natural products OC1=C(C(O)=O)C(=O)C(C)=C2C(C)C(C)OC=C21 CQIUKKVOEOPUDV-UHFFFAOYSA-N 0.000 title claims abstract description 81
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- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 2
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- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 1
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
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- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
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- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 1
- 239000000463 material Substances 0.000 description 1
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- 244000005700 microbiome Species 0.000 description 1
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- 235000013923 monosodium glutamate Nutrition 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
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- 235000019319 peptone Nutrition 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
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- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- 239000011686 zinc sulphate Substances 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
- B01D15/361—Ion-exchange
- B01D15/363—Anion-exchange
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/42—Selective adsorption, e.g. chromatography characterised by the development mode, e.g. by displacement or by elution
- B01D15/424—Elution mode
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- Life Sciences & Earth Sciences (AREA)
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- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
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- Genetics & Genomics (AREA)
- Biotechnology (AREA)
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- Biomedical Technology (AREA)
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- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention discloses a process for separating and removing citrinin in a nuclease liquid by utilizing a chromatographic technique, which comprises the steps of loading the nuclease liquid containing citrinin into a chromatographic column containing anion exchange resin for ion exchange, and collecting effluent liquid to obtain the nuclease liquid from which the citrinin is removed. The method utilizes an ion exchange mechanism, realizes the purpose of removing the citrinin through a fixed bed, ensures that the content of the citrinin in a product liquid is lower than the liquid phase detection limit, improves the enzyme activity of nuclease through a chromatographic separation process to 110 percent, and also has a certain decolorizing effect.
Description
Technical Field
The invention belongs to the technical field of biological separation, and particularly relates to a process for separating and removing citrinin in nuclease liquid by using a chromatographic technique.
Background
A nuclease is an enzyme capable of cleaving the phosphodiester bond of a polynucleotide chain. It belongs to a hydrolase and acts on the P-O position of a phosphodiester bond. A nuclease is a nucleic acid that acts to hydrolyze phosphodiester bonds between nucleotides in the first step of nucleic acid degradation. Nucleases have been reported to alter host cell metabolism, inhibit virus synthesis, inhibit influenza virus proliferation in vitro, and inhibit vaccinia and herpes virus formation in chicken embryos. Clinical nuclease is injected intramuscularly at 180 mg per day, which is beneficial to treating epidemic encephalitis.
Production of nuclease P1 methods of production mainly microbial fermentation methods, the microorganisms used generally include: penicillium citrinum, escherichia coli, yeast cells, most commonly penicillium citrinum. For example, Songwei and the like[1]Gelatin, agar and polyvinyl alcohol are respectively mixed with sodium alginate to prepare 3 composite carriers, the penicillium citrinum cells are subjected to immobilized fermentation, and the enzyme activity of the produced nuclease P1 is 468.3-501.4U/mL. Huge script and the like[2]The penicillium citrinum GX-K which is separated and screened from soil and has high nuclease P1 yield is taken as a production strain, and the best enzyme production conditions are researched as follows: bran is used as a returning state fermentation base material, 4 percent of cane sugar is added, the material-water ratio is 1:0.9, the culture is carried out for 72h at the temperature of 30 ℃, and the output of nuclease P1 reaches 5783.41U/g dry basis under the condition. However, the fermentation liquid obtained by the microbial fermentation method contains toxic substances such as citrinin besides the nuclease P1 and needs to be removed. Therefore, an efficient and energy-saving separation and extraction method is urgently needed to be found. The main separation methods are as follows: heating ofMethods, pH adjustment methods and adsorption methods. The heating method is a thermodynamic separation process which realizes separation by utilizing the different decomposition and inactivation temperatures of the citrinin and the nuclease P1 and firstly decomposing the citrinin at high temperature. The pH regulation method utilizes the difference of the resistance of citrinin and nuclease P1 to pH, wherein citrinin is partially decomposed at pH 4, and the enzyme activity of nuclease P1 is not reduced. The combination of the two methods can remove citrinin in nuclease, but the enzyme activity of nuclease P1 is reduced by 80% under the temperature and pH condition of completely removing citrinin. E.g., Kitabake N, etc[3]Research shows that citrinin can be degraded in 130 deg.c water condition, Trivedi AB, etc[4]Under the condition of 140 ℃ of water, citrinin can be degraded into citrinin H2, the toxicity is reduced, and Kang BY and the like[5]When ammonium sulfate or sodium glutamate is used as a nitrogen source, the fermentation environment can also be low in acidity, so that the production of citrinin is reduced. The adsorption chromatography based on the resin has the advantages of simple operation, low equipment requirement, low energy consumption, less environmental pollution and the like, and the adsorption method for separating the citrinin is only rarely reported so far.
The separation device comprises 1 resin column filled with adsorbent, a peristaltic pump and an automatic part collector, the whole system is divided into four steps of adsorption, impurity washing, desorption and regeneration by switching mobile phases, the steps are switched in sequence, pure water is switched to carry out impurity washing after the resin column adsorbs citrinin to be saturated, product liquid is collected at an outlet of an adsorption section, 1M HCl/NaCl solution is switched to carry out desorption, and finally the pure water is switched to carry out regeneration, the adsorbent can be reused, not only is all citrinin in the nuclease liquid removed, but also the enzyme activity is improved.
Disclosure of Invention
The purpose of the invention is as follows: the technical problem to be solved by the invention is to provide a process for separating and removing citrinin in nuclease liquid by using a chromatographic technique, so as to solve the problem of downstream separation in the nuclease industry, greatly reduce energy consumption in the citrinin separation process and realize large-scale clean production of nuclease, aiming at the defects of the prior art.
In order to solve the technical problem, the invention discloses a process for separating and removing citrinin in a nuclease liquid by utilizing a chromatographic technique, which comprises the steps of loading the nuclease liquid containing citrinin into a chromatographic column containing anion exchange resin for ion exchange, adsorbing citrinin on the resin, and collecting an effluent, wherein the obtained effluent is the nuclease liquid without citrinin.
Preferably, the nuclease is nuclease P1.
Wherein in the citrinin-containing nuclease solution, the enzyme activity of nuclease is 1000-3000U/mL; the content of the citrinin is 0.02-0.1 ppm.
Wherein the enzyme activity is defined as: the enzyme activity of the detoxified nuclease P1 was calculated using the following method: preparing a substrate solution (5 vt% RNA aqueous solution and 0.2M acetic acid buffer solution with the pH value of 5 are uniformly mixed according to the volume ratio of 1: 1), carrying out constant-temperature water bath at 70 ℃ for 10min, adding nuclease P1(1 mu L/mL), carrying out reaction at 70 ℃ for 15min, adding an equal volume of nucleic acid precipitator (the mass content is 2.5% of perchloric acid and 0.25% of ammonium molybdate) to terminate the reaction, carrying out ice bath for 10min, centrifuging after complete precipitation of macromolecular nucleic acid and enzyme, taking a supernatant, and measuring the nucleotide concentration of the supernatant by using HPLC (high performance liquid chromatography), wherein the nucleotide concentration is U. Wherein, the volume of the nucleotide concentration ratio of the added nuclease P1 is the enzyme activity U/mL of the nuclease P1.
Wherein, 1 ppm-1 x 10-3g/L。
Wherein, the nuclease liquid containing the citrinin is obtained by direct fermentation, and is required to be pretreated before being loaded on a column, namely, centrifuged, and supernatant is taken.
The anion exchange resin is prepared by taking styrene as a monomer and crosslinking through divinylbenzene, wherein the crosslinking degree is 3-10%, and is preferably 7%.
Further, the functional group of the anion exchange resin is a quaternary amino group, and the content of the quaternary amino group is 1.0-2.5 mmol/g of the anion exchange resin; exchangeable ions of the anion exchange resin are chloride ions, and the content of the chloride ions is 1.0-2.5 mmol/g of the anion exchange resin; the volume total exchange capacity is more than or equal to 1.35 mmol/mL.
Further, the particle size of the anion exchange resin0.4-0.7 mm, 42.00-48.00% water content, and 1.07-1.10g/cm wet density3。
Wherein the sample loading rate is 0.3-1.5 BV/h; the loading amount is just enough to saturate resin, and the resin is possibly adsorbed to saturation by continuously increasing the loading amount, so that citrinin remains in effluent liquid; wherein, whether the resin is saturated or not is calculated according to the saturated adsorption capacity of the resin to the citrinin, and the saturated adsorption capacity of the resin adopted in the invention is 7.504-10-2g/g resin.
Preferably, after the above process is finished, i.e., after the effluent is collected, the effluent is washed with water and the effluent is collected to obtain the nuclease solution from which citrinin is removed in the resin gaps.
Wherein the amount of water is 0.8-1 BV, and the flow rate is 0.3-1.5 BV/h.
Further preferably, after the washing, desorbing with a desorbent to remove the citrinin on the resin; the column was regenerated with water.
Wherein the desorbent is a mixed aqueous solution of HCl and NaCl, and the concentrations of the HCl and the NaCl are both 1M; the amount of the desorbent is 3-5 BV, and the flow rate is 0.3-1.5 BV/h.
Wherein, in the regeneration process, the using amount of water is 3-5 BV, and the flow rate is 0.3-1.5 BV/h.
In the process flow, the adsorption (the loading process of the nuclease liquid containing the citrinin), desorption (impurity washing and desorption) and regeneration operations are all carried out at normal temperature.
In the process flow, the device used by the invention consists of 1 resin column filled with anion exchange resin, a peristaltic pump and an automatic partial collector, the whole system is divided into four steps of adsorption, impurity washing, desorption and regeneration by switching mobile phases, the steps are switched in sequence, the advanced nuclease liquid is carried out until the resin column is full of citrinin, and the effluent liquid of the product is collected; then switching pure water to carry out impurity washing, and collecting effluent liquid, wherein the obtained effluent liquid is nuclease liquid containing the removed citrinin; then switching 1M HCl/NaCl solution (mixed aqueous solution of HCl and NaCl, concentration of HCl and NaCl is 1M) to desorb, and removing citrinin on the resin; and finally, switching pure water for regeneration, wherein the anion exchange resin can be repeatedly used.
After the treatment by the process route, the enzyme activity of the nuclease solution after removing the citrinin can reach 2200U/mL, which is about 10% higher than that of the original enzyme solution; meanwhile, after treatment, the color of the nuclease solution after removal of the citrinin is changed from original light yellow to milky white, which shows that the process has a certain decolorizing effect.
The method utilizes an ion exchange mechanism, realizes the purpose of removing the citrinin through a fixed bed, ensures that the content of the citrinin in a product liquid is lower than the liquid phase detection limit, improves the enzyme activity of nuclease through a chromatographic separation process to 110 percent, and also has a certain decolorizing effect.
Has the advantages that: compared with the prior art, the invention has the following advantages:
(1) the chromatographic separation operation is carried out at room temperature, so that the energy consumption is greatly reduced;
(2) the resin is easy to regenerate, can be repeatedly used, has low operation cost and can be directly used for process amplification;
(3) harmful impurities such as citrinin and the like are separated from nuclease of a product in one step by passing through a resin column, so that the purity and the quality of the nuclease product are ensured, and the enzyme activity of the nuclease is improved by a chromatographic separation process to 110%; meanwhile, the invention also has a certain decolorizing effect.
Drawings
The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.
FIG. 1 is a process flow diagram of the present invention.
FIG. 2 is a graph showing the decoloring effect of the present invention.
Detailed Description
In the following examples, the citrinin concentration in the feed liquid is detected by an external standard method, and the chromatographic conditions are as follows:
1) a detector: agilent model 1260 hplc-fluorescence detector;
2) a chromatographic column: agilent ZORBAX SB-C18 liquid chromatography column
3) Mobile phase: acetonitrile-isopropanol-0.08 mol/L phosphoric acid (volume ratio 35:10: 55);
4) flow rate: 0.6 mL/min;
5) column temperature: 28 ℃;
6) sample introduction volume: 20 μ L.
The detection method comprises the following steps:
1) equilibration of the chromatographic column: the prepared mobile phase acetonitrile-isopropanol-0.08 mol/L phosphoric acid (35:10:55) is filtered by a mixed microporous filter membrane with the pore diameter of 0.22 mu m, and then is subjected to ultrasonic treatment for 30 min. And (3) washing the chromatographic column by using the treated mobile phase at the flow rate of 0.5mL/min, simultaneously opening a column incubator, starting to collect a baseline, and finishing the balance when the baseline tends to be linear.
2) And (3) detection of the sample: and compiling a sample injection sequence and a method according to chromatographic conditions, placing the standard substance and the sample after membrane treatment on corresponding positions of an automatic sample injector according to the sample injection sequence, starting sample injection and collecting map information.
The enzyme activity of the detoxified nuclease P1 was calculated using the following method: preparing a substrate solution (5vt percent of RNA aqueous solution and 0.2M acetic buffer solution with the pH value of 5 are mixed uniformly according to the volume ratio of 1: 1), carrying out constant-temperature water bath at 70 ℃ for 10min, adding nuclease P1(1 mu L/mL), carrying out reaction at 70 ℃ for 15min, adding an equal volume of nucleic acid precipitator (the mass content is 2.5 percent of perchloric acid and 0.25 percent of ammonium molybdate) to stop the reaction, carrying out ice bath for 10min, centrifuging after complete precipitation of macromolecular nucleic acid and enzyme, and taking a supernatant to measure the nucleotide concentration of the supernatant by using HPLC.
Wherein, the concentration ratio of the nucleotide to the volume of the added nuclease P1 is the enzyme activity of the nuclease P1.
In the following examples, the ion exchange resin (Cl form 717) used was: styrene is taken as a monomer, and is crosslinked by divinylbenzene, the crosslinking degree is 7%, the functional groups are quaternary amino groups, and the content of the quaternary amino groups is 1.0-2.5 mmol/g of anion exchange resin; the exchangeable ions are chloride ions, and the content of the chloride ions is 1.0-2.5 mmol/g of anion exchange resin; the particle size of the ion exchange resin is 0.4-0.7 mm, the water content is 42.00-48.00%, and the wet density is 1.07-1.10g/cm3The volume total exchange capacity is more than or equal to 1.35 mmol/mL. Resin manufacturerCan be synthesized by itself according to the above conditions.
The following examples used a separation apparatus, consisting of 1 resin column filled with adsorbent, peristaltic pump and automatic partial collector, as shown in fig. 1, by switching mobile phase to divide the whole system into four steps of adsorption, impurity washing, desorption and regeneration, switching in order, after the resin column was saturated with citrinin, nuclease P1 solution was first switched to pure water for impurity washing, product solution was collected at the outlet of the adsorption section, then 1M HCl/NaCl solution was switched to desorb, and finally pure water was switched to regenerate, and the adsorbent could be reused. The 1M HCl/NaCl solution described in the following examples is a mixed aqueous solution of HCl and NaCl, both at 1M.
Example 1: obtaining nuclease P1 liquid.
Inoculating penicillium citrinum to 100mL of seed culture medium, and culturing at 30 ℃ for 24 h; then inoculating the seed culture solution into a fermentation culture medium according to the inoculation amount of 10vt percent, wherein the initial pH value is 6.50, the fermentation temperature is 30 ℃, and the culture time is 50 h. Centrifuging the obtained fermentation liquor to remove impurities such as protein and polysaccharide, and collecting the clear liquid for later use. Wherein the seed culture medium comprises the following components in percentage by mass: glucose 5%, peptone 0.5%, KH2PO40.05％，K2HPO2·3H2O 0.05％，MgSO40.04％，CaCl20.04% and pH 6.50; the mass percentages of all components in the fermentation medium are seed culture medium and ZnSO4·7H2O 0.04％。
Repeating the experiment for four times according to the method to respectively obtain four batches of clear liquid; in the first clear liquid, the enzyme activity of nuclease P1 in the obtained clear liquid is 2000U/mL, and the content of citrinin is 0.034 ppm; in the second batch of clear liquid, the enzyme activity of the nuclease P1 in the obtained clear liquid is 1950U/mL, and the citrinin is 0.051 ppm; in the third clear liquid, the enzyme activity of the nuclease P1 in the clear liquid is 2020U/mL, and the citrinin content is 0.043 ppm; in the fourth clear liquid, the activity of the nuclease P1 in the obtained clear liquid is 1970U/mL, and the citrinin content is 0.027 ppm.
Example 2: and (5) measuring the removal rate of the citrinin by a static shaking bottle.
0, 0.1, 0.5, 1, 5 and 10g of chlorine type 717 strong anion exchange resin are sequentially added into six 250mL conical flasks, 100mL of nuclease P1 liquid with higher citrinin content is sequentially poured (a citrinin standard is added into the non-centrifuged fermentation liquid prepared in example 1 to ensure that the final content of the citrinin is 3.752ppm), the bottles are sealed, and the bottles are placed in a shaking table at the temperature of 150r/min of 25 ℃ for 8 hours. The supernatant was collected and assayed for citrinin concentration by HPLC. The color of the nuclease P1 liquid with different resin addition amounts is changed differently, as shown in FIG. 2 (the addition amounts of 0, 0.1, 0.5, 1, 5 and 10g of resin are sequentially from left to right), the color of the nuclease P1 liquid is lighter when the resin addition amount is larger, and the treatment of the nuclease P1 liquid by using the Cl 717 resin has a certain decolorizing effect. The removal rate of citrinin from nuclease P1 solutions with different resin addition amounts is shown in Table 1.
Wherein, the removal rate is 1-the concentration of the citrinin in the supernatant after adding the resin shaker/the initial concentration of the citrinin in the nuclease P1 solution is 100%.
TABLE 1
Saturated adsorption capacity: 3.752*10-3*0.1/5＝7.504*10-2g/g wet resin.
In the following examples 3 to 6, the feed to the adsorption section was 1L, the impurity-washing sections were eluted with 1BV of pure water, the desorption section was desorbed with 3BV of 1M HCl/NaCl solution, the regeneration section was eluted with 3BV of pure water, and the flow rates of all sections were 0.3 BV/h.
Example 3: and (4) carrying out chromatographic separation on the nuclease P1 liquid.
A separation system consisting of 1 column of resin loaded with adsorbent, a peristaltic pump and an automatic partial collector was used. The resin column was packed with 0.6L of resin (Cl type 717), the diameter of the resin column was 5cm, and the height of the resin column was 70 cm. The first batch of nuclease solution P1 pretreated in example 1 was applied to the column at a concentration: nuclease P1 has enzyme activity of 2000U/mL, citrinin 0.034ppm, 1M HCl/NaCl as desorbent, and saturated adsorption capacity of 7.504 x 10-2g/g wet resin; the whole system is divided into four parts of adsorption, impurity washing, desorption and regeneration by switching the mobile phaseSequentially switching, namely switching pure water to wash impurities after the resin column is saturated after the citrinin is adsorbed by nuclease P1 liquid, collecting product liquid at an outlet of an adsorption section, switching 1M HCl/NaCl solution to desorb, and finally switching pure water to regenerate at the flow rate of 0.3 BV/h. And (3) detecting the concentration of the citrinin by using HPLC (high performance liquid chromatography) on the collected product effluent, wherein the concentration of the citrinin is lower than the liquid phase detection limit (0.01ppm), and the enzyme activity reaches 2196U/mL.
Example 4: and (4) carrying out chromatographic separation on the nuclease P1 liquid.
A separation system consisting of 1 column of resin loaded with adsorbent, a peristaltic pump and an automatic partial collector was used. The resin column was packed with 0.6L of resin (Cl type 717), the diameter of the resin column was 5cm, and the height of the resin column was 70 cm. The second batch of nuclease P1 after pretreatment in example 1 was applied to the column at a concentration: nuclease P1 has enzyme activity 1950U/mL, citrinin 0.051ppm, 1M HCl/NaCl as desorbent, and saturated adsorption capacity 7.504 x 10-2g/g wet resin; the whole system is divided into four steps of adsorption, impurity washing, desorption and regeneration by switching a mobile phase, the steps are switched in sequence, the nuclease P1 liquid with first amount is switched to pure water for impurity washing after the resin column adsorbs citrinin and is saturated, the product liquid is collected at the outlet of an adsorption section, then the HCl/NaCl solution with 1M is switched to carry out desorption, finally the pure water is switched to carry out regeneration, and the flow rate is 0.3 BV/h. And (3) detecting the concentration of the citrinin by using HPLC (high performance liquid chromatography) on the collected product effluent, wherein the citrinin concentration is lower than the liquid phase detection limit, and the enzyme activity reaches 2135U/mL.
Example 5: and (4) carrying out chromatographic separation on the nuclease P1 liquid.
A separation system consisting of 1 column of resin loaded with adsorbent, a peristaltic pump and an automatic partial collector was used. The resin column was packed with 0.6L of resin (Cl type 717), the diameter of the resin column was 5cm, and the height of the resin column was 70 cm. The pretreated third batch of nuclease P1 from example 1 was loaded on a column, and the loading concentration: nuclease P1 has enzyme activity of 2020U/mL, citrinin 0.043ppm, 1M HCl/NaCl as desorbent, and saturated adsorption capacity of 7.504 x 10-2g/g wet resin; the whole system is divided into four steps of adsorption, impurity washing, desorption and regeneration by switching the mobile phase, the steps are switched in sequence, the nuclease P1 liquid is firstly added, pure water is switched for impurity washing after the resin column is saturated with the citrinin, the product liquid is collected at the outlet of the adsorption section,then 1M HCl/NaCl solution is switched to desorb, and finally pure water is switched to regenerate with the flow rate of 0.3 BV/h. And (3) detecting the concentration of the citrinin by using HPLC (high performance liquid chromatography) on the collected product effluent, wherein the citrinin concentration is lower than the liquid phase detection limit, and the enzyme activity reaches 2245U/mL.
Example 6: and (4) carrying out chromatographic separation on the nuclease P1 liquid.
A separation system consisting of 1 column of resin loaded with adsorbent, a peristaltic pump and an automatic partial collector was used. The resin column was packed with 0.6L of resin (Cl type 717), the diameter of the resin column was 5cm, and the height of the resin column was 70 cm. The fourth batch of nuclease P1 after pretreatment in example 1 was loaded on the column, and the loading concentration: nuclease P1 enzyme activity is 1970U/mL, citrinin is 0.027ppm, 1M HCl/NaCl is used as a desorbent, and the saturated adsorption capacity is 7.504 × 10-2g/g wet resin; the whole system is divided into four steps of adsorption, impurity washing, desorption and regeneration by switching a mobile phase, the steps are switched in sequence, the nuclease P1 liquid with first amount is switched to pure water for impurity washing after the resin column adsorbs citrinin and is saturated, the product liquid is collected at the outlet of an adsorption section, then the HCl/NaCl solution with 1M is switched to carry out desorption, finally the pure water is switched to carry out regeneration, and the flow rate is 0.3 BV/h. And (3) detecting the concentration of the citrinin by using HPLC (high performance liquid chromatography) on the collected product effluent, wherein the citrinin concentration is lower than the liquid phase detection limit, and the enzyme activity reaches 2155U/mL.
Comparative example 1
In the same way as in example 2, the same batch of feed liquid and the same conditions of sample introduction amount, flow rate and the like are used, the Cl type 717 resin is respectively replaced by the weak anion exchange resin FPA53 and the strong cation exchange resin NH-1, and the presence of citrinin is detected in the product liquid collected at the outlet of the adsorption section, which indicates that the citrinin in the nuclease liquid cannot be removed by the two resins.
Reference documents:
[1] comparison research on production of nuclease Pl by fermentation of Songwei composite carrier immobilized cells.
[2] Solid-state fermentation conditions of Penicillium citrinum high-yielding nuclease P1 were studied.
[3]Kitabatake N,Trivedi AB,Doi E.Thermal decomposition and detoxification of citrinin under various moisture conditions[J].Journal of Agricultural and Food Chemistry,1991,39(12):2240-2244.
[4]Trivedi AB,Hirota M,Doi E,et al.ChemInform abstract:formation of a new toxic compound,citrinin H1,from citrinin on mild heating in water[J].Joumal of the Chemical Socitey,Perkin Transactions 1,1993,24(52):2167-2171.
[5]Kang BY,Zhang XH,Wu ZQ,et al.Production of citrinin-free Monascus pigments by submerged culture at low pH[J].Enzyme and Microbial Technology,2014,55:50-57.
The present invention provides a method and a concept for separating and removing citrinin from a nuclease solution by using a chromatographic technique, and a method and a way for implementing the method and the concept are numerous, and the above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and embellishments can be made without departing from the principle of the present invention, and these modifications and embellishments should be regarded as the protection scope of the present invention. All the components not specified in the present embodiment can be realized by the prior art.
Claims (6)
1. A process for separating and removing citrinin in a nuclease solution by utilizing a chromatographic technique is characterized in that the nuclease solution containing citrinin is loaded into a chromatographic column containing anion exchange resin for ion exchange, and effluent liquid is collected to obtain the nuclease solution from which the citrinin is removed;
wherein in the citrinin-containing nuclease solution, the enzyme activity of nuclease is 1000-3000U/mL; the content of the citrinin is 0.02-0.1 ppm;
wherein, the anion exchange resin takes styrene as a monomer, and is crosslinked by divinylbenzene, and the crosslinking degree is 3-10%; the functional group of the anion exchange resin is quaternary amino, and the content of the quaternary amino is 1.0-2.5 mmol/g of the anion exchange resin; exchangeable ions of the anion exchange resin are chloride ions, the content of the chloride ions is 1.0-2.5 mmol/g of the anion exchange resin, and the volume total exchange capacity is more than or equal to 1.35 mmol/mL; the particle size of the anion exchange resin is 0.4-0.7 mm, the water content is 42.00-48.00%, and the wet density is 1.07-1.10g/cm3。
2. The process according to claim 1, wherein the loading rate is 0.3 to 1.5 BV/h.
3. The process of claim 1, wherein the effluent is collected and then washed with water; wherein the amount of water is 0.8-1 BV, and the flow rate is 0.3-1.5 BV/h.
4. The process of claim 3, wherein the impurities are washed, desorbed with a desorbent, and the column is regenerated with water.
5. The process of claim 4, wherein the desorbent is an aqueous solution of HCl in combination with NaCl, both HCl and NaCl being at a concentration of 1M; the amount of the desorbent is 3-5 BV, and the flow rate is 0.3-1.5 BV/h.
6. The process according to claim 4, wherein the amount of water used in the regeneration process is 3 to 5BV, and the flow rate is 0.3 to 1.5 BV/h.
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| CN1808117A (en) * | 2006-01-05 | 2006-07-26 | 北京联合大学应用文理学院保健食品功能检测中心 | Method for detecting citrinin content in red koji fermentation product |
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