CN110172094B - Method for extracting high-purity metallothionein from oratosquillas - Google Patents

Abstract

The invention relates to the technical field of metallothionein extraction, and provides a method for extracting high-purity metallothionein from Oratosquillas, which aims to solve the problems of harsh extraction object requirement, long extraction period, high cost and low purity existing in a metallothionein extraction process, and comprises the following steps: (1) removing shell of Mantis shrimp, homogenizing edible part, centrifuging, extracting, and freeze drying; (2) performing gel filtration chromatography; (3) performing anion exchange chromatography; (4) desalting to obtain first and second sub-type metallothionein. The extraction process of high-purity metallothionein is designed by taking the edible part of the Oratosquilla as a raw material according to the specific matrix characteristics of the Oratosquilla, and is short in extraction period, large in extraction amount, low in cost, high in purity and suitable for industrial and large-scale production.

Description

Method for extracting high-purity metallothionein from oratosquillas

Technical Field

The invention relates to the technical field of metallothionein extraction, in particular to a method for extracting high-purity metallothionein from Oratosquilla tamariscina.

Background

Metallothionein (MT) is low molecular weight protein (6000-7000 Da) rich in cysteine (Cys), and can be combined with up to 20 monovalent or 7 divalent heavy metal ions through the binding capacity of cysteine thiol groups to heavy metals. MT plays an important physiological role in organisms, is mainly shown to participate in storage, transportation and metabolism of trace metal elements in the organisms and chelation and detoxification of non-essential heavy metal elements, has important significance in aspects of regulating homeostasis, scavenging free radicals, preventing tissue oxidative damage, antagonizing physiological and biochemical reactions and the like, and provides an active center for other metal proteins and metalloenzymes, so MT has very wide application in the fields of environmental monitoring, health-care food and medicine.

At present, MT is mainly extracted and purified from mammals (such as human beings, rats, mice, rabbits and the like) and some bacteria and fungi. But no effective technology is available at the present stage for mass production, so that the development and the application of the method are limited. The high-purity product has high selling price, the rabbit liver product produced by one company in China can only be bought in the market at present, the marked purity is more than 95%, the selling price is about 1500 yuan/5 mg, but a standard substance certificate is not provided, and the actual purity needs to be studied.

Oratosquilla (Oratosquilla orientalis), which is called as Clausena lansium, shrimp harrow and uroacia, belongs to Crustacea, is a perennial large benthic crustacean, is distributed in China, Japan and Philippines coastal areas, has large resource quantity, and the output of the Mantis oralis in China reaches 29.4 ten thousand tons in 2014. The mantis shrimps are always favored by consumers because of rich nutrition and delicious meat quality, and are representative high-value crustacean products in coastal areas of China.

In recent years, quality inspection mechanisms detect that the content of cadmium in the oratosquillas is over standard seriously, and the contents of elements such as copper, arsenic, zinc and the like are higher, which indicates that the oratosquillas are marine organisms with stronger enrichment capacity on trace elements in marine environment. The stress of metal elements can induce the expression of MT in organisms, so the Oratosquilla can be used as a good raw material for extracting MT. The metallothionein is prepared by extracting and purifying the oratosquillas as the raw material, so that on one hand, a new idea is provided for the development and utilization of marine biological resources, on the other hand, a foundation is laid for the subsequent development of cadmium enrichment mechanism of the oratosquillas, and the metallothionein has important significance for protecting ecological environment and similar health.

The Chinese patent literature discloses a method for simultaneously extracting two subtype metallothionein from Japanese scallops, and the application publication number is CN 109206507A, the method is used for extracting the metallothionein from the internal organs of the Japanese scallops by the methods of zinc chloride induction, multiple centrifugal extraction, primary purification by using a molecular sieve prepacked column with polymethacrylate as a filler and secondary purification by anion exchange chromatography. However, the extraction object of the invention only aims at the internal organs of the living patinopecten yessoensis, the internal organ part is a small part of the whole tissue of the patinopecten yessoensis, and zinc chloride is needed to induce the patinopecten yessoensis in the early stage, the living body is cultured for several days, and the production period is long; and a commercial molecular sieve prepacked column is adopted, and a corresponding automatic protein purification instrument is required to be matched, so that the equipment cost is high; the existing commercialized molecular sieve prepacked column has small capacity and high price, can purify the sample loading amount, greatly limits the production cost, and is not beneficial to industrialized and large-scale production; the extraction purity can only reach 87%. Therefore, the extraction process has the problems of strict extraction object requirement, long period, high cost and low purity.

Disclosure of Invention

The invention provides a method for extracting high-purity metallothionein from Oratosquilla tamariscina, aiming at overcoming the problems of rigorous extraction object requirements, long extraction period, high cost and low purity of the existing metallothionein extraction process.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for extracting high-purity Metallothionein (MT) from Oratosquilla tamariscina comprises the following steps:

(1) removing shell of Mantis shrimp, collecting edible part, homogenizing, centrifuging, extracting, and freeze drying to obtain Mantis shrimp metallothionein crude extract; (2) redissolving the crude extract of the mantis shrimp metallothionein, centrifuging, filtering by using a filter membrane, carrying out gel column chromatography, collecting eluent, and carrying out freeze drying to obtain a metallothionein mixture; the gel column layer is selected without column chromatography, so that the preparation chromatography equipment with small sample loading amount and high price is required in the column chromatography;

(3) redissolving the metallothionein mixture, carrying out anion exchange chromatography, respectively collecting two sub-type metallothionein eluents, and freeze-drying to obtain a first sub-type metallothionein crude product and a second sub-type metallothionein crude product;

(4) and respectively re-dissolving the first sub-type metallothionein crude product and the first sub-type metallothionein crude product, and desalting to obtain the first sub-type metallothionein and the second sub-type metallothionein.

The Oratosquilla is a marine organism with strong enrichment capacity on trace cadmium in marine environment. The stress of metal elements can induce the expression of MT in organisms, so the Oratosquilla can be used as a good raw material for extracting MT. The metallothionein is prepared by extracting and purifying the oratosquillas as the raw material, so that on one hand, a new idea is provided for the development and utilization of marine biological resources, on the other hand, a foundation is laid for the subsequent development of cadmium enrichment mechanism of the oratosquillas, and the metallothionein has important significance for protecting ecological environment and similar health. The method adopts the edible part of the shell-removed Oriental squids, namely the whole tissue as the raw material to directly extract the metallothionein, the raw material only needs to ensure freshness, is not necessarily living, and does not need later-stage metal solution induction culture, thereby reducing the complexity of the preparation process of the metallothionein, shortening the production period and lowering the production cost.

Because the processes and treatment parameters for extracting the metallothionein from different sources of aquatic organisms are different, the extraction method is only directed at extracting the metallothionein from the edible parts of the Oratosquillas, and is not applicable to extracting the metallothionein from other sources, and meanwhile, the extraction method of the metallothionein from other sources has no strong reference to the extraction method.

Preferably, in the step (1), the centrifugal extraction process comprises the following steps: adding Tris-HCl buffer solution, stirring and extracting at 45-55 ℃ for 1-2 h, freezing and centrifuging the extracting solution at high speed, collecting supernatant a, heating to 70-80 ℃, and preserving heat for 3-7 min; rapidly cooling to room temperature, then freezing at high speed and centrifuging again, collecting supernatant b, adding absolute ethyl alcohol, and precipitating at-18-25 ℃ overnight; and (3) performing high-speed freezing centrifugation again, adding ultrapure water into the precipitate to fully dissolve the precipitate, performing high-speed freezing centrifugation again, collecting supernatant c, and performing freeze drying to obtain a crude mantis shrimp metallothionein extract.

The meat quality of the mantis portulacae is relatively rich in water, the meat taste is fresh, sweet, tender and smooth, and the meat taste is light and soft. Firstly, extracting the edible part of the mantis shrimps for 1-2 hours at 45-55 ℃ by adopting Tris-HCl buffer solution, wherein the step is used for fully extracting metallothionein in tissues; heating the supernatant a obtained after the extracting solution is frozen at a high speed and centrifuged to 70-80 ℃ to remove heat-labile macromolecular protein, wherein the low treatment temperature can cause unclean removal of impure protein, and the high treatment temperature can cause denaturation and precipitation of metallothionein per se; the final precipitate is dissolved sufficiently by adding ultrapure water, and the advantage of not using Tris-HCl buffer solution is to avoid introducing excessive salt into the preparation.

Preferably, in the step (1), the pH value of the Tris-HCl buffer solution is 7.5-8.0, and the concentration is 0.02-0.05 mol/L; when the concentration of the Tris-HCl buffer solution is 0.02mol/L and the pH value is 7.8, the extraction effect on the metallothionein contained in the Oratosquilla tamariscina is best.

Preferably, in step (1), the absolute ethanol is precooled and kept at 4 ℃, and is added in an amount of 3 times of the volume of the supernatant b. The ethanol is used as an organic solvent to dehydrate and denature the metallothionein, and the ethanol is used for leading the metallothionein to generate reversible denaturation precipitation and further to be separated from other molecules such as other heteroproteins in the supernatant b. When the volume of the ethanol is 3 times of that of the supernatant b, the metallothionein is completely denatured and precipitated.

Preferably, in the step (1), the feed-liquid ratio of the oratosquillas to the Tris-HCl buffer solution (3-6): 1 g/mL. The dosage of Tris-HCl buffer solution is not suitable to be excessive, the excessive dilution factor is not beneficial to the extraction of the metallothionein, and the extraction effect is better when the ratio of the Tris-HCl buffer solution to the liquid is controlled.

Preferably, in the step (1), the temperature of the high-speed freezing centrifugation is controlled to be 4-10 ℃, the rotating speed is controlled to be 10000-15000 r/min, and the centrifugation time is controlled to be 20-30 min. The parameters of high-speed freezing centrifugation are key points of extraction effect, the rotating speed is too high, the energy consumption is increased, and poor centrifugation effect can be caused by too low rotating speed; too low a temperature increases energy consumption, while too high a temperature may have an effect on metallothionein structure and activity.

Preferably, in step (2), the conditions of gel filtration chromatography are as follows: the column chromatography packing is sephadex G50; Tris-HCl buffer with mobile phase of 0.02mol/L, pH ═ 8.0; the flow rate is 0.8 mL/min; the elution volume is 500mL, and the collection amount is 8 mL/tube; the absorbance values of the collected fractions from each tube were measured at 220nm and 270nm using an ultraviolet absorptiometer, together with the metal element signal in combination with ICP-MS.

The invention selects the traditional column chromatography separation operation mode of Sephadex G-50 self-packed column glass chromatographic column, peristaltic pump extraction mobile phase elution and ultraviolet detector auxiliary on-line monitoring, and can conveniently change the packing amount and the size of the glass chromatographic column according to the production requirement to produce as required. Compared with the traditional process, the method can greatly increase the preparation amount of one-time purification, reduce the production cost and is more suitable for industrial and large-scale production. The Tris-HCl buffer solution with the concentration of 0.02mol/L, pH-8.0 is selected as the mobile phase, so that the buffer solution has the advantages of relatively mild composition and good compatibility with biological tissues, the extraction effect is ensured, and the target object can be prevented from being damaged; the test wavelengths were chosen as 220nm and 270nm because the wavelengths are characteristic absorption peaks of proteins and metallothionein. By adopting the purification method and the parameters of the invention, two subtype metallothionein substances with the purity of more than 97 percent can be obtained.

Preferably, in step (3), the conditions for anion exchange chromatography are: the column chromatography packing is ion exchange agarose gel; the elution speed is 0.8mL/min, the total volume of elution is 450mL, the collection amount is 8 mL/tube, the ultraviolet absorption peak is monitored under 270nm, and simultaneously the ICP-MS is combined to determine the signal of the metal element; eluting with a first mobile phase to obtain a first subtype metallothionein crude product, wherein the first mobile phase is a Tris-HCl buffer solution with the concentration of 0.02mol/L, pH-8.0; and eluting with a second mobile phase, wherein the second mobile phase is a mixed solution with the pH value of 8.6 and containing 0.02mol/L Tris-HCl and 0.5mol/L NaCl, so as to obtain a second subtype metallothionein crude product.

The characteristics of metallothionein from different sources have certain difference, so that the mobile phase used in the separation process is different. Two different mobile phases are adopted for elution, so that the complete separation of two subtype metallothionein can be efficiently realized, and other impurities can be removed. Compared with the method for selecting a pure NaCl solution, the method for selecting the mixed solution of the pH 8.6, 0.02mol/L Tris-HCl and 0.5mol/L NaCl for the second mobile phase has the advantages that in the early stage, the pure water is used for elution, the ionic strength is not enough, and a certain amount of NaCl solution is added, so that the ionic strength and the elution capacity of the elution solution are increased, and the metallothionein with stronger binding force can be eluted. When the concentration and flow rate of the mobile phase are changed, although the two metallothionein can be separated, the separation degree of the two metallothionein is not high, and other impurities in the mixture of the two subtypes cannot be completely removed. Therefore, when the metallothionein is extracted from the edible part of the Oratosquilla esculenta, two subtype metallothionein pure products need to be obtained simultaneously by adopting the extraction and purification method and the process parameters provided by the invention, otherwise, two subtype metallothionein with ideal purity cannot be obtained.

Preferably, in the steps (2) and (3), Tris-HCl is adopted for redissolution, so that the consistency with the composition of a subsequent column chromatography solution can be ensured; in the step (4), the ultra-pure water is adopted for re-dissolving, so that new salt can be effectively prevented from being introduced into the final product.

Preferably, in step (4), the desalting conditions are: the column chromatography packing is sephadex; eluting with ultrapure water, and controlling the elution speed to be 1mL/min and 8 mL/tube; the UV absorption peak was monitored at 270nm while the metal element signal was monitored.

After the metallothionein is subjected to anion exchange chromatography, the metallothionein also contains more salt, so that further desalting treatment is required to ensure the purity of the Oratosquilla metallothionein. The desalting parameters also need to be strictly in accordance with the parameters of the present invention to ensure the purity of the finally obtained metallothionein.

Preferably, in the step (2), a filter membrane of 0.45 μm is adopted for filtration, and the filtrate obtained by the filter membrane with the aperture does not cause subsequent column chromatography blockage due to overlarge particle size of the impurity composition, thereby being beneficial to the smooth separation operation of the next step.

Therefore, the invention has the following beneficial effects: the extraction process of high-purity metallothionein is designed by taking the edible part of the Oratosquilla as a raw material according to the specific matrix characteristics of the Oratosquilla, and the high-purity metallothionein is separated, purified and prepared by adopting a glass chromatographic column to self-fill column chromatography packing, so that the process route is suitable for pilot-scale or expanded production of enterprises, and the production period and the production cost are reduced; by using the crustacean marine organism-oratosquillas with larger biological resource amount as a production raw material, the stable supply of raw materials for preparing metallothionein can be ensured, and the raw material cost is relatively lower; meanwhile, the added value of the primary marine products is improved, and a new technical means is provided for the fine and further processing and utilization of the primary marine products. The process has the advantages of short extraction period, large extraction amount, low cost, high purity, and suitability for industrial and large-scale production.

Drawings

FIG. 1 is the Sephadex G-50 chromatographic elution profile of example 1.

FIG. 2 is a DEAE Sepharose Fast Flow ion column chromatography elution curve and a metal element signal chart of example 1.

FIG. 3 is the desalting chart of the main peak of Mantis shrimp MT-1 in example 1.

FIG. 4 is a pictorial representation of MT obtained in example 1 after Sephadex G-50 gel column chromatography separation and lyophilization.

FIG. 5 is a schematic representation of the desalted fraction MT-1 from Sephadex G-25 of example 1.

FIG. 6 is a schematic representation of the desalted fraction MT-2 from Sephadex G-25 of example 1.

FIG. 7 is an SDS-PAGE electrophoresis of each sample in example 1: 1. lanes 2 and 3 are the small molecular weight Marker, Orodia squilla MT-1 and Orodia squilla MT-2 samples, respectively.

Detailed Description

The technical solution of the present invention is further specifically described below by using specific embodiments and with reference to the accompanying drawings.

In the present invention, all the equipment and materials are commercially available or commonly used in the art, and the methods in the following examples are conventional in the art unless otherwise specified.

For the sake of brief description, metallothionein in the following examples and comparative examples of the present invention is abbreviated as MT.

Example 1

(1) Removing shells of the lobster and taking an edible part, and mixing the following components in a liquid-material ratio of 4: 1, adding Tris-HCl buffer solution with the pH value of 7.8 and the concentration of 0.02mol/L, stirring and extracting for 1h in a water bath at the temperature of 51 ℃, freezing and centrifuging the extracting solution at a high speed for 25min at the temperature of 4 ℃ and at the speed of 13000r/min, and removing precipitates; collecting supernatant a, heating to 80 deg.C in water bath for 5min to remove heat-labile macromolecular protein, rapidly cooling to room temperature, and centrifuging at 4 deg.C and 13000r/min for 25 min; after collecting the supernatant b, 3 volumes of pre-cooled (temperature 4 ℃) absolute ethanol were added and precipitated overnight at-20 ℃. Centrifuging at 4 ℃ and 13000r/min for 25min, taking the precipitate, adding 2-3 mL of ultrapure water to fully dissolve, centrifuging at 4 ℃, collecting supernatant c, freeze-drying, and storing at-18 ℃ to obtain a crude mantis shrimp metallothionein extract, namely crude MT freeze-dried powder;

(2) sephadex G-50 gel filtration chromatography:

preparing a column: removing column head of Sephadex G-50 column (phi 2.5cm is multiplied by 60cm) and placing 1% HNO₃Soaking in an acid cylinder for one night, taking out, cleaning with ultrapure water, vertically fixing on an iron support, draining for later use;

swelling the gel: adding 1000mL of ultra-pure water subjected to ultrasonic degassing into Sephadex G-5035G, swelling at room temperature for 24h, removing upper floating gel, washing with clear water for 3-5 times, removing broken gel, vacuum degassing for 5-6h, and rinsing with pure water for 2-3 times to prepare for column packing;

column assembling: fixing the lower port of the column, adding 3mL of water, blocking the lower port, then gently stirring the swelled gel and mixing into thin slurry, pouring the thin slurry into the column along the tube wall at a constant speed through a funnel, paying attention to prevent bubbles from entering at the moment, marking the upper port of the column at about 5cm, closing a water outlet, adding 3cm of water, opening the water outlet, closing the lower port of the column when the liquid level reaches the marked position, and balancing overnight with ultrapure water after the gel is settled stably;

and (3) checking column effect: after the column is completely filled, the column layer is balanced by 0.02mol/L Tris-HCl (pH 8.0) buffer solution until the pH of the effluent eluent is consistent with the buffer solution and the height of a gel bed of the chromatographic column is stable;

sampling and collecting: dissolving 150mg of crude MT freeze-dried powder in 10mL of Tris-HCl, loading the obtained solution on a 0.45 mu m filter membrane, eluting the obtained solution by using 0.02mol/L of Tris-HCl (pH 8.0), measuring the absorbance of the collected components in each tube by using an ultraviolet absorptiometer at the flow rate of 0.8mL/min, 8 mL/tube and the elution volume of 500mL, collecting fractions with absorption peaks at 220nm and 270nm, measuring the absorbance of the collected components in each tube by using the ultraviolet absorptiometer, measuring a metal element signal in each tube by combining ICP-MS, finding that the metal signal can better correspond to the peak-off time at 270nm, mainly concentrating the obtained metal signal in a 16-38 tube, combining the liquids in the tubes, concentrating the liquids under reduced pressure, performing vacuum freeze drying, storing the obtained liquid at-18 ℃ for later use, wherein a Sephadex G-50 chromatographic elution curve is shown in figure 1, and an MT graph obtained after the separation and the freeze-drying of a;

(3) DEAE-Sepharose Fast Flow ion exchange chromatography:

the Sephadex G-50 gel filtration chromatography can not separate two subtypes of MT, ion exchange chromatography is further needed, and a DEAE-Sepharose Fast Flow anion exchange column (phi 2.5cm multiplied by 40cm) is used as a solid phase carrier to separate and purify the mantis shrimp MT so as to achieve the purpose of purification. Before column packing, the ethanol protective solution in the filler is cleaned by ultrapure water, then column packing is carried out, and the operation is carried out according to a Sephadex G-50 column packing method. Weighing 120mg of the component purified by Sephadex G-50, dissolving in 8mL of Tris-HCl, then loading, eluting at the speed of 0.8mL/min and 8 mL/tube with the total volume of 450mL, monitoring an ultraviolet absorption peak at 270nm, and simultaneously determining the signal of metal elements in each tube by combining ICP-MS. Eluting with 0.02mol/L Tris-HCl (pH 8.6) from the 0 th tube to the 25 th tube to obtain a component F1 (11 th-18 th tube), eluting with 0.02mol/L Tris-HCl +0.5mol/L NaCl (pH 8.6) from the 25 th tube after the F1 peak is completely removed to obtain a component F2 (33 th-40 th tube), collecting the 11 th-20 th tubes, combining, lyophilizing, and naming as a mouth mantis MT-1, collecting the 33 th-40 th tubes, lyophilizing, naming as a mouth mantis MT-2, and collecting a DEAE Sepharose Fast Flow ion column chromatography elution curve and a metal element signal diagram as shown in FIG. 2; (4) sephadex G-25 desalting:

after the MT is subjected to ion exchange chromatography, the MT also contains more salt, so that further desalting treatment is required to ensure the purity of the Oratosquilla shrimp MT. This patent uses Sephadex G-25 (phi 1.5cm is multiplied by 60cm) gel column to desalt the components MT-1 and MT-2 obtained by DEAE-Sepharose Fast Flow purification. The gel swelling and column packing in the concrete operation are carried out according to the method described by the Sephadex G-50 column, 80mg of MT-1 and MT-2 components are weighed and dissolved in 5mL of ultrapure water, the components are uniformly mixed and then loaded on the Sephadex G-25 gel column, the elution is carried out by using the ultrapure water, the flow rate elution speed is controlled to be 1mL/min and 8 mL/tube, 35 tubes are collected, the ultraviolet absorption peak is monitored under 270nm, and simultaneously the metal element signal is monitored. Combining and collecting the 15 th to 23 th tubes, and freeze-drying to obtain the desalted MT-1. The main peak desalting diagram of Mantis shrimp MT-1 is shown in FIG. 3, and the real diagrams of the two desalted components MT-1 and MT-2 are shown in FIGS. 5 and 6, respectively;

from the SDS-PAGE electrophoresis result of FIG. 7, it can be seen that the molecular weight of Oratosquilla shrimp MT-I is about 15KDa, the molecular weight of Oratosquilla shrimp MT-II is about 8KDa, the prepared Oratosla shrimp metallothionein electrophoresis has few impurity bands and high protein purity.

Example 2

(1) Removing shells of the lobster and taking an edible part, and mixing the following components in a liquid-material ratio of 3: 1 adding Tris-HCl buffer solution with pH 7.5 and concentration of 0.05mol/L, stirring and extracting for 2h in water bath at 45 ℃, freezing and centrifuging the extract at high speed for 20min at 10 ℃ and 10000r/min, and removing precipitate; collecting supernatant a, heating to 70 deg.C in water bath for 7min to remove heat-labile macromolecular protein, rapidly cooling to room temperature, and centrifuging at 10 deg.C and 10000r/min for 20 min; after collecting the supernatant b, 3 volumes of pre-cooled (temperature 4 ℃) absolute ethanol were added and precipitated overnight at-20 ℃. Centrifuging at 10 ℃ and 10000r/min for 20min, taking the precipitate, adding 2-3 mL of ultrapure water for fully dissolving, centrifuging at 4 ℃, collecting supernatant c, freeze-drying, and storing at-18 ℃ to obtain a crude mantis shrimp metallothionein extract, namely crude MT freeze-dried powder;

steps (2) to (4) were the same as in example 1.

Example 3

(1) Removing shells of the lobster and taking the edible part according to the liquid-material ratio of 6: 1 adding Tris-HCl buffer solution with pH of 8.0 and concentration of 0.04mol/L, stirring and extracting for 1.5h in water bath at 55 ℃, freezing and centrifuging the extract at high speed for 30min at 6 ℃ and 12000r/min, and removing precipitate; collecting supernatant a, heating to 75 deg.C in water bath for 3min to remove heat-labile macromolecular protein, rapidly cooling to room temperature, and centrifuging at 6 deg.C and 12000r/min for 25 min; after collecting the supernatant b, 3 volumes of pre-cooled (temperature 4 ℃) absolute ethanol were added and precipitated overnight at-20 ℃. Centrifuging at 6 ℃ and 12000r/min for 25min, taking the precipitate, adding 2-3 mL of ultrapure water to fully dissolve, centrifuging at 4 ℃, collecting supernatant c, freeze-drying, and storing at-18 ℃ to obtain a crude mantis shrimp metallothionein extract, namely crude MT freeze-dried powder;

steps (2) to (4) were the same as in example 1.

Comparative example

The comparative example is different from example 1 in that, in the DEAE-Sepharose Fast Flow ion exchange chromatography of step (3), a 0.5mol/L NaCl solution was used as a second mobile phase, and the rest of the process was completely the same.

The performance indexes of the two subtypes of MT extracted in examples 1 to 3 and the comparative example were measured, and the results are shown in Table 1:

TABLE 1 test results

Performance index	Example 1	Example 2	Example 3	Comparative example
					Oratosquilla MT-1 purity (%)	98.1	98.4	98.6	98.1
Oratosquilla MT-2 purity (%)	97.9	98.2	97.8	65.2

As can be seen from Table 1, the MT-1 of the two subtypes extracted by the method for extracting high-purity metallothionein from Orodia squilla has higher purity which can reach 98.1%. Only 0.5mol/L NaCl solution is used as the second mobile phase, so that the purity of MT-2 is only 65.2%, and the second elution peak has a tailing phenomenon, the analysis reason is that pH is a key factor influencing the stability of protein and the separation effect of column chromatography, and under the second mobile phase (pH 8.6, containing 0.02mol/L Tris-HCl and 0.5mol/L NaCl solution) used in the invention, MT-2 is good in stability, and the elution effect is better.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and other variations and modifications may be made without departing from the spirit of the invention as set forth in the claims.

Claims (6)

1. A method for extracting high-purity metallothionein from Oratosquilla tamariscina is characterized by comprising the following steps:

(1) removing shell of Mantis shrimp, collecting edible part, homogenizing, centrifuging, extracting, and freeze drying to obtain Mantis shrimp metallothionein crude extract; the centrifugal extraction process comprises the following steps: adding Tris-HCl buffer solution, stirring and extracting for 1-2 h at 45-55 ℃, freezing and centrifuging the extracting solution at a high speed, collecting supernatant a, heating to 70-80 ℃, preserving heat for 3-7 min, quickly cooling to room temperature, then freezing and centrifuging at a high speed, collecting supernatant b, adding absolute ethyl alcohol, and precipitating at-18-25 ℃ overnight; performing high-speed freezing centrifugation again, adding ultrapure water into the precipitate for full dissolution, performing high-speed freezing centrifugation again, collecting supernatant c, and performing freeze drying to obtain a crude mantis shrimp metallothionein extract;

(2) redissolving the crude extract of the mantis shrimp metallothionein, centrifuging, filtering with a filter membrane, performing gel filtration chromatography, collecting eluent, and freeze-drying to obtain a metallothionein mixture; the conditions of the gel filtration chromatography are as follows: the column chromatography packing is sephadex G50; Tris-HCl buffer with mobile phase 0.02mol/L, pH = 8.0; the flow rate is 0.8 mL/min; the elution volume is 500mL, and the collection amount is 8 mL/tube; measuring absorbance values of the collected components in each tube at 220nm and 270nm by using an ultraviolet absorption photometer, and simultaneously measuring metal element signals by combining ICP-MS;

(3) redissolving the metallothionein mixture, carrying out anion exchange chromatography, respectively collecting two sub-type metallothionein eluents, and freeze-drying to obtain a first sub-type metallothionein crude product and a second sub-type metallothionein crude product; the conditions for anion exchange chromatography were: the column chromatography filler is ion exchange Sepharose gel, and DEAE-Sepharose Fast Flow anion exchange column is adopted as a solid phase carrier; the elution speed is 0.8mL/min, the total volume of elution is 450mL, the collection amount is 8 mL/tube, the ultraviolet absorption peak is monitored under 270nm, and simultaneously the ICP-MS is combined to determine the signal of the metal element; eluting with a first mobile phase to obtain a first subtype metallothionein crude product, wherein the first mobile phase is a Tris-HCl buffer solution with the concentration of 0.02mol/L, pH = 8.0; eluting by using a second mobile phase to obtain a second subtype metallothionein crude product, wherein the second mobile phase is a mixed solution with the pH =8.0 and containing 0.02mol/L Tris-HCl and 0.5mol/L NaCl;

(4) respectively re-dissolving the first sub-type metallothionein crude product and the first sub-type metallothionein crude product, and desalting to obtain first sub-type metallothionein and second sub-type metallothionein; the desalting conditions were: the column chromatography filler is cross-linked Sephadex gel, and a Sephadex G-25 gel column is adopted for desalting; eluting with ultrapure water, and controlling the elution speed to be 1mL/min and 8 mL/tube; the UV absorption peak was monitored at 270nm while the metal element signal was monitored.

2. The method for extracting high-purity metallothionein from the Oratosquilla tamariscina according to claim 1, wherein in the step (1), the Tris-HCl buffer solution has a pH of 7.5-8.0 and a concentration of 0.02-0.05 mol/L; the addition amount of absolute ethanol is 3 times of the volume of the supernatant b.

3. The method for extracting high-purity metallothionein from Oratosquilla tamariscina according to claim 1, wherein in the step (1), the feed-liquid ratio of Oratosquilla tamariscina to Tris-HCl buffer (3-6): 1 g/mL.

4. The method for extracting high-purity metallothionein from Oratosquilla esculenta according to claim 1, wherein in step (1), the temperature of high-speed freezing centrifugation is controlled to be 4-10 ℃, the rotating speed is controlled to be 10000-15000 r/min, and the centrifugation time is controlled to be 20-30 min.

5. The method for extracting high-purity metallothionein from the Oratosquilla tamariscina according to claim 1, wherein in steps (2) and (3), Tris-HCl is adopted for reconstitution; and (4) redissolving by adopting ultrapure water.

6. The method for extracting high-purity metallothionein from Oratosquilla esculenta according to claim 1, wherein in step (2), the filtration is performed by using a 0.45 μm filter membrane.

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