CN107056935A - The preparation method of metallothionein - Google Patents
The preparation method of metallothionein Download PDFInfo
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- CN107056935A CN107056935A CN201610851991.3A CN201610851991A CN107056935A CN 107056935 A CN107056935 A CN 107056935A CN 201610851991 A CN201610851991 A CN 201610851991A CN 107056935 A CN107056935 A CN 107056935A
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- metallothionein
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/825—Metallothioneins
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Abstract
The invention discloses a kind of preparation method of metallothionein, belong to biological technical field.The present invention is handled through steam explosion again after plant is induced through aqueous metal salt, and the separating-purifying under certain pressure, temperature, inert atmosphere, is carried out enzymolysis with normal temperature type alpha amylase and is finally given metallothionein finished product.The present invention is from materials such as dungarunga, great Ye crops, melon seedlings, high content, high-quality metallothionein are prepared after induction, and the present invention is broken through cell membrane by impulsive force from inside to outside, is made cellular content using the steam explosion method under normal temperature, high pressure（Include metallothionein）All " broken wall and go out ", beneficial to the lock out operation of next step, and special biology enzyme is applied, it is ensured that high efficiency, high-quality, high yield that the present invention is utilized to induction plant.Simple production process, easily operated and Technique Popularizing.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of preparation method of metallothionein.
Background technology
Metallothionein（metallothionein）It is the metal-binding protein by microorganism and animal and plant generation, is one
Plant low molecule amount, high sulfhydryl content, can largely combine the protein of heavy metal ion, therefore referred to as metallothionein（Referred to as
MT）.
Root is it was found that the important physiological function of metallothionein shows that it is a kind of maximally effective freedom being currently known
Base scavenger.It removes hydroxyl free radical（•OH）Ability be about thousands of times of SOD, and scavenging activated oxygen（•O）Energy
Power is approximately glutathione（GSH）25 times, and with very strong antioxidation activity；And thus derive antitumor, the prevention heart
The physiological function such as cranial vascular disease and parkinsonism.Application of the metallothionein in fields such as pharmaceuticals, health products, cosmetics is got over
Come more extensive.
The main source of current metallothionein is rabbit liver.Due to worrying that people and animals laterally infect disease, the metal of animal sources
Sulfoprotein is progressively disabled in European and American areas.Therefore, the metallothionein that exploitation has more the plant source of security has been when business
It is anxious.
The content of the invention
In order to make up the deficiencies in the prior art, the invention provides a kind of preparation method of metallothionein.
The technical scheme is that：
A kind of preparation method of metallothionein, including step：
A raw materials prepare
1）Induce plant：Derivant is poured into root or sprayed in plant leaf surface, mean dose is controlled in 5-30 mg/kg butts
Plant；It is used in conjunction 5-20 days, every other day using 1 time, each dosage is more last high 0.3-3 times；
2）By the plant after above-mentioned induction through removal of impurities, rub silk, segment, drying, crushing after insert in steam-explosion jar, powder addition reaches
To 5-the 40% of tank volume, air rear enclosed in inert gas emptying tank is slowly passed through with air compressor machine；Constantly it is forced into 1-
5MPa, pressurize 20-120 minutes；Sealing jetting storehouse is spurted, steam explosion powder is obtained；Be filled with inert gas preserve, it is standby；
The preparation of B metallothioneins
3）Steam explosion powder enters in extractor, adds the Tris-HCl buffer solutions of 1-20 times of volumes, the quality of Tris-HCl buffer solutions
Concentration is 0.01 ~ 0.1mol/L, and inert gas is passed through with air compressor machine, after stirring evenly, and is extracted 10-60 minutes；
4）By step 3）Mixed solution in middle extractor is centrifuged, and takes supernatant；1-3 hours are stood, clarifying treatment, then
By ultrafiltration, nanofiltration separation system, metallothionein thick liquid of the molecular weight between 10000 to 15000 dalton is obtained；
5）The thick liquid of above-mentioned metallothionein is moved into enzymatic vessel, normal temperature type alpha-amylase is added, interval 1 minute is passed through indifferent gas
Body 1 minute；Enzymolysis 10-80 minutes；The normal temperature type alpha-amylase is by microwave induced gained variation bacillus licheniformis secretion institute
Alpha-amylase is obtained, the preference temperature of the normal temperature type alpha-amylase is 22-35 DEG C；
6）By step 5）Liquid after enzymolysis moves into refrigerated centrifuge high speed centrifugation, takes supernatant；
7）The directly upper Sephadex G-50 column chromatographies of supernatant are separated, buffered with 0.005 ~ 0.05 mol/L Tris-HCl
Liquid is eluted, and collects the eluent of the component containing metallothionein；
8）By step 7）The eluent of middle collection is concentrated in vacuo, and the remainder after concentration is washed with Tris-HCl buffer solutions
1 ~ 5 time, this step is repeated, metallothionein is obtained；Metallothionein finished product is obtained after drying, crushing.
Wherein, the induction plant of selection is shrub, dungarunga, great Ye crops, melon seedling etc.；Melon seedling includes its stem, leaf.
Further, step 1）Described in derivant be the metallic salt aqueous solution, derivant aqueous solution mass concentration be 10-
50%。
Step 1）Described in derivant include copper, iron, zinc, the sulfate of manganese, nitrate or one kind or many in chloride
Plant mixture.
Wherein, step 5）In, the obtaining step of the microwave induced gained variation bacillus licheniformis is specially：By lichens
The nutrient solution of bacillus is placed in microwave generator, and setting microwave power is 850-950W, and pulse frequency is 2300MHz, microwave
20s is handled, 20s is cooled down, it is reciprocal 25-35 times according to this；Nutrient solution after microwave treatment is coated on solid medium, 30 DEG C of bars
Cultivated 1-2 days under part, by screening the high bacillus licheniformis of alpha-amylase activity under four plants of normal temperature in the bacterium colony survived
Dissociant.
The dissociant for selecting alpha-amylase highest bacillus licheniformis under normal temperature expands culture, so as to obtain described
Normal temperature type alpha-amylase.
The addition of normal temperature type alpha-amylase described above meets the thick liquid 30-70U of every liter of metallothionein.
Further, the inert gas is nitrogen, helium or argon gas.
Further, the Tris-HCl pH of buffer 6 ~ 10.
Further, step 8）Described in dry be spray drying, heated-air drying, fluidized bed drying or freeze-drying.
Beneficial effects of the present invention are：
The present invention prepares high content, high-quality metal sulphur from materials such as dungarunga, great Ye crops, melon seedlings after induction
Albumen, and the present invention uses the steam explosion method under normal temperature, high pressure, breaks through cell membrane by impulsive force from inside to outside, makes
Cellular content（Include metallothionein）All " broken wall and go out ", beneficial to the lock out operation of next step, and apply it is special
Biology enzyme, it is ensured that high efficiency, high-quality, high yield that the present invention is utilized to induction plant.It is simple production process, easily operated
And Technique Popularizing.
Embodiment
Embodiment 1
1）High hainan holly leaf tree is selected as the material for preparing plant source metallothionein, from zinc sulfate as derivant, first
The solution of zinc sulfate of mass concentration 15% is prepared, then takes and sprays in the method on tea tree blade face, mean dose is controlled at 10 milligrams
Zinc/kilogram butt tea tree；It is used in conjunction 10 days, every other day using 1 time, each dosage is more last high 0.5 times；
2）Tealeaves after harvest induction, enters removal of impurities after factory, rubs silk, crushing；Insert in steam-explosion jar, addition reaches tank volume
25%, air rear enclosed in inert gas, such as helium, argon gas or nitrogen, emptying tank is slowly passed through with air compressor machine；Constantly pressurization
To 2MPa, pressurize 90 minutes；Sealing jetting storehouse is spurted, tealeaves steam explosion powder is obtained；Be filled with inert gas preserve, it is standby；
The impulsive force from inside to outside produced here with gases at high pressure during spurting breaks through cell membrane, makes cellular content
（Contain metallothionein）All " broken wall and go out ", beneficial to the lock out operation of next step；
3）Tealeaves steam explosion powder is entered in extractor, the Tris-HCl buffer solutions of 10 times of volumes are added, Tris-HCl buffer solutions
Mass concentration is 0.03mol/L, and inert gas is passed through with air compressor machine, and such as helium, argon gas or nitrogen are stirred evenly, and is extracted 30 minutes；
4）By step 3）Mixed solution in middle extractor is centrifuged, and takes supernatant；1.5 hours are stood, clarifying treatment, Ran Houtong
Ultrafiltration, nanofiltration separation system are crossed, metallothionein thick liquid of the molecular weight between 10000 to 15000 dalton is obtained；
5）The thick liquid of above-mentioned metallothionein is moved into enzymatic vessel, normal temperature type alpha-amylase is added, interval 1 minute is passed through indifferent gas
Body 1 minute；Enzymolysis 40 minutes；The normal temperature type alpha-amylase by microwave induced gained variation bacillus licheniformis secretion gained α-
Amylase, the preference temperature of the normal temperature type alpha-amylase is 22-35 DEG C；Normal temperature type alpha-amylase addition is that 40U/ rises metal
The thick liquid of sulfoprotein.
6）By step 5）Liquid after enzymolysis moves into refrigerated centrifuge high speed centrifugation, and rotating speed is 12000 revs/min, is taken
Clearly；
7）The directly upper Sephadex G-50 column chromatographies of supernatant are separated, with 0.015 mol/L Tris-HCl buffer solutions
（pH8.5）Eluted, collect the eluent of the component containing metallothionein；
8）By step 7）The eluent of middle collection is concentrated in vacuo, and the remainder after concentration is washed with Tris-HCl buffer solutions
3 times, this step is repeated, metallothionein is obtained；Metallothionein finished product is obtained after freeze-dried, crushing.
Step 5）Middle normal temperature type alpha-amylase is by microwave induced gained variation bacillus licheniformis secretion gained alpha-amylase；
The obtaining step of microwave induced gained variation bacillus licheniformis is specially：The nutrient solution of bacillus licheniformis is placed in microwave hair
Raw device, setting microwave power is 900W, and pulse frequency is 2300MHz, microwave treatment 20s, cools down 20s, according to this reciprocal 30 times；Will
Nutrient solution after microwave treatment is coated on solid medium, is cultivated 1-2 days under the conditions of 30 DEG C, is sieved in the bacterium colony survived
Select the dissociant of the high bacillus licheniformis of alpha-amylase activity under four plants of normal temperature.Select alpha-amylase activity highest under normal temperature
Bacillus licheniformis dissociant expand culture, so as to obtain normal temperature type alpha-amylase；Normal temperature type alpha-amylase is in 22-35
Expeditiously hydrolysis starch at a temperature of DEG C, it is not necessary to as the thermal-stable α-amylase used mostly at present needs high temperature（80-90℃）
Condition, thus reduce energy consumption and also reduce requirement to equipment；Herein starch can be mildly removed with enzyme process, it is to avoid form sediment
Influence of the powder to next step column chromatography；
After measured, the quality of tealeaves metallothionein obtained by the present embodiment is that the 0.084% of tealeaves butt quality, purity is 95.5%
（Fine work level is reached）.
Embodiment 2
1）Select highland watermelon seedling（Including leaf）It is used as the material for preparing plant source metallothionein；From copper sulphate as luring
Agent is led, the copper-bath of mass concentration 20% is prepared first, is sprayed in watermelon seedling（And blade face）Method, mean dose control
System is in 11 milligrams of copper/kilogram butt watermelon seedling；It is used in conjunction 12 days, every other day using 1 time, each dosage is more last high 0.6 times；
2）Watermelon seedling and leaf after harvest induction, enter removal of impurities after factory, rub silk, crushing；Insert in steam-explosion jar, addition reaches tank
The 30% of body volume, air rear enclosed in inert gas, such as helium, argon gas or nitrogen, emptying tank is slowly passed through with air compressor machine；
Constantly it is forced into 2.5MPa, pressurize 85 minutes；Sealing jetting storehouse is spurted, melon seedling steam explosion powder is obtained；Be filled with inert gas preserve,
It is standby；
The impulsive force from inside to outside produced here with gases at high pressure during spurting breaks through cell membrane, makes cellular content
（Contain metallothionein）All " broken wall and go out ", beneficial to the lock out operation of next step；
3）Melon seedling steam explosion powder is entered in extractor, the Tris-HCl buffer solutions of 8 times of volumes, the matter of Tris-HCl buffer solutions is added
Amount concentration is 0.025mol/L, inert gas is passed through with air compressor machine, such as helium, argon gas or nitrogen are stirred evenly, and is extracted 35 minutes；
4）By step 3）Mixed solution in middle extractor is centrifuged, and takes supernatant；2 hours are stood, then clarifying treatment passes through
Ultrafiltration, nanofiltration separation system, obtain metallothionein thick liquid of the molecular weight between 10000 to 15000 dalton；
5）The thick liquid of above-mentioned metallothionein is moved into enzymatic vessel, normal temperature type alpha-amylase is added, interval 1 minute is passed through indifferent gas
Body 1 minute；Enzymolysis 35 minutes；The normal temperature type alpha-amylase by microwave induced gained variation bacillus licheniformis secretion gained α-
Amylase, the preference temperature of the normal temperature type alpha-amylase is 22-35 DEG C；Normal temperature type alpha-amylase addition is that 50U/ rises metal
The thick liquid of sulfoprotein.
6）By step 5）Liquid after enzymolysis moves into refrigerated centrifuge high speed centrifugation, and rotating speed is 13000 revs/min, is taken
Clearly；
7）The directly upper Sephadex G-50 column chromatographies of supernatant are separated, with 0.012 mol/L Tris-HCl buffer solutions
（pH8.4）Eluted, collect the eluent of the component containing metallothionein；
8）By step 7）The eluent of middle collection is concentrated in vacuo, and the remainder after concentration is washed with Tris-HCl buffer solutions
3 times, this step is repeated, metallothionein is obtained；Metallothionein finished product is obtained after freeze-dried, crushing.
Step 5）Middle normal temperature type alpha-amylase is by microwave induced gained variation bacillus licheniformis secretion gained alpha-amylase；
The obtaining step of microwave induced gained variation bacillus licheniformis is specially：The nutrient solution of bacillus licheniformis is placed in microwave hair
Raw device, setting microwave power is 900W, and pulse frequency is 2300MHz, microwave treatment 20s, cools down 20s, according to this reciprocal 30 times；Will
Nutrient solution after microwave treatment is coated on solid medium, is cultivated 1-2 days under the conditions of 30 DEG C, is sieved in the bacterium colony survived
Select the dissociant of the high bacillus licheniformis of alpha-amylase activity under four plants of normal temperature.Select alpha-amylase activity highest under normal temperature
Bacillus licheniformis dissociant expand culture, so as to obtain normal temperature type alpha-amylase；Normal temperature type alpha-amylase is in 22-35
Expeditiously hydrolysis starch at a temperature of DEG C, it is not necessary to as the thermal-stable α-amylase used mostly at present needs high temperature（80-90℃）
Condition, thus reduce energy consumption and also reduce requirement to equipment；Herein starch can be mildly removed with enzyme process, it is to avoid form sediment
Influence of the powder to next step column chromatography；
After measured, the quality of watermelon seedling metallothionein obtained by the present embodiment is that the 0.097% of watermelon seedling butt quality, purity is
95.8%（Fine work level is reached）.
Embodiment 3
1）Selection maize leaves are used as the material for preparing plant source metallothionein；From zinc sulfate as derivant, prepare first
The solution of zinc sulfate of mass concentration 18%, then takes the method for pouring root, and mean dose is controlled in 9 milligrams of zinc/kilogram butt corn
Stalk；It is used in conjunction 14 days, every other day using 1 time, each dosage is more last high 0.7 times；
2）Maize leaves after harvest induction, enter removal of impurities after factory, rub silk, crushing；Insert in steam-explosion jar, addition reaches tank volume
20%, air rear enclosed in inert gas, such as helium, argon gas or nitrogen, emptying tank is slowly passed through with air compressor machine；Constantly add
It is depressed into 3MPa, pressurize 80 minutes；Sealing jetting storehouse is spurted, maize leaves steam explosion powder is obtained；Be filled with inert gas preserve, it is standby；
The impulsive force from inside to outside produced here with gases at high pressure during spurting breaks through cell membrane, makes cellular content
（Contain metallothionein）All " broken wall and go out ", beneficial to the lock out operation of next step；
3）Maize leaves steam explosion powder is entered in extractor, the Tris-HCl buffer solutions of 12 times of volumes, Tris-HCl buffer solutions is added
Mass concentration be 0.015mol/L, inert gas is passed through with air compressor machine, such as helium, argon gas or nitrogen are stirred evenly, extract 25 points
Clock；
4）By step 3）Mixed solution in middle extractor is centrifuged, and takes supernatant；2.5 hours are stood, clarifying treatment, Ran Houtong
Ultrafiltration, nanofiltration separation system are crossed, metallothionein thick liquid of the molecular weight between 10000 to 15000 dalton is obtained；
5）The thick liquid of above-mentioned metallothionein is moved into enzymatic vessel, normal temperature type alpha-amylase is added, interval 1 minute is passed through indifferent gas
Body 1 minute；Enzymolysis 30 minutes；The normal temperature type alpha-amylase by microwave induced gained variation bacillus licheniformis secretion gained α-
Amylase, the preference temperature of the normal temperature type alpha-amylase is 22-35 DEG C；Normal temperature type alpha-amylase addition is that 35U/ rises metal
The thick liquid of sulfoprotein.
6）By step 5）Liquid after enzymolysis moves into refrigerated centrifuge high speed centrifugation, and rotating speed is 15000 revs/min, is taken
Clearly；
7）The directly upper Sephadex G-50 column chromatographies of supernatant are separated, with 0.011 mol/L Tris-HCl buffer solutions
（pH8.6）Eluted, collect the eluent of the component containing metallothionein；
8）By step 7）The eluent of middle collection is concentrated in vacuo, and the remainder after concentration is washed with Tris-HCl buffer solutions
3 times, this step is repeated, metallothionein is obtained；Metallothionein finished product is obtained after freeze-dried, crushing.
Step 5）Middle normal temperature type alpha-amylase is by microwave induced gained variation bacillus licheniformis secretion gained alpha-amylase；
The obtaining step of microwave induced gained variation bacillus licheniformis is specially：The nutrient solution of bacillus licheniformis is placed in microwave hair
Raw device, setting microwave power is 900W, and pulse frequency is 2300MHz, microwave treatment 20s, cools down 20s, according to this reciprocal 30 times；Will
Nutrient solution after microwave treatment is coated on solid medium, is cultivated 1-2 days under the conditions of 30 DEG C, is sieved in the bacterium colony survived
Select the dissociant of the high bacillus licheniformis of alpha-amylase activity under four plants of normal temperature.Select alpha-amylase activity highest under normal temperature
Bacillus licheniformis dissociant expand culture, so as to obtain normal temperature type alpha-amylase；Normal temperature type alpha-amylase is in 22-35
Expeditiously hydrolysis starch at a temperature of DEG C, it is not necessary to as the thermal-stable α-amylase used mostly at present needs high temperature（80-90℃）
Condition, thus reduce energy consumption and also reduce requirement to equipment；Herein starch can be mildly removed with enzyme process, it is to avoid form sediment
Influence of the powder to next step column chromatography；
After measured, the quality of maize leaves metallothionein obtained by the present embodiment is that the 0.088% of maize leaves butt quality, purity is
95.9%（Fine work level is reached）.
Claims (10)
1. a kind of preparation method of metallothionein, it is characterised in that including step：
A raw materials prepare
1）Induce plant：Derivant is poured into root or sprayed in plant leaf surface, mean dose is controlled in 5-30 mg/kg butts
Plant；It is used in conjunction 5-20 days, every other day using 1 time, each dosage is more last high 0.3-3 times；
2）By the plant after above-mentioned induction through removal of impurities, rub silk, segment, drying, crushing after insert in steam-explosion jar, powder addition reaches
To 5-the 40% of tank volume, air rear enclosed in inert gas emptying tank is slowly passed through with air compressor machine；Constantly it is forced into 1-
5MPa, pressurize 20-120 minutes；Sealing jetting storehouse is spurted, steam explosion powder is obtained；Be filled with inert gas preserve, it is standby；
The preparation of B metallothioneins
3）Steam explosion powder enters in extractor, adds the Tris-HCl buffer solutions of 1-20 times of volumes, the quality of Tris-HCl buffer solutions
Concentration is 0.01 ~ 0.1mol/L, and inert gas is passed through with air compressor machine, after stirring evenly, and is extracted 10-60 minutes；
4）By step 3）Mixed solution in middle extractor is centrifuged, and takes supernatant；1-3 hours are stood, clarifying treatment, then
By ultrafiltration, nanofiltration separation system, metallothionein thick liquid of the molecular weight between 10000 to 15000 dalton is obtained；
5）The thick liquid of above-mentioned metallothionein is moved into enzymatic vessel, normal temperature type alpha-amylase is added, interval 1 minute is passed through indifferent gas
Body 1 minute；Enzymolysis 10-80 minutes；The normal temperature type alpha-amylase is by microwave induced gained variation bacillus licheniformis secretion institute
Alpha-amylase is obtained, the preference temperature of the normal temperature type alpha-amylase is 22-35 DEG C；
6）By step 5）Liquid after enzymolysis moves into refrigerated centrifuge high speed centrifugation, takes supernatant；
7）The directly upper Sephadex G-50 column chromatographies of supernatant are separated, buffered with 0.005 ~ 0.05 mol/L Tris-HCl
Liquid is eluted, and collects the eluent of the component containing metallothionein；
8）By step 7）The eluent of middle collection is concentrated in vacuo, and the remainder after concentration is washed with Tris-HCl buffer solutions
1 ~ 5 time, this step is repeated, metallothionein is obtained；Metallothionein finished product is obtained after drying, crushing.
2. the preparation method of metallothionein as claimed in claim 1, it is characterised in that：The induction plant of selection is shrub, small
Arbor, great Ye crops, melon seedling etc..
3. the preparation method of metallothionein as claimed in claim 1, it is characterised in that：Step 1）Described in derivant be metal
Salts solution, derivant aqueous solution mass concentration is 10-50%.
4. the preparation method of metallothionein as claimed in claim 1, it is characterised in that：Step 1）Described in derivant include
One or more mixtures in copper, iron, zinc, the sulfate of manganese, nitrate or chloride.
5. the preparation method of metallothionein as claimed in claim 1, it is characterised in that：Step 5）In, the microwave induced institute
The obtaining step of bacillus licheniformis of must making a variation is specially：The nutrient solution of bacillus licheniformis is placed in microwave generator, set
Microwave power is 850-950W, and pulse frequency is 2300MHz, microwave treatment 20s, cools down 20s, reciprocal 25-35 times according to this；Will be micro-
Nutrient solution after ripple processing is coated on solid medium, is cultivated 1-2 days under the conditions of 30 DEG C, is screened in the bacterium colony survived
The dissociant of the high bacillus licheniformis of alpha-amylase activity under four plants of normal temperature.
6. the preparation method of metallothionein as claimed in claim 5, it is characterised in that：Select under normal temperature alpha-amylase activity most
The dissociant of high bacillus licheniformis expands culture, so as to obtain the normal temperature type alpha-amylase.
7. the preparation method of metallothionein as described in claim 5 or 6, it is characterised in that：The normal temperature type alpha-amylase
Addition meets the thick liquid 30-70U of every liter of metallothionein.
8. the preparation method of metallothionein as claimed in claim 1, it is characterised in that：The inert gas is nitrogen, helium
Or argon gas.
9. the preparation method of metallothionein as claimed in claim 1, it is characterised in that：The Tris-HCl pH of buffer 6 ~
10。
10. the preparation method of metallothionein as claimed in claim 1, it is characterised in that：Step 8）Described in dry for spraying
Drying, heated-air drying, fluidized bed drying or freeze-drying.
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