CN105154501A - Technology for fermenting halostachys caspica metallothionein and application of halostachys caspica metallothionein - Google Patents
Technology for fermenting halostachys caspica metallothionein and application of halostachys caspica metallothionein Download PDFInfo
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Abstract
The invention provides a technology for fermenting halostachys caspica metallothionein and an application of the halostachys caspica metallothionein. The technology includes the steps that a recombination expression transformant in the prior art is cultivated at the temperature of 37 DEG C for staying overnight and then is inoculated to an improved fermentation medium at the proportion of 2%, fermentation is conducted in a full-automatic fermentation tank, and the liquid containing coefficient is 0.5; overall sterilization is carried out after an antifoaming agent is added, the ventilatory capacity is 1 L/min, the temperature is kept to be 37 DEG C, the rotating speed of stirring is 100 r/min, lactose with the concentration being 6 mmol/L is added for inducing after fermentation is conducted for 4 hours, and fermentation liquor is centrifuged for 10 minutes at the rotating speed of 8000 rpm after fermentation continues for 4 hours so that thalli can be obtained; then PBS is used for washing and centrifuging three times, the thallus dry weight of the halostachys caspica metallothionein is subjected to constant-weight weighing after drying is performed for 1.5 hours at the temperature of 105 DEG C, and the obtained thallus quantity is increased by 31% compared with shake-flask culture. By means of the technology for fermenting the halostachys caspica metallothionein and the application of the halostachys caspica metallothionein, the yield of the halostachys caspica metallothionein can be greatly increased, and the technology is suitable for large-scale industrial production and has wide significance to the application field of the metallothionein.
Description
Technical field
The present invention relates to technical field of biological fermentation, concrete, the present invention relates to the technical field of caspian halostachys metallothionein(MT) zymotechnique and application thereof.
Background technology
Metallothionein(MT) (
metallothionein, MT) and be that a class is extensively present in lower molecular weight in organism, is rich in the metal binding protein of halfcystine.If with the master metal cadmium of its combination, copper and zinc, be present in from microorganism to the mankind in various biology widely, its structure height is guarded.Nineteen fifty-seven Margoshes and Valles, when studying the biological action of cadmium, isolates this material first from the horse kidney of accumulation cadmium.MT has following feature: molecular weight is low, is generally 6 ~ 7kD; Energy chelated metal ions, albumen, by after thioester bond and melts combine, has special light absorption characteristics; Cysteine content is high, accounts for 23% ~ 33% of total amino acid residues, but does not form disulfide linkage, lacks die aromatischen Aminosaeuren and Histidine in molecule; Cysteine residues has comparatively conservative arrangement mode in aminoacid sequence.The sulfhydryl content that metallothionein(MT) enriches and heavy metal binding ability determine the diversity of its function.
Caspian halostachys (
halostachyscaspica) for Chenopodiaceae caspian halostachys belongs to, be that the important salt of growth on desert, half-desert saltings is raw, the raw rare salt succulence subshrub plant of drought, extremely strong to salinity adaptability, be one of salt desert main plant.Under high salt and heavy metal stress, the metallothionein gene expression level in its body is obvious ascendant trend.The producer of current domestic production MT, mainly extracts from rabbit liver, because MT raw materials requirement is special, and complex manufacturing, cause current MT output little and expensive.Existing document has disclosed a kind of caspian halostachys metallothionein gene and recombinant protein thereof and application (Liu Zhongyuan, number of patent application: 2013105667426), prior art discloses a kind of method being only applicable to laboratory and preparing caspian halostachys metallothionein(MT) on a small scale, caspian halostachys metallothionein(MT) is prepared in laboratory, and to have cost high, the limiting factors such as extraction yield is low, and in existing document, do not disclose a kind of technical scheme being suitable for suitability for industrialized production caspian halostachys metallothionein(MT), along with the purposes of caspian halostachys metallothionein(MT) product constantly expands, consumption constantly increases, the output of domestic and international caspian halostachys metallothionein(MT) can not meet market actual demand far away, so be badly in need of a kind of high yield, low cost, security is high, be suitable for the processing method of suitability for industrialized production caspian halostachys metallothionein(MT).
Summary of the invention
For the present Research of the method had no in prior art about suitability for industrialized production caspian halostachys metallothionein(MT), the object of the invention is intended to provide a kind of caspian halostachys metallothionein(MT) zymotechnique and application thereof, the fermention medium after improveing is obtained by condition optimizing, adopt fermentor tank to amplify to produce, thus improve caspian halostachys metallothionein(MT) output, overcome the low-producing present situation of high cost in current caspian halostachys metallothionein(MT) preparation field.The present invention changes the production method of conventional metals sulfoprotein, and the caspian halostachys metallothionein(MT) zymotechnique of the application of the invention, can increase substantially the output of caspian halostachys metallothionein(MT), be suitable for suitability for industrialized production.
The present invention is for realizing above technical purpose, and provide a kind of zymotechnique and application thereof of caspian halostachys metallothionein(MT), concrete fermentation step is as follows:
By the recombinant expressed transformant that obtains in prior art 37 DEG C of overnight incubation, the fermention medium after improvement is inoculated into again with the ratio of 2%, ferment at automatic fermenter, dress liquid coefficient is 0.5, overall sterilizing after interpolation defoamer, air flow is set to 1L/min, keep temperature 37 DEG C, mixing speed 100r/min, after fermentation 4h, adding concentration is that 6mmol/L lactose is induced, after continuing fermentation 4h, fermented liquid is obtained thalline with the centrifugal 10min of 8000rpm, again with centrifugal three times of PBS washing, after 105 DEG C of dry 1.5h, constant weight weighs the dry cell weight preparing caspian halostachys metallothionein(MT).
Concrete, the present invention also provides a kind of improved culture medium being suitable for the fermentation of caspian halostachys metallothionein(MT), obtains the fermention medium after improveing by condition optimizing, comprise two portions by weight: a part is the substratum of 900mL, containing 4g sucrose, 24g yeast leaching powder, 8g peptone; Another part is the phosphoric acid buffer of 100mL, wherein containing 2.31gKH
2pO
4, 12.54gK
2hPO
43H
2o, by the improved culture medium of mixed preparing 1L after two portions respectively autoclaving.
The present invention is based on existing prior art " a kind of caspian halostachys metallothionein gene and recombinant protein and application " thereof (Liu Zhongyuan, number of patent application: 2013105667426) disclosed caspian halostachys metallothionein gene and plasmid pET-32a (+) recombination to construct prokaryotic expression carrier, the prokaryotic expression carrier of restructuring is converted into E. coli BL21(DE3 again) obtain recombinant expressed transformant.
The present invention is based on existing prior art " a kind of caspian halostachys metallothionein gene and recombinant protein and application " thereof (Liu Zhongyuan, number of patent application: the caspian halostachys metallothionein(MT) 2013105667426) provided, described caspian halostachys metallothionein gene total length is 234bp, initiator codon is ATG, and terminator codon is TGA; This sequence intronless, has complete open reading frame, 78 amino acid of encoding; Coded product, namely the aminoacid sequence of caspian halostachys metallothionein(MT) of the present invention is as shown in SEQIDNO.2 in table; Molecular weight is 7.7kD, and iso-electric point is 4.78; Containing 14 halfcystines and 15 glycine.
The present invention is based on existing prior art " a kind of caspian halostachys metallothionein gene and recombinant protein and application " thereof (Liu Zhongyuan, number of patent application: the recombinant expression vector that the caspian halostachys metallothionein(MT) 2013105667426) provided adopts, comprises the nucleotide sequence of the caspian halostachys metallothionein gene that the present invention adopts; The preferred prokaryotic expression carrier of carrier framework of this recombinant expression vector, more preferably pET-32a (+), this recombinant expressed transformant preferred bacterium, more preferably E. coli BL21(DE3).
The present invention is based on existing prior art " a kind of caspian halostachys metallothionein gene and recombinant protein and application " thereof (Liu Zhongyuan, number of patent application: the caspian halostachys metallothionein(MT) 2013105667426) provided, its aminoacid sequence is as shown in sequence table SEQ IDNO.2.The restructuring caspian halostachys metallothionein(MT) obtained belongs to II shaped metal sulfoprotein, and the caspian halostachys metallothionein(MT) mature peptide coded by it is 78 amino acid, and molecular weight is 7.7kD, and iso-electric point is 4.78, containing 14 halfcystines and 15 glycine.
The invention provides the described application of fermentation caspian halostachys metallothionein(MT) in the fields such as medicine, health care of food, cosmetics additive, environmental protection.
By implementing the concrete summary of the invention of the present invention, following beneficial effect can be reached:
(1) the invention provides a kind of zymotechnique and application thereof of caspian halostachys metallothionein(MT), obtained the fermention medium of improvement by condition optimizing, its composition comprises two portions by weight: a part is the substratum of 900mL, containing 4g sucrose, 24g yeast leaching powder, 8g peptone; Another part is the phosphoric acid buffer of 100mL, wherein containing 2.31gKH
2pO
4, 12.54gK
2hPO
43H
2o, by the improved culture medium of mixed preparing 1L after two portions respectively autoclaving.After the lactose-induced 4h of 6mmol/L, tank amplifies fermentation by fermentation, makes biomass reach 2.68g/L, improves 31% than shake-flask culture, suitable amplification culture.
(2) zymotechnique of a kind of caspian halostachys metallothionein(MT) provided by the invention and application thereof, change the present situation that cost in caspian halostachys metallothionein(MT) preparation process is high, yield poorly, the output of caspian halostachys metallothionein(MT) can be increased substantially, and be suitable for industrialization scale operation, profound significance is embodied to metallothionein(MT) Application Areas.
Accompanying drawing explanation
Fig. 1 is shown as the mensuration figure of recombination bacillus coli growth curve.
Fig. 2 is shown as the effect diagram of different carbon source to biomass.
Fig. 3 is shown as the effect diagram of different concns sucrose to increment.
Fig. 4 is shown as the effect diagram of yeast powder concentration to biomass.
Fig. 5 is shown as the effect diagram of peptone concentration to biomass.
Fig. 6 is shown as the effect diagram of phosphate concn to Fungal biodiversity.
Fig. 7 is shown as the proof test figure of Optimal Medium.
Fig. 8 is shown as SDS-PAGE and detects expression of recombinant proteins situation map, and 2,4,6,8,10,12 represent the front total protein of different mono-clonal 1-6 recombinant bacterium induction, and 1,3,5,7,9,11 represent the rear total protein of different mono-clonal 1-6 recombinant bacterium induction.
Fig. 9 is shown as SDS-PAGE and detects expression of recombinant proteins situation map, 1,4,7,10,13,16 represent total protein before recombinant bacterium induction, 2-3,5-6,8-9,11-12,14-15,17-18:6,8,10,12,14 and 16mmol/L represent total protein after lactose-induced recombinant bacterium.
Figure 10 is shown as different induction time expression of recombinant proteins situation map, and 1,11 representatives lure front total protein; 2-4 representative induction 1h; 5-7 representative induction 2h; 8-10 representative induction 3h; 12-14 representative induction 4h; 15-17 representative induction 5h; 18-20 representative induction 6h.
Embodiment
, for embodiment, the present invention is described below, but the present invention is not limited to following embodiment.In addition, in following explanation, if no special instructions, % all refers to m/m mass percent.
The embodiment provided below the present invention is based on existing prior art " a kind of caspian halostachys metallothionein gene and recombinant protein and application " thereof (Liu Zhongyuan, number of patent application: 2013105667426) disclosed caspian halostachys metallothionein gene and plasmid pET-32a (+) recombination to construct prokaryotic expression carrier, the prokaryotic expression carrier of restructuring is converted into E. coli BL21(DE3 again) obtain recombinant expressed transformant, and adopting the zymotechnique existing recombinant expressed transformant basis being carried out following caspian halostachys metallothionein(MT).
The all reagent selected in the present invention and instrument are all well known selecting, but do not limit enforcement of the present invention, and other reagent more well known in the art and equipment are all applicable to the enforcement of the following embodiment of the present invention.
embodiment one: a kind of zymotechnique of caspian halostachys metallothionein(MT)
A zymotechnique for caspian halostachys metallothionein(MT), concrete fermentation process step is as follows:
(1) configuration of substratum and sterilizing
Obtained the fermention medium of improvement by condition optimizing, its composition comprises two portions by weight: a part is the substratum of 900mL, containing 4g sucrose, and 24g yeast leaching powder, 8g peptone; Another part is the phosphoric acid buffer of 100mL, wherein containing 2.31gKH
2pO
4, 12.54gK
2hPO
43H
2o, by the improved culture medium of mixed preparing 1L after two portions respectively autoclaving.
(2) fermentor tank enlarged culturing
By the recombinant expressed transformant that obtains in existing invention 37 DEG C of overnight incubation, the fermention medium after improvement is inoculated into again with the ratio of 2%, ferment at automatic fermenter, dress liquid coefficient is 0.5, overall sterilizing after interpolation defoamer, air flow is set to 1L/min, keep temperature 37 DEG C, mixing speed 100r/min, after fermentation 4h, adding concentration is that 6mmol/L lactose is induced, after continuing fermentation 4h, fermented liquid is obtained thalline with the centrifugal 10min of 8000rpm, again with centrifugal three times of PBS washing, after 105 DEG C of dry 1.5h, constant weight weighs the dry cell weight of caspian halostachys metallothionein(MT).
embodiment two: the selection of substratum in the zymotechnique of caspian halostachys metallothionein(MT)
The Preliminary fermentation substratum adopted is TB substratum, and initial fermentation condition is: inoculum size 1% (V/V), liquid amount 30/250mL, temperature 37 DEG C, and rotating speed 200r/min shaking table is cultured to OD
600during ≈ 0.6, add the IPTG of final concentration 1mmol/L, inducing culture 6h.
(1) selection in seed age
The LB substratum selecting recombination bacillus coli the most frequently used, as seed culture medium, measures the growth curve of seed liquor, see accompanying drawing 1.
From accompanying drawing 1, bacterial classification, within 0-1h, is in lag phase, enters logarithmic phase rapidly after 1h, and 6-10h is in stationary phase, and after 10h, thalline falls into a decline.The kind of fermented bacterium is advisable with the logarithmic growth middle and later periods age, and kind is too low for age, and the density of bacterial strain is low, and whole fermentation period can extend, and corresponding Product Expression amount also can reduce; Kind is excessively old for age, although bacterium amount is more, mostly bacterial strain is old and feeble bacterial strain, and vitality is lower, and plasmid also may be lost, thus affects the growth of recombinant bacterium and the expression of metallothionein(MT).Therefore according to the growth curve of seed, select fermented bacterium for planting 6h in age, OD
600the seed culture fluid of ≈ 1.5.
(2) different carbon source is on the impact of substratum
According to containing the equal principle of carbon mass fraction, substitute glycerine with dextrose plus saccharose two kinds of carbon sources respectively and carry out carbon source experiment of single factor, other components remain unchanged, and each CMC model 3 bottles is as repeating.Ferment by above-mentioned condition, sampling and measuring OD
600, experimental result is as accompanying drawing 2.
From accompanying drawing 2, the growing state of engineering bacteria in 3 kinds of carbon sources differs greatly, and engineering bacteria more easily utilizes sucrose and glycerine growth, relatively not easily utilizes glucose and maltose.This may be due to during using sucrose and glycerine as carbon source, effectively reduces the generation of acetic acid.The deleterious effect of acetic acid to fermenting process is many-sided, not only directly suppresses the expression of thalline breeding and metallothionein(MT), and also can cause catabiosis by changing medium pH, product stability reduces.To sum up, when taking sucrose as carbon source, it is better that engineering bacteria grows, and therefore, selects sucrose to originate as the carbon source of substratum.
(3) different carbon source concentration is on the impact of substratum
Take sucrose as carbon source, regulate sucrose concentration to be respectively 2,4,6 and 8g/L, other components remain unchanged, and each CMC model 3 bottles is as repeating.Ferment by above-mentioned condition, sampling and measuring OD
600, experimental result is as accompanying drawing 3.
From accompanying drawing 3, along with the rising of sucrose concentration, the growth of thalline finally reaches unanimity, but when sucrose concentration is higher than 4g/L, thalline more first reaches the highest biomass, and its lag phase is shorter, thalli growth speed, therefore selects to be that the sucrose of 4g/L is as sole carbon source with concentration.
(4) different yeast powder concentration is on the impact of substratum
With concentration be the sucrose of 4g/L as sole carbon source, regulate yeast powder concentration to be respectively 16,20,24,28 and 32g/L, other components remain unchanged, and each CMC model 3 bottles is as repeating.Ferment by above-mentioned condition, sampling and measuring OD
600, experimental result is as accompanying drawing 4.
From accompanying drawing 4, along with the increase of yeast powder concentration, Fungal biodiversity variation tendency is first rise to decline afterwards, wherein when yeast powder concentration reaches 24g/L, biomass reaches the highest, and thalli growth speed, therefore selects concentration to be that the yeast powder of 24g/L adds in substratum.
(5) different peptone concentration is on the impact of substratum
With concentration be the sucrose of 4g/L as sole carbon source, adding concentration is the yeast powder of 24g/L, and Function protein peptone concentration is respectively 8,12,16,20 and 24g/L, other components remain unchanged, and each CMC model 3 bottles is as repeating.Ferment by above-mentioned condition, sampling and measuring OD
600, experimental result is as accompanying drawing 5.
From accompanying drawing 5, peptone concentration does not show significant difference to Fungal biodiversity, and at various concentrations, the biomass of thalline and the speed of growth all show unanimously, and the 8g/L therefore selecting peptone concentration minimum is with conservation.
(6) different phosphate concn is on the impact of substratum
With concentration be the sucrose of 4g/L as sole carbon source, add that concentration is the yeast powder of 24g/L, concentration is the peptone of 8g/L, keep KH in initial medium
2p0
4, K
2hP0
4constant rate, using starting point concentration as benchmark, regulate two kinds of phosphoric acid salt total concns to be respectively 0.5,1 and 2 times of benchmark concentration, other components remain unchanged, and each CMC model 3 bottles is as repeating.Ferment by above-mentioned condition, sampling and measuring OD
600, experimental result is as accompanying drawing 6.
From accompanying drawing 6, unknown significance difference under three kinds of concentration, has certain decline 2 times of phosphate concn hypothallus amounts comparatively speaking, the phosphate concn that therefore to select with the phosphate concn of 1 times of i.e. original concentration be Optimal Medium.
(7) the optimization confirmatory experiment of substratum
The data obtained with above step are respectively improved Optimal Medium, take original culture medium as contrast, often organize three bottles of repetitions, and checking Optimal Medium, experimental result is as accompanying drawing 7.
From accompanying drawing 7, the substratum after improvement is really more favourable to the growth of thalline, and all comparatively original culture medium is higher for its colibacillary growth velocity and biomass.
Comprehensive above step, obtain the fermention medium after improveing by condition optimizing, its composition comprises two portions by weight: a part is the substratum of 900mL, containing 4g sucrose, 24g yeast leaching powder, 8g peptone; Another part is the phosphoric acid buffer of 100mL, wherein containing 2.31gKH
2pO
4, 12.54gK
2hPO
43H
2o, by the improved culture medium of mixed preparing 1L after two portions respectively autoclaving.
embodiment three: the abduction delivering of recombinant protein
Take IPTG as inductor, the expression of checking metallothionein(MT).Positive monoclonal is inoculated in the LB substratum of 5mL containing 50mg/LAmp, 37 DEG C, 220r/min incubated overnight, 1:100 transfers into 50mLLB(containing Amp by volume) fresh culture, continues under the same terms to cultivate, works as OD
600when reaching 0.4-0.6, adding isopropyl-β-D-thiogalactoside(IPTG) (IPTG) to final concentration is 1mmol/L, abduction delivering 4h, collect 2mL bacterium liquid centrifugal, precipitate resuspended with 100 μ l1xSDS sample-loading buffers, boil 15min, 15%SDS-PAGE electrophoretic analysis expression of results, experimental result is as accompanying drawing 8.
From accompanying drawing 8, different mono-clonal can go out metallothionein(MT) by abduction delivering, and therefore mono-clonal used is derivable correct recombinant bacterium, and the expression of metallothionein(MT) in order.
(1) different concns lactose as during inductor on the impact of Expression of Metallothionein situation
Positive monoclonal is inoculated in the LB substratum of 5mL containing 50mg/LAmp, 37 DEG C, 220r/min incubated overnight, 1:100 transfers into 50mLLB(containing Amp by volume) fresh culture, continues under the same terms to cultivate, works as OD
600during ≈ 0.8, add respectively final concentration be respectively 6,8,10,12 and 14mmol/L lactose inducement induce, abduction delivering 4h, sampling row SDS-PAGE, experimental result is as accompanying drawing 9.
From accompanying drawing 9, under above several lactose concn gradient, the expression amount of recombinant protein does not have significant difference, therefore, selects the 6mM of minimum lactose concn as its induced concentration.
(2) the different induction time of lactose is on the impact of Expression of Metallothionein situation
Positive monoclonal is inoculated in the LB substratum of 5mL containing 50mg/LAmp, 37 DEG C, 220r/min incubated overnight, 1:100 transfers into 50mLLB(containing Amp by volume) fresh culture, continues under the same terms to cultivate, works as OD
600during ≈ 0.8, adding final concentration is that 6mmol/L lactose inducement is induced, and induction starts sampling in latter continuous 6 hours each hour, and carry out SDS-PAGE by after sample preparation, experimental result is as accompanying drawing 10.
From accompanying drawing 10, after adding lactose, metallothionein(MT) expression amount in 1-4h presents the trend expressed and rise, and does not see and have significance expression amount to rise after 4h, therefore selects 4h as the right times of abduction delivering.
embodiment four: caspian halostachys metallothionein(MT) shake-flask culture compares with ferment tank
Shake flask fermentation system and fermentation tank tie up in condition larger difference, comprising: the whipping process in fermentor tank increases the shearing force to cell; Uppity key parameter in shaking flask can be controlled in fermentor tank and melt oxygen; In fermentor tank, tank pressure is higher causes CO
2concentration is more high.Optimization through early stage to metallothionein(MT) conditions of flask fermentation, shows that lactose can substitute the inductor of IPTG as fermentation.But whether shaking flask condition can be exaggerated, and it may be necessary amplification test and tests.Experimental result is in table 1.
Table 1: shake-flask culture and fermentor cultivation results contrast
| Shake-flask culture | Fermentor cultivation | |
| OD 600 | 1.186 | 1.390 |
| Dry weight (g/L) | 2.05 | 2.68 |
As can be seen from Table 1, in fermentor tank amplification culture process, in same time, the dry cell weight that fermentor cultivation obtains improves 31% than shake-flask culture.This result shows, the fermentation condition optimizing gained is suitable for being amplified further.
Confirm through above-mentioned serial experiment, caspian halostachys metallothionein(MT) fermentation manufacturing technique provided by the invention is applicable to scale operation.The caspian halostachys metallothionein(MT) of fermentation can be applied to fields such as comprising medicine, health care of food, cosmetics additive, environmental protection, and prospect is very wide.
As mentioned above; the present invention can be realized preferably; the above embodiments are only be described the preferred embodiment of the present invention; not scope of the present invention is limited; under not departing from the present invention and designing the prerequisite of spirit; the various distortion that those of ordinary skill in the art make technical scheme of the present invention and improvement, all should fall in protection domain that the present invention determines.
SEQIDNO.1
<110>OrganizationName: Xinjiang University, Liu Zhongyuan
<110>LastName: Liu Zhongyuan
<110>FirstName: Liu Zhongyuan
<120>Title: a kind of caspian halostachys metallothionein(MT) zymotechnique and application thereof
<130>AppFileReference:2
<213>OrganismName:Halostachyscaspica
<400>PreSequenceString:
atgtcttgctgtggtggtaactgtggttgtggagctggctgcaagtgcggcagtggctgc60
ggaggttgcaagatgttccctgactttggcgagaacacttctaaccccaccgttctcatc120
tccggcgttgcccccaagatctcatatgccgaaggatcagagatgggagtagctgttgag180
aacgatggatgcaagtgcggtcccaactgccaatgcaacccttgcacctgcaaa234
<212>Type:DNA
<211>Length:234
SequenceName:HcMT
SequenceDescription:
Feature
Sequence:HcMT:
<221>FeatureKey:gene
<222>LocationFrom:1
<222>LocationTo:234
OtherInformation:
CDSJoin:No
SEQIDNO.2
<213>OrganismName:Halostachyscaspica
<400>PreSequenceString:
MSCCGGNCGCGAGCKCGSGCGGCKMFPDFGENTSNPTVLISGVAPKISYAEGSEMGVAVE60
NDGCKCGPNCQCNPCTCK78
<212>Type:PRT
<211>Length:78
SequenceName:HcMT-pro
Sequence:HcMT-pro:
<221>FeatureKey:PEPTIDE
<222>LocationFrom:1
<222>LocationTo:78
OtherInformation:
CDSJoin:No
Claims (2)
1. the zymotechnique of a caspian halostachys metallothionein(MT), it is characterized in that, concrete fermentation process step is as follows: by the recombinant expressed transformant that obtains in prior art 37 DEG C of overnight incubation, the fermention medium after improvement is inoculated into again with the ratio of 2%, ferment at automatic fermenter, dress liquid coefficient is 0.5, overall sterilizing after interpolation defoamer, air flow is set to 1L/min, keep temperature 37 DEG C, mixing speed 100r/min, after fermentation 4h, adding concentration is that 6mmol/L lactose is induced, after continuing fermentation 4h, fermented liquid is obtained thalline with the centrifugal 10min of 8000rpm, again with centrifugal three times of PBS washing, after 105 DEG C of dry 1.5h, constant weight weighs the dry cell weight preparing caspian halostachys metallothionein(MT).
2. the zymotechnique of a kind of caspian halostachys metallothionein(MT) as claimed in claim 1, it is characterized in that, described fermention medium comprises two portions by weight: a part is the substratum of 900mL, containing 4g sucrose, 24g yeast leaching powder, 8g peptone; Another part is the phosphoric acid buffer of 100mL, wherein containing 2.31gKH
2pO
4, 12.54gK
2hPO
43H
2o, by the improved culture medium of mixed preparing 1L after two portions respectively autoclaving.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110368910A (en) * | 2019-07-25 | 2019-10-25 | 四川轻化工大学 | A kind of application of caspian halostachys metallothionein engineering bacteria adsorbent in sewage absorption |
| CN113957090A (en) * | 2021-11-12 | 2022-01-21 | 四川轻化工大学 | Halostachys chinensis metallothionein HcMT compound and application thereof in sunscreen cream |
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| CN113957090A (en) * | 2021-11-12 | 2022-01-21 | 四川轻化工大学 | Halostachys chinensis metallothionein HcMT compound and application thereof in sunscreen cream |
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