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A kind of fermenting process and application of salt ear wood metallothionein protein

A metallothionein and fermentation process technology, applied in biological fermentation, salt spike wood metallothionein fermentation process and its application fields, can solve the problems of high cost and low yield, and achieve the effect of increasing yield

Inactive Publication Date: 2018-12-28
XINJIANG UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In view of the fact that there is no research status in the prior art on the method of industrial production of Saltwood metallothionein, the purpose of the present invention is to provide a fermentation process of Saltwood metallothionein and its application, and obtain an improved fermentation process through condition optimization. The culture medium is amplified by fermentation tanks, thereby increasing the yield of metallothionein from Saltwood, which overcomes the current situation of high cost and low yield in the field of preparation of Saltwood metallothionein

Method used

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  • A kind of fermenting process and application of salt ear wood metallothionein protein
  • A kind of fermenting process and application of salt ear wood metallothionein protein
  • A kind of fermenting process and application of salt ear wood metallothionein protein

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Embodiment 1

[0029] Embodiment one: a kind of fermenting process of the metallothionein of Saltwood

[0030] A kind of fermenting technology of salt ear wood metallothionein, concrete fermentation process step is as follows:

[0031] (1) Configuration and sterilization of medium

[0032]The improved fermentation medium has been obtained through condition optimization, and its composition includes two parts by weight: a part is 900 mL of medium, containing 4g sucrose, 24g yeast extract powder, 8g peptone; the other part is 100mL of phosphate buffer, wherein Contains 2.31g KH 2 PO 4 , 12.54 g K 2 HPO 4 ·3H 2 O, the two parts were autoclaved separately and mixed to prepare 1 L of modified medium.

[0033] (2) Expanded culture in fermenter

[0034] Cultivate the recombinant expression transformant obtained in the prior invention at 37°C overnight, then inoculate the improved fermentation medium at a ratio of 2%, and ferment in a fully automatic fermenter with a liquid filling coefficien...

Embodiment 2

[0035] Embodiment two: the selection of culture medium in the fermentation technology of salt ear wood metallothionein

[0036] The initial fermentation medium used was TB medium, and the initial fermentation conditions were: inoculum size 1% (V / V), liquid volume 30 / 250 mL, temperature 37°C, speed 200 r / min Shaker cultivation to OD 600 When ≈0.6, add IPTG with a final concentration of 1 mmol / L, and induce culture for 6 h.

[0037] (1) Selection of seed age

[0038] Select the most commonly used LB medium for recombinant Escherichia coli as the seed medium, and measure the growth curve of the seed liquid, see the attached figure 1 .

[0039] attached by figure 1 It can be seen that the strains are in the lag phase within 0-1h, quickly enter the logarithmic growth phase after 1h, and are in the stable phase after 6-10h, and the bacteria begin to decline after 10h. The seed age of the fermented strains should be in the middle and late logarithmic growth period. If the seed ...

Embodiment 3

[0059] Example 3: Induced expression of recombinant protein

[0060] IPTG was used as an inducer to verify the expression of metallothionein. Inoculate positive single clones in 5 mL of LB medium containing 50 mg / L Amp, culture overnight at 37°C, 220 r / min, transfer to 50 mL of LB (containing Amp) fresh medium at a volume ratio of 1:100, and use the same conditions Continue to culture under, when OD 600 When it reaches 0.4-0.6, add isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mmol / L, induce expression for 4 h, collect 2 mL of bacterial liquid and centrifuge, and precipitate with 100 μl 1 x Resuspend in SDS loading buffer, boil for 15 min, and analyze the expression results by 15% SDS-PAGE electrophoresis. The experimental results are attached Figure 8 .

[0061] attached by Figure 8 It can be seen that the expression of metallothionein can be induced by different monoclones, so the monoclonal used is an inducible and correct recombinant bacter...

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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 [0001] The present invention relates to the technical field of biological fermentation, specifically, the present invention relates to the technical field of the metallothionein fermentation process and application thereof. Background technique [0002] Metallothionein ( Metallothionein , MT) are a class of low-molecular-weight, cysteine-rich metal-binding proteins widely present in organisms. The metals combined with it are mainly cadmium, copper and zinc, which widely exist in various organisms from microorganisms to humans, and their structures are highly conserved. In 1957, when Margoshes and Valles were studying the biological effects of cadmium, the substance was first isolated from horse kidneys that accumulated cadmium. MT has the following characteristics: low molecular weight, generally 6~7kD; it can chelate metal ions, and after the protein is combined with the metal through a thioester bond, it has special light absorption characteristics; the c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/02C12R1/19
Inventor 刘忠渊孟红恩张富春
Owner XINJIANG UNIVERSITY
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