Preparation method of recombinant bacillus subtilis and glutathione

A technology of Bacillus subtilis and glutathione, which is applied in the field of bioengineering, can solve the problems of low yield and unsuitability for mass production, and achieve the effects of high yield, high yield, and high yield

Pending Publication Date: 2021-04-16
SHANGHAI QINGPING PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Patents CN201810844388 and CN201680013630 exogenously express glutathione synthesis bifunctional enzyme genes in yeast and Escherichia coli, respectively, to achieve the purpose of fermentation and production of glutathione, but the yield is very low, which is not suitable for mass production

Method used

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  • Preparation method of recombinant bacillus subtilis and glutathione
  • Preparation method of recombinant bacillus subtilis and glutathione
  • Preparation method of recombinant bacillus subtilis and glutathione

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Construction of Recombinant Bacillus subtilis Engineering Bacteria for GSH Production

[0058] The bifunctional glutathione synthase gene (SEQ ID NO: 1) derived from Streptococcus thermophilus (gshFst) was selected, and the gene fragment of the bifunctional glutathione synthase was amplified using the recombinant plasmid pET28a-gshFst as a template, The linearized plasmid was obtained by double digestion with BamHI and SmaI, and the recombinant plasmid pHT-ST was obtained by ligation under the action of T4 ligase.

[0059] Preparation of DNA fragments: The recombinant plasmid pET28a-gshFst already in the laboratory was used as a template for DNA amplification. The reaction conditions were: pre-denaturation at 98°C for 5 min; denaturation at 98°C for 10 s; annealing at 60°C for 30 s; extension at 72°C for 5 min, a total of 30 cycles; after the end, final extension at 72°C for 10 min. The amplified product was verified by nucleic acid electrophoresis and purified by PCR ...

Embodiment 2

[0063] Production of GSH by Recombinant Bacteria BS-ST

[0064] (1) Inoculate the recombinant BS-ST engineered strains stored in glycerol tubes into LB medium, and culture them at 30°C for 11 hours as primary seeds.

[0065] (2) Transfer the primary seeds into LB liquid medium, and culture at 30° C. for 13 hours to obtain the secondary seed culture solution.

[0066] (3) Secondary seed culture liquid is inoculated in the fermentor tank of 2L by 10% inoculum size and carries out fed-batch fermentation, and fermentation medium adopts corn flour 10g / L, glucose 2g / L, industrial peptone 5g / L, Corn steep liquor 3g / L, urea 1g / L, calcium carbonate 0.5g / L, magnesium sulfate 0.8g / L, potassium dihydrogen phosphate 0.5g / L, dipotassium hydrogen phosphate 0.5g / L, feed solution containing glucose 500g / L L, peptone 2g / L, culture temperature 37°C, culture pH 6.8, fermentation 30h, OD600 is 46 at this time, add 1mM inducer IPTG to the medium, continue to culture for 5h, finally add glutamic ac...

Embodiment 3

[0069] Production of GSH by Recombinant Bacteria BS-ST

[0070] (1) Inoculate the recombinant BS-ST engineered strains stored in glycerol tubes into LB medium, and cultivate them at 30°C for 12 hours as primary seeds.

[0071] (2) The primary seeds were transferred into LB liquid medium, and cultured at 30° C. for 10 h to obtain the secondary seed culture solution.

[0072] (3) Secondary seed culture liquid is inoculated into the fermentor of 2L by 9.5% inoculum size and carries out fed-batch fermentation, and fermentation medium adopts corn flour 5g / L, glucose 3g / L, industrial peptone 4g / L, Corn steep liquor 3g / L, urea 0.5g / L, calcium carbonate 1.5g / L, magnesium sulfate 1.5g / L, potassium dihydrogen phosphate 0.4g / L, dipotassium hydrogen phosphate 0.4g / L, feeding solution containing 500g of glucose / L, peptone 1g / L, culture temperature 37°C, culture pH 6.8, fermentation 32h, OD600 is 48 at this time, add 1mM inducer IPTG to the medium, continue to culture for 5h, finally add ...

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Abstract

The invention relates to a preparation method of recombinant bacillus subtilis and glutathione, and the glutathione is produced by fermenting a bacillus subtilis gene engineering strain for overexpression of a bifunctional glutathione synthetase gene, so that the glutathione is high in yield, simple and convenient to operate. Meanwhile, the food-grade bacillus subtilis is used as a production strain, so that the method is safer and more reliable, and effective reference is provided for industrial green production of glutathione.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a method for preparing recombinant bacillus subtilis and glutathione. Background technique [0002] Glutathione (γ-L-glutamyl-cysteinyl-glycine, GSH) is an active tripeptide compound formed by the condensation of three amino acids, L-glutamic acid, L-cysteine ​​and glycine. in various organisms. Reduced glutathione has many important functions in living tissues. Glutathione can improve human immunity and has anti-aging effects. Its effect on the retarded cells of the elderly is greater than that of young people. Glutathione also has a very significant effect on the treatment of symptoms such as leukopenia caused by radiation and radiopharmaceuticals. [0003] At present, there are many preparation methods of glutathione, common ones include solvent extraction, chemical synthesis, biological fermentation and enzymatic method. At present, the production of glutathione at home and a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C12N1/21C12N15/52C12N15/75C12N15/77C12N15/66C12R1/125C12R1/15
Inventor 韦建国王德正李志敏刘世领
Owner SHANGHAI QINGPING PHARMA CO LTD
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