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Catalytic system coupled with ATP regeneration system and application of catalytic system in glutathione production

A catalytic system and regeneration system technology, applied in the direction of application, microbial-based methods, biochemical equipment and methods, etc., can solve the problems of unfriendly antibiotics, unfriendly antibiotic environment, low product yield, etc., to save time, cost and equipment Cost, save antibiotic use, good biosafety effect

Active Publication Date: 2019-10-25
INNOBIO CORP LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the prior art, there are still various problems hindering the industrial application of this technology. Patent CN201710543648 has obtained GshF-4 mutants with different amino acid sequences, and its synthetic activity, specific activity and optimum temperature have been improved, but there are The output is low; ATP consumption is large and the cost is high, which is not conducive to industrial production
[0005] Enzymatic synthesis of glutathione requires adenosine triphosphate (ATP) as an energy donor, but the price of ATP will greatly increase production costs, so the establishment of an ATP regeneration system is a key part of glutathione synthesis
Patent CN201310538982 overexpresses (exogenous) glutathione bifunctional enzyme (STH) and acetate kinase (ack) in E. coli cells to obtain recombinant expression cells. Although the purpose of ATP regeneration is achieved, Rosetta (DE3) is stable The sensitivity is not high, the cost of the required substrate dilithium acetyl phosphate is high, and the yield of the product is low, the broken enzyme solution increases the cost of separation, in addition, there are problems that the plasmid may be lost, and the use of antibiotics is not friendly to the environment It is difficult to carry out industrial promotion; patent CN201710452240 uses GshF enzyme, ATP regeneration enzyme (PPK enzyme and PAP enzyme) and AK enzyme to generate glutathione in the reactor, and uses adenosine to replace ATP or AMP, but the ATP in it is regenerated The enzyme system includes PPK enzyme, PAP enzyme and AK enzyme. There are problems of high cost caused by separate culture of multiple enzymes, cumbersome immobilization and separation steps, high energy consumption, and the possibility of loss of plasmids. The use of antibiotics is harmful to the environment. Unfriendly problems, etc., eventually lead to its failure to be well promoted in the industry

Method used

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  • Catalytic system coupled with ATP regeneration system and application of catalytic system in glutathione production
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  • Catalytic system coupled with ATP regeneration system and application of catalytic system in glutathione production

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Embodiment 1

[0032] Embodiment 1. Construction of recombinant Escherichia coli

[0033] 1. Construction of recombinant expression vectors

[0034] Codon optimization was performed on the target gene GshF (SEQ ID NO.1) derived from Streptococcus thermophilus and ppk2 (Gene ID: 3718134, VERSION: CP000143.2; SEQ ID NO.3) derived from Rhodobacter sphaeroides to obtain codon optimization The GshF gene sequence (SEQ ID NO.2) and the codon-optimized ppk2 gene sequence (SEQ ID NO.4) were used to design primers based on the codon-optimized sequence, that is, to introduce a linear The terminal sequence of the linearized vector (the terminal sequence of the linearized vector is the capital letter in the primer sequence), so that the 5' and 3' ends of the insert fragment have the consensus sequence (15-25bp) at both ends of the vector, as shown in Table 1 As shown, the primers and the template sequences were paired for high-fidelity PCR and gel-cut recovery; at the same time, the plasmid pET-30a was ...

Embodiment 2

[0051] Embodiment two, the optimization of the addition amount of glutathione synthesis reaction bacteria

[0052] In order to reduce the cost, the addition amount of BL21-GshF and BL21-ppk2 was optimized. The reaction system is as follows: L-glycine 160mM, L-sodium glutamate 160mM, L-cysteine ​​140mM, MgSO 4 ·7H 2O 70mM, ATP 2mM, 60mM sodium hexametaphosphate, 100mL deionized water. Wet cells were added to the reaction system, reacted at 42°C and pH 7 for 3 hours, then sampled, centrifuged at 8000rpm for 5 minutes, and the supernatant was detected by HPLC for the production of glutathione and the contents of ATP, ADP and AMP.

[0053] When the added amount of BL21-GshF wet cells was 50 mg, the effects of different addition amounts of BL21-ppk2 wet cells on the reaction results were compared. Such as Figure 4 As shown, 20-50 mg of BL21-ppk2 wet cells does not constitute a limiting factor for the synthesis of glutathione, and the conversion rate of glutathione in the syste...

Embodiment 4

[0056] Embodiment four, the optimization of mixed culture inoculum size

[0057] To obtain the mixed bacteria after mixing BL21-GshF and BL21-ppk2 at a ratio of 3:2, the inoculation ratio of the two must be controlled. After BL21-GshF and BL21-ppk2 were activated overnight, they were mixed according to different ratios (3:2, 2:1, 5:2, 3:1), and the mixed bacteria were inoculated with an inoculum volume of 10% by volume In the fermenter (TB medium), the temperature is maintained at 37°C±0.5°C, the pH is 6.7±0.1, by adjusting the ventilation rate (20-100L·h -1 ) and stirring speed (100-600rpm) to control the dissolved oxygen at 10%-40%, cultivate to the late logarithmic stage, put in the tank, centrifuge the bacteria, and use it as the reaction cells for later use.

[0058] Mix 50 mg of wet cells fermented with BL21-GshF and BL21-ppk2 at different inoculation ratios at 42°C and pH 7 with L-glycine 160 mM, L-sodium glutamate 160 mM, MgSO 4 ·7H 2 O 70mM, L-cysteine ​​hydrochlor...

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Abstract

The invention discloses a catalytic system coupled with an ATP regeneration system and application of the catalytic system in glutathione production. The application includes: building heterogenous expression / genome integrated Escherichia coli containing glutathione synthesis difunctional enzyme and ATP regeneration enzyme, performing glutathione synthesis reaction condition and catalytic system optimization, and using the mixed culture of the two kinds of Escherichia coli and strain proportion and catalytic reaction process optimization to achieve glutathione production, and raw material, equipment and synthesize reaction costs are saved to the maximum extent.

Description

technical field [0001] The invention belongs to the technical field of biocatalysis, and in particular relates to a catalytic system coupled with an ATP regeneration system and its application in the process of producing glutathione. Background technique [0002] Glutathione (γ-L-glutamyl-cysteinyl-glycine, GSH) is a small molecular polypeptide obtained by condensation of three amino acids, L-glutamic acid, L-cysteine ​​and glycine. In organisms, it is the most important small molecular peptide containing sulfhydryl groups in microorganisms and plant biological cells. Glutathione is an important antioxidant in the body. Reduced glutathione has many important functions in living tissues, and plays an important role in liver injury, eye diseases, substance metabolism, and regulation of cell apoptosis. In addition, it also plays an equally important role in the field of food preservation, so the demand for glutathione in the domestic and foreign pharmaceutical markets, food, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/54C12N15/52C12N15/70C12P21/02C07K5/037C12R1/19
CPCC12N9/93C12Y603/02003C12N9/1229C12Y207/04001C12N15/70C12P21/02C07K5/0215C12N2800/22Y02P20/584
Inventor 范超洪皓齐佳坤刘军吴文忠
Owner INNOBIO CORP LTD
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