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Recombinant escherichia coli KLUGIN73 and application thereof

A technology for recombining Escherichia coli and Escherichia coli, which is applied in the direction of recombinant DNA technology, applications, bacteria, etc., to achieve the effects of cost reduction, simple and single reaction conditions, and wide substrate spectrum

Pending Publication Date: 2022-06-10
董颖军
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] The present invention aims at the problems of low substrate tolerance, low space-time yield and high cost of industrial application in the production of icariin by enzymatic hydrolysis of icariin , provided a strain of recombinant Escherichia coli KLUGIN73, which expresses a thermophilic glycosidase, which rapidly hydrolyzes icariin to produce icariin at high temperature, effectively improving the space-time yield of the enzymatic hydrolysis reaction

Method used

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  • Recombinant escherichia coli KLUGIN73 and application thereof
  • Recombinant escherichia coli KLUGIN73 and application thereof
  • Recombinant escherichia coli KLUGIN73 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] The construction of embodiment 1 recombinant escherichia coli KLUGIN73

[0056] 1. Cloning of blg-XT1412 enzyme gene

[0057] 1.1 Primer design

[0058] According to the blg-XT1412 gene sequence, degenerate primers were designed online using GeneFisher software:

[0059] Upstream primer: 5'-ttaGCGGCCGCATGTCCCCCATCCTC-3';

[0060] Downstream primer: 5'-ggcGAATTCTTAGTGATGGTGATG-3'.

[0061] 1.2 Template preparation

[0062] With the liquid YPD medium of 100ml, activate Kluyveromyces lactis (Kluyveromyces lactis) XT1412 (Kluyveromyces lactis (Kluyveromyces lactis) XT1412 is disclosed in publication number and is CN10812936A, and the title of invention is " Kluyveromyces lactis mutant strain and its Glycosidase and application" Chinese invention patent), 28 ℃, 250rpm continuous culture for 2 days. After the cultivation, centrifuge at 5000rpm for 10min, collect the bacterial cells to extract genomic DNA, and store the product at -20°C until use.

[0063] 1.3 Gene ampli...

Embodiment 2

[0082] The production and fermentation process of embodiment 2 recombinant escherichia coli KLUGIN73

[0083] 2.1 Determination of fermentation medium The fermentation medium is composed of carbon source, nitrogen source and inorganic salts, suitable for 5-10000L fermenter, and the specific components are as follows:

[0084] Carbon source: glycerol 1-3%;

[0085] Nitrogen source: yeast extract powder 0.2-1.5%, tryptone 1-3%; inorganic salt: potassium dihydrogen phosphate 0.1-0.4%, dipotassium hydrogen phosphate 0.1-0.3%, ammonium sulfate 0.05-0.2%, pH: 6- 8 (ammonia adjustment).

[0086] 2.2 Determination of fermentation culture and induction enzyme production conditions

[0087] The fermentation medium is shown in 2.1, and the fermentation conditions are as follows:

[0088] Inoculation amount: 0.1-2% (volume ratio);

[0089] Culture time: 12-24 hours;

[0090] Cultivation temperature: 25-40°C

[0091] Stirring speed: 100-250r / min

[0092] After the cultivation, IPTG ...

Embodiment 3

[0098] Embodiment 3 recombinant escherichia coli KLUGIN73 produces the method for glycosidase

[0099] 1) Activating the recombinant Escherichia coli KLUGIN73,

[0100] 2) Place activated recombinant Escherichia coli KLUGIN73 in a fermentation medium for fermentation to obtain a fermentation product; wherein, the formulation of the fermentation medium is:

[0101] Carbon source: glycerin 1-3%; nitrogen source: yeast extract powder 0.2-1.5%, tryptone 1-3%; inorganic salts: potassium dihydrogen phosphate 0.1-0.4%, dipotassium hydrogen phosphate 0.1-0.3%, ammonium sulfate 0.05-0.2%; pH: 6-8 (adjusted by ammonia water);

[0102] Fermentation conditions: inoculum size: 0.1-2% (volume ratio); culture time: 12-24 hours; culture temperature: 25-40°C; stirring speed: 100-250r / min;

[0103] 3) Inducing enzyme production on the fermentation product to obtain Escherichia coli KLUGIN73 containing the recombinant protein XT1412; wherein,

[0104] Enzyme production conditions: add IPTG (i...

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Abstract

The invention discloses recombinant escherichia coli KLUGIN73 and application thereof. The preservation number of the recombinant escherichia coli KLUGIN73 is CCTCC (China Center For Type Culture Collection) NO: M 2022248. The thermophilic glycosidase generated by the escherichia coli KLUGIN73 has the capacity of hydrolyzing glucoside and rhamnoside, the substrate spectrum is wide, one-time hydrolysis of disaccharide of icariin can be achieved, the reaction condition is simple and single, and the cost of enzymatic hydrolysis of icariin is greatly reduced; according to the process for hydrolyzing icariin, the target product, namely icariin, is single, no by-product is generated, and the yield of the product is close to 100%; the recombinant glycosidase disclosed by the invention can completely hydrolyze 10mM icariin in a pure water phase, and the concentration of the recombinant glycosidase is far more than 1000 times of the solubility of the recombinant glycosidase in water, so that a process for producing icaritin by hydrolyzing icariin through an enzyme method has higher industrial application value.

Description

technical field [0001] The invention relates to the field of biological fermentation, in particular to a recombinant Escherichia coli KLUGIN73 and its application. Background technique [0002] On January 10, 2022, the State Drug Administration approved Icaritin (Akladin) Soft Capsules as a national first-class innovative drug, approved for patients who are not suitable or who refuse to accept standard treatment, and have not received Systemic therapy for unresectable hepatocellular carcinoma. Its core ingredient Icaritin (Icaritin, alias: 3,5,7-trihydroxy-2-(4-methoxyphenyl)-8-(3-methyl-2-buten-1-yl)- 4H-1-benzopyran-4-one, CAS number: 118525-40-9) comes from the traditional herbal medicine - Epimedium. [0003] [0004] However, in the herb Epimedium, icariin is almost absent, and it usually exists in the form of a diglycoside combined with a molecule of glucose and a molecule of rhamnose. The glycoside is called icariin (Icariin, alias: 4′-O-methyl-8-γ, γ-dimethylal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/56C12N9/24C12P17/06C12R1/19
CPCC12N9/2402C12N15/70C12P17/06Y02A50/30
Inventor 董颖军
Owner 董颖军
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