Engineered escherichia coli and method of whole cells of engineered escherichia coli to produce anhydroicaritin through catalysis

A technology of icariin and Escherichia coli is applied in the fields of bioengineering and biosynthesis of natural compounds, and achieves the effects of simple operation, omitting cumbersome steps and high industrialization potential.

Active Publication Date: 2021-01-15
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] After literature search, there is no report on the method o...

Method used

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  • Engineered escherichia coli and method of whole cells of engineered escherichia coli to produce anhydroicaritin through catalysis
  • Engineered escherichia coli and method of whole cells of engineered escherichia coli to produce anhydroicaritin through catalysis
  • Engineered escherichia coli and method of whole cells of engineered escherichia coli to produce anhydroicaritin through catalysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] 1.1 Exploration of the ratio of enzymes added to the purified dual-enzyme catalyzed icariin

[0034] 20°C, 0.5mM IPTG, 180rpm induction culture pQE-sprha2 for 20h, 30°C, 0.5mM, 200rpm IPTG induction culture pSE-pbgl 8h; pQE30-sprha2, pSE-pbgl cells were purified after induction Finally, the pure enzyme was obtained, and the purified enzyme was quantified for protein, and it was found that SPRHA2 was 2.2 μg / μL, and PBGL was 1.2 μg / μL. It is known that SPRHA2 reacts 0.3% (w / v) when the enzyme amount is 50 μg / mL The icariin 6h conversion rate of ) is more than 95%, and the enzyme amount of fixing SPRHA2 is 50 μ g / mL after taking two kinds of pure enzymes and diluting appropriate multiples by different enzyme amount ratios (1:1, 2:1, 3: 1, 4:1, 5:1) to react 0.3% (w / v) icariin; in order to observe the reaction speed of the two enzymes in the reaction process, samples were taken in 5 minutes and 5 hours of reaction, and the heat was terminated After 5min, the hydrolyzate wa...

Embodiment 2

[0048] Utilize Example 1 to construct the method for co-expressing Escherichia coli engineering bacteria pQE-sprha2-pbgl to catalyze icariin to generate icariin, the operation steps are as follows:

[0049] (1) 30°C, 0.5mM IPTG induced culture for 8 hours obtained in Example 1 co-expressed Escherichia coli engineered bacteria pQE-sprha2-pbgl, after the fermentation was completed, the bacteria were collected and the wet weight of the bacteria was weighed;

[0050] (2) Wash 23.8 mg wet weight thalline with 0.9% NaCl solution for 3 times, resuspend the thalline with boric acid borax buffer containing 0.2M boric acid and 0.05M borax, pH value 8, obtain 4.76mg / ml co-expression of Escherichia coli engineered bacteria pQE-sprha2-pbgl whole cell catalytic solution;

[0051] (3) Add the epimedium plant extract of 47.62g / L concentration to step (2) gained co-expression Escherichia coli engineering bacterium pQE-sprha2-pbgl whole cell catalytic liquid, 55 ℃, 220rpm react 10h; Epimedium ...

Embodiment 3

[0054] Utilize Example 1 to construct the method for co-expressing Escherichia coli engineering bacteria pQE-sprha2-pbgl to catalyze icariin to generate icariin, the operation steps are as follows:

[0055] (1) 30°C, 0.5mM IPTG induced culture for 8 hours obtained in Example 1 co-expressed Escherichia coli engineered bacteria pQE-sprha2-pbgl, after the fermentation was completed, the bacteria were collected and the wet weight of the bacteria was weighed;

[0056] (2) Wash 55.5 mg wet weight thalline with 0.9% NaCl solution for 3 times, resuspend the thalli with boric acid borax buffer solution containing 0.2M boric acid and 0.05M borax, pH value 8, obtain 11.1mg / ml co-expression of Escherichia coli engineered bacteria pQE-sprha2-pbgl whole cell catalytic solution;

[0057] (3) Add the epimedium plant extract of 44.4g / L concentration to step (2) gained co-expression Escherichia coli engineering bacterium pQE-sprha2-pbgl whole cell catalytic liquid, 55 ℃, 220rpm react 10h; Epim...

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Abstract

The invention discloses recombinant engineered escherichia coli pQE-sprha2-pbgl and a method of catalyzing icariin to produce anhydroicaritin by using the whole cells of the engineered escherichia coli. The strain co-expresses an alpha-L-rhamnosidase SPRHA2 and a beta-glucosidase PBGL through the form of polycistron, utilizes the method of whole-cell catalysis,takes an icariin crude extract as a substrate, and catalyzes the icariin to produce the anhydroicaritin through hydrolysis. Thus, the recombinant engineered escherichia coli strain containing the polycistron is constructed; and through the adoption of whole-cell catalysis, the conversion of the icariin to the anhydroicaritin can be directly realized, and the hydrolysis rate of the icariin is more than 98%.

Description

technical field [0001] The invention relates to the fields of bioengineering and biosynthesis of natural compounds, in particular to a strain of Escherichia coli engineering bacteria and a method for producing icariin from icariin by whole cells thereof. Background technique [0002] Icariin (dehydrated icariin, icariin) is a polyhydroxy flavonoid monomer component in Epimedium genus Berberidaceae, which has antitumor properties (Liu Song, Liu Chaoming, Lai Lijuan "Research Progress on the Pharmacological Effects of Icaritin[J]". Journal of Gannan Medical College, 2017,37(04):631-635.), anti-oxidation, anti-hepatic fibrosis, promotion of differentiation, mineralization, anti- Osteoporosis (Zheng Z G, Zhang X, Zhou Y P, et al. Anhydroicaritin, a SREBPs inhibitor, inhibits RANKL-induced osteoclastic differentiation and improves diabetic steoporosis in STZ-induced mice[J]. European Journal of Pharmacology, 2017, 809: 156 -162.), anti-inflammation, promotion of vascular endothe...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P17/06C12R1/19
CPCC12N9/2402C12N9/2445C12P17/06C12Y302/0104C12Y302/01021Y02A50/30
Inventor 杜丽琴丁波庞浩可丛雪黄日波刘家瑞闭海韦航韦宇拓
Owner GUANGXI UNIV
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