A strain of Escherichia coli and its whole cell catalytic production of icariin

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

Active Publication Date: 2022-07-05
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] After literature search, there is no report on the method of preparing icariin (dehydrated icariin, icariin) by whole cell catalysis

Method used

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  • A strain of Escherichia coli and its whole cell catalytic production of icariin
  • A strain of Escherichia coli and its whole cell catalytic production of icariin
  • A strain of Escherichia coli and its whole cell catalytic production of icariin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] 1.1 Exploration on the ratio of the amount of the purified double-enzyme catalyzed icariin

[0034] 20°C, 0.5mM IPTG, 180rpm to induce and culture pQE-sprha2 for 20h, 30°C, 0.5mM, 200rpm IPTG to induce and culture pSE-pbgl for 8h; the induced pQE30-sprha2, pSE-pbgl cells were cytolytically purified After obtaining the pure enzyme, protein quantification of the purified pure enzyme shows that SPRHA2 is 2.2 μg / μL, PBGL is 1.2 μg / μL, and it is known that SPRHA2 reacts 0.3% (w / v) when the amount of enzyme added is 50 μg / mL. ), the conversion rate of icariin for 6h was above 95%, and the amount of enzyme added to immobilized SPRHA2 was 50 μg / mL. After diluting the two pure enzymes at appropriate times, the ratio of the amount of enzyme added (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 during the reaction, samples were taken at 5 minutes and 5 hours of the reaction, respectively, and the heat was terminated...

Embodiment 2

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

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

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

[0051] (3) Add 47.62 g / L Epimedium plant extract to the whole cell catalytic solution of co-expressed Escherichia coli engineering bacteria pQE-sprha2-pbgl obtained in step (2), and react at 55°C and 220 rpm for ...

Embodiment 3

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

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

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

[0057] (3) Add 44.4 g / L Epimedium plant extract to the whole-cell catalytic solution of co-expressed Escherichia coli engineering bacteria pQE-sprha2-pbgl obtained in step (2), and react at 55°C and 220 rpm for 10 hours; The extract...

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PUM

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Abstract

The invention discloses a recombinant Escherichia coli engineering strain pQE‑sprha2‑pbgl and a method for using the whole cell catalyzed icariin to produce icariin. The strain co-expresses an α‑L‑ The rhamnosidase SPRHA2 and a β-glucosidase PBGL use a whole-cell catalysis method to catalyze the hydrolysis of icariin in crude icariin extract to produce icariin. The invention constructs a recombinant Escherichia coli strain containing polycistron, adopts the method of whole cell catalysis to directly realize the conversion of icariin to icariin, and the hydrolysis rate of icariin is over 98%.

Description

technical field [0001] The invention relates to the field of bioengineering and biosynthesis of natural compounds, in particular to a strain of Escherichia coli engineering bacteria and a method for catalyzing icariin to produce icariin in its whole cell. Background technique [0002] Icariin (dehydrated icariin, icariin) is a polyhydroxy flavonoid monomer component in the Berberidaceae Epimedium genus Epimedium, which has anti-tumor properties (Liu Song, Liu Chaoming, Lai Lijuan). et al "Research progress on the pharmacological effects of icariin [J]". Journal of Gannan Medical College, 2017, 37(04): 631-635.), antioxidant, anti-hepatic fibrosis, differentiation-promoting, mineralization, anti-oxidative Osteoporosis (Zheng Z G, Zhang X, Zhou Y P, et al. Anhydroicaritin, a SREBPs inhibitor, inhibits RANKL-induced osteoclastic differentiation and improves diabeticosteoporosis in STZ-induced mice[J]. European Journal of Pharmacology, 2017, 809:156 -162.), anti-inflammatory, p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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