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A kind of fermentation production method of salidroside

A fermentation broth and high-yield technology, applied in the fields of genetic engineering and microbial fermentation, can solve the problems of questioning economic value and low yield of secondary metabolites, and achieve the effects of good activity and stability, simple reaction method and good thermal stability.

Active Publication Date: 2021-06-15
孙敬方
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AI Technical Summary

Problems solved by technology

[0006] When using plant cell culture technology to obtain alpine salidroside, the production of secondary metabolites of Rhodiola rosea is usually low in cell culture, which makes its economic value questionable

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The acquisition of embodiment 1 high-yielding apple β-glucosidase genetically engineered bacteria

[0022] (1) According to the apple β-glucosidase gene sequence, base optimization and artificial synthesis of its gene mutant MBGLM (its amino acid sequence is shown in SEQ ID NO.1, the highest enzyme activity of this mutant is 198IU / ml, is wild 9 times the enzyme activity of the type strain), and loaded into the vector pUC57.

[0023] (2) The target gene was double-digested with NdeI and EcoRI and then recovered by tapping the gel, ligated with the pET-24d(+) vector digested with NdeI and EcoRI, and transformed into Escherichia coli BL21 (DE 3 );

[0024] Spread the transformation mixture on a 50 mg / L karamycin-resistant LB plate and pick the transformant, culture it at 37°C for 6-8 hours, pick a single colony, and obtain MBGLM-pET-24d(+) / E.coli BL21 (DE 3 ).

[0025] After culturing in LB / Kan liquid medium for 8-10 hours, the strains were preserved.

Embodiment 2

[0026] Embodiment 2 fermentation produces enzyme

[0027] Inoculate the genetically engineered bacteria MBGLM-pET-24d(+) / E.coli BL21(DE3) in the LB liquid medium containing Kan, and cultivate it at 37°C for 8-10 hours; Base, cultured at 37°C for 3h; induced with 0.4mM / LIPTG, cooled to 25°C and cultured at constant temperature for 40-48h to induce enzyme production. After fermentation, the bacteria were collected by centrifugation and ultrasonically disrupted. Centrifuge to obtain the supernatant after induction, which is the crude enzyme solution.

Embodiment 3

[0028] Embodiment 3 enzyme catalyzes the synthesis of salidroside

[0029] Weigh 250mM tyrosol, 50mM glucose, and 1.25U / ml crude enzyme solution and mix them in the buffer / n-hexane (1 / 1, V / V) mixed system. The reaction was shaken at 50° C. and 180 r / min for 24 hours. After the reaction, extract a certain amount of aqueous phase reaction solution, dilute it with 2 times the volume of distilled water, filter through a microporous membrane, concentrate the filtrate to dryness, and obtain a white solid powder. mp 156-159℃, IR(KBr)vcm - : 268, 1630, 1609; 13 CNMR (CDOD) 6: 35.0 (Ar-CH 2 -)69.0 (-CH 2 -O); 61.1(6), 70.7(4), 71.2(3), 73.6(2), 75.2(5), 103.6(Gal-1); 114.8(2), 129.5(3), 155.3(Ar) ,MSm / z:306.8(M + ), 324.8(M+H2O) 287.4(M-H 2 O+H + ). It is consistent with the data in the literature (Duan Jingyun, Zhang Dianzeng, Fan Yinke, etc. Pharmacodynamics Research of Compound Rhodiola Tablets [J]. Chinese Patent Medicine, 1999, 21(11):588-591).

[0030] Take the product ...

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Abstract

The invention provides a method for using apple β-glucosidase as a novel catalyst to produce and prepare salidroside through enzymatic transformation and fermentation. Using gene recombination technology to prepare high-efficiency apple β-glucosidase, add 50mM-1000mM tyrosol, 10mM-500mM glucose, and react at 37-50°C for 20-50h. Obtain salidroside.

Description

technical field [0001] The invention relates to a method for preparing salidroside by fermenting β-glucosidase mutants, which belongs to the field of genetic engineering and microbial fermentation. Background technique [0002] Salidroside (salidroside) is the main active ingredient of the sedum family Rhodiola plant. Rhodiola rosea is a perennial herbaceous plant, which mainly grows in areas with an altitude of 1600-4000 meters in high cold, dry, anoxic, strong ultraviolet radiation, and large temperature difference between day and night. It has strong environmental adaptability and vitality. Salidroside has a variety of physiological and pharmacological activities such as enhancing immune function, protecting cardiovascular and cerebrovascular, improving sexual function, improving memory, anti-cancer, and anti-depression. It can improve attention, mental and physical strength, and then affect the central nervous system. The state is particularly effective, it can signific...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N9/42C12N15/56C12N15/70C12P19/44C12R1/19
CPCC12N9/2445C12N15/70C12P19/44C12Y302/01021
Inventor 孙敬方
Owner 孙敬方
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