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Method for obtaining 5-hydroxytryptophane by fermenting engineering bacterial strain BL 21-DE3

A technology of BL21-DE3 and hydroxytryptophan, which is applied in the field of biogenetic engineering, can solve the problems of many reaction steps, unstable conditions, and high energy consumption, and achieve the effect of low cost and easy availability of raw materials

Inactive Publication Date: 2010-10-20
CHENGDU KAISHENG BIOLOGICAL SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The process has many reaction steps, the conditions for obtaining 5HTP are unstable, strict separation and purification conditions are required, and the energy consumption in the reaction process is huge, so the pilot-scale production has not been realized.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The enrichment fermentation of embodiment 1 engineering strain BL21-DE3

[0029] The initial strains were inoculated into the seed tank, TSB was used as the basic culture solution, aerated at 30°C for 5 hours, and then transferred to the production tank with TSB as the base culture solution, the pH value was adjusted to 7.0, and aerated at 30°C for 12 hours.

Embodiment 2

[0030] Secondary metabolism fermentation of embodiment 2 engineering strain BL21-DE3

[0031] After enrichment and fermentation for 12 hours, tryptophan solution dissolved in pH 7.8 phosphate buffer was added to the fermentation broth to make the final concentration of tryptophan reach 50% (w / w). The temperature is 60 to retard the proliferation of the bacteria, and the transformation is carried out in a fermenter for 1-2 hours, the ventilation rate is 1L / min, and the temperature is 35°C, and the reaction progress is detected by HPLC.

Embodiment 3

[0032] The monitoring of tryptophan / 5-hydroxytryptophan content in the fermented liquid of embodiment 3

[0033]With the reversed-phase chromatographic column DIMONDCl8 (5 μ m, 4.6 × 250 ram), the detection wavelength is 280 nm, the mobile phase is methanol: water=15: 85 (v / v), the flow rate is 1.0 mL / min, the injection volume is 20 μ l, and the sensitivity is 0.01 AUFS, column temperature is room temperature. The concentration is regressed with the integral value of the chromatographic peak area to obtain a linear equation Y=-258332+1.308413×106X (r=0.9991), RSD=0.68%, and the content of 5-hydroxytryptophan is within the range of 0.8-4g / ml. It has a good linear relationship with the peak area integral value; tryptophan peak area integral value / 5-hydroxytryptophan peak area integral value≤25%.

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Abstract

The invention provides a technology for obtaining 5-hydroxytryptophane by fermenting an engineering bacterial strain BL 21-DE. The engineering bacterial strain is prepared by utilizing a reported rabbit Oryctolagus cuniculus trptophan hydroxylase gene to build and reconstruct an expression vector and carrying out plasmid conversion. The bacterial strain is utilized for one-step fermentation process optimization; and under the condition of not purifying tryptophan hydroxylase, substrate tryptophan is added in the fermentation mature period to obtain the final product of 5-hydroxytryptophane. The invention has easily obtained raw material and low cost, conversion rate is more than 70%, and yield is more than 80%.

Description

technical field [0001] The invention belongs to the field of biogenetic engineering, and specifically relates to a method for transferring tryptophan hydroxylase gene into bacterial strain BL21-DE3 through plasmid transformation, and using the bacterial strain to ferment and obtain 5-hydroxytryptophan. Background technique [0002] Biotransformation uses microorganisms or enzymes as catalysts to replace non-renewable resources with renewable resources, and is an effective means for large-scale production of chemicals, medicines, energy, materials, etc. needed by humans. [0003] 5-hydroxytryptophan (5HTP) is the precursor of the important neurotransmitter 5-hydroxytryptamine in the human body. 5-hydroxytryptamine is a hormone secreted by the brain, which will affect our mood, sleep and appetite, and cause When serotonin is unbalanced, 5HTP is needed to maintain the body in a normal state. Therefore, if the 5HTP content is too low, it is easy to suffer from depression, anxie...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/04C12N15/53C12N15/70C12N1/21C12R1/19
Inventor 王建军
Owner CHENGDU KAISHENG BIOLOGICAL SCI & TECH
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