Preparation method of hydroxyectoine

A technology of hydroxy ectoine and ectoine, which is applied in the field of preparation of hydroxy ectoine, can solve the problems of decreased synthesis of ectoine, complex control process, high production cost, etc., and achieves the elimination of separation and purification process and process operation Simplicity and the effect of reducing production costs

Inactive Publication Date: 2015-12-23
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Abstract
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Problems solved by technology

[0003] The above-mentioned fermentation method has the following defects: (1) the synthesis of hydroxyectoine as a derivative of ectoine is induced by thermal stimulation, that is, it needs to increase the growth temperature to promote its synthesis, but increasing the growth temperature will seriously inhibit the halophilic bacteria The growth of ectoine leads to a serious decline in the synthesis of ectoine, thereby affecting the output of hydroxy ectoine; ...

Method used

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  • Preparation method of hydroxyectoine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] (1) Construction of ectoine hydroxylase bacterial strain

[0032] ①A pair of primers were designed according to the sequence of the ectD gene by PCR technology, using the genome of Halomonas elongatus (CGMCC1.6329) as a template, and the ectD gene fragment was amplified. The pair of primers respectively comprise enzyme cutting sites NheI and HindIII.

[0033] ② Use Takara restriction endonucleases NheI and HindIII to double-digest step ① to obtain the target fragment and pET-his vector plasmid, and obtain ectD and pET-his linear fragments with the same cohesive ends.

[0034] ③ Use TakaraT4 DNA ligase to connect the two target fragments obtained in step ② to obtain the target vector pET-his-ectD.

[0035] ④ Transform the vector obtained in step ③ into E.coliBL-21 (ACCC11171) to obtain a ectoine hydroxylase-producing strain.

[0036] (2) Cell preparation with ectoine hydroxylase activity

[0037] ① Inoculate the above-mentioned strains into a 500ml round-bottomed Erle...

Embodiment 2

[0047] (1) For the construction of a strain producing ectoine hydroxylase, refer to Example 1.

[0048](2) Preparation of bacterial cells with ectohydropyrimidine hydroxylase activity, refer to Example 1.

[0049] (3) Catalytic synthesis of hydroxy ectoine using ectoine fermentation broth

[0050] ①Centrifuge the fermentation broth containing 5g / L ectoine, collect the supernatant, add 44mmol / L HEPES as a buffer, and finally adjust the pH to 7.0 with NaOH.

[0051] ② Prepare the reaction system, add 0.035mol / L α-ketoglutaric acid to the above fermentation broth, adjust the pH to 7.0 with NaOH, and then add the above-mentioned bacteria with ectoine hydroxylase activity in an amount of 30g / L.

[0052] ③The above reaction system was placed in a constant temperature water bath shaker at 40°C, and reacted at 200rpm for 24h.

[0053] ④ Please refer to Example 1 for the liquid chromatography detection method. Detected by liquid chromatography, such as figure 1 and figure 2 Shown...

Embodiment 3

[0055] (1) For the construction of a strain producing ectoine hydroxylase, refer to Example 1.

[0056] (2) Preparation of bacterial cells with ectohydropyrimidine hydroxylase activity, refer to Example 1.

[0057] (3) Catalytic synthesis of hydroxy ectoine using ectoine fermentation broth

[0058] ①Centrifuge the fermentation broth containing 5g / L ectoine, collect the supernatant, add 44mmol / L HEPES as a buffer, and finally adjust the pH to 7.0 with NaOH.

[0059] ② Prepare the reaction system, add 0.035mol / L α-ketoglutaric acid to the above fermentation broth, adjust the pH to 7.0 with NaOH, and then add the above-mentioned bacteria with ectoine hydroxylase activity in an amount of 30g / L.

[0060] ③The above reaction system was placed in a constant temperature water bath shaker at 45°C, and reacted at 200rpm for 24h.

[0061] ④ Please refer to Example 1 for the liquid chromatography detection method. Detected by liquid chromatography, the hydroxyl in the reaction solution...

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Abstract

The invention relates to a preparation method of hydroxyectoine. According to the preparation method, an ectoine-containing feed liquid is adopted as a substrate, Escherichia coli with ectonine hydroxylase activity and alpha-ketoglutaric acid are added, and an enzymatic reaction is performed under conditions that the pH is 7.0-8.0 and the temperature is 37-45 DEG C to synthesize the hydroxyectoine. According to the method, the hydroxyectoine with the higher added value is synthesized with the ectoine-containing feed liquid, prepared with a fermentation method, used as the substrate, and the method has the advantages that the raw material source is wide, the production cost is low and the like.

Description

technical field [0001] The invention relates to the field of compound biotechnology production, in particular to a preparation method of hydroxy ectoine. Background technique [0002] In the prior art, hydroxytetrahydropyrimidine is mainly obtained by fermentation of Halomonas. Zhu Daochen and others studied the method of fermenting and producing hydroxyectoine by using the halophilic bacteria HalomonasventosaeDL7. In the fermentation process, it is necessary to culture bacteria first, and then within a short period of time (within 1 hour), the concentration of sodium chloride is increased from 1M to 3M and the culture temperature is increased from 30°C to 42°C to accumulate hydroxyectoine. The total concentration of intracellular and extracellular ectoine obtained in the final extraction was 13.8 g / L, and the total concentration of hydroxy ectoine was 6.2 g / L. [0003] The above-mentioned fermentation method has the following defects: (1) the synthesis of hydroxyectoine a...

Claims

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Application Information

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IPC IPC(8): C12P17/12C12R1/19
Inventor 谢希贤陈宁吴雪娇宁义科范晓光徐庆阳张成林
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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