阅读说明：本技术 一种促进嗜盐古菌Haloferax mediterranei合成甜菜碱的方法 (Method for promoting halophilic archaea Halofax mediterranei to synthesize betaine ) 是由 崔有为 陈思 于 2021-08-12 设计创作，主要内容包括：一种促进嗜盐古菌Haloferax mediterranei合成甜菜碱的方法属于生物发酵领域。其特征在于：提高H.mediterranei发酵液的盐度,并通过在外部施加特定强度的磁场,提高甜菜碱产量。即利用渗透冲击使H.mediterranei合成更多的甜菜碱以平衡胞内外渗透压,并将外源磁场作用于H.mediterranei发酵液,实现H.mediterranei甜菜碱产量的提高。(A method for promoting halophilic archaea Halofax mediterranei to synthesize betaine belongs to the field of biological fermentation. The method is characterized in that: the salinity of the H.mediterranei fermentation liquor is improved, and the yield of the betaine is improved by externally applying a magnetic field with specific strength. Namely, the H.mediterranei is used for synthesizing more betaine by osmotic shock so as to balance the osmotic pressure inside and outside cells, and an external magnetic field is acted on the H.mediterranei fermentation liquor, so that the yield of the H.mediterranei betaine is improved.)

1. A method for promoting halophilic archaea Halofax mediterranei to synthesize betaine is characterized in that a device used in the method comprises:

(1) the device comprises a fermentation tank, (2) a temperature control device, (3) a pH automatic controller, (4) an aeration pump, (5) a gas flowmeter, (6) an aeration head, and (7) a static magnetic field generating device;

the device connection process is as follows: the temperature control device (2) is arranged on the fermentation tank (1); the pH automatic controller (3) is connected with the fermentation tank (1); an aeration pump (4), a gas flowmeter (5) and an aeration head (6) are connected through a conduit for aeration; placing the fermentation tank (1) in a static magnetic field generating device (7);

halofax mediterranei is used as an inoculum for fermentation, and the steps are as follows:

(1) inoculating halophilic archaea Halofax mediterranei into a sterilized enrichment culture medium for enrichment culture, wherein the culture time is 72 h; the salinity of the enrichment medium is 150-200g NaCl/L;

(2) inoculating the enriched culture into a fermentation tank (1), and controlling the volume ratio of the enriched culture to the fermentation liquor to be 1: 20; the fermentation liquid is characterized in that the salinity is 250-300 gNaCl/L;

(3) adjusting the temperature control device (2) to control the temperature at 35-39 ℃, adjusting the pH automatic controller (3) to control the pH value at 6.9-7.3, and adjusting the gas flowmeter (5) to control the dissolved oxygen at more than 2mg/L and keep the dissolved oxygen consistent;

(4) adjusting the static magnetic field generating device (7) to control the magnetic field strength to be 10-60mT, wherein the optimal magnetic field strength is 50 mT;

(5) continuously fermenting for 18-30 h; stopping fermentation when the content of betaine cells is maximum; collecting cells to extract intracellular betaine.

Technical Field

The invention provides a method for promoting halophilic archaea Haloferax mediterranei (H.mediterranei) to synthesize betaine (glycine betaine). The invention belongs to the field of biological fermentation, and particularly relates to the field of betaine biosynthesis.

Background

Betaine is an alkaloid widely present in animals, plants and microorganisms. Betaine is a secondary product of metabolism, is a very important osmotic regulatory substance and can enhance the stress resistance of organisms. In recent years, betaine has been used as an additive in many applications such as food, feed, cosmetics, and medicines. At present, the betaine is produced by a chemical synthesis method in multiple uses, but the method has the disadvantages of large energy consumption, long time and more byproducts.

With the rapid development of molecular biology, metabolic engineering and other biological sciences, the production of target products by using microorganisms as cell factories has become a feasible means. Mediterranei is an extremely halophilic archaea, can survive at extremely high salinity, and betaine plays an important role in salt adaptation and short-term salt stress. Under high salt conditions, betaine can be absorbed by halophilic bacteria from the outside or synthesized by itself to maintain intracellular osmotic pressure, and simultaneously, it can stabilize cell structure and maintain the activity of intracellular enzymes and proteins. In recent years, the biological effects of Electric Fields (EFs), Magnetic Fields (MFs) or electromagnetic fields (EMF) on eukaryotes and prokaryotes have attracted intense interest to biologists because these physical conditions profoundly affect the metabolism, behavior and survival of cells. During the salt stress process of the H.mediterranei, an exogenous magnetic field can be used as a means for improving the cell proliferation of the H.mediterranei.

The patent provides a method for promoting synthesis of betaine from H.mediterranei by means of coupling hypertonic mutation with an exogenous magnetic field. The method can improve the content of H.mediterranei intracellular betaine and the yield of betaine.

Disclosure of Invention

The invention aims to improve the betaine yield of halophilic archaea H.

The invention regulates and controls the betaine yield of halophilic archaea H.mediterranei, and is characterized in that: the salinity of the H.mediterranei fermentation liquor is improved, and the yield of the betaine is improved by externally applying a magnetic field with specific strength. Namely, the H.mediterranei is used for synthesizing more betaine by osmotic shock so as to balance the osmotic pressure inside and outside cells, and an external magnetic field is acted on the H.mediterranei fermentation liquor, so that the yield of the H.mediterranei betaine is improved.

The invention provides a device for strengthening the synthesis of betaine by halophilic archaea H.mediterranei by utilizing a hypertonic mutation coupling exogenous magnetic field, which comprises the following components:

(1) the device comprises a fermentation tank, (2) a temperature control device, (3) a pH automatic controller, (4) an aeration pump, (5) a gas flowmeter, (6) an aeration head, and (7) a static magnetic field generating device.

The device connection process is as follows: the temperature control device (2) is arranged on the fermentation tank (1); the pH automatic controller (3) is connected with the fermentation tank (1); an aeration pump (4), a gas flowmeter (5) and an aeration head (6) are connected through a conduit for aeration; the fermentation tank (1) is placed in a static magnetic field generating device (7).

Based on the device, Halofax mediterranei (strain number ATCC 33500) is used as an inoculum for fermentation, and the main steps are as follows:

(1) halofax mediterranei (ATCC 33500) is inoculated into sterilized enrichment medium for enrichment culture, and the culture time is 72 h. The enrichment medium is characterized by salinity of 150-200 gNaCl/L;

(2) the enriched culture was inoculated in a fermentor (1) and the enriched culture fermentation broth was controlled at 1:20 (v/v). The fermentation liquid is characterized in that the salinity is 250-300 gNaCl/L;

(3) adjusting the temperature control device (2) to control the temperature at 35-39 ℃, adjusting the pH automatic controller (3) to control the pH value at 6.9-7.3, and adjusting the gas flowmeter (5) to control the aeration amount at 1.0-1.5L/min;

(4) adjusting the static magnetic field generating device (7) to control the magnetic field strength to be 10-60mT, wherein the optimal magnetic field strength is 50 mT;

(5) fermenting continuously for 18-30 h. Betaine content was measured continuously during the fermentation. The fermentation was stopped when the betaine cell content was maximal. Collecting cells to extract intracellular betaine.

It will be understood by those skilled in the art that various changes and modifications in form and detail may be made therein without departing from the spirit and scope of the invention, and these are to be considered as falling within the scope of the invention.

The technical principle of the invention is as follows:

similar compatible substances are key substances of the halophilic bacteria for resisting osmotic pressure, and a certain amount of similar compatible substances can help the halophilic bacteria to grow better in a high-salt environment. The major compatible solutes in different halophiles are different, and betaine is the major similar compatible substance of h. As the external osmotic pressure increases, h.mediterranei will synthesize more betaine to maintain intracellular osmotic pressure. Furthermore, the exogenous magnetic field enhances the synthesis of betaine from h.mediterranei via the choline oxidation pathway, i.e. choline is oxidized to betaine aldehyde by choline dehydrogenase and then to betaine aldehyde by betaine aldehyde dehydrogenase. Therefore, the invention promotes the activity of betaine aldehyde dehydrogenase in H.mediterranei by coupling hypertonic mutation with an external magnetic field, thereby promoting the synthesis of betaine.

The invention has the beneficial effects that:

the invention takes H.mediterranei as a cell factory by a fermentation mode, and promotes the synthesis of H.mediterranei intracellular betaine by hypertonic mutation coupling with an external magnetic field. The hypertonic condition can effectively promote the synthesis of betaine in H.mediterranei cells, the low-intensity magnetic field can promote the proliferation of H.mediterranei under the hypertonic condition, and the synergistic effect of the two can improve the total yield of betaine. The method is simple, safe, low in cost, reusable in magnetic equipment, nontoxic in physical facilities, and free from secondary pollution like other chemical methods.

Drawings

Fig. 1 is a schematic structural diagram of a device for synthesizing betaine by fermenting H.mediterranei under the condition of applying a static magnetic field. (1) The device comprises a fermentation tank, (2) a temperature control device, (3) a pH automatic controller, (4) an aeration pump, (5) a gas flowmeter, (6) an aeration head, and (7) a static magnetic field generating device.

FIG. 2 shows the growth of H.mediterranei and intracellular betaine content at 200g/L NaCl under no magnetic field.

FIG. 3 shows the growth of H.mediterranei and intracellular betaine content under the condition of coupling of 300g/LNaCl and an external magnetic field of 50 mT.

The specific implementation mode is as follows:

in order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, but the present invention is not limited to the following examples.

In the examples, the species used was Halofax mediterranei, species number ATCC 33500.

The enrichment medium formula of the strain is as follows: 200.0g/LNaCl, 2.0g/LKCl, 20g/LMgSO₄·7H₂O，0.01g/LFeSO₄，3.0g/LC₆H₅Na₃O₇·2H₂O, 7.5g/LCasein Hydrolysate, 10g/LYEAST EXTRACT. Meanwhile, the pH of the medium was adjusted to 7.2 with sodium hydroxide solution.

The formula of the fermentation culture solution of the strain is as follows: 4.0g/LKCl, 0.6917g/LCaCl₂·2H₂O，1.0g/LNH₄Cl，13.0g/LMgCl₂·6H₂O，20.0g/LMgSO₄·7H₂O，0.25g/LNaHCO₃，0.5g/LNaBr，0.4g/LKH₂PO₄，0.01g/LFeSO₄5.0g/LGlucose, 1.0ml/L SL-6 trace elements (SL-6 trace element solution prepared according to a known method and containing 0.1g zinc sulfate heptahydrate, 0.03g magnesium chloride tetrahydrate, 0.3g boric acid, 0.2g chlorodiamond hexahydrate, 0.01g copper chloride dihydrate, 0.02g nickel chloride hexahydrate and 0.03g sodium molybdate monohydrate per 100 ml), pH of the fermentation broth was adjusted to 7.2 with a sodium hydroxide solution.

Example 1:

(1) halofax mediterranei (ATCC 33500) is inoculated into sterilized enrichment medium for enrichment culture, and the culture time is 72 h. The enrichment medium is characterized in that the salinity is 200 gNaCl/L;

(2) the enriched culture was inoculated into a fermentor and 1L of fermentation broth was prepared, and the enriched culture, broth, was controlled to be 1:20 (v/v). The fermentation liquor is characterized in that the salinity is 200 gNaCl/L;

(3) adjusting the temperature control device to control the temperature at 37.0 ℃, adjusting the pH automatic controller to control the pH value at 7.2, adjusting the gas flow meter to control the aeration amount at 1.5L/min;

(4) and timing after the fermentation starts, sampling at 6h, 18h, 30h, 42h, 54h and 66h of the fermentation period in sequence, and detecting the dry cell weight of the bacterial liquid and the intracellular betaine content of H.mediterranei.

(5) The maximum intracellular betaine yield was reached at 18h, with a maximum yield of 188.27 mg.

Example 2:

(1) halofax mediterranei (ATCC 33500) is inoculated into sterilized enrichment medium for enrichment culture, and the culture time is 72 h. The enrichment medium is characterized in that the salinity is 200 gNaCl/L;

(2) the enriched culture was inoculated into a fermentor and 1L of fermentation broth was prepared, and the enriched culture, broth, was controlled to be 1:20 (v/v). The fermentation liquor is characterized in that the salinity is 300 gNaCl/L;

(3) adjusting the temperature control device to control the temperature at 37.0 ℃, adjusting the pH automatic controller to control the pH value at 7.2, adjusting the gas flow meter to control the aeration amount at 1.5L/min;

(4) adjusting the static magnetic field generating device to control the magnetic field strength to be 50 mT;

(5) and timing after the fermentation starts, sampling at 6h, 18h, 30h, 42h, 54h and 66h of the fermentation period in sequence, and detecting the dry cell weight of the bacterial liquid and the intracellular betaine content of H.mediterranei.

(6) The maximum intracellular betaine yield is reached at 30h, with a maximum yield of 441.20 mg.