阅读说明：本技术 一种生物合成乙醇胺的方法 (Method for biologically synthesizing ethanolamine ) 是由 周丽琴 方培华 王姝凝 杨美雪 邢晨光 刘刚 赵希景 于 2020-04-24 设计创作，主要内容包括：本发明公开了一种生物合成乙醇胺的方法,包括如下步骤：(1)在含有能够被酵母菌细胞代谢以促进生长和/或乙醇胺生产的碳源的发酵培养基中发酵培养经活化的德布尔有孢酵母Torulaspora delbrueckii OMK-71,获得发酵液；(2)将上述发酵液中加入含丝氨酸的反应缓冲液中,进行反应,获得反应液；(3)将步骤(2)所得的反应液经纯化后,获得乙醇胺。本发明中的德布尔有孢酵母Torulaspora delbrueckii OMK-71可用于大规模生物合成乙醇胺,乙醇胺的产量可以达到79g/L或更高。(The invention discloses a method for biologically synthesizing ethanolamine, which comprises the following steps: (1) fermentatively culturing the activated Torulaspora delbrueckii OMK-71 in a fermentation medium containing a carbon source capable of being metabolized by yeast cells to promote growth and/or ethanolamine production to obtain a fermentation broth; (2) adding the fermentation liquor into a reaction buffer solution containing serine for reaction to obtain a reaction solution; (3) and (3) purifying the reaction liquid obtained in the step (2) to obtain the ethanolamine. The Torulaspora delbrueckii OMK-71 in the invention can be used for large-scale biosynthesis of ethanolamine, and the yield of ethanolamine can reach 79g/L or higher.)

1. A method for biosynthesizing ethanolamine, comprising the steps of: the method comprises the following steps:

(1) transferring the activated Torulaspora delbrueckii OMK-71 into a fermentation culture medium containing a carbon source which can be metabolized by yeast cells to promote growth and/or ethanolamine production, and culturing to obtain a fermentation broth; the Debrella Torulaspora delbrueckii OMK-71 is preserved in the China center for type culture Collection in 2019, 12 months and 27 days, and the preservation numbers are as follows: CCTCC NO: m20191124;

(2) adding the fermentation liquor into a reaction buffer solution containing serine for reaction to obtain a reaction solution;

(3) and (3) purifying the reaction liquid obtained in the step (2) to obtain the ethanolamine.

2. The method of claim 1, wherein: the carbon source includes sucrose, fructose, xylose, ethanol, methanol, glycerol, glucose, cellulose, starch, and cellobiose.

3. The method of claim 2, wherein: the carbon source is glycerol.

4. The method of claim 1, wherein: the formula of the reaction buffer solution containing serine is as follows: glucose 18-22g/L, serine 1-150g/L, Tween 8090-110 mg/L, and potassium phosphate buffer solution to adjust pH to 7.1-7.3 to make potassium phosphate concentration 90-110 mM.

5. The method of claim 4, wherein: the formula of the reaction buffer solution containing serine is as follows: glucose 20g/L, serine 145g/L, Tween 80100 mg/L, and potassium phosphate buffer 1M was used to adjust the pH to 7.2, so that the final concentration of potassium phosphate was 100 mM.

6. The method of claim 1, wherein: the formula of the fermentation medium is as follows: 80-90% phosphoric acid 75-85mL, CaSO₄2-4g，K₂SO₄50-60g，KOH 10-15g，MgSO₄·7H₂40-50g of O, 110-130g of glycerol, 10-20g of yeast extract, 103-20g of peptone and 2-4mL of defoaming oil, and the volume is fixed to 3L by using purified water.

7. The method of claim 6, wherein: the formula of the fermentation medium is as follows: 85% phosphoric acid 80.1mL, CaSO₄2.79g，K₂SO₄54.6g，KOH 12.39g，MgSO₄·7H₂44.7g of O, 120g of glycerol, 15g of yeast extract, 15g of peptone and 3mL of defoaming oil, and the volume is adjusted to 3L by using purified water.

8. The method of claim 1, wherein: and (3) purifying, namely performing polyacrylamide flocculation, activated carbon decolorization, ultrafiltration, adsorption and elution of cation exchange resin and vacuum reduced pressure concentration in sequence.

9. A Debrella Torulaspora delbrueckii OMK-71, characterized in that: and is preserved in China center for type culture Collection in 2019, 12 months and 27 days, with the preservation numbers as follows: CCTCC NO: m20191124.

10. The use of Torulaspora delbrueckii OMK-71 as claimed in claim 9 for the biosynthesis of ethanolamine.

Technical Field

The invention belongs to the technical field of fermentation engineering, and particularly relates to a method for biologically synthesizing ethanolamine.

Background

Ethanolamine (Monoethanolamine), also known as 2-aminoethanol, 2-carboxyethylamine, Monoethanolamine, ETA or MEA, is a colorless, viscous liquid with an odor of ammonia, and its derivatives widely present in nature, such as phosphatidylethanolamine, are components of biological membranes, especially of prokaryotic biological membranes, and play an important role in blood coagulation; also, palmitoylethanolamide, a signaling molecule, may act on the CB1 receptor in the brain. The molecular formula is HOCH₂CH₂NH₂The molecule contains primary amines and alcohols, and therefore ethanolamine is hygroscopic and miscible with water, most alcohols and polyols. Which are weakly basic and can react with acids to form esters or salts.

Ethanolamine, which has many characteristics that make it widely used in industry, is an indispensable raw material for producing detergents, emulsifiers, polishing agents, medicines, corrosion inhibitors and chemical intermediates, and specifically, it is as follows:

ethanolamine is useful as a surfactant in personal care products, cosmetics, floor and tile cleaners, laundry detergents, and the like. In these types of products, ethanolamine removes dirt and oil from the skin by dissolving the oil and mixing other important ingredients. Ethanolamine is also a common ingredient in products such as hair dyes because it does not emit strong pungent odor.

Due to the emulsifying properties of ethanolamine, ethanolamine is also useful for industrial applications such as gas treatment, chemical manufacturing, and the like. Ethanolamine helps remove contaminants such as carbon dioxide and hydrogen sulfide from gasoline during gas processing in refineries and natural gas. Ethanolamine is very effective in washing carbon dioxide emitted from burning coal, methane and biogas. The ethanolamine washing reduces carbon emission, and enables the traditional coal and methane industry to be more modern, healthier and have higher market value. In the process of synthesizing ammonia in a chemical production plant, the carbon dioxide in the ammonia gas is also removed by utilizing the ethanolamine. Alternatively, reaction of ethanolamine with ammonia can yield ethylenediamine, which is a precursor of the common chelating agent EDTA.

Ethanolamine is also commonly used in the alkalization of water in the steam cycle of power plants, including nuclear power plants with pressurized water reactors, as a key component of the "all volatile processing of water (AVT)". Since ethanolamine does not accumulate in steam generators (boilers) and cracks due to its volatility, but is relatively uniformly distributed throughout the steam cycle, alkalization of water with ethanolamine can effectively reduce corrosion of metal components. Ethanolamine can also adjust the pH of personal care products and cosmetics to prevent degradation during storage and increase their useful life.

In pharmaceutical formulations, ethanolamine is used primarily to prepare emulsions and provide buffering. Ethanolamine is also an injectable sclerosing agent and may be a therapeutic option for hemorrhoids. 2-5mL ethanolamine oleate may be injected into the mucosa just above the hemorrhoid to cause ulceration and mucosal fixation, thereby preventing the hemorrhoid from descending from the anal canal. Ethanolamine can also be used as a plastic plasticizer, so that the toughness of the plastic can be increased, and the plastic is softer.

Worldwide, ethanolamine production is primarily concentrated in north america, europe, china, and asia (regions outside of china). In 2016, 64.7 million tons of ethanolamine were produced in North America, and 51.1 million tons and 39.6 million tons in Europe and China, respectively. In the same year, the consumption in North America accounts for about 28.85% of the total consumption in the world, and 39.4 ten thousand tons and 54.9 ten thousand tons of ethanolamine are consumed in Europe and China, respectively. Global consumption also increased from 181.1 ten thousand tons in 2012 to 191.9 ten thousand tons in 2017, with an annual growth rate of 1.17%. The worldwide ethanolamine market value in 2017 is $ 24.6 billion.

Currently, ethanolamine is commercially produced by the reaction of ammonia with ethylene oxide, which also produces diethanolamine and triethanolamine. In nature, all bacteria and eukaryotic cells contain the membrane lipid phosphatidylethanolamine, and when phosphodiesterase breaks down phosphatidylethanolamine, the natural ethanolamine is produced. However, large-scale biosynthesis of natural ethanolamine has not been achieved industrially so far.

Disclosure of Invention

The invention aims to provide a method for biosynthesizing ethanolamine.

Another object of the present invention is to provide Torulaspora delbrueckii OMK-71, a strain of Saccharomyces delbrueckii, which is used in the above method.

The technical scheme of the invention is as follows:

a method for biosynthesizing ethanolamine, comprising the steps of:

(1) transferring the activated Torulaspora delbrueckii OMK-71 into a fermentation culture medium containing a carbon source which can be metabolized by yeast cells to promote growth and/or ethanolamine production, and culturing to obtain a fermentation broth; the Debrella Torulaspora delbrueckii OMK-71 is preserved in the China center for type culture Collection (China, Wuhan university) in 2019 at 12 months and 27 days, and the preservation numbers are as follows: CCTCC NO: m20191124;

(2) adding the fermentation liquor into a reaction buffer solution containing serine for reaction to obtain a reaction solution;

(3) and (3) purifying the reaction liquid obtained in the step (2) to obtain the ethanolamine.

In a preferred embodiment of the present invention, the carbon source includes sucrose, fructose, xylose, ethanol, methanol, glycerol, glucose, cellulose, starch and cellobiose. The carbon source may be provided to the yeast throughout the cultivation period, or the yeast may be grown in the presence of another energy, such as a protein, for a period of time and then provided only in the fed-batch phase.

Further preferably, the carbon source is glycerol.

In a preferred embodiment of the present invention, the formulation of the serine-containing reaction buffer is: glucose 18-22g/L, serine 1-150g/L, Tween 8090-110 mg/L, and potassium phosphate buffer solution to adjust pH to 7.1-7.3 to make potassium phosphate concentration 90-110 mM.

Further preferably, the formula of the reaction buffer containing serine is as follows: glucose 20g/L, serine 145g/L, Tween 80100 mg/L, and potassium phosphate buffer 1M was used to adjust the pH to 7.2, so that the final concentration of potassium phosphate was 100 mM.

In a preferred embodiment of the invention, the formulation of the fermentation medium is: 80-90% phosphoric acid 75-85mL, CaSO₄2-4g，K₂SO₄50-60g，KOH 10-15g，MgSO₄·7H₂40-50g of O, 110-130g of glycerol, 10-20g of yeast extract, 10-20g of peptone and 2-4mL of defoaming oil, and the volume is fixed to 3L by using purified water.

Further preferably, the formula of the fermentation medium is as follows: 85% phosphoric acid 80.1mL, CaSO₄2.79g，K₂SO₄54.6g，KOH 12.39g，MgSO₄·7H₂O44.7g, glycerol 120g, yeast extract 15g, peptone 15g and defoaming oil 3mL, and the volume is adjusted to 3L by purified water.

In a preferred embodiment of the present invention, the purification in step (3) is sequentially performed by polyacrylamide flocculation, activated carbon decolorization, ultrafiltration, adsorption and elution by cation exchange resin, and vacuum concentration under reduced pressure.

The other technical scheme of the invention is as follows:

a Torulaspora delbrueckii OMK-71, deposited in the China center for type culture Collection (university of Wuhan, China) at 27 months 12 and 2019, with the collection numbers: CCTCC NO: m20191124.

The use of the above Debrella Torulaspora delbrueckii OMK-71 for the biosynthesis of ethanolamine.

The invention has the beneficial effects that:

1. the Torulaspora delbrueckii OMK-71 in the invention can convert serine to biosynthesize ethanolamine, and the yield of ethanolamine can reach 79g/L or higher.

2. The invention develops a route for biologically synthesizing the ethanolamine by converting serine by using the yeast Torulaspora delbrueckii OMK-71, has simple process, is different from other ethanolamine production methods, and can realize large-scale production.

Drawings

FIG. 1 is a high performance liquid chromatogram of ethanolamine in example 1 of the present invention, demonstrating that the resulting product is ethanolamine.

Detailed Description

The technical solution of the present invention will be further illustrated and described below with reference to the accompanying drawings by means of specific embodiments.

The invention is based on the discovery that all bacteria and eukaryotic cells contain the membrane lipid phosphatidylethanolamine, which when decomposed by phosphodiesterases produces the native ethanolamine. However, because ethanolamine has some inhibition on the growth of bacteria and eukaryotic cells, it is difficult for bacteria and eukaryotic cells to produce ethanolamine at high concentration, and even if produced, it is immediately used for glycerophospholipid metabolism.

The invention separates the yeast from the ocean mud bed in different areas, uses the ultra-fidelity Polymerase Chain Reaction (PCR) technology to amplify the 18S ribosomal RNA, 26S ribosomal RNA and Internal Transcribed Spacer (ITS) of each yeast and sequences. The target sequence obtained by sequencing is inquired on a nucleotide sequence database, BLAST, reciprocal (reciprocal) BLAST or PSI-BLAST analysis is carried out on a non-redundant database by using the target sequence as a reference sequence, and a yeast strain with high ethanolamine yield is bred by complex mutation breeding, preferably by complex mutation of the yeast strain through ultraviolet rays and lithium chloride, and the method for biosynthesizing the natural ethanolamine on a large scale by using the yeast strain. The method comprises the following steps: providing a yeast capable of producing ethanolamine in the presence of a carbon source; culturing the yeast in the presence of a carbon source; and purifying ethanolamine from the yeast or from a culture supernatant of the yeast.