阅读说明：本技术 大肠杆菌发酵生产氨基葡萄糖的方法 (Method for producing glucosamine by fermenting escherichia coli ) 是由 卢健行 马善丽 卢建智 韩宁 张建华 姚珊珊 于 2019-10-09 设计创作，主要内容包括：本发明公开了一种大肠杆菌发酵生产氨基葡萄糖的方法,将大肠杆菌、枯草芽孢杆菌、葡萄球菌进行种子活化后制得混合发酵剂,将混合发酵剂接种至含有葡萄糖和氮源的发酵培养基中恒温摇床培养,发酵过程采用恒速补料方式进行,用氨水控制发酵pH为6.8-7.0,得到含有氨基葡萄糖的发酵液,经分离纯化后得到氨基葡萄糖。本发明通过混合发酵剂提高大肠杆菌的发酵活力,半胱氨酸、丙氨酸的加入可以降低发酵过程中谷氨酸的产生速度,提高氨基葡萄糖的合成速率。(The invention discloses a method for producing glucosamine by fermenting escherichia coli, which comprises the steps of activating the seeds of escherichia coli, bacillus subtilis and staphylococcus to prepare a mixed leaven, inoculating the mixed leaven into a fermentation medium containing glucose and a nitrogen source to perform constant-temperature shaking table cultivation, performing the fermentation process in a constant-speed material feeding mode, controlling the fermentation pH to be 6.8-7.0 by ammonia water to obtain fermentation liquor containing the glucosamine, and separating and purifying to obtain the glucosamine. The fermentation activity of the escherichia coli is improved by the mixed starter, the production speed of the glutamic acid in the fermentation process can be reduced by adding the cysteine and the alanine, and the synthesis rate of the glucosamine is improved.)

1. A method for producing glucosamine by fermenting escherichia coli is characterized by comprising the following steps: the method comprises the following steps: activating the seeds of escherichia coli, bacillus subtilis and staphylococcus to prepare a mixed starter; inoculating the mixed starter to a fermentation medium containing glucose and a nitrogen source, and carrying out constant-temperature shaking culture; the fermentation medium consists of glucose, yeast extract powder, alanine, cysteine, sodium nitrate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, magnesium sulfate, ferrous sulfate, lactose and glycerol; the fermentation process is carried out by constant-speed feeding, ammonia water is used for controlling the fermentation pH to 6.8-7.0, fermentation liquor containing glucosamine is obtained, and the glucosamine is obtained after separation and purification.

2. The method for producing glucosamine by fermentation of escherichia coli according to claim 1, wherein the fermentation step comprises the following steps: and performing activation culture on the escherichia coli, the bacillus subtilis and the staphylococcus on a plate culture medium for 6-8 hours before performing seed activation.

3. The method for producing glucosamine by fermentation of escherichia coli according to claim 2, wherein: the plate culture medium comprises 11.5g/L of fish peptone, 6g/L of yeast extract powder, 5g/L of sodium chloride, 2g/L of ammonium sulfate and 15g/L of agar.

4. The method for producing glucosamine by fermentation of escherichia coli according to claim 1 or 2, wherein: the mixed starter is prepared by mixing Escherichia coli, Bacillus subtilis and Staphylococcus according to the volume ratio of 3 (1-2) to 0.8-1.

5. The method for producing glucosamine by fermentation of escherichia coli according to claim 1 or 2, wherein: the seeds of the escherichia coli, the bacillus subtilis and the staphylococcus are activated by inoculating strains in a seed culture medium, controlling the culture temperature to be 36-38 ℃, the shaking table speed to be 220-270rpm, and the culture time to be 12-14 h.

6. The method for producing glucosamine by fermentation of escherichia coli according to claim 5, wherein: the seed culture medium comprises 11.5g/L of fish peptone, 20g/L of yeast extract powder, 5g/L of sodium chloride, 2g/L of ammonium sulfate, 5.5g/L of glycerol and pH 6.8-7.0.

7. The method for producing glucosamine by fermentation of escherichia coli according to claim 1, wherein the fermentation step comprises the following steps: the fermentation medium comprises the following components in percentage by weight: 25g/L glucose, 20g/L yeast extract powder, 10g/L alanine, 10g/L cysteine, 3g/L sodium nitrate, 1.05g/L dipotassium phosphate, 0.45g/L potassium dihydrogen phosphate, 0.1g/L sodium chloride, 0.5g/L magnesium sulfate, 0.03g/L ferrous sulfate, 3.2g/L lactose and 5.5g/L glycerol.

8. the method for producing glucosamine by fermentation of escherichia coli according to claim 1, wherein the fermentation step comprises the following steps: the rotation speed of a shaking table is controlled to be 230 and 250rpm, the culture temperature is 35-38 ℃, and the culture time is 35-48 h.

9. The method for producing glucosamine by fermentation of escherichia coli according to claim 1, wherein the fermentation step comprises the following steps: the glucose feeding speed is controlled to be 3-5L/h in the fermentation process.

10. The method for producing glucosamine by fermentation of escherichia coli according to claim 1, wherein the fermentation step comprises the following steps: the mixed starter is inoculated on a fermentation medium according to the inoculation amount of 10%.

Technical Field

The invention belongs to the technical field of biological fermentation, and particularly relates to a method for producing glucosamine by fermenting escherichia coli.

Background

Glucosamine (GleN) is an important hexosamine formed by substituting one hydroxyl group of glucose with an amino group, and there are two main types of glucosamine on the market today, one is glucosamine hydrochloride and the other is glucosamine sulfate. D-Glucosamine Hydrochloride (D-Glucosamine Hydrochloride), molecular formula C₆H₁₃NO₅HCl, a white crystal, odorless, slightly sweet, easily soluble in water, slightly soluble in methanol, insoluble in organic solvents such as ethanol, has important physiological functions for human body, participates in liver and kidney detoxification, plays a role in anti-inflammation and liver protection, has good curative effect on rheumatic arthritis and gastric ulcer, and is a main raw material for synthesizing antibiotics and anticancer drugs; can also be used in food, cosmetic and feed additive. Glucosamine hydrochloride is extracted from natural chitin, is a marine biological agent, and is the main component of chondroitin sulfate. It can promote the synthesis of mucopolysaccharide, raise the viscosity of joint synovial fluid, improve the metabolism of joint cartilage, promote the repair of joint cartilage and has obvious antiphlogistic and analgesic effects. It has the effect of promoting the injection efficiency of antibiotics, and can be used as nutritional supplement for diabetic patients.

The current methods for producing glucosamine mainly comprise an acid hydrolysis method, an enzymolysis method and a microbial fermentation method. The production raw materials of the acid hydrolysis method and the enzymolysis method are from exoskeletons of fishes, shrimps, crabs and the like, and glucosamine is obtained by extracting chitin and chitosan and then carrying out acidolysis or enzymolysis; however, a large amount of concentrated hydrochloric acid is needed in the acid hydrolysis process, which can cause serious industrial pollution; the enzymolysis method is to degrade the exoskeleton of fishes, shrimps and crabs by using chitosan, and has low process efficiency and higher production cost, so that the search for a proper microbial fermentation method to realize the industrial production of glucosamine is the current environmental and market demand. Escherichia coli is a common glucosamine synthetase producing strain. The research shows that: in Escherichia coli, glucosamine is produced from glutamine as an amino donor and fructose-6-phosphate under the catalytic action of glucosamine synthetase (GlmS). However, in the prior art, the production amount of the glucosamine is low, and the industrial high-efficiency production is difficult to realize.

Disclosure of Invention

In order to make up the defects of the prior art, the invention provides the method for producing the glucosamine by fermenting the escherichia coli, which has the advantages of high production efficiency, low cost and simple operation.

The invention is realized by the following technical scheme:

A method for producing glucosamine by fermenting escherichia coli is characterized in that: the method comprises the following steps: activating the seeds of escherichia coli, bacillus subtilis and staphylococcus to prepare a mixed starter; inoculating the mixed starter to a fermentation medium containing glucose and a nitrogen source, and carrying out constant-temperature shaking culture; the fermentation medium consists of glucose, yeast extract powder, alanine, cysteine, sodium nitrate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, magnesium sulfate, ferrous sulfate, lactose and glycerol; the fermentation process is carried out by constant-speed feeding, ammonia water is used for controlling the fermentation pH to 6.8-7.0, fermentation liquor containing glucosamine is obtained, and the glucosamine is obtained after separation and purification.

Preferably, the Escherichia coli, the Bacillus subtilis and the staphylococcus are subjected to activation culture on a plate culture medium for 6-8h before seed activation.

Further, the plate culture medium comprises 11.5g/L of fish peptone, 6g/L of yeast extract powder, 5g/L of sodium chloride, 2g/L of ammonium sulfate and 15g/L of agar.

Preferably, the mixed starter is prepared according to the volume ratio of 3 (1-2) to 0.8-1) of escherichia coli, bacillus subtilis and staphylococcus.

Further, the seeds of the escherichia coli, the bacillus subtilis and the staphylococcus are activated by inoculating strains in a seed culture medium, controlling the culture temperature to be 36-38 ℃, the shaking table speed to be 220-270rpm, and the culture time to be 12-14 h.

Preferably, the seed culture medium comprises 11.5g/L of fish peptone, 20g/L of yeast extract powder, 5g/L of sodium chloride, 2g/L of ammonium sulfate, 5.5g/L of glycerol and pH 6.8-7.0.

Further, the fermentation medium comprises the following components in percentage by weight: 25g/L glucose, 20g/L yeast extract powder, 10g/L alanine, 10g/L cysteine, 3g/L sodium nitrate, 1.05g/L dipotassium phosphate, 0.45g/L potassium dihydrogen phosphate, 0.1g/L sodium chloride, 0.5g/L magnesium sulfate, 0.03g/L ferrous sulfate, 3.2g/L lactose and 5.5g/L glycerol.

Furthermore, the rotating speed of the shaking table is controlled to be 230-.

Further, the glucose feeding speed is controlled to be 3-5L/h in the fermentation process.

Further, the mixed starter is inoculated on the fermentation medium according to the inoculation amount of 10%.

The invention has the beneficial effects that: according to the invention, the fermentation activity of escherichia coli is improved by the mixed leaven, and the glucosamine is efficiently produced by fermentation, the production speed of glutamic acid in the fermentation process can be reduced by adding cysteine and alanine, the synthesis rate of the glucosamine is improved, the reaction cost is reduced, the yield of the glucosamine is improved, and the industrial production can be realized.

Detailed Description

The present invention will be described in further detail with reference to specific embodiments thereof to assist those skilled in the art in providing a more complete, accurate and thorough understanding of the inventive concept and aspects thereof, and the scope of the present invention includes, but is not limited to, the following examples, and any modifications in the details and form of the technical aspects thereof that fall within the spirit and scope of the present application are intended to be included therein.

The following examples all follow the following medium composition:

The plate culture medium comprises: 11.5g/L of fish peptone, 6g/L of yeast extract powder, 5g/L of sodium chloride, 2g/L of ammonium sulfate and 15g/L of agar.

The seed culture medium comprises: 11.5g/L of fish peptone, 20g/L of yeast extract powder, 5g/L of sodium chloride, 2g/L of ammonium sulfate and 5.5g/L of glycerol;

The fermentation medium comprises: 25g/L glucose, 20g/L yeast extract powder, 10g/L alanine, 10g/L cysteine, 3g/L sodium nitrate, 1.05g/L dipotassium phosphate, 0.45g/L potassium dihydrogen phosphate, 0.1g/L sodium chloride, 0.5g/L magnesium sulfate, 0.03g/L ferrous sulfate, 3.2g/L lactose and 5.5g/L glycerol.