阅读说明：本技术 微生物发酵生产n-乙酰氨基葡萄糖的方法 (Method for producing N-acetylglucosamine by microbial fermentation ) 是由 卢健行 韩宁 马善丽 赵鹏 吴祥舟 卢建功 于 2019-12-02 设计创作，主要内容包括：本发明公开了一种微生物发酵生产N-乙酰氨基葡萄糖的方法,将大肠杆菌、酿酒酵母菌制得混合发酵剂接种至发酵培养基中恒温摇床培养,控制摇床转速230-250rpm,培养温度为36-38℃,发酵过程采用恒速补料方式进行,控制葡萄糖补料速度为3-5g/L·h,用氨水控制发酵过程pH不小于6.9,当发酵液OD600nm大于30时,分批加入诱导剂IPTG和甲醇溶液,培养时间为35-72h。本发明通过混合发酵剂提高大肠杆菌活性,并通过多种手段降低谷氨酸的累积速度,有利于实现产品高产量规模化生产。(The invention discloses a method for producing N-acetylglucosamine by microbial fermentation, which comprises the steps of preparing a mixed leavening agent from escherichia coli and saccharomyces cerevisiae, inoculating the mixed leavening agent into a fermentation medium, carrying out constant-temperature shaking table cultivation, controlling the shaking table rotation speed to be 230 plus 250rpm, the cultivation temperature to be 36-38 ℃, carrying out the fermentation process in a constant-speed feeding manner, controlling the glucose feeding speed to be 3-5 g/L.h, controlling the pH value in the fermentation process to be not less than 6.9 by using ammonia water, and adding an inducer IPTG and a methanol solution in batches when the fermentation broth OD600nm is more than 30, wherein the cultivation time is 35-72 h. The invention improves the activity of the escherichia coli by mixing the leavening agent, reduces the accumulation speed of the glutamic acid by various means, and is beneficial to realizing the high-yield gauge production of the product.)

1. A method for producing N-acetylglucosamine by microbial fermentation is characterized in that: the method comprises the following steps: activating the seeds of escherichia coli and saccharomyces cerevisiae to prepare a mixed starter; inoculating the mixed starter to a fermentation medium for constant-temperature shaking table culture, and controlling the rotation speed of the shaking table to be 230-250rpm, wherein the culture temperature is 36-38 ℃; the fermentation process is carried out by adopting a constant-speed feeding mode, the feeding speed of glucose is controlled to be 3-5 g/L.h, the pH value of the fermentation process is controlled to be not less than 6.9 by using ammonia water, when the OD600nm of the fermentation liquor is more than 30, the inducer IPTG and the methanol solution are added in batches, and the culture time is 35-72 h.

2. The method for producing N-acetylglucosamine by fermentation of a microorganism according to claim 1, wherein: the fermentation medium comprises: 35g/L glucose, 20g/L yeast extract powder, 10g/L alanine, 10g/L cysteine, 3g/L sodium lactate, 1.05g/L dipotassium hydrogen phosphate, 0.45g/L potassium dihydrogen phosphate, 0.1g/L sodium chloride, 0.3g/L zinc sulfate, 0.5g/L magnesium sulfate, 0.03g/L ferrous sulfate, 3.2g/L lactose and 5.5g/L glycerol.

3. The method for producing N-acetylglucosamine by fermentation of a microorganism according to claim 1 or 2, wherein: the volume concentration of methanol added to the methanol solution was 0.5%.

4. The method for producing N-acetylglucosamine by fermentation of a microorganism according to claim 1 or 2, wherein: and performing activation culture on the escherichia coli and the saccharomyces cerevisiae on a plate culture medium for 6-8 hours before performing seed activation.

5. The method for producing N-acetylglucosamine by fermentation of a microorganism according to claim 4, wherein: the plate culture medium comprises 15g/L of peptone, 8g/L of yeast extract, 2g/L of galactose, 1g/L of sodium chloride, 1g/L of ammonium sulfate and 15g/L of agar.

6. The method for producing N-acetylglucosamine by fermentation of a microorganism according to claim 1 or 2, wherein: the seed activation of the escherichia coli and the saccharomyces cerevisiae is to inoculate the strain in a seed culture medium, the culture temperature is controlled to be 37 ℃, the shaking table speed is 220 and 270rpm, and the culture time is 12-14 h.

7. The method for producing N-acetylglucosamine by fermentation of a microorganism according to claim 6, wherein: the seed culture medium comprises 11.5g/L of peptone, 20g/L of yeast extract powder, 2g/L of galactose, 5g/L of sodium chloride, 2g/L of ammonium sulfate and 5.5g/L of glycerol.

8. The method for producing N-acetylglucosamine by fermentation of a microorganism according to claim 1 or 2, wherein: the mixed leaven is prepared by mixing the following components in a volume ratio of escherichia coli to saccharomyces cerevisiae (1-3): 1 to obtain the product.

Technical Field

The invention belongs to the technical field of biological fermentation, and particularly relates to a method for producing N-acetylglucosamine by microbial fermentation.

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.

N-acetylglucosamine is a white needle crystal, odorless, easily soluble in water, an important glucosamine derivative, is a basic composition unit of a plurality of important polysaccharides in biological cells, is a special monosaccharide with higher sweetness, has reducibility, is an important precursor for synthesizing bifidus factors, has important physiological functions in organisms, is clinically a medicament for treating rheumatic and rheumatoid arthritis, is also used as a food antioxidant and an infant food additive, is a sweetener for diabetics, is widely applied in the fields of food, medicine, chemical industry and the like, and has wide market prospect. At present, the domestic acquisition method of N-acetylglucosamine mainly comprises two methods, namely a chemical method and a biological method, wherein the biological method is used for synthesizing the N-acetylglucosamine by a microbial fermentation method, and has the advantages of short production time and high efficiency. However, glucosamine accumulated in the synthesis of N-acetylglucosamine by a fermentation method or N-acetylglucosamine can be transferred from the extracellular part into cells to be used as a carbon source, so that a large amount of N-acetylglucosamine cannot be accumulated in a fermentation liquid, and industrial 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 N-acetylglucosamine by microbial fermentation, 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 N-acetylglucosamine by microbial fermentation is characterized in that: the method comprises the following steps: activating the seeds of escherichia coli and saccharomyces cerevisiae to prepare a mixed starter; inoculating the mixed starter to a fermentation medium for constant-temperature shaking table culture, and controlling the rotation speed of the shaking table to be 230-250rpm, wherein the culture temperature is 36-38 ℃; the fermentation process is carried out by adopting a constant-speed feeding mode, the feeding speed of glucose is controlled to be 3-5 g/L.h, the pH value of the fermentation process is controlled to be not less than 6.9 by using ammonia water, when the OD600nm of the fermentation liquor is more than 30, the inducer IPTG and the methanol solution are added in batches, and the culture time is 35-72 h.

Preferably, the fermentation medium comprises: 35g/L glucose, 20g/L yeast extract powder, 10g/L alanine, 10g/L cysteine, 3g/L sodium lactate, 1.05g/L dipotassium hydrogen phosphate, 0.45g/L potassium dihydrogen phosphate, 0.1g/L sodium chloride, 0.3g/L zinc sulfate, 0.5g/L magnesium sulfate, 0.03g/L ferrous sulfate, 3.2g/L lactose and 5.5g/L glycerol; the addition of alanine and cysteine to the medium is advantageous to prevent the accumulation of glutamic acid in large amounts.

As a preferred scheme, the methanol solution is added, wherein the volume concentration of the methanol is 0.5 percent, and the methanol solution induces the saccharomyces cerevisiae to carry out protein expression.

Preferably, the escherichia coli and the saccharomyces cerevisiae are activated and cultured on a plate culture medium for 6-8 hours before being subjected to seed activation.

Preferably, the plate culture medium comprises 15g/L of peptone, 8g/L of yeast extract, 2g/L of galactose, 1g/L of sodium chloride, 1g/L of ammonium sulfate and 15g/L of agar.

Preferably, the seeds of the escherichia coli and the saccharomyces cerevisiae are activated by inoculating strains in a seed culture medium, controlling the culture temperature at 37 ℃, the shaking table speed at 220-270rpm, and the culture time at 12-14 h.

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

Preferably, the mixed starter is prepared by mixing the following components in a volume ratio of escherichia coli to saccharomyces cerevisiae (1-3): 1, preparing; inoculating the mixed starter into the fermentation medium according to the inoculation amount of 10-20%.

The invention has the beneficial effects that: according to the invention, the fermentation activity of escherichia coli is improved by mixing the leavening agent, and simultaneously, the saccharomyces cerevisiae is used as the leavening agent under the induction action of the methanol solution, so that the yield of the N-acetylglucosamine is increased, and finally, the high-efficiency fermentation production of the glucosamine is 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 raw materials used in the invention are common raw materials in the market, and the following examples are all composed of the following culture media:

the plate culture medium comprises: 15g/L of peptone, 8g/L of yeast extract powder, 2g/L of galactose, 1g/L of sodium chloride, 1g/L of ammonium sulfate and 15g/L of agar.

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

The fermentation medium comprises: 35g/L glucose, 20g/L yeast extract powder, 10g/L alanine, 10g/L cysteine, 3g/L sodium lactate, 1.05g/L dipotassium hydrogen phosphate, 0.45g/L potassium dihydrogen phosphate, 0.1g/L sodium chloride, 0.3g/L zinc sulfate, 0.5g/L magnesium sulfate, 0.03g/L ferrous sulfate, 3.2g/L lactose and 5.5g/L glycerol.