CN110724722A - Method for producing N-acetylglucosamine by microbial fermentation - Google Patents

Method for producing N-acetylglucosamine by microbial fermentation Download PDF

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Publication number
CN110724722A
CN110724722A CN201911210896.5A CN201911210896A CN110724722A CN 110724722 A CN110724722 A CN 110724722A CN 201911210896 A CN201911210896 A CN 201911210896A CN 110724722 A CN110724722 A CN 110724722A
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fermentation
acetylglucosamine
producing
culture
escherichia coli
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卢健行
韩宁
马善丽
赵鹏
吴祥舟
卢建功
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Shandong Runde Biotechnology Co Ltd
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Shandong Runde Biotechnology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/16—Preparation of compounds containing saccharide radicals produced by the action of an alpha-1, 6-glucosidase, e.g. amylose, debranched amylopectin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase

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  • Engineering & Computer Science (AREA)
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  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

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.

Description

Method for producing N-acetylglucosamine by microbial fermentation
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.
Example 1
Respectively performing activation culture on Escherichia coli and Saccharomyces cerevisiae on a plate culture medium for 7h, controlling the culture temperature to be 37 ℃, and controlling the shaking table speed to be 220 rpm; mixing the cultured escherichia coli and saccharomyces cerevisiae according to the volume ratio of 2:1, and inoculating the mixture into a seed culture medium for activation culture for 12 hours; culturing 3L of fermentation medium in a 5L fermentation tank, inoculating the activated mixed starter to the fermentation medium according to 12% of inoculation amount for fermentation culture, controlling the temperature at 37 ℃, controlling the feed supplement speed of glucose to be 3 g/L.h in the fermentation process, controlling the pH value of the fermentation liquid to be not less than 6.9 by using 20-30% ammonia water, adding an inducer IPTG and 0.5% methanol solution into the fermentation liquid when the OD600nm of the fermentation liquid is more than 30%, intermittently adding the inducer IPTG and the 0.5% methanol solution in four batches, controlling the final concentration of the IPTG in the fermentation liquid to be 0.1mmol/L, adding the 0.5% methanol solution in total 20ml, and performing fermentation culture for 48h, wherein the fermentation yield of the N-acetylglucosamine is 174 g/L.
Example 2
Respectively performing activation culture on Escherichia coli and Saccharomyces cerevisiae on a plate culture medium for 7h, controlling the culture temperature to be 37 ℃, and controlling the shaking table speed to be 220 rpm; mixing the cultured escherichia coli and saccharomyces cerevisiae according to the volume ratio of 2:1, and inoculating the mixture into a seed culture medium for activation culture for 12 hours; culturing 70L of fermentation medium in a 100L fermentation tank, inoculating the activated mixed starter to the fermentation medium according to 12% of inoculation amount for fermentation culture, controlling the temperature at 37 ℃, controlling the feed supplement speed of glucose to be 5 g/L.h in the fermentation process, controlling the pH value of the fermentation liquid to be not less than 6.9 by using 20-30% ammonia water, adding an inducer IPTG and 0.5% methanol solution into the fermentation liquid when the OD600nm of the fermentation liquid is more than 30%, intermittently adding the inducer IPTG and the 0.5% methanol solution in four batches, controlling the final concentration of the IPTG in the fermentation liquid to be 0.1mmol/L, adding the 0.5% methanol solution in 500ml, and performing fermentation culture for 48h, wherein the fermentation yield of the N-acetylglucosamine is 169 g/L.
Example 3
Activating and culturing Escherichia coli on a plate culture medium for 7h, controlling the culture temperature at 37 deg.C, and controlling the shaking table speed at 220 rpm; inoculating the cultured escherichia coli into a seed culture medium for activation culture for 12 hours; culturing 70L of fermentation medium in a 100L fermentation tank, inoculating the activated starter into the fermentation medium according to 12% of inoculation amount for fermentation culture, controlling the temperature to be 37 ℃, controlling the feed supplement speed of glucose to be 5 g/L.h in the fermentation process, controlling the pH value of the fermentation liquid to be not less than 6.9 by using 20-30% ammonia water, adding an inducer IPTG and 0.5% methanol solution into the fermentation liquid when the OD600nm of the fermentation liquid is more than 30%, adding the inducer IPTG and the 0.5% methanol solution intermittently in four batches, controlling the final concentration of the IPTG in the fermentation liquid to be 0.1mmol/L, adding the 0.5% methanol solution to be 500ml, and carrying out fermentation culture for 48h, wherein the fermentation yield of the N-acetylglucosamine is 104 g/L.
Example 4
Respectively performing activation culture on the saccharomyces cerevisiae on a plate culture medium for 7 hours, controlling the culture temperature to be 37 ℃, and controlling the shaking table speed to be 220 rpm; inoculating the cultured saccharomyces cerevisiae in a seed culture medium for activation culture for 12 h; culturing 70L of fermentation medium in a 100L fermentation tank, inoculating the activated starter into the fermentation medium according to 12% of inoculation amount for fermentation culture, controlling the temperature to be 37 ℃, controlling the feed supplement speed of glucose to be 5 g/L.h in the fermentation process, controlling the pH value of the fermentation liquid to be not less than 6.9 by using 20-30% ammonia water, adding an inducer IPTG and 0.5% methanol solution into the fermentation liquid when the OD600nm of the fermentation liquid is more than 30%, adding the inducer IPTG and the 0.5% methanol solution intermittently in four batches, controlling the final concentration of the IPTG in the fermentation liquid to be 0.1mmol/L, adding the 0.5% methanol solution to be 500ml, and carrying out fermentation culture for 48h, wherein the fermentation yield of the N-acetylglucosamine is 115 g/L.
Example 5
The difference between the fermentation preparation process and the example 1 is that no alanine is added into the fermentation medium, and the fermentation yield of the obtained N-acetylglucosamine is 126 g/L.
Example 6
The difference between the fermentation preparation process and the example 1 is that cysteine is not added into the fermentation culture medium, and the fermentation yield of the obtained N-acetylglucosamine is 131 g/L.

Claims (8)

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.
CN201911210896.5A 2019-12-02 2019-12-02 Method for producing N-acetylglucosamine by microbial fermentation Withdrawn CN110724722A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112608959A (en) * 2020-12-31 2021-04-06 河南巨龙生物工程股份有限公司 Method for improving acetylglucosamine fermentation unit
CN117646049A (en) * 2024-01-26 2024-03-05 山东润德生物科技有限公司 Method for preparing N-acetylglucosamine by microbial fermentation

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112608959A (en) * 2020-12-31 2021-04-06 河南巨龙生物工程股份有限公司 Method for improving acetylglucosamine fermentation unit
CN112608959B (en) * 2020-12-31 2024-04-23 河南巨龙生物工程股份有限公司 Method for improving fermentation unit of acetylglucosamine
CN117646049A (en) * 2024-01-26 2024-03-05 山东润德生物科技有限公司 Method for preparing N-acetylglucosamine by microbial fermentation
CN117646049B (en) * 2024-01-26 2024-04-26 山东润德生物科技有限公司 Method for preparing N-acetylglucosamine by microbial fermentation

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Application publication date: 20200124