CN114592020A - Method for producing N-acetylglucosamine by fermentation - Google Patents
Method for producing N-acetylglucosamine by fermentation Download PDFInfo
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- CN114592020A CN114592020A CN202011438798.XA CN202011438798A CN114592020A CN 114592020 A CN114592020 A CN 114592020A CN 202011438798 A CN202011438798 A CN 202011438798A CN 114592020 A CN114592020 A CN 114592020A
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Abstract
The invention discloses a method for producing N-acetylglucosamine by fermentation. The method comprises the steps of feeding glucose in the process of culturing escherichia coli, wherein the initial concentration of the glucose in the feeding process is 0.4-0.8%, the feeding speed is 9.8-10.5 g/L/h, and the percentage is the mass-to-volume ratio; the culture medium used during the culturing process comprises: KH (Perkin Elmer)2PO46.67g/L, 3.25g/L, CaCl g citric acid2·H2O0.05 g/L, etc.; the fermentation time of the method is more than 40 hours. The invention maintains higher thallus concentration, enables the thallus to achieve the capability of producing N-acetylglucosamine to the maximum extent, greatly improves the yield of the N-acetylglucosamine, shortens the fermentation period, and has larger influence on thallus growth and the generation of byproduct acetic acid.
Description
Technical Field
The invention relates to the field of biological fermentation, in particular to a method for producing N-acetylglucosamine by fermentation, and particularly relates to a method for improving N-acetylglucosamine in escherichia coli fermentation.
Background
Glucosamine is widely used in the production of nutritional health products, pharmaceuticals and foods. The united states is the world-wide country with the most use of glucosamine, and investigations have shown that 90% of the current glucosamine feedstocks on the us market are from china. With the increasing aging of the population and the increasing health care awareness of people, the market demand of products is still continuously increasing. At the end of the last 90 s, China has become the world's largest country for producing and exporting glucosamine as a raw material. At present, the main method for producing glucosamine in China is still a chitin extraction method, but with the enhancement of environmental awareness of people, the method is also rejected, more and more manufacturers aim at a microbial fermentation method, the construction of genetically engineered bacteria fermentation is a more advantageous method in the microbial method, the method produces N-acetylglucosamine by the fermentation of engineered bacteria, and then the glucosamine is hydrolyzed by hydrochloric acid to obtain glucosamine (Suare DC, Kilikian BV.acetic acid fermentation in aerobic growth of recombinant Escherichia coli. Process Biochemistry,2000,35: 1051. however, the problems of the genetically engineered bacteria fermentation which need to be overcome in the exploration stage are more, for example, the fermentation period is longer, the fermentation broth has low quality and brings difficulty to the subsequent separation and extraction, the phage pollution in the fermentation process of Escherichia coli (Qinqie, Qiu Lin, Liu, New Lily, the engineering bacteria for producing N-acetylglucosamine are constructed, Fermentation and application development [ J ] Chinese brewing, 2018(6):6-9.), these problems make the level of producing N-acetylglucosamine by domestic fermentation unable to be improved all the time, so this patent aims at improving the product quality, reducing the fermentation cost and reducing the pollution to the environment.
The existing N-acetylglucosamine fermentation process comprises the following steps: seeds in shake flasks were inoculated into seed medium [ ingredients per liter: k2HPO4 16g，KH2PO4 14g，MgSO4·7H2O 25g，CaCl2·H2O 0.015 g，(NH4)2SO4 7.5g]And sterilized at 121 ℃ for 20 minutes, and then 20g/L of glucose, 1g/L of a trace element solution and 200. mu.g/mL of kanamycin are added. The inoculation amount is 70mL/15L, the culture temperature is 36 ℃, the pH value of ammonia water is automatically adjusted to 6.9, the dissolved oxygen is 20-30%, the mixture is cultured in a 25L automatic control fermentation tank for 14-20h to logarithmic phase, and the mixture is inoculated into a fermentation culture medium according to the inoculation amount of 5% (the components are contained in each liter: KH (Perkin Elmer)2PO46.7g, 3g of citric acid, 5g of KCl and CaCl20.05g, 10mL of microelement mother liquor]After fermentation is started, dissolved oxygen is controlled to be more than 20% by adjusting ventilation volume and tank pressure, the temperature is 37 ℃, and the pH is controlled to be 6.8-7.2 by automatically feeding ammonia water in a flowing manner. Glucose fed-batch strategy: initial glucose is 0.4g/100mL, the fed-batch glucose concentration is 70%, the maximum sugar supplement amount is 13g/L every time, the culture is carried out for 72 hours, and the yield of the N-acetylglucosamine is 110 g/L.
Disclosure of Invention
Aiming at the defects in the prior art, the fermentation process for fermenting the N-acetylglucosamine by using the escherichia coli is provided. The yield of the N-acetylglucosamine is improved, the specific growth rate of thalli is controlled, the generation of acetic acid byproducts is reduced, the production capacity is improved, and the production cost is reduced by changing the initial glucose control amount and the maximum glucose supplement amount of glucose.
The invention is realized by the following technical scheme:
the invention provides a method for producing N-acetylglucosamine by fermentation, which comprises the steps of feeding glucose in the process of culturing escherichia coli, wherein the initial concentration of the glucose in the feeding process is 0.4-0.8%, the feeding speed is 9.8-10.5 g/L/h, and the percentage is the mass-volume ratio;
the culture medium used during the culturing process comprises:
KH2PO46.67g/L, 3.25g/L, CaCl g citric acid2·H2O 0.05g/L、MgSO4·7H2O 2.5 g/L、FeSO4·7H2O 5mg/L、ZnSO4·7H2O 3.8mg/L、MnSO4·H2O 0.33mg/L、 CuSO4·5H2O0.1 mg/L and CoCl6H2O 0.1mg/L；
The fermentation time of the method is more than 40 hours.
Preferably, the concentration of the fed glucose is 70%, and the percentage is mass volume percentage.
Preferably, the initial concentration is 0.6% to 0.8%, more preferably, the initial concentration is 0.65%.
Preferably, the feeding speed is 10 g/L/h.
In a specific embodiment of the invention, the fed-in glucose has an initial concentration of 0.65% and the fed-in rate is 10 g/L/h.
In the fermentation production process, the dissolved oxygen amount may be conventional in the art, and is preferably 20% or more.
In the fermentation production process, the temperature and the pH can be conventional in the field, preferably, the temperature is 37 ℃ and the pH is 6.8-7.2.
In a preferable embodiment, in the fermentation production process, the dissolved oxygen is more than 20%, the temperature is 37 ℃, and the pH is 6.8-7.2.
The invention achieves the technical effects that: on the basis of an optimized culture medium, the initial sugar concentration with a certain concentration is adopted to keep the thalli in a good growth state, meanwhile, the low-sugar fed-batch process is adopted in the later stage of fermentation to reduce the inhibition effect of glucose, maintain higher thalli concentration, enable the thalli to achieve the capability of producing N-acetylglucosamine to the maximum extent, greatly improve the yield of the N-acetylglucosamine, shorten the fermentation period, and have great influence on the growth of the thalli and the generation of byproduct acetic acid. The invention improves the fermentation process of the N-acetylglucosamine from a new angle, and has innovativeness, practicability and industrial application prospect.
Drawings
FIG. 1 is a schematic representation of the effect of different peak carbohydrate supplements on the bacterial concentration.
FIG. 2 is a graph showing the effect of different amounts of top-sugars on acetic acid content.
FIG. 3 is a graph showing the effect of different amounts of most sugar on the production of N-acetylglucosamine.
FIG. 4 is a graphical representation of the effect of different initial glucose concentrations on the concentration of bacteria.
FIG. 5 is a graphical representation of the effect of different initial glucose concentrations on acetic acid content.
FIG. 6 is a graph showing the effect of different initial glucose concentrations on N-acetylglucosamine production.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.
The culture method of the escherichia coli genetically engineered bacteria can be a conventional culture method in the field, and in the following examples, the strain used in the research is a recombinant bacterium E.coli-glmS-gna1 (Chengxin, Liulong, Lijiang Hua, Liujie, Guoguang, Chenjiangjian. Red homologous recombination knockdown of nagE and manX has an effect on the fermentation production of glucosamine by escherichia coli. The report of bioengineering. 2012,28(3):305 and 319.). Inoculating the strain into a slant culture medium, and culturing at 35 deg.C for 24 h; then inoculating the seeds into a primary seed culture medium, and culturing for 7 hours after the primary seeds are cultured for 6 hours at the rotation speed of a shaking table of 240rpm at the temperature of 36 ℃ and the pH value of 6.8-7.2.
Wherein the slant culture medium contains tryptone 10g/L, yeast powder 5g/L, NaCl 10g/L, agar powder 14g/L, kanamycin is added to make the final concentration 2mg/L, the pH value is 6.8-7.2, and sterilization is carried out for 15min at 121 ℃ before use.
Wherein the primary seed culture medium contains tryptone 10g/L, yeast powder 5g/L and NaCl 10g/L, kanamycin is added to the primary seed culture medium, the final concentration is 2mg/L, the pH value is 6.8-7.2, and sterilization is carried out for 20min at 121 ℃ before use.
Wherein the secondary seed culture medium contains K2HPO4 16g/L，KH2PO4 14g/L，MgSO4·7H2O 25g/L，CaCl·2H2O 0.015g/L，(NH4)2SO47.5g/L was sterilized at 121 ℃ for 20min before use, and after sterilization, 2% glucose and 0.1% trace element solution were added, and kanamycin was added to a final concentration of 200. mu.g/mL.
The microelement solution contains FeSO4·7H2O 0.5g/L，ZnSO4·7H2O 0.38g/L，MnSO4·H2O 0.033g/L，CuSO4·5H2O 0.01g/L，CoCl·6H2O 0.01g/L。
Example 1
Placing the strain subjected to slant culture and seed culture medium culture in a mechanical stirring fermentation tank containing a basic fermentation medium, stirring by using a stirring paddle containing blades in the fermentation process, and supplementing materials until the fermentation is finished. Wherein the fermentation tank is BMR-B series mechanical stirring fermentation tank (biological engineering equipment Co., Ltd., Shanghai) with a volume of 25L. Basic fermentation medium: KH (Perkin Elmer)2PO46.67g/L, 3.25g/L, CaCl g citric acid2·H2O 0.05g/L、MgSO4·7H2O 2.5g/L、FeSO4·7H2O 5mg/L、ZnSO4·7H2O 3.8mg/L、MnSO4·H2O 0.33mg/L、 CuSO4·5H2O0.1 mg/L and CoCl6H2O0.1 mg/L. Sterilizing at 121 deg.C for 20min before use. The method comprises the following steps of designing a glucose initial concentration of 0.4% (the glucose accounts for the mass-volume ratio of fermentation liquor) under the premise of consistent fermentation conditions by using three 25L fermentation tanks and 15L basic culture medium, feeding 70.0% of glucose solution at the beginning, and feeding the glucose with the highest glucose feeding amount per hour of 7g/L (the mass ratio of the fed-in glucose to the volume of the fermentation liquor).
Example 2
The specific operation details of this example are the same as those of example 1, except that the initial glucose concentration is set to 0.4% and 70.0% glucose solution is fed in under the condition of consistent fermentation conditions, and the maximum glucose feed per hour is 10 g/L.
Example 3
The specific operation details of this example are the same as those of example 1, except that the initial glucose concentration is set to 0.4% and 70.0% glucose solution is fed in under the condition of consistent fermentation conditions, and the maximum glucose feed per hour is 13 g/L.
Example 4
The specific operation details of this example are the same as those of example 1, except that the initial glucose concentration is set to 0.4% and 70.0% glucose solution is fed in under the condition of consistent fermentation conditions, and the maximum glucose feed per hour is 16 g/L.
Example 5
The specific operation details of this example are the same as those of example 1, except that the initial glucose concentration is set to 0.65% and 70.0% glucose solution is fed in under the condition of consistent fermentation conditions, and the maximum glucose feed per hour is 10 g/L.
Example 6
The specific operation details of this example are the same as those of example 1, except that the initial glucose concentration is set to 0.85% and 70.0% glucose solution is fed in under the condition of consistent fermentation conditions, and the maximum glucose feed per hour is 10 g/L.
Example 7
The specific operation details of this example are the same as those of example 1, except that the initial glucose concentration is 1.0% and 70.0% glucose solution is fed in under the condition of consistent fermentation conditions, and the maximum glucose feed per hour is 10 g/L.
Effects of the embodiment
The cell concentration, GlcNAc unit, and acetic acid content of examples 1 to 7 were measured to determine the optimum amount of added grape. The growth rate of the thallus is measured by the OD value of the thallus concentration; the GlcNAc units and acetic acid were determined by HPLC.
The specific test method is as follows:
fermentation unit determination: high performance liquid chromatography determination, and the chromatographic conditions are as follows: the instrument comprises the following steps: agilent 1200; a chromatographic column: c18 reverse phase chromatography column (250 mm. times.4.6 mm, 5 μm); flow rate: 0.6 mL/min; detection wavelength: 195 nm; sample introduction amount: 20 mu L of the solution; column temperature: 30 ℃; configuration of mobile phase: weighing 0.5g of sodium heptanesulfonate, adding 0.5mL of phosphoric acid, 4mL of 56g/L KOH and distilled water to dilute to 1000mL, adding 50mL of acetonitrile, filtering, and performing microwave ultrasonic treatment for 10min for later use.
Determination of acetic acid: HPLC method, chromatographic conditions: column ZORBAX SB-C18 (4.6 mm. times.250 mm, 5 μm); mobile phase: 0.5% KH2PO4Aqueous solution (with 85% H)3PO4Adjusted to ph2.7) -hexanenitrile (93: 13) (ii) a The flow rate is 0.8 ml/min; the column temperature is 45 ℃; the detection wavelength is 210 nm; the amount of the sample was 2.5. mu.l. The HPLC is a 1200 model HPLC (Agilent).
Measurement of OD: the optical density of the cell suspension (i.e., OD600 value) was measured using a spectrophotometer, and the cell concentration of E.coli was represented. The fermentation broth was diluted appropriately with distilled water to ensure an OD600 between 0.2 and 0.8, and read at a wavelength of 660nm using distilled water as a blank.
The test results of examples 1 to 4 are shown in FIGS. 1 to 3 and tables 1 to 3, and the test results of examples 5 to 7 are shown in FIGS. 4 to 6 and tables 4 to 6.
Examples 1-4 describe the effect of different glucose peak-to-peak sugar levels on fermentation. As can be seen from the results of FIGS. 1 to 3 and tables 1 to 3, the sugar supplement amount is less than 10g/L/h, the early growth rate of the cells is slow, the acetic acid content is low, but the yield of GlcNAc is also slow; when the sugar supplement amount is too high, the metabolic pathway of the thalli is changed, so that a large amount of acetic acid is accumulated, and the growth of the thalli and the yield of GlcNAc are influenced; when the highest sugar supplement amount is 10g/L/h, the thallus growth state is good, the concentration of the thallus is high, the content of a byproduct acetic acid is low, the yield of GlcNAc is improved to 131g/L, the fermentation period is prolonged to 48h, and the yield in the later fermentation period is stably increased.
TABLE 1
TABLE 2
TABLE 3
Examples 5-7 describe the effect of different initial glucose concentrations on fermentation. As can be seen from FIG. 4 and Table 4, different initial sugar concentrations had a large effect on the growth of the cells: the higher the initial sugar concentration is, the smaller the bacterial amount is; as is clear from FIG. 6 and Table 6, the final GlcNAc concentration in the fermentation broth was about 130g/L when the initial sugar concentrations were 0.4% and 0.8%; as can be seen from FIG. 5, the initial glucose concentration has a significant effect on acetic acid accumulation, and under the condition of 1.0% initial sugar concentration, the acetic acid content in the fermentation broth reaches 3g/L, while under the condition of 0.65% initial sugar concentration, the acetic acid content is only 1.6 g/L; when the initial concentration of glucose is 0.65%, the concentration of GlcNAc at the end of fermentation for 48h reaches 140g/L, which is 7% higher than the yield at other concentrations, so the initial concentration of glucose is selected to be 0.65%.
TABLE 4
TABLE 5
TABLE 6
Claims (7)
1. A method for producing N-acetylglucosamine by fermentation comprises feeding glucose in the process of culturing Escherichia coli, and is characterized in that the initial concentration of glucose in the feeding process is 0.4% -0.8%, the feeding speed is 9.8 g/L/h-10.5 g/L/h, and the percentages are mass-volume ratios;
the culture medium used during the culturing process comprises:
KH2PO46.67g/L, 3.25g/L, CaCl g citric acid2·H2O 0.05g/L、MgSO4·7H2O 2.5g/L、FeSO4·7H2O 5mg/L、ZnSO4·7H2O 3.8mg/L、MnSO4·H2O 0.33mg/L、CuSO4·5H2O0.1 mg/L and CoCl6H2O 0.1mg/L；
The fermentation time of the method is more than 40 hours.
2. The method of claim 1, wherein the concentration of glucose fed is 70% and the percentages are mass volume percentages.
3. The method of claim 1, wherein the initial concentration is between 0.6% and 0.8%.
4. The method of claim 3, wherein the initial concentration is 0.65%.
5. The method of claim 1, wherein the feed rate is 10 g/L/h.
6. The method according to any one of claims 1 to 5, wherein the dissolved oxygen amount is 20% or more in the fermentation production process.
7. The method of claim 6, wherein the temperature is 37 ℃ and the pH is 6.8 to 7.2 during the fermentation production.
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Citations (7)
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| CN104293724A (en) * | 2014-09-22 | 2015-01-21 | 上海工业生物技术研发中心 | Genetically engineered bacteria for efficiently producing N-acetylglucosamine |
| CN104988196A (en) * | 2015-07-30 | 2015-10-21 | 安徽丰原发酵技术工程研究有限公司 | Fermentation production method for N-acetylglucosamine |
| CN105463041A (en) * | 2015-12-17 | 2016-04-06 | 安徽丰原发酵技术工程研究有限公司 | Preparation method of glucosamine |
| WO2017174039A1 (en) * | 2016-04-05 | 2017-10-12 | 孙镧 | Method for producing n-acetyl-d-glucosamine and/or d-glucosamine salt by means of microbial fermentation |
| CN108103126A (en) * | 2016-11-25 | 2018-06-01 | 北大方正集团有限公司 | A kind of composition for improving Glucosamine fermentation unit yield and its application |
| US20190376102A1 (en) * | 2016-04-05 | 2019-12-12 | Lan Sun | Microbial fermentation method for production of n-acetyl-d-glucosamine and/or d-glucosamine salt |
| CN110885870A (en) * | 2019-12-09 | 2020-03-17 | 山东润德生物科技有限公司 | Fermentation production method of N-acetylglucosamine |
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- 2020-12-07 CN CN202011438798.XA patent/CN114592020B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104293724A (en) * | 2014-09-22 | 2015-01-21 | 上海工业生物技术研发中心 | Genetically engineered bacteria for efficiently producing N-acetylglucosamine |
| CN104988196A (en) * | 2015-07-30 | 2015-10-21 | 安徽丰原发酵技术工程研究有限公司 | Fermentation production method for N-acetylglucosamine |
| CN105463041A (en) * | 2015-12-17 | 2016-04-06 | 安徽丰原发酵技术工程研究有限公司 | Preparation method of glucosamine |
| WO2017174039A1 (en) * | 2016-04-05 | 2017-10-12 | 孙镧 | Method for producing n-acetyl-d-glucosamine and/or d-glucosamine salt by means of microbial fermentation |
| US20190376102A1 (en) * | 2016-04-05 | 2019-12-12 | Lan Sun | Microbial fermentation method for production of n-acetyl-d-glucosamine and/or d-glucosamine salt |
| CN108103126A (en) * | 2016-11-25 | 2018-06-01 | 北大方正集团有限公司 | A kind of composition for improving Glucosamine fermentation unit yield and its application |
| CN110885870A (en) * | 2019-12-09 | 2020-03-17 | 山东润德生物科技有限公司 | Fermentation production method of N-acetylglucosamine |
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