KR101745079B1 - Method for producing 2,3-butanediol comprising of adding acetate - Google Patents
Method for producing 2,3-butanediol comprising of adding acetate Download PDFInfo
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- KR101745079B1 KR101745079B1 KR1020150053256A KR20150053256A KR101745079B1 KR 101745079 B1 KR101745079 B1 KR 101745079B1 KR 1020150053256 A KR1020150053256 A KR 1020150053256A KR 20150053256 A KR20150053256 A KR 20150053256A KR 101745079 B1 KR101745079 B1 KR 101745079B1
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Abstract
The present invention relates to a process for the production of 2,3-butanediol comprising the step of adding metal acetate or ammonium acetate to a medium inoculated with a strain producing 2,3-butanediol. The production process of the present invention is characterized in that the addition of metal acetate or ammonium acetate to the strain improves the yield and productivity of 2,3-butanediol without affecting the growth conditions of the strain, , 3-butanediol can be produced.
Description
The present invention relates to a process for the production of 2,3-butanediol comprising the step of adding metal acetate or ammonium acetate to a medium inoculated with a strain producing 2,3-butanediol.
Bio refinery is a concept to replace the role played by petroleum in the existing industrial system with biomass, a renewable resource. So far, through the refinery of oil (Oil Refinery), fuel such as gasoline, light oil, It is an attempt to produce a variety of chemical products such as bio-ethanol, biodiesel and other fuels and bio-plastics through bio-refineries based on biomass. Recently, there have been many studies on the production of butanol (Butanol) which can be used not only as a chemical substance but also as a fuel material, butyric acid (butyric aicd) and butanediol which can be converted into various chemical substances and a biorefinery of butanediol (Korean Patent Publication No. 2012-0082141). In the case of 2,3-butanediol, it can be converted into diacetyl through a dehydration reaction, which is in the spotlight in the food industry since it has a buttery taste, 2,3-butanediol is a synthetic rubber Is known to be an industrially important chemical that can be converted to methyl ethyl ketone (MEK), which can be used as an additive for 1,3-butadiene and a fuel material, which are important raw materials in the manufacture of carbon black.
In the early 1900s, 2,3-butanediol production using microorganisms had already been developed on a pilot scale scale using microorganisms of the Klebsilla and Bacillus series. However, the production of 1,3-butadiene As the production process was developed, the study of 2,3-butanediol production using microorganisms was discontinued. However, research on the production of 2,3-butanediol using microorganisms has attracted attention again with the increase in interest in bio-refinery technology development, the development of microorganism improvement technology, fermentation technology, separation and purification technology through gene manipulation It is a situation.
It is an object of the present invention to provide a process for producing 2,3-butanediol in which the yield and productivity of 2,3-butanediol are improved.
The present invention provides a method for producing 2,3-butanediol comprising the step of adding metal acetate or ammonium acetate to a medium in which a strain producing 2,3-butanediol has been inoculated.
One embodiment is a metal acetate, potassium acetate may be at least one selected from the group consisting of - - (Na + CH 3 COO ) (
Another embodiment is 2,3-butane is Enterobacter aero jeneseu strain producing diol (E. aerogenes), keulrep when Ella pneumoniae (Klebsiella pneumonia) and keulrep when Ella oxy cytokine (Klebsiella oxytoca ) may be used.
In another embodiment, the medium may contain at least one carbon source selected from the group consisting of glucose, xylose, mannose, galactose and fructose.
Another embodiment may be that the metal acetate or ammonium acetate is added at 3 to 9 g / L relative to the medium.
Another embodiment may be that the metal acetate or ammonium acetate is added at 3 to 15 g / L to the medium.
Another embodiment may be that the carbon source is included in the medium in an amount of 60 to 200 g / L.
In another embodiment, the addition of metal acetate or ammonium acetate may increase the expression level of at least one 2,3-butanediol producing gene selected from the group consisting of budA , budB , budC and bdh .
Another embodiment may be to re-add the metal acetate or ammonium acetate after 12 hours and 24 hours after addition of the metal acetate or ammonium acetate to the medium in which the strain producing the 2,3-butanediol has been inoculated .
In another embodiment, the pH of the culture medium may be 6 to 7.
The production method of the present invention is to improve the yield and productivity of 2,3-butanediol without affecting the growth conditions of the strain including the pH condition of the medium by adding metal acetate or ammonium acetate to the strain And thus the continuous production of 2,3-butanediol is possible.
Fig. 1 is a graph showing the yield of 2,3-butanediol after 12 hours of culture.
Fig. 2 is a graph showing the yield of 2,3-butanediol after 24 hours of culture.
Fig. 3 shows the yields of 2,3-butanediol and the like as a result of culturing with different carbon sources.
Fig. 4 shows the expression amounts of budA , budB , budC and bdh , 2,3-butanediol producing genes, as electrophoresis results.
FIG. 5 shows changes in 2,3-butanediol production in media supplemented with potassium acetate by glucose concentration.
FIG. 6 is a graph showing the results of 2,3-butanediol production by addition of potash acetate at 12 hours in the medium supplemented with potassium acetate by glucose concentration.
The present invention relates to a process for the production of 2,3-butanediol comprising the step of adding metal acetate or ammonium acetate to a medium inoculated with a strain producing 2,3-butanediol. The production process of the present invention is characterized in that the addition of metal acetate or ammonium acetate to the strain improves the yield and productivity of 2,3-butanediol without affecting the growth conditions of the strain, , 3-butanediol can be produced.
Hereinafter, the present invention will be described in detail.
The present invention provides a process for the production of 2,3-butanediol, comprising the step of adding metal acetate or ammonium acetate to a medium inoculated with a strain producing 2,3-butanediol.
The metal acetate generally does not contain hydrogen ion (H + ) unlike acetic acid (CH 3 COO - H + ) to which acetate is added, so that it does not affect growth conditions, for example, pH conditions of the strain. The metal acetate is preferably an alkali metal acetate, and more preferably at least one selected from the group consisting of potassium acetate (CH 3 COO - K + ) and sodium acetate (CH 3 COO - Na + ).
When the metal acetate or ammonium acetate is added, the expression amount of the 2,3-butanediol-producing gene of the strain is increased to increase the 2,3-butanediol production amount. Specifically, 2,3-butanediol production Butanediol production amount is increased as the expression level of at least one 2,3-butanediol-producing gene selected from the group consisting of budA , budB , budC and bdh as a gene is increased.
The metal acetate or ammonium acetate may be added at 3 to 9 g / L to the medium. When metal acetate or ammonium acetate is added within the above range, a large amount of 2,3-butanediol is produced within 12 hours, and the process can be effectively applied to a process for producing 2,3-butanediol in a short time.
In addition, the metal acetate or ammonium acetate may be added at 3 to 15 g / L to the medium. When metal acetate or ammonium acetate is added within this range, it is characterized by an improved total yield of 2,3-butanediol.
In addition, the metal acetate or ammonium acetate may be added to the medium in which the strain producing the 2,3-butanediol is added, and then the metal acetate or ammonium acetate may be added again after 12 hours and 24 hours. After 12 hours and 24 hours, the metal acetate or ammonium acetate is re-added to continuously feed the acetate to continuously maximize the yield of 2,3-butanediol.
The 2,3-butanediol producing strain is a strain selected from the group consisting of Enterobacter aerogenes , Klebsiella pneumonia and Klebsiella oxytoca . And more preferably Escherichia coli. Especially, E. aerogenes is most preferable. If the addition of Enterobacter aero jeneseu (E. aerogenes) a metal acetate to the inoculated strains can maximize the production of all-2,3-dimethyl butane.
The medium may further contain a carbon source in order to maximize the production of 2,3-butanediol. The carbon source may be one or more selected from the group consisting of glucose, xylose, mannose, galactose and fructose, though it is not particularly limited as long as the production of 2,3-butanediol is maximized.
The carbon source is preferably contained in an amount of 60 to 200 g / L relative to the medium. When the concentration is in the above-mentioned range, if the concentration of acetate is kept within the range of 10 g / L or less, the production of 2,3-butanediol can be continued without affecting the growth of the strain, and the production can also be maximized.
The components of the medium other than the metal acetate or ammonium acetate and the carbon source may further comprise components known to promote the production of 2,3-butanediol and the growth of the strain at commercially viable levels. For example, the nitrogen source may include nitrate, urea, ammonium salt, and amino acid.
The production method can be carried out for a suitable time after the addition of the metal acetate or ammonium acetate, and 2,3-butanediol can be recovered after the cultivation.
The cultivation can be carried out at a specific life-sustaining temperature of the strain, and the pH condition can also be carried out under the desired conditions of the strain. Preferably, the pH of the culture medium is 6 to 7.
The 2,3-butanediol can be recovered by conventional means known to those skilled in the art, such as centrifugation, coagulation or filtration.
Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are provided to illustrate the present invention, and the present invention is not limited by the following examples, and various modifications and changes may be made.
Example 1. < RTI ID = 0.0 > 2,3- Butanediol production
The Enterobacter aerogenes strain was cultivated in a nutrient broth (2 g / L yeast extract, 1 g / L meat extract, 5 g / L peptone, 5 g / L NaCl) shaking incubator for 12 hours. The seed culture was inoculated into a production medium for the production of 2,3-butanediol, and 2,3-butanediol was produced in a shaking incubator at 37 ° C and 180 rpm. The production medium consisted of 3 g / L potassium hydrogen phosphate (KH 2 PO 4 ), 6.8 g / L sodium diphosphate (Na 2 HPO 4 ), 5 g / L yeast extract, 5.35 g / L ammonium sulfate ((NH 4) 2 SO 4 ), 10 g / L casamino acids (casamino acid), 0.75 g / L potassium chloride (KCl), 0.28 g / L sodium sulfate (Na 2 SO 4), 0.42 g / L Citric acid, 0.26 g / L Carbon source such as glucose, xylose, mannose, galactose and fructose was added to magnesium sulfate (MgSO 4 ), and to increase the production of 2,3-butanediol To the production medium was added sodium acetate at a concentration of 9-11 g / L. The initial pH of the production medium is in the range of 6.5 to 6.8 and the optimal pH range for 2,3-butanediol production is in the range of 6-7.
In addition, 2,3-butanediol was also produced in the production medium composition in which potash acetate was added at the same concentration instead of sodium acetate.
The production of 2,3-butanediol at various concentrations was compared using sodium acetate and potassium acetate as the acetate source in the production medium. Specifically, sodium acetate and potassium acetate were added to the production medium containing glucose (60 g / L) in the range of 0 to 19 g / L, respectively, and cultured in E. aerogenes strain at 37 ° C., Culturing was carried out for 12 hours or 24 hours under the same conditions of 180 rpm. The yield of consumed glucose, 2,3-butanediol and other by-products was measured using high performance liquid chromatography (HPLC), and the Aminex HPX-87H column (300 x 7.8 mm, Bio-Rad , And a refractive index detector (RID-10A, Shimadzu, Japan) at a column temperature of 50 ° C and 5 mN H 2 SO 4 mobile phase at a flow rate of 0.6 ml / min. 1 and Fig.
FIG. 1 is a graph showing the yield of 2,3-butanediol after 12 hours of culture, and FIG. 2 is a graph showing the yield of 2,3-butanediol after 24 hours of culture.
As shown in FIGS. 1 and 2, when sodium acetate and potassium acetate were added, the production of 2,3-butanediol was increased by about 2.4 and 2.5 times, respectively, as compared with the control group . Also, it was confirmed from FIG. 1 that the production amount of the initial 2,3-butanediol was remarkably high when sodium acetate and potassium acetate were added up to 11 g / L.
In place of glucose, xylose, mannose, galactose and fructose were used as the respective carbon sources (60 g / L) for 24 hours under the same conditions as above. Potassium acetate was used as the acetate, Respectively.
Fig. 3 shows the yields of 2,3-butanediol and the like as a result of culturing with different carbon sources.
As shown in FIG. 3, it was confirmed that the production amount of 2,3-butanediol was improved not only by glucose but also by using other carbon sources. Potassium acetate addition resulted in 2,3- Butanediol production was improved.
Example 2. 2,3- Butanediol Identify expression levels of genes related to production
To confirm the expression level of 2,3-butanediol-producing genes (budA, budB, bud C and bdh) in the acetate-added medium, 10 g / L of potash acetate was added to the production medium for 12 hours The cells were cultured to recover RNA, and a comparative experiment was carried out using cells cultured in a medium without acetate as a control. The cells were collected from the culture medium for 12 hours, adjusted to an OD value of 1 at a wavelength of 600 nm, and centrifuged at 13,000 rpm for 10 minutes using a centrifuge to recover the cells. RNA was extracted from the recovered cells using the easy-RED ™ BYF total RNA extraction kit (Intron, Cat. No. 17064, Korea). After that, cDNA extracted from the extracted RNA was used as template. In this case, Maxime RT premix (oligo dT primer) kit (Intron, Cat. No. 25081, Korea) was used. Finally, in order to confirm the expression level of the 2,3-butanediol producing gene, PCR was carried out using the Maxime PCR premix kit (Intron, Cat. No. 25025, Korea) with the synthesized cDNA under the following conditions :
Annealing 51-59 占 폚, 20 seconds,
Extension 72 ° C for 40 seconds (progress after final synthesis at 72 ° C for 5 minutes).
Finally, the PCR products were confirmed by electrophoresis, and the relative expression levels of 2,3-butanediol-producing genes were compared between the medium supplemented with potassium acetate and the culture medium without the addition of electrophoresis. The results are shown in Fig.
Fig. 4 shows the expression amounts of budA , budB , budC and bdh , 2,3-butanediol producing genes, as electrophoresis results.
As shown in FIG. 4, it was confirmed that 2,3-butanediol-producing gene was more expressed in the case of adding potassium acetate to the production medium than in the case of no addition of potassium acetate. In particular, in the case of budB , Respectively.
Example 3. Preparation of 2,3- Butanediol Production increase experiment
After 12 hours of fermentation, the amount of acetate fed through the addition of potash acetate (10 g / L) initially decreased and the production of 2,3-butanediol was stagnated. To solve this problem, 10 g / L of potash acetate was added to increase the acetate concentration in the medium again after 12 hours from the start of fermentation. In the same manner as in Example 1, the yield of 2,3-butanediol And the results are shown in Fig. 5 and Fig. 6.
FIG. 5 is a graph showing the results of experiments in which 2,3-butanediol was produced by adding 10 g / L of potash acetate to the culture medium. FIG. 6 shows that when the concentration of acetate Butane and 2,3-butanediol were stagnated, the productivity and yield of 2,3-butanediol were improved by adding 10 g / L of acetate to the medium again at 12 hours (Glucose concentration,?; 90,?; 110?; 130 g / L).
As shown in FIG. 5 and FIG. 6, productivity and yield of 3-butanediol were improved when acetate was added at 12 hours.
Claims (9)
Wherein the metal acetate is at least one selected from the group consisting of potassium acetate (CH 3 COO - K + ) and sodium acetate (CH 3 COO - Na + ).
Wherein the culture medium contains at least one carbon source selected from the group consisting of glucose, xylose, mannose, galactose and fructose.
Wherein said metal acetate or ammonium acetate is added in an amount of 3 to 9 g / L with respect to the culture medium.
Wherein said metal acetate or ammonium acetate is added at 3 to 15 g / L to the medium.
Wherein the carbon source is contained in an amount of 60 to 200 g / L with respect to the culture medium.
Wherein the expression level of at least one 2,3-butanediol-producing gene selected from the group consisting of budA , budB , budC, and bdh is increased by adding the metal acetate or ammonium acetate, 2,3-butanediol Diol production method.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101457211A (en) * | 2008-08-04 | 2009-06-17 | 山东大学 | Klebsiella pneumoniae and its application in preparing 2,3-butanediol |
| CN102226159A (en) * | 2010-11-29 | 2011-10-26 | 山东大学 | Strain of Enterobacter cloacae and its application in the preparation of 2,3-butylene glycol |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101457211A (en) * | 2008-08-04 | 2009-06-17 | 山东大学 | Klebsiella pneumoniae and its application in preparing 2,3-butanediol |
| CN102226159A (en) * | 2010-11-29 | 2011-10-26 | 山东大学 | Strain of Enterobacter cloacae and its application in the preparation of 2,3-butylene glycol |
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