CN112725233B - Strain for producing 2, 5-furandimethanol and application thereof - Google Patents
Strain for producing 2, 5-furandimethanol and application thereof Download PDFInfo
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Abstract
The invention provides a strain for producing 2, 5-fursA strain of pyran dimethanol and application thereof, belonging to the technical field of biology. A strain for producing 2, 5-furandimethanol, which is classified and named as Burkholderia cepacia (Burkholderia cepacia)Burkholderia contaminans) NJPI-15 strain, the preservation number is: CCTCC NO: M2020636. A method for converting 5-hydroxymethyl furfural into 2, 5-furandimethanol uses the strain as a biocatalyst, and converts 5-hydroxymethyl furfural into 2, 5-furandimethanol. The invention relates to a high 5-hydroxymethylfurfural tolerant strain separated from chemically polluted soilBurkholderia contaminansNJPI-15, which can produce 2, 5-furandimethanol with high yield.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a strain for producing 2, 5-furandimethanol and application thereof.
Background
The reasonable development and utilization of natural biomass resources is the main development direction of the social, economic and sustainable development in the 21 st century. 5-hydroxymethylfurfural (abbreviated as HMF) is one of the important bio-based platform compounds convertible from biomass resources, listed as "Top 10+4" bio-based platform chemicals. Two reactive functional groups, aromatic rings and formyl groups, are present in 5-hydroxymethylfurfural, which render biomolecules highly chemically reactive. Studies have shown that HMF can be converted to a variety of valuable products, such as 5-hydroxymethyl-2-furancarboxylic acid (HMFCA), 2, 5-Diformylfuran (DFF), 2, 5-furandimethanol (BHMF), and 2, 5-furandicarboxylic acid (FDCA). 2, 5-furandimethanol is a multifunctional diol having a wide range of applications including use in the field of molecular recognition for the synthesis of fine chemicals and artificial receptors, and as an important intermediate for synthetic resins, fibers, foams, pharmaceuticals, crown ethers, polymers, and the like.
The biocatalytic reaction can be completed in the environment of normal temperature and pressure, neutrality, water solution and the like, and the condition is very mild. Biocatalytic substrates are extremely selective, catalyzing only a specific class of substrates or reactions, whereas one substrate may be catalyzed by a number of different enzymes; in addition, the biological catalytic conversion has outstanding advantages for synthesizing chiral active pharmaceutical ingredients, and the conversion has low cost, little pollution and environmental protection, and is suitable for the requirements of sustainable development. In the prior art, 5-hydroxymethylfurfural is an inhibitor of microorganisms and enzymes, and the tolerance concentration of the microorganisms to the 5-hydroxymethylfurfural is low, so that the yield of 2, 5-furandimethanol is low, and the industrial production requirement is difficult to meet. Therefore, screening of 5-hydroxymethylfurfural-highly tolerant microorganisms is an important method for increasing the yield of 2, 5-furandimethanol.
Disclosure of Invention
The invention aims to provide a strain for producing 2, 5-furandimethanol, which can tolerate higher-concentration 5-hydroxymethylfurfural and produce 2, 5-furandimethanol with high yield.
The invention also aims to provide a method for producing 2, 5-furandimethanol by using the strain, which has the advantages of extremely short reaction time, mild conditions, no environmental pollution and high product selectivity.
The purpose of the invention is realized by adopting the following technical scheme.
A strain for producing 2, 5-furandimethanol is classified and named as Burkholderia cepacia (Burkholderia contineans) NJPI-15 strain, and the preservation number is as follows: CCTCC NO: M2020636.
A method for converting 5-hydroxymethyl furfural into 2, 5-furandimethanol uses the strain as a biocatalyst, and converts 5-hydroxymethyl furfural into 2, 5-furandimethanol.
In the invention, the strain is added into a reaction solution containing 5-hydroxymethylfurfural and converted at 15-45 ℃ to obtain 2, 5-furandimethanol.
In the present invention, the reaction solution further contains one or more of aspartic acid, nicotinic acid, glutamine, glycine, ribose, and xylose.
In the present invention, the pH of the reaction liquid is 5.0 to 10.0, and the content of 5-hydroxymethylfurfural is 50 to 150mM.
In the present invention, the concentration of glutamine in the reaction liquid is 20 to 100mM.
In a preferred embodiment, the concentration of 5-hydroxymethylfurfural in the reaction solution is 100mM.
In a preferred embodiment, the pH of the reaction solution is 7.0.
In a preferred embodiment, the conversion temperature is 35 ℃.
The invention also provides application of the strain in preparation of 2, 5-furandimethanol.
Has the advantages that: the invention provides a high 5-hydroxymethylfurfural tolerant strain Burkholderia continans NJPI-15 separated from chemically contaminated soil, which can produce 2, 5-furandimethanol with high yield.
Drawings
FIG. 1 shows the effect of different co-substrates on 2, 5-furandimethanol yield and 5-hydroxymethylfurfural conversion.
FIG. 2 shows the effect of different reaction temperatures on the yield of 2, 5-furandimethanol and the conversion of 5-hydroxymethylfurfural.
FIG. 3 shows the effect of different reaction pH on 2, 5-furandimethanol yield and 5-hydroxymethylfurfural conversion.
FIG. 4 shows the effect of different substrate concentrations on 2, 5-furandimethanol yield and 5-hydroxymethylfurfural conversion.
FIG. 5 production of 2, 5-furandimethanol by the fed batch process, with catalyst time on the abscissa (0 h for the start of cell catalysis) and concentration on the ordinate.
Detailed Description
Screening a solid culture medium: 5g/L of yeast powder, 10g/L of peptone, 10g/L of NaCl, 75mM of 5-hydroxymethylfurfural and 20g/L of agar.
Enrichment medium plate: 5g/L of yeast powder, 10g/L of peptone, 10g/L of NaCl and 20g/L of agar.
Liquid enrichment culture medium: 5g/L of yeast powder, 10g/L of peptone, 10g/L of NaCl and water as a solvent.
Liquid enrichment fermentation medium: 5g/L of yeast powder, 10g/L of peptone, 10g/L of NaCl, 2mM of 5-hydroxymethylfurfural and water as a solvent.
Detection methods of 5-hydroxymethyl furfural and 2, 5-furandimethanol: the content of 5-hydroxymethylfurfural and 2, 5-furandimethanol was measured by high performance liquid chromatography (Dionex P680) using a SymmetryShield RP 18C 18 column (4.6 mm. Times.250 mm) with an ultraviolet detector at 223 nm.
Example one
High-concentration 5-hydroxymethylfurfural is used as screening pressure to screen from soil samples around a chemical plant of a Nanjing large factory to obtain a 5-hydroxymethylfurfural tolerant strain Burkholderia continans NJPI-15.
(1) Adding deionized water into the soil sample, uniformly mixing, centrifuging, taking supernatant, coating the supernatant on a screening solid culture medium, and culturing at the temperature of 35 ℃ for 24-48 h. The method can screen the microorganisms resistant to high-concentration 5-hydroxymethylfurfural.
(2) And (2) selecting the single colony in the solid culture medium screened in the step (1) into a liquid enrichment culture medium for enrichment culture at the culture temperature of 35 ℃ for 12-14 h to obtain the enrichment culture solution.
(3) And (3) inoculating the enrichment culture solution obtained in the step (2) into a liquid enrichment fermentation culture medium according to the inoculation amount of 1% for culture. The culture temperature is 35 ℃, and the culture time is 16h. Centrifuging the fermentation liquor, collecting thallus cells, washing the thallus twice by using a phosphate buffer solution with the pH value of 7.0, and centrifuging to obtain microbial cells, namely the biocatalyst, wherein the weighed mass is called as the wet weight of the thallus.
(4) Preparing a reaction solution: pH 7.0, 50mM phosphate buffer solution containing 100mM 5-hydroxymethylfurfural. Adding 20mg of wet-weight bacteria into per milliliter of reaction liquid, adding the biocatalyst obtained in the step (3) into the reaction liquid, carrying out catalytic reaction for 6 hours at the temperature of 35 ℃ and the rotation speed of a shaking table of 200rpm, centrifuging, collecting supernate, and detecting the concentration of the 2, 5-furandimethanol in the supernate by using a high performance liquid chromatography (Dionex P680).
Through screening, a strain NJPI-15 which can tolerate high-concentration 5-hydroxymethylfurfural and produce 2, 5-furandimethanol in high yield is finally obtained. The 16S rDNA sequence (GenBank accession number: MW 165517) showed that the strain has 99% homology with Burkholderia cepacia (Burkholderia conttaminans), and thus belongs to the genus Burkholderia cepacia and is named as Burkholderia cepacia (Burkholderia conttaminans) NJPI-15.
Onion Burkholderia continans NJPI-15 is a gram-positive, immobile, rod-shaped, and strictly anaerobic bacterium with one end being branched, and the optimal growth temperature is 35-42 ℃. The physiological and biochemical characteristics of the compound are negative in experiments such as catalase reaction, gelatin liquefaction, nitrite reduction, indole generation and the like. Can ferment saccharide to produce acetic acid and lactic acid, and acetic acid as main components.
The deposit information of onion Burkholderia continans NJPI-15 is as follows: the culture medium is classified and named Burkholderia continans NJPI-15, the preservation date is 26 months and 10 months in 2020, the preservation unit is totally called China Center for Type Culture Collection (CCTCC) for short, and the preservation unit address is as follows: wuhan university, the preservation number is: CCTCC NO: m2020636.
Example two
This example illustrates the effect of different cosubstrates on 2, 5-furandimethanol yield and 5-hydroxymethylfurfural conversion
Onion Burkholderia NJPI-15 is inoculated in a liquid enrichment medium and cultured for 16h at 35 ℃. Centrifuging the fermentation liquor, collecting thallus cells, washing the thallus twice by using a phosphate buffer solution with the pH value of 7.0, and centrifuging to obtain the onion Burkholderia NJPI-15 thallus which is the biocatalyst.
Preparing a reaction solution: phosphate buffer solution, pH 7.0, 50mM, containing 100mM 5-hydroxymethylfurfural. According to the proportion of adding 20mg of wet-weight bacteria into each milliliter of reaction liquid, adding onion Burkholderia NJPI-15 bacteria into each reaction liquid respectively, then adding cosubstrates (aspartic acid, nicotinic acid, glutamine, glycine, ribose and xylose) with the final concentration of 50mM respectively, culturing for 6 hours at the temperature of 35 ℃ and the rotating speed of a shaking table of 200rpm, sampling respectively and detecting the concentration of 2, 5-furandimethanol by adopting a high performance liquid phase. No cosubstrate was added to the control, and the others were unchanged. As shown in FIG. 1, when the co-substrate was glutamine, the conversion rate of 5-hydroxymethylfurfural reached 100%, and the yield of 2, 5-furandimethanol reached the highest value, reaching 85.7%. Therefore, glutamine was chosen as a catalytic co-substrate for 5-hydroxymethylfurfural.
The effect of different concentrations of glutamine (25mM, 50mM,60mM,70mM,80mM, 100mM) on the yield of 2, 5-furandimethanol concentration was further compared. Preparing a plurality of parts of the following reaction liquid: phosphate buffer solution, pH 7.0, 50mM, containing 100mM 5-hydroxymethylfurfural. According to the proportion of adding 20mg of wet weight bacteria per milliliter of reaction liquid, adding onion Burkholdham NJPI-15 bacteria into each reaction liquid respectively, then adding glutamine with final concentration of 25mM,50mM,60mM,70mM,80mM and 100mM respectively, culturing for 6 hours at 35 ℃ and with the rotating speed of a shaking table of 200rpm, sampling respectively and detecting the concentration of 2, 5-furandimethanol by adopting a high performance liquid phase. As a result: when the concentration of glutamine is 70mM, the conversion rate of 5-hydroxymethylfurfural reaches 100%, and the yield of 2, 5-furandimethanol reaches the highest and reaches 95.2%.
EXAMPLE III
This example illustrates the effect of different temperatures on the yield of 2, 5-furandimethanol and the conversion of 5-hydroxymethylfurfural
Inoculating Burkholderia cepacia NJPI-15 into liquid enrichment fermentation medium, and culturing at 35 deg.C for 16h. Centrifuging the fermentation liquor, collecting thallus cells, washing the thallus twice by using a phosphate buffer solution with the pH value of 7.0, and centrifuging to obtain the onion Burkholderia NJPI-15 thallus which is the biocatalyst.
Preparing a reaction solution: phosphate buffered solution, pH 7.0, 50mM, containing 100mM 5-hydroxymethylfurfural and 70mM glutamine. According to the proportion of adding 20mg of wet-weight bacteria into each milliliter of reaction liquid, adding onion Burkholderia NJPI-15 bacteria into the reaction liquid, respectively culturing for 6 hours at different temperatures (20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃ and 45 ℃) under the condition that the rotation speed of a shaking table is 200rpm, respectively sampling and adopting a high performance liquid to detect the concentration of 2, 5-furandimethanol. As shown in FIG. 2, when the temperature was 35 ℃, the conversion of 5-hydroxymethylfurfural reached 100% and the yield of 2, 5-furandimethanol reached the highest 95.1%. Therefore, the catalytic reaction temperature of 5-hydroxymethylfurfural at 35 ℃ is selected.
Example four
This example illustrates the effect of different pH on the yield of 2, 5-furandimethanol and the conversion of 5-hydroxymethylfurfural
Inoculating Burkholderia cepacia NJPI-15 into liquid enrichment fermentation medium, and culturing at 35 deg.C for 16h. Centrifuging the fermentation liquor, collecting thallus cells, washing the thallus twice by using a phosphate buffer solution with the pH value of 7.0, and centrifuging to obtain the onion Burkholderia NJPI-15 thallus which is the biocatalyst.
Preparing reaction solutions with different pH values: 5-hydroxymethylfurfural and 70mM glutamine were added to buffer solutions having a pH of 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0, respectively, and a final concentration of 100mM were added to the buffer solutions, respectively, to obtain reaction solutions having different pH values. According to the proportion of adding 20mg of wet-weight bacteria into each milliliter of reaction liquid, adding onion Burkholderia NJPI-15 bacteria into each pH reaction liquid, culturing for 6 hours at 200rpm, sampling respectively, and detecting the concentration of 2, 5-furandimethanol by using a high performance liquid phase. As shown in FIG. 3, the conversion of 5-hydroxymethylfurfural reached 100% and the yield of 2, 5-furandimethanol reached the highest 96% at pH 7.0. Thus, a catalytic reaction pH of 5-hydroxymethylfurfural at pH 7.0 was selected.
EXAMPLE five
This example illustrates the effect of 5-hydroxymethylfurfural concentration on the yield of 2, 5-furandimethanol.
Inoculating Burkholderia cepacia NJPI-15 into liquid enrichment fermentation medium, and culturing at 35 deg.C for 16h. Centrifuging the fermentation liquor, collecting thallus cells, washing the thallus twice by using a phosphate buffer solution with the pH value of 7.0, and centrifuging to obtain the onion Burkholderia NJPI-15 thallus which is the biocatalyst.
Preparing a reaction solution: 5-hydroxymethylfurfural was added to a phosphate buffer solution at pH 7.0 and 50mM, which contained 70mM glutamine, to final concentrations of 0, 50mM, 75mM, 100mM, 125mM, 150mM and 200mM, respectively, to obtain reaction solutions at respective concentrations. According to the proportion of adding 20mg of wet-weight bacteria into each milliliter of reaction liquid, respectively adding onion Burkholderia NJPI-15 bacteria into the reaction liquid with each concentration, culturing for 6 hours at 35 ℃ at 200rpm, respectively sampling and detecting the concentration of 2, 5-furan dimethyl alcohol by adopting a high performance liquid chromatography. As shown in FIG. 4, when the concentration of 5-hydroxymethylfurfural in the reaction solution was less than 125mM, the conversion rates were all 100%, and when the concentration was 100mM, the total yield of 2, 5-furandimethanol was the highest, and the yield was 95%. Therefore, a reaction solution containing 100mM 5-hydroxymethylfurfural was selected.
EXAMPLE seven
This example illustrates the industrial application of a process for the preparation of 2, 5-furandimethanol.
When the consumption of the 5-hydroxymethylfurfural is gradually increased, the concentration of the product 2, 5-furandimethanol gradually approaches to saturation, so that the fermentation production is carried out in a fed-batch mode for improving the yield.
Inoculating Burkholderia cepacia NJPI-15 into liquid enrichment fermentation medium, and culturing at 35 deg.C for 16h. Centrifuging the fermentation liquor, collecting thallus cells, washing the thallus twice by using a phosphate buffer solution with the pH value of 7.0, and centrifuging to obtain the onion Burkholderia NJPI-15 thallus which is the biocatalyst.
Preparing a reaction solution: phosphate buffered solution containing 100mM 5-hydroxymethylfurfural and 70mM glutamine, pH 7.0, 50mM. 200mL of the reaction solution was charged into a 1L Erlenmeyer flask, and 20mg of wet-weight cells per mL of the reaction solution were added to the cell mixture, and the cell mixture was cultured at 200rpm and 35 ℃ while 20mL of a phosphate buffer solution of pH 7.0 and 500mM containing 1M 5-hydroxymethylfurfural and 700mM glutamine was added to the system every 6 hours. After 4 times of adding 5-hydroxymethylfurfural and glutamine, when the culture time is 33 hours from the time when the bacteria are added into the reaction solution, centrifuging and taking the supernatant. Then, a fresh reaction solution (a phosphate buffer solution of pH 7.0 and 50mM containing 100mM 5-hydroxymethylfurfural and 70mM glutamine) was added to the cell pellet, the cell pellet was cultured at 200rpm and 35 ℃, 20mL of a phosphate buffer solution of pH 7.0 and 500mM containing 1M 5-hydroxymethylfurfural and 700mM glutamine was added to the system every 6 hours, and the culture time was 15 hours from the time when a fresh reaction solution was added to the cell pellet, and then the supernatant was collected. And detecting the conversion rate of the 5-hydroxymethylfurfural and the yield change of the 2, 5-furandimethanol by high performance liquid chromatography. As a result: the total conversion rate of 5-hydroxymethylfurfural reaches 100%, the total yield of 2, 5-furandimethanol is 656.3mM, and the total yield exceeds 93% (figure 5).
Claims (10)
1. A strain for producing 2, 5-furandimethanol, which is classified and named as Burkholderia cepacia (B) (A)Burkholderia contaminans) NJPI-15 strain, the preservation number is: CCTCC NO: M2020636.
2. A method for converting 5-hydroxymethylfurfural into 2, 5-furandimethanol, characterized in that the strain as claimed in claim 1 is used as a biocatalyst, and the 5-hydroxymethylfurfural is converted into 2, 5-furandimethanol.
3. The method for converting 5-hydroxymethylfurfural into 2, 5-furandimethanol according to claim 2, wherein the strain according to claim 1 is added into a reaction solution containing 5-hydroxymethylfurfural, and the conversion is carried out at 15 to 45 ℃ to obtain 2, 5-furandimethanol.
4. The method according to claim 3, wherein the reaction solution further contains one or more of aspartic acid, nicotinic acid, glutamine, glycine, ribose and xylose.
5. The process according to claim 4 for converting 5-hydroxymethylfurfural into 2, 5-furandimethanol, characterized in that the reaction solution has a pH of 5.0 to 10.0 and a 5-hydroxymethylfurfural content of 50 to 150mM.
6. The method according to claim 5, wherein the concentration of glutamine in the reaction solution is 20 to 100mM.
7. The method of claim 6, wherein the concentration of 5-hydroxymethylfurfural in the reaction solution is 100mM.
8. The process of claim 7 for converting 5-hydroxymethylfurfural to 2, 5-furandimethanol, characterized in that the reaction solution has a pH of 7.0.
9. Process for the conversion of 5-hydroxymethylfurfural to 2, 5-furandimethanol according to claim 8, characterized in that the conversion temperature is 35 ℃.
10. Use of the strain of claim 1 for the preparation of 2, 5-furandimethanol.
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