CN114410517B - Bacillus amyloliquefaciens and application thereof in preparation of 2,5-furandicarboxylic acid - Google Patents
Bacillus amyloliquefaciens and application thereof in preparation of 2,5-furandicarboxylic acid Download PDFInfo
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Abstract
The invention relates to a bacillus amyloliquefaciens and application thereof in preparation of 2,5-furandicarboxylic acid, belonging to the technical field of biology. The invention screens out a bacillus amyloliquefaciens strain capable of being used for producing 2,5-furandicarboxylic acid, the bacillus amyloliquefaciens is screened from papermaking wastewater, has stronger 5-hydroxymethylfurfural tolerance, and the tolerance concentration range of 5-hydroxymethylfurfural is 0-20mM when the bacillus amyloliquefaciens is cultured in a culture medium. The bacillus amyloliquefaciens provided by the invention can realize the accumulation of 2,5-furandicarboxylic acid, the yield can reach 5mM, and a foundation is laid for further producing the 2,5-furandicarboxylic acid by using the bacillus amyloliquefaciens. The method for producing 2,5-furandicarboxylic acid by using bacillus amyloliquefaciens is simple and has good application prospect.
Description
Technical Field
The invention relates to a bacillus amyloliquefaciens and application thereof in preparation of 2,5-furandicarboxylic acid, belonging to the technical field of biology.
Background
2, 5-Furanedicarboxylic acid (FDCA) is the only aromatic ring-containing compound of the 12 bio-based platform compounds that have been highlighted by the U.S. department of energy, and is considered to be the most likely aromatic bio-based monomer to replace terephthalic acid (PTA). 2,5-furan dicarboxylic acid is used to replace terephthalic acid to synthesize a series of furan-based high molecular compounds or additives. The furyl polymer compound has great development potential on bio-based engineering plastics, bio-based fibers and bio-based packaging materials.
At present, the method for preparing 2,5-furandicarboxylic acid includes chemical catalysis method and biological enzyme catalysis method. The chemical catalysis method is the most widely used method at present, generally adopts noble metals as catalysts, often needs high-temperature and high-pressure reaction conditions, and often needs special treatment on the waste liquid after the reaction, for example, chinese patent document CN105037303A (application No. 201510395096.0) uses quaternary ammonium molybdate and tungstate as catalysts, oxygen, hydrogen peroxide or air as oxidants, heats to 80-120 ℃ in an alkaline environment, selectively oxidizes 5-hydroxymethylfurfural to prepare 2,5-furandicarboxylic acid; chinese patent document CN106749130A (application number 201710108023.8) adds a platinum-supported catalyst, 5-hydroxymethylfurfural and solvent water into a reactor, and reacts for 2-12 hours at 20-100 ℃ under an oxygen atmosphere to obtain 2,5-furandicarboxylic acid; chinese patent document CN107365287A (application number 201610308198.9) contacts an aqueous solution of 5-hydroxymethylfurfural with a noble metal catalyst and a solid basic auxiliary agent in the presence of air and/or oxygen, and carries out a catalytic oxidation reaction to synthesize 2,5-furandicarboxylic acid from 5-hydroxymethylfurfural. Different from a chemical catalysis method, the biological enzyme catalysis method can catalyze 5-hydroxymethylfurfural to oxidize and synthesize 2,5-furandicarboxylic acid at normal temperature and normal pressure, and has the advantages of stronger selectivity, simple process and the like. For example, chinese patent publication No. CN104371955A (application No. 201410606067. X) discloses a Raoultella terrestris for synthesizing 2,5-furandicarboxylic acid, the yield of 2,5-furandicarboxylic acid produced by applying the Raoultella terrestris to carry out whole-cell transformation can reach 7.95g/L; chinese patent document CN106554978A (application No. 201510644672.0) adopts lipase and 5-hydroxymethylfurfural oxidase to synergistically catalyze and oxidize 5-hydroxymethylfurfural in a "one-pot process" to prepare 2,5-furandicarboxylic acid; chinese patent document CN108977472A (application number 201710407324.0) adopts 5-hydroxymethyl furfural oxidase and 5-hydroxymethyl furfural oxidase mutase to carry out tandem catalytic oxidation on 5-hydroxymethyl furfural to prepare 2,5-furandicarboxylic acid; chinese patent document CN113388565A (application number 202110724944.3) uses two genetically modified Pseudomonas hophalla as biocatalysts to oxidize 5-hydroxymethylfurfural to prepare 2,5-furandicarboxylic acid.
Although the biological enzyme catalysis method has more advantages, 5-hydroxymethylfurfural has toxic effect on cells, and no report is found on the production of 2,5-furandicarboxylic acid by using bacillus amyloliquefaciens at present.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) and application thereof in preparing 2,5-furandicarboxylic acid. The screened bacillus amyloliquefaciens has higher tolerance of 5-hydroxymethyl furfural, is used for producing 2,5-furandicarboxylic acid, and has good application prospect.
The technical scheme of the invention is as follows:
a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) WTF1 is preserved in China center for type culture Collection in 2021, 7 months and 9 days at the preservation address: the preservation number of the Wuhan university in Wuhan, china is CCTCC NO: m2021847.
According to the invention, the nucleotide sequence of the 16sDNA of the Bacillus amyloliquefaciens is shown as SEQ ID NO. 1.
Preferably, the bacillus amyloliquefaciens has 5-hydroxymethylfurfural tolerance, and the tolerance concentration of the 5-hydroxymethylfurfural in the culture medium is 0-20mM.
The bacillus amyloliquefaciens is screened from paper making wastewater, the paper making wastewater is screened and cultured on a culture medium containing 5-hydroxymethylfurfural, and the yield of 2,5-furandicarboxylic acid in fermentation liquor is detected after fermentation to obtain a strain with higher yield of the 2,5-furandicarboxylic acid; the strain can still grow in a culture medium with the concentration of 5-hydroxymethylfurfural of 20mM, and is the strain with the highest 5-hydroxymethylfurfural tolerance among the reported strains in the same genus.
The method for culturing the bacillus amyloliquefaciens comprises the following steps:
selecting Bacillus amyloliquefaciens, streaking on a solid seed culture medium, culturing at 25-37 deg.C for 20-30h, selecting single colony, transferring into the seed culture medium, and culturing at 25-37 deg.C and 180-220rpm for 10-20h; inoculating into fermentation culture medium with 0.5-2% inoculum size, and culturing at 25-37 deg.C and 180-220rpm for 10-20h to obtain Bacillus amyloliquefaciens bacterial liquid.
Preferably, according to the invention, the fermentation medium or seed medium comprises: 10g/L of tryptone, 5g/L of yeast extract and 10g/L of sodium chloride; the solid medium was supplemented with 2% agar.
The application of the bacillus amyloliquefaciens in preparing 2,5-furandicarboxylic acid.
According to the invention, the application is preferably to produce the 2,5-furandicarboxylic acid by using 5-hydroxymethylfurfural as a substrate and adopting the whole-cell transformation of bacillus amyloliquefaciens.
A method for producing 2,5-furandicarboxylic acid by whole-cell transformation by using the bacillus amyloliquefaciens and taking 5-hydroxymethylfurfural as a substrate comprises the following steps:
at OD 600 Adding 5-hydroxymethylfurfural into 50-120 bacillus amyloliquefaciens suspension, wherein the final concentration of the 5-hydroxymethylfurfural is 30-125mM, and carrying out conversion production at 25-37 ℃ and 180-220rpm for 50-100h to obtain a conversion solution containing 2,5-furandicarboxylic acid.
When the bacillus amyloliquefaciens is directly cultured in a culture medium of a 5-hydroxymethylfurfural substrate, the bacillus amyloliquefaciens can tolerate 20mM of 5-hydroxymethylfurfural, and in the whole cell catalysis process, the cell concentration is high, 5-hydroxymethylfurfural with the substrate concentration of 30-125mM can be converted into 2,5-furandicarboxylic acid, although the high 5-hydroxymethylfurfural concentration can affect the cell activity of the bacillus amyloliquefaciens, most of the bacillus amyloliquefaciens can still play the role of a cell factory to generate the 2,5-furandicarboxylic acid.
According to the invention, the bacillus amyloliquefaciens suspension is obtained by resuspending bacillus amyloliquefaciens with a buffer solution with the pH value of 6-8.
More preferably, the bacillus amyloliquefaciens is obtained by culturing the bacillus amyloliquefaciens according to the culture method of the bacillus amyloliquefaciens and then centrifuging; or culturing Bacillus amyloliquefaciens in fermentation medium at 25-37 deg.C and 180-220rpm for 12-18 hr, and centrifuging.
Further preferably, the pH of the buffer is 7.
Further preferably, the buffer is a phosphate buffer.
Preferably, the OD of the Bacillus amyloliquefaciens bacterial suspension is 600 Is 90-110.
According to the invention, the final concentration of the 5-hydroxymethylfurfural is preferably 50 to 125mM.
Preferably, according to the invention, the temperature of the conversion production is between 28 and 32 ℃.
Preferably, according to the invention, the rotational speed of the conversion production is 190 to 210rpm.
Preferably, according to the invention, the conversion takes place over a period of 72 to 96 hours.
According to the invention, the preferable method for producing the 2,5-furandicarboxylic acid by using the bacillus amyloliquefaciens and using the 5-hydroxymethylfurfural as a substrate through whole-cell transformation specifically comprises the following steps:
selecting Bacillus amyloliquefaciens, streaking on a solid seed culture medium, culturing at 25-37 deg.C for 20-30h, selecting single colony, transferring into the seed culture medium, and culturing at 25-37 deg.C and 180-220rpm for 10-20h; inoculating into fermentation culture medium at 0.5-2%, culturing at 25-37 deg.C and 180-220rpm for 10-20 hr to obtain Bacillus amyloliquefaciens bacterial solution, centrifuging to obtain thallus, washing with buffer solution of pH6-8, and resuspending to obtain final concentration OD 600 50-120, then adding 5-hydroxymethylfurfural to make the final concentration of the 5-hydroxymethylfurfural 30-125mM, and carrying out conversion production for 50-100h at the temperature of 25-37 ℃ and under the condition of 180-220rpm, thus obtaining the conversion solution containing the 2,5-furandicarboxylic acid.
According to the invention, the preferable method for producing the 2,5-furandicarboxylic acid by using the bacillus amyloliquefaciens and using the 5-hydroxymethylfurfural as a substrate through whole-cell transformation specifically comprises the following steps:
culturing Bacillus amyloliquefaciens in a cell culture medium at 25-37 deg.C and 180-220rpm for 12-18h, centrifuging to obtain cell, washing with buffer solution of pH6-8, and suspending to obtain final cell concentration OD 600 50-120, then adding 5-hydroxymethylfurfural to make the final concentration of 5-hydroxymethylfurfural 30-125mM, and carrying out conversion production for 50-100h at 25-37 ℃ and 180-220rpm to obtain a culture solution containing 2,5-furandicarboxylic acid.
More preferably, the cells are washed with a buffer solution having a pH of 6 to 8 and then resuspended after washing the cells 2 times with the buffer solution.
The experimental procedures not specified in the present invention were carried out according to the prior art.
Has the advantages that:
the invention screens out a bacillus amyloliquefaciens strain capable of being used for producing 2,5-furandicarboxylic acid for the first time, the bacillus amyloliquefaciens is screened from papermaking wastewater, has stronger 5-hydroxymethylfurfural tolerance, and the tolerance concentration range of 5-hydroxymethylfurfural is 0-20mM when the bacillus amyloliquefaciens is cultured in a culture medium. The bacillus amyloliquefaciens provided by the invention can realize the accumulation of 2,5-furandicarboxylic acid, the yield can reach 5mM, and a foundation is laid for further producing the 2,5-furandicarboxylic acid by using the bacillus amyloliquefaciens. The method for producing 2,5-furandicarboxylic acid by using bacillus amyloliquefaciens is simple and has good application prospect.
Biological material:
the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) WTF1 is preserved in China center for type culture Collection in 2021, 7-9 months, with the preservation addresses: the preservation number of the Wuhan university in Wuhan, china is CCTCC NO: m2021847.
Drawings
FIG. 1 is a high performance liquid chromatogram of a standard 2,5-furandicarboxylic acid;
FIG. 2 is a high performance liquid chromatogram of the fermentation broth of example 1.
Detailed Description
The technical solution of the present invention is further illustrated by the following examples, but the scope of the present invention is not limited thereto. The experimental methods mentioned in the examples were carried out according to the prior art unless otherwise specified; the drugs and reagents mentioned in the examples are all common commercial products unless otherwise specified.
High Performance Liquid Chromatography (HPLC) determination method of 2,5-furandicarboxylic acid:
the instrument is Shimadzu LC-20A, and the detector is VWD; the detection wavelength is 268nm; the chromatographic column is Aminex HPX-87H (300X 7.8 mm); the mobile phase was 10mM H 2 SO 4 (ii) a The flow rate is 0.6mL/min; the column temperature is 60 ℃; the sample size was 10. Mu.L.
Example 1: screening of 2, 5-Furandicacid-producing Strain
Taking 100 mu L of papermaking wastewater in a centrifuge tube, diluting by 10 times, taking 200 mu L of diluted papermaking wastewater, coating the diluted papermaking wastewater on a solid screening culture medium, culturing at 30 ℃ for 24h, selecting a strain with larger colony morphology, inoculating the strain into a 250mL conical flask filled with 50mL of fermentation culture medium, culturing at 30 ℃ for 24h, taking 800 mu L of culture solution, transferring the culture solution into a storage tube containing 800 mu L of 40% glycerol, and storing in a refrigerator at-80 ℃.
Selecting a strain preserved at the temperature of minus 80 ℃, streaking the strain on a solid seed culture medium, culturing the strain at the temperature of 30 ℃ for 24h, selecting a single colony, inoculating the single colony in a 250mL triangular flask filled with 50mL fermentation medium, carrying out shake culture at the temperature of 30 ℃ and at the speed of 220rpm for 72h, centrifuging the fermentation liquor at the speed of 12000rpm for 10min, diluting the fermentation liquor by deionized water by a certain multiple, filtering the fermentation liquor by a filter membrane of 0.22 mu m, and carrying out quantitative analysis on the fermentation liquor by using a high performance liquid chromatograph, thus finally detecting the strain with higher yield of the 2,5-furandicarboxylic acid.
The culture medium involved in the screening process of the strain:
solid screening culture medium: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of sodium chloride, 20g/L of agar powder and 2.52g/L of 5-hydroxymethyl furfural;
fermentation medium or seed medium: 10g/L of tryptone, 5g/L of yeast extract and 10g/L of sodium chloride; if solid medium is needed, add 2% agar.
The high performance liquid chromatogram is shown in figures 1 and 2, and as can be seen from the high performance liquid chromatogram, a chromatographic peak with the same retention time as that of a 2,5-furandicarboxylic acid standard substance appears in a fermentation liquid, and the peak area of the chromatographic peak under the retention time is correspondingly increased after the 2,5-furandicarboxylic acid standard substance is externally added into the fermentation liquid, so that the compound is determined to be the 2,5-furandicarboxylic acid.
Example 2: molecular biological identification of 2,5-furandicarboxylic acid producing strains
The 16sDNA sequence of the selected strain is amplified from the genome by using a bacterial universal primer (27F/1492R), sequenced and compared for analysis. The 16s DNA sequence of the strain is shown in SEQ ID NO. 1. The sequence is subjected to Blast alignment in NCBI, and the similarity of the target sequence of the strain and Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is found to be 100%. Therefore, the strain was determined to be bacillus amyloliquefaciens.
The strain is preserved in China center for type culture Collection in 2021, 7 months and 9 days, and the preservation address is as follows: the preservation number of the Wuhan university in Wuhan, china is CCTCC NO: m2021847.
Example 3: production of 2,5-furandicarboxylic acid by whole cell transformation method
Streaking the bacillus amyloliquefaciens screened in the example 1 on a solid seed culture medium, culturing at 30 ℃ for 24 hours, picking out a single colony, transferring into the seed culture medium, and culturing at 30 ℃ and 220rpm for 15 hours; inoculating into fermentation medium with 1% inoculum size, culturing at 30 deg.C and 220rpm for 15 hr to obtain Bacillus amyloliquefaciens solution, centrifuging to obtain thallus, washing thallus with phosphate buffer solution of pH7.0 twice, and re-suspending to obtain final concentration OD 600 And then adding 5-hydroxymethylfurfural until the final concentration of the 5-hydroxymethylfurfural is 30 mM, 50 mM and 80mM respectively, and carrying out conversion production for 96 hours at the temperature of 30 ℃ and the rpm of 200 to obtain a conversion solution containing the 2,5-furandicarboxylic acid.
Wherein, the components of the fermentation medium or the seed culture medium are as follows: 10g/L of tryptone, 5g/L of yeast extract and 10g/L of sodium chloride; if necessary, the solid medium is added with 2% agar.
The yields of 2,5-furandicarboxylic acid in the conversion solution were 0.17g/L, 0.18g/L, and 0.23g/L, respectively, and the maximum yield of 2,5-furandicarboxylic acid was 0.23g/L, as analyzed by HPLC.
Example 4: production of 2,5-furandicarboxylic acid by whole cell transformation method
Streaking the bacillus amyloliquefaciens screened in the example 1 on a solid seed culture medium, culturing at 30 ℃ for 24 hours, picking out a single colony, transferring into the seed culture medium, and culturing at 30 ℃ and 220rpm for 15 hours; inoculating to fermentation medium at 1%, culturing at 30 deg.C and 220rpm for 15 hr to obtain Bacillus amyloliquefaciens bacterial solution, centrifuging to obtain thallus, washing with phosphate buffer solution of pH7.0 twice, and resuspending to obtain final concentration OD 600 Adding 5-hydroxymethylfurfural to make the final concentration of 5-hydroxymethylfurfural be 50, 75 and 10 respectively to be 1000mM, and carrying out conversion production for 96h at 30 ℃ and 200rpm, thus obtaining the conversion solution containing the 2,5-furandicarboxylic acid.
Wherein, the components of the fermentation medium or the seed culture medium are as follows: 10g/L of tryptone, 5g/L of yeast extract and 10g/L of sodium chloride; if necessary, the solid medium is added with 2% agar.
The yields of 2,5-furandicarboxylic acid in the conversion solution were 0.231g/L, 0.234g/L, and 0.392g/L, respectively, and the maximum yield of 2,5-furandicarboxylic acid was 0.392g/L, as determined by HPLC analysis.
Example 5: method for producing 2,5-furandicarboxylic acid by using bacillus amyloliquefaciens
Selecting Bacillus amyloliquefaciens preserved at-80 ℃ to streak on a solid seed culture medium, culturing at 30 ℃ for 24h, selecting a single colony, transferring into the seed culture medium, and culturing at 30 ℃ and 220rpm for 12h; inoculating into 500mL triangular flask containing 100mL fermentation medium at an inoculation amount of 1%, culturing at 30 deg.C and 220rpm for 16h, collecting bacterial liquid, centrifuging at low temperature for 30min (6000rpm, 4 deg.C) in refrigerated centrifuge, and collecting thallus.
Wherein, the components of the fermentation culture medium or the seed culture medium are as follows: 10g/L of tryptone, 5g/L of yeast extract and 10g/L of sodium chloride; if necessary, the solid medium is added with 2% agar.
The whole-cell catalysis steps are as follows: the pellet was washed 2 times with pH7.0 phosphate buffer solution, and then resuspended in pH7.0 phosphate buffer solution to make the final concentration of the cells OD 600 =100, and then 5-hydroxymethylfurfural is added to the bacterial suspension to give a conversion yield of 96h,2, 5-furandicarboxylic acid of 0.75g/L at 220rpm at 30 ℃ and about 5mM.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of Qilu Industrial science
<120> bacillus amyloliquefaciens and application thereof in preparation of 2,5-furandicarboxylic acid
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1448
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<213> Bacillus amyloliquefaciens
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tagcccaggt cataaggggc atgatgattt gacgtcatcc ccaccttcct ccggtttgtc 300
accggcagtc accttagagt gcccaactga atgctggcaa ctaagatcaa gggttgcgct 360
cgttgcggga cttaacccaa catctcacga cacgagctga cgacaaccat gcaccacctg 420
tcactctgcc cccgaagggg acgtcctatc tctaggattg tcagaggatg tcaagacctg 480
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gtcaattcct ttgagtttca gtcttgcgac cgtactcccc aggcggagtg cttaatgcgt 600
tagctgcagc actaaggggc ggaaaccccc taacacttag cactcatcgt ttacggcgtg 660
gactaccagg gtatctaatc ctgttcgctc cccacgcttt cgctcctcag cgtcagttac 720
agaccagaga gtcgccttcg ccactggtgt tcctccacat ctctacgcat ttcaccgcta 780
cacgtggaat tccactctcc tcttctgcac tcaagttccc cagtttccaa tgaccctccc 840
cggttgagcc gggggctttc acatcagact taagaaaccg cctgcgagcc ctttacgccc 900
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tcgtcctg 1448
Claims (13)
1. Bacillus amyloliquefaciens strain (A), (B), (C)Bacillusamyloliquefaciens) WTF1, deposited at the chinese type culture collection center on 9/7/2021 at the deposit address: the preservation number of the Wuhan university in Wuhan, china is CCTCC NO: m2021847.
2. The method for culturing Bacillus amyloliquefaciens according to claim 1, comprising the steps of:
selecting Bacillus amyloliquefaciens, streaking on a solid seed culture medium, culturing at 25-37 deg.C for 20-30h, selecting single colony, transferring into the seed culture medium, and culturing at 25-37 deg.C and 180-220rpm for 10-20h; inoculating into fermentation culture medium with 0.5-2% inoculum size, and culturing at 25-37 deg.C and 180-220rpm for 10-20h to obtain Bacillus amyloliquefaciens bacterial liquid.
3. The culture method of claim 2, wherein the fermentation medium or seed medium comprises: 10g/L of tryptone, 5g/L of yeast extract and 10g/L of sodium chloride; the solid medium was supplemented with 2% agar.
4. Use of the bacillus amyloliquefaciens of claim 1 for preparing 2,5-furandicarboxylic acid.
5. The use according to claim 4, wherein the use is for the production of 2,5-furandicarboxylic acid by whole-cell transformation with Bacillus amyloliquefaciens, using 5-hydroxymethylfurfural as a substrate.
6. A method for producing 2,5-furandicarboxylic acid by using the Bacillus amyloliquefaciens and performing whole-cell transformation by using 5-hydroxymethylfurfural as a substrate, which is characterized by comprising the following steps:
at OD 600 Adding 5-hydroxymethylfurfural into 50-120 Bacillus amyloliquefaciens suspension, wherein the final concentration of the 5-hydroxymethylfurfural is 30-125mM, and converting at 25-37 ℃ and 180-220rpm for 50-100h to obtain a conversion solution containing 2,5-furandicarboxylic acid.
7. The method of claim 6, wherein the Bacillus amyloliquefaciens bacterial suspension is obtained by resuspending Bacillus amyloliquefaciens bacteria in a buffer solution having a pH of 6 to 8.
8. The method according to claim 7, wherein the Bacillus amyloliquefaciens cell is obtained by centrifugation after culturing according to the method for culturing Bacillus amyloliquefaciens of claim 3; or culturing Bacillus amyloliquefaciens in a fermentation medium at 25-37 deg.C and 180-220rpm for 12-18 hr, and centrifuging.
9. The method of claim 7, wherein the buffer has a pH of 7.
10. The method of claim 7, wherein the buffer is a phosphate buffer.
11. The method of claim 6, wherein one or more of the following conditions are satisfied:
i. OD of the bacillus amyloliquefaciens bacterial suspension 600 Is 90 to 110;
the final concentration of the 5-hydroxymethylfurfural is 50-125mM;
the temperature of the conversion production is 28-32 ℃;
the rotational speed of the conversion production is 190-210rpm;
v. the time for the conversion production is 72-96h.
12. The method of claim 6, comprising the steps of:
selecting Bacillus amyloliquefaciens, streaking on a solid seed culture medium, culturing at 25-37 deg.C for 20-30h, selecting single colony, transferring into the seed culture medium, and culturing at 25-37 deg.C and 180-220rpm for 10-20h; inoculating into fermentation culture medium at 0.5-2%, culturing at 25-37 deg.C and 180-220rpm for 10-20 hr to obtain Bacillus amyloliquefaciens bacterial solution, centrifuging to obtain thallus, washing with buffer solution of pH6-8, and resuspending to obtain final concentration OD 600 50-120, then adding 5-hydroxymethylfurfural to make the final concentration of the 5-hydroxymethylfurfural be 30-125mM, and carrying out conversion production for 50-100h at the temperature of 25-37 ℃ and under the condition of 180-220rpm to obtain a conversion solution containing 2,5-furandicarboxylic acid;
or culturing Bacillus amyloliquefaciens in cell culture medium at 25-37 deg.C and 180-220rpm for 12-18h, centrifuging to obtain cell, washing with buffer solution of pH6-8, and resuspending to obtain final cell concentration OD 600 50-120, then adding 5-hydroxymethylfurfural to make the final concentration of the 5-hydroxymethylfurfural 30-125mM, and carrying out conversion production for 50-100h at 25-37 ℃ and 180-220rpm, thus obtaining a culture solution containing 2,5-furandicarboxylic acid.
13. The method of claim 12, wherein the cell is resuspended after washing with a buffer having a pH of 6 to 8 by washing the cell 2 times with the buffer.
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