CN110551639B - Aureobasidium pullulans strain and application thereof in synthesis of 2, 5-dihydroxymethylfuran - Google Patents
Aureobasidium pullulans strain and application thereof in synthesis of 2, 5-dihydroxymethylfuran Download PDFInfo
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- DSLRVRBSNLHVBH-UHFFFAOYSA-N 2,5-furandimethanol Chemical compound OCC1=CC=C(CO)O1 DSLRVRBSNLHVBH-UHFFFAOYSA-N 0.000 title claims abstract description 36
- 241000223678 Aureobasidium pullulans Species 0.000 title claims abstract description 26
- 238000003786 synthesis reaction Methods 0.000 title claims description 8
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- NOEGNKMFWQHSLB-UHFFFAOYSA-N 5-hydroxymethylfurfural Chemical compound OCC1=CC=C(C=O)O1 NOEGNKMFWQHSLB-UHFFFAOYSA-N 0.000 claims description 77
- RJGBSYZFOCAGQY-UHFFFAOYSA-N hydroxymethylfurfural Natural products COC1=CC=C(C=O)O1 RJGBSYZFOCAGQY-UHFFFAOYSA-N 0.000 claims description 77
- 239000000243 solution Substances 0.000 claims description 18
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
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- 239000004721 Polyphenylene oxide Substances 0.000 description 1
- MCMNRKCIXSYSNV-UHFFFAOYSA-N ZrO2 Inorganic materials O=[Zr]=O MCMNRKCIXSYSNV-UHFFFAOYSA-N 0.000 description 1
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- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention relates to an aureobasidium pullulans strain and application thereof in synthesizing 2, 5-dihydroxymethylfuran, belonging to the technical field of biochemistry, wherein the strain is aureobasidium pullulans F134 which is preserved in China Center for Type Culture Collection (CCTCC) for short, the preservation number is CCTCC No. M2019476, and the preservation date is 2019, 6 months and 20 days. The method utilizes aureobasidium pullulans F134 as a catalyst, can efficiently and selectively catalyze HMF to be converted into the target product BHMF, overcomes the defect that a chemical catalyst is not friendly to the environment, and has higher substrate concentration, more excellent reaction efficiency and better selectivity compared with the reported biological catalysis process. The method has the advantages of simple reaction process, no need of adding a culture medium, easy control, mild conditions and contribution to simplifying the subsequent separation and purification process of the target product.
Description
Technical Field
The invention belongs to the technical field of biochemistry, and particularly relates to an aureobasidium pullulans strain and application thereof in catalyzing selective reduction synthesis of 2, 5-dihydroxymethylfuran by 5-hydroxymethylfurfural.
Background
With the deepening of sustainable development concepts of green energy resources for replacing petroleum resources and environmental protection alternatives in the chemical industry, the biomass energy and the biotechnology occupy more and more critical positions in the current generation with increasingly severe resource and environmental problems. 5-Hydroxymethylfurfural (HMF) is considered to be the most valuable and potential bio-based platform chemical to replace the basic chemicals in the petrochemical industry. Is a byproduct of the lignin pretreatment process and is produced by the dehydration of hexoses (mainly glucose). The research on 5-hydroxymethylfurfural is mainly based on two important reasons: on the one hand, HMF in the biologically pretreated hydrolysate inhibits the growth of many microorganisms and has some toxic effects, thereby affecting the yield and efficiency of the subsequent fermentative synthesis of chemicals and fuels. On the other hand, because the HMF molecule is provided with a plurality of high-activity functional groups such as aldehyde group, hydroxymethyl and carbon-carbon double bond, a series of furan aromatic compounds can be generated by catalytic oxidation reduction. When the aldehyde group on the ring is reduced to hydroxyl, 2, 5-furandimethanol (2, 5-bis-hydroxymethyluran, BHMF) which is the reduction product of HMF is obtained. BHMF, as a diol with high added value, is a key bridge compound in organic synthesis, and has wide application in fine chemical synthesis, preparation research of novel functionalized polyether, polyurethane and multi-heterocyclic compounds of medicines. BHMF is also a new fuel oil which is raised in recent years, has high energy density and is equivalent to gasoline; the boiling point is high, the volatility is low, and the loss caused by storage and use is small; the water absorption in the air is stable and is not easy; the production raw material can be biomass such as cellulose and the like, can reduce the dependence on fossil energy and food, reduce the production cost and is beneficial to protecting the environment (Tetrahedron,2008,64(27): 6358-.
Currently, chemical conversion processes for preparing BHMF from HMF are still mainstream. Chemical conversion processes have made much progress, mainly using metal catalysts to catalyze selective reduction of HMF, and selective Catalytic Transfer Hydrogenation (CTH) under high pressure hydrogen. For example, Mingwei Ma et al teach the one-step preparation of catalysts for catalytic transfer hydrogenation by in situ reduction of Cu2(OH)2CO3The catalyst nano-Cu/AlOOH is prepared from AlOOH, and reacts for 9 hours at 160 ℃, the HMF conversion rate is more than 99%, and the BHMF yield also reaches 89.09%. The heterogeneous catalyst has higher catalytic activity and stability due to the stability of zero-valent copper in the catalyst and the stability of catalyst nanoparticles (Molecular Catalysis,2019,467, 52-60). Yanfu Ma et al adopt surfactant-assisted coprecipitation method/hydrothermal crystallization method to prepare doped ZrO2ZrO doped with rare earth metal2When the supported Co is used as a catalyst to prepare BHMF by selective hydrogenation of HMF in water, the conversion rate and the selectivity of alcohol both reach 100 percent. In the preparation process of the carrier, the addition of the surfactant improves the strong metal-carrier interaction, and the co-dispersion and stability are promoted. In the recycle test, the catalyst remained stable and the amount of deactivation was small (ACS Catalysis,2018,8(2), 1268-. However, the chemical reaction requiresHigher temperature and pressure are required, and the problems of poor product selectivity and poor substrate tolerance exist at the same time; the requirements on the air tightness, the material and the like of a reaction device are high; and the noble metal catalyst used in the catalytic process is expensive; because hydrogen has high dispersibility and inflammability, the potential safety hazard of preparing 2, 5-dimethylolfuran by hydrogenating 5-hydroxymethylfurfural by taking hydrogen as a hydrogen donor is large, and the hydrogen mainly comes from fossil resources at present, so that the hydrogen production cost is high.
The biological catalyst can be used for converting and generating the BHMF by taking the HMF as a substrate under mild reaction conditions, has high yield and high selectivity, and simultaneously keeps high chemical purity and low by-product. However, since 5-hydroxymethylfurfural has toxic effects on microbial cells, most microbes have low substrate tolerance concentration on HMF and low catalytic conversion rate of HMF. Therefore, the screening of the obtained microorganism which can tolerate high-concentration HMF, has high conversion speed and good selectivity is particularly important for establishing a biological catalysis path for synthesizing BHMF by taking HMF as a raw material.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide an aureobasidium pullulans strain which has high tolerance to a substrate HMF and can efficiently catalyze selective reduction of the HMF to synthesize BHMF.
The technical scheme of the invention is as follows:
an Aureobasidium pullulans strain is Aureobasidium pullulans (Aureobasidium basilicale) F134 which is preserved in China Center for Type Culture Collection (CCTCC) for short, the preservation number is CCTCC No. M2019476, the preservation date is 2019, 6 and 20 days, and the preservation address is Wuhan university in Wuhan, China.
The aureobasidium pullulans strain F134 is separated and screened from a radiation-polluted soil sample in a Xinjiang nuclear explosion test area. Through microorganism classification and identification, the strain is determined to belong to a new species of Aureobasidium, and the strain has strong tolerance to radiation and heavy metals.
The application of the aureobasidium pullulans strain F134 in the synthesis of 2, 5-dihydroxymethylfuran. The application method comprises the following steps:
(1) activating the aureobasidium pullulans strain F134 in a PDA culture medium, inoculating the activated aureobasidium pullulans strain F134 into a fresh PDA liquid culture medium according to the inoculation amount of 1 percent for culture, and collecting bacterial cells;
(2) adding 5-hydroxymethylfurfural into a buffer solution to prepare a 5-hydroxymethylfurfural solution with the concentration of 100-200 mM, adding the bacterial cells obtained in the step (1) into the 5-hydroxymethylfurfural solution, and reacting at the temperature of 15-45 ℃ and the pH of 5.0-10.0 to obtain 2, 5-dihydroxymethylfuran.
In the step (1), the PDA liquid culture medium formula is as follows: 200g of potato, 20g of glucose and 1000ml of distilled water.
Further, in the step (1), the activation conditions are as follows: activating at 28 ℃ for 36h at 200 r/min.
Further, in the step (1), the culture conditions are as follows: culturing at 30 deg.C and 200r/min for 72 h.
Further, in the step (2), the buffer solution is any one of a phosphate buffer solution, a Tris-HCl buffer solution or a glycine-NaOH buffer solution, and the pH value of the buffer solution is 5.0-10.0.
Further, in the step (2), the dosage of the bacterial cells is 20-400 mg/mL.
The reaction formula is as follows:
the invention has the beneficial effects that: compared with the prior art, the method has the following advantages:
1) the method utilizes the aureobasidium pullulans F134 as the catalyst, can efficiently and selectively catalyze the HMF to be converted into the target product BHMF, and overcomes the defect that a chemical catalyst is not environment-friendly.
2) The biological catalyst aureobasidium pullulans F134 used in the invention has high tolerance to HMF, can catalyze high-concentration substrate (180mM) to selectively reduce and synthesize a target product, and the yield reaches 70.4%. Compared with the reported biocatalysis process, the method has the advantages of higher substrate concentration, more excellent reaction efficiency and better selectivity.
3) The method has the advantages of simple reaction process, no need of adding a culture medium (the addition of the culture medium can make a reaction system more complicated), easy control, mild condition and contribution to simplifying the subsequent separation and purification process of the target product.
4) Aureobasidium pullulans F134 has obvious tolerance to extreme environments such as gamma rays, UV rays, salts, heavy metals and the like, and therefore, can not be limited by industrial production conditions.
Drawings
FIG. 1 is a colony morphology diagram of Aureobasidium pullulans strain F134 after being cultured on PDA solid medium for 72 h;
FIG. 2 is a liquid chromatogram of the synthesized product obtained after 5 hours of reaction in example 1.
Detailed Description
The invention is further described with reference to the following figures and specific embodiments. The present embodiments are to be considered as illustrative and not restrictive, and the spirit and scope of the invention is not to be limited to the details and modifications thereof.
The biological material Aureobasidium pullulans (Aureobasidium pullulans) F134 used in the embodiment is sourced from the institute of microorganism application of academy of agricultural sciences in Xinjiang, and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC No. M2019476, the preservation date of 2019, 6 and 20 days, and the preservation address of Wuhan university in Wuhan, China.
Example 1
Activation and culture of aureobasidium pullulans strain F134: inoculating aureobasidium pullulans strain F134 into a PDA liquid culture medium (200 g of potato, 20g of glucose and 1000ml of distilled water), and activating at 28 ℃ and 200r/min for 36 h; then, the cells were inoculated into a fresh PDA medium at an inoculum size of 1%, cultured at 30 ℃ and 200r/min for 72 hours, and the cells were collected. The morphological diagram of the colony of the aureobasidium pullulans strain F134 after being cultured on the PDA solid medium for 72 hours is shown in figure 1.
0.25mmoL 5-Hydroxymethylfurfural (HMF) is added into 2.5mL phosphate buffer (100mM, pH 7.4) to prepare a HMF solution with the concentration of 100mM, then somatic cells are added according to the concentration of 200mg/mL (based on the wet weight of the cells), the reaction is carried out at 15 ℃ and 850r/min, the reaction is monitored by liquid chromatography, after 5 hours, the conversion rate of the HMF is 40.93%, the selectivity of the BHMF product is 90.56%, and the liquid chromatogram is shown in figure 2, so that the retention time of the BHMF and the retention time of the HMF are 5.326min and 6.293min respectively.
Wherein, the method and conditions of the liquid chromatography detection are respectively as follows: the instrument is Thermo Fisher ultimate 3000, and the detector is a UV detector; the detection wavelength is 230 nm; the chromatographic column is Sepax GP-C18 column (4.6mm multiplied by 250mm,5 μm); the mobile phase is A, 20mM KH2PO 4; b, 100 percent acetonitrile; gradient elution (0min: 10% B; 7min: 24% B; 10min: 10% B); the flow rate was 1.0 mL/min-1(ii) a The column temperature was 25 ℃; the amount of sample was 5. mu.L.
Example 2
The activation and culture of the strain A. aureobasidium F134 were the same as in example 1.
0.25mmoL 5-Hydroxymethylfurfural (HMF) was added to 2.5mL of phosphate buffer (100mM, pH 7.4) to prepare a 100mM HMF solution, and then bacterial cells were added at a concentration of 200mg/mL (based on wet weight of cells) to react at 25 ℃ and 850r/min, and the reaction was monitored by liquid chromatography, and after 5 hours, the conversion of HMF was 83.15% and the selectivity of BHMF as a product was 97.29%.
Example 3
The activation and culture of the strain A. aureobasidium F134 were the same as in example 1.
0.25mmoL 5-Hydroxymethylfurfural (HMF) is added into 2.5mL phosphate buffer solution (100mM, pH 6.0) to prepare a HMF solution with the concentration of 100mM, then somatic cells are added according to the concentration of 200mg/mL (based on the wet weight of the cells), the reaction is carried out at 30 ℃ and 850r/min, the reaction is monitored by liquid chromatography, after 5 hours, the conversion rate of the HMF is 55.88 percent, and the selectivity of the BHMF product is 94.65 percent.
Example 4
The activation and culture of the strain A. aureobasidium F134 were the same as in example 1.
0.25mmoL 5-Hydroxymethylfurfural (HMF) was added to 2.5mL of phosphate buffer (100mM, pH 7.4) to prepare a 100mM HMF solution, and then bacterial cells were added at a concentration of 200mg/mL (based on wet weight of cells) to react at 45 ℃ and 850r/min, and the reaction was monitored by liquid chromatography, and after 5 hours, the conversion of HMF was 14.82% and the selectivity of BHMF as a product was 72.76%.
Example 5
The activation and culture of the strain A. aureobasidium F134 were the same as in example 1.
0.25mmoL 5-Hydroxymethylfurfural (HMF) was added to 2.5mL of phosphate buffer (100mM, pH 7.0) to prepare a 100mM HMF solution, and then bacterial cells were added at a concentration of 200mg/mL (based on wet weight of cells) to react at 30 ℃ and 850r/min, and the reaction was monitored by liquid chromatography, and after 5 hours, the conversion of 5HMF was 69.39% and the selectivity of BHMF as a product was 97.56%.
Example 6
The activation and culture of the strain A. aureobasidium F134 were the same as in example 1.
0.25mmoL 5-Hydroxymethylfurfural (HMF) is added into 2.5mL Tris-HCl buffer (50mM, pH 8.0) to prepare a HMF solution with the concentration of 100mM, then somatic cells are added according to the concentration of 200mg/mL (based on the wet weight of the cells), the reaction is monitored by liquid chromatography at 30 ℃ and 850r/min, and after 5 hours, the conversion rate of the HMF is 40.89% and the selectivity of the BHMF product is 91.69%.
Example 7
The activation and culture of the strain A. aureobasidium F134 were the same as in example 1.
0.25mmoL 5-Hydroxymethylfurfural (HMF) was added to 2.5mL of Gly-NaOH buffer (50mM, pH 9.0) to prepare a 100mM HMF solution, and then bacterial cells were added at a concentration of 200mg/mL (based on the wet weight of the cells) to react at 30 ℃ and 850r/min, and the reaction was monitored by liquid chromatography, and after 5 hours, the conversion of HMF was 64.30% and the selectivity of BHMF was 97.77%.
Example 8
The activation and culture of the strain A. aureobasidium F134 were the same as in example 1.
0.25mmoL 5-Hydroxymethylfurfural (HMF) was added to 2.5mL of phosphate buffer (100mM, pH 7.4) to prepare a 100mM HMF solution, and then bacterial cells were added at a concentration of 40mg/mL (based on wet weight of cells) to conduct a reaction at 30 ℃ and 850r/min, and the reaction was monitored by liquid chromatography, and after 12 hours, the conversion of HMF was 29.55% and the selectivity of BHMF as a product was 90.75%.
Example 9
The activation and culture of the strain A. aureobasidium F134 were the same as in example 1.
0.25mmoL 5-Hydroxymethylfurfural (HMF) is added into 2.5mL phosphate buffer solution (100mM, pH 7.4) to prepare HMF solution with the concentration of 100mM, then somatic cells are added according to the concentration of 80mg/mL (based on the wet weight of the cells), the reaction is carried out at 30 ℃ and 850r/min, the reaction is monitored by liquid chromatography, after 12 hours, the conversion rate of the HMF is 83.88 percent, and the selectivity of the BHMF product is 96.32 percent.
Example 10
The activation and culture of the strain A. aureobasidium F134 were the same as in example 1.
0.25mmoL 5-Hydroxymethylfurfural (HMF) was added to 2.5mL of phosphate buffer (100mM, pH 7.4) to prepare a 100mM HMF solution, and then bacterial cells were added at a concentration of 200mg/mL (based on wet weight of cells) to perform a reaction at 30 ℃ and 850r/min, and the reaction was monitored by liquid chromatography, and after 12 hours, the conversion of HMF was 99.63% and the selectivity of BHMF as a product was 93.21%.
Example 11
The activation and culture of the strain A. aureobasidium F134 were the same as in example 1.
0.375mmoL 5-Hydroxymethylfurfural (HMF) was added to 2.5mL of phosphate buffer (100mM, pH 7.4) to prepare a HMF solution with a concentration of 150mM, then bacterial cells were added at a concentration of 200mg/mL (based on wet weight of cells) and reacted at 30 ℃ and 850r/min, the reaction was monitored by liquid chromatography, and after 12 hours, the conversion of HMF was 62.60% and the selectivity of BHMF product was 93.99%.
Example 12
The activation and culture of the strain A. aureobasidium F134 were the same as in example 1.
0.5mmoL 5-Hydroxymethylfurfural (HMF) is added into 2.5mL phosphate buffer solution (100mM, pH 7.4) to prepare 200mM HMF solution, then somatic cells are added according to the concentration of 400mg/mL (based on the wet weight of the cells), the reaction is carried out at 30 ℃ and 850r/min, the reaction is monitored by liquid chromatography, after 12 hours, the conversion rate of the HMF is 55.33%, the selectivity of the BHMF product is 97.59%, after 36 hours, the conversion rate of the HMF is 61.66%, and the selectivity of the BHMF product is 96.13%.
Claims (6)
1. An application of an Aureobasidium pullulans strain in the synthesis of 2, 5-dihydroxymethylfuran is Aureobasidium pullulans F134 which is preserved in China center for type culture collection (CCTCC for short), the preservation number is CCTCC No. M2019476, the preservation date is 2019, 6 and 20 days, and the preservation address is Wuhan university in Wuhan, China.
2. Use according to claim 1, characterized in that the method of application comprises the steps of:
(1) activating the aureobasidium pullulans strain F134 in a PDA culture medium, inoculating the activated aureobasidium pullulans strain F134 into a fresh PDA liquid culture medium according to the inoculation amount of 1 percent for culture, and collecting bacterial cells;
(2) adding 5-hydroxymethylfurfural into a buffer solution to prepare a 5-hydroxymethylfurfural solution with the concentration of 100-200 mM, adding the bacterial cells obtained in the step (1) into the 5-hydroxymethylfurfural solution, and reacting at the temperature of 15-45 ℃ and the pH of 5.0-10.0 to obtain 2, 5-dihydroxymethylfuran.
3. The use according to claim 2, wherein in step (1), the activation conditions are: activating at 28 ℃ for 36h at 200 r/min.
4. The use of claim 2, wherein in step (1), the culture conditions are: culturing at 30 deg.C and 200r/min for 72 h.
5. The use of claim 2, wherein in the step (2), the buffer solution is any one of phosphate buffer solution, Tris-HCl buffer solution or glycine-NaOH buffer solution, and the pH of the buffer solution is 5.0-10.0.
6. The use according to claims 2 to 5, wherein in the step (2), the dosage of the bacterial cells is 20-400 mg/mL.
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