Preparation method for synthesizing 2, 5-furandimethanol from biomass saccharides through one-pot method based on aqueous phase catalysis system

Abstract

The invention discloses a preparation method for synthesizing 2, 5-furandimethanol (BHMF) from biomass saccharides through a one-pot method based on a water-phase catalysis system, and belongs to the technical field of chemical synthesis. According to the method, in a single water phase reaction system, efficient catalytic conversion is realized through two key steps of integrating biomass sugar dehydration to generate 5-hydroxymethylfurfural (HMF) and biologically reducing HMF to convert into BHMF. Wherein in the dehydration reaction stage, an acid catalyst is adopted, and a polyethylene glycol additive is introduced, so that the yield of HMF is remarkably increased, the generation of by-products levulinic acid and formic acid is effectively inhibited, and the toxic influence of the by-products on thalli in the subsequent reduction stage is reduced; meanwhile, due to the excellent biocompatibility, the inhibition effect on the thallus activity in the whole-cell catalytic reduction process is avoided. The innovative design ensures the continuity and system compatibility of the whole catalytic process, and realizes an efficient and stable catalytic conversion process.

Description

Technical Field

[0001] This invention relates to the field of chemical synthesis technology, specifically to a one-pot method for synthesizing 2,5-furandiethanol from biomass sugars based on an aqueous catalytic system. Background Technology

[0002] Biomass, as a carbon-containing renewable resource, can replace fossil fuels in the production of chemicals. Among these, 5-hydroxymethylfurfural (HMF), produced by the dehydration of hexose, is a core platform compound. Its furan ring, aldehyde group, and hydroxymethyl group endow it with high reactivity, enabling the derivatization of various high-value chemicals. 2,5-furandiethanol (BHMF), obtained by reducing HMF, is a multifunctional diol that can be used to synthesize pharmaceutical intermediates, crown ethers, and other fine chemicals, as well as serving as a monomer for biodegradable polyesters and polyamides.

[0003] Currently, BHMF production mainly relies on HMF reduction, but the high cost of HMF limits its large-scale application. A two-step process (hexose-HMF-BHMF) using inexpensive hexose as a raw material is more economical: the first step dehydrates hexose to HMF via chemical catalysis, and the second step reduces HMF via chemical or biocatalysis to achieve the final preparation of BHMF. Chemical catalysis requires noble metal (Pt / Pd, etc.) or non-noble metal (Cu / Ni, etc.) catalysts and a high-pressure hydrogen environment, posing problems such as high safety risks and high costs. Biocatalysis, on the other hand, has gained widespread attention in recent years due to its advantages of mild reaction conditions, high catalytic efficiency, and environmental friendliness. Biocatalysis typically utilizes whole microbial cells as catalysts, with intracellular cofactors replacing exogenous hydrogen as hydrogen donors, offering advantages such as mild conditions, high efficiency, and environmental friendliness.

[0004] The core challenge of the two-step method lies in the incompatibility of solvent systems. Specifically, in traditional cascade reactions, the dehydration step usually requires the use of non-aqueous media such as organic solvents or ionic liquids. This is because an aqueous environment generates excess hydrogen protons, triggering side reactions and producing more byproducts that inhibit whole-cell catalysts. However, the use of these non-aqueous solvents themselves can also significantly inhibit the activity of subsequent biocatalysts, forming a dual contradiction between solvent and catalyst. To mitigate the inhibitory effect of non-aqueous solvents, existing solutions mainly fall into two categories: one is to separate the non-aqueous solvent through methods such as distillation, but this increases operational complexity, leading to intermediate product loss and increased energy consumption; the other is to reduce the concentration of the non-aqueous solvent by diluting the reaction solution. For example, the AlCl3 / DMSO catalytic system reported in the literature "In-situ chemobiocatalytic conversion of glucose into 2,5-bis(hydroxymethyl)furan by AlCl3 / Enterobacter cloacae in biocompatible DMSO-water solvent" achieves dehydration in DMSO at 130℃, and then the reaction solution is diluted. Using *Enterobacter cloacae* HA188 as a whole-cell catalyst, in-situ catalysis of HMF to BHMF is performed in a 20% (v / v) DMSO solvent at 30℃. While dilution does alleviate inhibition to some extent, the inhibitory effect of non-aqueous solvents on microbial cells is quite strong. Even after dilution, the residual solvent still weakens the cellular catalytic activity, resulting in a final BHMF yield of only 47.2% for this method.

[0005] Therefore, the key to realizing the one-pot chemical-biocatalytic method for preparing BHMF lies in overcoming the bottleneck of solvent compatibility. It is necessary to design a novel reaction system that can maintain efficient dehydration reaction (avoiding the generation of byproducts) and protect cell activity, thereby eliminating the economic and efficiency losses caused by solvent separation or dilution, and promoting the development of the process towards green, low-consumption, and high-yield directions. Summary of the Invention

[0006] The purpose of this invention is to address the problems existing in the prior art and provide a one-pot method for synthesizing 2,5-furandimethyl from biomass sugars based on an aqueous phase catalytic system. This method achieves highly efficient catalytic conversion by integrating two key steps in a single aqueous phase reaction system: acidic dehydration of biomass sugars to generate HMF and neutral bioreduction to BHMF. Specifically, a polyethylene glycol (PEG) adjuvant is introduced during the dehydration reaction stage. This adjuvant significantly improves the HMF yield and effectively inhibits the formation of byproducts levulinic acid (LA) and formic acid (FA), reducing their toxic effects on the cells in the subsequent reduction stage. Furthermore, its excellent biocompatibility avoids the inhibitory effect on cell activity during whole-cell catalytic reduction. This innovative design ensures the continuity and system compatibility of the entire catalytic process, achieving a highly efficient and stable catalytic conversion.

[0007] To achieve the above-mentioned technical objectives, the present invention is implemented through the following technical solution: a one-pot synthesis method of 2,5-furandiethanol from biomass sugars based on an aqueous phase catalytic system, comprising the following steps:

[0008] 1) Using biomass sugar as raw material, 5-hydroxymethylfurfural is generated by dehydration reaction in water with acidic catalyst and polyethylene glycol auxiliaries;

[0009] 2) After adjusting the reaction solution obtained in step 1) to neutral, add a phosphate buffer system and a whole-cell biocatalyst, and use sugars as a co-substrate to carry out a reduction reaction to generate 2,5-furandiethanol.

[0010] Furthermore, the biomass sugar is selected from fructose, or at least one of inulin, sucrose and other biomass raw materials containing fructose units that can release fructose through hydrolysis, with fructose being preferred; the mass-volume ratio of biomass sugar to water is 0.2-0.6 g:1 mL.

[0011] Furthermore, the acidic catalyst is hydrochloric acid, sulfuric acid, phosphoric acid, or acetic acid; the molar ratio of the acidic catalyst to the biomass sugar is 0.25 to 2:1.

[0012] Furthermore, the dehydration reaction temperature is 75–95℃ and the reaction time is 0.5–4h.

[0013] Further, the polyethylene glycol additive is PEG-200, PEG-600, PEG-1000, PEG-2000 or PEG-4000, and the mass-volume ratio of the polyethylene glycol additive to water is 0.2-2.5 g:1 mL.

[0014] Preferably, the biomass sugar is fructose, and the mass-to-volume ratio of fructose to water is 0.2 g: 1 mL; the acidic catalyst is hydrochloric acid, and the molar ratio of hydrochloric acid to fructose is 0.5:1; the polyethylene glycol auxiliary agent is PEG-1000, and the concentration of PEG-1000 is 60% (w / v); the dehydration reaction temperature is 95°C, and the reaction time is 2 h.

[0015] Furthermore, the whole-cell biocatalyst used was Enterobacter cloacae HA188, which was deposited at the Guangdong Provincial Microbial Culture Collection Center on June 30, 2023, with the accession number GDMCC 1.4161. The final concentration of this bacterium in the reaction solution was 20–140 mg / mL, preferably 80 mg / mL.

[0016] Furthermore, the volume ratio of phosphate buffer to the reaction solution obtained in step 1) is 2 to 4:1, preferably 3:1.

[0017] Furthermore, the sugar used as a co-substrate is selected from at least one of glucose and fructose, with glucose being preferred; the final concentration of glucose in the reaction solution is 60-140 mM, and preferably, the glucose concentration is 100 mM.

[0018] Furthermore, the reduction reaction temperature is 20–35°C and the reaction time is 0.5–12 h; preferably, the reduction reaction temperature is 35°C and the reaction time is 6 h.

[0019] The beneficial effects of this invention are as follows:

[0020] 1. This application innovatively develops a one-pot continuous catalytic system in an aqueous phase. By adjusting the reaction environment in situ with phosphate buffer (pH 8.0±0.2), it efficiently integrates two key steps—acidic dehydration of fructose (generating HMF) and neutral bioreduction (converting to BHMF)—in a single aqueous phase. This effectively controls the excessive generation of byproducts and avoids the fatal impact of organic solvents on the microorganisms, achieving highly efficient synergy in the reaction process. This technology eliminates the multiphase separation or non-aqueous phase dilution steps in traditional processes, significantly reducing intermediate product loss and high energy consumption. The unique cascade reaction system design simultaneously solves the dual technical challenges of excessive aqueous dehydration byproducts inhibiting microbial activity during the reduction stage and organic solvents toxic to microorganisms. The integrated process achieves a BHMF yield of up to 0.71 (based on fructose moles) and a BHMF production intensity of 10.1 mmol*L. -1 *h -1 This provides an industrially feasible solution for the green and continuous production of bio-based platform compounds;

[0021] 2. This application uses water as a solvent system to achieve the efficient conversion of hexose to HMF under the action of an acidic catalyst. By introducing polyethylene glycol (PEG) as an auxiliary agent, PEG can form hydrogen bonds with water molecules, significantly binding water molecules and creating a localized dehydration microenvironment around the reactants (fructose, HMF, and intermediates). The reduction in free water concentration effectively inhibits side reaction pathways (such as HMF repolymerization or degradation), resulting in a significant increase in HMF yield.

[0022] 3. Compared with traditional organic solvents (such as DMSO) or non-aqueous solvents such as ionic liquids, PEG has excellent biocompatibility. While maintaining the integrity of microbial cell membranes, it effectively avoids the inhibition of cell activity in whole-cell catalytic reduction reactions (such as BHMF synthesis). More importantly, PEG can significantly inhibit the formation rate of by-products (LA and FA) in the dehydration reaction stage, thereby reducing the toxic effects of these by-products on the cells in the subsequent reduction stage. This dual protection mechanism not only ensures the smooth progress of each reaction step, but also achieves the organic compatibility and continuous optimization of the two-step reaction system.

[0023] 4. This application innovatively employs a composite catalytic scheme of hydrochloric acid and Enterobacter cloacae cells in a two-step reaction system. Hydrochloric acid, as the catalyst in the first stage, has the significant advantages of readily available raw materials and low cost. Meanwhile, Enterobacter cloacae cells, with their easy cultivation characteristics and the function of natural cofactors within the cells as hydrogen donors, achieve a mild and safe reduction reaction process in the second stage. The synergistic application of this dual-catalyst system not only significantly reduces the overall reaction cost, but also constructs an efficient, economical, and environmentally friendly reaction pathway through the complementary advantages of bio-chemical catalysts. Attached Figure Description

[0024] Figure 1 The effects of adding PEG-1000 on the conversion of substrate fructose, and the yields of products HMF, LA, and FA are shown in Figure A, which represents the effect without the addition of PEG-1000, and Figure B represents the effect with the addition of PEG-1000.

[0025] Figure 2 This is a chemical flow chart of the one-pot synthesis of BHMF using fructose as a raw material in an aqueous system.

[0026] Figure 3 The effect of relevant components in crude HMF solution on the conversion of HMF to BHMF catalyzed by Enterobacter cloacae HA188;

[0027] Figure 4These are high-performance liquid chromatograms of HMF conversion products catalyzed by whole cells. Sub-figure A is the spectrum before the reaction, sub-figure B is the spectrum after the reaction, sub-figure C is the spectrum of the negative control group using inactivated bacterial cells as catalyst, and sub-figure D is the spectrum of HMF and BHMF standards.

[0028] Figure 5 It is the 1H NMR spectrum of the purified HMF conversion product catalyzed by whole cell. Detailed Implementation

[0029] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Any modifications and substitutions made to the methods, steps, or conditions of the present invention without departing from the essence of the invention are within the scope of the present invention.

[0030] Example 1

[0031] This embodiment discloses a method for preparing 5-hydroxymethylfurfural (HMF) by aqueous-phase catalytic conversion of biomass sugars. The specific process is as follows:

[0032] Fructose (0.5 g, 2.77 mmol), water (2.5 mL), concentrated hydrochloric acid (1.38 mmol), and polyethylene glycol 1000 (PEG-1000, solid at room temperature, 1.7 g, 40% w / v)) were added to a glass test tube. The tube was sealed and reacted in a 95°C oil bath for 2 hours, yielding a total volume of 4.2 mL. The reaction was terminated by placing the test tube in an ice-water bath. An appropriate amount of the reaction solution was diluted, filtered, and the concentration of the product 5-hydroxymethylfurfural (HMF) was quantitatively analyzed by high-performance liquid chromatography (HPLC). The yield of HMF was calculated to be 0.69%.

[0033] Examples 2-23

[0034] Compared to Example 1, Examples 2-23 adjusted the molecular weight and amount of PEG, the type and amount of acidic catalyst, the oil bath reaction temperature, and the reaction time. The reaction parameters were systematically optimized (specific parameters are shown in Table 1), and the influence of each factor on the HMF yield was quantitatively analyzed. (Note: PEG-200 and PEG-600 are liquids at room temperature, and their volume fraction after addition is 40% (v / v); PEG-2000 and PEG-4000 are solids at room temperature, and their mass fraction after addition is 40% (w / v)).

[0035] Table 1. Optimized combinations of experimental parameters and corresponding HMF yields (Examples 2-23)

[0036]

[0037] Table 1 shows that the conversion of fructose to HMF in the aqueous phase is significantly affected by the molecular weight and amount of PEG, the type and dosage of acidic catalyst, reaction temperature, and time. In Example 8, when the PEG-1000 content was 60% (w / v), the HMF molar yield reached 0.80; further increasing the PEG-1000 content to 70% (w / v) increased the HMF molar yield to 0.82 (Example 9). Considering that the increase in HMF yield from 60% to 70% (w / v) of PEG-1000 content is limited, and that excessively high PEG-1000 concentration would inhibit the biocatalytic reaction in the second step, the preferred reaction conditions are as in Example 8: 2.77 mmol of fructose dissolved in 2.5 mL of water, 1.38 mmol of hydrochloric acid added as a catalyst, 3.7 g of PEG-1000 added as an adjuvant, reacted in an oil bath at 95°C for 2 h, with a mixed liquid volume of 6.1 mL, achieving an HMF molar yield of 0.80.

[0038] Comparative Example 1

[0039] The difference between Comparative Example 1 and Example 8 (preferred reaction conditions) is that no polyethylene glycol additive was added during the dehydration reaction stage. All other parameters were completely consistent with those of Example 8. After the reaction was terminated, the concentrations of substrate fructose, product HMF, and byproducts levulinic acid (LA) and formic acid (FA) were determined by HPLC, and the fructose conversion rate and the yield of each product were calculated to verify the regulatory effect of PEG-1000 on the reaction selectivity.

[0040] Figure 1 The graph shows the differences in fructose conversion, HMF yield, and byproduct LA and FA yields with and without PEG-1000 (Example 8). As can be seen from the graph, the addition of PEG-1000 increased the maximum HMF yield from 0.32 (sub-figure A) to 0.80 (sub-figure B), and the maximum fructose conversion from 0.59 (sub-figure A) to 0.98 (sub-figure B). Simultaneously, PEG-1000 effectively inhibited the formation of byproducts LA (decreasing from a maximum of 0.19 to a maximum of 0.11) and FA (decreasing from a maximum of 0.20 to a maximum of 0.05), confirming that PEG-1000 has a dual function of promoting conversion and inhibiting side reactions.

[0041] Example 24

[0042] Based on the optimal parameter system for fructose dehydration to prepare HMF established in Examples 1-23 (HMF yield reaching 0.80%), this example innovatively introduces *Enterobacter cloacae* HA188 as a biocatalyst to achieve in-situ reduction of crude HMF to 2,5-dihydroxymethylfuran (BHMF) at pH 8.0 and 35℃, constructing an aqueous one-pot continuous conversion process. The chemical flow is referenced from [reference missing]. Figure 2 .

[0043] The *Enterobacter cloacae* HA188 was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on June 30, 2023, with accession number GDMCC 1.4161.

[0044] 1) Expanding culture of Enterobacter cloacae HA188: The -80℃ glycerol-preserved bacterial strain was streaked onto YPD solid medium (20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract, 20 g / L agar) and incubated at 30℃ for 24 h. Single colonies were picked and inoculated into 5 mL of YPD liquid medium (20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract) and cultured at 30℃ with shaking at 200 rpm for 12 h. Then, the cells were transferred to YPD liquid medium at a 5% inoculum (v / v) and cultured under the same conditions until OD600≈2.0 (about 12 h). The cells were collected by centrifugation, washed twice with phosphate buffer, and the cells were collected.

[0045] 2) Evaluation of the tolerance of Enterobacter cloacae HA188 to key components of the biomass sugar conversion system:

[0046] Control group: HMF (100mM) and glucose (100mM) were added to Tris-HCl buffer (50mM, pH 8.0), and then E. cloacae HA188 cells (80mg / mL wet weight) were added.

[0047] Experimental group: The tolerance of Enterobacter cloacae HA188 to relevant components in crude fructose-converted HMF was investigated. The reaction solution was the control group with the addition of PEG-1000 (5, 10, 15, 20% (w / v)), NaCl (50, 100 mM), formic acid (5, 10 mM), levulinic acid (5, 10 mM), humic acid (0.5, 1.5 g / L) and a mixed inhibitor (PEG (15%, w / v) + NaCl (50 mM) + levulinic acid (5 mM) + formic acid (5 mM) + humic acid (1.5 g / L)).

[0048] A total of 14 combination schemes were used in the control and experimental groups. All reactions were carried out at 35℃ and 200 rpm for 6 hours with stirring. After the reaction, the supernatant was collected by centrifugation, and the formation of BHMF was detected by HPLC. The tolerance of whole cells to key components of the biomass sugar conversion system was evaluated by measuring the catalytic activity of whole cells under different conditions. The relative activity was defined as: Relative activity = (BHMF yield in experimental group) / (BHMF yield in control group) * 100.

[0049] When examining the tolerance of the strain, it is essential to ensure that the substrate HMF does not inhibit the catalytic activity of the strain. Our previous study (Sustainable Chemistry and Pharmacy, 2024, 42, 101813) showed that E. cloacae HA188 can efficiently reduce 100 mM HMF to BHMF, with a BHMF yield of 0.98. Therefore, in the control group, we set the substrate HMF concentration to 100 mM. In the preferred reaction conditions (Example 8), the initial concentration of the substrate fructose was 454 mM, and after 2 hours of reaction, the HMF concentration was 363 mM (yield 0.80). Therefore, diluting the crude HMF solution from Example 8 four-fold with phosphate buffer to a concentration of 91 mM ensured that the substrate HMF would not inhibit the catalytic activity of the strain.

[0050] Secondly, under the preferred reaction conditions, the crude HMF solution contains substances such as PEG-1000, FA, LA, NaCl, and humin, which may inhibit the whole-cell reduction of HMF. The concentrations of HMF, FA, and LA in the crude solution are 363, 7, and 18 mM, respectively. 1.38 mmol of HCl was used, and 1.38 mmol of NaOH was used to adjust the pH to neutral, generating 1.38 mmol of NaCl, resulting in a NaCl concentration of 226 mM in the reaction solution. Therefore, the concentrations of HMF, PEG-1000, FA, LA, and NaCl in the crude solution after a 4-fold dilution are 91 mM, 15% (w / v), 2 mM, 5 mM, and 57 mM, respectively. The concentration of humin could not be precisely measured, but it was estimated to be 0.5–1.5 g / L.

[0051] like Figure 3As shown, corresponding concentrations of NaCl, FA, LA, and humic acid did not significantly inhibit the catalytic reduction of HMF to BHMF by E. cloacae HA188. E. cloacae HA188 could tolerate 10% (w / v) PEG-1000 with a relative activity of 100%. However, when the PEG-1000 content increased to 15% and 20% (w / v), the relative activity of E. cloacae HA188 decreased to 91% and 82%, respectively. When the concentration of the mixed inhibitors simulated a 4-fold dilution of crude HMF, the relative activity of E. cloacae HA188 was 88%. These experiments confirm that high concentrations of PEG-1000 are the main inhibitor in diluted crude HMF. When 15% (w / v) PEG-1000 and other inhibitors were present simultaneously, E. cloacae HA188 maintained 88% of its biocatalytic activity. Therefore, it is feasible for E. cloacae HA188 to reduce crude HMF to BHMF.

[0052] 3) One-pot preparation of BHMF

[0053] Dehydration reaction stage: Fructose (0.5g, 2.77mmol), water (2.5mL), concentrated hydrochloric acid (1.38mmol) and 3.7g PEG-1000 (60%, w / v) were added to a glass test tube, sealed, and reacted in an oil bath at 95℃ for 2h to complete the conversion of HMF. The volume of the mixed liquid was 6.1mL, the HMF concentration was 363mM, and the yield was 0.80%.

[0054] Reduction stage: 1.38 mmol NaOH was added to the reaction solution to adjust the pH to 7.0 ± 0.2. Then, 3 volumes (18.3 mL) of phosphate buffer (50 mM, pH 8.0), *Enterobacter cloacae* HA188 wet bacterial cells (final concentration 80 mg / mL), and glucose (final concentration 100 mM) were added. The mixture was stirred at 35 °C and 200 rpm for 6 h. After the reaction, the supernatant was collected by centrifugation. The concentration of the target product BHMF was 81 mM, and the BHMF yield was 0.89 (based on the number of moles of HMF). The one-pot BHMF yield was 0.71 (based on the number of moles of fructose), and the BHMF production intensity reached 10.1 mmol / L. -1 *h -1 .

[0055] Product characterization: (1) High performance liquid chromatography (HPLC) analysis: After the reaction was completed, the reaction solution was appropriately diluted and filtered with a needle filter containing a 0.22 μm aqueous membrane. The filtrate was analyzed by HPLC. The analysis conditions were: Eclipse XDB-C18 column (4.6×250 mm, 5 μm, Agilent, USA) and DAD detector, mobile phase was 4 g / L (NH4)2SO4 solution (pH adjusted to 3.5 with phosphoric acid): acetonitrile = 90:10 (v / v), flow rate was 0.6 mL / min, column temperature was 30 ℃, and the maximum absorption wavelengths of HMF and BHMF were 283 nm and 223 nm, respectively. (2) Nuclear magnetic resonance (NMR) 1 ¹H NMR analysis: After the reaction was completed, the reaction solution was freeze-dried, the solid was resuspended in methanol, the precipitate was removed by filtration, the filtrate was distilled under reduced pressure, the obtained solid was reconstituted with water, filtered through a 0.22 μm aqueous membrane, and the filtrate was purified by preparative chromatography (NS4330, Hanbang Technology, China) with a C18 column. The initial flow rate was 0.5% acetic acid:acetonitrile = 98:2 (v / v), the flow rate was 2 mL / min, the detection wavelength was 223 nm, and after 5 min of injection, the acetonitrile ratio was gradually increased to 50% over 30 min. When the absorption peak appeared, the eluent was collected, the collected sample was distilled under reduced pressure to remove acetonitrile, and then freeze-dried to obtain a solid. After dissolving in deuterated water, NMR analysis was performed (Bruker AVANCE AV400, USA).

[0056] Figure 4 The HPLC chromatograms of the HMF conversion products catalyzed by whole cells are shown in sub-figures A and B. As can be seen, E. cloacae HA188 cells can efficiently convert HMF (10.7 min) to BHMF (8.7 min). The retention times of HMF and BHMF in the reaction solution are completely consistent with those of the standard (sub-figure D). The negative control group used inactivated bacterial cells as a catalyst, and HMF was not converted after the reaction (sub-figure C).

[0057] Figure 5 The purified product of whole-cell catalytic HMF conversion 1 The 1H NMR spectrum shows peaks with chemical shifts of 4.55 ppm and 6.35 ppm corresponding to hydrogen atoms on the hydroxymethyl and furan rings, respectively, and a peak with a chemical shift of 4.75 ppm representing deuterated water. 1 The H NMR spectrum is consistent with the BHMF structure.

[0058] Comprehensive HPLC and 1 The H NMR characterization results confirm that Enterobacter cloacae HA188 converts HMF to BHMF.

[0059] Examples 25-48

[0060] The difference between Examples 25-48 and Example 24 is that the co-substrate, pH, cell dosage, and reaction temperature of the whole-cell catalytic conversion of crude HMF to BHMF by E. cloacae HA188 were adjusted. The reaction parameters were optimized systematically (see Table 2 for specific parameters), and the influence of each factor on the yield of BHMF (in terms of the number of moles of HMF) was quantitatively analyzed.

[0061] Table 2. Optimized combinations of experimental parameters and corresponding BHMF yields (Examples 25-48)

[0062]

[0063]

[0064] As shown in Table 2, the optimal conditions for the conversion of crude HMF to BHMF by whole-cell catalysis of E. cloacae HA188 were as follows: in phosphate buffer (50 mM, pH 8.0), the substrate HMF concentration was 91 mM, and E. cloacae HA188 cells (80 mg / mL) and glucose (100 mM) were added. The reaction was carried out at 35 °C and 200 rpm for 6 h with stirring. The concentration of the product BHMF was 81 mM, and the yield of BHMF was 0.89 (based on the number of moles of HMF).

[0065] Examples 49-50

[0066] The difference between Examples 49 and 50 and Example 24 is that the substrate (biomass sugar) for the dehydration preparation of HMF was replaced with inulin and sucrose, respectively, instead of fructose. All other conditions remained unchanged. The specific process is as follows:

[0067] 0.5 g of inulin or sucrose was dissolved in 2.5 mL of water. 1.38 mmol of hydrochloric acid was added as a catalyst, and 3.7 g of PEG-1000 (60% w / v) was added as an adjuvant. The mixture was reacted in an oil bath at 95 °C for 2 h, resulting in a final volume of 6.1 mL. The reaction was terminated by placing the test tube in an ice-water bath. 1.38 mmol of NaOH was added, followed by 3 times the volume (18.3 mL) of phosphate buffer (50 mM, pH 8.0), wet E. cloacae HA188 bacterial cells (final concentration 80 mg / mL), and glucose (final concentration 100 mM). The mixture was stirred at 35 °C and 200 rpm for 6 h. The experimental results are shown in Table 3.

[0068] Table 3. Results of one-pot preparation of BHMF using inulin and sucrose as substrates (Examples 49-50)

[0069]

[0070] Data from Examples 49-50 and Example 24 confirm that the one-pot method for preparing BHMF using this technique yields the best results when fructose is used as the substrate.

[0071] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. However, the above description is merely a specific embodiment of the present invention, and the technical features of the present invention are not limited thereto. Any other embodiments derived by those skilled in the art without departing from the technical solution of the present invention should be covered within the scope of the present invention.

Claims

1. A method for the one-pot synthesis of 2,5-furandiethanol from biomass sugars based on an aqueous phase catalytic system, characterized in that, Includes the following steps: 1) Using biomass sugar as raw material, 5-hydroxymethylfurfural is generated by dehydration reaction in water with acidic catalyst and polyethylene glycol auxiliaries; 2) After adjusting the pH of the reaction solution obtained in step 1) to neutral, add phosphate buffer and whole-cell biocatalyst, and use sugars as co-substrate to carry out a reduction reaction to generate 2,5-furandiethanol.

2. The method for preparing 2,5-furandiethanol by one-pot synthesis of biomass sugars based on an aqueous phase catalytic system as described in claim 1, characterized in that, The biomass sugar is selected from fructose, or at least one of inulin, sucrose, and other biomass raw materials containing fructose units that can release fructose through hydrolysis; the mass-volume ratio of biomass sugar to water is 0.2-0.6 g:1 mL.

3. The method for preparing 2,5-furandiethanol by one-pot synthesis of biomass sugars based on an aqueous phase catalytic system as described in claim 1, characterized in that, The acidic catalyst is hydrochloric acid, sulfuric acid, phosphoric acid, or acetic acid; the molar ratio of the acidic catalyst to the biomass sugar is 0.25 to 2:

1.

4. The method for preparing 2,5-furandiethanol by one-pot synthesis of biomass sugars based on an aqueous phase catalytic system as described in claim 1, characterized in that, The dehydration reaction temperature is 75–95℃ and the reaction time is 0.5–4h.

5. The method for preparing 2,5-furandiethanol by one-pot synthesis of biomass sugars based on an aqueous phase catalytic system as described in claim 1, characterized in that, The polyethylene glycol additive is PEG-200, PEG-600, PEG-1000, PEG-2000 or PEG-4000, and the mass-to-volume ratio of the polyethylene glycol additive to water is 0.2-2.5 g:1 mL.

6. The method for preparing 2,5-furandiethanol from biomass sugars in a one-pot process based on an aqueous phase catalytic system as described in any one of claims 1-5, characterized in that, The biomass sugar is fructose, and the mass-to-volume ratio of fructose to water is 0.2 g: 1 mL; the acidic catalyst is hydrochloric acid, and the molar ratio of hydrochloric acid to fructose is 0.5:1; the polyethylene glycol auxiliary is PEG-1000, and the concentration of PEG-1000 is 60% (w / v); the dehydration reaction temperature is 95℃ and the reaction time is 2 h.

7. The method for preparing 2,5-furandiethanol by one-pot synthesis of biomass sugars based on an aqueous phase catalytic system as described in claim 1, characterized in that, The whole-cell biocatalyst used was Enterobacter cloacae HA188, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 30, 2023, with the accession number GDMCC1.4161. The final concentration of the bacteria in the reaction solution was 20–140 mg / mL.

8. The method for preparing 2,5-furandiethanol by one-pot synthesis of biomass sugars based on an aqueous phase catalytic system as described in claim 1, characterized in that, The volume ratio of phosphate buffer to the reaction solution obtained in step 1) is 2 to 4:

1.

9. The method for preparing 2,5-furandiethanol by one-pot synthesis of biomass sugars based on an aqueous phase catalytic system as described in claim 1, characterized in that, The sugar used as a co-substrate is selected from at least one of glucose and fructose; the final concentration of the co-substrate in the reaction solution is 60–140 mM.

10. The method for preparing 2,5-furandiethanol by one-pot synthesis of biomass sugars based on an aqueous phase catalytic system as described in claim 1, characterized in that, The reduction reaction temperature is 20–35℃ and the reaction time is 0.5–12 h.

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