CN114606278A

CN114606278A - Method for catalytically synthesizing 2,5-furandicarboxylic acid by using methylobacterium

Info

Publication number: CN114606278A
Application number: CN202210137972.XA
Authority: CN
Inventors: 于浩然; 王伦; 刘克
Original assignee: ZJU Hangzhou Global Scientific and Technological Innovation Center
Current assignee: ZJU Hangzhou Global Scientific and Technological Innovation Center
Priority date: 2022-02-15
Filing date: 2022-02-15
Publication date: 2022-06-10
Anticipated expiration: 2042-02-15
Also published as: CN114606278B

Abstract

The invention discloses a method for catalytically synthesizing 2,5-furandicarboxylic acid by utilizing methylobacterium, which comprises the following steps: taking 5-hydroxymethylfurfural as a substrate, and taking methylobacterium extorquens (AM 1) as a whole-cell catalyst to react to obtain 2,5-furandicarboxylic acid; methylobacterium extorquens (Methylorus extorquens) AM1 was deposited with the American Standard Biolabs Collection with accession number ATCC 14718. The method for producing 2,5-furandicarboxylic acid can directly convert the substrate 5-hydroxymethylfurfural with whole cells to obtain the 2,5-furandicarboxylic acid, has the characteristics of simple operation, low cost, pollution reduction and the like, and has good industrialization prospect.

Description

Method for catalytically synthesizing 2,5-furandicarboxylic acid by using methylobacterium

Technical Field

The invention relates to the technical field of biological engineering, in particular to a method for catalytically synthesizing 2,5-furandicarboxylic acid by utilizing methylobacterium.

Background

2,5-furandicarboxylic acid (FDCA) is an important bio-based platform compound, the most potential use of which is as a substitute for terephthalic acid (PTA) for the synthesis of renewable polyethylene-2, 5-furandicarboxylate (PEF), and is widely used in the synthesis of renewable polyethylene-2, 5-furandicarboxylate (PEF)Producing various bio-based high molecular polymers (such as polyamide, polyester, polyurethane and the like). At present, chemical synthesis is mainly adopted, 5-Hydroxymethylfurfural (HMF) is generally used as a raw material, and noble metal catalyst is used for O reaction at high temperature and high pressure₂And oxidizing to prepare FDCA. The method mainly comprises two steps: firstly, oxidizing side chain aldehyde group of HMF to generate carboxyl to obtain 5-hydroxymethyl-2-furancarboxylic acid (HFCA), and then oxidizing side chain hydroxymethyl of HFCA to generate aldehyde group (FFCA) and FDCA. Although 2,5-furandicarboxylic acid is a high-value bio-based material, the existing chemical production process needs an organic solvent and high-concentration alkali, and has high cost and serious pollution.

The whole-cell biotransformation method has the advantages of mild process, good substrate selectivity, small environmental pollution and the like, and has great development prospect. However, since 5-hydroxymethylfurfural has a toxic effect on cells, only a very small number of microorganisms can naturally utilize 5-hydroxymethylfurfural to produce 2,5-furandicarboxylic acid, and it has been reported that marine fungi Caldariomyces fumago, ralstonia solani, engineered pseudomonas putida S12 and pseudomonas hophatii can produce 2,5-furandicarboxylic acid in whole cells.

At present, no report on the production of 2,5-furandicarboxylic acid using methylobacterium and similar one-carbon microorganisms using 5-hydroxymethylfurfural as a substrate has been found. On the premise of no modification and optimization, the methylobacterium can be used for producing the 2,5-furandicarboxylic acid by natural whole-cell catalysis, and the methylobacterium is cultured by using a total synthetic culture medium with methanol as a carbon source, so that the culture cost is simple, the methylobacterium is not easy to contaminate, the requirement on equipment is low, and the methylobacterium has great research and development values.

Disclosure of Invention

The invention provides a method for catalytically synthesizing 2,5-furandicarboxylic acid by utilizing methylobacterium, which adopts demethylating bacillus (Methylorum extorquens) AM1 as a whole-cell catalyst to catalyze substrate 5-hydroxymethylfurfural for the first time to obtain 2,5-furandicarboxylic acid, overcomes the problems that 5-hydroxymethylfurfural has toxic action on cells and the production path of 2,5-furandicarboxylic acid is few in the prior art, and provides a new whole-cell biotransformation path for the preparation of 2,5-furandicarboxylic acid.

The specific technical scheme is as follows:

a method for catalytically synthesizing 2,5-furandicarboxylic acid by using methylobacterium comprises the following steps: taking 5-hydroxymethylfurfural as a substrate, and taking methylobacterium extorquens (AM 1) as a whole-cell catalyst to react to obtain 2,5-furandicarboxylic acid;

methylobacterium extorquens (Methylorus extorquens) AM1 was deposited with the American Standard Biolabs Collection with accession number ATCC 14718.

The invention discovers for the first time that the demethylated bacillus (methylrudum extorquens) AM1 can be used as a whole-cell catalyst to catalyze and obtain 2,5-furandicarboxylic acid.

Further, the reaction in the method adopts a liquid phase reaction system; in the reaction system, the concentration of 5-hydroxymethylfurfural is 1-500 mM, and the content of Methylorudium extorquens (AM 1) is OD₆₀₀10-500. Further, the concentration of 5-hydroxymethylfurfural was 500mM, and the content of Methylorudium extorquens (M.extorquens) AM1 was OD₆₀₀＝80。

Further, in the reaction system, the pH value is 6-9.5; further, the pH was 9.0.

Further, in the reaction system, the reaction temperature is 15-40 ℃, and the reaction time is 1-72 hours; further, the reaction temperature was 30 ℃ and the reaction time was 48 hours.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention directly applies the methylobacterium whole-cell catalyst to the catalytic generation of 2,5-furandicarboxylic acid by taking 5-hydroxymethylfurfural as a substrate, and can convert 500mM of the substrate into 1mM of 2,5-furandicarboxylic acid within 48 hours.

(2) The method for producing 2,5-furandicarboxylic acid can directly convert the substrate 5-hydroxymethylfurfural with whole cells to obtain the 2,5-furandicarboxylic acid, has the characteristics of simple operation, low cost, pollution reduction and the like, and has good industrialization prospect.

Drawings

FIG. 1 is a schematic diagram of the principle of producing 2,5-furandicarboxylic acid by using a methylobacterium whole cell catalysis substrate 5-hydroxymethylfurfural.

FIG. 2 is a HPLC chromatogram of 2,5-furandicarboxylic acid standard.

FIG. 3 is an HPLC chromatogram of 2,5-furandicarboxylic acid in example 1.

Detailed Description

The present invention will be further described with reference to the following specific examples, which are only illustrative of the present invention, but the scope of the present invention is not limited thereto. The present invention does not relate to the preservation of strains.

The experimental procedures in the following examples, unless otherwise specified, were carried out in a conventional manner according to the techniques or conditions described in the literature in this field or according to the product instructions. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. Unless otherwise stated, the quantitative experiments in the following examples were repeated three times and the results were averaged.

Example 1

Preparation of one-cell and whole-cell catalyst

Methylorudium extorquens AM1 is deposited in the American Type Culture Collection (ATCC) with the accession number ATCC 14718.

The schematic diagram of the 2,5-furandicarboxylic acid catalyzed by the whole cells of the methylobacterium is shown in figure 1, so that the high energy consumption and high pollution of the traditional chemical method are reduced, the process steps of enzyme purification and refining by the biological enzyme method are reduced, and the dependence on capital, energy consumption, equipment and environment is reduced.

The preparation method comprises the following steps:

1. activating demethylating bacillus methylorusextorquens AM1 on a plane culture medium, and culturing at 30 ℃ for 48 hours;

2. and (3) inoculating the single colony obtained in the step (1) into 5mL of liquid synthetic medium, and carrying out shaking culture at 30 ℃ and 220rpm for 36 hours to obtain a bacterial liquid. Each 1L of the culture medium contains: the carbon source is 0.1-1% by volume of methanol, 15-60 mM of piperazine-1, 4-diethylsulfonic acid, 0.7-3 mM of dipotassium phosphate, 1-4 mM of sodium dihydrogen phosphate, 0.2-1 mM of magnesium chloride, 3-20 mM of ammonium sulfate, 0.5-3 mu M of zinc sulfate, 0.5-3 mu M of manganese chloride, 0.5-40 mu M of ferrous sulfate, 0.5-5 mu M of ammonium molybdate, 0.2-3 mu M of copper sulfate, 0.5-5 mu M of cobalt chloride, 0.1-1 mu M of sodium tungstate, 0.1-100 mu M of calcium and rare earth ions, and/or the balance of water with pH of 6.8-7.2;

3. inoculating 4mL of the culture medium of the bacterial liquid obtained in the step 2 into 200mL of the liquid synthetic culture medium, and performing shaking culture at 30 ℃ and 220rpm until the OD600 nm value reaches 2;

4. and (3) after the step 3 is finished, centrifuging the whole fermentation system at room temperature and 4000rpm for 20 minutes, and collecting cell precipitates to obtain the whole-cell catalyst.

Two, whole cell catalysis

5. 1g (wet weight) of the whole-cell catalyst obtained in step 4 was taken, resuspended in 4mL of a buffer pH9.0100 mM Tris-HCl buffer, 5-hydroxymethylfurfural was added to the suspension at a final concentration of 500mM, and the mixture was reacted at 30 ℃ for 48 hours with shaking at 220 rpm.

Thirdly, detecting the yield of the 2,5-furandicarboxylic acid

6. After completion of step 5, centrifugation was carried out at 12000rpm for 2min at room temperature, and the supernatant was collected, followed by filtration through a 0.22 μm filter and collection of the filtrate, and 2,5-furandicarboxylic acid in the filtrate was detected by HPLC.

HPLC parameters:

a chromatographic column: aminex HPX-87H column (9 μm, 7.8X 300 mm);

mobile phase: 0.005M H₂SO₄；

Flow rate of mobile phase: 0.6 mL/min;

column temperature: 40 ℃;

sample injection amount: 20 mu L of the solution;

detection wavelength: 264 nm.

And (3) carrying out HPLC detection on the 2,5-furandicarboxylic acid standard substance according to the parameters, wherein the retention time is 17.948 min.

And (3) carrying out HPLC detection on the filtrate to obtain the peak area of a target peak, and calculating based on a standard curve to obtain the content of the 2,5-furandicarboxylic acid in the filtrate to be 1 mM. The chromatogram of the filtrate on HPLC is shown in FIG. 3.

The present invention has been described in detail, but the present application is not limited to the above embodiments and application fields, and the above embodiments are only illustrative and instructive, and are not restrictive. Those skilled in the art, having the benefit of this disclosure, may effect numerous modifications thereto and changes may be made without departing from the scope of the invention as defined by the appended claims.

Claims

1. A method for catalytically synthesizing 2,5-furandicarboxylic acid by using methylobacterium is characterized by comprising the following steps: taking 5-hydroxymethylfurfural as a substrate, and taking methylobacterium extorquens (AM 1) as a whole-cell catalyst to react to obtain 2,5-furandicarboxylic acid;

2. The process for catalytically synthesizing 2,5-furandicarboxylic acid by using Methylobacterium according to claim 1, wherein the reaction employs a liquid phase reaction system; in the reaction system, the content of 5-hydroxymethylfurfural is 1-500 mM, and the content of Methylorudium extorquens (AM 1) is OD₆₀₀＝10～500。

3. The method for catalytic synthesis of 2,5-furandicarboxylic acid using methylobacterium according to claim 2, wherein the pH in the reaction system is 6 to 9.5.

4. The method for catalytic synthesis of 2,5-furandicarboxylic acid by using methylobacterium according to claim 2, wherein the reaction temperature is 15 to 40 ℃ and the reaction time is 1 to 72 hours in the reaction system.