Method for producing 2,5-furandicarboxylic acid

Abstract

The invention relates to a method for producing 2,5-furandicarboxylic acid by oxidizing 5-formyl-2-furancarboxylic acid, which is present in an aqueous solution, by treatment with an aldehyde dehydrogenase in vitro to 2,5-furandicarboxylic acid, wherein the NAD(P)H formed during the oxidation is enzymatically oxidized back to NAD(P)+ by means of a dehydrogenase and D-fructose or acetone, after which the enzymes are removed.

Description

The next day, expression cultures with an optical density ODsso of 0.02 were inoculated and shaken at 37 °C until an ODsso of 0.3 was reached. The temperature was then lowered to 25 °C, and the cultures were induced with 0.1 mM IPTG upon reaching an OD550 of 0.5. After 22 h, the cultures were harvested (separated from the medium by centrifugation to form a cell pellet) and analyzed for expression of the recombinant enzyme using SDS-gel electrophoresis and an activity assay (use in a use test or an optical-enzymatic assay). Preparation of cell lysates by sonification For the preparation of the cell suspension, the cell pellet produced according to the above method was weighed into a suitable vessel and mixed with buffer and lysozyme (final concentration 0.5 mg / mL) (e.g., triethanolamine (TEA)-HCl) and dissolved under stirring. The mass fraction of biomass is typically 20%; the remainder consists of the buffer. A Branson Sonifier 450 was used for cell lysis. The suspension was treated three times with 15 ultrasonic pulses each (device settings: Timer = 15; Duty Cycle = 50; Output Control = 3-5). The resulting homogenate was centrifuged for 10 minutes at 4 °C and 16,000 rpm (Eppendorf Centrifuge 5417R) to separate the insoluble cell fragments and obtain the lysate. Table 1. Enzyme types and donor organisms for the enzymes used in the examples (ALDH = aldehyde dehydrogenase). Enzyme type (EC class) Catalyzed reaction Donor organism References ALDH I FFA^ FDCA Pseudomonas nitroreducens (NCBI Protein Database: WP_024766379.1); SEQ ID No. 2 ALDH II * FFA^ FDCA Saccharolobus soifataricus (NCBI Protein Database: WP_009990943.1); SEQ ID No. 4 ALDH III FFA^ FDCA Methylovorus glucosotrophus (NCBI Protein Database: WP_015829138.1); SEQ ID No. 6 Xylitol dehydrogenase (XDH) D-fructose -> D-sorbitol Galactocandida mastotermitis (Candida sp. HA167) (Habenicht et al., 1999); SEQ ID No. 8 Xylose reductase I D-glucose-^ D-sorbitol Rasamsonia emersonii (NCBI Protein Database: ACR78268.1); SEQ ID No. 12 Xylose reductase II L-Arabinose-^ L-Arabitol Kluyveromyces marxianus DMKU3-1042 (NCBI Protein Database: XP_022674194.1); SEQ ID No. 14 Alcohol dehydrogenase I (ADH I) Acetone^ 2-propanol (Geo-)Bacillus stearothermophilus NCA1503 (Sakoda & Imanaka, 1992); SEQ ID No. 10 Alcohol dehydrogenase II (ADH II) Dihydroxyacetone^ , glycerin Thermoanaerobacter brockii (NCBI Protein Database: CAA46053.1); SEQ ID No. 16 Mannitol dehydrogenase D-fructose ^ D-mannitol Rasamsonia emersonii SEQ ID No. 18 *This ALDH is classified in the NCBI Protein Database (entry WP_009990943.1) as 2,5-dioxopentanoate dehydrogenase (catalyzes the oxidation of 2,5-dioxopentanoate to a-ketoglutarate). Analytical Methods High-Performance Liquid Chromatography (HPLC) HPLC (High-Performance Liquid Chromatography) was used to quantify FFA and FDCA. Detection is performed using a UV detector. A Phenomenex Rezex ROA-Organic Acid H+ (8%) column with a corresponding precolumn is used for the measurement and eluted isocratically with 1 mM sulfuric acid. High-Performance Liquid Chromatography (HPLC) was used to quantify D-fructose and D-sorbitol. Detection is performed using a refractive index detector. A Phenomenex Rezex RCM-Monosaccharide Ca2+ column with a corresponding precolumn is used for the measurement, and the sample is eluted isocratically with 3.5% isopropanol. Determination of Enzyme Activities (Optical-Enzymatic Assay) Enzyme activities in the lysates were determined using a Shimadzu UV-1900 spectrophotometer. Therefore, the formation or consumption of NAD(P)H was monitored at a wavelength of 340 nm by measuring changes in absorbance. The measurements were performed using 0.2 mM cofactor (NAD(P)’or NAD(P)H). For this purpose, 20 ^l of a 10 mM stock solution of the cofactor was placed in a cuvette (Greiner bio-one semi-micro cuvette made of polystyrene), and the desired pH was adjusted with 100 mM TEA-HCl buffer (870 ^l). 10 ^l of lysate (diluted or undiluted) and 100 ^l of substrate solution were added to the cuvette, and the measurement was started immediately thereafter. The measurements were performed at 25 °C as standard. Using the extinction coefficient of NADH / NADPH at 340 nm (£ = 6220 L mol-1 cm-1) can be used to determine the enzyme activity of the lysate in U / ml (relative to the volume of the lysate), or U / g (relative to the biomass used for production). Here, 1 U represents 1 ^mol of substrate turnover per minute (1 U = 1 ^mol / min = 1.67 x 10-8 kat). The following examples describe preferred embodiments of the method according to the invention in greater detail. The lysates used in these examples were prepared according to the methods described above. Example 1 Oxidation of 5-formyl-2-furan carboxylic acid to 2,5-furan dicarboxylic acid - cofactor regeneration using XDH and D-fructose The reaction was carried out in a BioXplorer benchtop bioreactor with a Polyblock (H.E.L.). A stainless-steel reactor (max. volume 400 mL) equipped with a stirrer and a pH electrode was used as the vessel. pH control was achieved by adding 5 M NaOH or 1 M H2SO4. Initially, 5.5 g of FFA and 16 g of D-fructose (final concentration 600 mM) were added to 100 mL of a 100 mM potassium phosphate buffer (pH 7) and heated to 20 °C with stirring. Subsequently, 20 mL of ALDH I lysate, 13 mL of XDH lysate, and 3 mL of a 10 mM NAD+ solution (final concentration 0.2 mM) were added. The total volume of the reaction mixture was adjusted to 150 mL by adding deionized water. For analysis, 50 ^l of the mixture was combined with 200 ^l of acetonitrile and incubated in an Eppendorf Thermomixer at 85 °C and 1200 rpm for 15 min. The sample was briefly centrifuged in a centrifuge, mixed with 750 ^l of deionized water, vortexed, and then centrifuged for 5 min at max. g. 250 ^l of the supernatant was diluted in an HPLC vial with 750 ^l of an acetonitrile / water mixture (1 / 4 v / v) and analyzed by HPLC (UV detection). After 4 h, the FFA was completely oxidized to FDCA. For purification, the reactor contents were heated to 70 °C and stirred at this temperature for 1 h. After centrifuging out the denatured protein, the supernatant was filtered through a pleated filter. This yielded a clear solution, which was acidified to a pH < 2 using 10 mL of a 12 M H2SO4 solution. Upon cooling to 4 °C, a precipitate formed, which was filtered off. In this manner, 5.1 g of FDCA was isolated as a solid. Example 2 Oxidation of 5-formyl-2-furan carboxylic acid to 2,5-furan dicarboxylic acid - cofactor regeneration using ADH and acetone The reaction was carried out in a Labfors benchtop bioreactor (Infors AG). A glass reactor (volume 3.4 L) equipped with a stirrer and a pH electrode was used as the vessel. pH control was achieved by adding 5 M NaOH or 1 M H2SO4. Initially, 27.7 g of FFA was added to 550 ml of a 100 mM potassium phosphate buffer (pH 7) and heated to 20 °C with stirring. Subsequently, 67 mL of ALDH I lysate, 30 mL of ADH lysate, 10 mL of a 10 mM NAD+ solution (final concentration 0.2 mM), and 15 mL of acetone were added. Additionally, an overpressure of 320 mbar was applied. For analysis, 50 ^l of the sample was mixed with 200 ^l of acetonitrile and incubated in an Eppendorf Thermomixer at 85 °C and 1200 rpm for 15 min. The sample was briefly centrifuged in a centrifuge, mixed with 750 ^l of deionized water, vortexed, and then centrifuged for 5 min at max. g. 250 ^l of the supernatant was diluted in an HPLC vial with 750 ^l of an acetonitrile / water mixture (1 / 4 v / v) and analyzed by HPLC (UV detection). After 4.5 h, the FFA was completely oxidized to FDCA. FDCA can be isolated as a solid in the same manner as in Example 1. Example 3 Oxidation of 5-formyl-2-furan carboxylic acid to 2,5-furan dicarboxylic acid using aldehyde dehydrogenase II (ALDH II) and cofactor regeneration via XDH and D-fructose The following components were mixed in a glass vial: 300 ^l of an FFA solution (11.9 g / l), 10 ^l of deionized water, 50 ^l of ALDH II lysate, 50 ^l of a 1 M potassium phosphate buffer (pH 8), 50 ^l of a 1.5 M D-fructose solution, and 40 ^l of XDH lysate. The mixture was incubated with continuous shaking (Eppendorf Thermomixer; 20 °C, 800 rpm) for a total of 20 h. For analysis, 50 ^l of the mixture was combined with 200 ^l of acetonitrile and incubated in the Eppendorf Thermomixer at 85 °C and 1200 rpm for 15 min. The sample was briefly centrifuged in a centrifuge, mixed with 750 ^l of deionized water, vortexed, and then centrifuged for 5 min at max. g. 200 ^l of the supernatant was transferred to an HPLC vial with an insert and analyzed by HPLC (UV detection). In this manner, 40.8% of the FFA (7.1 g / l) was oxidized to FDCA. Example 4 Oxidation of 5-formyl-2-furan carboxylic acid to 2,5-furan dicarboxylic acid using aldehyde dehydrogenase III (ALDH III) and cofactor regeneration via ADH and acetone The following components were mixed in a glass vial: 5.2 mg FFA, 325 ^l deionized water, 35 ^l ALDH III lysate, 100 ^l of a 500 mM potassium phosphate buffer (pH 8), 15 ^l of acetone, 30 ^l of ADH lysate, and 5 ^l of a 10 mM NAD+ solution. The mixture was incubated under continuous shaking (Eppendorf Thermomixer; 20 °C, 800 rpm) for a total of 24 h. For analysis, 50 ^l of the mixture was combined with 200 ^l of acetonitrile and incubated in the Eppendorf Thermomixer at 85 °C and 1200 rpm for 15 min. The sample was briefly centrifuged, mixed with 750 ^l of deionized water, vortexed, and then centrifuged for 5 min at max. g. 200 ^l of the supernatant was transferred to an HPLC vial with an insert and analyzed by HPLC (UV detection). In this manner, 48.0% of the FFA (10.4 g / l) was oxidized to FDCA. Example 5 Oxidation of 5-formyl-2-furan carboxylic acid to 2,5-furan dicarboxylic acid using aldehyde dehydrogenase II (ALDH II) and various dehydrogenases for cofactor regeneration The following components were mixed in 3 glass vials (vials 1-3): 300 ^l of an FFA solution (final concentration 7.1 g / l), 50 ^l of a 1 M potassium phosphate buffer (pH 8), 50 ^l of ALDH II lysate, 5 ^l of a 10 mM NADP+ solution, 50 ^l of dehydrogenase lysate (see Table 2 below), 50 ^l of a 1.5 M substrate solution (see Table 2 below), and 5 ^l of deionized water. The mixture was incubated under continuous shaking (Eppendorf Thermomixer; 20 °C, 800 rpm) for a total of 24 h. For analysis, 50 ^l of the sample was mixed with 200 ^l of acetonitrile and incubated in an Eppendorf Thermomixer at 85 °C and 1200 rpm for 15 min. The sample was briefly centrifuged in a centrifuge, mixed with 750 ^l of deionized water, vortexed, and then centrifuged for 5 min at max. g. 200 ^l of the supernatant was transferred to an HPLC vial with an insert and analyzed by HPLC (UV detection). The results are shown in Table 2 below. Table 2. Vial Dehydrogenase Substrate for dehydrogenase FDCA yield [%] 1 Xylose reductase I D-glucose 79 2 Mannitol dehydrogenase D-fructose 71 3 Xylose reductase II L-arabinose 84 The results in Table 2 show that different dehydrogenases are suitable for cofactor regeneration (in this case, NADP+) using different substrates. Example 6 Oxidation of 5-formyl-2-furan carboxylic acid to 2,5-furan dicarboxylic acid using aldehyde dehydrogenase II (ALDH II) and cofactor regeneration via ADH and dihydroxyacetone The following components were mixed in a glass vial: 143 ^l of an FFA solution (final concentration 12 g / l), 125 ^l of a 250 mM potassium phosphate buffer (pH 7), 10 ^l of ALDH II lysate, 10 ^l of a 5 mM NADP+ solution, 10 ^l of dehydrogenase lysate (see Table 2 below), 50 ^l of a 300 g / l dihydroxyacetone solution, and 152 ^l of deionized water. The mixture was incubated under continuous shaking (Eppendorf Thermomixer; 30 °C, 800 rpm) for a total of 20 h. For analysis, 50 ^l of the mixture was combined with 200 ^l of acetonitrile and incubated in the Eppendorf Thermomixer at 85 °C and 1200 rpm for 15 min. 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Claims

1. A method for the preparation of 2,5-furan dicarboxylic acid by oxidizing 5-formyl-2-furan carboxylic acid, which is present in an aqueous solution, to 2,5-furan dicarboxylic acid by treating with an aldehyde dehydrogenase in vitro, wherein the NAD(P)H generated during the oxidation is enzymatically reoxidized to NAD(P)+, by the means of a dehydrogenase and D-fructose or acetone, as substate, after which the enzymes are removed, characterized by the combination of the steps:that the NAD(P)+-dependent aldehyde dehydrogenase for the oxidation of 5-formyl-2-furan carboxylic acid to 2,5-furan dicarboxylic acid has an amino acid sequence selected from the group consisting of:i) an amino acid sequence having at least 80% identity to SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6,ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, andiii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5.and that, as the dehydrogenase that oxidizes NAD(P)H to NAD(P)+,a) a xylitol dehydrogenase is used, which has an amino acid sequence selected from the group consisting of:i) an amino acid sequence having at least 80% identity to SEQ ID NO: 8,ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO. 7, andiii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID NO: 7,wherein D-fructose is used as the substrateorb) a NAD(P)H-dependent alcohol dehydrogenase is used, which has an amino acid sequence selected from the group consisting of:i) an amino acid sequence having at least 80% identity to SEQ ID NO: 10,ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO: 9, andiii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID NO: 9,wherein acetone is used as the substrate.

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